key: cord-255946-bcuivyku authors: kulkarni, prashanth; kodad, shruthi; mahadevappa, manjappa title: covid‐19 and namaste date: 2020-04-21 journal: influenza other respir viruses doi: 10.1111/irv.12746 sha: doc_id: 255946 cord_uid: bcuivyku nan to the editor-in-chief the novel coronavirus (sarscov-2) has emerged as a major pandemic stretching the healthcare resources of most countries of the world. in this context, it is imperative that social distancing and good hand hygiene is practised to stem the transmission of this highly conalternatively, other non-physical greeting forms can be explored like namaste, which is used in indian subcontinent since hundreds of years to greet people with folded hands, while maintaining a fair distance from each other [ figure 1 ]. an individual in addition to saying "namaste" presses his hands together in front of the chest and respectfully greets the other person. this form of greeting does not involve any physical touch between individuals and gives a sense of parity to all the parties. 3 in addition to following general principles of meticulous hand washing, rapid transmission of infections both in hospitals and the community can be overcome by adopting the no-touch salutation namaste and other such forms like bowing the head as done in some asian countries. world health organization guidelines on hand hygiene in health care namaste or handshake: time to ponder key: cord-304473-6aivjett authors: tenforde, mark w.; feldstein, leora r.; lindsell, christopher j.; patel, manish m.; self, wesley h. title: exposures in adult outpatients with covid‐19 infection during early community transmission, tennessee date: 2020-08-04 journal: influenza other respir viruses doi: 10.1111/irv.12792 sha: doc_id: 304473 cord_uid: 6aivjett nan the peer review history for this article is available at https://publo ns.com/publo n/10.1111/irv.12792. although not systematically asked, nineteen respondents reported working at a local meat processing plant; of these, only 47% (9/19) reported having known close contact with a person infected with covid-19 before their illness. in this cross-sectional survey, we generated insights into individual-level exposures and behaviors among non-hospitalized adults with covid-19 in tennessee in the early days following national social distancing guidelines. notably, only half of respondents could identify a close contact with a covid-19 case in the two-week period before illness onset. transmission of sars-cov-2 is likely facilitated through a pre-symptomatic or pauci-symptomatic shedding period in infected individuals, 2,3 which, along with limited early testing, could in part explain this common lack of a known case contact and highlights the need for robust testing infrastructure. respondents reported engaging in practical social distancing behaviors such as avoiding large gatherings. however, a sizeable majority reported working regularly in the two weeks before illness with few able to telework, suggesting workplace-related exposures were an important early source of transmission in tennessee. as the united states returns to the workplace, these early findings underscore the need for ongoing workplace safety measures to prevent transmission. if teleworking is not feasible, workplaces should employ practices to reduce transmission including identifying where and how workers could be exposed, establishing organizational policies and practices for social distancing more than 6 feet, performing at-work symptom screening, testing, and contact tracing once an infected employee is identified, and providing workplace education. 4 case-control studies should be considered to better understand workplace-associated risks. the findings and conclusions of this report are those of the authors and do not necessarily reflect the official position of the centers conceptualization (equal); formal analysis (lead) investigation (equal) supervision (equal) leora r feldstein: conceptualization (equal); formal analysis (supporting) investigation (equal) supervision (equal) writing-review & editing (equal) data curation (equal) investigation (equal) resources (equal) manish m patel: conceptualization (equal) investigation (equal) resources (equal) supervision (equal) wesley h self: conceptualization (equal); funding acquisition (equal) investigation (equal) resources (equal) supervision (equal) writing-review & editing (equal) tenforde 1 lindsell 2 self 2 for the ivy network investigators cdc covid-19 response team cdc covid-19 response team mailstop h24-7 coronavirus guidelines for america temporal dynamics in viral shedding and transmissibility of covid-19 serial interval of covid-19 among publicly reported confirmed cases self wh; for the ivy network investigators, cdc covid-19 response team. exposures in adult outpatients with covid-19 infection during early community transmission key: cord-268105-617qcgpe authors: refaey, samir; amin, marwa mohamed; roguski, katherine; azziz‐baumgartner, eduardo; uyeki, timothy m.; labib, manal; kandeel, amr title: cross‐sectional survey and surveillance for influenza viruses and mers‐cov among egyptian pilgrims returning from hajj during 2012‐2015 date: 2016-11-11 journal: influenza other respir viruses doi: 10.1111/irv.12429 sha: doc_id: 268105 cord_uid: 617qcgpe background: approximately 80 000 egyptians participate in hajj pilgrimage annually. the purpose of this study was to estimate influenza virus and mers‐cov prevalence among egyptian pilgrims returning from hajj. study: a cross‐sectional survey among 3 364 returning egyptian pilgrims from 2012 to 2015 was conducted. nasopharyngeal (np) and oropharyngeal (op) swabs were collected from all participants. sputum specimens were collected from participants with respiratory symptoms and productive cough at the time of their interview. specimens were tested for influenza viruses, and a convenience sample of np/op specimens was tested for mers‐cov. thirty percent of participants met the case definition for influenza‐like illness (ili), 14% tested positive for influenza viruses, and none tested positive for mers‐cov. self‐reported influenza vaccination was 20%. conclusions: high prevalence of reported ili during pilgrimage and confirmed influenza virus on return from pilgrimage suggest a continued need for influenza prevention strategies for egyptian hajj pilgrims. an evaluation of the ministry of health and population's current risk communication campaigns to increase influenza vaccine use among pilgrims may help identify strategies to improve vaccine coverage. pre-departure vaccination against influenza a(h1n1)pmd09 for all pilgrims during the 2009 season. 9 since then, the mohp has required seasonal influenza vaccination for all pilgrims as part of the saudi visa application process. although the requirement is not always enforced, seasonal influenza vaccine is available at local health offices for all egyptian pilgrims throughout most of year. the mohp has additionally conducted an annual survey among pilgrims returning from hajj to explore the risk of influenza virus transmission to the broader community. following the emergence of mers-cov in saudi arabia, the mohp expanded the survey to test for mers-cov. a cross-sectional survey was conducted at cairo international airport among egyptians returning from hajj during the week following the end of hajj each year from 2012 to 2015 (table 1) . cairo airport was selected for this survey as it is the main point of entry into egypt for returning hajj pilgrims. 9 cairo airport receives 7-8 flights during working hours (9am-9 pm) from saudi arabia, accounting for approximately 1500-2000 pilgrims per day. a team from the mohp sought to enroll a convenience sample of approximately 10% of pilgrims from each flight returning from hajj and congregating at the airport carousels, regardless of age, sex, and illness status. after providing verbal consent, participants were asked about demographic information, respiratory symptoms, and whether they received vaccines as part of their hajj visa application process. both nasopharyngeal (np) and oropharyngeal (op) swabs were collected from all participants regardless of the presence of respiratory symptoms. sputum specimens were collected from participants who presented with respiratory symptoms and a productive cough at the time of their interview. travelers who reported a history of subjective fever (a proxy for measured fever) and cough with symptom onset in the previous 10 days were categorized as having influenza-like illness (ili). 10 for minors, consent and survey responses were provided by accompanying parents. specimens positive for influenza a were subsequently tested for influenza a virus subtypes. sputum specimens and a convenience sample of np/op specimens were tested for mers-cov according to who guidelines. 11 the proportion of samples testing positive for influenza virus from participants was compared to those collected from ili casepatients through the national surveillance system during the same time period. t a b l e 1 the distribution of egyptian pilgrims surveyed by season according to gender, age group, presence of influenza-like illness (ili), vaccination status and influenza laboratory test result <5 y 0 (0) 1 (0.1) 2 (0.2) 6 (0.6) 9 (0.3) 5-<15 y 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 15-<50 hajj: infectious disease surveillance and control respiratory tract infections during the annual hajj: potential risks and mitigation strategies hajj-associated viral respiratory infections: a systematic review middle east respiratory syndrome coronavirus (mers-cov) -saudi arabia risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah-a link to health care facilities state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans pandemic (h1n1) 2009 and hajj pilgrims who received predeparture vaccination who surveillance case definitions for ili and sari world health organization. laboratory testing for middle east respiratory syndrome coronavirus, interim recommendations high prevalence of common respiratory viruses and no evidence of middle east respiratory syndrome coronavirus in hajj pilgrims returning to ghana respiratory viruses and bacteria among pilgrims during the respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices -united states cross-sectional survey and surveillance for influenza viruses and mers-cov among egyptian pilgrims returning from hajj during 2012-2015 key: cord-304569-o39kl5k4 authors: nguyen-van-tam, jonathan s title: from the editor's desk date: 2015-04-23 journal: influenza other respir viruses doi: 10.1111/irv.12311 sha: doc_id: 304569 cord_uid: o39kl5k4 nan it is now just over 1 year since i took over the editorship of influenza and other respiratory viruses (irv). in that time, there have been many changes to the journal and, of course, in the wider world of respiratory viruses. i'd like to begin by thanking my predecessor and the journal's founding editor, dr. alan hampson. alan set up, nurtured and grew irv into a vibrant and successful publication before officially handing over the reins in early 2014. he remains on the team as one of our senior editors. indeed my task as editor-in-chief would be impossible without the fantastic and dedicated team of senior editors, associate editors and professional staff at wiley, who support me, and all of whom deserve many personal thanks. the challenges posed by respiratory viruses remain as important as ever for global public health, and clinical practice. in the aftermath of the 2009 pandemic, governments and public health agencies are wrestling with 'pandemic fatigue', austerity programmes (that limit the appetite and ability to invest in pandemic preparedness), and a false impression among some politicians that since the 2009 pandemic was rather mild and 'not much to write home about', pandemic preparedness is in fact 'no big deal'. in amongst this mix are real issues pertaining to the ongoing controversy about the effectiveness of antiviral drugs, [1] [2] [3] ; and the fact that current vaccine manufacturing platforms can only offer commercial quantities of pandemic vaccine some four to 6 months after a novel virus has emerged, thus substantially reducing the overall public health benefits, even though vaccines themselves are effective. 4, 5 most public health agencies and individual experts, consider the potential pandemic threat posed by influenza a(h5n1) to be undiminished. but in addition, influenza a (h7n9) is now, if anything, considered a higher potential risk. 6 recent human cases of influenza a(h10n8), 7 and a (h5n6), 8 further remind us that the risk assessment landscape for influenza is constantly evolving and, in turn, this demands constant vigilance from the public health and scientific communities. if one shifts the focus away from influenza, the ongoing mers-cov outbreak in the middle east, is also of substantial concern, because despite its likely introduction into humans via close contact with dromedary camels, 9 nosocomial transmission appears to be a central concern, 10,11 case-fatality is high, household transmission is also described, 12 and there are currently no vaccines or specific therapies available. finally enterovirus d68 seems to be emerging as a potentially important respiratory pathogen in children. 13 seasonal influenza too, should not be overlooked as an ongoing problem. in the northern hemisphere winter of 2014-15 we have experienced substantial influenza a(h3n2) activity. unfortunately this has coincided with a poorly matched h3n2 vaccine component that has resulted in low effectiveness against the circulating h3n2 strains in the community. 14 this has recently been linked to excess winter mortality across europe in the population aged 65 years and over, 15 illustrating that we need more broadly protective vaccines, especially in the elderly. from the journal's perspective, to be in the best possible position to respond to these emerging and sometimes fastmoving threats, we have made some recent changes that enable us to publish findings with minimal delay. for papers of significant importance, we can offer a rapid peer-review scheme, which should enable us to make a decision on a manuscript within 14 days (or less). we have also improved our arrangements for all accepted articles (whether fasttracked or not). following acceptance, we can now publish all articles, as unedited manuscripts, online within 1 week of acceptance. we have also reconsidered the arrangements for review articles that we publish. we recognise that systematic reviews and meta-analyses have become a central part of evidence synthesis in modern science and medicine. with this has come the setting of standards for the conduct of such work: the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines; 16,17 and we now require any systematic reviews that we accept to conform to these principles. however, we shall continue to accept expert commentary articles alongside as these continue to be relevant and useful to our readers. hopefully these changes, along with our now wellestablished open access format will maintain the journal as a vibrant publication that is relevant, and highly accessible, to scientists and clinicians working in the field of respiratory virus infection for the foreseeable future. authors have the option to request rapid peer-review. papers considered for rapid peer-review will need to be of immediate relevance, interest, or importance to scientists, clinicians, public health practitioners or policy makers, usually in relation to a current or evolving event related to respiratory virus activity. in general, papers that report data more than 6 months old are unlikely to be considered eligible. if your paper qualifies for rapid peer-review, the journal will aim to have your paper turned round within 14 days. there is no additional charge for authors with rapid peer-review. step #1 -authors will need to contact the editorial office irv.eo@wiley.com, at least 1 week before online submission with an abstract; list of authors; please ensure a prisma checklist is completed if your paper is a systematic review article; details of research funding and disclosures of potential conflicts of interest should also be included. step #2 -the editors will confirm whether your paper will qualify for rapid peer-review. the decision is final and non-negotiable. step #3 -submit the paper via the scholarone system. accepted articles have been accepted for publication and undergone full peer review but have not been through the copyediting, typesetting, pagination and proofreading process. accepted articles are published online a few days after final acceptance, appear in pdf format only, are given a digital object identifier (doi), which allows them to be cited and tracked, and are indexed by pubmed. effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza a h1n1pdm09 virus infection: a meta-analysis of individual participant data oseltamivir for influenza in adults and children: systematic review of clinical study reports and summary of regulatory comments oseltamivir treatment for influenza in adults: a meta-analysis of randomised controlled trials effectiveness of h1n1 vaccine for the prevention of pandemic influenza in scotland, uk: a retrospective observational cohort study effects of vaccine program against pandemic influenza a(h1n1) virus, united states pandemic influenza viruses--hoping for the road not taken avian influenza a h10n8--a virus on the verge? evidence for camel-to-human transmission of mers coronavirus mers-cov outbreak in jeddah -a link to health care facilities hospital outbreak of middle east respiratory syndrome coronavirus transmission of merscoronavirus in household contacts high frequency of enterovirus d68 in children hospitalised with respiratory illness in norway low effectiveness of seasonal influenza vaccine in preventing laboratory-confirmed influenza in primary care in the united kingdom: 2014/15 mid-season results excess mortality among the elderly in european countries preferred reporting items for systematic review and meta-analysis protocols (prisma-p) 2015 statement key: cord-273620-gn8g6suq authors: szczawinska‐poplonyk, aleksandra; jonczyk‐potoczna, katarzyna; breborowicz, anna; bartkowska‐sniatkowska, alicja; figlerowicz, magdalena title: fatal respiratory distress syndrome due to coronavirus infection in a child with severe combined immunodeficiency date: 2012-11-30 journal: influenza other respir viruses doi: 10.1111/irv.12059 sha: doc_id: 273620 cord_uid: gn8g6suq please cite this paper as: szczawinska‐poplonyk et al. (2012) fatal respiratory distress syndrome due to coronavirus infection in a child with severe combined immunodeficiency. influenza and other respiratory viruses doi: 10.1111/irv.12059. coronaviruses have been demonstrated to contribute substantially to respiratory tract infections among the child population. though infected children commonly present mild upper airway symptoms, in high‐risk patients with underlying conditions, particularly in immunocompromised children these pathogens may lead to severe lung infection and extrapulmonary disorders. in this paper, we provide the first report of the case of a 15‐month‐old child with severe combined immunodeficiency and coronavirus hku1‐related pneumonia with fatal respiratory distress syndrome. severe combined immunodeficiency (scid) is a genetically and clinically heterogeneous group of the most severe primary immunodeficiencies, characterized by the absence of functional t lymphocytes resulting in profound impairment of the cellular and humoral adaptive immunity. depending on the genetic defect, b lymphocytes and natural killer (nk) cells may be present or absent and this feature constitutes the basis for the classical division into t-b + scid and t-b)scid, with further subdivisions into nk+ and nk) disorders. 1 respiratory tract infection is a common manifestation in children in question and may be present within the neonatal period or in early infancy. opportunistic pathogens may lead to rapidly progressive, fatal interstitial pneumonitis accompanied by hyperinflation resulting from small airway obstruction or to persistent bronchiolitic presentation. apart from pyogenic bacteria, such as pseudomonas aeruginosa, stenotrophomonas spp, burkholderia spp, as well as mycobacteria and fungi, in particular pneumocystis jiroveci, respiratory viruses like respiratory syncytial virus (rsv), adenovirus, parainfluenza virus, human metapneumovirus (hmpv) and other viruses -cytomegalovirus (cmv), varicella-zoster virus (vzv), and epstein-barr virus (ebv) are associated with severe pneumonia in scid children. 2 human coronaviruses (hcov) hcov-229e and hcov-oc43 and related new strains hcov-nl63 and hcov-hku1, identified after the epidemic outbreak of severe acquired respiratory syndrome (sars) coronavirus, are likely to be common respiratory viruses in otherwise healthy children and were not implicated in severe lung infections in immunocompromised patients thus far. 3 in this report we present the case of a child with delayed-onset scid and fatal respiratory coronavirus infection. a 15-month-old girl was referred to the university hospital due to persistent fever and interstitial pneumonitis for the purposes of diagnosis and treatment. she was the first child of young, non-consanguineous parents, born from the first pregnancy which was terminated in the 39th week of gestation using cesarian section surgery because of condylomata acuminata due to human papilloma *the institution to which the work should be attributed. virus (hpv) infection in the mother. in early infancy, mild eczema on the child's face, in the perioral and periorbital areas was observed. by the end of the first year of life the child had thrived and had not suffered from any severe infections. she received live bcg vaccine, tetanus and diphtheria toxoids, inactivated pertussis, inactivated poliomyelitis, recombinant hepatitis b and conjugated pneumococcal as well as haemophilus influenzae b vaccines without adverse reactions after the immunizations. on admission the child demonstrated paroxysmal nonproductive cough and clinical signs of respiratory insufficiency along with injected oral pharyngeal mucosa and dry erythematous lips. fine papular skin eruption affecting the face was observed. neither peripheral lymph nodes nor internal organs of the abdominal cavity were palpable. during hospitalization, lymphopenia, anemia, and thrombocytosis (the peak platelet count was 778 · 10 9 ⁄ l) as well as increased levels of inflammatory markers and coagulopathy were found in laboratory tests. dilation of the right coronary artery was revealed in echocardiography, giving rise to the suspicion of kawasaki disease and the instituting of treatment with aspirin and immunoglobulins. despite intensive pharmacotherapy with antibiotics, trimethoprime ⁄ sulfamethoxazole, acyclovir and antifungal agents, rapid deterioration of the child's clinical state and the exacerbation of respiratory insufficiency accompanied by progression in radiological features of the respiratory distress syndrome (rds) occured. on a chest x-ray, massive alveolar and interstitial infiltrations with bilaterally decreased aeration of the lung fields and blurred borders of the diaphragm and the heart shape (as shown on figure 1 ) were discernible. differential diagnostics that included examinations of the tracheal aspirate samples aimed at infectious agents were carried out. infections with viruses -rsv a and b, parainfluenza viruses 1,2, and 3, influenza a including h1n1 subtype and b, adenoviruses, cmv, ebv, mpv, rhinovirus, human immunodeficiency virus (hiv), with bacteria, such as streptococcus pneumoniae, haemophilus influenzae, legionella, bordetella pertussis, mycoplasma pneumoniae, chlamydophila pneumoniae, as well as with fungi -candida spp. and p. jiroveci were excluded based on negative pcr examinations, whereas the human coronavirus hku1 -rna proved positive. peripheral blood lymphocyte flow cytometric immunophenotyping revealed a total lack of expression of antigens characteristic for cd4 and cd8 t-cell subsets as well as nk cells along with the presence of functionally immature transitional and naïve b cells. this scid phenotype was subsequently confirmed by bone marrow flow cytometric evaluation. however, the presence of few nk cells was revealed, indicating for t-b + nk + scid. intensive therapy and mechanical ventilation conducted in the department of pediatric anesthesiology and intensive care did not contribute to either clinical or radiological improvement and the child died because of multiorgan failure. analysis of the available data concerning the effects of non-sars human coronaviruses (hcov) in children suggests that their clinical relevance in children is substantial, particularly in the hospital settings, even though the incidence of hcov airway infections are generally less frequent than with other viruses which have an established role in respiratory disease, such as rsv and influenza. 4 however, detailed epidemiological data on the prevalence of hcov infections in children are discordant, ranging from 2ae5% of nl63 strain in young children with bronchiolitis reported by ebihara et al. 5 to 18% in the study by vabret et al. 6 in different age groups of the child population. moreover, seroconversion with regard to hcov-229e and above-mentioned hcov-nl63 in young children was much higher and was estimated to 42ae9-50% and 75%, respectively. 7 the characteristics of clinical manifestation of coronavirus respiratory tract infection are predominantly reliant on case reports, and in otherwise, healthy children are comparable with bronchitis, bronchiolitis, and pneumonia due to other viral infections. the epidemiological study by kuypers et al. 8 indicated that a considerable proportion of coronavirusinfected children had underlying chronic central nervous system, cardiovascular, pulmonary, allergic, and renal or hepatic conditions and diseases. these authors also paid attention to immunocompromised pediatric patients with acute lymphocytic leukemia and organ transplant recipients as a high-risk group for the development of severe lung disfigure 1 . the chest x-ray of a 15-month-old child with severe combined immunodeficiency and respiratory distress syndrome due to coronavirus hku1 infection. note the massive alveolar and interstitial infiltrations, with bilaterally decreased aeration of the lung fields and blurred borders of the diaphragm and the heart shape. ease. however, it is worth noting that coronavirus respiratory infections have not been described in children with genetically determined immunodeficiencies thus far and this is the first report of a documented hcov-hku1-related pneumonia with the rds in a child with scid. it is also interesting to note that the preliminary clinical diagnosis in this patient was kawasaki disease, what is consistent with the hypothesis by esper et al. 9 regarding the association between kawasaki disease with hcov infection, supported by identification of the 'new heaven' coronavirus (hcov-nh) in 72ae7% of respiratory specimen from affected children. the identification of hcov-hku1 provides a novel insight into the epidemiology and clinical implications of coronavirus infections in severely immunocompromised children and indicates for consideration of this pathogen-related etiology of respiratory infection in scid. further, epidemiological studies are necessary to define the impact of hcov on lung disease in children with immunodeficiencies. the expanding clinical and immunological spectrum of severe combined immunodeficiency pneumonia in normal and immunocompromised children: an overview and update the widening scope of coronaviruses effects of coronavirus infections in children detection of human coronavirus nl63 in young children with bronchiolitis human coronavirus nl63 seroepidemiology of group 1 human coronaviruses in children clinical disease in children associated with newly described coronavirus subtypes association between a novel human coronavirus and kawasaki disease the assistance of dr husam samara from the department of immunology for the flow cytometric immunophenotyping of peripheral blood and bone marrow leukocytes is acknowledged. the authors have no competing interests. key: cord-292946-iggv9f0k authors: principi, nicola; scala, alessia; daleno, cristina; esposito, susanna title: influenza c virus–associated community‐acquired pneumonia in children date: 2012-12-07 journal: influenza other respir viruses doi: 10.1111/irv.12062 sha: doc_id: 292946 cord_uid: iggv9f0k to evaluate the impact of influenza c (icv) infection in children with community‐acquired pneumonia (cap), all of the children consecutively seen during 4 influenza seasons with respiratory symptoms and radiographically confirmed cap were prospectively evaluated. icv was identified in the respiratory secretions of five of 391 patients (1·3%). in children with icv‐associated cap, clinical data were similar to those observed in children with iav‐associated cap and worse than those observed in children with ibv‐associated. the phylogenetic tree showed that the sequenced strains clustered in two of the six icv lineages. these findings highlight that icv can be a cause of cap of children and that this can be severe enough to require hospitalization. despite its widespread circulation, influenza c virus (icv) is traditionally considered a scarcely virulent infectious agent because, unlike influenza a (iav) and b viruses (ibv), it is thought to be associated with infections that, when symptomatic, are mild and mainly involve the upper respiratory tract. [1] [2] [3] however, the frequency and clinical picture of icvassociated pediatric community-acquired pneumonia (cap) have not been defined, nor which virus lineages more frequently cause the disease. this prospective study was designed to evaluate the incidence and clinical relevance of icv infection in children with radiographically confirmed cap aged <15 years. the study was approved by the institutional review board of the fondazione irccs ca' granda ospedale maggiore policlinico, milan, italy, and was carried out in pediatric clinic 1 of the university of milan between 1 november and 31 march of four consecutive winter seasons (2008-2009, 2009-2010, 2010-2011, and 2011-2012) . the written informed consent of a parent or legal guardian was required, and children older than 8 years were asked to give their assent. all of the otherwise healthy children aged between 1 month and 14 years seen in our emergency room because of fever and signs and symptoms of lower respiratory tract involvement whose chest radiograph was consistent with cap were considered eligible for the study. the chest radiographs were evaluated by an independent expert radiologist who classified the findings as alveolar, interstitial, or no pneumonia in accordance with the world health organization (who) criteria. 4 upon admission, respiratory secretions were taken from each child using a pernasal flocked swab and stored in a tube of utm-rt (kit cat. no. 360c, copan italia, brescia, italy). viral tests for the identification of influenza viruses were performed for study purposes and were always available after the patient's discharge. upon enrollment, detailed information regarding the patients' demographics, clinical history, and disease characteristics was collected, together with a blood sample for the evaluation of laboratory variables including white blood cell (wbc) counts, c-reactive protein (crp) and procalcitonin (pct) levels, and blood cultures. first drug treatment as well as decision on when to hospitalize was chosen by attending pediatrician in charge on the basis of the guidelines of the italian society of pediatrics. 5 none of the children was treated with antivirals. all of the enrolled children (whether they were hospitalized or sent home immediately after enrollment) were reevaluated 15 ae 2 days later by means of interviews and clinical examinations carried out by trained investigators using standardized questionnaires. each sample underwent real-time polymerase chain reaction (pcr) to identify iav (and its subtypes) and ibv as previously described. 6 icv was identified by amplifying the non-structural protein (ns) gene using the method described by matsuzaki et al. 7 the laboratory-confirmed iav-, ibvand icv-positive samples were then tested for coinfections with respiratory syncytial virus (rsv)-a and rsv-b, parainfluenza virus 1, 2, 3, and 4, adenovirus, human metapneumovirus (hmpv), coronaviruses 229e, nl63, oc43, and hku1, enterovirus/rhinovirus, and human bocavirus using the luminex x tag respiratory virus panel (rvp) fast assay (luminex molecular diagnostics inc., toronto, on, canada) in accordance with the manufacturer's instructions. 6 a segment of the hemagglutinin-esterase (he) gene was sequenced in the icv-positive samples to determine the lineages of the circulating viruses. one segment was amplified using two published primers. 8 the sequences identified in this study, together with previously published 7, 9, 10 representative sequences of each lineage downloaded from the genbank database, were aligned using clustalx v.2.0.12 11 and default parameters. a phylogenetic tree based on the nucleotide sequences was reconstructed as previously described. 12 the five nucleotide sequences identified in this study were submitted to the genbank database and assigned accession numbers jx129180, jx129181, jx129182, jx129183, and jx129184. the study involved 391 children with radiographically confirmed cap (180 males; mean age ae sd 2á9 ae table 1 summarizes the characteristics of the five children with icv infection: four aged <3 years, the fifth aged 14 years. no other virus was found in any of these cases. all of them were hospitalized but a significant improvement in clinical signs and symptoms of the disease took place in few days in all the patients. post-discharge check-ups showed that all of the children were completely cured, and none experienced any recurrence. these characteristics were similar to those observed in children with iav-associated cap and worse than those observed in children with ibvassociated cap (none of the ibv-positive cases was hospitalized) ( table 2) . the phylogenetic tree constructed using the sequences of the five icvs identified in this study, and previously described icvs showed that the five sequenced strains clustered in two of the six classic icv lineages (figure 1 to the best of our knowledge, this is the first study specifically designed to evaluate the importance of icv in pediatric cap. the collected data seem to indicate that icv can be considered a possible cause of cap in children, particularly in younger subjects. our results also suggest the need for further prospective studies that should be carried out throughout the year to provide conclusive results. previous studies have shown that icv infection in temperate zones does not have the marked seasonal nature of iav or ibv infections as it has been in patients with respiratory infections during winter (concomitantly with other influenza viruses) as well as during late spring and early summer. 1 it is therefore possible that the global importance of icv as a cause of cap in children is greater than that found in our study, which only involved the winter months. the greater frequency of icv than ibv in patients with respiratory infections has previously reported by calvo et al. 2 and ant on et al. in spain, 3 who studied children and the general population and analyzed subjects with any type of respiratory tract infection. the clinical characteristics of icvassociated cap were not different from those found in cases of iav-associated cap, but our iav-and icv-positive cases required hospitalization significantly more often than the cases of ibv-associated cap. however, the number of patients with influenza viruses is too small to allow comprehensive comparisons. similarities in the clinical pictures of diseases due to iav and icv have been found by other authors in studies evaluating the global clinical features of influenza virus disease. 1 however, ours are the first data exclusively regarding cap and, given the importance of this disease in younger children, merit consideration. we found that icv was a possible cause of cap only during the first 2 years of enrollment. this may have been due to its different circulation rates year by year or to the relatively small number of children with cap enrolled in the last 2 years of the study. as the incidence of icv-associated cap in the 2 years with positive cases was 1á3% and 2á3%, respectively, it is possible that enrolling fewer than 100 cases of cap per year could have led to negative results even if icv was circulating. phylogenetic analysis of the he gene of the icvs identified in this study revealed the simultaneous co-circulation of c/ kanagawa/1/76-and c/sao paulo/378/82-related lineages in the influenza seasons 2008-2009 and 2009-2010. as there are no data concerning the circulation of icv in italy during previous or subsequent years, it is not possible to establish cap, community-acquired pneumonia; crp, c-reactive protein; pct, procalcitonin; wbc, white blood cell count. *p < 0á05 in differences in hospitalization rates between influenza a, influenza a/h1n1/2009, and influenza c versus influenza b; no other significant between-group differences groups. in conclusion, our findings suggest that icv can be an important cause of cap of children and could have considerable consequences. further studies aimed at defining the peak season of icv infection in different countries, the real role of icv in causing severe respiratory disease, and establishing which strains are more epidemiologically impor-tant in different countries and different years are urgently needed. figure 1 . phylogenetic tree of influenza c viruses based on a region of about 1000 bp of the he gene. the viruses isolated in this study (milan, italy) are marked by a black symbol. the other sequences have been described elsewhere. 7, [9] [10] [11] the values at the nodes are bootstrap supported on the basis of 500 replicates. clinical features of influenza c virus infection in children influenza c virus infection in children influenza c virus surveillance during the first influenza a (h1n1) 2009 pandemic wave in catalonia, spain world health organization, pneumonia vaccine trial investigators' group. standardization of interpretation of chest radiographs for the diagnosis of pneumonia in children. who/v&b/01.35. world health organization antibiotic therapy for pediatric community-acquired pneumonia: do we know when, what and for how long to treat? impact of viral infections in children with community-acquired pneumonia: results of a study of 17 respiratory viruses. influenza other respi viruses a nationwide epidemic of influenza c virus in japan in 2004 interspecies transmission of influenza c virus between humans and pigs frequent reassortment among influenza c viruses study of influenza c virus infection in france clustalwand clustal x version 2.0 mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods influenza c virus in pediatric pneumonia ª the authors would like to thank dr. yoko matsuzaki, department of infectious diseases, yamagata university faculty of medicine, japan, for providing the influenza cpositive cdna control and primer sequences for he amplification. this study was supported by a grant from the italian ministry of health (bando giovani ricercatori 2007). none of the authors has any potential conflict of interest. all of the authors have submitted the icmje form for disclosure of potential conflicts of interest. key: cord-279615-yne753y6 authors: jelley, lauren; levy, avram; deng, yi‐mo; spirason, natalie; lang, jurissa; buettner, iwona; druce, julian; blyth, chris; effler, paul; smith, david; barr, ian g. title: influenza c infections in western australia and victoria from 2008 to 2014 date: 2016-07-23 journal: influenza other respir viruses doi: 10.1111/irv.12402 sha: doc_id: 279615 cord_uid: yne753y6 background: influenza c is usually considered a minor cause of respiratory illness in humans with many infections being asymptomatic or clinically mild. large outbreaks can occur periodically resulting in significant morbidity. objectives: this study aimed at analyzing the available influenza c clinical samples from two widely separated states of australia, collected over a 7‐year period and to compare them with influenza c viruses detected in other parts of the world in recent years. patients/methods: between 2008 and 2014, 86 respiratory samples that were influenza c positive were collected from subjects with influenza‐like illness living in the states of victoria and western australia. a battery of other respiratory viruses were also tested for in these influenza c‐positive samples. virus isolation was attempted on all of these clinical samples, and gene sequencing was performed on all influenza c‐positive cultures. results and conclusions: detections of influenza c in respiratory samples were sporadic in most years studied, but higher rates of infection occurred in 2012 and 2014. many of the patients with influenza c had coinfections with other respiratory pathogens. phylogenetic analysis of the full‐length hemagglutinin–esterase–fusion (he) gene found that most of the viruses grouped in the c/sao paulo/378/82 clade with the remainder grouping in the c/kanagawa/1/76 clade. influenza c is often ignored compared with the other human pathogens in the orthomyxoviridae family (influenza a and influenza b). with respect to other influenza types, it causes a milder disease with fewer complications and, as it has not been included in routine testing, there is little new information being generated about its impact. in etiologic studies of respiratory illnesses that have included influenza c testing, it usually accounts for a low proportion of acute respiratory pathogens identified. for example, a canadian study 1 found 2.32% of respiratory samples tested from children identified influenza c, while a study in japan spanning 18 years 2 (1996-2013) found influenza c in 0.43%-1.73% of samples from children; this result was similar to a spanish study that reported influenza c in 0.7% of children's samples. 3 low rates were reported in a retrospective study scottish study that screened 3300 respiratory samples collected from children and adults during the period from august 2006 to june 2008 with only six positive influenza c-positive samples (0.2%) identified (with 4/6 from children ≤2 year) compared to 3.2% influenza a detections and 0.9% influenza b detections. 4 higher rates of influenza c infection have been reported in a nigerian study, where 4.8% of the respiratory samples from children were positive for influenza c 5 and finnish study among young adult male military recruits, 6 in which influenza c was identified in 4.2% of all samples. influenza c can also be identified in a significant proportion of children hospitalized with lower respiratory tract infections, as reported by shimizu et al. 7 2015, who found approximately 10% of children had influenza c present in samples collected from four japanese hospitals during 2009-2010. often these cases of influenza c also have other viruses or bacteria detected in the specimen making it difficult to attribute the contribution of influenza c infections to the clinical illness manifestations. 5, 8 in contrast to most viral etiology studies, serological studies often find high seroprevalence rates in the community which rise rapidly up to 10 years of age to around 50%-60% of the population, indicating that widespread transmission has occurred among children in many different countries. 9 unlike influenza a and b infections where early treatment with neuraminidase inhibitor drugs can be effective, there are no effective antiviral treatments for influenza c. this study analyzed influenza c viruses detected in respiratory samples collected from two influenza illness surveillance programs operating in the state of western australia (wa) from 2001 to 2014: one covering patients of all ages presenting to general practitioners with an influenza-like illness (ili), and the other covering young children presenting with a respiratory illness to a metropolitan pediatric hospital emergency department or a general practitioner. these were compared with influenza c viruses detected from routine screening of respiratory samples from melbourne, victoria from 2011 to 2014. this report is the first to isolate and characterize (including sequencing) influenza c viruses collected in australia. isolation of influenza c viruses was performed using madin-darby canine kidney (mdck) cells (atcc ccl-34) using maintenance media (dmem coon's basal media containing sodium bicarbonate (3%) with the addition of 2 mmol/l glutamine, 1% non-essential amino acids, 0.05% nahco3, 0.02 mol/l hepes, 4% penicillin and streptomycin, 2 μg/ml amphotericin b and 4 μg/ml trypsin). samples were incubated for up to 5 days at 33°c without co 2 , and virus growth was quantified by determining the hemagglutination titer (ha) with 0.5% fowl and turkey rbc. 8,12 prior to sequencing or culturing, due to the age of some of the clinical samples, a real-time pcr assay was designed and run so that samples could be retested at the who centre to confirm that viral rna was still detectable. primers and a minor groove binding probe were designed using an alignment of influenza c hemagglutinin-esterase-fusion (he) gene sequences from genbank. sequences were downloaded and aligned using the megalign program of dnastar (madison, wi, australia), and he primers and probes were obtained (geneworks, adelaide, sa, australia) and used for the real-time pcr of clinical samples and also for the confirmation of influenza c virus isolates (table 1) jelley et al. purified rna extracted from influenza c-positive samples was amplified using the bioline mytaq one-step rt-pcr kit (bioline, australia) using in-house-designed he gene-specific primers supplied by bioneer (melbourne, australia; table 1 ). rt-pcr was per for the 16 influenza c-positive samples from the spn(wa) gp surveillance, the age range was 1-40 years with the median age of 22 years (25% male, 75% female), while for the 63 influenza c-positive waive samples the age range was 0.6-5.1 years with a median age of 1.7 year (46% male, 54% female). note that the waive program only recruits children aged between 6 months and 59 months who attended the emergency department or were admitted to the princess margaret hospital, perth (wa) with an ili (defined by the presence or history of fever and a respiratory symptom). this study group was therefore made up exclusively of young children and they had higher rates of influenza c than the spn(wa) group (table 1) . during the waive study, the overall prevalence of influenza c cases was 1.6% and varied from 0% (2009) table 3 ). the most common multiple infections along with influenza c were with human enteroviruses/ rhinoviruses (13 samples), influenza a (seven samples; four with influenza a h1n1pdm09 and three with influenza a(h3n2)) followed by rsv (six samples) and adenovirus (six samples) ( hemagglutinin-esterase-fusion gene sequence was obtained for all samples that yielded influenza c virus isolates. thirty-five influenza c full he sequences (28 from perth/wa and seven from victoria) and one partial he sequence from perth were obtained from the virus isolates. these sequences were compared with publically available he sequences from reference viruses including those that represent the six antigenically distinct influenza c groups from recently circulating influenza c viruses (fig. 1) victorian samples were evenly distributed in both of these clades (fig. 1) . the bootstrap values for all of these clade assignments were high. the this is the first report characterizing influenza c viruses obtained from australia. influenza c viruses were detected sporadically in two widely in the waive study, 11.1% vs 16.8%, respectively. the frequency of large influenza c epidemics in australia is unknown, but looking at the waive data (table 2 ) there is some indication that they may occur approximately every 2 years, as the influenza c cases detected were the highest in 2010, 2012, and 2014. in japan, it also appears that influenza c epidemics could be detected every couple of years. 14 overall, we were moderately successful in isolating influenza c, with 51.5% of original clinical samples yielding isolates. these isolates and 2012 from children of less than 10 years of age. in a paediatric clinic of the university of milan, a study over 4 years (2008-9 to 2011-2) in children under 15 years of age with influenza c and radiographically confirmed community-acquired pneumonia, five cases were identified with two viruses of the c/kanagawa/1/76-lineage and three from the c/sao paulo/378/82-lineage. 18 clearly, viruses from these two influenza c lineages have been the dominant clades seen in many countries in recent years even though they appear to have changed very little based on their he phylogeny, since they were first reported. unfortunately, our sample size was not large enough to be able to gain an in depth understanding of influenza c within australia. nor can it determine the level of reassortment that has occurred as the six internal genes have yet to be sequenced. however, odagiri et al. detection of influenza c virus by real time rt-pcr assay. influenza other respir viruses epidemiological information regarding the periodic epidemics of influenza c virus in japan (1996-2013) and the seroprevalence of antibodies to different antigenic groups prospective study of influenza c in hospitalized children detection of influenza c virus but not influenza d virus in scottish respiratory samples specific viruses detected in nigerian children in association with acute respiratory disease influenza c virus infection in military recruits-symptoms and clinical manifestation influenza c virus and human metapneumovirus infections in hospitalized children with lower respiratory tract illness isolation and characterization of influenza c viruses in the philippines and japan influenza c virus high seroprevalence rates observed in 3 different population groups effectiveness of trivalent flu vaccine in healthy young children the detection and multiplication of influenza c virus in tissue culture new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml 3.0 the dominant antigenic group of influenza c infections changed from c/sao paulo/378/82-lineage to c/kanagawa/1/76-lineage in yamagata isolation of influenza c virus recombinants hemagglutinin-esterase-fusion (he) protein of influenza c virus phylogenetic analysis and seroprevalence of influenza c virus in mie prefecture, japan in 2012 influenza c virus-associated community-acquired pneumonia in children. influenza other respir viruses the melbourne who collaborating centre for reference and research on influenza is supported by the australian government department of health. the authors thank all staff from pathwest additional supporting information may be found online in the supporting information tab for this article. key: cord-309497-3v0asfa7 authors: asner, sandra; peci, adriana; marchand‐austin, alex; winter, anne‐luise; olsha, romy; kristjanson, erik; low, donald e.; gubbay, jonathan b. title: respiratory viral infections in institutions from late stage of the first and second waves of pandemic influenza a (h1n1) 2009, ontario, canada date: 2012-02-21 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2012.00336.x sha: doc_id: 309497 cord_uid: 3v0asfa7 please cite this paper as: asner et al. (2012) respiratory viral infections in institutions from late stage of the first and second waves of pandemic a (h1n1) 2009, ontario, canada. influenza and other respiratory viruses 6(3), e11–e15. we report the impact of respiratory viruses on various outbreak settings by using surveillance data from the late first and second wave periods of the 2009 pandemic. a total of 278/345(78·5%) outbreaks tested positive for at least one respiratory virus by multiplex pcr. we detected a(h1n1)pdm09 in 20·6% of all reported outbreaks of which 54·9% were reported by camps, schools, and day cares (csds) and 29·6% by long‐term care facilities (lcfts), whereas enterovirus/human rhinovirus (ent/hrv) accounted for 62% outbreaks of which 83·7% were reported by long‐term care facilities (lctfs). ent/hrv was frequently identified in ltcf outbreaks involving elderly residents, whereas in csds, a(h1n1)pdm09 was primarily detected. respiratory outbreaks are common in healthcare and community institutions such as long-term care facilities (ltcfs) and schools. 1, 2 the most commonly identified viruses have been influenza and respiratory syncytial virus (rsv). 1, 2 human rhinovirus (hrv) has more recently been identified as a major viral pathogen in ltcf outbreaks. 1 recent data reported by public health ontario (pho) indicated that pandemic influenza a (h1n1) 2009 [a(h1n1)pdm09] was a rare cause of ltcf respiratory outbreaks during the first period of wave i (april 20-june 12, 2009) of the 2009 pandemic. 3 we used surveillance data from the late stage of the first wave and the duration of the second wave periods (june 11-november 30, 2009) to ascertain the impact of a(h1n1)pdm09 and other respiratory viruses on different outbreak settings such as ltcfs and schools. for the purpose of this study, we considered the period from april 20 to august 31, 2009, as wave i and the period from september 1 to november 30, 2009, as wave ii, although wave ii activity continued until january 2010. 4 we investigated all respiratory outbreaks in ltcfs and camps, schools, day cares (csds) tested at pho laboratories from june 11 through november 30, 2009, in ontario, canada. a confirmed respiratory infection outbreak in a ltcf, as defined by ontario's ministry of health (moh), requires two cases of acute respiratory illness within 48 hours of which at least one has to be laboratory-confirmed, or three cases of acute respiratory illness occurring within 48 hours in a geographic area, none of which are laboratory-confirmed. 5 ]. an alternate multiplex naat kit (seeplex rv; seegene usa, rockville, md, usa) was used in conjunction with the luminex assay during periods of higher demand. an in-house assay specific for a(h1n1)pdm09 was also performed. 6 a total of 345 respiratory outbreaks in csds, ltcfs, and other facilities (hospitals, correctional facilities, and unknown) were reported to and tested at pho. molecular testing was performed on 1117 samples from different outbreak settings. the number of samples submitted per outbreak is shown in figure 1 . of the average of four samples (range 1-14) tested per outbreak, the median number of positive samples per outbreak was 2 (range 1-8). outbreak samples mostly originated from ltcfs (76ae8%) and csds (13ae9%). hospitals and correctional facilities comprised 17(4ae9%) and 1 (0ae3%) of remaining outbreaks, respectively. facility type was unknown for 4ae1% outbreaks tested. mean and median ages of all persons tested were 72ae1 and 83 years, respectively (range 1-106 years). mean and median ages of ltcf outbreak cases were 80ae9 and 85 years, respectively; csds had a much younger population with mean and median ages of 13ae4 and 12 years, respectively. at least one viral agent was identified in 278 (78ae5%) of the outbreaks tested. of these, 84 (30ae2%) outbreaks had only one sample with a virus identified, and the remaining 194 (69ae8%) had the same virus identified in two or more samples (range 2-8). ent ⁄ hrv and a(h1n1)pdm09 were the most common viruses detected in 214 (62%) and 71 (20ae6%) outbreaks, respectively. of the 71 outbreaks in which a(h1n1)pdm09 was detected, 39 (54ae9%) occurred in csds, 21 (29ae6%) in ltcfs, and 11 (15ae5%) in other facilities. of the 214 outbreaks in which ent ⁄ hrv was identified, 179 (83ae7%) occurred in ltcfs, 17 (8%) in csds, and 18 (8%) in other facilities ( figure 2 ). both viruses were identified throughout the entire study period, reaching a peak during october of wave ii (figure 2 ). two hundred and forty outbreaks (69ae6%) were caused by a single virus, the most common being ent ⁄ hrv (75%) or a(h1n1)pdm09 (17ae9%). one hundred and ninety of these single virus outbreaks occurred in ltcfs and 34 in csds. more than one etiological agent was identified in 38 (11%) of the outbreaks, of which 19 (50%) had two or more viruses detected within the same sample (coinfection). the most common coinfection was a(h1n1)pdm09 ⁄ ent ⁄ hrv, detected in 11 (58%) of the 19 outbreaks with coinfections. in the three outbreaks where two or more samples had viral coinfection (range 2-5 samples with coinfection), the same two viruses were found in all coinfection samples. there was an increased likelihood of identifying multiple viruses if 5 or more samples were tested (p < 0ae05; figure 1 ). nineteen outbreaks with multiple viruses detected were reported in ltcfs and 12 in csds. ent ⁄ hrv and a(h1n1)pdm09 were the most common co-circulating viruses reported in all outbreaks with multiple viruses detected, found in 7 of the 19 multiple virus outbreaks reported by ltcfs and 10 of the 12 multiple virus outbreaks reported by csds (table 1) . was detected in the majority of respiratory outbreaks in ltcfs, where the vast majority of residents were elderly. recently published reports 7-9 have already highlighted these findings. however, this study provides a larger sample size and is unique in that both institutional (predominantly elderly) and community outbreak (predominantly children) settings are described. we also found an increased likelihood of detecting viruses causing outbreaks as the number of specimens tested increased. viruses causing coinfections may be co-transmitted between patients as part of the same outbreak, rather than circulating independently of each other. additional bacterial analysis would be required to conclude that hrv is the sole etiology of individual outbreaks. in contrast, the pandemic virus was detected in almost all csd outbreaks, which mostly involved children and younger adults as recently highlighted in the literature. 10, 11 possible reasons for a lower prevalence of a(h1n1)pdm09 in ltcfs outbreaks include cross-protective antibodies from previous exposure to influenza a (h1n1) strains among elderly or minimal exposure to individuals more likely to be infected by the pandemic strain, such as children. 10, 11 from june 11 to november 30, 2009, we found that ent ⁄ hrv was frequently identified in ltcf outbreaks involving elderly residents, whereas in outbreak settings involving children and younger adults, a(h1n1)pdm09 was primarily detected. younger children were not well represented in the csd group, which had a median age of 13ae4 years. this likely reflects the age distribution of children that attend sleep away summer camps in ontario. limitations of our study include its observational design, which limits establishment of causality and also limits our ability to exclude the effect of measured or unmeasured confounders in our analysis. a distinction between upper and lower respiratory tract infections, the role of asymptomatic shedding, and comparison of severity of outbreaks could not be performed as clinical data were missing. in ontario, the local medical officer of health or designate determines whether an institutional outbreak meets the provincial case definition. pho laboratories do not receive sufficient outbreak information to make this determination. in this context, this study highlights the importance of submitting more than one sample to properly investigate an outbreak. other bacterial agents like mycoplasma pneumoniae or chlamydophila pneumonia could not be ruled out as the cause of outbreaks as samples were not routinely tested for them. a distinction could not be made between staff and resident cases in this study as staff information was not systematically reported. this highlights a current deficiency in the investigation and reporting of staff illness in ltcfs. 9 an increased rate of ent ⁄ hrv outbreaks may have been observed during the pandemic because of increased vigilance by individuals to report symptoms to their healthcare providers, who in turn had a lower threshold to report outbreaks to the public health units who coordinate laboratory investigations. therefore, we might have seen increased specimen collection and testing that may have influenced our findings. identification of specific non-influenza organisms associated with outbreaks can assist with outbreak management as the period of patient isolation and when to declare an outbreak over are dependent on the incubation period and period of communicability, which varies by organism. documentation of hrv as a cause of ltcf outbreaks is important, as recent studies suggest that hrv outbreaks can cause severe and fatal disease in ltcfs, especially among the elderly. 1 a study comparing viral outbreaks in different community ⁄ facility settings with prospective gathering of detailed clinical and epidemiological information, supported by comprehensive microbiological analysis, will help further understand the role of different respiratory viruses as etiologic agents. rhinovirus outbreaks in long-term care facilities influenza and respiratory syncytial virus in the elderly respiratory infection in institutions during early stages of pandemic (h1n1) ontario agency for health protection and promotion (oahpp) appendix b: provincial case definitions for reportable diseases analytical and clinical validation of novel real-time reverse transcriptase-polymerase chain reaction assays for the clinical detection of swine-origin h1n1 influenza viruses a rhinovirus outbreak among residents of a long-term care facility uncommon(ly considered) manifestations of infection with rhinovirus, agent of the common cold severe human rhinovirus outbreak associated with fatalities in a long-term care facility in ontario incidence of 2009 pandemic influenza a h1n1 infection in england: a cross-sectional serological study estimated epidemiologic parameters and morbidity associated with pandemic h1n1 influenza sandra asner, adriana peci, alex marchand-austin, anne-luise winter, romy olsha, and erik kristjanson have no conflicts of interest to declare.donald e. low participated in advisory board committee meetings for glaxosmithkline inc. and hoffman-la roche. he has also received research funding from both companies. jonathan b. gubbay has received a research grant from glaxosmithkline inc. to work on resistance to neuraminidase inhibitors. in june 2010, oahpp received a research grant from glaxosmithkline to study phenotypic resistance in influenza virus. key: cord-309518-seonrtn3 authors: alraddadi, basem m.; qushmaq, ismael; al‐hameed, fahad m.; mandourah, yasser; almekhlafi, ghaleb a.; jose, jesna; al‐omari, awad; kharaba, ayman; almotairi, abdullah; al khatib, kasim; shalhoub, sarah; abdulmomen, ahmed; mady, ahmed; solaiman, othman; al‐aithan, abdulsalam m.; al‐raddadi, rajaa; ragab, ahmed; balkhy, hanan h.; al harthy, abdulrahman; sadat, musharaf; tlayjeh, haytham; merson, laura; hayden, frederick g.; fowler, robert a.; arabi, yaseen m. title: noninvasive ventilation in critically ill patients with the middle east respiratory syndrome date: 2019-03-18 journal: influenza other respir viruses doi: 10.1111/irv.12635 sha: doc_id: 309518 cord_uid: seonrtn3 background: noninvasive ventilation (niv) has been used in patients with the middle east respiratory syndrome (mers) with acute hypoxemic respiratory failure, but the effectiveness of this approach has not been studied. methods: patients with mers from 14 saudi arabian centers were included in this analysis. patients who were initially managed with niv were compared to patients who were managed only with invasive mechanical ventilation (invasive mv). results: of 302 mers critically ill patients, niv was used initially in 105 (35%) patients, whereas 197 (65%) patients were only managed with invasive mv. patients who were managed with niv initially had lower baseline sofa score and less extensive infiltrates on chest radiograph compared with patients managed with invasive mv. the vast majority (92.4%) of patients who were managed initially with niv required intubation and invasive mechanical ventilation, and were more likely to require inhaled nitric oxide compared to those who were managed initially with invasive mv. icu and hospital length of stay were similar between niv patients and invasive mv patients. the use of niv was not independently associated with 90‐day mortality (propensity score‐adjusted odds ratio 0.61, 95% ci [0.23, 1.60] p = 0.27). conclusions: in patients with mers and acute hypoxemic respiratory failure, niv failure was very high. the use of niv was not associated with improved outcomes. middle east respiratory syndrome (mers) has emerged as a cause of severe respiratory illness in humans. 1, 2 as of march 1, 2019, 2279 cases of mers have been reported including 806 deaths. 3 the disease presentation ranges from asymptomatic infection to severe respiratory illness, multiorgan failure, and death. [4] [5] [6] acute hypoxemic respiratory failure (ahrf) develops in up to 70% of hospitalized patients with mers and is associated with high mortality. 7, 8 to date, there is no specific antiviral therapy for mers of proven effectiveness; supportive therapy remains the cornerstone of management. noninvasive ventilation has been increasingly used in the management of ahrf with variable success. [9] [10] [11] [12] while niv may initially avoid the need for intubation and invasive mechanical ventilation (mv) , several studies have reported high failure rates and the need for invasive ventilation among patients with severe acute respiratory distress syndrome (ards) and an association with increased mortality. 12 in a recent analysis from the lung safe study on unselected patients with ards, niv was associated with higher intensive care unit (icu) mortality in patients with the ratio of partial pressure of oxygen to the fraction of inspired oxygen (pao 2 /fio 2 ) lower than 150 mm hg. 12 the role of niv in ahrf secondary to viral respiratory infections is unclear. although some uncontrolled studies suggested that niv was effective and safe in management of patients with severe acute respiratory syndrome (sars), [13] [14] [15] others have highlighted concern of increased transmission risk to healthcare workers when patients with sars are treated with niv. 16 use of niv in ahrf caused by pandemic h1n12009 virus (pdmh1n1) infection has been reported from several countries, [17] [18] [19] with reported niv failure reaching up to 85%. 17 all studies were limited by their retrospective nature and, often, small sample size. noninvasive ventilation has been used in patients with mers, 5, 8 but its value in preventing intubation and impact on clinical outcomes has not been studied. the objective of this study was to assess the success of niv in mers patients with ahrf in avoiding intubation and its association with mortality and icu and hospital length of stay. our secondary objective was to identify factors associated with niv failure in mers patients. we conducted this analysis on a multicenter retrospective cohort of critically ill mers patients from 14 in this study, we included all patients with ahrf who required mechanical ventilation support in the icu, whether invasively or noninvasively. all patients who were managed initially with niv were compared to those who were managed with invasive mv without niv. for this analysis, we extracted baseline data including demographics, comorbidities, duration from onset of symptoms to emergency room admission, icu admission, and intubation. arterial blood gases, continuous variables were described as medians and interquartile ranges (q1, q3) or means and standard deviations and were tested we performed a secondary comparison of patients who had failed niv to those who had been successfully treated with niv. all statistical tests were two-sided with significance set at α < 0.05. analyses were conducted using sas version 9.2 (sas institute, cary, nc). in 105 patients who were managed initially with niv, niv was used for a median duration of 1 (1, 3) day and 97 patients (92.4%) eventually required intubation and invasive mv ( [11, 35] , p = 0.6). there was no significant difference in the duration of invasive mv and total duration of niv and invasive mv between the two groups, although invasive mv-free days and total niv and invasive mv-free days were significantly longer among niv patients compared to invasive mv patients (table 2, figure s2 ). overall, only 8/105 (7.6%) of the niv patients avoided subsequent intubation (table s1 ). these patients were significantly younger than tables s1 and s2). crude 90-day mortality was significantly higher in patients who failed niv compared with patients successfully treated only with niv (table s3 and figure s3 ). we have shown that among patients with mers-related ahrf, niv was commonly used, but nearly always resulted in subsequent transition to invasive ventilation. our results suggest that while the initial niv use in mers patients was not associated with reduction of mortality or length of icu or hospital stay, these patients had greater requirement for subsequent inhaled nitric oxide. a minority of patients were successfully managed with niv-those who were young and had less severe disease. these findings have important implications for early management of patients infected with mers, specifically, that there is little advantage to initial niv treatment for most patients with mers-related ahrf and that niv may be associated with greater subsequent need for oxygenation rescue therapy such as inhaled nitric oxide. noninvasive ventilation has been proven to be useful as a means to avoid intubation and improve clinical outcomes in certain conditions, generally, with the possibility for rather rapid reversal of respiratory failure-for example, pulmonary edema due to congestive heart failure, and respiratory failure due to copd exacerbations. 22, 23 for conditions that typically worsen or do not improve in the range of many hours (eg, most causes of pneumonia), there appears to be little advantage in using niv as a means to avoid intubation. 9, 24, 25 in choosing to use niv for as initial treatment for patients with hypoxemic respiratory failure, there is a practical risk of patients worsening on niv and requiring intubation at a time when they already have more advanced organ failure. few studies have assessed the effectiveness of niv in patients with ahrf secondary to ards and acute lung injury. the overall effectiveness of niv in reducing intubation rate or improving clinical outcome in these patients remains controversial. [26] [27] [28] post hoc analysis of the lung safe study found that niv was used in 15% of patients with ards and was associated with higher icu mortality in subset of patients with severe ards. 12 a randomized controlled trial of patients niv is generally not recommended for patients with hypoxia secondary to respiratory infections due to lack of efficacy and the potential for pathogen transmission. 16, 31, 32 it is also considered one of the aerosol-generating procedures that may increase risk of transmission to healthcare workers. 32 broad dispersion of exhaled air during niv via a face mask has previously been shown using a simulated patient encounter. 33 in conclusion, we report the results of niv use in mers patients from a large cohort of critically ill patients. we observed that there is little advantage to initial niv treatment for most patients with mers-related ahrf and that niv may be associated with greater subsequent need for oxygen rescue therapy. we would like to thank the international severe acute respiratory and emerging infection consortium (isaric) for their support in the database. the authors have no conflict of interest to disclose. bma: conception and design, data acquisition, analytical plan, interpretation of data for the work, drafting of the manuscript, critical revi middle east respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection mers-cov outbreak in jeddah-a link to health care facilities description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia a multiple-center survey on the use in clinical practice of noninvasive ventilation as a first-line intervention for acute respiratory distress syndrome use of noninvasive ventilation in patients with acute respiratory failure changing use of noninvasive ventilation in critically ill patients: trends over 15 years in francophone countries noninvasive ventilation of patients with acute respiratory distress syndrome. insights from the lung safe study effectiveness of noninvasive positive pressure ventilation in the treatment of acute respiratory failure in severe acute respiratory syndrome non-invasive versus invasive mechanical ventilation for respiratory failure in severe acute respiratory syndrome noninvasive positive pressure ventilation treatment for acute respiratory failure in sars transmission of severe acute respiratory syndrome during intubation and mechanical ventilation critically ill patients with 2009 influenza a(h1n1) infection in canada intensive care adult patients with severe respiratory failure caused by influenza a (h1n1)v in spain clinical findings and demographic factors associated with icu admission in utah due to novel 2009 influenza a(h1n1) infection critically ill patients with the middle east respiratory syndrome (mers): a multicenter retrospective cohort study infection prevention and control guidelines for patients with middle east respiratory syndrome coronavirus (mers-cov) infection noninvasive ventilation for acute exacerbations of chronic obstructive pulmonary disease non-invasive positive pressure ventilation (cpap or bilevel nppv) for cardiogenic pulmonary oedema predictors of failure of noninvasive positive pressure ventilation in patients with acute hypoxemic respiratory failure: a multi-center study non-invasive ventilation in community-acquired pneumonia and severe acute respiratory failure noninvasive ventilation for patients with acute lung injury or acute respiratory distress syndrome noninvasive ventilation for acute lung injury a meta-analysis of randomized controlled trials role of noninvasive ventilation in acute lung injury/acute respiratory distress syndrome: a proportion meta-analysis high-flow oxygen through nasal cannula in acute hypoxemic respiratory failure effect of noninvasive ventilation delivered by helmet vs face mask on the rate of endotracheal intubation in patients with acute respiratory distress syndrome: a randomized clinical trial clinical management of pandemic 2009 influenza a(h1n1) infection aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review exhaled air dispersion during noninvasive ventilation via helmets and a total facemask why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? additional supporting information may be found online in the supporting information section at the end of the article. how to cite this article the saudi critical care trials group noninvasive ventilation in critically ill patients with the middle east respiratory syndrome key: cord-269644-94yrkzns authors: cousin, mathias; molinari, nicolas; foulongne, vincent; caimmi, davide; vachier, isabelle; abely, michel; chiron, raphael title: rhinovirus‐associated pulmonary exacerbations show a lack of fev (1) improvement in children with cystic fibrosis date: 2016-01-29 journal: influenza other respir viruses doi: 10.1111/irv.12353 sha: doc_id: 269644 cord_uid: 94yrkzns background: respiratory viral infections lead to bronchial inflammation in patients with cystic fibrosis, especially during pulmonary exacerbations. the aim of this study was to determine the impact of viral‐associated pulmonary exacerbations in children with cystic fibrosis and failure to improve forced expiratory volume in 1 s (fev (1)) after an appropriate treatment. methods: we lead a pilot study from january 2009 until march 2013. children with a diagnosis of cystic fibrosis were longitudinally evaluated three times: at baseline (visit 1), at the diagnosis of pulmonary exacerbation (visit 2), and after exacerbation treatment (visit 3). nasal and bronchial samples were analyzed at each visit with multiplex viral respiratory pcr panel (qualitative detection of 16 viruses). pulmonary function tests were recorded at each visit, in order to highlight a possible failure to improve them after treatment. lack of improvement was defined by an increase in fev (1) less than 5% between visit 2 and visit 3. results: eighteen children were analyzed in the study. 10 patients failed to improve by more than 5% their fev (1) between visit 2 and visit 3. rhinovirus infection at visit 2 or visit 3 was the only risk factor significantly associated with such a failure (or, 12; 95% ci, 1·3–111·3), p = 0·03. conclusions: rhinovirus infection seems to play a role in the fev (1) recovery after pulmonary exacerbation treatment in children with cystic fibrosis. such an association needs to be confirmed by a large‐scale study because this finding may have important implications for pulmonary exacerbation management. despite conventional management with antibiotics and respiratory physiotherapy, pulmonary function undergoes a steady decline in cystic fibrosis (cf). pulmonary exacerbations (pex) are an important clinical outcome in cf, as they are associated with a faster rate of decline in forced expiratory volume in 1 s (fev 1 ) and with an increased morbidity and mortality in patients with cf. almost 25% of patients fail to recover to baseline fev 1 after a pex. 1, 2 the reasons why these patients fail to recover previous fev 1 after an appropriate treatment are not fully understood even though some risk factors have been identified (pancreatic insufficiency, low body mass index (bmi), chronic infection with cf pathogens, greater drop in fev 1 from baseline at treatment initiation). 3 viruses are frequent triggers of pex, and rhinoviruses are the most frequent viral agents found in patients with cf. 4 an impaired innate host defense may cause an increased susceptibility to viral respiratory infection in patients with cf, and some severe viral infections might be responsible for the exaggerated pulmonary inflammatory response. 5, 6 however, in the literature, there is a paucity of data regarding pex outcomes in cf children, with a concomitant viral respiratory infection. these infections seem to play a significant role in pulmonary morbidity and inflammation in patients with cf. indeed, patients presenting with concomitant bacterial and viral infections show a greater rate of both hospitalization and antibiotic prescription. 7 unfortunately, the consequences of viral respiratory infections on the evolution of cf lung disease are hard to study on a large scale, because, in order to evaluate such an issue, a close longitudinal follow-up with repeated viral swab assessment is needed. we could therefore speculate that the impact of respiratory viruses is probably underestimated in patients with cf. we hypothesized that respiratory viruses are major risk factors for maintaining and extending bronchial inflammation during the pex recovery period. the aim of this study was to determine the association between viral-associated pex and failure to improve fev 1 after pex treatment in cf children. this pediatric pilot study was conducted from january 2009 until march 2013 at the cf centers of montpellier and reims (france). patients aged from 7 to 18 years were recruited. pex was defined, according to european respiratory society criteria, as a change in respiratory status requiring antibiotic treatment, or by a cluster of symptoms, as indicated by eurocarecf working group. the study eudract (2008-00451-30) has been approved by our local ethic committee (ref 2008.07.06bis). all children and both parents signed the consent form. patients were longitudinally assessed three times in the cf university center: at baseline (visit 1, v1), at the diagnosis of pex (visit 2, v2), and after pex treatment (visit 3, v3). v3 was scheduled between 2 and 6 weeks after the beginning of pex treatment, because it has been shown that antibiotic response to exacerbation as assessed by pft is essentially complete 2 weeks after treatment initiation. 9 clinicians working in cf university centers managed pex, according to the guidelines. clinical data and pft were recorded at each visit. we sampled upper and lower airways by collecting two nasal swabs and two spontaneously expectorated sputa at each visit for viral and bacterial analysis. viral analysis was performed through a commercial multiplex pcr assay (anyplex-ii-rv16, seegene) providing a qualitative detection of 16 viruses (without genotype): human adenovirus; influenza a and b virus; human parainfluenza virus 1, 2, 3, and 4; human rhinovirus; human respiratory syncytial virus a and b; human bocavirus; human coronavirus 229e, nl63, and oc43; human metapneumovirus; and human enterovirus. clinicians were blinded to the results of virological tests until the end of the study. participants were categorized by their relative change in fev 1% predicted between visit 2 and visit 3 calculated as follows:([fev 1% predicted at visit 2-fev 1% predicted at visit 3] * 100)/fev 1% predicted at visit 2; as "responders" (≥5% of fev 1 improvement) and "non-responders" (<5% of fev 1 improvement) to pex treatment. this 5% threshold has been previously used in many studies. 10, 11 quantitative variables were expressed as means (standard deviation) and compared using the wilcoxon test as appropriate. qualitative variables were expressed as numbers (%), and the absolute numbers were compared using the chisquared test or the fisher's test as appropriate. logistic regressions were used to estimate or and 95% ci. in the present study, 34 patients were included (21 in montpellier center and 13 in reims center). among these patients, 18 completed the 3 visits and were analyzed. the other patients were lost to follow up because they did not go on exacerbation during the period of the study. eight patients were classified as responders and 10 patients as nonresponders to pex treatment. patient characteristics were similar between the two groups ( table 1) . patients did not present severe associated comorbidities: none had diabetes, one patient was undernourished, and one patient had a fev 1 below 60% at baseline. the delay between v2 and v3 varied from 13 to 50 days (median delay of 29 days), without any difference between the two groups (table 1 ). in the nonresponders' group, there were significantly more viral respiratory infections at v2 and/or v3 (10 versus 4, p = 0á0229) and more rhinovirus respiratory infections at v2 and/or v3 (8 versus 2, p = 0á0196) ( table 1) . at visit 2, we detected viruses in 50% of nasal swabs and in 67% of expectorated sputa. nasal and bronchial swabs were concordant in 78% of all visits. the proportion of viral respiratory infection was 72% at the diagnosis of pex (v2), 22% at baseline (v1), and 28% at v3. rhinoviruses accounted for 64% of identified viruses. we detected a viral co-infection in one patient (rhinovirus and metapneumovirus). influenza virus vaccination was recorded for 78% of the patients. pex did not occur during any specific respiratory viruses epidemic spread or a specific season. the only risk factor significantly associated with failure to improve fev 1 above 5% after pex was viral respiratory infection at v2 and/or v3 (or, 2á04; 95% ci, 1á57-2á67) p = 0á0088 and especially with rhinovirus infection: (or, 12; 95% ci, 1á29-111á32, p = 0á0288). other factors such as pancreatic insufficiency, atopic status, bmi, baseline treatment (azithromycin, inhaled corticosteroid therapy, and antibiotic therapy), persistent infection (with pseudomonas aeruginosa, staphylococcus aureus), detection of new bacteria at v2 (all pathogens included and for each bacteria species), spirometrics parameters (i.e., fev 1% drop between v1 and v2) and therapeutic features (i.e., adapted pex antibiotherapy according to ecfs guidelines 2014, 12 intravenous treatment, duration of antibiotic treatment) were not significantly associated with failure to improve fev 1 above 5% after pex treatment (table 2) . non-responder patients failed significantly to improve above 5% their forced vital capacity (fvc) between v2 and v3, (or, 0á07; 95% ci, 0á01-0á82), p = 0á0347 (table 2) . rhinovirus respiratory infection at v2 or v3 was significantly associated with failure to improve fvc above 5% after pex treatment (adjusted or, 15; 95% ci, 1á21-185á2), p = 0á0347. the results of this pilot study show that rhinovirus infection during pex is significantly associated with failure to improve fev 1 after pex treatment in children with cf. none of our subjects had new symptoms of a pulmonary exacerbation at the third visit. so we do not think that the non-responder's children were sick again but this failure in fev1 recovery might be the consequences of the exacerbation diagnosed at the second visit. to our knowledge, only one study assessed the relationship between respiratory viruses (sampled by bronchoscopy) and pft results after pex treatment in cf pediatric patients, and they observed a strong association between respiratory viruses and newly acquired common respiratory pathogens and a worse recovery of fev1 in the months following bronchoscopy for patients infected with respiratory viruses. 13 we performed a complete non-invasive assessment of viral respiratory infection in both upper and lower airways; such a method was useful because in 9% of the collected specimens, viral analysis was negative by nasal swab, but positive in sputum. virus and mainly rhinoviruses are associated with asthma exacerbations. in our study, one patient had asthma and was was treated with inhaled corticotherapy. the main limitation of our study is that we included a small number of patients and that we could have designed a fourth visit later to follow the fev1 of non-responder's children. moreover, our microbiological analysis was not exhaustive, because atypical mycobacteria, anaerobic bacteria, and fungi were not investigated. some studies show that, in the upper airways, rhinovirusassociated clinical symptoms are more likely the result of local and systemic immune responses than a consequence of direct cytopathogenic effects. 14 nevertheless, the pathophysiology of rhinovirus-associated pex remains unclear in cf, and two recent studies showed an exaggerated bronchial inflammation associated with rhinovirus infection in airways epithelial cells in patients with cf. 15 further large-scale analyses are required to study the impact of rhinoviruses on failure to recover to previous fev 1 levels in cf children. failure to recover to baseline pulmonary function after cystic fibrosis pulmonary exacerbation incidence and risk factors for pulmonary exacerbation treatment failures in patients with cystic fibrosis chronically infected with pseudomonas aeruginosa return of fev1 after pulmonary exacerbation in children with cystic fibrosis human rhinovirus infection in children with cystic fibrosis impaired innate host defense causes susceptibility to respiratory virus infections in cystic fibrosis role of respiratory viruses in pulmonary exacerbations in children with cystic fibrosis effect of respiratory virus infections including rhinovirus on clinical status in cystic fibrosis report from the eurocarecf working group on outcome parameters in clinical trials assessing time to pulmonary function benefit following antibiotic treatment of acute cystic fibrosis exacerbations susceptibility testing of pseudomonas aeruginosa isolates and clinical response to parenteral antibiotic administration: lack of association in cystic fibrosis heterogeneity of treatment response to azithromycin in patients with cystic fibrosis consensus study group. treatment of lung infection in patients with cystic fibrosis: current and future strategies respiratory viruses are associated with common respiratory pathogens in cystic fibrosis human rhinoviruses innate inflammatory responses of pediatric cystic fibrosis airway epithelial cells: effects of nonviral and viral stimulation the french association "vaincre la mucoviscidose" gave financial support to this study. the funding agency had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. none. key: cord-306923-eujbxdqi authors: ahmed, anwar e.; al‐jahdali, hamdan; alaqeel, mody; siddiq, salma s.; alsaab, hanan a.; sakr, ezzeldin a.; alyahya, hamed a.; alandonisi, munzir m.; subedar, alaa t.; ali, yosra z.; al otaibi, hazza; aloudah, nouf m.; baharoon, salim; al johani, sameera; alghamdi, mohammed g. title: factors associated with recovery delay in a sample of patients diagnosed by mers‐cov rrt‐pcr: a saudi arabian multicenter retrospective study date: 2018-04-25 journal: influenza other respir viruses doi: 10.1111/irv.12560 sha: doc_id: 306923 cord_uid: eujbxdqi background: research evidence exists that poor prognosis is common in middle east respiratory syndrome coronavirus (mers‐cov) patients. objectives: this study estimates recovery delay intervals and identifies associated factors in a sample of saudi arabian patients admitted for suspected mers‐cov and diagnosed by rrt‐pcr assay. methods: a multicenter retrospective study was conducted on 829 patients admitted between september 2012 and june 2016 and diagnosed by rrt‐pcr procedures to have mers‐cov and non‐mers‐cov infection in which 396 achieved recovery. detailed medical charts were reviewed for each patient who achieved recovery. time intervals in days were calculated from presentation to the initial rrt‐pcr diagnosis (diagnosis delay) and from the initial rrt‐pcr diagnosis to recovery (recovery delay). results: the median recovery delay in our sample was 5 days. according to the multivariate negative binomial model, elderly (age ≥ 65), mers‐cov infection, icu admission, and abnormal radiology findings were associated with longer recovery delay (adjusted relative risk (arr): 1.741, 2.138, 2.048, and 1.473, respectively). camel contact and the presence of respiratory symptoms at presentation were associated with a shorter recovery delay (expedited recovery) (arr: 0.267 and 0.537, respectively). diagnosis delay is a positive predictor for recovery delay (r = .421; p = .001). conclusions: the study evidence supports that longer recovery delay was seen in patients of older age, mers‐cov infection, icu admission, and abnormal radiology findings. shorter recovery delay was found in patients who had camel contact and respiratory symptoms at presentation. these findings may help us understand clinical decision making on directing hospital resources toward prompt screening, monitoring, and implementing clinical recovery and treatment strategies. laboratory-confirmed middle east respiratory syndrome coronavirus (mers-cov) has been documented in more than 2000 cases worldwide, causing 722 related deaths from september 2012 through september 2017. 1 much research evidence is available on factors associated with a poor prognosis in laboratory-confirmed mers-cov [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] and non-mers-cov 9-11 patients. a high mortality rate was systematically recognized in mers-cov patients of old age, 2,12-15 severe illness, 2,12-14 underlying condition, 2,12-14 and respiratory/gastrointestinal symptoms. 2 however, successful management of mers-cov such as clinical recovery and its predictors has not been given sufficient attention despite the virus having been in circulation since 2012. as per the authors' knowledge, two studies have so far addressed clinical improvement on laboratory-confirmed mers-cov patients. 16, 17 the first study, shalhoub et al, 16 was based on a case report in which their observations may not be generalized to a wider mers-cov population. the second study, al-turaiki et al, 17 utilized publicly available data from the saudi ministry of health. the major shortcomings in their study were several potential confounding factors such as underlying medical conditions and a primary or secondary mode of mers-cov transmission that had not been included in the analysis. in addition, the recovery delay was not reported in their study, and thus, factors related to the recovery delay were not examined. as of october 4, 2017, there was no available detailed data on recovery delay of laboratory-confirmed mers-cov and non-mers-cov patients. data on the time intervals between a patient's presentation or admission to a healthcare facility and the first specimen sample have been limited in patients suspected and screened for mers-cov by a real-time reverse-transcription polymerase chain reaction (rrt-pcr) test, as it might correlate with recovery delay intervals. early screening and diagnosis of mers-cov could greatly promote proper control and clinical management of cases, which may reduce the risk of transmission and increase the chance of successful outcomes. [18] [19] [20] this study provides more understanding of how long a period (in days) it may take to recover from mers-cov infection. the authors have studied, retrospectively, a cohort of survivorslaboratory-confirmed mers-cov and non-mers-cov patients-to estimate recovery delay intervals and identify possible associated factors in saudi arabia. the authors assessed whether the time interval between presentation and the initial rrt-pcr diagnosis (diagnosis delay) correlates with the time interval between initial rrt-pcr diagnosis and recovery (recovery delay). we hypothesized that older age, mers-cov infection, icu admission, and abnormal radiology findings might be associated with longer recovery delay. we hypothesized that diagnosis delay might positively correlate with a recovery delay. a multicenter retrospective study reviewed medical records of 829 patients from september 2012 to june 2016 who were admitted to the hospital and had been diagnosed by rrt-pcr assay for suspected mers-cov to have mers-cov and non-mers-cov infection. the study included patients who were admitted through emergency departments (pediatrics and adults) or patients who were admitted through outpatient clinics. screening referrals for mers-cov was made in accordance with the guidelines set by the saudi ministry of health in standard risk assessment algorithms for identifying and managing mers-cov infection. 21 in the study population, the rrt-pcr was used to detect mers-cov in multiple and/or different clinical specimens, including combined nasopharyngeal and throat swabs, sputum, blood, stool, and endotracheal aspirate (eta). the study gathered data from the two largest hospitals in saudi from patient charts, we collected demographic data: age and gender. elderly age was defined by classifying age into two groups using a cutoff of 65 years (≥65 years). the reason behind this classification was to assess the recovery delay for this vulnerable age group, as a previous study reported a high mortality rate in this group. 15 we collected data on route of transmission: camel contact and patient contact. the study authors collected various clinical data: fever (temperature ≥ 38°c); presence of any of the following respiratory symptoms: cough, bloody cough, shortness of breath, or chest pain; presence any of the following gastrointestinal symptoms: diarrhea, vomiting, or nausea; mers-cov infection; intensive care unit (icu) admission; hospital: kamc-riyadh or kfgh-jeddah; abnormal radiology findings; diabetes; renal disease; and hypertension. recovery delay was calculated as the number of days from the initial rrt-pcr diagnosis (±), which was the date found on the pathology report of the first specimen, to the clinical recovery (recovery delay), based on the date of hospital discharge or date of mers-cov or non-mers-cov infection was ruled out. in some cases, the clinical recovery was verified by taking a sample from different types of specimens at varying times. in all patients with initial rrt-pcr result, the medical records were reviewed from the date of presentation/hospital admission until 60 days after the initial rrt-pcr diagnosis. only patients who achieved recovery were analyzed. the study excluded patients with no available clinical recovery records and no discharge records within 60 days after the initial rrt-pcr diagnosis, as well as patients who had died. the final sample included 396 laboratory-confirmed mers-cov and non-mers-cov patients who had recovered and were identified by reviewing patient charts, hospital discharge records, and medical practitioner notes. the analysis was conducted using ibm statistical package for social sciences (spss) (24; spss, chicago, il). patients' characteristics were described by count and percent, and mean (± standard deviation) or median where appropriate. time intervals in days from presentation to initial rrt-pcr diagnosis (diagnosis delay) and from initial rrt-pcr diagnosis to recovery (recovery delay) were analyzed by spearman's correlation coefficient. the poisson and negative binomial models were used to model the frequency of recovery delay in days and identify unadjusted and adjusted factors associated with longer recovery delay. goodnessof-fit measures were used to compare and identify the best model. the model with the smaller deviance, larger log likelihood, smaller akaike information criterion, and smaller bayesian information criterion was considered the better model. in all analyses, a p-value of less than 5% was considered significant. relative risk (rr) and 95% confidence intervals (ci) were used to assess the strength of association between patients' characteristics and longer/shorter recovery delay. a total of 396 patients, suspected and screened for mers-cov by an rrt-pcr test, were analyzed. the average age was 46 years with age ranges between 1 and 95 years. the median recovery delay in our sample was 5 days. of the sample, 57.7% were male and 18.4% were admitted to icu. fever and respiratory symptoms were common presentations, occurring in 66.3% and 84.1% of the patients, respectively. the chest x-ray and/or ct scan were abnormal in almost half of the samples (48.4%). refer to table 1 for other sample parameters. the longer delays in diagnosis were positively correlated with longer recovery delay (r = .421; p = .001). according to univariate negative binomial regression analysis ( a multivariate negative binomial regression analysis (table 2) revealed six independent factors that affect the recovery delay. 22 and necessitate mechanical ventilation, 11 which is a risk factor for hospital mortality. 23 patients admitted to icu admission were at higher risk for longer recovery delay. a previous report showing similar findings, longer icu stay, and high mortality rate was reported in mers-cov patients who were admitted to the icu. 24 such patients would benefit from monitoring their responses to medical support and recognizing potential complications at an early stage. we found that camel contact was associated with shorter recovery delay. studies on recovery delay in patients with camel contact as compared to close contact exposure of a confirmed case or other exposure are lacking; however, camel contact has also been linked to lower 3-and 30-day mortality rates in mers-cov patients. 2 this important association requires more studies to identify whether camel contact is an independent protective factor of shorter recovery delay. in our study, patients with respiratory symptoms are more likely to experience shorter recovery delay than patients without respiratory symptoms. this finding is probably related to the shorter lag time between symptom onset and diagnosis, in which patients with presence of symptoms could be positively affected by an early diagnosis 18 and thus prompt medical support is deemed necessary. the time interval from presentation to initial rrt-pcr diagnosis (diagnosis delay) was positively correlated with the time interval from initial rrt-pcr diagnosis to recovery (recovery delay). early diagnosis is likely to improve clinical outcomes 18 and reduce the economic and physical burden of a disease. 25, 26 early diagnosis requires full utilization of hospital resources. individuals at high risk of mers-cov infection should be promptly screened after arrival at the healthcare facility, monitored for progression, and then having a prompt decision made for whether further rrt-pcr testing is needed. the authors noticed the following limitations. first, the study was based on chart reviews, and findings should be interpreted with caution. second, we did not collect information on the type of antiviral treatment or other supportive treatments given after diagnosis which may have affected clinical outcomes. 27, 28 third, despite this being the first investigation in this population, including a number of potential predictors for recovery delay, additional relevant predictors should be explored, such as the level of camel exposure, for example, hospital-acquired infections. fourth, patients with clinical recovery were identified by reviewing the medical records of the study sample within 60 days after the initial rrt-pcr diagnosis. studies with longer periods of follow-up in a larger population recovering from mers-cov are warranted to assess the long-term successful clinical outcomes. despite the mentioned limitations, data were aggregated directly from medical charts rather than public source databases. this chart review study was based on information from multicenters and a large sample size, and it provides valuable information on factors associated with prolonged or shorter recovery delay of patients suspected and screened for mers-cov by the rrt-pcr test. it is essential to develop interventional programs or guidelines to ensure early diagnosis, as this may reduce recovery delay intervals as well as improve patients' clinical outcomes. this research may enable identification of patients who require receiving appropriate medical support and care according to their illness progression. this also may prevent spread and transmission of the infection as individuals who are still severely ill can be appropriately isolated and managed apart from others who are responding to medical care. the study evidence supports that longer recovery delay was seen in patients with older age, mers-cov infection, icu admission, and abnormal radiology findings in a sample of patients diagnosed by rrt-pcr. recovery delay was significantly shorter in patients who had camel contact and respiratory symptoms at presentation. a prospective study is needed to evaluate the impact of camel exposure on recovery. evidence was found of an increasing recovery delay with a longer diagnosis delay. the findings may help understand clinical decision making as it directs hospital resources toward prompt screening, monitoring, and implementing clinical recovery and treatment strategies. there are no conflict of interests. middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment the predictors of 3-and 30-day mortality in 660 mers-cov patients estimating survival rates in mers-cov patients 14 and 45 days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study descriptive epidemiology and characteristics of confirmed cases of middle east respiratory syndrome coronavirus infection in the makkah region of saudi arabia estimation of merscoronavirus reproductive number and case fatality rate for the spring 2014 saudi arabia outbreak: insights from publicly available data an outbreak of middle east respiratory syndrome (mers) due to coronavirus in al-ahssa region, saudi arabia cd8 + t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome fatality risks for nosocomial outbreaks of middle east respiratory syndrome coronavirus in the middle east and south korea middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study spatial modelling of contribution of individual level risk factors for mortality from middle east respiratory syndrome coronavirus in the arabian peninsula risk factors for severity and mortality in patients with mers-cov: analysis of publicly available data from saudi arabia risks of death and severe disease in patients with middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus outbreak in the republic of korea successful recovery of mers cov pneumonia in a patient with acquired immunodeficiency syndrome: a case report building predictive models for mers-cov infections using data mining techniques diagnostic delays in 537 symptomatic cases of middle east respiratory syndrome coronavirus infection in saudi arabia the clinical and virological features of the first imported case causing mers-cov outbreak in south korea middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications case definition and surveillance guidance -updated early identification of pneumonia patients at increased risk of mers-cov infection in saudi arabia mechanical ventilation in patients in the intensive care unit of a general university hospital in southern brazil: an epidemiological study the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study accuracy and cost comparison in medical testing using sequential testing strategies reducing cost in sequential testing: a limit of indifference approach antibody response and disease severity in healthcare worker mers survivors recovery from the middle east respiratory syndrome is associated with antibody and t cell responses factors associated with recovery delay in a sample of patients diagnosed by mers-cov rrt-pcr: a saudi arabian multicenter retrospective study http://orcid.org/0000-0001-8743-6007 key: cord-285425-3v1bid02 authors: macintyre, chandini raina; chughtai, abrar ahmad; rahman, bayzidur; peng, yang; zhang, yi; seale, holly; wang, xiaoli; wang, quanyi title: the efficacy of medical masks and respirators against respiratory infection in healthcare workers date: 2017-08-30 journal: influenza other respir viruses doi: 10.1111/irv.12474 sha: doc_id: 285425 cord_uid: 3v1bid02 objective: we aimed to examine the efficacy of medical masks and respirators in protecting against respiratory infections using pooled data from two homogenous randomised control clinical trials (rcts). methods: the data collected on 3591 subjects in two similar rcts conducted in beijing, china, which examined the same infection outcomes, were pooled. four interventions were compared: (i) continuous n95 respirator use, (ii) targeted n95 respirator use, (iii) medical mask use and (iv) control arm. the outcomes were laboratory‐confirmed viral respiratory infection, influenza a or b, laboratory‐confirmed bacterial colonisation and pathogens grouped by mode of transmission. results: rates of all outcomes were consistently lower in the continuous n95 and/or targeted n95 arms. in adjusted analysis, rates of laboratory‐confirmed bacterial colonisation (rr 0.33, 95% ci 0.21‐0.51), laboratory‐confirmed viral infections (rr 0.46, 95% ci 0.23‐0.91) and droplet‐transmitted infections (rr 0.26, 95% ci 0.16‐0.42) were significantly lower in the continuous n95 arm. laboratory‐confirmed influenza was also lowest in the continuous n95 arm (rr 0.34, 95% ci 0.10‐1.11), but the difference was not statistically significant. rates of laboratory‐confirmed bacterial colonisation (rr 0.54, 95% ci 0.33‐0.87) and droplet‐transmitted infections (rr 0.43, 95% ci 0.25‐0.72) were also lower in the targeted n95 arm, but not in medical mask arm. conclusion: the results suggest that the classification of infections into droplet versus airborne transmission is an oversimplification. most guidelines recommend masks for infections spread by droplets. n95 respirators, as “airborne precautions,” provide superior protection for droplet‐transmitted infections. to ensure the occupational health and safety of healthcare worker, the superiority of respirators in preventing respiratory infections should be reflected in infection control guidelines. there is currently a lack of consensus around the efficacy of medical masks and respirators for healthcare workers (hcws) against influenza, with only five published randomised control trials (rcts) in hcws conducted to date. [1] [2] [3] [4] [5] while n95 respirators have been shown to be superior to medical masks in preventing clinical respiratory infection (cri), influenza illness (ili) and other outcomes, none of the studies were adequately powered to examine laboratory-confirmed influenza. in the smallest of the trials, involving only 32 hcws, there was no difference in the rates of respiratory illnesses between hcws who used medical masks and the control group. 1 a canadian study of 422 hospital nurses compared targeted use of n95 respirators and medical masks and found that the rate of serologically defined influenza was 25% in both arms. 2 however, in the absence of a control arm for comparison, the finding of no difference in outcomes between the intervention arms could represent either equal efficacy or equal inefficacy of the two interventions. the other two published hcw rcts used a more specific and less sensitive definition of influenza based on nucleic acid testing (nat) of respiratory specimens in symptomatic subjects. as such, even these substantially larger rcts were unable to demonstrate any significant difference in influenza infection between n95 respirators and medical masks. 3, 4 finally, a recent study examined the efficacy of cloth masks compared to medical mask and control groups, and found that cloth masks may increase the risk of infection in hcws. 5 guidelines for respiratory protection have been driven by presumed transmission mode alone, and under an assumption that influenza and other pathogens are spread by one mode alone. 6 however, the paradigm of unimodal droplet or airborne spread is based on outmoded experiments from the 1940s, which concluded that only large droplets are found at close proximity to the patient, while small droplet nuclei and airborne particles are found at a longer distance. [7] [8] [9] it has since been shown that both small and large particles can exist at short distances from the patient, and that aerosolised transmission can occur at close proximity. 9 in our two published rcts conducted in china, 3,4 we used the same outcomes, case definitions and measurement tools, and used the same testing methods for a range of different pathogens transmitted by different routes. this afforded an opportunity to pool the data from both trials for improved statistical power to examine the outcomes by pathogens and mode of transmission. the aim of this pooled analysis was to examine the efficacy of medical masks and respirators in hcws against respiratory infection. we pooled the results of our two rcts on mask and respirator use in hospital hcws in beijing, china. the first rct (trial 1) was conducted from december 2008 to january 2009, 3 hcws randomised to: medical mask arm (n = 492), n95 fit-tested arm (n = 461) and n95 non-fit-tested arm (n = 488). the rate of fit-test failure was very low (5/461) in this trial, so data from both n95 arms were combined for analysis. an additional 481 healthcare workers from nine hospitals were recruited to a control arm. these hospitals were purposefully selected as they indicated low levels of routine mask/respirator use during a pretrial assessment. participants in the control arms continued their usual mask wearing practices and were followed using the same protocol as applied to the other arms. 3 the second trial (trial 2) was conducted from 28 december 2009 to 7 february 2010, using the same design. 4 in trial 2, participants were randomised to three arms: medical masks at all times on shift (n = 572), continuous n95 respirators at all times on shift (n = 516) and targeted/intermittent use of n95 respirators only while doing high-risk procedures or barrier nursing of a patient with known respiratory illness (n = 521). fit testing was not performed in the second rct. in both trials, participants were followed for 4 weeks of wearing the medical masks or respirators, and an extra week of non-wearing of masks for the development of symptoms. demographic and clinical data were collected, including gender, age, smoking, vaccination status, pre-existing medical illnesses, hand hygiene and high-risk procedures. pharyngeal swabs were collected from symptomatic participants, and samples were tested at the laboratories of the beijing centers for disease control and prevention. there was no major difference in the products used in both clinical trials. in the first trial, we used medical masks (3m, catalogue number 1820) and n95 fit/ non-fit-tested respirator (3m, catalogue number 9132). the following products were used in the second trial: medical masks (3m, catalogue number 1817) and respirator (3m, catalogue number 1860). the interventions compared in the pooled analysis were as follows: (i) continuous use of n95 respirators (pooled data from both trials -1530 subjects); (ii) targeted n95 respirator use (data from trial 2-516 subjects); (iii) continuous use of medical masks (pooled data from both trials -1064 subjects) and (iv) and a control group (data from trial 1-481 subjects). only laboratory-confirmed outcomes were included in the analysis, which were defined and measured identically in both trials, and the laboratory testing has previously been described. 3, 4 laboratory-confirmed bacteria and viruses identified in participants were categorised according to droplet (n = 285), contact (n = 6) and airborne (n = 3) transmission modes (table s1a) . sixty-one coinfection cases with multitransmission were categorised separately. (table s1a) . as the largest number of confirmed infections was in the droplet category, we conducted a subgroup analysis of droplet-transmitted infections. given there were a large number of rsv cases (n = 33) in our data set and rsv is variously categorised as | 513 either "droplet" 11 or "contact" spread 12 in different guidelines, we performed a sensitivity analysis by including rsv into the droplet transmission category instead of contact. ethics approvals of two clinical trials were obtained from the institutional review board and human research ethics committee of the beijing center for disease prevention and control. we did not involve patients and their families in the design and conduct of the study. we have acknowledged the support of participants, and the results will be published in open access journal. the data sets from the two trials were pooled incorporating the common variables. we calculated the attack rate (proportion of outcome) of each of the four outcomes by the study arms. we conducted a fixed effect individual patient data (ipd) metaanalysis by fitting a multivariable log binomial model, using generalised estimating equation (gee) to account for clustering by hospital/ward. we used a fitted fixed effect model because there are only two trials. two studies were conducted in the same setting with similar participant characteristics, and they examined the same underlying effect. in the analysis, relative risk (rr) was estimated using the control arm as the referent category after adjusting for potential confounders and their interaction terms with a trial id number. the overall rates of seasonal infection were higher in the second trial than the first. the consistency assumption (ie between study homogeneity) for the ipd meta-analysis was tested by fitting an interaction term between trial id and trial arms where a significant interaction is indicative of inconsistency. 13 any interaction term (between trial id and covariates other than trial arm) that was not a confounder was subsequently excluded from the model using backward elimination approach. this approach is described in detailed elsewhere. 4 we repeated the above-described methods for each of the outcomes. after combining the data sets from the two trials, 3591 cases were in the ipd meta-analysis, none of the interaction terms between trial arm and trial id was significant for any of the outcome variables. thus, the consistency assumption for the ipd meta-analysis was satisfied. however, a significant interaction was observed between trial id and hand washing for laboratory-confirmed bacterial colonisation only; therefore, we estimated the rr for trial id stratified by hand washing. in the similar analysis, the risk of influenza was also lower in medical mask arm compared to control; however, the difference was not statistically significant (rr 0.81 and 95% ci 0.25-2.68) arm. table 5 compares the results of this analysis with the individual studies. we demonstrated superior clinical efficacy of continuous use of n95 respirator (also known as "airborne precautions") against infections presumed to be spread by the droplet mode, including influenza. this suggests that transmission is more complex than assumed by traditional classifications, and supports the fact that both large and small droplets are present close to the patient, and that aerosol transmission may occur for presumed "droplet" infections. respirators are designed to provide respiratory protection through filtration and fit, and properly fitted respirators provide better protection compared to medical masks. 3, 4 we could not demonstrate efficacy of medical masks against any outcome, but the non-significant trend appeared to be towards protection. medical masks may well have efficacy, 5 but if so, the degree of efficacy was too small to detect in this study, and larger studies are needed, given the widespread use of these devices in health care. a diagnosis of influenza requires the detection of virus from respiratory specimens, or a fourfold rise in serological titres, both of which are highly resource-intensive and depend on daily subject follow-up and on optimal timing of specimen collection. for all these reasons, the published studies to date have been unable to determine whether there is a difference in efficacy against influenza infection between medical masks and n95 respirators. this study can therefore usefully inform policies for prevention of influenza. in the first rct, compared to medical masks, n95 respirators were found to be protective against cri, but not against ili or laboratoryconfirmed influenza. 3 when compared with the control arm, rates of laboratory-confirmed virus and bacterial colonisation were significantly lower in n95 arm (table 5 ). in the second rct, continuous use of n95 respirators was associated with lower rates of cri and laboratory-confirmed bacterial colonisation compared to the medical mask use. 4 pooled analysis of these studies improved the power to analyse other infectious outcomes by intervention and to allow analysis by mode of transmission. an important finding of this analysis was the efficacy of n95 respirators against droplet-transmitted infections. generally, medical masks are considered sufficient for droplet-transmitted infections such as influenza. 18 however, this study has demonstrated a clear benefit of using n95 respirators (both continuous and targeted) to protect hcws against droplet infections and does not show significant protection of medical masks. in the light of these findings, it may be prudent to use respirators when the transmission mode of a disease is unknown or when hcws exposed to droplet-transmitted infections with a high-case fatality rate. 6 this study has some limitations. firstly, the reporting of the results included in figure 1 is different from the ipd meta-analysis results. this is due to the uneven distribution of randomisation arms and differing seasonal attack rates between the trials. in figure 1 , these between-trial differences were not taken into account. the ipd meta-analysis takes into account of these and gives an unbiased association. secondly, the control arm in trial 1 was not randomised; however, the risk of bias is less due to similar study setting, outcome measures and participant characteristics. moreover, whether infection was acquired in the community or the hospital cannot be determined, but the rct design should result in community exposure being distributed equally across all arms. finally, we categorised pathogens according to various transmission modes, while certain viruses are transmitted via multiple routes. the pooled data were suggestive of an effect of respirators against influenza, but probably did not have enough statistical power for this outcome. the major strength of this study is the use of the same endpoints, measurements and methods in the two trials, which allowed valid pooling of the data. it is a long-held belief in hospital infection control that a mask is adequate for droplet-transmitted infections. we showed that the use of respirators provides better protection against respiratory infections, even those presumed to be spread predominantly by the droplet mode. the targeted use of a respirator was also effective, whereas no efficacy was demonstrated for medical masks alone. however, the trends suggest some degree of protection from medi3. for many infections, more than one mode of transmission is possible, and our data suggest that transmission of infections is more complex than suggested by these paradigms. clinical efficacy data are a higher level of evidence than theoretical paradigms of transmission, and show better protection afforded by respirators. use of surgical face masks to reduce the incidence of the common cold among health care workers in japan: a randomized controlled trial surgical mask vs n95 respirator for preventing influenza among health care workers: a randomized trial a cluster randomized clinical trial comparing fit-tested and non-fit-tested n95 respirators to medical masks to prevent respiratory virus infection in health care workers. influenza other respir viruses a randomized clinical trial of three options for n95 respirators and medical masks in health workers a cluster randomised trial of cloth masks compared with medical masks in healthcare workers respiratory protection for healthcare workers treating ebola virus disease (evd): are facemasks sufficient to meet occupational health and safety obligations? bacteriologic procedures in the evaluation of methods for control of air-borne infection transmission of ebola viruses: what we know and what we do not know health workers need optimal respiratory protection for ebola public health agency of canada. pathogen safety data sheets and risk assessment center for disease control and prevention (cdc). respiratory syncytial virus infection (rsv): transmission and prevention public health agency of canada indirect and mixed-treatment comparison, network, or multiple-treatments meta-analysis: many names, many benefits, many concerns for the next generation evidence synthesis tool prevention strategies for seasonal influenza in healthcare settings infection prevention and control of epidemic-and pandemic-prone acute respiratory infections in health care exposure to influenza virus aerosols during routine patient care measurements of airborne influenza virus in aerosol particles from human coughs guideline for isolation precautions: preventing transmission of infectious agents in health care settings guidance on personal protective equipment to be used by healthcare workers during management of patients with ebola virus disease in u.s. hospitals, including procedures for putting on (donning) and removing (doffing) interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) uncertainty, risk analysis and change for ebola personal protective equipment guidelines ebola and marburg virus disease epidemics: preparedness, alert, control, and evaluation infection prevention and control during health care for probable or confirmed cases of novel coronavirus (ncov) infection centers for disease control and prevention and world health organization. infection control for viral haemorrhagic fevers in the african health care setting facemasks for the prevention of infection in healthcare and community settings availability, consistency and evidence-base of policies and guidelines on the use of mask and respirator to protect hospital health care workers: a global analysis this is pooled analysis of two clinical trials. key: cord-002257-30s14h9j authors: ratnamohan, vigneswary m.; zeng, frank; donovan, linda; macintyre, chandini r.; kok, jen; dwyer, dominic e. title: phylogenetic analysis of human rhinoviruses collected over four successive years in sydney, australia date: 2016-08-09 journal: influenza other respir viruses doi: 10.1111/irv.12404 sha: doc_id: 2257 cord_uid: 30s14h9j background: human rhinoviruses (hrv) cause a wide spectrum of disease, ranging from a mild influenza‐like illness (ili) to severe respiratory infection. molecular epidemiological data are limited for hrv circulating in the southern hemisphere. objectives: to identify the species and genotypes of hrv from clinical samples collected in sydney, australia, from 2006 to 2009. methods: combined nose and throat swabs or nasopharyngeal aspirates collected from individuals with ili were tested for hrv using real‐time reverse‐transcriptase polymerase chain reaction (rt‐pcr). sequencing data of 5′utr and vp4/vp2 coding regions on rt‐pcr‐positive specimens were analysed. results: human rhinoviruses were detected by real‐time pcr in 20.9% (116/555) of samples tested. phylogenetic analysis of 5′utr and vp4/vp2 on hrv‐positive samples was concordant in the grouping of hrv a and b species but not hrv c species. eighty per cent (16/20) of sequences that grouped as hrv c in the vp4/vp2 tree clustered as hrv a, alongside some previously described c strains as subspecies c/a. discordant branching was seen within hrv a group: two sequences clustering as a in the vp4/vp2 tree branched within the c/a subspecies in the 5′utr tree, and one sequence showed identity to different hrv a strains in the two genes. the prevalence of hrv c and c/a species was greater in paediatric compared to adult patients (47.9% vs 25.5%, p = .032). conclusion: human rhinoviruses are a common cause of respiratory infections, and hrv c is present in the southern hemisphere. sequencing of multiple hrv regions may be necessary to determine exact phylogenetic relationships. human rhinoviruses (hrv) are a diverse virus group, currently known to contain 167 serotypes. 1 along with human enteroviruses (hev), hrv belong to the picornaviridae family, 2 although they are phylo genetically unrelated to hev despite similarities in genome organization and structure. human rhinoviruses are generally associated with the common cold and mild upper respiratory infections, 3 but can also cause severe respiratory infections in immunocompromised hosts (including lung and hematopoietic stem cell transplant recipients) or patients with chronic pulmonary diseases. [4] [5] [6] [7] unlike respiratory syncytial virus (rsv) and influenza viruses, hrv can cause respiratory illness throughout the year, but peak incidence occurs in early autumn and spring in temperate climates. 6 early molecular analyses of the hrv capsid protein coding regions clustered different serotypes into two distinct species, hrv a and hrv b. 8 in 2007, a new hrv genetic variant (subsequently designated hrv c) was identified in patients with severe pneumonia from the united states of america (usa), germany, hong kong, australia and china. [9] [10] [11] [12] [13] severe respiratory disease and asthma exacerbations in children were observed. 11, 14 hrv c has been further divided into two subspecies, hrv cc and hrv ca. 15 hrv c strains are difficult to grow in cell lines known to support the growth of other rhinoviruses, 11 although two hrv c isolates have been propagated in nasal epithelial cell cultures. 16 there are limited molecular epidemiological data on hrv circulating in the southern hemisphere, including australia. this study aimed to identify the species and genotypes of hrv from clinical samples collected in sydney, australia, over four consecutive years by analysing the nucleotide homology in the 5′utr, vp4 and part of the vp2 capsid protein coding regions. primers used to amplify all picornaviruses, 18 the hrv-specific probes 5′utr and vp4-vp2 gene sequencing primers are listed in table 1 . the rt-pcr probe sequences were designed to include most hrv sequences available in genbank ® in 2007, and sequences generated from the present study. rna was prepared directly from combined nts or npa using roche high pure rna kits (roche, mannheim, germany). the cdna was reverse-transcribed from 10 μl of specimen rna using 100 units of superscript iii reverse transcriptase (invitrogen, carlsbad, ca, usa) and 4 μl used for real-time pcr as described previously. 19 amplification was performed in glass capillaries on human rhinoviruses rt-pcr-positive samples were then tested using utr primers ev140 and ev170 to amplify a 395-bp product. 20 where rna was available, samples were amplified with primers rhi3a and evp4r, generating a 638-bp fragment that included part of 5′utr, all of vp4 and part of vp2 regions. 18 cs07-7-a24 a a a cs09-5-a24 a a a cs09-22-a24 a a a cs09-3-a33 a a a cs09-12-a33 a a a •cs09-2-a67 a branches with hrv89 aa ms07-20-a57 a a a ms07-21-a57 a a a cs09-6-a21 a a a cs09-17-a21 a a a cs09-21-a21 a a a ms06-4-a61 a a a ms06-6-a61 a a a cs09-15-a41 a a a ms08-8-a55 a a a cs09-11-a58 a a a cs09-26-a58 a a a ms06-2-a89 a a a cs09-28-a8 a a a cs07-9-1b a a a cs07-12-1b a a a cs07-13-1b a a a cs07-14-1b a a a cs07-18-1b a a a ms08-6-a43 a a a cs09-4-a43 a a a ms07-16-a31 a a a cs09-13-a31 a a a cs09-20-a47 a a a cs07-24-a56 a a a cs09-23-a98 a a a ms08-2-a15 a a a ms08-3-a15 a a a cs07-6-a15 a a a cs09-27-a15 a a a cs07-4-a22 a a a cs09-1-a23 a a a cs09-8-a23 a a a (continues) using blast ® , nearly all the sequences showed >98% similarity to one or more hrv sequences with partial cds available in genbank ® . these were approximately 390-420 bp and did not cover the full sequence from 5′utr to vp2 of our study samples, and were not included in the construction of the ml tree. as the data size was large, it was not possible to include each one of the hrv prototype sequences in the tree construction. figure 1 shows the ml tree for the 5′utr for all 93 sequences, and figs 2 and 3 show the ml trees, respectively, of vp4/vp2 and 5′utr/vp4/vp2 regions of the 65 samples. in group c with vp4/vp2 analysis also clustered in group c in the 5′utr along with reference strain c-eu840728; 16 of the 20 hrv c samples grouped as hrv a, along with reference strains c-x2-ef077280, c-eu840952, c-dq875932, c-jq994498, c-ef077279, c-ef582387 and c-gq223227. four clades that grouped as c species in the vp4/vp2 analysis segregated into the a genogroup in the 5′utr analysis (figs 1 and 2) . discordant branching was seen in the following and indicated by a circle figure 1 ). samples with asterisk (*) denote strains that grouped as c in the vp4/vp2 region, but grouped with hrv a species. samples with only 5′utr sequences are denoted by ^. discordant branches in the vp4/vp2 and 5′utr sequences are indicated by • sample ms06-3 and hrv a12 (ef173415) clustered in a clade more related to c-dq875932 (subspecies ca). the two samples showed between 80% and 82% id to c-dq875932 strain and samples in that cluster and similar identity to a15 and its cluster in the utr tree, but in the vp4/vp2 tree the two samples showed 61% identity to c-dq875932 and lower to other c strains and 62% and 80% identity to a 15 and 1b, respectively. maximum-likelihood tree of the 5′utr/vp4/vp2 region, showing relationships of clinical strains, newly described strains and prototype strains of hrv (constructed as described for fig. 1 ). samples with asterisk (*) denote strains that grouped as c in the vp4/vp2 region, but grouped with hrv a species in 5′utr analysis. discordant branches in the vp4/vp2 and 5′utr sequences are indicated by • the 65 sample sequences segregated into three phylogenetically distinct species: 44 (67.7%) hrv a, 20 (30.8%) hrv c and one (1.5%) hrv b. several clades were represented within hrv a and c (fig. 2) . within hrv a, identity at > 86% was seen with known hrv reference strains as shown in table 2 the grouping of samples as c and ca in the 5′utr/vp4/vp2 analysis was very similar to that seen in the 5′utr analysis. the discordant branching pattern seen with samples ms06-3 and hrv a12 (ef173415), ms07-18 and ms08-7 and cs09-2 (indicated with •) in the 5′utr ml tree was not seen; it was in agreement with that seen in the vp4/vp2 ml tree. in the distribution of the different hrv subtypes is shown in table 3 . both hrv a and c or c/a variant were detected in higher numbers than hrv b. human rhinoviruses c or c/a variant was detected more in our study, the sequences grouped into three phylogenetically distinct species: a (52.6%), c/ca (37.6%) and b (9.6%). however, there was discordance between proposed phylogeny groups when sequences from the 5′utr and vp4/vp2 coding regions were analysed. sixteen of the 20 samples that clustered as hrv c in the vp4/ vp2 ml tree segregated as a in the 5′utr analysis, as did some of the early, well-characterized hrv c strains from new york (eu840952 and dq875932), 23, 26 san francisco (ef077279 and ef077280), 25 hong kong special administrative region (ef582387) 11 and china (gq223227). 15 twelve of 28 sequences with only utr sequences also segregated along with the above-mentioned reference c strains, branching in two major subgroups within hrv a. these have been labelled in this study as c/a, clustering with c strains, but grouping as a in the 5′utr region: these formed 30% of the clinical sequences. huang et al. 15 reported similar clustering of field strains in their study and designated the above c strains (eu840952, dq875932, ef077279, ef077280, gq223227 and ef582387) as ca, a subspecies of hrv c that clustered differently to hrv a, hrv b and hrv c in the utr. they further showed that hrv ca subspecies were formed from interspecies recombination in the 5′utr region. similar inconsistent clustering of field strains as compared to vp4/vp2 was also reported in the 3d polymerase-coding region as well as 5′utr region. 30 three discordant branching events were seen in our analyses. ms07-18 (identity to a65) in one branch and ms06-3 along with hrv a12 in another branch segregated as a in the vp4/vp2 analysis, but localized within the ca subspecies in the 5′utr analysis. sample cs09-2 clustered with hrv a89 with 96% relatedness in the utr region but differed in the vp4/vp2 region. these three samples may represent recombinants as reported by palmenberg et al. and kim et al. 31, 32 there is no designated region within the hrv genome that is 26, 34 the clustering of hrv c as c and c/a when 5′utr region was included in the analysis (figs 1 and 3 ) in our study may suggest that they are true c species that showed recombination with a in the 5′utr. in this study, the 5′utr primers and the primers chosen to amplify the entire vp4 and partial vp2 region produced amplicons with overlapping sequences that resulted in an approximately 900bp continuous sequence. we used a single set of primers for each of the two pcr assays but did not use cloning, which has been used in other studies. 24 were collected from patients with respiratory illness who were sick enough to warrant testing or in many cases hospitalization, but our data are insufficient to attribute clinical severity to any of the hrv it is possible that hrv c or the c/a variants may cause exacerbation of respiratory infections in infants that require presentation to ed compared to hrv a or b, and this may contribute to the higher percentage of hrv c or c/a infection in the paediatric population. one of the early reports of hrv c-qpm variant severity was from australian samples collected in 2003 from children with lower respiratory infection. 37 other reports have observed the association between hrv c variants and asthmatic wheeze and severe lower respiratory infections. 10, 11, 23, 27, 38 xiang et al. 35 reported that the clinical manifestations of hrv a and c are similar, and co-infection with rsv and hrv in infants increases the severity of infection. both hrv a and c are more virulent than hrv b in infants and hrv virulence is greater in winter, although peak infection rates occur in spring and fall. 9 in conclusion, the present study shows that sequencing of one region alone is insufficient for determining the lineage of the hrv variants. the presence of many diverse strains has become apparent, and it is likely that more will emerge. genotypic assignment and identification of hrv types will facilitate monitoring of emerging novel variants, and investigations into type-associated differences in disease epidemiology, transmission and outcomes. samples used in this study were not collected for this study per se, but as part of a study assessing the use of face mask in controlling respiratory virus transmission in households following approval by the local institutional review board or for laboratory diagnosis of patients with an influenza-like illness. the present study does not involve the reporting of patient data, and no patient intervention occurred with the obtained results. virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses a collaborative report: rhinoviruses-extension of the numbering system from 89 to 100 viruses and bacteria in the etiology of the common cold rhinovirus infections in hematopoietic stem cell transplant recipients with pneumonia detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients human rhinoviruses human rhinovirus c in adult hematopoietic stem cell transplant recipients with respiratory illness genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70 human rhinovirus species and season of infection determine illness severity a recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in germany clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus c (hrv c) human rhinovirus group c infection in children with lower respiratory tract infection assay for 5′ noncoding region analysis of all human rhinovirus prototype strains evidence of recombination and genetic diversity in human rhinoviruses in children with acute respiratory infection molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus c face mask use and control of respiratory virus transmission in households detection of human picornaviruses by multiplex reverse transcription-pcr and liquid hybridization pandemic clinical case definitions are non-specific: multiple respiratory viruses circulating in the early phases of the 2009 influenza pandemic in new south wales enterovirus 68 is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses detection and differentiation of picornaviruses in clinical samples following genomic amplification a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood masstag polymerase-chainreaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity new respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses human rhinovirus c: age, season, and lower respiratory illness over the past 3 decades proposals for the classification of human rhinovirus species a, b and c into genotypically assigned types analysis of complete genome sequences of human rhinovirus molecular characterization of human rhinovirus field strains isolated during surveillance of enteroviruses sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution identification of recombinant human rhinovirus a and c in circulating strains from upper and lower respiratory infections clinical and molecular features of human rhinovirus c analysis of genetic diversity and sites of recombination in human rhinovirus species c human rhinovirus c infections mirror those of human rhinovirus a in children with community-acquired pneumonia phylogenetic analysis of rhinovirus isolates collected during successive epidemic seasons molecular characterization and distinguishing features of a novel human rhinovirus (hrv) c, hrvc-qce, detected in children with fever, cough and wheeze during a novel group of rhinoviruses is associated with asthma hospitalizations we thank ms. gordana nedeljkovic for compiling patient information from the laboratory information system network. funding: none.competing intesrests: none declared. vmr participated in the study design, analysed the results and drafted the manuscript. fz and ld performed nucleic acid testing. crm was the chief investigator of the study on the use of face masks to reduce household transmission of respiratory viruses. jk had input in the preparation and editing of the manuscript. ded participated in the design of the study and was involved in manuscript editing. all authors have read and accepted the manuscript. additional supporting information may be found online in the supporting information tab for this article. key: cord-259368-k8t8brjy authors: ren, xiang; li, yu; yang, xiaokun; li, zhili; cui, jinzhao; zhu, aiqin; zhao, hongting; yu, jianxing; nie, taoran; ren, minrui; dong, shuaibing; cheng, ying; chen, qiulan; chang, zhaorui; sun, junling; wang, liping; feng, luzhao; gao, george f.; feng, zijian; li, zhongjie title: evidence for pre‐symptomatic transmission of coronavirus disease 2019 (covid‐19) in china date: 2020-08-07 journal: influenza other respir viruses doi: 10.1111/irv.12787 sha: doc_id: 259368 cord_uid: k8t8brjy background: between mid‐january and early february, provinces of mainland china outside the epicentre in hubei province were on high alert for importations and transmission of covid‐19. many properties of covid‐19 infection and transmission were still not yet established. methods: we collated and analysed data on 449 of the earliest covid‐19 cases detected outside hubei province to make inferences about transmission dynamics and severity of infection. we analysed 64 clusters to make inferences on serial interval and potential role of pre‐symptomatic transmission. results: we estimated an epidemic doubling time of 5.3 days (95% confidence interval (ci): 4.3, 6.7) and a median incubation period of 4.6 days (95% ci: 4.0, 5.2). we estimated a serial interval distribution with mean 5.7 days (95% ci: 4.7, 6.8) and standard deviation 3.5 days, and effective reproductive number was 1.98 (95% ci: 1.68, 2.35). we estimated that 32/80 (40%) of transmission events were likely to have occurred prior to symptoms onset in primary cases. secondary cases in clusters had less severe illness on average than cluster primary cases. conclusions: the majority of transmissions are occurring around illness onset in an infected person, and pre‐symptomatic transmission does play a role. detection of milder infections among the secondary cases may be more reflective of true disease severity. a novel coronavirus named "severe acute respiratory syndrome coronavirus 2" (sars-cov-2) was first identified in january 2020 as the pathogen responsible for a cluster of cases of atypical pneumonia in wuhan, a large city located in hubei province in central china. 1, 2 genetic analysis of the virus indicates that it originated from a bat coronavirus. 2 sars-cov-2 is considered distinct from sars-cov or mers-cov, and coronavirus disease 2019 caused by it has rapidly become a global health concern. 3 incidence of infections slowly increased through january 2020 with a reproductive number estimated to be in the range 2.2-3.6. [4] [5] [6] [7] starting in mid-january 2020, covid-19 cases began to be identified in other cities in china and also in other countries. 8, 9 a number of studies suggested the probable phenomenon of covid-19 transmissions during incubation period, [10] [11] [12] these studies used cluster cases in limited number of families for analysis, and the relative frequency of pre-symptomatic transmission was not quantified. moreover, there was an analysis on publicly available data indicating the existence of negative serial intervals, also implying pre-symptomatic transmission. 13 here, we retrospectively analyse data on cases identified outside of hubei province through the chinese public health event surveillance system at the early stage of transmission in china, in order to provide insights on the transmission dynamics of covid-19. data were extracted from the epidemiological reports of the chinese public health event surveillance system, through which the first confirmed case or potential outbreaks leading to clusters of suspected cases for each county were required to be investigated and reported. the extracted variables included demographic data, possible exposure and travel history, and clinical data using a structured form. the events reported by 29 january 2020 were included for data extraction. for events by 23 january 2020, which was the date of "locking down" wuhan city, all events (168 cases) in the system were extracted, and we used these data to construct an epidemic curve and estimate the incubation period distribution. of the events (281 cases) reported between 24 january and 29 january 2020, we focused on those events which included probable human-to-human transmission or epidemiologically linked cases, so that we could capture a larger number of these clusters for analyses of serial intervals, transmission events and comparative severity between primary cases and secondary cases. all cases included in our analyses were laboratory-confirmed cases, and the case definitions followed we drew the epidemic curve by illness onset for cases who reported that they had been in wuhan in the 14 days of preceding onset, and a separate epidemic curve for the other cases. cases with onset dates closed to the end of epidemic curve may not be reported due to delays in seeking medical attention and consequent delays in laboratory testing. to allow for onset-to-reporting delays, we used the nowcasting approach described by van de kassteele et al 15 to jointly estimate the augmented case number on the most recent dates and the onset-to-reporting distribution. based on 1000 augmented epidemic curves, then we fitted exponential growth models to obtain estimates of the growth rate and doubling time. we examine the characteristics of confirmed cases and compared the demographics, onset symptoms and results of some clinical tests between confirmed sporadic and cluster primary cases and cluster secondary cases. we also obtained information on secondary cases that had not been to wuhan but had close contact with another case that had been to wuhan and were presumed to be infected by that particular case. in these pairs of primary and secondary cases, we fitted a normal distribution to the serial intervals between illness onset dates, allowing for negative and zero serial intervals, and correcting for growth rates in the early stage of an epidemic. specifically, the parameters of the fitted normal distribution were corrected to normal (µ′,σ) where µ′ = µ + σ 2 ρ for the epidemic growth rate r estimated below (personal communication, neil ferguson). because information was available on the periods during which each secondary case had been exposed to their presumptive infector, we were able to identify cases where exposure is likely to have occurred before or after illness onset in the primary case infector. when the exposure window overlapped the onset date, we used our fitted incubation period distribution to resample 1000 infection times at random, excluding any that fell outside the exposure window, and counted the proportion of resampled times that fell before symptom onset in the infector. we collected detailed information on 168 confirmed cases, including 145 and 23 reporting that they had or had not, respectively, been in wuhan in the past 14 days. the demographics of the 145 and 23 cases were very similar (data not shown). the median age was 48 f i g u r e 1 panel a: occurrence by date of illness onset of cases identified outside of hubei province in persons with a history of travel from wuhan in the 14 d prior to onset. panel b: occurrence by date of illness onset of cases identified outside of hubei province in persons without a history of travel from wuhan in the 14 d prior to onset. panel c: augmented occurrence (yellow bars) by date of illness onset of cases identified outside of hubei province in persons with a history of travel from wuhan in the 14 d prior to onset with back filled cases considering delays between illness onset and seeking care and being tested (range 10-81) and 91 (54%) were male. only three (2%) of the cases who had visited wuhan also reported visiting the huanan seafood wholesale market. figure 1 shows the epidemic curve by illness onset of the cases in persons that had been to wuhan compared to cases in other persons that were presumed to represent onwards transmission. we used data augmentation to correct for reporting delays in cases with recent onset ( figure 1c ) and estimated that the growth rate was 0.14 per day (95% ci: 0.11, 0.17), and the doubling time was 5.3 days (95% ci: 4.3, 6.7). in this analysis, the mean onset to reporting delay was estimated to be 5.7 days (95% ci: 5.3, 6.1). we obtained data on exposure windows for 98 cases in individuals that had been in wuhan, by assuming that they had been infected while they were in wuhan. we fitted a lognormal distribution to the data on exposure periods and onset dates, correcting for epidemic growth, and estimated that the incubation period had mean 5. (14%) occurred in those who had a meal together, 2 (3%) were colleagues working together, 2 (3%) were those who took the same vehicle, and 1 (1%) occurred among neighbours. the identified 80 observations of transmission events were used for estimation of the serial interval ( figure 2b ). we fitted a normal distribution to these data, with allowing 4 negative serial intervals and correcting for epidemic growth, and estimated that the serial interval distribution had mean 5.7 days (95% ci: 4.7, 6.8) and standard deviation 3.5 days ( figure 2b ). in detailed investigations of contact patterns, we obtained information on the period when the secondary infection could have occurred, and related this to the illness onset date of the infector. figure 3 shows that of 80 pairs identified, pre-symptomatic transmission occurred in 9 pairs, post-symptomatic transmission occurred in 16 pairs, and in the remaining 55 pairs, transmission could have occurred either before or after the corresponding infector's illness onset date. in these 55 pairs, we used monte carlo simulations based on the incubation period distribution described above to estimate that 23/55 of transmission events had greater than 50% chance to be pre-symptomatic transmissions. thus, in total we infer that 32/80 (40%) of the observed transmission events were likely or very likely to have occurred prior to the onset of symptoms in the infector. we examined demographic and clinical characteristics of all the confirmed sporadic and clusters cases ( table 1) . age distributions were similar between the two groups of patients, while less male cases were identified in cluster secondary cases (43% vs 60%). cluster primary and sporadic cases were generally more severe, with 26% and 7% in severe and critical conditions, respectively, compared with 12% and 1% in cluster secondary cases. nearly all the primary and sporadic cases (96%) had a radiologic indication of pneumonia, compared with 74% in the secondary cases. among 80 cluster secondary cases, 87% had been classified as mild cases, while 66% (110/166) of the cluster primary and sporadic cases were mild. the primary and sporadic cases showed largely similar onset symptoms as the secondary cases. fever is the most common symptom, followed by headache, fatigue, dry cough and myalgia. generally higher proportions of the cluster primary cases and sporadic cases reported an onset of the symptoms than cluster secondary cases (table 1 ). in addition to systemic and respiratory presentations, a small proportion of the cases also reported gastrointestinal symptoms, including vomiting and diarrhoea. in this study, we report estimates of the transmission dynamics of covid-19 based on cases identified outside of hubei province. importantly, we examine quantitative evidence for pre-symptomatic infectiousness, a feature which complicates control strategies. we transmission events used to infer the occurrence of pre-symptomatic transmission. dots indicate the dates of onset of primary and secondary cases, and the shaded area in each row indicates the period of exposure of the secondary case to the primary case during which the secondary infection is thought to have occurred. brackets indicate the exposure window when the primary case was thought to have been infected. data were resolved to the nearest day, and so transmission windows are plotted from the start of the first date to the end of the last date, onset dates are plotted in the middle of the corresponding day, and if the secondary onset date is the same as the primary onset date, then the former is offset slightly so that both can be seen note: for patients having clinical test results of both lymphocyte count and proportion, a normal lymphocyte count refers to both the lymphocyte count within the range of 1-4 × 10 9 /l and the lymphocyte proportion to be 20%-40%. patients with either lymphoycyte count or lymphocyte proportion lower or higher than the normal range will be classified as "decreased" or "increased," respectively. a category of lymphocyte count is determined by both the count and proportion of lymphocyte (the proportion is derived as lymphocyte count divided by white blood cell count) in the blood test. demonstrate that the phenomenon of pre-symptomatic transmission is not uncommon, having occurred in a minimum of 9/80 transmission events ( figure 3 ). using statistical inference based on our estimated incubation period distribution, we estimated that pre-symptomatic transmission was likely to have occurred in up to 40% of the 80 transmission events in our data set. this observation should be interpreted in the context of isolation of some cases after illness onset, reducing the amount of post-symptomatic transmission that might otherwise have occurred. this also implies that social distancing measures may be some of the most important strategies to reduce transmissibility, for example closing schools and encouraging, or facilitating working at home, in addition to control of onwards transmission by sequestering symptomatic persons at home or in isolation facilities. our finding was consistent with other reports that the serial interval distribution was similar to, or much lower than the incubation period distribution, 13,18-21 consistent with the occurrence of pre-symptomatic transmission in case reports. [22] [23] [24] [25] [26] pre-symptomatic infectiousness is generally not thought to occur for most respiratory viruses, but measles is a well-known example of a respiratory infection that can be spread before symptom onset, 27 and viral shedding during the incubation period has also been reported for influenza. 28 viral shedding has been reported during the incubation period for covid-19, 29 and there were also asymptomatic cases for covid-19 across all age groups, in varying proportions depending on the intensity of surveillance and testing. 30, 31 in conducting investigations of clusters of cases, it is often found that secondary cases have on average milder illnesses than primary cases; for example, this was noted for middle east respiratory syndrome coronavirus infections. 32 here, we found that secondary cases were more likely to have milder disease (table 1) , which would be consistent with the existence of some milder covid-19 infections, which were not laboratory confirmed. the cluster investigations that we report here are not likely to represent the full spectrum of mild infections, and better information would be provided by prospective studies of close contacts with repeated collection of respiratory swabs and sera. our estimates of the growth rate of infections in wuhan in early january 2020 of around 0.14 per day (95% ci: 0.11, 0.17) are very consistent with previous reports. 4, 6 our estimate of the effective reproductive number in wuhan is now slightly lower than the previous estimate of r 0 4 and some other estimates of r 0 5-7 but that is because of the shorter serial interval. we included a limited number of cases in other cities in china that were detected in persons that had not been to wuhan ( figure 1b ), but the sample size was insufficient at the time of analysis to determine whether there has been sustained transmission in any other cities. the incubation period averaged 4.6 days and up to 11.1 days in 95% of infections (figure 2a) , which is important for specifying quarantine periods and also for understanding transmission dynamics. the city of wuhan was put under lockdown on 23 january 2020, and other nearby cities were also locked down on the following days. this stopped exportation of infections to other chinese cities, and the effect of this intervention has become more apparent in early february, given the delays that occur between infection, illness onset, admission to hospital and then laboratory testing. we restricted the study participants to covid19 analysis were also subject to ascertainment of symptom onset date, which was self-reported by the patient and could be less reliable especially at the early stage of symptom presentation. in conclusion, our analysis showed evidence indicative of pre-symptomatic transmission of covid-19. quarantine of exposed persons having close contact with infected cases before symptom onset could reduce the risk of further transmission. we gratefully thank benjamin j. cowling, peng wu, jessica y. wong, yiu chung lau, tim k. tsang from school of public health, university of hong kong for their guidance and support on study design, data analysis and interpretation. we appreciate the staff members of the county-, city-and province-level centers for disease control and prevention of china for their fieldwork on case investigation, data collection and validation. clinical features of patients infected with 2019 novel coronavirus in wuhan a novel coronavirus from patients with pneumonia in china a novel coronavirus outbreak of global health concern early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia pattern of early human-to-human transmission of wuhan nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak journey of a thai taxi driver and novel coronavirus first case of 2019 novel coronavirus in the united states potential presymptomatic transmission of sars-cov-2 covid-19 transmission within a family cluster by presymptomatic carriers in china presumed asymptomatic carrier transmission of covid-19 serial interval of covid-19 among publicly reported confirmed cases new coronavirus pneumonia prevention and control program (second edition) 2020. in chinese nowcasting the number of new symptomatic cases during infectious disease outbreaks using constrained p-spline smoothing alternative methods of estimating an incubation distribution: examples from severe acute respiratory syndrome how generation intervals shape the relationship between growth rates and reproductive numbers serial interval of novel coronavirus (covid-19) infections temporal dynamics in viral shedding and transmissibility of covid-19 estimating the generation interval for coronavirus disease (covid-19) based on symptom onset data quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing presymptomatic transmission of sars-cov-2 -singapore delivery of infection from asymptomatic carriers of covid-19 in a familial cluster asymptomatic and presymptomatic sars-cov-2 infections in residents of a long-term care skilled nursing facility a covid-19 transmission within a family cluster by presymptomatic infectors in china transmission of covid-19 in the terminal stages of the incubation period: a familial cluster evidence base of incubation periods, periods of infectiousness and exclusion policies for the control of communicable diseases in schools and preschools the dynamic relationship between clinical symptomatology and viral shedding in naturally acquired seasonal and pandemic influenza virus infections viral load of sars-cov-2 in clinical samples estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship a well infant with coronavirus disease 2019 (covid-19) with high viral load middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility evidence for pre-symptomatic transmission of coronavirus disease 2019 (covid-19) in china key: cord-263927-hnsyas9q authors: peci, adriana; winter, anne‐luise; gubbay, jonathan b.; skowronski, danuta m.; balogun, elizabeth i.; de lima, cedric; crowcroft, natasha s.; rebbapragada, anu title: community‐acquired respiratory viruses and co‐infection among patients of ontario sentinel practices, april 2009 to february 2010 date: 2012-08-09 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2012.00418.x sha: doc_id: 263927 cord_uid: hnsyas9q please cite this paper as: peci et al. (2012) community‐acquired respiratory viruses and co‐infection among patients of ontario sentinel practices, april 2009 to february 2010. influenza and other respiratory viruses 7(4), 559–566. background respiratory viruses are known to cocirculate but this has not been described in detail during an influenza pandemic. objectives to describe respiratory viruses, including co‐infection and associated attributes such as age, sex or comorbidity, in patients presenting with influenza‐like illness to a community sentinel network, during the pandemic a(h1n1)pdm09 in ontario, canada. methods respiratory samples and epidemiologic details were collected from 1018 patients with influenza‐like illness as part of respiratory virus surveillance and a multiprovincial case–control study of influenza vaccine effectiveness. results at least one virus was detected in 668 (65·6%) of 1018 samples; 512 (50·3%) had single infections and 156 (15·3%) co‐infections. of single infections, the most common viruses were influenza a in 304 (59·4%) samples of which 275 (90·5%) were influenza a(h1n1)pdm09, and enterovirus/rhinovirus in 149 (29·1%) samples. the most common co‐infections were influenza a and respiratory syncytial virus b, and influenza a and enterovirus/rhinovirus. in multinomial logistic regression analyses adjusted for age, sex, comorbidity, and timeliness of sample collection, single infection was less often detected in the elderly and co‐infection more often in patients <30 years of age. co‐infection, but not single infection, was more likely detected in patients who had a sample collected within 2 days of symptom onset as compared to 3–7 days. conclusions respiratory viral co‐infections are commonly detected when using molecular techniques. early sample collection increases likelihood of detection of co‐infection. further studies are needed to better understand the clinical significance of viral co‐infection. a novel influenza virus, a(h1n1)pdm09 emerged in april 2009 and spread rapidly, primarily through human-tohuman transmission. several million people were infected globally. 1 an important feature of this virus was that it mostly affected younger people with 60% of patients under 18 years of age, suggesting possible pre-existing immunity in the elderly due to previous exposure to antigenically related influenza strains. 2, 3 the assumption made in most pandemic plans before 2009 was that the pandemic virus would be the dominant circulating respiratory virus. 4 few studies performed extensive respiratory testing beyond influenza during the pandemic, and fewer still focused on community cases. casalegno et al. documented cocirculation and co-infection of a(h1n1)pdm09 with rhinovirus during the pandemic. 5 watanabe et al. found a wide range of etiologic agents were identified among respiratory samples that were influenza negative, highlighting the need to diagnose other viral organisms that can co-circulate with influenza. 6 louie et al. investigated samples from laboratory-confirmed fatal a(h1n1)pdm09 cases during the pandemic and bacterial pathogens were identified in 22 of 77 samples. 7 prior to the 2009 pandemic, respiratory viral co-infection was reported in 7-27% of respiratory samples submitted for viral diagnosis. [8] [9] [10] [11] [12] [13] higher proportions of influenza a, respiratory syncytial virus (rsv), parainfluenza viruses, and rhinovirus, compared with other circulating viruses have been detected in patients with co-infections. [14] [15] [16] [17] [18] co-infection has not been fully explored due to limitations of several studies. some studies focused on younger age groups, hospitalized patients or deceased individuals, which does not represent the general population. [8] [9] [10] 12, 15, 19, 20 others have utilized a small sample size or limited their focus to certain viral pathogens, underestimating the role of other viruses in co-infection. 9, 15, 16, 20, 21 this study enrolled community patients presenting with (ili) to a community sentinel network, during the influenza pandemic a(h1n1)pdm09 in ontario, canada and documented the profile of respiratory viruses causing ili symptoms. this study aimed to describe respiratory viruses including co-infections and host-associated attributes such as age, sex, and comorbidity. data were collected as part of a multiprovincial case-control sentinel network study that has been described elsewhere. 21 the sentinel network included 117 sentinels across the province of ontario (with a population of 13ae4 million) who volunteered to participate in the study. it was anticipated that each sentinel would submit an average of 1-2 samples per week from their clinical practice during the study period, april 21, 2009 to february 25, 2010 . this period was chosen to span the full pandemic in ontario. eligible patients were ontario residents, who presented to a sentinel's office with influenza-like illness (ili) within seven days of symptom onset; number and selection of eligible patients was at the sentinel's discretion. ili was defined as acute onset of fever and cough and one or more of the following: sore throat, myalgia, arthralgia, headache or prostration. standard information was collected including date of birth, sex, chronic conditions, symptom onset, and sample collection date. the main outcome was the number of respiratory viruses detected per sample. samples were categorized as negative, single infection or co-infection when no virus, one virus, or at least two viruses were detected, respectively. age was determined as age at symptom onset and categorized as 0-4, 5-14, 15-29, 30-54, 55-64, and 65+years. time to sample collection was calculated as the difference between sample collection and symptom onset dates and categorized as less than or equal to twodays or 3-7 days. chronic condition was defined as heart ⁄ lung ⁄ renal ⁄ metabolic ⁄ blood ⁄ immune conditions or conditions that compromise the management of respiratory secretions and increase risk of aspiration and categorized as yes ⁄ no. this study was approved by the university of toronto's ethics board and all patients gave verbal consent to participate. a nasal or nasopharyngeal sample was collected from each patient using starswabò multitrans collection swab and transported at 4°c for testing at public health ontario laboratory (phol)-toronto; in this study, each sample represents one patient. viral rna was extracted directly from samples using nuclisens ò easymag ò (biomérieux, inc., marcy l'etoile, france). samples were tested for influenza a and influenza b by influenza real-time reverse transcriptase (rrt)-pcr and also for influenza a, influenza b, enterovirus ⁄ rhinovirus, rsv, parainfluenza, adenovirus, coronaviruses, and metapneumovirus by a commercial multiplex pcr method [luminex respiratory viral panel (luminex molecular diagnostics, toronto, on, canada) or seeplex rv (seegene usa, rockville, md, usa)]. in the event of discrepant results between the two methods, positive results for influenza a by either method were considered positive. rrt-pcr was used for subtyping of influenza a samples; all influenza a specimens were subtyped, but not all attempts were successful. statistical analyses were performed using stata software version 10.0 (statacorp, college station, tx, usa). descriptive analyses were conducted to derive the proportion of single, co-infection and no infection as well as describe patient characteristics using chi-square. crude and adjusted multinomial logistic regression were employed to evaluate any association of single, co-infection and noinfection with patient characteristics including age, sex, chronic condition, and time to sample collection. odds ratios (or) with 95% confidence intervals (ci) were calculated. a total of 1018 respiratory samples from 1018 patients with influenza-like illness were included in this study after excluding 102 (9ae1%) samples that did not meet study inclusion criteria (figure 1 ). at least one respiratory virus was detected in 668 (65ae6%) of the samples. of the 831 detected viruses, influenza a was the most frequent accounting for 452 (54ae4%) followed by enterovirus ⁄ rhinovirus 194 (23ae3%) and rsv 120 (14ae4%) ( table 1) . of 452 influenza a viruses, 408 (90ae3%) were a(h1n1)pdm09, two (0ae4%) were h3, and 42 (9ae3%) could not be subtyped presumably due to low viral load. peaks in detection for influenza a occurred in june and october 2010, for enterovirus ⁄ rhinovirus in september 2010, and for rsv in october 2010 ( figure 2 ). a single virus was detected in 512 (50ae3%) samples. of these, 304 (59ae4%) were influenza a and 208 (40ae6%) were other respiratory viruses, the most common being enterovirus ⁄ rhinovirus, detected in 149 (29ae1%) samples (table 2) . peaks for single infection occurred in june and september 2009, which were mainly due to the increase in influenza a and enterovirus ⁄ rhinovirus, respectively ( figure 3 ). viral co-infection was detected in 156 (15ae3%) of the samples of which 149 (95ae5%) were dual infections and seven (4ae5%) triple infections. one hundred and fortyeight (94ae9%) of the co-infections were combination of a(h1n1)pdm09 and another respiratory virus and eight (5ae1%) were non-influenza combinations. the most common co-infections were influenza a ⁄ rsv b and influenza a ⁄ enterovirus ⁄ rhinovirus, responsible for 64ae1% and 21ae8% of co-infections, respectively ( table 3 ).the highest proportion of co-infection was detected in october, corresponding with peak activity of influenza a and rsv ( figure 3 ). the median age of patients in the study was 23 years with a range of 3 months-96 years of age ( table 4 ). the highest proportion of single and co-infections was observed in children 5-14 and 0-4 years of age, respectively. the proportion of those with no infection detected steadily increased with age, peaking at the elderly, aged 65 years and over ( figure 4 ). females were overrepresented, comprising 599 (58ae8%) of the patients included in this study. patients with no-infection, single infection, and co-infection did not differ with regards to sex. two hundred and nineteen (21ae6%) patients had a chronic condition. of these, 37ae4% had no virus detected, 44ae8% had single infections, and 17ae8% had co-infections, whereas among the 795 participants without comorbidities, the distribution was 33ae6%, 51ae8%, and 14ae6%, respectively; however, that was not statistically significant ( table 4 ). the median number of days from symptom onset to sample collection was two with a range of 0-7 days. five hundred and eighty-two (57ae2%) and 436 (42ae8%) samples were collected within 2 days and 3-7 days, respectively. of the 582 samples collected within 2 days of onset, 32ae9% had no virus detected, 48ae5% had single infections, and 18ae6% had co-infections, whereas among those collected within 3-7 days, the distribution was 36ae3%, 52ae8%, and 11%, which was statistically significant. in crude and adjusted multinomial logistic regression, patients with single and co-infections were compared to those with no infection. compared to the elderly, patients under 65 years of age were more likely to have a single infection; the highest likelihood was observed in children 5-14 years of age (table 5) . patients under 30 years of age were more likely to have co-infections compared with patients 65 and over; this was most evident in the 0-4 age group. presence of a chronic condition did not increase the likelihood of single infection but increased the likelihood of co-infection; this did not achieve significance. co-infection was more likely detected in patients who had samples collected within 2 days as compared to 3-7 days; this did not apply for those with single infections. there was no sex difference. in this study, 66% of samples tested during the 2009 pandemic in ontario had at least one virus detected and 15% had co-infections. these findings are consistent with reports from other studies with the range of co-infection reported from 7-27%. [8] [9] [10] [11] [12] [13] however, positivity and co-infection rates vary widely between studies. there are various reasons for this finding: firstly, detection methods differ notably between studies, which impacts sensitivity, specificity and other technical parameters; secondly, viruses targeted differ from one study to another as does the study population. 21 this study was conducted during the influenza pandemic a(h1n1)pdm09 which was associated with an increased number of samples submitted and high detection of figure 2 ). this demonstrates the importance of monitoring circulating respiratory viruses when advising clinicians to prescribe antivirals empirically during a pandemic. 2 (0-7) 2 (0-7) 2 (0-7) 2 (0-7) *p-value <0ae05 is considered significant. **time to specimen collection = collection date-symptom onset date. despite the higher prevalence of enterovirus ⁄ rhinovirus (23ae4%) than rsv (14ae5%), co-infection with a(h1n1) pdm09/rsv was more common than a(h1n1)pdm09 ⁄ enterovirus ⁄ rhinovirus, accounting for 64ae1% and 22ae8% of the co-infections, respectively. this may reflect the younger age of patients infected by a(h1n1)pdm09, who were therefore also at greater risk of rsv. in addition, rsv cocirculated with a(h1n1)pdm09 more than enterovirus ⁄ rhinovirus, which peaked before the second wave (figure 2 ). there may also be preferential interactions among certain pathogens; viral interactions were not assessed in this study. 23 when other respiratory samples positive for enterovirus ⁄ rhinovirus were further evaluated at our laboratory, they all were confirmed as rhinovirus, not enterovirus. 24 single infection was more commonly detected in those less than 65 years of age. it is known that respiratory infections are more common in children for several reasons, including an immature immune system, lack of preexisting immunity particularly to new emerging viruses, and greater viral exposure opportunities. 11, 18, 25 younger patients shed higher levels of virus when infected and also may be brought for medical care earlier than older patients, facilitating detection in these groups. 26 in addition, lower detection of single and co-infection in elderly may be explained by pre-existing immunity to a(h1n1)pdm09 and other respiratory viruses. 2 co-infection was more common in persons less than 30 years of age as compared to older adults. these data are congruent with findings from a previously published study where co-infection was more likely in younger than older adults. 18 the combined effect of predominance of a(h1n1)pdm09 and the greater likelihood of infection with other respiratory viruses among younger ages likely explains our age-related findings of co-infection during the pandemic, which may not be generalizable to a typical influenza season. the presence of comorbidities did not increase the likelihood of having a single infection but increased the likelihood of co-infections; this did not achieve statistical significance. patients with chronic conditions are at higher risk of severe disease and consequently may be more likely to seek medical care. 26 selection bias is unlikely to influence these results as the proportion of patients with comorbidities was similar (21ae5%) to that in ontario's population (20ae3%). 27 sample collection within 2 days of symptom onset was found to independently increase the likelihood of detecting a viral co-infection but not single infection. long et al. reported an inverse relationship between duration of symptoms and viral detection rate due to greater viral shedding earlier in the disease process. 11 this study was designed to examine circulating viruses and co-infection. the presence of more than two viruses in the same sample may not always indicate clinical infection. as viruses may be detected in asymptomatic patients, it is impossible to determine which viruses caused symptoms. previous studies suggest co-infection may manifest higher disease severity, which may shorten the time to medical care and viral detection; disease severity was not assessed in the current study. 19, 28 as viral-bacterial co-infections also occurred during the pandemic, it will be interesting for further studies to investigate their characteristics and impact on disease severity. 28, 29 in summary, a(h1n1)pdm09 was frequently detected among community patients with ili. however, other respiratory viruses cocirculated with a(h1n1)pdm09 during the pandemic, reinforcing the need to test for other viral agents even during a pandemic to appropriately guide clinical treatment decisions. viral diagnosis, primarily a(h1n1)pdm09, was made more often in patients less than 65 years of age. viral co-infection was commonly detected in this study and was most likely detected in individuals less than 30 years of age. earlier sample collection improves the detection of viral co-infections. understanding the contribution of other circulating respiratory pathogens during a pandemic may lead to improved individual diagnosis and recommendations for community-based clinicians, and more effective prevention and treatment of respiratory infections, including use of influenza antivirals. novel swine-origin influenza a (h1n1) virus in humans a (h1n1) influenza virus pandemic: a review complications of seasonal and pandemic influenza who checklist for influenza pandemic preparedness planning 2005. department of communicable disease surveillance and response global influenza programme. available online: http: ⁄ ⁄ a (h1n1)v, rvs: the race for hivernal pandemics respiratory virus infections among hospitalized patients with suspected influenza a h1n1 2009 virus during the first pandemic wave in brazil bacterial coinfections in lung tissue specimens from fatal cases of the association of newly identified respiratory viruses with lowers respiratory tract infections in korean children association of rhinovirus infection with increased disease severity in acute bronchiolitis single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-y response prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens. duration of symptoms significantly affects detection rate frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction multipathogen infections in hospitalized children with acute respiratory infections development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay low mortality rates related to respiratory virus infections after bone marrow transplant incidence of common respiratory viral infections related to climate factors in hospitalized children in hong kong respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology prevalence of human respiratory viruses in adults with acute respiratory tract infections in beijing correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection impact of respiratory virus infections on persons with chronic underlying conditions human metapneumovirus and respiratory syncytial virus in hospitalized danish children with acute respiratory tract infection association between the 2008-09 seasonal influenza vaccine and pandemic h1n1 illness during spring-summer 2009: four observational studies from canada evidence from multiplex molecular assays for complex multipathogen interaction in acute respiratory infections rhinovirus outbreaks in long-term care facilities viruses in communityacquired pneumonia in children aged less than 3 years old; high rate of viral confection correlation of pandemic (h1n1) 2009 viral load with disease severity and prolonged viral shedding in children self-reported ph1n1 influenza vaccination coverage for ontario streptococcus pneumonia coinfection is correlated with the severity of h1n1 pandemic influenza invasive group a streptococcal infection concurrent with 2009 h1n1 influenza authors wish to acknowledge the staff of molecular diagnostics and virus detection departments at public health ontario laboratory-toronto, the ontario and national sentinel influenza vaccine effectiveness study and the college of family physicians of canada funded by the canadian institute of health research. dms was principal investigator on a clinical trial of pediatric influenza vaccine dose response for which influenza vaccine was provided free by sanofi pasteur. jbg has received research grants from glaxosmithkline inc. and hoffman-la roche ltd to study antiviral resistance in influenza. key: cord-276363-m8di6dpt authors: holm, majbrit v.; blank, patricia r.; szucs, thomas d. title: influenza vaccination coverage rates in europe – covering five consecutive seasons (2001–2006) in five countries date: 2008-06-28 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2008.00036.x sha: doc_id: 276363 cord_uid: m8di6dpt objective to understand potential drivers and barriers to influenza vaccination in the general population. methods 47 982 household surveys were conducted in five european countries between 2001 and 2006. results overall influenza vaccination coverage increased over the years and reached 26·2% in 2005/06. among the elderly ≥65 years, the rate increased significantly to 67·8% (2005/06). the most common reason for being vaccinated over the 5 years was the perception of influenza as a serious illness, which people want to avoid. the main reason for not getting vaccinated among those never previously vaccinated was feeling that they were unlikely to catch influenza. a recommendation by the family physician was the most encouraging factor for vaccination. the severity of influenza and the efficacy of vaccination are well documented in the medical literature. 1,2 eradication of influenza is impossible but continuous immunization of the population can minimize the impact of the disease. 3 in addition to providing substantial health benefits, vaccination may also be associated with significant economic benefits, not only among the elderly but also among healthy working adults and children. despite this knowledge and ongoing efforts by policy-makers, physicians and other healthcare providers, influenza vaccination rates in the five european countries surveyed remain limited, with the additional effect that manufacturing capacity may be too low for producing a sufficient amount of an appropriate monovalent vaccine when a pandemic occurs. 3 the who states that the risk of a new pandemic is at its highest level since the last pandemic in 1968. 4 this situation might influence the immunization coverage rates in the population. published literature evaluating vaccination coverage rates in europe shows that importance placed on influenza vaccination varies greatly between countries. 3 two recent studies covering several european countries have been published. 5, 6 this report is an update of the earlier work by szucs and muller. we now have data available for five consecutive influenza seasons which allows us to go beyond the usual cross-sectional approach to analyzing vaccination rates. the main focus of this paper is on high-risk group coverage. a second objective is to understand the determinants for being or not being vaccinated, and to describe the populations' opinions regarding influenza and vaccination. in this context, we examine whether the threat of avian influenza had an impact on recent changes in vaccination coverage in the different countries. this survey is an ongoing assessment of influenza coverage rates in france, great britain, italy, spain, and germany. during [7] [8] [9] [10] [11] four target groups were specified. 1. individuals aged ‡65 years 2. individuals who suffer from a chronic illness 3. individuals who work in the medical field 4. combined group of individuals aged ‡65 years or who suffer from a chronic illness or who work in the medical field. for example, in germany the group of chronic illness sufferers is defined according to the german standing commission on immunization, as children, adolescents and adults suffering from chronic diseases of respiratory organs, chronic cardiovascular or liver diseases, as well as nephropathies and diabetes, or other metabolic disorders. in our study, people suffering from heart diseases, pulmonary diseases, diabetes, or other chronic illnesses were included in the chronic illness group. the survey questions have been published previously. 6 the questions covered reasons to get vaccinated this winter, reasons for not getting vaccinated against influenza, and options that would encourage persons to get vaccinated against influenza. for the 2005 ⁄ 06 survey, questions on influenza pandemics and avian influenza were added. the survey populations were representative of the adult population from age 14 years (germany, italy, spain); from age 15 (france), or from age 16 (great britain). in spain, persons above age 75 were not covered. sample weights were applied to correct for small deviations from the applicable age and gender quota and the annual datasets were pooled. statistical evaluation used spss ò version 13 for windows. bivariate associations of categorical variables were assessed using the chi-squared test. a chi-squared test for trend was used to assess time trends. in the case of continuous variables, differences of means were tested using oneway anova. for all statistical tests, two-sided p = 0ae05 was used as the level of statistical significance. ninety-five percent confidence intervals (ci) were reported as appropriate. due to the descriptive nature of these data, no correction for multiple testing was made. covariates identified as predictors of influenza vaccination in univariate analysis were considered as candidates for multivariable analysis. logistic regression was used to identify independent correlates of the outcome of interest, i.e. vaccination coverage. the overall sample consisted of 47 982 persons. in table 1 an overview of the sample is given for the year 2005 ⁄ 06 only. in an earlier publication, similar data can be found for the years 2002 ⁄ 03 and 2003 ⁄ 04. 6 spain was expected to show a lower number of people over 65 years of age, as the survey covered only persons £75 years old. the reason for the great deviation in the number of chronic ill persons in germany compared to other countries remains unclear as there is no difference in the way the question was asked in the five countries. the overall vaccination coverage across countries, based on an average of the country samples, decreased from 22ae5% (95% ci: 21ae6-23ae4%) in season 2001 ⁄ 02 to 21ae3% (95% ci: 20ae5-22ae1%) in season 2002 ⁄ 03. thereafter, it increased to 23ae4% (95% ci: 22ae6-24ae2%) in season 2003 ⁄ 04, to 23ae6% (95% ci: 22ae7-27ae1%) in season 2004 ⁄ 05, and to 26ae2% (95% ci: 25ae3-27ae1%) in season 2005 ⁄ 06 ( figure 2 ). the increase between season 2004 ⁄ 05 and season 2005 ⁄ 06 was statistically significant (p < 0ae001). this was mainly due to significant increases in immunization uptake in germany and italy, where the coverage increased to 32ae5% and 24ae1%, respectively, in 2005 ⁄ 06. adjusting the overall vaccination rate in europe (weighting the population sizes) resulted in an average vaccination rate of 26ae8% in season 2005 ⁄ 06. vaccination rates were highly age-dependent. older age was associated with higher vaccination rates. in season 2005 ⁄ 06 the immunization uptake across all countries was holm et al. ª 2008 the authors higher for all age groups compared to the previous seasons ( figure 1 ). across all five seasons, vaccination rates appeared to be associated with gender in great britain, italy, and spain. in great britain a higher vaccination rate was observed in women, whereas in italy and spain, the majority of the vaccinated were men (details not shown). in the year 2005 ⁄ 06, 39ae0% of the respondents expressed the intention to get vaccinated against influenza in the coming winter of 2006 ⁄ 07. over the years, the proportion of those expressing such an intention was on average 36%, about 13% higher than the actual vaccination rate. the gap was highest in germany (between 15% and 20% over the years) and almost non-existent in italy in season 2005 ⁄ 06. the overall vaccination coverage rate in persons aged ‡65 increased over time ( figure 2 ). the increase between season 2004 ⁄ 05 and season 2005 ⁄ 06 was statistically significant (p < 0ae001). coverage in the elderly was highest in great britain (79%) and lowest in germany and italy (63ae4%). it was significantly different from the population under 65 years of age. since season 2003 ⁄ 04, data on health status in terms of chronic illness were collected. persons with a chronic illness showed significantly higher vaccination coverage than those not suffering from a chronic disease (figure 2) . the highest coverage among the chronically ill persons was found in great britain (66ae4%) and the lowest in france (51ae8%). working in the medical field did not seem to be a driving factor for vaccination as the vaccination coverage rate in this sub-population was not significantly different from the rest of the sample (figure 2) , at the unadjusted level. however, adjustment for age and other covariates revealed the presence of an association ( table 2 ). for persons in the combined target group a significant difference in coverage was found compared to the non-target group population. the vaccination rate in this group increased over the years and the increase in season 2005 ⁄ 06 was significantly different from the previous season. the coverage rate in the combined target group was highest in great britain (60%) and lowest in germany (49%). however, this result could be influenced by the observed difference in the proportion of chronically ill respondents in germany (table 1) . table 2 shows unadjusted odds ratios for the target groups for the year 2005 ⁄ 06. odds ratios across all seasons did not greatly differ from the 2005 ⁄ 06 results. adjusted odds ratios were investigated in logistic regression models. the adjustment took into account gender, age over 65 years, work in medical field, and chronic illness. for years where data on chronic illness were not available, data were only adjusted for the remaining covariates. the odds ratios for the combined target group were only adjusted for age. multivariate adjustment showed significantly higher vaccination rates for healthcare workers in great britain (or: 1ae8, 95% ci: 1ae5-2ae2), france (or: 1ae7, 95% ci: 1ae4-2ae1), italy (or: 1ae4, 95% ci: 1ae1-1ae9), and spain (or: 2ae1, 95% ci: 1ae6-3ae6). the impact of chronic illness on the vaccination rate was significantly lower after multivariate adjustment, mainly due to taking into account the effect of age (germany or: 2ae3, 95% ci: 2ae0; 2ae6, italy or: 5ae0, 95% ci: 4ae2; 6ae0, france or: 3ae4, 95% ci: 2ae7; 4ae2 and spain or: 3ae3, 95% ci: 2ae8; 4ae0). all other odds ratios were not substantially changed by multivariate adjustment (details not shown). for those who reported to have been vaccinated in season 2005 ⁄ 06, the most frequently stated reasons were that influenza is a serious illness that people want to avoid and that they have received a recommendation from their family physician or nurse (table 3 ). in france the most commonly stated reason for vaccination was that the vaccine is provided free. over the 5-year period, the ranking of the cause for getting vaccinated did not change substantially. the proportion of respondents whose decision to get vaccinated was influenced by the recent attention given to avian influenza or a possible influenza pandemic varied from 13% in germany to 1ae3% in spain. it was 8ae6% in great britain, 4ae1% in italy, and 2ae0% in france. across all countries, the persons who gave the threat of avian influenza as a reason for vaccination were not found to be statistically different from other vaccinated persons (table 3) . only the proportion of those vaccinated for first time was statistically higher among the group influenced by the attention given to avian influenza (p < 0ae001). for those of the total survey population who had never been vaccinated, the reasons for not being vaccinated varied across countries over the 5 years of observation. overall, the most frequently stated reasons were no expectation of catching influenza, not having considered vaccination, and absence of a family physician's recommendation ( table 3) . the level of knowledge about influenza and the vaccine among the general population was similar across countries. seventy-nine percent of the respondents agreed with the statement that you can catch influenza even if you are vaccinated against it. sixty-eight percent agreed with the statement that if you catch influenza after having had the vaccine, the infection is less severe. fifty-eight percent said that it is important to get the influenza vaccine each year and 52% agreed that the side effects associated with the vaccine (fever, headache...) are acceptable. most of the participants did not agree with the following statements: the vaccine is not useful if you are in good health and if you have the vaccine, you will not catch influenza. a recommendation by the family physician or nurse, and receiving more information regarding the vaccine being efficacious and well tolerated were regarded as the most important factors that might encourage vaccination (table 3) . data on this question were not available for france. the vaccination coverage rate in the total sample is currently 26ae2% (season 2005 ⁄ 06). a statistically significant increment of 2ae5% was observed between season 2004 ⁄ 05 and season 2005 ⁄ 06. this was mainly due to significant increases in immunization uptake in germany and italy. in germany reimbursement of vaccination for all age groups has been implemented in several federal states to encourage vaccination rates. 12 this may explain the high coverage rates in germany. a sub-analysis of the data obtained in great britain showed that wales reached a vaccination rate of 33ae3% in season 2005 ⁄ 06, higher than the german coverage rate (32ae5%). the immunization rates in the defined highrisk target groups were also increased in season 2005 ⁄ 06. in particular, higher age and suffering from chronic illness were important predictors of vaccination. in the elderly ‡65 years, the lowest coverage was observed in germany and italy and the highest in great britain. in great britain or, odds ratio; ci, confidence interval; p-value, pearson chi-squared. *reference category. holm et al. ª 2008 the authors general practitioners are encouraged to recommend vaccination to eligible high-risk patients, which may have contributed to the high vaccination rates in the target groups. 9 in spain, vaccination coverage increased in those aged ‡60 after the age threshold of vaccination recommendations was reduced to age 60 in some communities. as healthcare workers are in contact with patients, it is critical for this group to be vaccinated in order to reduce transmission of the disease. additionally, they play an important role in the communication and motivation of the public to get vaccinated. however, the vaccination rate among healthcare workers remained low compared to the other high-risk groups. a recent published literature review identified low coverage rates in this professional group to be especially a problem in europe, 13 with vaccination rates between 12% and 25%. 5 our observation of coverage rates increasing from 19% to 25% over the years is consistent with earlier findings. considering the entire population, 39% of the 2005 ⁄ 06 respondents expressed the intention to get vaccinated in season 2006 ⁄ 07. the gap between those who intended to get vaccinated and those who actually received vaccination was stable over the years, at 13% on average. there was, however, substantial variation between countries. the persistence of this gap indicates the potential to increase vaccination coverage rates in europe. however, realizing this potential, activating the correct drivers, and dealing with the barriers to vaccination remains a challenge. only italy seems to have been able to diminish the gap. in season 2003 ⁄ 04 the italian vaccination campaign was intensified by the ministry of health due to the increased focus on severe acute respiratory syndrome. 14 a realistic vaccination coverage rate target in europe could be set at the level of vaccination intentions (39%). in the general population, the characteristics of those who gave the attention to avian influenza -a possible influenza pandemic as a reason for vaccination -were not found to be statistically different from the rest of the vacci-nated group. nonsignificant trends hinted at a larger proportion of women and a slightly lower mean age of this relatively small subgroup. the majority of persons influenced by the attention given to avian influenza were those who were vaccinated for the first time. in the general population, a recommendation from the family physician is the most important encouragement for vaccination. this confirms findings from several previous studies. 5, 15, 16 it was also stated that more information on the vaccines regarding tolerability and efficacy would motivate persons to get vaccinated. we did not cover the vaccination rates of schoolchildren in our article. however, high vaccination coverage in children and subsequent positive external effects will be difficult to achieve at least in some countries. this reason makes high vaccination rates in the risk populations even more important. telephone surveys are an appropriate method to investigate influenza vaccination uptake at the population level. telephone interviews have been used on several occasions to study vaccination rates in europe. 6, 17, 18 the main advantage of telephone interviews is a potentially high response rate obtained in an affordable and fast manner. the selection process based on random dialing of telephone numbers has been shown to be of high quality. 19 in france, the questionnaire was a self-administered mail survey. in mailed questionnaires, there is a high risk of respondents omitting questions and of a low return rate. on the other hand, mailed questionnaires are an even more affordable option for large-scale surveys. 19 the limitations of the present data collection are described in greater detail in an earlier publication. 6 an increasing problem is the use of wireless telephones. in the usa people with landlines had a higher odds (1ae27) of being vaccinated than those with only access to wireless telephones. 20 if this is believed to be similar in europe, we might have slightly overestimated the vaccination rate. the different methodological approach used in france may have affected the reliability of the comparison across countries. this is supported by the fact that the french gave different reasons for and against vaccination compared to the other countries. the who considers the current influenza pandemic risk to be at its highest level since the last pandemic. 4 hence, efforts should be made at all national and international levels to increase vaccination coverage according to the who objectives (i.e. 50% vaccination coverage to be reached in the elderly in 2006 and 75% in 2010). 21 among elderly ‡65 years, a vaccination rate of 50% or higher was reached in all the countries studied. so far, only great britain has reached the 2010 target of 75% with an immunization rate of 79% in season 2005 ⁄ 06. the existence of national targets may provide a partial explanation for this success. 22 vaccines for preventing influenza in the elderly individual and community impact of influenza control of influenza. public health policies influenza vaccination in europe: an inventory of strategies to reach target populations and optimise vaccination uptake influenza vaccination coverage rates in five european countries-a population-based cross-sectional analysis of two consecutive influenza seasons rki-ratgeber infektionskrankheiten -merkblä tter fü r ä rztezielgruppen der impfung the influenza immunisation programme [www document protocole de mise en place de la chimio-prophylaxie dans une collectivité de personnes à risque lors d'une é pidè mie de grippe, en pé eriode de circulation du virus grippal influenza coverages in spain and vaccination-related factors in the subgroup aged 50-64 years zahlt meine kasse fü r die grippeschutzimpfung? influenza vaccination of healthcare workers: a literature review of attitudes and beliefs seasons determinants of adult influenza and pneumonia immunization rates cross-sectional study on influenza vaccination influenza vaccination coverage in elderly people influenza vaccination coverage and reasons to refrain among high-risk persons in four european countries health measurement scales. a practical guide to their development and use telephone coverage and health survey estimates: evaluating the need for concern about wireless substitution prevention and control of influenza pandemics and annual epidemics, 56th wha, 10th plenary meeting department of health. summary of flu immunisation policykey points about flu immunisation policy in england this study was made possible by an unrestricted research grant from aventis-pasteur msd, lyon, france. we thank the geig for making the french data available for analysis. furthermore, we would like to thank bertrand verwee, christine pilet from sanofi pasteur, and matthias schwenkglenks from the european center of pharmaceutical medicine, basel, switzerland, for their comments on the study and on the data analyses. key: cord-002137-j5sfiyz8 authors: ward, kirsten; seale, holly; zwar, nicholas; leask, julie; macintyre, c. raina title: annual influenza vaccination: coverage and attitudes of primary care staff in australia date: 2010-10-12 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2010.00158.x sha: doc_id: 2137 cord_uid: j5sfiyz8 please cite this paper as: ward et al. (2011) annual influenza vaccination: coverage and attitudes of primary care staff in australia. influenza and other respiratory viruses 5(2), 135–141. background annual influenza vaccination is recommended for all australian health care workers (hcws) including those working in primary health care. there is limited published data on coverage, workplace provision, attitudes and personal barriers to influenza vaccination amongst primary health care staff. the aim of this study was to contribute to the limited literature base in this important area by investigating these issues in the primary health care setting in new south wales (nsw), australia. methods a postal survey was sent to general practitioners (gps) and practice nurses (pns) from inner city, semi‐urban and rural areas of nsw, australia. there were 139 responses in total (response rate 36%) from 79 gps (response rate 30%) and 60 pns (response rate 46%). results reported influenza vaccination coverage in both 2007 and 2008 was greater than 70%, with gps reporting higher coverage than pns in both years. the main barriers identified were lack of awareness of vaccination recommendations for general practice staff and concern about adverse effects from the vaccine. conclusions rates of influenza vaccination coverage reported in this study were higher than in previous studies of hospital and institutional hcws, though it is possible that the study design may have contributed to these higher results. nevertheless, these findings highlight that more needs to be done to understand barriers to vaccination in this group, to inform the development of appropriate strategies to increase vaccination coverage in primary health care staff, with a special focus on pns. influenza is a serious respiratory virus which costs the australian healthcare system $115 million annually. 1 primary health care workers (hcws) like general practitioners (gps) and practice nurses (pns) have found to be at higher risk for influenza than the general population. 2 this may be because of: (i) exposure to influenza infection in both the general community and the workplace; and (ii) close proximity to visitors and patients. 1, 3 vaccines remain the cornerstone of influenza prevention in many countries worldwide 4 and are considered to be 50-80% effective in healthy persons aged 16-65 years. 5 annual influenza vaccination of australian hcws is recommended by the national health & medical research council (nhmrc), 3 the australian committee on safety and quality in healthcare 6 and various jurisdictional health departments, including new south wales (nsw). 7 the royal australian college of general practitioners 8 recommend that general practice staff members are offered immunisation appropriate to their duties. whilst there have been numerous australian studies on influenza vaccine uptake amongst hospital and institutional hcws 6, [9] [10] [11] [12] [13] and some studies on attitudes of primary care clinicians to influenza vaccination for their patients 14, 15 , there has been limited published studies to date on influenza vaccination coverage, barriers and enablers amongst primary health care staff in australia. influenza vaccination coverage amongst gps in australia was 50% in 1998. 16 in neighbouring new zealand, coverage amongst gps was 68% and 64% in pns in 2001. 17 other countries have reported lower coverage estimates, with only 35% of canadian family physicians from québec 18 and 36% of gps from the netherlands 19 vaccinated in 1996 and 2008, respectively. a study across all primary health care professions in israel reported an average of 30% coverage across physicians, nurses, pharmacists and administration staff. 20 factors associated with influenza vaccination status have been examined in primary health care clinicians in other countries. [17] [18] [19] [20] [21] amongst this group of hcw's, significant predictors for vaccine acceptance include the following: agreement that hcw's have professional responsibility to be vaccinated, on-site access to free vaccine, workplace recommendation for staff influenza vaccination, desire for self-protection and belief that the benefits of vaccination outweigh the risk of vaccine side effects. 17, 21 furthermore, previous influenza vaccination has been significantly associated with current vaccine acceptance in both hospital hcws and primary care physicians. 20 factors significantly associated with lack of vaccine acceptance include the following: no medical indication for vaccination, belief that regular medical exposure will protect against the disease, low risk of contracting influenza, fear of vaccine side effects and lack of time or priority. 17, 19, 20 some of these factors are similar to those cited by hospital hcws whilst others differ. in a review of attitudes and predictors to influenza vaccination of hospital hcw's, lack of convenient access to vaccine and poor knowledge about influenza infection were prominent reasons for lack of vaccination with the desire for self-protection and belief in the vaccine's effectiveness the most prominent reasons for vaccine acceptance in this group. 22 to the best of our knowledge, there have been limited studies which specifically examine: influenza vaccination coverage, workplace provision of vaccination, knowledge, attitudes and personal barriers to influenza vaccination amongst gps and pns in australia. the aim of this study therefore was to contribute to the limited literature base in this important area by investigating this in the primary health care setting in nsw, australia. a paper-based survey was developed based on pilot work undertaken by the authors and commonly identified barriers to vaccination from the literature. 6, 23, 24 it elicited demographic data about the respondents, their influenza vaccination status from 2007 to 2009 and identified barriers to being immunised. it also posed questions intended to determine the respondent's attitude towards vaccination, based on seven statements about efficacy, safety, adverse events and recommended target groups for influenza vaccine and was part of a wider survey that incorporated questions on pandemic influenza. the survey was piloted with four gps and pns from outside the study area. feedback from this process contributed to enhanced content, altered survey structure and modified wording. our sample was drawn from divisions of general practice (dgp) in nsw. dgps are government-funded organisations that provide support to a defined geographical catchment of general practices in australia. they are classified by population and locality into five categories based on rural, remote metropolitan areas (rrma). 25, 26 nsw has the highest number of dgps, with 32. purposive sampling by the authors was used to select four dgps in nsw to represent a diverse sample from metropolitan, semiurban and rural areas. the final sample size for each participating division was weighted according to how many gps and pns were practicing in the area. the study was undertaken from the 1st february to 1st april 2009, prior to the pandemic (h1n1) influenza 2009 which was identified in late april 2009. 27 authors were blinded to participant selection, as they were randomly selected from de-identified dgp databases of gps and pns. surveys were posted by dgps and were accompanied by personalised explanatory letter and a reply-paid envelope. non-responders were sent a second letter and survey by the dgps within 4 weeks. quantitative data was entered into microsoft access and was analysed using microsoft excel. responses to the geographical location question were categorised into inner city, semi-urban and rural areas. questions about influenza vaccine-related barriers, attitudes and beliefs were categorised into either agree, disagree or uncertain. the categories were compared with demographic characteristics and selfreported vaccination status of respondents using categorical data analysis. ethical approval was granted by the university of new south wales ethics committee. of the 390 staff that was sent a survey, 139 completed and returned it, giving an overall response rate of 36%. fifteen surveys were returned uncompleted as the staff member was no longer at the practice. response rates were higher amongst pns (46%) than gps (30%). the demographic and occupational characteristics of the respondents are summarised in table 1 . there was some variation in geographical location of respondents, with 58ae3% (81 ⁄ 139) located in semi-urban areas, 36% (50 ⁄ 139) in the inner city and the remaining 5ae7% (8 ⁄ 139) in rural locations. of the respondents, 45% (62 ⁄ 139) were working in practices with £4 gps. our sample parallels the findings of the 2007-2008 bettering the evaluation and care of health (beach) survey in terms of the spread in gp age, years worked in general practice and geographical location. the beach program is a continuous national study of general practice activity in australia. it provides a reliable, ongoing, representative description of general practice activity nationwide. 28 just over 70% of respondents were vaccinated against influenza in 2007 (70ae5%, 98 ⁄ 139, 95% ci: 62ae9-78ae1) and in 2008 (72ae7%, 101 ⁄ 139, 95% ci: 65ae3-80ae1). differences in vaccination coverage between gps and pns for both 2007 (p = 0ae74) & 2008 (p = 0ae22) (see table 1 participants indicated that free influenza vaccine was most commonly provided at the practice for gps (79ae1%, 110 ⁄ 139, 95% ci: 72ae3-85ae9), administration staff (74ae8%, 104 ⁄ 139, 95% ci: 67ae6-82ae0) and pns (72ae7%, 101 ⁄ 139, 95% ci:65ae3-80ae1). of the gps working at a practice which provided free influenza vaccine for gps, 84ae5% (49 ⁄ 58) were vaccinated in 2008 in contrast to 58ae3% (7 ⁄ 12) coverage in gps from practices that did not provide the vaccine free of charge for them. for pns, 69ae8% (37 ⁄ 53) vaccinated in 2008 worked at a practice where the vaccine was provided free and 66% (2 ⁄ 3) were vaccinated despite the vaccine not being provided free for pns at their practice. respondents' knowledge, attitudes and perceptions of influenza vaccination are summarised in table 2 . over 90% of the participants believe that the influenza vaccine is attitudes towards vaccination barriers amongst the respondents are presented in table 3 . while there was a low level of agreement with all of the statements provided (<10%), the most commonly identified barriers were lack of awareness of any recommendation for general practice staff to receive influenza vaccination (10ae1%, 14 ⁄ 139, 95% ci: 5ae1-15ae1) and unacceptable nature of vaccination side effects (7ae2%, 10 ⁄ 139, 95% ci: 2ae9-11ae5). having to pay for the vaccine was identified as a barrier to getting vaccinated by 25ae2% (35 ⁄ 139) of respondents. five of the twelve gps, who worked in a practice that did not provide free vaccine, felt that paying for a vaccine was a barrier. our study of primary health care staff found much higher influenza vaccination coverage than hospital hcws in australia. overall reported coverage of gps and pns was markedly higher than those for australian institutional and hospital hcws which have been found to range from 18% to 58%. 6, [9] [10] [11] [12] 23 self-reported vaccination coverage for gps in our study (for 2007) was higher than the percentage reported for australian hospital-based doctors from the northern territory (72ae7% versus 28%) 12 and western australia (72ae7% versus 51ae9%). 9 pns in our study had higher coverage when compared to nurses in residential aged care facilities (racf) (68ae3% versus 62%) 11 , and hospital-based nurses (68ae3% versus 49ae6%) yet had lower coverage than pns from the australian capital territory (act) (68ae3% versus 79%). 29 the australian national influenza & pneumococcal survey provides the earliest available influenza vaccination coverage estimates for gps in australia. 16 results of this survey found coverage for nsw gps was the lowest of any jurisdiction, with 29% vaccinated in 1998, and just over 20% for the preceding 3 years. comparing these rates to those observed in our study, influenza vaccination coverage amongst gps in nsw appears to have risen substantially from 1998 to 2008. more recently, a national survey from the australian general practice network (agpn) 23 assessed influenza vaccination coverage in gps and pns in the same years as our study (2007 ⁄ 2008) with similar response rates (34% versus 36%). comparing vaccine uptake between the studies for both gps and pns in nsw only, the agpn study reported slightly lower coverage (5%) across both years in both groups. 23 as data was collected at a practice level in the agpn study, individual vaccination status may have been incorrectly reported and may be underestimating the actual coverage rate. however, our results may overestimate actual coverage because of a number of reasons including the low response rate and use of self-reported vaccination status. studies into gp influenza vaccination coverage have been performed in other countries. [17] [18] [19] [20] [21] 30 cowen et al. 21 in the united states (us) had a similar response rate to our study (30% versus 38%), but reported a much higher vaccination coverage rate for us family physicians (84%). semaille et al. 30 and brunton et al. 17 reported influenza vaccination coverage as 67% amongst french gps and 68% amongst new zealand gps, respectively. in contrast, studies from the netherlands 19 and israel 20 found lower coverage rates for gps with 36% and 40%, respectively. even though the majority of our respondents worked in a practice that provided free vaccine for their staff, many felt that paying for the vaccine was a barrier to getting vaccinated. the wording of the question may have impacted on the result, as respondents could have taken it to mean for gps in general and not for them personally. however, it is interesting to note, of the gps who said their practice does not provide free vaccine, 40% stated that paying for the vaccine was a barrier to receiving it. further qualitative research would assist in addressing these gaps in understanding. the gps and pns in our study largely disagreed with the common vaccination myths presented in the survey (see table 3 ). in contrast, the most frequently reported reason amongst dutch gps for not being vaccinated was having no medical indication for influenza vaccination 19 and in israel; physicians were much less frequently influenced by the fear that vaccination would cause influenza when compared to other practice staff. 20 there was almost no consistency of agreement with both these misconceptions in our sample or that of litt et al., 16 who found that the main reasons indicated by australian gps for being vaccinated against influenza were concern about getting influenza or its complications and to prevent having time off work because of influenza. live viruses in the influenza vaccine was the most common myth supported by respondents (21ae6%, 30 ⁄ 139) yet a decade ago, less than 4% of australian gps gave this as a reason for not getting influenza vaccine. 16 the reasons behind this shift in belief is unknown; however, technology could play a part, with increased access to a variety of information sources. in the light of this, gp education should continue to focus on dispelling this myth through use of evidence-based information. other barriers to influenza vaccination identified in our study were lack of awareness of any recommendations for general practice staff to receive the influenza vaccine (10%) and unacceptable side effects of vaccination (7%). these are similar to those commonly cited by hcws in other countries. [19] [20] [21] for hospital doctors, being too busy has been identified as a major barrier to getting vaccinated against influenza. 6, 12, 24 in contrast, only a small number of participants, all of whom were gps, identified lack of time as a barrier as did approximately 25% of gp respondents the australian influenza and pneumococcal vaccination survey (2003) in the elderly. 16 a previous investigation of general practice staff across 53 dgps in australia found a significant association between workplace influenza vaccination policy and staff vaccination. 23 we found that interrelationship between staff beliefs and practice policies may be an important determinant of hcw immunization behaviours in these practices. office policies demanding immunisation and operating within an effective hierarchy can lead staff members to re-evaluate their beliefs about influenza immunization in the light of their own experience. 31 continued efforts at the practice level to make formal commitments to staff health by developing policies for influenza vaccination of staff may assist in increasing coverage in this group. provision of the influenza vaccine to patients along with consistent, direct, exposure to influenza like illness by this population 32 may further impact on their decision to be vaccinated. pandemic (h1n1) influenza 2009 is also likely to increase awareness of influenza vaccination and instigate new policies and practices surrounding vaccination of primary health care staff as has been seen in other countries during heightened awareness of an impending influenza pandemic threat. 33 there are a number of limitations to this study including sample size, generalisability and use of self-report for vaccination status. although the sample size was small, compared to other general practice-based surveys, it would be considered reasonable in the light of the challenges with surveying this population. 34, 35 there is potential for selection bias in this study towards those who are particularly concerned about influenza and ⁄ or vaccination or those who accept vaccination. furthermore, using self-reported vaccination status in adults has been shown to overestimate coverage. 36, 37 there may also be limitations with generalisability because our study was conducted only in one state of australia. this study did not collect any data on nonrespondents or outcomes for those who intended to be vaccinated in 2009. in addition, the barriers in our survey may not have covered all possible options, thus may have influenced participants response. qualitative research is needed to further explore these findings. despite these limitations, influenza vaccination coverage was found to be relatively high amongst the gps and pns in our study; however, there is still room for improvement. understanding barriers to vaccination is the first step to developing effective strategies to overcome them. for institution-based hcws, there is now an extensive literature base around knowledge, attitudes and practices towards influenza vaccination, whereas, there are only a few studies exploring these topics in the primary health care setting in australia. to enhance development and targeting of strategies to increase coverage, a more complete and current understanding of coverage in this group is needed to build on the estimates presented here. we believe this study to be a basis for future investigations and interventions to increase influenza vaccination rates in primary health care staff in australia. influenza-related disease: the cost to the australian healthcare system the role of influenza vaccine in health care workers in the era of severe acute respiratory syndrome national health and medical research council (nhmrc). the australian immunisation handbook, 9th edn. canberra: australian government world health organization. influenza vaccines vaccines for preventing influenza in healthy adults discussion paper: influenza vaccination amongst health care workers policy directive: occupational assessment, screening & vaccination against specified infectious diseases royal australian college of general practitioners. infection control standards for office based practices co-ordinated approach to healthcare worker influenza vaccination in an area health service influenza vaccine coverage among health care workers in victorian public hospitals influenza vaccination of staff in aged care facilities in the act: how can we improve the uptake of influenza vaccine? influenza immunisation of doctors at an australian tertiary hospital: immunisation rate and factors contributing to uptake attitudes amongst australian hospital healthcare workers towards seasonal influenza and vaccination barriers and facilitators to influenza vaccination among high-risk groups aged less than 65 years -views from general practitioners and practice nurses differences in attitudes, beliefs and knowledge of hospital health care workers and community doctors to vaccination of older people australian national influenza and pneumococcal survey in the elderly knowledge and attitudes about influenza vaccination amongst general practitioners, practice nurses, and people aged 65 and over vaccination practices of quebec family physicians. influenza vaccination status and professional practices for influenza vaccination influenza immunization of dutch general practitioners: vaccination rate and attitudes towards vaccination influenza vaccination among primary healthcare workers influenza vaccination status and influenza-related perspectives and practices among us physicians influenza vaccination of health care workers in hospitals -a review of studies on attitudes and predictors general practice network influenza survey report national foundation of infectious diseases. improving influenza vaccination rates in health care workers. strategies to increase protection for workers and patients. atlanta: centre for disease control and prevention australian institute of health & welfare (aihw). rural, regional and remote health: a guide to remoteness classifications fast facts: rural remote metropolitan area (rrma) classification initial human transmission dynamics of the pandemic (h1n1) 2009 virus in north america general practice series no. 22. (cat. no.gep22). canberra: australian government influenza vaccination in act nurse immunisers evaluation of the vaccine coverage of the general practitioners in the french community organizational culture influences health care workers' influenza immunization behaviour annual report of the national influenza surveillance scheme high coverage of influenza vaccination among health care workers can be achieved during heightened awareness of impending threat response rates of victorian general practitioners to a mailed survey on miscarriage: randomised trial of a prize and two forms of introduction to the research the effect of cash and other financial inducements on the response rate of general practitioners in a national postal study sensitivity and specificity of patient self-report of influenza and pneumococcal polysaccharide vaccinations among elderly outpatients in diverse patient care strata validity of self-reported influenza and pneumococcal vaccination status among a cohort of hospitalized elderly inpatients influenza vaccine coverage and attitudes in australian primary care staff ª thanks to the following for their support in this study; central sydney general practice network, albury wodonga regional gp network shire gps (sutherland division of general practice) and went west ltd. j leask is an investigator on a grant which is part funded by sanofi pasteur. c. r. macintyre receives funding from influenza vaccine manufacturers gsk and csl biotherapies for investigator-driven research. k ward has received funding from wyeth to attend an immunisation conference. all other authors of this manuscript have no conflicts of interest to declare. national centre for immunisation research and surveillance is supported by the australian government department of health and ageing, the nsw department of health and the children's hospital at westmead. key: cord-289358-4abypk6o authors: asner, sandra a.; petrich, astrid; hamid, jemila s.; mertz, dominik; richardson, susan e.; smieja, marek title: clinical severity of rhinovirus/enterovirus compared to other respiratory viruses in children date: 2014-05-07 journal: influenza other respir viruses doi: 10.1111/irv.12255 sha: doc_id: 289358 cord_uid: 4abypk6o background: human rhinovirus/enterovirus (hrv/ent) infections are commonly identified in children with acute respiratory infections (aris), but data on their clinical severity remain limited. objectives: we compared the clinical severity of hrv/ent to respiratory syncytial virus (rsv), influenza a/b (flu), and other common respiratory viruses in children. patients/methods: retrospective study of children with aris and confirmed single positive viral infections on mid‐turbinate swabs by molecular assays. outcome measures included hospital admission and, for inpatients, a composite endpoint consisting of intensive care admission, hospitalization >5 days, oxygen requirements or death. results: a total of 116 hrv/ent, 102 rsv, 99 flu, and 64 other common respiratory viruses were identified. children with single hrv/ent infections presented with significantly higher rates of underlying immunosuppressive conditions compared to those with rsv (37·9% versus 13·6%; p < 0·001), flu (37·9% versus 22%; p = 0·018) or any other single viral infection (37·9% versus 22·5%; p = 0·024). in multivariable analysis adjusted for underlying conditions and age, children with hrv/ent infections had increased odds of hospitalization compared to children with rsv infections (or 2·6; 95% ci 1·4, 4·8; p < 0·003) or flu infections (or 3·0; 95% ci 1·6, 5·8; <0·001) and increased odds of severe clinical disease among inpatients (or 3·0; 95% ci 1·6,5·6; p = 0·001) when compared to those with flu infections. conclusions: children with hrv/ent had a more severe clinical course than those with rsv and flua/b infections and often had significant comorbidities. these findings emphasize the importance of considering hrv/ent infection in children presenting with severe acute respiratory tract infections. human rhinovirus/enterovirus (hrv/ent) has been recently identified as the leading pathogen in acute asthma exacerbations, bronchiolitis, and viral pneumonia, although the clinical severity of respiratory illnesses attributed to hrv/ent remains uncertain. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] studies conducted among younger children admitted for bronchiolitis reported conflicting information about the clinical severity of hrv/ent infections, possibly as a result of different age groups studied. 11, 12 a study by papadopulos et al., 4 reported that hrv/ent was a significant predictor for severe disease, another study by midulla et al. 12 reported that hrv/ent bronchiolitis resulted in less severe disease compared to rsv bronchiolitis among infants. conversely, studies involving older hospitalized children 1, 5, 13 consistently reported equivalent disease severity between respiratory illnesses caused by hrv/ent and those caused by other common respiratory viruses. explanations for the discrepancies observed among these studies 4, 12, 13 include the use of different clinical criteria to assess clinical severity (clinical severity scores versus clinical outcomes) and the focus on different patient populations and age groups (inpatients and young children). furthermore, the contribution of hrv/ent infection versus host-related factors such as underlying immunosuppression to the severity of the disease was not reported in most studies. in summary, there is growing evidence that hrv/ent is the most commonly identified virus in lrtis in both adults and children and thus needs to be regarded as a pathogen responsible for much more than just the common cold. the objective of this study was to compare the clinical characteristics and the severity of illness of hrv-/ent-positive children to those positive for rsv a/b, flu-a/ flu-b, and other respiratory viruses by (i) using a large dataset with a broad pediatric inpatient and outpatient population; (ii) considering several clinical endpoints to assess disease severity, and (iii) conducting multivariable analysis to adjust for potential confounding variables. we conducted a single-center retrospective study of children presenting with an acute respiratory illness and any single viral infection documented by molecular assays from midturbinate swabs to the hospital for sick children, toronto, canada. multiplex pcr was conducted in a random sample of all patients presenting with arti in whom the treating physician ordered a mid-turbinate swab. testing for respiratory viruses relied on clinical assessment as routinely done in children with respiratory illnesses. these included common cold, pharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, and community-acquired pneumonia (cap). we used a dataset from the microbiology laboratory at the hospital for sick children, which originally aimed to compare the sensitivities of four different multiplex assays: resplex ii v2.0 (qiagen, mississauga, on, canada); seeplex rv15 kit (seegene inc., seoul, korea); xtag-rvp and xtag-rvp fast, (luminex, austin, tx, usa) in addition to dfa and viral culture. the results from the original study were recently published elsewhere. 14 the dataset was augmented through retrospective chart review. children with documented viral coinfections (i.e., detection of more than one viral pathogen in the same sample) were excluded as their inclusion would not allow single viral infection comparisons as a result of not being able to determine which of the viruses detected was the leading pathogen. data were collected from november 2007 to april 2008 and january 2009 to march 2009, the times when multiplex pcr testing was routinely utilized in randomly selected patients under 18 years of age presenting with acute respiratory tract infections (artis). urti was defined as the detection of any single respiratory viral infection together with symptoms involving the upper respiratory tract (nose and pharynx). 15 lrti was defined as any patient with cough, tachypnea, and/ or any respiratory distress or wheezing. communityacquired pneumonia (cap) was defined as any of the above lrti symptoms with pulmonary infiltrates diagnosed on a chest x-ray by a radiologist. severe clinical disease was defined by hospital admission for outpatients and by a composite endpoint including intensive care (icu) admission, hospitalization >5 days, oxygen requirements or death in patients admitted to the hospital. ethics approval was obtained from the research ethics board at the hospital for sick children. information on patient demographics, relevant baseline characteristics such as underlying comorbidities and outcomes were all extracted from health records. relevant underlying comorbidities were grouped into three mutually exclusive categories: cardiorespiratory, prematurity, and any immunosuppressive/metabolic conditions. one or more of the following immunodeficiency states were included in the latter group: hematopoietic stem cell transplant (allogeneic and autologous) and solid organ transplant (sot) recipients, recipients of cancer chemotherapy or long-term immunosuppression for any chronic disease, and congenital immunodeficiency states. metabolic conditions included any inherited metabolic diseases such as cystinuria, phenylketonuria (pku), gout, and thyroid disease. in case of multiple comorbidities, patients were referred to the group considered as the most significant comorbidity: an underlying immunocompromised/metabolic condition was considered most significant comorbidity, followed by a cardiorespiratory comorbidity. outcomes consisted of hospital admission, duration of hospitalization, admission length >5 days, intensive care (icu) admission, any supplemental oxygen requirements, presence of urti, cap, and all-cause mortality. from november 2007 to april 2008 and january 2009 to march 2009, 5345 mid-turbinate swabs (floqswabs, copan italia, brescia, italy) were tested for respiratory viruses of which 750 (14%) were randomly selected each week among all specimens collected from children with acute respiratory tract infections (artis)) and submitted to the clinical laboratory for routine virology testing including molecular assays. neither laboratory staff nor clinicians were aware in advance which patient would be selected for viral detection by molecular assays. swabs were assayed by four different nucleic acid amplification-based assays: resplex ii v2.0 , a true positive result was defined as two or more positive test results from among the four molecular assays. 14 bacterial coinfection was defined as the presence of any bacterial pathogen, identified by culture from blood cultures or respiratory samples (bronchoalveolar lavage, tracheal secretions, sputum samples or pleural fluid) upon initial consultation with respiratory symptoms or within 30 days of their initial consultation. staphylococcus epidermidis positive blood cultures were considered as pathogens if drawn from more than one peripheral blood culture, from one blood culture drawn from a central line, or from one peripheral line in high-risk patients such as those with underlying immunosuppressive conditions, prosthetic devices, or newborns according to recent guidelines. 16 in addition, a positive urinary antigen for streptococcus pneumonia retrieved upon admission or within 30 days of admission was also considered as bacterial coinfection. positive bacterial urinary or stool cultures and bacterial pathogen identified from skin or wound swabs were not considered as bacterial coinfections. standard descriptive and comparative statistics were performed on data categorized by viral pathogen, where hrv/ ent was used as the reference and compared to rsv, flu, or category consisting of all other common viruses including piv 1-4, hmpv, hbov, adv, and coronaviruses. the chisquare test or fisher's exact test was used to compare categorical variables between groups as appropriate. multivariable logistic regression was used to compare the clinical correlates of clinical disease severity between children infected with hrv/ent and those infected with either rsv, flu, or other common single respiratory viruses. we derived medians, used the mann-whitney nonparametric method for comparisons of non-normally distributed continuous data, and used a transformation using the natural logarithm (ln) of the outcome variable when performing multivariable linear regression. a two-sided test was performed, and -a p value <0á05 was considered to be statistically significant. data were analyzed using spss statistical software (version 20.0, spss inc, chicago, il, usa). a total of 742 children evaluated for any respiratory illness were screened for respiratory viruses from mid-turbinate swabs by molecular assays during the study period. of these, 462 (62á3%) children were detected positive for any respiratory virus. among these 462 patients, 205 (53á8%) were admitted, and among these inpatients, 143 (37á5%) presented with severe disease. a total of 81 (17á5%) had two or more respiratory viruses and were excluded from this analyses. the remaining, 381 (82á5%), tested positive for a single respiratory virus: 116 (30á4%) hrv/ent, 102 (26á8%) rsv, 99 (26á0%) flu, and 64 (16á8%) other respiratory viruses. our rate of overall bacterial coinfection was low (6á4%). among inpatients, children with single hrv/ent infections were significantly older compared to those with single rsv infections (median age 1á9 years versus 0á4 years, p < 0á002). children with hrv/ent infections presented with a significantly higher rate of underlying cardiorespiratory comorbidities compared to children with rsv (37á6% versus 14á6%; or 3á5; 95% ci 1á3, 9á3; p = 0á01), flu (37á6% versus 6á5; or 9; 95% ci 4á0, 38á0; p = 0á005), or any other single viral infection (37á6% versus 14á6%; or 3á3; 95% ci 1á4, 9á0; p = 0á007) ( table 1) . children with hrv/ent infections had significantly higher rates of pneumonia compared to those with rsv (24á1% versus 9á7%; or 2á3; 95% ci 1á1-5á3; p = 0á044). when compared to children with rsv infections or flu infections, those with hrv/ent infections had significantly higher admission rates (73á3% versus, respectively, 39á8% and 37á8%; both p < 0á001). among children who were admitted to hospital, those with hrv/ent infections had an increased length of stay compared to those with flu infections (8 days, iqr 3-19á5 versus 4 days; iqr 2-11 days; p = 0á035). of six children who died, four presented with hrv/ent infections; one presented with a progressive acute respiratory distress syndrome (ards) after adv infection and died 4 weeks after flu-a infection; and the remaining one presented with an hbov infection. of the four fatalities with hrv/ent infections, three presented with underlying immunosuppressive conditions and one with a cardiac comorbidity. two of the patients died from a sepsis-like picture with respiratory failure and possible underlying pneumonia with no other pathogens identified, thus suggesting that hrv/ent may have potentially contributed to the mortality. the remaining two patients likely died of their underlying disease ( table 1) . in multivariable analysis adjusted for age and comorbidities, we identified that children with hrv/ent infections were significantly more likely to be hospitalized compared to those with rsv infections (or 2á6; 95% ci 1á4, 4á8; p < 0á003) or flu infections (or 3á0; 95% ci 1á6-5á8; <0á001) (tables 2). furthermore, age (or 1á1 per year; 95% ci 1á0, 1á2; p = 0á009) and underlying immunosuppressive/metabolic conditions (or 6á5; 95% ci 3á4, 12á5; p < 0á001) were significant predictors of hospital admission. an interaction term measuring the combination of hrv-/ent-positive status and underlying immunocompromised condition was not significant, and its inclusion did not affect the risk estimates above. post hoc analyzes conducted among patients with an underlying immunosuppressive condition did not affect the risk estimates above although our results were not human rhinovirus-/enterovirus-infected children were significantly more likely to present with severe clinical disease in multivariable analysis compared with children admitted to hospital with flu infections (or 3á0; 95% ci 1á6, 5á6; p = 0á001) (tables 3). children admitted with hrv/ent infections had an 80% increase in the length of stay compared to those admitted with flu infections (b coefficient 0á6, 95% ci 0á1, 1á0; p = 0á012) in multivariable analysis, where the logarithm of admission length was used as an outcome variable. children with underlying immunosuppressive or metabolic states had a 100% increase in the length of admission compared to those without underlying immunosuppressive or metabolic conditions (b coefficient 0á7; 95% ci 0á3, 1á0; p < 0á001) ( table 4 ). three important observations made in our study were that hrv/ent acute respiratory infections were very commonly detected in our patient population; children with hrv/ent infections had more severe outcomes compared to those with other common respiratory viruses; and children with underlying cardiorespiratory or immunocompromised/metabolic conditions were more likely to present with hrv/ent infections. the proportion of single hrv/ent infections reported in our study (30á4%) was significantly higher than in studies which used conventional diagnostic methods (5%), 6,17-19 but comparable to those in which molecular tests were used, including studies of children with artis (27á2%) 2,11 and hrv/ent, enterovirus/rhinovirus; rsv, respiratory syncytial virus; flu, influenza; others: piv, parainfluenza; hmpv, human meta-pneumovirus; adv, adenovirus, coronaviruses, hbov. values indicated in bold were considered as significant p < 0.05. hrv/ent, enterovirus/rhinovirus; rsv, respiratory syncytial virus; flu, influenza; others: piv, parainfluenza; hmpv, human meta-pneumovirus; adv, adenovirus, coronaviruses, hbov. values indicated in bold were considered as significant p < 0.05. infants with bronchiolitis (29%). 4 these discrepancies can be attributed to the higher sensitivity and broader range of virus detection by molecular-based methods. similar to others studies, 2,4,5,20 children infected by hrv/ ent alone were younger compared to those with flu infections but older compared to those with rsv infections. in our study, children with hrv/ent infections presented with higher rates of underlying cardiorespiratory and immunosuppressive conditions compared to those with any other single viral infection. our findings are similar to those described in adults admitted with hrv/ent respiratory infections, in whom high rates of underlying immunosuppressive conditions were identified, thus suggesting that these patients may be at higher risk of hrv/ent infections. 7, [21] [22] [23] the extent to which severity of respiratory illness is attributed to hrv/ent or to other underlying conditions is yet unclear. notwithstanding the high proportion of underlying comorbidities identified among our hrv/ent population, hrv-/ ent-positive status was identified as a significant independent predictor for hospital admission and resulted in an increased clinical disease severity among inpatients compared with either rsv or flu, while controlling for underlying comorbidities and age. furthermore, we found significantly longer duration of hospitalization among children admitted with hrv/ent infections compared to those admitted with flu infections. also, higher rates of pneumonia were observed among children with single hrv/ent infections compared to those with rsv single viral infections as suggested by a previous study by malcolm et al. 7 finally, half of the deaths observed among the four hrv-/ent-infected children with underlying conditions may have resulted from their hrv/ ent respiratory illnesses as no other viral, bacterial, or fungal pathogens were documented. all these findings contrast with recently published studies 1, 5, 13 which reported equivalent disease severity between subjects with hrv/ent infections and those with other common viral infections. these studies, however, had major limitations, which may have resulted in not detecting a true difference. firstly, the studies were conducted in inpatients only. one can speculate that hospitalized patients share a level of comorbidity leading to more similar outcomes regardless of the virus involved. 24 furthermore, most of these studies compared hrv/ent infections to all other viruses combined and did not adjust for underlying comorbidities. while treatment of flu-a-positive children with oseltamivir might have been expected to improve their prognosis, no children included in this study received oseltamivir, as specimens were collected before the implementation of guidelines advocating for oseltamivir treatment of high-risk children 25 thus, the difference in outcomes between hrv/ent and treated flu-a may be even more pronounced than found in our study. an important strength of our study was the inclusion of outpatients, thus enabling the use of hospital admission as a measure of clinical severity. furthermore, the adjustment for underlying comorbidities in multivariable analysis reinforced the association between hrv/ent and clinical outcomes. given our low rate of bacterial coinfections, no adjustment for this variable was made in multivariable analyses. finally, we had adequate sample size to compare hrv/ent to a number of different viruses. potential limitations of our study relate to its retrospective design. however, we believe that most of our patient-related important outcomes could be well assessed though chart review. similarly, the observational nature of our study may have led to selection bias as not all consecutive patients were tested for respiratory viruses as a result of limited resources. random sampling ensured an unbiased selection of samples tested by molecular assays. furthermore, the criteria for using virologic diagnostics as part of routine patient care may have varied during the study. for instance, viral culture was only routinely used from november 2007 to april 2008, which may have affected the comparisons of illness severity between different viruses. third, we assessed the presence or absence of viruses, but did not measure their viral load; higher viral loads in acutely ill subjects compared with asymptomatic children might have further strengthened the association between hrv/ent and severe disease as rhinoviruses are also commonly identified in community-based controls. 6, 26 future research should measure viral loads and use sequencing for genotyping analysis to differentiate hrv from enterovirus and to speciate hrv. recent studies suggest that human rhinovirus species a and c (hrv-a and hrv-c) may be associated with greater clinical severity compared with hrv-b species. 25, 27, 28 finally, our estimation of bacterial coinfection may have been underestimated as adequate respiratory samples such as bal are rarely performed in children and pneumonia rarely result in positive blood cultures. also, urt samples are 441 not necessarily representative of lrt disease. this limitation inherent to studies assessing the severity of respiratory illnesses in children may be overcome in future prospective studies, conducted among children with specific underlying comorbidities (e.g., immunocompromised children), which would include validated molecular assays for detection of both viral and bacterial pathogens from urt samples in all children with arti. in conclusion, our findings provide new insight into the burden and severity of hrv/ent infections and reinforce the need for routine diagnosis in hospital settings. hrv/ent infections were very common and associated with more severe disease than other common viruses such as flu or rsv highlighting the need for development and testing of specific antiviral drugs. human rhinovirus species associated with hospitalizations for acute respiratory illness in young us children role of rhinovirus c respiratory infections in sick and healthy children in spain a novel group of rhinoviruses is associated with asthma hospitalizations association of rhinovirus infection with increased disease severity in acute bronchiolitis rhino/enteroviruses in hospitalized children: a comparison to influenza viruses viral pneumonia clinical features of patients with acute respiratory illness and rhinovirus in their bronchoalveolar lavages community study of role of viral infections in exacerbations of asthma in 9-11 year old children role of viral respiratory infections in asthma and asthma exacerbations viruses in asthma exacerbations role of rhinovirus in hospitalized infants with respiratory tract infections in spain respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants characterization of 2009 pandemic influenza a (h1n1) virus specimens with a positive hemagglutinin 1 signal in the luminex xtag respiratory viral panel assay evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children respiratory syncytial virus infection in patients with hematological diseases: single-center study and review of the literature clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 update by the infectious diseases society of america infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis guidelines for the management of adult lower respiratory tract infections induced sputum in the diagnosis of childhood community-acquired pneumonia population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children editorial response: rhinovirus pneumonia-a clinical entity? recovery of viruses other than cytomegalovirus from bronchoalveolar lavage fluid rhinovirus infections in myelosuppressed adult blood and marrow transplant recipients community controls were preferred to hospital controls in a case-control study where the cases are derived from the hospital association between human rhinovirus c and severity of acute asthma in children acute lower respiratory tract infection novel human rhinoviruses and exacerbation of asthma in children detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study in-kind contribution of resplex ii v2.0 kits was provided by qiagen. dr susan richardson has received honoraria from qiagen and luminex molecular diagnostics for a study of multiplex respiratory pcr in children. the funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. dr. health sciences foundation (jack hirsh fellowship). the authors had no financial or other forms of conflict of interests. key: cord-293354-55nawxos authors: kenmoe, sebastien; tchendjou, patrice; vernet, marie‐astrid; moyo‐tetang, suzie; mossus, tatiana; njankouo‐ripa, mohamadou; kenne, angeladine; penlap beng, véronique; vabret, astrid; njouom, richard title: viral etiology of severe acute respiratory infections in hospitalized children in cameroon, 2011–2013 date: 2016-05-09 journal: influenza other respir viruses doi: 10.1111/irv.12391 sha: doc_id: 293354 cord_uid: 55nawxos background: severe acute respiratory illness (sari) is recognized as an important cause of morbidity, mortality, and hospitalization among children in developing countries. little is known, however, in tropical countries like cameroon about the cause and seasonality of respiratory infections, especially in hospitalized settings. objectives: our study investigates the viral etiology and seasonality of sari in hospitalized children in yaounde, cameroon. methods: prospective clinic surveillance was conducted to identify hospitalized children aged ≤15 years presenting with respiratory symptoms ≤5‐day duration. demographic and clinical data, and respiratory specimens were collected. nasopharyngeal samples were tested for 17 respiratory viruses using a multiplex polymerase chain reaction. the viral distribution and demographic data were statistically analyzed. results: from september 2011 through september 2013, 347 children aged ≤15 years were enrolled. at least one virus was identified in each of 65·4% children, of which 29·5% were coinfections; 27·3% were positive for human adenovirus (hadv), 13·2% for human respiratory syncytial virus (hrsv), 11·5% for rhinovirus/enterovirus (rv/ev), 10·6% for human bocavirus (hbov), 9·8% for influenza virus (inf), 6·6% for human parainfluenza virus (hpiv), 5·7% for human coronavirus (hcov), and 2·3% for human metapneumovirus (hmpv). while hrsv showed seasonal patterns, hadv and rv/ev were detected throughout the year and no evident temporal patterns were observed for the remaining viruses. conclusion: respiratory viruses were associated with a high burden of hospitalizations among children in cameroon. nevertheless, additional studies evaluating asymptomatic cameroonian children will be important in understanding the relationship between viral carriage and disease. severe acute respiratory illness (sari) is the leading cause of hospitalization, morbidity, and mortality in children younger than 5 years throughout the world. the burden of these infections is particularly important in developing countries with a meta-analysis estimating that 265 000 (95% ci 160 000-450 000) in-hospital deaths took place in 2010 with 99% of those deaths occurring in developing countries. 1 although there is little information on the causes of these respiratory illnesses in developing countries, available data indicate that viral agents play an important role globally. 2 a meta-analysis estimates that hrsv causes 33á8 million infections each year among children younger than 5 years. about 3á4 million of these cases require hospital admission, and an estimated 66 000-199 000 of children die with 99% of deaths occurring in developing countries. 3 many other viruses such as influenza viruses, rhinoviruses (rv), human parainfluenza viruses (hpiv), human adenoviruses (hadv), human coronaviruses (hcov-oc43, hcov-229e, hcov-nl63, and hcov-hku1), and enteroviruses (ev) have also been implicated worldwide. 2, 4, 5 discovered recently, human metapneumoviruses (hmpvs) and human bocaviruses (hbovs) are viruses resulting in similar symptoms. other emergent viruses such as sars-cov and mers-cov are responsible for more severe symptoms like respiratory distress syndrome. [6] [7] [8] real-time reverse-transcription polymerase chain reaction (rrt-pcr) assays have been shown to be a sensitive and specific tool for the detection of these viruses. furthermore, the introduction of multiplex rrt-pcr enables the set-up of assays detecting two or more targets in single clinical specimen. 9, 10 in 2007, the centre pasteur du cameroun (cpc) began implementing sentinel surveillance for influenza in cameroon. this surveillance system has enabled a better understanding of the epidemiology of influenza in the country. 11, 12 even though our recent publication has demonstrated that those respiratory viruses play an important role in patients consulting our sentinel sites, 13 there remains a lack of information regarding patients hospitalized with sari. this study was conducted to gain new insights into the timeliness of virus circulation and viral etiology among children aged ≤15 years, hospitalized for sari. this prospective observational study was conducted in the pediatric service of 'centre hospitalier d'essos' in yaounde, cameroon. this central region has an equatorial climate with a defined moderate rainfall during march-june and intense precipitation during september-november. children aged less than or equal to 15 years hospitalized for sari were daily recruited in our study. a total of 347 patients were enrolled by experienced nurses or physicians during a 2-year period between september 2011 and september 2013. sari was defined according to a previously suggested who case definition 14 with a history of symptoms of <5 days. these criteria of hospitalized patient inclusion were as follows: fever >38°c and cough and/or sore throat. children were enrolled only after an informed written consent was obtained from their parents or legal guardians. this study was part of a global study aimed at assessing risk factors associated with severe influenza in cameroon (immi project), which was reviewed and approved by the national research ethics committee and the ministry of health of cameroon. demographic and clinical information was obtained from participants using a standardized questionnaire. nasopharyngeal swab specimens were collected from hospitalized children and transported to the cpc as previously described. 13 viral rna and dna were extracted using qiaamp viral rna mini kit and qiaamp dna mini kit (qiagen, hilden, germany), respectively, following the manufacturers' instructions. a final elution volume of 60 ll of rna and 200 ll of dna were stored at -80°c till testing by multiplex pcr/rt-pcr. testing for influenza a and b viruses were conducted, and influenza a virus-positive specimens were subtyped according to the method developed by us centers for disease control and prevention (cdc). 15 then, the detection of 14 non-influenza respiratory viruses, including hmpv, hrsv, hpiv (1-4), ev, rv, hcov-oc43, hcov-229e, hcov-nl63, hcov-hku1, hadv, and hbov were performed by 4 duplex rrt-pcr, using a commercially available respiratory multi well system r-gene tm (biom erieux, lyon, france) following the manufacturer's instructions. pcr amplifications were performed on abi 7500 fast (applied biosystems, foster city, ca, usa). a patient was considered to have a single viral infection if only one pathogen was detected in the specimens. in case of more than one viral pathogen detected, patients were considered to have viral coinfections. the data obtained and the laboratory results were anonymized and entered into a database prepared with microsoft â excel (microsoft, washington, dc, usa). the database was then checked and cleaned for abnormal wrongful entries. we analyzed the demographics of the study subjects and the positive cases, as well as the clinical characteristics of all respiratory viruses. proportions were compared using a chisquared or the fisher exact test. we analyzed the clinical and coinfection characteristics of positive according to the negative patients for respiratory viruses using a stepwise logistic regression model. uncertainty was expressed as 95% confidence intervals (cis). statistical analysis was performed with the r program version 2á15á1, and p-values of 0á05 or less were considered statistically significant. during the study period, 374 hospitalized children with sari aged 0-15 years were enrolled. as 27 patients were excluded due to insufficient material for analysis of all viruses, 347 (92,8%) subjects were analyzed. the median age of the patients was 1á5 years (iqr: 0á75-3á0 years). most patients (88á4%) were below 5 years of age, with 131 (37á8%) children ≤1 year, 100 (28á8%) children aged [1] [2] years, 76 (21á9%) children aged [2] [3] [4] [5] years, and 40 (11á5%) children aged [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] years. half of patients (180 [51á9%]) were male. among the 347 hospitalized participants with sari, the most common respiratory symptoms were cough (85á9%, 298/ 347), rhinorrhea (78á4%, 272/347), fatigue (57á9%, 201/347), and wheezing (41á8%, 145/347). demographic data and clinical characteristics of the 347 cases studied are summarized in table 1 . a majority of sari was observed among children aged ≤5 years (90á7%, 206/227), and the proportions infected were significantly negatively associated with age (odds ratio [or], 0á91; 95% ci, 0á84-0á99, p = 0á03). there were no significant differences in terms of gender between patients infected with different viruses. influenza virus were more frequently detected in the individuals over 1 year (p = 0á04). for the remaining viruses, there was no difference between rate of infection and age (table 2) . among positive samples, single viral infection accounted for 70á5% (160/227) of cases, whereas infections with multiple viruses were observed in 29á5% (67/227), including 53 double infections, 13 triple infections, and one quadruple infection. specific viruses detected in the study population of 347 hospitalized children are listed in table 3 , in descending detection rate. the mean age and age group distribution of patients with multiple viral infections were not significantly different from those of group with single infection (table 2) . hadv, hrsv, influenza and hmpv were more frequent in single infections whereas rv/ev, hbov, hpiv, and hcov were more frequent in mixed infections ( figure 2 ). in a binary logistic regression, positive samples was negatively associated only with headaches (p = 0á003, odds ratio [or], 0á08; 95% ci, 0á01-0á3). no other clinical symptoms were associated with the positive patients, nor with any other specific viruses detected or coinfections.seasonality. the study also investigated the monthly distribution of viruses. the seasonal variations of the viral respiratory infections are shown in figure 2. in the first period from september 2011 to november 2012, the average number of samples collected monthly was 18á7, whereas in the second period from december 2012 to september 2013 the number of enrolled cases was much lower, the average number of samples collected monthly being 6á6. however, hrsv showed a pronounced seasonality, with peaks during the rainy season from september to december. the circulation of influenza viruses was similar to hrsv although a few cases were detected outside the rainy season peak. rv/ev was detected almost year-round with peak activity in the rainy season between september and december corresponding to a higher number of samples collected during that period. on the other hand, hadv occurred throughout the study period with no evident temporal patterns. hbov occurred intermittently all over the study period, while the prevalence of remaining viral etiologies was low, ranging from 0 to 5 positive samples per month for each virus, numbers which appeared insufficient to allow for the description of seasonal patterns. coinfections were observed throughout the study period. the aim of this study has been to determine the viral panorama of sari in 374 hospitalized children during a 2year period in cameroon. the data presented here are unique, to the best of our knowledge. it represents viral etiologies (17 respiratory viral targets) in children aged ≤15 years hospitalized for sari in cameroon. more importantly, this study describes the incidence of adv and the recently discovered hbov in cameroon for the first time. 8 our results demonstrate the potential importance of respiratory viruses in the etiology of hospitalized children with sari in cameroon. of 347 samples tested, at least one respiratory virus was detected in 227 (64á5%). this detection rate is consistent with results of other etiologic studies of hospitalized children conducted in similar settings. [16] [17] [18] [19] [20] nonetheless, this proportion appears to vary largely between countries. kwofie et al. recorded a lower percentage of 25á7% in ghana 21 and khor et al. recorded 26á4% in malaysia. 22 higher percentages 94á2% in china, 23 91% in france, 24 and 90% in alaska 25 were reported by zhang et al., singleton et al., and huguenin et al., respectively. comparison among published studies is always difficult. possible reasons for these varying outcomes among studies include true differences in epidemiology, differences in the inclusion criteria for study population, diagnostic methods used, panel of viral agents tested, climate, study duration, and patients' ages. consider that all the above studies included hospitalized children; lower prevalence recorded by khor et al. might have been due to their 27-year study duration and laboratory methods (direct if and viral isolation) which are known to be less sensitive than molecular techniques. 25 the most prevalent virus detected in our study was adv, representing 27á3% of the total number of viruses observed. this value is higher than that (0á8-13á3%) reported in most other similar studies. 16, 17, 22, [26] [27] [28] it is nonetheless similar to what was noted in alaska 24 particularly. this finding shows that adv could play a more significant role than originally thought. accordingly, additional efforts describing our associated serotypes will help to further understand how much this virus contributes to disease severity. unlike our results, most similar studies showed that hrsv was the most prevalent viral pathogen. 22, [31] [32] [33] in our study, hrsv was the second most frequent pathogen after hadv with merely 13á2% of 347 hospitalized children testing positive. however, this proportion was similar to that reported in other studies (5á7-15%). 16, 28, 29, 34 unlike previous similar studies, rv/ev accounted for only 11á5% of positivity in our study. 17, 18, 24, 26, 29 hbov was the fourth most common virus with a prevalence of 10á6% in all cases. this result is in line with the results of others authors, who have reported a worldwide detection rate of hbov ranges between 2 and 19% in nasopharyngeal samples. 35 the remaining viral etiologies were identified in 9á8%, 6á6%, 5á7%, and 2á3% for influenza, hpiv, hcov, and hmpv, respectively. these results correspond with those reported by other authors. 23, 27, 32, 33, 36, 37 as previously shown, we also found that virus-associated hospitalization figures were significantly higher among children (p = 0á03). 27, 28, 38 according to reports, there is always a higher incidence of viral respiratory infection in children than in adults. this may be partially explained by the lower detection rate of respiratory viruses in the elderly due to reduced viral shedding in older age groups. 39 hrsv, the second most commonly virus detected in this study, has been reported to be the leading cause of sari in young children. 19, 22, 36 nevertheless, we did not find a significant age-specific prevalence in hrsv detection during this study. this was probably due to the fact that our statistical analysis was hampered by the limited size of study participants older than 5 years. headaches were negatively associated with positives samples (p = 0á003). however, as a majority of our study population was made up of children unable to report headaches, only 46 patients (13á3%) were taken into account in the analysis.multiple viral infections were observed in 29á5% of the patients with positive viral detection. this is a very similar percentage to the one found by others researchers. 22, 26, 37 bocavirus has been frequently implicated in coinfections. 17, 19, 25 our results reflect those of other studies in which hbov was associated (35á8% versus 8á1%, p < 0á001) with multiple infections. surprisingly, adv and picornaviruses were also most frequently involved in coinfections, which probably resulted from the fact that they were detected throughout the year and were among the most prevalent. in spite of our intensive surveillance strategy with daily recruitment, the number of inclusions was not stable over the study period. notwithstanding this lack of uniformity in number of samples collected during the first (september 2011 to november 2012) and second (december 2012 to september 2013) periods, we were able to detect a seasonal pattern thanks to the sufficient number of enrolled patients during two consecutive years. we found that rsv cases were detected mainly during the major rainfalls from september to november, which is consistent with the data in the literature. 22, 23, 31 the seasonality of influenza virus is very uncertain in tropical areas. the current study did not confirm that influenza circulated between september and november, as shown in our previous studies. 13 this fact might have contributed to the reduction in the inclusion rate during the second period due to a true change in the epidemiology of sari. indeed, that would have contributed to reduce the numbers eligible patients in the second period of the study. rv/ev and adv circulated year-round, a finding reported in other studies. 17, 23, 28 for the remaining viruses, no evident temporal patterns were observed. this study is ongoing, however, and any changes in the seasonal circulation will be detected. several points must be taken into consideration when looking at our findings. firstly, our study was conducted at only one site, which might have underestimated the overall detection rate for selected viruses. likewise, the enrolled cases may not be generalizable for the entire population of children in cameroon. subsequently, as discussed by broor et al., 38 there is no denial that some respiratory viruses can be detected in asymptomatic children 24, 40, 41 or shed for long periods of time after infection. 24, 42 hence, the role played by the viruses detected in these sari cases is less clear considering that we did not evaluate respiratory specimens from asymptomatic children without respiratory symptoms. otherwise, despite tests for a large panel of respiratory viruses we did not evaluate bacterial etiologies, due to the difficulty in obtaining adequate samples for culture. it has been shown that bacterial pathogens may have been responsible for sari symptoms 16 ; further studies will thus be required to specify their role in sari in cameroon. lastly, although nurses in charge of recruitment were well trained, they did not systematically record severe symptoms such as convulsion, stridor, chest pain, inability to drink or breastfeed, and outcomes of study participants. this limited our ability to assert the clinical case severity and outcome of our sari cases. this situation also added a limitation to this study regarding the representativeness of children included. notwithstanding its limits, this is the first study in cameroon to characterize 17 common respiratory viruses in pediatrics with sari during a 2-year consecutive period. using a multiplex rrt-pcr, we were able to identify a wide variety of viruses and their seasonal patterns. in our resource-limited setting, this process is particularly useful for selecting only the major contributors to sari and thereby enhancing our existing surveillance systems. additional studies evaluating asymptomatic cameroonian children will be important to understanding the relationship between viral carriage and disease. with this new method, we will also be able to evaluate the role of viral loads in respiratory infections in future research. global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis viral etiology of hospitalized acute lower respiratory infections in children under 5 years of age -a systematic review and meta-analysis viral etiology of acute lower respiratory tract infections in hospitalized young children in northern taiwan viral infections of the lower respiratory tract emerging respiratory agents: new viruses for old diseases? cloning of a human parvovirus by molecular screening of respiratory tract samples use of a multiplex pcr/rt-pcr approach to assess the viral causes of influenza-like illnesses in cambodia during three consecutive dry seasons advances in the laboratory diagnosis of viral respiratory disease circulation of human influenza viruses and emergence of oseltamivir-resistant a(h1n1) viruses in cameroon, central africa spatiotemporal circulation of influenza viruses in 5 african countries during 2008-2009: a collaborative study of the institut pasteur international network viral etiology of influenza-like illnesses in cameroon strategy to enhance influenza surveillance worldwide cdc protocol of realtime rtpcr for swine flu influenza a (h1n1) the role of respiratory viral infections among children hospitalized for community-acquired pneumonia in a developing country respiratory viruses from hospitalized children with severe pneumonia in the philippines respiratory viruses in hospitalized children with influenza-like illness during the h1n1 frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction viral pathogens associated with acute respiratory infections in central vietnamese children respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of 27 years viral etiology and clinical profiles of children with severe acute respiratory infections in china viral respiratory infections in hospitalized and community control children in alaska broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex rt-pcr dna microarray system human bocavirus and other respiratory viral infections in a 2-year cohort of hospitalized children respiratory virus infections in hospitalized children and adults in lao pdr respiratory viral coinfections identified by a 10-plex real-time reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa etiology and epidemiology of viral pneumonia among hospitalized children in rural mozambique adenoviruses in the immunocompromised host detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study lower respiratory tract infection in hospitalized children viral etiology of lower respiratory tract infections in hospitalized children in mexico rna viruses in young nepalese children hospitalized with severe pneumonia human bocavirus-the first 5 years viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city viral etiology of acute respiratory tract infections in children presenting to hospital rates of respiratory virusassociated hospitalization in children aged <5 years in rural northern india performance of diagnostic tests to detect respiratory viruses in older adults detecting respiratory viruses in asymptomatic children viral and bacterial causes of severe acute respiratory illness among children aged less than 5 years in a high malaria prevalence area of western kenya persistence of rhinovirus and enterovirus rna after acute respiratory illness in children viral etiology of sari in cameroon the authors. influenza and other respiratory viruses published by we are grateful to all the nurses and other healthcare workers who collected specimens, data, and information and provided their support with clinical evaluations of patients during this study. 'this work was funded by the international network of pasteur institutes (ptr 351) and the "institut de microbiologie et de maladies infectieuses (immi)" in france'. key: cord-286096-h275nner authors: huijskens, elisabeth g. w.; van erkel, adriana j. m.; palmen, fernand m. h.; buiting, anton g. m.; kluytmans, jan a. j. w.; rossen, john w. a. title: viral and bacterial aetiology of community‐acquired pneumonia in adults date: 2012-08-22 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2012.00425.x sha: doc_id: 286096 cord_uid: h275nner please cite this paper as: huijskens et al. (2012) viral and bacterial aetiology of community‐acquired pneumonia in adults. influenza and other respiratory viruses 7(4), 567–573. background modern molecular techniques reveal new information on the role of respiratory viruses in community‐acquired pneumonia. in this study, we tried to determine the prevalence of respiratory viruses and bacteria in patients with community‐acquired pneumonia who were admitted to the hospital. methods between april 2008 and april 2009, 408 adult patients (aged between 20 and 94 years) with community‐acquired pneumonia were tested for the presence of respiratory pathogens using bacterial cultures, real‐time pcr for viruses and bacteria, urinary antigen testing for legionella and pneumococci and serology for the presence of viral and bacterial pathogens. results pathogens were identified in 263 (64·5%) of the 408 patients. the most common single organisms in these 263 patients were streptococcus pneumoniae (22·8%), coxiella burnetii (6·8%) and influenza a virus (3·8%). of the 263 patients detected with pathogens, 117 (44·5%) patients were positive for one or more viral pathogens. of these 117 patients, 52 (44·4%) had no bacterial pathogen. multiple virus infections (≥2) were found in 16 patients. conclusion in conclusion, respiratory viruses are frequently found in patients with cap and may therefore play an important role in the aetiology of this disease. community-acquired pneumonia (cap) is a common disorder and a major medical problem. in 30-50% of the patients with cap, no specific organism is identified, despite the extensive use of diagnostic tests. [1] [2] [3] the most common causative pathogen of bacterial cap is streptococcus pneumoniae. 4 mycoplasma pneumoniae and chlamydophila pneumoniae are among the most common 'atypical' pathogens. potential viral causes of cap are often not explored because of the lack of antiviral agents and the relative unfamiliarity with viral pneumonia. however, it is well known that viral infections of the respiratory tract are the cause for significant mortality and morbidity all over the world, particularly in children and elderly adults. viral respiratory pathogens that are commonly found include rhinoviruses, coronaviruses, influenza viruses, respiratory syncytial viruses, parainfluenza viruses and adenoviruses. over the past decade, analysis of clinical specimens of the respiratory tract through different diagnostic methods have led to the discovery of new viruses, such as human metapneumovirus, 9 human severe acute respiratory syndrome coronavirus, 10 coronaviruses nl63 11 and hku1, 12 human bocavirus 13 and the recently described polyomaviruses kipyv 14 and wupyv. 15 in this study, we tried to reveal the aetiology of community-acquired pneumonia in patients admitted to the hospital with community-acquired pneumonia using extensive molecular testing for viral and bacterial pathogens. community-acquired pneumonia was defined as suspicion of acute respiratory tract infection with a new or progressive infiltrate on a chest radiograph and one of the following criteria: fever (temperature ‡38ae0°c) or hypothermia (temperature <35°c), new cough with or without sputum production, abnormal percussion and altered breath sounds on auscultation, dyspnoea or tachypnea or hypoxia, and leucocytosis or leucopenia. we excluded patients with recent hospitalization (<2 weeks) and those residing in long-term care facilities, patients with known bronchial obstruction or a history of post-obstructive pneumonia other than copd, patients with primary lung cancer or another malignancy metastatic to the lungs, and patients with aids, patients with known or suspected pneumocystis jirovecii pneumonia or patients with known or suspected active tuberculosis. the study was approved by the local medical ethics committee. written informed consent was obtained from all participants in the study. at the emergency ward a throat swab was taken, and two sets of blood samples were obtained and cultured according to standard microbiological procedures. if available, a sputum sample was evaluated by use of gram staining and culture. urinary antigen detection tests for streptococcus pneumoniae and legionella pneumophila were performed with the binaxnow pneumococcal urinary antigen test and the binaxnowlegionella urinary antigen test (both from binax, me, usa). paired serum samples were obtained during the acute and convalescent phases of infection (separated by at least 2 weeks) for serological studies. a case report form was obtained for every patient. all samples were tested using real-time pcr for the presence of respiratory viruses and bacteria including adenovirus (adv), human bocavirus (hbov), ki-and wu polyomaviruses (kipyv and wupyv), human metapneumovirus (hmpv), human rhinovirus (hrv), human coronaviruses (hcov) (oc43, nl63, hku and 229e), parainfluenza viruses (piv), 1-4 influenza viruses a and b (infa, infb), respiratory syncytial virus (rsv), legionella pneumophila, mycoplasma pneumoniae, chlamydophila psittaci, chlamydophila pneumoniae, coxiella burnetii and streptococcus pneumoniae. real-time pcr procedures were performed as described in reference [16] [17] [18] [19] [20] [21] [22] . briefly, nucleic acids were extracted from the throat swabs with the magna pure lc using the total nucleic acid isolation kit system according to the manufacturer's protocol (roche diagnostics, basel, switzerland). each sample was eluted in 1000 ll of buffer sufficient to perform all the real-time pcrs. cdna was synthesized by using multiscribe reverse transcriptase (rt) and random hexamers (both from applied biosystems, carlsbad, ca, usa). the reaction was performed in 100 ll of reaction mixture consisting of 10 ll rt-buffer (10·), 22 ll mgcl 2 (25 mm), 20 ll dntp-mix (10 mm), 5 ll random hexameer (50 lm), 2,5 ll multi-scribe rt (50 u ⁄ ll), 2 ll rnase inhibitor (20 u ⁄ ll) (ez rt-pcr kit; applied biosystems) and 40 ll of the isolated sample. after incubation for 10 minutes at 25°c, rt was carried out for 30 minutes at 48°c, followed by rt inactivation for 5 minutes at 95°c. an aetiological agent for cap was considered present, if any of the following criteria were met: a pathogenic microorganism was cultured from blood samples; the urinary antigen test was positive for s. pneumoniae or l. pneumophila; pcr of the throat swab or sputum samples yielded a positive result; sputum samples (presence of >25 polymorphonuclear leucocytes and <10 squamous cells per field) with a predominant organism and compatible results from gram stain; the presence of igm antibodies for m. pneumoniae, or a fourfold increase in igg antibody titres for m. pneumoniae, l. pneumophila, chlamydophila psittaci and coxiella burnetii. groups were compared by a chi-squared test with a significance level of p < 0ae05. analyses were conducted using pasw statistics 18 (ibm company, chicago, il, usa). from april 2008 through march 2009, 408 patients were included. the demographic characteristics of the patients are presented in table 1 . patients ranged in age from 20 to 94 years (mean 65 years; median 68 years), 61ae3% of the patients were men and 38ae7% were women. of the 408 patients, we collected 408 throat swabs, 408 pneumococcal urinary antigen tests, 405 legionella urinary antigen tests, 203 sputum samples for bacterial culture, 163 sputum samples for molecular detection, 329 blood cultures, 90 serum samples for m. pneumoniae, 66 serum samples for l. pneumophila, 44 serum samples for chlamydophila psittaci and 104 serum samples for coxiella burnetii. all samples were taken from unique patients, that is, none of the patients had duplicate samples taken. aetiology was identified in 263 (64ae5%) of the 408 patients, and more than one pathogen was isolated in 106 patients (26ae0%). results are shown in table 2 . of the 263 patients identified with a pathogen, 117 (28ae7%) patients were positive for one or more viral pathogens. a bacterial pathogen was detected in 65 (55ae5%) of these 117 patients, whereas in 52 (44ae4%) of these patients, only respiratory viruses were detected. s. pneumoniae, coxiella burnetii and infa were identified as the only micro-organism in 22ae8%, 6ae8% and 3ae8% of the 263 patients, respectively. in 139 patients with cap, s. pneumoniae was detected. in 60 (43ae2%) patients, s. pneumoniae was the only identifiable pathogen. of those 60 patients, 17 were diagnosed by the urinary antigen assay alone, 14 were only detected by culture or molecular detection of s. pneumoniae in sputum samples and six had only positive blood cultures. in addition, in five patients, both blood culture and the urinary antigen test were positive, in three patients both blood culture and sputum samples were positive, in seven patients sputum samples and the urinary antigen test were positive and in eight patient all three tests were positive. of the remaining 79 s. pneumoniae patients having other pathogens as well, 11 patients had only a positive urinary antigen assay, and 53 patients had a positive pcr from sputum, two patients had only a positive blood culture and urinary antigen test, three patients had a positive blood culture and a positive pcr from sputum samples and nine patients had a positive pcr from the sputum sample and a positive urinary antigen test. in 53 patients diagnosed with pneumococcal cap, the following viruses were found: hrv 19 times, infa 11 times, hcov oc43 eight times, piv1 seven times. rsv, hcov 229e three times, hmpv, hcov nl63, and infb all two times and kipyv, wupyv and hbov were each detected one time. haemophilus influenzae was found in 21 of the 203 sputum samples and in one blood culture. in 11 of these patients, one or more respiratory viruses were detected as well. hrv was detected three times, piv1, infa, hcov oc43 and rsv were each detected two times, and hbov, kipyv and wupyv were each detected one time. statistical analysis showed a significant association between patients with h. influenzae and viral pathogens (p = 0ae023) and a significant association between patients with s. pneumoniae and viral pathogens (p = 0ae003). legionella was found in 15 patients. of two patients the legionella urinary antigen tests, serum samples and respiratory samples were positive for l. pneumophila. three patients had a positive legionella urinary antigen test and positive respiratory sample, in four patients only the urinary antigen test was positive, in three patients only the respiratory sample and in one patient only the serum samples for l. pneumophila were positive. mycoplasma pneumoniae-specific igm was found in only one of the 90 patients tested, and in two respiratory samples, m. pneumoniae dna was detected. coxiella burnetii, causing q fever, was found in 37 patients (in 12 serum samples and in 25 respiratory samples). chlamydophila psittaci was found in seven patients and chlamydophila pneumoniae was found in two patients. in total, 117 (28ae7%; 69 male and 48 female) of the 408 patients were positive for one or more viral pathogens. there was no significant difference between viruses infecting men and women. all common respiratory viruses were detected. hrv was detected at the highest frequency, in 34 (8ae3%) of the 408 patients. the detection rates for the other viruses were 5ae6% for infa, 4ae4% for piv1, 2ae9% for hcov oc43, 2ae2% for infb, 2ae0% for rsv, 1ae5% for hcov nl63, 1ae2% for piv3 and hcov 229e. for hmpv, piv 2, hcov hku, hbov, kipyv, wupyv and adv, the detection rates were <1%. piv4 was not detected. in the 117 virus-positive patients, a total of 136 viruses were detected. multiple virus infections ( ‡2) were found in 16 patients (table 3) . thirteen (3ae2%) of the 408 patients had two respiratory viruses and 3 (0ae7%) patients had three viruses present in their respiratory samples. pivs were the most frequently found viral agents in the multiple infected patients and were found in eight patients [piv 1 (six times), piv 2 (one time), piv 3 (three times)], followed by coronaviruses in seven patients [hku (one time), oc43 (two times), 229e (one time), nl63 (four times)] and infb in four patients, infa in three patients. hrvs, hbov, rsv, kipyv and wupyv were co-detected in two patients. the period of the year in which viruses were detected varied per virus as presented in figure 1 . whereas infa had one major peak lasting from january to february 2009, infb was detected throughout the whole study period be it at a low rate. also hrvs were found during the entire year, although a small peak was observed in may this study revealed the viral and bacterial aetiology in 263 (64ae5%) of 408 patients with community-acquired pneumonia. in spite of using sputum samples, blood cultures, urine for antigen assays, throat swabs and serum samples, no causative agent was found in 35% of the subjects. this is in line with the literature, where levels of identification of the causa-tive micro-organisms in cap vary from 46% to 83%. this variation is attributable to differences in detection techniques and in selection of patients who are included. we improved the yield by testing sputum samples for s. pneumoniae, mycoplasma pneumoniae, chlamydophila pneumoniae, chlamydophila psittaci and l. pneumophila. we identified s. pneumoniae (22ae8%) as the most common single organism in 263 patients, followed by coxiella burnetii (6ae8%) and influenza a virus (3ae8%). this was similar to other studies, which also reported s. pneumoniae as a predominant causative agent. 1 on the other hand, we did not find many mycoplasma pneumoniae infections in our study, even though in the literature m. pneumoniae is among the most common 'atypical' pathogens. this might be due to the fact that m. pneumoniae infections occur in cyclic epidemics every 3-5 years and infections are generally mild. many adult cases may be asymptomatic and not in need of medical attention. 25 in the netherlands, cap affects about 5-10 persons per 1000 inhabitants, and only 5-20% of these cases are admitted to hospitals. furthermore, the distribution of pathogens causing cap may vary by country, owing to geographic differences. in our study, chlamydophila pneumoniae was detected in throat samples of two patients only. some studies indicated c. pneumoniae as one of the most common 'atypical' pathogens. however, a recent study by wellinghausen et al., 29 found a low prevalence (<1%) of c. pneumonia which is in accordance with our findings. it is unclear what the reasons are for these different detection rates. the most important limitation in our study was that we did not include a control group to determine the prevalence of viral respiratory pathogens and bacterial pathogens. in a case-control study by van gageldonk-lafeber et al., 30 the incidence and aetiology of acute respiratory tract infections in patients visiting their general practitioners was studied and the researchers detected pathogens, mostly viruses, in approximately 30% of the subjects with no respiratory complaints. another limitation was the incomplete sputum sample collection. the reason was the inability of patients to produce sputum. therefore, throat swabs were taken of all patients. as the sensitivity of throat swabs may be lower than the sensitivity of sputum, nasopharyngeal sampling or washings, it is possible that we underestimated the prevalence of viruses in our population. respiratory viruses were found as the only detectable pathogen in 52 patients who had been included throughout the year, covering all the seasons. year-round inclusion is important to cover the complete spectrum of respiratory virus infections, because several viruses are known to be found only in particular months of the year. infa has been found as the second most frequent pathogen in cap patients and the most common viral pathogen in all the age groups. in adults, infa, rsv, rhinoviruses and adenoviruses are recognized as important causes for cap. 32 however, viruses that cause community-acquired pneumonia are often overlooked by clinicians. it still remains unclear whether some respiratory virus can cause pneumonia by itself or whether it needs the help of other respiratory pathogens. in our study, viral and bacterial pathogens were found in 65 (16%) patients. co-infection rates have been described in 5ae7-22ae5% of cap in other studies. the most common bacterial co-pathogens were h. influenzae and s. pneumoniae. in agreement with other studies, we found an association between s. pneumoniae and viruses. we also found an association between h. influenzae and viruses. in general, blood samples for bacterial culture are relatively easily obtainable, and if positive, they provide a microbiological diagnosis. in our study, 13ae1% of the blood cultures revealed a pathogen, which is similar to the results of other studies. in the literature, s. pneumoniae pcr on sputum samples as a diagnostic tool for pneumococcal disease has had mixed results because distinguishing colonization from infection using s. pneumoniae pcr is difficult even by quantifying the load. [35] [36] [37] [38] culture has important limitations as well. prior antibiotic therapy is of great influence on the growth of s. pneumoniae in sputum samples and blood cultures. several studies found that during antibiotic treatment sputum samples became rapidly negative for s. pneumoniae in contrast to the s. pneumoniae pcr that remained positive. in our study, s. pneumoniae was detected in all culture-positive sputum samples and in many culture-negative sputum samples by pcr, presumably reflecting the increased sensitivity of molecular technique above traditional culture methods. of the 60 patients with s. pneumoniae as causative organism, the pneumococcal antigen assay was positive in 62%. the results of the pneumococcal antigen assay showed a lower sensitivity compared with data reported by others. 42 the reason for this difference in sensitivity is unclear but could be explained by the influence of prior antibiotic therapy, not concentrating the urine before executing the assay, and the fact that the pneumococcal antigen assay is more sensitive in patients who are bacteraemic than in patients without a bacteraemia. legionella pneumophila was diagnosed in 15 cases (3ae8%), which is in agreement with results obtained by previous studies. 44 finally, in our study population, a relatively large number of cap cases were caused by coxiella burnetii. this was owing to a q fever outbreak in our area with over 4000 notified cases in the netherlands between 2007 and 2010. 45 in 408 adult patients presenting at the hospital with cap, a pathogen was demonstrated in 64ae5%. s. pneumoniae, influenza a virus and coxiella burnetii were the three most frequent pathogens. mixed viral and bacterial infections were frequently observed, and in 29% of the patients with cap, a virus was detected, including 13% of the patients in which only viruses were detected. further investigations are warranted to elucidate the importance of viruses as causative agents in the pathogenesis of cap in adult patients. etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods the etiology of communityacquired pneumonia in australia: why penicillin plus doxycycline or a macrolide is the most appropriate therapy antibiotics for community acquired pneumonia in adult outpatients pathogenesis, treatment, and prevention of pneumococcal pneumonia epidemiology and etiology of community-acquired pneumonia a worldwide perspective of atypical pathogens in community-acquired pneumonia global burden of acute respiratory infections in children: implications for interventions community-acquired viral pneumonia a newly discovered human pneumovirus isolated from young children with respiratory tract disease a novel coronavirus associated with severe acute respiratory syndrome identification of a new human coronavirus cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a third human polyomavirus identification of a novel polyomavirus from patients with acute respiratory tract infections increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time pcr in samples from patients with respiratory symptoms human bocavirus and ki ⁄ wu polyomaviruses in pediatric intensive care patients diagnostic value of real-time polymerase chain reaction to detect viruses in young children admitted to the paediatric intensive care unit with lower respiratory tract infection utility of real-time pcr for diagnosis of legionnaires' disease in routine clinical practice quantitative detection of streptococcus pneumoniae in nasopharyngeal secretions by realtime pcr development of an internally controlled real-time pcr assay for detection of chlamydophila psittaci in the lightcycler 2.0 system interlaboratory evaluation of different extraction and real-time pcr methods for detection of coxiella burnetii dna in serum community-acquired pneumonia incidence and characteristics of viral community-acquired pneumonia in adults mycoplasma pneumoniae and its role as a human pathogen swab/nvalt (dutch working party on antibiotic policy and dutch association of chest physicians) guidelines on the management of community-acquired pneumonia in adults time trends in primary-care morbidity, hospitalization and mortality due to pneumonia respiratory viruses in adults with community-acquired pneumonia low prevalence of chlamydia pneumoniae in adults with community-acquired pneumonia a casecontrol study of acute respiratory tract infection in general practice patients in the netherlands identification of respiratory viruses in adults: nasopharyngeal versus oropharyngeal sampling viral pneumonia aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (lrti) in primary care bts guidelines for the management of community acquired pneumonia in adults: update is quantitative pcr for the pneumolysin (ply) gene useful for detection of pneumococcal lower respiratory tract infection? quantitative detection of streptococcus pneumoniae, haemophilus influenzae, and moraxella catarrhalis in lower respiratory tract samples by realtime pcr evaluation of a pcr assay for detection of streptococcus pneumoniae in respiratory and nonrespiratory samples from adults with community-acquired pneumonia association between pneumococcal load and disease severity in adults with pneumonia specificity of a quantitative real-time polymerase chain reaction assay for the detection of invasive pneumococcal disease: identifying streptococcus pneumoniae using quantitative polymerase chain reaction quantitative detection of streptococcus pneumoniae from sputum samples with real-time quantitative polymerase chain reaction for etiologic diagnosis of community-acquired pneumonia real-time polymerase chain reaction assay for detection of streptococcus pneumoniae in sputum samples from patients with community-acquired pneumonia rapid urinary antigen test for diagnosis of pneumococcal communityacquired pneumonia in adults novel approaches to the identification of streptococcus pneumoniae as the cause of communityacquired pneumonia detection of respiratory viruses and legionella spp. by real-time polymerase chain reaction in patients with community acquired pneumonia bleeker-rovers cp. q fever in the netherlands from 2007 to 2010 we would like to acknowledge marcel peeters, who died during the course of this study. he was very much involved in the initiation of this study and his enthusiasm and knowledge was indispensable. furthermore, we thank lotte broers and petra van esch for their technical assistance. the authors declare no conflict of interest. key: cord-002852-m4l2l2r1 authors: munyua, peninah m.; githinji, jane w.; waiboci, lilian w.; njagi, leonard m.; arunga, geoffrey; mwasi, lydia; murithi mbabu, r.; macharia, joseph m.; breiman, robert f.; kariuki njenga, m.; katz, mark a. title: detection of influenza a virus in live bird markets in kenya, 2009–2011 date: 2012-04-19 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2012.00365.x sha: doc_id: 2852 cord_uid: m4l2l2r1 please cite this paper as: munyua et al. (2013) detection of influenza a virus in live bird markets in kenya, 2009–2011. influenza and other respiratory viruses 7(2), 113–119. background surveillance for influenza viruses within live bird markets (lbms) has been recognized as an effective tool for detecting circulating avian influenza viruses (aivs). in sub‐saharan africa, limited data exist on aivs in animal hosts, and in kenya the presence of influenza virus in animal hosts has not been described. objectives this surveillance project aimed to detect influenza a virus in poultry traded in five lbms in kenya. methods we visited each market monthly and collected oropharyngeal and cloacal specimens from poultry and environmental specimens for virological testing for influenza a by real time rt‐pcr. on each visit, we collected information on the number and types of birds in each market, health status of the birds, and market practices. results during march 24, 2009–february 28, 2011, we collected 5221 cloacal and oropharyngeal swabs. of the 5199 (99·6%) specimens tested, influenza a virus was detected in 42 (0·8%), including 35/4166 (0·8%) specimens from chickens, 3/381 (0·8%) from turkeys, and 4/335 (1·2%) from geese. none of the 317 duck specimens were positive. influenza was more commonly detected in oropharyngeal [33 (1·3%)] than in cloacal [9 (0·4%)] specimens. none of the 485 environmental specimens were positive. virus was detected in all five markets during most (14/22) of the months. ducks and geese were kept longer at the market (median 30 days) than chickens (median 2 days). conclusions influenza a was detected in a small percentage of poultry traded in lbms in kenya. efforts should be made to promote practices that could limit the maintenance and transmission of aivs in lbms. influenza a viruses are zoonotic pathogens that infect a variety of domestic poultry such as chickens, turkeys, ducks, and geese. [1] [2] [3] from the mid-1970s, investigations have revealed reservoirs of influenza viruses present in wild bird populations and domestic poultry. 2, 4, 5 surveillance for influenza viruses within live bird markets (lbms) has been recognized as an effective tool for detecting circulating influenza subtypes in the poultry population. 6 live bird markets are ideal sites for virus mixing and transmission because of their nature of congregating birds from various farms coupled with the practices of mixing newly arrived birds with those that have been in the market for extended periods. since the 1970s, influenza viruses have been isolated from birds in lbms in multiple countries. from 7% to 30% of fecal swabs from ducks were positive for circulating h3, h4, h5, h6, h7, and h9 influenza virus subtypes in lbms in taiwan, vietnam, and hong kong in the 1980s before the onset of the h5n1, h7n2, and h9n2 poultry epidemics in southeast asia. 7, 8 influenza viruses have also been detected in various environmental specimens collected in contaminated areas in lbms including drinking water troughs, and surfaces in the delivery, holding and slaughter areas in markets. 9, 10 in a study in hong kong, influenza a (h9n2) was isolated in 1% of fecal swabs and 7% of drinking water samples collected in eight live poultry markets. 11 most (56%) of lbms in indonesia were found to have ‡6 sites contaminated with avian influenza virus (h5n1) by real-time reverse transcription polymerase chain reaction (rt rt-pcr) with slaughter and sale areas being the most heavily contaminated. 9 although avian influenza viruses (aivs) in the poultry population have not been described in kenya, qualitative risk assessment studies carried out in 2007-2008 following the 2005 threat of introduction of highly pathogenic avian influenza (hpai) h5n1 in the country suggested a significant risk of transmission of aivs if the virus were introduced into the poultry population. 12 the risk assessment identified complex marketing chains of poultry involving multiple middle men and markets coupled with unsatisfactory levels of biosecurity along the poultry chain as important factors that could contribute to the spread and transmission of influenza viruses through the poultry population and potentially to the human population. 12 in march 2009, the kenya medical research institute ⁄ us centers for disease control and prevention -kenya (ke-mri ⁄ cdc-k) in collaboration with the kenya department of veterinary services (dvs) initiated surveillance to assess the presence of avian influenza viruses in birds traded in lbms in kenya. additionally, we investigated market practices that could contribute to mixing and transmission of virus within the market. between march 24, 2009 and february 28, 2011, we conducted surveillance in five lbms in kenya: kariokor, burma, and kawangware markets, located within the capital city of nairobi; nyambari market, located 40 km north of the city along a major highway; and nakuru market, located in a major urban center in the rift valley province, about 150 km north of nairobi ( figure 1 ). we did not collect samples for the months of december because the staff were unavailable to visit the markets. we chose these markets because they are among the largest poultry markets in the country, and they trade primarily in chickens. the nyambari market trades in multiple avian species, including turkeys, geese, ducks, and doves. the kariokor market is housed in an enclosed building, while the other four are outdoor markets. in four of the five markets, birds were kept in wire mesh cages, each housing 40 birds during the day and night. in the fifth market, nyambari, birds were not kept in cages during the day but they stayed close to the feeding and watering troughs. at night, the birds at nyambari market were driven to a shelter located 500 m from the market. the five lbms receive poultry from districts across the country. the range of birds sold at the markets included chickens (indigenous chickens, spent layers and broilers), ducks, turkeys, and geese. the markets sell live poultry for restocking to farmers and for slaughter to individual homes and hotels. at kariokor and kawangware markets, poultry slaughter is carried out within the market premises. no slaughtering occurs at the other three markets. each market was visited once a month, and an oropharyngeal (op) and a cloacal (cl) swabs collected from 25 birds on every visit. birds that had stayed the longest in the market were preferentially sampled. for the market where there were multiple species, we sampled from all the poultry species. in the chicken markets where the birds were confined in cages, birds from all of the cages were sampled; on average, 3-5 birds were sampled from each cage. in addition, five environmental specimens were collected by swabbing fecal droppings on the floor of the bird cages during each monthly market visit. plastic-shafted polyester-tipped swabs were used to collect op and cl swabs from birds and to collect environmental specimens. the swabs were each placed in cryovials containing 2 ml of freshly prepared viral transport media (vtm) containing bovine serum albumin and veal infusion broth supplemented with amphotericin b and gentamycin (http://www.who.int/csr/resources/publications/surveillance/ annex8.pdf). specimens were labeled and transported at 4°c to the kemri ⁄ cdc-k laboratory and frozen at )80°c within 24 hours after collection until testing. we administered a standardized questionnaire to the poultry traders during each visit. the questionnaire included questions about the number and types of birds in each market, whether the markets had been cleaned using disinfectants, the presence of rodents and wild birds, the number of days the birds had been in the market, the source of the birds, and the health status of the birds. all specimens were tested by real-time reverse transcription polymerase chain reaction (rt rt-pcr) at the biosafety level 3 kemri ⁄ cdc-k laboratory in kisumu using the cdc protocol for influenza a virus detection. 13 briefly, total rna was isolated from 100 ll of the oropharyngeal specimens using the qiaamp rna extraction kit (qiagen inc, valencia, ca, usa) according to the manufacturer's instructions. total rna was extracted from 100 ll of each cloacal and environmental specimen using the magmax viral rna isolation kit (ambion inc, applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. one step rt rt-pcr was carried out using the agpath-id rt rt-pcr kit (applied biosystems). 13 the rt rt-pcr machine was set to run at 10 minutes at 45°c for reverse transcription, 10 minutes at 95°c to activate the taq polymerase, and a typical 45 cycle pcr with denaturation at 95°c for 15 seconds and annealing ⁄ extension at 55°c for 1 minute. fluorescence was read at the annealing ⁄ extension step. the results were collected as cycle threshold (c t ) values. specimens with c t values of <40ae0 were considered positive. data were entered and stored in an ms access database and analyzed using sas version 9.1 (cary, nc, usa). descriptive statistics on number of birds sold, suppliers, and length of stay of birds were calculated. we used chisquare test for all the bivariate analysis. the average number of chickens present at the market on the day of the monthly visit was 641, 629, 381, and 218 for kawangware, burma, nakuru, and kariokor market, respectively. at the nyambari market, the average number of ducks, geese, and turkeys was 45, 52, and 60, respectively. additionally in nyambari market, doves, rabbits, and guinea fowl were occasionally present for sale and were housed in the same cages. the source of the poultry traded varied greatly for all species and markets. overall, birds were sourced by traders and middlemen from districts across five of the eight provinces in kenya. over half (55%) of the chickens traded in all the markets originated from rift valley province [bomet (33%), baringo (16%), and kericho (5ae5%) district] (figure 1 ). half (51%) of the ducks traded originated from the western province districts of bungoma, busia, kakamega, and malaba, while 16% originated from the neighboring country of uganda. forty-three percent of the geese originated from the rift valley province (nakuru district) and 22% from central province (nyandarua district). a majority (68%) of the turkeys originated from the western province [bungoma (25%), kakamega (17%), malaba (9ae4%), busia (14%), and teso (2ae6%) districts]. the five markets were open for trading for 7 days every week. rodents were reported to be present in kariokor, burma, kawangware, and nakuru markets, and disinfection was rarely carried out in any of the five markets. in all five markets, wild birds were observed mixing and feeding with the poultry. the majority of the birds sampled [2322 (88ae9%)] were supplied to the market traders by middlemen (table 1) . a small percentage (5ae0%) was bought directly from a farm by the traders, and 2ae5% of the poultry were bought from other markets (table 1) . ducks, geese, and turkeys stayed on average 15 times longer than chickens in the market ( table 2 ). over half of the ducks, geese and turkeys had been in the markets for 30 days at the time of sampling. we collected 5221 cloacal and oropharyngeal swabs. of these, 4176 (80%) were from chickens, 321 (6ae1%) from ducks, 382 (7ae3%) from turkeys, and 342 (6ae6%) from geese (table 3) . most (99ae5%) of the specimens were collected from healthy birds, but 0ae5% of samples were collected from clinically sick birds that mainly had diarrhea, difficulty in breathing, and nasal discharges. of the 5199 (99ae6%) specimens tested that could be linked to individual bird data, influenza a virus was detected in 42 (0ae8%). influenza was detected in 35 ⁄ 4166 (0ae8%) chicken op ⁄ cl specimens, 3 ⁄ 381 (0ae8%) turkey op ⁄ cl specimens, and 4 ⁄ 335 (1ae2%) geese op ⁄ cl specimens (table 3) . no virus was detected in 317 duck op ⁄ cl specimens [2ae3% upper limit at 95% confidence level (ci)] ( table 3) . the mean c t value of the 42 specimens that were positive for influenza a by rt rt-pcr was 37ae2 (standard error 0ae29); the median c t value was 38ae1 (range 33ae1-39ae6). test results for 22 (0ae4%) specimens could not be linked to individual bird data and were excluded from further analysis. all of these 22 specimens were negative for influenza a. none of 485 environmental specimens collected and tested for influenza a virus were positive (upper limit 95% ci 0ae8%) ( table 4 ). in total, we collected specimens for 22 months and influenza virus was detected in the poultry in 14 (63ae64%) of detection of influenza a virus in live bird markets ª 2012 blackwell publishing ltd these months. we did not observe any seasonal or monthly differences in influenza detection ( figure 2 ). the median monthly detection rate was 0ae4%; the highest detection rate (4ae1%) was in january 2011. influenza virus was detected in both op and cl specimens in chickens (table 3 ). in turkeys and geese, influenza a was detected in op but not in cl specimens (table 3) . in all species, virus detection was significantly higher in op [33 (1ae2%)] than in cloacal [9 (0ae4%)] specimens (p-value < 0ae01). overall, influenza virus prevalence was highest in geese 4 ⁄ 168 (2ae4%) and lowest in turkeys 3 ⁄ 191 (1ae6%). there was no significant difference in the observed prevalence of influenza among chickens, turkeys, geese, and ducks (p-value 0ae29). all the influenza positive specimens were from healthy birds. influenza a was detected in all the five markets (table 4 ). in the four markets trading primarily in chickens, the detection rate varied from 0ae5% in nakuru to 1ae2% in kariokor. the detection rate in nyambari market, which traded in mixed species (turkeys, geese and ducks), was 0ae7%. there was no significant difference in the influenza detection rate in the five markets during this period. in total, the 35 influenza a-positive specimens in chicken were distributed in 13 administrative districts. ten of 35 (28ae6%) influenza a-positive chicken specimens were from birds sourced from one district (bomet district) where 1 ⁄ 3 of the chickens sold in the four markets originated from. influenza a detection by district was variable and ranged from 0ae3% to 12ae5% for this period. however, there was no significant difference in the influenza a detection rate of the chicken, geese, or turkey specimens by district or source of the birds. we detected influenza a viruses in poultry traded in all five lbms in kenya. the influenza a viral rna was detected in geese, turkeys, and chicken. to our knowledge, this is the first time influenza a rna has been detected in poultry traded in lbms in kenya. in our study, the overall influenza a rna detection rate among the birds sampled was 1ae6%. surveillance studies on influenza viruses have recorded variable prevalence of aivs in poultry traded in lbms in different countries around the world. in a study conducted in korea in 2003, 6% of chicken specimens were positive for aivs, 14 whereas 31% and 6ae1% of duck and geese specimens, respectively, were positive for aivs in vietnamese markets in 2001. 7 in the vietnam study, aivs were not detected from chicken specimens. 7 we did not detect influenza a in any of the duck specimens. this finding was unexpected, because ducks are associated with maintenance of influenza virus in domestic birds. in fact, many studies conducted in lbms and farms reported a higher prevalence of influenza in ducks compared with other poultry species. 7,15-17 it is not clear why influenza a virus was not detected in ducks in the markets in kenya, particularly in light of the fact that the ducks stayed for longer periods of time in the market and were housed in the market together with geese and turkeys. however, all of the ducks sampled were adults of market age, and it is possible that they had already been exposed to influenza viruses early in life and therefore may have developed some immunity to the circulating viruses. screening for anti-influenza a antibodies in these species would have helped to clarify this, but this was beyond the scope of our surveillance project. in our surveillance, in all avian species, influenza a virus was more commonly detected in oropharyngeal specimens than in cloacal specimens by rt rt-pcr. in a study of ai in backyard poultry in mali in 2007, 2ae2% of tracheal swabs and 1ae3% of cloacal swabs tested positive for influenza a by rt rt-pcr. 16 experimental studies in ducks showed that viruses replicated to higher levels in the trachea than in the cloaca of both inoculated and contact birds, suggesting that the digestive tract is not the main site of h5n1 influenza virus replication in ducks. 18, 19 likewise, experimentally inoculated geese and chicken shed higher virus titers in oropharyngeal swabs than in cloacal swabs. 20, 21 naturally occurring inhibitors present in cloacal and environmental swabs have been shown to limit the sensitivity of rt rt-pcr in detection of influenza a. 22 to minimize this effect in our study, we used the magmax extraction kit, which has been shown to be more effective in removing inhibitors, though the effect of inhibitors cannot be completely ruled out. 22 in the five markets, we observed several practices that could promote influenza transmission among birds. these included keeping markets open for 7 days a week, limited cleaning and disinfection of the market, mixing of new and old birds, trading multiple poultry species in the same market, and mixing with wild birds. these factors were found to be associated with transmission of low pathogenic avian influenza viruses in markets in north america. 23 environmental sampling, where specimens are collected from contaminated areas of the market, has been suggested as an effective surveillance method for influenza virus circulation. as part of our surveillance, we collected specimens from fecal droppings on the ground in the markets. however, we did not detect influenza a viruses in any of the specimens. one study in hong kong detected aivs in up to 1% of fecal swab specimens. 11 the reason for lack of detection in the environmental specimens is not clear, but we suspect that the high environmental temperatures in kenya may limit the survival of any virus shed in feces by the birds. reduced viability of several aiv subtypes has been shown to be associated with increases in temperature. [24] [25] [26] the presence of rt rt-pcr inhibitors could also have limited influenza a viral rna detection in these samples. 22 our surveillance was subject to certain limitations. we did not carry out subtyping of the influenza a specimens or virus isolation; hence, we are not able to report influenza subtypes from the birds sampled. we used rt rt-pcr for screening of the specimens for influenza a virus to determine positivity. although this method has high sensitivity and specificity for detection of type a influenza matrix gene, we may have missed some infections; in one study, virus isolation in embryonated chicken eggs was found to detect an additional 2ae3% of specimens that were negative by rt rt-pcr. 27 the authors attributed the reduced sensitivity of rt rt-pcr in part to the presence of rt rt-pcr inhibitory substances in the samples and the less volume used in rt rt-pcr assays compared with virus isolation. 27 additionally, virological studies only establish the prevalence of active infections. serology testing would have provided more information about the extent of previous exposure at the farms and markets. however, in our case, we sampled poultry in the market destined for sale, and bleeding of the birds would have been undesirable for the traders. our results show that influenza a viruses circulate regularly in lbms in kenya. continued monitoring of influenza viruses in poultry in lbms would help in detecting new introductions of aivs in the poultry population that would be of public health and socioeconomic significance to the poultry industry in the country. early detection of new potentially dangerous influenza viruses could lead to early application of control measures that could minimize the public health impact of outbreaks of hpai viruses and decrease the impact on the livelihoods along the poultry value chain. position of the us centers for diseases control and prevention. ecology, epidemiology and human health implications of avian influenza viruses: why do we need to share genetic data? evolution and ecology of influenza a viruses influenza viruses: transmission between species avian influenza in birds and mammals pathobiology of avian influenza virus infections in birds and mammals wet markets -a continuing source of severe acute respiratory syndrome and influenza? isolation and characterization of avian influenza viruses, including highly pathogenic h5n1, from poultry in live bird markets in hanoi molecular and biological characteristics of h5 and h7 avian influenza viruses in live-bird markets of the northeastern united states environmental sampling for avian influenza virus a (h5n1) in live-bird markets evaluation of routine depopulation, cleaning, and disinfection procedures in the live bird markets poultry drinking water used for avian influenza surveillance an overview of the poultry sector and status of highly pathogenic avian influenza (hpai) in kenya-background paper collaborative research on pro-poor hpai risk reduction rtpcr (rrtpcr) protocol for detection and characterization of influenza (version avian influenza viruses in korean live poultry markets and their pathogenic potential characterization of low-pathogenic h5 subtype influenza viruses from eurasia: implications for the origin of highly pathogenic h5n1 viruses avian influenza in backyard poultry of the mopti region the influenza virus gene pool in a poultry market in south central china role of domestic ducks in the propagation and biological evolution of highly pathogenic h5n1 influenza viruses in asia are ducks contributing to the endemicity of highly pathogenic h5n1 influenza virus in asia? experimental infection of swans and geese with highly pathogenic avian influenza virus (h5n1) of asian lineage virus shedding and potential for interspecies waterborne transmission of highly pathogenic h5n1 influenza virus in sparrows and chickens removal of realtime reverse transcription polymerase chain reaction (rt-pcr) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of avian influenza virus by rt-pcr description of live poultry markets in the united states and factors associated with repeated presence of h5 ⁄ h7 low-pathogenicity avian influenza virus tenacity of avian influenza viruses the effect of temperature and uv light on infectivity of avian influenza virus (h5n1, thai field strain) in chicken fecal manure avian influenza virus h9n2 survival at different temperatures and phs development of real-time rt-pcr for the detection of avian influenza virus detection of influenza a virus in live bird markets ª the authors wish to express gratitude to the staff of the virology laboratory, central veterinary laboratory, kabete for their tireless participation in sampling of the birds and mr dennis odhiambo of cdc-k ieip laboratory for his excellent technical support during the surveillance, period which made this work a success. the authors thank joshua mott for his helpful advice. addendum p. munyua contributed to the overall design of the study, coordinated the field work, analyzed the data, and wrote the manuscript. l. waiboci and l. mwasi were responsible for all the rna extractions and testing and reviewed the manuscript. j. githinji, l. njagi, r. murithi and j. macharia were responsible for the field work, approval for the surveillance work, and reviewed the manuscript. g. arunga coordinated the data entry, analyzed the data, and reviewed the manuscript. r. breiman, k. njenga, m. katz contributed to the overall design of the study and reviewed the manuscript. the findings and conclusions in this report are those of the authors and do not necessarily represent the official key: cord-003298-vojbn0p4 authors: hsieh, ying‐hen title: pandemic influenza a (h1n1) during winter influenza season in the southern hemisphere date: 2010-07-08 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2010.00147.x sha: doc_id: 3298 cord_uid: vojbn0p4 please cite this paper as: hsieh ying‐hen. (2010) pandemic influenza a (h1n1) during winter influenza season in the southern hemisphere. influenza and other respiratory viruses 4(4), 187–197. background countries in the southern hemisphere experienced sizable epidemics of pandemic influenza h1n1 in their winter season during may–august, 2009. methods we make use of the richards model to fit the publicly available epidemic data (confirmed cases, hospitalizations, and deaths) of six southern hemisphere countries (argentina, brazil, chile, australia, new zealand, and south africa) to draw useful conclusions, in terms of its reproduction numbers and outbreak turning points, regarding the new ph1n1 virus in a typical winter influenza season. results the estimates for the reproduction numbers of these six countries range from a high of 1·53 (95% ci: 1·22, 1·84) for confirmed case data of brazil to a low of 1·16 (1·09, 1·22) for ph1n1 hospitalizations in australia. for each country, model fits using confirmed cases, hospitalizations, or deaths data always yield similar estimates for the reproduction number. moreover, the turning points for these closely related outbreak indicators always follow the correct chronological order, i.e., case–hospitalization–death, whenever two or more of these three indicators are available. conclusions the results suggest that the winter ph1n1 outbreaks in the southern hemisphere were similar to the earlier spring and later winter outbreaks in north america in its severity and transmissibility, as indicated by the reproduction numbers. therefore, the current strain has not become more severe or transmissible while circulating around the globe in 2009 as some experts had cautioned. the results will be useful for global preparedness planning of possible tertiary waves of ph1n1 infections in the fall/winter of 2010. the pandemic influenza h1n1 2009 (ph1n1) was first identified by the centers for disease control in two children in april. 1 as of 14 february 2010, worldwide more than 212 countries and overseas territories or communities have reported laboratory-confirmed cases of pandemic influenza h1n1 2009, including at least 15921 deaths. 2 as predicted earlier by many experts (e.g., 3) , the southern hemisphere also witnessed a sizable epidemic of ph1n1 in its winter season during may-august, with attack rates exceeding those in a typical influenza season in these countries. moreover, it is important to monitor changes in antigenicity, severity, transmissibility, and antiviral resistance in the southern hemisphere to compare with those of the spring and winter outbreaks in the northern hemisphere. we construct simple mathematical models using the epidemiologic data for these southern hemisphere countries to draw timely and useful conclusions regarding the role played by the new ph1n1 strain under the setting of a typical winter influenza season. data we obtain the 2009 ph1n1 data for argentina, chile, and brazil used in this study from the respective ministries of health of argentina, 4 chile, 5 and brazil 6 websites. the new zealand data were accessed from the institute of environmental science and research of new zealand website. 7 the australian data were obtained from department of health and ageing website. 8 the south africa data were accessed from the national institute for communicable diseases (nicd) website. 9 methods richards 10 proposed the following model to study the growth of biologic populations: here, the prime '¢' denotes the derivative or time rate of change and the time unit is in days or epidemiological weeks (or e-weeks that start on sunday and end on saturdays), depending on the time unit of the data used. c(t) is the cumulative number of cases (confirmed, hospitalization, or death) at time unit (day or week) t, k is the 'maximum' case number or the final outbreak size over a single wave of outbreak, r is the per capita growth rate of the cumulative number of cases, while a is an exponent of deviation of the epidemiologic curve. the solution of richards model can be explicitly given in terms of its model parameters and the initial value c(0) as cðtþ ¼ k½1 þ e àrðtàt m þ à1=a . the parameter t m is related to the turning point t i (defined as the time when the rate of case accumulation changes from increasing to decreasing or vice versa) of the epidemic by the simple formula t m = t i + (ln a) ⁄ r, where ln denotes the natural logarithm function. by fitting cumulative case data of a particular outbreak, these four quantities, namely, the growth rate r, carrying capacity k, inflection point t i , and exponent of deviation a of the model, can be obtained simultaneously using standard software such as matlab or sas. in this model formulation, the basic reproduction number r 0 is given by the formula r 0 ¼ exp ðrtþ, where t is the disease generation time (or generation interval) defined as the average time interval from one person being infected to the time when an infection by this infected individual occur. the estimation of mean generation time could be difficult because it is often impractical to report observed generation time, and sophisticated statistical procedures have been developed to attain this goal (see e.g., 11, 12 ) . moreover, it has been shown mathematically 11 that, given the growth rate r, the expression r 0 ¼ exp ðrtþ provides the upper bound of basic reproduction number regardless of the distribution of the generation interval that is being used. in the case where there is little prior human immunity for the virus under consideration, the estimate for r 0 approximates the number of infections caused by an infectious individual entering an immunologically naïve population. readers are referred to [13] [14] [15] for more technical details regarding the richards model. the basic premise of the richards model is that the incidence curve of a single wave of infections consists of a single peak of high incidence, resulting in an s-shaped cumulative epidemic curve with a single turning point for the outbreak. the turning point t i can be easily pinpointed by locating the inflection point of the cumulative case curve, i.e., the moment at which the trajectory begins to decline. this quantity in time has obvious epidemiologic importance, indicating either the beginning of a new wave of infections (i.e., moment of acceleration after deceleration) or the end of the current wave of infections (i.e., moment of deceleration after acceleration). one of the advantages in using the richards model for mathematical modeling is to fit the accumulative case number, which helps to smooth out the stochastic variations in epidemic curve owing to variations in data collection. when there are indeed two or more waves of infections, a variation of the s-shaped richards model has been proposed, 14 which distinguishes between two types of turning points: one in the initial s curve that signifies the first turning point that ends initial exponential growth (or a downturn in case number) and a second type of turning point in the epidemic curve where the growth rate of the number of cumulative cases begin to increase again (or a upturn in case number), signifying the beginning of the next wave of infections. this variation of the richards model provides a systematic method of determining whether an outbreak is 1-wave or multi-wave in nature, and is designed to isolate the main turning points from those peaks and valleys resulting from the data stochasticity. more details on the multi-wave richards model can be found in, [14] [15] [16] where 2003 sars incidence curves for the great toronto area and singapore are shown to contain two peaks (local maximum or turning point of the first type) and one valley (local minimum or turning point of second type). the readers are also referred to 14 for its application to 2-wave dengue outbreak in taiwan in 2007. we note that the estimates of r 0 should be valid for the new h1n1 strain, although discrepancy might exist because of prior immunity. 17 moreover, no mass intervention measures (e.g., community-level isolation ⁄ quarantine ⁄ closure) were implemented at any time during the course of the outbreaks, although some measures were taken in each country with varying degrees of impact. to be conservative in our conclusions, we will henceforth denote the reproduction number obtained here as the effective reproduction number r of the outbreak in question. we fit the cumulative pandemic influenza a (h1n1) 2009 case data from various southern hemisphere countries described earlier to the richards model and its multi-wave variants. the country-wise results of model parameter estimation, using least squares techniques, are detailed in the following text. for the purpose of computing r 0 , we make use of the mean estimated generation interval (and its 95% ci) of t = 1ae91 days (95% ci: 1ae30-2ae71) as given in, 18 which was estimated from early mexico novel h1n1 data before april 30. the daily 2009 ph1n1 confirmed case, hospitalization [of all severe respiratory infections or sever respiratory infection (sri)], and ph1n1 death data of argentina 4 during may 19 to august 23 are fitted to the richards model. the results of model parameter estimation for argentina are given in tables 1 and 2. table 1 shows that the daily confirmed ph1n1 case data fit both 1-wave and 2-wave richards models. using the daily confirmed case data, the 1-wave model fit gives a turning point of june 26, while the 2-wave yields 3 turning points, one 'downturn' tuning point for each wave around june 1 and june 24, and an 'upturn' turning point on june 4 signifying the start of the second wave. for comparison, both the daily sri hospitalization data (6 ⁄ 1-7 ⁄ 25) and daily ph1n1 death data (6 ⁄ 12-8 ⁄ 20) fit 1-wave richards models ( table 2 ). the turning point using daily sri hospitalization data is june 28, while the turning point for the daily h1n1 death data is july 1. for the purpose of comparison with the estimates for the final case number k for each wave of outbreak, we also provide the actual case numbers in the table legends. the estimates for the reproduction number, using the daily confirmed ph1n1 case data, are r = 1ae41 (95% ci: 1ae23, 1ae60), and 1ae84 (95% ci: 0ae86, 2ae82) and 1ae34 (95% ci: 1ae19, 1ae48), respectively, for the 2-wave model. the corresponding estimates are r = 1ae27 (95% ci: 1ae16, 1ae38) and 1ae28 (95% ci: 1ae16, 1ae39), respectively, using sri hospitalization and ph1n1 death data. the graphs of the theoretical curves are shown in figure 1a -d. the weekly ph1n1 hospitalization data by onset week of eweek 20-33 (may 10 to august 15) and daily death data of june 1 to august 18 in chile 6 fit 1-wave richards model (table 3 and figure 1e -f). the turning point for weekly ph1n1 hospitalization data is e-week 26, whereas the turning point for the daily death data is july 11. the estimated reproduction numbers are 1ae24 (95% ci: 1ae14, 1ae35) for weekly ph1n1 hospitalization data and 1ae29 (95% ci: 1ae17, 1ae41) for the daily death data. note that the confirmed ph1n1 case data for chile did not converge for earlier ministry of health of chile pandemic virus report reported in july, most likely attributable to the transient state of data collection during earlier stages of the outbreak (e.g., changing case definitions and level of surveillance), and was not given in the pandemic influenza report in august 5 and onward. the weekly ph1n1 confirmed case data by onset week of e-week 21-33 (may 17 to august 15) in brazil 6 also fits both 1-wave and 2-wave richards models (table 4 ). the turning point for 1-wave model is e-week 31, while the two respective 'downturn' turning points for the two waves are e-week 26 and e-week 31. the estimated reproduction numbers are 1ae53 (95% ci: 1ae22, 1ae84) for 1-wave model, and 1ae66 (95% ci: 1ae23, 2ae09) and 1ae39 (95% ci: 0ae99, 1ae79) for the respective two waves of the 2-wave model. the graph for model fit is given in figure 1g . we first note that the new zealand data are given in calendar weeks ending on sundays, 7 as opposed to e-weeks that end on saturdays. the weekly ph1n1 confirmed case data by onset week of weeks ending on april 12-september 20 and weekly ph1n1 hospitalization data by hospitalization, onset, or report week of weeks ending on june 7-september 13 in new zealand 7 fit 1-wave richards models (table 5 and figure 2a-d) . moreover, the sentinel and non-sentinel surveillance data by reporting week of weeks ending on june 14-september 20 and may 3-september 20, respectively, also fit 1-wave richards models. the turning point for the weekly ph1n1 confirmed case data is the week ending july 5 and for the hospitalization data is the succeeding week ending july 12, giving strong evidence that the turning point for the better had occurred by early july. the turning point for the sentinel and non-sentinel surveillance data are the weeks ending july 19 and july 12, respectively. the estimated reproduction numbers are 1ae22 (95% ci: 1ae13, 1ae31) for the weekly confirmed case data; 1ae29 (95% ci: 1ae16, 1ae41) for weekly ph1n1 hospitalization data; 1ae18 (95% ci: 1ae10, 1ae25) for the sentinel; and 1ae22 (95% ci: 1ae13, 1ae31) for the non-sentinel surveillance data. the weekly ph1n1 confirmed case and hospitalization data by onset week in australia 8 fit the 1-wave richards model (table 6 and figure 2e -f). the turning point for both the weekly ph1n1 confirmed case data and the hospitalization data is e-week 30, showing that the down turning point of case number had occurred by late july. the estimated reproduction numbers are 1ae19 (95% ci: 1ae11, 1ae26) for the weekly confirmed case data; 1ae16 (95% ci: 1ae09, 1ae22) for weekly ph1n1 hospitalization data. the weekly ph1n1 confirmed case data of weeks ending on july 5 to september 20 in south africa 9 fit the 1-wave richards model (table 7 and figure 2g ). the turning point for the weekly ph1n1 confirmed case data is the week ending august 16. the estimated reproduction number is 1ae22 (95% ci: 1ae11, 1ae33). several alternate estimates of the generation time t have been reported. hence, we perform sensitivity analysis on r for argentina and new zealand data using estimates of t obtained from ph1n1 data in us: t = (2ae6, 3ae2) 19 and t = 2ae5 20 ; in netherlands: t = 2ae7 (sd = 1ae1); 21 and in japan: t = 2ae83 (variance = 1ae26) [nishiura, personal communications]. the resulting estimates for r are given in tables 8 and 9 . reproduction number 23 as well as numerous estimates for spring ⁄ summer outbreaks of other countries in literature (see table 2 , 23). the estimates for r in argentina and new zealand obtained by using different estimates of generation interval (t) for 2009 ph1n1 in the literature are also in close agreement (tables 8 and 9 ). the results indicate that higher values of t will raise all the estimates for r, as can be expected from the formula for r given earlier. for the argentina hospitalization data with t = 1ae91 18 , r = 1ae27 is obtained; whereas using a t value of around 2ae7 21 we have r = 1ae40 (see table 8b ). both estimates are within reasonable range. however, the discrepancy becomes larger for larger values of r (see next to last row, table 8a ), thus indicating the dependence of r on accurate estimate of t. the corresponding estimates for other countries are similar and hence are omitted for brevity. the estimates for new zealand are substantially lower than the range of 1ae49-2ae55 for reproduction number obtained in, 24 using the case data of 585 confirmed cases and 38 probable cases from 28 may to 28 june. we note, however, that 63 imported cases were first removed for the data in. 24 this omission most likely resulted in an overestimation of the initial growth rate, by ignoring these imported cases that must have collectively played a significant role in the early local transmissions in the island nation. we note that the role of imported cases is problematic to quantitate when estimating transmissibility, as there are no 'local' source of infection for these imported cases. however, by discounting the imported cases, which in the case of new zealand data accounts for more than 10% of total cases, one ignores the infections caused by the imported infective and hence inevitably overestimates the transmissibility of the remaining local cases. there are several factors relating to imported cases that might impact the estimation of r, such as the timing and the size of the imported cases, the nature of the resulting local infections (e.g., household, community, nosocomial), and not the least of all the epidemiological characteristics of the disease under consideration. for example, a few imported cases at the early stages that set off a fast-spreading disease locally (e.g., pandemic influenza) would have a decidedly different effect when compared to the situation with imported cases scattered over a long period of time for a less transmissible disease with no apparent asymptomatic infections such as sars, unless there are very fastdeveloping local clusters of infections or nosocomial spread, as in the 2003 sars outbreak. a study on ph1n1 epidemic in victoria, australia, indicates that, by using methods designed to account for the undetected transmission caused by unidentified imported cases, the mean estimate for r reduces dramatically from 2ae4 to 1ae6. 26 the reproduction number obtained for brazil is only slightly higher than other southern hemisphere countries studied (see figure 3 ), but with a much larger confidence interval. in the cases of argentina and brazil where both 1-wave and 2-wave richards models fit the cumulative case data, r is always larger in the first phase in may to june, perhaps indicative of the initial severity or lack of immunity ⁄ intervention during early stages of a novel virus outbreak. we note that higher r for earlier data was also observed in, 22 after correction for ascertainment bias. it is particularly interesting to note that for countries such as argentina, chile, new zealand, and australia, where multiple estimates were obtained from distinct epidemic data (case, hospitalization, and deaths), the estimates of r are highly comparable. therefore, in events where the disease epidemic curve is not readily available or does not converge (as in the case of chile), the hospitalization and deaths data, while not necessarily accurate for detecting the among the south american countries, the downturn turning point of the outbreak occurred earlier in argentina and chile in june, while brazil did not experience its downtown until late july. new zealand (early july) had its turning point slightly ahead of australia (late july). for south africa the outbreak did not turn for the better until mid-august. moreover, in countries where multiple epidemic data (cases, hospitalizations, and deaths) are available for model fit, the resulting turning points for reporting of cases, hospitalizations, and deaths always follow the proper chronological order of disease progression (table 10 and figure 4) , including that of australia where the turning points for reporting of cases and hospitalizations fall on the same e-week. this gives further credence to the detection of turning points of an outbreak, not only for disease incidence but also for the temporal changes in disease progression and treatment of the infected individuals. we note that in general the turning points of an outbreak correspond to peaks and valleys in the epidemic curve. however, as a result of stochastic variations, multiple spikes often appear around the peaks. moreover, some temporary deceleration or acceleration in the cumulative rate might not be related to a declining epidemic. the richards model has the advantage of isolating a small range of time period during which the turning points (up turns and down turns) may have occurred. [13] [14] [15] [16] 25 we also note that the estimates for the outbreak sizes, k, also closely approximate the actual case numbers for the periods of table 9 . estimates of effective reproduction number r for new zealand data using estimates of t obtained from ph1n1 data of: us with (a) t = (2ae6, 3ae2) 19 14, 16 and hence are not repeated here. in addition to existing pre-immunity, estimates of r may also be influenced by factors such as social mixing patterns, access to medical care, and level of intervention measures in each country. to address this issue, model specific for each country is required to account for the social mixing and healthcare infrastructure of each country, as well as any specific interventions that were implemented, but is beyond the scope of this work. numerous sophisticated models have been proposed (e.g., 27-30) that give time-dependent reproduction number r, which could change with time owing to interventions and ⁄ or susceptible depletion but requires more detailed and high-quality data. nevertheless, the richards model provides a swift and viable method to quick estimate of r as well as identifying the important turning points of the outbreak, with a minimal requirement of epidemic data that are typically available to the public. it is interesting to note that, for argentina, the hospitalization data for confirmed ph1n1 cases are not available on the web; therefore, we used the hospitalization data for severe respiratory infections, which include but are not limited to the confirmed ph1n1 cases. however, the resulting estimate for reproduction number is surprisingly similar to the estimates using the case data or the death data. moreover, the estimated turning point for hospitalizations is also consistent with those of cases and deaths. most of the southern hemisphere countries studied are of substantial size, and thus the epidemic had different timings in different regions. for example, in australia, the epidemic in victoria was a full month ahead of some other states might well have impacted the epidemic in other states. in such cases, instead of using national totals, the detailed data for each region are needed to account for the regional heterogeneity. however, other factors such as the level of mobility between regions must then be considered, which would call for a complicated model with spatial elements, which is again beyond the scope of this study. in summary, our results suggest that the winter pandemic a (h1n1) outbreak in the southern hemisphere, as indicated by its reproduction number, was not dissimilar to the earlier spring epidemics 23 or the fall ⁄ winter outbreaks in the northern hemisphere based on preliminary results of an ongoing study of the fall ⁄ winter outbreak in taiwan using the same modeling approach. therefore, the current strain, while circulating around the globe, has not become more severe or transmissible either in the southern or in the northern hemisphere, as some experts had cautioned. the results, which corroborate earlier findings that the 2009 pandemic had not been severe in the southern hemisphere, 31 will be useful for global preparedness planning of possible tertiary waves of ph1n1 infections in the fall ⁄ winter of both southern and northern hemispheres in 2010. swine influenza a (h1n1) infection in two children -southern california pandemic (h1n1) 2009 -update 88 managing and reducing uncertainty in an emerging influenza pandemic influenza pandé mica (h1n1) brazilian ministry of health influenza surveillance report no. 19, for the week ending 18 the national institute for communicable diseases (nicd) of south africa. nicd weekly situation report a flexible growth function for empirical use how generation intervals shape the relationship between growth rates and reproductive numbers the ideal reporting interval for an epidemic to objectively interpret the epidemiological time course real-time forecast of multi-wave epidemic outbreaks richards model: a simple procedure for real-time prediction of outbreak severity turning points, reproduction number, and impact of climatological events on multi-wave dengue outbreaks understanding influenza transmission, immunity and pandemic threats pandemic potential of a strain of influenza a (h1n1): early findings the transmissibility and control of pandemic influenza a (h1n1) virus estimation of the reproductive number and the serial interval in early phase of the 2009 influenza a ⁄ h1n1 pandemic in the usa epidemiology and control of influenza a(h1n1)v in the netherlands: the first 115 cases initial human transmission dynamics of the pandemic (h1n1) 2009 virus in north america transmission dynamics and impact of pandemic infl uenza a (h1n1) 2009 virus estimating the reproduction number of the novel influenza a virus (h1n1) in a southern hemisphere setting: preliminary estimate in new zealand intervention measures, turning point, and reproduction number for dengue early transmission characteristics of influenza a(h1n1)v in australia: victorian state different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures estimating in real time the efficacy of measures to control emerging communicable diseases real time bayesian estimation of the epidemic potential of emerging infectious diseases a likelihood-based method for real-time estimation of the serial interval and reproductive number of an epidemic pandemic h1n1 influenza lessons from the southern hemisphere yhh is supported by grants (nsc 97-2314-b-039-013-my3, nsc 97-2118-m-039-004, and cmu 97 323). the author is grateful to the reviewers for their insightful comments and suggestions, which significantly improved this manuscript. key: cord-279716-kxfc4npg authors: blachere, francoise m.; lindsley, william g.; slaven, james e.; green, brett j.; anderson, stacey e.; chen, bean t.; beezhold, don h. title: bioaerosol sampling for the detection of aerosolized influenza virus date: 2007-10-22 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2007.00020.x sha: doc_id: 279716 cord_uid: kxfc4npg background influenza virus was used to characterize the efficacy of a cyclone‐based, two‐stage personal bioaerosol sampler for the collection and size fractionation of aerosolized viral particles. methods a collison single‐jet nebulizer was used to aerosolize the attenuated flumist® vaccine into a calm‐air settling chamber. viral particles were captured with bioaerosol samplers that utilize 2 microcentrifuge tubes to collect airborne particulates. the first tube (t1) collects particles greater than 1.8 μm in diameter, while the second tube (t2) collects particles between 1.0 and 1.8 μm, and the back‐up filter (f) collects submicron particles. following aerosolization, quantitative pcr was used to detect and quantify h1n1 and h3n2 influenza strains. results based on qpcr results, we demonstrate that aerosolized viral particles were efficiently collected and separated according to aerodynamic size using the two‐stage bioaerosol sampler. most viral particles were collected in t2 (1‐1.8 μm) and on the back‐up filter (< 1 μm) of the bioaerosol sampler. furthermore, we found that the detection of viral particles with the two‐stage sampler was directly proportional to the collection time. consequently, viral particle counts were significantly greater at 40 minutes in comparison to 5, 10 and 20 minute aerosol collection points. conclusions due to a lack of empirical data, aerosol transmission of influenza is often questioned. using flumist®, we demonstrated that a newly developed bioaerosol sampler is able to recover and size fractionate aerosolized viral particles. this sampler should be an important tool for studying viral transmission in clinical settings and may significantly contribute towards understanding the modes of influenza virus transmission. influenza infections are a public health concern accounting for more than 30 000 deaths and 100 000 hospitalizations annually. 1 the primary populations at risk for infection include young children, elderly adults, and immunocompromised subjects. during influenza outbreaks or pandemics, healthcare workers are at an elevated risk for acquiring influenza infection due to the prolonged periods of exposure to influenza viruses within healthcare settings. the mechanisms of influenza virus dissemination and transmission are poorly understood. experimental studies have shown that influenza viruses can be transmitted via contact with respiratory secretions, large droplets, and aerosolized small particles ⁄ droplet nuclei. 2 mucous membrane contact of large droplets expelled from the respiratory tract are thought to be the predominant mode by which influenza infection is transmitted, but small particle aerosols and droplet nuclei are also of concern due to the potential for prolonged airborne suspension. 3, 4 given the epidemic potential and public health concern of newly emerging diseases such as avian influenza (h5n1) and severe acute respiratory syndrome (sars), 5, 6 it is important to develop methods to study aerosolized viral particles. such measures would not only enable improved monitoring and detection of viruses, but more importantly, would help prevent widespread transmission. to date, several bioaerosol samplers are available that use impaction or impinger sampling methodologies for viral aerosol detection. 7, 8 however, limitations exist within each of these methods such as poor collection efficiency, limited sampling time, and inability to separate particles based on size. further complicating the study of viral aerosols is that in many environments where it would be desirable to sample for viral aerosols (e.g., hospitals, airports), the level of other biologically relevant particles such as bacteria, fungi, and pollens can also be very high. based on a one-stage, cyclone-based bioaerosol sampler developed at the national institute for occupational safety and health (niosh), 9 a two-stage, cyclone-based bioaerosol sampler was recently fabricated. 10 the two-stage bioaerosol sampler is a lightweight device that can be used either as an area sampler (e.g., in a hospital room) or as a personal breathing zone air-sampling device worn on the lapel of the subject (e.g., a healthcare worker). this bioaerosol sampler contains two microcentrifuge tubes and a back-up filter, which separate airborne particulates based on their aerodynamic diameter. 10 the first tube (t1) of the sampler collects particles that are greater than approximately 1.8 lm in diameter, while the second tube (t2) collects particles from 1.0 to 1.8 lm in diameter and the back-up filter (f) collects submicron particulates. because the sample is deposited directly in microcentrifuge tubes, the sampler facilitates the direct processing of samples for downstream diagnostic applications such as quantitative polymerase chain reaction (qpcr) and enzyme-linked immunosorbent assay. the bioaerosol sampler potentially eliminates several limitations associated with other air sampling devices such as sample loss, contamination, and degradation. in this proof of concept study, we characterized the utility of the two-stage bioaerosol sampler for fractionating viral particles and separating them from other particulates. the influenza virus vaccine flumist ò 2005-2006 formula was purchased from medimmune vaccine, inc. (gaithersburg, md, usa). flumist ò is a live, trivalent vaccine, composed of the a ⁄ new caledonia ⁄ 20 ⁄ 99 (h1n1), a ⁄ california ⁄ 7 ⁄ 2004 (h3n2), and b ⁄ jiangsu ⁄ 10 ⁄ 2003 (b ⁄ shanghai ⁄ 361 ⁄ 2002-like) strains. these strains are genetically altered to attenuated, cold-adapted, and temperature-sensitive phenotypes, which limits viral replication to the nasal pharynx. each 0.5 ml dose has been formulated to contain approximately 10 7 tcid 50 (10 6.5 -10 7.5 median tissue culture infectious dose) per viral strain. viral aerosols (flumist ò ) were collected using 4 two-stage bioaerosol samplers and two vertical reference samplers placed at the bottom of a calm air aerosol settling chamber, as described previously. 11 the bioaerosol samplers were connected to personal air sampling pumps (model 224-pcxr4; skc, eighty four, pa, for aerosolization experiments, one dose (0.5 ml) of flu-mist ò was diluted with 49.5 ml of saline (0.9% nacl). one milliliter of this solution was initially drawn for assay, and the remainder placed in a collison single-jet nebulizer (bgi incorporated, waltham, ma, usa). the solution was aerosolized at 138 kpa (20 lbs ⁄ in 2 ) air pressure, passed through a diffusion drier (model 3062; tsi incorporated, shoreview, mn, usa), and mixed with 26 l ⁄ minute dry filtered air. the dry aerosol then flowed into the top of the calm air chamber. to avoid possible degradation of the virus by ozone, an unavoidable by-product, the bipolar ionizer employed in earlier studies 10 was not used. instead, all conducting lines and the settling chamber itself were metal and grounded. previous tests with the calm air chamber indicated that these precautions were sufficient to avoid electrostatic aerosol deposition. 11 the concentration and size distribution of the diluted aerosol was recorded using an aerodynamic particle sizer (aps model 3321; tsi), which drew air from a vertical probe placed at the same height as the bioaerosol sampler inlets. at the start of each experiment, the nebulizer was operated for 10 minutes to allow the aerosol concentration to reach equilibrium. after 10 minutes, the sampling pumps and vacuum source were activated simultaneously for all samplers. for aerosol collection studies, sampler pumps were switched off after 5, 10, 20, and 40 minutes, while reference samplers were switched off after 20 and 40 minutes. after aerosol collection, the exterior of each sampler was disinfected (conflikt; decon labs, king of prussia, pa, usa) and the collection tubes and filters removed for analysis. a 1 ml aliquot of the solution remaining in the nebulizer was also removed for analysis. the particle size-segregation characteristics of the twostage sampler were reported previously for a range of particle sizes. 10 using this information, the particle size and number data from the aps, an estimate was made of the relative amounts of viral material that should have collected in the first and second tubes and on the back-up filter. this estimate was compared to the actual proportions of viral material found in each stage of the sampler as measured by qpcr (below) to indicate if the two-stage sampler was separating the aerosol particles based on size as expected. viral rna was extracted using the magmax tm viral rna isolation kit (ambion, austin, tx, usa). briefly, the supplied lysis ⁄ binding solution supplemented with carrier rna was used in the extraction of viral rna from either the neat flumist ò (qpcr standards) or experimental samples (t1 and t2). the back-up filters (f) from the bioaerosol samplers were aseptically removed, immersed in 1 ml of the supplemented lysis ⁄ binding solution and vortexed at moderate speed for 2 minutes. viral lysis, rna binding, magnetic capture, washing, and drying of the rna-bound beads were all carried out according to the manufacturer's instructions. cdna was generated by reverse transcription of the isolated viral rna using taqman tm reverse transcription reagents (applied biosystems, foster city, ca, usa). in brief, 40 ll of viral rna was added to the rt-pcr mixture containing 10 ll of 10x rt buffer, 22 ll of 25 mm mgcl 2 , 20 ll of 10 mm dntp mix, and 5 ll of 50 mm random hexamers. rnase inhibitor (2.0 ll, 20 u ⁄ ml) and 2.5 ll of multiscribe reverse transcriptase (50 u ⁄ ml) were added finally, for a final volume of 100 ll. samples were briefly centrifuged, placed in a thermo hybaid thermocycler (ashford, uk) and run under the following conditions: 25°c for 10 minutes, 48°c for 30 minutes, and 95°c for 5 minutes. a control without template was run for each experiment. as a pilot study to simulate the more biologically complex aerosols that one might encounter in field situations, flu-mist ò was mixed with fungal spores and then aerosolized and collected. aspergillus versicolor (atcc 44408, american type culture collection) was grown on malt extract agar for 16 days at 24.9°c and the spores isolated as previously described. 10 spore concentrations were determined by hemacytometer count. for co-aerosolization, one dose of flumist ò was diluted with 49.5 ml of 0.9% nacl containing 10 7 a. versicolor spores and 2 ml of rnasecure reagent (ambion) to inhibit viral rna degradation. the aerosol was generated using a collison single-jet nebulizer, passed through a diffusion drier and mixed with dry filtered air using the same experimental procedure as described above. the aerosol was collected with 4 two-stage samplers and two reference samplers. two of the two-stage samplers and one reference sampler collected the aerosol for 10 minutes, while the remaining samplers collected the aerosol for 40 minutes. genomic dna was extracted from a. versicolor-containing samples as described by griffin et al. 12 briefly, 400 ll of ap1 buffer supplemented with 4 ll rnase a (dneasy plant mini kit; qiagen, valencia, ca, usa) was added to 10 7 spores (qpcr standard) or experimental samples. filters were eluted in 1 ml of the supplemented ap1 buffer, vortexed for 2 minutes, and transferred to a microcentrifuge tube. approximately 250 mg of 0.1 mm zirconia ⁄ silica beads (biospec products, inc., bartlesville, ok, usa) was added to each sample. samples were bead-beaten at maximum speed in a mini-beadbeater-8 (biospec products, inc.) for 1 minute and chilled on ice for 2 minutes. the bead-beating ⁄ cooling steps were repeated twice. samples were centrifuged for 10 minutes at 11000 g and protein precipitation, washing, and drying of fungal dna was carried out according to the manufacturer's instructions. primer ⁄ probe design and optimization 13 real-time pcr primers and probes were specifically targeted against influenza surface glycoproteins and designed using primer express software (applied biosystems). sequence information for a. versicolor was obtained from the u.s. environmental protection agency (http://www.epa.gov/microbes/moldtech.htm). synthesis of all primers and probes was performed by applied biosystems. table 1 lists the sequence and dye labels for primers and probes used in qpcr detection. to determine the optimal primer concentrations for qpcr analysis, an optimization qpcr matrix was performed for each primer ⁄ probe combination (singleplex). optimal primer and probe concentrations used in qpcr detection ranged from 250 to 900 nm. for detection of influenza or a. versicolor in aerosol samples, 10 ll of the generated viral cdna or fungal genomic dna, respectively, was added to 25 ll applied biosystem's taqman tm universal pcr master mix. the appropriate concentration of primers and tamra-labeled probes was added, and brought to the final reaction volume of 50 ll with pcr-grade water. all reactions were run using the applied biosystem's 7500 real-time pcr system at the following conditions: 50°c for 2 minutes, 95°c for 10 minutes, 40 cycles at 95°c for 15 seconds, and 60°c for 1 minute. in order to quantify the relative amount of viral particles or fungal spores collected at each stage of the bioaerosol sampler, qpcr was performed in parallel using either serial 10-fold dilutions of cdna generated from a single dose of non-aerosolized flumist ò containing approximately 10 7 tcid 50 per influenza strain or genomic dna isolated from 10 7 spores. a negative control without template was included in all qpcr reactions. statistical analysis was conducted on the experimental setup as repeated-measures anova, with main effects of stage and time. stage is a three-level categorical variable with factors tube 1, tube 2, and back-up filter. to determine the effect of time, measurements were taken at 5, 10, 20, and 40 minutes. proc mixed in sas v9.1 (sas institute, cary, nc, usa) was used to analyze the data for significance and to calculate regression parameters. proc mixed was also used to insure similarity of experiments by testing variability between replicates. results were considered significant if p < 0.05. standard curves were generated for influenza strains h1n1 and h3n2 using serial 10-fold dilutions of cdna isolated from a single dose of flumist ò (10 7 tcid 50 ⁄ viral strain). for qpcr detection of a. versicolor, a standard curve was generated using genomic dna isolated from 10 7 spores and serially diluted 10-fold. the standard curves for h1n1, h3n2, and a. versicolor were linear over a 4-log range of 1.0 · 10 3 -1.0 · 10 6 particles. we were unable to generate a reproducible signal for the b strain virus using the specific primers that were designed. standard curves were used to extrapolate relative viral or spore numbers in unknown samples using the 10 7 titer reported by the manufacturer or hemacytometer counts, respectively. when flumist ò was aerosolized the virus-laden aerosol in the calm air chamber typically contained about 17 000 particles ⁄ cm 3 with a median aerodynamic diameter of 0.8 lm and a geometric standard deviation of 1.33. as part of methods development, the optimal back-up filter composition for virus collection was determined. four bioaerosol samplers were fitted with different types of filters -gelatin, glass fiber, ptfe, and polyvinyl chloride. following a 40 minute collection, several gelatin filters were found to be fractured. during rna extraction, the glass fiber filters released a high number of particulates that presumably interfered with qpcr detection. based on an initial experimental run, the ptfe filter was found to be optimal for the recovery of viral particles ( figure 1 ). all subsequent aerosol experiments were performed with the ptfe filters. to characterize the collection efficiency of the bioaerosol sampler, three identical experiments were performed. figure 2(a, b) illustrates the collective results for viral aerosols collected at 5, 10, 20, and 40 minutes. for both h1n1 and h3n2, collection of viral particles with the two-stage sampler was linear up to 40 minutes. viral particle counts were significantly greater at 40 minutes in comparison with the 5, 10, and 20 minute collection times (p < 0.05). furthermore, as predicted by the aerodynamic particle size data, the greatest proportions of the viral particles were detected in t2 and the back-up filter f, regardless of the collection time. for strain h1n1 at 40 minutes, 75% of the viral particles were detected in stage t2 and f, while only 25% of particles were detected in t1. likewise, with strain h3n2, 77% of the particles were detected in stages t2 and f with 23% of the particles detected in stage t1. these results were consistent throughout all experiments and demonstrate the ability of the bioaerosol sampler to separate particles based on aerodynamic size. while a similar distribution of h3n2 particles was recovered, for unknown reasons the absolute particle numbers were significantly lower. to examine the effectiveness of the bioaerosol sampler to separate a mix of aerosolized particles, we co-aerosolized flumist ò with a. versicolor spores (aerodynamic diameter 2.3 lm). following 40 minutes of aerosolization (figure 3 ), the bioaerosol sampler was able to separate these particles of differing size. both qpcr and spore counts confirmed that stage t1 retained 96% of the a. versicolor spores while stage t2, with 2% of the spores, was found to contain the greatest amount of viral particles (48%). an overall shift in the deposition of viral particles toward t1 was observed when co-aerosolized with a. versicolor spores (24%-42%). significantly fewer viral particles were detected on the back-up filter f (10%) in comparison with the back-up filter f using flumist ò alone (38%), suggesting some interaction or aggregation of the particles during aerosolization. the need for rapid and accurate methods for detecting airborne viruses has increased in recent years, particularly following the reported outbreaks of avian influenza (h5n1) and sars. various aerobiological monitoring studies have shown a high degree of variability with capturing, recovering, and detecting aerosolized viral particles in environmental and clinical samples. 7,14-16 using the niosh bioaerosol sampler, we were able to overcome some common viral particle sampling limitations and fractionate aerosolized influenza particles from an artificially generated aerosol. bioaerosols vary in size, concentration, composition, and settling times. 3,17 an aerodynamic particle sizer was used to monitor the concentration and size distribution of the flumist ò aerosol within the settling chamber. using the bioaerosol sampler, it could be demonstrated that capture, recovery, and subsequent detection for each sampler stage (t1, t2, f) were consistent with the expected values based on particle size. as anticipated, qpcr results confirmed that a significant number of the aerosolized viral particles were localized within stages t2 and f. these results are in agreement with the sampler cutoff size of 1.0 and <1 lm for stages t2 and f, respectively. the small number of aerosolized particles detected in stage t1 can be attributed to the collection efficiency of the bioaerosol sampler. the engineering design of the bioaerosol sampler is critical in the size-fractionation of bioaerosols, but sampling time must also be taken into consideration. qpcr results of aerosolized viral samples collected at 5, 10, 20, and 40 minutes confirms that a sampling time of 40 minutes yields the highest quantity of viral particles; however, this does not necessarily represent an ideal environmental sampling time as the quantities of aerosolized influenza virus expelled from patients have not yet been evaluated. in aerobiological studies using different viral strains, 18, 19 it was found that prolonged sampling periods resulted in decreased viral recovery. likewise, because influenza viruses are stress sensitive, the possibility exists that lengthier sampling times may result in the desiccation of viral-laden aerosol and consequently compromise stability. future studies will aim at addressing the effects of prolonged sampling time on viral recovery and stability. using the bioaerosol sampler, the collection of aerosols within disposable microcentrifuge tubes limits sample loss and contamination. moreover, samples are directly processed within the respective tubes and further analyzed by different diagnostic methods including immunoassays or molecular detection techniques such as qpcr. currently qpcr is the preferred methodology for the rapid and sensitive detection of viruses; 20 however, several limitations still exist. the sensitivity and specificity of qpcr detection is limited to the targeted template. in this study, primers and probes were designed to selectively amplify the ha and na genes from strains h1n1 and h3n2, respectively. interestingly, the results showed a considerable difference in the number of viral particles that were detected for strains h1n1 and h3n2. the flumist ò vaccine is formulated so as to contain an equivalent concentration of each viral strain using the median culture infectious dose (tcid 50 ) assay 21 but does not account for non-viable viral particles. qpcr can detect viral cdna from both viable and non-viable viral particles, and perhaps explain this discrepancy. as for the qpcr detection of strain b, erratic results were obtained. as qpcr detection was successful for both strains h1n1 and h3n2, this may not be due to the presence of inhibitors in the reaction but instead is more likely due to poor primer or probe design or the presence of secondary (10 7 ) and flumist ò were co-aerosolized together into the calm air chamber. samples were collected at 40 minutes and analyzed for influenza viruses using qpcr and a. versicolor using qpcr and hemacytometer counts. data for each stage is presented as the percentage of total number of particles collected in the three stages. spore counts were the average of eight replicate hemacytometer counts. values for the flumist ò with no fungal spores were taken from the previous experiment presented in table 2 and represent the combined average values for h1n1 and h3n2 strains. structures in the influenza b rna, resulting in poor reverse transcription and insufficient target template. environmental bioaerosols vary considerably and may consist of a number of different microorganisms including viruses, bacteria, and fungi. recent findings by lindsley et al 10 demonstrated that the two-stage bioaerosol sampler was effective at collecting and separating aerosolized fungal spores and fragments. results of the current study provide supporting evidence that the bioaerosol sampler is able to successfully recover and size-fractionate viral-laden aerosols. the culmination of these results suggests that the bioaerosol sampler would be an ideal air-sampling device for the aerobiological monitoring of various microorganisms within occupational environments. however, preliminary findings from a co-aerosolization study suggest fractionation of environmental samples may be more complex. namely, an overall shift in the viral particle deposition in the presence of a. versicolor spores was observed. the shift in viral particle deposition may be attributed to the attachment of viral particles to a. versicolor spores. aps data (not shown) further suggest that particles adhere to one another. given that the viral and spore-laden solution is pre-mixed prior to aerosolizing, it remains unclear as to whether the binding may occur before, during or after aerosolization within the calm-air settling chamber. future studies will further elaborate on the use of the bioaerosol sampler to capture and effectively size-fractionate co-aerosolized particles of different species. currently, there are conflicting views as to whether influenza viruses are spread by direct contact with secretions, large droplet transmission, or aerosol transmission. due to the lack of virtually any environmental data, aerosol transmission of influenza viruses is often overlooked as a possible mode of transmission. in this study, by aerosolizing flumist ò , we demonstrate the recovery of aerosolized viral particles using the bioaerosol sampler and detection of influenza by qpcr. whether aerosolized influenza particles are a significant contributor to influenza transmission in work environments and the community remains to be determined. future experiments will focus on studying the dissemination of viral-laden aerosols utilizing an artificial cough generator to simulate cough dispersal of influenza viruses within a room-sized aerosol chamber. these findings would significantly contribute toward understanding the transmission of aerosolized influenza viral particles. furthermore, we will test the bioaerosol sampler in a healthcare setting to monitor the prevalence of airborne influenza viral particles and to study the transmission of influenza via the inhalation of aerosolized viral particles. such studies will help to further elucidate the routes of transmission of influenza and would ultimately contribute to better patient management as well as improve infection control guidelines and decrease worker health risk. avian flu to human influenza transmission of influenza: implications for control in health care settings toward understanding the risk of secondary airborne infection: emission of respirable pathogens review of aerosol transmission of influenza a virus the origins of pandemic influenza-lessons from the 1918 virus the severe acute respiratory syndrome sampling methodologies and dosage assessment techniques for submicronmetre and ultrafine virus aerosol particles new personal sampler for viable airborne viruses: feasibility study development of a personal sampler for collecting fungal spores a two-stage cyclone using microcentrifuge tubes for personal aerosol sampling design and use of a settling chamber for sampler evaluation under calm-air conditions a rapid and efficient assay for extracting dna from fungi the value of a database in surveillance and vaccine selection long-term sampling of viable airborne viruses collection efficiencies of aerosol samplers for viruscontaining aerosols optimization of a sampling system for recovery and detection of airborne porcine reproductive and respiratory syndrome virus and swine influenza virus on-line monitoring and identification of bioaersosols air sampling for the detection of exotic newcastle disease virus in poultry houses spincon based air sampler foot and mouth disease virus test report. cambridge: lares corporation molecular diagnosis of influenza a simple method of estimating fifty percent end points this work was supported by internal funds from the health effects laboratory division. the findings and conclusions in this report have not been formally disseminated by the niosh and should not be construed to represent any agency determination or policy. key: cord-299790-vciposnk authors: ho, zheng jie marc; zhao, xiahong; cook, alex r; loh, jin phang; ng, sock hoon; tan, boon huan; lee, vernon j title: clinical differences between respiratory viral and bacterial monoand dual pathogen detected among singapore military servicemen with febrile respiratory illness date: 2015-06-09 journal: influenza other respir viruses doi: 10.1111/irv.12312 sha: doc_id: 299790 cord_uid: vciposnk background: although it is known that febrile respiratory illnesses (fri) may be caused by multiple respiratory pathogens, there are no population-level studies describing its impact on clinical disease. methods: between may 2009 and october 2012, 7733 fri patients and controls in the singapore military had clinical data and nasal wash samples collected prospectively and sent for pcr testing. patients with one pathogen detected (mono-pathogen) were compared with those with two pathogens (dual pathogen) for differences in basic demographics and clinical presentation. results: in total, 45.8% had one pathogen detected, 20.2% had two pathogens detected, 30.9% had no pathogens detected, and 3.1% had more than two pathogens. multiple pathogens were associated with recruits, those with asthma and non-smokers. influenza a (80.0%), influenza b (73.0%) and mycoplasma (70.6%) were most commonly associated with mono-infections, while adenovirus was most commonly associated with dual infections (62.9%). influenza a paired with s. pneumoniae had higher proportions of chills and rigors than their respective mono-pathogens (p = 0.03, p = 0.009). h. influenzae paired with either enterovirus or parainfluenzae had higher proportions of cough with phlegm than their respective mono-pathogens. although there were observed differences in mean proportions of body temperature, nasal symptoms, sore throat, body aches and joint pains between viral and bacterial mono-pathogens, there were few differences between distinct dual-pathogen pairs and their respective mono-pathogen counterparts. conclusion: a substantial number of fri patients have multiple pathogens detected. observed clinical differences between patients of dual pathogen and mono-pathogen indicate the likely presence of complex microbial interactions between the various pathogens. febrile respiratory illnesses (fri) are caused by a wide range of pathogens, most commonly by viruses and bacteria, 1,2 some of which cause more serious clinical disease and morbidity. 3, 4 it may also be due to multiple pathogens co-existing in a microenvironment of complex interactions, 5 which is not unexpected as the respiratory mucosa has abundant resident flora to begin with. for instance, one study showed that 15.3% of ambulatory patients with influenza-like illness had two viruses detected, 6 and another found that in 28.2% of children with community-acquired pneumonia, the illness was due to mixed viral-bacterial infections. 7 others also previously described respiratory viral 8, 9 and bacterial co-infections 10, 11 in various settings, although most focus on specific pathogen combinations, especially of the synergism between influenza and streptococcus pneumoniae (s. pneumoniae). [12] [13] [14] [15] however, there are no population-level studies describing multiple pathogens among persons with upper respiratory tract infections and their impact on clinical disease. such information is of particular importance to countries within the tropical belt where there is a predilection towards multiple pathogens due to the year-round circulation of respiratory pathogens. 16, 17 a previous study documented the clinical characteristics and epidemiology of viral mono-pathogens gleaned from the respiratory disease sentinel surveillance programme of the singapore military. 18 here, we analyse additional data from the programme, compare patients with one (mono-pathogen) and two pathogens detected (dual pathogen), and describe observed differences in clinical characteristics. all singaporean males enter national service for 2 years after high school or equivalent. during this period, the majority spend most of their time in communal living and training quarters in military camps and return home on weekends, resulting in a semi-closed environment with community interaction. sentinel surveillance for febrile respiratory patients were performed at five major sites. the period of study was from may 2009 to oct 2012, and servicemen who sought primary health care at these camps during regular consultation hours were recruited. the fri inclusion criterion was having a body temperature of 37.5°c and above with cough or sore throat. after obtaining informed consent, a standardised questionnaire was administered and nasal wash sampling performed by trained personnel followed by routine clinical assessment by an attending physician. repeat consultations were excluded if the patient was deemed to not have recovered from the first episode of illness. two weeks after the initial consultation, patients were reviewed (through case records and phone calls to patients, if necessary) to determine the number of patients who eventually required referral to hospitals for further evaluation, were diagnosed with pneumonia and/or were admitted for further treatment. randomly selected unmatched controls (at a rate of 5-10 persons per week) were also obtained across the year for comparative purposes of baseline commensal rates: these are soldiers from the same camps who were reporting sick at the medical centre for reasons other than respiratory symptoms or acute infections (e.g. those with muscle sprains were selected as controls). this is to prevent mild respiratory infections from being selected and confounding the baseline rates. informed consent was also sought from controls before recruitment. nasal wash samples were obtained from trained medical staff from each side of the nose and placed in universal transport media. these were stored in fridge at 4°c and transported to the laboratory using carriers with ice packs within 24 h. an iso15189-accredited laboratory that regularly takes part in qcmd eqa programmes was used to perform molecular diagnostic testing. detailed laboratory methods have been described in the previous publication. 18 briefly, this was done by the extraction of nucleic acids using the dna mini kit (qiagen, inc, valencia, ca, usa) and then tested using multiplex pcr assays coupled with bead array detection technology (resplex i and ii, version 2.0, qiagen, inc, valencia, ca, usa) which can simultaneously detect and subtype 18 different pathogens. first, pathogens of the same genus were grouped (e.g. 'influenza a' includes its various subtypes, and 'enterovirus' also includes coxsackievirus, echovirus and rhinovirus). demographic characteristics for controls, mono-pathogens, dual pathogens and patients with more than two pathogens were analysed and compared using descriptive statistics. analyses on the prevalence of co-existing pathogens were then performed. interval/ratio variables were compared using oneway analysis of variance (one-way anova). comparison of nominal variables with expected frequencies less than or equal to 5 was done using fisher's exact test, while comparison of nominal variables with expected frequencies more than 5 was done using pearson's chi-square test. pearson's chi-square test was conducted to identify trend in proportions. further analysis focussed on comparing patients with one and two pathogens. in this regard, i) controls, ii) patients with more than two pathogens as well as iii) mono-and dual pathogens with sample sizes of less than 15 observations (considered too small for analysis) were excluded. as a result, a total of 11 mono-pathogens and 18 dual-pathogen pairs were available for comparison. permutation tests were conducted to compare the number of symptoms observed between mono-pathogen and dualpathogen patients for each pathogen as a proxy for severity of infection. to assess differences in symptom expression, dual pathogens were compared against mono-pathogens for mean proportions of 16 symptoms (or signs). empirical proportions of symptoms with 95% confidence intervals (cis) for both mono-pathogens and dual pathogens were calculated and compared using pearson's chi-square test at a significance level of 0.05. symptoms with onsets in at least 30% of patients for a minimum of one pathogen or combination were described in detail. in particular, dual infections with statistically different results from their respective viral monoinfections were highlighted. r statistical software (version 3.0.3) was used to perform all statistical analyses. 19 ethics approval was given by the singapore military joint medical committee for research and the national university of singapore's ethics review committee. of 7733 samples of patients tested, 45.8% had monopathogens and 20.2% had dual pathogens detected. no pathogens were picked up in 30.9% samples, while 3.1% samples had more than two pathogens. among dual pathogens, virus-bacterial pairs were most common at 76.0%, followed by bacteria-bacteria (15.2%) and virusvirus pairs (8.8%). demographics for patients and controls are detailed in table 1 . gender and the prevalence of heart disease were similar across all groups. mean age was slightly higher in controls, and the number of persons with asthma was higher among patients. multiple pathogens were also more commonly detected among recruits and in those not currently smoking. table 2 shows the differences in detection of pathogens between patients and controls. there were no significant differences in rsv, m. pneumonia, s. pneumonia and n. meningitidis between the two groups. among dual pathogens, there were 13 virus-bacteria, 2 bacteria-bacteria and 3 virus-virus combinations with more than 15 observations each. the most common virus-virus pair was that of influenza a with enterovirus; and of bacteria-bacteria pairs, it was haemophilus influenzae (h. influenzae) with s. pneumoniae. the top three virus-bacteria observations were h. influenzae, paired with adenovirus, enterovirus and coronavirus, respectively. figure 2 depicts the incidence of dual-pathogen pairs, with further details in table s1 . of the 238 samples with more than 2 pathogens detected, h. influenzae, s. pneumoniae, adenovirus and enterovirus were most commonly involved. the most common trio was adenovirus with s. pneumoniae and h. influenzae, which accounted for 15.1% of samples with more than 2 pathogens. number of symptoms mono-and dual-pathogen patients had similar symptom loads (with 8.3 symptoms on average). however, among dual-pathogen patients, those involving s. pneumoniae (p = 0.02), neisseria meningitidis (n. meningitidis) (p = 0.01) and h. influenzae (p = 0.02) displayed a higher number of symptoms than corresponding mono-pathogen patients. nine common symptoms, not ranked by severity, are presented in figure 3 , with further details in table s2 and figure s1 . mean body temperature of viral mono-pathogen patients was slightly higher than that of bacterial mono-pathogen patients ( mean proportion of viral mono-pathogen patients with chills and rigors was lower than that of bacterial monopathogen patients (0.737 vs 0.793; p < 0.001). dual pathogens with both s. pneumoniae and influenza a were associated with high proportions of chills and rigors (1.00, 95%ci 0.805, 1.00). this was significantly more than s. pneumoniae (0.767, 95%ci 0.701, 0.825; p = 0.03) and cough with sputum mean proportions of viral and bacterial mono-pathogen patients having cough with sputum were similar, at 0.681 and 0.677, respectively, although mean proportion of dual pathogens with the symptom was higher, at 0.738 (p < 0.001). specific dual pathogens with a higher proportion of cough with sputum than both respective bacterial and viral mono-pathogen patients were h. influenzae paired with enterovirus (p = 0.002; p = 0.001), or parainfluenzae (p = 0.02; p = 0.002). mean proportion of viral mono-pathogen patients having dry cough was higher than that of bacterial mono-pathogen patients (0.249 vs 0.194; p < 0.001). h. influenzae with enterovirus, with higher mean proportions of cough with phlegm as described above, showed a corresponding decrease in dry cough. the proportion among dualpathogen patients was also lower than the patients infected with the virus alone or the bacteria alone (p = 0.005, p = 0.03, respectively). mean proportion of viral mono-pathogen patients having nasal symptoms (sneezing, blocked nose and running nose) was higher than that of bacterial mono-pathogen patients (0.790 vs 0.670, p < 0.001). mean proportion for dual infections with nasal symptoms lay in between at 0.748, statistically different from both viral (p = 0.002) and bacterial (p < 0.001) mono-pathogen levels. however, no specific dual-pathogen pairs had statistically different levels than their respective viral mono-pathogens. mean proportion of viral mono-pathogen patients with sore throat was only slightly higher than that of bacterial monopathogen patients (0.868 vs 0.837; p = 0.01). the mean proportions for dual pathogens were similar to viral monopathogen levels (0.865) and likewise statistically higher than bacterial mono-pathogen levels (p = 0.03). interestingly, however, dual pathogens of coronavirus with s. pneumoniae (p = 0.007) or h. influenzae (p = 0.001) were instead found to be statistically lower than patients with coronavirus alone. mean proportions of viral mono-pathogen patients with headache were similar to that of bacterial mono-pathogen patients (0.727 vs 0.747). for dual pathogens (0.765), the mean proportion were only slightly higher than viral monopathogen patients (p = 0.01). however, no dual-pathogen pairs had statistically different levels than their respective viral mono-pathogens. mean proportions of viral mono-pathogen patients with body aches were similar to that of bacterial mono-pathogen patients (0.638 vs 0.611). mean proportion of dual pathogens with body aches (0.603) was slightly lower than viral monopathogen patients (p = 0.03). however, no dual-pathogen pairs had statistically different levels than their respective viral mono-pathogens. mean proportion of viral mono-pathogen patients with joint pains were higher than that of bacterial monopathogen patients (0.298 vs 0.228; p ≤ 0.001). mean proportions of dual infections with joint pains (0.263) were in between these two levels, being statistically different from both viral (p = 0.02) and bacterial (p = 0.03) monopathogen patients. however, no dual-pathogen pairs had statistically different levels than their respective viral monopathogens. patients were reviewed 2 weeks after the first consultation to ascertain whether any complications had developed in the interim. the proportion of patients referred to hospitals (for further evaluation), as well as the proportion diagnosed with pneumonia, were found to increase significantly with the number of pathogens detected (p = 0.001 and p = 0.04, respectively) (table 3) . however, there were no clear trends in the number of patients eventually requiring inpatient treatment, possibly as a result of relatively small numbers. much emphasis in respiratory illness research that is based on clinical presentations has thus far centred on monoinfections, although in reality a substantial portion of patients may actually have two or more potential pathogens. our study shows that the prevalence of patients with two or more pathogens in a tropical setting was 23.3%, most commonly due to virus-bacteria pairs. often, it seems that the role of 'less pathogenic' co-detected microbes are casually disregardedperhaps for ease of data interpretation. yet such assumptions are questionable especially because the impact of multiple pathogens on clinical characteristics has not been well studied. this formed the impetus for our analysis of the distribution of dual pathogens in ambulatory fri patients, and comparing associated clinical presentations between mono-and dual-pathogen patients. although we cannot conclude cause-effect relationships from the study, we noted a few interesting trends. the association between new recruits and multiple pathogens is likely due to the ease of transmission within the communal environment (of increased population density) on entry into military service, as described in clinical studies among similar cohorts. 20,21 these conditions also promote shifts in predominant circulating respiratory pathogens with time, as had been previously described, 18 sometimes culminating in outbreaks of respiratory disease. 21, 22 to prevent the occurrence of such incidents, mitigating measuressuch as appropriate education on hand and respiratory hygienehave been implemented. the higher prevalence of asthma in patients and the decreased number of pathogens among current smokers may also reflect the effects of the two on the upper respiratory tract. 23, 24 for example, previous studies describe the effect of cigarette smoke in causing reduced competitive commensal organisms in the respiratory tract. 25, 26 among dual infections, virus-virus pairs constitute only 3.0% of the entire data set, within the lower end of range of viral co-infection studies in ambulatory settings (1.73-15.3%). 6, 8, 9 this may be due to local interactions between immune and microbial mechanisms preventing the occurrence of co-existing viral respiratory pathogens. such negative correlations have been previously described, 27 including the replacement of one virus with another when the former is removed from the general population through vaccinations. 18 the genus enterovirus was most prevalent (56.6%) among viral-viral pairs, similar to two other viral coinfection studies reporting rhinovirus rates of 49.3% and 69.5%. 9, 28 virus-bacterial pairs were most common, with a significant proportion involving adenovirus, particularly paired with h. influenzae (59.8%). such a finding had also been previously observed among hospitalised children, where 45% of those with adenovirus were co-infected with various bacteria. 29 previous chinchilla models on experimental otitis media also point towards possible synergisms between adenovirus and h. influenzae, 30 although further studies are needed to conclusively determine whether such interactions exist in the upper respiratory tract. when it came to symptoms, the increased incidences of chills and rigors and elevated body temperatures in influenza a and influenza b, respectively, when paired with s. pneumoniae correspond to previous studies showing the disposition to superinfection caused by the influenza virus on respiratory epithelium, in both laboratory and hospital studies. [31] [32] [33] [34] [35] our results show that these apply to ambulatory patients as well. however, we also noted that these systemictype symptoms appeared to be distinct from localised upper respiratory tract symptoms (such as running nose and cough), which were not found to be significantly different from patients with influenza alone. next, a higher prevalence of cough with phlegm was correlated with a number of dual-pathogen combinations, all of which involved the bacteria h. influenzae. although there are microbiological studies on the bacteria's interactions with rhinovirus, 36, 37 there is insufficient information to conclusively explain the observations noted with parainfluenzae, warranting further studies. finally, diversity in the impact of dual pathogens on clinical manifestations, as seen through the results of other symptoms, is likely indicative of complex and diverse microbial interactions between respiratory pathogens in the upper respiratory tract. bosch et al. have detailed a number of known microbiologic mechanisms, including various modalities of synergisms and competition between species. 6 these include pathogens that are usually associated with asymptomatic colonisation in healthy individuals (e.g. s. pneumoniae and h. influenzae), which are potentially pathogenic with shifts in the respiratory tract microenvironmentfor instance, the introduction of new microbes. [38] [39] [40] many of these are not yet fully understood, and it is hoped that such epidemiological data may spur greater interest in co-pathogen microbiology research. our study does not explore patients infected with more than 2 pathogens and co-pathogen pairs with <15 observations. although it identifies observed correlations between pairs and symptoms, it does not determine sequence of pathogens in relation to onset of symptoms or prove causality, which require further microbiological or case-control epidemiological research. severity of symptoms other than fever was not determined, actual diagnoses by doctors were not analysed, and further differences in the actual clinical impact could not be observed. although statistically significant differences have been described, the clinical significance of these findings have to be considered alongside as small differences may not be easily translatable to clinical practice and the large number of statistical comparisons increase the chances of type i (i.e. false-positive) errors. the study predominantly involved young adult males, limiting the generalizability to other populations. it is also conducted in a tropical setting with a fairly constant climate; thus, the effect of such changes on symptomology (e.g. in a temperate country) cannot be determined. by grouping pathogens of the same genus together in analysis, it is also not possible to determine whether specific subtypes are the cause for the observations made. we are unable to detect the presence of dual-pathogen patients involving two or more viruses from the same genus, especially within enteroviruses. although we compared differences in the detection of organisms between patients and controls, we are unable to conclude on whether certain organisms (such as n. meningitidis and adenovirus) are actually commensals, and pcr is not the optimal method for diagnosis of bacterial infections. we have described the aetiology of dual pathogens causing fri in the tropical setting and compared differences with monopathogens with regard to observed clinical manifestations. the presence of higher incidences of certain symptoms with specific pathogen pairs is indicative of underlying complex microbial interactions and affirms existing microbiological co-pathogen studies. however, many of these processes are still not well explored in existing literature, opening many opportunities for further research into this area. additional supporting information may be found in the online version of this article: figure s1 . proportions of each clinical symptom between dual pathogens and their mono-pathogen counterparts. table s1 . checkerboard of dual pathogens detected among cases. the 18 dual-pathogen pairs for further analysis of symptoms (i.e. 15 observations or more) are in bold. table s2 . mean proportions and comparisons between viral and bacterial mono-pathogens and dual pathogens. acute respiratory illness in the community. frequency of illness and the agents involved etiology and incidence of viral and bacterial acute respiratory illness among older children and adults in rural western kenya acute respiratory symptoms in adults in general practice viral agents responsible for febrile respiratory illnesses among military recruits training in tropical singapore viral and bacterial interactions in the upper respiratory tract community-acquired respiratory viruses and co-infection among patients of ontario sentinel practices epidemiology and microbiological investigations of community-acquired pneumonia in children admitted at the emergency department of a university hospital co-infection cases of human common respiratory viruses in beijing dual respiratory virus infections impact of bacterial and viral coinfection on mycoplasmal pneumonia in childhood community-acquired pneumonia pulmonary bacterial coinfection in infants and children with viral respiratory infection complications and associated bacterial co-infections among children hospitalized with seasonal or pandemic influenza bacterial co-infection with h1n1 infection in patients admitted with community acquired pneumonia the impact of bacterial and viral coinfection in severe influenza epidemiology and clinical characteristics of hospitalized patients with pandemic influenza a (h1n1) 2009 infections: the effects of bacterial coinfection seasonal trends of viral respiratory tract infections in the tropics epidemiology and seasonality of respiratory tract virus infections in the tropics respiratory viral pathogens among singapore military servicemen 2009-2012: epidemiology and clinical characteristics r: a language and environment for statistical computing. r foundation for statistical computing association between barracks type and acute respiratory infection in a gender integrated army basic combat training population h1n1 outbreak in a swiss military boot camp-observations and suggestions outbreak of h3n2 influenza at a us military base in djibouti during the h1n1 pandemic of airway allergy and viral infection increased h1n1 infection rate in children with asthma cigarette smoking and mechanisms of susceptibility to infections of the respiratory tract and other organ systems recovery of potential pathogens and interfering bacteria in the nasopharynx of smokers and nonsmokers broad spectrum respiratory pathogen analysis of throat swabs from military recruits reveals interference between rhinoviruses and adenoviruses single, dual and multiple respiratory virus infections and risk of hospitalization and mortality mixed infection is common in children with respiratory adenovirus infection synergistic effect of adenovirus type 1 and nontypeable haemophilus influenzae in a chinchilla model of experimental otitis media immune dysfunction and bacterial coinfections following influenza insights into the interaction between influenza virus and pneumococcus influenza virus neuraminidase contributes to secondary bacterial pneumonia influenza a virus facilitates streptococcus pneumoniae transmission and disease interactions between streptococcus pneumoniae and influenza virus: a mutually beneficial relationship? influenzae potentiates airway epithelial cell responses to rhinovirus by increasing icam-1 and tlr3 expression human rhinovirus-induced isg15 selectively modulates epithelial antiviral immunity dynamics of nasopharyngeal colonization by potential respiratory pathogens human polymicrobial infections the ecology of nasal colonization of streptococcus pneumoniae, haemophilus influenzae and staphylococcus aureus: the role of competition and interactions with host's immune response the authors would like to acknowledge the staff from dso national laboratories for their tireless efforts in the collection of data and testing of laboratory samples, as well as staff from hq medical corps, singapore armed forces, and the centre for infectious disease epidemiology research (cider) at the saw swee hock school of public health, national university of singapore for their support. the programme was supported by a singapore ministry of defence-funded operational research programme and the centre for infectious diseases epidemiology and research in the saw swee hock school of public health of the national university of singapore and national university health system. the funders had no role in the study design, data collection, analysis, decision to publish or preparation of the manuscript. the authors declare that they have no competing interests. key: cord-297609-6g39lu1y authors: wertheim, heiman f l; nadjm, behzad; thomas, sherine; malik, suhud; nguyen, diep ngoc thi; vu, dung viet tien; van nguyen, kinh; van nguyen, chau vinh; nguyen, liem thanh; tran, sinh thi; phung, thuy bich thi; nguyen, trung vu; hien, tran tinh; nguyen, uyen hanh; taylor, walter; truong, khanh huu; ha, tuan manh; chokephaibulkit, kulkanya; farrar, jeremy; wolbers, marcel; de jong, menno d; van doorn, h rogier; puthavathana, pilaipan title: viral and atypical bacterial aetiologies of infection in hospitalised patients admitted with clinical suspicion of influenza in thailand, vietnam and indonesia date: 2015-10-13 journal: influenza other respir viruses doi: 10.1111/irv.12326 sha: doc_id: 297609 cord_uid: 6g39lu1y background: influenza constitutes a leading cause of morbidity and mortality worldwide. there is limited information about the aetiology of infection presenting clinically as influenza in hospitalised adults and children in south-east asia. such data are important for future management of respiratory infections. objectives: to describe the aetiology of infection presenting clinically as influenza in those hospitalised in south-east asia. methods: respiratory specimens archived from july 2008 to june 2009 from patients hospitalised with suspected influenza from indonesia, thailand and vietnam were tested for respiratory viruses and atypical bacteria by polymerase chain reaction. results: a total of 1222 patients’ samples were tested. of 1222, 776 patients (63·5%) were under the age of 5. viruses detected included rhinoviruses in 229 of 1222 patients (18·7%), bocaviruses in 200 (16·4%), respiratory syncytial viruses in 144 (11·8%), parainfluenza viruses in 140 (11·5%; piv1: 32; piv2: 12; piv3: 71; piv4: 25), adenovirus in 102 (8·4%), influenza viruses in 93 (7·6%; influenza a: 77; influenza b: 16) and coronaviruses in 23 (1·8%; oc43: 14; e229: 9). bacterial pathogens were mycoplasma pneumoniae (n = 33, 2·7%), chlamydophila psittaci (n = 2), c. pneumoniae (n = 1), bordetella pertussis (n = 1) and legionella pneumophila (n = 2). overall, in-hospital case fatality rate was 29 of 1222 (2·4%). conclusion: respiratory viruses were the most commonly detected pathogens in patients hospitalised with a clinical suspicion of influenza. rhinovirus was the most frequently detected virus, and m. pneumoniae, the most common atypical bacterium. the low number of detected influenza viruses demonstrates a low benefit for empirical oseltamivir therapy, unless during an influenza outbreak. influenza is a common reason for primary care consultation and constitutes a leading cause of hospitalisation, morbidity and mortality worldwide. 1 non-influenza viruses are the most common causes of illness that clinically resemble influenza, particularly in children. data on the epidemiology and disease burden of influenza-related disease in south-east asia (sea) are emerging, and this, in turn, is shedding more light on the epidemiology of other viral and bacterial aetiologies of influenza-like illnesses (ilis) in hospitalised adults and children in this region. 2 the insight provided by this information is important not only for future prevention strategies, treatment and clinical management of respiratory infections, but also to guide future studies in this region. recently, many diagnostic advances have been made in order to help to determine the aetiology of acute respiratory infections. these advances have been made possible by molecular diagnostic techniques that are now in widespread use. furthermore, research in this field has gained much attention and funding due to the outbreaks of severe acute respiratory syndrome coronavirus (sars-cov) and avian influenza viruses a/h5n1 and a/h7n9 in sea and middle eastern respiratory syndrome coronavirus (mers-cov) in the middle east. generally, the causative agents of ili remain undiagnosed in routine clinical practice due to the expense of testing and the slow turnaround time for most diagnostic polymerase chain reactions (pcrs), considered too long to impact the management of usually self-limiting respiratory tract infections. however, with the increasing use of antiviral drugs, such as neuraminidase inhibitors for influenza, testing for ili pathogens may become more important for rational antiviral drug treatment and to prevent overuse of antibiotics, thereby helping to control costs and prevent the emergence and spread of antimicrobial drug resistance. during the past few years, several novel causative agents of ili have been identified in respiratory specimens, including human metapneumovirus, new human coronaviruses (nl63, hku1, sars-cov, mers-cov), rhinovirus c and continuously emerging novel lineages and subtypes of influenza virus a capable of infecting humans (h5n1, h5n6, h1n1pdm09, h3n2v, h7n7, h7n9, h9n2, h6n1, h10n8). viruses for which disease causation has not (yet) been established have also been identified, including human bocavirus and respiratory polyomaviruses, and likely there are many more to come. [3] [4] [5] [6] the most comprehensive data on the causes of ili originate from developed countries, with some data available from sea. recently, studies have been published from asian countries, [7] [8] [9] [10] [11] [12] providing much needed, valuable information and showing a similar spectrum of viral pathogens. this study was designed to determine the viral and atypical bacterial aetiologies of ilis in patients in sea using molecular techniques. the detection of influenza viruses and respiratory pathogens other than influenza viruses in specimens that were sent for routine influenza testing can provide valuable insights into the epidemiology of ili across all age groups in three sea countries over the same time period. furthermore, this study could address the potential occurrence of multiple simultaneous infections in the same patient. we performed a laboratory-based surveillance study, in which we tested all archived respiratory specimens from hospitalised patients over 1 year (july 2008 to june 2009) with clinically diagnosed ili meeting the following criteria: age ≥1 year, signs of ili according to treating physician, duration of ili symptoms ≤10 days and respiratory specimens sent for influenza testing. clinicians at all participating hospitals received regular training in who protocols for the management and recognition of influenza/ili. archived respiratory specimens (nose swab, throat swab, nasopharyngeal aspirate, nasal wash, tracheal aspirate, bronchoalveolar lavage) were obtained from 12 sites in three countries across sea (three sites in indonesia, four sites in thailand, five sites in vietnam). influenza testing capacity was created for a randomised clinical study comparing two dosages of oseltamivir for severe influenza by sites in the south east asia infectious diseases clinical research network, as described elsewhere. 13 the specimens used for this study were stored at à80°c until further testing. where more than one specimen type was available for each patient, they were prioritised in the sequence endotracheal aspirate/bronchoalveolar lavage > nasopharyngeal aspirate > throat swab > nasal swab/ washing. this retrospective study for additional testing on archived specimens after full anonymisation of specimens was approved by the institutional review boards of each site. commercially available multiplex polymerase chain reaction (pcr)/gel electrophoresis assays (seeplex â rv12 ace detection and seeplex â pneumobacter ace; seegene, south korea [14] [15] [16] ) were used to detect 12 major respiratory viruses and six respiratory bacterial pathogens: influenza viruses a and b, respiratory syncytial viruses (rsvs) a and b, rhinoviruses a/b, coronaviruses oc43/hku1 and 229e/ nl63, adenovirus, parainfluenza viruses 1-3, human metapneumovirus, mycoplasma pneumoniae, chlamydophila pneumoniae, legionella pneumophila serotype 1 and bordetella pertussis. additional in-house real-time [reverse-transcriptase (rt)] pcrs were used to detect the following viruses: rhinoviruses a, b and c, enteroviruses, parainfluenza virus 4, bocavirus, parechoviruses and chlamydophila psittaci, as described elsewhere. [17] [18] [19] basic demographic data (country, sex, age) were collected as well as date of disease onset, date of admission, type of specimen and intensive care unit (icu) admission. for the analysis results of streptococcus pneumoniae and haemophilus influenzae, pcrs were not taken into account, because of the high positivity rates resulting from nasopharyngeal carriage of 25á4% and 31á4%, respectively, in this study (data not shown). the baseline demographic characteristics (age category, sex, country of admission and proportion of icu admissions) were summarised as frequency and proportion. the frequencies of different pathogens were summarised per country, sex and age category to determine the most frequent viruses and bacteria in each stratum. disease outcomes including severity (severe defined as icu admission), death, duration of hospitalisation and duration of illness were examined by regression analysis. the dependence of binary outcomes (severe disease and death) on the presence of pathogens was analysed by logistic regression, adjusted for age and sex. kaplan-meier survival analysis was used to visualise the number of hospitalisation days between each age category (patient deaths were censored). cox regression was used to analyse whether viruses were found more often if the duration of illness before presentation to hospital was shorter. regression analysis was based on the data from vietnam and thailand only as relevant clinical data were missing from indonesia. the statistical software sas version 9.2 (sas institute inc., cary, nc, usa) was used for all computations. a two-sided p value of 0á05 or less was interpreted as statistically significant. a total of 1222 patient samples were collected at participating sites. table 1 summarises the demographic data. the median number of days from illness onset to specimen collection was 4 (iqr: 3-6 days). for 155 patients, no exact admission date was known. the majority of patients in this study were under the age of 5 (776, 63á5%). most patients were enrolled in vietnam (n = 826, 67á6%). the median age was higher in indonesian patients as compared to those from vietnam and thailand (16 year, versus 4 year and 2 year, respectively). one hundred and 67 patients (13á7%) were admitted to the intensive care unit (icu); icu admission data from 232 patients were unknown (indonesia: 223; thailand: 2; vietnam: 7). sixtyseven patients (5á5%) were mechanically ventilated and the overall reported in-hospital mortality was 2á4% (n = 29). detected bacterial pathogens were m. pneumoniae (n = 33, 2á7%), c. psittaci (n = 2), c. pneumoniae (n = 1), b. pertussis (n = 1) and l. pneumophila (n = 2). fluctuations in frequencies were found in particular months for several viruses, with rsv occurring from july to november 2008 and rhinovirus peaking in march 2009 ( figure 2 ). due to differences in age group representation, the number of viral agents detected per patient was lower in indonesia (60/225, 0á27) than in vietnam (800/826, 0á97) and thailand (151/171, 0á88). likewise, the proportion of identified viral agents detected that were rsv was higher in thailand and vietnam than in indonesia (24/151, 15á9%; 117/800, 14á6%, respectively, compared with 3/60, 5%) whilst coronavirus was a greater proportion of identified viral agents in indonesia (7/60, 11á7%) than thailand (3/151, 2á0%) and vietnam (11/800, 1á4%). see table s1 for pathogens detected by country. there were 234 of 1222 patients (19á2%) who had at least two pathogens detected (bacterial and/or viral). of these, 170 patients were found to have two pathogens, 54 patients had three pathogens, nine patients had four pathogens, and one patient had five pathogens. the frequency of each pathogen is shown in table s2 . the pathogens most commonly detected in patients with two or more pathogens were bocavirus (n = 132) and rhinovirus (n = 123). the proportion of patients who had either a virus or a bacterium detected appeared to decrease with age, with the highest proportion of pathogens detected in the 0-4 age group, but increased again in those older than 65. most viral pathogens were more common in children, with parechovirus and parainfluenza virus 4 only found in children (table 2) . m. pneumoniae was found only in patients younger than 45 years, whilst both cases of c. psittacii occurred in patients 45 years and above. the icu admissions for each country are shown in table 1 . the number of patients who were mechanically ventilated was 42 of 826 (5á1%) in vietnam and 21 of 171 (12á3%) in thailand (data not available for indonesia). for the vietnamese data set, where data were most complete, icu admission was related to age; 15 of 616 children (2á4%) aged under 5 years, three of 36 (8á3%) aged 5-14 years, 69 of 106 (65á1%) aged 15-44 years, 33 of 45 (73á3%) aged 45-64 years and 20 of 23 (87%) aged over 65 years were admitted to icu (v 2 for trend p < 0á0005). logistic regression was performed on this data set including the covariates: pathogen type (virus, bacterial, both and neither), age and sex. the analysis showed that only higher age was associated with icu admission (adjusted or for icu admission in those aged >64 years compared to those aged <5 years: 243á79, p < 0á001, table 3 ). no specific pathogen was associated with icu admission. there were only sufficient data available to analyse the duration of hospitalisation for patients in vietnam. the median duration of hospitalisation was 5 days (iqr: 3-7 days). multivariate cox regression analysis for hospitalisation days by pathogen type (virus, bacterial, both and neither), sex and age was performed, and both pathogen type (p = 0á0004) and age category (p = 0á015) had a significant effect. it was found that patients in the >65-year group were hospitalised longer, but did not differ by sex. influenza a virus-positive patients were admitted for a median of 4 days (iqr: 3-8 days) versus 5 days (iqr: 3-8 days) for patients where influenza a was not detected. a similar pattern was seen for coronavirus e229: median of 3 days (iqr: 2-4 days) for positive patients versus 5 days (iqr: 3-8 days) in the negative group. patients who were m. pneumoniae-positive were admitted longer: a median of 9 days (iqr: 7-12 days) versus a median of 5 days (iqr: 3-8 days). there were 29 deaths and a viral pathogen was detected in 12 (41%) of these: influenza virus a (n = 3), rhinovirus (n = 3), bocavirus (n = 1), coronavirus (n = 2), parainfluenza virus (n = 1), rsv (n = 1) and one combination of influenza a with adenovirus and rhinovirus. data concerning the cause of death were not collected in this study; consequently, conclusions about causation cannot be drawn. influenza-like illnesses are a major cause of mortality and morbidity worldwide, especially in younger and elderly age groups. it has been suggested that a large proportion of iliattributable deaths globally occur in africa and sea. 20 data regarding the aetiology of respiratory infections in sea are limited, with some of the available data suggesting that viruses account for a large proportion of these infections. 21 in this 1-year study, we used molecular techniques to detect viruses and atypical bacteria from samples collected from patients hospitalised with ili. the majority of patients enrolled in this study were enrolled in vietnam and were under the age of 5. for ili diagnostics and surveillance, nose and throat swabs are considered the sampling methods of choice, whereas for the diagnostics of h. influenzae and s. pneumoniae, gram-staining and bacterial culture of representative purulent sputum, blood culture or urinary antigen tests (for s. pneumoniae) would be the testing method of choice, which were not done. detection rates of these two pathogens were therefore not included in the analysis. in this study, a potential pathogen was identified in 60á6% of patients. this rate is higher than the previously published rates of between 35% and 47% of organisms identified, in studies looking at the aetiology of respiratory tract infections in different asian countries. 9, 22 a total of 58á6% of the study cohort were found to be positive for viruses compared to just 3á2% of the cohort who had an atypical bacterial pathogen alone detected. more recent studies form asian countries using molecular techniques have shown the rates of virus detection reaching 72%. 10 the most common viruses detected in our study were rhinoviruses, accounting for 32% of the patients in whom viral pathogens were detected and 18á7% of the cohort as a whole, similar to other studies. 1, 23 variation between the three countries in the proportion of patients in whom viral pathogens were detected, and variations in the relative importance of different viruses, largely represents the differing age groups sampled, with the younger patients in vietnam and thailand having higher a proportion of rsv detected. bocaviruses were first described in 2005, and since then, numerous studies have described their detection in the human respiratory tract 3 and their possible association with disease, but causation has not convincingly been proven. 24 in this study, this virus was detected in 16á4% of patients, which is similar to other studies where detection rates ranged from 1á5% to 21á5%. 25, 26 these previous studies as well as this study have detected bocavirus, predominantly from young children rather than from adults. in this study as in other studies, bocaviruses were commonly (136/200, 68% bocavirus isolates in our study, table s2 ) detected in combination with other pathogens, most commonly rhinovirus. atypical bacteria were rarely (3á2%) detected. this rate of detection is lower than previously published data, where detection varied from 13% to 26%. 27, 28 however, as we did not include serological diagnostic methods for atypical bacteria, the true prevalence in our patients may have been higher. the most commonly detected atypical bacteria in this study were m. pneumoniae, followed by c. psittaci and l. pneumophila. the results from our study are similar to the aetiological studies of community-acquired pneumonia in singapore and malaysia, in which m. pneumoniae was the commonest atypical pathogen detected. 29 very little is known about the epidemiology of c. psittaci in sea, and indeed this pathogen has only very recently been described as causing human disease in vietnam. 30 the type of pathogen detected was significantly associated with the duration of hospitalisation. atypical bacteria and in particular infection with m. pneumoniae was associated with longer duration of hospitalisation. the age of the patients was also significantly associated with longer duration of hospitalisation, with the >65 years age group requiring longer admissions. this probably corresponds with increasing comorbidities and decreasing immunity with age, similar to other regions in the world. no clear seasonality was seen with the various pathogens detected, but rates of infection appeared to be higher in the winter and spring months and a study over a single year may miss seasonal trends. rates of influenza virus a increased in indonesia and vietnam in april 2009 coinciding with an epidemic of seasonal h3n2 influenza in vietnam, prior to the first sporadic cases of h1n1pdm09 detected at the end of may 2009. 31 the strengths of this type of study include the large numbers of patients who were enrolled prospectively from three different countries in south-east asia allowing an overview of the viral and atypical bacterial causes of ilis in the region. this study also used rt-pcr techniques; however, an important limitation was the lack of microscopy and sputum cultures for common bacteria associated with ilis such as s. pneumoniae and h. influenzae type b and serology for atypical pathogens. also, limited clinical data were recorded for these cases, making it difficult to assess the clinical significance of the various pathogens detected and the virulence of each pathogen. although swabs from all patients meeting our eligibility criteria were analysed, we have no data concerning the number of patients who may have been admitted with ili but not swabbed. the lack of clinical data also limits comparison between sites as differing admission criteria may impact on the distribution of pathogens. similarly, both the lack of clinical data and the absence of data concerning overall admission limit our ability to interpret the findings relating to icu admission; the finding that adult patients were overrepresented amongst those admitted to icu could represent differential routine sampling of adults and children on such units; however, we feel this is unlikely. there was a lack of data related to patients and co-morbidities, such as immunosuppression and copd, which could also have had an impact in the severity of infection seen. furthermore, a lack of suitable control patients also limits our ability to determine the attributable fraction of ili that is likely to be caused by each virus and also prevents useful analysis of the relevance of the high rates of nasopharyngeal carriage of s. pneumoniae and h. influenzae type b. establishing the aetiology of ili is becoming increasingly important, not only to establish the burden of various pathogens in order to improve management and prevention, but also to guide antiviral treatment and to try to limit the inappropriate use of antimicrobials to reduce the emergence and spread of antibiotic resistance. our study, along with overwhelming evidence from many other studies from these and other countries, suggests that viral causes of ili are much more common than bacterial causes, but that the viral aetiology may vary in space and time. in recent years, the emergence of sars-cov, mers-cov and avian influenza viruses a/h5n1 and a/h7n9 has highlighted the risks posed by respiratory viral infections in humans and sea has become an area of increasing interest for monitoring pathogens. very little has been published from this region regarding the incidence and prevalence of viral or atypical organisms that can contribute to ilis, making studies looking at this increasingly important. additional supporting information may be found in the online version of this article: table s1 . frequency of detected pathogens by country. table s2 . frequency of pathogens found in a combination with another pathogen. prospective study of the incidence, aetiology and outcome of adult lower respiratory tract illness in the community etiology and management of community-acquired pneumonia in asia cloning of a human parvovirus by molecular screening of respiratory tract samples viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis respiratory infections unique to asia masstag polymerasechain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during viral pathogens associated with acute respiratory infections in central vietnamese children association between nasopharyngeal load of streptococcus pneumoniae, viral coinfection, and radiologically confirmed pneumonia in vietnamese children identification of viral and atypical bacterial pathogens in children hospitalized with acute respiratory infections in hong kong by multiplex pcr assays viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city a prospective three-year cohort study of the epidemiology and virology of acute respiratory infections of children in rural india fatal respiratory infections associated with rhinovirus outbreak effect of double dose oseltamivir on clinical and virological outcomes in children and adults admitted to hospital with severe influenza: double blind randomised controlled trial use of the seeplex rv detection kit for surveillance of respiratory viral outbreaks in comparison of the seeplex reverse transcription pcr assay with the r-mix viral culture and immunofluorescence techniques for detection of eight respiratory viruses detection of 12 respiratory viruses with two-set multiplex reverse transcriptase-pcr assay using a dual priming oligonucleotide system rapid detection of human parechoviruses in clinical samples by realtime pcr development of an internally controlled real-time pcr assay for detection of chlamydophila psittaci in the lightcycler 2.0 system development and evaluation of a four-tube real time multiplex pcr assay covering fourteen respiratory viruses, and comparison to its corresponding single target counterparts epidemiology and etiology of childhood pneumonia non-flu aetiology of ili in 3 se asian countries ª 2015 the authors. influenza and other respiratory viruses published by viral etiology of acute lower respiratory tract infections in hospitalized young children in northern taiwan respiratory viral infections detected by multiplex pcr among pediatric patients with lower respiratory tract infections seen at an urban hospital in delhi from aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (lrti) in primary care human bocavirus-the first 5 years high frequency of human bocavirus 1 dna in infants and adults with lower acute respiratory infection human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand atypical bacterial pathogen infection in children with acute bronchiolitis in northeast thailand acute bronchitis in the community: clinical features, infective factors, changes in pulmonary function and bronchial reactivity to histamine an asian study on the prevalence of atypical respiratory pathogens in communityacquired pneumonia first report of human psittacosis in vietnam a clinical virological and epidemiological analysis the investigators were involved in all aspects including protocol design, study execution and oversight and the writing of this publication. richard molenkamp and bob de wever, dept of medical microbiology, academic medical center, amsterdam, the netherlands, are kindly acknowledged for sharing of protocols and positive controls for setting up in-house real-time (rt-) pcrs for respiratory viruses and atypical bacteria. the authors declare that they have no conflict of interests. the study was conducted by the seaicrn (http://www.seaicrn.org/) and sponsored by the national institute of allergy and infectious diseases. site support was also provided by the wellcome trust (london, uk) through its major overseas programmes. h wertheim, md de jong, hr van doorn and p puthavathana contributed to study design, study implementation, analysis and interpretation of the data, writing of the manuscript, and reading and approval of the final version. b nadjm involved in analysis and interpretation of the data, writing of the manuscript, and reading and approval of the final version. s thomas, dung vtv and uyen hn participated in analysis and interpretation of the data and reading and approval of the final version. n agustiningsih and diep ntn contributed to study implementation, analysis and interpretation of the data, and reading and approval of the final version. s malik, kinh vn, chau vvn, liem tn, sinh tt, thuy btp, trung vn, tran th, khanh ht, tuan mh and kulkanya c involved in study implementation and reading and approval of the final version. w taylor and j farrar contributed to study design, study implementation and reading and approval of the final version. m wolbers involved in study design, study implementation, analysis and interpretation of the data, and reading and approval of the final version. key: cord-002138-uywz5nqw authors: boëlle, pierre‐yves; ansart, séverine; cori, anne; valleron, alain‐jacques title: transmission parameters of the a/h1n1 (2009) influenza virus pandemic: a review date: 2011-03-31 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2011.00234.x sha: doc_id: 2138 cord_uid: uywz5nqw please cite this paper as: boëlle p‐y et al. (2011) transmission parameters of the a/h1n1 (2009) influenza virus pandemic: a review. influenza and other respiratory viruses 5(5), 306–316. background the new influenza virus a/h1n1 (2009), identified in mid‐2009, rapidly spread over the world. estimating the transmissibility of this new virus was a public health priority. methods we reviewed all studies presenting estimates of the serial interval or generation time and the reproduction number of the a/h1n1 (2009) virus infection. results thirteen studies documented the serial interval from household or close‐contact studies, with overall mean 3 days (95% ci: 2·4, 3·6); taking into account tertiary transmission reduced this estimate to 2·6 days. model‐based estimates were more variable, from 1·9 to 6 days. twenty‐four studies reported reproduction numbers for community‐based epidemics at the town or country level. the range was 1·2–3·1, with larger estimates reported at the beginning of the pandemic. accounting for under‐reporting in the early period of the pandemic and limiting variation because of the choice of the generation time interval, the reproduction number was between 1·2 and 2·3 with median 1·5. discussion the serial interval of a/h1n1 (2009) flu was typically short, with mean value similar to the seasonal flu. the estimates of the reproduction number were more variable. compared with past influenza pandemics, the median reproduction number was similar (1968) or slightly smaller (1889, 1918, 1957). in april 2009, a new influenza virus a ⁄ h1n1 (2009) was isolated in mexico and has rapidly spread over the world, being reported in 214 countries 1 year after its first identification. 1 the spread of the virus was extremely fast worldwide. 2 as soon as the new virus was identified, a major issue was to estimate the transmissibility of the new virus. in the guidance document 'global surveillance during an influenza pandemic' released by the world health organization, three parameters were highlighted that should be documented quickly in this respect: the incubation period (time between infection and symptoms), the serial interval (time between symptoms onset in primary case and secondary case), and the reproduction ratio ⁄ number (average number of secondary cases per primary case). 3 these parameters are instrumental to assessing the feasibility and efficacy of intervention strategies against pandemic influenza. 4 information regarding the serial interval and the reproduction number from past pandemics has been limited. for the serial interval, the best information concerned sea-sonal influenza infections 5, 6 and no information was available regarding past pandemics. there was comparatively more information regarding reproduction numbers, with estimates obtained in the last four pandemics (1889, 1918, 1957, 1968) . [7] [8] [9] [10] [11] [12] estimates have ranged between 1 and 6 depending not only on place, time, wave, but also on the methods and assumptions used in estimation. as we have now entered the post-pandemic period for h1n1(2009), 13 it is timely to review the results of all studies regarding the serial interval or generation time for the a ⁄ h1n1 flu, as well as the reproduction numbers, to allow comparison with previous pandemics and help in planning. here, we present the results of a systematic review of published estimates concerning the first wave of a ⁄ h1n1 (2009) concerning the serial interval and the reproduction number. the generation time (gt) is the time interval between the date of infection in one case and that in its infector. 14 it is difficult to measure in practice, as the actual time of infection is not observed. the serial interval (si), i.e., the time interval between the date of symptoms onset in one case and that in its infector, is therefore often considered instead of the gt because it has the same mean. 14 the gt or si informs on the speed of transmission of the disease. it is not an intrinsic property of the disease, but a combination of biology (how much and when is a person infectious) and behavior (how many and when contacts leading to infection occur). 15 a random sample of pairs of secondary case and their infector would allow unbiased estimation of the si but is seldom available. in practice, various designs are used to observe pairs of infectee ⁄ infector, and this may impact the observed distribution. 16 for example, cases may be observed in households, where common exposure may have led to coprimary cases and ongoing transmission to an overlap of secondary and tertiary cases. statistical modeling is therefore required to recover the true si distribution. the reproduction number (or reproduction ratio), denoted r, is defined as the average number of secondary cases caused by one index case. 17 a reproduction number may be calculated at any time during an outbreak, a value larger than 1 corresponding to epidemic spread of the disease. in practice, additional qualifiers are often used when reporting a reproduction number: 'initial' in the beginning of an epidemic; 'basic' when the whole population is initially susceptible to the disease -r is in this case denoted r 0 ; 'effective' when the natural course of the outbreak is altered, for example, by interventions. several methods are available to estimate reproduction numbers: using attack rates, 18 the exponential growth rate, 19 averaging over transmission chains. 20 an assumption regarding the gt distribution may be required to estimate the reproduction number; in this case, a shorter mean gt will likely lead to a smaller reproduction number estimate. for all studies, we abstracted the date of publication, the place and date where the data were collected, the estimate of the reproduction number and of the mean si or gt and its confidence interval when reported; we summarized how the data were collected and the method for analysis. we focused on reproduction number estimates described as 'basic' or 'initial'. in studies where the reproduction number was estimated as a function of time, we reported the range of r(t) values. seventeen independent estimates of the mean si or gt during the 2009 h1n1 pandemic were reported in sixteen studies. details are reported in table 1 and figure 1 . the data were collected early in the pandemic, between april and august 2009. in a first group of 13 studies, estimation was based on the analysis of observed time intervals between cases and their close contacts, especially in households. cases and their households or contacts were included as part of the local health authorities response to the pandemic, except in one study where households had been included in a prospective clinical trial. 21 whether the data were prospective or retrospective was not reported except in two retrospective cases. 22, 23 household contacts only were used in eight studies, 21,23-29 yielding mean sis in the range of 2ae6-4ae4 days. in all but one study, the index case was the first case in the household. household observed serial intervals were defined as the difference in date of symptoms onset between incident cases and the index case. in the study where the index case could be different from the first, all cases after the index were considered as secondary cases of the index case. 27 the other five studies included contacts not limited to the household, 22, [30] [31] [32] [33] with mean sis in the range of 2ae5-3ae5 days. here, pairs of infector ⁄ infectee were identified where the infector was the only, or most probable, source of infection. the largest estimate (4ae4 days; range = [1, 9] ) was obtained from only five serial intervals observed in three households. 29 one estimate (2ae6 days, range = [1, 3] ) was based on eight observed household si; however, only the first non-index-case in the household was used. 28 in four instances, only the median si was reported, 22 23 very similar to the reported medians. in these calculations, coprimary cases (same day as index case) and those occurring more than 7 days after the index case were excluded. in all but two cases, the mean si was calculated as the empirical mean of observed intervals, excluding the possibility of tertiary transmission. more sophisticated modeling allowing for several generations of transmission was carried out in the two other cases. in the first case, a householdbased study, the empirical mean si (excluding coprimary cases and cases >7 days after) was 2ae9 days (95% ci [2ae7, 3ae1]). 24 after modeling, the reported mean si decreased to 2ae6 days (95% ci [2ae2, 3ae5]). in the second case, derived from the ff100 cohort in the uk, the empirical mean si of observed serial intervals was 3ae4 days (95% ci [2ae9, 3ae9]) and the reported mean si decreased to 2ae5 days (95% ci [2ae1, 2ae9]) after modeling. 31 an overall estimate of the mean si derived from these studies (excluding 28 ), weighting by the number of observed sis used for estimation in each study, was 3ae0 days (ci 95% [2ae4, 3ae6]). no correlation was found between the reported si and the size of the study (p = 0ae3) or the date of report (p = 0ae3). household-based studies did not yield different estimates of the mean si than close-contact studies (p = 0ae15). a second group of four studies reported the si or gt estimated by modeling epidemic curves. these included the smallest estimate of all, a mean gt of 1ae9 days (ci 95% [1ae3, 2ae7]) in a mexican village, 34 and the largest, a mean gt of 4-5 days in ontario. 35 the two other estimates used epidemic curves in the united states and mexico, with results in the range of 2ae6-3ae2 days for the mean si or gt. white estimated the mean si at 2ae6 days (ci 95% [1ae9, 3ae3]), but accounting for increased case ascertainment in time reduced this estimate to 2ae2 days. 36 in yang, the mean gt was 2ae7 or 3ae2 days depending on the assumed parametric form (weibull or gamma). 29 reproduction numbers were reported in 24 studies (see table 2 and figure 2 ) for 20 countries. all studies focused on the first few months of the pandemic, with data obtained between march and october 2009. overall, the estimates at the community level (town, region or country) varied between 1ae1 and 3ae1, with a median value of 1ae6. in the netherlands, as in other european countries (except the uk), the reproduction number was smaller than 1 (r = 0ae5) in the period considered. 32, 37 the largest estimate (r = 3ae3) was obtained from the analysis of a school outbreak. 22 ten papers qualified the reproduction number as 'basic', with a range from 1ae3 to 2ae3, all assuming that the whole population was susceptible at first; this range was not significantly different from that of the otherwise reported r values. in 15 analyses, the exponential growth rate estimated from the initial epidemic curve was used to estimate the reproduction number, with a range of values between 0ae5 and 3ae1. the method for estimating the exponential growth rate was variable (for example, poisson regression, 38 birth and death process, 39 least squares, 40 modified logistic growth 41 ), as was the gt distribution (mean gt between 1ae9 and 4ae1) and the formula linking the exponential growth rate to the reproduction number. other methods of estimation included fitting the output of transmission models to the data. 34, 35, 42 in two instances, the reproduction number was estimated using cases seen in tourists returning from mexico, yielding 1ae4 (95% ci [1ae2, 1ae8]) 34 when comparing the number of cases in 9 countries to model predictions and 1ae7 (ci 95% [1ae6, 1ae9]) using only the date of first introduction in 12 countries. 43 no correlation was found in the reported reproduction number and the gt used in its computation (r = )0ae04; p = 0ae83). there was a decreasing trend in the reported values with time (r = )0ae5, p = 0ae004), large estimates being more frequent at first. a first explanation was the inclusion of correction for under-reporting: while no correction was applied for an early estimate in mexico (r = 2ae2 38 ) and another analysis (r = 2ae3 29 ), accounting for an increasing trend in case reporting led to large differences, from 2ae4 to 1ae6 after such correction in australia, 44 from 2ae2 to 1ae7 in the united states, 36 and from 2 to 1ae4 in mexico. 42 another issue was the importance of school outbreaks in the early epidemic curve, so that the estimated reproduction number was not representative of transmission in the community. for example, the r estimate was 2ae3 in japan with a gt of 1ae9 days, 39 but the reproduction number was approximately 1ae3 when transmission was later established in the community. 45 considering only estimates for which underdeclaration was taken into account and the generation time was close to 3 days, the reproduction number was between 1ae2 and 2ae3, with median value 1ae5. using all published information as of july 2010 regarding the a ⁄ h1n1 (2009) pandemic shows that the mean si was <3 days and the reproduction number typically close to 1ae5. a striking feature of the household ⁄ close-contact studies for the mean si was that most estimates were in the range of 2ae5-3ae5 days, although the sampling as well as the methfraser (34) white (36) yang (29) yang (29) tuite (35) yang (29) suess (28) cowling (21) leung (25) lessler* (22) hahné (32) sg-spain* (30) mcbryde (33) ghani (31) cauchemez (24) france (23) pedroni* (27) morgan (26) epidemic curve modelling ods of analysis was different. overall, the weighted mean si of all estimates was 3ae0 days (ci 95% [2ae7, 3ae3]), but this reduced to 2ae6 days in the studies where tertiary transmission was accounted for. studies in households may have provided the best framework to estimate the si, as potential contacts could be more easily identified. the only truly prospective study yielded little information (8 events), 21 so that the best evidence remains that from american households. 24 the number of studies documenting the serial interval in the 2009 influenza pandemic contrasts with the relative absence of information for past pandemics or seasonal influenza. indeed, before 2009, the two best documented values for the mean si concerned seasonal influenza, with two estimates obtained in household-based studies: 2ae6 days (ci 95% 2ae1, 3ae0) 6 and 3ae6 days (ci 95% [2ae9, 4ae3]) 5 ; no information was available for past pandemics. the current estimate for a ⁄ h1n1 (2009) was somewhat closer to the first estimate; it was also in good agreement with the 2ae8 days obtained by using the profile of viral excretion as a indicative of the gt distribution. 46 as reported mean sis are rather short, it is worth examining whether the mean si could have been biased downwards. changes in behavior and interventions may make long serial intervals unlikely. there was little information regarding behavior change and interventions in the reported studies: household members were, for example, instructed in simple hand-hygiene, 21 some index cases received antiviral treatment, 26 but neither isolation nor quarantine was reported. as all studies concerned the early phase of the pandemic, differences in attitude toward the a ⁄ h1n1 flu may have been limited. a second issue is that the combination of rapid transmission and limited number of contacts in households may lead to small intervals. 16 the secondary attack rates were modest (13%, 24 11ae2%, 31 11ae3%, 23 8% 21 ), arguing against a large effect in this respect because of susceptible depletion. a short follow-up could also limit the possibility of observing long serial intervals. in most studies when this was reported, the duration of follow-up in households was approximately 1 week so that few secondary cases should have been missed. finally, some cases counted as secondary may have been attributed to common exposure (coprimary cases), leading to a downward bias. however, in most cases, cases occurring on the same date as the index case were excluded from the calculations, therefore limiting this bias. upward bias could occur because of successive generations of influenza overlapping in small time periods. 15 indeed, some observed serial intervals in close-contact studies may be between primary and tertiary cases rather than secondary cases. two studies used modeling to explicitly account for such phenomenon: in both approaches, the modeled mean si was shorter than the empirical mean of observed values (2ae6 vs. 2ae9 24 ; 2ae5 vs. 3ae4 31 ). this suggests that tertiary transmission is always an issue for estimating the serial interval of influenza, and further implies that estimates reported in the other 11 studies could have been biased upwards. the gt or si estimates obtained by modeling epidemic curves were more variable. in such approaches, the mean gt depends on the structure of the model 14 : in the classical seir model, it is l + i, where l and i are the average durations of the latent and infectious period, so that the mean gt should have been 6 days instead of 4-5 days in canada 35 ; when the e and i stages are split into two (as in 34, 46 ) , the mean gt is l + 3 ⁄ 4 · i, so that it should have been 1ae6 days rather than 1ae9 days in the la gloria epidemic in mexico. 34 the two other modeling approaches yielded estimates similar to those in households ⁄ closecontact studies. reproduction numbers for the a ⁄ h1n1 2009 pandemic varied according to place, methods, and hypotheses, with a reported range from 1ae1 to 3ae3. while the most used approach relied on determining the initial exponential growth, the formulas and fitting methods changed with authors and how the initial exponential growth period was chosen was little documented. when provided, the sensitivity analyses illustrated that somewhat arbitrary hypotheses (choice of the gt, correction for under-reporting, exponential growth period,…) had a large effect on the reported value. in mexico alone, estimates ranged between 1ae2 and 3ae1 during the same period. 29, 34, 38, 40, 42 factors explaining these differences were numerous. the first is differences in data, either by nature (travelers back from mexico or suspected ⁄ confirmed cases in mexico or local epidemics or genetic sequence) or by collection time. for example, cases were added to the epidemic curve in retrospect, so that early estimates were biased upwards. 38 a second factor was the choice of the gt distribution, shorter mean gts leading to smaller reproduction number estimates: from 3ae1 (mean gt = 4 days) to 2ae2 (mean gt = 3 days), 38 from 3ae4 (mean gt = 5ae5 days) to 1ae9 (mean gt = 2ae3 days; supplementary material 29 ), and from 2ae0 (mean gt = 2ae6days) to 1ae4 (mean gt = 1ae9 days 34 ). when similar mean gts were allowed (approximately 3 days), less variability was present (2ae2, 38 2ae0, 34 2ae3 29 ). a third factor was underdeclaration in the initial period of the pandemic, leading to smaller estimates: from 2 to 1ae4 42 and from 2ae6 to 2ae4. 29 methods less dependent on the completeness of the data (genetic sequences, travelers out of mexico) consistently led to lower estimates, from 1ae2 to 1ae7. the short mean gt (1ae9 days 34 ) estimated early in mexico may have led to underestimation when it was used to estimate the reproduction number in later studies. the impact was moderate: for example, the reproduction number in several countries from the southern hemisphere ranged between 1ae2 to 1ae6 using a gt of 1ae9 days 41 and increased by approximately 10% when a mean gt of 2ae8 days was used. in practice, collecting data that allow the joint estimation of the serial interval and reproduction ratio should be encouraged to limit these uncertainties. 48 in approximately one report of two, the authors described the estimated reproduction ratio as 'basic' (i.e., r 0 ), while others used 'initial', 'effective', several qualifiers or none. estimating r 0 requires an additional assumption on the initial susceptibility of the population, and all authors reporting r 0 assumed, often implicitly, that the whole population was initially susceptible. it is now known that adults over 50 years of age were less susceptible to the disease, 24,49 making this assumption incorrect. practically, this means that it is unlikely that any of the reported estimates were truly 'basic' and that accounting for differential susceptibility will be required to obtain r 0 estimates. for public health purposes, however, it is the initial r which is the most relevant estimates as it informs on the required strength of interventions and is useful to calibrate mathematical models. in this respect, the reproduction number may have been poorly estimated at the start of the pandemic, as a result of poor case ascertainment; how imported cases in the course of the outbreak were accounted for in estimation; and the over-representation of places like schools where transmission was large. 45 several new methods have been proposed to estimate the reproduction number during the pandemic, 34, 36, 42, 50, 51 which should now be compared in terms of data requirements, applicability, and how they deal with the issues listed earlier to help select best practice. overall, the initial reproduction number estimates of a ⁄ h1n1 (2009) pandemic ranged from 1ae2 to 2ae3 with median value 1ae5 when correction for underdeclaration was applied and the mean gt was approximately 3 days. this was lower than the median for 1889, 1918, and 1957, but compared with 1968 (see figure 2 ). for example, the reproduction number (using a mean gt of 2ae8 days) was between 1ae7 and 3ae0 (median 2ae1) in 96 cities in 1889, 7 between 1ae3 and 2ae5 in 1918 (using estimates obtained with gts approximately 3 days), 8, 11, 12, [52] [53] [54] lower than 2 in 1957, 10, [55] [56] [57] and in the range of 1-2 in 1968. 9, 57 a large number of studies have documented transmission parameters for the a ⁄ h1n1 (2009) pandemic almost in real time. short generation times and low reproduction number were characteristic in the first year of introduction of the virus. the a ⁄ h1n1 (2009) pandemic led to less mortality than previous pandemics, compared with past flu pandemics regarding transmission. pandemic (h1n1) 2009 -update 109 spread of a novel influenza a (h1n1) virus via global airline transportation world health organization. global surveillance during an influenza pandemic modeling targeted layered containment of an influenza pandemic in the united states estimation of the serial interval of influenza strategies for containing an emerging influenza pandemic in southeast asia transmissibility and geographic spread of the 1889 influenza pandemic epidemiologic characterization of the 1918 influenza pandemic summer wave in copenhagen: implications for pandemic control strategies estimates of the transmissibility of the 1968 (hong kong) influenza pandemic: evidence of increased transmissibility between successive waves potential impact of antiviral drug use during influenza pandemic transmissibility of 1918 pandemic influenza estimation of the reproductive number of the spanish flu epidemic in world health organization. h1n1 in post-pandemic period a note on generation times in epidemic models some model based considerations on observing generation times for communicable diseases the interval between successive cases of an infectious disease infectious diseases of humans the estimation of the basic reproduction number for infectious diseases how generation intervals shape the relationship between growth rates and reproductive numbers different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures comparative epidemiology of pandemic and seasonal influenza a in households outbreak of 2009 pandemic influenza a (h1n1) at a new york city school household transmission of 2009 influenza a (h1n1) virus after a school-based outbreak household transmission of 2009 pandemic influenza a(h1n1) virus in the united states a school outbreak of pandemic (h1n1) 2009 infection: assessment of secondary household transmission and the protective role of oseltamivir household transmission of pandemic (h1n1) outbreak of 2009 pandemic influenza a(h1n1) shedding and transmission of novel influenza virus a ⁄ h1n1 infection in households -germany the transmissibility and control of pandemic influenza a(h1n1) virus surveillance group for new influenza a(h1n1) virus investigation and control in spain. new influenza a(h1n1) virus infections in spain the early transmission dynamics of h1n1pdm influenza in the united kingdom epidemiology and control of influenza a(h1n1)v in the netherlands: the first 115 cases early transmission characteristics of influenza a(h1n1)v in australia: victorian state pandemic potential of a strain of influenza a(h1n1): early findings estimated epidemiologic parameters and morbidity associated with pandemic h1n1 influenza estimation of the reproductive number and the serial interval in early phase of the 2009 influenza a ⁄ h1n1 pandemic in the usa estimating the effective reproduction number for pandemic influenza from notification data made publicly available in real time: a multi-country analysis for influenza a ⁄ h1n1v a preliminary estimation of the reproduction ratio for new influenza a(h1n1) from the outbreak in mexico transmission potential of the new influenza a(h1n1) virus and its age-specificity in japan modelling of the influenza a(h1n1)v outbreak in mexico city pandemic influenza a(h1n1) during winter influenza season in the southern hemisphere initial human transmission dynamics of the pandemic (h1n1) 2009 virus in north america multiscale mobility networks and the spatial spreading of infectious diseases early transmission characteristics of influenza a(h1n1)v in australia: victorian state pros and cons of estimating the reproduction number from early epidemic growth rate of influenza a(h1n1) time lines of infection and disease in human influenza: a review of volunteer challenge studies vaccination against pandemic influenza a ⁄ h1n1v in england: a realtime economic evaluation real-time estimates in early detection of sars real-time comparative monitoring of the a ⁄ h1n1 pandemic in france seasonal transmission potential and activity peaks of the new influenza a(h1n1): a monte carlo likelihood analysis based on human mobility real time bayesian estimation of the epidemic potential of emerging infectious diseases time variations in the transmissibility of pandemic influenza in prussia quantifying social distancing arising from pandemic influenza estimates of the reproduction numbers of spanish influenza using morbidity data analyses of the 1957 (asian) influenza pandemic in the united kingdom and the impact of school closures real-time epidemic forecasting for pandemic influenza transmissibility and mortality impact of epidemic and pandemic influenza, with emphasis on the unusually deadly 1951 epidemic epidemiological and transmissibility analysis of influenza a(h1n1)v in a southern hemisphere setting: peru a preliminary analysis of the epidemiology of influenza a(h1n1)v virus infection in thailand from early outbreak data school closure and mitigation of pandemic (h1n1) 2009, hong kong a clinical virological and epidemiological analysis community-based measures for mitigating the 2009 h1n1 pandemic in china estimating the reproduction number of the novel influenza a virus (h1n1) in a southern hemisphere setting: preliminary estimate in new zealand pandemic influenza 2009 on reunion island: a mild wave linked to a low reproduction number transmissibility of 2009 pandemic influenza a(h1n1) in new zealand: effective reproduction number and influence of age, ethnicity and importations pandemic (h1n1) 2009 influenza community transmission was established in one australian state when the virus was first identified in north america partial support by fp7 project flumodcont (n°20160). key: cord-267531-tqqj4cy0 authors: he, ying; lin, guang-yu; wang, qiong; cai, xiao-ying; zhang, yin-hui; lin, chuang-xing; lu, chang-dong; lu, xue-dong title: a 3-year prospective study of the epidemiology of acute respiratory viral infections in hospitalized children in shenzhen, china date: 2014-05-14 journal: influenza other respir viruses doi: 10.1111/irv.12257 sha: doc_id: 267531 cord_uid: tqqj4cy0 background: the epidemiology of local viral etiologies is essential for the management of viral respiratory tract infections. limited data are available in china to describe the epidemiology of viral respiratory infections, especially in small–medium cities and rural areas. objectives: to determine the viral etiology and seasonality of acute respiratory infections in hospitalized children, a 3-year study was conducted in shenzhen, china. methods: nasopharyngeal aspirates from eligible children were collected. influenza and other respiratory viruses were tested by molecular assays simultaneously. data were analyzed to describe the frequency and seasonality. results: of the 2025 children enrolled in the study, 971 (48·0%) were positive for at least one viral pathogen, in which 890 (91·7%) were <4 years of age. the three most prevalent viruses were influenza a (iav; 35·8%), respiratory syncytial virus (rsv; 30·5%) and human rhinovirus (hrv; 21·5%). co-infections were found in 302 cases (31·1%), and dual viral infection was dominant. rsv, hrv and iav were the most frequent viral agents involved in co-infection. on the whole, the obvious seasonal peaks mainly from march to may were observed with peak strength varying from 1 year to another. conclusions: this study provides a basic profile of the epidemiology of acute respiratory viral infection in hospitalized children in shenzhen. the spectrum of viruses in the study site is similar to that in other places, but the seasonality is closely related to geographic position, different from that in big cities in northern china and neighboring hong kong. acute respiratory tract infections (artis) are a persistent and pervasive public health problem in both developed and developing countries. they cause a great burden of disease worldwide. especially in developing countries including china, artis, mainly pneumonia, are the leading cause of death among children under the age of 5 years. 1,2 a great variety of pathogens can cause artis, and viruses have been considered as the predominant pathogens in this children population. 3, 4 the most frequently reported viruses include respiratory syncytial virus (rsv), influenza viruses a and b (iav, ibv), parainfluenza viruses (pivs), human rhinovirus (hrv) and adenovirus (adv), which are responsible for most episodes of artis in children. 1 in the past decade, several new viruses associated with artis such as human metapneumovirus (hmpv), novel strains of coronaviruses (sars-cov, hcov-nl63 and hkui), human bocavirus (bov), wu polyomavirus (wupoyv) and ki polyomavirus (kipoyv) have been discovered in human respiratory tract specimens. among them, some have been identified to be causative pathogens of artis. 1, 4, 5 currently, there are no approved vaccines or medications available for most of the respiratory viruses. 1 a better understanding of the epidemiology of viral respiratory tract infections in children plays a key role for the prevention, control and treatment of artis. studies showed that many viral respiratory infections exhibited predictable seasonal variations. however, the epidemiological profiles of viral respiratory infections from different climate zones or different countries in the same climate zone may be varied. [6] [7] [8] [9] [10] [11] [12] china is a large country crossing three climate zones, and great differences in climate are found from region to region. a better understanding of the epidemiology of artis in different regions could be helpful to develop effective surveillance, prevention and treatment strategies. although some studies on the epidemiology of artis have recently been reported in big cities such as beijing, shanghai and hong kong, [13] [14] [15] [16] the epidemic characteristics of viruses in artis are still not well established all around china, especially in other cities and rural areas. shenzhen is the largest migratory city of china with high population density and population mobility. it is located in southern china at 22°27 0 -22°52 0 n and 113°46 0 -114°37 0 e, immediately north of hong kong, with a typical subtropical monsoon climate. the annual average temperature and relative humidity of shenzhen are about 23°c (12-33°c) and 77%, respectively. the purpose of this study is to investigate the prevalence, seasonality and clinical characteristics of acute viral respiratory infections in hospitalized children in shenzhen and to provide insights into etiologies of artis in local infants and children. a consecutive 3-year prospective study from july 2007 to june 2010 was conducted in shenzhen, a coastal city neighboring hong kong. four hospitals including a children's hospital were chosen for the study. selected patients with artis admitted to the pediatric wards were enrolled. the inclusion criteria were as follows: less than 14 years old, acute fever (t ≥ 38°c), with any one of respiratory symptoms (such as sore throat, cough, wheezing and dyspnoea/ tachypnoea), normal or low leukocyte count, the onset of illness within 3 days before hospitalization. the diagnosis of pneumonia was based on the guideline of the management of childhood community acquired pneumonia (cap) issued by the chinese medical association in 2006. 17 in the guideline, the clinical symptoms and signs for the diagnosis of childhood cap include fever, cough, tachypnoea (defined according to different age), difficulty breathing and/or lower chest wall indrawing. x-ray evaluation has been carried out when necessary. the study protocol was approved by the medical ethical committees of the hospitals. written informed consent was obtained from the parents or legal guardians of the children. nasopharyngeal aspirates (npa) were obtained by trained personnel following standard operating procedures within 24 hour after admission. the specimens were transported immediately to the laboratory by sterile viral transport media, then divided into aliquots and immediately frozen at à80°c until further processing. total viral nucleic acids (dna and rna) were extracted from 200 ll of npa specimen using the axyprep body fluid dna/rna miniprep kit (axygen, union city, ca, usa), according to the manufacturer's instructions. purified dna and rna were stored at à80°c in aliquots for further pcr analysis. for each specimen, assays for ten common and newly identified viruses were performed. briefly, wupoyv and bov were tested using monoplex pcrs described previously. 18, 19 other viruses were tested using the luminex platform and multiplex xtag tm respiratory viral panel assay (rvp assay) according to the manufacturer's instructions. 20 all multiple infection samples were retested. if there was discordance between two tests, the sample was confirmed by monoplex pcr. statistical package for the social sciences (spss) for windows version 11.0 (spss inc. chicago, il, usa) was used. for comparison of categorical data, chi-square or fisher's exact test was used. all tests were two-tailed, and a p value below 0á05 was considered statistically significant. a total of 2025 specimens were obtained from 2025 eligible patients ranging from 15 days to 14 years old with a median age of 12 months, in which 89á6% of patients were < 4 years old. there were 964 (47á6%) females and 1061 (52á4%) males included. of all hospitalized children enrolled in this study, 84á0% involved lower respiratory infection and 16á0% had upper respiratory tract infection (table 1) . among 971 positive cases, 572 (58á9%) were diagnosed as pneumonia. about 971 of the 2025 cases (48á0%) were positive for at least one viral pathogen. among them, iav, rsv, hrv and pivs were detected in 348 (35á8%), 296 (30á5%), 209 (21á5%) and 169 (17á4%) cases, respectively. single infection was observed in 669 (68á9%) cases, and multiple infection was found in 302 (31á1%). our results also showed that rsv, iav and hrv were the main pathogens in single viral infection cases ( table 1) . the monthly positive rates varied from 32á5% to 75á0% with a mean of 45á1% ( figure 1 ). in the year 2009, when influenza a (h1n1) was pandemic worldwide, the positive rate started to increase in march and the highest positive rate 75á0% was observed in may. among the 971 positive cases, a total of 1335 viral pathogens were detected. the most frequently detected pathogen was iav (26á1%, 348/1335), followed by rsv (22á2%, 296/1335), hrv (15á7%, 209/1335), piv1 and piv3 (12á7%, 169/1335) ( about 302 co-infection cases were identified, accounting for 14á9% of all 2025 hospitalized children. during the h1n1 outbreak from march to august 2009, co-infection cases and co-infection rate increased significantly ( figure 2 ). 143 of 302 (47á4%) co-infection cases were detected during that time. among them, 121 cases were involved in iav infection, including 90 dual infection cases. of all co-infection cases, 247 (81á8%), 49 (16á2%) and 5 (1á7%) were infected with two, three and four potential viral pathogens, respectively. one multiple infection with five viruses was detected in a rsv iav hrv bov piv3 adv hmpv wupoyv piv1 ibv total a total cases 163 172 85 55 51 49 37 26 24 7 669 bronchitis 12 19 7 7 5 2 2 3 5 0 62 bronchiolitis 45 19 20 7 13 9 8 2 5 0 128 pneumonia 93 97 55 28 24 28 25 18 14 5 387 urti 13 37 3 13 9 10 2 3 0 2 *case number and percentage in all enrolled children. **incidence rate in this age group significantly higher than the other age groups. ***no significant difference between these three age groups. †no significant differences between these two age groups, but significantly lower than the other age groups. 6-month-old infant. iav, rsv and hrv were the three most frequently found viruses in co-infection and detected in 176, 133 and 124 cases with co-infection rates of 50á6%, 44á9% and 59á3%, respectively ( table 2) . various multiple infection patterns were observed in the study. a total of 152 (50á3%) co-infection cases involved at least two viruses of rsv, hrv and iav. co-infection rate of each individual virus detected varied significantly. the lowest and highest co-infection rates were observed in wupoyv (33á3%) and ibv (66á7%), respectively. 91á4% (276/302) of co-infection cases were tested in the age group of 4 years old or younger ( table 2) , but among all age groups, no statistical difference in co-infection rate was found (v 2 = 1á83, p = 0á8721). gender-specific difference in co-infection rate was not observed (v 2 = 2á17, p = 0á1404). there was no significant difference in co-infection rates between picu and non-picu cases. similarly, no significant difference in clinical symptoms was observed between co-infection and single cases (data not shown). in general, respiratory viruses were detected more often in the period of march to may than in other months (55á4% and 40á6%, respectively, v 2 = 28á06, p = 0á0000), and obvious seasonal peaks were observed during those months with peak strength varying from 1 year to another. a weaker seasonal peak could also be distinguished in some winter months in different years ( figure 1) . the seasonality profile of each individual virus detected was diversified. a seasonal distribution of iav can be observed from late spring to summer (mainly march to may) and sometimes in fall (october, november or december). a wide seasonal peak of iav infection was detected from march to august 2009 ( figure 3a ). although rsv was tested almost a whole year, two yearly peaks were identified. one was found in november and/or december and the other stronger one was found in march to may of the year. the peak duration in 2009 was longer than those in other years. the seasonal trends of hrv and pivs were similar to that of rsv, but the peaks of these three viruses fluctuated and shifted mildly ( figure 3b ). although ibv and adv had a low detection rate in the study, similar seasonality was observed and their infection peaks were mainly in midwinter. peaks in spring and summer were also observed in some years ( figure 3c ). our investigation did not find regular seasonality in bov infections. a sudden increase in bov infection was recorded in april and may 2010. although the positive rate of hmpv infection was only 4á8%, regular seasonality was observed from march to may of each year. of 39 patients with wupoyv infection, 36 were detected after july 2008. our data implied that peak months of wupoyv infection were from march to may ( figure 3d ). the positive rates of viral infections in male and female were 52á5% and 47á5%, respectively. no significant gender difference was revealed (v 2 = 0á012, p = 0á9118). the distribution of viral agents and infection patterns in different age groups are shown in table 2 . of all 971 positive children, 890 (91á7%) were 4 years old or younger. the positive rate in this age group was significantly higher than that in children more than 4 years old (v 2 = 8á26, p = 0á0041). children under 6 months were the most susceptible to respiratory viral pathogens with a positive rate of 14á8% (table 2) . very few long-term prospective studies were performed for viral etiologies of artis among hospitalized children. in this present study, the infection frequency, seasonality, co-infection pattern and clinical features of viral respiratory infections were investigated based on prospective analysis of three consecutive year's data from hospitalized children with artis. our results provided a distinctive epidemiological profile of viral respiratory infections in hospitalized children with artis in the study areas, which was different from those in the big cities in northern china such as beijing and shanghai and also different from that in adjacent hong kong. overall, 48á0% of our cases were positive for respiratory virus infections, which resembled the latest study in the same city. 21 a similar incidence rate has been obtained in neighboring regions 13, 22 and other cities such as rome 23 and milan, 24 but it was different from other studies. [10] [11] [12] in china, the overall positive rate reported varied from 27á3 to 74á8% depending on different areas and detection methods. 15, 16, [25] [26] [27] [28] [29] [30] [31] the rate of respiratory viral infections varied worldwide, and many factors such as geographic distribu-tion, study design and detection protocols could lead to these variations. 1, 7, 8, 32 in our study, leukocyte count was used as an indicator of inclusion criteria and it probably affected the positive rate. viruses not considered in the study, for example coronaviruses, would underestimate the positive rate. most studies showed that rsv or hrv was the most prevalent viruses in children with viral respiratory tract infection. 1 in this study, iav was the most frequently detected respiratory virus, followed by rsv and hrv. iav (h1n1) outbreak in 2009 could explain this shift. data showed that about 60% of iav infections were detected during the outbreak period. studies showed that the h1n1 outbreak could change viral distribution patterns. 24, 29, 33 regardless of the iav (h1n1) outbreak, rsv and hrv were the two most common viral pathogens in artis, which was consistent with most previous studies. 1, 10, 15, 16, 22, [25] [26] [27] [28] [29] our study further confirmed the importance of rsv and hrv in children with artis, especially in children < 4 years of age. 10, 14, 23 our results also showed that 12á7% of viral pathogens detected were piv1 and piv3, which implied that pivs played an important role in children with artis. similar findings were obtained in the studies conducted in shanghai, 14, 34 changsha, 26 harbin, 30 hong kong 13 and rome. 23 the prevalence of piv3 was twofold higher than that of piv1, particularly in infants, which was similar with other reports, 25, 26, 30, 35 implying that infants could be more vulnerable to infection with piv3 than piv1. hmpv has been proven to be one of the main viral pathogens responsible for artis in children. 5 the positive rate found in the study was consistent with previously published results. 10, 36, 37 in china, the infection rate of hmpv varied from 3á2 to 10á6%. 22, 26, 28, 29, 31 the seasonality of hmpv in this study was mainly from march to may, similar to that in hong kong, 36 but different from other places. 5, 37 in our study, 4á9% of cases were positive for bov, which coincided with 5á0% in hong kong 38 and higher than guangzhou 39 and eastern guangdong. 22 our result suggested that bov might be present throughout the year with no seasonal distribution. however, seasonal distribution was noted from september to february in hong kong 38 and may and june in guangzhou. 39 the use of multiple pcr made it possible to simultaneously detect a broad spectrum of viruses with excellent sensitivity, at the same time, with increased viral detection rate and co-infection rate for artis. 12,40 among our positive cases, co-infection rate was 31á1%, which was similar to 27á9% reported by do et al. 10 co-infection rate reported elsewhere varied widely from 25á4 to 47á9%. 40 the relatively lower co-infection rates ranging from 0á24 to 26á9% were reported in the studies conducted in various cities of china. 22, [25] [26] [27] [28] [29] [30] [31] in most of these studies, immunofluorescence kits were used to test a lower number of respiratory viruses. it was worth to note that in the study by peng et al. 32 in wuhan, china, 69á5% of co-infection rate was reported with immunofluorescence kit. these variations might be attributed to geographic differences, diagnostic methods for viral agents and study design. 12, 32, 34, 41, 42 pathogens in those negative patients need to be further investigated as only ten common and newly identified viruses were included in our study, which might underestimate positive rate or coinfection rate. it was notable that the correlation between co-infection rate and positive rate was not observed. of multiple infections, dual infection was predominant in this study whether or not considering the iav (h1n1) outbreak in 2009, which was consistent with previous studies. 28, 32, 42, 43 similar with the studies conducted in the cities of guangzhou and wuhan, china, 28, 29 our study showed that iav, rsv and hrv were the main viruses involved in multiple infections. high co-infection rate between these three viruses could be explained from the overlap of their seasonal distributions. a variety of predominant multiple infection patterns between respiratory viruses were observed in different studies. 12, 32, 42, 43 for example, it was shown in martin et al.'s study 43 that adv and coronaviruses were the most common co-infection pattern. our study showed that rsv and hrv were the two most viruses involved in multiple infection, followed by iav and pivs, regardless of iav infection in the h1n1 outbreak period. it was difficult to explain the variations of coinfection patterns based only on seasonal distribution. a recent study suggested that co-infection patterns were not random and certain pathogens had higher frequency of coinfection. 41 as molecular assays only detect nucleic acid and positive result does not mean the presence of the pathogen, when studying co-infection patterns of respiratory viruses, the ability to differentiate the real causative pathogens needs to be solved first. viral load detection could provide some clues for solving this issue. 43, 44 although high co-infection rates have been reported in various studies, the associations among multiple infections, hospitalization rate and severity of artis were still not clear with inconsistent results in different studies. 42, 43, 45 our data suggested that multiple infection had less association with the severity of disease, consistent with peng et al.'s study. 32 the relationship between co-infection rates and age group was also investigated in our study, and little correlation was observed. several previous studies observed that co-infection rates were more frequent in a certain age group, but results were varied. 32, 43 in contrast to temperate region, where most viruses had winter-spring seasonality, the respiratory viral infections in tropical and subtropical regions appeared mainly to be spring-summer seasonality. 9 in this study, due to the high detection rate and similar seasonality of rsv, hrv, iav, piv and hmpv, an overall spring-summer seasonality of viral respiratory infections in children was concluded. studies conducted in hong kong showed that a clear seasonal peak was from april to september, 36, 46 with a longer duration than our study. the overall seasonality in this study was also different from the studies conducted in northern or central cities of china, in which the seasonality of most viruses presented in autumn-winter and/or winter-spring. 15, [25] [26] [27] 30 the winter-spring seasonality was also observed in guangzhou, a city about 150 kilometers north of shenzhen. 28 different seasonal onset and duration were observed in various studies conducted in (sub-) tropical regions. in these studies, ambient temperature, humidity and rainfall were widely used to explain these differences in seasonality, but inconsistent results were observed. 9, 46, 47 although most studies demonstrated that the seasonality of viral respiratory infections was correlated with increased rainfall, effects of climate factors such as humidity and temperature on the seasonality were complex and interactive. 9, 46, 48 the study areas have four indistinct seasons, and the coldest month usually emerges in january (average 12°c). during the period from march to may, the weather featured warm ambient temperature (average 18-25°c), high relative humidity (average 85%-90%) and increasing rainfall. these meteorological conditions were perhaps conducive to viral survival. 9, 48 in addition, intensive temperature fluctuations during seasonal alternation could increase the susceptibility to infections. 49 as reported in other studies in temperate, tropical and subtropical regions, viral infection rates in children population showed an inverse correlation with age, with younger individuals experiencing higher viral infection rates. 3, 4, 6, 9, 24 our results suggested that children younger than 4 years of age, particularly <6 months, were at higher risk of hospitalization for artis, compared with older children. this was particularly substantiated in rsv infection. our presumption was supported by other studies. 14,25-28 of course, this speculation needed to be validated by the population-based study. the findings reported elsewhere suggested that more males than females were affected by artis, which were not observed in our study. notably, our study occurred over a span of 3 years, which included the iav (h1n1) outbreak in 2009. the impact of the outbreak on the results should be considered. data showed that the detection rate of iav increased significantly and co-infection rate during outbreak months was much higher than average co-infection rate. unfortunately, we did not type these influenza strains based on the original study design. it was most likely that these strains contributed to the relatively high proportion of iav. relatively higher single and multiple infections of rsv, hrv and pivs were also observed during the outbreak of iav. increased susceptible population and awareness, intensive testing and altered patient and physician behavior could lead to these increases. these factors could partly explain the relatively high proportion of pneumonia cases in the study. furthermore, studies showed that the outbreak of iav (h1n1) could increase the risk of other viral infections such as rsv and hrv. 24, 33 other limitations also existed in this study. first, molecular methods allowed the detection of only viral nucleic acid even without virus replication, which complicates the interpretation of positive detection results. second, the subtype identification of some common respiratory viruses such as iav and hrv was not performed in our study, particularly during the iav (h1n1) outbreak in 2009. in summary, despite those aforementioned limitations, this three consecutive years' surveillance would provide a basic profile of the spectrum, seasonality, age and gender distribution, co-infection patterns as well as clinical association of viral respiratory infections in hospitalized children in the study sites. it could help the prediction, prevention and control of artis in children. respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology causes of deaths in children younger than 5 years in china in 2008 respiratory virus infection in infants and children emerging respiratory agents: new viruses for old diseases? ten years of human metapneumovirus research occurrence of respiratory virus: time, place and person epidemiology of respiratory syncytial virus infection in northern taiwan variation in timing of respiratory syncytial virus outbreaks: lessons from national surveillance epidemiology and seasonality of respiratory tract virus infections in the tropics viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city influenza and other respiratory viruses in three central american countries contribution of common and recently described respiratory viruses to annual hospitalizations in children in south africa identification of viral and atypical bacterial pathogens in children hospitalized with acute respiratory infections in hong kong by multiplex pcr assays molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in shanghai three years surveillance of viral etiology of acute lower respiratory tract infection in children from detection for respiratory viruses in shanghai with multiplex pcr from guidelines for management of childhood community acquired pneumonia (for trial implementation) (i) cloning of a human parvovirus by molecular screening of respiratory tract samples wu polyomavirus infection among children in south china principles of the xtag respiratory viral panel assay (rvp assay) respiratory virus multiplex rt-pcr assay sensitivities and influence factors in hospitalized children with lower respiratory tract infections viral pathogens of acute lower respiratory tract infection in hospitalized children from east guangdong of china detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy epidemiological and clinical features of respiratory viral infections in hospitalized children during the circulation of influenza virus a(h1n1) viral pathogens of acute lower respiratory tract infection in china viral etiology of 1165 hospitalized children with acute lower respiratory tract infection comparative analysis of clinical features of common respiratory tract viral infection and its trends in hospitalized children of chongqing during investigation on prevalence and mixed infection of childhood respiratory virus infection in guangzhou the epidemiology and etiology of influenza-like illness in chinese children from 2008 to 2010 respiratory viruses in hospitalized children with acute lower respiratory tract infections in harbin, china viral pathogens of acute respiratory infection in hospitalized children from suzhou multipathogen infections in hospitalized children with acute respiratory infections the impact of pandemic influenza a(h1n1) 2009 on the circulation of respiratory viruses high incidence of multiple viral infections identified in upper respiratory tract infected children under three years of age in human parainfluenza virus type 4 infection in chinese children with lower respiratory tract infections: a comparison study children with respiratory disease associated with metapneumovirus in hong kong seasonal distribution and epidemiological characteristics of human metapneumovirus infections in pediatric inpatients in southeast china pediatric hospitalization of acute respiratory tract infections with human bocavirus in hong kong detection of human bocavirus from children and adults with acute respiratory tract illness in guangzhou, southern china coinfections with influenza and other respiratory viruses evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections mixed respiratory virus infections multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection evaluation of viral co-infections in hospitalized and nonhospitalized children with respiratory infections using microarrays epidemiology of respiratory syncytial virus infection among paediatric patients in hong kong: seasonality and disease impact incidence of common respiratory viral infections related to climate factors in hospitalized children in hong kong the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus exposure to cold and respiratory tract infections we are most grateful to the clinicians and nurses for their assistance in sample collection. we also thank dr. yi-wei tang for reviewing this manuscript and dr. linda chui, dr leo su for their help in improving english in this paper. none of the authors has a conflict of interest. key: cord-278554-rg92gcc6 authors: aoyagi, yumiko; beck, charles r; dingwall, robert; nguyen-van-tam, jonathan s title: healthcare workers' willingness to work during an influenza pandemic: a systematic review and meta-analysis date: 2015-04-23 journal: influenza other respir viruses doi: 10.1111/irv.12310 sha: doc_id: 278554 cord_uid: rg92gcc6 to estimate the proportion of healthcare workers (hcws) willing to work during an influenza pandemic and identify associated risk factors, we undertook a systematic review and meta-analysis compliant with prisma guidance. databases and grey literature were searched to april 2013, and records were screened against protocol eligibility criteria. data extraction and risk of bias assessments were undertaken using a piloted form. random-effects meta-analyses estimated (i) pooled proportion of hcws willing to work and (ii) pooled odds ratios of risk factors associated with willingness to work. heterogeneity was quantified using the i(2) statistic, and publication bias was assessed using funnel plots and egger's test. data were synthesized narratively where meta-analyses were not possible. forty-three studies met our inclusion criteria. meta-analysis of the proportion of hcws willing to work was abandoned due to excessive heterogeneity (i(2) = 99·2%). narrative synthesis showed study estimates ranged from 23·1% to 95·8% willingness to work, depending on context. meta-analyses of specific factors showed that male hcws, physicians and nurses, full-time employment, perceived personal safety, awareness of pandemic risk and clinical knowledge of influenza pandemics, role-specific knowledge, pandemic response training, and confidence in personal skills were statistically significantly associated with increased willingness. childcare obligations were significantly associated with decreased willingness. hcws' willingness to work during an influenza pandemic was moderately high, albeit highly variable. numerous risk factors showed a statistically significant association with willingness to work despite significant heterogeneity between studies. none of the included studies were based on appropriate theoretical constructs of population behaviour. although variable in severity, 1,2 one consistent feature of pandemic influenza is a surge in demand for health care. 3, 4 hospitalization due to influenza a(h1n1)pdm09 in the usa was estimated at approximately 274 000 cases between april 2009 and april 2010 5 contrasting with 95 000 annual influenza-associated primary hospitalizations from 1979 to 2001. 6 in 2009-10, the availability of intensive care unit beds came under pressure in most national health systems. 1, 7 healthcare workers (hcws) play key roles during an influenza pandemic, but a serious shortage of personnel may occur at peak times or in severe pandemics because of absenteeism due to illness, caring for family members who are ill, or refusal to work. 8 effective preparation for the next pandemic requires estimates of hcws' willingness to work and an understanding of influencing factors. the available data are highly variable. one nigerian study found only one quarter of hcws stating they would be willing to work in a unit treating patients with influenza a(h1n1)pdm09, 9 whilst an australian qualitative study of family physicians found 100% of participants willing to work. 10 chaffee 11 first reviewed willingness to work during disasters and reported that the following factors would be influential: type of disaster, concern for close family, friends and pets, responsibility for dependants, the perceived value of one's response, belief in a duty of care, access to personal protective equipment (ppe), provision of basic needs (water, food, rest, shelter and communication tools) and prolonged working hours. three published reviews reported that similar factors would be associated with willingness to work during an influenza pandemic, [12] [13] [14] but the data were not summarized quantitatively. we addressed this evidence gap by conducting a systematic review and meta-analysis in accordance with the preferred reporting items for systematic review and meta-analyses (prisma) statement. the review questions sought to elucidate the proportion of hcws willing to work during an influenza pandemic, and to identify risk factors associated with willingness to work. our findings are interpreted with reference to sociological understandings of population behaviour, which have to date largely been absent from the peer-reviewed literature, but are highly relevant to the development of appropriate interventions to minimize refusal to work. the study protocol was registered with the national institute for health research international prospective register of scientific reviews (prospero; #crd42013004865) prior to executing the literature search strategy. 15 the prisma checklist is available as supporting information. we sought to analyse data collected exclusively from hcws including doctors, nurses, hospital workers, emergency healthcare service workers, public health workers, medical and nursing students, non-clinical support staff and retirees. the outcome measures of interest were the proportion of hcws reporting willingness to work during an influenza pandemic, and odds ratios or case counts allowing the derivation of odds ratios pertaining to factors associated with willingness to work. we included study manuscripts written in english reporting original quantitative research derived from a cross-sectional design, studies pertaining to a prior or hypothetical influenza pandemic, and studies reporting data pertaining to the aforementioned outcome measures, with no limitations on the time and place of publication. the following databases were searched from their inception to april 2013: medline, embase, web of knowledge, scopus, amed, assia, bioethicsweb, cinahl, cochrane library and psycinfo. google scholar and opengrey were also searched. search terms were 'pandemic + influenza + willingness to work/report to work' to avoid including studies on willingness to accept vaccination. these terms were used in both keyword and mesh searches as appropriate for each database as follows: #1. pandemics (mesh); #02. influenza, human (mesh); #03. 'attitude of health personnel' (mesh) or willingness (keyword); #04. hospital administration (mesh) or report to work (keyword); #05. willing* adj5 work (keyword); #06. respon* adj5 work (keyword); #07. would come (keyword); #08. #03 or #04 or #05 or #06 or #07; #09. #01 and #02 and #08 (see also table s1 ). reference lists in eligible articles were also searched. all identified records were imported to endnote software x4 (thomson reuters, toronto, ca, usa) and duplicate entries removed. the remaining records were screened by a single researcher (ya) against the protocol eligibility criteria following a sequential assessment of the study title, abstract and full-text article. where this was unclear, agreement on eligibility of each study was achieved through discussion with a second researcher (rd or jsn-v-t). data extraction was performed by a single researcher (ya) using a piloted form collecting details of study characteristics {title, author, publication year, place, study period, study design, participants, subject [pandemic of avian influenza origin/influenza a(h1n1)pdm09/non-specified, hypothetical influenza pandemic]}; definition of outcome measures; questionnaire type; validation; statistical analysis and any stated limitations; percentage of willingness to work; and risk factors association with willingness. odds ratios (ors) of factors both unadjusted and adjusted were extracted to estimate the association with willingness to work. crude case counts and the percentage of people in each risk factor stratum were extracted where available. risk of bias was assessed for each study using a newcastle-ottawa assessment scale modified for crosssectional studies by herzog et al. 16 descriptive statistics were calculated using microsoft â office excel â 2010 (microsoft corporation, richmond, va, usa). random-effects meta-analysis estimated the proportion of hcws (including 95% confidence intervals [cis]) who reported willingness to work during an influenza pandemic. random-effect meta-analysis of pooled odds ratios (including 95% cis) estimated the association of factors with willingness to work. 17 heterogeneity between studies was assessed using the i 2 statistic. 18 we considered it statistically inappropriate to perform meta-analysis where i 2 exceeded 85%. 19 to explore sources of heterogeneity, we planned to conduct subgroup analyses according to the type of influenza pandemic; geographical region; survey time period; type of questionnaire; type of participants; sex of participants; and newcastle-ottawa assessment scale score. we used galbraith plots to detect those studies that contributed substantial heterogeneity and conducted sensitivity analyses excluding them from our pooled estimates. 20 for each meta-analysis, publication bias was assessed graphically using a funnel plot of effect size versus standard error and statistically using egger's regression test. 21 meta-analysis of pooled proportions was conducted using statsdirect version 2.7.9 (statsdirect ltd., cheshire, uk), and meta-analysis of pooled odds ratios was conducted using we identified a total of 1133 unique records of which 43 studies met protocol eligibility criteria (see figure 1 ). two the included studies comprised entirely of cross-sectional surveys including two pre-/post-intervention studies and are summarized in table 1 . the participant population sizes ranged from 60 to 4306 with a median of 725 (interquartile range [iqr] 308-1711). the earliest publication was in 2006, and the majority of articles were published in 2009 (11; 25á6%) and 2010 (13; 30á2%). 28 of 43 (67á4%) studies used a hypothetical influenza pandemic as the subject, 21 (48á8%) were conducted in the usa, and 21 (48á9%) investigated both clinical and non-clinical staff within hospital settings. assessments using the modified newcastle-ottawa scale showed that 23 of 43 studies were at moderate risk of bias (2-3 of five stars) for the selection domain, whilst 10 studies were at low risk (4-5 stars) and ten studies were at high risk (0-1 stars); many studies used convenience sampling and few justified the study sample size, appropriately considered nonresponders and used a validated measurement tool. for the comparability domain, 24 were at high risk (0 of two stars), eight at moderate risk (one star) and 11 at low risk of bias (two stars). many studies did not clarify how statistical adjustment for confounding variables was carried out, or reported unadjusted estimates only. for the outcome domain, 39 studies were at moderate risk of bias (two of three stars) and four were at high risk (one star). willingness to work was self-reported in all 43 studies although the statistical test used was clearly described in only 39 studies (see figure s1 ). the percentage of participants who expressed a willingness to work ranged from 23á1% (community nurses during the influenza a(h1n1)pdm09 pandemic in hong kong in 2009) 22 to 95á8% (a study of us medical students targeting a hypothetical influenza pandemic). 23 we abandoned metaanalysis to estimate a pooled mean proportion of hcws willing to work due to very high statistical heterogeneity between studies (i 2 = 99á2%). our planned subgroup analyses were unable to adequately explain the sources of heterogeneity between studies as this remained above our threshold of 85% in each analysis. the percentage of willingness to work seemed to depend on the particular context of the study. studies of hypothetical influenza pandemics, which did not include detailed conditions such as virulence of the strain and availability of protective equipment, tended to show a high level of willingness to work. however, studies of precise scenarios or those which investigated willingness during the relatively mild influenza a (h1n1)pdm09 pandemic tended to present relatively low levels of willingness. this finding may correspond with earlier work by syrett et al. 24 which showed that willingness factors associated with willingness to work data were extracted from 33 studies. pooled estimates from meta-analyses of individual factors associated with willingness to work are summarized in table 2 . overall, females were one-third less likely to be willing to work compared with males. by occupational group, physicians were most likely to be willing to work, followed by nurses, then other health workers. urban or metropolitan area workers were less likely to be willing to work than rural area workers. full-time workers were more likely to be willing to work than parttime employees. respondents living with children or having childcare obligations were one-third less likely to be willing to work compared with those without these obligations. one study identified that pregnancy in a family member reduced willingness to work. 25 marital status (not meta-analysed) did not influence willingness to work. perceived personal safety at work and perception of pandemic risk (aware that a pandemic was likely) were both associated with increased willingness to work. likewise, the provision of protective measures (mainly personal protective equipment) increased willingness to work, although metaanalysis was abandoned due to high heterogeneity (i 2 = 97á8%). training in pandemic preparedness, general and specific role knowledge, confidence in personal skills, good communication skills and perception of role importance all had positive effects on willingness to work. confidence in employers as judged by 'belief that the employer can provide timely information' also positively influenced willingness to work, although meta-analysis was abandoned due to high heterogeneity. the funnel plot of the percentage of hcws willing to work did not present a clear funnel shape, appeared to scatter widely without any detectable association with the standard error and overflowed the false 95% ci range. egger's regression test reached statistical significance and showed that studies reporting a lower percentage were more likely to be published (p = 0á004). funnel plots and egger's regressions tests pertaining to meta-analyses of factors associated with willingness to work revealed no evidence of publication bias except for previous training and comparison of physicians and nurses (see table 2 ), which suggested possible underreporting of studies with an adverse result. this study advances knowledge from previous reviews on willingness to work during influenza pandemics by adding further new studies and subjecting the findings to statistical evaluation where possible. the search was conducted comprehensively and yielded 43 studies from 11 countries. however, quality of the included studies was not uniformly high and excessive statistical heterogeneity prevented metaanalysis of the primary outcome measure. whilst it was not possible to identify a single clear source of the heterogeneity encountered, almost certainly the wide variation in settings, scenarios and respondents contributed significantly. metaanalysis suggested that sex and job category would affect willingness to work although studies varied greatly in the composition of their samples. hypothetical scenarios varied in virulence, stage and the amount of information provided to respondents. studies of influenza a(h1n1)pdm09 were conducted at different junctures during the evolution of the 2009-10 pandemic. there was no consistency in terms of how respondents were asked about their willingness to work, and the design of questionnaires used to collect outcome data from respondents varied between studies. remarkably, despite such high heterogeneity, some factors emerged showing a consistent association with willingness to work. whilst previous reviews suggested these from a narrative approach, this study has confirmed them statistically. being male, a physician or nurse (especially the former), and a full-time worker were all positively associated with willingness to work. these factors are essentially nonmodifiable; without access to the raw data, we could not disentangle any potential confounding between being male and the likelihood of being a physician or full-time worker in studies providing only unadjusted ors. nevertheless these were consistent findings across most studies and firm knowledge that these are reliable and statistically proven influencers of willingness to work is important information for both policy makers and healthcare service managers, even though they are difficult factors to influence. childcare obligation was a consistent barrier to hcws' willingness to work. the importance of this factor may be an artefact of the high participation of women in the hcw workforce in most countries, combined with traditional cultural expectations that they will take primary responsibility for childcare. it is, nevertheless, an important finding for managers. it is not clear whether this is driven mainly by practicality, that is the need to provide childcare at home, or by concerns about whether the safety of children might be compromised by infection brought in from the parental workplace. paradoxically, the evidence that hcws are at increased risk of influenza infection is rather mixed and somewhat inconsistent, 26 whereas the evidence that children (rather than adults) are usually the introducers of influenza infection into households is firmly established. 27 this question should be further investigated because it has implications for appropriate organizational responses. if it is simply a practical matter, then managers need to consider what help could be given in emergencies through the expansion of onsite or community childcare provision. if it is a concern about cross-infection, then appropriate education and information programmes may resolve the problem. in either case, it is unlikely that simple disciplinary sanctions will be effective, because of the social force of parental obligations. indeed, these may well be counterproductive, if other workers perceive them to have been unreasonably applied by managers unsympathetic to real personal dilemmas. confidence in safety, risk perception, prior training, general and role knowledge and confidence in skills were statistically proven facilitators for willingness to work. these are all addressable by detailed pandemic preparedness educational activities at healthcare unit level. importantly, one message arising from assessments of pandemic planning activities prior to the 2009-10 pandemic was that whilst national level pandemic planning was generally successful, the level of planning at local level was insufficient, including training on pandemic influenza for hcws. 28 a particular feature of pandemics is the level of anxiety provoked by the disruption of 'business as usual' and the destabilization of usually stable organizational environments. 29 whilst it is not necessary to retrain hcws frequently, this is a topic that should be addressed in their basic education and managers should ensure that updating materials are readily available, and regularly revised, so that programmes can rapidly be rolled out when a pandemic is identified. evidence of organizational preparedness will contribute to the confidence of hcws that they will not be placed at undue risk by being asked to work in different ways or in different environments from those that they are accustomed to. a number of limitations with the present study warrant discussion. our literature search was limited to records published in english. therefore, we cannot exclude the possibility of having omitted outcome data published in other languages. many of the included studies were at moderate or high risk of bias. moreover, only a small number were available for analysis in relation to some risk factors; these results should be interpreted cautiously. the possibility of publication bias might also be a limitation. however, considering that the percentage of willingness was relatively high in most studies, this suggests that unpublished data may not have found statistically significantly higher percentages of willingness to work. whilst some studies used questionnaires based on recognized psychological theories, these were commonly 'fear-appeal' theories. unfortunately, this may not be appropriate as the preferable behaviour (working during an influenza pandemic) would not result in release from personal fear. 30 we did not identify any studies that investigated the interaction between individual and organizational responses, which biased the findings towards individual fears rather than the social conditions that might provoke or alleviate these. as important as our specific results themselves, is the fact that we identified a multiplicity of approaches to studying the issue of hcw willingness to work during a pandemic; mainly small, ad hoc enquiries, not based on any consistent scenarios or theoretical approaches. to solve this, a consistent methodological framework is needed before any further studies are undertaken. the outbreaks of ebola virus disease in west africa and mers-cov in the middle east offer two very different settings in which to improve study designs and understanding of hcws' willingness to work where infectious disease creates appreciable personal risk. in the meantime, policy makers should recognize that hcw willingness to work during an influenza pandemic is likely to be improved by practical measures to support childcare responsibilities and by the timely provision of relevant and high-quality training and information as a pandemic develops. whilst the above would hold true for influenza, the actual risks and perceptions are not consistent across all novel respiratory viruses. for example, 5% of nurses in ontario refused to work during the sars crisis when the risk to hcws was almost exclusively nosocomial (compared with pandemic influenza where the risk is community-wide). 31 similarly, in the ongoing mers-cov epidemic, the risk of nosocomial infection is presently greater than in wider community settings. 32, 33 conclusions hcws' willingness to work during an influenza pandemic is moderately high although highly variable, and substantial statistical heterogeneity precluded formal meta-analysis. numerous risk factors are associated with willingness of hcws to work during an influenza pandemic, revealing potential points of intervention to increase willingness to work. we identified a wide variety of approaches to the study of willingness to work. for improved future understanding, we advocate a coordinated global approach with standardized protocols and based on appropriate theoretical constructs; and the evaluation of packages of intervention through controlled studies. additional supporting information may be found in the online version of this article: table s1 . full electronic search strategy (medline). figure s1 . summary of risk of bias of included studies using the modified newcastle-ottawa scale (n = 43). data s1. prisma 2009 checklist. pandemic influenza preliminary estimates of mortality and years of life lost associated with the a/h1n1 pandemic in the us and comparison with past influenza seasons world health organization. pandemic influenza preparedness and response: a who guidance document. geneva: world health organization america's forgotten pandemic estimating the burden of 2009 pandemic influenza a (h1n1) in the united states influenza-associated hospitalizations in the united states complications among adults hospitalized with influenza: a comparison of seasonal influenza and the 2009 h1n1 pandemic pandemic influenza and hospital resources pandemic 2009): how prepared are healthcare providers in calabar, nigeria? the gp's response to pandemic influenza: a qualitative study willingness of health care personnel to work in a disaster: an integrative review of the literature healthcare workers willingness to work during a pandemic factors associated with the willingness of health care personnel to work during an influenza public health emergency: an integrative review healthcare workers' willingness to report to work during an influenza pandemic: a systematic literature review an international registry of systematic-review protocols are healthcare workers' intentions to vaccinate related to their knowledge, beliefs and attitudes? a systematic review meta-analysis in clinical trials measuring inconsistency in meta-analyses influenza vaccination for immunocompromised patients: systematic review and meta-analysis from a public health policy perspective graphical display of estimates having differing standard errors bias in meta-analysis detected by a simple, graphical test will the community nurse continue to function during h1n1 influenza pandemic: a cross-sectional study of hong kong community nurses? perspectives of future physicians on disaster medicine and public health preparedness: challenges of building a capable and sustainable auxiliary medical workforce will emergency health care providers respond to mass casualty incidents? how would australian hospital staff react to an avian influenza admission, or an influenza pandemic incidence of influenza in healthy adults and healthcare workers: a systematic review and metaanalysis estimating household and community transmission parameters for influenza who regional office for europe. recommendations for good practice in pandemic preparedness: identified through evaluation of the response to pandemic (h1n1) 2009. copenhagen: who regional office for europe introduction: why a sociology of pandemics? putting the fear back into fear appeals: the extended parallel process model spring of fear, chapter 6: the nurses' survey hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description hospital outbreak of middle east respiratory syndrome coronavirus gauging us emergency medical services workers' willingness to respond to pandemic influenza using a threat-and efficacy-based assessment framework assessment of local public health workers' willingness to respond to pandemic influenza through application of the extended parallel process model assessment of medical reserve corps volunteers' emergency response willingness using a threat-and efficacy-based model determinants of emergency response willingness in the local public health workforce by jurisdictional and scenario patterns: a cross-sectional survey nurses' ability and willingness to work during pandemic flu health care workers' ability and willingness to report to work during public health emergencies hospital health care workers' understanding of and attitudes toward pandemic influenza in beijing who will show up? estimating ability and willingness of essential hospital personnel to report to work in response to a disaster will they just pack up and leave?"-attitudes and intended behaviour of hospital health care workers during an influenza pandemic characterizing hospital workers' willingness to report to duty in an influenza pandemic through threat-and efficacy-based assessment ethical planning for an influenza pandemic survey study of the knowledge, attitudes, and expected behaviors of critical care clinicians regarding an influenza pandemic hospital disaster staffing: if you call, will they come? survey of alberta family physicians' reactions to the spring and summer phase of ph1n1 (pandemic swine flu) outbreak willingness of university nursing students to volunteer during a pandemic preparing for an influenza pandemic: healthcare workers' opinions on working during a pandemic willingness of frontline health care workers to work during a public health emergency factors associated with the ability and willingness of essential workers to report to duty during a pandemic will the nhs continue to function in an influenza pandemic? a survey of healthcare workers in the west midlands healthcare workers' perceptions of the duty to work during an influenza pandemic evaluation of a pandemic preparedness training intervention of emergency medical services personnel pre-pandemic planning survey of healthcare workers at a tertiary care children's hospital: ethical and workforce issues anticipated behaviors of emergency prehospital medical care providers during an influenza pandemic assessing public health department employees' willingness to report to work during an influenza pandemic local public health workers' perceptions toward responding to an influenza pandemic senior clinical nurses effectively contribute to the pandemic influenza public health response pandemic-related ability and willingness in home healthcare workers mitigating absenteeism in hospital workers during a pandemic knowledge and anticipated behavior of health care workers in response to an outbreak of pandemic influenza in georgia a national survey of emergency nurses and avian influenza threat are belgian senior medical students ready to deliver basic medical care in case of a h5n1 pandemic ensuring adequate human medical resources during an avian influenza a/h5n1 pandemic nurses' fears and professional obligations concerning possible human-to-human avian flu survey of hospital healthcare personnel response during a potential avian influenza pandemic: will they come to work knowledge and attitudes of healthcare workers in chinese intensive care units regarding 2009 h1n1 influenza pandemic perception, attitudes and knowledge regarding the 2009 swine-origin influenza a (h1n1) virus pandemic among health-care workers in australia influenza vaccination and intention to receive the pandemic h1n1 influenza vaccine among healthcare workers of british columbia, canada: a cross-sectional study nurses' perspectives and concerns towards an infectious disease epidemic in egypt influenza a h1ni (pandemic 2009): how prepared are healthcare providers in calabar, nigeria? factors associated with motivation and hesitation to work among health professionals during a public crisis: a cross sectional study of hospital workers in japan during the pandemic (h1n1) we thank the authors of the articles cited in this paper. we also thank nicola darlington (university of nottingham) for assistance with developing the search terms and john mair jenkins (health education east midlands) and roshni joshi (university of nottingham) for help with manuscript preparation. this research was supported by the university of nottingham as a master of public health dissertation project. jsn-v-t and crb are respectively editor-in-chief and associate editor for influenza and other respiratory viruses; however they played no role whatsoever in the editorial process for this paper, including decisions to send the manuscript for independent peer-review or about final acceptance of a revised version. all of the above functions were handled alone by dr john wood, senior editor (reviews). key: cord-002136-mkl89qkt authors: nunes, sandro f.; murcia, pablo r.; tiley, laurence s.; brown, ian h.; tucker, alexander w.; maskell, duncan j.; wood, james lionel n. title: an ex vivo swine tracheal organ culture for the study of influenza infection date: 2009-12-09 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2009.00119.x sha: doc_id: 2136 cord_uid: mkl89qkt background the threat posed by swine influenza viruses with potential to transmit from pig populations to other hosts, including humans, requires the development of new experimental systems to study different aspects of influenza infection. ex vivo organ culture (evoc) systems have been successfully used in the study of both human and animal respiratory pathogens. objectives we aimed to develop an air interface evoc using pig tracheas in the study of influenza infection demonstrating that tracheal explants can be effectively maintained in organ culture and support productive influenza infection. methods tracheal explants were maintained in the air interface evoc system for 7 days. histological characteristics were analysed with different staining protocols and co‐ordinated ciliary movement on the epithelial surface was evaluated through a bead clearance assay. explants were infected with a swine h1n1 influenza virus. influenza infection of epithelial cells was confirmed by immunohistochemistry and viral replication was quantified by plaque assays and real‐time rt‐pcr. results histological analysis and bead clearance assay showed that the tissue architecture of the explants was maintained for up to 7 days, while ciliary movement exhibited a gradual decrease after 4 days. challenge with swine h1n1 influenza virus showed that the evoc tracheal system shows histological changes consistent with in vivo influenza infection and supported productive viral replication over multiple cycles of infection. conclusion the air interface evoc system using pig trachea described here constitutes a useful biological tool with a wide range of applications in the study of influenza infection. influenza a virus is the most significant worldwide respiratory pathogen, infecting a range of species that includes humans, pigs, horses, sea mammals and birds. 1 influenza viruses are divided into different subtypes based on the antigenic reactivity of their two surface glycoproteins, the haemagglutinin and the neuraminidase. so far, 16 different haemagglutinin (h1-h16) and nine neuraminidase (n1-n9) subtypes have been identified among all influenza a viruses. 2 waterfowl, shorebirds and gulls are considered the natural hosts of all influenza subtypes, from which mammalian influenza viruses are directly or indirectly derived. 1, 3 influenza infection in humans and pigs is primarily restricted to the upper and lower respiratory tract with viral replication occurring in the epithelial cells present on the surface of the respiratory mucosa. 4 the acute phase of infection is characterized by a multifocal destruction of the epithelium with cellular desquamation to the luminal space, which may often lead to patches along the mucosa with few or no epithelial cells over the basement membrane. it is also associated with the presence of oedema, vascular congestion, hyperaemia and infiltration of inflammatory cells into the submucosa underlying the epithelial layer. 4, 5 the presence of both n-acetylneuraminic acid-a2,3-galactose (neu-aca2,3gal) and neuaca2,6gal receptors in the pig respiratory tract makes this species susceptible to most human and avian influenza viruses. 6, 7 for this reason, pigs have been proposed as a 'mixing-vessel' for reassortment between human and avian viruses. 3, [6] [7] [8] the significance of cross-species transmission of swine influenza viruses to humans is underscored by the recent human influenza outbreak with an h1n1 of possible swine origin. 9, 10 in addition to being important natural hosts, pigs constitute a valuable animal model for the study of influenza infection, 5, 11 as virus may be transmitted experimentally to in-contact animals and they can exhibit clinical signs, histopathology and cytokine expression profiles comparable to influenza infection in other species. [3] [4] [5] 11, 12 in vivo studies have provided valuable information on clinical course, viral pathogenesis, innate and acquired immune responses, vaccine efficacy and antiviral treatment. 4, 5, [13] [14] [15] however, such studies are greatly affected by animal welfare considerations and logistical constraints. to overcome such constrains, in vitro systems that involve either established or primary cell lines have been widely used, 16, 17 but such systems usually lack the natural physiological context present in the respiratory mucosa, such as cell polarization, coordinated ciliary activity, mucus production and apical contact with air. ex vivo organ cultures (evoc) of tracheal explants with an air interface system have been successfully developed and used in the study of both human and animal respiratory pathogens. [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] such systems reproduce, to a great extent, the physiological conditions and the cellular complexity of the respiratory mucosa of the host in a highly controlled setting. the three-dimensional structure and cell diversity of the respiratory mucosa is preserved together with important physiological features like normal expression levels of cell receptors, effective mucus production and ciliary activity. 18, 23, 27 moreover, evoc systems allow a quantitative analysis of the effects of pharmacological agents (i.e. antiviral drugs) and ⁄ or the host innate response on infection dynamics. 23, 27, 31 in addition, tissue collected from a single animal can be used in numerous replicas, which reduces the experimental variability and the number of animals used, following the principles of (reduction, replacement and refinement (3rs) in animal experimentation. 32 in this study, we developed a swine tracheal evoc system for the study of influenza virus infection. swine tracheal explants infected with a contemporary avian-like swine h1n1 virus supported productive infection and exhibited histopathological changes consistent with in vivo influenza infection. overall, this system constitutes a useful physiologically relevant model for the study of influenza infection in a scalable, well-defined and controlled experimental setting. tissue collection and preparation weaned pigs (6-8 weeks old), sourced from a specific pathogen free (spf) closed herd serologically negative against influenza virus, were killed by intravenous administration of sodium pentobarbitone (0ae8 mg ⁄ kg i.v. to effect). whole tracheas were aseptically collected and transported in pre-warmed culture medium consisting of a 1:1 mixture of dulbecco's modified eagle's medium (dmem) and roswell park memorial institute (rpmi) 1640 medium supplemented with penicillin (100 u ⁄ ml; sigma), streptomycin (50 lg ⁄ ml; sigma, dorset, uk), l-glutamine (10 mm; sigma) and amphotericin (2ae5 lg ⁄ ml; sigma). once in the laboratory, tracheas were kept at 37°c in a 5% co 2 -95% air mixture in a humidified incubator. four to six washes were performed, over a period of 3 hours, by immersing the tissue in fresh, warm dmem ⁄ rpmi medium. the presence of bacterial flora was evaluated by inoculation of the final washing medium in brain heart infusion broth (oxoid, hampshire, uk) with further incubation at 37°c for 72 hours. after the washing steps, the surrounding connective tissue present exterior to the tracheal cartilage was removed and tracheas were opened and cut lengthways into four strips. each segment, incorporating the respiratory mucosa and underlying cartilage, was then cut into approximately 1ae0 · 0ae5 cm explants and placed on a square section of sterile filter paper, which was in turn placed onto agarose plugs in 6-well plates with the medium bathing the filter paper and the basal portion of the explants, as described previously. 29 explants were maintained at 37°c in a 5% co 2 -95% air mixture in a humidified incubator for up to 7 days. tissue samples were fixed in 10% (v ⁄ v) buffered formalin and paraffin-embedded. five micrometre sections from across each explant were subject to haematoxylin-eosin, periodic acid-schiff and van gieson staining. haematoxylin-eosin stained slides were used to measure the thickness of the epithelial layer using imagej software (http://rsbweb.nih.gov/ij/). five randomly selected fields across each section were measured and statistical significance was assessed by analysis of variance using prism 5 (graphpad software, la jolla, ca, usa). five microlitres of an emulsion of 1 lm diameter polystyrene microsphere beads (polybead microspheres, polysciences inc., northampton, uk) were pipetted onto the epithelial surface of the explants, and bead clearance was evaluated at 10, 20 and 30 minutes post-inoculation. the assay was considered positive if the microbeads had been completely cleared to one edge of the tissue. virus a stock of avian-like swine h1n1 influenza virus (a ⁄ swine ⁄ england ⁄ 453 ⁄ 06) was grown in embryonated chicken eggs, aliquoted and stored at )80°c. the virus was isolated at the veterinary laboratories agency (vla) from pigs suffering a respiratory disease outbreak in england in 2006. viral titres were determined by haemagglutination assays using adult chicken erythrocytes and plaque assays on madin-darby canine kidney (mdck) cells. 33 organ culture inoculation viral inoculations were performed in triplicate in three independent experiments, with doses ranging from 2ae5 · 10 1 to 2ae5 · 10 6 plaque forming units (pfu). five microlitres of viral suspension was applied onto the epithelial surface of the explants. culture medium was used for mock-infected controls. inoculated explants were sampled every 24 hours for histology, bead clearance assays and viral quantification. re-hydrated sections were treated with 3% h 2 o 2 in methanol for 15 minutes at 22 ± 3°c for endogenous peroxidase blocking, washed twice in tap water and incubated with pronase xxiv (menapath, wokingham, uk) for 10 minutes at 22 ± 3°c for antigen retrieval. after two washes with distilled water, sections were blocked with 1ae5% donkey serum in tris base saline buffer (tbs) for 20 minutes and incubated overnight at 4°c with a chicken anti-swine influenza polyclonal antibody (1:4000) kindly provided by vla-weybridge. slides were washed twice for 5 minutes with 0ae05% tween 20 in tbs (tbs ⁄ t) and incubated for 1 hour at 22 ± 3°c with a donkey anti-chicken igy biotin-labelled antiserum (1:200 in blocking buffer; jackson immunoresearch europe ltd, newmarket, uk). sections were washed twice in tbs ⁄ t, incubated for 30 minutes with vectastain elite abc reagent (vectastain elite abc kit, vector laboratories peterborough, uk) prepared following the manufacturer's instructions, then washed twice with tbs ⁄ t (5 minutes each). colour development was carried out by incubation with sigma-fastô solution (sigma) for 10 minutes. slides were counterstained with mayer's haematoxylin, dehydrated and mounted with dpx mounting medium. inoculated explants were immersed in 1ae5 ml of sterile phosphate-buffered saline (ph 7ae4) and shaken in a mixer mill mm300 (retsch, leeds, uk) at 20 hz for 10 minutes at 4°c, followed by centrifugation at 16 200 g for 10 minutes. the collected supernatant was used in plaque assays (performed in triplicate) in mdck cells. results were expressed as arithmetic mean pfu ⁄ ml. quantification of viral copy number by real-time rt-pcr rna extraction and reverse transcription. inoculated explants were disrupted in 1 ml of trizol ò reagent (invi-trogen) using a mixer mill ò mm300 (retsch, leeds, uk). rna extractions were performed according to the manufacturer's instructions, with slight modifications: after the phase separation step, the aqueous phase containing the rna fraction was transferred to a new tube and purified with rneasy mini kit (qiagen, crawley, uk). cdna synthesis from 50 ng of total rna was performed with superscriptô iii reverse transcriptase (invitrogen, paisley, uk), according to the manufacturer's instructions, using the specific primer for the influenza matrix segment described in hoffmann et al. 34 real-time rt-pcr. viral copy numbers were estimated using a real-time quantitative rt-pcr (qpcr) assay 35 run in a corbett rotor gene 3000 (corbett research, mortlake, australia). cycle parameters were: 15 minutes at 95°c, followed by 45 cycles of 15 seconds at 94°c and 60 seconds at 60°c, with fluorescence acquisition in the last step. standard curves were generated using 10-fold dilutions of a plasmid containing the target sequence of the influenza matrix segment, ranging from 1 · 10 2 to 1 · 10 8 copies per microlitre. for each run, all samples, as well as the no-template controls, plasmid standards, and positive and negative controls were run in triplicate and expressed as the arithmetic mean number of vrna copies per microlitre of cdna. tracheal explants maintain normal histological architecture for up to 7 days, with progressive loss of ciliary activity uninfected tracheal explants preserved their normal histology without major alterations for up to 7 days after removal from the animal ( figure 1a -e), with signs of sporadic cell degeneration in the transitional layers between the epithelium and the submucosa. the overall structure of both the pseudostratified columnar epithelium, and the underlying lamina propria were maintained. a progressive increase in the amount of mucus on the epithelial apical surface was observed from day 3 onwards (figure 1d,e) . periodic acid-schiff staining showed that explants maintained overall carbohydrate levels (figure 2a-d) . van gieson staining showed that the elastic fibres present in the submucosa had a wave-like form after day 4 indicating a small level of fibre relaxation ( figure 2e-h) . the average thickness of the epithelium did not change significantly (p = 0ae4) for the duration of the organ culture ( figure 3a) . a bead clearance assay was used to evaluate co-ordinated ciliary activity. tracheal explants were tested immediately after collection and every 24 hours thereafter. beads were cleared in less than 30 minutes in all pieces examined. however, a progressive increase in the time for complete tracheal explants were infected with avian-like swine h1n1 influenza virus (a ⁄ swine ⁄ england ⁄ 453 ⁄ 06). a high dose (2ae5 · 10 6 pfu) was initially used to attempt to ensure viral infection of the majority of susceptible cells. mock-infected pieces displayed normal histological appearance as described above ( figure 4a ,c). infected explants ( figure 4b ,d) exhibited histopathological changes compatible with in vivo influenza infection, 4,36 including loss of cilia at the apical surface, shrinking and vacuolization of epithelial cells with signs of destruction and desquamation and the presence of cellular debris in the mucus of the epithelial surface. furthermore, a significant difference in the thickness of the epithelial layer between mock and virus-infected was observed (p < 0ae0001) with a marked reduction at day 1 post-infection (pi) (figure 4e ). ciliary-mediated bead clearance was reduced 24 hours pi and completely absent at day 5 ( figure 4f ). viral antigen was detected in infected explants by immunohistochemistry. the distribution of positive cells was relatively widespread throughout the whole section and was limited to the epithelial layer ( figure 5a ). positive staining was predominantly seen in the nucleus, but with some also in the cytoplasm ( figure 5b ). viral antigen was also detected between the cilia and in aggregates of mucosal material present at the surface of the epithelium. in subsequent infections with a lower dose of virus (2ae5 · 10 2 pfu) the pattern of cells with positive staining remained evenly distributed across the tissue section (figure 5c,d) . to determine if the swine tracheal explants supported productive viral replication, explants were infected with 2ae5 · 10 2 pfu of swine influenza virus and maintained in organ culture for 5 days. infectious extracellular virus extracted from the epithelial surface was quantified in plaque assays in mdck cells. a marked increase in the amount of infectious virus was observed 24 hours pi, when the number of infectious units increased by approximately 10-fold when compared with virus recovered immediately after infection. viral titres consistently increased by 3 log 10 on day 2 and remained stable up to day 4 pi (at c 6ae4 log 10 ) followed by a small decline by day 5 pi (figure 5e) , showing that tracheal explants support productive viral infection. a qpcr assay to measure intracellular and extracellular virus was used as a complementary method to assess infection kinetics. to this end, explants were inoculated with 2ae5 · 10 1 , 2ae5 · 10 2 or 2ae5 · 10 4 pfu, and maintained in organ culture for 5 days. viral quantification showed that infection kinetics was dose dependent (figure 5f-h) . in explants inoculated with 2ae5 · 10 4 pfu, the peak viral load was observed at day 1 pi. this was reduced at day 3 pi but then there followed a resurgence in copy number by day 5 pi ( figure 5f ). inoculation with 2ae5 · 10 2 pfu showed a different pattern of infection in which the peak shifted to day 3 pi, with a higher viral copy number than the one observed when a higher dose was used ( figure 5g ). viral rna was detected at day 2 pi in explants inoculated with the lowest dose (2ae5 · 10 1 pfu) and a marked increase in viral copy number was observed at days 4 and 5 pi (figure 5h) . we have established an ex vivo air interface tracheal organ culture for the study of influenza viruses which could also be used to study other respiratory pathogens. evoc systems have been developed previously for the study of respiratory pathogens using human, swine, bovine, canine and horse respiratory tissue. 18, 20, 23, 25, 27, 29, 30 the experimental value of the tracheal explants described here is illustrated by the preservation of good levels of both histological and functional features for seven consecutive days, and by showing productive infection with an avian-like swine h1n1 virus. a vast body of knowledge in influenza biology has been generated using diverse experimental systems, each possessing advantages and disadvantages. in vivo studies have provided valuable data on influenza pathogenesis, although detailed information about quantitative infection dynamics and the host innate immune response is still scarce. 1, [3] [4] [5] 15, 37, 38 for respiratory pathogens in particular, samples are usually collected by nasal swabs or bronchoalveolar lavage, which may not fully reflect the tissue-specific bacterial and viral infection dynamics occurring throughout the respiratory tract, making it difficult to dissect regional tissue-specific effects from generalized systemic responses. cell culture systems, using epithelial cells from the respiratory mucosa, may lack key physiological features such as cell polarization, reliable expression levels of cellular surface receptors or the intricate relationships between the different cell types present in the respiratory tract. 39 primary cell cultures are difficult to establish, possess an inadequate distribution of the different cell types and become altered after a limited number of passages. established continuous cell lines are by definition aberrant and usually belong to a cell type different to the natural target cells and, in some cases, are derived from a species different from the natural host. ex vivo organ cultures provide an alternative solution. nasal mucosa 27 and tracheal explants 39 immersed in cell culture medium have been used previously for the study of influenza infection. however, using this technique cells are subjected to submersion-related stress and to a continuous source of infecting pathogen as the inoculum remains in the cell culture medium. 22 the tracheal organ culture described here exhibits several advantages compared with submerged organ cultures, cell cultures or in vivo studies. first, in an air interface system the explants are exposed basolaterally to culture medium and the apical cell surface is exposed to the air. the threedimensional structure of the tissue as well as the interactions among cells is preserved. in particular, the cells of the epithelial lining of the trachea maintain the same morphological appearance, differentiation and polarity as described in the living host. in contrast to submerged systems, once explants are inoculated, re-infection from the culture medium or other components of the system is rare. the absence of a blood supply constitutes a weakness of the air interface evoc system as recruitment of cells of the immune system to the infection site is abrogated. however, such a potential pitfall might be turned to advantage and used to dissect the effects of innate and intrinsic immunity. 40 in this study, the overall histology of the explants was preserved for 7 days with no significant differences in the thickness of the uninfected epithelium. this is consistent with previous reports of similar systems in which explants of respiratory tissue from different species have been maintained for variable periods of time, ranging from 60 hours to 5 days. 23, 25, 27 mucus production provides an important nonspecific defence against viral infections. 41, 42 during the initial washing steps in setting up the explants, a majority of the mucus is removed from the epithelial surface. however, mucus is produced de novo by goblet cells throughout the duration of the organ culture, and thus the physiological conditions present in the living host are maintained in this regard. we also evaluated the function of the mucociliary escalator system, and thus also of co-ordinated ciliary function of the tracheal explants, using a simple bead clearance assay. for beads to be cleared from the surface of the organ culture piece, individual cilia must beat in a coordinated manner across the epithelial surface with mucus acting as a vehicle to transport unwanted particles away from where the alveoli would have been in a wave-like motion towards where the larynx would have been. 43 by using the bead clearance assay, we were able to distinguish between small patches of ciliary movement and truly coordinated ciliary activity, an important measure of the robustness of the system. the increase in the clearance time observed at later time points in uninfected explants may be associated with changes in ph, or energy limitations of the tissue. although the supplemented culture medium contains most of the essential components required for survival of the explants, it may not constitute a complete substitute for the nutrients available from the living host. infection of swine tracheal explants with a ⁄ swine ⁄ england ⁄ 435 ⁄ 06 (h1n1) resulted in productive infection with histopathological and immunohistochemical patterns consistent with those observed in influenza infections of both humans and pigs. 3, 4, 13, 36 as expected, infection kinetics was dose dependent: when using a high dose of virus we observed a mild infection that peaked bimodally at day 1 and day 5 pi. with an intermediate dose, a single peak occurred at day 3 pi, whereas the lowest infection dose resulted in detectable virus at day 2 pi, followed by a subsequent rise in copy number at later time points. such influenza infection in a pig tracheal evoc system ª 2009 blackwell publishing ltd, influenza and other respiratory viruses, 4, 7-15 distinct patterns might be related to differential levels of innate immune response, as well as to the initial number of susceptible target cells. a large inoculum would be expected to infect most of the susceptible cells and viral replication may also trigger a massive innate immune response, resulting in a small number of susceptible cells available for a second round of viral replication. once the antiviral signalling molecules are depleted, the remaining infectious particles can infect the susceptible cells, and this would be consistent with the second rise in virus titres observed at day 5 ( figure 5f ). although a second round of infection could not be ruled out, the peaks are too separated in time for this to be likely. a smaller dose of virus might trigger a milder innate immune response, restricted to neighbouring cells, allowing the virus to infect a larger number of cells over multiple rounds of infection. accordingly, the kinetics of infection with the lowest viral dose displayed a marked shift to the right because more replication cycles were required to infect all or most of the susceptible cells. further experiments that incorporate the quantification of cytokine expression will help to test this hypothesis. in our study, an influenza virus of swine origin was used in the validation of the pig trachea evoc system. future studies, including viruses of different subtypes isolated from diverse hosts, should be performed to evaluate the susceptibility of pig trachea to influenza infection and to study the mechanisms underlying any possible host-specific specificity. in summary, the evoc system constitutes a useful biological tool with a wide range of possible applications for the study of influenza infection, including the evaluation of mutants obtained by reverse genetics, the study of natural variability and reassortment events, the effects of antiviral drugs in natural target cells, investigation of host susceptibility and range of influenza viruses and the development of mathematical models of infection dynamics. when possible, evoc systems should be considered as an alternative to in vivo experiments, applying the principles of 3rs in animal experimentation. evolution and ecology of influenza a viruses characterization of a novel influenza a virus hemagglutinin subtype (h16) obtained from black-headed gulls the epidemiology and evolution of influenza viruses in pigs the pathology of influenza virus infections pathogenesis of emerging avian influenza viruses in mammals and the host innate immune response molecular basis for the generation in pigs of influenza a viruses with pandemic potential potential for transmission of avian influenza viruses to pigs the nucleoprotein as a possible major factor in determining host specificity of influenza h3n2 viruses triple-reassortant swine influenza a (h1) in humans in the united states antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans animal models for the study of influenza pathogenesis and therapy correlations between lung proinflammatory cytokine levels, virus replication, and disease after swine influenza virus challenge of vaccination-immune pigs cytokines in the pathogenesis of influenza in vivo studies on cytokine involvement during acute viral respiratory disease of swine: troublesome but rewarding the pathogenesis of influenza in humans infection of human airway epithelium by human and avian strains of influenza a virus h5n1 avian influenza virus induces apoptotic cell death in mammalian airway epithelial cells ciliostasis is a key early event during colonization of canine tracheal tissue by bordetella bronchiseptica adherence of bordetella bronchiseptica 276 to porcine trachea maintained in organ culture a human respiratory-tissue organ culture incorporating an air interface development of rhinovirus study model using organ culture of turbinate mucosa tracheal organ cultures as a useful tool to study felid herpesvirus 1 infection in respiratory epithelium quantification of mrna encoding cytokines and chemokines and assessment of ciliary function in canine tracheal epithelium during infection with canine respiratory coronavirus (crcov) interaction of pneumolysin-sufficient and -deficient isogenic variants of streptococcus pneumoniae with human respiratory mucosa interaction of bordetella periitussis with human respiratory mucosa in vitro interaction of pseudomonas aeruginosa with human respiratory mucosa in vitro in vitro culture of porcine respiratory nasal mucosa explants for studying the interaction of porcine viruses with the respiratory tract in vitro culture of equine respiratory mucosa explants critical assessment of an in vitro bovine respiratory organ culture system: a model of bovine herpesvirus-1 infection mutation of the maturase lipoprotein attenuates the virulence of streptococcus equi to a greater extent than does loss of general lipoprotein lipidation the use of human nasal in vitro cell systems during drug discovery and development the principles of humane experimental technique new low-viscosity overlay medium for viral plaque assays universal primer set for the full-length amplification of all influenza a viruses development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h5 and h7 hemagglutinin subtypes localization of swine influenza virus in naturally infected pigs influenza type a in humans, mammals and birds: determinants of virus virulence, host-range and interspecies transmission realities and enigmas of human viral influenza: pathogenesis, epidemiology and control cultures of equine respiratory epithelial cells and organ explants as tools for the study of equine influenza virus infection intrinsic immunity: a front-line defense against viral attack defense mechanisms against influenza virus infection in the respiratory tract mucosa effective mucus clearance is essential for respiratory health efficient mucociliary transport relies on efficient regulation of ciliary beating we thank yvonne spencer, sara marsh, alejandro nunez (vla-weybridge) and carina ferrari for their assistance with the immunohistochemistry protocol and also t. j.mckinley for his assistance in the statistical analysis of data. this work was supported by a grant from defra and hefce under the veterinary training and research initiative to the cambridge infectious diseases consortium (cidc) through a fellowship awarded to s. f. nunes. j.l.n.w. is funded by the alborada trust and p.r.m. is funded by the wellcome trust. the swine influenza virus was obtained through a programme of influenza surveillance in gb pigs funded by defra (project ed1204). key: cord-000114-f0vud3gu authors: kim, jeong‐ki; negovetich, nicholas j.; forrest, heather l.; webster, robert g. title: ducks: the “trojan horses” of h5n1 influenza date: 2009-05-31 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2009.00084.x sha: doc_id: 114 cord_uid: f0vud3gu abstract wild ducks are the main reservoir of influenza a viruses that can be transmitted to domestic poultry and mammals, including humans. of the 16 hemagglutinin (ha) subtypes of influenza a viruses, only the h5 and h7 subtypes cause highly pathogenic (hp) influenza in the natural hosts. several duck species are naturally resistant to hp asian h5n1 influenza viruses. these duck species can shed and spread virus from both the respiratory and intestinal tracts while showing few or no disease signs. while the hp asian h5n1 viruses are 100% lethal for chickens and other gallinaceous poultry, the absence of disease signs in some duck species has led to the concept that ducks are the “trojan horses” of h5n1 in their surreptitious spread of virus. an important unresolved issue is whether the hp h5n1 viruses are maintained in the wild duck population of the world. here, we review the ecology and pathobiology of ducks infected with influenza a viruses and ducks’ role in the maintenance and spread of hp h5n1 viruses. we also identify the key questions about the role of ducks that must be resolved in order to understand the emergence and control of pandemic influenza. it is generally accepted that wild duck species can spread hp h5n1 viruses, but there is insufficient evidence to show that ducks maintain these viruses and transfer them from one generation to the next. avian influenza is caused by type a viruses of the family orthomyxoviridae. the influenza a viruses infect primarily free-living aquatic birds, and they are classified by their hemagglutinin (ha) and neuraminidase (na) surface glycoproteins. all 16 ha and 9 na subtypes have been isolated from aquatic birds; wild ducks are the main reservoir. the viruses cause asymptomatic or low pathogenic infection in these natural hosts. 1 however, certain strains of influenza a virus have crossed the host range barrier and infected other species, including humans. these viruses are the source of the influenza pandemics that emerge at irregular intervals. 1, 2 the h5 and h7 subtypes are of particular concern because they can become highly pathogenic (hp), causing systemic illness and death in both avian and mammalian species, including humans. 2 the h5n1 virus that emerged in asia in 1996 is unique among the hp avian influenza (hpai) viruses in that it has continued to circulate in avian species for more than a decade and has spread to more than 60 countries in eurasia (http://www.who.int/csr/ disease/avian_influenza/en/). while the h5n1 hpai viruses are 100% lethal to chickens and gallinaceous poultry, they often cause asymptomatic infection in some species of domestic and wild ducks. these ''silent spreaders'' of h5n1 hpai viruses are therefore referred to as ''trojan horses''. [3] [4] [5] clearly, ducks play a complex and vital role in the biology and the overall natural history of influenza, including h5n1 hpai viruses. ducks are members of the subfamily anatinae, which contains most species of anserine birds. this subfamily is nearly cosmopolitan in distribution, and its members occupy almost all aquatic habitats. the ecology of these birds, summarized in figure 1 , facilitates the maintenance and spread of avian influenza viruses. although human influenza a isolates and the currently circulating h5n1 hpai viruses typically infect the upper respiratory tract, the primary site of infection in ducks is the intestine. 6 avian influenza viruses enter the environment when the host defecates or drools, and they then infect susceptible hosts as they feed and drink. avian influenza virus replication has been observed in the respiratory tract, 6 but the contribution of this site to maintenance of infection in the population is unresolved. specifically, fecal shedding of h4n7, h7n3, and h11n9 virions from experimentally infected mallard ducks persists longer and at higher titers than tracheal shedding. 6 when a large number of birds roost on a small pond (for example, in the staging ⁄ marshalling areas), as many as 10 10 eid 50 •g )1 •d )1 infectious virions are estimated to enter the environment in the fecal matter of each infected duck. 6 further, avian influenza viruses are stable in water 1,7 and have been isolated from the surface of ponds containing a large number of waterfowl. 8, 9 although aerosol transmission cannot be dismissed, the larger number of positive cloacal than tracheal swabs, the high fecal virus titer, and the stability of the virions in water suggest that lowpathogenic avian influenza (lpai) viruses persist in duck populations through fecal-oral transmission. 1 this mechanism could partially explain the higher prevalence of infection in surface-feeding (dabbling) ducks than in diving ducks that typically feed in deeper water. 10 surveillance data suggest year-round transmission of avian influenza viruses within duck populations. the prevalence of infection exhibits an annual cyclical pattern in both north american 1,11 and eurasian 12 duck populations (figure 1 ), peaking before and during the fall migration as a result of the influx of immunologically naïve juveniles. 1, 9, 10, 13 experi-mentally infected white pekin ducks have shed virus for more than 3 weeks after inoculation. 3, 14 coupled with limited morbidity and serum antibody response, 3 infected birds are likely to shed virus during the first few weeks of the fall migration, dispersing it along their numerous migration corridors. however, the prevalence of infection is much lower along the migration routes and at the wintering grounds than at the marshalling areas. 9, 12, 15, 16 this disparity may reflect the development of immunity to circulating virus subtypes within the duck population or a decline in transmission because of population dispersal. 13 in general, prevalence of infection is higher at the wintering grounds and spring nesting sites in duck populations from europe than in north american populations (figure 1 ). the most likely explanation for this difference is random variation, since surveillance studies from multiple areas in north america and in europe often obtain slightly different prevalence values in the duck populations. many factors can affect prevalence including, but not limited to, the size of the duck population, sampling location, and time of collection. thus, the few multi-year studies that exist likely exemplify the variation that one would observe if additional sampling sites were included in the studies, and not the differences in geography between europe and north america. 16 prevalence is at its lowest figure 1 . overview of the annual movement and behavior of migratory ducks and their role in interspecies transmission. during spring and fall migration, the ducks rest and feed for a few days to weeks at numerous stopover sites (wetlands, lakes, or ponds) along the migration route. the length of stay and the aquatic habitat allows the transmission of influenza viruses to and from the domestic duck populations. domestic ducks that become infected are likely to maintain the virus locally and increase the probability of its spread to other species. in the diagram, solid arrows indicate confirmed routes of transmission of lpai and ⁄ or hpai viruses between species. the dashed line represents a probable but unconfirmed route of transmission. the graphs indicate the average prevalence of low-pathogenic avian influenza in north american and european duck populations during 3 stages of the annual migration. 10, 16 during the spring migration but increases again after the breeding season, when the ducks have moved to the molting and staging areas. 11, 13, 16 it is not clear how the duck population acquires avian influenza viruses during the spring of every year. infectious virions may persist through the winter in the frozen waters of the breeding areas and reinfect the ducks when they return in the spring. 1, 6 alternatively, the duck populations may carry the viruses throughout the entire migration. year-round prevalence in the ducks supports the latter, although persistence in the frozen habitats could play a role in the perpetuation of the viruses. 10 some virus subtypes are isolated more frequently than others. 10, 11 three ha subtypes, h3, h4, and h6, are common in both north american and european ducks, 11, 12 and the most prevalent subtype combinations in both areas are h4n6 and h6n2. 16 explanations vary for why certain ha and na subtypes (and combinations) are common or rare in wild birds. the general hypothesis is that these subtypes likely have the highest fitness, with replication rates balanced by a level of virulence that sufficiently increases transmission probability to the level where infection in the next cohort of birds is almost guaranteed. it is speculated that this could be largely influenced by the functional balance between ha binding affinity and na enzymatic activity. 17 although the h6 gene is of eurasian origin and is widely distributed in north american ducks, genomic analysis of viruses suggests limited intercontinental exchange between eurasia and the americas. 18 therefore, novel viral genotypes must arise via mutation and reassortment of the genomes in circulation within a specific geographic area. the marshalling areas provide such an opportunity by attracting populations of ducks from various breeding areas and migration corridors, with each population harboring a potentially different combination of subtypes. 16 coinfection of ducks with two or more virus subtypes is common, 19 as is reassortment, 14 and emergent strains that are most virulent to gallinaceous poultry can have low pathogenicity in the duck hosts. 20 however, the role of ducks in the maintenance and spread of influenza viruses, and especially in the emergence of novel genotypes, appears to depend on their migratory behavior. specifically, ducks that migrate annually are likely to spread influenza viruses along the migration routes, primarily by exposing the resident and domestic duck populations at the numerous stopover sites. 10, 16, 21 in contrast, domestic and resident ducks maintain the viruses in close proximity to other species and have been implicated in the spread of both lpai and hpai viruses to domestic poultry and terrestrial birds. 20, 22, 23 low-pathogenic avian influenza lpai viruses can pass through the upper digestive tract of ducks and replicate in the lower intestinal tract without causing clinical manifestations of disease. 3, 6 further evidence that the intestinal tract is the target organ of lpai viruses in ducks includes the replication of virus in the lower intestinal tract, but not in the lungs, after direct inoculation into the crop and rectum 6 and high fecal virus titers after intravenous inoculation. 3 the specific site of lpai virus replication is believed to be the crypts of lieberkühn in the large intestine. 3 the other potential target organ for lpai viruses in ducks is the respiratory tract. lungs of mallard ducks intranasally inoculated with lpai viruses showed mild pneumonia, and lymphocyte and macrophage infiltration within 2 days. immunostaining for viral nucleoprotein revealed intermittent staining of respiratory epithelial cells but no viral replication in the lung tissue. 24 this evidence indicates that the respiratory tract and not the lung tissue itself is the primary target of infection. the species diversity of ducks may also play an important role in the pathogenicity of influenza viruses. mallard duck embryos inoculated with lpai virus have significantly lower mortality rates than inoculated muscovy duck embryos; however, in regards to replication, the lpai viruses behave in a different way. viral antigens were found in the internal organs (nasal sinuses, pharynx, trachea, bronchi, lung, and air sacs) of the mallard duck embryos, but not in those of the muscovy duck embryos. the reason for this mortality ⁄ virus replication paradox in mallard ducks is unclear but is in keeping with the evidence that mallard ducks are considered to be the main reservoirs of lpai viruses in nature. 25 although several studies have examined the serum antibody response in both naturally and experimentally infected ducks, knowledge of the avian immune response to influenza viruses is very limited. 26 white pekin ducks inoculated with an h7n2 lpai virus developed negligible serum hemagglutination inhibition (hi) antibody titers despite fecal shedding of virus until day 7 post-inoculation. animals reinoculated 46 days later with the same virus strain had a marked antibody response, but virus could not be isolated from any of the organs. these results and the lack of a secondary immune response after inoculation with formalin-inactivated virus suggested that the rapid immune response in re-infected birds may restrict influenza infection to short time scales. 3 it is noteworthy that prior infection does not protect ducks against subsequent infection with other virus subtypes. for example, ducks infected with an h4n6 subtype are protected from reinfection with the same virus, but they shed virions for 8 days after challenge with an h11n3 isolate. 27 these data have applications in the field, where isolation of influenza virus from migratory waterfowl is infrequent during the winter, potentially indicating the existence of a significant level of immunity in wintering ducks acquired from previous influenza infections. further illustration of this is seen from a study of wild waterfowl in italy over six winter seasons, in which 17 of the 20 viruses isolated were of the h1n1 subtype, suggesting that the wintering ducks had some degree of immunity to the other subtypes of circulating influenza strains. 28 highly pathogenic avian influenza several experimental studies have investigated the pathogenicity of h5n1 hpai viruses (isolated since 2002) in ducks. cherry valley pekin ducks inoculated with a 2003 hpai h5n1 strain isolated from duck meat at a quarantine inspection station in china developed neurologic signs, including blindness and head shaking, although none died. high virus titers were detected in the respiratory organs (lung and trachea), brain, liver, kidneys, and colon, and microscopic lesions were observed in the brain (viral encephalitis), heart (myocarditis with degeneration and necrosis of myocytes), and bursa (mild lymphoid follicular hyperplasia). 29 viral neurotropism and pancreatotropism have been observed in multiple other studies of recent hpai virus isolates. ducks lethally challenged with these h5n1 hpai viruses showed severe neurologic signs, including torticollis, incoordination, tremors, and seizures. 30, 31 immunohistochemistry positivity was recorded in the cerebrum and brain stem, and in situ hybridization detected virus in the neurons and glial cells of the cerebral gray matter, further confirming the strong neurotropism of post-2002 isolates. 30, 31 although the route of entry of virus into the central nervous system has not been determined, at least two different hypotheses have been proposed, including ascending transmission of virus via vagal, olfactory, and trigeminal nerve fibers, and penetration of the blood-brain barrier. 32, 33 another recurring characteristic of recent h5n1 hpai viruses in ducks is that virus titers are frequently higher in oropharyngeal swabs than in cloacal swabs. 30, 34 pharyngeal excretion of h5n1 hpai viruses has been suggested to originate from the lung and ⁄ or air sac, as only these tissues have shown immunohistochemical evidence of virus replication. preferential pharyngeal excretion suggests that pharyngeal swabs, as well as the customary cloacal swabs, should be taken when conducting surveillance of avian influenza viruses in wild ducks. 34 otherwise, the prevalence of h5n1 hpai may be underestimated. additional studies of the role and rates of respiratory transmission of h5n1 hpai viruses in ducks are needed, especially as they relate to cloacal excretion. in 1996, the parental virus (a ⁄ goose ⁄ guangdong ⁄ 1 ⁄ 96; a ⁄ gs ⁄ gd ⁄ 1 ⁄ 96) of currently circulating h5n1 hpai viruses emerged in southern china. genetic evidence revealed that this virus originated from h5 lpai viruses carried from northern japan by wild ducks or other migratory birds. 15, 35 a reassortant h5n1 hpai virus subsequently emerged in poultry at farms and live animal markets in hong kong in 1997. genetic analyses showed that the h5 ha gene of the reassortant virus was derived from an a ⁄ gs ⁄ gd ⁄ 1 ⁄ 96-like virus, while the remaining gene segments were derived from low-pathogenic viruses: the n1 na gene came from a ⁄ teal ⁄ hong kong ⁄ w312 ⁄ 97 (h6n1) virus, and the internal genes from a ⁄ quail ⁄ hong kong ⁄ g1 ⁄ 97 (h9n2) virus. 36 the reassortant virus caused the first lethal infection in humans (6 deaths among 18 known cases) by direct bird-to-human transmission. 37 between 1999 and 2002, h5n1 hpai viruses with the h5 ha gene of a ⁄ gs ⁄ gd ⁄ 1 ⁄ 96-like viruses but with a diversity of genotypes in the other genes, re-emerged multiple times in hong kong. 37 the first indication of the spread of h5n1 hpai viruses from domestic to wild species of aquatic birds occurred in kowloon and penfold park in hong kong, 38 where 19 different duck species were infected. some species, including the red-crested pochard (netta rufina), were highly susceptible (19 ⁄ 20 died), whereas others, including the bahama pintail (anas bahamensis), were less susceptible (4 ⁄ 21 died). the next major event in nature was the massive die-off of waterfowl at qinghai lake in china. [39] [40] [41] four different genotypes of h5n1 hpai viruses co-circulated in the waterfowl there; one of these became dominant and spread westward to india, europe, and africa. notable features of the dominant qinghai lake h5n1 hpai isolates were the acquisition of a lys627 mutation in the pb2 gene and the absence of pathogenicity in mallard ducks. 42 the lys627 mutation has been found to be associated with pathogenicity in mammalian species, 43, 44 suggesting that it may have been generated while the virus was replicating in a mammal. the virus was likely transmitted from domestic ducks to wild ducks en route to qinghai lake. the role of duck species in the westward spread of the qinghai-h5n1 virus remains controversial. circumstantial evidence from global wildlife surveillance supports the hypothesis that migratory birds, including wild ducks, have contributed to the current eurasian endemic of h5n1 hpai viruses. 45 surveillance studies in thailand in 2004 showed that most domestic grazing ducks infected with h5n1 hpai viruses were asymptomatic 4 and that the initial spread of h5n1 hpai viruses to chickens and humans corresponded to the movement of grazing ducks. 4, 46 in fact, infected domestic ducks grazing on man-made wetlands (e.g., harvested rice fields and irrigation canals) may maintain the infection and spread it to wild birds that feed at the same sites. if these wild birds are migratory and experience limited morbidity, they in turn can disperse hpai viruses widely (figure 2) , as suggested by the high genetic similarity of hpai isolates from africa, europe, and the middle east to the qinghai-h5n1 virus. 37 the evolution of h5n1 hpai viruses by reassortment with lpai viruses in the aquatic bird reservoir played an important part in the genesis of the multiple genotypes, clades, and subclades of asian h5n1 hpai viruses and is ongoing. 37 however, it is the ever-increasing poultry industry that provides the reassortment interface between wild and domestic avian species. the number of domestic ducks, chickens, and other poultry continues to increase, but biosecurity and separation of species is not always taken into account. ducks raised in a closed high-biosecurity system in thailand were protected from infection while h5n1 hpai viruses were circulating among backyard ducks, open house ducks, and grazing ducks. 4 therefore, biosecurity can prevent the spread of influenza viruses from wild to domestic ducks. live poultry markets (wet markets) have been identified as a risk factor in the genesis of novel influenza viruses 49 and were identified as the source of the human outbreak of hp h5n1 viruses in hong kong. the ban on ducks, geese, and later, quail, together with improved biosecurity (clean days), have markedly reduced the influenza virus diversity in the hong kong wet markets. 37 live poultry markets are being phased out in hong kong, and in the interim no live poultry can be carried over from 1 day to the next. taiwan plans to close all live poultry markets by 2009, and shanghai authorities are reducing the number of wet markets. overall, however, the role of live poultry markets in the emergence and control of pandemic influenza has been largely ignored. universal closure of live markets would make biological sense but is difficult in regions where refrigeration is not widely available. vaccination has been accepted as an option for the control of hpai by the food and agriculture organization of the united nations and the world organization for animal health. emphasis is placed on the use of vaccine to facilitate eradication. the continued use of poultry influenza vaccines without an eradication plan has immediate benefits but also long-term consequences. the difficulty with continued vaccine usage is that it promotes genetic variation and allows shedding of virus in the absence of disease signs, thus creating the potential for epicenters of virus spread. further, while both inactivated oil emulsion whole-virus h5 vaccines and recombinant ndv vaccines containing h5 has are highly efficacious in chickens, the recombinant ndv vaccines are less efficacious in ducks. the experience in vietnam illustrates these points. in 2005, after 61 human cases of hp h5n1 virus infection and 19 deaths, universal poultry vaccination and reduction of duck populations were implemented, with dramatic results. in 2006, there were no human cases of h5n1 influenza. however, in 2007-2008, there were 14 human cases of hp h5n1 virus infection and 10 deaths (http://www.who.int/ csr/disease/avian_influenza/country/cases_table_2009_03_11/ en/index.html). the difficulty of controlling h5n1 hpai viruses in ducks by vaccination and the enormous task of vaccinating sufficient poultry to maintain ''herd immunity'' remain daunting obstacles. influenza in humans is considered a non-eradicable disease due to periodic introduction of viruses from their natural reservoir, wild migratory birds -mainly ducks. the culling of wild birds is not an option. the sole current option is biosecurity and eradication of hp influenza from domestic poultry. the longer-term goal will be to understand the genetics of natural resistance in ducks and to introduce these traits into domestic animals. there is consensus that the migratory waterfowl of the world (predominantly wild ducks) serve as the natural reservoirs of all influenza a viruses, which cause asymptomatic infection in these birds. influenza viruses have probably co-evolved with ducks over millennia, establishing an equilibrium between hosts and parasites so that neither suffers a significant loss of biological fitness; the evidence being minimal signs of disease in the hosts and the annual isolation of common subtypes. the unanswered question is whether these migratory bird species are the reservoirs of the currently circulating h5n1 hpai viruses. until the emergence of the asian h5n1 hpai viruses, the available data indicated that each new outbreak of hp h5 or h7 virus died out or was stamped out and that subsequent hp strains emerged from the low-pathogenic h5 and h7 virus reservoir. all species of birds tested to date support replication of some hp h5n1 strains and, providing they are not killed rapidly, could contribute to the spread of h5n1 hpai viruses. the present review has concentrated on ducks, some species of which are susceptible to h5n1 hpai virus-caused disease and death while others (e.g., mallards) are quite resistant. 34, 50 therefore, ducks that are infected but are naturally resistant to disease could have contributed to the spread of h5n1 hpai viruses westward from qinghai lake in 2005 to europe, africa, india, and the middle east. an unanswered question is whether the h5n1 hpai viruses are being carried back to the duck breeding areas and are infecting the next generation. extensive surveillance in the migratory pathways in europe and asia has provided no evidence of h5n1 hpai viruses in the new generation of birds after the breeding season. while all duck species tested to date are susceptible to lethal infection with h5n1 hpai viruses, some species, including the mallard and pintail ducks, are less susceptible and many survive. it is in these relatively resistant species that the h5n1 hpai viruses could be maintained. however, surveillance studies to date in these species have detected no h5n1 hpai viruses in breeding or juvenile birds. experimental studies show that some mallard ducks continue to shed virus for up to 17 days, allowing the development of humoral immunity and subsequent selection of antigenic variants within the same bird. 23 if this occurs, it could be argued that a limited number of ducks would be sufficient to maintain the virus in nature. continued surveillance is needed to determine whether h5n1 hpai viruses are maintained in nature by a small number of naturally resistant ducks that are long-term virus shedders. while naturally resistant ducks can be argued to have been involved in the spread of h5n1 hpai viruses from qinghai lake to the rest of eurasia, it is difficult to explain why h5n1 hpai viruses have not spread to susceptible species in the philippines, australia, and the americas, which are on the direct flyways of migratory waterfowl. more than 6ae6 million birds migrate from eastern asia to alaska yearly (alaska.usgs.gov ⁄ science ⁄ biology ⁄ avian_influenza ⁄ migrants_tables.html). despite intense surveillance in alaska, no h5n1 hpai viruses have been detected to date, and influenza viruses of eurasian lineage are introduced into the americas only rarely. 18 the major spreaders of influenza in domestic poultry are humans. as described by , from the molecular epidemiology data, transmission of h5n1 influenza in domestic poultry is perpetuated largely through movement of poultry and poultry products rather than continued reintroduction of viruses from migrating birds. 42 the alternative reservoir, the domestic duck population, has a higher likelihood of perpetuating h5n1 hpai viruses. prospective surveillance continued to isolate h5n1 hpai viruses from apparently healthy ducks, geese, and chickens in southeast asian poultry markets during 2004-2006. naturally resistant ducks might not be expected to show disease signs, but the absence of morbidity in highly susceptible geese and chickens is surprising. the widespread use of vaccine in chickens may explain this observation, but vaccine has been used less in geese and ducks. an alternative possibility is that the susceptible poultry had cross immunity as the result of exposure to co-circulating influenza viruses. experimental studies have demonstrated that chickens previously infected with h9n2 virus and then inoculated with h5n1 hpai virus become infected and shed virus but do not show disease signs. 51 the continuing co-circulation of multiple subtypes of lpai viruses in domestic poultry could explain why a small percentage of susceptible domestic species can appear healthy while shedding transmissible levels of h5n1 hpai virus. to provide answers to these unresolved questions about the role of domestic species, it will be necessary to establish long-term prospective surveillance in domestic poultry in the hypothetical ''epicenter zones,'' including china, indonesia, vietnam, egypt, and nigeria. it is noteworthy that in these regions, control of h5n1 hpai virus is attempted by the continuing use of vaccines. an area that has been surprisingly neglected is the genetics of ducks, the ultimate reservoir species of all influenza a subtypes. the wild duck reservoir contributes some or all of the genes of future pandemic strains in humans and future panzootic strains in domestic poultry. immune mechanisms in ducks are currently understudied, and the molecular basis of resistance of some duck species to lethal infection is unresolved. sequencing of the genome of the mallard duck is warranted, as it could provide insight into the factors that contribute to markedly reduced influenza virus pathogenicity. because wild ducks are the main reservoir of all influenza a viruses and the ultimate source of future pandemics, members of the scientific community who are interested in understanding the emergence and control of pandemic influenza should direct their attention to the following questions: • do ducks (wild or domestic) serve as the reservoirs of the asian h5n1 hpai viruses? • what genomic characteristics of ducks are associated with natural resistance in some species? • is antigenic diversity driven naturally in ducks or is it the consequence of vaccine usage? • what dose of vaccine antigen is required to prevent transmissible levels of excretion of h5n1 hpai viruses by ducks of different species (and geese and swans)? • is eradication of asian h5n1 hpai viruses achievable? • can the use of transgenic animals containing the natural resistance gene(s) of mallard ducks prevent pathogenic influenza virus infection? evolution and ecology of influenza a viruses highly pathogenic avian influenza duck influenza lacking evidence of disease signs and immune response domestic ducks and h5n1 influenza epidemic the immunogenicity and efficacy against h5n1 challenge of reverse genetics-derived h5n3 influenza vaccine in ducks and chickens intestinal influenza: replication and characterization of influenza viruses in ducks persistence of avian influenza viruses in water water-borne transmission of influenza a viruses? the perpetuation of orthomyxoviruses and paramyxoviruses in canadian waterfowl global patterns of influenza a virus in wild birds influenza a viruses of migrating wild aquatic birds in north america spatial, temporal, and species variation in prevalence of influenza a viruses in wild migratory birds circulation of influenza viruses and paramyxoviruses in waterfowl originating from 2 different areas of north america genetic reassortment of influenza a viruses in the intestinal tract of ducks precursor genes of future pandemic influenza viruses are perpetuated in ducks nesting in siberia surveillance of influenza a virus in migratory waterfowl in northern europe functional balance between haemagglutinin and neuraminidase in influenza virus infections influenza in migratory birds and evidence of limited intercontinental virus exchange coinfection of wild ducks by influenza a viruses: distribution patterns and biological significance are ducks contributing to the endemicity of highly pathogenic h5n1 influenza virus in asia? ecology of spring-migrating anatidae: a review free-grazing ducks and highly pathogenic avian influenza role of domestic ducks in the propagation and biological evolution of highly pathogenic h5n1 influenza viruses in asia pathological lesions in the lungs of ducks infected with influenza a viruses avian embryo susceptibility to italian h7n1 avian influenza viruses belonging to different lineages immunology of avian influenza virus: a review the isolation of influenza a viruses and paramyxoviruses from the feral ducks in new zealand circulation of influenza viruses in wild waterfowl wintering in italy during the 1993-99 period: evidence of virus shedding and seroconvcrsion in wild ducks pathogenicity of h5 influenza viruses for ducks reemerging h5n1 influenza viruses in hong kong in 2002 are highly pathogenic to ducks pathologic findings of highly pathogenic avian influenza virus a ⁄ duck ⁄ vietnam ⁄ 12 ⁄ 05 (h5n1) in experimentally infected pekin ducks, based on immunohistochemistry and in situ hybridization the invasion routes of neurovirulent a hong kong 483 ⁄ 97 (h5n1) influenza virus into the central nervous system after respiratory infection in mice enhanced neuropathogenicity of avian influenza a virus by passages through air sac and brain of chicks wild ducks as long-distance vectors of highly pathogenic avian influenza virus (h5ni) characterization of low-pathogenic h5 subtype influenza viruses from eurasia: implications for the origin of highly pathogenic h5n1 viruses h5n1 influenza viruses isolated from geese in southeastern china: evidence for genetic reassortment and interspecies transmission to ducks avian influenza virus (h5n1): a threat to human health investigation of outbreaks of highly pathogenic h5n1 avian influenza in waterfowl and wild birds in hong kong in late h5n1 virus outbreak in migratory waterfowl properties and dissemination of h5n1 viruses isolated during an influenza outbreak in migratory waterfowl in western china highly pathogenic h5n1 influenza virus infection in migratory birds establishment of multiple sublineages of h5n1 influenza virus in asia: implications for pandemic control molecular basis for high virulence of hong kong h5n1 influenza a viruses a single amino-acid in the pb2-gene of influenza-a virus is a determinant of host range duck migration and past influenza a (h5n1) outbreak areas highly pathogenic avian influenza h5n1 spring migration of northern pintails from california's central valley wintering area tracked with satellite telemetry: routes, timing, and destinations highly pathogenic avian influenza virus (h5n1) isolated from whooper swans wet markets -a continuing source of severe acute respiratory syndrome and influenza? susceptibility of north american ducks and gulls to h5n1 highly pathogenic avian influenza viruses modulation of the severity of highly pathogenic h5n1 influenza in chickens previously inoculated with israeli h9n2 influenza viruses this work was supported by the national institute of allergy and infectious diseases (contract number hhsn266200700005c) and by the american lebanese syrian associated charities (alsac). we thank sharon naron for scientific editing, betsy williford and elizabeth stevens for illustrations, and james knowles for manuscript preparation. key: cord-319129-o1kzb6dm authors: peng, jianhui; su, dongwei; zhang, ziwei; wang, mingke title: identification and management of asymptomatic carriers of coronavirus disease 2019 (covid‐19) in china date: 2020-06-08 journal: influenza other respir viruses doi: 10.1111/irv.12768 sha: doc_id: 319129 cord_uid: o1kzb6dm nan to the editor, we read with interest the recent article by yu transmission through asymptomatic carriers has been a huge challenge for covid-19 prevention and control. 1 there are two types of asymptomatic carriers: those who never develop symptoms and those who are detected during the incubation period (pre-symptomatic detection) prior to symptom onset. 3 here, we discussed the identification and management of asymptomatic carriers of covid-19 in china. so far, asymptomatic carriers of covid-19 may be found by the following ways. 3 since april 1, 2020, the number of asymptomatic infectors has been reported online by national health commission of the people's republic of china on a daily basis, and the following management measures were required to be carried out to minimize the risk of their transmission in china. 3 first, persons detected as asymptomatic infectors would be isolated for 14 days, which may prevent them from becoming contagion sources. those can be lifted from isolation by negative nucleic acid tests on two consecutive samples at least 24 hours apart. 3 second, epidemiological investigation of asymptomatic infectors would be strengthened, and strict disinfection would be implement in their living places such as homes, medical institutions, isolation wards, transport tools, and medical observation places. third, since early detection of asymptomatic carriers is critical to contain their transmission, current screening methods also need to be strengthened. nucleic acid screening is practical and quick for the population. however, due to specimen collection, testing methods, product stability, false-negative results have been frequently reported, which will hamper case detection and disease control. 8 therefore, multiple screening and monitoring of nucleic acid combining with antigens and antibodies in blood and other body fluids are recommended. 8 at present, persons who are significant epidemiological associations with covid-19 patients (eg, close contacts) will be put under 14-day centralized medical observation in china. 3 as the epidemic enters a new stage, in order to consolidate the previous anti-epidemic achievements and prevent the epidemic from rebounding, we should further strengthen the monitoring of asymptomatic carriers and some special populations who may play a greater role in the spread of covid-19, including front-line medical staff, disease control personnel, street epidemic prevention and control point staff, and delivery personnel. since asymptomatic carriers have no clinical symptoms, they are difficult to identify, diagnose, and isolate. this can lead to loopholes in prevention and control measures, resulting in increased difficulty in controlling the spread of covid-19. 9 the public health education should be strengthened, and formation of good hygiene habits is important. in particular, awareness of self-protection, self-supervision and administration, and pre-service training of above special populations are critical to reducing the spread of asymptomatic infections. in future, further definition of high-risk populations and development of effective screening strategies and programs will support rapid identification and management of asymptomatic carrier transmission of covid-19. 9 further study is needed on asymptomatic carriers including their frequency relative to symptomatic infections, their disease course, and factors associated with having an asymptomatic rather than symptomatic infection. 8 since there can be a gray area between asymptomatic and symptomatic infections, verification: all authors have seen the manuscript and agree to the content. all the authors played a significant role in the paper. covid-19 transmission through asymptomatic carriers is a challenge to containment. influenza other respir viruses covert coronavirus infections could be seeding new outbreaks joint taskforce on covid-19 prevention and control, china state council. protocol for the management of asymptomatic persons infected with covid-19 virus clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing presumed asymptomatic carrier transmission of covid-19 transmission of 2019-ncov infection from an asymptomatic contact in germany sars-cov-2 viral load in upper respiratory specimens of infected patients screening and management of asymptomatic infection of corona virus disease 2019 (covid-19) advances on presymptomatic or asymptomatic carrier transmission of covid-19 key: cord-273861-sg7esn4p authors: yi, lina; zou, lirong; lu, jing; kang, min; song, yingchao; su, juan; zhang, xin; liang, lijun; ni, hanzhong; ke, changwen; wu, jie title: a cluster of adenovirus type b55 infection in a neurosurgical inpatient department of a general hospital in guangdong, china date: 2017-06-26 journal: influenza other respir viruses doi: 10.1111/irv.12457 sha: doc_id: 273861 cord_uid: sg7esn4p background: human adenovirus type 55 is a re‐emerging human respiratory pathogen that is associated with several respiratory infections outbreaks in military and school populations. in this study, we describe the first hadv55‐associated hospital outbreak documented in guangdong, china. methods: active surveillance was conducted in the involved neurosurgical inpatient department. all staff and patients in the involved neurosurgical department were surveyed for any symptoms of fever (≥38°c) and enlarged tonsils during the outbreak period. throat swabs and demographic information were collected for all cases. for each specimen, assays for common respiratory viruses were performed using one‐step reverse transcription‐polymerase chain reaction. hadv‐positive samples were inoculated onto hep‐2 cells for isolation. hexon genes, fiber genes, penton genes, and whole genomes were sequenced. a phylogenetic tree was constructed. results and conclusions: forty‐three cases, including 24 laboratory‐confirmed cases and 19 possible cases, were identified. nurses had the highest attack rate of infection, with a rate of 36.4%. the attack rate for doctors and inpatients was 20.0% and 16.7%, respectively. hadv55 was the sole pathogen identified during this outbreak. the hexon, fiber, and penton genes from seven isolated hadv55 stains were sequenced. all these genes showed 100% homology and fell into the hadv55 [p14h11f14] cluster, indicating that hadv55 was the single viral strain for the outbreak. while not conclusive, the epidemic investigation revealed that the outbreak was introduced by nurses from sources outside the hospital. it was likely that a transmission from staff to inpatients had occurred. human adenovirus (hadv) is a common pathogen among children and adults. infections of hadv can cause a variety of clinical diseases, ranging from asymptomatic and self-limited conditions to pneumonia and even death. 1 as of now, at least 68 hadv genotypes have been identified and are classified into seven species (a-g). 2 furthermore, viruses in species b, c, and e are more commonly associated with symptomatic respiratory infections. 1, [3] [4] [5] [6] [7] human adenovirus type 55 (hadv55), a member of hadv-b family, was first identified from a military outbreak in spain in 1969. 8 this virus used to be recognized as hadv-b11a by partial characterization of its hexon and fiber epitopes. [9] [10] [11] in 2006, michael et al. revealed that hadv55 was an emergent respiratory pathogen. 12 it had evolved from a homologous recombination between hadv-b14 and hadv-b11 in its hexon gene, which conferred changes in viral serotype and immune activity. in recent decades, hadv55 has been associated with several respiratory infections outbreaks. 9, 11, 13, 14 crowed communities, military training camps, and schools are common settings for hadv infections. here, we describe a hadv55 outbreak occurred in a neurosurgical inpatient department of a jiangmen hospital in guangdong province, china. on june 30, 2016, one local center for disease control and prevention of guangdong was informed that several staff members, including nurses and doctors working in a general hospital, fell ill with symptoms of a fever and sore throat. the hospital is a district general hospital. the by reviewing patients' medical records, we identified the first case and determined the investigation period began on june 11, 10 days before the onset of the first case. all staff and patients in this neurosurgical department were surveyed for any symptoms of fever (≥38°c) and enlarged tonsils during the outbreak period. laboratory-confirmed cases were defined as persons with positive hadv55 pcr assays. those not confirmed by the laboratory, but with symptoms and signs (fever (≥38°c) and enlarged tonsils), were defined as clinical-confirmed cases (possible cases). throat swabs from all cases were collected and tested. information on demographic characteristics, onset of illness, and clinical symptoms were collected. for the use of all above-mentioned specimens, written informed consent from all participants involved in the research was obtained (or from their parents or legal guardians in the case of a minor). this study was approved by the ethics committee of the guangdong provincial center for disease control and prevention, and was in compliance with the helsinki declaration. the hexon, fiber, and penton genes of hadv were amplified. the primers we used are listed as follows: hexon-f multiple sequence alignments were performed with clustalw, 15 and alignments were manually edited with aliview. 16 for whole genome sequences, mafft software was used for alignment with default parameters (http://mafft.cbrc.jp/alignment/server/). maximum likelihood (ml) trees were estimated in raxml 17 using the generalized time-reversible (gtr) nucleotide substitution model with gamma distribution among site rate heterogeneity. 18 data analysis was performed with spss 15.0 (spss inc., chicago, il, usa). differences with an error probability of p<.05 were regarded as significant. for categorical data, we used chi-square testing and fisher exact testing as appropriate. the outbreak ranged from june 21 to july 8, 2016. the first cases were two nurses working at neurosurgical unit a. our investigation identified 43 cases, including 24 laboratory-confirmed cases and 19 possible cases. the onset of illness in all cases is shown in figure 1 . two peaks were observed. the first one was from june 21-28. it included 25 cases, among which 21 were nurses. the second was from june 29 to july 5. during this period, 13 of 15 infected inpatients were identified. it was observed that, within each unit, the first cases were all nurses. (table 3 ). most cases (41 cases) reported upper respiratory symptoms. fever was the most common symptom, followed by sore throat, cough, expectoration, headache, and runny nose. twelve of these 43 cases developed pneumonia. no mechanical ventilation was needed. two cases developed conjunctivitis. most clinical symptoms and signs between infected inpatients and infected medical workers did not differ (table 3) . however, there were significantly more inpatients who developed pneumonia (p<.001). we reviewed all inpatients' medical records and surveyed their visitors. neither the inpatients nor their visitors had respiratory symptoms before the outbreak. we also interviewed all the chief doctors and nurses working in these three units and enquired about the staff's routine practice in the involved neurosurgical department. there are separated doctor offices, nurse stations, and nurse resting rooms within each unit. the staff had basically followed the standard infection control procedures when they cared for the patients. in the nurse stations and the resting rooms, the nurses preferred not to wear any protective equipment. they shared most office supplies, such as computers, medical records, and examination records. more often, it was the nurses who visited the doctors' offices for daily work communication, while the doctors rarely visited the nurse stations and their resting rooms, except for the acupuncturist, who was responsible for the entire department and was used to drinking water in nurse station a. there was a dispensing counter in nurse station a from which nurses from unit b and the nsicu sometimes pick up medicines. other interactions between units were fewer. one day before the outbreak, nurses from unit a had a party in a karaoke bar. control measures were implemented from june 23, when the hospital infection control committee was informed of this possible nosocomial infection. environmental cleaning was enhanced. self-protections, including hand hygiene and surgical mask wearing, were re-enforced. all cases and their close contacts were followed up. on june 27, due to the increasing number of cases, further control measures were implemented. staff with symptoms were either given medical leave or treated in isolation, as what had been implemented for the infected inpatients. the personnel entering and leaving the involved neurosurgical department were restricted. visitors to the department were required to wear surgical masks. in addition, screenings for similar symptoms were performed in the entire hospital. specimens from cases and the environment were collected and sent to the local cdc for pathogen detection. the fiber gene (1048 nucleotides (nt)), hexon gene (1603 nt), and penton gene (1206 nt) were obtained from eight hadv strains isolated in the outbreak. sequences of each gene were completely identical, suggesting the outbreak was caused by the single viral introduction. phylogenetic trees of fiber, hexon, and penton genes were constructed by integrating all closely related adenovirus sequences from this study identified a cluster of adenovirus type b55 infection in the neurosurgical inpatient department of a general hospital. through targeted surveys, the possibility of inpatients as the source of this outbreak was excluded. we suggested that the pathogen of this outbreak may have been introduced into the hospital by nurses in unit a. they had a party in a karaoke bar 1 day before the outbreak. one or some our study has limitations. first, several respiratory viruses could cause respiratory infections. 25 previously published data show that it is difficult to make a reliable distinction among these pathogens based on clinical signs and symptoms. 26 to ensure case findings, we used a sensitive definition of possible cases in this outbreak, which may include both adenovirus and non-adenovirus infections, as well as infected patients who may not have been involved in this infection cycle. our investigation found 27 throat swab specimens with a hadvpositive status, while pathogens for the remaining cases were not fully clarified. therefore, the attack rate may have been overestimated. more laboratory tests should be carried out to understand pathogenic characteristics. second, based on our investigation, we implicated that the virus could be intruded from outside of the hospital by the nurse or clinical features and treatment of adenovirus infections molecular identification and epidemiological features of human adenoviruses associated with acute respiratory infections in hospitalized children in southern china emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study severe community-acquired pneumonia caused by human adenovirus in immunocompetent adults: a multicenter case series severe pneumonia due to adenovirus serotype 14: a new respiratory threat? adenovirus infection as possible cause of acute liver failure in a healthy child: a case report adenovirus pneumonia in an immunocompetent adult occurrence of respiratory illness due to an atypical strain of adenovirus type 11 during a large outbreak in spanish military recruits outbreak of acute respiratory disease in china caused by b2 species of adenovirus type 11 respiratory disease caused by a species b2 adenovirus in a military camp in turkey outbreak of febrile respiratory illness associated with adenovirus 11a infection in a singapore military training camp computational analysis identifies human adenovirus type 55 as a re-emergent acute respiratory disease pathogen an outbreak of acute respiratory disease in china caused by human adenovirus type b55 in a physical training facility epidemiology of human adenovirus and molecular characterization of human adenovirus 55 in china clustal w and clustal x version 2.0 aliview: a fast and lightweight alignment viewer and editor for large datasets raxml-vi-hpc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models the general stochastic model of nucleotide substitution incubation periods of acute respiratory viral infections: a systematic review emergence of community-acquired adenovirus type 55 as a cause of community-onset pneumonia cross-sectional study of the relationship of peripheral blood cell profiles with severity of infection by adenovirus type 55 outbreak of adenovirus type 55 infection in israel disseminated adenovirus disease in immunocompromised and immunocompetent children intravenous ribavirin treatment for severe adenovirus disease in immunocompromised children a 3-year prospective study of the epidemiology of acute respiratory viral infections in hospitalized children in shenzhen, china. influenza other respir viruses molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in shanghai a cluster of adenovirus type b55 infection in a neurosurgical inpatient department of a general hospital in guangdong, china. influenza other respi viruses [a2016538]. the authors declare no conflict of interest. clinical data and patient samples were provided by jw, ck, and mk. all authors read and approved the final manuscript. key: cord-269665-byuv48wi authors: macintyre, chandini raina; wang, quanyi; cauchemez, simon; seale, holly; dwyer, dominic e.; yang, peng; shi, weixian; gao, zhanhai; pang, xinghuo; zhang, yi; wang, xiaoli; duan, wei; rahman, bayzidur; ferguson, neil title: a cluster randomized clinical trial comparing fit‐tested and non‐fit‐tested n95 respirators to medical masks to prevent respiratory virus infection in health care workers date: 2011-01-27 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2011.00198.x sha: doc_id: 269665 cord_uid: byuv48wi please cite this paper as: macintyre et al. (2011) a cluster randomized clinical trial comparing fit‐tested and non‐fit‐tested n95 respirators to medical masks to prevent respiratory virus infection in health care workers. influenza and other respiratory viruses doi: 10.1111/j.1750‐2659.2010.00198.x. background we compared the efficacy of medical masks, n95 respirators (fit tested and non fit tested), in health care workers (hcws). methods a cluster randomized clinical trial (rct) of 1441 hcws in 15 beijing hospitals was performed during the 2008/2009 winter. participants wore masks or respirators during the entire work shift for 4 weeks. outcomes included clinical respiratory illness (cri), influenza‐like illness (ili), laboratory‐confirmed respiratory virus infection and influenza. a convenience no‐mask/respirator group of 481 health workers from nine hospitals was compared. findings the rates of cri (3·9% versus 6·7%), ili (0·3% versus 0·6%), laboratory‐confirmed respiratory virus (1·4% versus 2·6%) and influenza (0·3% versus 1%) infection were consistently lower for the n95 group compared to medical masks. by intention‐to‐treat analysis, when p values were adjusted for clustering, non‐fit‐tested n95 respirators were significantly more protective than medical masks against cri, but no other outcomes were significant. the rates of all outcomes were higher in the convenience no‐mask group compared to the intervention arms. there was no significant difference in outcomes between the n95 arms with and without fit testing. rates of fit test failure were low. in a post hoc analysis adjusted for potential confounders, n95 masks and hospital level were significant, but medical masks, vaccination, handwashing and high‐risk procedures were not. interpretation rates of infection in the medical mask group were double that in the n95 group. a benefit of respirators is suggested but would need to be confirmed by a larger trial, as this study may have been underpowered. the finding on fit testing is specific to the type of respirator used in the study and cannot be generalized to other respirators. trial registration australian new zealand clinical trials registry (anzctr), actrn: actrn12609000257268 (http://www.anzctr.org.au). background we compared the efficacy of medical masks, n95 respirators (fit tested and non fit tested), in health care workers (hcws). methods a cluster randomized clinical trial (rct) of 1441 hcws in 15 beijing hospitals was performed during the 2008 ⁄ 2009 winter. participants wore masks or respirators during the entire work shift for 4 weeks. outcomes included clinical respiratory illness (cri), influenza-like illness (ili), laboratoryconfirmed respiratory virus infection and influenza. a convenience no-mask ⁄ respirator group of 481 health workers from nine hospitals was compared. findings the rates of cri (3ae9% versus 6ae7%), ili (0ae3% versus 0ae6%), laboratory-confirmed respiratory virus (1ae4% versus 2ae6%) and influenza (0ae3% versus 1%) infection were consistently lower for the n95 group compared to medical masks. by intentionto-treat analysis, when p values were adjusted for clustering, nonfit-tested n95 respirators were significantly more protective than medical masks against cri, but no other outcomes were significant. the rates of all outcomes were higher in the convenience no-mask group compared to the intervention arms. there was no significant difference in outcomes between the n95 arms with and without fit testing. rates of fit test failure were low. in a post hoc analysis adjusted for potential confounders, n95 masks and hospital level were significant, but medical masks, vaccination, handwashing and high-risk procedures were not. interpretation rates of infection in the medical mask group were double that in the n95 group. a benefit of respirators is suggested but would need to be confirmed by a larger trial, as this study may have been underpowered. the finding on fit testing is specific to the type of respirator used in the study and cannot be generalized to other respirators. the current influenza a h1n1 2009 virus pandemic, 1 the ongoing zoonotic transmission of influenza a h5n1 and the emergence of oseltamivir-resistant seasonal influenza a h1n1 are threats to human health. hospital health care workers (hcws) are key to effective pandemic response and the capacity of health care systems. respiratory protec-tion is one of the key non-pharmaceutical interventions for protection of hcws. nosocomial influenza and other outbreaks result in significant morbidity and costs 2,3 and can occur in the absence of community epidemics. 4 during outbreaks of infectious diseases, hospitals may amplify virus transmission, as demonstrated during severe acute respiratory syndrome (sars). 5 furthermore, anticipated antiviral shortages and original article delays in vaccine development make non-pharmaceutical interventions crucial. there are gaps in knowledge about prevention of influenza by medical masks and respirators. there are several prospective, randomized controlled trials on the use of handwashing, [6] [7] [8] but only two trials on the use of medical masks ⁄ respirators in households. 9, 10 in one of these studies, we showed that medical masks ⁄ respirators in compliant users in the household setting were associated with reductions in the risk of influenza-like illness (ili)associated infection. 10 to date, there is one small randomized controlled trial (rct) of medical masks compared to respirators in hcws 11 which found no difference, but lacked a control arm. medical masks are not designed to provide respiratory protection. 12 they have consistently lower filtration efficiency when compared to respirators, which are designed specifically for respiratory protection. [13] [14] [15] medical masks were designed to prevent wound contamination when worn by the surgeon; however, three rcts failed to show efficacy against their intended design. [16] [17] [18] the aim of this study was to determine the efficacy of medical masks compared to fit-tested and non-fit-tested n95 respirators in hcws in the prevention of disease because of influenza and other respiratory viruses. a prospective, cluster randomized trial of medical mask and respirator use in frontline hcws was conducted from december 2008 to january 2009 in beijing, china. we initially aimed to determine the efficacy of two different kinds of respiratory protection (n95 respirators and medical masks) during the influenza season compared to each other and compared to a no-mask group. however, although we intended to have a randomized control group, this was not acceptable to the chinese irb, who felt it would be unethical to assign hcws randomly to not wear a mask, given mask use was widespread in chinese hospitals that were included in the randomization. as such, we studied a convenience-selected no-mask group of hcws who did not wear a mask. these hcws were selected from other hospitals where mask wearing was not routine during the study period. absence of randomization in the no-mask group meant that we eventually had to restrict the primary analysis of the trial to the comparison of the efficacy of n95 respirators and medical masks with each other. participants were hospital hcws aged ‡18 years from the emergency departments and respiratory wards of 15 hospitals. these wards were selected as high-risk settings in which repeated and multiple exposures to respiratory infections are expected. we also monitored all participating wards by active surveillance for clinically compatible illness, including in the no-mask group, for outbreaks of respiratory infection in patients during the study period, and none was detected. all hospitals were large, tertiary hospitals in urban beijing, and there was no variation in the start of the influenza season within this geographic area. recruitment commenced on the 1 december 2008 and final follow-up was completed on 15 january 2009. the study protocol was approved by the institutional review board and human research ethics committee of the beijing ministry for health. verbal informed consent was provided by participants, and they were provided written information about the study. the nine hospitals in the convenience no-arm group were not part of the randomization, but hcws in those hospitals were selected from the same type of wards as the intervention arms (emergency departments and respiratory wards). they were followed up in the same way as the trial participants for development of infections. the unit of randomization was hospitals. hospitals were randomized to one of three intervention arms: (i) medical masks (3mô medical mask, catalogue number 1820, st paul, mn, usa); (ii) n95 fit-tested mask (3mô flat-fold n95 respirator, catalogue number 9132) and (iii) n95 nonfit-tested mask (3mô flat-fold n95 respirator, catalogue number 9132). figure 1 outlines the recruitment and randomization (using a secure computerized randomization program) process. a pre-study assessment of hospital infection control levels determined that the hospitals had sufficient diversity to warrant stratified randomization by size of hospital and level of infection control. this assessment measured ventilation, spatial dimensions, bedding configuration, handwashing facilities and personal protective equipment use. the ministry of health in 1989 categorizes hospitals in china into three levels (level 3 is the highest) depending on their level of sophistication, equipment and staff ⁄ bed numbers. fifteen hospitals were randomized -five level 2 and ten level 3. (i) clinical respiratory illness (cri), 19 defined as two or more respiratory or one respiratory symptom and a systemic symptom; (ii) ili, defined as fever ‡38°c plus one respiratory symptom (i.e. cough, runny nose, etc.); (iii) laboratory-confirmed viral respiratory infection (detection of adenoviruses, human metapneumovirus, coronavirus 229e ⁄ nl63, parainfluenza viruses 1, 2 and 3, influenza viruses a and b, respiratory syncytial virus a and b, rhinovirus a ⁄ b and coronavirus oc43 ⁄ hku1 by multiplex pcr); (iv) laboratory-confirmed influenza a or b and (v) adherence with mask ⁄ respirator use. the choice of a relatively broad cri definition was dictated by our interest in interrupting transmission of a wide range of respiratory viruses, which in adults may or may not be accompanied by fever. also, all respiratory pathogens share a similar transmission mechanism namely aerosol, droplet and fomite spread, although the relative role of these factors may vary between different viruses and in different clinical situations. other endpoints included adverse effects, measured using a semi-structured questionnaire and adherence. any nurse, doctor or ward clerk who worked full time in the emergency or respiratory wards at the hospital were eligible. hcws were excluded if they: (i) were unable or refused to consent; (ii) had beards, long moustaches or long facial hair stubble; (iii) had a current respiratory illness, rhinitis and ⁄ or allergy and (iv) worked part-time or did not work in the aforementioned wards ⁄ departments. in all participating wards, 100% of eligible health workers participated. participants wore the mask or respirator on every shift for 4 consecutive weeks after being shown when to wear it and how to fit it correctly. participants were supplied daily with either three masks for the medical mask group or two n95 respirators. participants were asked to store the mask in a paper bag every time they removed it (for toilet breaks, tea ⁄ lunch breaks and at the end of every shift) and place the bagged mask or respirator in their locker. all participants were instructed on the importance of hand hygiene prior to ⁄ after the removal of medical masks and respirators. participants in arm two underwent a fit-testing proce-dure using a 3mô ft-30 bitrex fit test kit according to the manufacturers' instructions (3mô, st paul, mn, usa). detailed demographic and clinical details of all participants were collected. this included age, sex, smoking history, comorbidities, seasonal influenza vaccination status, medications, conduct of high-risk procedures (defined as suctioning, intubation, nebulized medications, chest physiotherapy and other aerosol generating procedures), handwashing practices, use of other personal protective equipment (gowns, gloves, eye shields and hair ⁄ foot covers) and results of laboratory tests. use of specific interventions for influenza such as antivirals was also measured. participants were followed for 4 weeks of wearing the masks or respirators and an extra week of non-wearing for development of respiratory symptoms. all participants received a mercury thermometer to measure their temperature at the beginning of each day and at the onset of any symptoms. diary cards were provided for the duration to record daily the (i) number of hours worked; (ii) mask ⁄ respirator usage and (iii) recognized cri encounters. participants were contacted daily by phone or face-to-face contact to actively identify incident cases of respiratory infection. at each ward, the head nurse actively followed up all participants and identified incident illness. staff members from the district cdc also undertook daily monitoring of the sites. if participants were symptomatic, swabs of both tonsils and the posterior pharyngeal wall were collected. we also monitored adherence with mask or respirator use over the 4-week time course by: (i) observation: the head ward nurse observed compliance on the ward on a daily basis and recorded the information on a structured form, (ii) self-report: a diary card with tick boxes was given to each subject, to be carried during the day. adherence to wearing the masks or respirators was monitored by these diary cards and returned to researchers on a weekly basis. exit interviews with participants were conducted after the 4 weeks to gain further insights into adherence and other issues around the use of masks ⁄ respirators including adverse effects. participants with symptoms had two pharyngeal swabs collected by a trained nurse or doctor. double rayon-tipped, plastic-shafted swabs were used to scratch both tonsilar areas and the posterior pharyngeal wall. these were transported immediately after collection to the laboratory, or at 4°c within 48 hours if transport was delayed. pharyngeal swabs were tested with at the laboratories of the beijing centers for disease control and prevention. viral dna ⁄ rna was extracted from 300 ll of each respiratory specimen using the viral gene-spin tm kit (intron biotechnology, inc., seoul, korea) according to the manufacturer's instructions. reverse transcription was performed on 8 ll of rna in a final reaction volume of 20 ll for 1ae5 hours at 37°c, using the revertaid tm first strand cdna synthesis kit (fermentas, burlington, on, canada) to synthesize cdna. multiplex polymerase chain reaction (pcr) was carried out using the seeplex ò rv12 detection kit (seegen, inc., seoul, korea) to detect adenoviruses, human metapneumovirus, coronavirus 229e ⁄ nl63, parainfluenza viruses 1, 2 or 3, influenza viruses a or b, respiratory syncytial virus a or b, rhinovirus a ⁄ b and coronavirus oc43 ⁄ hku1. three microlitres of synthesized first-strand cdna, 4 ll of multiplex primers, 10 ll master mix (hot start taq dna polymerase and dntp are included in the reaction buffer) and 3 ll of 8-methoxypsoralen (8-mop) were added (8-mop, accompanied by uv irradiation for 20 minutes, prevents amplification of contaminated dna). a mixture of 12 viral clones was used as a positive control template, and sterile deionized water was used as a negative control. after preheating at 95°c for 15 minutes, 40 amplification cycles were carried out under the following conditions in a thermal cycler (geneamp pcr system 9700, foster city, ca, usa): 94°c for 30 seconds, 60°c for 1ae5 minutes and 72°c for 1ae5 minutes. amplification was completed at the final extension step at 72°c for 10 minutes. the multiplex pcr products were visualized by electrophoresis on an ethidium bromidestained 2% agarose gel. viral isolation by mdck cell culture was undertaken for some of the influenza samples which were positive by nuclei acid detection. specimen processing, dna ⁄ rna extraction, pcr amplification and pcr product analyses were conducted in different rooms to avoid cross-contamination. the primary endpoints of interest as described above were analysed by intention-to-treat analysis. the two n95 arms were also combined and compared to the medical mask arm, given that there was no significant difference between them and rates of fit test failure were extremely low in the fit-tested arm (5 ⁄ 461 fit test failures). differences in proportions between the trial arms were tested by calculation of pearson's chi-square using sas 9.2 software (cary, nc, usa). the distribution of key potentially confounding variables between study arms was compared. to estimate the odds ratio while adjusting for the clustering effects, we used a random effect logistic regression model. in the model, we added a hospital-specific random intercept in the linear predictors, and maximum likelihood was estimated using adaptive quadrature. 20 the model was fitted using 'xtlogit' command in stata (college station, tx, usa). 21 we also conducted multivariable analysis to adjust for the potential confounders. in the initial model, we included all the variables along with the main exposure variable those were significant (p < 0ae05) in the univariate analysis. we then used a backward elimination method to remove the variables that did not have any confounding effect, that is, could not make meaningful (roughly 10%) change in the effect measure with the main exposure variable. 22 in case of high multi-collinearity because of strong correlation among the potential confounders, we chose the more relevant ones having the highest confounding effect on the association of interest. we analysed compliance as wearing the mask for >80% of the shift. to obtain 80% power at 2-sided 5% significant level for detecting a significant difference of attack rate between the intervention arms, and for an assumed 5% attack rate in the n95 arm and 12% in the medical mask arm, a sample size of 488 participants or five clusters (hospitals) per arm was required for cluster size (m) 100 and intra-cluster correlation coefficient (icc) 0ae01. 23 the design effect (deff) for this cluster randomization trial was 2 (deff = 1 + (m)1) · icc = 1 + (100)1) · 0ae01 = 2). as such, we aimed to recruit a sample size of 500 per arm. a total of 1441 nurses and doctors in 15 beijing hospitals were recruited into the randomized arms and 481 nurses and doctors in nine hospitals were recruited into the convenience no-mask group. figure 1 shows the recruitment process. the distribution of demographic variables was generally similar between arms (table 1) , but was significantly different for anyone smoking in the family, four or more people in family, four or more adults in family, influenza vaccination in 2008 and 2007, public transport, handwashing, hospital level and high-risk procedures. in regards to hand hygiene, 83% (382 ⁄ 461), 87ae8% (428 ⁄ 488) and 88ae6% (435 ⁄ 492) of participants from the n95 fit test arm, n95 non-fit test arm and medical mask arm stated that they washed their hands between patients, respectively. for all outcomes, non-fit-tested n95 respirators had lower rates of infections compared to fit-tested n95s (for all n95 versus medical masks, the rates were 3ae9% versus 6ae7% for cri, 0ae3% versus 0ae6% for ili, 1ae4% versus 2ae6% for laboratory-confirmed virus and 0ae3% versus 1% for influenza) but these differences were not significant. all infection outcomes were consistently higher (approximately double) in the medical mask group compared to the n95 group ( figure 2 ). there were no cases of influenza in the non-fit-tested n95 arm, three in the fit-tested n95 arm and five in the medical mask arm. after adjustment for clustering, non-fit-tested n95 masks were significantly protective compared to medical masks against cri, but other outcomes were not significant between n95 and medical masks ( table 2) . when compared to the convenience no-mask group and adjusted for clustering, n95 non-fit-tested was significantly protective against cri, and all n95 was protective against laboratory-confirmed virus and laboratoryconfirmed influenza (table 3 ). in a post hoc analysis carried out to adjust for potential confounders which were unevenly distributed between arms, all n95 and hospital level remained significant for cri and laboratory-confirmed viral infection, but handwashing, vaccination and high-risk procedures were not significant (table 4) . fit-testing failure rate was very low (5 ⁄ 461, 1ae08%). rates of adherence in all arms of the study were high (figure 3 ). table 5 shows adverse events associated with medical mask or n95 use, and that n95 respirators were associated with higher rates of adverse events. adherence with mask or respirator wearing was high and not significantly different in all arms, with 74% adherence (95% ci 70-78%) in the n95 fit-tested arm, 68% in the n95 nonfit-tested arm (95% ci 64-73%) and 76% in the medical mask arm (95% ci 72-79%). the duration of mask wearing in these arms, respectively, was 5ae2 hours (95% ci 5ae1-5ae4 hours), 4ae9 hours (95% ci 4ae8-5ae1 hours) and 5 hours (95% ci 4ae9-5ae2 hours; figure 3 ). we found that rates of respiratory tract infection were approximately double in the medical mask group compared to the n95 group in health workers who wore masks throughout their shift. however, only the n95 non-fittested arm was significantly protective against cri, and there were no other significant differences between n95 respirators and medical masks for the four primary outcomes in the adjusted analysis. however, it should be noted that under the null hypothesis where there is no difference between groups, the probability that we wrongly find at least one significant difference given the 12 tests undertaken is 46%. the trial may also be underpowered because observed attack rates were lower than expected. the rates of all outcomes were higher in the convenience no-mask group than in the masks groups. *ili definition using fever >38 -note, this is less sensitive than laboratory-confirmed infection. **any respiratory virus. ***odds ratio -medical group as reference. a random effect logistic model accounting for clustering was used to compute odd ratios. p m : p value adjusted for clustering of hospitals using random effect logistic regression model. 29 cri, clinical respiratory illness; ili, influenza-like illness. intention-to-treat analysis, n95 respirators but not medical masks had significantly lower rates of infection compared to no masks. however, the convenience no-mask group was not a randomized control arm and hospitals in this group were actually selected on the basis that most of their staff did not wear masks (which is not the norm in hospitals in beijing), suggesting that conditions in those hospitals were different than those in hospitals from the masks groups. as a consequence, it is not possible to make any definitive judgement on the efficacy of masks on this basis. one possible bias would be if those hospitals had differentially higher risk of infection compared to the intervention hospitals, for example because of the occurrence of outbreaks. however, we monitored all hospitals involved in the study for outbreaks which may have increased apparent attack rates, and none were documented. other than that, possible sources of bias that could have plausibly increased the infection rate in the control arm (namely vaccination, handwashing, hospital level and high-risk procedures) were measured. in a post hoc adjusted analysis, only hospital level and the n95 arm were significant against cri and laboratory-confirmed viral infection. respiratory protection is a key strategy for pandemic control and key to sustaining the health care workforce. the fact that rates of all outcomes were consistently lower in the n95 group suggest that n95 respirators might offer better protection for hcws; but a larger trial is needed to make a definitive judgment about the relative efficacy of respirators and medical masks. a recent, smaller trial found no difference between n95 and medical masks, but was *ili definition using fever >38 -note, this is less sensitive than laboratory-confirmed infection. **any respiratory virus. ***odds ratio -no-mask convenience group as reference. a random effect logistic model accounting for clustering was used to compute odd ratios. cri, clinical respiratory illness; ili, influenza-like illness. bold text signifies statistical significance. probably underpowered to detect any differences. 11 further, the intervention in that study was use of respiratory protection only during care of identified febrile patients with ili or high-risk procedures. this is different from the intervention in our study, which comprised wearing the mask for the entire shift. in addition, that study measured serological evidence of influenza as an outcome, which comprised the majority of outcomes, but did not exclude influenza-vaccinated participants, a flaw that would have resulted in falsepositive cases of 'influenza'. the finding that fit testing did not improve the efficacy of n95 respirators is important, although it could be explained by a lack of power. the value of fit testing varies with the quality of the respirator, and our study used a high-quality respirator. these results would not be generalizable to other respirators, where fit testing may be more important. as such, we still recommend that fit testing be part of the process of using respirators. the small number of randomization units along with the small numbers of cases means that estimation of multivariate models would not necessarily converge. in the post hoc multivariable analysis, we could not adjust for all of the factors because of high correlation among some of them. other limitations of the study include the generalizability of our results to other types of respirators and to other hcw populations in other countries. scoping work with australian hcws showed compliance of 10% with continual mask wearing during a severe influenza season. 24 beijing was selected to maximize the power of the study because of the strong culture of mask wearing among hcws. another limitation of the study is that cluster rcts can be impacted by heterogeneity of behaviours, meaning that we cannot exclude such effects caused by behaviours we did not measure. the cluster design is also strength, as interventions against infectious diseases can have herd effects. in infectious diseases which can spread from person to person, the 'herd effect' is a real and documented phenomenon where protecting some individuals with an intervention (most commonly vaccination, but also applicable to other interventions) can also protect individuals who were not protected by the intervention. therefore, if some individuals are randomized to masks on a ward, the individuals who do not wear masks may also be protected because of the effect the masks have on interrupting the transmission of disease from person to person. this is why it is preferable to use cluster design, where everyone in the cluster gets the same intervention. in our study, masks or respirators were worn during the entire shift. some policies recommend mask ⁄ respirator use only when hcws are conducting high-risk procedures or entering an isolation room. whether masks ⁄ respirators will be protective when used only when an identified episode of exposure occurs depends on whether hcws accurately identify all episodes of risk, whether most transmission occurs after clearly identified exposures and whether there is transmission from asymptomatic or pre-symptomatic infections. there is currently no evidence on how much of a hcws' risk is unidentified or unrecognized. in our study, hcws who conducted high-risk procedures had higher rates of cri, but not of laboratory-confirmed pathogens or influenza. further clinical research is required to determine the efficacy of continuous versus targeted mask use. until now, public health policy for dealing with pandemics has relied heavily on data from a modest number of often old and inadequate studies. data from the sars outbreak showed that masks reduced transmission of sars and other viral respiratory infections. 25, 26 during sars, the use of n95 respirators and medical masks was the major protective infection control measure. 27 however, the relative contribution of each type or the difference between n95 respirators and medical masks cannot clearly be determined from observational data. problems with adherence to mask ⁄ respirator use are also a potential problem. we showed that in australia, less than half of parents who were randomized to wear a medical mask or respirator while their child was ill adhered with mask wearing. 10 there may be adverse effects of wearing masks, which can reduce adherence. [28] [29] [30] our study showed significantly higher reported adverse effects of n95 respirators compared to medical masks, consistent with other studies. 28 interestingly, this population of chinese hcws reported overall similar rates of discomfort with masks as parents in our household study, 10 with higher rates in the n95 group, but it did not affect their adherence with mask ⁄ respirator wearing. this suggests that discomfort is not the primary driver of adherence, and rather, cultural acceptability and other behavioural factors may be the main reason for non-adherence. the past experience of beijing health workers with sars may also be a factor in the high adherence. this level of adherence may not translate to western cultural contexts in a normal winter season, especially for n95 respirators; however, adherence can change with perception of risk. during a pandemic, we would expect hcws to have higher adherence to infection control measures. in summary, our study adds evidence on the use of respiratory protection for hcws, but highlights the need for larger trials and comparison of different policy options. sanofi-pasteur msd on the modelling of varicella zoster virus. the remaining author(s) declare that they have no competing interests. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. prior to the start of this study, nmf acted as a consultant for roche, novartis and gsk biologicals (ceasing in 2007). pandemic potential of a strain of influenza a (h1n1): early findings nosocomial influenza infection among post-influenza-vaccinated patients with severe pulmonary diseases disruption of services in an internal medicine unit due to a nosocomial influenza outbreak a nosocomial outbreak of influenza during a period without influenza epidemic activity epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong short-and long-term effects of handwashing with antimicrobial or plain soap in the community effect of antibacterial home cleaning and handwashing products on infectious disease symptoms: a randomized, double-blind trial handwashing and risk of respiratory infections: a quantitative systematic review facemasks and hand hygiene to prevent influenza transmission in households: a randomized trial face mask use and control of respiratory virus transmission in households surgical mask vs n95 respirator for preventing influenza among health care workers: a randomized trial novel h1n1 influenza and respiratory protection for health care workers do n95 respirators provide 95% protection level against airborne viruses, and how adequate are surgical masks? comparison of performance of three different types of respiratory protection devices aerosol penetration and leakage characteristics of masks used in the health care industry is a mask necessary in the operating theatre? surgical face masks in modern operating rooms-a costly and unnecessary ritual? postoperative wound infections and surgical face masks: a controlled study influenza burden of illness: estimates from a national prospective survey of household contacts in france multilevel and longitudinal modeling using stata regression methods in biostatistics: linear, logistic, survival, and repeated measures models an introduction to categorical data analysis design and analysis of cluster randomization trials in health research feasibility exercise to evaluate the use of particulate respirators by emergency department staff during the 2007 influenza season respiratory infections during sars outbreak evaluation of control measures implemented in the severe acute respiratory syndrome outbreak in beijing effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) headaches and the n95 face-mask amongst healthcare providers the physiological impact of wearing an n95 mask during hemodialysis as a precaution against sars in patients with end-stage renal disease a stepwise resampling method of multiple hypothesis-testing we acknowledge the guidance and support we received from the staff at 3m china. this comprised assistance with fit testing for the study, but not financial support. thanks also go to the trial staff at the beijing centre for disease control and surveillance and their affiliated public health units. thanks to the staff from the 15 beijing hospitals which participated. professor raina macintyre designed the study, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. dr simon cauchemez thanks the research council, uk, and neil ferguson thanks the medical research council for centre funding. professor c. raina macintyre: as the lead investigator raina macintyre was responsible for conception and design of the trial, overseeing the whole study, analysing data and writing the report; professor quanyi wang: implementation, contribution to design, analysis and drafting of paper; dr simon cauchemez: statistical analysis and drafting of paper; dr holly seale: study design, form ⁄ database development, monitoring and review of paper; professor dominic e dwyer: study design, clinical and laboratory technical assistance and drafting of paper; dr peng yang: project manager; dr weixian shi: laboratory testing in china; dr zhanhai gao: statistical analysis and drafting of paper; dr xinghuo pang: recruitment and training; dr yi zhang: database management and analysis; dr xiaoli wang: database management and analysis; dr wei duan: recruitment and training; dr bayzidur rahman: statistical analysis and drafting of paper; professor neil ferguson: statistical analysis and drafting of paper. professor raina macintyre: raina macintyre receives funding from influenza vaccine manufacturers gsk and csl biotherapies for investigator-driven research. she has also been on advisory boards for wyeth, gsk and merck. dr simon cauchemez received consulting fees from key: cord-311884-3lkkpg9g authors: paz–bailey, gabriela; quandelacy, talia m.; adams, laura e.; olsen, sonja j.; blanton, lenee; munoz-jordan, jorge l.; lozier, matthew; alvarado, luisa i.; johansson, michael a. title: recent influenza activity in tropical puerto rico has become synchronized with mainland us date: 2020-07-02 journal: influenza other respir viruses doi: 10.1111/irv.12744 sha: doc_id: 311884 cord_uid: 3lkkpg9g background: we used data from the sentinel enhanced dengue surveillance system (sedss) to describe influenza trends in southern puerto rico during 2012‐2018 and compare them to trends in the united states. methods: patients with fever onset ≤ 7 days presenting were enrolled. nasal/oropharyngeal swabs were tested for influenza a and b viruses by pcr. virologic data were obtained from the us world health organization (who) collaborating laboratories system and the national respiratory and enteric virus surveillance system (nrevss). we compared influenza a and b infections identified from sedss and who/nrevss laboratories reported by us department of health and human services (hhs) region using time series decomposition methods, and analysed coherence of climate and influenza trends by region. results: among 23,124 participants, 9% were positive for influenza a and 5% for influenza b. influenza a and b viruses were identified year‐round, with no clear seasonal patterns from 2012 to 2015 and peaks in december‐january in 2016‐2017 and 2017‐2018 seasons. influenza seasons in hhs regions were relatively synchronized in recent years with the seasons in puerto rico. we observed high coherence between absolute humidity and influenza a and b virus in hhs regions. in puerto rico, coherence was much lower in the early years but increased to similar levels to hhs regions by 2017‐2018. conclusions: influenza seasons in puerto rico have recently become synchronized with seasons in us hhs regions. current us recommendations are for everyone 6 months and older to receive influenza vaccination by the end of october seem appropriate for puerto rico. influenza is a significant public health problem globally. seasonal influenza has a high disease burden, reducing school attendance, increasing worker absenteeism and impacting daily productivity. 1 since 2010, cdc estimates that influenza is associated with between 9.3-49 million illnesses, 140 000-960 000 hospitalizations and 12 000-79 000 deaths each year in the united states (us). 2 though antivirals treat infection, vaccination remains the primary tool to prevent influenza-associated morbidity and mortality. 3 the us advisory committee on immunization practices (acip) recommends routine annual influenza vaccination for individuals, aged ≥6-months who do not have contraindications. 4 influenza immunization programmes must consider disease seasonality to most effectively use immunization. 5 in high-income temperate countries, influenza has been well described. most seasonal epidemics in temperate regions occur during the winter months, between november and march in the northern hemisphere and between april and september in the southern hemisphere. 6 these seasonal patterns are thought to be driven by annual changes in climate, contact rates, human immunity and other factors. 1, 7, 8 some tropical and subtropical regions experience annual epidemics coinciding with local rainy seasons, 9 whereas others have semi-annual epidemics or yearround influenza activity without well-defined influenza seasons. 10, 11 influenza seasonality in tropical and subtropical regions is less understood. 9 few studies examined influenza in puerto rico, and none figure s1 ). we also explored coherence between climate and influenza trends for puerto rico and hhs regions, to determine whether climatic differences explained differences in influenza trends. figure s1 ). 1 we compared the number of confirmed influenza a and b cases in sedss to virologic surveillance data collected by us who and to explore if differences in seasonality between puerto rico and hhs regions were due to differences in climate, we obtained regional climate data (absolute humidity, temperature and precipitation) from 2012 to 2018 from the north american regional reanalysis data set from the national oceanic and atmospheric administration (noaa). 18 daily climate data were temporally and spatially aggregated to weekly means for each hhs region and puerto rico. weekly mean time series data were normalized. in order to analyse the possible relationship to climate, we extended the timeframe considered for the seasonal component to 40-80 weeks to include potential climatic trends that might occur outside the typical seasonal periodicity. for each region, we used the morlet wavelet decomposition to extract the major seasonal component (period: 40-80 weeks) for weekly influenza cases and the weekly mean absolute humidity, temperature and precipitation. the us cdc funded the study, and participated in the study design, data analysis, data interpretation and preparation of the manuscript. all authors had full access to study data, and all authors had final responsibility for the decision to submit for publication. (table 1) . overall, 14% of participants were positive for influenza; 9% for influenza a and 5% for influenza b viruses. although there were no differences by gender, the percentage of participants with positive results for influenza a or b viruses differed by age (p < .001) and days after symptom onset (p < .001; table 2 ). we compared the time series and peak timing of overall influenza and type-specific patterns in puerto rico to the hhs regions. in puerto rico (sedss), influenza peaks varied substantially be(table s1 ). similar coherence patterns are also observed when looking at influenza a and b. to investigate possible climatic drivers of influenza a and b epidemics patterns within each geographic region, we assessed the coherence and phase differences between location-specific climate variables and type-specific influenza incidence (figure 2a -i). in terms of the overall time series climatic trends, puerto rico was consistently warmer and more humid over the time series compared with weekly average absolute humidity and temperature in hhs regions (figure 2a 19, 20 and epidemiological studies showed lower humidity is associated with the onset of influenza epidemics in the united states. 21 increased proximity between susceptible and infected hosts associated with cold weather is also frequently suggested as an important driver of influenza seasonality. 8 we acknowledge funding provided by the centers for disease control and prevention. we would like to thank the contribution of all study participants. we would also like to acknowledge the work of olga lorenzi and her support to this project. the authors report no conflict of interest. gpb designed the study, contributed to the analyses, participated in the interpretation of results and wrote the first draft of the paper. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. additional supporting information may be found online in the supporting information section. global influenza seasonality: reconciling patterns across temperate and tropical regions disease burden of influenza a review of the evidence to support influenza vaccine introduction in countries and areas of who's western pacific region prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices-united states epidemiological, antigenic and genetic characteristics of seasonal influenza a(h1n1), a(h3n2) and b influenza viruses: basis for the who recommendation on the composition of influenza vaccines for use in the 2009-2010 northern hemisphere season global epidemiology of influenza: past and present influenza seasonality: lifting the fog influenza seasonality: underlying causes and modeling theories influenza in the tropics virologically confirmed population-based burden of hospitalization caused by influenza a and b among children in hong kong timing of influenza epidemics and vaccines in the american tropics enhanced surveillance for fatal dengue-like acute febrile illness in puerto rico differential diagnosis of respiratory viruses by using real time rt-pcr methodology detection of influenza by real time rt-pcr is not affected by delays in respiratory specimen processing world health organization. the use of pcr in the surveillance and diagnosis of influenza world health organization a practical guide to wavelet analysis synchrony, waves, and spatial hierarchies in the spread of influenza earth system research laboratory's physical sciences division (psd) infectivity of influenza virus aerosols influenza virus transmission is dependent on relative humidity and temperature absolute humidity modulates influenza survival, transmission, and seasonality seasonal trends of viral respiratory tract infections in the tropics estimating the impact of school closure on influenza transmission from sentinel data seasonality of respiratory viruses causing hospitalizations for acute respiratory infections in children in nha trang, vietnam urbanization and humidity shape the intensity of influenza epidemics in u correction: continental synchronicity of human influenza virus epidemics despite climatic variation divergent evolutionary trajectories of influenza b viruses underlie their contemporaneous epidemic activity national, regional, and state level outpatient illness and viral surveillance centers for disease control and prevention. 2013-14 influenza season vaccination coverage estimates for local areas and territories key: cord-317533-xpfqdeqv authors: smuts, heidi title: human coronavirus nl63 infections in infants hospitalised with acute respiratory tract infections in south africa date: 2008-07-24 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2008.00049.x sha: doc_id: 317533 cord_uid: xpfqdeqv background human coronavirus nl63 (hcov‐nl63) is a novel respiratory virus which is associated with respiratory tract infections in children. objective to determine the role of hcov‐nl63 in infants and young children hospitalised with acute respiratory tract infections (ari) in cape town, south africa. methods respiratory specimens were collected from 1055 infants and young children hospitalised with ari in 2003–2004. samples were screened by rt‐pcr to detect hcov‐nl63 and human metapneumovirus (hmpv). standard shell vial culture and immunofluoresence was used to detect the common respiratory viruses including rsv, influenza a and b viruses, parainfluenza viruses 1, 2, 3, adenovirus and cmv. results a respiratory virus was found in 401/1055 (38·0%) samples. hcov‐nl63 was detected in 9/1055 (0·85%) with peak activity during autumn (67%). most patients had a diagnosis of pneumonia or lower respiratory tract infection (6/9; 67%). conclusions this is the first report of hcov‐nl63 infections in hospitalised children in africa. during the 2‐year period hcov‐nl63 played a minor role in ari in children. a number of respiratory viruses including influenza viruses, respiratory syncytial virus (rsv), parainfluenza viruses, adenovirus and the recently described human metapneumovirus (hmpv) play an important role in acute respiratory tract infections (ari) in children. infections with these viruses may often lead to hospitalisation. however, in a substantial portion of respiratory infections the aetiological agent is not known. there has been renewed interest in human coronaviruses (hcov) as a cause of some of these infections. coronaviruses are large enveloped single-stranded rna viruses that can infect both humans and a variety of domestic animals causing respiratory and enteric illness. until recently human coronavirus (hcov) 229e and oc43, identified in the 1960s, 1 were the only known coronaviruses to infect humans. although primarily responsible for mild infections including the common cold 2 reports of more severe upper and lower respiratory tract infections associated with hcov-229e and hcov-oc43 have been documented. 3, 4 the identification of a coronavirus, sars-cov, as the causative agent of severe acute respiratory syndrome in 2003 5 has resulted in an increased interest in this group of viruses. subsequently two new human coronaviruses, hcov-nl63 6,7 and hcov-hku1, 8 have been described. both infect young children, the elderly and immunocompromised and can lead to severe respiratory tract infections requiring hospitalisation. the prevalence and clinical importance of hcov-nl63 in the south african hospital setting is not known. in this retrospective study 1055 nasopharyngeal, tracheal aspirate and bronchoalveoloar lavage samples were taken from children (age 13 days to 5 years) hospitalised with respiratory tract infections in 2003 and 2004 in the red cross war memorial children's hospital in cape town. croup as a specific diagnosis was not reported for any of these children. samples had previously been screened by using an indirect immunofluorecence assay (light diagnostics, chemicon international, temecula, ca, usa) for the common respiratory viruses including rsv, influenza viruses a and b, parainfluenza viruses 1, 2, 3, adenovirus and cmv. hmpv was also tested for using reverse-transcription-pcr (rt-pcr). in the south african setting, where the prevalence of hiv is high, all infant respiratory samples are routinely screened for cmv as in our setting this virus is a major cause of pneumonia in hiv-infected children. for the detection of hcov-nl63, rna was extracted from 200 ll of sample using the seek viral rna kit according to the manufacturer's instructions (talent, trieste, italy). ten microlitres rna was reverse transcribed into cdna using random primers (roche diagnostics gmbh, penzberg, germany) and the iscript kit (bio-rad, hercules, ca, usa). pcr amplification of a region of the 1b gene of hcov-nl63 was used for screening and positive samples were confirmed by amplification of a portion of the 1a gene. briefly 10 ll of cdna was added to a 50 ll of pcr mix containing 2 iu supertherm polymerase (jmr holdings, kent, uk), 1ae5 mm mgcl 2 , 200 lm each dntp and 0ae2 lm primers. pcr was performed with two sets of outer primers, one set to the 1b gene and one targeting the 1a gene from the study of van der hoek et al. 6 to improve sensitivity a nested pcr was performed using 2ae5 ll outer product and inner primers described by smuts et al. 9 the 1b and 1a pcr products were 169 and 520 bp respectively. the 1a amplicons were sequenced directly and the nucleotide sequences were deposited into genbank (eu477762-eu477770). a respiratory virus was detected in 401 ⁄ 1055 (38ae0%) samples collected over the 2-year period from 2003 to 2004. the detection rate was higher in 2004, 248 ⁄ 559 (44ae4%), compared with 2003, 153 ⁄ 496 (30ae8%). cmv was most frequently found (158 ⁄ 1055; 15ae0%) followed by adenovirus (n = 65; 6ae2%), rsv (42; 4ae0%), parainfluenza 3 (n = 32; 3ae0%), hmpv (n = 28; 2ae6%), influenza virus a (n = 8; 0ae76%), parainfluenza virus 1 (n = 6; 0ae57%), parainfluenza virus 2 (n = 4; 0ae38%) and influenza virus b (n = 1; 0ae09%). in 48 (4ae5%) samples a known respiratory virus was grown in shell vial culture but could not be further identified. of cmv-positive respiratory samples 44ae9% (71 ⁄ 158) were from hiv-infected children, 24ae0% (38 ⁄ 158) from hiv-negative children and for the remainder the hiv status was unknown. in both hiv-positive and hiv-negative groups the rates of co-infection with another known respiratory virus were similar, 12 ⁄ 71 (16ae9%) and 7 ⁄ 38 (18ae4%) respectively. hcov-nl63 was detected in 4 ⁄ 496 (0ae81%) and 5 ⁄ 559 (0ae89%) samples from 2003 and 2004 respectively. all hcov-nl63-infected children, with the exception of one aged 30 months, were under 2 years ( table 1 ). the majority, 6 ⁄ 9 (66ae7%), were <6 months. the hiv status of only those children from 2004 was known and 4 ⁄ 5 hcov-nl63-infected children from this year were hiv-positive. in two instances a co-pathogen was identified, hmpv and adenovirus. in 2003 3 ⁄ 4 positive samples were collected in march (autumn) while in 2004 hcov-nl63-positive samples were found in march, may, august and september (table 1) . a diagnosis of pneumonia or lower respiratory tract infection was made in six (67%) children. two hcov-nl63-positive infants aged 50 and 71 days respectively, required admission to the intensive care unit; both were hiv-positive. to determine the role hcov-nl63 plays in respiratory illness in infants, respiratory samples that had previously been screened for rsv, influenza viruses a and b, parainfluenza viruses 1, 2, 3, adenovirus, hmpv and cmv were also tested by rt-pcr for hcov-nl63. due to limited resources, screening for other viruses such as enteroviruses, rhinoviruses and the other hcovs was not undertaken. this may be considered a limitation of the study as there is accumulating evidence that these viruses, in particular rhinoviruses, may play a more significant role in lower respiratory tract infections than previously recognised. 10 the role and clinical significance of the recently identified human bocavirus 11 and polyomaviruses 12, 13 in respiratory disease is still under investigation. ideally comprehensive screening of respiratory samples for most if not all respiratory viruses and relevant respiratory bacteria should be undertaken in order to obtain greater insight into the epidemiological significance of these pathogens in respiratory disease in the local setting. further this would be beneficial to the clinical management of patients, including the administration of appropriate antiviral drugs and antibiotics. cmv was the most prevalent virus detected in the study samples. cmv-pneumonia is an important life-threatening complication in hiv-infected infants in south africa. a post-mortem study of hiv-infected children in kwazulu-natal, south africa showed frequent (52%) cmv detection in lung tissue compared with uninfected controls (4%). 14 the significance of cmv detection in respiratory samples from uninfected children is not known but probably represents viral shedding from either a recently acquired primary infection or reactivation. this study is the first to report the presence of hcov-nl63 in children hospitalised with respiratory illness in africa. hcov-nl63 was found to circulate in infants and young children in both 2003 and 2004 with similar low prevalence rates of 0ae8%. this finding is lower than that reported in previous studies where detection of hcov-nl63 ranged from 1% to 7ae3% 6,7,15-20 although higher prevalences of 8ae8% and 9ae3% have also been reported. 21, 22 most hcov-nl63-positive children (75%) were under 6 months, indicating that this younger age group may be more susceptible to severe infections requiring hospitalisation; a finding supported by other studies where 38 ⁄ 76 (50%), 4 ⁄ 5 (80%) and 11 ⁄ 12 (92%) of hcov-nl63 infections occurred in this age group respectively. 7, 19, 21 further, immunosuppression may also contribute to increased susceptibility to severe hcov-nl63 infection. in this study 4 ⁄ 5 hcov-nl63-infected whose hiv status was known, were hiv infected. all four were <4 months of age and two required admission to icu. it is not known whether maternal antibodies, if present, would provide protection or reduce the severity of infection. this protection is likely to be most effective in infants under 2 months. in this study very few infected infants were under this age, indicating a possible protective advantage. this observation is supported by the findings of other studies. 7, [15] [16] [17] in a very recent study by dijkman et al., hcov-nl63 specific maternal antibodies were found in all newborns studied. these antibodies disappeared within 3 months providing further evidence of their possible protective role in early life. in a recently published study 9 of ambulatory children presenting with acute wheezing at the outpatient's department of the same hospital in 2004, 3ae6% (3 ⁄ 83) showed evidence of hcov-nl63 infection. this is significantly different (p = 0ae0186) from the prevalence in the hospitalised group. this indicates that the virus is circulating in the community, probably causing mild symptoms which may trigger a wheezing episode requiring medical attention. in contrast the lower rate of hcov-nl63 infection identified in the hospitalised children suggests that the virus rarely causes severe lower respiratory tract infections. however, all hcov-infected children in the ambulatory group were over 12 months of age supporting the possibility that younger children are more susceptible to severe infections requiring hospitalisation. hcov-nl63 infections appear to be seasonal; 67% of infections occurred during autumn. no hcov-nl63 infection was detected during the winter months in either 2003 and 2004, a finding also noted in the ambulatory population 9 this pattern differs from that previously reported where hcov-nl63 infection was predominantly found during the winter season. 6, [15] [16] [17] [18] [19] 21, 22 detection during early spring and summer indicates that hcov-nl63 may circulate at low levels throughout the year. in this study the clinical symptoms of hcov-nl63infected infants were similar to those previously reported with lower respiratory tract infections including pneumonia predominating in this group. 6, 7, 15, 17, 20, 21 in this study only specimens from hospitalised children were examined resulting in a bias towards children with more severe respiratory illness. mild hcov-nl63 symptoms have also been reported 9, 16, 23 indicating that hcov-nl63 infections are probably more severe in the very young and immunocompromised. the role of hcov-nl63 in enteric disease has not been established but reports of gastroenteritis associated with coronavirus infection have been documented with frequencies ranging from 6% to 33%. 15, 17, 20, 22 in this study one child had a history of gastroenteritis and vomiting. a significant association with hcov-nl63 infection and croup has been made 23 but in this study it could not be determined if any of the samples were from children with croup. in conclusion these findings suggest that although hcov-nl63 is circulating in the community it plays a minor role in severe respiratory tract infections in young children who require hospitalisation. ethical approval (018 ⁄ 2004) was granted by the research ethics committee of the faculty of health sciences, university of cape town, south africa. coronaviridae: the viruses and their replication isolation of rhinoviruses and coronaviruses from 38 colds in adults frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infections by use of a novel real-time reverse-transcription polymerase chain reaction coronavirus hku1 and other coronavirus infections in hong kong a novel coronavirus associated with severe acute respiratory syndrome identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans characterisation and complete genome sequence of a novel coronavirus hku1 from patients with pneumonia role of human metapneumovirus, human coronavirus nl63 and human bocavirus in infants and young children with acute wheezing rhinovirus-associated hospitalisations in young children cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a third polyomavirus identification of a novel polyomavirus from patients with acute respiratory tract infections pneumocystis carnii and cmv infections in severely ill hiv-infected african children new human coronavirus, hcov-nl63, associated with severe lower tract disease in australia human coronavirus nl-63 infections in children: a 1-year study a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl63 in children hospitalised with respiratory tract infections in belgium human coronavirus nl63 and 229e seroconversion in children detection of human coronavirus nl63, human metapneumovirus and respiratory syncytial virus in children with respiratory tract infections in southwest sweden clinical disease in children associated with newly described coronavirus subtypes evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children human coronavirus nl63 croup is associated with the novel coronavirus nl63 the author thanks the staff of the virology diagnostic laboratory for performing the shell vial culture and immunofluorescence and dr diana hardie (division medical virology ⁄ national health laboratory service, university of cape town) for critical reading of the manuscript. this study was funded by the poliomyelitis research foundation (grant number 05 ⁄ 09). key: cord-316956-nnqi0dj1 authors: gamiño‐arroyo, ana e.; moreno‐espinosa, sarbelio; llamosas‐gallardo, beatriz; ortiz‐hernández, ana a.; guerrero, m. lourdes; galindo‐fraga, arturo; galán‐herrera, juan f.; prado‐galbarro, francisco j.; beigel, john h.; ruiz‐palacios, guillermo m.; noyola, daniel e. title: epidemiology and clinical characteristics of respiratory syncytial virus infections among children and adults in mexico date: 2016-08-18 journal: influenza other respir viruses doi: 10.1111/irv.12414 sha: doc_id: 316956 cord_uid: nnqi0dj1 background: respiratory syncytial virus (rsv) is a leading etiological agent of acute respiratory tract infections and hospitalizations in children. however, little information is available regarding rsv infections in latin american countries, particularly among adult patients. objective: to describe the epidemiology of rsv infection and to analyze the factors associated with severe infections in children and adults in mexico. methods: patients ≥1 month old, who presented with an influenza‐like illness (ili) to six hospitals in mexico, were eligible for participation in the study. multiplex reverse‐transcriptase polymerase chain reaction identified viral pathogens in nasal swabs from 5629 episodes of ili. patients in whom rsv was detected were included in this report. results: respiratory syncytial virus was detected in 399 children and 171 adults. rsv a was detected in 413 cases and rsv b in 163, including six patients who had coinfection with both subtypes; 414 (72.6%) patients required hospital admission, including 96 (16.8%) patients that required admission to the intensive care unit. coinfection with one or more respiratory pathogens other than rsv was detected in 159 cases. young age (in children) and older age (in adults) as well as the presence of some underlying conditions were associated with more severe disease. conclusions: this study confirms that rsv is an important respiratory pathogen in children in mexico. in addition, a substantial number of cases in adults were also detected highlighting the relevance of this virus in all ages. it is important to identify subjects at high risk of complications who may benefit from current or future preventive interventions. respiratory syncytial virus (rsv) is the major infectious cause of lower respiratory tract illness in infants and young children around the world. 1,2 it has also been recognized as an important etiologic agent of pneumonia and other respiratory tract infections in adults and elderly patients. 3, 4 the clinical presentation of this infection varies widely, from mild upper respiratory tract disease to bronchiolitis and pneumonia. 5 this virus is responsible for the majority of bronchiolitis cases and causes approximately 50% of pneumonia cases during the first years of life. 6 in children, host factors such as young age, prematurity, and chronic cardiopulmonary diseases have been associated with severe disease. in addition, other factors such as lower socioeconomic status, exposure to cigarette smoke, air pollution, crowded households, and the lack of breastfeeding have also been associated with severe disease. 7 viral factors associated with virulence leading to severe disease are not sufficiently understood. 8 human rsv is a member of the paramyxoviridae family. outbreaks of rsv infections occur between fall and spring in temperate climates and tend to last up to 5 months. 9,10 rsv isolates can be divided into two groups: group a and group b based on antigenic and genetic characteristics. 8 these two groups cocirculate in the human population, with group a being more prevalent. several studies have compared the severity of disease between infants infected with rsv group a and group b with mixed results. most studies have not found significant clinical differences between both subtypes. 8 despite the recognized importance of rsv as a cause of respiratory illness, the information regarding the epidemiology of this virus in latin america, particularly among adults, is limited. 11 in the present study, 570 cases of rsv infection identified during four epidemic years in mexico were evaluated to clarify the epidemiology of this infection and to assess the possible variations in demographic and clinical characteristics according to viral groups. results from patients enrolled during the first year of the study have previously been described. 6 in this report, we analyzed the characteristics of 570 patients (399 children and 171 adults) with confirmed rsv infection included in the study during a 4-year period. patients ≥1 month old who presented with an ili to any of the participating hospitals were eligible for participation in the study. ili was defined by the presence of at least one respiratory symptom (e.g., shortness of breath, nasal congestion, and cough) and one of the two following criteria: (i) fever ≥38°c on examination, or self-reported fever, or feverishness in the past 24 hours; (ii) one or more nonrespiratory symptoms (e.g., malaise, headache, myalgia, or chest pain). in order to rule out a nosocomial infection, patients who had been hospitalized for more than 48 hours at the time of symptom onset were excluded from the study. subjects were interviewed and examined at the time of enrollment, a nasopharyngeal swab was obtained for the detection of respiratory pathogens, and a blood sample for the complete blood counts and chemistry analysis were obtained. when available, the results of other tests obtained for standard clinical care were extracted from medical records. subjects were evaluated again on day 28 after enrollment, and follow-up information was also obtained by a telephone call on day 14 after the enrollment. at each follow-up visit, clinical information (symptoms, rehospitalizations, and death) was assessed. these visits allowed the ascertainment of the final disposition of the study patients (outpatient, emergency room, hospital ward, or intensive care unit [icu]). nasopharyngeal swab specimens were collected from enrolled patients, placed in a tube with viral transport media, and maintained under refrigeration until they were sent to the molecular biology verbal consent was obtained from parents/legal tutor and subjects before screening to determine eligibility. once a patient was determined to be eligible for study participation, written informed consent and assent (for children older than 8 years) were obtained. the protocol was approved by the institutional review board of each hospital. means and standard deviation were used to summarize the quantitative variables, while frequencies and percentages were used for the qualitative variables. comparisons between groups were made using student's t-test for quantitative variables and the chi-squared or fisher's exact test for qualitative variables. multivariate logistic regression analysis was used to determine the factors associated with hospitalization and icu admission. data were analyzed with the use of pspp and openepi. a p value <.05 was considered as statistically significant. between 2010 and 2014, there were 5662 subjects enrolled in the ili-002 study; 33 patients did not have respiratory samples available f i g u r e 1 respiratory syncytial virus infections in subjects included during four seasons in the ili-002 study f i g u r e 2 respiratory syncytial virus seasonality according to the viral subtype and presence of coinfection for viral testing, and therefore, the final sample size for analysis was 5629 subjects. of these subjects, there were 570 (10.7%) cases with rsv infection: 407 with rsv a infection, 157 with rsv b, and six in whom both rsv a and b were detected (fig. 1) . rsv cases were detected throughout the study period. however, a marked seasonality was observed with a high number of cases during the fall and winter and a decrease in detections during spring and summer (fig. 2) . rsv a was the predominant virus between 2010 and 2012, but in seasons between 2012 and 2014, both rsv subtypes circulated simultaneously. the characteristics of patients with rsv infection were compared to those of all other patients enrolled in the ili-002 study ( table 1) . the age group affected more frequently by rsv was that of children 0-5 years of age; 66.84% of rsv infections occurred in children in this age group, while 30% occurred in adults and 3.16% in children 6-17 years of age; 24.6% (381/1550) of respiratory tract infections in children <5 years were caused by rsv as compared to 4.7% (18/379) of children older than 5 years and 4.6% (171/3700) of adults. also, the proportion of cases seen as outpatients was lower in patients with rsv infection compared to those in which this virus was not detected, while the proportion of icu admissions was higher (table 1) . in 159 (28%) patients, another pathogen was detected in addition to rsv. in 136 of these patients, there was one additional pathogen detected, while in 22 cases there were two, and in one case, there were three; these included rhinovirus (n=61), coronaviruses (n=30), parainfluenza viruses (n=17), influenza a (n=17), influenza b (n=12), adenovirus (n=12), metapneumovirus (n=11), bordetella pertussis (n=11), mycoplasma (n=6), and bocavirus (n=6). the proportions of infections caused by each rsv subtype and the presence of coinfections, according to the age group, are shown in fig. 3 . we compared the characteristics of infections caused by rsv a and rsv b to assess whether there were significant clinical differences between them (table 2) ; the six cases with coinfection with rsv a and b were excluded from this analysis. we did not find any differences in the medical history between patients with rsv a and rsv b infections. we also compared the frequency of each symptom between patients with rsv a and rsv b infections; no significant differences were observed for any of the symptoms that were recorded (including fever, cough, sore throat, fatigue, headache, myalgias, eye symptoms, sneezing, rhinorrhea, shortness of breath, nausea, vomiting, diarrhea, confusion, malaise, and irritability) (data not shown). in addition, we compared the clinical characteristics of patients with and without coinfection with other pathogens (table 3) . differences in the age group distribution were noted between cases with and without coinfection with other pathogens: the proportion of pediatric cases was higher for those infections caused by rsv only, while a larger proportion of coinfections was noted in adults. most symptoms were as frequent in patients with infections caused by rsv only compared to those with coinfections (data not shown); however, there were differences in the frequencies of some symptoms: we present the characteristics of rsv infections detected in a prospective, multicenter study of children and adults seeking clinical care. our data obtained over four seasons provide a description of the epichildren with rsv infection in whom other pathogens were detected were hospitalized less frequently than those infected by rsv only. however, among hospitalized children, the presence of another respiratory pathogen was associated with admission to the icu. in order to interpret these results, it is important to take into account that there was a wide variety of coinfecting pathogens and that in some cases two and even three viruses, in addition to rsv, were detected. one of the main objectives of the study was to identify the factors associated with severe disease leading to rsv hospitalization in our country. approximately 40% of subjects with rsv infection that were enrolled in the study had at least one underlying disorder. the most frequent conditions included congenital malformations/congenital syndromes, cardiovascular disorders, chronic lung disease, and asthma. we observed significant differences in the factors associated with hospitalization between children and adults; while multivariate logistic regression analysis indicated that young age was the main variable associated with hospitalization in children, adults showed an association between additional factors and the requirement of hospitalization. in this age group, multivariate logistic regression analysis showed an association between rsv infection and age, the presence of any underlying condition, and asthma. in their global study of respiratory tract viral infections in elderly adults, falsey et al. 15 found that congestive heart failure, other cardiopulmonary diseases, and noninflammatory cerebrovascular/neurological disorders appeared to be associated with rsv infection; however, on multivariate analysis, no specific factors were associated with this virus. in the group of children, multivariate logistic regression analysis showed an association between the risk of icu admission and the presence of coinfection with other respiratory pathogens, cardiovasother limitations include the fact that the presence of underlying conditions (including history of prematurity and other chronic diseases) was based on patient self-report, which might not be totally reliable. also, we did not collect data on palivizumab use which might be relevant in infants; however, access to this prophylaxis is limited in mexico because of the high cost and, therefore, the number of patients that may have received it is likely to be low. on the other hand, strengths of our study include the participa instituto nacional de pediatría: beatriz llamosas-gallardo hospital infantil de méxico federico gómez juana del carmen báez national institute of allergies and infectious disease (niaid): cliff lane, mary smolskis, dean follmann, sally hunsberger la red is funded by the mexico ministry of health and the u.s. national institute of allergy and infectious diseases viral etiology of hospitalized acute lower respiratory infections in children under 5 years of age -a systematic review and meta-analysis respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults rates of hospitalizations for respiratory syncytial virus, human metapneumovirus and influenza virus in older adults respiratory syncytial virus infection: immune response, immunopathogenesis, and treatment clinical characteristics and outcomes of influenza and other influenza-like illnesses in mexico city performance of different mono-and multiplex nucleic acid amplification tests on a multipathogen external quality assessment panel viral and host factors in human respiratory syncytial virus pathogenesis global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis respiratory syncytial virus epidemics: the ups and downs of a seasonal virus systematic review and meta-analysis of respiratory syncytial virus infection epidemiology in latin america world health organization. cdc protocol of realtime rtpcr for swine influenza a(h1n1) viral infections in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation respiratory syncytial virus-and human metapneumovirus-associated emergency department and hospital burden in adults respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness respiratory syncytial virus pneumonia among the elderly: an assessment of disease burden respiratory syncytial virus: how, why and what to do associations between co-detected respiratory viruses in children with acute respiratory infections viral coinfection in childhood respiratory tract infections pre-existing disease is associated with a significantly higher risk of death in severe respiratory syncytial virus infection epidemiological and clinical data of hospitalizations associated with respiratory syncytial virus infection in children under 5 years of age in spain: five multicenter study respiratory syncytial virus genotypes and disease severity among children in hospital correlation between respiratory syncytial virus genotype and severity of illness sequencing and analysis of globally obtained human respiratory syncytial virus a and b genomes epidemiology and clinical characteristics of respiratory syncytial virus infections among children and adults in mexico ili-002 study principal investigators, coprincipal investigators, study staff, niaid, leidos and westat staff include the following: instituto nacional de ciencias médicas y nutrición salvador zubirán: guillermo d. e. noyola has participated as a member of the speakers' bureau of abbvie. the rest of the authors declare that they have no relevant conflicts of interest to report. key: cord-324012-q2ilk6gs authors: inui, ken; nguyen, tung; tseng, hsin‐jou; tsai, chuanfu mark; tsai, yun‐long; chung, simon; padungtod, pawin; zhu, huachen; guan, yi; kalpravidh, wantanee; claes, filip title: a field‐deployable insulated isothermal rt‐pcr assay for identification of influenza a (h7n9) shows good performance in the laboratory date: 2019-09-05 journal: influenza other respir viruses doi: 10.1111/irv.12646 sha: doc_id: 324012 cord_uid: q2ilk6gs background: avian influenza a (h7n9) remains circulating in china. for countries at risk of introduction of h7n9, such as vietnam, early detection of h7n9 virus is essential for the early containment of the virus. insulated isothermal reverse transcriptase pcr (iirt‐pcr) is a portable pcr system that can be deployed under field conditions to identify pathogens at the sampling site. applying pcr at the sampling site will greatly reduce the time to obtain a diagnostic result which allows the veterinary authority to take immediate action to contain disease spreading. objective: to determine analytical and diagnostic sensitivity and specificity of the portable iirt‐pcr for h7n9 virus detection. methods: a panel of 59 virus isolates, including h7n9, avian influenza viruses of subtype h1 to h13, swine and human influenza viruses, newcastle disease virus, and infectious bursal disease virus, were tested by h7 and n9 iirt‐pcr reagents, using probes and primers specific to h7 or n9, in comparison with laboratory‐based real‐time rt‐pcr assays to determine analytical sensitivity and specificity. fifty oropharyngeal samples from experimentally infected chicken and ducks with h7n9 and 50 non‐infected control swabs were tested by the h7 iirt‐pcr to determine diagnostic sensitivity and specificity. results: the h7 and n9 iirt‐pcr reagents yielded comparable levels of analytical sensitivity and specificity with real‐time rt‐pcr for the detection of h7n9 virus. diagnostic sensitivity and specificity of h7 iirt‐pcr were 98% and 100%, respectively. conclusion: the observed high sensitivity and specificity of iirt‐pcr for h7n9 detection show its potential for early detection of h7n9 in risk‐based surveillance. the current avian influenza surveillance in vietnam relies on the testing of samples at regional animal health offices (raho's) or the national centre for veterinary diagnosis (ncvd). the average time to obtain a laboratory result is 2.5 days, with the majority of this time being attributed to sample shipment and transport to the laboratories (6-24 hours on average). novel technologies, such as the insulated isothermal pcr (iirt-pcr), are portable pcr systems that can be applied under field conditions by the fast pcr reaction (in 42 minutes). the iirt-pcr portable system is a miniature portable device for field test, using freeze-dried thermostable reagents and powered by rechargeable lithium batteries. the iirt-pcr applies the concept of rayleigh-benard convection to drive a pcr reaction in capillary tubes. the primers/probe design rules are the same as for rt-pcr. while conventional pcr requires multiple cycles of heating and cooling, iirt-pcr is performed through the creation of a temperature gradient in a capillary tube with a single heating source at the bottom of a capillary tube and establishment of thermal convection within the tube, mimicking the cycles of conventional real-time pcr. this system has recently been developed to rapidly detect pathogenic viruses, including white spot syndrome virus, classical swine fever, foot and mouth disease, equine influenza, bluetongue virus, and mers-cov. [5] [6] [7] [8] [9] [10] taking advantage of the iirt-pcr portable system, the aim of this study was to validate the performance of the iirt-pcr portable system for h7n9 detection. this study used a panel of 59 virus isolates for analytical specificity that included 28 h7n9 aivs, 7 h7 aivs of eurasian lineage but not in the cluster of recent h7n9 virus in china, 15 non-h7 aivs, seven non-h7 influenza viruses of swine and human origin, two poultry viruses, newcastle disease virus, and infectious bursal disease virus (table 1) . the h7 and n9 iirt-pcr reagents yielded comparable levels of analytical sensitivity and specificity with real-time rt-pcr for the detection of h7n9 virus. diagnostic sensitivity and specificity of h7 iirt-pcr were 98% and 100%, respectively. the observed high sensitivity and specificity of iirt-pcr for h7n9 detection show its potential for early detection of h7n9 in risk-based surveillance. diagnostic, influenza a (h7n9), pcr, sensitivity, specificity a total of 50 oropharyngeal swab samples were collected from 25 chickens and 25 ducks experimentally infected with six different strains of aiv subtype h7n9. they were confirmed to be positive for aiv h7n9 by virus isolation and used as positive samples. another set of 50 oropharyngeal swabs were collected from the same chickens and ducks before infection. they were negative for aiv h7n9 by virus isolation and used as negative samples. in vitro transcribed rna (ivt rna) was generated from a plasmid con pockit™ iirt-pcr assay for aiv subtype h7 was performed by using pockit™ influenza h7 reagent set (genereach usa). the n9 iirt-pcr was developed by using the primers and probes in the rrt-pcr for avian influenza a (h7n9) described by who without any modifications. 11 the primers and probes used are listed in table 2 . iirt-pcr reagent is a single dose lyophilized format which can be shipped under ambient temperature for one week. briefly, the lyophilized reagent was reconstituted with 50 μl premix buffer b and mixed with 5 μl nucleic acid extract. subsequently, 50 μl of the final mixture was transferred to an r-tube limit of detection 95% (lod95%) of a reaction was determined by probit analysis at 95% confidence interval by spss v14 (spss). the 2 × 2 contingency tables were analyzed by kappa statistic using spss to determine the inter-assay agreement. the analytical sensitivity of aiv h7 iirt-pcr was determined by analysis. the hit rates of the aiv n9 iirt-pcr for 300, 100, 33, 10, and 0 were 100% (5/5), 58.33% (7/12), 57.14% (4/7), 0% (0/7), and 0% (0/12), with an estimated lod95 95% of 171 copies/ reaction. the analytical sensitivity of aiv h7 iirt-pcr was further analyzed using 10-fold serial dilutions (up to 10 −8 ) of representative viruses from four groups of aiv h7n9 viruses. the detection endpoint (all triplicates positive) was compared to that of rrt-pcr assays using primers and probe set of h7 coda and h7 cnic. the sensitivity of aiv h7 iirt-pcr was shown to be comparable to that of h7 coda rrt-pcr assay in detecting the 4 representative h7n9 viruses, while having at least one log higher sensitivity when compared to that of h7 cnic rrt-pcr assay ( table 3 ). the sensitivity of aiv n9 iirt-pcr was shown to be about one log lower than that of h7 coda rrt-pcr assay in detecting the four representative h7n9 viruses, and sensitivity similar to that of h7 cnic rrt-pcr assay (table 3) . the analytical specificities of the aiv h7 and n9 iirt-pcr assay a total of 50 virus isolation-positive and 50 virus isolation-negative reference samples from chickens and ducks experimentally infected with different subtype h7n9 aivs were tested by the h7 iirt-pcr and compared in a 2 × 2 table (table 4 ). using virus isolation as the gold standard, the iirt-pcr showed a sensitivity of 98% and a specificity of 100%. results from field pilot studies (unpublished data) showed that it was feasible to install the system rapidly at any given sites and that personnel in the flied could be trained in the use of the iirt-pcr system within 2 days. one of the challenges of the system is how it will be incorporated into current ongoing active surveillance designs. iirt-pcr can be complementary to current surveillance designs, yet it needs to be clear if and how results will be confirmed by standard laboratory-based tests (real-time rt-pcr, virus isolation, or sequencing) and how data sharing will occur from local levels to province or central levels. in conclusion, this study showed that field-based portable pcr (iirt-pcr) can be used for the early diagnosis of h7n9 as an alternative approach to laboratory-based real-time pcr. using field-based test will reduce the time to obtain a result and will enable possible quick response measures in the field, reducing the risk of further spread and human infections with h7n9 in currently non-infected countries. a7 evolution of influenza a(h7n9) viruses from waves i to iv reverse transcription-insulated isothermal pcr (rt-iirt-pcr) assay for rapid and sensitive detection of foot-and-mouth disease virus a rapid field-deployable reverse transcription-insulated isothermal polymerase chain reaction assay for sensitive and specific detection of bluetongue virus rapid detection of equine influenza virus h3n8 subtype by insulated isothermal rt-pcr (iirt-pcr) assay using the pockit nucleic acid analyzer evaluation and clinical validation of two field-deployable reverse transcription-insulated isothermal pcr assays for the detection of the middle east respiratory syndrome-coronavirus insulated isothermal reverse transcriptase pcr (iirt-pcr) for rapid and sensitive detection of classical swine fever virus validation of a commercial insulated isothermal pcr-based pockit test for rapid and easy detection of white spot syndrome virus infection in litopenaeus vannamei key: cord-310956-qwe4ndvb authors: qian, yan‐hua; su, jing; shi, ping; he, en‐qi; shao, jie; sun, na; zu, rong‐qiang; yu, rong‐bin title: attempted early detection of influenza a (h1n1) pandemic with surveillance data of influenza‐like illness and unexplained pneumonia date: 2011-04-18 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2011.00248.x sha: doc_id: 310956 cord_uid: qwe4ndvb please cite this paper as: qian et al. (2011) attempted early detection of influenza a (h1n1) pandemic with surveillance data of influenza‐like illness and unexplained pneumonia. influenza and other respiratory viruses 5(6), e479–e486. background to collect disease information and provide data for early detection of epidemics, two surveillance systems were established for influenza‐like illness (ili) and unexplained pneumonia (up) in wuxi, people’s republic of china. objectives the current study aims to describe the performance of these surveillance systems during 2004–2009 and to evaluate the value of surveillance data in detection of influenza epidemics. methods two national ili sentinel hospitals and three up sentinel hospitals provided data to the surveillance systems. the surveillance data from hospital‐based outpatient clinics and emergency rooms were compared by year. the ili data of 2009 were further modeled based on previous data using both a control chart method and a moving average regression method. alarms of potential epidemics would be raised when the input surveillance data surpassed a threshold. results in 2009, the proportions of ili and respiratory illness with fever (one surveillance syndrome of the up system) to total patient visits (3·40% and 11·76%, respectively) were higher than the previous years. the surveillance data of both systems also showed developing trends similar to the influenza a (h1n1) pandemic in 2009. when the surveillance data of 2009 were fitted in the two detection models, alarms were produced on the occurrence of the first local case of influenza a (h1n1), outbreaks in schools and in general populations. conclusions the results indicated the potential for using ili and up surveillance data as syndromic indicators to detect and provide an early warning for influenza epidemics. influenza, commonly known as the flu, is a severe infectious disease accounting for between 250 000 and 500 000 deaths worldwide every year. millions of people died in the influenza pandemic of 1918, one of the worst epidemic disasters in recorded human history. 1 the life style of modern society, including overcrowded cities and rapid global transportation, has resulted in dissemination of influenza and generation of novel virus strains. in the past year, the world health organization (who) announced a level-6 alert for the influenza a (h1n1) pandemic, the highest level of disease outbreak, because of the sustained spread of this new virus on a global scale. 2 the world was fortunate not to have experienced another devastating influenza epidemic, because the fatality rate of the new virus strain was similar to seasonal influenza virus. 3 however, there remains an inevitability of a new influenza pandemic, which could be highly infectious and as deadly as the 1918 influenza pandemic. thus, it is necessary, although challenging to detect the onset of a new influenza epidemic, to allow the government to make timely and proper interventions to reduce the social and economic impact from influenza epidemics. the influenza surveillance system long has been playing an essential role in collecting disease information and providing evidence for prevention and control strategies and measures. two surveillance systems were established in wuxi for influenza-like illness (ili) and unexplained pneumonia (up) after the severe acute respiratory syndrome (sars) outbreak. the ili system was implemented for influenza surveillance, and the up system was designed to track lower respiratory illness with pneumonia symptoms. although the disease surveillance systems appeared decades later than in developed countries, their performance is by no means inferior to define the distribution of circulating strains in the community and detect disease aberration. here we described and compared the accumulated ili data from 2004 to 2009 and up data from 2007 to 2009. to further evaluate the effectiveness of these surveillance systems in early warning of influenza epidemics, we monitored ili data between 2004 and 2008 by both a control chart method and the serfling method and tested goodness of fit using influenza a (h1n1) data of 2009. wuxi is a prefecture-level city of jiangsu province, located in eastern china, with a population of 6ae2 million. wuxi has seven districts and two county-level cities (jiangyin and yixing). wuxi has a typical subtropical monsoon climate and seasonal influenza usually peaks once a year. 4 there are two surveillance systems in wuxi. one is ili surveillance system and the other is up surveillance systems. both systems collected data from sentinel hospital-based outpatient clinics and emergency rooms. all the selected sentinel hospitals are general hospitals and available to every resident in wuxi city including jiangyin and yixing county. the participating doctors in referral sentinel hospitals were provided with detailed diagnosis instructions for sample selection. as required by the chinese center for disease control and prevention (cdc), two hospitals (children's hospital and the no. 3 people's hospital of wuxi) were selected as the national sentinel hospitals for ili surveillance. the number of patient visits ranged from 8000 to 12 000 ⁄ week. ili was defined as follows: fever ‡38°c, either cough or sore throat, and lack of evidence of other laboratory-confirmed diagnosis. pharyngeal swab specimens collected from patients with ili were sent to cdc laboratories for subsequent isolation and identification. the proportion of patient visits for ili was calculated as the number of patients with ili divided by the total number of patients and was reported through the influenza surveillance system of china. the up surveillance was performed in three hospitals (the no. 2 people's hospital of wuxi, the people's hospital of jiangyin county, and the people's hospital of yixing county). this system was built with special attention to lower respiratory illness, especially pneumonia caused by unknown reagent like sars. routinely collected data included respiratory illness with fever (riwf, also known as acute respiratory illness) and classified pneumonia. emergency measures would be taken in case of any up. pneumonia was diagnosed according to international classification of diseases (10th version, j00-j99). unexplained pneumonia was defined as follows: fever ‡38°c, pneumonia-like characteristics in diagnostic imaging, decreased white blood cell count or decreased lymphocyte differential count in early clinical stage, and no improvement in or even worsening of patient's condition after regular antibiotic treatment for 3-5 days. the total number of patient visits ranged from 11 000 to 14 000 ⁄ week, among whom an average of 3ae5-3ae7% were diagnosed as classified pneumonia. weekly numbers of riwf and classified pneumonia were reported to wuxi center for disease prevention and control. we retrospectively studied ili data from 2004 to 2009 and up data from 2007 to 2009, respectively. all the data were organized by microsoft office excel 2007. statistical analysis was performed by using spss software (version 17.0, spss inc., chicago, il, usa). the kolmogorov-smirnov test was used to check the normality of the data. the chisquare test was used for the comparison of different ratios and proportions. the ili data during 2004-2008 were used in statistical modeling for influenza detection. both a control chart method 5-7 and a moving average regression method 8, 9 were applied to produce the threshold value. alarms of potential epidemics were raised when the input surveillance data of 2009 surpassed the threshold. the control chart method was originally developed to determine and control the situation for manufacturing processes and was later implicated in surveillance analysis. 10 in this study, a long-term average weekly proportion of ili (p) and its standard deviation (sp) were calibrated using all the surveillance data. the data higher than p + 2sp were referred to as being out of statistical control and were excluded. such data were usually observed in epidemics of seasonal flu and could not generally represent a stable and predictable situation over time. adjusted average weekly proportion of ili (p') and standard deviation (sp') were calculated with those data in the state of statistical control. the threshold was defined as p' + 2sp'. the moving average regression method was originally proposed by serfling and was used in our study to generate a time-varying threshold for influenza. 8, [11] [12] [13] [14] a linear secular trend was estimated from the surveillance data through 2004-2008 by using the regression analysis tool of excel. the secular trend was then removed from the original surveillance data, so the adjusted data represented only seasonal change and could be compared by year. the time-varying threshold was calibrated as the 5-year moving average plus 1ae6 sd (standard deviation) (p = 0ae05) or 2ae0 sd (p = 0ae01) from the mean. the first imported case of the influenza a (h1n1) was reported on june 25, 2009 (the 26th week of the year). the first local case was reported on august 16 (week 33). the first school case was reported on september 3 (week 36), and transmission spread quickly when students came back to school in september. the influenza epidemic spread widely during september to december in the general population, with 132 confirmed cases in september, 89 cases in october, 146 cases in november, and 113 cases in december. the last identified patient was reported on february 12, 2010. the epidemic timeline is shown in figure 1 . the surveillance data collected from the two sentinel hospitals showed an earlier peak of the proportion of ili in 2009 when compared with the same period in 2007 and 2008 ( figure 2 ). the ili proportion aberrantly increased to 3ae72% in the 22nd week of 2009, higher than 3ae61% in 2007 and 2ae74% in 2008. this increase in ili proportion appeared 4 weeks earlier before the first imported case of influenza a (h1n1) and started to decline after week 28. simultaneously with the appearance of the first local case, the proportion of ili started to increase rapidly beginning in week 34, indicating the early stage of the influenza epi-demic. the proportion of ili peaked in the general population at week 39 (4ae74%), when the schools also experienced a peak of illness. figure 3 ). the proportion of riwf to total patient visits (11ae76%) was also significantly higher than that in the previous 2 years (9ae91% and 10ae14%, respectively), while the proportion of pneumonia to riwf was similar in all 3 years (3ae53-3ae69%) ( table 1 and figure 3 ). the proportion of riwf began to increase in week 32, around the time of the first imported influenza a (h1n1) case. when we examined the 2009 epidemic season, the trend of up surveillance data was similar to the trend of ili data (figures 2 and 3) . therefore, the data observed from up surveillance system may also reflect the epidemic situation of influenza. the basic design of outbreak detection is that an alert is generated when the current data surpass a threshold. therefore, determining a threshold is essential and crucial for the performance of an early warning system. to obtain the optimal sensitivity and specificity, both control chart method and moving average regression method were used to calibrate the threshold. the control chart method was used to determine an influenza threshold by week. a centerline threshold value was drawn based on the weekly proportion of ili from 2004 to 2008. when the 2009 data were fitted into the chart, alarms of aberrant higher proportions of illness appeared in week 18, week 20 to week 29, week 31 to week 41, week 45 to week 47, and week 49 (figure 4) . these results were consistent with the true observations, including the first imported case in week 26, the first local case in week 33, the outbreak in school beginning in week 36, and the outbreak peak in general population in week 45. in the moving average method, a time-varying threshold for influenza was generated. the ili counts for 2009 were higher than the moving average baseline in week 18, week 20 to week 21, week 24, and week 32 to week 50 ( figure 5 ). more precisely, the ili counts were higher than mean + 1ae6 sd in week 34 to week 37, week 39, and week 45 to week 47, and the periods from week 34 to week 37 and week 46 to week 47 showed the highest ili counts (above mean + 2ae0 sd). the aberration points were consistent with the reported date of the first local case, the outbreak in schools, and the peak in the general population. the early detection of disease outbreaks has long been important to public health. the importance of a surveillance network at the global level has been unprecedentedly emphasized because of the emergence of newly infectious diseases, pandemics, and the threats of bioterrorism. 15 the monitoring of surveillance data plays an essential role in detecting signals for a potential outbreak. for diseases or illnesses with enormous social impact, timeliness of detection is extremely valuable. with these types of diseases or illnesses, indicators like hospital consultations rather than mortality or morbidity are used in modeling, because they tend to provide earlier outbreak indication. 16, 17 for influenza, ili is commonly used as an indicator to predict the occurrence of epidemics. 13, 18, 19 unlike other diseases, the value of routinely collected ili data for influenza detection has been carefully studied and confirmed. 17, 18, 20 in this current study, weekly counts of consultations for ili and up were monitored for influenza detection. the distribution of sentinel ili data in 2009 ( figure 2) were consistent with the real spread of the pandemic and showed aberrant increase close to or earlier than the reference data. these results confirmed the capability of ili surveillance system to detect influenza. although the up surveillance system was designed to detect sars instead of influenza, the data are useful because ili and pneumonia usually have similar symptoms such as fever, cough, and breathing difficulty. compared with the reference data (figure 1 ), the sentinel up data (table 1 and figure 3 ) also showed similar epidemic trend. however, the efficiency of early detection was not high using the up surveillance system as significantly increased proportion of riwf was observed in sentinel hospitals only after the first local case was reported. a reasonable explanation might be because of the diagnostic criteria. ili was used specifically to indicate influenza, while riwf includes pneumonia and many other kinds of respiratory illnesses besides ili. the nature of the up system could not distinguish between the respiratory complaints related to influenza and those caused by other pathogens. the population at risk could be another reason for not being able to detect outbreaks because the ili sentinel sites and up sentinel sites cover different areas. nevertheless, the sentinel up data provide additional information to detect influenza epidemic and can add confidence to declare an epidemic alarm. at the beginning of the 2009 pandemic flu period, asia was in especially high alert of epidemic influenza and china implemented aggressive policies trying to delay illness spread by early identification and isolation of every confirmed h1n1 patient. the increased proportion of ili and riwf before the first imported h1n1 case could be explained as a surveillance artifact because of the heightened concern of individuals. with the development of the epidemic, control strategies were changed to intensive treatment of the seriously ill and self-isolation of mild cases. people who had mild flu symptoms were encouraged to take anti-viral medicines and stay at home instead of seeking medical care in hospitals. all the sentinel hospitals collected data as usual without any strengthened effort. the higher proportion of ili and riwf observed after week 29 was more likely due to the high incidence of h1n1 influenza in general population rather than surveillance artifact. a variety of statistical methods have been developed to monitor surveillance data, including time series, regression, cumulative sum (cusum), and hidden markov models. 9, 11, 14, [21] [22] [23] [24] the performance of these models is usually evaluated from the following characteristics: sensitivity, specificity, and timeliness. 21, 25 sensitivity is the ability to find a real outbreak alert; specificity is the ability to exclude false alarm; timeliness is the ability to raise an alarm within the shortest amount of time from the onset of the peak of the season. most approaches of early detection produce alerts based on the theory of signal detection. 25 the optimal sensitivity and specificity depend on a proper level of threshold, but there is always a trade-off between a high threshold for good specificity and a low threshold for good sensitivity. the calibration of a threshold is a complex determination for each system. both the control chart method and the serfling method are time series approaches and have their own advantages in reducing the variance caused by epidemic data or secular trend. 14, 21 the control chart method optimizes the threshold calibration by excluding the unexpected data that are beyond the controlled scope. 6 the serfling method provides reasonably accurate estimates by adjusting the threshold line with secular regression trend. 8 the us centers for disease control and prevention (atlanta, ga, usa) developed an early aberration reporting system (ears) based on cusum method, which can use only recent data and is supposed to incorporate the threshold calculation by regression method. 21, 22 however, a study in hong kong showed that the regression method and the time series method are superior to the cusum method in a small city with fewer sentinel sites, more variable data, and a more abrupt peak in activity. 14 as a result, the cusum method was not used in our study. the control chart method detected all four remarkable signals, namely the occurrence of the first imported case, the first local case, outbreaks in schools, and the peak in the general population ( figure 4) . it also produced two false-positive alarms, one in the beginning and the other at the end of the epidemic period. when using mean + 1-ae6 sd as the threshold, the serfling method detected the outbreaks in schools and the peak in the general population, with a false-positive signal in the interval; when raising the threshold to mean + 2ae0 sd, only the outbreaks in schools could be detected in a timely manner, and the second alarm was 1 week after the peak in the general population ( figure 5 ). the comparison of the two methods indicated that the control chart method had better sensitivity and timeliness and the serfling method had better specificity. nevertheless, considering that the false-positive alerts of the control method appeared outside the epidemic period, this fact might be taken in favor of a more sensitive detection. the same opinion was seen in gault's paper. 18 a final decision probably should be made combining the results of both methods. the control chart method can generate sensitive and timely alerts, and serfling method can offer secular trend to increase specificity and avoid wasting medical resources. in developing countries like china, surveillance systems like the ili and up systems have only recently been implemented. we performed this study after the 2009 pandemic flu in attempt to make full use of these routinely collected background data. the results are theoretically helpful for healthcare workers to take prevention and control measures when influenza epidemic occur, including preparing antiviral drugs and other medical supply needs, and sending health education messages. so far no such actions, which we already took during the epidemic period, have been applied based on our study. future studies will be carried out to improve the performance of these surveillance systems, such as adding more sentinel sites, covering larger population, improving the quality of indicators, and trying different detection algorithms. studies of decision making are also needed before these approaches could be used in practice. the decision theory provides mathematical methods to estimate the benefits of true alarms and the costs of false alarms for getting an optimal threshold. 25 this analysis was beyond the scope of our current study, and we will investigate such methods in our future work. in conclusion, this study described the surveillance data of ili and up during 2007-2009 and the epidemic progress of influenza a (h1n1) pandemic in wuxi, china. the surveillance data of both systems were correlated with the influenza epidemic and therefore valuable in monitoring influenza activity and generating early warning methods. the control chart method and serfling method had their respective merits and faults in data analysis. further studies could be considered to optimize the threshold of warning. the principle is to improve performance of surveillance systems and allow timely precautionary measures to be implemented in vulnerable populations. emerging infections: pandemic influenza world now at the start of 2009 influenza pandemic mortality from pandemic a ⁄ h1n1 2009 influenza in england: public health surveillance study guideline for prevention and control of influenza statistical quality control methods in infection control and hospital epidemiology, part ii: chart use, statistical properties, and research issues statistical quality control methods in infection control and hospital epidemiology, part i: introduction and basic theory study on early warning based on influenza surveillance data in guangdong province methods for current statistical analysis of excess pneumonia-influenza deaths time-series forecasting use and interpretation of statistical quality control charts influenza-associated hospitalizations in the united states impact of influenza vaccination on seasonal mortality in the us elderly population can syndromic thresholds provide early warning of national influenza outbreaks? methods for monitoring influenza surveillance data trends and directions of global public health surveillance the impact of influenza epidemics on hospitalizations value of icd-9-coded chief complaints for detection of epidemics performance of a syndromic system for influenza based on the activity of general practitioners, france situational uses of syndromic surveillance sensitivity, specificity and predictive values of health service based indicators for the surveillance of influenza a epidemics comparing aberration detection methods with simulated data a simulation model for assessing aberration detection methods used in public health surveillance for systems with limited baselines review of an influenza surveillance system, beijing, people's republic of china disease surveillance using a hidden markov model the emerging science of very early detection of disease outbreaks key: cord-316217-ynh8d853 authors: yoshihara, keisuke; le, minh nhat; toizumi, michiko; nguyen, hien anh; vo, hien minh; odagiri, takato; fujisaki, seiichiro; ariyoshi, koya; moriuchi, hiroyuki; hashizume, masahiro; dang, duc anh; yoshida, lay‐myint title: influenza b associated paediatric acute respiratory infection hospitalization in central vietnam date: 2019-02-28 journal: influenza other respir viruses doi: 10.1111/irv.12626 sha: doc_id: 316217 cord_uid: ynh8d853 background: influenza b is one of the major etiologies for acute respiratory infections (ari) among children worldwide; however, its clinical‐epidemiological information is limited. we aimed to investigate the hospitalization incidence and clinical‐epidemiological characteristics of influenza b‐associated paediatric aris in central vietnam. methods: we collected clinical‐epidemiological information and nasopharyngeal swabs from ari children hospitalized at khanh hoa general hospital, nha trang, vietnam from february 2007 through june 2013. nasopharyngeal samples were screened for 13 respiratory viruses using multiplex‐pcrs. influenza b‐confirmed cases were genotyped by haemagglutinin gene sequencing. we analyzed the clinical‐epidemiological characteristics of influenza b lineages (victoria/yamagata) and who groups. results: in the pre‐a/h1n1pdm09 period, influenza b‐associated ari hospitalization incidence among children under five was low, ranging between 14.7 and 80.7 per 100 000 population. the incidence increased to between 51.4 and 330 in the post‐a/h1n1pdm09. influenza b ari cases were slightly older with milder symptoms. both victoria and yamagata lineages were detected before the a/h1n1pdm09 outbreak; however, victoria lineage became predominant in 2010‐2013 (84% victoria vs 16% yamagata). victoria and yamagata lineages did not differ in demographic and clinical characteristics. in victoria lineage, group1 ari cases were clinically more severe compared to group5, presenting a greater proportion of wheeze, tachypnea, and lower respiratory tract infection. conclusions: the current results highlight the increased incidence of influenza b‐related ari hospitalization among children in central vietnam in the post‐a/h1n1pdm09 era. furthermore, the difference in clinical severity between victoria lineage group1 and 5 implies the importance of influenza b genetic variation on clinical presentation. influenza viruses belong to the family orthomyxoviridae, which possess a segmented negative-stranded rna genome. 1,2 among three influenza types, namely, type-a, b and c, influenza a and b often cause seasonal acute respiratory infections (ari) epidemics and impose a high socioeconomic burden, particularly among children and the elderly population. 3, 4 in temperate climate regions, peaks of influenza-associated aris may appear in early autumn through winter, 5 whereas year-round circulation with no apparent seasonal trend may be seen in tropical climate regions. [6] [7] [8] [9] influenza b differs from type-a in terms of genetic composition and its ability to infect only humans and seals. 10, 11 two surface glycoproteins, haemagglutinin (ha) and neuraminidase (na), play pivotal roles during pathogenesis. 12 unlike influenza a, influenza b does not possess multiple subtypes. instead, influenza b obtains its genetic variation mainly through genetic drift, including nucleotide substitutions, insertions, and deletions, which explains the slower molecular evolutionary rate and smaller capacity to cause seasonal ari outbreaks compared to influenza a. 13, 14 genetic reassortment of influenza b genetic components was previously documented 15 ; however, its frequency is not as high as that of influenza a due to its limited animal reservoir. despite the clinical importance of influenza b among ari cases, majority of previous literature focused on influenza a. therefore, clinical and molecular epidemiological information on influenza b is relatively limited worldwide. the first influenza b strain b/lee/40 was isolated in 1940. 16 in the 1970s, influenza b diverged into two genetically and antigenically distinct lineages, victoria-like (b/victoria/2/87) and yamagata-like (b/yamagata/16/88) lineages. 17, 18 since then, the two lineages have been co-circulating in many regions of the world. 19 previous studies from cambodia, china, india, and taiwan have reported the increase of influenza b-associated ari cases in the following seasons after the emergence of the pandemic a/ h1n1pdm09 strain. 4, 17, 18, 20, 21 furthermore, some studies presented the circulation of victoria lineage as a dominant type in the post-a/h1n1pdm09 period, 17, 18, [20] [21] [22] [23] whereas others showed cocirculation of both lineages. 3, 4 regardless of a few epidemiological studies on influenza b lineage-specific clinical presentation, 21, 23 the clinical aspect of two genetically distinct lineages has not been clearly understood up to date. in this study, we investigated the incidence and clinicalepidemiological characteristics of paediatric hospitalized influenza b ari cases in vietnam. population-based prospective paediatric ari surveillance in khanh hoa province, nha trang, vietnam was established in february 2007. 24 all children from 16 communes admitted to the paediatric ward of khanh hoa general hospital (khgh), which is the only hospital in the region, due to ari symptoms (cough and/or difficulty breathing) from february 2007 to june 2013 were enrolled in the current study. nha trang city in south central region of vietnam has a hot and dry season or tropical climate with a short rainy or wet season from september to december. the detailed characterization of the target population was described in the previous study. 24 written informed consent from the parents or guardians of all the enrolled ari children were obtained. nasopharyngeal (np) swab, clinical-epidemiological information, chest radiograph, and laboratory test data were collected from each enrollee. currently, vaccination against influenza type a and b is not included in the nationwide immunization program in vietnam. its availability in our study site is limited and not commonly used among children. lower respiratory tract infection (lrti) was defined based on the modified world health organization (who) integrated management of childhood illnesses (imci) algorithms. 25 the presence of tachypnea (respiratory rate >60/min for children ≦1 month, >50/min for 2-11 months and >40/min for 12-59 months of age) was categorized as mild lrtis. furthermore, children with a general danger sign (the situation in which children were either unable to drink, under convulsion, or lethargy), chest-wall indrawing, or stridor were categorized as severe lrtis. radiologically-confirmed pneumonia was defined as the presence of either substantial alveolar consolidation or pleural effusion in chest x-ray result following the standardized interpretation method established by who vaccine trial investigators group. 26 ari cases with abnormal shadow but neither substantial alveolar consolidation nor pleural effusion were categorized into chest x-ray abnormality or other lower respiratory infections. 26 adenovirus, and bocavirus as previously described. 24 for the cur confirmed that there was statistical evidence of an increase in influenza b-associated paediatric ari hospitalization in "post-a/ with regard to the demographic and clinical characterization of overall paediatric ari hospitalization cases enrolled in the current study (n = 4,429), 2,602 were male (58.8%), and the median age (in months) was 16.6 (iqr: 8.6-27.3). other demographic information, including socioeconomic status, medical history, as well as detailed clinical information, was summarized in table 2 . the demographic and clinical characteristics between influenza b (n = 133) and non-influenza b ari groups (n = 4296) were compared ( table 2 ). the male proportion was higher in the non-influenza b ari group (50.4%, influenza b vs 59.0%, non-influenza b, p = 0.046). regarding the median age (in month), the influenza b ari group was older (22.9, influenza b vs 16.5, non-influenza b, p < 0.001), and the greater proportion of ari children were 3 years or older in the influenza b ari group. furthermore, family smoking was more frequent in the influenza b ari group (p = 0.003). with respect to the clinical information, the respiratory rate (per min) was faster in the non-influenza b ari group (32.0, influenza b vs 33.0, non-influenza b, p = 0.012) ( table 2) . furthermore, ari children with wheeze (p = 0.005) and breathing difficulty (p = 0.022) were more common in the non-influenza b ari group. the proportion of paediatric ari hospitalizations with chest x-ray abnormal findings was also greater in the non-influenza b ari group ( furthermore, in the comparison between influenza a and b, influenza b-associated ari hospitalizations were slightly older with different age distribution pattern (p = 0.002) (table s2) . parental smoking was more common in influenza b ari group (p = 0.011). regarding the clinical information, influenza a group was associated with higher body temperature (p = 0.003) as well as frequent presence of chest x-ray abnormality (p < 0.001). (table s3) . on the contrary, the overall number of yamagata lineage-related ari hospitalizations was limited throughout the investigation period in both pre-and post-a/ h1n1pdm09 periods. in the demographic characteristics comparison between influenza b table s3 . firstly, demographic information among who groups (1, 4, and 5) of victoria lineage was compared (table 4 ). there were no significant differences in sex distribution and median age among the three groups. daycare attendance was more common in group 5 com(table s4) . with regard to demographic characterization, no statistically significant differences were detected between groups 2 and 3. both group 2-and group 3-associated ari hospitalizations were commonly observed among children younger than 2 years. the results of a comparison in clinical manifestations did not reveal any significant differences. interestingly, we also observed unusual split of rsv seasonal peak in 2010 as we previously described. 37 further studies would be necessary to understand the effect of a/h1n1pdm09 on respiratory virus circulation trend. among all the paediatric ari hospitalization cases enrolled in the current study, 133 were influenza b positive ( table 2 ). the median age in influenza b ari group was older than non-influenza b aris, and influenza b group presented the higher proportion of family smoking, which is known to be one of the major risk factors for influenza morbidity and mortality. 38 we also have to take the lineage-specific sero-epidemiological information into account to gain a better understanding of influenza b lineage circulation pattern. a study from taiwan suggested switching of dominantly circulating lineage may occur every 2-3 years. 18 in the future study, we will continue monitoring the lineage-specific circulation trend of influenza b strains in order to investigate the lineagespecific viral transmissibility based on the viral genetic variation. regarding the demographic information of influenza b lineages, median age was slightly older in victoria lineage (table 3) , which contradicted the reports from china and slovenia that presented victoria lineage aris were younger than yamagata lineage. 21, 45 the difference in age of infection has been controversial due to the differences in inclusion criteria among studies. furthermore, clinical aspect of influenza b lineages is poorly understood. in the current study, the clinical data did not present a significant difference between lineages (table 3) , which was in line with previous reports from china, france, serbia, and slovenia. 41, [45] [46] [47] [48] although the body temperature was slightly higher in yamagata lineage aris, it was probably due to the difference in age distribution. in our study, the hospitalization duration did not differ between lineages, which con male sex (%) 41 we would like to express our sincere gratitude to the medical doctors, nurses, and laboratory technicians at khanh hoa general hospital and staffs in khanh hoa health service for their kind support on clinical sample collection and ari clinical data management. we would like to express our special appreciation for administrative staff and professors in leading program at nagasaki university, graduate school of biomedical sciences for their kind support during the research. this study was conducted in part at the joint usage/research center on tropical disease, institute of tropical medicine, nagasaki university, japan. the authors have declared that no competing interests exist. lay-myint yoshida https://orcid.org/0000-0002-7567-3248 evolutionary characteristics of influenza b virus since its first isolation in 1940: dynamic circulation of deletion and insertion mechanism the gene structure and replication of influenza virus influenza b lineage circulation and hospitalization rates in a subtropical city surveillance and molecular characterization of human influenza b viruses during 2006-2010 revealed co-circulation of yamagata-like and victoria-like strains in eastern india epidemiology and seasonality of respiratory tract virus infections in the tropics influenza surveillance in indonesia influenza viruses in thailand: 7 years of sentinel surveillance data epidemiological and virological characteristics of influenza in the western pacific region of the world health organization influenza b virus genome: assignment of viral polypeptides to rna segments influenza b virus in seals epidemiological, antigenic and genetic characteristics of seasonal influenza a(h1n1), a(h3n2) and b influenza viruses: basis for the who recommendation on the composition of influenza vaccines for use in the 2009-2010 northern hemisphere season reassortment and insertion-deletion are strategies for the evolution of influenza b viruses in nature the evolutionary dynamics of human influenza b virus evolution of influenza a and b viruses: conservation of structural features in the hemagglutinin genes molecular epidemiologic and clinical characteristics of influenza b-associated complications among hospitalized patients during an outbreak in taiwan phylogenetic and evolutionary history of influenza b viruses cocirculation of two distinct evolutionary lineages of influenza type b virus since 1983 epidemiological and virological characteristics of influenza viruses circulating in cambodia from differing epidemiological dynamics of influenza b virus lineages in guangzhou, southern china local persistence and global dissemination play a significant role in the circulation of influenza b viruses in leyte island molecular epidemiology and phylogenetic analyses of influenza b virus in thailand during viral pathogens associated with acute respiratory infections in central vietnamese children integrated approach to child health in developing countries burden of disease caused by haemophilus influenzae type b in children younger than 5 years: global estimates genetic analysis of influenza b viruses isolated in uganda during the 2009-2010 seasons kakusan4 and aminosan: two programs for comparing nonpartitioned, proportional and separate models for combined molecular phylogenetic analyses of multilocus sequence data mega6: molecular evolutionary genetics analysis version 6.0 interrupted time series regression for the evaluation of public health interventions: a tutorial time series regression studies in environmental epidemiology impact of 2009 pandemic influenza among vietnamese children based on a population-based prospective surveillance from 2007 to 2011. influenza other respir viruses influenza a and b virus infections in children incidence of influenza-related hospitalizations in different age groups of children in finland: a 16-year study evolutionary pattern of reemerging influenza b/victoria lineage viruses in sao paulo, brazil, 1996-2012: implications for vaccine composition strategy the 2009 pandemic influenza a(h1n1) coincides with changes in the epidemiology of other viral pathogens causing acute respiratory tract infections in children association of rsv-a on1 genotype with increased pediatric acute lower respiratory tract infection in vietnam is smoking a risk factor for influenza hospitalization and death? cigarette smoking as a risk factor for influenza-associated mortality: evidence from an elderly cohort. influenza other respir viruses differences in clinical features between influenza a h1n1, a h3n2, and b in adult patients clinical characteristics are similar across type a and b influenza virus infections the burden of influenza b: a structured literature review a comparison of the clinical and epidemiological characteristics of adult patients with laboratoryconfirmed influenza a or b during the 2011-2012 influenza season in korea: a multi-center study the contrasting phylodynamics of human influenza b viruses a comparison of the demographic and clinical characteristics of laboratory-confirmed influenza b yamagata and victoria lineage infection influenza b viruses in the population of province of vojvodina during the season: differentiation of b/yamagata and b/victoria lineages by real-time rt-pcr, antigenic and phylogenetic characterization outcomes and susceptibility to neuraminidase inhibitors in individuals infected with different influenza b lineages: the influenza resistance information study comparative epidemiology of influenza b yamagata-and victoria-lineage viruses in households clinical features of children infected with different strains of influenza b in southern taiwan structural basis for receptor specificity of influenza b virus hemagglutinin role of receptor binding specificity in influenza a virus transmission and pathogenesis virulence-associated substitution d222g in the hemagglutinin of 2009 pandemic influenza a(h1n1) virus affects receptor binding observed association between the ha1 mutation d222g in the 2009 pandemic influenza a(h1n1) virus and severe clinical outcome avian-type receptorbinding ability can increase influenza virus pathogenicity in macaques the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis multipathogen infections in hospitalized children with acute respiratory infections dual respiratory virus infections dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis insights into the interaction between influenza virus and pneumococcus key: cord-326039-pnf2xjox authors: seale, holly; dwyer, dominic e.; cowling, benjamin j.; wang, quanyi; yang, peng; macintyre, c. raina title: a review of medical masks and respirators for use during an influenza pandemic date: 2009-08-18 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2009.00101.x sha: doc_id: 326039 cord_uid: pnf2xjox nan to the editor: on 11 june 2009, the world health organisation (who) raised the influenza pandemic alert to level 6 (defined as 'sustained community level outbreaks in at least one other country in another who region') because of the emergence of a novel influenza a ⁄ h1n1 subtype. 1 australia's first laboratory confirmed case of pandemic (h1n1) 2009 virus, a nsw woman who had visited los angeles, was reported in the second week of may 2009. within a month, 1336 laboratory confirmed cases had been identified, the majority of them in victoria, rising to nearly 20 000 australia-wide by the end of july. given the lack of a specific vaccine against the pandemic (h1n1) 2009 virus, mitigation measures in australia have so far focused on identifying, treating, and isolating people who have the disease, and educating the public about the steps that individuals can take to reduce transmission. antiviral medications have been deployed as both treatment and prophylaxis. clinical trials of the pandemic (h1n1) 2009 vaccine are currently underway; however, it is unclear when and to whom the vaccine will be made available. healthcare workers (hcws) and those on the 'front line' will be the first to be vaccinated; there will be a lag time for members of the general public. non-pharmacological public health interventions including use of face masks are therefore likely to play a vital role in mitigating disease spread, particularly in developing countries. medical masks are unfitted devices worn by an infected person, hcw or member of the public to reduce transfer of potentially infectious respiratory tract material between individuals. they are designed to be disposable. surgical masks are specifically designed to protect patients from contamination of wounds during surgical procedures. in contrast, a respirator is a fitted device that protects the wearer against inhalation of harmful contaminated material. respirators can be disposable or reusable and are recommended for use in high-risk activities (e.g. aerosolgenerating procedures) in healthcare settings. the national institute for occupational safety and health (niosh) regulates the testing and certification of respiratory protection equipment. 2 the niosh tests filters for the effects of loading (particle burden), temperature, and relative humidity and requires a minimum filtration efficiency of 95%, 99% or 99ae97% using neutralized 0ae075-mm count median diameter solid aerosols at 85 l ⁄ min. filters can be certified for a range of efficiency classes (e.g. 95%, 99% or 100%) as well as for their ability to withstand degradation as a result of loading or oil mist exposures. n95 filters are not permitted to have more than 5% of the challenge aerosol concentration penetrate the filter, and would be expected to have less aerosol penetration with either larger or smaller particles than the size used in certification testing. in 1973, a letter to the editor of the new england journal of medicine from jack resnick, md, 3 suggested '…perhaps the ancient oriental custom of wearing gauze or cloth, surgical-type masks during a cold has some merit? perhaps western society has another lesson to learn by observing the oriental customs besides acupuncture.' he proposed that this matter be studied in a rigorous manner. since then, there have been many studies on the filtration efficiency under controlled laboratory settings, but until recently there has been limited study as to whether masks or respirators will provide clinically relevant protection in healthcare settings. most data have been derived from at best observational settings and frequently has been based on anecdotal rather than controlled trials evidence. 4 focused specifically on n95 masks. 8, 9 although the authors concluded from the pooled estimate of effect that the intervention effectiveness was 91%, this evidence was thin as the studies included showed inconsistencies and failed to adequately describe the use of controls. jefferson and colleagues concluded that more experimental studies were needed to identify the effectiveness of wearing face masks or respirators in reducing exhaled infectious viral particles. 7 in 2009, we reported the first prospective cluster-randomized trial comparing surgical masks, non-fit-tested p2 masks (n95 equivalent) and no masks in prevention of influenza-like illness (ili) in households. 10 intention to treat analysis showed no significant difference in the relative risk of ili in the mask groups compared with the control group. however, less than half of the subjects wore masks 'most of the time'. adherence to mask use significantly reduced the risk of ili-associated infection, with a hazard ratio of 0ae26 (95% ci 0ae09-0ae77; p = 0ae015). a recently reported randomized trial showed a significant benefit of both hand hygiene and face masks (worn by the index case and contacts) in preventing influenza transmission in households, although adherence to the face mask intervention was low among household contacts. 11 surgical masks and n95 respirators have been recognized as an important non-invasive technology to use during this new pandemic period. however, there are many factors which may compromise the overall effectiveness of these measures. poor training, lack of guidance and consistency in the use of masks, improper use and for n95 respirators, the need for fit-testing, may limit their usefulness. data from the sars experience in toronto illustrated the need for training and monitoring; loeb and colleagues (2004) found that nine of 32 (28%) nurses entering a sars patient's room did not consistently wear appropriate respiratory protection. 12 there is also the problem of workplace acceptance. in a logistics exercise undertaken during the peak of seasonal influenza activity in 2007 in australia, compliance by emergency department staff, with n95 mask wearing was found to be low, with only 36ae1% of participants wearing the mask 'occasionally' in week one and only 18ae8% by week four. many staff reported that they found the mask hot and hard to breathe through, and others reported that they had problems both communicating with patients and storing the mask between uses. 13 much time and effort has been devoted to developing an optimal strategy for the use of pandemic vaccines and antivirals, in addition to non-pharmaceutical measures. however, comprehensive assessments of the literature to date recognize the generally poor quality of evidence on which to base non-pharmaceutical pandemic planning decisions. despite the lack of high level evidence, recommendations on the use of face masks and respirators for hcws are made by many health authorities. to ensure that hcws wear face masks to protect themselves during this time, cultural attitudes and the physical discomfort and mechanical issues associated with long-term respirator use must be addressed. other factors that affect the use of personal protective equipment, such as staff and management attitudes about the value of respirator use, fatigue and the availability of replacement masks, also need to be considered. director-general of the world health organization, editors national institute for occupational safety and health. respiratory protective devices; final rules and notice, 42 cfr part 84 rationale for chinese gauze masks? effect of hand hygiene on infectious disease risk in the community setting: a meta-analysis non-pharmaceutical public health interventions for pandemic influenza: an evaluation of the evidence base interventions for the interruption or reduction of the spread of respiratory viruses physical interventions to interrupt or reduce the spread of respiratory viruses: systematic review sars transmission among hospital workers in hong kong effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) face mask use and control of respiratory virus transmission in households facemasks and hand hygiene to prevent influenza transmission in households: a randomized trial sars among critical care nurses feasibility exercise to evaluate the use of particulate respirators by emergency department staff during the 2007 influenza season all authors contributed to the writing and revising of the text. there are no conflicts of interest. key: cord-323066-tvguutak authors: praznik, ajda; vinšek, neža; prodan, ana; erčulj, vanja; pokorn, marko; mrvič, tatjana; paro, darja; krivec, uroš; strle, franc; petrovec, miroslav; žnidaršič eržen, marta; grosek, štefan title: risk factors for bronchiolitis severity: a retrospective review of patients admitted to the university hospital from central region of slovenia date: 2018-08-09 journal: influenza other respir viruses doi: 10.1111/irv.12587 sha: doc_id: 323066 cord_uid: tvguutak aim: study's objective was to identify risk factors associated with bronchiolitis severity. methods: a retrospective chart review of all children <2 years old diagnosed with bronchiolitis at the university medical centre ljubljana between may 2014 and april 2015, who were treated as outpatients (paediatric emergency department, ped group) or as inpatients in the standard hospital setting (ward group) or in the paediatric intensive care unit (picu group). detection of respiratory viruses in nasopharyngeal swab was accomplished by rt‐pcr. severity was assessed by wang respiratory score and hospitalization longer than 24 hours. results: the study included 761 children. the three most frequently detected viruses were respiratory syncytial virus (rsv), human rhinovirus (hrv) and human bocavirus (hbov) (57.5%, 272/473; 25.6%, 121/473; 18.4%, 87/473). patient groups differed in wang respiratory score for the severity of bronchiolitis (p < 0.001). no differences regarding the causative viruses were found. there was a lower proportion of children with the presence of more than one virus in picu group compared to other two groups (p = 0.017). the three groups significantly differed in age, birthweight, comorbidities, bronchodilator treatment and antibiotic usage. however, multiple regression analysis revealed that younger age and the use of antibiotics were associated with bronchiolitis severity defined as hospitalization for >24 hours. conclusions: respiratory syncytial virus, hrv and hbov were the most frequently detected viruses. the majority of patients admitted to the picu had only one virus detected. younger age and the use of antibiotics were associated with bronchiolitis severity. bronchiolitis is a potentially life-threatening viral respiratory infection that affects children under 2 years of age. 1 it is common and occurs worldwide. the bronchiolitis mortality rate is approximately 2 per 100 000 infants and is higher in developing than in developed countries. 2 the most commonly identified causative agent is respiratory syncytial virus (rsv). 2 other viruses implicated in the aetiology of bronchiolitis include human metapneumovirus (hmpv), parainfluenza virus (piv), influenza virus a and b virus (infv), human rhinovirus (hrv), human coronavirus (hcov), adenovirus (adv), enterovirus (ev) and human bocavirus (hbov). 3 the majority of children with bronchiolitis can be managed as outpatients; approximately 2%-3% of patients younger than 1 year are hospitalized, usually during the seasonal rsv epidemics that occur in cold months. 1 treatment of bronchiolitis is symptomatic and focuses on the maintenance of hydration, oxygenation and antipyresis. in severe cases, intensive care management may be needed with continuous positive airway pressure (cpap), intubation and mechanical ventilation. some infants with secondary bacterial infection need antibiotic treatment. [4] [5] [6] in infants with pre-existing medical conditions and immunodeficiency, complications such as acute respiratory distress syndrome (ards), bronchiolitis obliterans, congestive heart failure and secondary bacterial infection may develop. [4] [5] [6] [7] [8] although there is no universally accepted measure for the assessment of bronchiolitis severity, the wang respiratory score, using clinical observation (general appearance, respiratory rate, presence of wheezing and retractions), is widely used. 9 children with bronchiolitis and wang respiratory score 3 or less can be managed on an outpatient basis, while children with scores 4-8 usually require hospitalization and those with even higher scores are admitted to the picu. 10 the objective of our study was to ascertain demographic characteristics, clinical findings and presumptive aetiologic agents (respiratory viruses demonstrated in nasopharyngeal swab) associated with bronchiolitis severity defined as length of hospitalization for >24 hours. the study was approved by the national medical ethics committee of the republic of slovenia (kme 37/09/15) (nmec rs). the study was conducted at the university medical centre ljubljana (umcl), a university hospital serving the central region of slovenia with a population of around 700 000 inhabitants (approximately one-third of all slovenian population). we performed a retrospective chart review of all patients with bronchiolitis <2 years of age, referred to the three paediatric departments, that is umcl children's hospital, department of infectious diseases and paediatric intensive care unit (picu) between 1 may 2014 and 30 april 2015. we excluded patients that had been previously diagnosed with asthma, and all previous episodes if a patient had more than one episode of bronchiolitis in studied time span. patients were identified through a query of the electronic medical record based on a bronchiolitis diagnosis (icd-10 code j21.0-9). electronic medical records of patients included in the study were reviewed, and statistical analysis was performed for the following clinical and laboratory data: gender, chronological age at admission, prematurity (defined as birth before 37 weeks of gestation), birthweight, history of allergies, number of previous bronchiolitis episodes, clinical manifestations of bronchiolitis using the wang respiratory score, 9 comorbidities (chronic lung disease, congenital heart disease, immune deficiency or neuromuscular diseases), body temperature at admission, treatment with bronchodilators, antibiotics or supplemental oxygen and respiratory virus detected in the nasopharyngeal swab. only the first swab taken from individual patient was included. swabs were routinely taken from patients in two departments, while in another one sampling was sparse. high season and low season of incidence were also included as variables. using rrt-pcr, the sensitivity of the nasopharyngeal swab is above 90% compared to a composite gold standard. 13, 14 for 288 patients, no swab analysis was performed. bronchiolitis severity was assessed by wang respiratory score and as a duration of hospitalization for >24 hours. numerical data were presented as median (range) and categorical as frequencies (percentages). the differences in categorical variables among the three groups were tested using chi-square test or likelihood ratio test; kruskal-wallis test was performed for numerical variables. the clinical and laboratory findings were compared between the three groups of patients, defined according to the site of management: patients treated as outpatients (paediatric emergency department, ped group), patients treated in the standard hospital setting (ward group) and children admitted to the paediatric intensive care unit (picu group). multiple logistic regression analysis was used to identify key variables associated with severe bronchiolitis, defined with the duration of hospitalization for >24 hours. a p-value < 0.05 was considered statistically significant. all analyses were performed using spss 23.0. the study group comprised 761 children with bronchiolitis. there was a male predominance with 468 (61.5%) boys. of 761 children, 138 were treated as outpatients (ped group), 599 were hospitalized at a regular paediatric unit (ward group), and 24 were treated in a paediatric intensive care unit (picu group). nasopharyngeal swabs were taken in 473/761 (62%) children. of 623 patients admitted to hospital, 413 (54%) were hospitalized for >24 hours. the median length of hospital stay was 12 days in picu group and 2 days in ward group (p < 0.001). the three study groups differed in wang's score for particular clinical sign parameter (p < 0.001) apart from wheezing, and in the need for supplemental oxygen treatment (p < 0.001)-only a few patients required oxygen treatment in the outpatient ped group, while almost all patients needed oxygen treatment in picu group. the three study groups differed in wang score (p < 0.001) ( figure 1 ). they also differed in several clinical and microbiological characteristics ( table 1) analysis of the presence of more than one virus in nasopharyngeal swab revealed that rsv and hbov were the two most frequently simultaneously detected viruses and that the proportion of patients with more than one of detected viruses in individual swab was statistically significantly lower in picu than in ped or ward (p = 0.017) ( table 1) . multiple logistic regression analysis showed that out of several factors only younger chronological age (p < 0.001) and treatment with antibiotics (p = 0.003) were associated with severe bronchiolitis defined as hospitalization >24 hours. (table 2 ). in the present study, we compared characteristics of children with bronchiolitis treated in the outpatient clinic, admitted to the ward or admitted to the paediatric intensive care unit. age, prematurity, comorbidity and treatment with bronchodilators and antibiotics, differed in the three groups suggesting these factors relate to disease severity. the type of virus did not correlate with treatment group (table 1 ). further analyses revealed that chronological younger age and treatment with antibiotics independently were associated with hospitalization longer than 24 hours (table 2) . chronological age is known as the most important predictor of severe bronchiolitis. similar to study, several others have found a significant association between the age of less than 6 months and a higher risk of hospitalization and severe bronchiolitis. [15] [16] [17] [18] however, grimwood et al did not find a significant correlation between children aged less than 2 months and bronchiolitis severity in a multivariate analysis. 19 hospitalization rates that are attributable to rsv bronchiolitis are usually the highest between 30 and 90 days after birth. 20 this age coincides with the declining concentration of transplacentally acquired maternal anti-rsv immunoglobulin, which protects infants against disease. 20, 21 certain studies have shown that prematurity is independently associated with more severe bronchiolitis, 15, 16, 22 while the others have not found significantly higher rates of hospitalization and severe bronchiolitis among premature compared to full-term infants. 17, 20, 23 in the present study, the proportion of preterm children was highest in the picu group and lowest in the ped group, which suggested a more severe bronchiolitis. however, multiple logistic regression model did not show a correlation between prematurity and in the present study, antibiotic treatment was significantly associated with severe bronchiolitis, which is in accordance with some previous reports. 24, 25 although the design of our study did not enable analysis of the appropriateness of antibiotic therapy, the finding that almost all patients in the picu group received antibiotic treatment compared to only 3.6% in the ped group might suggest that bacterial superinfections lead to a more severe course of bronchiolitis. another frequently mentioned risk factor for severe bronchiolitis is comorbidity. 23, [26] [27] [28] in our study, one-third of children hospitalized in picu had at least one comorbidity, while in the ped group the corresponding proportion was significantly lower. however, again, in the multiple logistic regression analysis comorbidities did not correlate with the severe course of bronchiolitis (table 2) as shown also in some other studies. 1, 16 one should take into account that our study did not analyse the association of comorbidities with specific virus types as done in mentioned studies. 5, 15, 26, 29 another limitation is that we did not analyse specific comorbidities, but only as a group determined by previous studies. 24, 30, 31 this is because there were only 31 children with comorbidity and we believe that dividing the comorbidity group into smaller groups would not give reliable information. the present study showed that rsv, hrv and hbov were the most frequently present viruses in nasopharyngeal swab of children with bronchiolitis. the predominance of rsv is in agreement with several previous reports. 3, 5, 32 the second most common pathogen is usually hrv, followed by infv or piv 5, 19, 33 while in our study hbov ranked the third. some reports indicated that hbov is rarely detected as a single agent in hospitalized children with bronchiolitis, leading to speculation that this virus is more likely to be an innocent bystander than a true pathogen. 5 however, uršič et al have recently reported the first fatal case of an extremely severe bronchiolitis caused by hbov in an immunocompetent child. 16 the absence of association between virus type and characteristics of patients is in agreement with the findings of several previous articles. 6, 17, 18 nevertheless, in some reports, differences in the severity of the disease caused by various types of viruses or viral co-infections were established. 1, 34, 35 according to our study, multiple viruses detected in the nasopharyngeal swab were moderately associated with a more severe course of the bronchiolitis, although not significant (p = 0.091). other studies proved that children with viral coinfection were less likely to be admitted to intensive care unit than children with single virus infection. 21, 22 possible explanation is that as one or more viral respiratory pathogens can be detected in the upper respiratory tract of as many as 30% of asymptomatic young children, 36, 37 it is likely that in several patients with bronchiolitis and more than one virus detected in nasopharyngeal swab, one of the viruses is responsible for the acute infection while the others are innocent bystanders, possibly as a result of prolonged shedding after an already resolved infection. it is not unusual that a child has a history of more than one episode of bronchiolitis. 38 studies suggest that repeated infections by rsv can be in part due to variability of the virus strains. 39 acute bronchiolitis can cause (transitory) anatomical and histological changes in lower respiratory tract that may prone to a more severe new episode. 29 however, in accordance with some previous reports 40 in our study, the number of previous episodes of bronchiolitis did not correlate with bronchiolitis severity. our study also showed that the proportion of children who received bronchodilators was statistically different in the ped, ward and picu group (table 1) . this is probably due to the common practice in several countries (such as united states, switzerland and belgium) that children exhibiting a moderately severe bronchiolitis are more often given bronchodilators than those with mild or severe disease. [41] [42] [43] the data on the efficiency of bronchodilators in bronchiolitis are conflicting. 41 many studies agree that bronchodilators may improve clinical symptom scores, but they do not affect disease resolution, need for hospitalization, or length of stay. 5 interpretation. in addition, children with performed viral diagnosis were younger and were treated in the high seasonal months. comparison of the three groups regarding virus repartition should be taken with precaution as the sample of children treated in the standard hospital setting with viral data available seems to be biased. data are not missing at random, but are available to higher extent for younger children and for children treated in the high season months. in conclusion, our study revealed that rsv, hrv and hbov were the most frequently detected viruses in children with bronchiolitis. chronological younger age and the use of antibiotics were associated with severe bronchiolitis defined as hospitalization longer than 1 day. further prospective studies are needed to assess the importance of various respiratory viruses and host factors in this common paediatric illness. we thank the institute of microbiology and immunology and the national institute of public health who provided us with data necessary for statistical analysis. the authors have no competing interests. ana prodan http://orcid.org/0000-0002-1116-2844 the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis risk factors for bronchiolitis-associated deaths among infants in the united states impact of human rhinovirus types and viral load on the severity of illness in hospitalized children with lower respiratory tract infections ljubljana: združenje za infektologijo, slovensko zdravniško društvo ljubljana viral bronchiolitis in children diagnosis and management of bronchiolitis clinical findings and severity of acute bronchiolitis bronchodilators for treatment of mild bronchiolitis: a factorial randomised trial observer agreement for respiratory signs and oximetry in infants hospitalized with lower respiratory infections e-newsletter on communicable diseases and environmental health, enboz -elektronske novice s področja nalezljivih bolezni in okoljskega zdravja the role of human coronaviruses in children hospitalized for acute bronchiolitis, acute gastroenteritis, and febrile seizures: a 2-year prospective study comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction comparison of nasal and nasopharyngeal swabs for influenza detection in adults the burden of respiratory syncytial virus infection in young children incidence and risk factors of hospitalization for bronchiolitis in preterm children: a retrospective longitudinal study in italy respiratory syncytial virus infection in navajo and white mountain apache children comparison of risk factors for human metapneumovirus and respiratory syncytial virus disease severity in young children risk factors for respiratory syncytial virus bronchiolitis hospital admission in new zealand respiratory syncytial virus-associated hospitalizations among children less than 24 months of age respiratory syncytial virus transplacental antibody transfer and kinetics in mother-infant pairs in bangladesh risk factors in children hospitalized with rsv bronchiolitis versus non-rsv bronchiolitis rates of hospitalization for respiratory syncytial virus infection among children in medicaid risk factors for respiratory syncytial virus hospitalisation in children with heart disease variation in inpatient diagnostic testing and management of bronchiolitis pre-existing disease is associated with a significantly higher risk of death in severe respiratory syncytial virus infection updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection respiratory syncytial virus and parainfluenza virus evaluation of risk factors for recurrent wheezing episodes risk factors for hospital admission with rsv bronchiolitis in england: a population-based birth cohort study chronic diseases, chromosomal abnormalities, and congenital malformations as risk factors for respiratory syncytial virus hospitalization: a population-based cohort study respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus multicenter study of viral etiology and relapse in hospitalized children with bronchiolitis viral etiologies of infant bronchiolitis, croup and upper respiratory illness during 4 consecutive years frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia bronchiolitis (and rsv) in infants and children molecular epidemiology of respiratory syncytial virus infections among children with acute respiratory symptoms in a community over three seasons respiratory syncytial virus disease in preterm infants in the u.s. born at 32-35 weeks gestation not receiving immunoprophylaxis bronchiolitis management preferences and the influence of pulse oximetry and respiratory rate on the decision to admit current management of acute bronchiolitis in switzerland bronchiolitis management by the belgian paediatrician: discrepancies between evidence-based medicine and practice clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis efficacy of bronchodilator therapy in bronchiolitis. a meta-analysis risk factors for bronchiolitis severity: a retrospective review of patients admitted to the university hospital from central region of slovenia key: cord-328086-ji2emajn authors: zhou, jie‐ying; peng, ying; peng, xiao‐you; gao, han‐chun; sun, ya‐ping; xie, le‐yun; zhong, li‐li; duan, zhao‐jun; xie, zhi‐ping; cao, you‐de title: human bocavirus and human metapneumovirus in hospitalized children with lower respiratory tract illness in changsha, china date: 2018-01-11 journal: influenza other respir viruses doi: 10.1111/irv.12535 sha: doc_id: 328086 cord_uid: ji2emajn background: lower respiratory tract illness is a major cause of morbidity and mortality in children worldwide, however, information about the epidemiological and clinical characteristics of lrtis caused by hmpv and hbov in china is limited. objectives: human bocavirus (hbov) and human metapneumovirus (hmpv) are two important viruses for children with lower respiratory tract infections (lrti). we aimed to assay the correlation between viral load and clinical characteristics of hbov and hmpv with lrti in changsha, china. methods: nasopharyngeal aspirates (npas) from children with lrti were collected. real‐time pcr was used to screen hbov and hmpv. analyses were performed using spss 16.0 software. results: pneumonia was the most frequent diagnosis. there was no significant difference between hbov‐ and hmpv‐positive patients in age (p = .506) or hospitalization duration (p = .280); 24.1% and 18.2% were positive for hbov and hmpv. hbov infections peaked in summer (32.2%), and hmpv infections peaked in winter (28.9%). the hbov‐positive patients had a shorter hospitalization duration than the hbov‐negative patients (p = .021), and the hmpv‐positive patients had a higher prevalence of fever than the hmpv‐negative patients (p = .002). the hbov viral load was significantly higher among patients aged <1 year (p = .006). the mean hbov and hmpv viral loads were not significantly different between patients with single infections and coinfections. patients infected with hbov only were older than those coinfected with hbov and other respiratory viruses (p = .005). no significant difference was found in the clinical characteristics of patients infected with hmpv only and those coinfected with hmpv and other respiratory viruses. conclusion: pneumonia was the most frequent diagnosis caused by hbov and hmpv. neither hbov nor hmpv viral load was correlated with disease severity. hmpv, which was originally isolated in the netherlands 1 and is closely related to avian metapneumovirus, was the first human disease-causing pathogen identified in the genus metapneumovirus. 2 hmpv causes various clinical symptoms, such as cough, wheezing, and fever. 2 young children and the elderly are more susceptible to hmpv infection. 2, 3 indeed, hmpv mainly infects children under 5 years of age and causes upper respiratory tract and severe lower respiratory tract infections. [4] [5] [6] [7] [8] hbov was discovered in sweden and classified in the family parvoviridae, 9 and causes various clinical symptoms, including cough, wheezing, and fever. 2 hbov infections are largely confined to infants and young children (<24 months). globally, the average prevalence of hbov in respiratory tract samples ranges from 1.0 (ci: 0.0-2.0) to 56.8% (ci: 46.9-66.8). 10 however, information about the epidemiological and clinical char(table 1) . to standardize quantification of hbov and hmpv, known numbers of dna or rna transcripts containing the primer targets were used in 10-fold serial dilutions (10 0 to 10 7 copies/μl). any amplification detected before 40 cycles was considered positive. quantification of >10 0 copies/μl (10 3 copies/ml) was considered a positive result. an internal positive control gene (gapdh), positive control, and negative (water) control were included in all reactions. viral loads were expressed as initial copy numbers per real-time pcr reaction, and quantitative results were transformed as the log number of viral copies/μl. continuous variables are expressed as means ± standard deviation (sd) and were compared by independent samples t test. categorical variables are expressed as frequencies or percentages. comparisons were performed by chi-squared or fisher's exact test. analyses were performed using spss 16.0 software. two-sided p-values <.05 were considered to indicate statistical significance. in total, we analyzed 1092 samples collected from lrti patients patients were divided into two groups: those with and without hbov or hmpv infections (table 2) (table 5 ). according to the british thoracic society guidelines for the management of community-acquired pneumonia in children 11 and the clinical diagnosis, lrtis were classified as mild to moderate or severe. the mean hbov and hmpv viral loads did not differ significantly between the two groups (p = .053 and p = .231) (figure 3 ). in this study, we used real-time pcr to explore the etiology and exwith lrtis. this is higher than previous study (62.34%) in changsha that used traditional pcr, 18 and lower than that a study in lanzhou that used real-time pcr. 6 on the one hand, the high detection rate in our study could be caused by the greater sensitivity of real-time pcr compared with traditional pcr. on the other hand, it is possible that the detection rate differs geographically. rsv was the most frequently virus detected in patients with respiratory infections, followed by piv3, hbov, adv, and hmpv. our data support previous reports of rsv, piv3, hbov, adv, and hmpv as the major agents associated with lrtis among children in a hospital setting. 12, [19] [20] [21] [22] the hbov infection rate (24.1%) in the present study is consistent with earlier reports (1.9%-24.6% 8, 9, 16, [23] [24] [25] [26] , and the incidence of hmpv (18.2%) in patients with respiratory tract infections is similar to that in other regions (1.5%-18% 2, 9, 17, 23, [26] [27] [28] . therefore, hmpv and hbov are major causes of lrtis worldwide. the seasonal peaks of hbov and hmpv infections vary among countries because of differences in climatic and geographic factors. in this study, hbov activity peaked in summer, in agreement with the report by jiang. 29 in contrast, detection of hbov in lanzhou peaked in december and april, 30 possibly due to the dry, cold weather in lanzhou and the warm, humid weather in changsha. hmpv detection peaked in winter, which is in line with previous studies. 2, 7, 18, 31 in contrast, in hong kong, hmpv detection peaks in spring/summer. 32 the seasonality of hbov differed geographically, possibly due to climatic factors. the most frequent symptoms of hbov-and hmpv-positive patients were cough and fever, in accordance with previous reports. 6, 18, 33 however, deng reported that in chongqing, wheezing was the most frequent symptom exhibited by hbov-positive patients with severe lrtis. 16 there was no difference between the hbov-and hmpv-positive patients in the incidence of fever, tachypnea, cyanosis, o 2 therapy, or wbc count. the hbov-positive patients had a shorter hospitalization duration than hbov-negative patients (p = .021). in contrast, deng reported that hbov-positive patients had a longer hospitalization duration. the longer hospitalization duration of hbov-negative patients in our study may have been caused by the presence of other viruses (such as rsv and piv3) in many of them. also, hospitalization duration was significantly associated with age (≤6 months), maternal smoking during pregnancy, and a family history of asthma. 19 a high hmpv viral load contributes to development of fever. 4, 5 indeed, hmpv-positive patients had a higher incidence of fever than hmpv-negative patients (p = .002) in our study. we assessed the relationship between viral load and clinical features (age, gender, respiratory rate, temperature, cyanosis, hospitalization duration, and wbc count). the only significant association of hbov-or hpmv-positive patients was a higher viral load in <1-year-old hbov-positive patients. this is in agreement with jiang's report that patients with a high viral load were significantly younger. 29 in contrast, the duration of wheezing and hospitalization was longer in children with a high than a low hbov viral load in the study by deng, 16 possibly due to inclusion of only patients with severe lrtis. hmpv and hbov coinfections with other viruses are common in this study has the following strengths: a considerable duration, large number of patients, and comparison of the most common viruses. its limitation includes the lack of a control group without clinical evidence of illness. further studies should compare a symptomatic group with a control group and a symptomatic period with an asymptomatic period. in summary, we present a prospective study of lrtis caused by hbov and hmpv. a further comprehensive and in-depth study of the role of hbov and hmpv in lrtis in china is warranted. a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus: insights from a ten-year molecular and epidemiological analysis in germany outbreak of human metapneumovirus infection in a severe motor-and-intellectual disabilities ward in japan viral load and acute otitis media development after human metapneumovirus upper respiratory tract infection clinical disease and viral load in children infected with respiratory syncytial virus or human metapneumovirus clinical characteristics and viral load of respiratory syncytial virus and human metapneumovirus in children hospitaled for acute lower respiratory tract infection detection of human metapneumovirus and respiratory syncytial virus by real-time polymerase chain reaction among hospitalized young children in iran incidence, morbidity, and costs of human metapneumovirus infection in hospitalized children high human bocavirus viral load is associated with disease severity in children under five years of age human bocavirus: current knowledge and future challenges british thoracic society guidelines for the management of community acquired pneumonia in children: update 2011 etiology, seasonality and clinical characterization of viral respiratory infections among hospitalized children in beirut, lebanon infections and coinfections by respiratory human bocavirus during eight seasons in hospitalized children abdel baset rw. respiratory syncytial virus and human metapneumovirus in severe lower respiratory tract infections in children under two human metapneumovirus infection in jordanian children: epidemiology and risk factors for severe disease high viral load of human bocavirus correlates with duration of wheezing in children with severe lower respiratory tract infection burden of human metapneumovirus infection in young children clinical features of human metapneumovirus genotypes in children with acute lower respiratory tract infection in changsha severe lower respiratory tract infection in infants and toddlers from a non-affluent population: viral etiology and co-detection as risk factors viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection associations between co-detected respiratory viruses in children with acute respiratory infections multiple viral respiratory pathogens in children with bronchiolitis viral etiologies of hospitalized acute lower respiratory infection patients in china bocavirus in children with respiratory tract infection high prevalence of human bocavirus detected in young children with severe acute lower respiratory tract disease by use of a standard pcr protocol and a novel real-time pcr protocol viral coinfection in acute respiratory infection in mexican children treated by the emergency service: a cross-sectional study comparison of severe pneumonia caused by human metapneumovirus and respiratory syncytial virus in hospitalized children detection of human bocavirus and human metapneumovirus by real-time clinical significance of different virus load of human bocavirus in patients with lower respiratory tract infection human bocavirus infection in young children with acute respiratory tract infection in lanzhou the prevalence of human bocavirus, human coronavirus-nl63, human metapneumovirus, human polyomavirus ki and wu in respiratory tract infections in kuwait children with respiratory disease associated with metapneumovirus in hong kong human metapneumovirus (hmpv) infection in immunocompromised children human metapneumovirus and human bocavirus associated with respiratory infection in apulian population real-time pcr for diagnosis of human bocavirus infections and phylogenetic analysis human metapneumovirus viral load is an important risk factor for disease severity in young children association between high nasopharyngeal viral load and disease severity in children with human metapneumovirus infection development and assay of rna transcripts of enterovirus species a to d, rhinovirus species a to c, and human parechovirus: assessment of assay sensitivity and specificity of real-time screening and typing methods application of taqman lowdensity arrays for simultaneous detection of multiple respiratory pathogens human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays impact of human coronavirus infections in otherwise healthy children who attended an emergency department real-time quantitative pcr detection of four human bocaviruses human bocavirus and human metapneumovirus in hospitalized children with lower respiratory tract illness in changsha, china we thank li-li li, na-liu, jie-mei yu, li-li pang, and other staff members in department of viral diarrhea (national institute for viral disease control and prevention) for detection of virus pathogens. the authors have no competing interests to report. the process obtained families' informed consent, and the study protocol was approved by the first affiliated hospital of hunan normal university, changsha, china. http://orcid.org/0000-0002-5152-3093 key: cord-315949-7id5mitl authors: sentilhes, anne‐charlotte; choumlivong, khamla; celhay, olivier; sisouk, thongchanh; phonekeo, darouny; vongphrachanh, phengta; brey, paul; buchy, philippe title: respiratory virus infections in hospitalized children and adults in lao pdr date: 2013-06-25 journal: influenza other respir viruses doi: 10.1111/irv.12135 sha: doc_id: 315949 cord_uid: 7id5mitl background: acute respiratory infections are an important cause of morbidity and mortality worldwide, with a major burden of disease in developing countries. the relative contribution of viruses in acute lower respiratory infections (alri) is, however, poorly documented in lao pdr. objective: the objective of this study is to investigate the etiology of alri in patients of all ages in two hospitals of laos. methods: multiplex pcr/rt‐pcr methods were used to target 18 major common respiratory viruses. between august 2009 and october 2010, samples from 292 patients presenting with alri were collected. results and conclusion: viruses were detected in 162 (55%) samples. in 48% (140/292) of the total alri cases, a single virus was detected while coinfections were observed in 8% (22/292) of the samples. the most frequent viruses were rhinovirus/enterovirus (35%), human respiratory syncytial virus (26%), and influenza viruses (13%). parainfluenza viruses were detected in 9%, adenovirus in 6%, human metapneumovirus in 4%, coronaviruses (229e, nl63, oc43, hku1) in 4%, and bocavirus in 3% of alri specimens. most viral infections occurred in patients below 5 years of age. the distribution of viruses varied according to age‐groups. no significant correlation was observed between the severity of the disease and the age of patients or the virus species. this study provides the description of viral etiology among patients presenting with alri in lao pdr. additional investigations are required to better understand the clinical role of the different viruses and their seasonality in laos. acute respiratory infections are a leading cause of morbidity and mortality worldwide. 1 they represent around 2 million deaths per year, especially in infants. 2 the burden of these infections is particularly important in developing countries. 3 during the last decade, south east asia received much attention from the international scientific community due to the emergence of respiratory viruses with pandemic potential (sars-cov, avian influenza a/ h5n1 virus). 4 respiratory infections can be caused by numerous viruses, including influenza viruses, parainfluenza viruses, human respiratory syncytial virus (hrsv), human metapneumovirus (hmpv), human coronaviruses (hcov), adenoviruses, human bocavirus, and human enteroviruses. molecular techniques have become more and more popular to detect these viruses. multiplex reverse transcription-polymerase chain reaction (rt-pcr) has been shown to be a sensitive tool and allows identification of a majority of respiratory viruses, as well as coinfections. [5] [6] [7] in lao pdr, the etiology of respiratory infections is still poorly documented. to improve the clinical management of the patients, limit unnecessary antibiotic use, and prevent opportunistic secondary infections, it appears important to develop surveillance and tools to assess the etiology of acute respiratory infections in this country. 8, 9 the purpose of this study was to describe during a limited period of time the viral etiology of acute lower respiratory infections (alri) in patients hospitalized in two lao hospitals by using a set of five multiplex rt-pcr/pcr targeting 18 common respiratory viruses. this study was part of the surveillance and investigation of epidemic situations in south-east asia (sisea) project. this project was implemented by the international network of pasteur institutes in asia to improve the surveillance and management of epidemic situations in the region. the study included children and adults hospitalized for alri. for patients below 5 years of age, alri was defined as cough or dyspnea on admission with a duration of symptoms <14 days and polypnea at more than 50 breaths/min for children aged below 1 year and more than 40 breaths/min for children between 1 and 5 years old. in infants and young children, fever is sometimes absent and was therefore not considered as an inclusion criteria. for the children between 5 and 15 years old, the inclusion criteria in addition to the cough, was fever >38°c on admission (or history of fever) with a history of symptoms of <14 days. for the adults (>15-years-old), alri definition consisted of cough and fever for <14 days and one of the following symptoms of lower respiratory infection: dyspnea, chest pain, and abnormal auscultatory findings. patients with known tuberculosis (tb), known acquired immunodeficiency such as hiv/aids, and cancer were excluded. severity was assessed according to the world health organization criteria. 10 the surveillance of alri was conducted in two laotian hospitals: setthathirath hospital in vientiane capital (between august 2009 and october 2010) and in the provincial hospital of luang prabang province (between january and june 2010). in setthathirath hospital, the patients enrolled in the study were from all ages and were recruited from three wards: pediatric, intensive care unit, and internal medicine. in luang prabang provincial hospital, only children below 5 years of age hospitalized in the pediatric ward were included. for each patient who met the alri case definition, nasopharyngeal and throat swabs were collected and immediately placed into a sterile tube containing viral transport medium (vtm). 11 the samples were transported at 4°c to the national center for laboratory and epidemiology. they were then aliquoted and stored at à80°c prior to testing. nucleic acids were extracted using qiagen viral rna mini kit (qiagen, ca, usa). for each sample, 140 ll of vtm was processed according to the manufacturer's instructions, eluted in 60 ll of qiagen ave buffer, and then stored at à80°c until testing by multiplex pcr/rt-pcr. five multiplex pcr/rt-pcr were used to screen for eighteen common respiratory viruses: influenza a (ia); influenza b (ib); influenza c (ic); hmpv; hrsv; parainfluenza viruses 1-4 (piv1-4); human rhinovirus (hrhv); enteroviruses; severe acute respiratory syndrome-associated coronavirus (sars-cov); hcovs oc43, 229e, hku1, and nl63; human bocavirus, and adenoviruses. these tests were performed as described by buecher et al. 6, 7, 12 influenza a viruses subtyping influenza a strains were subtyped according to the method developed by the two french national influenza centres (northern and southern france) and described in the who information for laboratory diagnosis of pandemic (h1n1) 2009 virus. 13, 14 clinical data hospital physicians filled out a standard case report form for each participant, including information on patient's medical history, clinical features, treatment, laboratory and radiological results, and status at the time of discharge. medical records and chest x-rays were retrospectively reviewed by an expert pulmonologist. severity was assessed according to the world health organization criteria. 10 clinical data were anonymized and entered into a database by two persons who did not have knowledge of virus identities. the protocol was approved by the national ethical committee of lao pdr and by the clinical research committee (corc) of institut pasteur in paris. samples were collected after an informed written consent was obtained from the patients or their guardians/parents (in children below 15 years of age). proportions were compared using a chi-squared or contingency table randomization test as appropriate. p-values < 0á05 were considered significant. the mann-whitney u-test was applied to compare continuous or ordinal measures. analyses were performed using stata/se version 11.1 (statacorp., college station, tx, usa) and r statistical software (r 2.8.1, r foundation for statistical computing, vienna, austria). for analysis purposes, the following pathogens were grouped together: hcov oc43, hcov hku1, hcov 229e, and hcov nl63 (hcovs); influenza a, influenza b, and influenza c viruses (influenza); parainfluenza viruses 1-4 between august 2009 and october 2010, 292 nasopharyngeal swabs were collected including 27 samples from luang prabang provincial hospital and 265 samples from setthathirath hospital (vientiane). of the 292 patients, 71% were hospitalized in pediatric ward, 24% in internal medicine department, and 5% in intensive care unit. the median duration of hospitalization was 4 days. of the study population, 145 (49á7%) were male. the median age of the patients was 2á2 years (range: 12 days-86 years) with 187 patients (64%) below 5 years of age. the different age-groups are described in figure 1 . to allow comparisons with statistical significance, the 5 age-groups were merged into two main groups: infants and children aged ≤5 years (n = 187, 64%), and adults and children above 5 years of age (n = 105, 36%). (table s1 ). among these multiple viral infections, the most frequent pathogens were rhinoviruses, hrsv, and adenoviruses. a majority of alri were observed among children aged ≤5 years (187/292). the positivity rate was also the highest (i.e., 70%) in children aged ≤5 (figure 1 ). human respiratory syncytial virus was found in patients aged from 15 days to 31 years but was more frequent in young children aged ≤5 years (p = 0á0097) ( figure 3 ). influenza viruses were detected in all age-groups (median age = 12á3 years; range: 3á6 months-66 years), but were more frequent in the age-group >5 years (p < 10 e-6). rhinoviruses were mainly detected in young patients and in few occasions in elderly (median age = 1á2 years; range: 12 days -86 years). bocavirus was exclusively detected in children below 5 years of age (range = 6 months -3á5 years). for the other viruses, there was no difference statistically significant between both the age-groups. the monthly distribution of the viral respiratory infections as well as of the rhinoviruses/enteroviruses and hrsv is shown in figure 4 . the average number of samples collected monthly was 19á5 (range: 8-32). we did not observe a clear seasonality for the alri associated with a respiratory virus. rhinovirus was detected all year round. human respiratory syncytial virus circulation seemed more important during the rainy season, between july and october. the prevalence of the other viruses was low during the study period, ranging from 0 to 5 positive samples per month for each virus. these numbers appeared too low to allow the description of seasonal patterns. however, pivs were detected only between march and august 2010 with a peak in may. coinfections were observed all year long, but with only one or two cases per month. as for pivs, there was a peak in may 2010, which was most probably only a bias corresponding to a higher number of samples collected during that month. the clinical classification of the 162 patients who tested positive is summarized in the table 1 . bronchitis and exacerbation of asthma were the most frequent clinical presentations recorded in patients who tested positive for respiratory viruses (59/162, 36%). bronchiolitis and pneumonia with or without pleurisy were identified in respec-tively 20% and 19% of the patients with viral infections. the expert pulmonologist could not review 39 clinical records, which were excluded from the clinical analysis and labeled as unspecified alri. there was no significant difference in clinical presentation between patients with single infections and those with multiple viral infections. a total of 64 viruses were detected in the 59 patients presenting with bronchitis, 52 39 viruses in the 33 patients who experienced a bronchiolitis, and 37 viruses in the 31 patients with pneumonia. in bronchiolitis, the most common viruses detected were hrsv (16 patients) and rhinovirus (15 patients). in patients presenting with bronchitis and pneumonia, all the respiratory viruses tested were detected with an approximately similar frequency. the proportion of patients from whom no virus was detected was higher in patient hospitalized for pneumonia (46%) than in those presenting with bronchitis or asthma (37%) or bronchiolitis (23%). bronchiolitis was diagnosed almost exclusively in patients aged <2 years (31 of 33), whereas pneumonia was more frequent in patients aged >5 years. three deaths occurred during the study period. for two of them, no virus was identified, and for one patient, a rhinovirus was detected. these patients were respectively 5, 18 and 60 years old. of the 162 patients for which respiratory viruses were detected, 39 (24%) presented symptoms of severity. among these, 45 viruses were identified. the most common virus observed was the rhinovirus (21), followed by hrsv (7), influenza viruses (6), and bocavirus (4). in this study, we report for the first time in lao pdr the viral etiologies in patients hospitalized for alris. we identified 186 respiratory viruses in 162 (55%) patients of all ages using 5 multiplex pcr/rt-pcr. rhinovirus and hrsv were the viruses the most frequently detected, representing 35% and 26% of the total number of viruses observed, respectively. these results are consistent with other studies conducted in the region previously. 6, 15, 16 the majority of the patients included in the study were aged <5 years (64%), and 48% were <2 years old. human respiratory syncytial virus is frequently defined as the predominant virus associated with hospitalizations for alri in children aged ≤5 years. [17] [18] [19] however, in our study, we detected more rhinoviruses (hrhvs) and enteroviruses (35%) than hrsv (26%) among the 292 patients included. even if hrhvs are typically associated with the common cold, 20 recent studies suggest that these viruses may also be associated with more severe illness, including lower respiratory disease and asthma exacerbations. 21, 22 in this study, hrhv was detected in respiratory specimens from 33% of patients with bronchitis or asthma, in 25% of patients with bronchiolitis, and in 16% of those presenting with pneumonia. rhinoviruses/enteroviruses were often implicated in coinfections (73% of all the coinfections detected). however, the clinical significance of the detection of a hrhv by a highly sensitive rt-pcr method has been questioned as table 1 . these viruses can also be detected in asymptomatic children. 23, 24 rhinoviruses and enteroviruses seem to circulate all year round, without clear seasonality. human respiratory syncytial virus was the second most common virus detected in this study with a total of 49 cases (26% of patients with a positive rt-pcr result). this virus is recognized as the leading cause of hospitalizations in children aged ≤5 years for respiratory illness in industrialized countries. [25] [26] [27] similarly to other countries, we demonstrated a substantial burden of hrsv-associated alri in lao pdr. the infants and children aged <5 years were significantly more frequently infected, and then the incidence of hrsv infections decreased with age, probably because of the development of anti-hrsv immunity which is boosted during each subsequent reinfection. [28] [29] [30] during the study period, the peak of hrsv activity occurred from june to october, which corresponds to the rainy season ( figure 4) . similar observations were also reported in neighboring countries. 17, 25, 31 the overall incidence of influenza viruses infection was relatively low (12%), and the majority of the cases detected (69á6%) were among patients older than 5 years with a median age of 12á3 years. these results are in line with those of a preliminary study on influenza-like illness in lao pdr in which the incidence of influenza was 10á4%. 32 the influenza a virus strains detected during the study period were exclusively 2009 pandemic h1n1 viruses. as expected and as reported in other studies, the serotypes 1 and 3 were the most frequent pivs detected in laos. 12, 17, 33 however, piv-4 was also identified only in four cases and accounted for 24á5% of all the pivs detected. piv-4 is usually uncommon, 17,34,35 but has been identified in severe respiratory illnesses, 5,36 and its role is probably more important than originally thought. 37, 38 the overall prevalence of hmpv was 4%, which is comparable to the results reported in greece, 39 the usa, 40 or thailand. 41 the detection rate of the human bocavirus, another recently discovered respiratory virus, was 3%. this prevalence appears to vary largely between countries: 0á4% in cambodia 6 where a similar study was conducted, 3á9% in thailand, 42 16% in vietnam, 17 and 24á5% in china. 16 bocavirus is often implicated in coinfections. 12, 19, 43 in this study, 66% of the bocavirus strains detected were observed during multiple infections. human coronaviruses were detected in 4% of the alri patients in lao pdr, which is comparable to other countries (china: 5%; vietnam: 8%, cambodia: 8%). 6, 16, 17 hcov-oc43, hcov-nl63, and hcov-229e were identified only in patients aged <4 years while the only case of hcov-hku1 infection was observed in a 75 years old patient hospitalized for pneumonia. of the 162 patients infected by a respiratory virus, we detected 13á6% of coinfections. the majority of these multiple infections were identified in patients <5 years of age (90%) and associated a rhinovirus (15/22) . as rhinoviruses were detected all year round, coinfection cases were also observed regularly each month. respiratory virus coinfections being frequent, 5, 19, 44 it demonstrates the usefulness of the multiplex rt-pcr approach, which allows the detection of the most important viruses in only few reactions while multiple infections are often undetected in viral culture or by direct immunofluorescence. nevertheless, the difficult question of the clinical significance of these multiple infections remains unanswered. in our study, we did not see any association between coinfection and severity of the disease, which is in line with other reports, 5, 19, 24, 45 but this has been subjected to much controversy. [46] [47] [48] we also did not find any significant association between any virus and disease severity. in this study, bronchitis and pneumonia were the most frequent clinical presentations observed among all the agegroups of patients hospitalized for alri. bronchiolitis was observed almost exclusively in patients <2 years of age (40/43 cases). the viruses that were most frequently detected in patients <2 years of age with bronchiolitis were hrsv (15) and hrhvs (14) , which is consistent with previous observations. [49] [50] [51] fifty-seven percent of pneumonia occurred in children below 5 years of age. a better understanding of the roles of the different viruses is of great importance as pneumonia is responsible for approximately 19% of all deaths in children aged <5 years, of which more than 70% take place in sub-saharan africa and south-east asia. 1 the two main viruses observed in pneumonia in lao pdr were hrhvs and influenza a viruses. even if lao pdr is significantly less populated than neighboring thailand, vietnam, and china, the viral etiologies observed in laotian patients hospitalized with alri demonstrate some similarities to those of other south-east asian countries. however, this study has several limitations. indeed, it was conducted in only two sites (vientiane capital and luang prabang), and the second site was included only during the last 6 months of the study. moreover, our sample size is limited, especially when we stratify by age and viral etiology. finally, it was difficult to collect sputum, particularly in young children. thus, identification of bacteria was not possible. this study aimed at determining the main viral etiologies of patients hospitalized in lao pdr with alri. rhinoviruses, hrsv, and influenza virus were the more common viruses detected in the patients. bronchitis and pneumonia accounted for the majority of the hospitalizations for alri. these data are consistent with those of the literature. this study demonstrated also the usefulness of multiplex pcr/ rt-pcr to detect viral infections and to expand our knowledge of respiratory infections in such country where the data are still sparse. although the low numbers of some viruses do not allow drawing clear conclusions and considering that bacterial infections cannot be dismissed, this study provides some important preliminary data that can be used for other more focused surveys in a larger population, for instance to better describe the seasonality of the respiratory viruses. the frequency of viral infection should be taken into account by pediatricians to avoid unnecessary use of antibiotics. additional supporting information may be found in the online version of this article: table s1 . multiple infections detected in 292 patients presenting with alri. epidemiology and etiology of childhood pneumonia estimates of world-wide distribution of child deaths from acute respiratory infections lung infection-a public health priority emerging infectious diseases in southeast asia: regional challenges to control development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses use of a multiplex pcr/rt-pcr approach to assess the viral causes of influenza-like illnesses in cambodia during three consecutive dry seasons simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-pcr assays the role of respiratory viral infections among children hospitalized for community-acquired pneumonia in a developing country pneumonia research to reduce childhood mortality in the developing world pocket book of hospital care for children: guidelines for the management of common illnesses with limited resources highly pathogenic influenza a(h5n1) virus survival in complex artificial aquatic biotopes the association of newly identified respiratory viruses with lower respiratory tract infections in korean children information for laboratory diagnosis of pandemic (h1n1) pandemic a(h1n1)2009 influenza virus detection by real time rt-pcr: is viral quantification useful? viral pathogens associated with acute respiratory infections in central vietnamese children molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in shanghai viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city incidence of respiratory pathogens in persons hospitalized with pneumonia in two provinces in thailand viral and atypical bacterial detection in acute respiratory infection in children under five years rhinovirus and the lower respiratory tract rhinovirus-associated hospitalizations in young children rhinovirus associated with severe lower respiratory tract infections in children human rhinovirus infections in rural thailand: epidemiological evidence for rhinovirus as both pathogen and bystander viral respiratory infections in hospitalized and community control children in alaska the burden of hospitalized lower respiratory tract infection due to respiratory syncytial virus in rural thailand the burden of respiratory syncytial virus infection in young children global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis two distinct subtypes of human respiratory syncytial virus genetic variability of group a human respiratory syncytial virus strains circulating in germany from 1998 to seroprevalence of anti-rsv igg in thai children aged 6 months to 5 years a study of the genetic variability of human respiratory syncytial virus (hrsv) in cambodia reveals the existence of a new hrsv group b genotype an early report from newly established laboratory-based influenza surveillance in lao pdr parainfluenza virus type 3: seasonality and risk of infection and reinfection in young children specific viruses detected in nigerian children in association with acute respiratory disease progress in the development of human parainfluenza virus vaccines parainfluenza virus type 4 infections in pediatric patients human parainfluenza virus type 4 infection in chinese children with lower respiratory tract infections: a comparison study detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-pcr contribution of human metapneumovirus to influenza-like infections in north greece population-based incidence of human metapneumovirus infection among hospitalized children human metapneumovirus in infants and young children in thailand with lower respiratory tract infections; molecular characteristics and clinical presentations human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand evidence of human coronavirus hku1 and human bocavirus in australian children detection of nine respiratory rna viruses using three multiplex rt-pcr assays incorporating a novel rna internal control transcript detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy single versus dual respiratory virus infections in hospitalized infants respiratory viral infections detected by multiplex pcr among pediatric patients with lower respiratory tract infections seen at an urban hospital in delhi from impact of human metapneumovirus and respiratory syncytial virus co-infection in severe bronchiolitis viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis respiratory syncytial virus and human rhinoviruses are the major causes of severe lower respiratory tract infections in kuwait role of rhinovirus in hospitalized infants with respiratory tract infections in spain vientiane climate guide to the average weather & temperatures this study was supported by surveillance and investigation of epidemic situations in south-east asia (sisea) project, a grant from the french agency for development (afd). we gratefully thank dr anne mornand for her precious expertise in pediatric pulmonology and review of the clinical data and dr jean-jacques bernatas for the coordination of the project. we thank the team of the virology unit at institut pasteur in cambodia for their assistance in implementing the multiplex pcr/rt-pcr techniques in lao pdr. key: cord-324432-k0g3r1lw authors: maykowski, philip; smithgall, marie; zachariah, philip; oberhardt, matthew; vargas, celibell; reed, carrie; demmer, ryan t.; stockwell, melissa s.; saiman, lisa title: seasonality and clinical impact of human parainfluenza viruses date: 2018-08-29 journal: influenza other respir viruses doi: 10.1111/irv.12597 sha: doc_id: 324432 cord_uid: k0g3r1lw background: widespread availability of rapid diagnostic testing for respiratory viruses allows more in‐depth studies of human parainfluenza viruses (hpiv). objectives: this study aimed to assess seasonality of hpiv types 1‐4, clinical outcomes by hpiv type, and risk factors for illness severity. patients/methods: this retrospective study was performed from january 2013 to december 2015 in children and adults with hpiv, detected by multiplex reverse transcription polymerase chain reaction, participating in a community surveillance study of acute respiratory infections (aris) in new york city and patients admitted to a tertiary care center in the same neighborhood. seasonality trends by hpiv type were compared between the community and hospital groups. the associations between hpiv type, demographics, clinical characteristics, and illness severity were assessed. results: hpiv was detected in 69 (4%) of 1753 community surveillance participants (median age 9.2 years) and 680 hospitalized patients (median age 6.8 years). seasonality for hpiv types 1‐3 agreed with previously described patterns; hpiv‐4 occurred annually in late summer and fall. in the community cohort, 22 (32%) participants sought medical care, 9 (13%) reported antibiotic use, and 20 (29%) reported ≥1 day of missed work or school. among hospitalized patients, 24% had ≥4 chronic conditions. multivariable ordinal logistic regression demonstrated that increased severity of illness was significantly associated with hpiv‐4 and chronic cardiovascular and respiratory conditions in children and with age ≥65 years and chronic respiratory conditions in adults. conclusions: hpiv‐4 presented late summer and early fall annually and was associated with increased severity of illness in hospitalized children. human parainfluenza viruses (hpiv) types 1-4 are common respiratory pathogens that cause both upper and lower respiratory tract illnesses, especially in young children. 1 hpiv-1 and hpiv-2 are more common in children and more often associated with croup. 1 hpiv-3 is more frequently associated with bronchiolitis, bronchitis, and pneumonia. hpiv-4, while least well-characterized, has been described as associated with both mild illness in children and lower respiratory tract disease. 2 hpiv types have varying seasonality, prevalence, and clinical manifestations. in the united states, hpiv-1 usually occurs in the fall of odd numbered years, hpiv-2 occurs every fall, and hpiv-3 occurs in spring and summer. 1, 3 the seasonality of hpiv-4 has not been as well defined, often due to its low prevalence. 4 a recent study conducted in colorado found a year-round prevalence of hpiv-4 with peaks in the fall of odd numbered years. 5 however, estimates of hpiv-4 prevalence are increasing, potentially due to improved and increased diagnostic testing, but few recent studies have assessed this. 6, 7 widespread availability of multiplex reverse transcription polymerase chain reaction (rt-pcr) assays allows for more in-depth studies of the epidemiology and impact of hpiv types. the population of this study included both a community-based cohort and hospitalized children and adults with laboratory-detected hpiv. the inclusion of the community cohort allowed for a broader evaluation of the impact of hpiv than has been previously assessed. the study's objectives were to (a) assess the seasonality of hpiv types 1-4, (b) determine clinical outcomes by hpiv type, and (c) identify risk factors for increased severity of illness. a retrospective study of participants in a community surveillance cohort and hospitalized patients with hpiv types 1-4 detected from january 1, 2013 to december 31, 2015 was performed. the community cohort was derived from the mobile surveillance for acute respiratory infections (aris) and influenza-like illness (ili) in the community (mosaic) study, a 5-year community-based surveillance ordinal logistic regression demonstrated that increased severity of illness was significantly associated with hpiv-4 and chronic cardiovascular and respiratory conditions in children and with age ≥65 years and chronic respiratory conditions in adults. conclusions: hpiv-4 presented late summer and early fall annually and was associated with increased severity of illness in hospitalized children. epidemiology, parainfluenza, respiratory, seasonality, viruses f i g u r e 1 flowcharts depicting the overall number of respiratory viral panel (rvp) tests ordered which yielded the final number of human parainfluenza virus (hpiv) types in the community cohort (1a) and in hospitalized patients (1b) study in new york city (nyc) that includes 250 households annually. 8, 9 households were identified by contacting a random sample of participants who had taken part in a large population-based survey of an urban, primarily immigrant latino community. for this study, eligible households had 3 or more members with at least 1 member under 18 years of age, were spanish-or english-speaking, and had a cellular telephone with text messaging. 8 text messages were sent twice weekly inquiring about possible respiratory illness among household members. nasal swabs were collected from ill participants by research staff if at least two of the following were reported to be present: fever/feverishness, runny nose/congestion, sore throat, cough, and/or myalgia. for infants, either runny nose or congestion prompted collection of a swab. hospitalized adults and children with laboratory-detected hpiv admitted to a university-affiliated medical center in nyc located in the same neighborhood as the community cohort were included. testing was requested by treating clinicians, and as per the standard of care, is generally recommended for all hospitalizations to evaluate acute respiratory symptoms and guide transmission precautions. patients hospitalized within 2 calendar-days of hpiv detection were included. those with hpiv detected >2 days before admission, >2 days after admission and those with a second positive test for the same hpiv type within 4 weeks of the first positive test were excluded. nasopharyngeal swabs for both the community cohort and the hospitalized patients were tested for respiratory pathogens using multiplex rt-pcr (biofire diagnostics, inc. salt lake city, utah) which has been reported to have 91%-100% sensitivity and 98%-100% 10, 11 swabs from the community were tested in a research laboratory at columbia university medical center (cumc). swabs from hospitalized patients were tested in the clinical microbiology laboratory at newyork-presbyterian hospital at cumc. blood, urine, and respiratory tract cultures for bacteria were sent by treating clinicians as per the standard of care for suspected infections and also processed by the clinical microbiology laboratory. for the community cohort, days of missed school or work; medical care in primary care clinics, urgent care, or emergency departments; hospitalizations; and use of antibiotics were collected by follow-up calls at least 15 days after the resolution of respiratory illness. for hospitalized patients, demographic and clinical characteristics were obtained from electronic medical records (emr). primary icd-9 diagnoses were categorized as respiratory or nonrespiratory. patients' primary and secondary diagnoses were categorized into groups of chronic conditions previously described as risk factors for severe respiratory disease in adults and children with respiratory viruses. [12] [13] [14] conditions were excluded if they could not be classified within a chronic condition category, had <15 occurrences across all patients, or have not been associated with severe respiratory disease, for example bipolar disorder. manual chart review of readmitted patients was performed to determine whether readmissions within 4 weeks were due to respiratory illness. hospital course and healthcare utilization data were obtained from the emr including radiographs within 2 days of hpiv detection; all antibiotics within 2 days prior to or 7 days after hpiv detection; icu admission and 30-day mortality. respiratory support including use of continuous positive airway pressure (cpap), mechanical ventilation, or extracorporeal membrane oxygenation (ecmo) was determined by billed procedure codes. use of bilevel positive airway pressure (bipap) or oxygen supplementation was not assessed, as accurate usage data were unavailable in structured electronic sources. ta b l e 1 comparison of demographic characteristics, symptoms, and outcomes of participants in a community cohort, by human parainfluenza viruses (hpiv) type to assess seasonality, epidemiologic curves that modeled the detection of each hpiv type in the community cohort versus hospitalized patients were created. demographic and clinical characteristics of the two groups were analyzed as proportions, means, and/or medians; associations between hpiv type and clinical course were examined using chi-square, fisher's exact, and anova tests, as appropriate. ta b l e 2 comparison of demographic and clinical characteristics of hospitalized patients with different human parainfluenza viruses (hpiv) in the community cohort, 1805 swabs were obtained, of which 69 (4%) of 1753 participants were positive for at least one hpiv type ( figure 1a ). the 69 hpiv-positive swabs were from 59 households; hospital community at least one hpiv type ( figure 1b) the mean length of hospitalization was 5.3 (sd ± 8.4) days and varied by hpiv type ( were associated with higher odds of increased severity of illness. among the 290 adults, 12 (4%) had severe, 41 (14%) had moderate, and 237 (82%) had mild illness (table 5 ). in the final adjusted model for adults, age ≥65 years and respiratory conditions were associated with higher odds of increased severity of illness. we had a unique opportunity to compare the epidemiology and clinical impact of hpiv types among individuals in a communitybased surveillance study and among hospitalized patients during the same time and in the same geographic area. the seasonality of hpiv types 1-3 was consistent with published trends, 1,3 but we found that hpiv-4 occurred annually in late summer and fall, suggestive of a local epidemiologic trend that has not been previously described. we found that hpiv-3 was most common among hospitalized patients (52%) while hpiv-4 was most common in the community cohort (36% we modified a previously validated severity of illness score to further explore the predictors of illness severity. in children, increased severity of illness was associated with respiratory conditions, cardiovascular conditions and hpiv-4 while in adults, increased severity of illness was associated with age ≥65 years and cardiovascular conditions. the association of comorbid conditions and severe illness in those with hpiv is consistent with other respiratory viruses, most notably rsv and influenza, which both can exacerbate underlying cardiac and pulmonary conditions. [12] [13] [14] 21, 22 this may also explain why 21% of hospitalized patients had a nonrespiratory primary diagnosis. the rate of co-detection was similar across hpiv strains and as others have shown, viral co-detections did not impact severity of disease. 23 however, due to limited sample size, we could not assess the impact of specific co-infections. while only 17% of hpiv-positive patients had a positive bacterial culture, 73% received at least one dose of an antibiotic between 2 days before and 7 days after hpiv detection. thus, most antibiotic usage represented empiric therapy, suggesting an opportunity for antibiotic stewardship. this study had limitations. the community cohort was 100% hispanic/latino, reducing the study's generalizability, and small, limiting our ability to find differences in severity of illness or symptomatology associated with hpiv types. our hospital is a referral center and cares for many patients with underlying conditions. furthermore, its referral status could impact accurate interpretation of "local" epidemiology as only 32% of the hospitalized patients lived in the same zip codes as the mosaic study participants (m. stockwell, personal communication). testing of hospitalized patients relied on clinicians' judgement. we did not review radiographic findings or capture bipap use which may have biased assessment of severity of illness. the severity of illness score was developed for children and not previously tested in adults. use of administrative data restricted our ability to assess patients' symptomatology. although most patients had a respiratory diagnosis, we may have misclassified hpiv infections that actually represented prolonged hpiv shedding from previous illnesses. we did not classify positive bacterial cultures as consistent with infection vs. colonization vs. contamination. in this study, each hpiv type demonstrated seasonality, but each was similar in the community and among hospitalized patients. we found that hpiv-4 had distinct epidemiology, not previously described, and was associated with increased severity of illness in hospitalized children. as demonstrated for other respiratory viruses, older age and underlying conditions, particularly respiratory and cardiac conditions, were associated with increased severity of illness. additional studies exploring hpiv incidence and severity in both children and adults could help reinforce the need for vaccine development. the authors thank alexandra hill-ricciuti for analytic assistance. seasonal trends of human parainfluenza viral infections: united states estimates of parainfluenza virus-associated hospitalizations and cost among children aged less than 5 years in the united states epidemiology and clinical presentation of parainfluenza type 4 in children: a 3-year comparative study to parainfluenza types 1-3 epidemiology of parainfluenza infection in england and wales, 1998-2013: any evidence of change? human parainfluenza type 4 infections mosaic: mobile surveillance for acute respiratory infections and influenza-like illness in the community community -and hospital laboratory-based surveillance for respiratory viruses. influenza other respir viruses filmarray respiratory panel information sheet multicenter evaluation of the eplex respiratory pathogen panel for the detection of viral and bacterial respiratory tract pathogens in nasopharyngeal swabs respiratory syncytial virus infection in elderly and high-risk adults children with complex chronic conditions in inpatient hospital settings in the united states pediatric deaths attributable to complex chronic conditions: a population-based study of washington state predicting severe pneumonia outcomes in children human parainfluenza virus types 1-4 in hospitalized children with acute lower respiratory infections in china a prospective study of parainfluenza virus type 4 infections in children attending daycare severe pneumonia after cardiac surgery as a result of infection with parainfluenza virus type 4 human parainfluenza virus 4 outbreak and the role of diagnostic tests defining the risk and associated morbidity and mortality of severe respiratory syncytial virus infection among infants with chronic lung disease influenza virus infections among patients attending emergency department according to main reason to presenting to ed: a 3-year prospective observational study during seasonal epidemic periods single-and multiple viral respiratory infections in children: disease and management cannot be related to a specific pathogen seasonality and clinical impact of human parainfluenza viruses key: cord-349330-akwxwfg3 authors: wabe, nasir; lindeman, robert; post, jeffrey j.; rawlinson, william; miao, melissa; westbrook, johanna i.; georgiou, andrew title: cepheid xpert(®) flu/rsv and seegene allplex(™) rp1 show high diagnostic agreement for the detection of influenza a/b and respiratory syncytial viruses in clinical practice date: 2020-08-20 journal: influenza other respir viruses doi: 10.1111/irv.12799 sha: doc_id: 349330 cord_uid: akwxwfg3 background: molecular assays based on reverse transcription‐polymerase chain reaction (rt‐pcr) provide reliable results for the detection of respiratory pathogens, although diagnostic agreement varies. this study determined the agreement between the rt‐pcr assays (xpert(®) flu/rsv vs allplex(™) rp1) in detecting influenza a, influenza b, and respiratory syncytial viruses (rsvs) in clinical practice. methods: we retrospectively identified 914 patient encounters where testing with both xpert(®) flu/rsv and allplex(™) rp1 was undertaken between october 2015 and september 2019 in seven hospitals across new south wales, australia. the diagnostic agreement of the two assays was evaluated using positive percent agreement, negative percent agreement, and prevalence and bias‐adjusted kappa. results: the positive percent agreement was 95.1% for influenza a, 87.5% for influenza b, and 77.8% for rsv. the negative percent agreement was 99.4% for influenza a, 99.9% for influenza b, and 100% for rsv. the prevalence and bias‐adjusted kappa was 0.98 for influenza a, 0.99 for influenza b, and 0.97 for rsv. in a sensitivity analysis, the positive percent agreement values were significantly higher during the non‐influenza season than the influenza season for influenza b and rsv. conclusions: the xpert(®) flu/rsv and allplex(™) rp1 demonstrated a high diagnostic agreement for all three viruses assessed. the seasonal variation in the positive percent agreement of the two assays for influenza b and rsv may have been due to lower numbers assessed, variability in the virology of infections outside the peak season, or changes in the physiology of the infected host in different seasons. acute respiratory infections due to influenza and respiratory syncytial viruses (rsvs) have a significant health and economic burden in australia 1,2 and internationally. 3, 4 worldwide, influenza is implicated in approximately two percent of all respiratory deaths, 5 with an official world health organization estimate of 290 000-650 000 seasonal influenza-associated deaths globally each year. 6 in 2017, the influenza virus was implicated in an estimated 11.5% of episodes of care for lower respiratory tract infection, including 9.5 million hospitalizations and 81.5 million hospital days worldwide. 4 viruses and provides real-time influenza a subtyping. 7 it comprises three viral panels and one bacterial panel. the first of these viral panels (the allplex ™ rp1) detects influenza a and subtypes (h1, h1pdm09, and h3), influenza b, and rsv (types a and b). although the allplex ™ rp offers comprehensive testing for respiratory viruses, it is undertaken by a central laboratory and specimens are tested in batches (processing multiple specimens simultaneously) rather than through continuous needs-based testing. therefore, it has a test turnaround time of 1-4 days depending upon the frequency of batch testing and location of the hospital. 8 the xpert flu/rsv xc is an automated, multiplex real-time, rt-pcr assay for the detection of influenza a, influenza b, and rsv. 9 it has a faster turnaround time of 1-4 hours, 8,10 but unlike the allplex ™ rp1, xpert ® flu/rsv cannot discriminate among influenza a virus subtypes. nevertheless, the rapid identification of respiratory viruses has the potential to improve patient outcomes by supporting clinical decision-making around antimicrobial use. 11 more rapid identification could also improve clinical processes, including optimizing bed management and infection control 12 as well as minimizing unnecessary ancillary test utilizations, 8, 10, 13 with subsequent reductions to healthcare costs. 14 all molecular assays based on rt-pcr provide reliable results for the detection of respiratory viruses but are not always in diagnostic agreement. previous studies reported the performance of xpert ® flu/rsv and allplex ™ rp1 against other reference assays. 7, 9, [15] [16] [17] [18] [19] [20] however, to the best of our knowledge, no prior published studies have compared the xpert ® flu/rsv and allplex ™ rp1 in clinical practice. the objective of the study was to determine the diagnostic agreement between xpert ® flu/rsv vs allplex ™ rp1 for the detection of influenza a, influenza b, and rsv in the hospital emergency department (ed) or inpatient settings. we conducted a retrospective observational study utilizing 4 years of data from october 2015 to september 2019 extracted from seven public hospitals (hospitals a-g) in new south wales, australia (six general hospitals and one children's hospital [hospital d]). xpert ® flu/rsv was introduced to four of the study hospitals (hospitals a-d) in july 2017, while hospitals e-g have used the test since october 2015. therefore, the data before july 2017 were only from the other three hospitals (hospitals e-g). eligibility criteria included patients for whom both xpert ® flu/rsv and allplex ™ rp1 tests were ordered at the same time for the same episode of care. exclusion criteria specified patients for whom: (a) only one of the assays was ordered, (b) both assays were ordered but at different times (eg, xpert ® flu/rsv while the patient was in the ed and allplex ™ rp1 while the patient was in the inpatient ward), and (c) both assays were ordered but the result of one of the assays was not reported or missing due to unacceptable specimens ( figure 1 ). the data used for this study were sourced from the laboratory information system (lis) of each hospital. the lis contains laboratory test order information including, but not limited to, patient age and sex, patient medical reference number, test order episode identification, type of tests ordered, location of the order, test results and specimen types, as well as the date and time a specimen is collected, received at the laboratory and a verified result available. all study hospitals used one lis and thus had similar test catalogs and configurations. detailed information regarding the use of the two assays has been reported in our previous studies. 8, 10, 21 briefly, allplex ™ rp has been used as a referral test at a large central laboratory located at hospital b and all other hospitals sent samples to this laboratory for analysis. testing was performed in batches once or twice depending upon demand. 8 as allplex ™ rp was offered as three viral panels, in addition to panel 1, clinicians had the option of ordering panel 2 (which detects adenovirus, metapneumovirus, enterovirus, and parainfluenza viruses) and panel 3 (which detects bocaviruses, coronaviruses, and rhinoviruses) depending on the clinical scenario. unlike allplextm rp, testing using the xpert ® flu/rsv was performed at local hospital laboratories avoiding the need for sending the specimen to the central laboratory. while both assays were available to clinicians during the study period, the use of xpert ® flu/rsv was recommended to be used by the laboratory service for patients at high risk of influenza, where a result was required more urgently. this included intensive care patients with influenza-like illness, immunocompromised patients with influenza-like illness, ed patients with a significant respiratory infection and isolation requirement. nasopharyngeal swabs and nasopharyngeal aspirates were the two main types of specimens that have been used for both xpert ® flu/ rsv and allplextm rp1. sputum or bronchial lavages were also used in very few cases. a two-by-two contingency table comparing the results of xpert ® flu/rsv against allplex ™ rp1 was created. the diagnostic agreement of the two assays was evaluated using positive percent agreement (ppa), negative percent agreement (npa), kappa, and prevalence and bias-adjusted kappa (pabak) along with their 95% confidence intervals (ci). ppa (which is analogous to sensitivity) was calculated by dividing the number of allplex ™ rp1 + and xpert ® + cases by total allplex ™ rp1 + cases. npa (which was analogous to specificity) was calculated by dividing the number of allplex ™ rp1− and xpert ® − cases by total allplex ™ rp1− cases. the values of kappa can range from −1 to 1 and can be interpreted as <0.2 as none to slight; 0.21-0.4 as fair; 0.41-0.6 as moderate; 0.61-0.8 as substantial; and 0.81-1.0 as almost perfect agreement. 22 because kappa can be affected by disease prevalence and potential bias between the assays, pabak was reported to account for these influences. 23 this is particularly important given the low prevalence of respiratory viruses in our sample. bias in the context of this study is said to occur when the two assays differ in the frequency of the detection of a given virus in the study sample. 23 baseline factors associated with discordant results between the two assays were determined using penalized logistic regression as it reduces the small-sample bias and is thus suitable for modeling low prevalence binary outcomes. 24 a discordant result (yes/no) was defined as discrepancies between the results of the two assays in at least one of the three viruses (ie, allplex ™ rp1 + and xpert ® − and vice versa). ethical approval for the study was granted by the human research ethics committee of the south eastern sydney local health district (reference, hrec/16/powh/412). a total of 914 patient encounters fulfilled the inclusion criteria ( figure 1 ). the median age was 28 years, and 55.2% (n = 505) were male. a nasopharyngeal swab was the most common specimen type (table 1 ). the median turnaround time from specimen receipt to authorized result was 3.3 hours (ranged from 1. 5 the diagnostic agreement measures are shown in overall, 23 specimens showed a discordant result. one of these specimens showed discrepancies in results for more than one virus (table s1 ). gender, age category, setting where tests were ordered or the type of specimen was not associated with a discordant result. in other words, there were no significant differences in the results of the two tests across these characteristics. however, influenza season status was significantly associated with a discordant result. the difference between the two assays was higher during influenza season compared to the non-influenza season. xpert ® flu/rsv and allplex ™ rp1 were 3.38 times more likely to have discordant results during the influenza season than the noninfluenza season (table 3 ). sensitivity analysis by influenza season status was conducted as it was associated with the discrepancy between the results of the we retrospectively evaluated the agreement of two pcr-based assays ( the main clinical implication of our finding is that there is no need to use both assays at the same time in clinical practice, given the abbreviations: ed, emergency department; np, nasopharyngeal. a np swab (allplex) and np aspirate (xpert) or np aspirate (allplex) and np swab (xpert) or the use of sputum or bronchial lavages in one of the assays. to the best of our knowledge, this is the first study to compare the xpert ® flu/rsv and allplex ™ rp1. the inclusion of diverse study populations from multiple sites including six general and one children's hospitals can be considered as the main strength of this study. the key limitation of this study was that, in the case of discrepant results between the two assays, a three-way comparison using another confirmatory method has not been conducted to verify the results. this study utilized retrospective data from hospitals where xpert ® flu/rsv and allplex ™ rp1 were the main laboratory tests for the diagnosis of influenza and rsv. it was not possible to determine which of the two assays was correct in the case of discrepancies between the assays. the allplex ™ rp1 assay flexibility in assessing other non-respiratory viruses at a lower unit cost, also means assessment of other causes of a patient's symptoms are potentially assessed. according to the local guideline for the use of xpert ® flu/ rsv, its use was mainly reserved for patients at high risk of influenza where an urgent result was needed. the study population selected for this study may, therefore, differ in disease severity from other patients presenting to the hospitals with influenza-like illnesses but were not eligible to receive the test, potentially introducing bias into the study. as this study was not designed to assess test cost or health economic outcomes, no conclusions around this can be drawn. in conclusion, xpert ® flu/rsv xc and allplex ™ rp1 demonstrated a high diagnostic agreement for all three viruses assessed. the seasonal variation in the ppa of the two assays for influenza b and rsv may have been due to lower numbers assessed, variability in the virology of infections outside the peak season, or changes in infected host physiology in different seasons. the study is part of a partnership project funded by a national the authors declare that they have no conflict of interest. https://orcid.org/0000-0002-9740-6319 incidence of acute respiratory infections in australia epidemiology of viral respiratory infections in australian working-age adults (20-64 years global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study mortality, morbidity, and hospitalisations due to influenza lower respiratory tract infections, 2017: an analysis for the global burden of disease study global mortality associated with seasonal influenza epidemics: new burden estimates and predictors from the glamor project estimates of global seasonal influenza-associated respiratory mortality: a modelling study performance evaluation of allplex respiratory panels 1, 2, and 3 for detection of respiratory viruses and influenza a virus subtypes impact of rapid molecular diagnostic testing of respiratory viruses on outcomes of adults hospitalized with respiratory illness: a multicenter quasi-experimental study prospective and retrospective evaluation of the performance of the fda-approved cepheid xpert flu/rsv xc assay the impact of rapid molecular diagnostic testing for respiratory viruses on outcomes for emergency department patients rapid molecular panels: what is in the best interest of the patient? a review of patient outcome studies for multiplex panels used in bloodstream, respiratory, and neurological infections rapid testing for respiratory syncytial virus in a paediatric emergency department: benefits for infection control and bed management emergency department management of febrile respiratory illness in children cost analysis of multiplex pcr testing for diagnosing respiratory virus infections evaluation of seegene allplex respiratory panel 1 kit for the detection of influenza virus and human respiratory syncytial virus clinical performance of the allplextm respiratory panel 1 test compared to simplexatm flu a/b and rsv for detection of influenza virus and respiratory syncytial virus infection including their subtyping evaluation of allplex respiratory panel 1/2/3 multiplex real-time pcr assays for the detection of respiratory viruses with influenza a virus subtyping performance characteristics of xpert flu/rsv xc assay prospective and retrospective evaluation of the cepheid xpert ® flu/rsv xc assay for rapid detection of influenza a, influenza b, and respiratory syncytial virus charnot-katsikas a. comparison of cepheid xpert flu/rsv xc and biofire filmarray for detection of influenza a, influenza b, and respiratory syncytial virus timing of respiratory virus molecular testing in emergency departments and its association with patient care outcomes: a retrospective observational study across six australian hospitals interrater reliability: the kappa statistic bias, prevalence and kappa firth's logistic regression with rare events: accurate effect estimates and predictions sensitivity, specificity, and predictive values: foundations, pliabilities, and pitfalls in research and practice how to cite this article rp1 show high diagnostic agreement for the detection of influenza a/b and respiratory syncytial viruses in clinical practice key: cord-332057-1vvlzuue authors: pourbohloul, babak; ahued, armando; davoudi, bahman; meza, rafael; meyers, lauren a.; skowronski, danuta m.; villaseñor, ignacio; galván, fernando; cravioto, patricia; earn, david j. d.; dushoff, jonathan; fisman, david; edmunds, w. john; hupert, nathaniel; scarpino, samuel v.; trujillo, jesús; lutzow, miguel; morales, jorge; contreras, ada; chávez, carolina; patrick, david m.; brunham, robert c. title: initial human transmission dynamics of the pandemic (h1n1) 2009 virus in north america date: 2009-08-18 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2009.00100.x sha: doc_id: 332057 cord_uid: 1vvlzuue background between 5 and 25 april 2009, pandemic (h1n1) 2009 caused a substantial, severe outbreak in mexico, and subsequently developed into the first global pandemic in 41 years. we determined the reproduction number of pandemic (h1n1) 2009 by analyzing the dynamics of the complete case series in mexico city during this early period. methods we analyzed three mutually exclusive datasets from mexico city distrito federal which constituted all suspect cases from 15 march to 25 april: confirmed pandemic (h1n1) 2009 infections, non‐pandemic influenza a infections and patients who tested negative for influenza. we estimated the initial reproduction number from 497 suspect cases identified prior to 20 april, using a novel contact network methodology incorporating dates of symptom onset and hospitalization, variation in contact rates, extrinsic sociological factors, and uncertainties in underreporting and disease progression. we tested the robustness of this estimate using both the subset of laboratory‐confirmed pandemic (h1n1) 2009 infections and an extended case series through 25 april, adjusted for suspected ascertainment bias. results the initial reproduction number (95% confidence interval range) for this novel virus is 1·51 (1·32–1·71) based on suspected cases and 1·43 (1·29–1·57) based on confirmed cases before 20 april. the longer time series (through 25 april) yielded a higher estimate of 2·04 (1·84–2·25), which reduced to 1·44 (1·38–1·51) after correction for ascertainment bias. conclusions the estimated transmission characteristics of pandemic (h1n1) 2009 suggest that pharmaceutical and non‐pharmaceutical mitigation measures may appreciably limit its spread prior the development of an effective vaccine. influenza a of the h1n1 subtype caused the 1918 pandemic, was replaced in 1957 by the h2n2 subtype, reemerged in 1976 ⁄ 1977, and has since variously contributed to influenza illness (alongside the dominant h3n2 subtype that emerged in 1968). 1 the a ⁄ h1n1 subtype was present in swine populations by 1930, and separate north american and eurasian lineages are now considered endemic in these animals. 2 during the fourth week of april 2009, pandemic (h1n1) 2009 virus consisting of north american and eurasian components was identified as the cause of sporadic but mild human illness in california and texas and a substantial and severe outbreak in mexico. this novel influenza variant is believed to be the result of a recent reassortment event among three distinct swine influenza virus lineages, resulting in a novel hybrid h1n1 virus including north american swine hemagglutinin (h1), eurasian neuraminidase (n1) and matrix proteins, and contribution of remaining segments from the classic triple reassortant swine virus. 3 the human index case of pandemic (h1n1) 2009 appears to have occurred in the town of la gloria in the state of veracruz, a region containing large-scale industrial pig farms. spread to mexico city occurred by 15 march 2009. 4 between 15 march and 19 april, sporadic cases in mexico city increased erratically, with person-to-person transmission becoming sustained and amplified after mass population return to the city following the holy week holiday (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) . a public health emergency was decreed in mexico on 23 april. after laboratory confirmation of pandemic (h1n1) 2009 infection on 23 april, direccion general de epidemiologia, secretaria de salud, mexico (dge) developed case definitions. a suspected case was defined as severe respiratory illness with fever, cough and difficulty breathing. a probable case was defined as a suspected case in a patient from whom a specimen had been collected and tested positive for influenza a. a confirmed case was defined as a probable case that tested positive for pandemic (h1n1) 2009 by real-time reverse-transcription polymerase chain reaction. health-care officials were contacted and asked to provide retrospective and ongoing data for persons having illness consistent with these case definitions and seeking care on or after 1 march. as of 15 june, 6241 cases including 108 deaths have been reported as laboratory-confirmed because of pandemic (h1n1) 2009 in mexico. returning travelers have also seeded cases in 74 other countries, including 35 928 confirmed cases globally as of 15 june with 55 deaths reported outside of mexico to date. 5 on 11 june, the world health organization (who) declared the outbreak to be the first global influenza pandemic in 41 years. 6 the initial community outbreak in mexico city, a metropolitan urban area with about 20 million inhabitants, provides a critical glimpse into the further epidemic potential of this virus. early transmission in mexico city was punctuated by two pivotal sociological events. first, from 5 to 19 april, schools were closed and approximately 10% of the population left the city for the holy week holiday. 7 this may have temporarily reduced transmission within the city while increasing the risk of spread to other communities. second, on 23 april, the government of mexico city declared a public health emergency that may have increased hospital visits for milder illness that might otherwise have gone unreported. mexico as a whole experienced a mild and delayed 2008-2009 influenza season, with other human subtypes of influenza viruses; the extent to which human influenza and other respiratory viruses contributed to illness reports during that period is unknown. using the time series of the first 497 suspect cases, we have estimated the initial rate of spread -the reproduction number r -of pandemic (h1n1) 2009, using methods of contact network epidemiology that incorporates extrinsic sociological drivers and uncertainties in disease progression and underreporting. [8] [9] [10] to date, it is unknown whether prior infection with human a ⁄ h1n1 subtypes provides cross immunity to the novel variant; if not, our estimate reflects its basic reproduction number r 0 . we analyzed four data sets provided by public health officials in mexico city. the first time series consists of early reports of suspect influenza infection from all hospitals in the mexico city metropolitan area between 15 march and 30 april (hereafter s) ( figure 1 ). this data included dates of self-reported onset of symptoms and hospitalization for approximately 3798 suspect cases of pandemic (h1n1) 2009 variant. while it is likely that these cases ranged from mild to fatal and that treatment ranged from home-based self-care to hospitalization, such individual-level clinical information was not available. however, prior to laboratory testing, it was unknown which of these infections were pandemic (h1n1) 2009 rather than typical influenza a or other respiratory infectious agents. after laboratory results from suspect cases were released on 4 june 2009, we received three additional epidemiological time series provided by the mexico city ministry of health (secretaria de salud del distrito federal) from mexico city (distrito federal) from 1 march to 30 may. these mutually exclusive data sets include symptom onset dates for cases where laboratory tests were negative for influenza a virus (hereafter n), cases where laboratory tests were positive for any influenza a strain, excluding pandemic (h1n1) 2009 (hereafter a), and cases with laboratory confirmation for pandemic (h1n1) 2009 (hereafter c) (figure 1 ). using a network-based statistical approach, we estimated the initial reproduction number (r) of the novel north american a ⁄ h1n1 variant during this period of spread. 11 this method initially estimates the time series of infection dates and the rates of removal through recovery, death or hospitalization, and then incorporates these values into a stochastic, network-based model to estimate the reproduction number. the estimate is refined with every additional day of time series data. for typical unmitigated influenza epidemics, values from this method typically converge to the best estimate of r within a few days, before the acceleration of the outbreak, and remain stable throughout the epidemic period. therefore, any deviations from the converged value likely reflect external influences including social events and ⁄ or intervention [please refer to the supplementary material for more details]. the estimation method requires information about disease progression, specifically the duration of the incubation (t l ), asymptomatic infectious (t a ) and symptomatic infectious (t s ) periods, as well as the contact patterns underlying disease transmission (specifically the expected number of contacts for each new case). at the time of this study, little was known about these parameters for the pandemic (h1n1) 2009 outbreak in mexico city. empirical data from contact investigations of confirmed cases of pandemic (h1n1) 2009 in the canadian province of ontario suggest that the median interval between contact with a confirmed symptomatic case and development of symptoms is 6 days (95% ci 5-7 days). as such, we used a 5-day latent period and 2-day asymptomatic infectious period as a plausible upper bound for time from infection until development of symptomatic disease. thus, we considered a wide range of possible values estimated for other strains of influenza and large urban centers, respectively ( table 1 ). the infectious period of a case may be curtailed by hospitalization or selfisolation prior to recovery or death. we therefore directly estimated the distribution of removal rates via either natural causes or intervention by calculating the numbers of days between the onset of symptoms and hospitalization (type ii and iii triage protocol) or self-isolation (type i triage protocol) for all reported cases. we assumed that unreported asymptomatic or mild cases 12 remained infectious until recovery. the early time series of suspect cases in mexico city (s) revealed a 1-month period in which daily case counts remained low (never surpassing twenty) followed by a fairly sharp climb and descent ( figure 1 ). the large pulse, from 78 to 407 cases occurred from 19 april -the last day of the holy week period -to 26 april, shortly after initial pronouncements by mexico's public health secretary. the number of laboratory confirmed cases of pandemic (h1n1) 2009 (epidemic curve c) was significantly lower than the initial curve of suspected patients (s). many of the original suspect cases were not infected with influenza a at all (n), and a number were infected by influenza a strains other than pandemic (h1n1) 2009 (a). based on suspect cases prior to 20 april, we estimate a reproduction number of 1ae51 (95% ci 1ae32-1ae71); for confirmed cases, it is 1ae43 (95% ci 1ae29-1ae57). (table 2 ) to assess the reliability of estimates made from early epidemic data, we estimated the reproduction number of pandemic (h1n1) 2009 using both early data (figure 1 , curve a) and later data (figure 1, other curves) , and compared the results of these two analyses. for each day, we estimate r using the full time series preceding and including that day. the estimates stabilize approximately one week before the end of holy week. this value likely represents the intrinsic rate of transmission that would ultimately drive the exponential growth of epidemic (the so-called 'epidemic curve'). the analysis of seasonal influenza incidence in mexico from 1990 to 2005 suggests that the bulk of seasonal influenza transmission occurs between november and february, declining in march and subsiding in april. 13 therefore, the apparent peak (figure 1 , curve a) between 19 april and 10 may likely does not correspond to a seasonal influenza epidemic peak. the surprisingly synchronous changes in non-influenza a cases (curve n) suggests that some of the fluctuations may be caused by extrinsic factors rather than underlying transmission dynamics. assuming that there were no other respiratory epidemics in the same period, it is plausible that the marked increase in reported cases after 19 april is attributable to a surge in notification spurred by the end of holy week and the public health decrees. assuming that the improved surveillance and public health alerts led to increased case ascertainment of both a and c in a similar manner, one can use the rates of a to 'deflate' the number of confirmed cases with date of symptoms onset later than 17 april to estimate the underlying transmission-driven incidence curve (figure 2a,b) . this adjusted time series should represent the number of cases identified if the public health measures described had not been implemented. of note, the peak of the adjusted epidemic curve shifts from 27 april to 23 april, the date the public alert took place and when the social distancing interventions were implemented. this suggests that the public health interventions may have significantly mitigated the epidemic. adjustment using both the patients without influenza (curve n) and those with non-pandemic influenza a (curve a) has similar impacts ( figure 2 ). looking at the time period immediately before 23 april, one may also use either curve a or n to 'inflate' the number of confirmed cases to reflect the hypothetical scenario of early heightened surveillance and public awareness (from the start of the epidemic) ( figure 3a ,b). this approach increases confirmed cases closer to the 'true' prevalence. estimates of r based on the four adjusted curves are also given in table 2 . unlike the estimates based on the raw data, these estimates remain relatively stable throughout the period of heightened awareness and surveillance. to test the sensitivity of the estimates to underlying assumptions, we repeatedly re-estimated r using random choices of parameters from the ranges given in table 1 . figure 4 shows the distribution of r for case series s, c, and both a-and n-deflated c, with each series run with data from before and after the surge (to 19 april and to 25 april, respectively). each panel summarizes the estimates of r using 388 080 combinations of parameter values. the parameter with greatest impact on estimated reproduction number over the range assessed is the latency period (data not shown). the results suggest that the estimates of r for the second segment of the adjusted time series (after public health alerts) are in agreement with the first segments of both time series s and c. figure 4 also illustrates that if the time series representing confirmed cases is not adjusted for extrinsic factors, a wider confidence interval will be achieved. with a reproduction number of approximately 1ae5, the pandemic (h1n1) 2009 virus appears to exhibit community transmissibility similar to the 1957 and 1968 pandemics, or the recently emerged respiratory pathogen sars coronavirus (sars-cov) but less than the fall wave of the 1918 influenza pandemic. 10, 14, 15 early global dissemination of both sars and pandemic (h1n1) 2009 illustrates the complex inter-connectivity of human populations globally and the epidemiological significance of individual and social behavior. our analysis shows that even noisy and potentially biased early epidemic outbreak data may form the basis for stable estimates of biological characteristics of emerging pathogens. our analytical method enables us to evaluate the impact of variability in disease serial interval and contact network structure on the temporal progression of an outbreak -and the related epidemiological parameter r; additionally, we can use this method to evaluate the effect of external social drivers, while respecting the pattern observed in time series data in mexico city (for more details, see the supplementary material). although most discussions of pandemic (h1n1) 2009 will likely focus on how it compares with the three 20th century influenza pandemics, we believe that comparison with sars-cov will also yield critical policy implications for public health intervention. the sars-cov had a relatively long 4-to 6-day incubation period and a peak infectious period that was delayed until the tenth day of severe illness. 16 sars was transmitted predominantly in the healthcare settings, with a case fatality rate exceeding 10%. these features made sars relatively easy to detect and readily amenable to individual-based control measures such as case isolation and contact tracing before substan-tial spread could occur beyond initial seeding and local hospital-based outbreaks. influenza viruses, in contrast, are characterized by shorter incubation (typically 1-4 days), pre-clinical virus shedding and peak infectiousness shortly after illness onset. 17, 18 influenza illness comprises a spectrum including mild or asymptomatic infection with overall case fatality below 2% even during the worst pandemic on record (1918) and 10-fold lower still during subsequent pandemics. 19 in keeping with this classic influenza profile, pandemic (h1n1) 2009 shows a larger proportion of mild infections, community-based propagation and a lower case fatality than sars. 20 thus, while the reproduction numbers of the two infections are not dramatically different, they likely will require a different set of population-based social distancing and mitigation measures. general reinforcement of voluntary self-isolation, cough etiquette, handwashing and self-monitoring by contacts combined with social strategies to disrupt complex contact networks and lessen virus amplification and adaptation at the community level are needed. national health authorities in north america and europe have implemented varying school closure policies (e.g. broadly in mexico, targeted in the uk and minimal in the us) in an attempt to contain viral transmission; as of this writing, these measures are being scaled back, but further interventions to change social contact patterns may be important as the outbreak progresses. 21 pandemics are classically defined as the emergence of novel influenza a subtypes that have not previously been experienced by human populations, or at least not for a 2009 remains a worthwhile goal until a safe and effective vaccine can be developed and administered. furthermore, global inequalities in social and economic conditions amplify the impacts of infectious diseases, including influenza. 22 as our estimates account for important sociological anomalies and are based on multiple data sources from communities rather than closed settings, they are likely to be broadly applicable. however, our optimistic prognosis relies on several critical assumptions about disease progression, virulence, and reporting rates, and it is clear that worse scenarios could evolve. vigilant surveillance, self-isolation, adherence to social distancing and hygiene measures, strategic school closures, and other community measures to mitigate spread, as directed by national policy, may be paramount in the months to come. preparation of the mexico city data (collection, data processing, interpretation and verification for accuracy, statistical analysis) and report on public health response a. ahued, i. villaseñor, f. galván, p. cravioto, j. trujillo, m. lutzow, j. morales, a. contreras, c. chávez. influenza pandemics of the 20th century olsen cw influenza in pigs and their role as the intermediate host genbank sequences from 2009 h1n1 influenza outbreak outbreak of swine-origin influenza a (h1n1) virus infection -mexico influenza a (h1n1) -update 49, global alert and response (gar), world health organization statement by who director-general, dr margaret chan a comparative analysis of influenza vaccination programs spread of epidemic disease on networks network theory and sars: predicting outbreak diversity earn djd early real-time estimation of infectious disease reproduction number. arxiv pandemic potential of a strain of influenza a (h1n1): early findings mortality due to influenza and pneumonia in mexico between sars outbreaks in ontario, hong kong and singapore: the role of diagnosis and isolation as a control mechanism transmission dynamics ª transmissibility of 1918 pandemic influenza severe acute respiratory syndrome (sars): a year in review containing pandemic influenza at the source strategies for containing an emerging influenza pandemic in southeast asia potter cw chronicle of influenza pandemics swine-origin influenza a (h1n1) virus infections in a school update on school (k•12) and child care programs: interim cdc guidance in response to human infections with the novel influenza a (h1n1) virus global burden of disease 2005: call for collaborators available at modeling control strategies of respiratory pathogens predicting epidemics on directed contact networks spread of infectious disease through clustered populations we would like to thank the generous support of the secretaria de salud del distrito federal, for providing epidemiological data and insight. this work was supported by the canadian additional supporting information may be found in the online version of this article:initial human transmission dynamics of the pandemic (h1n1) 2009 virus in north america. please note: wiley-blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. any queries (other than missing material) should be directed to the corresponding author for the article. key: cord-320592-wnrzv4hg authors: giuffrida, maría j; valero, nereida; mosquera, jesús; alvarez de mon, melchor; chacín, betulio; espina, luz marina; gotera, jennifer; bermudez, john; mavarez, alibeth title: increased cytokine/chemokines in serum from asthmatic and non-asthmatic patients with viral respiratory infection date: 2013-08-21 journal: influenza other respir viruses doi: 10.1111/irv.12155 sha: doc_id: 320592 cord_uid: wnrzv4hg background: respiratory viral infections can induce different cytokine/chemokine profiles in lung tissues and have a significant influence on patients with asthma. there is little information about the systemic cytokine status in viral respiratory-infected asthmatic patients compared with non-asthmatic patients. objectives: the aim of this study was to determine changes in circulating cytokines (il-1β, tnf-α, il-4, il-5) and chemokines (mcp1: monocyte chemoattractant protein-1 and rantes: regulated on activation normal t cell expressed and secreted) in patients with an asthmatic versus a non-asthmatic background with respiratory syncytial virus, parainfluenza virus or adenovirus respiratory infection. in addition, human monocyte cultures were incubated with respiratory viruses to determine the cytokine/chemokine profiles. patients/methods: patients with asthmatic (n = 34) and non-asthmatic (n = 18) history and respiratory infections with respiratory syncytial virus, parainfluenza, and adenovirus were studied. healthy individuals with similar age and sex (n = 10) were used as controls. cytokine/chemokine content in blood and culture supernatants was determined by elisa. monocytes were isolated by hystopaque gradient and cocultured with each of the above-mentioned viruses. results: similar increased cytokine concentrations were observed in asthmatic and non-asthmatic patients. however, higher concentrations of chemokines were observed in asthmatic patients. virus-infected monocyte cultures showed similar cytokine/chemokine profiles to those observed in the patients. conclusions: circulating cytokine profiles induced by acute viral lung infection were not related to asthmatic status, except for chemokines that were already increased in the asthmatic status. monocytes could play an important role in the increased circulating concentration of cytokines found during respiratory viral infections. background respiratory viral infections can induce different cytokine/chemokine profiles in lung tissues and have a significant influence on patients with asthma. there is little information about the systemic cytokine status in viral respiratory-infected asthmatic patients compared with non-asthmatic patients. objectives the aim of this study was to determine changes in circulating cytokines (il-1b, tnf-a, il-4, il-5) and chemokines (mcp1: monocyte chemoattractant protein-1 and rantes: regulated on activation normal t cell expressed and secreted) in patients with an asthmatic versus a non-asthmatic background with respiratory syncytial virus, parainfluenza virus or adenovirus respiratory infection. in addition, human monocyte cultures were incubated with respiratory viruses to determine the cytokine/ chemokine profiles. patients/methods patients with asthmatic (n = 34) and nonasthmatic (n = 18) history and respiratory infections with respiratory syncytial virus, parainfluenza, and adenovirus were studied. healthy individuals with similar age and sex (n = 10) were used as controls. cytokine/chemokine content in blood and culture supernatants was determined by elisa. monocytes were isolated by hystopaque gradient and cocultured with each of the abovementioned viruses. viral respiratory tract infections can have profound effects on asthma. 1 respiratory viral infections can have a significant influence on asthmatic patients, where viral respiratory infections are found in association with asthma exacerbations. 1, 2 the infections associated with these wheezing events are multiple and include infections by respiratory syncytial virus (rsv), human rhinovirus, metapneumovirus, parainfluenza, coronavirus, and other viruses. previous studies have shown differences in the bronchoalveolar lavage cytokine profiles between non-asthmatic and asthmatic patients related to the type of cellular infiltrate observed, 3 suggesting that immune response is related to atopic status. however, circulating cytokine response after viral respiratory infection in asthmatic and non-asthmatic status has been little studied. in this regard, circulating blood levels of several cytokines have been reported to be increased during viral respiratory tract infections. 2 these cytokines may represent inflammatory markers with different profiles in asthmatic and nonasthmatic patients. several cytokines have been described to play an important role in the pathogenesis of asthma: il-4, il-5, il-8, il-10, il-12 (p40), il-13, il-17, tnfa, il-1, mcp-1, rantes, gm-csf, eotaxin, and ifnc. asthma is one of the most heterogeneous respiratory diseases and may also demonstrate systemic patterns beyond the respiratory system. determination of serum cytokine levels in asthmatic patients could have potential utility in the diagnosis of asthma and certain phenotypes, in prediction of attacks and choice of treatment. in addition to cytokine/chemokine profiles, the increased systemic concentration of those molecules could activate circulating leukocytes with further deleterious effects in the lung. 1,2 therefore, the aim of this study was to describe changes in several inflammatory cytokines (il-1b, tnf-a), th2 cytokines (il-4, il-5), and chemokines as mcp-1 (monocyte chemoattractant protein-1) and rantes (regulated on the activation normal t cell expressed and secreted) in the systemic circulation during acute viral infection in patients with an asthmatic and a non-asthmatic background and their relationship with the respiratory infection type (upper and lower) and type of virus infection. in addition, to determine the role of monocytes in circulating cytokine profiles, the cytokine/chemokine profiles after viral-monocyte interaction were studied. male and female patients (n = 52) presenting clinical diagnosis of acute respiratory infection (ari; 34 asthmatic patients and 18 without pre-existing asthma) were studied. the inclusion criteria were those individuals who had at least one respiratory symptom, such as cough, wheeze, running nose, or sneeze, and who were suspected by a professional physician to have viral infection. asthmatic patients were selected according to the criteria of the global initiative for asthma (gina) program. 4 asthmatic crisis was not present (at least 1 month previous) when blood samples were taken. in addition to suggestive clinical diagnosis (pneumonia or bronchitis), viral infection (rsv, parainfluenza and adenovirus) was confirmed by the presence of virus in specimens from nasopharynx and bronchoalveolar lavage. viral replication was demonstrated in hep-2 cell cultures (protocol -520-i; national institute for health, usa). 5 healthy individuals with similar age and sex (n = 10) were studied as controls. blood samples were obtained from patients and controls, and serum was stored at à70°c until use. we excluded individuals who had cardiac disease, immunodeficiency, and chronic inflammatory disease. no patients were treated with antibiotics, anti-alergics, or steroid when blood samples were obtained. the ethics committee of instituto de investigaciones cl ınicas dr am erico negrette and fonacit (caracas, venezuela) approved this study, and written informed consent was obtained from all patients and controls prior to blood collection. nasopharynx and bronchoalveolar samples from patients were sonicated and centrifuged, and supernatants were added to hep-2 cell cultures. previously, cells were grown to 50% confluence in eagle's minimum essential medium (mem) containing 7% fbs and 1% antibiotic/antimycotic. after two washes with pbs, 200 ll of supernatant was added to cell cultures. cultures were incubated for 1 hour, and then, 300 ll of mem containing 10% fbs and 1% antibiotic/ antimycotic was added. cultures were then incubated at 37°c in 5% co 2 for 96 to 120 hours. supernatants from cultures were centrifuged and stored at à70°c as the source of virus. viral cell culture infection (rsv, parainfluenza 1, 2, and 3 and adenovirus) was determined by direct immunofluorescence using a commercial kit (light diagnostics simulfluor respiratory screen kit; chemicon internacional, temecula, ca, usa). quantification mononuclear leukocytes were obtained from heparinized venous blood from five healthy adult donors. cells were isolated through density gradient centrifugation in hystopaque 1á077 (sigma chemical co., st. louis mo, usa). cells were suspended at 2 9 10 6 cell/ml in rpmi 1640 supplemented with 100 u/ml penicillin, 10 lg/ml streptomycin, and 10% fbs and then incubated at 37°c in a humidified atmosphere with 5% co 2 . after 3-hour incubation, adherent cells were enriched by washing away unattached cells twice. after washing, a mean of 3 9 10 5 adherent cells/well was obtained. monocyte cultures from each donor were infected (moi: 1) by virus obtained from each infected hep-2 cell culture supernatants (rsv, parainfluenza and adenovirus). 6 monocytes cultured in hep-2 cell culture supernatants without virus were used as negative controls. after 24 hours, supernatants were collected and cytokine and chemokine determinations were performed using elisa. results were expressed as pg/mg of cellular protein. monocyte virus infection was determined by direct immunofluorescence (light diagnostics simulfluor respiratory screen kit; chemicon internacional, ca, usa). values were expressed as mean ae standard deviation. the significance of differences was tested by anova and the bonferroni post hoc test. correlation analysis was performed using pearson's correlation. two-tailed p values <0á05 were considered statistically significant. age and laboratory findings in studied groups are shown in table 1 . increased levels of ige were observed in asthmatic patients when compared with non-asthmatic patients and controls. in general, the serum levels of il-1b, tnf-a, il-4, il-5, mcp-1, and rantes were increased in patients with acute viral respiratory infections ( figure 1 ). cytokine expression was similar in asthmatic and non-asthmatic patients; only chemokines (mcp-1 and rantes) were significantly increased in asthmatic patients when compared with control and non-asthmatic patients. asthmatic patients with bronchitis were not found (figure 1 ). in general, serum chemokines (mcp-1 and rantes) had lower levels in bronchitis than in pneumonia ( figure 2 ). the infection by rsv, parainfluenza virus, and adenovirus had similar cytokine induction; however, only rsv induced significant increased expression of chemokines ( figure 3) . in vitro infection of normal monocyte cultures by rsv, parainfluenza virus, or adenovirus induced increased cytokine concentrations in culture supernatants; only rsv and parainfluenza virus were capable of inducing significant increase of mcp-1 expression (figure 4) . no significant increased expression of il-8 was found in patient's serum (control: 29á55 ae 5á81; viral ari: 73á58 ae 60á30 pg/ml) or in viral-infected monocyte cultures (control: 648á1 ae 164á4; rsv: 978á0 ae 239á4; parainfluenza: 1133á0 ae 250á5 and adenovirus: 961á3 ae 130á2 pg/ mg protein). expression of il-4 was positively correlated with il-5 expression (r = 0á4662; p < 0á001). there was no correlation between the studied serum cytokines and serum ige levels. there was no association between the age of the donors and cytokine/chemokine production. the respiratory tract is a frequent primary site of viral infections and may result in the development of acute respiratory distress syndrome (ards). the current understanding of the pathogenesis of ards suggests that the degree of inflammatory response and its sustained leukocyte activation may determine the clinical evolution of ards. during the lung inflammatory events, the alveolar compartmentalization could be lost, allowing the passage of cytokines released to the bloodstream by any other organ to the pulmonary endothelium or inversely from the lung to the bloodstream. these cytokines, especially il-1, tnf-a, and il-8, have important roles in the lung dysfunction. 7 in this study, patients with viral ari had increased serum levels of proinflammatory cytokines (il-1b and tnf-a), th2 cytokines (il-4 and il-5), and chemokines (mcp-1 and ran-tes), suggesting complex immune interactions during viral respiratory infection. the source of those cytokines may be cells localized in lung tissue. in this regard, epithelial cells, mast cells, basophils, monocyte/macrophages, and lymphocytes could secrete diverse types of cytokines and chemokines (il-1, tnf, il-4, il-5, il8, mcp-1, rantes) during lung tissue-virus interactions. [8] [9] [10] however, in this study, monocyte cultures infected by rsv, adenovirus, or parainfluenza virus were capable of reproducing a similar cytokine and chemokine profiles to those observed in virus-infected patients, suggesting that in part, circulating monocytes could be the source of serum cytokines and chemokines. accordingly, the chemokine and chemokine receptor inducer effect of rsv on monocyte/macrophage cultures has been reported. 11 as alveolar compartmentalization could be lost during viral-induced lung injury, 7 a combination of both lung and circulating monocyte cytokine and chemokine productions can contribute to the increased serum levels found in this study. patients with ari were mainly infected by rsv. human respiratory syncytial virus is a ubiquitous respiratory virus causing serious lower respiratory tract disease in infants and young children worldwide. previous studies have shown that rsv infection modulates cytokine (il-6, il-1, tnf-a, il-8, and gm-csf), chemokine (il-8, ip-10, mcp-1, and rantes), and chemokine receptor expression in vivo and in vitro, suggesting that particular cytokine expression profiles may be an indicator of disease severity. [11] [12] [13] accordingly, in this study, serum from rsv-infected patients and rsv-infected monocyte cultures had increased levels of il-1b, tnf-a, il-4, il-5, mcp-1, and rantes. these effects probably are a common feature of respiratory virus infection, because similar serum cytokine/chemokine levels and viral cytokine inducer effect on monocyte cultures were observed in adenovirus and parainfluencia virus infections. in addition, infection with rsv could be a risk factor for further lung diseases, as the persistence of cytokines and chemokines in fully recovered rsv-infected patients can provide a substratum for the development of subsequent asthma. 14 regardless of cytokine production sources, they can have harmful effects on the lungs. the initial injury in lung tissue could release factors that contribute to inflammation, including chemokines (mcp-1), interleukins (il-1b, il-2, il-4, il-13), and prostaglandins (pge 2 ). 15 this study found increased the expression of il-1b, tnf-a, il-4, il-5, mcp-1, and rantes in the serum of patients with viral respiratory infection. these cytokines could have a deleterious effect in the lung and other tissues. tnf-a can induce apoptosis in lung tissues with resulting in emphysema 16 and activation of several proinflammatory events. 17-20 il-1 can induce neutrophilic inflammatory responses in respiratory disorders 21 and release many other cytokines and chemokines. 22, 23 in addition, il-1b induces eosinophil accumulation and it is a th2 and b cell growth factor. 24 in this regard, during this study the activation of th2 profile was observed as shown by the increased production of il-4 and il-5. these cytokines increase allergic airway inflammation. [25] [26] [27] increased concentration of circulating chemokines found in asthmatic patients may be important in leukocyte recruitment to lung tissues. rantes is involved in the chemoattraction of eosinophils, monocytes, and t-lymphocytes, and therefore, it has high relevance to asthma. 10 mcp-1 is involved in the recruitment of regulatory and effector leukocytes (monocytes, lymphocytes, and basophils) into tissues. 9 therefore, chemokines could play an important role in asthma pathogenesis. ige has a multifunctional role in the pathogenesis of allergic inflammation. beside its involvement in ige-mediated degranulation of mast cells and basophils, it is also involved in the activation of macrophage/monocytes and the stimulation of th2 cells. both involve local infiltration of il-4 and il-5 secreting th2-like cells, and both show pathologic evidence of epithelial damage, which likely serves to amplify tissue inflammation. in the case of asthma, bronchial epithelial damage (e.g., damage caused by viruses or eosinophil cationic proteins) and cytokine release from airway epithelium are believed to play an important role in the pathogenesis of airway inflammation. 28 in this study, increased levels of ige was found in patients with an asthmatic background, associated with increased production of circulating il-4 and il-5, suggesting a possible role of ige in the increased levels of those cytokines and in the pathogenesis of asthma; however, correlation analysis failed to demonstrate statistical significance between il-4 or il-5 and ige values. previous reports have shown differences in the bronchoalveolar lavage cytokine profiles between chronic obstructive pulmonary disease (copd) and asthma. in this regard, the profile of cytokine expression in copd is different from that in asthma, probably related to the type of cellular infiltrate observed in the two diseases. 3 thus, in asthma, an infiltration of eosinophils and th2-cells involving il-4, 5 and 13 production is usually found. in copd, the neutrophil chemokine il-8 and proinflammatory cytokines il-1 and tnf-a play a more prominent role. 3 in our study, the bronchoalveolar lavage cytokine profile was not analyzed; however, circulating cytokine profiles showed no differences in the expression of proinflammatory and th2 cytokine profiles in both asthmatic and non-asthmatic patients, suggesting that the response during virus infection was not related to atopic status. in addition, levels of ige were not correlated with th2 (il-4 and il-5) expression. only the serum levels of chemokines were observed to be increased in asthmatic patients, suggesting an increased cellular infiltration (monocytes, neutrophils, or eosinophils) at tissue level. accordingly, the important role of chemokines in virusassociated asthma exacerbations has been reported. 13 previous reports have also shown different inflammatory response to rsv of asthmatic epithelium cultures compared with nonasthmatic epithelium. 12 comparative studies between asthmatic and non-asthmatic monocyte cultures were not performed in this study. however, cytokine/chemokine inducer effect of rsv and other viruses on monocyte cultures remained similar to the circulating cytokine profiles in asthmatic and non-asthmatic patients, suggesting a possible similar response of asthmatic and non-asthmatic monocyte cultures to virus infection. nevertheless, we cannot rule out a different cellular response of viral-infected asthmatic monocyte cultures, because a different chemokine (mcp-1 and rantes) profile was observed in rsv-treated monocyte cultures from asthmatic and non-asthmatic patients. 29 as an unexpected finding in this study, we found similar levels of il-4 in asthmatic and non-asthmatic patients, as asthmatic patients have a th2 profile and rsv is a th2-associated virus. we have not a clear explanation for this finding; however, previous exposure to rsv can sensitize non-asthmatic airways with further increased cytokine response. in this regard, rsv infection in neonates leads to inflammatory airway disease characterized by airway hyperreactivity, peribronchial, and perivascular inflammation, and subepithelial fibrosis in adults. 30 in conclusion, the present study demonstrates the main characteristics of serum cytokine profiles in healthy, asthmatic, and non-asthmatic patients. while serum cytokine patterns were not different in non-asthmatic and asthmatic individuals, higher values of chemokines were observed in asthmatic patients. furthermore, cytokine profiles observed in virus-monocyte cultures suggest a role of these cells in the increased circulating cytokines/chemokines in patients. the precise mechanism regarding how those systemic cytokines might be involved in driving viral respiratory infection processes remains elusive. understanding the mechanisms of viral induced asthma: new therapeutic directions viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing cytokines in chronic obstructive pulmonary disease global initiative for asthma (gina) program. the global burden of asthma: executive summary of the gina dissemination committee report diagnostic of respiratory syncytial virus infection: comparison of reverse transcription-pcr to viral culture and serology in adults with respiratory illness respiratory syncytial virus infection of human lung endothelial cells enhances selectively intercellular adhesion molecule-1 expression the lung in sepsis: guilty or innocent? interleukin-8 mrna synthesis and protein secretion are continuously up-regulated by respiratory syncytial virus persistently infected cells significant involvement of ccl2 (mcp-1) in inflammatory disorders of the lung mcp-1 and rantes are mediators of acute and chronic inflammation chemokine-receptor upregulation and disease severity in respiratory syncytial virus infection intrinsic phenotypic differences of asthmatic epithelium and its inflammatory responses to rsv and air pollution the role of chemokines in virus-associated asthma exacerbations the role of cytokines and chemokines in severe respiratory syncytial virus infection and subsequent asthma waters cm: epithelial repair mechanisms in the lung alveolar epithelial and endothelial cell apoptosis in emphysema: what we know and what we need to know balance of matrix metalloprotease-9 and tissue inhibitor of metalloprotease-1 from alveolar macrophages in cigarette smokers: regulation by interleukin-10 circulating cell adhesion molecules in bronchial lavage and serum in copd patients with chronic bronchitis potential role of soluble trail in epithelial injury in children with severe rsv infection tumor necrosis factor-alpha decreases surfactant protein b mrna in murine lung autoinflammatory diseases: new insights into clinical aspects and pathogenesis inflammasome and il-1b-mediated disorders proinflammatory activation pattern of human umbilical vein endothelial cells induced by il-1b, tnf-a, and lps cytokine-regulated accumulation of eosinophils in inflammatory disease the role of th2 immune pathway modulation in the treatment of severe asthma and its phenotypes: are we getting closer? regulation of inflammation by interleukin-4: a review of "alternatives role of innate lymphocytes in infection and inflammation atopic dermatitis: the skin as a window into the pathogenesis of chronic allergic diseases respiratory syncytial virus infection increases regulated on activation normal t cell expressed and secreted and monocyte chemotactic protein 1 levels in serum of patients with asthma and in human monocyte cultures exposure of neonates to respiratory syncytial virus is critical in determining subsequent airway response in adults this work was supported by grants from fondo de investigaci on de la seguridad social (spain), consejer ıa de educaci on, comunidad de madrid, mitic-cm (s-2010/ bmd-2502), instituto de salud carlos iii, mec (pio51871, ciberehd) and condes (maracaibo, venezuela). key: cord-338390-v4ncshav authors: moghadas, seyed m.; pizzi, nick j.; wu, jianhong; yan, ping title: managing public health crises: the role of models in pandemic preparedness date: 2009-03-02 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2009.00081.x sha: doc_id: 338390 cord_uid: v4ncshav background given the enormity of challenges involved in pandemic preparedness, design and implementation of effective and cost‐effective public health policies is a major task that requires an integrated approach through engagement of scientific, administrative, and political communities across disciplines. there is ample evidence to suggest that modeling may be a viable approach to accomplish this task. methods to demonstrate the importance of synergism between modelers, public health experts, and policymakers, the university of winnipeg organized an interdisciplinary workshop on the role of models in pandemic preparedness in september 2008. the workshop provided an excellent opportunity to present outcomes of recent scientific investigations that thoroughly evaluate the merits of preventive, therapeutic, and social distancing mechanisms, where community structures, priority groups, healthcare providers, and responders to emergency situations are given specific consideration. results this interactive workshop was clearly successful in strengthening ties between various disciplines and creating venues for modelers to effectively communicate with policymakers. the importance of modeling in pandemic planning was highlighted, and key parameters that affect policy decision‐making were identified. core assumptions and important activities in canadian pandemic plans at the provincial and national levels were also discussed. conclusions there will be little time for thoughtful and rapid reflection once an influenza pandemic strikes, and therefore preparedness is an unavoidable priority. modeling and simulations are key resources in pandemic planning to map out interdependencies and support complex decision‐making. models are most effective in formulating strategies for managing public health crises when there are synergies between modelers, planners, and policymakers. influenza pandemics have historically been devastating to humanity with significant morbidity, mortality, and socioeconomic costs. 1 the 1918-1919 pandemic, the so-called ''mother of all pandemics,'' 2 was responsible for over 50 million deaths among countless infections worldwide. today, 40 years after the last pandemic in 1968, the world may be on the brink of another major global pandemic, with a toll that could exceed that of the 1918-1919 pandemic. 3 while the nature of the next influenza pandemic cannot be predicted with certainty, the identification of strategies to effectively curtail the spread of disease is an unavoidable priority in responding to this global threat. in light of this, the university of winnipeg hosted a multidisciplinary workshop on the role of models in pandemic preparedness. 4 the workshop brought together public health experts, key decision makers, and infectious disease modelers to: (i) identify the strengths and weaknesses of mathematical models, and suggest ways to improve their predictive ability that will ultimately influence policy effectiveness; and (ii) provide an opportunity for the discussion of priority components of a pandemic plan and determine key parameters that affect policy decision making. the first day of this workshop consisted of several outstanding presentations by modelers with the purpose of forging strong links between theory, policy and practice. these included evaluations and model predictions for antiviral strategies and their implications for drug stockpiling; the role of population contact networks in the emergence and spread of drug-resistance; targeting influenza vaccination at specific age groups; optimal control of pandemic outbreaks; and the usefulness of non-pharmaceutical interventions in disease mitigation. dr. chris bowman (institute for biodiagnostics, national research council canada) presented the findings of two modeling studies for the management of drug-resistance in the population, 5,6 especially when concerning the scarcity of antiviral supplies. these studies suggest that an adaptive antiviral strategy with conservative initial treatment levels, followed by a timely increase in the scale of drug-use, can minimize the final size of a pandemic while preventing the occurrence of large resistant outbreaks. dr. bowman emphasized that the strategic use of drugs may involve decisions for rationing of limited stockpiles and prioritizing high-risk individuals, and therefore ethical considerations should be taken into account for maximum protection of community health. a comparative evaluation of antiviral strategies in homogeneous and heterogeneous population interactions was presented by dr. murray alexander (institute for biodiagnostics, national research council canada). he underscored the importance of prolonging the effectiveness of antiviral drugs through an adaptive treatment strategy, in particular for heterogeneous community structure in which the wide-spread of resistance is more likely to take place. these presentations also provided a brief overview of some recent studies carried out by canadian modelers in the subject of pandemic preparedness. [7] [8] [9] [10] [11] dr. babak pourbohloul (director, mathematical modeling, bc centre for disease control) proposed an important question regarding ''a forced marriage'' or ''necessity for integration'' between mathematical models and public health policy. in his summary of the day, dr. pourbohloul acknowledged that the talks were very encouraging and pointed towards integration and development of modeling platforms that could inform policy in canada. he also highlighted the significant progress evident since the first pandemic meeting in vancouver, 2005, during which very little could be communicated to policymakers regarding the value of modeling perspectives. dr. pourbohloul drew attention to various models presented in the workshop, which attest to the fact that we are not lagging behind the current methodology in canada, but rather are in the forefront. [5] [6] [7] [8] [9] [10] [11] however, the central issue is not this, but integration with public health, which is the approach taken by us and uk colleagues for disease modeling and management. a major drawback for canadian modelers is the lack of appropriate infrastructure, and this calls for investments from healthcare departments and government organizations that could provide modelers with the impetus to continue development of more realistic models. with regard to models used for pandemic planning, we need to critically evaluate their implications for policy implementation. there are two major reasons underlying this evaluation: first, data are limited and prior to the emergence of a novel pandemic strain, it is not possible to study the epidemiological impact of disease or interventions in a real world environment; second, public health authorities would need to be prepared for all the likely scenarios that could influence the outcome of preparedness strategies. models, by definition, are not supposed to be perfect; approximations are necessary and predictions are made on this understanding. however, a more important question is how much of the knowledge of canadian modelers has been employed to support policy decision-making? is it all based upon experience of other countries? perhaps in canada, there has not been much communication between modelers and policymakers and therefore modeling results have not been translated into the context of public health. the time has now come to build a pandemic consortium in canada to have a unified voice from modelers, and close the gaps with infectious disease experts and public health colleagues. dr. susan tamblyn (co-chair, canadian pandemic antivirals working group) also emphasized the importance of making progress on linking the modeling with decision making within canada. these enterprises are still really separate in canada, whereas the value of modeling groups working very closely with the government and health departments is clearly evident in a few countries. we seem to have this linkage in a couple of provinces in canada, but it is not elevated to the national level. as planners, they understand that modeling can help formulate pandemic policies; however, the lack of collaboration with canadian modelers obliged them to turn to outside results from published models. hopefully, the two groups can work closer together to have beneficial impact with regards to pandemic preparedness. dr. tamblyn also expressed her concern about public health questions, which often are not amenable to modeling, and about modeling studies that use unrealistic assumptions and scenarios. therefore, modelers should also be fully engaged in the process of formulating the questions that policymakers need to address in planning for a pandemic. the point was highlighted by dr. ping yan (centre for communicable disease and infection control, public health agency of canada) that models should be based on realistic assumptions to create fundamental knowledge in all aspects of pandemic research. on the second day, the workshop comprised several presentations by participants from the public health domain. these included unanswered questions concerning the emergence of novel infectious diseases; understanding the space-time dynamics of influenza spread; influenza mortal-ity in pandemics and seasonal outbreaks; the impact of global air transportation on the spread of diseases; the role of models in public health planning and decision making; the evolution of pandemic influenza viruses; and the potential for novel means to prevent these pandemics. dr julien arino (university of manitoba) outlined the objectives of an ongoing data-driven project that aims to draw out the likely patterns of disease spread through the network of all international airports in the world with direct and indirect connections. this investigation can have important implications for heading off a global pandemic, with a particular focus on the optimal allocation of containment resources in the most probable ports of disease introduction and spread in canada. this presentation was followed by an overview of the ontario government's pandemic preparedness plan (allison stuart, assistant deputy minister of the ontario ministry of health and long-term care), which provides the most comprehensive provincial plan in canada, having undergone five iterations developed over a 5-year period. this plan details guidance to local planners and specific strategies for health sector sub-groups (critical care, pediatrics, laboratories, long-term care, persons with chronic diseases, mental health settings), first responders, faith groups, private sector organizations, and first nations communities. this presentation also included a list of concerns which modeling should address relating to acute care services (e.g., estimated hospital surge capacity for a given jurisdiction during a pandemic); local implementation (e.g., identification of the tipping point when primary care will not be able to meet the 24-48 hour standard of care); and antivirals (e.g., identifying the optimal use of drugs and distribution methods for treatment and prophylaxis to decelerate the spread of a pandemic). dr. joanne langley (co-chair, canadian pandemic vaccine working group) presented a detailed analysis of the potential benefits and uncertainties relating to the standard pillars of pandemic influenza contingency plans, covering antiviral drugs; healthcare delivery planning; vaccines; public health measures; and infection control practices. this included the importance of personal protective equipment such as the n95 mask in the healthcare setting, the need for regular and frequent hand washing, and a risk analysis of potential amantadine resistance. dr. langley also stressed the need for ''real time'' modeling to provide a rapid analysis of alternative tactical decisions following the onset of a pandemic. dr. mark walderhaug (associate director, us center for biologics evaluation and research, fda) discussed a stock-and-flow model used for simulating the impact of an influenza pandemic on the us blood supply. the model assumes that susceptibility to the pandemic virus will be universal; multiple waves of infection can occur and each wave adversely impacts infected communities for 6-8 weeks; and absenteeism may reach as high as 40% dur-ing the peak periods. model simulations for the entire us blood supply were presented, and the need for acquiring detailed data of inter-regional flow of blood was emphasized. these data are essential for projecting various scenarios, including run-out for hospitals despite adequate national supplies and time frames for elective surgery cancellations while the blood supply recovers, which highlight the significant challenges involved in supply distribution. dr. paul gully (senior advisor, world health organization) emphasized the fact that models are essential for guiding public health, but may also raise more questions for policymakers. he expressed growing concerns about being able to fulfill the requirements for pandemic containment that come from modeling studies: ''models lead to policy but have to confront political reality''. previous work suggests that a nascent influenza pandemic can be contained at the source if antiviral therapy for a sizable proportion of affected individuals (80-90%) is accompanied by a rapid implementation of non-pharmaceutical measures (such as movement restriction) over a very short period of time (days to 3 weeks). 12, 13 on serious discussions from a political standpoint, dr. gully demonstrated the significant challenges involved in building the capacity for a timely response to meet the condition for averting a global pandemic. despite these challenges, he acknowledged that models are invaluable tools for making assumptions explicit and for best using limited data, highlighting key factors determining policy needs, and providing quantitative predictions. discussions of the day were then expanded to the implementation of various strategies from a transmission dynamic standpoint. in their capacity, what models offer should be taken along with other health and economic factors to guide sound public health policies. they are not meant to make decisions on managing public health crises, but rather provide recommendations to policymakers. however, for rapid decision making, one would need to consider the interface between simple, interactive, and relatively complex models that may encapsulate population demographics pertaining to the location of a pandemic outbreak. dr. tamblyn chaired the summary and discussion session of the workshop on day 3, and acknowledged the true interdisciplinary nature of the meeting, enriched discussions, very interesting and relevant presentations, with kudos for planning long health-breaks that allowed for interactions and flow of emerging ideas. she distinguished the meeting as the one that has met its objectives and provided an opportunity for effective communications between modelers and public health authorities on the subject of pandemic preparedness in canada. dr. ying-hen hsieh (china medical university, taiwan) offered his perspectives on the workshop with great potential for expanding collaboration with canadian colleagues in future work. the meeting highlighted important aspects of canadian public health that will be useful for creating an effective venue to communicate with public health in taiwan. dr. hsieh, as a prominent modeler in taiwan, shared his experience with sars (severe acute respiratory syndrome) and exemplified the opportunities missed by public health to engage modelers: ''by the time they called me in, it was 2 weeks before the end of sars outbreaks''. in 2005, there was a cabinet agreement to promote an influenza vaccine r&d program in taiwan, partly for the economic opportunities it offers; he was brought in after the decision was made with the hope that ''modelling results will be in line with government policy''. he depicted that in public health in taiwan, a highly challenging task has been to establish collaborative efforts, but the important lesson from this workshop is to understand the process of making decisions, identify its key parameters, and determine effective ways to communicate with policymakers. dr. benjamin ridenhour (us center for disease control) acknowledged that the workshop had been successful in bringing together the communities involved in pandemic preparedness, to share their various viewpoints and expertise in modeling and public health, in a very congenial and friendly environment. the us center for disease control has made substantial efforts to co-ordinate pandemic activities through synergism between public health officials and modelers, which has led to benefits for planning strategies in the united states. as modelers, we need to strengthen our ties to public health, and exploit our potential for developing models that can inform and optimize health policy decisions. this workshop has demonstrated that strong networking is required to adequately prepare for the pressure of real time crises, and cope with surging demands in a pandemic-related emergency. in closing the workshop, dr. seyed moghadas (institute for biodiagnostics, national research council canada) valued the time and efforts of participants and appreciated their contributions to the success of this event. key points inferred from presentations and discussions include: 1. in canada, the pandemic goals are to (i) minimize serious illness and overall deaths; and (ii) minimize social disruption. pandemic containment has not been a priority to date and may not be feasible. development of a pandemic vaccine may take up to 6 months following pandemic detection. however, as novel influenza strains most often emerge in asia, strong surveillance leading to early detection there can increase our lead time for pandemic vaccine production. 3. immunization of children can result in significant changes in contact patterns and attack rates. age is a surrogate for individual behavior that influences pathogen transmission in the population; vaccine efficacy may also vary in different age groups. 4. antiviral therapy is the cornerstone of the pandemic response in canada until vaccine is available; however, implementation of the strategy is determined by pandemic planners at the provincial level. the meeting provided an opportunity for modelers to engage in detailed discussions about modeling strategies that can be employed for gaining new insight into disease processes at the population level and making findings of public health significance. while models serve to synthesize data and suggest optimal scenarios in public health, they can also promote dialogue between modelers and policymakers about alternatives, uncertainties, and assumptions that underlie critical decisions. the workshop revealed that pandemic planning requires involvement of communities across disciplines with firm commitment to the notion that research must ultimately influence policy. a history of influenza influenza: the mother of all pandemics avian influenza h5n1: is it a cause for concern? workshop on managing public health crises population-wide emergence of antiviral resistance during pandemic influenza antiviral resistance during pandemic influenza: implications for stockpiling and drug use emergence of drug-resistance: implications for antiviral control of pandemic influenza a delay differential model for pandemic influenza with antiviral treatment simple models for containment of a pandemic the impact of prophylaxis of healthcare workers on influenza pandemic burden management of drug-resistance in the population: influenza as a case study strategies for containing an emerging influenza pandemic in southeast asia containing pandemic influenza at the source the workshop was funded by the mathematics of information technology and complex systems (mitacs), public health agency of canada (phac), international centre for infectious diseases (icid), national research council canada's institute for biodiagnostics (nrc-ibd), and the university of winnipeg. we wish to express our appreciation to all the participants for their significant contribution to the workshop. the authors, as the organizing committee, would like to thank margaret montague, sarah dietrich, and justyna swistak for assistance with meeting logistics. the authors declare that they have no competing interests. sm, np, jw, and py proposed and organized the workshop. sm summarized and drafted the preliminary version of this manuscript based on presentations and round-table discussions. all the authors have contributed to this manuscript, and approved its final version. key: cord-337372-y43prnko authors: bin‐reza, faisal; lopez chavarrias, vicente; nicoll, angus; chamberland, mary e. title: the use of masks and respirators to prevent transmission of influenza: a systematic review of the scientific evidence date: 2011-12-21 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2011.00307.x sha: doc_id: 337372 cord_uid: y43prnko please cite this paper as: bin‐reza et al. (2012) the use of masks and respirators to prevent transmission of influenza: a systematic review of the scientific evidence. influenza and other respiratory viruses 6(4), 257–267. there are limited data on the use of masks and respirators to reduce transmission of influenza. a systematic review was undertaken to help inform pandemic influenza guidance in the united kingdom. the initial review was performed in november 2009 and updated in june 2010 and january 2011. inclusion criteria included randomised controlled trials and quasi‐experimental and observational studies of humans published in english with an outcome of laboratory‐confirmed or clinically‐diagnosed influenza and other viral respiratory infections. there were 17 eligible studies. six of eight randomised controlled trials found no significant differences between control and intervention groups (masks with or without hand hygiene; n95/p2 respirators). one household trial found that mask wearing coupled with hand sanitiser use reduced secondary transmission of upper respiratory infection/influenza‐like illness/laboratory‐confirmed influenza compared with education; hand sanitiser alone resulted in no reduction. one hospital‐based trial found a lower rate of clinical respiratory illness associated with non‐fit‐tested n95 respirator use compared with medical masks. eight of nine retrospective observational studies found that mask and/or respirator use was independently associated with a reduced risk of severe acute respiratory syndrome (sars). findings, however, may not be applicable to influenza and many studies were suboptimal. none of the studies established a conclusive relationship between mask/respirator use and protection against influenza infection. some evidence suggests that mask use is best undertaken as part of a package of personal protection especially hand hygiene. the effectiveness of masks and respirators is likely linked to early, consistent and correct usage. personal protective equipment to help reduce transmission of influenza is generally advised according to the risk of exposure to the influenza virus and the degree of infectivity and human pathogenicity of the virus. the paucity of scientific evidence upon which to base guidance for the use of masks and respirators in healthcare and community settings has been a particularly vexing issue for policymakers. the health protection agency (hpa) undertook a scientific evidence-based review of the use of masks and respirators in an influenza pandemic to inform relevant guidance following the emergence of pandemic a (h1n1) 2009 influenza. the department of health commissioned the hpa to update the review in support of the revision of the united kingdom (uk) influenza pandemic preparedness strategy. 1 the review was published on-line at: http:// www.dh.gov.uk/prod_consum_dh/groups/dh_digitalassets/ documents/digitalasset/dh_125425.pdf. a further update of the evidence base subsequently was performed in january 2011 and described herein. we generally followed the approach detailed in the university of york's systematic reviews: crd's guidance for undertaking reviews in health care. the original search of the pubmed database was conducted on 7 november 2009; subsequent updates of the pubmed database search were undertaken on 23 june 2010 and 12 january 2011. 1 the november 2009 search also included the following scientific databases: bandolier, the cochrane library database of systematic reviews, the database of abstracts of reviews of effects, the health technology assessment database, the national health service (nhs) economic evaluation database, the uk database of uncertainties about the effects of treatments, the nhs centre for reviews and dissemination and the cumulative index to nursing and allied health literature. 2 no additional publications resulted from these databases. the initial search in november 2009 had no time period restrictions. a limited effort was made to identify additional studies: reference lists of review articles were examined; the european centre for disease prevention and control's (ecdc) antimicrobial resistance and health care associated infection programme was consulted; and mec's and an's hardcopy literature files were hand-searched. we included the following types of studies listed in the hierarchical order of study design quality: randomised controlled trials (i.e. randomised cross-over trial and cluster randomised trial); quasi-experimental studies (i.e. non-randomised controlled study, before-and-after study and interrupted time series); and observational studies (cohort study and case-control study). only human studies published in english which had an abstract were included (table 1) . infection with pandemic strains, seasonal influenza a or b viruses and zoonotic viruses such as swine or avian influenza were included because mask ⁄ respirator guidance is needed for all types of influenza. studies that evaluated the effect of masks ⁄ respirators on transmission of other respiratory viruses were included as a proxy for influenza. a two-stage selection process was used to identify studies that appeared to meet the inclusion criteria. firstly, fb-r or vlc scanned and excluded papers on the basis of the 'title' for relevance; in the second and third searches, some relevant titles were excluded because they had been selected for review during a prior search. secondly, to enhance the reliability of the selection process, fb-r, vlc, mec and an independently reviewed the abstracts for the remaining papers. fb-r or vlc used a pre-designed form to perform an initial data extraction of the full article and make an initial determination regarding its eligibility. mec or an subsequently reviewed all of the papers, supplemented fb-r's and vlc's initial abstraction as necessary and re-assessed each paper for inclusion in the review. any differences were resolved by mutual agreement. mec and an assessed the quality of the eligible studies using the critical appraisal skills programme tools 3 for randomised controlled trials, case-control studies and cohort studies. the three separate database searches yielded a total of 6015 titles; five articles were identified by scanning the reference lists of review articles and three articles were from mec's hard copy collection ( figure 1 ). full papers were obtained for 76 articles; of these, 17 studies were eligible for inclusion. descriptions, findings and comments for these studies are detailed in tables 2-4. three of the randomised trials were hospital-based studies, 4-6 and five were conducted in community settings. [7] [8] [9] [10] [11] two of these studies compared n95 respirators (designed to seal tightly to the wearer's face and filter out very small particles or aerosols that may contain viruses) and surgical masks (used to block large droplets from coming into contact with the wearer's mouth or nose) amongst healthcare workers; one trial found a lower rate of clinical respiratory illness associated with the use of non-fit-tested n95 respirators compared with medical masks, 6 whilst a non-inferiority trial found that masks and respirators offered similar protection to nurses against laboratory-confirmed influenza infection. 5 a trial conducted amongst crowded, urban households found that, despite poor compliance, mask wearing coupled with hand sanitiser use, reduced secondary transmission of upper respiratory infection ⁄ influenza-like illness ⁄ laboratory-confirmed influenza compared with education; hand sanitiser alone resulted in no reduction in this aggregated outcome. 11 although the remaining five trials found no significant differences between control and intervention groups, there were some notable findings. household contacts who wore a p2 respirator (considered to have an equivalent rating to an n95 respirator) 'all' or 'most' of the time for the first 5 days were less likely to develop an influenza-like illness compared with less frequent users in one study. 9 another study found a significant reduction in laboratory-confirmed influenza amongst household contacts that began hand hygiene or hand hygiene plus a mask within 36 hours of the index case's illness. 8 a trial conducted amongst resident university students detected significant reductions in influenza-like illness during weeks 4-6 in the mask and hand hygiene group after adjusting for vaccine receipt and other potential confounders. 10 the requirements for mask ⁄ respirator wearing and subsequent compliance varied by study ( table 2) ; for example, in macintyre's study of healthcare workers in china in december 2008 through january 2009 6 'participants wore the mask or respirator on every shift for 4 consecutive weeks after being shown when to wear it', whilst nurses in canada wore a mask or respirator during the 2008 ⁄ 09 influenza season when caring for patients with febrile respiratory illness and during aerosol-generating procedures. 5 observational studies all of the observational studies evaluated mask and respirator use following the outbreaks of severe acute respiratory syndrome (sars) in 2003; [12] [13] [14] [15] [16] [17] [18] [19] [20] seven studies were conducted amongst healthcare workers and two were community-based. all but two 12,13 of the case-control studies in healthcare workers reported that wearing masks and ⁄ or respirators appeared to protect workers from acquiring sars. 14-17 a retrospective cohort study of nurses who worked in two toronto hospital intensive care units found that the relative risk of sars for nurses who consistently wore a n95 respirator was half that for nurses who consistently wore a surgical mask; however, the difference was not significant because of a small sample size. 18 none of the studies we reviewed established a conclusive relationship between mask ⁄ respirator use and protection against influenza infection. some useful clues, however, could be gleaned. subanalyses performed for one of the larger randomised controlled studies in a household setting found evidence of reduced rates of influenza-like illness if household contacts consistently wore the mask or respirator. 9 the authors of a randomised trial of mask plus alcohol-based sanitiser and mask-only group amongst u.s. university students living in residence halls noted that their study may have been better positioned to identify a protective effect because participants initiated the interventions at the beginning of the influenza season. 10 cowling's 8 finding that there was a significant reduction in the secondary attack ratio if the hand hygiene and mask plus hand hygiene interventions were begun within 36 hours of the index case lends support to this hypothesis. anticipating the paucity of studies that focused solely on influenza, we included the effect of masks ⁄ respirators on respiratory viruses other than influenza. such studies have often been used to support infection control guidance for influenza. however, the difficulties in interpreting the observational studies of sars suggest that they are of limited use for guiding policy on influenza. firstly, sars is an unusual acute viral respiratory infection with a very different epidemiology to almost all other respiratory viral infections. it is fundamentally different from human influenza: it rarely infects children, has a long incubation period, transmits little early on, mostly transmits in cannot distinguish relative contributions of hand hygiene and mask as they were combined. bin-reza et al. masks and respirators to prevent influenza ª 2011 blackwell publishing ltd healthcare settings, is not prone to extensive global spread and has only appeared once. secondly, the studies were poorly designed, had many weaknesses and so were very difficult to interpret. issues of concern include the use of a non-specific definition for exposure to a sars patient (e.g. coming within one metre of a patient), inconsistency in providing information about the comparability of cases and controls and collection of data after a lengthy period following the outbreak. several lacked microbiological confirmation of cases or controls and it would seem likely that a number of the sars cases were not cases at all. because all the cases knew they were cases, recall bias was highly likely. the single case-control study that tried to address some of these limitations did not find that inconsistent use of masks or respirators was associated with sars infection. 13 it is important to note three considerations when assessing the practical implications of the review's findings. firstly, development of evidence-based guidance about mask ⁄ respirator use is inextricably linked to what is known about how influenza is spread and specific risk factors that can affect transmissibility (e.g. host factors, pathogen factors, environmental factors and particle size). however, this is an area equally fraught with uncertainty; there are limited and conflicting evidence regarding the relative importance and frequency of direct contact, indirect contact, droplet and aerosol modes of transmission. 21, 22 historically, transmission has been thought to occur principally through respiratory droplets and masks have been used as a barrier against droplets emitted by coughing and sneezing. in the last decade, there has been increasing interest in a possible role for aerosol transmission of influenza and the advisability of filtering respirators to block such transmission. for example, studies have found that infected patients can produce aerosol particles containing influenza virus 23 and that hospital airflow patterns can influence influenza transmission via aerosols. 24 secondly, although the focus of this review has been on masks and respirators, limiting transmission of influenza in both healthcare and community settings requires a multifaceted approach, of which masks and respirators are but one component. in the healthcare setting, this 'hierarchy of controls' includes administrative controls help to reduce the introduction and spread of infection (e.g. policies to restrict entrance of ill visitors and workers, vaccination of healthcare workers); environmental ⁄ engineering controls (e.g. adequate ventilation); and lastly, use of personal protective equipment and hand hygiene. 25 in the community setting, a similarly structured approach is advised. however, during both the planning for an eventual pandemic and the subsequent public health response to the h1n1 pandemic, concern over policy and guidance related to mask ⁄ respirator use has at times seemed to overshadow almost all hcws wore n95 respirator or surgical mask in all patient settings. unadjusted univariate analysis found inconsistent use of masks or respirators not associated with higher risk of sars in any of the 3 contact settings; multivariate analysis found inconsistent use of >1 type of ppe during direct contact independent risk for sars. no serological testing of controls; reporting bias possible. nishiura ⁄ viet nam (14) period 1: time from admission of index case to occurrence of secondary cases in one hospital: 25 laboratory-confirmed sars cases compared with 90 controls (hcws and relatives of patients). period 2: during a nosocomial outbreak in the hospital with strict isolation procedures, quarantine of hcws and increased use of ppe: 4 laboratory-confirmed sars cases compared with 26 controls with only physicians and nurses in both groups. period 1: univariate analysis found masks (or 0ae3, 95%ci 0ae1-0ae7) and gowns (or 0ae2, 95%ci 0ae0-0ae8) protective; in logistic regression analyses, only masks protective (or = 0ae29, 95% ci 0ae11-0ae73) period 2: use of masks (or < 0ae1, 95% ci 0ae0-0ae3) and gowns (p = 0ae010, or and ci not calculable) associated with non-infection for doctors and nurses. possible recall bias; exposures imprecisely quantified; no serological testing of controls. nishiyama ⁄ viet nam (15) risk factors for serologicallyconfirmed sars infection assessed for 85 case and control hcws who had direct contact with sars patients. multivariate logistic regression analysis found significant risk for sars amongst hcws who never wore mask compared with those who always wore a mask (or 12ae6, 95% ci 2ae0-80ae0, p < 0ae01) possible reporting bias as interview conducted 7 months after outbreak; nature of exposures to sars not specified; community exposures not assessed. seto ⁄ china -hong kong (16) 13 sars-infected hcws with no community exposures compared with 241 hcws without clinical sars; all reported direct contact with 11 sars patients in 5 hospitals. univariate analysis found hcws who used surgical masks or n95 respirators, gowns or hand washing less likely to develop sars; logistic regression analysis found use of any mask significant (or 13, 95% ci 3-60). no serological testing of controls; reporting bias possible as interviews conducted a month after cases identified; community exposures not assessed. teleman ⁄ singapore (17) evaluated risk factors for serologically-confirmed sars amongst 36 ill case-hcws exposed to 3 highly infectious source patients and 50 well control-hcws that came within 1 m of serologically-confirmed sars patients. adjusted logistic regression analyses found that wearing n95 respirator during each patient contact (adj or 0ae1, 95% ci 0ae02-0ae86, p = 0ae04) and hand washing after patient contact (adj or 0ae07, 95% ci 0ae008-0ae66, p = 0ae02) protective. small sample size; no serological testing of the controls; limited recall of precise exposure data; no assessment of community ⁄ household exposures. masks and respirators to prevent influenza ª 2011 blackwell publishing ltd other important controls. 26 it is somewhat paradoxical that whilst continued effort and resources are needed to assess the independent effect of masks and respirators on influenza transmission, their use would always be recommended in combination with other control measures. thirdly the practical implications of policy, guidance and recommendations on mask ⁄ respirator use and other infection control measures must be considered. the only two studies that compared mask and respirators to protect healthcare workers from influenza infection essentially reached different conclusions 5, 6 illustrating the difficulties facing policymakers. 27 further, a simulation study found that strict adherence to guidance about personal protective equipment (which included masks and respirators) compromised normal ward functioning in a uk hospital setting. 28 this review had a prescribed narrow focus that permitted us to examine a relatively small number of studies. we considered employing quantitative techniques, but on analysis found the studies comprised a range of study designs, pathogens, participants, interventions and opportunities for bias and confounding would render any metaanalysis findings open to criticism. a review that included interventions other than mask ⁄ respirator use, experimental laboratory and ⁄ animal-human studies on mask ⁄ respirator efficacy, cost-effectiveness studies and the occurrence of adverse events would present a more comprehensive picture. several systematic reviews of interventions to limit the transmission of respiratory viral infections and ⁄ or specifically influenza have been undertaken. most have considered a range of interventions; 29-33 one focused specifically on respiratory protection. 34 within the boundaries established by our inclusion criteria, our search strategy captured essentially the same studies on masks and respirators that others have identified. jefferson et al derived pooled estimates of the effectiveness of wearing an n95 respirator (91%) and wearing a mask (68%) for any respiratory viral infection; 29 however, these estimates were derived from the analyses of six sars studies whose methodology was problematic. we carefully noted how well exposures in various studies were detailed and if cases and controls were laboratory-confirmed to avoid misclassification bias. we did not feel that such a heterogeneous group of studies could be combined even for sars. in conclusion, there is a limited evidence base to support the use of masks and ⁄ or respirators in healthcare or community settings. mask use is best undertaken as part of a package of personal protection, especially including hand hygiene in both home and healthcare settings. early initiation and correct and consistent wearing of masks ⁄ respirators may improve their effectiveness. however, this remains a major challenge -both in the context of a formal study and in everyday practice. continued research on the effectiveness masks ⁄ respirators use and other closely associated considerations remains an urgent priority with emphasis being on carefully designed observational studies and trials best conducted titles and abstracts identified and screened n = 5351 (1st search) n = 317 (2nd search) n = 347 (3rd search) figure 1 . diagram of search strategy results and article selection for three searches. 1 includes 3 papers that were sought for review and abstraction in the first search. 2 includes 6 papers that were sought for review and abstraction in the second search. 3 one of these papers (reference no. 6) became available on-line on 27 january 2011. 4 reasons for exclusion included an inability to distinguish the effect of mask use from other personal protective equipment or lack of quantitative data. masks and respirators to prevent influenza ª 2011 blackwell publishing ltd outside a crisis situation. 35 however, examination of the literature has highlighted that well-designed studies in this field are challenging. 27 studies need to be adequately powered to assess potentially small differences between interventions and the independent effect of mask ⁄ respirator wearing when a second intervention (e.g. hand hygiene) is employed; an appropriate control group must be identified (e.g. no use of masks ⁄ respirators). most of the studies we examined were too small to reliably detect what would be anticipated to be moderate effects. perhaps, one solution is to fund large multi-centre trials with similar protocols in different sites for multiple years to achieve sufficient power. protocols should include the collection of detailed exposure data, objective monitoring of compliance and assessment of potential confounders. it may be difficult to design studies employing a control group that does not use any protective equipment (including masks ⁄ respirators), particularly in healthcare settings, as such precautions are routinely recommended. finally, there is a striking paucity of published studies with microbiologically proven influenza infection as an outcome; inclusion of laboratory outcomes is essential in any future study of masks ⁄ respirators on transmission of influenza. uk influenza pandemic preparedness strategy 2011: strategy for consultation crd's guidance for undertaking reviews in health care use of surgical face masks to reduce the incidence of the common cold among health care workers in japan: a randomized controlled trial surgical mask vs n95 respirator for preventing influenza among health care workers: a randomized trial a cluster randomized clinical trial comparing fit-tested and non-fit-tested n95 respirators to medical masks to prevent respiratory virus infection in health care workers preliminary findings of a randomized trial of non-pharmaceutical interventions to prevent influenza transmission in households facemasks and hand hygiene to prevent influenza transmission in households: a cluster randomized trial face mask use and control of respiratory virus transmission in households mask use, hand hygiene, and seasonal influenza-like illness among young adults: a randomized intervention trial impact of non-pharmaceutical interventions on uris and influenza in crowded, urban households which preventive measures might protect health care workers from sars? sars transmission among hospital workers in hong kong rapid awareness and transmission of severe acute respiratory syndrome in hanoi french hospital, vietnam risk factors for sars infection within hospitals in hanoi, vietnam effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) factors associated with transmission of severe acute respiratory syndrome among health-care workers in singapore sars among critical care nurses sars transmission, risk factors, and prevention in hong kong risk factors for sars among persons without known contact with sars patients transmission of influenza a in human beings aerosol transmission of influenza a virus: a review of new studies measurements of airborne influenza virus in aerosol particles from human coughs possible role of aerosol transmission in a hospital outbreak of influenza pandemic (h1n1) 2009 influenza -a summary of guidance for infection control in healthcare settings respiratory protection against influenza (editorial) respirators versus medical masks: evidence accumulates but the jury remains out (editorial) personal protective equipment in an influenza pandemic: a uk simulation exercise physical interventions to interrupt or reduce the spread of respiratory viruses: systematic review physical interventions to interrupt or reduce the spread of respiratory viruses: systematic review physical interventions to interrupt or reduce the spread of respiratory viruses non-pharmaceutical public health interventions for pandemic influenza: an evaluation of the evidence base protecting health care workers from sars and other respiratory pathogens: a review of the infection control literature face masks to prevent transmission of influenza virus: a systematic review research findings from nonpharmaceutical intervention studies for pandemic influenza and current gaps in the research we gratefully acknowledge the librarians at the health pro mary e chamberland provided assistance to the health protection agency, u.s. centers for disease control and prevention and the world health organization in the development of infection control recommendations for pandemic influenza. angus nicoll helped develop the ecdc infection control guidance for pandemic, seasonal and avian influenza. fb-r, vlc, an and mec analysed the data. fb-r and mec were the principal writers of the manuscript with contributions from an and vlc. key: cord-351008-p0n1fdxw authors: monge, susana; duijster, janneke; kommer, geert jan; van de kassteele, jan; krafft, thomas; engelen, paul; valk, jens p.; de waard, jan; de nooij, jan; riezebos‐brilman, annelies; van der hoek, wim; van asten, liselotte title: ambulance dispatch calls attributable to influenza a and other common respiratory viruses in the netherlands (2014‐2016) date: 2020-05-14 journal: influenza other respir viruses doi: 10.1111/irv.12731 sha: doc_id: 351008 cord_uid: p0n1fdxw background: ambulance dispatches could be useful for syndromic surveillance of severe respiratory infections. we evaluated whether ambulance dispatch calls of highest urgency reflect the circulation of influenza a virus, influenza b virus, respiratory syncytial virus (rsv), rhinovirus, adenovirus, coronavirus, parainfluenzavirus and human metapneumovirus (hmpv). methods: we analysed calls from four ambulance call centres serving 25% of the population in the netherlands (2014‐2016). the chief symptom and urgency level is recorded during triage; we restricted our analysis to calls with the highest urgency and identified those compatible with a respiratory syndrome. we modelled the relation between respiratory syndrome calls (rsc) and respiratory virus trends using binomial regression with identity link function. results: we included 211 739 calls, of which 15 385 (7.3%) were rsc. proportion of rsc showed periodicity with winter peaks and smaller interseasonal increases. overall, 15% of rsc were attributable to respiratory viruses (20% in out‐of‐office hour calls). there was large variation by age group: in <15 years, only rsv was associated and explained 11% of rsc; in 15‐64 years, only influenza a (explained 3% of rsc); and in ≥65 years adenovirus explained 9% of rsc, distributed throughout the year, and hmpv (4%) and influenza a (1%) mainly during the winter peaks. additionally, rhinovirus was associated with total rsc. conclusion: high urgency ambulance dispatches reflect the burden of different respiratory viruses and might be useful to monitor the respiratory season overall. influenza plays a smaller role than other viruses: rsv is important in children while adenovirus and hmpv are the biggest contributors to emergency calls in the elderly. surveillance of respiratory viruses is mainly centred on influenza, for which robust systems have been developed in most countries, generally based on sentinel networks of general practitioners (gps, primary care). 1 comparable surveillance systems do not exist for other respiratory viruses, despite the increasing interest and leadership of the world health organization (who) in expanding surveillance for respiratory syncytial virus (rsv) now that a vaccine may become available. 2 for most viruses, surveillance is limited to laboratory-based counts, often with unknown denominator, low representativeness or lack of standard sampling criteria. who encourages surveillance of severe acute respiratory infections (sari) in the context of the pandemic influenza severity assessment program. 1 this is fundamental to determine the severity of circulating viruses, their pressure on healthcare services and the groups most at risk of severe outcomes. surveillance of severe infections requiring secondary care is much less developed than surveillance in primary care. in europe, a few countries have established hospital-based surveillance based on syndromic sari, laboratory confirmed cases or a combination of both. 3, 4 in the netherlands, a pilot involving three hospitals has been running since 2015. 3 syndromic surveillance using ready-to-use data has also been explored, mainly in emergency rooms. [5] [6] [7] few initiatives have used ambulance data 5, 8, 9, 10 or ambulance dispatch centre data. 5, 8, 10, 11 ambulance dispatch centres could be an alternative source of readily available data to monitor the occurrence of severe respiratory infections. during the triage process, information is collected and recorded in real time, including the chief symptom in very broad categories, as their objective is to rapidly assign an urgency level and prioritize resources. a recent study in the netherlands has shown how the variability in respiratory syndromes is correlated with ili from sentinel gp surveillance, 12 making it a potential source for syndromic surveillance. however, not all respiratory viruses will result in ili, and although the ili case definition focuses on detecting influenza infections, ili can be caused by a wide range of viruses. in this study, we aimed to assess to what extent ambulance dispatches reflect the activity of different respiratory viruses in order to advance our understanding of their use for the surveillance of severe acute infections by different respiratory viruses. specifically, we evaluated the association of syndromes compatible with respiratory infections in ambulance dispatches with trends in detections of influenza a, influenza b, rsv, rhinovirus, adenovirus, coronavirus, parainfluenza and human metapneumovirus (hmpv). the netherlands is divided into 25 regional ambulance services we focused our analysis to a1 urgency calls, as we previously found these to have a stronger association with ili. 12 these calls may better capture variations in acute severe infections by respiratory viruses and be a valid source for their surveillance. calls with triage codes that were potentially compatible with respiratory infections (table 1) year, and hmpv (4%) and influenza a (1%) mainly during the winter peaks. additionally, rhinovirus was associated with total rsc. high urgency ambulance dispatches reflect the burden of different respiratory viruses and might be useful to monitor the respiratory season overall. influenza plays a smaller role than other viruses: rsv is important in children while adenovirus and hmpv are the biggest contributors to emergency calls in the elderly. adenovirus, ambulance, coronavirus, influenza, respiratory syncytial virus, rhinovirus the number of respiratory virus identifications was obtained from the weekly sentinel surveillance system of the dutch working group on clinical virology. twenty-one virological laboratories voluntarily provide aggregated weekly number of diagnoses; individualized information such as age or sex is not provided. also, no distinctions are made between primary or secondary care, or different diagnostic methods, although currently the majority use molecular methods or rapid tests. 13 we included weekly reports of influenza a, influenza b, rhinovirus, rsv, adenovirus, coronavirus, parainfluenza and hmpv. we analysed weekly rsc as a proportion of the total number of calls overall, by age group and by time of the day: office hours because the trends in rsc might coincide, precede or lag behind the trends in viruses reports, we considered virus reports either in the current week, or lagged up to 4 weeks to the right, that is future in time (+lags), or 4 weeks to the left, that is backwards in time (−lags), for a total of 9 time-lagged variables of each virus. when building the models, the time lag with the lowest p-value was selected, and only one time lag per virus was allowed. because one of the viruses with the highest interest in monitoring its severity is influenza a (due to shifts, drifts and its pandemic potential), we forced it into the model, unless its coefficient was negative due to biological implausibility. subsequently, other viruses were added if statistically significant, had a positive coefficient and did not revert to negative the coefficients of viruses previously added to the model. finally, because the influenza epidemic size and severity varies by season, an interaction between influenza a and an indicator variable for the epidemiologic year (from week 27 to week 26 of the following year) was tested and retained if p < .05. the indicator variable itself was not included, as we wanted to attribute differences between years to influenza a. model assumptions and absence of remaining seasonality and autocorrelation were assessed by residuals diagnostics. we used r, version 3.4.0. table 2 ). the most frequent triage code among rsc was "abnormal breathing, troubles speaking between two breaths" (table 1) . weekly average number of rsc was 98 (range 58-138), which corresponds to 2.3 calls per 100 000 inhabitants every week. the proportion of rsc showed a periodic pattern peaking in winter, with lower interseasonal peaks (figure 1 ). the periodicity was evident in out-of-office hours and people ≥65 years, but the pattern was less clear in other groups and, in children <15 years, the peak occurred earlier. among the included respiratory viruses, the most frequently reported were rhinovirus and influenza a, followed by rsv (table 3) . most viruses had a periodic pattern similar to rsc, peaking in winter, except for rhinovirus and parainfluenza, which had a less distinct pattern with peaks in the autumn or the spring (figure 1 ). adenovirus reports showed smaller interseasonal peaks in addition to winter peaks. associations between respiratory viruses and the proportion of rsc are reported in table 4 and figure 2 . in children <15 years, only rsv was associated with rsc, explaining part of the rsc winter e adjusted by sine and a cosine term with periodicity of 1 y. f +lags mean that the rsc from the current week are best associated with viruses from x weeks in the past (ie trend in viruses precedes rsc); -lags mean that they are best associated with viruses from x weeks in the future (ie trend of rsc precedes the viruses). *calculated applying the model coefficient to the average weekly number of virus reports, and multiplied by the annual number of ambulance calls by epidemiologic year, age group, office or out-of-office hours, as appropriate; for the overall effects, this represents the average per epidemiologic year; for epidemiologic year-specific effects, the numbers for incomplete epidemiologic years are extrapolations to represent complete epidemiologic years if the average weekly ili incidence and ambulance calls were similar in non-observed weeks than in observed weeks. **calculated dividing the number of rsc attributable to each virus (from the previous column) by the number of observed rsc by age group, epidemiologic year, office or out-of-office hours, as appropriate. our results show that trends in rsc from highest urgency ambulance dispatches are associated with trends in the activity of common respiratory viruses. depending on the subgroup 0%-20% of rsc was attributable to a combination of respiratory viruses. the specific viruses contributing to rsc varied by age group, with estimates of 1%-11% of rsc being attributable per individual virus. their burden on these 4 call centres covering a 4 million population was 948 of highest urgency calls per year (22/100 000 inhabitants), although this varied by virus and age group. in emergency departments, 25% of all acute respiratory diseases are attributable to respiratory pathogens, 14 up to 80% in children. 15 in our study, the majority of rsc were incorporated into the unexplained baseline. this is not an unexpected finding, since the categories of symptoms included in ampds triage codes are very broad, resulting in high background noise. 16 nevertheless, variability in rsc above this high baseline was associated with trends of common respiratory viruses, pointing at their potential usefulness to monitor the respiratory season overall (ie irrespective of the causative pathogen), as previously shown by its association with ili. 12 the different viruses potentially involved in rsc, their individual trends, and their seasonal variation in severity would make it challenging to design indicators and models that will allow us to prospectively use rsc data for situational awareness for specific viruses separately. conversely, large or unexpected increases in a specific respiratory virus might be reflected to a certain extent in rsc. influenza a is a leading cause of acute lower respiratory tract infection, particularly in the elderly. 15, 16 by contrast, in our study its contribution to rsc was low, especially among the elderly. in children, influenza was not associated to rsc, consistently with its low to marginal role in sari in this age group. 13, [17] [18] [19] the effect of influenza a on rsc (1%-3%) is lower than what we found for ili, which was attributed 4%-34% of rsc. 12 influenza b did not show association with rsc in any group, in line with our understanding of its lower, less severe impact and lower clinical burden. lower representativeness of the laboratory data in our current study may have underestimated the association for influenza, or oppositely, its effect estimated through ili may be overestimated because ili is caused also by other viruses. the effect of influenza a on rsc was found to vary by season only for the overall sample. this is fundamental to assess whether causing severe infections, second to rsv in children, [17] [18] [19] and after influenza in adults. 15, [21] [22] [23] its presentation year-round, with peaks in autumn and winter, 24 also contributes to its high overall impact. adenovirus explained a significant proportion of rsc, especially among the elderly. adenovirus is rarely detected in cases of severe respiratory infection, 15, 22 although in a study in finland, it was the second aetiology in mechanically ventilated patients with community-acquired pneumonia. 21 hmpv had a similar relative effect as adenovirus, although its impact on number of ambulance calls was smaller, since it was less frequent. in children <15 years the peak in rsc developed earlier in the year, and our model associated this to rsv, consistent with its earlier presentation in the season. 13, 16, 25 rsv is the leading cause of sari in young children 13, [17] [18] [19] 23 and has been highly associated to sari syndromes in emergency departments 6, 14 and ambulances. 10, 18 the differences between office and out-of-office hours likely reflect that ambulance calls in these two time frames are distinct populations, probably with a different share of clinical pictures and severity. however, we cannot totally rule out a lack of statistical power during office hours, since the number of calls was smaller. ambulance dispatches are convenient for syndromic surveillance because they reflect events that are perceived as urgent (and thus potentially severe), they are recorded continuously and they have a virtually universal coverage. 8, 26 moreover, triage algorithms are increasingly standardized internationally. 5, 11 however, the true usefulness and added value of ambulance dispatches for infectious disease surveillance needs to be studied and piloted prospectively. some challenges for routinely using ambulance dispatch data prospectively include establishing data sharing routines and complying with data protection regulations. there are limitations to our data. because we did not include a2urgency level calls in our analysis, our results cannot be interpreted as the burden of respiratory viruses in ambulance services as a whole, but only in the highest urgency services. since all associations are evaluated ecologically, spurious attribution of rsc trends to respiratory viruses cannot be ruled out. sentinel laboratory surveillance has several limitations: it is passive and reported trends can include surveillance artefacts; it does not provide information on age, so overall number of virus detections was used; and while often biased to secondary care, it captures patients from both primary and secondary care, and the pathogens underlying their symptoms may differ from patients in ambulance dispatches. our study covered only 6 ravs, 25% of the population in the netherlands, but we do not believe these are fundamentally different from non-included ravs. however, because the sentinel laboratory surveillance is widespread throughout the country, it could be possible that intensity or timeliness of circulation of the different viruses nationally is different from specific regional patterns in ravs included in our study. finally, as the netherlands has a comprehensive primary care system where gps that have a strong gate-keeping role (including out-of-office services), our study results cannot be directly compared to health systems with higher use of emergency medical services. because of its ability to capture variations in respiratory virus circulation, ambulance dispatch data might be useful to signal events and to monitor the respiratory season as a whole, specifically reflecting severe infections and thus complementing existing surveillance systems. it will probably have less potential for drawing conclusions about the separate effect of specific individual viruses when not combined with information from other data sources, due to the low magnitude of some associations, the different viruses reflected in rsc and their proportional variation throughout the year. the true utility of ambulance dispatch data needs to be tested prospectively and faces potential challenges regarding timely data sharing and data protection. we appreciate the contribution of the four regional ambulance none declared. https://orcid.org/0000-0003-1412-3012 liselotte van asten https://orcid.org/0000-0002-4123-7595 wileyonlinelibrary.com/journal/irv 1. who. global epidemiological surveillance standards for influenza. world health organization world health organization. who strategy to pilot global respiratory syncytial virus surveillance based on the global influenza surveillance and response system (gisrs) severe acute respiratory infections: the missing block in the surveillance pyramid] (dutch) developing a system to estimate the severity of influenza infection in england: findings from a hospital-based surveillance system between validity and timeliness of syndromic influenza surveillance during the autumn/winter wave of a (h1n1) influenza 2009: results of emergency medical dispatch, ambulance and emergency department data from three european regions emergency department syndromic surveillance providing early warning of seasonal respiratory activity in england syndromic surveillance for influenza in the emergency department-a systematic review utility of emergency call centre, dispatch and ambulance data for syndromic surveillance of infectious diseases: a scoping review a concept for routine emergency-care data-based syndromic surveillance in europe utility of ambulance data for real-time syndromic surveillance: a pilot in the west midlands region, united kingdom assessment of ambulance dispatch data for surveillance of influenza-like illness in melbourne use of ambulance dispatch calls for surveillance of severe acute respiratory infections annual report surveillance of influenza and other respiratory infections in the netherlands: winter validation of syndromic surveillance for respiratory infections prevalence of respiratory viruses among adults, by season, age, respiratory tract region and type of medical prevalence and seasonality of six respiratory viruses during five consecutive epidemic seasons in belgium respiratory viruses in children admitted to hospital intensive care units: evaluating the clart(r) pneumovir dna array clinical evaluation of viral acute respiratory tract infections in children presenting to the emergency department of a tertiary referral hospital in the netherlands viral aetiology and clinical outcomes in hospitalised infants presenting with respiratory distress what is the real role of respiratory viruses in severe community-acquired pneumonia? lower respiratory tract virus findings in mechanically ventilated patients with severe community-acquired pneumonia clinical characteristics and outcome of respiratory syncytial virus infection among adults hospitalized with influenza-like illness in france frequency of respiratory viruses among patients admitted to 26 intensive care units in seven consecutive winter-spring seasons (2009-2016) in northern italy prevalence of rhinoviruses in young children of an unselected birth cohort from the netherlands seasonality and geographical spread of respiratory syncytial virus epidemics in 15 european countries clinical evaluation of the emergency medical services (ems) ambulance dispatch-based syndromic surveillance system ambulance dispatch calls attributable to influenza a and other common respiratory viruses in the netherlands key: cord-354151-psog34u3 authors: van asten, liselotte; bijkerk, paul; fanoy, ewout; van ginkel, annemarijn; suijkerbuijk, anita; van der hoek, wim; meijer, adam; vennema, harry title: early occurrence of influenza a epidemics coincided with changes in occurrence of other respiratory virus infections date: 2015-12-11 journal: influenza other respir viruses doi: 10.1111/irv.12348 sha: doc_id: 354151 cord_uid: psog34u3 background: viral interaction in which outbreaks of influenza and other common respiratory viruses might affect each other has been postulated by several short studies. regarding longer time periods, influenza epidemics occasionally occur very early in the season, as during the 2009 pandemic. whether early occurrence of influenza epidemics impacts outbreaks of other common seasonal viruses is not clear. objectives: we investigated whether early occurrence of influenza outbreaks coincides with shifts in the occurrence of other common viruses, including both respiratory and non‐respiratory viruses. methods: we investigated time trends of and the correlation between positive laboratory diagnoses of eight common viruses in the netherlands over a 10‐year time period (2003–2012): influenza viruses types a and b, respiratory syncytial virus (rsv), rhinovirus, coronavirus, norovirus, enterovirus, and rotavirus. we compared trends in viruses between early and late influenza seasons. results: between 2003 and 2012, influenza b, rsv, and coronavirus showed shifts in their occurrence when influenza a epidemics occurred earlier than usual (before week 1). although shifts were not always consistently of the same type, when influenza type a hit early, rsv outbreaks tended to be delayed, coronavirus outbreaks tended to be intensified, and influenza virus type b tended not to occur at all. occurrence of rhinovirus, norovirus, rotavirus, and enterovirus did not change. conclusion: when influenza a epidemics occured early, timing of the epidemics of several respiratory winter viruses usually occurring close in time to influenza a was affected, while trends in rhinoviruses (occurring in autumn) and trends in enteral viruses were not. it has been suggested that annual epidemics of different viral infections can interfere with each other, but clear trends over long time periods and underlying mechanisms are not known. [1] [2] [3] [4] a few population-level studies in europe were based on observations in one respiratory season only (the 2009 h1n1 pandemic) in which the annually recurring influenza epidemic occurred relatively early. with the occurrence of several early influenza a seasons in recent years, an exploration of longer time trends of different viruses was considered useful to gain more insight in the suggested relationship between circulating viruses. understanding viral shifts and potential drivers thereof is relevant for further understanding whether certain viruses might promote or inhibit (pandemic) influenza spread and whether influenza vaccination could potentially affect trends in other respiratory viruses. in europe, influenza epidemics generally occur in winter, with the official start of the epidemic when influenza-like illness (ili) incidence in primary care sentinel surveillance exceeds an epidemic threshold (in combination with influenza a virus detection in clinical specimens collected from a subset of those ili patients). 5 in the netherlands, the influenza epidemic threshold has been calculated at an ili incidence of 5á1/10 000 for minimally two consecutive weeks which is usually not exceeded before the turn of the year, [5] [6] [7] [8] but the timing of the first exceedance (i.e., start of the epidemic) can vary between november and march (based on data from 1970 to 2006). 9 an extremely early influenza season was the 2009/2010 season when the influenza a (h1n1)pdm09 pandemic strain appeared and the ili epidemic threshold was reached by early october (week 41). such early occurrence may affect the circulation of other seasonal pathogens, and theories on possible interference between outbreaks of different respiratory viruses have been postulated to be a possible cause of delays in expected seasonal outbreaks of other respiratory viruses. 1-3 while those earlier population-level studies focused mainly on the possible interaction between influenza a virus and rhinovirus circulation, there may also be a relationship between influenza a and other prevalent viruses. therefore, we investigated trends in several common viruses for which laboratory data were available from national surveillance in the netherlands for a longer time period of up to 10 years including both respiratory and enteral viruses. we investigated trends in the reporting of common infectious respiratory and enteral viruses for which laboratory data were available for the 10the data were available from the "weekly virological records system" of the dutch working group on clinical virology giving the number of positive laboratory diagnoses by year and week, but not providing data on the denominator nor on age or gender of the patient. submitting laboratories are associated with either hospitals or regional laboratories to which both gps and hospitals submit samples. the estimated proportion of all positive diagnostics captured by this national surveillance varies between the monitored pathogens and was estimated between 38% (for rotavirus) and 73% (for influenza virus) in a 2002 study of five pathogens. 10 the types of tests used can differ between the submitting laboratories and over time. respiratory viruses were detected in throat swabs mainly by pcr-based methods in respiratory specimens from patients with respiratory disease symptoms. enteric viruses were detected in fecal samples by eia-based methods (rotavirus, norovirus) and by pcr-based methods (norovirus). enterovirus was detected mainly by pcr and in the early years also by culturing, in throat swabs, cnf, and in fecal specimens collected from patients suspected for an enterovirus infection. cross-reaction between rhinovirus and enterovirus pcrs for throat swab samples cannot be excluded completely. when collected from patients suspected for enterovirus infection, typing of enteroviruses indicated that this was a minority. when collected from patients with respiratory symptoms, enteroviruses might be misidentified as rhinovirus, as occurred during enterovirus d68 outbreaks. 11 to determine whether an influenza a season occurred relatively early, we used both the influenza sentinel surveillance data and the laboratory data from the weekly virological records system. as general practitioner (gp) influenza sentinel surveillance is the current gold standard for influenza surveillance in the netherlands (with an epidemic threshold), early influenza a seasons were first identified using published dates of the influenza epidemics. we combined this information with visual inspection (as there is no threshold available) of trends in influenza a laboratory diagnoses reported in the weekly virological records system for confirmation of the early ili epidemics and for identification of any additional early influenza a seasons according to those laboratory data. a pre-defined cutoff point for the identification of early influenza seasons is not available. using the published ili epidemic periods, we identified two relatively early influenza a seasons wherein influenza epidemics started before the turn of the year (i.e., before week 1): the 2003/2004 season (epidemic starting in week 49 lasting to week 4) and the 2009/2010 season (epidemic starting in week 41 lasting to week 51). 12, 13 these two early epidemics were also confirmed by visible early increases in influenza laboratory data reported in the weekly virological records system ( figure 1 ). after visual inspection of the laboratory data, the 2010/2011 season was additionally selected as an early season as influenza a diagnoses were clearly on the rise before week 1 ( figure 1 ). while the influenza epidemic in the 2008/2009 season did not start early (week 1) 8 according to the ili sentinel surveillance system, in the laboratory surveillance data this season seemed neither clearly early nor clearly late and was therefore excluded (influenza a laboratory diagnoses in this season are represented in appendix 1). as most of the viruses under study peak in winter, we defined year-long seasons running from july to june rather than using calendar years (january to december). for comparability between the seasons, we presented the number of detected viruses per week as a percentage of the total number of detections during the study period per each respective virus. due to the intensified testing that occurred during the a (h1n1)pdm09 pandemic, the total number of influenza a virus laboratory diagnoses in the pandemic season was 13á1 times higher than the average number of influenza a virus laboratory diagnoses in the 2003-2012 seasons, excluding the pandemic season. to facilitate the inclusion of such a high peak into the presented graphs, the number of influenza a virus laboratory diagnoses during the 2009/ 2010 season was scaled down (reduced by a factor 13á1, changing the height but not the shape of the epidemic in that year). as the number of influenza b virus laboratory diagnoses was very low during the pandemic, such downscaling was not performed for the influenza b virus laboratory diagnoses. smoothing of time series was performed by calculating 3-week moving averages (average of current, previous, and next week's value). correlation coefficients between different viruses were calculated using spearman's rank correlations for non-normally distributed data. we also shifted the time series forwards and backwards in time (à10 to +10 weeks) and compared whether the time shift with maximal correlation differed between early and late influenza seasons. the highest numbers of laboratory reports were available for rsv and the lowest for influenza b (20 282 and 1309 reported diagnoses during the total study period, table 1 ). time series of all considered pathogens are shown in figure 1 . almost all of the included respiratory viruses (influenza a and b virus, rsv, coronavirus) except rhinovirus showed very clear seasonality in their reporting over time. the numbers increase, peak, and are elevated during winter season (december-february) or occasionally in very early spring (march-april) ( figure 1 and appendix 2a-d). however, while influenza b virus diagnoses also displayed such winter seasonality, outbreaks did not occur each winter (five of the ten observed years showed clear influenza b epidemics, while the other 5 years showed very low levels or even near-absence). in our data, rhinovirus levels showed less clear seasonality than the other respiratory viruses. they tended to be elevated over broad periods ( figure 1 ) and seemed to be present year-round with less pronounced elevations during autumn/winter/spring. generally, rsv van asten et al. epidemics preceded influenza a virus epidemics, which in turn preceded influenza b epidemics (in the years that influenza b epidemics occurred). increases in coronavirus also usually preceded increases in influenza a (but not in the 2009/2010 season). the timing of rhinovirus levels relative to influenza a virus in laboratory diagnoses was less clear (due to seemingly year-round presence of rhinoviruses in specimens submitted for laboratory diagnoses). the two gastrointestinal viruses in the study (norovirus and rotavirus) both also presented with winter seasonality, often with norovirus preceding rotavirus epidemics ( figure 1 and appendix 2c). norovirus and influenza a virus epidemics usually overlapped, but the norovirus epidemics had broader peaks, and numbers tended to start increasing earlier than influenza a virus diagnoses levels and decreased after the disappearance of influenza a virus. also, rotavirus outbreaks usually occurred before influenza a outbreaks started. enteroviruses, of which there are many types, can cause respiratory illness, but they also cause other illness. 14 enterovirus data were sparse until 2005, but in the 2006-2012 time period, enterovirus levels generally peak in summer (june-august). visually, we assessed whether seasons with early influenza a virus epidemics (as reported in the weekly virological records system) coincided with shifts in the reporting of other common pathogens or shifts in temperature and humidity trends. occurrence of seasons with relatively early influenza a virus circulation compared to seasons with later circulation of influenza a virus is shown in figure 2 (early seasons in color, late seasons in gray). for the other viruses, the same grouping was made (based on early and late influenza seasons), with their trend during early influenza a seasons also given in color ( the correlation coefficients between influenza and other viruses were stratified by timing of the occurrence of the influenza epidemic (early versus late, table 3 ). as the different viruses might differ in the timing of their occurrence relative to influenza a occurrence, the correlation coefficients were calculated at different time lags (shifted between 10 weeks earlier up to 10 weeks later) to determine the time shift with the maximum correlation with influenza a. when comparing seasons with early versus later occurrence of influenza epidemics, the maximum correlation coefficient was observed at different lags for all viruses. this difference in time lag relative to influenza a occurrence was smallest for influenza b, indicating that influenza b circulation relative to influenza a tends to occur at a more stable delay than for the other viruses. this suggests that changes in timing of influenza a circulation will be accompanied by changes in timing of influenza b circulation. however, such an overall correlation coefficient (calculated over multiple seasons combined) obscured the visual observation that in van asten et al. two of the three early influenza seasons, influenza b virus was almost absent (figure 2 ). viruses that showed a shifted trend of reporting during years with early influenza a epidemics were of respiratory nature with clear winter seasonality and with epidemics occurring relatively close in time to influenza a virus epidemics. although per individual virus the shifts were not consistently of the same type or direction, generally said, in years when influenza a hit early, rsv tended to be delayed, coronavirus outbreaks tended to be intensified, and influenza b virus tended not to circulate at all. viruses that act on other organ systems (enteral) seemed not to be affected by the timing of influenza a outbreaks. in our data, rhinovirus showed little seasonality, but rather year-round presence, and therefore, no association with timing of influenza a could be observed. changes in seasonal patterns of respiratory pathogen laboratory reports have been observed in other, shorter, studies. in hong kong, after the early occurring a(h1n1) pdm09 pandemic, increases were observed in adenovirus and parainfluenza virus detections, while the usual rsv summer peak disappeared in data from hospitals and clinics. 15 rhinovirus and coronavirus were not included. in another study in beijing, all respiratory virus epidemics, except rhinovirus, were delayed after the pandemic influenza epidemic (data came from fever clinics which screen patients prior to being assigned to a specific hospital department). 16 however, both regions have different climates from the netherlands (and from each other) complicating comparisons. other european observations have also been reported regarding the effect of rhinovirus on influenza virus circulation. while those reports hypothesize on viral interference between influenza virus and rhinovirus circulation (with rhinovirus delaying influenza spread), 1-3,17 we (like the beijing study 16 ) did not observe clear-cut trends in rhinovirus reports in our laboratory diagnoses data. the relationship previously reported between rhinovirus and influenza virus might perhaps be age specific because in one study, the reduced likelihood of a(h1n1)pdm09 detection in rhinovirus-positive samples 3 was observed in a pediatric population, as was the reduced likelihood of detecting eight viruses in rhinovirus-positive samples in another study in children, 17 while our data were not age specific and we did not have such information on concurrent infections. besides the reported possible interaction between rhinovirus and influenza a virus, much less is known about interaction between and with other (respiratory and nonrespiratory) viruses. our data suggest that respiratory viruses may impact each other's seasonality (in this study focused on the relationship between influenza a virus and other respiratory viruses), albeit through unknown mechanisms, as also hinted at by the beijing and hong kong data. 15, 16 virtually all research suggesting interaction between respiratory viruses in human populations has focused on observational (ecological) studies such as ours, most including only one or a few seasons of data while our study included a 10-year period. only one prospective cohort study has been published (based on one season) which showed rhinovirus and coronavirus to interrupt the a(h1n1)pdm09 pandemic. 18 our results could not confirm their finding of coronavirus inhibiting influenza a virus circulation, but we observed more coronavirus laboratory reports when influenza a virus circulated early. in contrast to most of the other studies, we could investigate whether patterns recurred due to our longer study period. this revealed that coronavirus laboratory reporting was more intense in 2009/2010 when it did not overlap with influenza a virus circulation. however, it was also more intense in the early influenza a year thereafter (2010/2011) when it actually completely overlapped with the influenza a epidemic hinting that direct biological inhibition of either virus by the other might be unlikely and thus apparent interaction reported by others may have depended on indirect factors or may be spurious. these variations by year in our data illustrate how results from shorter observational studies have to be interpreted with caution, especially when they focus only on 1 year or one season. however, also in our longer study we have to remain cautious; to refute biological inhibition between coronavirus and influenza, it would be helpful to know whether the coronavirus and influenza virus specimens were from the same age group. unfortunately, as in other observational studies, surveillance artifacts could not be ruled out for any of the viruses in our study. these artifacts concern increases and decreases in laboratory testing over time that may not necessarily be related to changes in virus circulation in the population. like previous studies, we did not have information on issues potentially underlying such changes, such as shifts in laboratory testing policy, availability and use of new diagnostic tools, variations in testing intensity due to previous or new outbreaks, and possible outsourcing of certain tests. furthermore, information on age was not available in our data, possibly diluting trends that might have been visible were data investigated age specifically, or alternatively creating trends and shifts that might otherwise have been absent in certain age groups. if certain age groups are more likely to be hospitalized (such as infants with rsv infection) and/or to be sampled than other age groups and thus overrepresented in the laboratory data, our observed trends might not be representative for the total population if virus circulation trends actually differ between age groups. future ecological analyses should preferably include age-specific data as this may further clarify the true occurrence of shifted seasonality. comparison between the different studies (including ours) is also made difficult by unknowns other than age, such as illness severity and influenza vaccination status. as other ecological-type studies have suggested, also our observed shifts in the occurrence of influenza b virus, rsv, and coronavirus during early influenza a seasons suggest that viral interaction might play a role in the occurrence of virus epidemics. [1] [2] [3] 17 with our data, the precise direction of potential interactions is not clear, that is, which virus(es) impacts which. there are several types of virus-virus interactions, described in a review by da palma, 19 ranging from (i) direct virus-virus interactions (e.g., when nucleic acids or proteins of one virus physically interact with the genes or gene products of a coinfecting virus); (ii) indirect interactions resulting from alterations in the host environment; to (iii) immunological interactions. however, with the current data we cannot determine whether the viruses impacted each other biologically. further, there are other issues that can impact the timing and magnitude of seasonal epidemics such as environmental, social, and behavioral phenomena that may also be the potential drivers of observed shifts in reporting patterns instead of viral inter-action (alone). 20 to further attempt to disentangle these issues, future ecological studies should preferably include age-specific virological data over multiple seasons and better yet, cohort studies could be performed that (i) address the order of infection by different viruses (by serial sampling of subjects during the study period regardless of symptoms) as proposed by others; 17, 21 (ii) include persons from the same household as was done by pascalis et al.; 18 and (iii) include multiple years and thus multiple seasons per virus. there are several examples highlighting the importance of understanding drivers of viral shifts. as suggested by greer et al., further studies are relevant in light of the discussion whether rhinovirus promotes (pandemic) influenza spread through coughing and sneezing 22 or whether it may paradoxically provide natural protection due to inhibition of influenza infection in those already infected with rhinovirus. 17 further studies may also provide input to the question whether influenza vaccination can render persons more susceptible to other respiratory viruses due to the lack of temporary nonspecific immunity induced by actual influenza infection. 23 canadian and australian researchers hypothesize that through this same mechanism of induced temporary non-specific table 3 . correlation coefficients between number of weekly laboratory diagnoses of influenza a and weekly counts of other pathogens at different time lags (10 weeks later to 10 weeks earlier)* *bolded numbers indicate the largest correlation coefficient (with a negative correlation for enterovirus due to opposite seasonality with influenza a virus). immunity, the circulation of a seasonal influenza strain preceding a pandemic strain might decrease the susceptibility to that ensuing pandemic strain. 24 not evaluated in our study but possibly also playing a role in virus shifts or virus-virus interactions is the variation in the circulation of influenza a subtypes from year to year. evaluation of such an effect might require evaluation of longer time series as the first general impression from our data was that there was no clear consistent association with subtype as the three early influenza years were not all dominated by the same influenza a type ( studying the association of climatic factors (an example of environmental phenomena potentially affecting virus circulation) with influenza [26] [27] [28] was beyond the scope of this study, although covering the timespan of the study, a visual assessment of temperature and humidity trends showed no clear visual association between early influenza occurrence and early occurrence of low temperatures or low humidity. although two of the three early ili seasons (2009/2010, 2010/ 2011) seem to have longer and colder cold spells (average weekly temperatures below 0°c) that also occur earlier in the winter season than in the other years, the first mentioned season (the 2009/2010 pandemic year) actually showed the cold spell occurring after the influenza season instead of at the beginning or during the influenza season (appendix 2e). unlike earlier studies, we included several non-respiratory pathogens, for which the results showed no shifts in trends, and therefore, interaction with influenza a virus seems unlikely. as norovirus, rotavirus, and enterovirus affect other organ systems, direct interaction may not be expected. however, sporadic examples do not rule out such unexpected interactions as others have suggested that live polio vaccine (against an enteral virus) might prevent otitis media 29 (a respiratory infection), and decrease infantile diarrhea mortality 30 (gastro-intestinal mortality). another issue is that, like others, we investigated shifts in virus circulation within the same seasons. whether early occurrence of influenza a may affect the circulation of seasonal pathogens in seasons thereafter is not known. in conclusion, when influenza hit early we observed shifts in patterns of several respiratory pathogens that occur close in time with influenza a. further research is needed to understand whether this was caused by (in)direct biological interaction between viruses or other underlying mechanisms such as human behavior and environmental factors or whether these observed shifts were just random occurrences. understanding these phenomena is of value in understand-ing or predicting the timing and magnitude of viral epidemics, providing knowledge which can be used for early warning and the allocation of healthcare resources. does viral interference affect spread of influenza? interference between outbreaks of respiratory viruses rhinoviruses delayed the circulation of the pandemic influenza a (h1n1) 2009 virus in france impact of the 2009 influenza a(h1n1) pandemic wave on the pattern of hibernal respiratory virus epidemics influenza surveillance in europe: establishing epidemic thresholds by the moving epidemic method 08 in the netherlands: antigenic variation, oseltamivir resistance and vaccine composition for the continue morbiditeits registratie peilstations nederland de beste tijd voor griepvaccinatie multi-centre study in the netherlands examining laboratory ability to detect enterovirus 68, an emerging respiratory pathogen de mexicaanse grieppandemie van 2009: een overzicht met focus op nederland the enteroviruses: problems in need of treatments the impact of pandemic influenza a (h1n1) 2009 on the circulation of respiratory viruses 22 ª 2015 the authors. influenza and other respiratory viruses published by influenza a/h1n1 2009 pandemic and respiratory virus infections do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections? intense co-circulation of noninfluenza respiratory viruses during the first wave of pandemic influenza ph1n1/2009: a cohort study in reunion island a systematic approach to virus-virus interactions seasonality of viral infections: mechanisms and unknowns respiratory viruses in hospitalized children with influenza-like illness during the h1n1 2009 pandemic in sweden coinfection can trigger multiple pandemic waves increased risk of noninfluenza respiratory virus infections associated with receipt of inactivated influenza vaccine seasonal influenza vaccination and the risk of infection with pandemic influenza: a possible illustration of non-specific temporary immunity following infection recent developments with regard to influenza; flu-watchers in action] actuele ontwikkelingen betreffende influenza; griepspotters in actie driving factors of influenza transmission in the netherlands environmental role in influenza virus outbreaks spatiotemporal characteristics of pandemic influenza viral interference induced by live attenuated virus vaccine (opv) can prevent otitis media effect of the administration of oral poliovirus vaccine on infantile diarrhoea mortality we would like to thank the contributing laboratories and the dutch working group for clinical virology (nwkv) for virological surveillance. key: cord-336335-spap39b7 authors: silva, denise r; viana, vinícius p; müller, alice m; livi, fernando p; dalcin, paulo de tarso r title: respiratory viral infections and effects of meteorological parameters and air pollution in adults with respiratory symptoms admitted to the emergency room date: 2013-08-26 journal: influenza other respir viruses doi: 10.1111/irv.12158 sha: doc_id: 336335 cord_uid: spap39b7 background: respiratory viral infections (rvis) are the most common causes of respiratory infections. the prevalence of respiratory viruses in adults is underestimated. meteorological variations and air pollution are likely to play a role in these infections. objectives: the objectives of this study were to determine the number of emergency visits for influenza-like illness (ili) and severe acute respiratory infection (sari) and to evaluate the association between ili/sari, rvi prevalence, and meteorological factors/air pollution, in the city of porto alegre, brazil, from november 2008 to october 2010. methods: eleven thousand nine hundred and fifty-three hospitalizations (adults and children) for respiratory symptoms were correlated with meteorological parameters and air pollutants. in a subset of adults, nasopharyngeal aspirates were collected and analyzed through ifi test. the data were analyzed using time-series analysis. results: influenza-like illness and sari were diagnosed in 3698 (30·9%) and 2063 (17·7%) patients, respectively. thirty-seven (9·0%) samples were positive by ifi and 93 of 410 (22·7%) were ifi and/or pcr positive. in a multivariate logistic regression model, ifi positivity was statistically associated with absolute humidity, use of air conditioning, and presence of mold in home. sunshine duration was significantly associated with the frequency of ili cases. for sari cases, the variables mean temperature, sunshine duration, relative humidity, and mean concentration of pollutants were singnificant. conclusions: at least 22% of infections in adult patients admitted to er with respiratory complaints were caused by rvi. the correlations among meteorological variables, air pollution, ili/sari cases, and respiratory viruses demonstrated the relevance of climate factors as significant underlying contributors to the prevalence of rvi. respiratory tract infections are the most common causes of infection, and viruses account for the majority of these infections, leading to significant levels of morbidity and mortality. 1 emergency rooms (ers) serve as the frontline for patients at highest risk for respiratory infection diseases, especially because of the acute nature of these illnesses. 2 the prevalence of respiratory viral infection (rvi) in adults admitted to the er is largely unexplored, as most relevant data concern infants and children. 3, 4 rvi can be severe in elderly patients, especially in those with underlying respiratory or cardiac disease. during winter months, rvi can account for many of the admissions to hospitals. 5, 6 the etiology of respiratory infections in adults remains undetermined in more than 50% of cases. 7, 8 in a study with 510 adults hospitalized with pulmonary diseases, an overall prevalence of respiratory viruses (rvs) in the lower respiratory tract was of 42á2%, with rhinoviruses and influenza a virus being the most common. 9 in adults with acute asthma admitted to the er, a prevalence of 12á2% of rvi was found. 10 in another study, with adults admitted to hospital with respiratory symptoms, viruses accounted for 15% of hospital admissions for respiratory infections. 11 seasonality of certain acute respiratory tract infection pathogens can be explained by meteorological variations. in a study, temperature was highly inversely correlated with respiratory syncytial virus (rsv), influenza a, and adenovirus frequency; rhinovirus was also associated with relative humidity (rh). climatic factors may influence the interaction among the host, pathogen, and environment, increasing the probability of exposure, susceptibility, and infection. 12 in addition, experimental data have shown that air pollutants affect lung immune responses and inflammatory reactions and that these effects may underlie the increased risk for respiratory infections. 13, 14 because of the large impact respiratory virus infections have on morbidity and even mortality, it is important to understand whether and how meteorological factors and exposure to air pollutants could influence respiratory virus infections. the aims of this study were to determine the number of emergency room visits for influenza-like illness (ili) and severe acute respiratory infection (sari) and to evaluate the association between ili/sari frequency, respiratory virus prevalence, and meteorological factors/air pollution, especially in adult population, in a humid subtropical climate. the present study was divided into two parts: in the first one, we characterized the symptomatic respiratory subjects attending the er at hospital de cl ınicas de porto alegre (hcpa), during 1 year (november 2008 to october 2009). in the second part, we included patients with respiratory symptoms ≤5 days to determine the prevalence of rvi; this part was conducted during 2 years (november 2008 to october 2010). in the first year of the study, we also collected climate and air pollution data. the study was conducted at hcpa, in the city of porto alegre, southern brazil. the hcpa is a general, tertiary care, university-affiliated hospital with 750 beds and approximately 30 000 hospitalizations/year. with a population of 1 360 590 inhabitants, porto alegre is surrounded by a metropolitan area that encompasses 31 municipalities (3 717 430 inhabitants). porto alegre has a humid subtropical climate, with the hallmark of the great variability (classification cfa in k€ oppen-geiger). 15 the ethics committee at hcpa has approved access to patient records. all subjects selected for the study gave written informed consent to participate. in the first part of the study, the clinical records of all daily visits (adults ≥18 years and children) to the er were reviewed by the research team. patients with respiratory symptoms (cough, coryza, nasal obstruction, odynophagia, dyspnea, chest pain, dysphonia, wheezing, and fever), regardless of the onset time, were included in the study. patients presenting only with fever were not included. demographic, clinical, and laboratorial characteristics were registered in a standardized questionnaire: sex, age, race, years of schooling, smoking status, symptoms at admission, duration of symptoms, presence of comorbidities, admission vital signs (temperature, heart rate, respiratory rate, and peripheral oxygen saturation measured by a digital oximeter), breath sounds, radiological findings, length of hospital stay, intensive care unit (icu) admission, hospitalization outcome (death or discharge). in the second part, adults ≥18 years with respiratory symptoms ≤5 days were included, and nasopharyngeal aspirates were collected. every day, a daily shift (morning, afternoon, or evening) was randomized for inclusion of patients. the patients were interviewed by a member of the research team, and the following data were registered in a standardized questionnaire (in addition to the data already registered in the first part): family income, influenza vaccine, previous antibiotic use, and occupational exposure, flu symptoms in the family, presence of smokers at home, family history of respiratory disease, air conditioning at home or at work, use of wood stoves, mold in home. the total cases of ili (fever >38°c and cough or sore throat) and sari (fever >38°c and cough or sore throat and shortness of breath or difficulty breathing) were registered. nasopharyngeal aspirates were obtained according to a standard protocol for the second part of study. an indirect immunofluorescence assay (ifi) was carried out using the respiratory daily meteorological parameters, like temperature (maximum, average, and minimum, in°c), relative (%) and absolute (g/kg) humidity, rainfall (mm), and sunshine duration (number of sunshine hours per day), were obtained from the climate laboratory at the federal university of rio grande do sul. also, the mean concentration of pollutants (lm/m 3 ) was recorded. the methodology used by this laboratory informs automatically the pollutant that reached the highest concentration in the last 24 hours. in the city of porto alegre, it is probable that the pollutant that is responsible for more than 90% of the days is ozone, and in the rest of days, particulate matter of <10 lm dynamic diameter (pm 10 ). data were presented as number of cases, mean ae standard deviation (sd), or median with interquartile range (iqr). categorical comparisons were carried out by chi-square test using yates's correction if indicated or by fisher's exact test. continuous variables were compared using the t-test or wilcoxon test. odds ratios (ors) and nominal 95% confidence intervals (ci) were presented. a two-sided p value <0á05 was considered significant for all analyses. data analysis was performed using spss 18á0 (statistical package for the social sciences, chicago, il, usa) and the free statistical software r (http://www.r-project.org). the data were analyzed using time-series analysis, through a generalized linear model (glm) to examine the association between ili or sari and air pollution/meteorological variables, using logistic regression. an autoregressive integrated moving average (arima) model was developed for ili or sari. pollution and meteorological parameters were inserted as explanatory variables into these arima models. for the second part of the study, further models were explored to include ifi and/or pcr results as outcome variables. volatility of the mean model variance error(s) was addressed using autoregressive conditional heteroscedasticity (arch) models within an arima modeling framework. an arima was developed for ili and sari. arima (p, d, q) are useful tools for analyzing time-series data containing non-stationary common trends, and these models were proposed by box and jenkins in 1976. the arima (p, d, q) allow to make predictions from their parties ar (autoregressive) and ma (moving average). for both sg and sars, we made the identification of the order of the parts and autoregressive moving average following the box-jenkins approach. first we identified the need for differentiation, for ili and sari, the graphical analysis of time-series and autocorrelation functions, as well as for testing the unit root (dickey-fuller) who did not reject the hypothesis of non-stationarity for ili and sari. in a second moment, it was noted that there was no seasonality for ili and sari, by analyzing the periodograms and autocorrelation functions and partial autocorrelation. subsequently, variable selection models arima (p, d, q) for ili and sari followed the approach backward. the akaike information criterion (aic) which acts to penalize the number of parameters in the modeland the schwarz information criterion (sic) (or bayesian information criterion, bic) were also used to select models. sample size requirements were estimated from the literature review. 10, 11 using a prevalence of rvi in adults of 15%, with a significance level of 99%, and total confidence interval amplitude of 0á10, we calculated that 339 patients would be needed in the second part of the study. during the 12-month study period, there were 37 059 admissions to the er (24 189 adults and 12 870 children), of which 11 953 (32á3%) presented with respiratory symptoms. the most common symptoms were cough (73á4%), fever (56á1%), dyspnea (40á9%), chest pain (24á5%), and coryza (20á9%). the median duration of symptoms before admission was 3 days (iqr: 1-6 days). a total of 2205 (18á5%) patients admitted to er needed to be hospitalized; of these patients, 242 (2á0%) required icu admission. ili and sari were diagnosed in 3698 (30á9%) and 2063 (17á7%) patients, respectively. the overall mortality rate among all study participants was 280 of 11 953 (2á3%). demographic and clinical characteristics of the study population are shown in table 1 . according to the selection criteria of the study, 425 patients met the inclusion criteria and were invited to participate, and 15 patients declined to participate. then, 410 adults were enrolled for virological investigation, 255 in the first year and 155 in the second one. there were no differences in age, sex, race, years of schooling, and symptoms between selected sample and all adults admitted to the er in the same period. however, as expected, the median duration of symptoms was lower in the selected seven samples considered to be unsatisfactory for ifi were positive by pcr. another 50 samples that were negative in ifi were positive by pcr. of the 37 samples positive in ifi, 18 were sent for pcr too, and in nine, the result was positive. in seven of these cases, the results of ifi and pcr were concordant. in one patient, adenovirus was identified on ifi and influenza a was found in pcr. in another case, ifi detected piv type 2, and pcr identified piv types 1 and 3. the characteristics of patients with pcr positive and negative were shown in table 3 . figure 1 shows timesseries graph for virus percent positive by ifi and by pcr by month. table 4 shows the descriptive statistics corresponding to the environmental variables considered in this study. figure 2 shows the modeled and observed values for ili and sari cases. figures 3 and 4 show the daily number of patients with ili and sari and meteorological parameters. we checked the autocorrelation between the covariates of climate data and observations from previous days, and we considered the following lags (in days) for each variable: average temperature (5), rainfall (2), sunshine duration (6), relative humidity (5), mean concentration of pollutants (3), and absolute humidity (2) . the number of ili and sari cases tends to be higher between july 5, 2009, and august 22, 2009 . in this period, the mean temperatures (tmin: 9á52 ae 3á89°c and tmax: 18á9 ae 4á92°c) and the sunshine duration (4á13 ae 3á31 hours of sun per day) were lower, as expected in winter. the rainfall tends to be higher than the median calculated for the entire year (0 mm, iqr: 0-5á4 mm). in addition, the absolute humidity (ah; 7á8 ae 2á1 g/kg) and mean concentration of pollutants (20á0 ae 7á0 lm/m 3 ) were lower in this period compared with the annual values. table 5 shows multivariate logistic regression model for ifi and pcr. we included 255 patients in logistic regression for ifi and 180 patients in logistic regression for pcr, because we have climate data only for the first year of study. ifi positivity was statistically associated with ah (or: 0á72; 95% ci 0á59-0á86), use of air conditioning (or: 4á16; 95% ci 1á45-11á83), and presence of mold in home (or: 2á95; 95% ci 1á10-8á29). on the other hand, pcr positivity was statistically associated with use of air conditioning (or: 2á27; 95% ci 1á04-4á97), average temperature (or: 0á92; 95% ci 0á86-0á98), and mean concentration of pollutants (or: 1á04; 95% ci 1á00-1á08). the multivariate time-series models for ili and sari cases are summarized in table 6 . sunshine duration was the only independent covariate that was significantly associated with the frequency of ili cases. the b-coefficient for this parameter was negative, indicating increasing ili frequency with decreasing sunshine duration. in the model for sari cases, the following variables proved to be significant: mean temperature (b = 0á399; p = 0á025), sunshine duration (b = à0á392; p = 0á007), rh (b = à0á098; p = 0á05), and mean concentration of pollutants (b = à0á079; p = 0á018). acute rvis are responsible for causing significant levels of morbidity and mortality. the most common respiratory syndrome caused by these pathogens is ili. a more severe presentation, named sari, was also related to some rvs. 16, 17 in this study, we have examined the relationship between ili and sari cases, meteorological variables, and air pollution using multivariate time-series analyses. we found that ili cases were inversely correlated with sunshine duration. in addition, sari cases were significantly associated with mean temperature, sunshine duration, rh, and concentration of pollutants. seasonal cycles of infectious diseases have been attributed to changes in atmospheric variables, the prevalence or virulence of the pathogen, or the behavior of the host. 18 earlier investigations have demonstrated that lower temperatures and sunshine duration, conditions usually encountered in winter, were associated with admissions for rvi. 12, 19 temperature was found to be highly inversely correlated with rsv, influenza a, and adenovirus frequency. 12 interestingly, we found a positive correlation between temperature and sari cases. one possible explanation is that it was demonstrated that for every one degree celsius rise in temperature, the risk of premature death and acute morbidity especially among respiratory patients is up to six times higher than in the rest of the population. second, evidence is emerging that increasing temperature is associated with increases in air pollution, especially ground-level ozone, and can amplify the adverse effects of poor air quality. 20 taking this evidence into account, we could expect that higher temperatures may have increased concentration of pollutants, leading to more sari cases. however, our data showed a decrease in air pollution during the months with a higher prevalence of sari. the third hypothesis to explain the relationship between higher temperatures and sari cases was related to el niño southern oscillation (enso) phenomenon. enso undergoes cycles between warm phases (el niño episodes) and reverse cold phases (la niña episodes). in the southern region of brazil, this phenomenon is associated with elevated temperatures and rainfall, especially in spring and in the period between may and july. previous reports have determined that el niño events were associated with increased hospitalizations and more severe influenza epidemics. 21, 22 severe acute respiratory infection cases were found to be negatively related to rh in our study. previous studies have demonstrated that higher rh decreases the survival of lipidenveloped virus, like influenza a, influenza b, rsv, and piv. [23] [24] [25] the use of indoor heating in winter lowers the rh; breathing dry air could cause desiccation of the nasal mucosa, epithelial damage, and reduced mucociliary clearance, increasing the host susceptibility to rvis. 19 however, even in tropical regions with humid climate (rh >70%), a higher activity of influenza can be found. this observation could be explained by the variation of viral stability in different rh levels. the stability of aerosolized influenza virions is maximal at lower rh (20-40%), moderate at higher rh (60-80%), and minimum at a mid-range rh (50%). 23 in a multivariate logistic regression model for ifi-positive patients, we found that ah was a protect factor for rvi. a recent study suggested that ah may better correlate with influenza virus survival and transmission. unlike rh, ah measures the actual water vapor content of air irrespective of temperature and has a prominent wintertime low, both indoor and outdoor. such findings suggest that humidification measures could be helpful decreasing survival and transmissibility of influenza. 26 air pollution has been associated with adverse health outcomes. studies have suggested acute effects causing respiratory symptoms, cardiovascular events, hospital admissions, and mortality. although the available evidences indicate associations between exposure to pollutants and increased risk of rvi, potential mechanisms mediating these effects are largely unexplored. 27, 28 surprisingly, our results showed that sari cases were associated with a decrease in mean concentration of pollutants. in fact, this could be a reflection of higher rainfall in the same period, as rain acts washing out or scattering pollutants from atmosphere. 29 on the other hand, we cannot exclude an effect of indoor pollution. in the last years, indoor pollution has been recognized as an emerging health problem, as about 90% of our time is spent indoors where we are exposed to chemical and biological contaminants. 30 we estimated indoor pollution indirectly in our study, questioning patients about the use of wood stoves and air conditioning, and the presence of mold in home. our findings suggested that ifi-positive patients were more prone to live in a residence with mold growth. dampness and mold are two important sources of indoor pollution, consistently associated with respiratory symptoms. home dampness may be a marker for mold growth, dust mites, endotoxins, and reduced ventilation, which could increase concentrations of indoor pollutants. 31 cough, wheezing, and upper respiratory symptoms were associated with dampness and mold in a metaanalysis. 32 according to these results, the prevalence of cough and wheezing was higher in patients with mold in home and ifi positive. air conditioning was also positively related to ifi test in this study. air conditioning use was associated with fewer hospital admissions for cardiovascular diseases, chronic obstructive pulmonary disease, and pneumonia on days with high concentrations of pm 10 , 33 as individuals are less exposed to outdoor pollutants. nevertheless, the majority of virus transmission occurs within indoor, air-conditioned (i.e., cooler, lower humidity) environments that favor airborne virus survival and transmission. 24, 25 in hot and humid conditions, indoor transmission in air conditioning environments may account for most of the transmission. 34 we found a prevalence of 22% of rv, which is higher than that previous studies have demonstrated (between 12% and 15% in adults). 12, 13 moreover, the length of stay was lower in our ifi-and/or pcr-positive patients. this finding is consistent with existing knowledge that virus identification allows the prompt initiation of therapy when indicated and avoids the unnecessary use of antibiotics, decreasing the length of hospital stay. the present study has some limitations. first, it was based on data collected from a single center, which may have potential biases because of the characteristics of the catchment population, like vaccination coverage. second, it is also important to note that this investigation was performed in a group of hospitalized patients, which is a bias toward the most severe disease cases. additionally, we do not have the concentrations of individual air pollutants, but it is implausible to reliably separate the effects of air pollutants because they frequently react with each other, sometimes potentiating individual effects. 10, 35 the short study period should also be considered a limitation. finally, the use of molecular techniques (pcr) in all study patients could be useful, increasing the number of viruses detected, as limited sensitivity of ifi method is well known. 10, 36 despite these limitations, this is the first study, to our knowledge, to analyze the relationship between rv, meteorological parameters, and air pollution in an adult population. in conclusion, we found that in adult patients admitted to er with respiratory complaints, at least 22% of infections were caused by rv. the correlations found among meteorological variables, air pollution, ili/sari cases, and rv demonstrated the relevance of climate factors as significant underlying contributors to the prevalence of rvi in a temperate region. there is still a need of additional investigations to clarify and confirm these data, perhaps using longer time-series observations. fundo de incentivo a pesquisa -sbpt; fipe-hcpa; fapergs. communicable respiratory threats in the ed: tuberculosis, influenza, sars, and other aerosolized infections role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy the contribution of influenza to combined acute respiratory infections, hospital admissions, and deaths in winter excess hospital admissions for pneumonia and influenza in persons > or = 65 years associated with influenza epidemics in three english health districts: 1987-95 community-acquired pneumonia prospective epidemiologic survey of patients with community-acquired pneumonia requiring hospitalization in switzerland frequency of detection of respiratory viruses in the lower respiratory tract of hospitalized adults incidência de infecc ßão viral do trato respirat orio em asma aguda atendida em sala de emergência surveillance of respiratory virus infections in adult hospital admissions using rapid methods are meteorological parameters associated with acute respiratory tract infections? increased susceptibility to rsv infection by exposure to inhaled diesel engine emissions ultrafine carbon black particles enhance respiratory syncitial virus-induced airway reactivity, pulmonary inflammation, and chemokine expression updated world map of the k€ oppen-geiger climate classification influenza activity-united states and worldwide, 2007-08 season influenza pandêmica (h1n1) 2009 -an alise da situac ßão epidemiol ogica e da resposta no ano de seasonal variation in host susceptibility and cycles of certain infectious diseases influenza virus transmission is dependent on relative humidity and temperature climate change and respiratory disease: european respiratory society position statement association of normal weather periods and el nino events with hospitalization for viral pneumonia in females: california, 1983-1998 respiratory virus and the effects of climate ª 2013 the authors. influenza and other respiratory viruses published by survival of airborne influenza virus: effects of propagating host, relative humidity, and composition of spray fluids the effect of environmental parameters on the survival of airborne infectious agents aerosol transmission of influenza a virus: a review of new studies absolute humidity modulates influenza survival, transmission, and seasonality air pollution and health ambient air pollution and health climate change and human health the index project: executive summary of a european union project on indoor air pollutants variations of formaldehyde and voc levels during 3 years in new and older homes meta-analyses of the associations of respiratory health effects with dampness and mold in homes air conditioning and source-specific particles as modifiers of the effect of pm(10) on hospital admissions for heart and lung disease incidence of common respiratory viral infections related to climate factors in hospitalized children in hong kong air pollution and cardiovascular disease: a statement for healthcare professionals from the expert panel on population and prevention science of the american heart association advances in the laboratory diagnosis of viral respiratory disease the authors have no competing interests to disclose. key: cord-350928-vj5qlzpj authors: arnott, alicia; vong, sirenda; rith, sareth; naughtin, monica; ly, sowath; guillard, bertrand; deubel, vincent; buchy, philippe title: human bocavirus amongst an all‐ages population hospitalised with acute lower respiratory infections in cambodia date: 2012-04-25 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2012.00369.x sha: doc_id: 350928 cord_uid: vj5qlzpj please cite this paper as: arnott et al. (2013) human bocavirus amongst an all‐ages population hospitalised with acute lower respiratory infections in cambodia. influenza and other respiratory viruses 7(2) 201–210. background human bocavirus (hbov) is a novel parvovirus that is associated with respiratory and gastrointestinal tract disease. objectives to investigate the prevalence and genetic diversity of hbov amongst hospitalized patients with acute lower respiratory infection (alri) in cambodia. study design samples were collected from 2773 patients of all ages hospitalised with symptoms of alri between 2007 and 2009. all samples were screened by multiplex rt‐pcr/pcr for 18 respiratory viruses. all samples positive for hbov were sequenced and included in this study. results of the samples tested, 43 (1·5%) were positive for hbov. the incidence of hbov did not vary between the consecutive seasons investigated, and hbov infections were detected year‐round. the incidence of hbov infection was highest in patients aged <2 years, with pneumonia or bronchopneumonia the most common clinical diagnosis, regardless of age. a total of 19 patients (44%) were co‐infected with hbov and an additional respiratory pathogen. all isolates were classified as hbov type 1 (hbov‐1). high conservation between cambodian np1 and v1v2 gene sequences was observed. conclusions human bocavirus infection can result in serious illness, however is frequently detected in the context of viral co‐infection. specific studies are required to further understand the true pathogenesis of hbov in the context of severe respiratory illness. human bocavirus (hbov) is a novel parvovirus that was first identified in pools of nasopharyngeal aspirates obtained from individuals with respiratory tract infections in 2005. 1 the hbov genome has three open reading frames encoding the 2 non-structural proteins ns1 and np1, and the structural viral capsid proteins vp1 and vp2. 2, 3 there are four closely related hbov genotypes, hbov-1, hbov-2 (consisting of hbov-2a and hbov-2b strains), hbov-3 and hbov-4, all of which share the same genomic organisation. 3, 4 hbov-1, the genotype initially identified by schildgen et al. 5 in sweden, has since been detected worldwide. hbov-2-4 were mainly identified in stool samples. 6, 7 homology at the protein level between the four hbov genotypes is high, between 70 and 90%. 3 hbov-3 is thought by some to be the result of a recombination event between hbov-1 and hbov-2, owing to the similarity of the hbov-3 ns1 and v1v2 genes to those of hbov-1 and hbov-2, respectively. 6 indeed, hbov-3 is likely detected along with hbov-1 during routine diagnostic screening, as the conserved ns1 genes regularly targeted for testing are genetically similar. in patients where hbov is the only virus detected, the clinical symptoms reported are similar to those occurring as a result of infection with respiratory syncytial virus (rsv) and human metapneumovirus (hmpv), including bronchiolitis, bronchitis, pneumonia and exacerbation of asthma. 5 predominantly hbov-1, but also hbov-2 dna, has been detected in respiratory samples, 8 whereas all four genotypes have been detected in stool samples, demonstrating that hbov replication is not limited to the respiratory tract as first thought. 4, 8 indeed, up to 25% of patients with hbov infection report gastrointestinal symptoms. [8] [9] [10] [11] hence, the tropism of strains belonging to hbov genotypes 1-4 remains unknown. the seasonality of hbov infection is also unclear, with seasonal [12] [13] [14] and year-round [15] [16] [17] transmission reported. the clinical significance of hbov infection is also yet to be fully elucidated, as hbov is frequently detected in association with additional respiratory pathogens. in the absence of an established cell culture system or animal model, the pathogenicity and replication mechanisms of hbov remain unclear. 5, 18, 19 a recent report suggests that hbov is, however, a true respiratory pathogen capable of causing sometimes severe, and even life-threatening, illness. 20 here, we report the findings of the first study investigating the prevalence, seasonality, clinical characteristics and the molecular epidemiology of hbov in amongst an all-ages population of patients hospitalized for acute lower respiratory illness (alri) in cambodia over 3 consecutive years. during april 2007-december 2009, all patients admitted with symptoms of alri to takeo (southern cambodia) and kampong cham (central-north cambodia) provincial hospitals were recruited into this study. 21 the age-specific criteria used to diagnose alri cases and severe alri cases were defined previously. 21 samples were collected, transported and stored as described. 21 additional clinical specimens and data, including sputum samples for bacteria identification and chest x-rays, were collected from patients where possible. samples were screened by multiplex rt-pcr ⁄ pcr at the institut pasteur in cambodia for 18 respiratory viruses including rsv, human metapneumovirus (hmpv), hbov, influenza a and b viruses, coronaviruses oc43, 229e, hku1 and nl63, severe acute respiratory syndrome-associated coronavirus (sars-cov), parainfluenza viruses 1-4, adenoviruses, rhinovirus and enterovirus, as previously reported. 21, 22 samples that tested positive for hbov were included in this study. the national ethics committee of cambodia approved this study. all patients ⁄ parents of sick children who participated provided written informed consent. viral dna from nasopharyngeal samples was extracted using the qiaamp dna mini kit (qiagen, valencia, ca, usa) as per the manufacturer's instructions. dna was eluted in a final volume of 200 ll qiagen ae buffer and stored at )80°c until required. both hbov and human albumin dna were amplified in parallel from each sample. amplification of human albumin dna was performed as described. 23 to identify strains belonging to hbov groups 1, 2 and 3, strain-specific primers were used. to identify hbov-1 strains, a 354-bp fragment of the conserved hbov-1 np-1 gene was targeted using published primers. 1 the forward and reverse primers correspond to positions 2281 and 2634 of the np-1 gene of the hbov prototype strain st1 (genbank accession number dq000495). thermocycling was performed using go-taq flexi dna polymerase (promega, madisson, wi, usa) under the following conditions: pcr activation at 94°c for 2 minutes, followed by 40 cycles of denaturation at 94°c for 30 seconds, annealing at 59ae5°c for 30 seconds, extension at 72°c for 1 minute and a final extension cycle of 72°c for 10 minutes. all samples were also screened by pcr for the presence of hbov2 and hbov3 dna, as described. 8 for phylogenetic analysis, a 819-bp region of the variable hbov-1 v1 ⁄ v2 gene was amplified using published primers. 9 primers correspond to positions 4370-4387, and 5172-5189 of the hbov prototype strain st1 genome (genbank accession number dq000495). thermocycling was performed using go-taq flexi dna polymerase (promega) under the following conditions: pcr activation at 94°c for 2 minutes, followed by 40 cycles of denaturation at 94°c for 1 minute, annealing at 54°c for 1 minute, extension at 72°c for 2 minutes and a final extension cycle of 72°c for 10 minutes. each pcr contained the appropriate positive and negative controls. all pcr products were visualised using ethidium bromide under uv light on a 1ae5% agarose gel. when required, pcr products were purified using the qiaquick gel extraction protocol of the qiaquick pcr purification kit (qiagen) as per the manufacturer's instructions, prior to sequencing. single-pass sequencing reactions were performed at a contract sequencing facility using the abi big-dye terminator cycle sequencing kit on an abi 3730xl automatic dna analyser (macrogen, seoul, korea). consensus sequences were generated using clc main workbench software, version 5ae6ae1 (http://www.clcbio.com). nucleotide sequences of reference hbov strains were obtained from genbank and used to construct alignments and phylogeny (table 1) . cambodian and reference hbov nucleotide sequences were aligned using the clustal w alignment program of mega software, version 4ae0. 24 the average pairwise jukes-cantor distance was found to be 0ae01 for both of the hbov np1 and vp1 ⁄ vp2 alignments, indicating that the data were suitable to generate neighbour-joining trees. 25 neighbour-joining trees were constructed using the p-distance nucleotide substitution model, with 1000 bootstrap replicates, using mega 4ae0 software. pairwise nucleotide distances (p distance), the proportion of sites at which nucleotide sequences differ divided by the total number of nucleotides compared, were calculated using the pairwise distance function of mega software version 4ae0. pairwise amino acid distances (p distance), the proportion of sites at which amino acid sequences differ divided by the total number of residues compared, were calculated using the poisson model in the pairwise distance function of mega software version 4ae0. deduced partial amino acid sequences of cambodian np1 and vp1 ⁄ vp2 strains were generated by translating nucleotide sequences with the standard genetic code using mega software version 4ae0. potential n-glycosylation sites were predicted by the nxt motif, where x is not a proline; potential o-glycosylation sites were predicted by the kpx n motif (where x is any amino acid). [26] [27] [28] [29] nucleotide sequence accession numbers the nucleotide sequences of hbov strains obtained in this study were deposited in genbank under the accession numbers jq618248 to jo618263 (np1 sequences) and jq618264 to jq618278 (vp1 ⁄ vp2 sequences). a total of 2773 patients presenting with alri participated in the study (no refusals). nasopharyngeal swabs were collected from all patients, of which 43 (1ae5%) tested positive for hbov. hbov infections were detected year-round, and the annual incidence amongst the study patients did not vary significantly (1ae5-1ae7%) during 2007-2009 ( figure 1 ). the median age of the hbov-infected patients was 1 year (range, 0ae3-56 years), and 46ae5% were female. the incidence of hbov infection was highest amongst children aged <2 years ( table 2) . whereas bronchiolitis was only observed in patients aged <2 years, pneumonia or bronchopneumonia was the most common clinical diagnosis, regardless of age (37ae2%; table 2 ). only respiratory specimens were available for testing in this study, and no clinical information regarding gastrointestinal symptoms was recorded. nineteen (44%) patients were co-infected with an additional respiratory virus (table 3) . co-infection with hrhv was most frequently detected (21%), followed by parainfluenza virus type 3 (piv3, 4ae6%) and respiratory syncytial table 1 . reference human bocavirus (hbov) strains used in this study to construct phylogeny. shown are the name of the strain, genbank accession number, year of isolation, country of isolation and genotype. whether strains were included in figure 2 (hbov np1 phylogeny), figure virus (rsv, 4ae6%). one patient was co-infected with both rsv and hrhv (table 3 ). detection and distribution of hbov sequences were successfully obtained from 19 (44ae2%) samples: 16 np-1 gene and 15 vp1 ⁄ vp2 gene sequences. both np-1 and vp1 ⁄ vp2 sequences were successfully amplified from 12 (63%) samples. all cambodian hbov strains were classified as hbov-1 (figures 2 and 3 ). of the samples from which hbov-1 sequences were obtained, one was collected in 2007, nine in 2008 and nine in 2009 (figures 2 and 3) . twelve (63%) samples were collected in takeo province, and seven (37%) were collected in kampong cham province, but the patient recruitment was also higher in takeo than in kampong cham hospital (data not shown). analysis of hbov-1 np1 gene sequences pairwise distance (p distance) analysis revealed very high conservation of np-1 gene sequences amongst the cambodian hbov-1 strains. homology at the nucleotide (nt) level varied between 98ae9 and 100%. identity at the amino acid (aa) level was slightly lower, between 96ae7 and 100%. the np-1 gene is highly conserved between strains, and however, we identified 5 different non-synonymous mutations present in cambodian np-1 sequences relative to the prototype hbov-1 strain st1 ( figure 4 ). the observed nt mutations resulted in the following 5 aa substitutions: ser 92 (100% conservation amongst cambodian np-1 sequences), asn 44 (65%), asn 59 (12%), ser 47 (6%), arg 53 (6%) (figure 4) . we observed 1 potential n-glycosylation site, starting at amino acid position 86, which was conserved amongst all reference and cambodian hbov strains analysed (figure 4 ). analysis of hbov-1 vp1 ⁄ vp2 gene sequences homology at the nt level was also very high amongst the partial vp1 ⁄ vp2 gene sequences, between 97ae1 and 100%. homology at the aa level was slightly lower, between 96ae6 and 100%. present on the surface of the virion, the vp1 ⁄ vp2 protein is potentially subjected to strong selective pressure from the host immune response. relative to the reference strains st1 and st2, 8 non-synonymous nt mutations resulting in the following aa substitutions were observed amongst cambodian vp1 ⁄ vp2 gene sequences: his 546 (66%), leu 631 (26%), tyr 540 (26%), thr 650 (13%), ser 466 (6%), pro 490 (6%), gln 493 (6%) and lys 545 (6%) (figure 5 ). one additional nt mutation resulting in the aa substitution ser 590 , present in reference strain st2 but not st1, was conserved amongst all of the cambodian vp1 ⁄ vp2 sequences investigated ( figure 5) . a stop codon was present at position 672 in all reference and cambodian vp1 ⁄ vp2 sequences included in this study ( figure 5 ). potential n-and o-glycosylation sites amongst hbov-1 vp1 ⁄ vp2 gene sequences two potential n-glycosylation sites, located at amino acid positions 519 and 638, were conserved amongst the reference isolates and all of the cambodian vp1 ⁄ vp2 sequences investigated ( figure 5 ). one potential o-glycosylation site was identified, located at amino acid position 552, which was also conserved amongst the reference isolates and all of the cambodian vp1 ⁄ vp2 sequences analysed ( figure 5 ). here, we report the results of the first study to investigate the incidence and genetic diversity of hbov amongst globally, the incidence of hbov infection has been reported to range from 1ae5 to 19%. 30 overall, the incidence of hbov infection amongst the all-ages population of cambodian patients hospitalised with alri was 1ae5%, which did not vary annually and was lower than that reported regionally. [31] [32] [33] [34] amongst the same population, the incidence of hbov was similar to that of hmpv (1ae7%), 21 but lower than rsv (8ae2%). 35 a number of factors can influence incidence estimates, including disease severity cam2008-e244 ...........................................s.....r......................................s. cam2009-p280 ........................................n...............................................s. cam2008-e374 ........................................................................................s. cam2009-e532 ........................................n........................................... cam2008-e446 ........................................n...............................................s. cam2008-e473 ........................................n...............................................s. cam2008-e474 ........................................n............................................. amongst the population investigated, study design, testing protocols and sensitivity of diagnostic tests used, especially as low viral loads are common in hbov infections. 5 in this study, alri patient samples were screened for hbov infection using a highly sensitive multiplex pcr assay previously shown to have a lower limit of detection of 4 copies of hbov dna ⁄ ll of viral transport medium. 22 therefore, the low incidence of hbov infection observed amongst the cambodian alri population was not a result of limited sensitivity of the diagnostic test used. the proportion of hbov infections detected amongst hospitalised cambodian alri patients was significantly higher compared to that of cambodian outpatients with ili (1ae5% versus 0ae4%, p = 0ae02). 22 the higher incidence of hbov infection amongst alri patients was not thought to be a result of a high carriage rate within the population. in parallel, the incidence of hbov infection amongst cambodian ili outpatients and asymptomatic controls was investigated previously, with only one asymptomatic individual testing positive for hbov dna. 22 however, persistent and prolonged shedding is a characteristic of parvoviruses, and it is thought that low-level asymptomatic shedding can persist following hbov infection. 20, 36, 37 two independent stud-ies reported the hbov carriage rate to be 43% amongst asymptomatic children, 38 and that the incidence of hbov amongst asymptomatic children was higher than amongst symptomatic children. 39 however, these two studies were conducted amongst children aged <2 years. hence, the high carriage rate may have been biased towards the very young age of the sample population with hbov incidence highest amongst children aged <2 years. 1, 9, 40 amongst a population of slightly older children, up to 5 years of age, brieu et al. 36 reported that hbov dna was not detected following testing of asymptomatic children. in the absence of an established continuous culture system, it is currently unclear to what extent shedding occurs following hbov infection, whether shedding and carriage rates amongst children and adults are equivalent, and whether shedding is innocuous or infectious. however, the higher incidence of hbov infection amongst hospitalised cambodian alri patients observed in this study relative to outpatients with ili suggests that hbov infection can result in severe illness. twenty-four (56%) of the hbov-infected individuals tested positive for infection with hbov only, of which 14 patients were classified as severe respiratory infection, including three patients aged <1 year who presented with ..........n................................................................................................ cam2009-p280 .....s.......................p..q....................................................h........................ cam2009-p232 .....................................................................................h........................ cam2009-p231 .....................................................................................h........................ cam2009-e585 ...............................................................................y.............................. cam2009-e569 .....................................................................................h........................ cam2009-e532 ....................................................................................kh........................ cam2008-p125 .............................................................................................................. cam2008-p0202 .....................................................................................h........................ cam2008-e474 .....................................................................................h........................ cam2008-e473 .....................................................................................h........................ cam2008-e446 .....................................................................................h........................ cam2008-e374 .....................................................................................h........................ cam2008-e244 ...............................................................................y.............................. cam2008-e216 ...............................................................................y.............................. cam2007-e141 ...............................................................................y............................. . 43 recently, a severe case of hbov infection resulting in acute respiratory failure was reported, in which hbov was the only pathogen detected. 20 the possibility that clinical illness amongst the cambodian hbov-infected patients was exacerbated because of bacterial co-infection cannot, however, be excluded as testing for bacterial co-infection could not be performed because of a lack of suitable specimens obtained from young participants. whether hbov represents a true pathogen, or an opportunistic co-pathogen, has been the source of much debate. 2, 5 a primary reason that the role of hbov as a sole pathogenic agent is questioned is that hbov is commonly detected as a co-infection. globally, the frequency of hbov-1 co-infection with other respiratory viruses amongst children with lower respiratory tract infection is reported to be as high as 83%. 4, 5, 44 furthermore, it has been reported that hbov coinfection may be higher amongst hospitalised individuals compared with those with mild illness as shedding is potentially enhanced by airway inflammation resulting from coinfection with an additional respiratory pathogen, or that hbov may play a role in enhancing or aggravating symptoms of existing infections. 5 mechanisms proposed to explain the high rate of co-infection with other respiratory viruses include that hbov is either a helper virus, facilitating replication of other respiratory pathogens or that hbov may require a helper virus to facilitate productive infection. 45 it must also be considered that the more recent widespread use of highly sensitive multiplex assays enabling simultaneous detection of multiple pathogens in a single patient sample may also contribute to the high rate of coinfection with hbov and additional respiratory pathogens detected. in this study, 44% of patients were co-infected with hbov and an additional respiratory virus. regionally, the reported rate of hbov co-infections varies and does not appear to be particularly associated with hospitalisation: 42% amongst hospitalized children in singapore, 32 90% amongst out-patient children aged <5 years in rural thailand, 37 9% amongst hospitalised children in vietnam. 31 in this study, co-infection with hbov and hrhv was most frequently detected (table 3) , analogous to the findings of a similar study conducted amongst paediatric patients in rural thailand. 37 whether the high frequency of hbov and hrhv co-infection is a reflection of the high incidence of hrhv circulating in the general population 2 or whether hrhv co-infection is beneficial to hbov replication and pathogenesis remains unknown. in the absence of an established cell culture system or animal model, much remains unclear regarding the pathogenesis of hbov. all of the hbov strains obtained from respiratory specimens for analysis in this study were classified as hbov-1. globally, hbov-1 strains have been reported to show low nucleotide and protein diversity; mean genetic diversity <1% at nt and <0ae5% at aa levels. 44 comparatively, the diversity of hbov-2, -3 and -4 strains, all identified in stool species, is far greater. 8 the low level of diversity reported worldwide amongst hbov-1 strains has resulted in speculation that hbov-1 evolved recently from the enteric bocavirus species, acquiring tropism for cells of the respiratory tract. 8, 46 the organisation of the hbov genome is similar to that of other parvoviruses, namely that the non-structural proteins are encoded by conserved genomic regions, whereas diversity is concentrated in regions encoding the capsid proteins. 45 analysis of amino acid sequences obtained from cambodian hbov-1 strains revealed 0-2ae9% nt diversity and 0-3ae4% aa diversity amongst vp1 ⁄ vp2 sequences. greater variation amongst sequences encoding the vp1 ⁄ vp2 capsid proteins was anticipated as vp1 ⁄ vp2 is under pressure from host immune responses. the level of diversity amongst cambodian np1 sequences was 0-1ae1% at the nt level, which is similar to that recently reported by kapoor et al. 46 surprisingly, the level of diversity at the aa level amongst np1 sequences was equivalent to that observed for vp1 ⁄ vp2 sequences, at 0-3ae3%. five amino acid substitutions were observed amongst cambodian np1 aa sequences, including asn 599 that has been reported previously by ma et al., 47 following analysis of hbov-1 np1 sequences obtained from japanese children with alri. the observed diversity amongst cambodian np-1 sequences may have occurred as a result of immune pressure, as it was recently reported that the hbov non-structural proteins, including np1, are targeted by humoral responses. 30 the potential impact, if any, of non-synonymous mutations within this conserved region of the hbov-1 genome remains unclear and warrants further investigation. to the best of our knowledge, we present the results of the first study to investigate the circulation and diversity of hbov infection amongst hospitalised patients in cambodia. we demonstrate that similar to the findings of other studies, hbov infection can result in serious illness, however is frequently detected in the context of viral co-infection. ongoing studies are required to further understand the true pathogenesis of hbov-1 in the context of severe respiratory illness. cloning of a human parvovirus by molecular screening of respiratory tract samples the human bocaviruses: a review and discussion of their role in infection human bocavirus capsid structure: insights into the structural repertoire of the parvoviridae human bocavirus-the first 5 years human bocavirus: passenger or pathogen in acute respiratory tract infections? a novel bocavirus associated with acute gastroenteritis in australian children a newly identified bocavirus species in human stool newly recognized bocaviruses (hbov, hbov2) in children and adults with gastrointestinal illness in the united states human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus human bocavirus in febrile children human bocavirus: prevalence and clinical spectrum at a children's hospital clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in hong kong detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study correlation between bocavirus infection and humoral response, and co-infection with other respiratory viruses in children with acute respiratory infection frequent detection of bocavirus dna in german children with respiratory tract infections viral and atypical bacterial detection in acute respiratory infection in children under five years human bocavirus infection bocavirus episome in infected human tissue contains non-identical termini detection of head-to-tail dna sequences of human bocavirus in clinical samples human bocavirus as the cause of a life-threatening infection genetic variability of human metapneumovirus amongst an all ages population in cambodia between use of a multiplex pcr ⁄ rt-pcr approach to assess the viral causes of influenza-like illnesses in cambodia during three consecutive dry seasons taqman pcr-based gene dosage assay for predictive testing in individuals from a cancer family with ink4 locus haploinsufficiency mega4: molecular evolutionary genetics analysis (mega) software version 4.0 molecular evolution and phylogenetics genetic variability of group a human respiratory syncytial virus strains circulating in from 1998 to genetic variability among group a and b respiratory syncytial viruses in mozambique: identification of a new cluster of group b isolates identification of variable domains of the attachment (g) protein of subgroup a respiratory syncytial viruses do n-glycoproteins have preference for specific sequons? antibodies against structural and nonstructural proteins of human bocavirus in human sera viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city the incidence of human bocavirus infection among children admitted to hospital in singapore detection and quantitative analysis of human bocavirus associated with respiratory tract infection in osaka city human bocavirus (hbov) in thailand: clinical manifestations in a hospitalized pediatric patient and molecular virus characterization a study of the genetic variability of human respiratory syncytial virus (hrsv) in cambodia reveals the existence of a new hrsv group b genotype human bocavirus infection in children with respiratory tract disease human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand frequent and prolonged shedding of bocavirus in young children attending daycare clinical and epidemiologic characteristics of human bocavirus in danish infants: results from a prospective birth cohort study evidence of human bocavirus circulating in children and adults severe pneumonia and human bocavirus in adult serologically verified human bocavirus pneumonia in children human bocavirus: a cause of severe asthma exacerbation in children human bocavirus infections in hospitalized children and adults does human bocavirus infection depend on helper viruses? a challenging case report human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections detection of human bocavirus in japanese children with lower respiratory tract infections we would like to thank all of the technical staff in the virology and epidemiology units at the institut pasteur in cambodia for their assistance. we are also grateful to all the staff from the hospitals for their collaboration in the sisea project. key: cord-307338-4nta9b6w authors: slomka, marek j.; densham, anstice l. e.; coward, vivien j.; essen, steve; brookes, sharon m.; irvine, richard m.; spackman, erica; ridgeon, jonathan; gardner, rebecca; hanna, amanda; suarez, david l.; brown, ian h. title: original article: real time reverse transcription (rrt)‐polymerase chain reaction (pcr) methods for detection of pandemic (h1n1) 2009 influenza virus and european swine influenza a virus infections in pigs date: 2010-08-17 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2010.00149.x sha: doc_id: 307338 cord_uid: 4nta9b6w please cite this paper as: slomka et al. (2010) real time reverse transcription (rrt)‐polymerase chain reaction (pcr) methods for detection of pandemic (h1n1) 2009 influenza virus and european swine influenza a virus infections in pigs. influenza and other respiratory viruses 4(5), 277–293. background there is a requirement to detect and differentiate pandemic (h1n1) 2009 (h1n1v) and established swine influenza a viruses (sivs) by real time reverse transcription (rrt) pcr methods. objectives first, modify an existing matrix (m) gene rrt pcr for sensitive generic detection of h1n1v and other european sivs. second, design an h1 rrt pcr to specifically detect h1n1v infections. methods rrt pcr assays were used to test laboratory isolates of siv (n = 51; 37 european and 14 north american), h1n1v (n = 5) and avian influenza virus (aiv; n = 43). diagnostic sensitivity and specificity were calculated for swabs (n = 133) and tissues (n = 116) collected from field cases and pigs infected experimentally with sivs and h1n1v. results the “perfect match” m gene rrt pcr was the most sensitive variant of this test for detection of established european sivs and h1n1v. h1 rrt pcr specifically detected h1n1v but not european sivs. validation with clinical specimens included comparison with virus isolation (vi) as a “gold standard”, while field infection with h1n1v in swine was independently confirmed by sequencing h1n1v amplified by conventional rt pcr. “perfect match” m gene rrt pcr had 100% sensitivity and 95·2% specificity for swabs, 93·6% and 98·6% for tissues. h1 rrt pcr demonstrated sensitivity and specificity of 100% and 99·1%, respectively, for the swabs, and 100% and 100% for the tissues. conclusions two rrt pcrs for the purposes of (i) generic detection of siv and h1n1v infection in european pigs, and for (ii) specific detection of h1n1v (pandemic influenza) infection were validated. the pandemic (h1n1) 2009 influenza a virus (h1n1v) emerged in april 2009 and was first detected in a cluster of human respiratory cases in mexico and the usa; initially, the virus could not be subtyped using available molecular tests. [1] [2] [3] genetic and antigenic characterisation of the h1n1v virus showed it to be distinct from current seasonal h1n1 influenza a viruses circulating in humans. 4, 5 rapid spread of h1n1v to other continents and frequent escalating human transmission resulted in an official declaration of a new human influenza a pandemic by the world health organization (who) on 11 june 2009. 6, 7 regardless of their host of origin, influenza a viruses usually have the potential to infect and, during dual infections, re-assort in other host species. pigs are particularly susceptible to infections from other hosts because receptors for influenza a viruses of both human and avian origin are present in their upper respiratory tract. 8, 9 swine influenza viruses (sivs) circulating in pig herds include h1n1, h3n2 and h1n2 subtypes and these have been observed globally. [10] [11] [12] [13] h1n1 sivs were first isolated in the 1930s, and phylogenetic studies have shown that these ''classical swine'' isolates were originally closely related to the human h1n1 ''spanish 'flu''' pandemic influenza virus of 1918, but subsequently these classical h1n1 sivs evolved into a unique genetic lineage. 11 h3n2 sivs have also evolved distinctly in pigs following the human ''asian 'flu''' pandemic of 1968. during the early 1990s, ''avian-like'' h1n1 sivs became common in european farmed pigs and have replaced classical swine h1n1. 10, 11 however, classical swine h1n1 remained present in the usa and many parts of asia. [10] [11] [12] [13] evolution of sivs in north america has included emergence of a triple re-assortment h3n2 in 1998 that contained genes of human, avian, and classical h1n1 swine influenza origin genes. 12, 13 these triple reassortant viruses have become endemic in north america, and, through additional reassortment events, triple reassortant variants of the h1n1 and h1n2 subtypes have been detected. 5, 12, 13 in the case of h1n2 sivs isolated in europe, the h1 gene is of human h1 seasonal influenza origin. 11, 14 the first instance of infection of pigs with h1n1v was reported in may 2009 in canada where it was suspected that pigs were infected through contact with infected humans. 15, 16 sivs are known to infect humans 17, 18 and poultry, [19] [20] [21] [22] while human influenza a viruses are also known to transmit to pigs. 23 continuing human cases of h1n1v during 2009 sustained veterinary concerns that this virus may become established in pigs, with a degree of concern that this host may then serve as a source for further influenza a reassortment and future zoonotic transmission. 24 although swine influenza is not listed as a notifiable disease by the world organisation for animal health (oie), 25, 26 siv surveillance programmes have been carried out in europe and north america and have provided valuable epidemiological information. [10] [11] [12] [13] these surveillance programmes have been based on conventional testing as recommended by the oie, 27 using attempted virus isolation (vi) in cell culture and embryonated fowls' eggs (efes), followed by typing with defined antisera to identify the haemagglutinin (h) and neuraminidase (n) subtypes using well-established haemagglutination and neuraminidase inhibition tests (hi and ni), respectively. 27 in recent years, highly sensitive and specific real time reverse transcription polymerase chain reaction (rrt pcr) technology has been exploited by veterinary institutes and reference laboratories to develop and validate appropriate tests for avian influenza viruses (aivs). 28 these include validated rrt pcr tests for notifiable ai caused by h5 and h7 subtype viruses, following concerns resulting from the spread of h5n1 highly pathogenic (hp) ai in poultry in the eastern hemisphere, together with a number of accompanying zoonotic cases. many veterinary laboratories have already embraced generic ai rrt pcrs that detect all six-teen h subtypes of influenza a, typically through amplifying within the highly conserved m gene, and also as specific assays for aivs of h5 and h7 subtypes. 28 while public health institutions have recently described rrt pcrs for the detection of h1n1v in the context of the current human pandemic, [29] [30] [31] [32] in this study, we describe and validate similar approaches for the detection of h1n1v in pigs. full genome sequence analysis of the current h1n1v reveals the virus to be putatively of swine origin. 4, 5 segment 7, which includes the matrix (m) gene that encodes the matrix m1 and m2 proteins, appears to be of eurasian siv origin, while segment 4, which encodes the h1 haemagglutinin (ha), is related to american classical siv h1 genes. 4, 5 this study outlines the adaptation of an existing m gene rrt pcr assay for the generic detection of sivs and h1n1v, plus a novel rrt pcr that amplifies within the h1 gene for the specific detection of h1n1v in european pigs. viruses ninety-nine laboratory isolates of influenza a viruses were obtained from the influenza virus repository at the veterinary laboratories agency (vla-weybridge), grown in 9-to 10-day-old specific-pathogen-free embryonated fowls' eggs (efes) and typed using standard protocols. 27, 33 these included 51 swine influenza virus (siv) isolates, 43 avian influenza virus (aiv) isolates plus five h1n1v isolates, which included two from humans and three from pigs (table 1) . these efe-grown influenza a viruses were diluted at least 100-to 1000-fold prior to rna extraction to give levels of virus that approximate to those present in clinical specimens. 34 biological infectivity of efe-grown influenza a viruses was determined as the median egg infectious dose (eid 50 ) per ml. 35 ninety-seven frozen ()70°c) archived respiratory tissues were originally obtained during 1991-2009 from the routine swine influenza surveillance programme in united kingdom, in which acute respiratory disease of a suspected viral aetiology forms the primary submission selection criterion. all were tissue pools that included varying proportions of trachea and lung specimens. virus isolation (vi) in efes at the time of submission divided these into 31 and 66 tissue specimens from pigs that were infected with siv (table 2 ) and uninfected, respectively. in addition, 104 nasal swabs from pigs in united kingdom were collected by the vla regional laboratories at thirsk and bury st edmunds during the summer of 2009. these were obtained from swine that had been submitted for endemic disease investigations. thirty-nine clinical specimens were received between september and november 2009 from nine pig herds at nine different locations in six countries and included 24 respiratory swabs and 15 respiratory tissues ( table 3) . fifteen of these specimens (six swabs and nine tissues) were shown to be avian influenza viruses, with highly pathogenic (hp) h5 and h7 isolates indicated positive for h1n1v by non-rrt pcr approaches (table 3) , i.e. amplification of rna extracted from the clinical specimen by conventional rt pcr using primers that had been designed specifically for the ha gene of current h1n1v isolates, available at: http://www.who.int/csr/resources/publications/swineflu/genomeprimers_20090512.pdf amplicons were electrophoresed in 2% agarose and stained with redsafeô (intron biotechnology, kyungki-do, korea) for visualisation, excised and purified from agarose using the qiaquick ò gel extraction kit (qiagen, crawley, uk). nucleotide sequencing was performed using the big dye ò terminator v3ae1 cycle sequencing kit (applied biosystems, warrington, uk), and checked by blast analysis at: http://blast.ncbi.nlm.nih.gov/blast.cgi?program= blastn&blast_programs=megablast&page_type= blastsearch&show_defaults=on&link_loc=blast home=blasthome, which confirmed these 15 specimens as h1n1v positive. in the case of four of these 15 h1n1v specimens, virus was grown in efes (table 3 ) and rna extracted from infective allantoic fluid, then similarly pcr amplified and sequenced to provide confirmation of h1n1v infection. uk pigs positive for avian-like swine h1n1 archived tissues were pools that contained varying proportions of respiratory tissues, including trachea and ⁄ or lung, except for a ⁄ swine ⁄ england ⁄ 3 ⁄ 09 where two distinct tissues were tested from the same pig. these samples were used to assess (i) m gene rrt pcrs (original avian protocol and two variants) and (ii) h1-118 rrt pcr. ct values for the most sensitive m gene rrt pcr for a given tissue specimen is shown in normal type, while ct values for the two less sensitive m gene rrt pcr variants are shown in italic type. clinical specimens from pigs infected experimentally six landrace hybrid pigs (age 4-5 weeks) were each infected experimentally by inoculation with 2 ml h1n1v (a ⁄ california ⁄ 07 ⁄ 09), which contained 10 5ae8 eid 50 per animal via the intranasal route. 36 twenty-three nasal swabs were obtained by swabbing the infected pigs daily from 1 to 7 days postinfection (dpi), and 10 pig tissues (lung n = 5, thoracic trachea n = 5) were obtained from animals killed humanely for post-mortem examination on 2,4 and 7 dpi (table 4) . swabs one hundred and ten swabs from the field (104 from vla regional laboratories during summer 2009; six from proven field cases of h1n1v infection, table 3 ) and 23 swabs from pigs infected experimentally (table 4 ) were stored at )70°c in 1 ml brain heart infusion broth containing antibiotics (bhib). swabs from the vla regional laboratories and h1n1v experimentally infected pigs (n = 127) were tested by vi in 9-to 10-day-old efes by means of double inoculations via the allantoic and amniotic cavities, followed by a second efe passage in the event of the first passage being vi negative. influenza a virus growth was detected by a positive haemagglutination assay (ha) result obtained by testing harvested amnio ⁄ allantoic fluid with chicken red blood cells. 27 in the case of six swabs from h1n1v-field-infected pigs, infection with this pandemic virus was independently proven by conventional pcr amplification, sequencing and blast searching as described earlier (table 3) . organ homogenates (approximately 10% w ⁄ v in bhib) were prepared from 97 archived uk tissue specimens (31 from siv-positive pigs ( table 2 ) and 66 siv-negative pigs by vi) and 15 tissues from h1n1v-field-infected pigs (september-november 2009; table 3 ) by grinding with sterile sharp sand. for nine of 15 tissues from h1n1v-fieldinfected pigs, infection with this pandemic virus was independently proven by conventional pcr amplification, sequencing and blast searching as described earlier (table 3 ). ten tissue specimens from the experimentally infected pigs (table 4 ) were disrupted in bhib using a general laboratory homogenizer (omni international, marietta, ga, usa) to provide a similar 10% w ⁄ v suspension. all tissue homogenates were clarified by centrifugation for 1 minute prior to vi and rna extraction. vi was carried out by inoculating clarified tissue homogenates into 9-to 10-day-old efes as described earlier for pig swabs. the mini viral rna kit (qiagen) was used to extract rna from allantoic fluids, bhib swabs fluids and clarified tissue homogenates. this was performed either manually by the ''spun column'' method in accordance with the manufacturer's protocol, or by robotic rna extraction by the same kit chemistry adapted to a universal biorobot (qiagen). 34 comparison of the m gene primer and probe sequence were performed on the influenza research database website http://www.fludb.org. several programmes available on the site were used for the comparison of available swine influenza sequence information including the snp analysis, blast and alignment viewer. 37 the primers and hydrolysis probe used initially were those from the m gene rrt pcr originally described by spackman et al. 38 (table 5) for global and generic detection of aivs at final concentrations of 0ae4 and 0ae3 lm, respectively. 34 the reverse primer was modified in two variations of the m gene rrt pcr assay to investigate the detection of sivs and h1n1v isolates. the first variant (''perfect match'' m gene rrt pcr) included modification of the aiv reverse primer used by spackman et al. 38 to provide a perfect match primer (reverse modified, i.e. ''rev-mod'') with the corresponding region in the m gene of h1n1v isolates ( the second variant (''combo'' m gene rrt pcr) included an equimolar mix of the original aiv reverse primer plus the above rev-mod primer, with each included at 0ae2 lm final concentration. otherwise cycling conditions, temperatures and chemistry details for both variants were as outlined for the m gene rrt pcr, 34 in which the original m gene rrt pcr and the two variants produced the same 101-bp product. two microlitres of extracted rna were tested in each m gene rrt pcr in a final 25 ll volume using mx 3000 realtime pcr instruments (stratagene, amsterdam, the netherlands). 34 fluorescence thresholds for all rrt pcr experiments were determined using default settings in the supplied stratagene mxpro software, and these were inspected visually after every experiment to ensure consistency. the antisense hydrolysis probe (31 nucleotides long) was 118 pro-rev: fam-5¢-ctg gct tct tac ctt tt*c ata taa gtt ctt c-3¢ [1377-1407], which was labelled with the fam fluorophore at the 5¢ end and with black hole quencher (bhq1) at an internal t nucleotide (indicated as t*), while the 3¢ terminal c nucleotide was modified to a dideoxy c to prevent probe extension by polymerase activity (eurogentec, liège (luik), belgium). primers and probe were included at final concentrations of 0ae4 and 0ae3 lm, respectively, and 2 ll of extracted rna were tested in each h1-118 rrt pcr in a final 25 ll volume. other chemistry details and cycling conditions were as outlined for the h7 ha2 rrt pcr 34 where stratagene mx 3000 realtime pcr instruments were used. occasionally, ''h1-118'' rrt pcr products were visually checked by electrophoresis in 3% (w ⁄ v) agarose gels according to standard procedures. 40 table 5 . these were designed to investigate any uk clinical specimens that may be positive by m gene rrt pcr but negative by ''h1-118'' rrt pcr and negative by vi. three ha gene alignments that included european siv isolates of the h1n1 (avian-like swine), h1n2 and human-like swine h3n2 subtypes were constructed using the megalign software from the lasergene package. the primerselect programme was then used to design appropriate primers to generate small (i.e. <230 bp) amplicons that were of sufficient size for sequencing and identification by blast searching. five clinical specimens (one uk archived swine tissue submitted in january 2009 and four nasal swabs from analysis of the m gene primers and probe to available swine influenza sequences shows several different patterns based on geographical and temporal origins of the viruses (table 5 ). m gene sequences are available from over 500 siv isolates from north america, europe and asia, with different sequence patterns in each region (data not shown). the sequence for the forward m gene rrt pcr primer and the probe remains highly conserved for most isolates (table 5 ). however, the reverse primer has extensive variability at primarily five different positions for h1n1v and siv isolates compared to the ''avian'' m gene rrt pcr primers and probes originally designed by spackman et al. 38 current sivs and h1n1v isolates have from zero to four nucleotide mismatches in the reverse primer ( table 5 ) that could potentially contribute to a decrease in sensitivity for some viruses. this influenced the design of the ''perfect match'' m gene rrt pcr ''rev-mod'' primer for h1n1v detection, which is located at the same corresponding position as the ''m)124 r'' primer for the ''avian'' m gene rrt pcr (table 5) . h1n1v isolate a ⁄ california ⁄ 07 ⁄ 09 and siv isolate a ⁄ swine ⁄ england ⁄ 742104 ⁄ 95 (h3n2) were grown in efes and eid 50 titres determined. rna was extracted from these influenza a preparations and used to construct 10fold dilution series, and both were tested by the three m gene rrt pcr assays, namely the ''avian'', 38 ''combo'' and ''perfect match''. the 10-fold dilution series from a ⁄ california ⁄ 07 ⁄ 09 (h1n1v) was also tested by the ''h1-118'' rrt pcr. the detection limit corresponded to a viral titre (pre-rna extraction) of 1 · 10 1 eid 50 ⁄ ml for the ''perfect match'' m gene and ''h1-118'' rrt pcrs, which typically registered at ct values 34-36. when a dilution series of quantified t7 in vitro rna transcripts were tested, the detection limit of the ''perfect match'' m gene rrt pcr at these ct values was 200 rna copies and 2000 rna copies for the h1n1v and h3n2 targets, respectively. this discrepancy in sensitivity may relate to differences in the m gene reverse primer binding sequence for the two influenza a viruses ( fifty-one laboratory isolates of established sivs were divided into five categories based on combinations of subtype and temporal and geographical origin (table 1) . for the two classical h1n1 sivs isolated in united kingdom in 1986 and 1996, the ''avian'' and ''combo'' m gene variant rrt pcr assays appeared to be the most sensitive. however, when 35 contemporary uk and european sivs were tested, the ''perfect match'' m gene rrt pcr was the most sensitive for detecting 34 of the isolates in the avian-like swine h1n1, h1n2 and human-like swine h3n2 categories (table 1) . these three categories are representative of the sivs that have been circulating in europe for up to 30 years. 11 it should be noted that the ''combo'' m gene rrt pcr assay was slightly less sensitive, detecting the 35 contemporary european sivs at ct values that were higher, but by no more than 2 for the majority of these isolates ( table 1 ). the one exception was a ⁄ swine ⁄ eire ⁄ 89 ⁄ 96 (h1n1 avian-like), which was most sensitively detected by the original ''avian'' m gene rrt pcr (table 1) . in contrast, the 14 north american sivs were most sensitively detected by the ''combo'' m gene rrt pcr assay in which the lowest ct values were obtained, followed by the ''avian'' m gene rrt pcr. the ''perfect match'' m gene rrt pcr assay was the least sensitive for detection of the 14 north american sivs as the ct values obtained were consistently higher than in the other two assays (table 1) . it can be inferred from these findings that the ''perfect match'' m gene rrt pcr assay would be the most sensitive to use for generic testing of contemporary european sivs as well as any pig infections caused by h1n1v. however, for north american pigs, the ''combo'' m gene rrt pcr provided the most sensitive approach to generic siv testing for 13 of 14 north american isolates, the one exception being a ⁄ swine ⁄ nebraska ⁄ 02013 ⁄ 08 (h1n1) where the ''avian'' m gene rrt pcr registered a slightly lower ct value compared to the ''combo'' m gene rrt pcr (table 1) . representative isolates of all sixteen h-types were included in the 43 aivs tested (table 1) . forty-two of 43 were detected most sensitively by the ''avian'' m gene rrt pcr as this test consistently gave the lowest ct values, the one exception being a ⁄ duck ⁄ belgium ⁄ 10157 ⁄ 07 (h12n5) ( table 1 ). this was followed by the ''combo'' m gene rrt pcr, while the ''perfect match'' m gene rrt pcr assay proved to be the least sensitive for the detection of the aiv isolates. the low sensitivity of the ''perfect match'' m gene rrt pcr assay resulted in ''no ct'' being recorded for six aiv isolates, possibly because their rna had been extracted from highly diluted virus preparations. one aiv rna (a ⁄ mallard ⁄ sweden ⁄ 08, h7n7) was so highly diluted that a high ct value (36ae86) was obtained with the ''avian'' m gene rrt pcr, but ''no ct'' registered by both the ''perfect match'' and ''combo'' m gene rrt pcr assays (table 1 ). the ''h1-118'' rrt pcr assay was used to test 94 siv and aiv isolates plus five h1n1v isolates (table 1 ). in the tests, comparable ct values were obtained for the five h1n1v isolates to those observed in the ''perfect match'' m gene rrt pcr assay ( table 1) . none of the 37 european siv laboratory isolates, including the 22 h1n1 (20 avian-like swine and two classical) and eight h1n2 sivs, were detected by the ''h1-118'' rrt pcr assay. the ''h1-118'' rrt pcr assay was designed specifically for differential detection of h1n1v influenza a viruses. non-detection of contemporary, established european h1 sivs by the ''h1-118'' rrt pcr as well as h1 aivs indicated that it fulfils this condition (table 1) . however, positive fluorescence signals were obtained in ''h1-118'' rrt pcr tests with two of the north american h1n1 sivs, namely a ⁄ swine ⁄ wisconsin ⁄ h04ys2 ⁄ 04 and a ⁄ swine ⁄ nebraska ⁄ 02013 ⁄ 08 (table 1) . tests with the five other north american h1n1 sivs produced negative fluorescence signals, but gel electrophoresis in 3% w ⁄ v agarose showed that all seven of the north american h1n1 isolates tested produced a band of the predicted 118 -bp size ( table 1) . h1 gene sequences are available in the public databases for two of these north american h1n1 isolates, namely a ⁄ swine ⁄ ontario ⁄ 11112 ⁄ 04 (accession no. dq280250) and a ⁄ swine ⁄ indiana ⁄ 1726 ⁄ 88 (accession no. m81707 or cy039925). a degree of conservation was observed in the primer binding sequences for the ''h1-118'' rrt pcr, while mismatches in the probe-binding region appeared to account for the failure to generate fluorescence signals (data not shown). ''h1-118'' rrt pcr testing of both north american h1n2 isolates also produced the 118-bp band of predicted size. a ⁄ swine ⁄ indiana ⁄ 9k035 ⁄ 99 (h1n2) (af250124) produced fluorescence in this test, but a ⁄ swine ⁄ ontario ⁄ 48235 ⁄ 04 (h1n2) (dq280236) was fluorescence negative ( table 1 ). the a ⁄ swine ⁄ indiana ⁄ 9k035 ⁄ 99 h1 gene sequence has a perfect match with the primer and probe-binding regions for the ''h1-118'' rrt pcr, while the corresponding h1 gene sequence in a ⁄ swine ⁄ ontario ⁄ 48235 ⁄ 04 included significant mismatches in its probe-binding region (data not shown). the 14 non-influenza a viruses were tested by the three m gene rrt pcr assays and by the ''h1-118'' rrt pcr assay and only negative results were obtained. ninety-seven frozen archived respiratory tissues that had been obtained from pigs in the united kingdom since 1991 were tested. these were divided into two groups, those that had been obtained from pigs that were (i) siv positive (n = 31) and (ii) siv negative (n = 66) based on vi at the time of sample submission. all archived tissue specimens from positive pigs were retested by vi in the course of this study, as well as by the three m gene rrt pcr approaches and ''h1-118'' rrt pcr ( table 2 ). the 31 tissues from siv-positive pigs included 18 tissues from pigs infected with avian-like swine h1n1 viruses between 1992 and 2008, six from pigs with h1n2 virus infections from 1994 to 2009 and seven from pigs infected with human-like swine h3n2 virus during 1991-1994 ( table 2 ). three of these archived tissue samples from vi-positive pigs gave no ct value with any of the m gene rrt pcr assays. no ct value was obtained by the ''avian'' m gene rrt pcr with three further samples, although these were positive by the other two m gene assays ( table 2 ). one tissue specimen was negative by the ''combo'' m gene rrt pcr but gave high ct's by the other two m gene rrt pcrs ( table 2) . as in the case of the more contemporary siv laboratory isolates from united kingdom and european origin (table 1) , the ''perfect match'' m gene rrt pcr was shown to be the most sensitive of the m gene rrt pcr variants for the majority of the archived siv-positive tissue specimens, as for 27 of 28 tissues the m gene ''perfect match'' rrt pcr registered the lowest ct compared to the two other m gene rrt pcrs ( table 2 ). the one exception was the oldest tissue specimen, from which a ⁄ swine ⁄ england ⁄ 119404 ⁄ 91 (h3n2) had been isolated; in this case, the original avian m gene rrt pcr was most sensitive ( table 2) . these test results with archived tissue specimens obtained from the field reinforced the observation that the ''perfect match'' m gene rrt pcr is the most sensitive for detecting contemporary european and uk sivs. all 31 archived uk tissue samples from siv-positive pigs were negative by the ''h1-118'' rrt pcr assay (table 2) . only four of these archived tissues were vi positive during the current validation study, this may reflect the age and ⁄ or storage history of the tissues, plus the use of only two egg passages for vi. sixty-six archived tissue specimens from siv-negative pigs from united kingdom (organ pools of lung, trachea and tonsil, or lung only) were tested by the ''perfect match'' m gene rrt pcr assay to assess its specificity. all gave ''no ct'' results except one, which was submitted in january 2009 and produced a ct value of 25ae45 by the ''perfect match'' m gene rrt pcr. repeat vi testing during the current validation study failed to recover viable influenza a virus from this specimen. however, investigation of this specimen by conventional rt pcr and amplicon sequencing identified an h1 gene that was highly suggestive of an h1n2 siv. all 66 of these archived tissues from siv-negative pigs were ''no ct'' by the ''h1-118'' rrt pcr. nasal swabs collected in the field by vla regional laboratories one hundred and four nasal swabs obtained from pigs in united kingdom during the summer of 2009 were tested by vi in efes, the ''perfect match'' m gene rrt pcr assay and the ''h1-118'' rrt pcr assay. one hundred of these swabs were negative by all three tests. however, four swabs that originated from two uk pig herds gave negative results by vi and ''h1-118 rrt'' pcr but resulted in positive amplifications by ''perfect match'' m gene rrt pcr, with ct values of 27ae56 and 30ae77 for one herd and 34ae54 and 38ae01 for the second herd. investigation by conventional rt pcr and sequencing revealed an h1 gene that was suggestive of h1n2 siv. of the 39 specimens (24 respiratory swabs and 15 respiratory tissues) received between september and november 2009 from nine geographically diverse pig herds (table 3) , fifteen (six swabs and nine tissues) originating from eight pig herds had been shown to be h1n1v positive by non-rrt pcr approaches (table 3) . these and a further 24 specimens (18 swabs and six tissues) that included two lung specimens from an epidemiologically related ninth herd in northern ireland (ni ''c''; table 3 ) were tested by both the ''perfect match'' m gene and ''h1-118'' rrt pcrs. twenty-two of the 24 swabs were positive by both rrt pcrs, with ct value ranges of 20ae64-34ae14 and 20ae74-34ae66 for the ''perfect match'' m gene and ''h1-118'' rrt pcr assays, respectively ( table 3 ). the submission derived from herd ''c'' in ni that included two lung tissues gave positive results by both rrt pcrs for one specimen, but the second gave high ct values (37ae50 and 37ae93 by ''perfect match'' m gene and ''h1-118'' rrt pcrs, respectively) that were just above the positive cutoff for the two tests (table 3) . however, the clear positive result for the first lung specimen had identified h1n1v infection in this pig herd. one swab from the norwegian herd gave ''no ct'' by both rrt pcr assays, but the other 11 nasal swabs from the same herd gave similar positive ct values by both methods. the 15 tissues were all positive by both tests, with ct value ranges of 20ae84-33ae17 and 20ae89-32ae84 for the ''perfect match'' m gene and ''h1-118 rrt'' pcr assays, respectively (table 3) . thirty-three clinical specimens collected from pigs infected experimentally with h1n1v 36 were tested by vi, the ''perfect match'' m gene rrt pcr assay and the ''h1-118'' rrt pcr assay (table 4 ). these samples included 23 nasal swabs taken between 1 and 7 dpi, and post-mortem tissues (lung, n = 5; thoracic trachea, n = 5). twentytwo of the 23 swabs gave positive results by vi and both rrt pcr assays, while one swab was weakly positive by the two rrt pcrs and vi negative (7 dpi, pig 2; table 4 ). the ct ranges for these swabs were 22ae20-34ae48 and 24ae23-36ae52 for ''perfect match'' m gene and ''h1-118'' rrt pcr assays, respectively. the 10 post-mortem tissues from the experimentally infected pigs gave concordant results by all three tests, i.e. seven were positive and three were negative ( table 4 ). the ct ranges for these positive tissues were 20ae33-34ae28 and 21ae17-34ae42 for the ''perfect match'' m gene and ''h1-118'' rrt pcr assays, respectively. the sensitivity and specificity were calculated for the ''perfect match'' m gene and ''h1-118'' rrt pcr assays from the clinical specimens (respiratory swabs and tissues) used in this validation study. for 249 clinical specimens, confirmatory results were available either as vi, or in the case of 15 h1n1v field specimens, through independent confirmation by amplicon sequencing (table 3) . for 133 swabs, a diagnostic sensitivity of 100% and a specificity 95ae2% were calculated for the ''perfect match'' m gene rrt pcr. with this assay, there were five results that were vi negative, but ''perfect match'' m gene rrt pcr positive (table 6a ). evidence presented elsewhere in the results strongly suggests that these five were genuine positives. when the swabs were tested by ''h1-118 rrt'' pcr, a diagnostic sensitivity of 100% and specificity of 99ae1% was determined (table 6b) . there was only one swab, obtained from an experimentally infected pig at 7 dpi, that was vi negative but positive by ''h1-118 rrt'' pcr (table 6b) , this swab from pig 2 was also positive by ''perfect match'' m gene rrt pcr (table 4 ). in the case of the 97 archived pig tissues collected in united kingdom between 1991 and 2009, the specimens were divided into those from siv-positive (n = 31; table 2 ) and siv-negative (n = 66) pigs that had been identified by vi at the time of the original submission. by inclusion of 19 additional tissues from h1n1v-fieldinfected (table 3 ) and experimentally infected (table 4) pigs, a diagnostic sensitivity of 93ae6% and a specificity of 98ae6% were determined for the ''perfect match'' m gene rrt pcr (table 7a) . three archived tissues from pigs that were vi positive gave negative results by ''perfect match'' m gene rrt pcr (table 2) , and one archived tissue was positive by ''perfect match'' m gene rrt pcr but vi negative (table 7a ). when the same tissues were tested by ''h1-118'' rrt pcr assay, the diagnostic sensitivity and specificity were both 100%, respectively (table 7b ). in recent years, the m gene rrt pcr assay 38 has gained widespread acceptance as a validated method for the global detection of all aiv subtypes, and this method is referred to as a suitable screening assay in the oie and eu diagnostic manuals for ai. 33, 43 although no international recommendations have been made concerning rrt pcr testing for sivs, the highly conserved nature of the m gene in all influenza a viruses led to the m gene rrt pcr being considered as a candidate for an initial screening assay for detecting pigs infected with all sivs, including any infections because of h1n1v. in this study, we have investigated the application of the ''avian'' m gene rrt pcr, 38 but sequence differences observed in the m gene of h1n1v isolates also prompted investigation of two new variations of the original m gene rrt pcr. the original ''avian'' m gene rrt pcr reverse primer (m)124 r), 38 with four nucleotide differences to h1n1v viruses (table 5) , clearly resulted in a significantly lower sensitivity when testing h1n1v laboratory isolates (table 1) . consequently, the original avian m gene rrt pcr 38 should not be used for detection of h1n1v, and this deficiency of the diagnostic test needed to be addressed. among the three variants of the m gene rrt pcr that were evaluated, the ''perfect match'' with the revised reverse primer (''revmod'') provided the most sensitive results for many of the european sivs (tables 1 and 2 ). m gene sequence analysis showed that the ''rev-mod'' primer compensated for two or three nucleotide mismatches in the corresponding region of the m gene from contemporary european sivs (table 5) . however, experiments with t7 in vitro rna transcripts corresponding to the amplified m gene region of a ⁄ swine ⁄ england ⁄ 742104 ⁄ 95 (h3n2) suggested that the ''perfect match'' m gene rrt pcr may not be maximally sensitive for detection of all contemporary european sivs, which again reflected sequence mismatches with the ''revmod'' primer. nevertheless, the ''perfect match'' m gene rrt pcr demonstrated successful detection of contemporary european sivs in archived tissues from infected pigs ( table 2) , plus five specimens (four nasal swabs from uk herds and one archived tissue, collected in 2009, highly likely to be h1n2), that were negative by vi (tables 6 and 7) . importantly, the ''perfect match'' rrt pcr successfully detected both h1n1v laboratory isolates (table 1 ) and clinical specimens from pigs that had been infected with h1n1v in the field (table 3) and experimentally (table 4) . 36 the ''combo'' m gene rrt pcr assay was slightly less sensitive for detection of contemporary european sivs and h1n1v (tables 1 and 2) . however, for the testing of most north american sivs that were included in this study, the ''perfect match'' m gene rrt pcr suffered from reduced or even poor sensitivity (table 1) . for these sivs, improved sensitivity was restored by employing the ''combo'' m gene rrt pcr, where inclusion of the original ''avian'' m)124 r reverse primer reduced the number of nucleotide mismatches within the corresponding sequence of the north american sivs' m gene (tables 1 and 5 ). it must also be emphasised that the ''avian'' m gene rrt pcr remains the most sensitive of the three m gene rrt pcrs for the detection of aivs (table 1) . the diagnostic sensitivity and specificity of the ''perfect match'' m gene rrt pcr assay were 100% and 95ae2%, respectively, from the testing of 133 swabs (table 6a ). the five discrepant results included a vi-negative nasal swab obtained from a h1n1v-experimentally infected pig at 7 dpi when viral shedding appeared to be waning (ct 34ae48), and this low titre was mirrored by the corresponding h1-118 rrt pcr (ct 34ae08; table 4 ). the other four discrepant results were vi-negative swabs collected by uk regional laboratories during summer 2009 that were shown to be likely h1n2 siv positives, so affirming these ''perfect match'' m gene rrt pcr results. this inferred a greater sensitivity for the rrt pcrs compared to vi. as regards ''perfect match'' m gene rrt pcr testing of the 116 swine tissues, however, there were four discrepant results, where a diagnostic sensitivity and specificity of 93ae6% and 98ae6%, respectively, were observed (table 7a ). the 96 uk archived swine tissues were classified as siv positive or negative at the time of original submission based on vi. three of the 31 tissues from siv-positive pigs were negative (no ct) by the ''perfect match'' m gene rrt pcr (table 2 ). it was speculated that at the time of the original submission these pigs may have been classified as siv positive based on a vi result from another localised tissue portion or clinical specimen that was no longer available for the current validation. it was also observed that only 4 of 31 tissues obtained from siv-positive pigs were positive when vi was repeated through two egg passages during the current validation (table 2 ). this may be a consequence of the long-term storage history of these archived tissues, where infectivity may have been compromised by repeated freeze-thawing, or even because of limiting vi to two egg passages. one archived tissue from an siv-negative pig collected in 2009 was positive by ''perfect match'' m gene rrt pcr, negative by ''h1-118'' rrt pcr and vi (table 7) , but additional investigations suggested that this was a non-viable h1n2 specimen. the h1-118 rrt pcr assay was designed for the differential detection of h1n1v infections of pigs. this method successfully detected five h1n1v laboratory isolates (table 1 ) and the presence of h1n1v in 48 clinical specimens (29 swabs and 19 post-mortem tissues) in fieldinfected and experimentally infected pigs (tables 3 and 4) . it was also shown that the h1 genes from nine h1 aivs, 30 european h1 sivs (i.e. classical h1n1, contemporary avian-like swine h1n1 and h1n2) were not detected ( table 1 ) by this assay. in addition, no positive signal was obtained when 24 archived tissue samples obtained from pigs infected with european h1n1 and h1n2 sivs were tested with the h1-118 rrt pcr assay ( table 2 ). this affirmed the ability of the h1-118 rrt pcr assay to differentiate h1n1v infections from those caused by the sivs endemic in europe. however, this test did show some cross-reactivity with north american h1 sivs (table 1) . this was not unexpected in view of the evolutionary relationship between the h1 gene of north american triple reassortment sivs and h1n1v. 5 consequently, it would not be an appropriate test for differentiating h1n1v in north american pig infections. the diagnostic sensitivity and specificity of the ''h1-118'' rrt pcr assay for 133 swabs were 100% and 99ae1%, respectively (table 6b) ; the one discrepant result was the swab obtained at 7 dpi from pig 2 infected experimentally with h1n1v when the titre of viral shedding was declining (table 5) . for the ''h1-118'' rrt pcr using tissue samples, both the diagnostic sensitivity and specificity of this assay were 100% (table 7b) . fifteen clinical specimens from h1n1v-field-infected pigs ( table 3) were independently proven as h1n1v positive by conventional rt pcr and amplicon sequencing because vi was not consistently successful for the identification of h1n1v. this is because h1n1v does not consistently haemagglutinate chicken red blood cells, which may not be observed until the third passage in efes (data not shown). a further 24 clinical specimens (18 swabs and six tissues) from h1n1v-field-infected herds were not included in the diagnostic sensitivity and specificity calculations, but testing by both rrt pcrs gave highly concordant results as judged by a comparison of ct values (table 3) . public health laboratories have already described rrt pcr protocols for the detection of h1n1v in humans, [29] [30] [31] [32] and in this study, we have validated rrt pcr testing for detection of h1n1v for veterinary purposes, specifically for detecting h1n1v in pigs. this included evaluation of a generic approach to detect known sivs as well as h1n1v. this was achieved for european sivs by the ''perfect mach'' m gene rrt pcr assay, which sensitively detected all known types of european sivs in addition to h1n1v. however, it was noted that contemporary irish sivs may be the exception to this observation in europe (table 1) . testing of recent irish sivs by the various m gene rrt pcr assays suggests that the original ''avian'' m gene rrt pcr may be the more sensitive approach (raleigh and flynn, personal communication). in the case of north american sivs, the ''combo'' m gene rrt pcr assay was most sensitive for generic detection, and this method was slightly less sensitive for the detection of h1n1v than the ''perfect match'' m gene rrt pcr assay. the ''h1-118'' rrt pcr assay was shown to be able to differentiate h1n1v from siv infections in clinical samples derived from field-infected and experimentally infected pigs relevant to a european setting. validation of the generic and differential rrt pcr assays involved not only testing of a range of laboratory-grown influenza a virus isolates, but also crucially included testing of the same set of clinical specimens from pigs infected with sivs or h1n1v. primers and probe for the differential ''h1-118'' rrt pcr were deliberately designed within the relatively conserved ha2 region of the ha gene of h1n1v. however, continuing molecular evolution the ha gene may yet result in nucleotide changes within these primer and probe-binding sequences. vigilance for relevant nucleotide changes by sequencing the ha genes of new h1n1v swine isolates may identify potential future modifications to the ''h1-118'' rrt pcr. validation of the rrt pcr assays was conducted from the perspective of veterinary contingency planning for the eventuality of h1n1v outbreaks in the european pig population. to date, h1n1v infections have been already documented in pigs in the americas, eurasia and australasia 15, 16, 44 and examples of field cases have been included in this study ( table 3) . as the human pandemic caused by h1n1v continues to spread globally, it is anticipated that further pig infections with h1n1v will be reported, and precedent suggests that these will be followed by sustained transmission within pig populations. 36 analogous contingency planning was conducted in 2005, when the westward spread of h5n1 highly pathogenic aiv prompted the validation of a h5 rrt pcr for the detection of both this h5 virus and other eurasian h5 aivs. 45 in conclusion, it is possible to present these approaches as sensitive and specific methods for use in surveillance for siv and current h1n1v isolates in european pig populations. pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico swine influenza a (h1n1) infection in two children -southern california novel swine-origin influenza a (h1n1) virus investigation team. emergence of a novel swine-origin influenza a (h1n1) virus in humans antigenic and genetic characteristics of swine-origin 2009 a (h1n1) influenza viruses circulating in humans origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic pandemic potential of a strain of influenza a (h1n1): early findings world now at the start of 2009 influenza pandemic. who statements evolving complexities of influenza virus and its receptors the role of swine in the generation of novel influenza viruses the epidemiology and evolution of influenza viruses in pigs the role of pigs in interspecies transmission the emergence of novel swine influenza viruses in north america swine influenza viruses: a north american perspective multiple genetic reassortment of avian and human influenza a viruses in european pigs, resulting in the emergence of an h1n2 virus of novel genotype oie disease notification in pigs canada. (early may). available at pigs destroyed following flu outbreak laboratory characterization of a swine influenza virus isolated from a fatal case of human influenza human infection by a swine influenza a (h1n1) virus in switzerland isolation of viruses antigenically related to the swine influenza virus from an outbreak of respiratory disease in turkey farms in israel interspecies transmission and reassortment of influenza a viruses in pigs and turkeys in the united states the nucleotide sequence of the ha1 of the haemagglutinin of an h1 avian influenza virus isolate from turkeys in germany provides additional evidence suggesting recent transmission from pigs isolation from turkey breeder hens of a reassortant h1n2 influenza virus with swine, human, and avian lineage genes further evidence for infection of pigs with human-like h1n1 influenza viruses in china avian and swine influenza viruses: our current understanding of the zoonotic risk diseases of swine oie (world organisation for animal health) swine influenza: manual of diagnostic tests and vaccines for terrestrial animals, chapter 2.8.8. paris: oie a review of rt-pcr technologies used in veterinary virology and disease control: sensitive and specific diagnosis of five livestock diseases notifiable to the world organisation for animal health available at evaluation of four real-time pcr assays for detection of influenza a(h1n1)v viruses molecular detection of a novel human influenza (h1n1) of pandemic potential by conventional and real-time quantitative rt-pcr assays detection of novel (swine origin) h1n1 influenza a virus by quantitative real-time rt-pcr avian influenza: manual of diagnostic tests and vaccines for terrestrial animals validated realtime reverse transcriptase pcr methods for the diagnosis and pathotyping of eurasian h7 avian influenza viruses a simple method of estimating fifty percent endpoints replication, pathogenesis and transmission of pandemic (h1n1) 2009 virus in non-immune pigs biohealthbase: informatics support in the elucidation of influenza virus host pathogen interactions and virulence development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h5 and h7 haemagglutinin subtypes the influenza virus resource at the national center for biotechnology information the condensed protocols from ''molecular cloning: a laboratory manual a method for the rapid construction of crna standard curves in quantitative real-time reverse transcription polymerase chain reaction real-time rt-pcr, chapter 9 report reference: nswpigs1_2009, ref oie: 8332 validated h5 eurasian real-time reverse transcriptase-polymerase chain reaction and its application in h5n1 outbreaks in the authors thank all laboratories that supplied specimens of swine influenza and pandemic (h1n1) 2009 influenza used in this study, in particular the vla regional laboratories at bury st edmunds and thirsk for their long-standing contribution to swine influenza surveillance in united kingdom. the contribution of dr dennis alexander to the checking of this manuscript is also gratefully acknowledged, as is the funding that was provided from the uk department of the environment, food and rural affairs (defra). key: cord-343050-1pfqgvie authors: huang, qiu sue; turner, nikki; baker, michael g; williamson, deborah a; wong, conroy; webby, richard; widdowson, marc-alain title: southern hemisphere influenza and vaccine effectiveness research and surveillance date: 2015-06-09 journal: influenza other respir viruses doi: 10.1111/irv.12315 sha: doc_id: 343050 cord_uid: 1pfqgvie the 2009 influenza a(h1n1)pdm09 pandemic highlighted the need for improved scientific knowledge to support better pandemic preparedness and seasonal influenza control. the southern hemisphere influenza and vaccine effectiveness research and surveillance (shivers) project, a 5-year (2012–2016) multiagency and multidisciplinary collaboration, aimed to measure disease burden, epidemiology, aetiology, risk factors, immunology, effectiveness of vaccination and other prevention strategies for influenza and other respiratory infectious diseases of public health importance. two active, prospective, population-based surveillance systems were established for monitoring influenza and other respiratory pathogens among those hospitalized patients with acute respiratory illness and those enrolled patients seeking consultations at sentinel general practices. in 2015, a sero-epidemiological study will use a sample of patients from the same practices. these data will provide a full picture of the disease burden and risk factors from asymptomatic infections to severe hospitalized disease and deaths and related economic burden. the results during the first 2 years (2012–2013) provided scientific evidence to (a) support a change to nz's vaccination policy for young children due to high influenza hospitalizations in these children; (b) contribute to the revision of the world health organization's case definition for severe acute respiratory illness for global influenza surveillance; and (c) contribute in part to vaccine strain selection using vaccine effectiveness assessment in the prevention of influenza-related consultations and hospitalizations. in summary, shivers provides valuable international platforms for supporting seasonal influenza control and pandemic preparedness, and responding to other emerging/endemic respiratory-related infections. the 2009 influenza a(h1n1)pdm09 pandemic highlighted the need for improved scientific knowledge to support better pandemic preparedness and seasonal influenza control. the southern hemisphere influenza and vaccine effectiveness research and surveillance (shivers) project, a 5-year (2012-2016) multiagency and multidisciplinary collaboration, aimed to measure disease burden, epidemiology, aetiology, risk factors, immunology, effectiveness of vaccination and other prevention strategies for influenza and other respiratory infectious diseases of public health importance. two active, prospective, population-based surveillance systems were established for monitoring influenza and other respiratory pathogens among those hospitalized patients with acute respiratory illness and those enrolled patients seeking consultations at sentinel general practices. in 2015, a sero-epidemiological study will use a sample of patients from the same practices. these data will provide a full picture of the disease burden and risk factors from asymptomatic infections to severe hospitalized disease and deaths and related economic burden. the results during the first 2 years (2012-2013) provided scientific evidence to (a) support a change to nz's vaccination policy for young children due to high influenza hospitalizations in these children; (b) contribute to the revision of the world health organization's case definition for severe acute respiratory illness for global influenza surveillance; and (c) contribute in part to vaccine strain selection using vaccine effectiveness assessment in the prevention of influenza-related consultations and hospitalizations. in summary, shivers provides valuable international platforms for supporting seasonal influenza control and pandemic preparedness, and responding to other emerging/endemic respiratory-related infections. keywords disease burden, epidemiology, immunology, influenza, risk factors, vaccine effectiveness. the 2009 influenza a(h1n1)pdm09 pandemic provided a test of global preparedness to assess the epidemiology of a pandemic and to respond appropriately and rapidly. the world was illprepared to respond to a severe influenza pandemic or to any similarly global, sustained and threatening public health emergency. 1 one fundamental constraint highlighted during the pandemic was the limited understanding of the epidemiology and severity of the pandemic which in turn hampered international efforts to mount an appropriate response. 2 rapid assessment of the epidemiologic, virologic and clinic features of a pandemic is essential to provide critical information to decision-makers on how to minimize morbidity and mortality, and mitigate potential economic and societal disruption. soon after the pandemic virus emerged in april 2009 in mexico and spread globally, public health leaders, anxious to understand the full breadth of influenza epidemiology, turned their attention to countries in southern temperate areas with an upcoming influenza season. this demonstrated the absence of an established real-time system in the southern hemisphere to provide more complete surveillance of an influenza pandemic. also, such a system would help monitor the epidemiology of new strains of seasonal influenza and the effectiveness of vaccination, both for the southern hemisphere and for upcoming northern hemisphere seasons. in december 2010, the us centers for disease control and prevention (us-cdc) made a funding opportunity announcement for a temperate southern hemisphere site to conduct research on the disease burden, epidemiology, and prevention of influenza and other respiratory diseases of public health importance. new zealand (nz) is a temperate southern hemisphere country with a population of 4á4 million people. the influenza season mainly occurs from june to september. [3] [4] [5] [6] nz's predominantly public-funded healthcare system with associated integrated health information systems is a strong asset in conducting population-based research. all new zealanders are assigned a unique health identifier allowing tracking of healthcare utilization over time and confidential record linkage to multiple databases including hospitalization and surveillance data. additionally, patients are registered with primary care providers who maintain highly computerized records with detailed demography, immunization status and clinical information. the nz population is well characterized in terms of demographic structure, particularly by ethnicity and socio-economic status. indigenous maori and pacific peoples (collectively about 20% of the population) appear particularly vulnerable to influenza and other respiratory infections. 3, 7 in in this article, we describe the objectives and study designs of shivers. we also describe lessons learned from the first 2 years and planned future studies as well as international applications. the overarching aim of shivers is to comprehensively investigate the disease burden, epidemiology, aetiology, risk factors, immunology, effectiveness of vaccination and other prevention strategies for influenza and other respiratory viral diseases of public health importance. the project consists of 9 objectives as detailed in table 1 . they can be divided into five main streams: influenza disease burden data are essential to allocate limited health resources, assist influenza vaccination policy development and improve vaccine uptake, particularly for subpopulations at risk. however, the evidence to support valid and precise estimates of influenza disease burden globally remains weak with low quality, partly due to the short duration of studies and the heterogeneity of study settings and methods (statistical modelling, active versus passive case findings, virological versus clinical detection). [8] [9] [10] [11] [12] [13] [14] in addition, there is scarce information on sero-epidemiologic investigation of seasonal influenza at a population level. serology can detect both symptomatic and asymptomatic infections, thus estimating the true incidence of influenza infection. this parameter cannot be determined by either disease surveillance programmes or detection of virologically confirmed cases as they would vastly underestimate influenza incidence and overestimate severity. [15] [16] [17] [18] shivers allows calculation of rates of infection and different clinical presentations in the same population at the same time for an accurate picture of the relative severity of influenza infection in the population and vulnerable subpopulations at four levels: (a) severe hospitalized disease; (b) moderate disease requiring a general practice visit; (c) mild disease not requiring a general practice visit; (d) incidence of infection (symptomatic and asymptomatic). aetiology shivers provides an integrated platform for the study of respiratory diseases caused by influenza and other common and emerging respiratory pathogens. the aetiological component allows us to (1) monitor antigenic drift of seasonal influenza viruses, contributing to who's annual vaccine strain selection; (2) support pandemic preparedness including surveillance for new subtypes of influenza a viruses (e.g. a(h7n9)); (3) monitor common non-influenza respiratory pathogens to understand their impact on the disease and epidemiology; and (4) provide early detection for emerging respiratory viruses (e.g. mers-cov). there is increasing evidence in the literature for the importance of polymicrobial infections. however, there remain gaps in our understanding of respiratory virus codetection and whether this represents co-infection and affects clinical disease manifestations and severity. there are contradictory reports with some suggesting that co-infections increase the severity of respiratory disease, 19-23 while others have found either no association [24] [25] [26] [27] [28] or that co-infections may actually be protective. 29 additionally, bacterial coinfections associated with cases of influenza are a leading cause of severe morbidity and mortality: bacterial coinfections complicated nearly all influenza deaths in the 1918 pandemic and up to 34% of the 2009 a(h1n1)pdm09 infections managed in intensive care units worldwide. 30, 31 shivers will help our understanding of the potential role of pathogen co-detection in patient outcome, severity, aetiology, demography and underlying risk conditions. influenza vaccine strain selection requires annual consultations and frequent updates to match the antigenic drift of the circulating viral strains, and ample evidence indicates that influenza vaccine effectiveness (ve) varies not only by virus type (subtype) but also from year to year. 32, 33 robust and timely vaccine effectiveness estimates are important to measure the public health benefit of seasonal influenza control strategies, pandemic preparedness and vaccine strain selection. 34 many ve estimates derive from observational studies with existing data collecting systems which often have multiple limitations and biases, and there are international calls for more rigorous ve studies. [34] [35] [36] [37] [38] [39] shivers is providing robust and timely estimations of the protective effect of seasonal influenza vaccine in the prevention of hospitalizations and general practice consultations for laboratory-confirmed influenza using case test-negative control methods. 40, 41 immune response an individual's immune response to influenza infection can vary depending on many factors (e.g. age, underlying conditions and ethnicity). clinical observation during the 2009 pandemic indicated that the unexpected low morbidity and mortality rates in the elderly were in part due to their crossreactive immunity. 3, 42, 43 there are knowledge gaps regarding each component of adaptive immune responses in determining an individual's risk of acquiring influenza virus infection and the severity of the resulting disease: antihemagglutinin (ha) antibodies, antineuraminidase (na) antibodies, isotypes of responding antibodies, influenza-specific cd4+, cd8+ t cells, surface markers and key cytokines. 44, 45 additionally, there are scarce data on the correlation of cellular immunologic and neuraminidase targeted antibody responses in individuals with serologically (anti-ha antibodies) defined influenza infection. 46, 47 by interconnecting the epidemiological and immune studies in severe and moderate disease cases and high-risk subgroups (e.g. pacific and maori ethnic groups) and individuals with serologically defined influenza infection, shivers will facilitate our understanding of host immune responses that determine an individual's risk of acquiring influenza infection or developing severe disease. identification and quantification of risk factors for influenza infection and poor outcomes (hospitalization, icu treatment, death) provides evidence to inform decisions on targeted pharmaceutical (vaccinations, antivirals), healthcare (e.g. improved treatment of comorbidities) and non-pharmaceutical (e.g. exposure to infections) interventions to reduce the risk of seasonal and pandemic influenza. elderly people have a significantly higher risk of influenza-associated death compared with non-elderly people. 48 additionally, the 2009 pandemic in nz revealed that the risk of hospitalization and death was markedly higher for maori and pacific people, and those from the most deprived socioeconomic groups. 3, 42, 49 however, it is not clear whether these sociodemographic factors are independent risk factors for influenza. furthermore, some chronic health conditions (high body mass index, asthma and pregnancy) have been shown to increase the risk of having a poor outcome from influenza infection. [50] [51] [52] [53] in nz, household crowding has been identified as a risk factor for transmission of meningococcal disease, 54 rheumatic fever 55 and tuberculosis 56 and may also be contributing to higher rates of influenza for some populations. the household setting (crowding, housing conditions) may influence transmission of influenza, but these effects remain poorly understood. 57-59 shivers will provide a multifaceted understanding of influenza risk that considers organism, host and environmental factors and opportunities for intervention. this comprehensive and quantitative approach will include detailed consideration of the independent contributions of host ethnicity, socioeconomic position, chronic illness status, obesity, household environment exposures and infecting virus. study sites shivers study sites are located within two district health boards of the auckland region of nz: adhb and cmdhb ( figure 1 ). this is a predominantly urban population of 906 000 people, with a wide spectrum of socio-economic, cultural, ethnic and demographic groups broadly similar to the new zealand population. 60 we established two surveillance platforms (hospital and sentinel general practice) in the two dhbs: four publicly funded hospitals serve the secondary healthcare needs for all residents of the two dhbs: auckland city hospital and the associated starship children's hospital (adhb), and middlemore hospital and the associated kidz first children's hospital (cmdhb). in 2012, we began active, prospective, continuous, population-based surveillance for influenza and other respiratory pathogens among persons residing in the two dhbs hospitalized for respiratory disease (figure 2 ). research nurses reviewed daily records of all overnight acutely admitted inpatients to identify any inpatient with a suspected acute respiratory illness (ari). they interviewed these patients by applying the world health organization (who) interim sari case definition: 'an acute respiratory illness with a history of fever or measured fever of ≥38°c, and cough, and onset within the past 7 days, and requiring inpatient hospitalization'. 61 since 2013, the who final sari case definition has been used with onset changed from '7 days' to '10 days'. the patients were differentiated into sari cases (those who met the sari case definition) and non-sari patients (those with ari who did not meet the sari case definition). a case report form captured information on demography, history of presenting illness, comorbidities, influenza vaccination history, disease outcome and risk factors. 60 if a patient met the sari case definition, a respiratory specimen (nasopharyngeal swab or aspirate) was collected and tested simultaneously for influenza and other respiratory viruses by real-time reverse transcription (rt) polymerase chain reaction (pcr) techniques: influenza virus, respiratory syncytial virus (rsv), rhinovirus, parainfluenza virus types 1-3, adenovirus and human metapneumovirus ( figure 3) . 60 a systematic sample of about 50% of non-sari patients were also interviewed and provided a respiratory sample, in addition to those from whom a specimen was collected for clinical purposes. some sari cases and non-sari patients were also tested for clinical purposes for a range of respiratory bacteria (e.g. streptococcus pneumonia, in 2013, we began active, prospective, population-based surveillance for influenza and other respiratory pathogens among persons enrolled in sentinel general practices who seek medical consultations (figure 4 ). eighteen sentinel general practices situated within adhb and cmdhb were recruited. these practices have a combined total of 103 752 enrolled patients, covering approximately 12% of the adhb and cmdhb population. the participating general practitioners (gp) and practice nurses (pn) assessed all consultationseeking patients. if a patient met the influenza-like illness (ili) case definition: 'an acute respiratory illness with a history of fever or measured fever of ≥38°c, and cough, and onset within the past 10 days, and requiring consultation in that general practice', a respiratory specimen (nasopharyngeal swab or throat swab) was collected to test for influenza and other respiratory viruses by real-time rt-pcr ( figure 3 ). gp/pn documented the components of the case definition that were present and recorded patients who met the ili case definition in an advanced electronic form designed for the practice management system (pms). patient information already captured in the pms was automatically retrieved, including demography, comorbidities, vaccination history and regular medication list. further data were captured by interviewing ili patients regarding influenza vaccination obtained elsewhere, pregnancy and a clinician's judgement of obesity. to more fully understand the epidemiology of influenza, these two platforms will be leveraged for other studies: (a) sero-epidemiological study: to obtain rates of mild influenza illness that do not trigger gp visits as well as symptomatic/asymptomatic infections, the enrolled patients in those sentinel general practices will be used to randomly select a cohort of persons (stratified by age and ethnicity) and followed through one influenza season. the serologic surveys will measure preand post-season antibodies to circulating influenza strains using relevant vaccine reference strains as antigens; (b) immunology study: a subset of samples from severe, moderate influenza cases, related risk groups and individuals with serologically defined influenza infection are selected for the study of humoral and cellular immune responses ( figure 3) ; and (c) remaining studies: the combined laboratory testing results and metadata collected from these platforms are used to study vaccine effectiveness, interaction between respiratory pathogens, respiratory mortality, risk factors and economic burden and vaccine cost-effectiveness. our innovative study design interconnecting multiple objectives, in addition to exploiting nz's unique healthcare structure, will maximize efficiency and study power. the two surveillance platforms provided specimens and data to serve the nine objectives of shivers. figure 5 shows how each of the objectives is linked to each other and maps data and specimen flows between them. since its conception in 2006, sari surveillance has become a recognized international standard for monitoring hospitalized severe respiratory disease related to influenza and other pathogens. 61 occurred over this time. [65] [66] [67] it was designed to monitor trends in severe influenza disease and to best capture the majority of influenza respiratory disease to estimate the burden of influenza-associated respiratory hospitalizations, and risk factors for severe disease. 63 the initial sari case definition included the symptom onset of acute respiratory illness within 7 days. 61 the shivers results in 2012 showed that a small proportion (7%) of influenza cases had specimens collected 8-9 days after the symptom onset. these specimens only consisted of a small proportion (9%) of total specimens; thus, the cost of testing was minimal (manuscript in preparation). this result was shared with the who global influenza programme. it contributed to a change in the final who sari case definition, with onset shifting from 'within 7 days' to 'within 10 days'. 63 burden and epidemiology shivers allows the estimation of influenza disease burden and risk factors at various levels of severity. firstly, the disease burden of severe influenza is estimated from the hospital surveillance platform. it measured population-based incidence for sari-associated influenza hospitalizations including icu admissions and in-hospital deaths as it provided reliable numerators and denominators, thus without a need for additional healthcare utilization surveys. [68] [69] [70] [71] our first-year findings (30 april 2012 to 28 april 2013) showed that the sari-associated influenza hospitalization rate was substantial with the overall adjusted annual incidence of 54/ 100 000 persons (manuscript in preparation). this rate was similar to us data on influenza-associated hospitalizations during 1979-2001, with an average annual incidence of 36á8/ 100 000. 12 the very young (0-4 years) and elderly (≥65 years) had the highest sari-associated influenza hospitalization rates, consistent with trends identified in international literature, particularly those from developed countries with temperate climates. 10, [72] [73] [74] [75] a high rate of influenza-related hospitalizations and low vaccine uptake (6%) in young children (6 months to 4 years) from shivers led the nz government to change vaccination policy by extending free influenza vaccination to those in this age group who have been hospitalized or have a history of significant respiratory illness. 76 sari surveillance is likely to underestimate the true burden of severe influenza resulting in hospitalization. some patients will present with non-respiratory symptoms or respiratory disease that does not meet the sari case definition, or stay briefly in emergency department. 63, 66 shivers has begun to address this gap; a pilot study in 2012 testing persons with respiratory disease who did not meet the sari case definition showed that a small proportion (6%) of non-sari patients were positive for influenza viruses, compared with 18% of sari cases (manuscript in preparation). future work to expand the case definition to all acute hospital admissions in a sample of very young children will further expand our knowledge of the burden of influenza in this important group potentially protected by maternal immunization. additionally, sari and associated influenza cases will be linked to the hospital discharge data to determine the accuracy and validity of the discharge data by determining proportions of the principal discharge diagnosis code categories that are sari and influenza cases. this will help inform modelling studies of icd-coded data and help provide some validation of these with laboratory-confirmed data. sari surveillance is also likely to grossly undercount the actual number of influenza-associated deaths because only a minority of influenza-related sari deaths are correctly diagnosed, tested and recorded as such. additional influenza deaths resulting from secondary bacterial infections and exacerbation of pre-existing chronic conditions and atypical clinical presentations are not captured. 77 this limitation presents a challenge in accurately measuring influenzarelated mortality. future work on statistical modelling may allow for indirect estimation of 'excess' mortality attributable to influenza in those broad categories such as pneumonia, respiratory or circulatory deaths during influenza seasons. 78 secondly, the disease burden of moderate influenza is estimated using data from shivers ili surveillance. our findings from the 2013 season (29 april to 29 september) showed that the ili-associated influenza consultation rate was about 14 times higher than sari-associated influenza hospitalization rate (manuscript in preparation). additionally, ili-associated influenza consultations and sari-associated influenza hospitalizations showed contrasting socio-demographic patterns: higher rates of ili-associated influenza consultations were shown in preschoolers (aged 1-4 years), school-age children and adults (<65 years), those of asian ethnicity and those from least deprived socio-economic status (ses) groups. this was a different picture from sari surveillance where sari-associated influenza hospitalizations were more frequent in the very young (under 1 year), the elderly, m aori and pacific peoples and those from most deprived ses groups. 79 these results provided insights into the interplay between healthcare access opportunities and related health-seeking behaviours and the differential effect of the predominant strains on various age groups. thirdly, the disease burden of mild influenza not requiring medical consultation (e.g. school/work-related absenteeism) and influenza infection (symptomatic/asymptomatic) will be estimated from the shivers serosurvey. additionally, we will also conduct severity assessment using true numbers of infections as the denominator to calculate case/fatality and case/hospitalization ratios. furthermore, the data on influenza disease burden will allow us to estimate direct medical costs and indirect societal costs (e.g. loss of productivity, loss of earning and loss of life) for the study population and subpopulations. 80, 81 aetiology preliminary results in 2013 identified an under-recognized burden of non-influenza respiratory viruses, particularly rsv and rhinoviruses, in sari and ili cases in nz as we have never had active population-based study on these viruses previously although substantial burden of rsv and rhinovirus has been described elsewhere. 82, 83 ili-associated consultations and sari-associated hospitalizations for rsv and rhinovirus show different socio-demographic patterns (age, ethnicity and ses) from that of influenza. for example, both ili-and sari-associated rsv incidences were similarhigh rates for very young (<1 year and 1-4 years) followed by elderly (≥65 years). this presented a very different agespecific incidence profile from that of influenza (indicated above). this result, together with subsequent multiyear surveillance data, may provide insights on differential effects of various respiratory viruses on the age distribution of the host and disease severity. shivers surveillance platforms provided a systematic opportunity to estimate ve for the prevention of general practice visits and hospitalizations for rt-pcr-confirmed influenza from the same population in the same influenza season. 40, 41 a case test-negative control design is used to estimate annual propensity-adjusted vaccine effectiveness in both hospital and community settings. the data in 2013 showed moderate effectiveness of influenza vaccine against medically attended and hospitalized influenza in nz with 56% (95% ci 34,70) against influenza presenting to general practice and 52% (95% ci 32,66) protection against laboratory-confirmed influenza hospitalization. 41 immune responses shivers surveillance platforms also provided sera and whole-blood samples during acute and convalescent phases of infection to study humoral and cellular immune responses from a subset of severe (n = 39) and moderate (n = 29) influenza cases in 2013. with these samples, and using a combination of serological and immunological assays, we were able to (a) estimate the relative contribution of early adaptive and cellular immune responses to disease severity; (b) identify differences in the immune profiles between these diseases groups; and (c) identify immunological correlates of disease severity in subpopulations. data acquired so far indicate that sari cases may experience a more robust immunologic response during infection (i.e. greater increases in ha-and na-specific antibody titres as well as magnitude of t-cell response). the ability to parse out immunological differences between severe and moderate influenza cases in this pilot cohort highlights the value of adding the active immunology study to the shivers platforms. as nz has a well-characterized socio-demographic distribution (age, sex, ethnicity, deprivation) from population census data, socio-demographic risk factors can be characterized quite easily. shivers will use the results obtained from hospital and sentinel general practice surveillance to disentangle the effect of ethnic and socio-economic gradients. for other more specific risk factors (e.g. host factors such as comorbidities, and environmental factors such as housing conditions), there are limited data available on their distribution in the population. consequently, it is difficult to assess the importance of the risk factor data collected. there are several comparison/control groups such as hospital-based control populations without respiratory illness, serosurvey participants as a control group and sari/ili testnegative controls. these controls could be compared with sari/ili cases to estimate the importance of specific risk factors for influenza infection and related severe/moderate diseases including socio-economic, underlying medical conditions, health intervention, health service utilization, and environmental and behavioural factors. we have established active, prospective, population-based surveillance systems for a wide range of respiratory disease presentations and designed a portfolio of influenza studies based on these platforms. the shivers results during the first 2 years (2012-2013) provided scientific evidence to support change to nz's vaccination policy for young children due to high sari-associated influenza hospitalizations in these children; contribute to the finalization of the world health organization's sari case definition for global influenza surveillance; and contribute in part to vaccine strain selection with vaccine effectiveness assessment in the prevention of general practice visits and hospitalizations for laboratoryconfirmed influenza. in the next 3 years (2014-2016), this project will continue to help us to understand (1) the burden of influenza infection including symptomatic/asymptomatic infection and mild disease not requiring medical consultation; (2) influenza risk that considers organism, host and environmental factors; (3) the impact of viral-viral and viral-bacteria co-infections on clinical disease and severity; and (4) the nature of responding adaptive immune responses in determining individual's risk of acquiring influenza virus infection. over 5 years, we hope this project will shed more light on the burden of influenza and other respiratory pathogens in our study population and subgroups and estimate key epidemiologic parameters such as relative rates of infection, clinical disease, general practice visits and hospitalizations as well as risk factors for illness, effectiveness of vaccination, mechanisms of immunity and monitoring for new influenza viruses with pandemic potential such as a(h7n9) and other emerging viruses (e.g. mers-cov) and provide a framework for timely assessment of severity which is essential in an event of emergence of these pathogens. the platforms established here are relevant not only for new zealand policy, but also for the region and the world. it will provide robust systematic virologic, epidemiologic and vaccine effectiveness data on circulating pandemic or seasonal influenza viruses at a time when circulation in the northern hemisphere is low. this could provide valuable information on all emergent respiratory pathogens that have some winter seasonality. moreover, the data elements on a range of disease severities collected in the same population at the same time will generate epidemiologic parameters that maybe broadly generalizable and translatable to similar developed, southern and northern temperate countries worldwide. this will help enormously to better understand more basic surveillance data and to extrapolate those data in models to plan and predict influenza behaviour, generate burden estimates, model the impact of seasonal influenza vaccination to support more global use and better prepare for the next pandemic. in summary, shivers is expected to provide extensive data to guide improved methods for disease surveillance; improve clinical case management, early detection and optimization of laboratory diagnosis; inform vaccine strain selection and vaccine development; guide targeted vaccination strategies for population and subgroups; understand host immune responses and identify better immune diagnostic markers. korrapadu, louise optland, cecilia dela cruz. special thanks to it staff and sari surveillance participants. also, a special thanks to dr dean erdman from gastroenteritis and respiratory viruses laboratory branch, the u.s. centers for disease control and prevention who provided the real-time pcr assay for non-influenza respiratory viruses. support in kind is provided by the nz ministry of health. report of the review committee on the functioning of the international health regulations (2005) in relation to pandemic (h1n1) pandemic preparedness and response-lessons from the h1n1 influenza of 2009 surveillance for the 2009 pandemic influenza a (h1n1) virus and seasonal influenza viruses -new zealand influenza surveillance and immunisation in new zealand influenza surveillance in new zealand in 2005 pandemic influenza a(h1n1)v in new zealand: the experience from increasing incidence of serious infectious diseases and inequalities in new zealand: a national epidemiological study incidence of influenza in healthy adults and healthcare workers: a systematic review and metaanalysis pandemic influenza a (h1n1) in pregnancy: a systematic review of the literature global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis background paper on influenza vaccines and immunization influenza-associated hospitalizations in the united states mortality associated with influenza and respiratory syncytial virus in the united states assessment of the burden of influenza in children influenza infection rates, measurement errors and the interpretation of paired serology comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study influenza-related deaths-available methods for estimating numbers and detecting patterns for seasonal and pandemic influenza in europe epidemiological characteristics of 2009 (h1n1) pandemic influenza based on paired sera from a longitudinal community cohort study human metapneumovirus in hospitalized children in amman, jordan viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection dual respiratory virus infections the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis severity of viral coinfection in hospitalized infants with respiratory syncytial virus infection simultaneous multiple viral infections in childhood acute lower respiratory tract infections in southern taiwan broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex rt-pcr dna microarray system pandemic (h1n1) 2009 and seasonal influenza a (h1n1) co-infection rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcription-pcr dna microarray systems multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children bacterial coinfection in influenza: a grand rounds review bacterial and viral infections associated with influenza influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses efficacy and effectiveness of influenza vaccines: a systematic review and metaanalysis new strategies are needed to improve the accuracy of influenza vaccine effectiveness estimates among seniors vaccines for preventing influenza in healthy adults the case test-negative design for studies of the effectiveness of influenza vaccine the test-negative design for estimating influenza vaccine effectiveness vaccine effectiveness in older individuals: what has been learned from the influenza-vaccine experience challenges in evaluating influenza vaccine effectiveness and the mortality benefits controversy the effectiveness of seasonal trivalent inactivated influenza vaccine in preventing laboratory confirmed influenza hospitalisations in auckland the effectiveness of seasonal trivalent inactivated influenza vaccine in preventing laboratory confirmed influenza hospitalisations and primary care visits in auckland risk factors and immunity in a nationally representative population following the 2009 influenza a(h1n1) pandemic severe pandemic 2009 h1n1 influenza disease due to pathogenic immune complexes the lower serum immunoglobulin g2 level in severe cases than in mild cases of pandemic h1n1 2009 influenza is associated with cytokine dysregulation association between severe pandemic 2009 influenza a (h1n1) virus infection and immunoglobulin g(2) subclass deficiency host adaptive immunity deficiency in severe pandemic influenza the role of polymorphisms in host immune genes in determining the severity of respiratory illness caused by pandemic h1n1 influenza populations at risk for severe or complicated influenza illness: systematic review and meta-analysis the second wave of 2009 pandemic influenza a(h1n1) in new zealand a novel risk factor for a novel virus: obesity and 2009 pandemic influenza a (h1n1) critical care services and 2009 h1n1 influenza in australia and new zealand risk factors and outcomes among children admitted to hospital with pandemic h1n1 influenza pandemic influenza a (h1n1) in pregnancy: a systematic review of the literature household crowding a major risk factor for epidemic meningococcal disease in auckland children acute rheumatic fever associated with household crowding in a developed country tuberculosis associated with household crowding in a developed country household transmission of the 2009 pandemic a/h1n1 influenza virus: elevated laboratory-confirmed secondary attack rates and evidence of asymptomatic infections comparative epidemiology of pandemic and seasonal influenza a in households impact of pollution, climate, and sociodemographic factors on spatiotemporal dynamics of seasonal respiratory viruses implementing hospitalbased surveillance for severe acute respiratory infections (sari) caused by influenza and other respiratory pathogens in new zealand interim global epidemiological surveillance standards for influenza generic protocol for influenza surveillance global epidemiological surveillance standards for influenza who regional office for europe guidance for influenza surveillance in humans validity of clinical case definitions for influenza surveillance among hospitalized patients: results from a rural community in north india what are the most sensitive and specific sign and symptom combinations for influenza in patients hospitalized with acute respiratory illness? results from western kenya lessons from a one-year hospital-based surveillance of acute respiratory infections in influenza study in southern hemisphere ª 2015 the authors. influenza and other respiratory viruses published by berlin-comparing case definitions to monitor influenza incidence of influenza-like illness and severe acute respiratory infection during three influenza seasons in bangladesh estimation of the national disease burden of influenza-associated severe acute respiratory illness in kenya and guatemala: a novel methodology the cost of influenza in thailand the substantial hospitalization burden of influenza in central china: surveillance for severe, acute respiratory infection, and influenza viruses the underrecognized burden of influenza in young children burden of seasonal influenza hospitalization in children adult hospitalizations for laboratory-positive influenza during the virologically confirmed populationbased burden of hospitalization caused by influenza a and b among children in hong kong high influenza hospitalisations lead to extension of free influenza vaccination to young children estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study influenza-associated primary care consultations and hospitalisations show contrasting socio-demorgraphic patterns medical and economic burden of influenza in the elderly population in central and eastern european countries influenza cost and cost-effectiveness studies globally-a review disease burden of the most commonly detected respiratory viruses in hospitalized patients calculated using the disability adjusted life year (daly) model acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden the shivers investigation team (listed in an alphabetic order) the shivers project is funded by us cdc (1u01ip000480-01). the project is a 5-year research cooperative agreement between the authors declare that they have no competing interests. key: cord-335948-qkfxfmxb authors: ampofo, william k.; baylor, norman; cobey, sarah; cox, nancy j.; daves, sharon; edwards, steven; ferguson, neil; grohmann, gary; hay, alan; katz, jacqueline; kullabutr, kornnika; lambert, linda; levandowski, roland; mishra, a. c.; monto, arnold; siqueira, marilda; tashiro, masato; waddell, anthony l.; wairagkar, niteen; wood, john; zambon, maria; zhang, wenqing title: improving influenza vaccine virus selectionreport of a who informal consultation held at who headquarters, geneva, switzerland, 14–16 june 2010 date: 2011-08-08 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2011.00277.x sha: doc_id: 335948 cord_uid: qkfxfmxb • for almost 60 years, the who global influenza surveillance and response system (gisrs) has been the key player in monitoring the evolution and spread of influenza viruses and recommending the strains to be used in human influenza vaccines. the gisrs has also worked to continually monitor and assess the risk posed by potential pandemic viruses and to guide appropriate public health responses. • the expanded and enhanced role of the gisrs following the adoption of the international health regulations (2005), recognition of the continuing threat posed by avian h5n1 and the aftermath of the 2009 h1n1 pandemic provide an opportune time to critically review the process by which influenza vaccine viruses are selected. in addition to identifying potential areas for improvement, such a review will also help to promote greater appreciation by the wider influenza and policy‐making community of the complexity of influenza vaccine virus selection. • the selection process is highly coordinated and involves continual year‐round integration of virological data and epidemiological information by national influenza centres (nics), thorough antigenic and genetic characterization of viruses by who collaborating centres (whoccs) as part of selecting suitable candidate vaccine viruses, and the preparation of suitable reassortants and corresponding reagents for vaccine standardization by who essential regulatory laboratories (erls). • ensuring the optimal effectiveness of vaccines has been assisted in recent years by advances in molecular diagnosis and the availability of more extensive genetic sequence data. however, there remain a number of challenging constraints including variations in the assays used, the possibility of complications resulting from non‐antigenic changes, the limited availability of suitable vaccine viruses and the requirement for recommendations to be made up to a year in advance of the peak of influenza season because of production constraints. • effective collaboration and coordination between human and animal influenza networks is increasingly recognized as an essential requirement for the improved integration of data on animal and human viruses, the identification of unusual influenza a viruses infecting human, the evaluation of pandemic risk and the selection of candidate viruses for pandemic vaccines. • training workshops, assessments and donations have led to significant increases in trained laboratory personnel and equipment with resulting expansion in both geographical surveillance coverage and in the capacities of nics and other laboratories. this has resulted in a significant increase in the volume of information reported to who on the spread, intensity and impact of influenza. in addition, initiatives such as the who shipment fund project have facilitated the timely sharing of clinical specimens and virus isolates and contributed to a more comprehensive understanding of the global distribution and temporal circulation of different viruses. it will be important to sustain and build upon the gains made in these and other areas. • although the haemagglutination inhibition (hai) assay is likely to remain the assay of choice for the antigenic characterization of viruses in the foreseeable future, alternative assays – for example based upon advanced recombinant dna and protein technologies – may be more adaptable to automation. other technologies such as microtitre neuraminidase inhibition assays may also have significant implications for both vaccine virus selection and vaccine development. • microneutralization assays provide an important adjunct to the hai assay in virus antigenic characterization. improvements in the use and potential automation of such assays should facilitate large‐scale serological studies, while other advanced techniques such as epitope mapping should allow for a more accurate assessment of the quality of a protective immune response and aid the development of additional criteria for measuring immunity. • standardized seroepidemiological surveys to assess the impact of influenza in a population could help to establish well‐characterized banks of age‐stratified representative sera as a national, regional and global resource, while providing direct evidence of the specific benefits of vaccination. • advances in high‐throughput genetic sequencing coupled with advanced bioinformatics tools, together with more x‐ray crystallographic data, should accelerate understanding of the genetic and phenotypic changes that underlie virus evolution and more specifically help to predict the influence of amino acid changes on virus antigenicity. • complex mathematical modelling techniques are increasingly being used to gain insights into the evolution and epidemiology of influenza viruses. however, their value in predicting the timing and nature of future antigenic and genetic changes is likely to be limited at present. the application of simpler non‐mechanistic statistical algorithms, such as those already used as the basis of antigenic cartography, and phylogenetic modelling are more likely to be useful in facilitating vaccine virus selection and in aiding assessment of the pandemic potential of avian and other animal influenza viruses. • the adoption of alternative vaccine technologies – such as live‐attenuated, quadrivalent or non‐ha‐based vaccines – has significant implications for vaccine virus selection, as well as for vaccine regulatory and manufacturing processes. recent collaboration between the gisrs and vaccine manufacturers has resulted in the increased availability of egg isolates and high‐growth reassortants for vaccine production, the development of qualified cell cultures and the investigation of alternative methods of vaccine potency testing. who will continue to support these and other efforts to increase the reliability and timeliness of the global influenza vaccine supply. • the who gisrs and its partners are continually working to identify improvements, harness new technologies and strengthen and sustain collaboration. who will continue in its central role of coordinating worldwide expertise to meet the increasing public health need for influenza vaccines and will support efforts to improve the vaccine virus selection process, including through the convening of periodic international consultations. • for almost 60 years, the who global influenza surveillance and response system (gisrs) has been the key player in monitoring the evolution and spread of influenza viruses and recommending the strains to be used in human influenza vaccines. the gisrs has also worked to continually monitor and assess the risk posed by potential pandemic viruses and to guide appropriate public health responses. • the expanded and enhanced role of the gisrs following the adoption of the international health regulations (2005) , recognition of the continuing threat posed by avian h5n1 and the aftermath of the 2009 h1n1 pandemic provide an opportune time to critically review the process by which influenza vaccine viruses are selected. in addition to identifying potential areas for improvement, such a review will also help to promote greater appreciation by the wider influenza and policy-making community of the complexity of influenza vaccine virus selection. • the selection process is highly coordinated and involves continual year-round integration of virological data and epidemiological information by national influenza centres (nics), thorough antigenic and genetic characterization of viruses by who collaborating centres (whoccs) as part of selecting suitable candidate vaccine viruses, and the preparation of suitable reassortants and corresponding reagents for vaccine standardization by who essential regulatory laboratories (erls). • ensuring the optimal effectiveness of vaccines has been assisted in recent years by advances in molecular diagnosis and the availability of more extensive genetic sequence data. however, there remain a number of challenging constraints including variations in the assays used, the possibility of complications resulting from non-antigenic changes, the limited availability of suitable vaccine viruses and the requirement for recommendations to be made up to a year in advance of the peak of influenza season because of production constraints. • effective collaboration and coordination between human and animal influenza networks is increasingly recognized as an essential requirement for the improved integration of data on animal and human viruses, the identification of unusual influenza a viruses infecting human, the evaluation of pandemic risk and the selection of candidate viruses for pandemic vaccines. • training workshops, assessments and donations have led to significant increases in trained laboratory personnel and equipment with resulting expansion in both geographical surveillance coverage and in the capacities of nics and other laboratories. this has resulted in a significant increase in the volume of information reported to who on the spread, intensity and impact of influenza. in addition, initiatives such as the who shipment fund project have facilitated the timely sharing of clinical specimens and virus isolates and contributed to a more comprehensive understanding of the global distribution and temporal circulation of different viruses. it will be important to sustain and build upon the gains made in these and other areas. • although the haemagglutination inhibition (hai) assay is likely to remain the assay of choice for the antigenic characterization of viruses in the foreseeable future, alternative assays -for example based upon advanced recombinant dna and protein technologies -may be more adaptable to automation. other technologies such as microtitre neuraminidase inhibition assays may also have significant implications for both vaccine virus selection and vaccine development. • microneutralization assays provide an important adjunct to the hai assay in virus antigenic characterization. improvements in the use and potential automation of such assays should facilitate large-scale serological studies, while other advanced techniques such as epitope mapping should allow for a more accurate assessment of the quality of a protective immune response and aid the development of additional criteria for measuring immunity. • standardized seroepidemiological surveys to assess the impact of influenza in a population could help to establish well-characterized banks of age-stratified representative sera as a national, regional and global resource, while providing direct evidence of the specific benefits of vaccination. • advances in high-throughput genetic sequencing coupled with advanced bioinformatics tools, together with more x-ray crystallographic data, should accelerate understanding of the genetic and phenotypic changes that underlie virus evolution and more specifically help to predict the influence of amino acid changes on virus antigenicity. • complex mathematical modelling techniques are increasingly being used to gain insights into the evolution and epidemiology of influenza viruses. however, their value in predicting the timing and nature of future antigenic and genetic changes is likely to be limited at present. the application of simpler non-mechanistic statistical algorithms, such as those already used as the basis of antigenic cartography, and phylogenetic modelling are more likely to be useful in facilitating vaccine virus selection and in aiding assessment of the pandemic potential of avian and other animal influenza viruses. • the adoption of alternative vaccine technologies -such as live-attenuated, quadrivalent or non-ha-based vaccines -has significant implications for vaccine virus selection, as well as for vaccine regulatory and manufacturing processes. recent collaboration between the gisrs and vaccine manufacturers has resulted in the increased availability of egg isolates and high-growth reassortants for vaccine production, the development of qualified cell cultures and the investigation of alternative methods of vaccine potency testing. who will continue to support these and other efforts to increase the reliability and timeliness of the global influenza vaccine supply. • the who gisrs and its partners are continually working to identify improvements, harness new technologies and strengthen and sustain collaboration. who will continue in its central role of coordinating worldwide expertise to meet the increasing public health need for influenza vaccines and will support efforts to improve the vaccine virus selection process, including through the convening of periodic international consultations. the historic initiative to establish a global network to detect and identify new and potentially dangerous influenza viruses predates the adoption of the who constitution in 1948. with memories of the 1918-1919 influenza pandemic still vivid, and the ever-evolving threat posed by influenza recognized, the who global influenza surveillance and response system (gisrs) was formally established in 1952. influenza thus became one of the first diseases to highlight the importance of international monitoring and collaboration in protecting human health. following the re-emergence of human cases of highly pathogenic avian h5n1 influenza in 2003 and the adoption of the international health regulations (2005), the gisrs was strengthened and its role in protecting public health enhanced. in addition to tracking the course and impact of annual influenza epidemics and monitoring the evolution of seasonal influenza viruses, the gisrs also acts as a global alert mechanism for the emergence of influenza viruses with the potential to cause a human pandemic. the network provides support to both seasonal and pandemic influenza preparedness and response activities in areas such as diagnostics, vaccine development, virological surveillance and risk assessment. it also acts as the focus of who efforts to former who global influenza surveillance network (gisn), which has been renamed as who global influenza surveillance and response system (gisrs) since 24 may 2011, when the world health assembly resolution wha 64.5 was adopted. assist member states in strengthening their national capacity for the surveillance, diagnosis, characterization and sharing of influenza viruses. as a key player in global influenza risk assessment and response, the gisrs continues to evolve and expand, and as of december 2010 consisted of 135 national influenza centres (nics) in 105 countries, six who collaborating centres (whoccs), 11 who h5 reference laboratories and four who essential regulatory laboratories (erls). the gisrs also works to ensure the successful coordination of who activities with those of external agencies such as the global outbreak alert and response network (goarn), national regulatory authorities, academic and veterinary institutes, and the pharmaceutical industry. the first formal who recommendations on influenza vaccine composition were issued in 1971. since 1998, separate and appropriately timed recommendations for the northern and southern hemispheres have been issued each year in february and september, respectively. these biannual recommendations are based upon the virological and epidemiological information generated by the gisrs and play a crucial role in the development, production and availability of effective influenza vaccines. the continuing threat posed by avian h5n1, the aftermath of the 2009 h1n1 pandemic, the increased knowledge of influenza, and the development and availability of new technologies provide a timely opportunity to review the complex processes and issues involved in influenza vaccine virus selection and to identify potential areas for improvement. this who informal consultation represents the latest step in an ongoing process of gisrs strengthening and was convened with the following objectives: • to review the current vaccine virus selection process, including its constraints and limitations; • to identify opportunities for improving influenza surveillance and representative virus sharing; • to assess the potential for improving the assays and technologies used for vaccine virus selection; and • to assess the potential impact of new vaccine technologies on the vaccine virus selection process. participants were drawn from a broad and highly diverse range of institutes and sectors including the following: whoccs, nics, who erls, who h5 reference laboratories, national regulatory authorities, public health agencies, academia, influenza vaccine manufacturers, and veterinary laboratories and organizations. the primary goal of the gisrs vaccine virus selection process (annex 1) is to generate and analyse the data needed to recommend the influenza vaccine viruses that will most closely match the influenza viruses likely to be circulating during forthcoming influenza seasons. current vaccine technologies and production schedules mean that decisions on vaccine composition have to be made almost a full year in advance of the peak of seasonal influenza activity. as a result, the process relies upon the earliest possible detection of emerging antigenic variants and the most up-to-date information on their potential future epidemiological significance. information must therefore be collected year round on the continuous evolution and global circulation of human influenza viruses to provide a sound basis for the biannual who recommendations on the composition of influenza vaccines for use in the northern and southern hemispheres. for countries in equatorial regions, epidemiological considerations influence which recommendation (february or september) individual national and regional authorities consider more appropriate. national influenza centres (nics) play a vital role in this complex process. their core activities include collating epidemiological information, diagnosing cases of influenza a and b infection, and identifying the subtype or lineage of the viruses responsible. the primarily molecular diagnosis of infection using rt-pcr techniques is based upon standardized primers and probes provided by the gisrs. viruses must also be isolated to allow their antigenic identification using the type-and subtype-specific reference reagents provided in annually distributed who kits. further detailed characterization may include sequence analyses to monitor genetic changes and assessment of virological traits such as resistance to antiviral drugs. sequence data are shared within the gisrs using public databases such as genbank and gi-said epiflu. nics in some settings then attempt to relate potentially important virological changes observed with clinical and epidemiological information and trends and may even conduct serological studies to evaluate the immune status of the population. weekly reports on the virological characteristics and epidemiology of circulating viruses are submitted to the who flunet -an internet-based data-query and reporting tool. information on the virus subtypes and lineages is collated, together with observations of potential clinical or epidemiological importance, and regular summaries of the geographical spread, intensity and impact of influenza are produced by who. if human infection with an avian or other animal influenza virus is suspected, a suitably equipped nic or other national influenza reference laboratory can conduct preliminary diagnostic testing using rt-pcr protocols and ⁄ or reagents for h5, h7 and h9 subtypes provided by who. such rt-pcr testing does not require high-level biocontainment facilities. however, it is expected that the detection of any unusual influenza a virus distinct from known circulating viruses, especially one suspected to be of animal origin or unsubtypable using current who reagents, will immediately be reported to who and collaboration urgently initiated with a who collaborating centre (whocc). if the required laboratory biosafety facilities and procedures are not available, then virus isolation should not be attempted in the national laboratory and the sample should be promptly sent to a whocc. the routine and timely sharing of representative circulating influenza viruses and unusual viruses with a whocc is an essential step in the vaccine virus selection process. the criteria for forwarding viruses include their temporal, geographical and age-group distribution, severity of cases and virological characteristics such as unidentified subtype and antiviral drug resistance. whoccs are then responsible for the systematic antigenic characterization of the thousands of viruses forwarded each year by nics and other laboratories, and for the detailed genetic characterization of a selected subset. such detailed antigenic and genetic characterization is a necessary step in monitoring virus evolution and detecting any distinct antigenic variants that may necessitate updating the seasonal vaccine composition. the process also allows for the identification and characterization of animal viruses causing sporadic human infections, assessment of the risk they pose and the potential development of candidate vaccine viruses as part of pandemic preparedness. of prime importance in immunity to influenza is the production of antibodies to the virus haemagglutinin (ha) protein. such antibodies can neutralize the infectivity of viruses, and their level in the blood has been shown to correlate with the level of protection against infection with a homologous virus. as a result, influenza vaccine virus selection has primarily been based upon the antigenic characterization of virus ha using the haemagglutination inhibition (hai) assay. hai tests provide a visual readout of the ability of specific antibodies to prevent the attachment of ha to red blood cells (rbcs) and thus prevent their agglutination. antigenic drift in the ha of circulating viruses in response to host immunity reduces the effectiveness of vaccines and is therefore the major consideration when recommendations are made on the composition of influenza vaccines. the hai test is likely to remain the assay of choice for the antigenic characterization of virus ha for the foreseeable future. strain-specific antisera are produced by infecting previously unexposed ('naive') ferrets with either vaccine viruses, reference viruses representative of circulating viruses or viruses that appear in hai tests to be potential antigenic variants. the resulting sets of reference viruses and antisera are then used to evaluate the antigenic characteristics of the has of recent isolates. where antigenic differences are detected, these are likely to affect human immunity against the new variants. the hai test is a surrogate for the more complicated and time-consuming virus neutralization assay used to clarify antigenic relationships when observed variations in hai titre reflect, for example, changes in receptor binding rather than differences in antigenicity. a subset of between 10% and 20% of all viruses received is selected for genetic sequencing and more detailed analysis -principally of their ha and na components. this subset is selected to include representative circulating viruses, as well as apparent antigenic variants and viruses from severe or fatal cases. phylogenetic analyses are carried out to better understand the evolution of circulating viruses, their degree of genetic heterogeneity and the emergence of new genetic clades. antigenic or other phenotypic variants may thus be defined in terms of separate genetic clades with distinct amino acid signatures. relating the locations of amino acid substitutions to antigenic, receptor-binding or glycosylation sites on the 3d structure of the ha molecule then helps to identify the individual substitutions associated with phenotypic (antigenic) changes. identifying such amino acid signatures also facilitates global monitoring of the emergence, distribution and impact of different genetic variants. this is particularly helpful when data on emergent variants are limited at the time of a who vaccine consultation. comparisons of the sequences found in clinical specimens and virus isolates are also useful in revealing amino acid substitutions which result from passage in different substrates, mainly mdck cells and eggs. up-todate sequence data are shared within gisrs and made publically available via the gisaid epiflu database. complete genome sequencing is necessary to identify animal (including avian) viruses causing human infection and is important in detecting the emergence of reassortant viruses among co-circulating human viruses or between human and animal viruses. whoccs maintain panels of reference reagents for all influenza a subtypes. these include h5 (especially h5n1), h9 and h7 avian viruses and various h1n1 and h3n2 swine viruses, as well as viruses present in other animals such as horses and dogs. whoccs also collaborate with the who erls in serological studies of representative human sera from previously vaccinated individuals. sera are provided by vaccine manufacturers and are used in hai tests to assess whether or not the antibodies induced by current vaccines are likely to be effective against currently circulating viruses. the results the principal criteria used to decide whether or not to recommend changes to influenza vaccine components include: • the emergence of an antigenically and genetically distinct variant among circulating viruses (including a novel influenza a virus with the potential to cause a pandemic); • evidence of the geographical spread of such a distinct variant and its association with outbreaks of disease, indicating its future epidemiological significance; • the reduced ability of existing vaccine-induced antibodies to neutralize the emergent variant; and • the availability of suitable candidate vaccine viruses. to facilitate collaborative studies by the whoccs and who erls and ensure that appropriate potential candidate vaccine viruses are identified in advance of the who vaccine composition consultation, the most recent virological and epidemiological data are shared and discussed via teleconferences held 6 and 2 weeks before the who consultation. a summary of each teleconference is promptly distributed to keep all nics and vaccine manufacturers informed of the developing situation. in addition, potential candidate vaccine viruses are provided to manufacturers. during the formal biannual consultations, the technical advisory group considers the cumulative antigenic and genetic data on the viruses characterized by whoccs. the data are set against the broader epidemiological context collated by who and are supported by serological data from whoccs and who erls, as well as by additional information provided by nics. hai data obtained in the different centres using a wide variety of reference viruses and ferret antisera are correlated using common reference reagents. in recent years, antigenic cartography has been used to collate and statistically visualize the degree of antigenic variation. the interpretation of hai data may, however, be complicated by the influence of changes in the receptor-binding properties of natural viruses or by the selection of variants during isolation and passaging in different cell or egg substrates. comparisons with sequence data are made to relate any differences in antigenicity with specific ha genetic clades and to more precisely define the identity of antigenic variants. the results of virus neutralization tests, which usually correspond to those of hai tests, are used to clarify the true antigenic relationships between different viruses. if the antigenic data, supported by genetic and serological data, indicate that a new antigenic variant is spreading globally, then a change in that component of the seasonal vaccine is considered to be warranted. the implementation of a rec-ommendation to update a vaccine component is, however, contingent upon the availability of suitable vaccine viruses. only after all the factors have been taken into account is a decision taken on whether or not to recommend a change in influenza vaccine virus composition. the decision is announced at an information meeting immediately following each who consultation and published on the who web site and in the who weekly epidemiological record. since the re-emergence of human cases of highly pathogenic h5n1 avian influenza in 2003, who has also regularly reviewed the available antigenic and genetic data on human and avian viruses in relation to the epidemiology of h5n1 influenza among birds. to support the development of safe and effective human h5n1 vaccines, who has coordinated the development of a number of candidate attenuated vaccine viruses (annex 1) and made them available to vaccine producers. clinical trials have been conducted to evaluate the immunogenicity of different h5n1 vaccine formulations and the breadth of antibody responses elicited. in addition, as part of pandemic preparedness, who has coordinated the ongoing development and updating of an inventory of h2, h7 and h9 candidate vaccine viruses. important constraints on the vaccine virus selection process include the tight timelines involved (annex 1), particularly in the northern hemisphere, where since recent years seasonal influenza activity tends to start increasing in middle or late january in general. as a consequence, decisions often have to be made relatively early in the influenza season. in addition, post-infection ferret antisera against potential antigenic variants are urgently required to define their antigenic relationships to previously circulating viruses. panels of recent isolates must also be prepared to assess the degree to which they are neutralized by antibodies in the sera of previously vaccinated individuals. finally, potential new candidate vaccine viruses must be prepared and evaluated for their suitability in vaccine production. ensuring the timely availability of viruses with suitable growth properties is a crucial step in ensuring that sufficient quantities of vaccine can be produced in time for administration prior to the next influenza season. although cell culture has steadily replaced the use of embryonated eggs for the primary isolation of viruses, candidate vaccine viruses must still be isolated directly in eggs according to current regulatory requirements. the limited availability of egg isolates, particularly of recent h3n2 viruses which generally grow poorly in eggs, has led to the establishment of cooperative research and development agreements (cra-das) and similar agreements between the vaccine industry and a number of whoccs to increase the availability of egg isolates for vaccine use. the gisrs vaccine virus selection process necessarily involves a series of collaborative steps, including the selection of prototype antigenic variants and suitable vaccine viruses, and the provision of standardizing reagents by the who erls. the process thus impacts directly upon the subsequent authorizing of vaccine composition by national and regional regulatory authorities and upon the large-scale production of vaccine by manufacturers. mismatches have occasionally occurred as a result of the emergence of variant strains shortly after the recommendations have been made, highlighting one of the unavoidable consequences of current vaccine development and production constraints. nevertheless, retrospective studies have shown that with very few exceptions who vaccine virus recommendations have closely matched the influenza viruses that have circulated during the following influenza season. in addition, following the out-of-season emergence of the pandemic a(h1n1) 2009 virus, this closely integrated system demonstrated its unique ability to very rapidly orchestrate the development and provision of appropriate (suitably attenuated) candidate vaccine viruses for pandemic vaccine production. global influenza surveillance has always presented a major challenge as it is a highly demanding public health need with a significantly uneven distribution of surveillance capacity worldwide. since the outbreak of severe acute respiratory syndrome (sars) in 2003, the re-emergence of h5n1 infection in humans and the 2009 h1n1 pandemic, it has become ever clearer that surveillance and the prompt sharing of viruses and information are central to the broad range of influenza preparedness and response activities. although the known impact and the awareness of seasonal influenza vary in different parts of the world, the threat posed by avian h5n1 viruses has galvanized influenza surveillance efforts in all countries. improving surveillance and acquiring the capacity to detect and report unusual cases of influenza are essential components of global pandemic planning and are enshrined in the international health regulations (2005) . successful efforts to increase the capacity of nics and other laboratories have been made, and in a number of settings the development, revision and adoption of guidelines on strengthened national, regional and global surveillance and collaboration is under way. global influenza surveillance has also been strengthened through expanded geographical coverage and the collection of more data of better quality. for example, in africa there are now 25 influenza laboratories in 21 countries, including 12 recognized nics, almost all of which have the capacity to conduct rt-pcr diagnosis of influenza infection. in less than two years, the percentage of african countries with an nic increased from 17% to 26% with the number of countries with no influenza laboratory markedly decreasing. global, regional and national training workshops, assessments and donations have all led to significant increases in trained personnel, equipment procurement and laboratory capacity, resulting in the increasingly widespread use of molecular techniques such as real-time rt-pcr and genetic sequencing. recent who capacity-building activities have included bsl-3 training courses for nics to promote safe practices when working with highly pathogenic influenza viruses, and courses on virus isolation, gene sequencing and antiviral resistance detection. increased participation in both internal and external quality assurance programmes such as the who external quality assessment project (eqap) has contributed to marked improvements in laboratory proficiency. these and other efforts enabled a more effective response to the emergence of the 2009 h1n1 pandemic in many countries. however, the pandemic also revealed significant limitations in the analysis and integration of epidemiological and virological surveillance data. in addition, few early seroprevalence surveys were conducted to allow for the timely assessment of the extent and impact of the pandemic. the pandemic also revealed significant gaps in laboratory infrastructure and personnel, equipment procurement and funding, particularly in developing countries. improvements and training in areas such as web-based integration and analyses of clinical, epidemiological and virological data are being implemented but care must be taken to ensure that such activities are not conducted at the expense of detection, characterization and virus-sharing activities in less well-resourced settings. identified research priorities in influenza surveillance and response include evaluation of the temporal and geographical circulation of influenza viruses and of the burden of influenza. in all settings, establishing a sound evidence base will support the development or updating of national, regional and global policies, plans and guidelines. this in turn could lead to greater acceptance of the use of influenza vaccines, particularly seasonal vaccines, and assist in the development of vaccination policies. the primary requirement of nics will remain the prompt diagnosis of influenza infection and the timely sharing of clinical specimens and virus isolates -especially those obtained from unusual, severe or fatal cases -backed up by appropriate epidemiological and clinical information. procedures should be in place to ensure that the increasingly predominant use of molecular diagnostic techniques, particularly real-time rt-pcr, does not adversely affect the timely isolation and for-improving influenza vaccine virus selection ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 6, 147-152 warding of viruses. improved communication between nics and whoccs on how best to facilitate prompt virus sharing, including discussion of the constraints faced, could improve coordination and avoid potential delays. a more systematic approach to engaging nic information and expertise would also lead to significant benefits. such an approach is likely to be facilitated by a number of developments in the use of who web-based tools. for example, nics with enhanced capabilities currently strengthen the collaborative characterization of viruses and aid early assessment of the significance of genetic and antigenic changes by sharing detailed virological information (especially ha sequences) on selected viruses, either directly or via public databases. as technologies advance, national patterns of seropositivity to circulating influenza viruses may also become available on a more timely basis and could thus guide vaccine use. this is particularly important given the increasing emphasis now placed on assessing vaccine effectiveness. comprehensive nic summary reports forwarded just prior to each who consultation also provide highly beneficial additional data to inform who recommendations on vaccine composition. to overcome logistical and other obstacles to the safe and efficient shipping of clinical specimens and virus isolates to whoccs, a who shipment fund project was established. the project provides support to nics and other influenza laboratories in all countries by arranging the transport of specimens and isolates along a guaranteed cold chain, especially in settings where there are severe financial and infrastructural constraints. as a direct result of the project, and associated 'infectious substances shipping' workshops conducted in all who regions, there has been a significant increase in the number of countries sharing specimens and isolates, especially following the outbreak of the 2009 h1n1 pandemic. furthermore, the expansion and harmonization of the information currently provided in the accompanying standard shipping form to include information such as clinical outcome, patient vaccination status or recent travel history would greatly enhance understanding of the epidemiological context associated with the spread of viruses. a better understanding of the diversity and evolution of animal influenza viruses is essential for evaluating the pandemic risk posed by subtypes currently causing sporadic human infections (such as h5n1 and h9n2) and informing the selection of candidate vaccine viruses. the emergence of h5n1 in particular led to the establishment in 2005 of the oie-fao network of expertise on animal influenza (of-flu) -a worldwide network of approximately 20 laboratories and institutions that coordinates the global surveillance of animal influenza. a number of joint who-offlu tech-nical initiatives on influenza at the human-animal interface have been conducted (including successful collaboration during the 2009 h1n1 pandemic) and reciprocal participation in annual meetings has taken place. there remains, however, considerable scope for improved coordination and collaboration with the animal influenza surveillance sector, especially in the collection and analysis of antigenic and genetic data, the timely exchange of representative viruses and reference reagents, and the conducting of serological studies of human exposure to zoonotic infection. influenza is an important disease of many avian and mammalian species with serious economic consequences for livestock industries and has potential adverse impacts on human food supplies. despite this, animal influenza surveillance coverage is limited with a shortage of epidemiological data on the circulation of various viruses in different countries. efforts are now under way to establish triggers for initiating enhanced surveillance that go beyond animal disease notification and sporadic human infections. although there is increasing understanding of the interrelationships between animal and human influenza and the need for 'integrated' surveillance, full collaboration at both national and global levels is currently constrained by a number of practical, funding, regulatory and policy issues. maintaining a regular dialogue based upon the mutual interests of the different networks will be an important public health activity and may also help to enhance the sustainability of animal influenza surveillance in particular settings. a more formal collaborative mechanism might allow for the improved integration of animal virus data into the who candidate vaccine virus selection process. increased awareness of the content and extent of use of animal influenza vaccines would also aid understanding of their impact on virus evolution. a range of laboratory assays and other techniques provide the complementary information on changes in the antigenic and genetic characteristics of influenza viruses needed to select the most appropriate influenza vaccine viruses. however, inherent limitations in the biological assays used and significant variations in the results obtained by different laboratories complicate the collation and definitive interpretation of data. because the hai test outlined previously is a simple, rapid and reproducible surrogate assay for virus neutralization, it is widely used to measure the antigenic relationships between different viruses as well as antibody responses to infection or vaccination. in addition, the test provides the basis of the only current quantitative correlate of protection against infection (serum hai antibody titre ‡40) used to standardize inactivated vaccines. however, variations in the physical characteristics of rbcs obtained from different species and differences in the receptor-binding properties of different viruses influence both the sensitivity and the utility of the assay. furthermore, changes in receptor-binding affinity or specificity associated with adaptation, antigenic drift or the isolation and passage of viruses in eggs and cell culture may also affect hai titres. standardization between laboratories has also proved difficult, and the assay is currently not suitable for use in a fully automated system. a range of practical refinements such as attempts to develop 'synthetic' rbcs (for example using glycan-coated beads) have been unsuccessful. given the currently limited knowledge of the principal natural receptors for influenza viruses, such approaches are unlikely to circumvent the virus-dependent shortcomings of assays based upon natural rbcs which are therefore likely to remain the primary approach to antigenic characterization for the foreseeable future. recent developments based on the use of panels of recombinant ha do offer alternative or supplementary microtitre or microarray binding-assay formats for assessing antibody specificity and antibody inhibition of the ha-glycan receptor interaction. although such approaches are relatively expensive and require a high degree of skill to implement, they are potentially highly suited to automation and in time may reduce the need for virus isolates. in addition, such formats can readily be adapted to incorporate biosensor technologies to provide more quantitative analyses of binding characteristics. a number of such assays are currently being validated using ferret and human antisera. the contribution of antibodies against virus na in conferring protection following natural infection or vaccination is still not well understood. studies of na antigenic variation have been limited, and the na content of influenza vaccines is not currently standardized. although neuraminidase inhibition (nai) assays were conducted more routinely in the past, these were cumbersome to perform and were complicated by the relatively low levels of antibodies against na in post-infection ferret sera and by interference from antibodies against ha. a number of different nai microtitre assay formats have recently been developed. these have been used to correlate antigenic changes with sequence variations in the na component, provide more precise information on the evolution of na and assess na antibody responses following vaccination. improved understanding of antigenic drift in na and of the role of anti-na antibodies in conferring immunity might have significant implications for both vaccine virus selection and vaccine development. microneutralization (mn) assays -based on measuring virus replication, cell viability or na activity -provide an important adjunct to hai tests in antigenic characterization. mn assays are generally more sensitive and measure a broader repertoire of functional antibodies that neutralize viral replication, with potential advantages in the evaluation of human serological responses. in addition, comparisons of mn and hai tests for measuring antibody responses in vaccinated individuals have shown a consistent degree of correlation and have confirmed the utility of mn assays in analyses of human antibody responses to h3 vaccine components. techniques for simplifying assay formats and making them more readily applicable to the routine testing of low-titre viruses are under investigation, and efforts are under way to use mn assays for h1 and b viruses. this should facilitate the use of mn assays to overcome the variable nature of interactions between viruses and rbcs, and hence in interpreting 'anomalous' hai results which complicate vaccine virus selection. pseudotype virus neutralization assays may also offer some advantages in scale and standardization over conventional mn assays for measuring serological responses to particular viruses, especially highly pathogenic viruses. furthermore, ongoing improvements in automation will potentially enable the more labour-intensive mn assay to be applied to large-scale serological analysis. epitope mapping using genome fragment phage display libraries provides another powerful technique for further dissecting the fine specificity of antibody responses to vaccination and infection and should allow for a better assessment of the quality of a 'protective' immune response and aid the development of additional correlates of immunity. to encourage the performance of seroepidemiological surveys to assess the impact of influenza in a population, countries should be supported in establishing well-characterized serum banks of age-stratified representative sera as a national, regional and global resource. current advantages of the gisrs serological activities undertaken in support of vaccine virus selection include the use of shared serum panels and common antigens, with frequent consensus obtained from participating whoccs and who erls. limitations include the large variability of hai data, a requirement for antibody standards and a need for mn or other assays to resolve inconsistencies. the availability of antibody standards would not only enhance the comparability of serological data generated in different laboratories and countries but also facilitate the comparison of antibody responses to different vaccines. increasing attention to influenza vaccine effectiveness studies will lead to the availability of more real-time data for comparing clinical benefit with the degree of antigenic improving influenza vaccine virus selection ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 6, 147-152 relatedness of vaccine and circulating viruses. such studies, especially those based upon laboratory-confirmed outcomes, should provide evidence of the specific benefits of vaccination. consistent studies providing estimates of vaccine efficacy over successive influenza seasons should improve understanding of the effects of small rather than major antigenic differences between vaccine and circulating viruses on clinical outcomes and should help to allay concerns arising from a perceived vaccine mismatch caused by the emergence of virus clades exhibiting little or no antigenic drift. recent advances in high-throughput genetic sequencing could potentially lead to a greatly enhanced understanding of the genetic changes occurring in influenza viruses and the evolutionary interactions that occur between co-circulating viruses. in-depth analyses of the precise mechanisms involved in the evolution and epidemiology of influenza would require advanced bioinformatics tools to comprehensively mine the data produced. such an approach should reveal, for example, the broader genetic changes that underlie antigenic variation in ha and thus allow for a better understanding of the relationship between genetic evolution and antigenic drift. increased information from x-ray crystallography on the structural features of the has of recent viruses and specific mutants, together with developments in computer modelling, should assist in attempts to predict the likely influence of amino acid substitutions on the antigenic and receptor-binding properties of new variants. further development of high-throughput laboratory systems for integrated and automated genetic and phenotypic analyses -from initial sample accession to data management -offers the intriguing prospect of a futuristic standardized virtual network for virus characterization in an epidemiological context. as such systems will have broad implications, not only for vaccine virus selection, but also for the organization and conduct of global influenza surveillance, it is extremely important that their development and deployment are integrated with the activities of the who gisrs. numerous mathematical modelling techniques have now been used to gain insights into the mechanisms that underlie both the evolution and the epidemiology of influenza viruses. for example, exploratory models have been developed to generate and test various hypotheses to explain the relatively restricted diversity of influenza viruses in terms of constrained antigenic repertoire, and to explore the underlying nature of immunity. they have also been used to improve understanding of the extent of between-subtype and between-type competition and of the potential conse-quences of such interactions for trends in the incidence of seasonal influenza viruses. phylogenetic models have also been used to identify changes in selective constraints in relation to antigenic drift and inter-species transmission. when based upon the amino acid substitutions associated with mammalian host adaptation, such models may aid assessment of the pandemic potential of avian and other animal viruses. phylodynamic modelling based upon available sequence data, supplemented with antigenic data, has already been successfully used to trace the emergence of new antigenic and genetic variants and track their geographical spread. however, in the absence of greatly improved understanding of the underlying evolutionary and biological mechanisms and other processes involved, the capacity of current mathematical modelling techniques to predict the timing and nature of future antigenic and genetic changes is limited. the intrinsically stochastic nature of influenza evolution may make such predictive modelling extremely challenging. where changes occur over short time scales, the application of simpler non-mechanistic statistical algorithms, such as those used as the basis of antigenic cartography, is likely to be more useful in facilitating vaccine virus selection than attempts to develop predictive models from the existing complex dynamical models of influenza evolution and transmission. such predictive models might presently be better suited for use in understanding the possible long-term effects of vaccination, optimizing the timing and location of focused surveillance efforts and predicting the possible consequences of the emergence of a novel virus. eventually, these models should be able to take advantage of integrated immunological and antigenic surveillance data to develop predictions of short-term dynamics in specific locations. all new influenza vaccine technologies have implications for vaccine virus selection and for regulatory and manufacturing processes. however, any potential requirement to tailor the virus selection process to specific types of vaccine is unlikely to be a crucial issue, especially if advances in vaccine technology and speed of production lead to greater flexibility in the timing of recommendations. although live-attenuated vaccines are not yet universally licensed, the current vaccine composition recommendation process is used. however, antibody response is not a good correlate of protection for such vaccines and the identification of a true correlate might affect the requirement for annual updating. several quadrivalent vaccines are also now under development that contain representative strains of the two influenza b virus lineages (b ⁄ victoria and b ⁄ yamagata) together with influenza a(h1n1) and a(h3n2) viruses. this raises a number of issues that could affect vaccine supply, including the possibility of two poorly growing vaccine viruses; the likely variable impact of a fourth component on vaccine yields and timing of manufacture; the prioritization of influenza b lineage viruses in the context of both trivalent and quadrivalent vaccine production; and the need for a fourth set of reagents. adjuvanted vaccines have been licensed with the primary aims of inducing better immune responses in certain age groups and allowing 'antigen sparing'. although there has been no specific intention to provide a broader spectrum of immunity to circumvent the need for annual vaccine updates, different products are likely to show a different breadth of response. providing recommendations in relation to product-specific cross-reactivity over successive influenza seasons is unlikely to be a feasible option for the who gisrs. in addition, various types of recombinant vaccines are now under development, including protein subunit, dna, vector and vlp vaccines -none of which are presently licensed. in the case of non-ha-based vaccines, different guidelines will apply and all such vaccines are likely to impact the current vaccine virus selection process in various ways depending upon their precise type and mechanism of protection. the level of protection afforded by immunity to na is receiving continued interest. currently, this component is included as part of the candidate vaccine virus and is selected on the basis of its sequence but not antigenicity. standardization of the na component would require antigenic characterization during the virus selection process, while antigenic changes in na in the absence of a corresponding change in ha antigenicity may on its own necessitate the updating of vaccine composition. for all such vaccines, ha variant selection may become less crucial than it is for current vaccines. although high-growth reassortants have been used to manufacture influenza a vaccine components for many years, their yields have been variable and there is continued need to identify the molecular determinants of high yield to engineer a more reliable and reproducible production process. reverse genetics, now used in the united states to produce virus reassortants for live-attenuated vaccines, has also been used to produce attenuated candidate h5n1 vaccine viruses suitable for inactivated vaccine manufacture. this approach was, however, less successful than classical reassortment in obtaining a suitable 2009 h1n1 pandemic vaccine virus, emphasizing the need for further investigation of the applicability of reverse genetics in the routine provision of suitable vaccine viruses. following the licensing of cell culture vaccines, the feasibility of isolating seasonal vaccine viruses in qualified 1 cell lines is being evaluated in a collaboration involving a number of whoccs and who erls under cradas with vaccine manufacturers. these studies should provide the basis for the introduction of a universal qualified cell culture system for providing mammalian cell-derived seasonal influenza candidate vaccine viruses. this would result in a greater choice of candidates, especially for recent h3n2 viruses, and may provide greater flexibility in responding to the 'late' emergence of a variant necessitating a vaccine composition change. such virus isolates would not be subject to undesirable egg-selected changes and would potentially provide a better match to the natural virus. however, the relative merits of egg and cell culture candidate vaccine viruses have still to be rigorously evaluated. guidance on quality assurance aspects has already been published by the european medicines agency (ema). the finalization of new ema regulatory guidelines may be accompanied by a who technical document on harmonizing regulatory approaches worldwide and the engagement of other regulatory authorities in vaccine-manufacturing nations. vaccine manufacturers and the who erls are also collaborating in an evaluation of cell culture-based reagents for use in single radial immunodiffusion (srid) potency testing, due for completion in early 2011. in addition, despite international consensus on the key quality specifications for 2009 h1n1 pandemic influenza vaccines, reagents to calibrate the majority of candidate vaccines using conventional potency tests only became available immediately prior to the initiation of clinical trials. in some cases, candidate vaccines were available ahead of the reagents. although national authorities proved flexible in accepting the use of validated alternative potency tests to allow clinical trials to proceed, newer methods such as high-performance liquid chromatography (hplc) and mass spectrometry are now being evaluated. the gisrs has a long history of success in recommending influenza vaccine compositions that have closely matched the combination of viruses circulating during subsequent influenza seasons. based upon the voluntary participation of its many constituent partners, the gisrs enjoys strong institutional and governmental support. global influenza surveillance is the foundation of the vaccine virus selection process. efforts to enhance and strengthen national, regional and global laboratory capacity for virological surveillance and representative virus sharing must continue. as part of this, improved integration of virological and disease surveillance data will be a key aim and will help to build the foundations for future studies of the impact and burden of influenza worldwide. to strengthen the pandemic influenza preparedness, collaboration between the gisrs and veterinary laboratories and organizations such as offlu in relation to zoonotic influenza infection has been greatly enhanced and has included the development of appropriate candidate human vaccine viruses from animal viruses. however, there remains considerable scope for improvement in this area, including the more timely exchange of information, viruses and reagents, and strengthened technical collaboration at all levels. although antigenic characterization of promptly forwarded virus isolates will remain the central criterion for selecting influenza vaccine viruses in the foreseeable future, technological developments (such as advanced recombinant dna and protein technologies, and highthroughput sequencing and advanced bio-informatics tools) will inevitably impact current gisrs surveillance and virus selection activities. in the interests of global public health, it will be important to integrate into the gisrs system appropriate information and data generated by various networks using emerging technologies. antigenic cartography has been adopted by the gisrs in recent years as a means of integrating hai data from different laboratories to allow for statistical comparison and visual display. the development of new statistical algorithms to complement the use of antigenic cartography may further facilitate vaccine virus selection. greater emphasis should be placed on conducting human serological studies which incorporate the use of antibody standards to improve the comparability of results. such studies would improve current understanding of the prevalence and spread of influenza, and complement the development of improved epidemiological models. greater collaborative effort is needed to generate randomly sampled, representative and integrated serological, epidemiological and evolutionary data that provide snapshots of host and viral populations suitable for modelling hypotheses on virus evolution and host immunity. the application of advanced techniques for dissecting the fine specificity of antibody responses to vaccination and infection should also lead to improvements in understanding the quality of a 'protective' immune response and aid in the development of additional correlates of immunity. recent collaboration between the gisrs and external partners including academic institutions and vaccine manufacturers has resulted in the increased availability of egg isolates and high-growth reassortants. new approaches to the generation of high-growth vaccine viruses involving the use of reverse genetics and qualified cell cultures will continue to be evaluated and developed, as will alternative methods of vaccine potency testing. who will continue to support these and other efforts to increase the reliability and timeliness of global influenza vaccine supply. new vaccine types currently under development may allow more flexibility in the timing of recommendations on vaccine virus composition. conversely, alterations to the virus selection process and additional information may be needed in relation to new-generation vaccine types with different compositions and mechanisms of protection. the who gisrs vaccine virus selection process lies at the heart of global efforts to address the constantly evolving threat posed by influenza. for decades, this highly collaborative and complex process has ensured a continued supply who writing group of effective seasonal vaccines and was able to respond very rapidly to the emergence of the 2009 h1n1 pandemic. if the current limitations and constraints inherent in the process are to be overcome, ongoing efforts by the who gisrs and its partners must continue to identify improvements, harness new technologies and strengthen collaboration. who will continue in its central role of developing and coordinating worldwide expertise to meet the increasing public health need for influenza vaccines and will support this process through the convening of periodic international consultations on improving influenza vaccine virus selection. annex 1: process of influenza vaccine virus selection and development 2 the diagram shows that the individual steps in the selection of candidate vaccine viruses and development of standardizing reagents for seasonal influenza and for a potential h5n1 influenza pandemic are essentially equivalent. for seasonal vaccines the timelines are: • steps 1-4: the collection, isolation and thorough antigenic and genetic characterization of recent virus isolates continues throughout the year; • step 4a: comparisons of the recognition of representative recent viruses by vaccine-induced antibodies in human sera are conducted 2-3 weeks before the biannual who vaccine consultation meetings; • steps 5, 6a and 7a: candidate viruses for vaccine use are reviewed and selected, and high-growth reassortants prepared and characterized following identification of (potential) antigenic variants -these steps are not solely dictated by the recommendations of the who biannual vaccine virus consultations. • step 8: evaluation of their growth properties is conducted in a timely manner around the time of the who vaccine virus consultations and prior to authorization of vaccine composition by national authorities. • step 9a: preparation of the standardizing reagents for new vaccine components is initiated once the particular vaccine virus has been selected following the who recommendation. the who informal consultation for improving influenza vaccine virus selection, 14-16 june 2010, was organized by the virus monitoring and vaccine support (vmv) unit of who, with participation from who collaborating centres on influenza, erls, national influenza centres, national control laboratories, national regulatory authorities, academic and veterinary institutions, influenza vaccine manufacturers and other collaborating organizations. in accordance with who policy, all members of the writing group that assisted who in the development of this meeting report had completed the who declaration of interests for who experts. these declarations were then evaluated by the who secretariat prior to the consultation. the members of the writing group declared the following personal current or recent (within the last 4 years) financial or other interests relevant to the subject area: group on data needs). plos curr influenza 2009 (revised 23 december 2009). rrn1135. northern hemisphere season. writing committee of the world health organization consultation on northern hemisphere influenza vaccine composition for public workshop on immune correlates of protection against influenza a viruses in support of pandemic vaccine development use of antigenic cartography in vaccine seed strain selection influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses who. a description of the process of seasonal and h5n1 influenza vaccine virus selection and development. draft version antigenic and genetic characteristics of influenza a(h5n1) and influenza a(h9n2) viruses and candidate vaccine viruses developed for potential use in human vaccines strategies to improve global influenza surveillance: a decision tool for policymakers global influenza surveillance network: laboratory surveillance and response to pandemic h1n1 improving the process of vaccine virus selection determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the octet rapid semiautomated subtyping of influenza virus species during the 2009 swine origin influenza a h1n1 virus epidemic in milwaukee, wisconsin a practical influenza neutralization assay to simultaneously quantify hemagglutinin and neuraminidase-inhibiting antibody responses development of rapid, automated diagnostics for infectious disease: advances and challenges high-throughput dna sequencingconcepts and limitations high-throughput laboratories for homeland and national security current challenges in implementing cell-derived influenza vaccines: implications for production and regulation establishment of retroviral pseudotypes with influenza hemagglutinins from h1, h3, and h5 subtypes for sensitive and specific detection of neutralizing antibodies new tests for an old foe: an update on influenza screening ecological and immunological determinants of influenza evolution population dynamics of rapid fixation in cytotoxic t lymphocyte escape mutants of influenza a epochal evolution shapes the phylodynamics of interpandemic influenza a (h3n2) in humans rambaut a et al. the genomic and epidemiological dynamics of human influenza a virus the generation of influenza outbreaks by a network of host immune responses against a limited set of antigenic types h1n1 influenza pandemic: insights from modelling. the who informal network for mathematical modelling for pandemic influenza h1n1 2009 (working ne win immunization, vaccines and biologicals (ivb) global influenza programme (gip) who regional office for south-east asia (searo) global influenza programme (gip) who regional office for europe (euro) global influenza programme (gip) global influenza programme (gip) keiji fukuda, health, safety and environment (hse) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) immunization, vaccines and biologicals (ivb) global influenza programme (gip) global alert and response (gar) global influenza programme (gip) global influenza programme (gip) who regional office for the americas (amro) laszlo palkonyay, immunization, vaccines and biologicals (ivb) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) ludy suryantoro, health, safety and environment (hse) global influenza programme (gip) immunization, vaccines and biologicals (ivb) global influenza programme (gip) acknowledgements ª 2011 blackwell publishing ltd. the world health organization retains copyright and all other rights in the manuscript of this article as submitted for publication. key: cord-001521-l36f1gp7 authors: nan title: oral and poster manuscripts date: 2011-04-08 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2011.00209.x sha: doc_id: 1521 cord_uid: l36f1gp7 nan pandemic influenza h1n1 (h1n1pdm) virus of swine-origin causes mild disease, but occasionally is associated with acute respiratory distress syndrome and death. 1, 2 it is important to understand the pathogenesis of this new disease. previously we showed a comparable virus tropism and host innate immune responses between h1n1pdm and seasonal h1n1 influenza virus in the human respiratory tract, 3 however h1n1pdm virus differed from seasonal h1n1 influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection. we now compare the tropism and host responses elicited by pandemic h1n1 with that of related swine influenza viruses and a pandemic-swine reassortant virus in ex vivo and in vitro cultures of the human respiratory tract and conjunctiva. we have used recombinant virus to investigate the role of the hemagglutinin (ha) and neuraminidase (na) of h1n1pdm virus in its conjunctival tropism. these findings are relevant for understanding transmission and therapy. fragments of human conjunctiva, bronchi, and lung tissues were cut into 2-3 mm fragments within 2 h of collection and infected with influenza a viruses at a titer of 10 6 tcid 50 ⁄ ml. viruses investigated included h1n1pdm (a ⁄ hk ⁄ 415742 ⁄ 09), swine h1n2 virus (a ⁄ swine ⁄ hk ⁄ 915 ⁄ 04), which shares a common derivation for seven genes with h1n1pdm, a natural swine reassortant h1n1 (a ⁄ swine ⁄ hk ⁄ 201 ⁄ 10), which has acquired the na gene from h1n1pdm and other swine influenza h1n1 viruses. reverse genetics derived recombinant viruses with ha and na gene segments of seasonal h1n1 and pandemic h1n1 swapped were also studied. lung fragments were cultured at 37°c in culture plates; conjunctival and bronchial biopsies were cultured in air-liquid interface at 33 and 37°c respectively. tissue fragments were infected for 1 h and incubated for 1, 24, and 48 h post infection. infectious viral yield was assessed by titration in mdck cells. the infected tissues were fixed with formalin and analyzed by immunohistochemistry for influenza antigen. cytokines profiles induced by influenza virus infected respiratory epithelial cells in vitro were measured by quantitative rt-pcr and elisa. we found comparable replication in seasonal and pandemic h1n1 viruses in human respiratory tract, while the swine influenza a ⁄ swine ⁄ hk ⁄ 4361 ⁄ 99 (h1n1) virus and a ⁄ swine ⁄ hk ⁄ 915 ⁄ 04 (h1n2) virus failed to infect and replicate in human lung ex vivo culture, but it replicated productively in human bronchus ex vivo. interestingly, the swine reassortant influenza h1n1 (a ⁄ swine ⁄ hk ⁄ 201 ⁄ 10) virus (with the na from h1n1pdm) infected and productivity replicated in lung ex vivo and in vitro. pandemic h1n1pdm virus, but not seasonal h1n1 virus, was able to infect ex vivo cultures of human conjunctiva, suggesting subtle differences in receptor binding profile in h1n1pdm, seasonal viruses, and the swine related h1n2 viruses. using reverse genetics derived recombinant viruses, we were able to demonstrate that the ha and na segments of h1n1pdm, but not the polymerase genes, were required for the conjunctival tropism of h1n1pdm ( figure 1 ). in contrast with highly pathogenic influenza h5n1 virus, which induced high cytokine and chemokine decretion, the related swine viruses, a ⁄ swine ⁄ hk ⁄ 915 ⁄ 04 (h1n2), as well as the swine pandemic reassortant virus, a ⁄ swine ⁄ hk ⁄ 201 ⁄ 10 (h1n1) we studied were similar to h1n1pdm and seasonal influenza viruses in their intrinsic capacity for cytokine dysregulation. collectively, our results suggest that pandemic h1n1pdm virus differs in modest but subtle ways from seasonal h1n1 virus in its intrinsic virulence for humans, findings that are in accord with the epidemiology of the pandemic to date. the ha and na gene segments are key to the conjunctival tropism manifested by the h1n1pdm virus. the pandemic reassortant influenza h1n1 (a ⁄ swine ⁄ hk ⁄ 201 ⁄ 10) virus isolated from swine with the na from h1n1pdm shares with h1n1pdm the capacity for productive replication in lung ex vivo and in vitro. these findings are relevant for understanding transmission and therapy. isolation of influenza viruses from specimens is traditionally performed in two classical systems: embryonated chicken eggs and mdck cell culture. nevertheless, several publications are dedicated to the theme of alternative cell culture systems, which may be used for influenza virus isolation and cultivation. [1] [2] [3] this is in part because mdck cells are of animal origin, which means that they cannot be used as a proper model for estimating interactions between a human virus and a human cell culture as a host. a variety of human monolayer and suspension cell cultures have been tested on their capability to support influenza virus replication. among them, some support influenza a virus growth as well as mdck cells do, 4 others support replication of a virus, but do not enable the formation of mature viral particles, 5 whereas others show only a weak level of replication or are not permissive at all. 2 caco-2 cells, for example, represent a good substitute for mdck cells, because it has been shown that the rate of viral isolation in caco-2 cells is as effective as in mdck, and sometimes is even better. 6 the success of viral replication is determined not only by the cell culture type, but also by the virus itself. despite the accepted view that it is the type of receptor that defines the interaction between the virus and the host cell, there is evidence that it is not the only factor that predetermines the fate of the cell. 7 the fate of the infected cell can also differ. a series of articles show that apoptosis is the most probable mechanism of cell killing by influenza viruses. 8, 9 influenza a viruses of different subtypes induce apoptosis to a different extent (e.g. h3 viruses provoke more strong apoptotic response than h1 viruses do 10 ). nevertheless, it has been demonstrated that caco-2 cells do not follow the apoptotic pathway and die through necrosis. 11 the sjpl cell line also dies through necrotic pathway and not apoptosis. 12 the aim of our work was to compare growth characteristics of different flu viruses (e.g. avian, swine, and human) in various human and animal cell cultures and to evaluate their influence on cell culture growth. the parameters measured in the study were as follows: cytopathic changes of cell cultures following virus infection, hemagglutinin production, np synthesis, the dose-dependent effect of infection on cell proliferation, and the ability of viruses to induce apoptosis. influenza viruses used included: highly pathogenic avian h5n1 a ⁄ kurgan ⁄ 5 ⁄ 05, low pathogenic avian h5n1 a ⁄ gull ⁄ kostanai ⁄ 7 ⁄ 07, swine h1n1 a ⁄ swine ⁄ 1976 ⁄ 31, human h1n1v a ⁄ california ⁄ 07 ⁄ 09, human h1n1v a ⁄ saint-petersburg ⁄ 5 ⁄ 09, human h1n1 a ⁄ brisbane ⁄ 59 ⁄ 07, and human h3n2 a ⁄ brisbane ⁄ 10 ⁄ 07. the viruses were propagated in 10-days embryonated chicken eggs, the allantoic fluid was collected, the aliquots were made and stored at )70°c for further use. to evaluate tcid50 for each virus on all cell cultures, 96-well plates were used. the cells were seeded 0ae2 ml per well (concentration of 1-1ae5 · 10 cells ⁄ ml). the confluent 24-h old monolayer was used for viral inoculation. the cells were washed twice with serum-free medium, then 0ae05 ml of tenfold viral dilutions from viral aliquots were added and left for 45 min for contact at 37°c. the cells were then washed to remove the non-attached particles, and the wells were filled with tpck-trypsin (2 lg ⁄ ml)-containing medium without bovine fetal serum. the plates were observed daily for cytopathic effect, and the results were evaluated at 72 h after infection for cytopathic effect and by reaction of hemagglutination with suspension of chicken erythrocytes (0ae75%). infection of suspension cell cultures was done in centrifuge tubes. cells (concentration 3-5 · 10 5 ) were inoculated with viral dilutions (moi = 1-10). after 45 min of contact, cells were washed, resuspended in rpmi with trypsin and fetal serum, and seeded in 24-well plates (1 ml in each well). the results were fixed after 72 h, calculating the number of cells grown and estimating the rate of apoptosis by hoechst-33258 staining. 13 cells were grown in 24-well plates with seeding concentration 1 · 10 cells ⁄ ml. one millilitre of cell suspension was placed in each well, inoculated with viral dilution (moi = 1-1000) and left for 72 h. after, the cells were detached from plastic with versene and calculated in fuks-rosental camera to evaluate the number of cells. the monoclonal antibodies obtained in research institute of influenza towards viral nucleoprotein np were used following the standard protocol described in. 13 for all viruses tested, mdck turned out to be more permissive than sp cell culture. avian viruses, independently of their pathogenicity, replicated efficiently on both animal cultures tested. human h1n1 and h3n2 viruses demonstrated weaker replication in sp cells. the most significant differences were seen for swine influenza and pandemic h1n1v viruses which replicated in mdck cells at the rates comparable with other viruses, but showed poorer growth in sp cell line (see table 1 ). human cell lines displayed clear differences in their susceptibility to viruses of various origins. avian influenza viruses replicated in all cell lines except girardi heart, and the most intense replication rate was observed for ecv-304, l-41, and rd lines. a-549 and a-172 were poorly infected, as well as all suspension cell lines tested. seasonal human h1n1, as well as h3n2 viruses, replicated in all cell cultures tested, but the rate of infectivity was rather low in practically all cultures tested with the exception of rd and t-98g cell lines. strikingly, swine influenza virus and human pandemic h1n1v viruses didn't replicate well in any of human lines tested. a weak replication rate was observed in ecv-304, rd, and t-98g, but in general, human cell lines were the titers produced by swine and pandemic influenza viruses are shaded in grey. *low-pathogenic avian influenza virus; **highly-pathogenic avian influenza virus poorly susceptible to pandemic h1n1v. swine influenza virus differed because it infected weakly a-172 and girardi heart cell cultures, which was not the case for h1n1v viruses. our study has shown that all influenza viruses were able to induce apoptosis in the cell cultures tested. the degradation of chromatin found in the nucleus with hoechst-33258 staining was seen before the first symptoms of cytopathic effect (cpe) in monolayer of cells. in cell cultures where the cpe was not visible, high doses of virus still induced apoptotic response. the process of apoptosis is rather well studied in mdck cells and some other cell types, so we've focused on three human monolayer cell cultures that are relatively poorly studied: a-549, ecv-304, and flech. these cell cultures are less susceptible to viral infection, and besides, it was interesting to find out whether the viruses that do not cause any cpe do infect these cultures. a-549 turned out to be most sensitive to apoptotic response, while flech turned out to demonstrate weak reaction. time needed for apoptosis induction by different flu viruses also varied. the earliest apoptosis was noted for h5n1 and h3n2 viruses and h1n1 viruses induced apoptosis at about 20 h postinfection. it is well-known that apoptosis can be induced only by a reproducing virus, and that uv-kills viruses that are not capable of it. we tested whether swine and pandemic h1n1v viruses (that do not show cpe in these cultures) do replicate in them and induce apoptosis with the help of monoclonal antibodies against viral np. the obtained data show that they indeed do replicate in these cell cultures, as we observed np fluorescence, and that they also induce apoptosis (see table 2 ). we've shown earlier 13 thus, we've tested the ability of swine and pandemic h1n1v viruses in this aspect. it was shown that these viruses were comparable with the effect seen for seasonal h1n1 virus. moreover, swine influenza virus induced stronger apoptotic response in hemablastoid cell lines in comparison with pandemic h1n1v viruses, which also have a swine origin. we also checked the ability of flu viruses to influence monolayer cell cultures growth. the data clearly indicated that only ecv-304 endothelial line and t-98g glioblastoma line displayed cell proliferation in response to low moi. apoptosis wasn't registered in these stimulated cultures, apparently because the moi was very low. all the other monolayer cultures didn't respond to low moi by stimulation of their proliferation. interaction between an influenza virus particle and a host cell can follow several scenarios. cpe seen in infected cells is accompanied with high rates of viral particles production and leads to cell death. the death itself may be through apoptotic or necrotic pathways. 4, 8 also, infection process in low doses can stimulate cell proliferation -the effect seen for hemablastoid lines, histiocytes, peripheral blood cell lines, 14, 15 and in glioblastoma and endothelial cell lines as it was described here. considering the origin of ecv line, 16 these cells bear all the antigenic, biochemical, and physiological traits of umbilical cord and are actively used in pharmacological tests as well as glioblastoma cells; they also are of special interest for oncogenesis studies. table 2 . replication, apoptosis induction, and np synthesis of influenza viruses in a-549, ecv-304 and flech cell cultures. the numbers represent the log 10 tcid 50 ⁄ 0ae2 ml calculated by reed-muench method as described in. 17 the ()) symbol means that no cpe could be observed in any dilution and no hemagglutination could be registered. the (+) symbol means that apoptosis was observed with hoechst-33258 staining though the productive replication and production of progeny viruses in human cell lines was generally low, it is evident that viral infection does occur in these cells, even for swine and h1n1v viruses. it can be demonstrated by the presence of np de novo synthesis and by stimulation of virus-induced apoptosis. in fact, we observe a contradiction: avian influenza viruses actively reproduce in human cell lines, but we do not see their vast spreading in human population, while h1n1v viruses that hardly replicate in all human cultures tested have caused the latest pandemic. influenza viruses continue to cause problems globally in humans and their livestock, particularly poultry and pigs, as a consequence of antigenic drift and shift, resulting frequently and unpredictably in novel mutant and reassortant strains, some of which acquire the ability to cross species barriers and become pathogenic in their new hosts. long-term surveillance of influenza in migratory waterfowl in north america and europe have established the importance of anseriformes (waterfowl) and charadriiformes (gull and shorebird) in the perpetuation of all known subtypes of influenza a viruses. the available evidence suggests that each of the 16 hemagglutinin (ha) and nine neuraminidase (na) subtype combinations exist in harmony with their natural hosts, cause no overt disease, and are shed predominantly in the feces. 1, 2 in this study we determined the subtypes and prevalence of low-pathogenic influenza a viruses present on the territory of kazakhstan in 2004-2006 and further analysed the ha and na genes of these isolates in order to obtain a more detailed knowledge about the genetic variation of influenza a virus in their natural hosts. (institute for biological safety problems, gvardeiskiy, zhambyl oblast, kazakhstan)). samples that were identified as influenza a virus positive by matrix rrt-pcr were thawed, mixed with an equal volume of phosphate buffered saline containing antibiotics (penicillin 2000 u ⁄ ml, streptomycin 2 mg ⁄ ml, and gentamicin 50 lg ⁄ ml), incubated for 20 minutes at room temperature, and centrifuged at 1500 g for 15 minutes. the supernatant (0ae2 ml ⁄ egg) was inoculated into the allantoic cavity of four 9-day old embryonated hens' eggs as described in european union council directive 92 ⁄ 40 ⁄ eec. 3 embryonic death within the first 24 hour of incubation was considered as non-specific, and these eggs were discarded. after incubation at 37°c for 3 days the allantoic fluid was harvested and tested by haemagglutination (ha) assay as describe in european union council directive 92 ⁄ 40 ⁄ eec. in the cases where no influenza a virus was detected on the initial virus isolation attempt, the allantoic fluid was passaged twice in embryonated hens eggs. the number of virus passages in embryonated eggs was limited to the maximum two to limit laboratory manipulation. a sample was considered negative when the second passage ha test was negative. the subtypes of the virus isolates were determined by conventional haemagglutination inhibition (hi) test and neuraminidase inhibition (ni) test, as describe in european union council directive 92 ⁄ 40 ⁄ eec. 3 rna extraction and pcr with specific primers rna was extracted from infective allantoic fluid using rneasy mini kit (qiagene, gmbh, germany) according to the manufacturer's instructions. the rna was converted to full-length cdna using reverse transcriptase. the rt mix comprised 2ae5 ll of dmpc water, 5 ll of 5· first strand buffer (invitrogen), 0ae5 ll of 10 mm dntp mix (amersham biosciences), 2 ll of 50 mm uni12 primer, 32 u of rnaguard (amersham biosciences), 200 u of mmlv reverse transcriptase (invitrogen) and 5 ll rna solution in total volume of 25 ll. the reactions were incubated at 42°c for 60 minutes followed by inactivation of the enzyme at 95°c for 5 min. pcr amplification with ha and na gene specific primers was performed to amplify the product containing the full length ns gene. twenty-five microliter pcr-mix contained 1· platinum taq buffer (invitrogen), 200 lm dntp, 2ae5 mm mgcl 2 , 240 nm each of fw primer and rw primer, 1 u platinum taq dna polymerase (invitrogen) and 3 ll cdna. reactions were placed in a thermal cycler at 95°c for 2 min, then cycled 35 times between 95°c 20 seconds, annealing at 58°c for 60 seconds, and elongation at 72°c for 90 seconds and were finally kept at 8°c until later use. sequences of the purified pcr products were determined using gene specific primers and bigdye terminator version 3ae1 chemistry (applied biosystems, foster city, ca), according to the manufacturer's instructions. reactions were run on a abi310 tm dna analyzer (applied biosystems). sequencing was performed at least twice in each direction. after sequencing, assembly of sequences, removal of low quality sequence data, nucleotide sequence translation into protein sequence, additional multiple sequence alignments, and processing were performed with the bioedit software version 7ae0ae4ae1 with an engine based on the custal w algorithm. the phylogenetic analysis, based on complete gene nucleotide sequences were conducted using molecular evolutionary genetics analysis (mega, version 4ae0) software using neighbor joining tree inference analysis with the tamura-nei c-model, with 1000 bootstrap replications to assign confidence levels to branches. [4] [5] [6] [7] ha and na sequences obtained from genbank the ha and na gene was analyzed both with selected number of influenza isolates and in comparison with virus genes obtained from genbank were used in phylogenetic studies [22] . the nucleotide sequence data obtained in this study has been submitted to the genbank database and is available under accession numbers fj434373, fj436942ae1, fj434369, fj434370, gu982281-gu982284 for ha and fj434374, fj436943ae1, fj434371, fj434372, gu982285-gu982288 for na. avian influenza prevalence in our study h4, h5, and h13 influenza a virus subtypes were found to circulate at the same time, in the same geographic region in the kazakhstan. this finding most likely indicates the existence of a large reservoir of different influenza a viruses in kazakhstan. we analyzed the ha and na gene sequences of the eight influenza a viruses isolated in kazakhstan together with selected number of isolates, reported between year 1941 to 2008, and previously published in the genbank. 8 phylogenetic analysis of the h4 ha gene showed that all viruses separated into the american and eurasian lineages ( figure 1 ). an evolutionary tree suggests that north american isolates have diverged extensively from those circulating in other parts of the world. geographic barriers which determine flyway outlay may prevent the gene pools from extensive mixing. the lack of correlation between date of isolation and evolutionary distance suggests that different h4 ha genes co circulate in a fashion similar to avian h3 ha genes and influenza c genes, implying the absence of selective pressure by antibody that would give a significant advantage to antigenic variants. analysis of phylogenetic relationships among the ha5 ha genes reported in this study clearly shows that viruses belong to the western pacific flyway, one of the major migratory flyways in this region that have subsequently spread throughout eurasia. 9 these findings provide further evidence of the dynamic influenza virus gene pool in this region. along the western pacific migratory flyway, the influenza virus gene pool in the domestic waterfowl of southern china has 'mixed' longitudinally with viruses isolated from japan, mongolia, and siberia. however, it appears that there has also been 'mixing' latitudinally through overlapping migratory flyways, thereby facilitating interaction between the influenza virus gene pool in domestic waterfowl in the eastern and western extremities of the eurasian continent. this helps to explain the latitudinal spread of the qinghai-like (clade 2ae2) h5n1 virus in the last 2 years, while h5n1 outbreaks in korea and japan may represent the longitudinally transmitting pathway. 10 ha of subtype h13 so far has been found exclusively in shorebirds, such as gulls, and in a pilot whale (potentially a spillover from shorebirds), but not in other avian species that are natural hosts of influenza a virus, such as ducks and geese; therefore the study of the evolution of these viruses is very interesting. phylogenetic analysis h13 ha gene revealed three significantly different evolutionary lines: an american line, a european line, and a line comprising the isolates from america and eurasia. 11 further we analyzed na genes of influenza viruses (figure 2) . the na gene is important both because of its functional role in promoting the dissemination of the virus during infection, and because, like ha, it is a principal target of the immune system. it was shown that phylogeny of na genes of influenza have the same properties as hemagglutinin. na genes of kazakhstanian viruses belong to eurasian lineage of virus evolution. obtained data are important for surveillance and diagnostics because some of the lpai viruses examined in this study can infect and be shed by chickens and turkeys and may have epidemiology potential during further recombination with other influenza viruses. influenza virus is divided into different subtypes based on hemagglutinin (ha) and neuraminidase (na) on the virus surface. within each subtype, ha continues to mutate and produce immunologically distinct strains, as antigenic drift. the continuous mutation of influenza virus (iv) is important for annual epidemics and occasional pandemics of disease in humans. antigenic drift requires vaccines to be updated to correspond with the dominant epidemic strains. in humans, ivs show both antigenic drift frequently. in contrast, ivs from birds are in evolutionary stasis, 1 and they show little amino acid changes. 2, 3 the reason is that ivs in bird intestine are not subjected to strong immune selection. hemagglutinin (ha) gene of influenza a virus encodes the major surface antigen, which is the target for the protective neutralizing antibody response that is generated by infection or vaccination. in humans, influenza a viruses show antigenic drift with amino acid changes in the globular head of the ha so as to evade herd immunity of the population. on the contrary, avian influenza a viruses show evolutionary stasis in wild birds. h6 aivs have occurred frequently in chicken farms in the world. 4 although vaccination is not permitted, h6n1 aivs have circulated in taiwan for a time. 5 the seroprevalence in chicken flocks reaches about 50% in the field. h6n1 aivs invades internal organs, such as kidney and lung. 6 thus, viruses in chicken flocks are pressured into antibody selection. here, we report that h6n1 aivs in the field have showed evolutional changes instead of evolutional stasis. in response to requests from poultry farmers for diagnostic investigations of illness in poultry flocks, the authors did necropsy at the pen-site. after careful examination, tracheae were taken and kept in cold for virus isolation in the laboratory. for avian influenza virus isolation, trachea was homogenized 1:10 in tpb with antibiotics. the homogenate was frozen and thawed three times and then centrifuged at 1157 g for 15 minutes. the supernatant was passed through a 0ae45 lm filter. the homogenate was examined for the presence of virus by inoculation into five 9-to 11-day-old specific-pathogen-free (spf) chicken eggs for two passages. thirteen h6n1 aivs were isolated in this laboratory during 2000 and 2008 from different parts of taiwan. besides the viruses isolated in this laboratory, the ha sequences of 27 chicken h6n1 aivs were from the genbank. the accession numbers of hemagglutinin of aiv reference strains included in this study were as the following: g2 ⁄ 87, dq376619; g23 ⁄ 87, dq376620; 0824 ⁄ 97, dq376621; na3 ⁄ 98, dq376622; 165 ⁄ 99, dq376626; 0705 ⁄ 99, dq376624; ns2 ⁄ 99, dq376623; sp1 ⁄ 00, dq376628; 0329 ⁄ 01, dq376633; 1205 ⁄ 01, dq376630; 1212 ⁄ 01, dq376631; 1215 ⁄ 01, dq376632; 0208 ⁄ 02, dq376641; 0408 ⁄ 02, dq376642; pf1 ⁄ 02, dq376635; pf2 ⁄ 02, dq376636; pf3 ⁄ 02, dq376637; a37 ⁄ 02, dq376639; 0320 ⁄ 02, dq376638; 0107 ⁄ 02, dq376640; 0222 ⁄ 02, dq376634; 0706 ⁄ 03, dq376644; 1203 ⁄ 03, dq376645; ch1006 ⁄ 04, dq376649; 0114 ⁄ 04, dq376647; a342 ⁄ 05, dq376653 and 0204 ⁄ 05, dq376652. the viruses isolated were propagated in the allantoic cavities of 10-day-old embryonated spf eggs for 72 hour. the virus rna was extracted using qiaamp viral rna miniprep kit (qiagen) . six-week-old balb ⁄ c mice were injected emulsion intraperitoneally with 100 lg of purified and concentrated a ⁄ chicken ⁄ taiwan ⁄ 2838v ⁄ 00 (h6n1) virion with complete freund's adjuvant. every two weeks, the mice were boosted supplementary five times with 50 lg of virion in incomplete freund's adjuvant. when the mice were boosted, blood was collected from tail vein and tested by the western blot assay to check the antibody titers. the mice were then injected intraperitoneally with 50 lg of virion at week 8. five days after the last injection, the splenocytes in the mice were fused with myeloma cells (sp2 ⁄ 0-ag14). one week before fusion, the myeloma cell line was expended in dmem medium (hyclone laboratories, logan, ut) with 10% fetal bovine serum at 37°c to ensure they were in the exponential growth phase. the spleen cells from immunized mice were washed, harvested, and mixed with the previously prepared myeloma cells and fused by gradually adding 50% polyethylene glycol-1500. the resulting pellet was plated into 96 well tissue culture plates. only the fused cells grew in medium with hypoxanthine-aminopterin-thymidine (hat). with fresh medium replacement over 2 weeks, the hybridomas were ready for screening. hightiter monoclonal antibody (mab) preparations were obtained from the ascetic fluid of mice injected with the selected hybridoma clones. the antibody from mouse ascetic fluids was purified by precipitation with ammonium sulfate, then aliquoted and frozen at )70°c, avoiding repeated freezing and thawing. eventually, six mabs were obtained and named ch11-d10, eb2-b3, eb2-e5, eb2-f9, ff9-f5, and ff9-f7, respectively. the hi test was performed following a standard method. all the viruses were diluted twofold and reacted with 1% chicken erythrocytes in the v-bottomed microtiter plate by the hemagglutination test. after agglutination, four hemagglutinating units of a ⁄ chicken ⁄ taiwan ⁄ 2838v ⁄ 00 (h6n1) and ascetic fluids from the immunized mice of the six mabs were prepared for hi test. hi titers of 2 4 or more were regarded as positive. the cases submitted for diagnosis from chicken farms had respiratory signs, increase in mortality, or drop in egg production (e.g. egg production dropped from 10% to 40%). the extent of drop in egg production depended on the chicken ages. for example, the age of case 3511 was 27 weeks, a stage of increasing egg production. however, after h6n1 aiv infection, the egg production decreased 1% instead of increasing and then stayed at 65% for a week. the infected chickens showed signs of decreasing activity, anorexia from 150 g per bird to 90 g per bird, and respiratory signs. case 2829 showed infection in the second floor first and then transmitted to third and fourth floor, indicating that the virus transmitted by air or human movement. however, most cases showed air borne transmission from one flock to another in spite of enforcing restrictions of persons entering the poultry pens and changing clothes and booths. in most cases, males' mortality was higher than that of female pen mates. by comparing the sequences of ha of those h6n1 viruses, we found that amino acid changes in ha1 were higher than those in ha2, showing that antigenic changes on the globular head of ha molecule rather than randomly on the whole ha protein, indicating that h6n1 viruses in taiwan had been selected in the presence of antibody pressure. the aa residues and changes that showed yearly trends were the followings: a-5s, i19s, v30i, n35s, e39k, l45m, e54d, q66k, a94v, or t, s127n, s128r, k133n, y138d, n139t, s140i, g141d, l149v, i152v, g155e, t188n, g226s, a285v, k304e, d386n, i533m, and m535i. however, their significance on antigenic variation was previously unknown. by hemagglutinination inhibition (hi) assays, except mab ch11-d10, all other monoclonal antibodies elicited from 2838v ⁄ 00 showed different hi titers with the different h6n1 viruses (table 1 ). however, those 5 mabs showed negative hi to 2829 and 2831, the early h6n1 strains. this indicated that the epitopes recognized by those mabs were undergoing antigenic drift. introduction aquatic birds are recognized as the natural reservoirs of the influenza a virus as all known subtypes (h1-h16, n1-n9) have been found in them. phylogenetic analyses of influenza viruses found in other animals revealed that all were directly or indirectly derived from viruses resident in aquatic birds. 1 however, the prevalence, movement, and evolutionary dynamics of influenza viruses in these avian hosts have not been well defined. southern china was hypothesized to be an 'epicenter' for the generation of human pandemic influenza viruses as all major influenza pandemic viruses in the 20th century emerged from this region. 2 the ecological background that facilitates the occurrence of these pandemic influenza strains has not been fully explored. in the past two decades, four lineages, belonging to h5n1, h9n2, and h6n1 viruses, have become established and long-term endemic in different types of poultry in this region. [3] [4] [5] some of these viruses were disseminated to many countries in eurasia and africa and have continued to cause sporadic human infection, posing a persistent pandemic threat to the world. 6 in the mean time, the endemic influenza lineages have undergone extensive genetic reassortment events giving rise to many variants, dramatically increasing the genetic diversity of the influenza virus in this region. questions remain as to how and where these viruses emerged, and what were the sources of the gene segments incorporated within the novel reassortant variants of the h5n1, h9n2, and h6n1 virus lineages. to address these questions, surveillance of influenza in migratory and domestic (sentinel) ducks has been conducted since 2002 at poyang lake, the biggest fresh-water lake and the major migratory bird aggregation site in southern china. the aim of this study is to identify the prevalence, seasonality, and movement of virus between migratory and domestic ducks. migratory ducks were captured during over-wintering, from november to march. cloacal swabs and blood samples were collected from each individual bird. all birds were released after sampling. to observe the interaction between migratory ducks and domestic birds, we also sampled domestic ducks from two duck farms (designated as sentinel ducks) surrounded by rice fields and inaccessible to other types of poultry, but accessible to migratory birds. that is, the sentinel ducks share the same water body with migratory ducks and have the chance to spread viruses to each other. for sentinel ducks, sampling was conducted fortnightly, all year-round, on the two farms from august 2002 onwards. cloacal swabs and fresh fecal droppings were taken. about 200 birds were randomly sampled fortnightly from these farmed ducks. all swabs were soaked in vials containing 1ae5 ml transport medium with antibiotics and kept on ice-packs during sampling and immediately stored in )80°c freezers for further use. blood samples from migratory ducks were treated according to methods previously described. 7 serological survey and virus subtyping in migratory and sentinel ducks used hemagglutination inhibition (hi) and neuraminidase inhibition (ni) tests as previously described. 3 for isolates that were not identified by reference antisera, subtypes were determined by rt-pcr using subtype specific ha and na diagnostic primers. prevalence and seasonal patterns of influenza virus in migratory and sentinel ducks during 2002 during -2007 a total of 11 508 cloacal swabs from migratory ducks and 36 475 cloacal or fecal swabs from sentinel ducks were collected at poyang lake. from these specimens, 90 influenza isolates were obtained from migratory ducks and 1681 from sentinel ducks; isolation rates of 0ae78% and 4ae61%, respectively (table 1) . it was noted in sentinel ducks that virus occurrence formed a seasonal peak from november to february, which completely overlapped the over-wintering months of migratory ducks. this suggests that virus movement or transmission between migratory and sentinel ducks occurred during this period at poyang lake. thirty positive samples (hi titer ‡20) were identified from 715 blood samples collected during november and december in 2004. among these, 15 samples were positive to h6, 8 were positive to h9, 5 were positive to h1, and 2 were positive to h4. one serum sample was positive to both h4 and h6, which suggested co-infection of influenza virus in migratory ducks might occur in natural conditions. poyang lake, which is located in the northeastern part of jiangxi province, is the largest freshwater lake in china and is part of the eastern asia-australia migration route. 9 every year, hundreds of thousands of migratory ducks congregate at poyang lake during the migration season. 9 recent farming practice involves raising domestic waterfowl in dense populations in the poyang lake region. farmraised domestic waterfowl are allowed to feed in and share the same water body with migratory birds, thereby facilitating direct interactions between domestic waterfowl and freeranging migratory birds. this makes poyang lake an ideal site to observe the dynamics of influenza virus interactions between migratory and sentinel ducks in southern china. in our longitudinal surveillance during [2002] [2003] [2004] [2005] [2006] [2007] , the overall virus isolation rate from migratory ducks was less than 1%, which suggests a low prevalence of viral infection during the birds' southern migration. similar results have been observed in taiwan, which is also an important stopover site for migratory birds along the eastern asia-australia migration route during 10 years of surveillance. 10 the overlap in seasonal patterns of virus infection between migratory and sentinel ducks found in our study suggests that virus movement or transmission between migratory and sentinel ducks occurred during the period of time migratory birds were at poyang lake. the ha subtypes harbored in migratory and sentinel ducks were similar in our study. for migratory ducks, h4, h6, h10 were the predominant subtypes, while h3, h4, and h10 were the major subtypes in sentinel ducks. hpai h5n1 was only detected from migratory ducks in early 2005 on two sampling occasions. from phylogenetic analyses the h5n1 viruses isolated from migratory ducks were closely related to the viruses endemic in domestic poultry in southern china. 8 therefore, it appears that h5n1 viruses endemic in domestic poultry could be transmitted to migratory ducks via close contact in southern china. only lp h5 viruses were detected from sentinel ducks at poyang lake during this period. whether h5n1 virus infection was absent from sentinel ducks at poyang lake needs further investigation. serological surveys provided further evidence for the prevalence of aiv in migratory ducks at poyang lake. the serological results in 2004 did not match well with the epidemiological results during [2002] [2003] [2004] [2005] [2006] [2007] , which suggests that influenza virus infection in migratory birds could be influenced by multiple factors, such as host immune status, population size, spatial and temporal variations, and migration routes. southern china has the biggest domestic duck population in the world. our study demonstrates that dynamic interactions between migratory ducks and sentinel ducks occurred frequently throughout the surveillance period. thus, sentinel ducks could be treated as intermediate hosts between the ''real gene pool'' from migratory ducks and domestic poultry in the whole influenza virus ecosystem. a sentinel duck sampling system may be a feasible method to represent the viruses in the natural gene pool and a baseline for virus or gene interactions between migratory and domestic ducks. 11 further investigations and surveillance are required to better understand the role of the domestic duck population in facilitating virus interactions and the generation of genetic diversity. two distinct lineages of h9n2 influenza viruses represented by a ⁄ chicken ⁄ beijing ⁄ 1 ⁄ 94 (ck ⁄ bei-like) and a ⁄ quail ⁄ hong kong ⁄ g1 ⁄ 97 (g1-like) have become established and endemic in poultry in southern china. 1 these established h9n2 lineages continue evolving to generate many different reassortant variants (or genotypes) 1,2 and are causing sporadic cases of human infection. 3, 4 studies of h9n2 viruses isolated from pigs in hong kong and shandong province have also raised the possibility of reassortment with human-like viruses from pigs. 5, 6 in addition, h9n2 viruses isolated beyond the late 1990s had preferential binding with a-2,6-neuacgal human-like receptors. 7 these observations suggest that the h9n2 influenza viruses still have pandemic potential. unlike highly pathogenic h5n1 influenza viruses that have been rarely detected in the live-poultry markets in hong kong since 2002, h9n2 viruses are still frequently isolated in our surveillance program. therefore, we try to understand the continuing evolution of h9n2 viruses through genetic characterization and phylogenetic analyses of the viruses isolated in hong kong live-poultry markets from 2005 to 2009. a total of 47 421 terrestrial poultry were sampled at different live-poultry markets in the hong kong sar between january 2005 and december 2009. of those samples, 29 691 were from chickens and the others were from minor poultry species including chukar, pheasant, guinea fowl, silky chicken, and pigeon. fecal droppings, cloacal and tracheal swabs, drinking water, and environmental samples from cages were collected into transport medium. viruses were isolated in 9-to 11-day old embryonated eggs as described previously. 8 virus isolates from positive sampling occasions were selected for sequence analysis. rna extraction, cdna synthesis, and pcr were carried out as described previously. 8 dna sequencing was performed using bigdye terminator v3ae1 cycle sequencing kit on an abi 3730 dna analyzer (applied biosystems) following manufacturer's instructions. all sequences were assembled and edited with lasergene 7ae0 (dnastar, madison, wi) software. sequence alignment and residue analysis were performed with the bioedit sequence alignment editor, version 7ae0. 9 all eight gene segments of sequenced viruses were characterized and analyzed phylogenetically together with virus sequence data available in public databases. maximum-likelihood trees were constructed using garli 0ae96. 10 estimates of the phylogenies were calculated by performing 1000 neighbor-joining bootstrap replicates using paup*4ae0. 11 systematic surveillance of live-poultry in hong kong from 2005 to 2009 resulted in 1088 h9n2 isolates from 47 421 samples (overall isolation rate, 2ae3%) ( table 1 ). there were 966 strains isolated from 42 253 chicken samples (isolation rate, 2ae3%). of these viruses, four were isolated from 1556 tracheal swabs (isolation rate, 0ae3%), while 541 isolates were isolated from 29 030 cloacal or fecal swabs (isolation rate, 1ae9%). an additional 421 isolates were collected from 6926 drinking water samples (isolation rate, 6ae1%). there were 122 strains of h9n2 viruses isolated from 5168 minor poultry samples (isolation rate, 2ae4%) ( table 1) . of these viruses, only one was isolated from 1209 tracheal swabs (isolation rate, 0ae1%), whereas 72 strains of viruses were isolated from 3610 cloacal or fecal swabs (isolation rate, 2ae0%). the isolation rate in drinking water in minor poultry was again higher when compared with other sampling methods with 49 strains isolated from 297 drinking water samples (isolation rate, 16%). taken together, these findings suggest that the h9n2 viruses mainly replicated in the intestinal tract of chickens and minor poultry species. also, the high isolation rate in drinking water samples could be a sensitive indicator for monitoring the prevalence of h9n2 viruses in the field. to better understand the evolutionary pathway of h9n2 viruses in southern china, 50 representative viruses, isolated from hong kong live-poultry markets from 2005 to 2009, were sequenced and genetically characterized. phylogenetic analysis of the h9 ha gene revealed that ck ⁄ bei-like viruses were predominant and one chicken isolate had a g1-like ha gene ( figure 1 ). this is the first time the g1like h9 ha gene has been detected in chickens from livepoultry markets in hong kong. the ck ⁄ bei-like lineage is further divided into two subgroups as previously described. 1 subgroup 1 is represented by qa ⁄ st ⁄ 243 ⁄ 00 and subgroup 2 is represented by dk ⁄ hk ⁄ y280 ⁄ 97. all h9n2 viruses in this study belonged to subgroup 2 of the ck ⁄ bei-like lineage except for the virus with the g1-like ha gene. phylogenetic analysis of the na gene also showed a similar evolutionary pattern to the ha gene with all viruses clustered within the ck ⁄ bei-like lineage. these results revealed that ck ⁄ bei-like viruses are predominant in both chickens and minor poultry. all of the pb1, pa, np, ns and m genes clustered with those of h9n2 lineage viruses previously prevailing in terrestrial poultry in southern china. phylogenetic analysis of the pb2 gene revealed three different lineages; g1-like (n = 1), ck ⁄ sh ⁄ f ⁄ 98-like (n = 15), and unknown avian (n = 34). the sh ⁄ f ⁄ 98-like lineage (or f ⁄ 98-like) was previously reported in eastern china 12 and was used previously for vaccine production in an intensive vaccination program. 13 this pb2 gene lineage was also distinguishable from the ck ⁄ bei-like lineage and its presence in the viral genome may be due to reassortment between the vaccine strain and field isolates, followed by selective establishment in terrestrial poultry. gene constellation analyses of the 50 viruses revealed six genotypes. thirty-four of the viruses analyzed belonged to two genotypes, b14 and b15, which were also the prevailing reassortants found in other provinces in southern china since 2005. 2 the remaining sixteen viruses belonged to four novel genotypes that have not been identified before in this region. characterization of h9n2 influenza viruses isolated from live poultry in hong kong markets from a 5 year surveillance program revealed that ck ⁄ bei-like viruses were predominant in southern china and were continuing to evolve. two recognized and four novel genotypes were identified in this study. one characterized virus, ck ⁄ hk ⁄ nt499 ⁄ 07, had a g1like ha gene (the first time this has been detected in hong kong poultry markets) that showed a close relationship with two human h9n2 strains isolated in 2009. g1-like viruses were usually detected and caused outbreaks in chickens of middle eastern and european countries, [14] [15] [16] and minor poultry, mainly quail, in southern china. 1 whether the g1-like virus was transmitted from china to middle eastern and european countries, as the highly pathogenic h5n1 virus did in the last five years, or vice versa, is still unknown. since the ck ⁄ hk ⁄ nt499 ⁄ 07 strain clustered with other g1-like strains isolated previously in minor poultry in southern china, the g1-like viruses in chicken may be due to interspecies transmission from minor poultry species. genetic studies demonstrated that reassortants with genotypes b14 and b15 persistently occurred in either chickens or other minor poultry species from 2005 to 2009. other genotypes that were prevalent in southern china might be being gradually replaced and four novel genotypes were identified in this study. these novel genotypes were generated through reassortment of viruses with different lineages. a newly emerged f ⁄ 98-like lineage originating from eastern china is responsible for generation of some of the novel genotypes found in this study. 12 the ck ⁄ bei-like lineage is gradually being replaced by f ⁄ 98-like lineages which are becoming dominant in northern and eastern china. 12, 17 animal experiments have also demonstrated that f ⁄ 98-like viruses are more effective in replication and transmission in chickens compared with ck ⁄ bei-like viruses. 18 since the f ⁄ 98-like lineage of the pb2 gene has been introduced into southern china, this newly emerged lineage may have a higher tendency to replace the rnp genes in the circulating ck ⁄ bei-like viruses and subsequently become the endemic virus in terrestrial poultry. in vietnam, the modelling of the pandemic h1n1 progression estimates that 460 000 (260 000-740 000) pigs might be exposed to the virus on the basis of 410 000 cases among swine owners (220 000-670 000). 1 a poor level of biosecurity, high animal densities, and a mix of species could increase the risk of influenza virus flow, persistence, and emergence on swine and poultry farms. this study was set up in the red river delta, where a third of the national pig husbandry is produced. 2 the aims are to give preliminary information of the epidemiological state of swine influenza and in order to further assess the risk of infection of swiv, through cross-species transmissions from poultry to pigs. this paper will present the preliminary results on swiv and the risk factors of pig seropositivity in vietnam. a cross-sectional study was conducted in two provinces of the red river delta in april 2009. pig farms were randomly selected from nine communes representative of at risk area of avian h5n1. in each farm, pig and poultry were sampled and collected to virological and serological analyses. interviews were conducted in all farms by trained interviewees. questionnaires included closed and open questions on ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 60-78 livestock husbandry ⁄ management and household characteristics, such as herd size and structure, health history and vaccination, pig housing, watering and feeding system, reproduction, purchasing of animals, biosecurity measures, pig contact with poultry, and environmental factors. the virological detection assay was performed on pools of nasal swab specimens from pigs. we investigated whether real-time rt-pcr assay could detect gene m on pools of nasal swab specimens before attempting virus isolation from individual nasal swab specimens. the poultry and pig sera were tested against influenza type a with an enzyme-like immunosorbant assay (elisa) competition test idvetª. this commercial kit is designed to specifically detect antibodies directed against the np protein antigen of influenza type a viruses. the positive serum samples were examined in hemagglutination inhibition (hi) to determine antibody titers and subtypes. the hi test was tailored for h1, h3, and h9 subtypes in pigs and h6 and h9 subtypes in poultry. seroneutralization tests by pseudo particles were used to test the presence of antibodies directed against h5 subtype. we analysed the data for relationships between influenza a serological status (the outcome variable) and possible risk factors using r version 2ae11ae1 (r development core team). the statistical unit was the individual. initially, the quantitative variables were encoded into categorical variables according to the quartiles or median. descriptive statistics (e.g., means or medians, proportions, standard deviations) were calculated for all herd-level and commune level predictors to assist in the subsequent modeling process. we also performed the independence test among all variables to determine if variables were dependant. then, univariate analysis of potential risk factors for the pigs being positive for swiv and estimation of odds ratios were performed using generalised linear mixed models with binary outcome and logit link function for each herd-level and commune-level variable to determine which variables were individually associated with influenza a seropositivity at a significance level of p < 0ae30. herd and commune of residence were included as a random effect to account for the correlation of observations at the herd level. the third stage of the analyses included the four herdlevel variables found to be significantly (p < 0ae30) associated with influenza a seropositivity. an automatic process using all possible associations between the selected variables was computed into a mixed logistic regression models, with random effects. when two variables were collinear, as determined before, only one variable was likely to enter the multivariable model, and therefore, the selection of which collinear variable to enter the model was guided by biological plausibility and statistical significance. all of the 146 pools of nasal swabs were rt-pcr negative. the maximal possible prevalence considering perfect diagnostic tests would be of 2ae03% at a confidence level of 95%, in an infinite population within these regions (win-episcope 2ae0). six hundred-and-nine pig sera were tested in 76 nonvaccinating farms. the herd seroprevalence of swine influenza in the commune previously infected by the avian h5n1 in the red river delta raised by 17ae1% [8ae7; 25ae6] in april 2009. but among 13 seropositive farms, only four had at least two seropositive pigs. the within-herd seroprevalence is very low, and no seropositivity was detected in the majority of farms. estimates had large confidence intervals due to small sample sizes. the individual seroprevalence raised 3ae62% [1ae98; 5ae27]. the subtyping of seropositive sera is still in process. descriptive statistical analyses on five major risk factors of swiv: farm size, breeding vs. fattening, purchasing, percentage of family income, and poultry production, were conducted. based on this analysis, three types of farming systems were identified and included in mixed models ( table 1) . percentage of family income by pig production and poultry production were not differentiating factors for this typology. whereas types 1 and 2 seem to be specialized in fattening, the type 3 produces and might sell piglets on the farm site. the exploration of the different variance components indicated that the random effect variances were mainly associated with the herd, while the commune did not seem to have any effect. therefore we included in all models only the herd as a random effect. the random effect term for herd was modelled, assuming a normal distribution with a table 1 . typology of farming system type 1: large fattening farms largest scale production, with more than 40 pigs per year specialized in fattening, and purchase more than 20 pigs per year type 2: small fattening farms small scale of production, with less than 20 pigs per year specialized in fattening, and purchase less than 20 pigs per year type 3: medium breeding-fattening farms medium scale of production, with less than 40 pigs per year breeding and fattening piglets, with rare purchase common variance [$n(0,r2herd)]. 3 the univariate analyses were conducted on 22 variables and typology variables, with herd as random effect. some coefficient or confidence intervals were inconsistent because of small effectives, especially for the percentage of self-product culture or the pig freegrazing because of the lack of positive results in the dataset. the only one significant (p value < 0ae1) parameter was the percentage of pig sales in the familial annual income. surprisingly, common risk factors of swine influenza infection, such as farm size, animal movements, and sanitary parameters got low odds ratio individually (without being significant); the typology provides the hypothesis of complex interactions effects that increase the risk of infection. as shown in table 2 , the farming system type 3 got a higher seroprevalence of 6ae47% [3ae00-11ae94] and a higher risk indicator, with or = 5ae26 (p-value = 0ae18) in comparison with type 1. this finding was not significant. in the multivariate mixed model, the percentage of familial income provided by pig production was the only one significant variable, with or = 0ae22 [0ae04-1ae25]. the focus on diseased animals in the winter-time is usually required in order to increase the likelihood to isolate the virus, although the isolation rate on healthy or clinical samples never exceed 6%. 4 the season and the lack of disease reports might explain the difficulties to detect influenza viruses. additionally, the pooling method tends to decrease the isolation rate because of a dilution effect, potential presence of pcr assay inhibitors, or uneven distribution of virus in the sample. 5 our seroprevalence results must be confirmed and the subtypes identified, especially because we found only one positive animal in a few farms that could be attributed to false positive results of the elisa test (performances are not known). these preliminary results are in favor of a virus circulation at low level in the spring, but must be completed by further surveys in the winter and before the new year (têt celebration) when pig production, trade, and movement increase at their maximum. no clear prior information on the expected prevalence of swine influenza in vietnam, tests sensitivity, and speci-ficity could be obtained from literature or reliable sources. bayesian methods will be carried out in the future in order to compute prevalence and ⁄ or to estimate the probabilities of freedom. the risk factors analysis was limited by the lack of positive results. further studies are necessary to identify the at-risk season and type of farming systems at risk of swine influenza infection. however, this investigation of risk factors leads to the hypothesis that medium size breeding-fattening farms had a higher risk than large or small size fattening farms. further investigation are needed to precise this typology. the risk of swiv infection increases with a combination of three major factors. poultry production does not seem to play any role on swine infection. the generalized linear mixed model afforded to take into account all the non investigated parameters at the herd level. although we investigated the most common risk factors of swine influenza infection covering different kind of fields, the herd random effect might explain risk variations. mixed models have become a frequently used tool in epidemiology. due to software limitations, random effects are often assumed to be normally distributed. since random effects are not observed, the accuracy of this assumption is difficult to check. 6 further studies, such as case-control or cohort studies could help to identify more precisely risk factors of swine influenza seropositivity, as these study designs are more adapted than cross-sectional studies. the concept that swine are a mixing-vessel for the reassortment of influenza viruses and for the emergence of pandemic influenza viruses has been re-enforced by the emergence of the recent pandemic. 1 the pandemic h1n1 virus of 2009 (h1n1pdm) is believed to have emerged through the reassortment of north american triple reassortant and eurasian avian-like swine influenza viruses. 2 since the immediate precursor of this pandemic virus has not yet been identified, it is not possible to be definite whether the reassortment leading to the pandemic occurred in swine, but swine influenza viruses are the nearest known ancestors of each gene segment of h1n1pdm. 1, 2 the mechanisms of pandemic emergence are not clear. it is believed that the pandemics of 1957 and 1968 arose through reassortment of the pre-existing human seasonal influenza virus with avian influenza viruses, and swine have been proposed to be a possible intermediate host where such reassortment between human and avian viruses may take place. 4 the 2009 pandemic was the first to arise for over 40 years and the first to occur after the understanding that pandemics arise from animal influenza viruses. 3 systematic studies of influenza virus ecology and evolution in swine are, therefore, important in order to understand the dynamics of pandemic emergence. furthermore, since swine are the likely host within which h1n1pdm virus originated, it was predicted that this virus would readily infect swine and may reassort with endemic swine influenza viruses. these predictions have now been confirmed with reports of h1n1pdm being detected in pigs in many countries and reassortment with endemic swine influenza virus being confirmed. 5 while h1n1pdm has been genetically and antigenically stable in humans, reassortment between h1n1pdm, which is well adapted to transmission in humans, and other avian or swine viruses may lead to the origin of novel viruses posing a threat to public health. in addition to endemic swine virus lineages, avian influenza viruses such as h9n2 and highly pathogenic avian influenza (hpai) h5n1 have also been occasionally identified in pigs in parts of asia. 6, 7 it has been shown that h1n1pdm readily reassorts with h5n1 to generate viable progeny in vitro. 8 it is therefore essential to monitor the ecology, evolution, and biological characteristics of swine influenza viruses so that their continued evolution and zoonotic and pandemic potential can be monitored. there is however, a paucity of surveillance data on swine influenza viruses worldwide. this is in part related to the negative commercial consequences that may arise from detection of influenza in a swine herd leading to a major economic loss to the producer. here we outline a surveillance system that has been in place in hong kong for the last decade, based on sampling animals arriving at an abattoir in hong kong. we demonstrate the feasibility of such surveillance in an abattoir setting and compare methods used for detection influenza viruses in swine. virus isolation was carried out by inoculation into mdck cells and by allantoic inoculation in embryonated eggs as previously described. 6 virus isolates were subtyped by haemagglutination inhibition tests using specific antisera and genetically characterized by sequencing and phylogenetic analysis of the haemagglutin gene. 2, 6 virus detection by rt-pcr a subset of 1154 recent specimens was tested in parallel by real time pcr using the biorobot universal system (qiagen) that enables fully-automated viral nucleic acid extraction and downstream reaction setup in a 96-well plate format. total viral nucleic acids were extracted in a 96-well plate format with the qiaamp virus biorobot mdx kit (qiagen) on the biorobot universal system (qiagen) according to the manufacturer's instructions. briefly, 140 ll of sample was lysed in 280 ll buffer al, supplemented with 5ae6 lg carrier rna in a s block (qiagen), which placed the samples into a 96 well plate format. after protease digestion, samples were transferred to silica based membrane in 96 well plate format for binding. following two washing steps, rna was eluted in 80 ll of elution buffer (buffer ave) into a 96 well elution microplate cl (qiagen) . for the synthesis of cdna, 10 ll of purified rna was used in a 20 ll reaction containing 4 ll of 5· buffer, 0ae75 nm of each deoxynucleotide triphosphate (dntp), 10 mm dithiothreitol, 3 lg random primer, 40 u of rnaseout recombinant ribonuclease inhibitor, and 200 u of superscript iii reverse transcriptase (all from invitrogen). reactions were performed in the geneamp 9700 thermocycler (applied biosystems) with the following parameters: 60 minutes at 50°c, 15 minutes at 72°c, and soak at 4°c. subsequent to the reactions, 20 ll of cdna was diluted 1 ⁄ 10 by adding 180 ll of ae buffer (qiagen) . real-time pcr was performed using the power sybrò green pcr master mix (applied biosystems) according to the manufacturer's instructions. briefly, 5 ll of 1⁄ 10 diluted cdna was amplified in a 25 ll reaction containing 12ae5 ll of 2· power sybr green pcr master mix, 800 nm of forward primer m52c (5¢-ctt cta acc gag gtc gaa acg-3¢) and 800 nm of reverse primer m253r (5¢-agg gca ttt tgg aca aag ⁄ t cgt cta-3¢). the primers have been designed to amplify the sequences in the conserved region of influenza a virus matrix gene, thereby detecting viruses from different species including swine influenza viruses. 8 real-time pcr was performed in the abi 7500 fast system (applied biosystems) with the following cycling conditions: 10 minutes at 95°c once, 30 seconds at 95°c, and 1 minutes at 50°c for 40 cycles, followed by melting curve analysis with 15 seconds at 95°c, 1 minutes at 50°c, and 15 seconds at 95°c. in each assay, serially diluted plasmids containing the full length m gene cloned from a ⁄ vietnam ⁄ 1204 ⁄ 2004 (h5n1) were included as standards to perform absolute quantification. a manual baseline was set from cycles 3-15 and a manual cycle threshold (ct) was set at 0ae2. samples that were positive or unequivocal results from the real-time pcr were confirmed by performing gel electrophoresis on the pcr products. positive visual identification was made in the presence of the target pcr product at 244 bp in length. a total of 16 566 tracheal and 11 100 nasal swabs were processed during the years january 2002-april 2010 and yielded 393 influenza virus isolates, an overall virus isolation rate of 1ae4%. of these, 352 were subtype h1 (classical swine, eurasian avian-like swine, and triple-reassortant), 25 were human-like h3 viruses, and 16 were eurasian avianlike swine h3n2 viruses. culture in mdck cells yielded 95% of h1 subtype viruses, 100% of the human seasonal-like h3n2 viruses, and 68ae8% of the avian-like eurasian swine h3n2 viruses. culture in embryonated eggs yielded 17ae9% of the h1 subtype viruses, 0% of the human seasonal-like h3n2 viruses, and 62ae5% of eurasian avian-like swine h3n2 viruses ( figure 1 ). tracheal and nasal swabs each gave comparable overall virus isolation rates (1ae4%). however, isolation rates for human-like h3n2 viruses were 4ae8 fold higher in nasal swabs (0ae17% versus 0ae036% respectively; p = 0ae002) ( figure 1) . a parallel evaluation of rt-pcr and culture was carried out in 1154 specimens. rt-pcr detected 10 ⁄ 12 (83%) of the culture positive specimens. rt-pcr was also positive in 8 ⁄ 1154 (0ae7%) culture negative specimens, but all these specimens had very low virus load in the rt pcr tests. virus could not be cultured from these culture negative specimens even by attempts at virus re-isolation from the frozen specimen. surveillance in an abattoir setting provides an acceptable yield of influenza viruses and is a feasible method of swine influenza surveillance. sampling in a large abattoir setting allows surveillance to be carried out anonymously with no negative consequences to the supplier. the supply-chain of pigs to the hong kong abattoir involves pigs being trucked in over long distances and may provide opportunity for virus amplification during transport. thus, virus isolation rates may be lower in more vertically integrated and homogenous production and slaughter systems where less mixing of pigs occurs. our results indicate that mdck cell culture is essential for optimizing virus isolation during swine influenza surveillance. allantoic inoculation of embryonated eggs by itself is sub-optimal for isolation of swine influenza viruses. it is however possible that inoculation of embryonated eggs by the amniotic route may lead to better isolation rates than allantoic inoculation. rt-pcr detection is an alternative method for virus detection. but the additional specimens detected by rt-pcr did not yield culturable virus, even following attempts at re-isolation and sequential passage. the rt-pcr positive ⁄ virus isolation negative specimens had very low virus load, and this may be the explanation for the inability to isolate such viruses. in addition, rt-pcr did not detect all viruses isolated by culture. tracheal and nasal swabs gave comparable isolation rates with the exception of human-like h3n2 viruses which were more frequently isolated from nasal swabs. this may suggest that, in contrast to endemic swine influenza virus lineages, these human-like h3n2 viruses are less adapted to replication in the lower respiratory tract. in summary, collection of nasal or tracheal swabs in an abattoir setting together with virus isolation in mdck cells provides a feasible approach to surveillance of swine influenza viruses. kong, 2003 kong, -2010 introduction wild waterfowl are the natural reservoir of influenza a viruses (aiv), and they play an important role in the genesis of pandemic influenza. it is suggested that the 1918 pandemic virus was purely derived from avian virus, which adapted to humans and caused efficient human-to-human transmission, 1 while the pandemics of 1957 and 1968 had acquired the viral haemagglutinin, pb1 polymerase, and in 1957, the neuraminidase gene segments from the avian gene pool. 2 the major regional outbreaks of highly pathogenic avian influenza (hpai) h5n1 in asia, europe, and africa highlight the potential role played by migratory waterfowl in disseminating highly pathogenic influenza viruses. 3 therefore defining the influenza virus gene-pool in wild birds is of vital importance. surveillance was carried out 2-3 times weekly from 2003 to 2010 during the winter months of october to april in the hong kong mai po nature reserve and lok ma chau, hong kong. the hong kong mai po nature reserve and lok ma chau are along the east asia-australian flyway where a peak of more than 30 000 ducks and grebes congregate every winter. 4 fecal droppings were collected and transported in vials containing 1ae0 ml of vtm, which was prepared from m199 (9ae5 g ⁄ l), penicillin g (2 · 10 6 u ⁄ l), polymyxin b (10 · 10 6 u ⁄ l), gentamicin (250 mg ⁄ l), nystatin (0ae5 · 10 6 u ⁄ l), ofloxacin hcl (100 mg ⁄ l), and sulfamethoxazole (1 g ⁄ l). an aliquot of 100 ll from each swab sample was inoculated into the allantoic cavity of a 9-to 11-day-old chicken embryonated egg, and incubated for 3 days at 37°c. positive ha isolates were subtyped using standard antisera 5, 6 and rt-pcr was performed with the used of one-step rt-pcr assay (invitrogen) described earlier, 7 followed by sequencing on abi prism 3730xl dna analyzer. the determination of species of origin was performed by dna barcoding of the mitochondrial cyto-chrome oxidase i gene from dna extracted from the fecal droppings. 8 during the 7-year surveillance period, a total of 193 influenza viruses were isolated from 39 354 samples collected, an overall isolation rate of 0ae49%. a total of 192 isolates were obtained from 39 152 specimens collected during the winter period coinciding with the southern migration of waterfowl along the east asian flyway and one isolate obtained from 202 samples collected in spring during the period when northern migration of waterfowl took place along the east asian-australasian flyway. the isolation in hong kong was slightly lower than a similar study conducted in south korea in which the isolation rate of migratory birds was 0ae8% in 2003-2008. 9 this suggested a slightly lower prevalence of influenza virus present in hong kong as the birds migrated southwards. the viruses isolated in hong kong, representing hemagglutinin (ha) subtypes of h1-h12 and neuramidinase (na) subtypes of n1-n9, were all from wild waterfowl ( table 1) . out of the twelve ha subtypes isolated, h10 and h6 were the two subtypes that were isolated frequently every year for h10 and in six out of seven years for h6, respectively. h10 and h6 viruses accounted for 16ae7% and 22ae4% of all virus isolated, respectively. on the other hand, h3, h9, and h12 were the least prevalence (0ae5%) and were only isolated once in 7 years. of the na subtypes, n1 and n2 were isolated most often (24ae4% and 19ae2% of all isolates, respectively) and n5 was the least (0ae5%). november was the month that had the highest prevalence of influenza virus (0ae83% of samples being positive) compared to only 0ae3% in march. the subtype's variation was the most diverse in december during our 7 years of surveillance. this suggested that more of these wild migratory birds may be carrying influenza virus when they arrive in hong kong. however the continued isolation of viruses suggests continued circulation of these viruses in the vicinity of mai po. the study of dna barcoding for the mitochondrial cytochrome oxidase i gene retrieved from fecal droppings revealed that the isolates originated mainly (89ae2%) from birds of the order anseriform, family anatidae including eurasian wigeon, northern shoveler, northern pintail, common teal, and garganey. non-anseriformes which were found to have shed aiv viruses were cormorant, grey heron, and stint. none of the water samples collected from the ponds where these birds congregate were found to be positive for the virus. phylogenetic analyses of the ha gene of the lpai h5 viruses isolated in this study clustered with that of the other lpai h5 viruses isolated from hokkaido, mongolia, and siberia and were not closely related to the hpai h5n1. satellite tracking of 47 eurasian wigeons and northern pintails in dec 2008 and 2009 revealed their flyway from hong kong to as far north as eastern russia, eastern mongolia, and northern china. no hpai h5n1 viruses were isolated in this study from apparently healthy birds. however, as part of the surveillance of dead wild birds carried out by the department of agricultural, fisheries and conservation of the government of hong kong during this same period, over 50 dead wild birds were tested positive for hpai h5n1 and has been reported elsewhere. 10 our influenza surveillance in hong kong has revealed a diversity of influenza virus subtypes the migratory waterfowl infected within the region. the result of the phylogenetic analysis correlated with the findings from satellite tracking that viruses isolated in hong kong were closely related to those isolated in areas along the migratory route. no healthy bird was isolated with hpai h5n,1 although dead wild birds have been regularly found to have hpai h5n1 virus, suggesting that infected birds might not live for a long period. introduction a novel swine-origin h1n1 influenza virus emerged in mexico in april 2009 and rapidly spread worldwide, causing the first influenza pandemic of the 21st century. 1 most confirmed human cases of h1n1 ⁄ 2009 influenza have been uncomplicated and mild, but the increasing number of cases and affected persons worldwide warrant optimal prevention and treatment measures. today, almost all of the pandemic h1n1 ⁄ 2009 viruses tested are resistant to m2blockers. 2 therefore, only the neuraminidase (na) inhibitors are currently recommended for treatment of this pandemic influenza. for the control of influenza infection, the clinical use of oseltamivir has increased substantially during the 2009 pandemic. to date, the majority of tested clinical isolates have remained susceptible to na inhibitors, oseltamivir and zanamivir, 2 but oseltamivir-resistant variants with h275y na mutation (n1 numbering) have been isolated from individuals taking prophylaxis, from immunocompromised patients, and from a few community clusters. 3, 4 in view of the high prevalence of oseltamivirresistant seasonal h1n1 influenza viruses in 2007-2008, the isolation of resistant h1n1 ⁄ 2009 viruses without known oseltamivir exposure raised great concern about the transmissibility and fitness of these resistant viruses. here we studied the transmissibility of a closely matched pair of pandemic h1n1 ⁄ 2009 clinical isolates, one oseltamivir-sensitive and one resistant, in both direct contact and respiratory droplets routes among ferrets. viral fitness was evaluated by co-infecting a ferret with both the oseltamivir-sensitive and -resistant viruses. the viruses were also characterized by full genome sequencing, susceptibility to na inhibitors, and growth in mdck and mdck-siat1 cells. oseltamivir-resistant influenza a ⁄ denmark ⁄ 528 ⁄ 09 (h1n1) virus (a ⁄ dm ⁄ 528 ⁄ 09) was isolated from the throat swab of a patient who had influenza-like symptoms and received post-exposure oseltamivir prophylaxis (75 mg once daily). 5 wild-type influenza a ⁄ denmark ⁄ 524 ⁄ 09 (h1n1) virus (a ⁄ dm ⁄ 524 ⁄ 09) was isolated from a patient in the same cluster of infection as the a ⁄ dm ⁄ 528 ⁄ 09 virus. to assess growth kinetics of viruses, confluent mdck or mdck siat1 cell monolayers were infected with viruses at a multiplicity of infection (moi) of approximately 2ae0 pfu ⁄ cell (single-step) or 0ae001 pfu ⁄ cell (multi-step). supernatants were collected every 2 h or 12 h p.i. for 6 time points. a modified fluorometric assay using the fluorogenic substrate 2¢-(4-methylumbelliferyl)a-d-n-acetylneuraminic acid (munana) was used to determine viral na activity. 6 the drug concentration required to inhibit 50% of the na enzymatic activity (ic 50 ) was determined by plotting the percent inhibition of na activity as a function of compound concentration calculated in the graphpad prism 4 (la jolla, ca) software from the inhibitor-response curve. na enzyme kinetics were determined by measuring na activity every 60 seconds for 60 minutes under the same conditions as above, when all viruses were standardized to an equivalent dose of 10 6ae0 pfu ⁄ ml. the k m and v max were calculated by fitting the data to the appropriate michaelis-menten equations using nonlinear regression in the graphpad prism 4 software. young adult ferrets (4-5 months of age) were obtained from the ferret breeding program at st. jude children's research hospital. all ferrets were seronegative for influenza a h1n1 and h3n2 viruses and for influenza b viruses. for transmission studies, the donor ferrets were lightly anesthetized with isoflurane and inoculated intranasally with 10 6 tcid 50 virus in 1ae0 ml sterile pbs . after the donor ferrets were confirmed to shed virus on day 2 p.i., each donor was then housed in the same cage with two naïve direct-contact ferrets. two additional recipient ferrets were placed in an adjacent cage isolated from the donor's cage by a two layers of wire mesh (approximately 5 cm apart) that prevented physical contact but allowed the passage of respiratory droplets. ferret weight and temperature were recorded daily for 21 days. nasal washes were collected from donors and recipients on day 1, 2, 4, 6, 8, 10, 12, and 14 p.i. by flushing both nostrils with 1ae0 ml pbs, and tcid 50 titers were determined in mdck cells. serum samples were collected 3 weeks after virus inoculation, and were tested for seroconvention by hi assay. full genome sequencing revealed that the pair of h1n1 ⁄ 2009 viruses differed only at na amino acid position 275, where the pandemic a ⁄ dm ⁄ 528 ⁄ 09 virus had an h275y amino acid mutation caused by a single t-to-c nucleotide substitution at codon 275. the wild-type a ⁄ dm ⁄ 524 ⁄ 09 was susceptible to oseltamivir carboxylate (mean ic 50 : 5ae0 nm), but the a ⁄ dm ⁄ 528 ⁄ 09 carrying the h275y na mutation had ic 50 values approximately 200-300 times of the wild-type viruses (mean ic 50 : 972 nm). the ic 50 of zanamivir was comparable for both viruses and were uniformly low (mean ic 50 £ 1ae3 nm). the h275y na mutation confers resistance to oseltamivir carboxylate but did not alter susceptibility to zanamivir. to understand the impact of the h275y mutation on the na enzymatic properties, na enzyme kinetics was determined. the na of the oseltamivir-resistant virus had a slightly higher k m (mean = 55 lm) and lower v max (mean = 101 u ⁄ sec) than na of the sensitive virus (k m , mean = 80 lm; vmax, mean = 86 u ⁄ sec). the results suggested that the h275y na mutation reduced na affinity for substrate and na catalytic activity, although the function of na was not severely impaired. to further evaluate the impact of the h275y na mutation on virus growth in vitro, single-and multi-cycle growth studies of both viruses were performed in mdck and mdck-siat1 cells. in the both single-and multiple-cycle growth curves, the two viruses reached comparable levels eventually, but the initial growth of the resistant virus was significantly delayed by at least 1-2 logs in comparison to that of wild-type virus (p < 0ae05). the donor ferrets inoculated with wild-type a ⁄ dm ⁄ 524 ⁄ 09 or oseltamivir-resistant virus shed virus productively until day 6 or day 8 p.i., with a peak virus titer comparable to that of a ⁄ dm ⁄ 524 ⁄ 09 virus (table 1 ). in a ⁄ dm ⁄ 524 ⁄ 09 virus group, two of 2 direct-contact ferrets the weight loss in ferrets is the maximum percentage loss compared with the initial weight. virus shedding is indicated as number of virus-shedding animals ⁄ total number; mean peak virus titer (log 10 tcid 50 ⁄ ml) in nasal wash samples is indicated in parentheses. serum hemagglutination inhibition (hi) titer to homologous virus in ferret serum was determined on day 21 p.i. duan et al. and 1 of 2 respiratory droplet-contact ferrets were infected through virus transmission, as indicated by the virus titers and inflammatory cell counts in their nasal washes and also by sero-conversion. under identical conditions, in a ⁄ dm ⁄ 528 ⁄ 09 group, only 2 of 2 direct-contact ferrets were infected through virus transmission, but neither respiratory droplet-contact ferrets was infected, as confirmed by the absence of sero-conversion (table 1) . virus shedding in the direct-contact ferrets was lower and peaked after a longer interval in this group than in the oseltamivir-sensitive a ⁄ dm ⁄ 524 ⁄ 09 group (table 1) , but the resistant viruses appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. these results showed that an oseltamivir-resistant h275y mutant of pandemic h1n1 virus, a ⁄ dm ⁄ 528 ⁄ 09 virus could be only transmitted efficiently by direct contact. to compare the relative fitness, growth capability, and transmissibility of the sensitive and resistant h1n1 ⁄ 2009 viruses within host, a donor ferret was co-inoculated with a 1:1 ratio of the sensitive and resistant viruses, and another two naive ferrets were housed with the donor to test direct contact. during co-infection, the pattern of virus shedding and the clinical signs were similar to those in ferrets inoculated with either a ⁄ dm ⁄ 524 ⁄ 09 or a ⁄ dm ⁄ 528 ⁄ 09 virus (table 1 ). in the inoculated donor ferret, the virus population in the nasal washes remained mixed but wild-type viruses outgrew the resistant virus progressively ( figure 1 ). two of 2 direct-contact ferrets were infected through virus transmission, but only wild-type virus was detected in both direct-contact ferrets ( figure 1 ). in summary, oseltamivir-sensitive a ⁄ dm ⁄ 524 ⁄ 09 virus possessed better growth capability in the upper respiratory tract than did resistant a ⁄ dm ⁄ 528 ⁄ 09 virus, and thus had an advantage in directcontact transmission. our study determined the comparative transmissibility of two naturally circulating oseltamivir-sensitive and -resistant pandemic h1n1 ⁄ 2009 viruses; we demonstrated inefficient respiratory-droplet transmission of an oseltamivir-resistant h275y mutant of pandemic h1n1 virus among ferrets, although it retained efficient direct-contact transmission. we suggest that the lower fitness of resistant virus within the host along with its reduced na function and delayed growth in vitro may in part explain its less efficient transmission. notably, the h275y mutant of h1n1 ⁄ 2009 used in this study was the first oseltamivir-resistant h1n1 ⁄ 2009 isolate from a patient on oseltamivir prophylaxis to be characterized for transmissibility. our observation in the animal model is consistent with the epidemiological data collected from humans, which showed no evidence of predominant or continued circulation of oseltamivir-resistant viruses. 4 as this study was undertaken, additional h275y mutants of h1n1 ⁄ 2009 viruses have emerged in the absence of oseltamivir use. 7, 8 the emergence of these viruses should raise concerns as to whether resistant h1n1 ⁄ 2009 viruses will acquire greater fitness and spread worldwide as the naturally resistant h1n1 viruses did during the 2007-2008 season. two independent studies have evaluated the pathogenecity and transmission of other oseltamivir-resistant pandemic h1n1 ⁄ 2009 clinical isolates in the animal models. 9, 10 one of the studies, 9 which also used an oseltamivir-resistant virus isolated from a patient under oseltamivir prophylaxis, observed similar results as ours: although the respiratory-droplet route of transmission was not investigated, it was shown that the resistant isolate was transmitted though direct-contact route and was as virulent as wild-type virus in ferrets. 9 in another study, 10 two oseltamivir-resistant isolates were transmitted through the respiratory-droplet route in ferrets, and the dynamics of transmission were different between the two isolates. apparently, these two oseltamivir-resistant isolates were still unequal in their transmissibility and were disparate from the resistant isolate in our study. the isolation history of the two resistant isolates was unclear in this study, and this would be an important factor to understand the fitness of drug-resistant viruses. further studies with more clinical isolates of diverse isolation background are warranted to identify how these novel h275y mutants of pandemic h1n1 ⁄ 2009 virus have changed to retain their full transmissibility. taken together, all these related studies underline the necessity of continuous monitoring of drug resistance and characterization of potential evolving viral proteins. this study was supported by contract hhsn266200700005c from the national institute of allergy and infectious diseases, national institutes of pigs have been considered as hypothetical ''mixing vessels'' facilitating the genesis of pandemic influenza viruses. 1, 2 the pandemic h1n1 ⁄ 2009 virus (ph1n1 ⁄ 09) contained a very unique genetic combination and was thought to be of swine origin, as each of its eight gene segments had been found to be circulating in pig populations for more than a decade. 3 however, such a gene constellation had not been found previously in pig herds all around the world. only after its initial emergence in humans has this virus been repeatedly detected in pigs, and found to further reassort with other swine influenza virus. [3] [4] [5] a primary question remaining to be answered is whether the ph1n1 ⁄ 09-like and their genetically related viruses could become established in pig populations, thereby posing novel threats to public health. despite the fact that ph1n1 ⁄ 09 first appeared in mexico and the united states, and six of its eight gene segments were derived from the established north american triple reassortant swine influenza virus (trig), its neuraminidase (na) and matrix protein (m) genes belonged to the eurasian avian-like swine lineage (ea), which had never been detected in north america previously. 3, 6 likewise, the trig-like viruses were never reported in europe. 6 in contrast, both lineages of virus were frequently detected in asia, and reassortants between them have also been documented in recent years. 3, 5 this has given rise to a complicated ecological situation, i.e. the simultaneous prevalence of multiple genotypes of h1n1 and h1n2 viruses in pigs. 3, 5 among them, two representative reassortants showed the most similar genotypic characterization to the ph1n1 ⁄ 09 virus, the sw ⁄ hk ⁄ 915 ⁄ 2004 (h1n2) and sw ⁄ hk ⁄ 201 ⁄ 2010 (h1n1), which respectively harbor seven and six gene segments closely related to the pandemic strains. 3 , 5 to understand their in vivo characteristics and zoonotic potential, these two viruses, together with a human prototype strain and a swine ph1n1 ⁄ 09-like isolate, were chosen for a study of their pathogenicity and transmissibility in domestic pigs, ferrets, and mice. the prototype ph1n1 ⁄ 09 virus, a ⁄ california ⁄ 04 ⁄ 2009 (ca04), was provided by the world health organization collaborating centers for reference and research on influenza (atlanta, ga, usa). three ph1n1 ⁄ 09-related swine influenza viruses were isolated through our surveillance program in south china as previously described. 3, 5 the a ⁄ swine ⁄ guangdong ⁄ 106 ⁄ 2009 (h1n1, gd106) virus was a ph1n1 ⁄ 09-like swine isolate. a ⁄ swine ⁄ hong kong ⁄ 915 ⁄ 2004 (h1n2, hk915), the closest pandemic ancestor known to date, possesses an m gene derived from the ea lineage, with the other gene segments from trig viruses. 3 a ⁄ swine ⁄ hong kong ⁄ 201 ⁄ 2010 (h1n1, hk201), a recent pandemic reassortant progeny, had a ph1n1 ⁄ 09like na gene (also belonging to the ea lineage), an ea-like hemagglutinin (ha) gene, and six trig-like internal genes. 5 all viruses were propagated in madin-darby canine kidney (mdck) cells for three passages, and their titers were determined by plaque assays. all experiments with live viruses were conducted in biosafety level 3 (bsl-3) containment laboratories. pigs (4-6 week old, n = 5-6) and ferrets (5 month old, male, n = 3) were intranasally infected with 10 6 pfu of each virus, and mice (8) (9) week old, female balb ⁄ c, n = 10) with a dose of 10 4 pfu. naïve uninfected pigs (n = 2) were co-housed in the same cage with the inoculated ones from each group. body weights and clinical signs were recorded daily. virus replication was determined by titration of the virus in nasal and rectal swabs (pigs), nasal washes (ferrets), as well as from lungs and other organs (pigs and mice). seroconversion was tested by hemagglutination inhibition (hi) assays. histopathological and immunohistochemical analysis were performed as previously described. 7 statistical analysis was performed by mean analysis with pasw statistics 18 (spss inc., chicago, il, usa). the probability of a significant difference was computed using anova (analysis of variance). results were considered significant at p < 0ae05. the pathogenicity of the four viruses tested differed significantly in inoculated mice. animals infected with 10 4 pfu of hk915 experienced the most severe body weight loss (25ae1 ± 4ae7%) but started to recover after 8 days post-infection (dpi). hk201 caused similar peak body weight loss (16ae9 ± 4ae6% on 8 dpi) in mice as did ca04 (17ae3 ± 2ae4%, on 7 dpi), but the onset of clinical signs and weight loss (on 4 dpi) was 1 day later than those caused by the other three viruses. the gd106-infected group suffered the least body weight loss (6ae9 ± 1ae9%, 5 dpi) and was the earliest to recover. although all four viruses were detected in the lungs with comparable virus titers on 3 dpi (p > 0ae05), mice inoculated with gd106 consistently showed the lowest lung index (lung weight ⁄ body weight, %) on 3, 6, and 14 dpi (p < 0ae01), suggesting the slightest injury and consolidation of the lungs. in concordance with the body weight change, the lung index from the hk915 group was higher than that from any other groups on 6 and 14 dpi, indicating the marked virulence of hk915 in mice. notably, virus titer of hk201 in the nasal turbinate was lower than the other groups both on 3 and 6 dpi (p < 0ae01), but virus replication in the lower respiratory tract was either higher (in the trachea) or similar (in the lungs). observations of the body weight changes caused by infection of ph1n1 ⁄ 09 or its genetically related swine viruses in ferrets have come to a similar conclusion as that for the mouse experiment. after nasal inoculation with 10 6 pfu of each virus, all groups of ferrets experienced transient body weight loss for 2-3 days, except for those infected with gd106, which showed no significant weight loss (p > 0ae05). although ferrets from the ca04-infected group reached their peak weight loss (6ae2 ± 0ae8%, 2 dpi) one day earlier than those from the hk201 and hk915 groups, they began to regain body weight quickly thereafter. hk201-infected ferrets also recovered rapidly and their body weights reached the same level as those of the gd106-infected group at 6 dpi. comparatively, ferrets inoculated with hk915 had the most retarded body weight recovery, which did not get back to the baseline level until 11 dpi. hk201 was only detectable in the nasal wash on 2 dpi, whereas the duration of virus shedding for gd106, hk915, and ca04 was 4-6 days. by combining the data obtained from the virus titration in the mouse turbinate and ferret nasal washes, a possible conclusion can be made that hk201 may have lower transmissibility than the other three viruses. after inoculation or exposure by direct contact (physical contact) with the ph1n1 ⁄ 09 virus and its close relatives, most pigs experience no or mild symptoms, such as slight loss of appetite and inactivity. body weight loss was only recorded in pigs inoculated with hk915 during the second week post-inoculation, but not in their contact pigs or in the other groups. diarrhea was observed intermittently in each of the inoculated or contact groups throughout the experiment, and viruses could be recovered in the rectal swabs, saliva, drinking water, and environmental swabs (inner cage walls accessible to the pigs) at various time points. however, virus titers in the positive rectal swabs were just slightly above the detection limit, while those from the environment sometimes could be higher. whether these viruses can replicate in the digestive tract or were just carried-over by contaminated foods and water requires further investigation. although virus could be detected in the nasal swabs of all infected or contact animals, the lowest peak titer was from pigs inoculated or in contact with hk201 (0ae5-1ae5 log tcid50 ⁄ ml lower than the other groups), suggesting unfavorable replication in the nasal cavity for this virus. postmortem examination on 4 and 7 dpi revealed that pigs infected with hk915 had the most extensive gross lesions in the lungs, and histochemical staining of viral nucleoprotein (np) in lung tissues on 4 dpi also suggested the best replication for hk915 in the lower respiratory tract. on 11 days post-contact (dpc), all pigs exposed to the inoculated animals developed sero-conversions (hi = 80-160) except for one from the gd106 contact group. however, on 17 dpc, its hi titer reached 40, indicating slower seroconversion. this study revealed that both the 2009 pandemic h1n1 and its genetically related swine viruses could readily infect mice, ferrets, and pigs causing mild to moderate clinical symptoms. they could also transmit efficiently between pigs. when compared with the pandemic stains and its reassortant progeny (hk201), the hk915 (h1n2) virus containing the ea-like m gene in the genetic context of the trig virus showed consistently higher virulence in all three mammalian models tested, but it is still unknown what might happen if such a virus further reassorts to obtain the pandemic-like or ea-like na gene. however, our findings suggest that pigs could likely maintain the prevalence of different genotypes of pandemic-related influenza viruses, and highlight the zoonotic potential of multiple strains of swine influenza virus. pandemic influenza viruses emerge from the animal reservoirs. 1 among the three pandemics that occurred in the last century, we learned that the 1957 h2n2 and the 1968 h3n2 pandemic viruses emerged by reassortment between circulating human virus and avian-origin influenza virus(es). 1 studies on the emergence of the catastrophic 1918 spanish h1n1 virus suggest that the virus may have obtained all of its eight gene segments from the avian reservoir, 2,3 or alternatively is a reassortant between mammalian and a previously circulating human influenza virus. 4 over 40 years since the last pandemic, the first pandemic in the 21st century arose in 2009 and was caused by a swine-origin influenza virus containing a unique gene combination, with gene segments derived from the circulating north america ''triple reassortant'' (pb2, pb1, pa, ha, np, and ns) and the ''eurasian'' (na and m) swine influenza viruses. 5, 6 analysis of the pandemic h1n1 viruses failed to identify known molecular markers predictive of adaptation to humans. 6 the ''triple reassortant'' swine influenza viruses emerged in late 1990s in north america is a reassortant between classical swine (descendent of the 1918 virus after adaptation in swine population), avian, and human influenza viruses. 7 the eurasian influenza virus was originally an avian influenza virus that was introduced into the european swine population in the late 1970s. 8, 9 while incidents of zoonotic infection with triple reassortant or eurasian influenza in humans have been reported, 10, 11 sustained human-to-human transmission has never been established. these results suggest that the unique gene combination seen with the pandemic h1n1 viruses may confer its transmissibility among humans. we have carried out systematic prospective surveillance of swine influenza in southern china over that last 12 years through samples routinely collected at an abattoir in hong kong. during this time, the surveillance results suggest co-circulation of classical swine h1n1, triple reassortant h1n2, eurasian swine h1n1, and a range of reassortants between these three virus lineages. 4, 12 ferrets have been reported as a suitable model for the study of influenza transmission as they are naturally susceptible to influenza infection, exhibit similar clinical signs (including sneezing), and possess receptor distribution in the airway similar to that of humans. [13] [14] [15] to identify molecular determinants that enable sustained human-to-human transmission, we compared the pandemic virus with genetically related swine influenza viruses obtained from this surveillance program for their ability to transmit from ferret to ferret by direct contact or aerosol transmission. viruses human h3n2 influenza virus [a ⁄ wuhan ⁄ 359 ⁄ 95 (wuhan95)] and pandemic h1n1 influenza viruses [a ⁄ california ⁄ 4 ⁄ 09 (ca04)] were included for the study. swine influenza viruses that are genetically related with the pandemic h1n1 virus were selected from our surveillance system, including classical swine-like influenza virus a ⁄ sw ⁄ hk ⁄ 4167 ⁄ 99 (h1n1) (swhk4167), triple reassortant-like a ⁄ sw ⁄ arkansas ⁄ 2976 ⁄ 02 (h1n2) (swar2976), and one reassortant between triple reassortant and eurasia swine influenza viruses [a ⁄ sw ⁄ hk ⁄ 915 ⁄ 04 (h1n2) (swhk915)]. swhk915 contains seven gene segments (pb2,pb1,pa,ha,np,m,ns) closely related to the pandemic h1n1 viruses. transmissibility was tested in 4-to 6-month-old male ferrets obtained from triple f farm (sayre, pa); all ferrets were tested to have hi titer £40 against human seasonal influenza h1n1 (a ⁄ tennessee ⁄ 560 ⁄ 2009), h3n2 (a ⁄ brisbane ⁄ 10 ⁄ 2007), and influenza b (b ⁄ florida ⁄ 4 ⁄ 2006) prior the experiments. in each virus group, three ferrets were inoculated with 10 5 tcid 50 of the virus. at 1 day postinoculation (dpi), we introduced one naïve direct contact ferret to share the cage with inoculated ferret, and one naïve aerosol contact ferret into the adjacent compartment of the cage separated by a double-layered perforated divider. nasal washes were collected every other day and tested for influenza virus antigen and to determine viral titers (tcid 50 ). weight changes, temperature, and clinical signs were monitored daily. transmission is defined by detection of virus from nasal washes and ⁄ or by seroconversion (>4fold rise in the post-sera collected after 16-18 days post contact). experiments were performed in the p2+ laboratory at st. jude children's research hospital. all studies were conducted under applicable laws and guidelines and after approval from the st. jude children's research hospital animal care and use committee. at 10 5 tcid 50 inoculation dose, all viruses replicated efficiently in the ferret upper respiratory tract with peak titers detected from inoculated ferrets at 2 dpi. lower peak titers were detected from swhk4167 and swhk915 inoculated ferrets, however, the differences were not statistically significant (table 1) . tissues collected from inoculated ferrets at 3 dpi showed that pandemic h1n1 and swine influenza viruses replicated both in the upper and lower respiratory tract of the ferrets, while the replication of human seasonal influenza wuhan95 was restricted in the upper respiratory tract. direct contact transmission from inoculated donor ferrets to their cage-mates was observed for all viruses studied, albeit at different efficiency. human seasonal influenza (wuhan95) and pandemic h1n1 viruses (ca04) transmitted most efficiently via direct contact route as the virus can be detected on 2 dpi from direct contact ferrets, and the peak titers were detected on 4 dpi from direct contacts. moderate direct contact transmission efficiency was detected from swar2976 and swhk915 viruses as the virus can be detected from direct contact ferrets at 4 dpi, with peak titers detected at 4 dpi or 6 dpi. classical swine-like swhk4167 showed least efficient contact transmission as virus could be detected from all direct contacts only at 6 dpi, and the peak titer detected on 8 dpi. aerosol transmission was detected in groups of human seasonal influenza virus wuhan95 (2 ⁄ 3), pandemic h1n1 influenza virus ca04 (3 ⁄ 3), as well as swine precursor virus swhk915 (1 ⁄ 3). transmission of wuhan95 and ca04 to aerosol contacts was detected at 4 dpi or 6 dpi, while transmission of swhk915 was detected later at 8 dpi, suggesting that the swhk915 virus possessed aerosol transmission potential, but may require further adaptation to acquire efficient aerosol transmissibility. in addition to viral detection from nasal washes, we also detected viruses from the rectal swabs of ferrets inoculated or infected with pandemic h1n1 viruses (ca04) or classical swine-like virus (swhk4167), which share the common origin for the ha, np, and ns gene segments. while many of the swine influenza viruses studied were able to transmit via the direct contact route, swhk915, which shares a common genetic derivation for seven genes with h1n1pdm, possessed capacity for aerosol transmission, albeit of moderate efficiency. swhk915 differed from swine triple reassortant viruses in the origins of its m gene. it is possible that the m gene derived from eurasian avianlike swine viruses also contributes to the transmissibility of h1n1pdm influenza viruses. outbreaks of highly pathogenic avian influenza (hpai) of the h5n1 subtype are of extreme concern to global health organisations as human infection can result in severe acute respiratory distress syndrome, multi-organ failure, and coma. hpai viruses of either h5 or h7 subtypes contain a characteristic multi-basic cleavage site in the hemagglutinin glycoprotein 1 as well as other virulence factors 2 that expand the viral tropism beyond the respiratory tract of poultry. there is also emerging evidence of viral rna or antigen in multiple organs and the cns of humans infected with h5n1 that is consistent with systemic infection 3, 4 and raises the question of the role of the cleavage site in dissemination of the virus in this species. the majority of human cases with h5n1 have involved contact with sick or contaminated poultry and exposure to respiratory secretions of birds that can be inhaled and ingested. particular risk factors for h5n1 infection include bathing with sick birds, improper hand washing after handling sick birds, or slaughtering poultry. 5 viral inoculum may also be consumed directly during a variety of religious and cultural practices, such as drinking contaminated duck blood and kissing of merit release birds. h5n1 infection is lethal in 60% of human cases, and the pathogenetic mechanisms leading to this level of mortality are unclear. to date 505 cases have been reported to the who, although many more people have potentially been exposed to h5n1 through contact with infected bird populations. 6 some studies have suggested that genetic factors may predispose an individual to severe h5n1 disease, 7 but little is known about the influence of route of virus exposure on morbidity and mortality. in ferrets, an animal model frequently used to study influenza because of its similar disease profile to humans, 8 swayne et al. 9 observed that exposure to a virulent h5n1 strain a ⁄ vietnam ⁄ 1203 ⁄ 2004 by intra-gastric gavage did not lead to disease and did not generate an antibody response, whereas ferrets that experienced a more natural exposure by being fed contaminated meat developed severe signs of infection. in this study we further assessed the disease profile of h5n1 following a natural oral exposure in the ferret model. to achieve this inoculation condition, conscious ferrets voluntarily consumed a liquid inoculum of h5n1 hpai strain a ⁄ vietnam ⁄ 1203 ⁄ 2004. as a comparison anesthetised ferrets were exposed by intranasal administration of inoculum and the ensuing disease profiles of the different routes of infection were compared. eight ferrets per group were inoculated with 10 6 egg infectious dose 50 of a ⁄ vietnam ⁄ 1203 ⁄ 2004 in a volume of 500 ll that was given to the nares of anaesthetized ferrets to establish a total respiratory tract (trt) infection or voluntarily consumed by conscious ferrets to establish an oral infection. ferrets were culled at a predetermined humane endpoint that was defined as either a > 10% weight loss and ⁄ or evidence of neurological signs, discussed in 10 ; animals that did not reach the humane endpoint were euthanased on day 14 after challenge. nasal washes and oral swabs collected during the course of infection and organ homogenates were assessed for the presence of replicating virus by growth in embryonated-chicken eggs; viral loads were determined by titration on vero cells and expressed as tcid 50 . tissue samples were fixed with formalin and embedded in paraffin for sectioning. viral lesions were identified by hematoxylin and eosin staining of the sections and the presence of viral antigen in the sections was determined by staining with antibody to influenza a nucleoprotein. pre-and post-exposure antibody responses were assessed by hemagglutination-inhibition assays using irradiated a ⁄ vietnam ⁄ 1203 ⁄ 2004 virus. the majority (75%) of ferrets infected by the trt route rapidly became inactive, developed severe disease, and were euthanased at the humane endpoint following infection ( figure 1 ). ferrets infected orally had an improved chance of survival, as only 25% of animals developed severe disease (figure 1 ), and the surviving ferrets were more active than ferrets infected by the trt throughout the stage of acute infection (data not shown). the improved survival rate and wellbeing of ferrets infected orally was not a result of poor infection rates by this route, as 5 of 6 surviving ferrets developed h5 specific antibodies by day 14 post-infection, and they did not have pre-existing antibodies to h5n1 (data not shown). the two ferrets that developed severe disease after oral infection had similar disease profiles to ferrets infected by the trt route; they both progressed to a > 10% weight loss and exhibited neurological signs (data not shown). viral loads in organs of these two ferrets confirmed dissemination to extra-pulmonary sites (table 1) : replicating virus was detected at high titres in the spleen, pancreas, liver, and brain. similar findings were recorded in ferrets with trt infections in this study (not shown) and elsewhere. 10 viral load in nasal washes and oral swabs taken at days 3, 5, and 7 post-infection by the oral route did not correlate with the development of severe disease, and virus was isolated only sporadically and at low titre from the nasopharynx of these animals (data not shown). interestingly, the two ferrets with severe disease after being infected orally had no detectable viral antigen or lesions in the olfactory epithelium and bulb (table 1) , whereas 5 of 6 ferrets culled after infection by the trt route had lesions and viral antigen in both the olfactory epithelium and bulb (data not shown). trt oral 50 100 figure 1 . percentage of ferrets that survived infection after oral or trt infection. ferrets were exposed to a ⁄ vietnam ⁄ 1203 ⁄ 2004 by the total respiratory tract (trt) route (circles) or the oral route (triangles). the percentages of ferrets that survived infection are indicated at each day following challenge. ferrets exposed orally were more likely to survive h5n1 infection than ferrets exposed to the same dose of virus by the trt. the improved survival rates that were observed after an oral infection could be a consequence of low-level viral replication in the upper respiratory tract in combination with delivery of a substantial portion of the inoculum directly to the stomach where it may have been inactivated by the harsh environment of the gastro-intestinal tract. 11 most ferrets infected orally developed an h5-specific antibody response which differs from the studies of swayne et al. 6 in which ferrets gavaged with a liquid inoculum neither developed signs of disease nor an antibody response. however swayne et al. administered virus to anaesthetized ferrets by gastric gavage that would have bypassed the oropharynx. in our study virus was administered to the oral cavity directly and would have had access to the oropharynx. low level of replication at this site may have been sufficient to trigger an antibody response. the two ferrets that developed severe disease following oral infection had a similar profile of viral dissemination as ferrets infected by the trt route. differences were seen in the olfactory epithelium and bulb as lesions, and viral antigen did not occur in these sites following oral infection, although cerebral involvement was identified. one route of dissemination of h5n1 into the cns may be by transport within nerves through the olfactory bulb into the cerebrum. 12 due to the absence of lesions and antigen in these sites following oral infection the spread of virus into the brain in these two animals may be occurring through involvement of other cranial nerves or the hematagenous routes. nasal turbinates ) 3ae00 ) ) ) ) pharyngeal lymph node interactions of oseltamivir-sensitive and -resistant highly pathogenic h5n1 influenza viruses in a ferret model <2ae5 b ) + + + + olfactory epithelium nd a nd ) ) ) ) olfactory bulb nd nd ) ) ) ) trachea <2ae5 ) ) nd ) nd lung ) <2ae5 + + ) + spleen 3ae75 ) + + ) + small intestine ) ) ) ) ) + pancreas ) 3ae50 + ) + + the pandemic potential of highly pathogenic h5n1 influenza viruses remains a serious public health concern. while the neuraminidase (na) inhibitors are currently our first treatment option, the possibility of the emergence of virulent and transmissible drug-resistant h5n1 variants has important implications. clinically derived drug-resistant viruses have carried mutations that are na subtype-specific and differ with the na inhibitor used. 1 the most commonly observed mutations are h274y and n294s in the influenza a n1 na subtype (n2 numbering here and throughout the text); e119a ⁄ g ⁄ d ⁄ v and r292k in the n2 na subtype; and r152k and d198n in influenza b viruses. 2 h5n1 influenza viruses isolated from untreated patients are susceptible to the na inhibitors oseltamivir and zanamivir, 3 although oseltamivir-resistant variants with the h274y na mutation have been reported in five patients after 4,5 or before 6 drug treatment; and the isolation of two oseltamivir-resistant h5n1 viruses with n294s na mutation from an egyptian girl and her uncle after oseltamivir treatment were described. 6 the impact of drug resistance would depend on the fitness (i.e., infectivity in vitro, virulence, and transmissibility in vivo) of the drug-resistant virus. if the resistance mutation only modestly reduces the virus' biological fitness and does not impair its replication efficiency and transmissibility, the effectiveness of antiviral treatment can be significantly impaired. the recombinant wild-type h5n1 influenza a ⁄ vietnam ⁄ 1203 ⁄ 04 (vn-wt), a ⁄ turkey ⁄ 15 ⁄ 06 (tk-wt) viruses, and oseltamivir-resistant viruses with h274y na mutation (vn-h274y and tk-h274y) were generated by using the 8-plasmid reverse genetics system. susceptibility to na inhibitors was tested by using a fluorescence-based na enzyme inhibition assay with munana substrate at a final concentration of 100 lm. viral fitness was studied in vivo in a ferret model: groups of three ferrets were lightly anesthetized with isoflurane and inoculated intranasally with vn-wt, vn-h274y, or mixtures of the two at a different ratios at a dose of 10 2 pfu in 0ae5 ml pbs; they were inoculated with tk-wt, tk-h274y, or mixtures of the two at a different ratios at a dose of 10 6 pfu in 0ae5 ml pbs. respiratory signs (labored breezing, sneezing, wheezing, and nasal discharge), neurologic signs (hind-limb paresis, ataxia, torticollis, and tremor), relative inactivity index, weight, and body temperature were recorded daily. virus replication in the upper respiratory tract (urt) was determined on days 2, 4, and 6 p.i. the competitive fitness (i.e., co-inoculation of ferrets with different ratios of oseltamivir-resistant and -sensitive h5n1 viruses) was evaluated by the proportion of clones in day-6 nasal washes that contained the h274y na mutation. na mutations were analyzed by sequence analysis of individual clones ($20 clones ⁄ sample) created by ligation of purified pcr products extracted from nasal wash samples into a topo vector. introduction of the h274y na mutation conferred high resistance to oseltamivir carboxylate in vitro; the mean ic 50 of the vn-h274y and tk-h274y viruses was 3375 and 1208 times, respectively, that of the corresponding wildtype viruses. the oseltamivir ic 50 of the tk-wt virus was $16 times that of the vn-wt virus. all four recombinant h5n1 viruses were susceptible to zanamivir. introduction of the h274y na mutation reduced $90% and 60% of the na activity of vn-h274y and tk-h274y viruses, respectively, as compared to the wild-type virus activity (p < 0ae01; two-tailed t-test). all ferrets inoculated with either vn-wt or vn-h274y virus exhibited acute disease signs (high fever, marked weight loss, anorexia, extreme lethargy), rapid progression, and death by day 6-7 p.i., and no differences in clinical signs and replication in the urt of ferrets were observed between wild-type and oseltamivir-resistant viruses ( table 1) . both of the tk viruses caused milder illness than did the vn viruses, despite a much higher dose (10 6 pfu ⁄ ferret), and the tk-h274y virus caused less weight loss and fever than the tk-wt virus (table 1) . however, competitive fitness experiments revealed a disparity in the growth capacity of vn-h274y and tk-h274y viruses as compared to their wild-type counterparts: clonal analysis established the uncompromised fitness of vn-h274y virus and the impaired fitness of tk-h274y virus (table 2) . although, the trend towards an increase ⁄decrease in the frequency of the h274y na mutation relative to the wild-type was statistically significant (p > 0ae05) for two studied groups only. mutations within the na catalytic (r292k) and framework (e119a ⁄ k, i222l, h274l, n294s) sites or near the na active enzyme site (v116i, i117t ⁄ v, q136h, k150n, a250t) emerged spontaneously (without drug pressure) in both pairs of viruses (results not shown). the na substitutions i254v and e276a could exert compensatory effect on the fitness of vn-h274y and tk-h274y viruses. the lethality and continuing circulation of h5n1 influenza viruses warrants an urgent search for an optimal therapy. our results showed that the h274y na mutation affects the fitness of two h5n1 influenza viruses differently: the oseltamivir-resistant a ⁄ vietnam ⁄ 1203 ⁄ 04-like virus outgrew its wild-type counterpart, while the oseltamivir-resistant a ⁄ turkey ⁄ 15 ⁄ 06-like virus showed less fitness than its wild-type counterpart. we used a novel approach to compare the fitness of oseltamivir-sensitive and -resistant influenza viruses that included analysis of virus-virus interactions within the host (competitive fitness) during co-infection with these viruses. although mixed populations were present in the urt of ferrets on day 6 p.i., the fitness of vn-h274y virus was uncompromised as compared to that of its drug-sensitive counterpart, while that of tk-h274y virus was impaired. a minor population of na inhibitor-resistant variants may gain a replication advantage under suboptimal therapy in two ways: (i) preexisting variants less sensitive to the drug are selected from the quasispecies population, leading to an increase of the number of resistant clones, and (ii) outgrowing variants may acquire additional compensatory mutations that enhance their fitness. it is possible that use of antiviral drugs (particularly at suboptimal concentration) against mixtures of oseltamivir-resistant and sensitive viruses will promote the spread of drug-resistant variants * ferrets in all groups inoculated with a ⁄ vietnam ⁄ 1203 ⁄ 04 virus died by day 6-7 p.i. and were observed once daily for 7 days. ** results obtained from one ferret. *** by inhibiting drug-sensitive variants that are competing with them for the dominance in the infected host. the influence of multiple genes on the fitness of viruses carrying h274 na mutation cannot be excluded. in our study we focused on additional na mutations, and sequence analysis of individual na clones 7 was done to identify potential host-dependent and compensatory na mutations. we found that the na mutations e119a and n294s, which confer cross-resistance to oseltamivir and zanamivir, 1,2 can emerge spontaneously in clade 2.2 h5n1 influenza virus in ferrets. further, we observed that mutations at na catalytic (r292k) and framework (i222l and n294s) sites and in close proximity to the na enzyme active site (v116i, i117t ⁄ v, q136h, k150n, a250t) emerged without drug pressure in both pairs of h5n1 viruses. compensatory mutations in na or other genes may mitigate any fitness cost imposed by resistance mutations. our study identified six potential compensatory na changes (d103v, f132s, i254v, e276a, h296l, and f466s) that may affect the fitness of viruses with the h274y na mutation. we suggest that na mutations at residues i254v and e276a are of importance. interestingly, we observed differences in predominance of i254v and e276a na mutations in different genetic backgrounds: i254v mutation was identified in a ⁄ vietnam ⁄ 1203 ⁄ 04 (h5n1)-like and e276a in a ⁄ turkey ⁄ 15 ⁄ 06 (h5n1)-like genetic background. moreover, i254v na mutation was identified only when ferrets were inoculated with the mixtures of vn-wt and vn-h274y viruses, but not in ferrets inoculated with vn-h274y virus. none of the potential compensatory na mutations was identified in the original inoculum used to infect ferrets. the h274y na mutation causes a large shift in the position of the side chain of the neighboring e276 residue, 8 which must form a salt bridge with r224 to accommodate the large hydrophobic pentyl ether group of oseltamivir. residue i254 is located near the na active site, and although it does not alter polarity, it results in a shorter side-chain and, thus, may indirectly affect the residues in the na active site. we suggest that antigenic and genetic diversity, virulence, the degree of na functional loss, and differences in host immune response and genetic background can contribute to the observed differences in the fitness of h5n1 influenza viruses. therefore, the risk of emergence of drugresistant influenza viruses with uncompromised fitness should be monitored closely and considered in pandemic planning. this study was supported by contract hhsn266200700005c from the national institute of allergy and infectious diseases, national institutes of health, and by the american lebanese syrian associated charities (alsac). the data presented in the manuscript have been published at: govorkova ea, ilyushina na, marathe bm, mcclaren laninamivir (r-125489) is a strong na inhibitor against various influenza viruses, including oseltamivir-resistant viruses. [1] [2] [3] [4] [5] [6] we discovered a single intranasal administration of laninamivir octanoate (cs-8958), a prodrug of laninamivir, showed a superior anti-virus efficacy in mouse and ferret infection models compared to repeated administra-tion of oseltamivir and zanamivir. [2] [3] [4] 7 this suggested that cs-8958 works as a novel long-acting na inhibitor of influenza virus in vivo. a single inhalation of cs-8958 proved noninferiority in adult patients 8 and significantly superior in child patients, 9 compared to an approved dosage regimen of oseltamivir for treatment. cs-8958 has been commercially available as an inhaled drug, inavir ò , for the treatment of influenza in japan since october 2010. the long-acting characteristics of cs-8958 are explained by several reasons. first, cs-8958 was quickly hydrolyzed to an active metabolite, laninamivir, after an intranasal administration to mice, and was retained for a long time as laninamivir in target organs, such as lung and trachea. 10 however, with an intranasal administration of laninamivir, it disappeared quickly and did not demonstrate its longlasting characteristics. 10 another reason is a strong binding of laninamivir to nas of seasonal influenza viruses compared to other three na inhibitors, oseltamivir carboxylate, zanamivir, and peramivir. 3 in the following, the tight-binding ability of laninamivir to pandemic (h1n1)2009 na, as well as to the seasonal influenza virus nas, was demonstrated. in addition, we present a hypothesis of the mechanism of the long-lasting property of cs-8958 in mouse based on a localization of an enzyme that hydrolyzes cs-8958 to laninamivir. the influenza viruses, pandemic(h1n1)2009 (inf139), a ⁄ new caledonia ⁄ 20 ⁄ 99 (h1n1), a ⁄ panama ⁄ 2007 ⁄ 99 (h3n2), and b ⁄ mie ⁄ 1 ⁄ 93 were treated with excess na inhibitors, such as oseltamivir carboxylate, zanamivir, peramivir, and laninamivir, and then unbound na inhibitors were removed from the mixtures with a bio-spin column bio-gel p-6 (bio-rad laboratories, hercules, ca, usa). the na substrate, 4-methylumbelliferyl-n-acetyl-a-d-neuraminic acid (nacalai tesque, japan) was added to the virus-na inhibitor complex, and the na activities were followed for 6 hours at room temperature by measuring the fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. the enzyme which hydrolyzes cs-8958 to laninamivir was partially purified from rat lungs using ion exchange column chromatography, and almost all bands separated by an sdspolyacrylamide gel electrophoresis were identified by mass spectrometry. the gene expression profiles of the enzyme were investigated by the bioexpress database (genelogic inc., gaithersburg, md, usa). the enzyme gene cloned from mouse lung mrna was transiently expressed in cos cells. antiserum to the esterase was prepared by immunizing rabbits, and immunostaining was done using histomouse-tm-max kit (invitorgen corp., carlsbad, ca, usa) according to the manufacturer's manual. binding stability of na inhibitors to the four viruses are shown in figure 1 the enzyme that hydrolyzes cs-8958 to laninamivir in rat lungs was identified as carboxyesterase. this esterase was shown to be expressed in epithelial cells of rat lung by in situ hybridization. 11 the mouse homolog of the rat esterase was carboxylesterase 3 (ces3). the mrna of the mouse ces3 was shown to be highly expressed in lung and liver by the gene expression profile, and ces3 was also found to contain signal sequences for retention in endoplasmic reticulum (er) and golgi at the c-terminus. the cloned ces3 gene and the ces3 gene lacking the signal sequence were exogenously expressed in the cos cells. the cs-8958-hydrolyzing activity associated with the cos cells expressing ces3 was recovered from the culture sup of the cos cells expressing ces3 lacking the retention signal sequence. localization of ces3 was immunohistologically confirmed inside the airway epithelium cells of mice, which are the target cells for influenza virus infection. the long acting property of intranasal administration of cs-8958 in mice can be explained both by the long retention of laninamivir in the respiratory tract and by the stable binding of laninamivir to influenza virus na. again, stable binding of laninamivir to na of pandemic (h1n1)2009 virus was also observed similar to that of seasonal h1n1 virus. the following are speculated as the mechanisms for the long-lasting characteristics of cs-8958 in mice. we explain the mechanism by clarifying a cs-8958 hydrolyzing enzyme and its localization inside cells. the hypothesis of the mechanism is presented in figure 2 . briefly, hydrophilic laninamivir may not enter easily inside cells, whereas hydrophobic cs-8958 may enter inside cells. ces3 with er ⁄ golgi retention signal hydrolyzes octanoate of cs-8958 figure 1 . difference of binding stabilities of various na inhibitors to influenza virus neuraminidases. the na substrate was added to the influenza virus-na inhibitor complex (oseltamivir carboxylate, n; zanamivir, h; peramivir, s; laninamivir, •; distilled water, ¤), and the na reaction was followed for 360 minutes. the background (only the na substrate [d] ) is also shown. a part of data from. 3 to generate the hydrophilic drug, laninamivir, and then it is trapped inside er ⁄ golgi because of its high hydrophilicity. the glycoprotein, na, which matures in er ⁄ golgi, meets laninamivir there and efficiently makes a stable complex with it. there are some questions that remain. how does cs-8958 move from the cell membrane to er ⁄ golgi? is laninamivir indeed trapped inside er ⁄ golgi, and does it make a complex with na in mice? we are now making an attempt to clarify these concerns. in our study, we have explored the antiviral potential of two newly synthesized compounds to provide protection against the novel pandemic influenza virus h1n1 (2009) strain. the compounds were reconstituted in dimethylsulphoxide (dmso), and so the initial studies began with cytotoxicity determination of solvent on uninfected and untreated madin-darby canine kidney (mdck) cells. on obtaining an upper limit for dmso, the compounds were tested for estimation of their maximum non-toxic dose to the mdck cells. thereafter, the effective dose of the compounds was evaluated and validated by a number of assays and gene expression profiling at both nucleic acid and protein level. we found that these newly synthesized compounds possess potent inhibitory activity towards the novel pandemic influenza h1n1 (2009) virus. these findings are being evaluated in vivo for a better understanding of their inhibitory capabilities and also their effect on the host metabolism. this will be required in the course of development of new drugs for use in the prophylaxis and treatment against the influenza virus. the mdck cell line (from nccs, pune) was maintained in 1· dmem media (sigma, st. louis, mo, usa) supplemented with 10% fetal calf serum and antibiotics viz. 100 unit ⁄ ml penicillin and 100 lg ⁄ ml streptomycin at 37°c ⁄ 5% co 2 . 5 the synthesized compounds used in this study were kindly provided by the department of chemistry, university of delhi, delhi, india. the pandemic influenza h1n1 (2009) virus was isolated and propagated in the allantoic cavities of embryonated chicken eggs during the pandemic period. the virus stocks were prepared and stored at )80°c. plaque assay was performed as previously described by hui et al., 2003. 6 briefly, 0ae5 · 10 6 mdck cells ⁄ ml were seeded in six-well plates and maintained in dmem for 24 hours at 37°c ⁄ 5%co 2 . the monolayer of the cells was inoculated with serially diluted virus samples for 45 minutes at 37°c ⁄ 5%co 2 . subsequently, a mixture of agar overlay was added, and the plates were incubated at 37°c for 5 days or until formation of plaques. the plaques were visualized after removal of the agar plug and staining with 0ae1% crystal violet or neutral red solution. the virus titre was expressed as plaque forming unit (pfu) per milliliter. the in vitro cytotoxicity analysis was performed to determine the 50% cytotoxic concentration (cc 50 ) of the compounds on mdck cells. the compounds were dissolved in dimethylsulfoxide (dmso), and so a prior cytotoxicity analysis was performed to determine the toxic concentration of dmso on the cells. various concentrations of compounds were mixed with dmem containing 1% fcs before addition to the preformed monolayer of mdck cells in 96-well plates. a series of suitable controls for in vitro cc 50 determination was included in every plate, and the plates were incubated in the optimum environment for mdck cell culture. the cc 50 of test compounds was analyzed by estimation of percentage cell viability of the compound-and mocktreated mdck cells by performing a colorimetric assay using tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyl tetrazolium bromide (mtt) at end-point of 48 hours post-incubation. the assay was performed as described by mosman 1983. 7 briefly, mtt stock at a concentration of 0ae5 mg ⁄ ml was prepared in 1· pbs. the media was aspirated from the wells and 100 ll of mtt dye from the stock was added to each well. following incubation at 37°c ⁄ 5% co 2 for 2-4 hours, the dye was very carefully removed from the wells, and the cells were incubated with 100 ll of stop solution (dmso) per well at 37°c ⁄ 5% co 2 for 1 hour. the absorbance of the supernatants from each well was measured at 540 nm, and the percentage cell viability was calculated. madin-darby canine kidney cells were maintained overnight in a 12-well tissue culture plate at 37°c ⁄ 5% co 2 . the cells were inoculated with various virus dilutions at 37°c ⁄ 5% co 2 for 45 minutes and observed for cytopathic effect (cpe). the media from the experimental wells were aspirated after 48-72 hours of infection and were subjected to plaque assay. the percentage cell viability was determined by performing mtt assay. the results of both these tests were used to assess tcid 50 of the virus. the pre-formed monolayer of mdck cells was inoculated with the 10-fold dilution corresponding to tcid 50 of the virus for 1 hour at 37°c ⁄ co 2 . the experimental setup included control wells for the cells, virus, and compound. meanwhile, the concentrated stocks of the synthesized compounds were diluted with dmem (with1% fcs) to various concentrations within their respective cc 50 ranges. one hour post-infection, the cells were incubated with these diluted solutions. the cells were observed at various time intervals post-inoculation for cpe, and 200 ll media was collected from each experimental well for performing hemagglutination test. after 48 h, the media was collected for plaque assay and the cells were subjected to mtt cell viability assay. preformed monolayers of mdck cells were infected with virus and treated with the respective inhibitory concentration of the compounds. forty-eight to 72 hours post-incubation, total cellular rna was isolated using ribozol (amresco, solon, oh, usa) and treated with 50 lg ⁄ ml of dnase (promega, madison, usa). the concentration and quality of the rna from each well were determined by measuring their absorbance at 260 and 280 nm. one microgram of the cdna synthesized from each rna sample was used for sybr green-based real-time pcr detection of the ha gene of pandemic influenza h1n1 (2009) virus. as a control, human glyceraldehyde-3-phosphate dehydrogenase (hgapdh) was also amplified using gene specific primers. 8, 9 immunoblotting immunoblotting was performed to further validate the antiviral potential of the compounds. the experimental protocol was the same as for real time rt-pcr analysis. the cells were harvested 48 hours post-treatment with the compounds to prepare whole cell lysates in mammalian cell lysis buffer [0ae1 m nacl, 0ae01 m tris cl (ph 7ae6), 0ae001 m edta (ph 8ae0), 1 m m protease inhibitor cocktail, 100 lg ⁄ ml pmsf]. the protein concentration was determined by bca protein assay. the cell lysates were fractionated on 12% polyacrylamide for western blotting. the blot was developed using sheep monoclonal antibody (santa cruz biotechnology, ca, usa) against ha protein of influenza virus and horseradish peroxide conjugated rabbit-anti sheep igg (1:1000 dilutions) as secondary antibody. 10 the median cytotoxic concentration for compound meuh came out to be 150 lm, and that for flh was 160 lm. compounds showing potent antiviral effect on the pandemic influenza h1n1 (2009) virus propagation in madindarby canine kidney cells ( figure 1 ). the viral titres remained constant in cells treated with the compounds, while they increased in the untreated virus infected cells. ed 50 for the compounds meuh and flh were 150 and 160 lm, respectively. fifty-two percent (meuh) and 45% (flh) inhibition against the pandemic influenza h1n1 (2009) virus was achieved using ed 50 of the test compounds. both the compounds were able to reduce the rna levels of the ha gene by approximately 40-45%, whereas approximately 50% inhibition was seen when both the compounds were used in combination. similar results were obtained by the immunoblotting analysis ( figure 2 ). antiviral therapy has shown to be a promising tool in the management of various respiratory diseases, including those caused by influenza viruses. we have already shown inhibition of influenza virus replication in our earlier studies using catalytic nucleic acids, 11 which can be used as an approach in the development of new therapeutic strategy. these therapies are very useful as the influenza virus vaccines need annual renewals due to frequent genetic drifts in the viral surface proteins. in pandemic situations the existing vaccines do not provide complete protection against the novel virus as the population generally remains naïve for the newly mutated surface antigens. the antiviral drugs play an important role in the control of novel viral strains for which there are no vaccines available. however, the key obstruction in the extensive use of antiviral drugs is their cost and relative therapeutic efficacy provided. two classes of drugs were being used for treatment and control of the influenza virus infection in humans, the m2 ionchannel blockers 12, 13 (amantadine and rimantadine), which prevent viral uncoating, and the neuraminidase inhibitors 14, 15 (zanamivir and oseltmivir), which prevent the release of influenza virions from the cytoplasmic membrane. but widespread resistance to these antiviral drugs 16, 17 has limited their use. thus, novel drugs are required for the effective therapy against the emerging strains of influenza virus. the novel chemical compounds used in our study were tested for their antiviral efficacy against the pandemic influenza h1n1 (2009) virus. a reduction in the cpe in compound treated virus infected mdck cells indicated presence of antiviral activity in chemical compounds. the persistence of constant viral titers in the compound treated cells provided evidence for the interference posed by the compounds in the replication of influenza virus. inhibition in the ha gene expression further validated our hypothesis for the antiviral effect of compounds. the efficacy of these compounds in animal models is currently being validated in our laboratory. further, molecular studies are required to ameliorate the awareness regarding the mode of action of these chemical compounds against the viruses. 4 and is now licensed in japan, 5 while another, laninamivir, is being developed as an inhaled prodrug. 6 resistance to nais among circulating influenza viruses was previously low (<1% worldwide). [7] [8] [9] however, the 2007-2008 influenza season was marked by a worldwide emergence of oseltamivir-resistant seasonal influenza a (h1n1) viruses with the h275y (h274y in n2 numbering) in the na. [9] [10] [11] [12] [13] [14] the prevalence of oseltamivir resistance was even higher in the subsequent 2008-2009 influenza season with many countries reporting up to 100% oseltamivir resistance, 15 seasonal and pandemic influenza viruses collected globally between october 1, 2008 and september 30, 2009 were submitted to the who collaborating center for surveillance, epidemiology and control of influenza at the centers for disease control and prevention (cdc) in atlanta, ga, usa, and propagated in madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa). reference viruses representative of oseltamivir-sensitive and -resistant seasonal and pandemic viruses were also propagated in mdck cells. susceptibilities of virus isolates to the nais oseltamivir carboxylate (hoffman-la roche, basel, switzerland) and zanamivir (glaxosmithkline, uxbridge, uk) were assessed in the chemiluminescent ni assay using the na-star tm kit (applied biosystems, foster city, ca, usa) as previously described. 9 additionally, subsets of virus isolates were tested for susceptibility to peramivir (biocryst pharmaceuticals, birmingham, al, usa). fifty percent inhibitory concentration (ic 50 ) values were calculated using jaspr curve fitting software, an in-house program developed at cdc. curve fitting in jaspr was done using the equation: v = vmax · (1 ) ([i] ⁄ (ki + [i]))), where vmax is the maximum rate of metabolism, [i] is the inhibitor concentration, v is the response being inhibited, and ki is the ic 50 for the inhibition curve. box-and-whisker plot analyses 7 of log-transformed ic 50 s were performed for each virus type ⁄ subtype and nai using sas 9.2 software (sas institute, cary, nc, usa) to identify viruses with extreme ic 50 values (outliers). outliers were characterized based on a statistical cutoff of ic 50 greater than three interquartile ranges from the 75th percentile. outliers were subjected to genetic analysis by pyrosequencing 17 and ⁄ or conventional sequencing 18 to detect known or novel markers of nai resistance. those harboring previously characterized mutations in the na associated with nai resistance were considered drug-resistant; their descriptive statistics were determined separately from naisusceptible viruses. descriptive statistics to compute the mean, median, and standard deviation (sd), and a one-way analysis of variance were performed on original scale ic 50 data, using sas 9.2 software (sas institute) for each nai and virus among seasonal influenza a (h1n1) viruses tested for oseltamivir susceptibility (n = 1533), 1431 (93ae3%) were outliers for the drug (table 1 ) and harbored the oseltamivir-resistance conferring h275y mutation in the na. by contrast, only a small proportion (0ae7%) of tested h1n1pdm viruses (n = 2259) were resistant to oseltamivir. all influenza a (h3n2) viruses (n = 834) were sensitive to oseltamivir except for one outlier, a ⁄ ontario ⁄ rv0442 ⁄ 2009 with d151v mutation in the na, whose ic 50 of 2ae50 nm was beyond the statistical cut-value off and >10-fold the mean ic 50 for the drug (0ae24 nm). all influenza b viruses (n = 914) were sensitive to oseltamivir with exception of an outlier b ⁄ texas ⁄ 38 ⁄ 2008, with d197e (d198e in n2 numbering) mutation in the na, whose ic 50 was beyond the cut-off, but only fourfold greater than the mean ic 50 for the drug. all virus types ⁄ subtypes tested for zanamivir were sensitive to the drug (table 1) , except for some outliers among seasonal influenza a (h1n1) and a (h3n2) outliers. the seasonal influenza a (h1n1) outliers included a ⁄ thailand ⁄ 1035 ⁄ 2008 (h1n1) and a ⁄ hawaii ⁄ 20 ⁄ 2008 (h1n1), both with combined h275y and d151d ⁄ g mutations in their na. the presence of concurrent mutations at na residues h275 and d151 in seasonal influenza a (h1n1) virus isolates substantially enhances resistance to oseltamivir and peramivir and ⁄ or zanamivir, however, the changes at d151 are typically cell-derived and not present in clinical specimens. 19 influenza a (h3n2) outliers for zanamivir included a ⁄ ontario ⁄ rv0442 ⁄ 2009 with d151v mutation in the na, as well as a ⁄ maryland ⁄ 02 ⁄ 2009 and a ⁄ vladivostok ⁄ 53 ⁄ 2009 with d151g and mixed d151d ⁄ g mutations, respectively. some mild outliers for zanamivir among a (h3n2) viruses with ic 50 beyond the statistical cutoff but <10-fold mean ic 50 for the drug were also identified; their genetic analysis revealed presence of wildtype and mutant sequences at residue 151 namely, d151d ⁄ g, d151d ⁄ n, or d151d ⁄ a. mutations at residue d151 of the na are associated with reduced susceptibility to zanamivir in a (h3n2) viruses, 9 but were reported to be cell-culture derived in recent h3n2 viruses. 20 all virus isolates tested for peramivir (n = 1058) were sensitive to the drug, except for h275y variants among seasonal influenza a (h1n1) and h1n1pdm viruses, which exhibited reduced susceptibility to the drug. in addition, one influenza a (h3n2) isolate, a ⁄ ontario ⁄ rv0442 ⁄ 2009 with d151v mutation in the na, showed reduced susceptibility to peramivir. the ic 50 values determined in functional ni assays provide valuable information for detection of resistant viruses, but should not be used to draw direct correlations with drug concentrations needed to inhibit virus replication in the infected human host, as clinical data to support such inferences are inadequate. 21 nevertheless, combining elevated ic 50 values with the presence of established molecular markers of resistance in the na of virus isolates and their matching clinical specimens provides a reliable and reasonably comprehensive approach of identifying nai-resistant isolates for surveillance purposes. in this study, outliers with elevated ic 50 values for oseltamivir among seasonal influenza a (h1n1) and h1n1pdm viruses were confirmed to be oseltamivir-resistant based on the presence of the h275y mutation in the na. outliers for oseltamivir and ⁄ or zanamivir among influenza a (h3n2) viruses in this study were shown to harbor mutations at d151, which were earlier associated with reduced susceptibility to zanamivir, 9 and were cell-culture derived. 20 the effects of d151 mutations on nai susceptibility appear to be strain-specific; however, there are no conclusive supporting data and further investigations are required. outliers among the influenza a viruses in this study exhibited changes in the na, derived naturally or through cell-culture, which altered their susceptibility to nais. however, mild outliers for oseltamivir and ⁄ or zanamivir among influenza a viruses with slightly elevated ic 50 s, but without apparent changes in the na are sometimes identified. in such instances it is imperative to exclude the potential presence of influenza b among such outliers, using conclusive genetic tests such as real time pcr, since influenza b viruses exhibit higher ic 50 values for oseltamivir and zanamivir than influenza a viruses. 21 viruses exhibiting such mixes are typically excluded from statistical analyses of ic 50 s for respective drugs and virus type ⁄ subtype. establishment of a clinically relevant ic 50 cutoff value which could be used to differentiate statistical outliers from truly resistant viruses is imperative. global surveillance for nai susceptibility of influenza viruses circulating globally should be sustained to reflect the impact of seasonal and pandemic of influenza, given the limited pharmaceutical options available for control of influenza infections. nasopharyngeal swab specimens from patients with acute respiratory infection were collected at 164 influenza sentinel surveillance units (outpatient and hospital-based) all over mongolia. specimens were transported to the virology laboratory, nccd, ulaanbaatar, and rt-rt pcr positive samples were grown in a mdck cell culture according to the protocol developed by cdc. 5 6 and influenza virus gene segment 7 (m genes) sequencing (7 strains-genbank accession numbers: cy053364, cy053365, cy054547, cy054549, cy055171, cy065990, and cy065998) and influenza virus gene segment 6 (na gene) sequencing (2 strains genbank accession numbers: cy073447 and cy073448) by the standard methods with applied biosystems 3130xl genetic analyzer using primers supplied by who collaboration centers. a chemiluminescent na inhibition assay was performed with veritas microplate luminometer using the commercially available kit, na-star (applied biosystems, foster city, ca, usa), according to the manufacturers protocol. the na inhibitor susceptibility of influenza virus isolates was expressed at the concentration of na inhibitor needed to reduce na enzyme activity by 50% (ic 50 ). oseltamivir carboxylate, was provided by f. hoffman-la roche ltd (basel, switzerland). na inhibition assay data were analyzed using robosage software comparing test data with the data produced by the reference na inhibitor sensitive and resistance strains, which were provided by the who influenza collaboration center, melbourne, australia. all viruses tested were sensitive to oseltamivir with two exceptions: a seasonal influenza virus a ⁄ ulaanbaatar ⁄ 1735 ⁄ 2009(h1n1) with 137ae1 nm ic 50 value and a pandemic influenza virus a ⁄ dundgovi ⁄ 381 ⁄ 2010(h1n1) with 65ae6 nm ic 50 value ( figure 1 ). there was oseltamivir resistance detected in 2ae7% (37 ⁄ 1) of seasonal a (h1n1) and in 0ae4% (262 ⁄ 1) of a (h1n1) pdm viruses. the oseltamivirresistant viruses were collected from untreated patients. in total, 18 influenza b viruses were analyzed by na inhibition assay and all were sensitive to oseltamivir. the na of both oseltamivir-resistant strains contained h275y mutation based on the sequencing analysis. the difference in the na amino-acid sequences between the mongolian oseltamivir-resistant viruses and the respective oseltamivir-sensitive reference viruses is shown in table all 37 a(h1n1) viruses analyzed for m2 channel inhibitor resistance by pyrosequencing contained the s31n mutation and, thus, were resistant to this class of anti-influenza drugs. the segment 7 sequencing revealed that 6 seasonal a(h1n1) viruses possess the common s31n mutation. of note, a single strain a ⁄ zavkhan ⁄ 8299 ⁄ 2009(h1n1) contained an unusual s31d change in the m2 protein. our study shows that the same prevalence [2ae7% (1 ⁄ 37)] of seasonal a(h1n1) viruses with h275y mutation in 2008 ⁄ 2009 season in mongolia with the published data for 2007 ⁄ 2008 season from japan. 7, 8 however the prevalence of oseltamivir resistance in japan has dramatically increased in 2008 ⁄ 2009 season to 100% (77 ⁄ 77). the observed double mutations: h275y and d354g in a ⁄ ulaanbaatar ⁄ 1735 ⁄ 2009(h1n1) strain, which have been also found in japan in 2008 ⁄ 2009 season. 7 the patient from whom the oseltamivir resistant seasonal influenza h1n1 virus has been isolated was a 1-year-old boy, living in ulaanbaatar, the capital city, without history of using oseltamivir. the patient from whom the oseltamivir resistant a(h1n1)pdm virus was isolated was a 59 year-old man, residing in the dundgovi, the southern province, also without history of antiviral treatment. according to the who data, isolation of the pandemic viruses carrying h275y change from untreated patients has been uncommon. circulation of amantadine-resistant seasonal a (h1n1) viruses has been increasing in mongolia since 2006 ⁄ 2007 influenza season. 4 all pandemic influenza a(h1n1) strains (16) tested were resistant to m2 channel inhibitors due to the presence of the s31n mutation in the m2 protein. among seasonal a(h1n1) viruses, one contained a s31d change whereas the others had s31n, the well established marker of resistance to both amantadine and rimantadine. this is the first report of detecting the s31d change in the seasonal a(h1n1) viruses. according to the cdc data (unpublished), the s31d change conferred the drug resistance in the a(h3n2) viruses according to the virus yield reduction assay. it is essential to continue the antiviral resistance surveillance of influenza virus strains circulating in mongolia to ensure the efficiency of a proper clinical management of influenza patients. (conferred by the s31n mutation). of note, the genotype 2 and genotype 3 dual resistant viruses from asia appear to be genetically similar to those previously reported dual resistant viruses from hong kong, sar. 4, 5 the genotype 4 virus was the only dual resistant virus with a nearly complete 2c genome. oseltamivir-resistance for this virus appears to be the result of a reassortment as demonstrated by the presence of the oseltamivir-resistant clade 2b na gene. although the detection of dual resistant seasonal influenza a (h1n1) viruses is still rare, there has been an increased prevalence of dual resistance viruses during the last three seasons: 0.06% (1 of 1753 tested in 2007-2008), 1.5% (21 of 1426 in 2008-2009) , and 28% (7 of 25 in 2009-2010) (v 2 p < 0.001). while the continued circulation or co-circulation of seasonal a (h1n1) viruses is uncertain, the emergence of dual resistant influenza viruses in five countries does present a public health concern, especially since dual resistant viruses would limit the options for antiviral treatment to a single licensed antiviral drug: zanamivir. moreover, the markers of resistance seen in seasonal a (h1n1) viruses also confer resistance in the more widely circulating 2009 pandemic a (h1n1) virus. and, since the acquisition of mutations in influenza a viruses typically occur through drug selection, spontaneous mutation, or genetic reassortment with another drug resistant influenza a viruses, the detection of influenza a (h1n1) viruses that are resistant to both adamantanes and oseltamivir warrants close monitoring, even if only detected at low frequency. new antiviral agents and strategies for antiviral therapy are likely to be necessary in the future. 1 heightening concern that drug resistance will likewise become prominent in pandemic viral strains and highlighting the need for antiviral drug resistance surveillance. the h275y mutation in h1n1 neuraminidase is the most common mutation conferring resistance. however, due to the high mutation rates of viruses, new mutations can be expected that will also render viral neuraminidase less sensitive to antiviral drugs. pcr methods can be used to detect previously identified mutations; however, functional neuraminidase enzyme activity inhibition testing is necessary for detecting drug resistance that results from novel mutations. the two neuraminidase enzyme inhibition assays using either the fluorescent munana or chemiluminescent na-star ò substrate are robust tools for ni susceptibility testing. the munan-a-based assay is broadly used by many groups, including many regional health organizations for ni susceptibility testing, yet no standardized protocol or dedicated kit has been in place for this assay, making comparison of data generated between different laboratories difficult. borrowing from multiple neuraminidase inhibitor susceptibility network (nisn)-published munana-based neuraminidase assay protocols, 2 we have developed a kit-based fluorescent neuraminidase assay that offers both standardization and off-the-shelf quality-controlled reagents for ni susceptibility testing and other neuraminidase assay applications. the na-fluor tm influenza neuraminidase assay reagents and protocols were optimized in comparison to published nisn protocols according to the criteria of assay performance, ease-of-use, consideration of historically used assay conditions, reagent storage stability, and environmental impact. our optimized assay conditions consists of 100 lm munana, 33ae3 mm mes, 4 mm cacl 2 , and ph 6ae5 in a 100 ll assay volume, and performing the assay for 60 minutes at 37°c following a 30 minutes preincubation of drug with the virus. these conditions are consistent with the majority of published influenza ni screening data in publication. 2 the standard na-fluor tm assay workflow for screening viral isolates for sensitivity to nis includes first titering the viral sample by neuraminidase activity to determine optimal virus concentration to be used in subsequent ic 50 determination assays. the na-fluor tm assay is an ideal tool for titering virus based on neuraminidase activity in the viral coat. titering of viral samples prior to running the ic 50 determination assays insured that assays would be performed within the fluorescence detection dynamic range of both the assay and the fluorometric instrument being used. viral titers giving rfus in the range of 2000-5000 were used for subsequent assays. comparison to traditional munana assays a primary goal of developing a standardized munana assay was to provide a standardized protocol and set of reagents that would allow for comparison of ni surveillance data between laboratories and over time. in addition, the assay should provide data comparable to historical data sets based on traditional munana-based protocols. to insure that our newly developed na-fluor tm assay met these criteria we performed side-by-side comparisons of the na-fluor tm assay to munana-based nisn protocols, as well as our na-xtd tm and na-star ò chemiluminescent neuraminidase assays to compare assay sensitivity and dynamic range and for ni ic50 determination with multiple viral isolates. for all assay comparisons, assays were performed according to respective published protocols. for direct comparison of results, an equivalent amount of virus (and concomitant neuraminidase activity) was used for each assay. the na-fluor tm assay provides low-end sensitivity (by signal to noise ratio) and dynamic range similar to nisnpublished, munana-based protocols (data not shown). these assays all show a low-end detection of approximately 0ae002 u ⁄ well and dynamic range of 2-3 orders of magnitude when performed simultaneously side-by-side using serial dilutions of bacterial (clostridium perfringes) neuraminidase. these assays show approximately onefold less dynamic range and approximately fivefold less low-end sensitivity than chemiluminescent assays under these conditions. given the large amount of archived ni inhibition data for viral isolates over the past decade, it is very important for a standardized assay to generate data similar to established protocols so that data can be compared in relative terms. when run side-by-side, na-fluor tm assay provided oseltamivir carboxylate and zanamivir ic 50 values similar to nisn-published, munana-based protocols. ic 50 values vary somewhat for munana assays versus chemiluminescent assays depending on the viral isolate, as previously described. 3 the na-fluor tm assay also exhibited similar sensitivity for detecting ni sensitive virus compared to nisn-published fluorescent assays as shown in figure 1 . the large shift in ic 50 values between oseltamivir-sensitive and resistant virus using the na-fluor tm assay enables detection of mutant virus in mixed viral samples ( figure 2 ). this capability is critical for identifying resistant virus in clinical isolates presenting mixed populations of resistant and sensitive virus during ni susceptibility surveillance. several characteristics of the na-fluor tm assay make it an ideal assay for processing large numbers of viral isolates for ni sensitivity surveillance or for using the assay for high throughput screening for lead discovery of new antiviral reagents. the na-fluor tm assay signal was found to remain stable for up to 4 hours after stop solution addition when stored at room temperature and for several days when stored at 4°c (data not shown). ic 50 values did not change over these times, indicating that the assay is compatible with processing many samples in a short time frame. the na-fluor tm assay was also found to be highly reproducible giving a z' of 0ae78 or above indicating that the assay can be used confidently to identify nis in high throughput screening mode. 4 the assay can tolerate up to 5% dmso, a common compound delivery reagent used in high throughput screens (data not shown). we have developed a standardized na-fluor tm assay suggested protocol that gives data similar to established mun-ana protocols. however, we have also found that several protocol adaptations can be made that generate comparable data while allowing the user more flexibility in assay mode, use of additional reagents, and to meet user-specified assay time requirements. the na-fluor tm assay can be run in either the standard 60 minutes ⁄ 37°c endpoint mode described above or as real-time kinetic assay 5 with repeated reads taken over time without the addition of stop solution, which both serves to terminate neuraminidase activity and to enhance the fluorescence of the product. for typical ni-sensitive viral strains, the rate of munana substrate turnover at 37°c is linear for at least 4 hours (data not shown). as would be expected, rates of substrate turnover decrease in the presence of nis reflected in a decreased slope exhibited by real-time kinetic reads. real-time acquired rfus are typically 5-6 fold lower than rfus acquired after addition of stop solution at the same time point. ic 50 values obtained using slope analysis for real-time assays are similar to values obtained by endpoint analysis. whether run in real-time or end-point mode, the linear rate of substrate turnover allows the user to run the assay for shorter or longer assay times than the standard protocol without compromise to assay performance. the na-fluor tm assay is also compatible with standard methods used in many laboratories to inactivate virus. 6 we have shown that ni ic 50 values for multiple viral strains remain unchanged when the assay is performed in the presence of 0ae1% np-40 or 1% triton x-100 (data not shown). similar results are also obtained by adjusting the na-fluor tm stop solution to 40% ethanol prior to addition for assay termination. the assay is unaffected by phenol red concentrations present in cell culture media. we have developed a standardized munana-based fluorescent neuraminidase assay, the na-fluor tm influenza neuraminidase assay kit, which has been optimized for ni susceptibility screening. the assay provides data that can be compared to data generated using traditional munanabased protocols. the assay is economical, highly reproducible, easy to use, and environmentally friendly. the assay is flexible and amendable to user-specific adaptations including assay mode, assay timing, and reagent compatibility. trademarks ⁄ licensing ª 2010 life technologies corporation. all rights reserved. relenza is a registered trademark of glaxo to test the prophylactic potency of h5-vhhb, mice were treated intranasally with pbs, 100 lg of h5-vhhb, or negative control rsv-vhhb at 4, 24, or 48 hours before infection with one ld 50 of nibrg-14ma virus. body weight loss was monitored daily, and on day 4 mice were sacrificed to determine the viral load in the lungs. all mice that received h5-vhhb retained their original body weight, whereas those receiving pbs or rsv-vhhb gradually lost weight (data not shown). intranasal administration of h5-vhhb at 4 or 24 hours before challenge resulted in undetectable lung virus titers. when animals were treated with h5-vhhb 48 hours before challenge, virus titers were 50fold lower compared to pbs and rsv-vhhb treated mice, and three out of seven animals still had undetectable virus titers ( figure 1 ). we next determined if h5-vhhb nanobody ò could be also be used therapeutically. we administered 60 lg of this nanobody ò intranasally to mice up to 72 hours after chal-lenge with 1 ld 50 of nibrg-14ma virus. four days after challenge, animals that received h5-vhhb 4, 24, or 48 hours after challenge had significantly higher body weight (data not shown) and lower lung virus loads than control mice. although mice treated with h5-vhhb nanobody ò 72 hours after challenge were not clinically protected compared to control mice, they had significantly lower lung virus titers (figure 1 ). to identify the ha amino acid residues that are potentially involved in h5-vhh binding, escape viruses were selected by growth and plaque purification of nibrg-14ma virus in the presence of h5-vhhm or h5-vhhb nanobodies ò . the ha sequences of six independently isolated h5-vhhm escape viruses revealed substitution of a lysine by a glutamic acid residue at position 189 in ha1 (h5 numbering). in addition, two h5-vhhm escape mutants carried an n154d and four carried an n154s substitution. the three-dimensional structure of nibrg-14 ha shows that n154d ⁄ s and k189e are close to each other as part of the corresponding antigenic site b in h3 ha. 4, 5 interestingly, the n154d ⁄ s mutations remove an n-glycosylation site, which is surmised to have evolved in h5n1 ha as a strategy to mask an antigenic site. 6 escape viruses selected in the presence of h5-vhhb carried k189n (n = 4) or k189e (n = 2) substitutions. these results indicate that residues in antigenic site b, at the top of ha and very close to the receptor binding domain (rbd), are essential for neutralization of the virus by h5-vhhm ⁄ b nanobodies ò (figure 2 ). the virus titer was measured in lung homogenates prepared on day 4 after challenge. the x axis refers to the time points in hours relative to the challenge (time = 0 hours) when ha-specific nanobodies (h5-vhhb), control nanobodies (rsv-vhhb) or pbs was administered to the mice. # below detection limit, n not determined [n = 4-7 mice per condition: p values < 0ae05 (*)]. here we demonstrated that prophylactic and therapeutic treatment with llama-derived immunoglobulin single variable domain fragments is effective to control infection with h5n1 influenza virus in a mouse model. we demonstrate that pulmonary delivery is a highly effective route of administration to treat or prevent influenza virus infection. in addition, we demonstrate that a homobivalent h5-vhhb has powerful h5n1-neutralizing activity in vivo. it is important to note that we used a mouse-adapted derivative of the non-highly pathogenic nibrg-14 virus in our challenge model. nevertheless, this virus induces severe morbidity and lethality in mice. compared to conventional neutralizing monoclonal antibodies, vhhs offer the advantage that they are easy to produce in escherichia coli, typically with high yield. in addition, their small size (15 kda for a monovalent vhh) and high folding capacity allow the generation of oligovalent vhh derivatives. in vitro escape selection revealed that a k189e substitution in ha1 abolished the neutralizing effect of h5-vhhm ⁄ b. a lys or arg residue at this position is conserved in all human h5n1 virus isolates. of note, all selected escape mutants contained a glutamic acid or serine residue at position 189, which suggests that the conserved positively charged amino acid is important for neutralization by h5-vhh nanobodies ò . interestingly, escape mutants selected with h5-vhhm also carried an n154d ⁄ s co-mutation that removes an n-glycosylation site in this antigenic site of ha. the predicted n-glycosylation site at n154 in a ⁄ hong kong ⁄ 156 ⁄ 97 ha was shown to be glycosylated and may have evolved to mask an antigenic site near the rbd. 7, 8 the selected amino acid changes are located near the receptor binding site of ha. therefore, it is possible that enhanced receptor binding properties of these escape viruses contribute to or are responsible for the loss of neutralizing activity of h5-vhh nanobodies ò . 9, 10 we conclude that influenza virus neutralizing nanobodies ò have considerable potential for the treatment of h5n1 virus infections. although we focused on vhhs that presumably recognizes an epitope near the rbd, it is possible to select vhh molecules that bind to other epitopes in ha, including more conserved domains. more, a novel na (i117m) substitution was discovered in a series of specimens from a patient. for the amantadine resistance, 1113 samples were tested, and all of them were confirmed to be resistant. we collected respiratory specimens from patients who had been clinically refractory to antiviral treatment since october 2009 upon ethical approval from the relevant institutions. to investigate the resistant pattern, sequence analysis to the na and matrix (m2) genes were conducted by reverse transcription (rt)-pcr and sequencing reaction. the obtained sequences were analyzed by the influenza sequences and epitopes database, which was developed in korea. eleven patients were found to be having oseltamivir-resistant pandemic (h1n1) 2009 viruses with the h275y substitution in the viral na genes (tables 1 and 2 ). some cases were associated with oseltamivir treatment on the basis of h275y change from the oseltamivir-sensitive genotypes to oseltamivir-resistant genotypes in consecutive samples from the same patient. furthermore, a novel na (i117m) substitution that may be associated with oseltamivir resistance was detected in specimens from one patient (patient g) who had myelodysplasia and received oseltamivir and peramivir (tables 1 and 2 ). in addition, we obtained viruses from clinical specimens (patients a and c) and evaluated antiviral susceptibility by measuring the dose of oseltamivir and zanamivir required for 50% inhibition (ic 50 ) of na activity. these viruses (from patients a and c) were resistant only to oseltamivir (ic 50 713ae2 and 359ae4 nmol ⁄ l, respectively). susceptibility to zanamivir was not altered whether na contained y275 or h275 (ic 50 0ae13 and 0ae78 nmol ⁄ l, respectively). one isolate of pandemic (h1n1) 2009 virus with an oseltamivir-sensitive genotype (h275 in its na) was susceptible to oseltamivir (ic 50 1ae18 nmol ⁄ l) and zanamivir (ic 50 0ae42 nmol ⁄ l). patients with oseltamivir-resistant pandemic (h1n1) 2009 were treated during hospitalization with oseltamivir alone or with a combination of other antiviral drugs ( we found 11 patients of oseltamivir resistance with h275y mutation in the na gene of pandemic (h1n1) 2009 virus through the surveillance of patient refractory to antiviral treatment. in addition, novel amino acid change (i to m) at position 117 in the na gene, which might influence oseltamivir susceptibility, was detected in sequential specimens of a patient. these data showed that generation of oseltamivir resistance could be associated with oseltamivir treatment. therefore, it needs to strengthen the antiviral monitoring by supplementation of the clinical data including antiviral treatment. during the pandemic, oral oseltamivir was the primary antiviral medication used for treatment of hospitalized patients with ph1n1 infection. many physicians worried that clinical deterioration or failure to respond to treatment with oseltamivir was due to either oseltamivir resistance or oseltamivir failure. in the united states, two investigational intravenous (iv) nais were available during 2009-2010: peramivir through emergency use authorization and zanamivir by investigational new drug application. peramivir would be an option for patients with oseltamivir failure, but would not be appropriate for patients infected with h275y oseltamivir resistant mutants. iv zanamivir was available in limited supply, but would be appropriate for severely ill patients infected with an oseltamivir-resistant ph1n1 virus. during the pandemic, clinicians had few options for antiviral resistance testing in the united states. to respond to this need, the us centers for disease control and prevention (cdc) offered antiviral resistance testing for patients suspected to have clinical failure due to oseltamivir resistance. we describe the methods that cdc used to prioritize patients for testing during the pandemic and to detect markers for oseltamivir resistance, as well as the results from this testing. to facilitate decisions on which patients to test, we developed testing algorithms that were shared with state labora-tories, epidemiologists, and the emergency operation center at cdc. we prioritized patients who might benefit the most from antiviral testing given the inherent delay in providing antiviral results, e.g. patients who might have prolonged ph1n1 shedding. patients that were critically ill [intensive care unit (icu) admission] or patients with severe immunocompromising conditions with clinical evidence for oseltamivir treatment failure (persistent detection of virus and clinical unresponsiveness to the drug) were prioritized. in addition, we tested specimens from patients that failed oseltamivir chemoprophylaxis. standard forms with information regarding specimen and minimal clinical information were collected on all patients. all protocols were validated and approved by clinical laboratory improvement amendments, e.g. quality standards to ensure accuracy, reliability, and timeliness of patient test results. information collected on patients was deemed public health response, not research, at cdc. clinical specimens, confirmed as 2009 pandemic influenza a (h1n1), were tested for the h275y mutation in the na using pyrosequencing. 5 results were returned to sender within 24-48 hours of specimen receipt. from october 2009 until july 2010, a total of 116 specimens from 73 patients were submitted for testing. viruses from 26 (36%) of 73 patients had h275y mutation in the na in at least one submitted specimen. clinical information was available for 66 patients (table 1) . most patients had received oseltamivir for treatment prior to obtaining the specimen sent for antiviral testing. four patients received oseltamivir for chemoprophylaxis, all were immunosuppressed, and all had the h275y mutant; duration of chemoprophylaxis until ph1n1 infection was detected varied (4-39 days). among the patients with an h275y mutant who were treated with oseltamivir, the median time on oseltamivir prior to collection of specimen with h275y mutation was 18 days (range 3-36 days). three patients were part of a hospital cluster of oseltamivir-resistant virus infections and were infected with h275y mutants prior to oseltamivir treatment. 6 patients with immunocompromising conditions accounted for almost half of all patient specimens tested, but they accounted for the majority of oseltamivir-resistant ph1n1 virus infections (table 1) ; among 32 individuals with severe immunocompromising conditions and clinical failure while on oseltamivir therapy, 23 (72%) had the h275y mutant detected. among the immunosuppressed patients with an oseltamivir-resistant virus, 16 (70%) had hematologic malignancies reported. in contrast, among the subset of icu patients without immunocompromising conditions and clinical failure while on oseltamivir therapy, we found little resistance: 2 (5ae9%) of 34 icu patients had oseltamivir resistance detected. during the 2009 pandemic, we were able to provide timely and useful information to clinicians regarding suspected cases of oseltamivir resistance. our testing algorithm limited the number of specimens to specimens from the highest risk patients that would benefit the most from antiviral treatment. such an approach allowed us to offer this service without compromising our public health duties. in addition, the information we collected on patients from this service complimented our data on the national surveillance for antiviral resistance. we also performed national antiviral resistance surveillance from april 2009 to july 2010. overall, 67 resistant ph1n1 viruses were identified from april 2009 to july 2010 in the united states among 6812 tested samples, including specimens described above, surveillance specimens, and resistant viruses reported in the literature. 7 further studies to understand risk factors for oseltamivir-resistant ph1n1 infection in patients with severe immunocompromising conditions are needed. while efforts to provide antiviral testing technology and materials to state laboratories are ongoing, clinicians still have limited options for such testing. rapid and inexpensive assays that could be performed by clinical laboratories, especially those caring for immunosuppressed patients, would be useful to inform patient care. the applied biosystems ò na-xtd tm influenza neuraminidase assay kit provides the next-generation na-xtd tm 1,2-dioxetane chemiluminescent neuraminidase (na) substrate, together with all necessary assay reagents and microplates, to quantitate sensitivity of influenza virus isolates to neuraminidase inhibitors. like the na-star ò influenza neuraminidase inhibitor resistance detection kit, the na-xtd tm influenza neuraminidase assay provides highly sensitive detection of influenza neuraminidase activity. in addition, the na-xtd tm assay provides extended-glow light emission that eliminates the need for reagent injection and enables signal measurement either immediately or up to several hours after assay completion. the na-xtd tm assay is also used to quantitate influenza na activity directly in cellbased virus cultures to monitor viral growth or inhibition. global monitoring of influenza strains for resistance to neuraminidase inhibitors (nis) is essential for understanding their efficacy for seasonal, pandemic, or avian influenza, and studying the epidemiology of viral strains and resistance mutations. functional neuraminidase inhibition assays enable detection of any resistance mutation, making them extremely important for global monitoring of virus sensitivity to nis. the first-generation chemiluminescent na-star ò influenza neuraminidase inhibitor resistance detection kit has been widely used for virus ni sensitivity assays, 1-8 including identification of a ⁄ h1n1 pandemic virus resistant to oseltamivir. 9, 10 in addition, this assay has been used for identification of new ni compounds, 11 ni characterization, 12 studies of virus transmission, 13 drug delivery, 14 na quantitation of virus-like particles, 15 and cell-based virus quantitation. 16 neuraminidase assays performed with chemiluminescent 1,2-dioxetane substrates, including na-star ò and na-xtd tm substrates, typically provide 5-to-50-fold higher sensitivity by signal-to-noise ratio than assays performed with the fluorescent munana substrate. in addition, chemiluminescent assays provide linear results over 3-4 order of magnitude of neuraminidase concentration compared to 2-3 orders of magnitude with the fluorescent assay. 2 the high assay sensitivity achieved with chemiluminescent assays enables use of lower concentrations of viral stocks, and the wide assay range minimizes the need to pre-titer virus stocks prior to ic 50 determination. chemiluminescent reactions result in conversion of chemical energy to light energy, as light emission. the na-xtd tm substrate is a 1,2-dioxetane structure bearing a sialic acid cleavable group. to perform the na-xtd assay, virus dilutions (from cell culture supernatant) are pre-incubated in the presence of neuraminidase inhibitor. then na-xtd substrate is added and incubated for 30 minutes for substrate cleavage to proceed. finally, light emission is triggered upon addition of na-xtd accelerator, which provides a ph shift and a proprietary polymeric enhancer, both required for efficient light emission. chemiluminescent assays are performed in solid white microplates, and light emission is measured in a luminometer. the na-xtd tm substrate has a single structural difference from the na-star ò substrate that provides a much longer-lasting chemiluminescent signal, with a signal half-life of approximately 2 hours (not shown), compared to $10 minutes with the na-star assay, eliminating the need for luminometer instruments equipped with reagent injectors and enabling more convenient batch-mode processing of assay plates. the na-xtd tm assay kit also provides a new accelerator solution, containing a next-generation polymer enhancer, and a triton ò x-100-containing sample prep buffer providing enhanced na activity. read-time flexibility is demonstrated by determination of oseltamivir ic 50 values using data collected over 3 hours after addition of na-xtd tm accelerator. although signal intensity slowly decreases over time, the ic 50 curves and values are identical at each time point, shown using influenza b ⁄ lee ⁄ 40 ( figure 1) . triton x-100 detergent at 1% has been shown to inactivate flu virus while increasing neuraminidase activity. 17 the addition of na sample prep buffer (containing 10% triton x-100) to virus stocks (at 1 ⁄ 10 volume, achieving a final concentration of 1%) provides increase in na activity up to fourfold, but is not consistently observed, and seems to be most effective with more concentrated virus stocks. ic 50 values are unaffected by the addition of triton x-100 to the virus stock prior to virus dilution (not shown), so the assay is compatible with known virus inactivation reagents. assay sensitivity and ic 50 values determined with the na-xtd assay have been compared to those obtained with both the chemiluminescent na-star assay and the fluorescent na-fluor assay (not shown). the chemiluminescent assays provide 2-to 50-fold higher sensitivity by signal-to-noise ratio, depending on the virus strain, wider assay dynamic range, and better low-end detection limit than the fluorescent assay. the wide assay range with the chemiluminescent assays enables determination of ic 50 values over a range of virus concentrations, eliminating the need to titer virus prior to performing ic 50 determination assays. ic 50 values obtained with the na-xtd assay are nearly identical to those obtained with the na-star assay, with both oseltamivir and zanamivir neuraminidase inhibitors, and tend to be slightly lower than ic 50 values obtained with the fluorescent assay. viral na quantitation provides a convenient read-out to measure viral growth or inhibition, including inhibition in the presence of inhibitory compounds or antibodies, described as accelerated viral inhibition with na as readout assay (avina). 16 bation in the presence of varying concentrations of oseltamivir carboxylate. samples of culture media were assayed 24 hours later. quantitation of na activity with the na-xtd tm assay demonstrates inhibition of viral growth by oseltamivir carboxylate in cell culture ( figure 2 ). different volumes of culture media were assayed with the na-xtd assay, either in the culture plate or in a separate assay plate (not shown). performing the assay using the entire well contents (100 ll) reduces assay sensitivity due to the high concentration of phenol red. assaying a smaller volume of culture medium (either in culture plate or a separate assay plate) provides higher sensitivity, and enables temporal monitoring or use of remaining culture medium for other assays. the applied biosystems ò na-xtd tm influenza neuraminidase assay kit is a next-generation chemiluminescent neuraminidase assay providing high assay sensitivity and ''glow'' light emission kinetics for improved ease-ofuse. the applied biosystems ò na-fluor tm influenza neuraminidase assay kit, based on the fluorescent mun-ana substrate, has also been developed to complement the na-xtd tm and na-star ò chemiluminescence assays, for users lacking luminometer instrumentation or choosing to use fluorescence assay detection. together these kits offer: • standardized reagents and protocols • choice of detection technology • simple instrumentation requirements • high sensitivity for use with low virus concentrations • compatibility with batch-mode processing and largescale assay throughput • broad specificity of influenza detection • flexibility in assay format • additional na assay applications -cell-based viral assays, screening for new nis, detection of na from other organisms functional neuraminidase inhibition assays enable detection of any resistance mutation and are extremely important in conjunction with sequence-based screening assays for global monitoring of virus isolates for ni resistance mutations, including known and new mutations. together, these assays provide highly sensitive, convenient and versatile assay systems with standardized assay reagents, and simple assay protocols for influenza researchers. 15 over 200 000 hospitalizations and 36 000 deaths in the us annually are attributable to seasonal influenza, primarily in chronically ill persons and the elderly. 1-3 following the emergence of pandemic 2009 h1n1 influenza, severe illnesses have also been observed in children and young healthy adults. 4 the occurrences of staphylococcal and pneumococcal pneumonia complicating influenza pandemics are well described. [5] [6] [7] although temporal associations of bacterial pneumonia and influenza circulation have been reported, there is little precise data on rates of bacterial complications of seasonal or pandemic influenza. the study of bacterial lung infection has been hampered by insensitive tests for invasive disease and the difficulty of interpreting routinely obtained sputum culture results. 8, 9 procalcitonin (proct), the prohormone of calcitonin, can discriminate viral and bacterial infections. 10 this 116-aminoacid precursor protein normally produced by neuroendocrine cells of the lungs and thyroid gland was first shown to be elevated in bacterial infections in patients with pulmonary injury and pneumonitis. 11 stimuli of proct include tnf-a, endotoxin, and other bacterial products. 12 several studies indicate that bacterial infections commonly induce hyperprocalcitonemia, but that viral infections, including 2009 h1n1, are associated with only minimal increases. 10, 12, 13 of note, proct induction is attenuated by viral-induced interferon-c. 14 a meta-analysis of studies comparing proct and crp as markers for bacterial infection found that proct was more sensitive and specific than crp for differentiating bacterial from other causes of inflammation. 15, 16 therefore, we measured proct levels in patients with seasonal and pandemic influenza and compared results with conventional methods for bacterial diagnosis. adults ‡21 years of age admitted to rochester general hospital (rgh) from november 1st to june 30th for two winter seasons (2008-2010) with an admitting diagnosis compatible with acute respiratory tract infection were recruited for the study. patients were screened within 24 hours of admission, and those with prior antibiotic use, immunosupression, or pregnancy were excluded. subjects or their legal guardian provided written informed consent. the study was approved by the university of rochester and rgh research subjects review board. at enrollment demographic, clinical and laboratory information was collected. influenza testing included nosethroat swabs (nts) for rapid antigen, viral culture, and reverse transcription-polymerase chain reaction (rt-pcr) and serology. testing for bacterial pathogens included blood cultures, sputum for culture and gram stain, nts for mycoplasma pneumoniae and chlamydophila pneumoniae pcr, s. pneumoniae antigen testing, and pneumococcal serology. if patients were unable to expectorate, sputum was induced with normal saline and bronchodilators. specimens were considered adequate by the standard criteria of >25 neutrophils (pmns) and <10 epithelial cells per high power field. serum was collected at admission and hospital day 2 for proct measurements. influenza infection was defined a positive result for any of the following tests: . cloned proteins were coated on eia plates at 2 ug ⁄ ml in bicarbonate buffer. after overnight incubation, plates were washed and two-fold dilutions of serum were incubated overnight at room temperature. plates were washed and incubated with alkaline phosphatase conjugate for 3 hours, followed by substrate. a greater than or equal to fourfold rise in titer was considered evidence of infection with s. pneumoniae. urinary antigen for s. pneumoniae samples were assayed for antigen using the binax now kit. (binax inc, scarborough, me, usa). the proct was measured using time resolved amplified cryptate emission technology (kryptor pct; brahms, henningsdorf, germany). functional sensitivity is 0ae06 ng ⁄ ml (normal levels are 0ae033 ± 0ae003 ng ⁄ ml). 18 mycoplasma and chlamydia pcr real-time pcr targeting the p1 adhesion gene for m. pneumoniae and the ompa gene for c. pneumoniae was used to detect atypical bacteria. 19 results fifty-one of 529 (9ae6%) illnesses evaluated tested positive for influenza virus. of these, 20 were due to ''seasonal influenza'' (16 influenza a ⁄ h3n2 and 4 influenza b), and 31 were identified as ''pandemic influenza'' (2009 h1n1). demographics of both groups were similar: mean ages 55 ± 18 and 50 ± 11 years, respectively, and equivalent sex and racial characteristics. other than a higher incidence of underlying lung disease in the seasonal group (75% versus 45%, p = 0ae05), pre-existing medical conditions including obesity were similar. symptoms, physical findings, and discharge diagnoses did not differ, and chest radiographs (cxr) showed infiltrates in 20% and 13% of seasonal and pandemic subjects, respectively. two pandemic and one seasonal influenza patient developed respiratory failure, and none died. overall, bacterial infections were diagnosed in 8 (16%) subjects (4-seasonal and 4-pandemic), and none were bacteremic. bacterial infections included: 5-s. pneumoniae, 1-m. pneumoniae, 1-s. aureus, and 1-h. influenzae. all seasonal patients were diagnosed with asthma or bronchitis, whereas three pandemic patients had pneumonia. mean serum proct (ng ⁄ ml) levels in seasonal versus pandemic patients on admission and day 2 were: 0ae14 ± 0ae17 versus 1ae05 ± 2ae86 and 0ae17 ± 0ae17 versus 1ae39 ± 4ae37, respectively, and were not significantly different (table 1) . several patients in the pandemic group had high proct levels, and there was a trend toward more pandemic patients having admission proct values ‡0ae5 ng ⁄ ml than seasonal subjects [1 (5%) versus 6 (19%), p = 0ae22] ( figure 1a , b). of the four patients with proc-t > 1ae5 ng ⁄ ml, two had dense infiltrates on cxr, one had a peripheral wbc of 17 200 ⁄ ml with a threefold increase in s. pneumoniae antibody, and one developed respiratory failure associated with copd exacerbation. reliable sputum samples (within 6 hours of antibiotics) were collected in only 22 (43%) subjects. of these, proct was ‡0ae5 ng ⁄ ml in two with influenza alone and three associated with bacterial infection, and <0ae5 ng ⁄ ml in 12 with influenza alone and five associated with bacterial infection. in the 22 with reliable sputa and accepting the conventional bacterial diagnosis, sensitivity of a proc-t ‡ 0ae5 ng ⁄ ml for bacterial infection was 38%, specificity 86%, positive predictive value 60%, and negative predictive value 71%. notably, one patient considered to have influenza alone (proct -0ae87 ng ⁄ ml) had group a streptococcus and s. aureus in a contaminated sputum and bilateral infiltrates on cxr. three of five patients with bacterial infections and proct < 0ae25 ng ⁄ ml had a clinical diagnosis of bronchitis. mean proct values were significantly higher in patients with infiltrates versus those with atelectasis or no acute disease on cxr (2ae4 ± 4ae8 ng ⁄ ml versus 0ae36 ± 1ae2 ng ⁄ ml, p = 0ae02). combining patients with proct values ‡0ae5 ng ⁄ ml with those having positive bacterial tests, rates of bacterial infection associated with seasonal and pandemic influenza were 20% and 26%, respectively. notably, antibiotics were administered to 88% of subjects despite 73% having no acute disease on cxr. in our study, bacterial infections were diagnosed in approximately 25% of adults hospitalized with influenza with no significant difference in rates noted between seasonal and pandemic influenza infected subjects. previous reports of bacterial infection rates of 4-15% with seasonal influenza are difficult to compare with recent studies of pandemic influenza, because the latter tended to focus on more severely ill patients. [20] [21] [22] bacterial pneumonia has been suspected or diagnosed in 13-24% of patients in intensive care associated with 2009 h1n1 infection and up to 38% of patients who died. 23, 24 despite aggressive pursuit of specimens for bacterial testing, diagnoses could be confirmed in only 8 (15ae6%) of patients using conventional methodology. given the difficulty in establishing a diagnosis of bacterial infection, elevated proct values may be helpful to identify patients at high risk for invasive disease. in a study of 25 patients with severe 2009 h1n1 or bacterial infection necessitating intensive care, a threshold proct level of 0ae8 ng ⁄ ml, demonstrated 100% sensitivity and 62% specificity for bacterial infection. 25 among 38 patients with 2009 h1n1 associated pneumonia, many of whom had respiratory failure, a threshold proct value of 0ae5 ng ⁄ ml provided a sensitivity of 100% and specificity of 53% for bacterial infection. 26 access to samples from lower airways in ventilated patients in these studies may have improved recovery of bacteria and account for the different results we observed. it should be noted that none of our patients were bacteremic, which is a very strong stimulus for proct release. proct levels have been used successfully to guide therapy in community acquired pneumonia, and our data showing high proct levels in patients with infiltrates on cxr suggests proct may be most useful for excluding invasive disease. 27, 28 elevated proct levels were not observed in patients with purulent sputum and clear cxr. it is notable that a proct level of <0ae25 ng ⁄ ml did not exclude patients with bacterial bronchitis since proct has been used to guide antibiotic therapy in copd exacerbations. 29 while it could be argued that healthy patients with bacterial bronchitis do not require antibiotic treatment, physician behavior in our study indicates antibiotics are frequently prescribed. combining patients with proct values ‡0ae5 ng ⁄ ml and those with a positive bacterial test, approximately 25% in patients in our study had bacterial complications associated with influenza infection. efforts should be made to curtail antibiotic use in hemodynamically stable patients with clear cxrs. given physician discomfort regarding discontinuing antibiotics, proct measurements in combination with routine bacterial cultures should be useful tools to guide therapy. influenza, mrsa, cytokines: diagnosis, treatment, prevention -a possible strategy for outpatient care we started the antiviral treatment of influenza in humans using neuraminidase inhibitors on january 29, 1999 in a successful attempt to cure a 96-year-old patient. since then, we have used the inhalant antiviral drug zanamivir, and later (october 1, 2002) changed to the use of oseltamivir with systemic bioavailability for treating patients with influenza. after 10 years of experience with antiviral treatment of outpatients, we highlight the importance of early diagnosis and early treatment. 1 the necessity of an earliest possible diagnosis was confirmed in the pandemic of 2009. large hospitals reported that patients with an h1n1 ⁄ 09 infection had to be treated with extracorporeal membrane oxygenation. we are convinced this is due to delayed recognition of infection in most cases. valuable time is lost when the patient with a sudden onset has to be brought to a hospital for emergency treatment. the point at which the patient goes to the doctor is decisive, and this problem of timing and the delivery of early treatment is not specific to germany. in our medical office, we assessed patients with suspected influenza (to date 262 seasonal infections, and in 2009, 25 h1n1 ⁄ 09) through clinical diagnosis, 2 and then proven by point of care rapid test (quickvue; quidel, san diego, ca, usa) followed by pcr. all of the patients undergo concomitant lab tests: leukopenia, serum iron level, and the humoral inflammation status [sum of the c-reactive protein (crp) and fibrinogen levels]. because of the constant threat of a bacterial superinfection, a bacterial swab and antibiogram is carried out on every patient. in all cases positive for influenza, oseltamivir was given immediately. nowadays it is important that a double infection with influenza and mrsa must be recognized immediately and treatment started at once with antivirals and, when appropriate, with a suitable antibiotic. we pay particular attention to an extremely low iron level (signum mali ominis). in addition we monitor oxygen saturation and the course of the humoral inflammation status every 1-2 hours for every of our outpatients. among our patients with seasonal influenza, we saw 132 within 12 hours, 103 within 36 hours, and 27 within 48 hours after disease onset. for pandemic influenza, it was 12 patients within 12 hours, 11 within 24 hours, and two within 36 hours. for all patients, we measured crp < 3ae5 mg and fibrinogen < 350 mg ⁄ dl (12 hours), crp < 10 mg and fibrinogen < 450 mg ⁄ dl (36 hours), and crp > 10 mg and fibrinogen < 500 mg ⁄ dl (48 hours, only seasonal cases). antibiotics were necessary in 130 cases, heparin and oxygen administration in 27 cases. one hundred forty-eight patients had a superinfection following influenza. the most common strains were haemophilus parainfluenzae and staphylococcus aureus. the subsequent use of a suitable antibiotic was only necessary in 50% of the patients. in all cases diagnosed, treatment (including heparin and oxygen administration) and monitoring were conducted in our medical office. none of our patients (seasonal and pandemic) had to be admitted to hospital. the early decision of whether or not antiviral and antibacterial treatment is taking effect is the only way the threat of a cytokine storm can be averted. not only does the primary care physician have to be aware of the pathophysiology involved, but also the necessary diagnostic and therapeutic options have to be made available to him. the result will lead to a saving of both lives and healthcare costs. this applies both in epidemic as well as in pandemic times. today we know that influenza leaves behind a defenceless immune system, and that the proteases of s. aureus contribute to influenza associated pneumonia. mark von itzstein, who discovered neuraminidase inhibitors, emphasized the synergistic cooperation of viruses and bacteria (personal communication, 2009). mrsa and influenza viruses are posing problems worldwide. the case of a 17-year-old boy with h1n1 ⁄ 09 infection demonstrates how fatal developments can be prevented. due to his constantly recurring colds, we had already detected the mrsa colonization years earlier and had always worked on boosting his general health and resistance. both the patient and his family were included in dealing with the problem. the patient was, and is, always vaccinated early with a virosomal vaccine (baxter). during the oktoberfest in munich in september 2009, when h1n1 infections were increasingly occurring, we learned that our patient had come down with an extremely acute feverish illness. with the help of the rapid test, we diagnosed an h1n1 ⁄ 09 virus infection and started treatment with oseltamivir immediately. the humoral inflammation status, which had increased very rapidly to more than 4ae5 mg ⁄ dl within hours, was treated with the effective cotrimoxazol from the antibiogram. at the same time, the patient was heparinized. the following day the patient had no fever and was symptom-free. it was only through our early knowledge of what could develop pathophysiologically that we were in a position to make the right decision at the right time. every doctor treating outpatients can follow this procedure if he is familiar with the pathophysiology of the disease and has the available tests on hand: virus rapid test, additional laboratory parameters (leukopenia, iron), and the humoral inflammation status. the decisive factor, however, is the constant clinical alertness towards the course of every acute feverish cold with acute onset. the patient has to remain in the care of the attending physician, and the chosen treatment has to be administered and monitored. this means constant spo2 measurements and checking the humoral inflammation status every 2 hours. if a clinical worsening occurs during monitoring, the treatment regime has to be changed immediately, which means the administration of an appropriate antibiotic. this outpatient care on the part of the doctor has to be available 7 days a week so that no time will be lost. reports from the netherlands and denmark show that, with the help of this preventive strategy under the motto 'search and destroy,' the dangerous, fatal course of infections reported in germany with at least four deaths a day, can be avoided. however, the doctor has to be adequately remunerated for the elaborate amount of time this intensive outpatient care requires. with our strategy, we have moved from divergence to convergence in the care of our patients. we reported on our 10 years of clinical experience with this approach at the antivirals congress in peking. our main message was early diagnosis and early treatment. we were able to demonstrate this in 287 outpatients with seasonal influenza and 25 h1n1 ⁄ 09 outpatients. our creed is: as much outpatient care as possible and as little hospitalization as possible. virological and autopsy findings in suspected and confirmed fatal cases of 2009 h1n1 pandemic influenza in the czech republic -preliminary results influenza viruses cause substantial morbidity and mortality. pandemic influenza may have a serious impact on certain (mainly younger) age groups in comparison with seasonal flu. influenza is one of few viral infections capable of causing a pneumonia that is difficult to cure and ⁄ or leads to sudden death. the aim of this study was to analyze and compare virological and autopsy findings in patients who died with suspected or confirmed 2009 h1n1 pandemic influenza virus infection. there were 2477 virologically confirmed cases of pandemic influenza and 102 deaths in the czech republic during pandemic wave. more than 400 influenza strains belonging to the new pandemic variant were isolated in the national influenza reference laboratory. postmortem biological samples were collected from any patient who died with suspected influenza infection to test for respiratory viruses. the samples were screened for 2009 h1n1 pandemic influenza virus by real-time pcr (rt pcr), and when rt pcr positive, by virus isolation assay. no immunohistochemical staining for influenza antigen was done on the rna pcr positive cases. other important respiratory viruses such as respiratory syncytial virus, parainfluenza viruses, and adenoviruses were detected by virus isolation assay in a suitable cell culture. epidemiological analysis of postmortem histopathologic findings in the airway tissue was carried out in 57 of 61 fatal cases. virological findings were subsequently correlated with histological changes and available demographic and clinical data. statistical analysis was performed by t-test using spss software. sixty-one deaths (34 males, 27 females) were analyzed. the rna of the 2009 h1n1 pandemic influenza virus was detected by pcr in 38 cases, while 23 cases remained negative. five respiratory syncytial viruses and two adenoviruses were detected in the influenza negative group. the mean age of 38 confirmed 2009 h1n1 pandemic influenza victims was 46ae0 years, age range 7-74 years and median 47ae5 years. the mean age of 23 influenza negative victims was 55ae1 years, age range 1-89 years and median 57ae0 years. the 95% ci for the difference in the age between the two groups is )1ae7; 19ae9. the test is statistically significant at the 10% level. the obtained significance (p = 0ae07) can be explained by the relatively small size of the study group. the most common postmortem histopathologic finding in the lung tissue of the 2009 h1n1 pandemic influenza virus-positive victims was diffuse alveolar damage (often bilateral) and ⁄ or hyaline membrane formation, possibly with signs of respiratory distress syndrome (in 18, i.e., 51ae4%, of 35 autopsied patients). in the 2009 h1n1 pandemic influenza virus negatives, the most common finding was pneumonia or bronchopneumonia with the detection of various bacterial species (in 12, i.e., 54ae5% of 22 autopsied patients). the cause might be either primary bacterial infection or superinfection following primary infection with influenza virus that remained undetected. the 2009 h1n1 pandemic influenza victims were younger than the patients who died with suspected but undetected 2009 h1n1 pandemic influenza. the majority of deaths were primarily linked to rapidly developing respiratory failure. this result supports the previous reports of severe respiratory outcomes in younger age groups that are typically linked to the spread of a pandemic strain of influenza. 1 due to limited amount of pandemic vaccine, especially at the beginning of pandemic, it is advisable to assess experiences with antiviral treatment, mainly dosing, and way of antiviral administration. primers specific for each of the eight genes of pandemic h1n1 ⁄ 2009 were adopted from assays as described previously to discriminate against seasonal human h1n1 and h3n2 viral segments (table 1) . 9 the primers were allowed to cross-react specifically with the sister clade viral segments of pandemic h1n1 ⁄ 2009. 2 the method we employed in this study was a 2-step singleplex sybr green-based real-time rt-pcr. this approach helped lower the running cost of the assays and facilitated downstream molecular analyses (e.g., sequencing) by using screened cdna samples. viral rna was extracted from viral cultures or clinical samples as described 3, 10 and was converted to cdna in a universal rt-pcr. each 10 ll rt reaction containing 5ae5 ll of purified rna, 2 ll of 5· firststrand buffer (invitrogen), 100 u of superscript ii reverse transcriptase (invitrogen), 0ae1 lg of uni12 (5¢-ag-caaaagcagg-3¢), 11 0ae5 mm of deoxynucleoside triphosphates and 10 mm of dithiothreitol was incubated at 42°c for 50 minutes, followed by 72°c for 15 minutes for heat inactivation. for each segment-specific real-time pcr, the 20 ll reaction contained 1 ll of a 10-fold diluted cdna samples, 10 ll of fast sybr green master mix (applied biosystems), and 0ae5 lm of the corresponding primer pair. the thermocycling conditions of all eight segment-specific pcrs were optimized as 95°c for 20 seconds, followed by 30 cycles of 95°c for 3 seconds and 62°c for 30 seconds, and all eight assays were performed simultaneously in a 7500 sequence detection system (applied biosystems). at the end of the amplification step, pcr products went through a melting curve analysis to determine the specificity of the assay (60-95°c; temperature increment: 0ae1°c ⁄ seconds). cdna of a ⁄ california ⁄ 04 ⁄ 2009 virus was used as a positive control. robust and specific amplification was achieved in all eight segment-specific real-time rt-pcr reactions. 9 pcr product for each segment of pandemic h1n1 ⁄ 2009 yielded unique melting curve pattern with distinctive melting temperature (tm), which was not observed in negative and water controls ( figure 1 ). reactions with tm value within 2 sds of the mean tm were determined as positive. we evaluated the assays with a number of serologically confirmed human clinical samples. all pandemic h1n1 ⁄ 2009 samples (n = 67) were positive in all eight assays, while all seasonal samples (h1n1 = 38; h3n2 = 66) were negative in all assays, as expected ( figure 1 and data not shown). these results showed that no reassortant of pandemic h1n1 and seasonal viruses was present in the tested human isolates. we applied these assays to our on-going influenza virus surveillance program in swine. nasal and tracheal swab samples were collected at an abattoir in hong kong and cultured in madin darby canine kidney cells or embryonated eggs as described. 12 positive viral cultures in hemagglutination assays were tested with the established segmentspecific real-time rt-pcr assays. among 59 swine viral isolates collected from 2009 to september 2010, 10 of them were recognized as pandemic h1n1 ⁄ 2009 in all eight segments. they were confirmed to be of pandemic h1n1 ⁄ 2009 origin by subsequent full genome sequencing analyses, showing that there were interspecies transmissions of the virus from humans to pigs. 13, 14 the remaining 49 viruses had one to seven gene segments positive in the segment-specific real-time rt-pcrs. thirty of them were selected as representative samples for full genome sequencing analyses based on the genotyping data generated in our assays. they were swine h1n1 or h1n2 viruses with their gene segments derived from tr or eurasian avian-like swine lineages. it should be highlighted that all of their positive gene segments in our assays belonged to the sister groups of pandemic h1n1 ⁄ 2009. their melting curve patterns were very similar to those derived from segments of pandemic h1n1 ⁄ 2009, except for ha of tr lineage. 9 our results successfully demonstrated the use of these segment-specific real-time rt-pcrs to recognize gene segments of contemporary tr (pb2, pb1, pa, ha, np, and ns) and ea (na and m) swine viruses. 2 the ha-specific assay was able to discriminate pandemic h1n1 ⁄ 2009 from other contemporary swine viruses in the same lineage. nevertheless, to confirm the identity and to examine all the genetic variations in the viruses of interest, full genome sequencing analyses were necessary. in this study, the biggest obstacles in primer design were sequence similarity and diversity of influenza viruses. we attempted to use degenerated primers, but they were highly non-specific. the finalized non-degenerated primers crossreacted with genes from pandemic h1n1 ⁄ 2009 and its sister clade tr (pb2, pb1, pa, ha, np, and ns) and ea (na and m) swine viruses with some minor sequence mismatches. three avian (h5n1, h7n7, and h9n2) and 1 classical swine (h1n1) were also tested with our assays. all of these animal viruses were negative, except for ns gene of the classical swine virus. our segment-specific real-time rt-pcr assays might be used in high throughput genotyping. they detected pandemic h1n1 ⁄ 2009 viruses and acted as a preliminary screen-ing tool to select virus reassortants of interesting genotypes for further sequencing analyses. in fact, we identified a novel reassortant in january 2010 during the course of this study. this sw ⁄ hk ⁄ 201 ⁄ 2010 has a previously unidentified viral gene combination as shown in figure 1 . it was confirmed to be a reassortant between pandemic h1n1 ⁄ 2009 and other swine viruses in full genome sequencing characterization. it has a pandemic h1n1-like n1 gene, an ea-like h1, and the other six internal genes derived from tr swine viruses. 13, 14 the eight established real-time rt-pcrs can rapidly reveal the gene-origins of influenza viruses. we are currently using these assays in influenza surveillance in humans and other animals. it is believed that similar strategy might be applied to detect and genotype other influenza viruses and possible reassortants in the future. pandemic influenza a ⁄ h1n1 ⁄ 2009 infects millions of people around the world. a significant fraction of the world's population may also already have been exposed to the virus and, although asymptomatic, may be at least partially immune to the disease. a precise assessment of the number of people exposed to the influenza a ⁄ h1n1 ⁄ 2009 virus is epidemiologically relevant. however, assays typically used to estimate antibody titers against a particular influenza strain, namely hi and neutralization, require use of the actual virus. this seriously limits broad implementation, particularly in regions where high biosafety facilities are unavailable. we developed an elisa method for the evaluation of presence of specific 2009 h1n1 influenza virus-antibodies in serum samples. mouse anti-histidine tagged antibodies (100 ll; 5 lg ⁄ ml; abd serotec ò , uk) in pbs (ph 7ae2) were dispensed into standard 96-well plates and incubated for 12-16 hour at room temperature. excess antibody was removed by at least two successive alternate washings with pbs-tween 0ae05% and pbs. commercial blocking solution (300 ll, superblock ò t20; pierce ò , usa) was added and incubated for at least 2 hour at room temperature. after successive washing steps with pbs-tween 0ae05%, non-glycosylated histidine-tagged recombinant protein (100 ll; 10 lg ⁄ ml) was added to each well. this protein consisted of the receptor-binding domain of the hemagglutinin of the influenza a ⁄ h1n1 virus. 1,2 after 1 hour incubation, wells were washed for at least two alternating 5 minutes cycles with pbs-tween and pbs. a 1:50 dilution of the serum or plasma sample to be assayed (100 ll) was added to each well and incubated at room temperature for 1 hour. after repeated alternating 5 minutes pbs-tween 0ae05% and pbs washes, anti-human igg antibody solution (100 ll ⁄ well; 1:30 000 dilution in pbs-tween 0ae05%) marked with horse radish peroxidase (pierce ò , usa) was added and incubated for 1 hour at room temperature. after repeated alternate washes with pbs-tween 0ae05% and pbs), substrate solution (100 ll; 1-step ultra tmb-elisa; pierce ò ) was added to each well. after incubation for 15 minutes at room temperature in darkness, the enzymatic reaction was stopped by addition of 1 m h 2 so 4 (50 ll ⁄ well). yellow color produced by the enzymatic reaction was evaluated by absorbance at 450 nm in a biotek ò microplate reader (usa). blank assays using albumin in place of human sera established the elisa background signal, which was subtracted from sample absorbance signals: abs serum sample ¼ abs serum sample before correction à abs albumin sample : absorbance values were normalized based on the average signal of 103 non-exposed subjects (uninfected subjects), and expressed as normalized absorbance (abs norm ): where abs serum ample is the sample absorbance signal, abs albumin sample is the albumin control absorbance signal, abs non exposed subjects is the average absorbance signal of non-exposed subject samples. for ferret serum samples, the same basic protocol was followed, with minor modifications. an anti-igg anti-ferret polyclonal antibody preparation was used at a dilution of 1:30 000 in pbs-tween 0ae05%. 2 a recombinant receptor-binding domain of the ha of the influenza a ⁄ h1n1 ⁄ 2009 virus, expressed in escherichia coli strains, 2 was used as the elisa antigen. this 27 kda protein, designated here as ha 63-286 -rbd, contained amino acids 63-286 of the influenza a ⁄ mexico ⁄ indre4114 ⁄ 2009(h1n1) hemagglutinin. a sequence coding for a series of six histidines at the n-terminus of the protein was included in the genetic construct to allow purification using immobilized metal affinity chromatography (imac) and attachment to assay surfaces treated with anti-histidine antibodies (or alternatively co +2 or ni +2 ). a panel of four samples (kindly provided by st. jude from ferrets exposed to different influenza strains, namely h3n2, h1n1 swine, and h5n1, was also tested by the elisa method using 1:50 dilutions. protein ha 63-286 -rbd specifically and selectively recognizes antibodies from serum samples from convalescent h1n1 ⁄ 2009 influenza subjects. dubois et al. 4 demonstrated that this protein, produced in e. coli, folds properly into a 3-d structure practically indistinguishable from the analogous region in the ha of the influenza a ⁄ h1n1 ⁄ 2009 virus. ha 63-286 -rbd preserves three of the conformational immunogenic epitopes (sa, sb, and cb) described for influenza a ⁄ h1n1 hemagglutinins. 5 the recombinant protein was used as the antigen, attached through histidine tags to microplate surfaces treated with anti-histidine antibodies to discriminate between serum samples from subjects exposed and non-exposed to influenza a ⁄ h1n1 ⁄ 2009. samples collected before the pandemic onset, and therefore presumed to exhibit low specific antibody titers against influenza a ⁄ h1n1 ⁄ 2009, were analyzed by elisa using the antigen ha 63-286 -rbd. the histogram of normalized absorbance values from this sample set displayed a normal behavior with a standard deviation of 0ae57 units. only 12ae5, 9ae7, and 3ae88% of these samples exhibited normalized absorbance values higher than 1ae5, 1ae75, and 2ae0, respectively. no sample from non-exposed individuals presented an absorbance value higher than 2ae5. variability among samples from non-exposed subjects was much lower than in samples with high specific serum antibody titers from convalescent h1n1 ⁄ 2009 patients. exposure to the h1n1 ⁄ 2009 influenza virus with this elisa method can be predicted by absorbance values normalized to those of abs norm ¼ ðabs serum ample à abs albumin sample þ=ðabs non exposed subjects à abs albumin sample þ ð1þ serum from uninfected subjects. consequently, for reliable results, inclusion of samples from non-exposed subjects on every assay microplate is necessary. figure 1 shows the analysis of 20 human serum samples, including 17 samples from convalescent patients with positive diagnosis by rt-pcr. three positive (dark gray bars) and two negative controls (light gray bars) were included in the same microplate. all serum samples corresponding to convalescent subjects exhibited absorbance values 1ae75-4ae5 times higher than negative samples ( figure 1 ). normalized absorbance values above 1ae5 suggested exposure to the virus, although, a more conservative threshold value of 1ae75 units is proposed for discrimination between exposed and non-exposed subjects. the elisa method described here yields adequate reproducibility and a high signal ⁄ noise ratio within determinations in the same microplate and among different microplates. using a normalized absorbance value of 1ae75, the method was able to discriminate samples from convalescent patients, preferably after the third week of infection, and at least up to the twentyfourth week of exposure. assay sensibility was further validated against results from hi assays. a previously reported study showed that all members in a pool of fourteen samples diagnosed as positive by hi exhibited normalized absorbance values higher than 1ae5, and 85% of them exhibited normalized absorbance values higher than 2ae0. 1 in general, high hi titers (>320) were correlated with normalized absorbance values higher than 4ae0. figure 2a shows results using the ha-rbd elisa method and the hi assay on a pool of seventeen known positive serum samples corresponding to convalescent h1n1 ⁄ 2009 patients. all samples determined as positive by hi (10 samples) were also positive by elisa. while sensitivity of the hi assay was 10 ⁄ 17 = 58ae88%, the elisa method recognized all samples correctly as positive (100% sensitivity) when a threshold of 1ae5 or 1ae75 was used. figure 2b shows that sera from ferrets infected with other influenza strains (h3n2, h1n1 swine, and h5n1) showed no cross-reactivity when analyzed by elisa. in summary, the ha-rbd elisa method presented here consistently distinguished influenza a ⁄ h1n1 ⁄ 2009 infected and non-infected individuals, particularly after the third week of infection ⁄ exposure. since no actual viral particles are required, this assay can be readily implemented in any basic laboratory. in addition, should sufficient vaccine be unavailable, this elisa could determine the level of specific antibodies against the virus and presumably the extent of partial protection in a subject. therefore, the elisa protocol might allow better administration of vaccination programs during pandemic or seasonal influenza outbreaks. in april 2009, a novel h1n1 influenza virus emerged in north america and caused the first influenza pandemic of the 21st century. [1] [2] [3] [4] the 2009 pandemic h1n1 (pdmh1n1) has a unique gene constellation that was not previously identified in any species or elsewhere. it is genetically related to the triple reassortant swine h1n1 influenza viruses currently circulating in north america, with the exception of the neuraminidase (na) and matrix (m) genes, which are derived from a eurasian swine influenza virus. swine h1n1 influenza viruses were first isolated in 1930 and continued to circulate in north america with very little antigenic changes (classical swine h1n1) until 1998. since 1999, however, the antigenic make up of swine h1 viruses has shown increased diversity due to multiple reassortment events and the introduction of h1n1 genes from human influenza viruses. currently, four swine h1 clusters (a, b, c, d) are found endemic in the north american swine population. 5, 6 these swine h1 viruses show substantial antigenic drift compared to the classical swine h1 viruses. cluster d swine h1 is derived from current human h1 viruses, and there is a substantial antigenic divergence between classical swine h1 and human seasonal h1 viruses. epidemiological evidence shows a two-way transmission of influenza viruses between swine and humans, and such events lead to the emergence of the pdmh1n1 virus. 5, 7, 8 phylogenetic analysis have suggested that possible ancestors of the eight genes of pdmh1n1 were circulating in the swine population for at least 10 years prior to the emergence of the pdmh1n1 virus in humans, although the pdmh1n1 virus itself was not isolated from pigs until after the pandemic. interestingly, pdmh1n1 infections have been reported not only in humans and pigs, but also in other animal species such as turkeys, cats, ferrets, cheetahs, and dogs. [9] [10] [11] after the first report of pdmh1n1 infection in swine in canada, other countries, including argentina, australia, singapore, northern ireland, finland, iceland, england, united states, japan, and china reported outbreaks of pdmh1n1 in swine as well. 9, [12] [13] [14] the ample geographic range of pdmh1n1 outbreaks in swine, its apparent broad host range, and the possibility of two-way transmission between swine and humans poses a tremendous challenge for controlling the virus. therefore, to differentiate pdmh1n1 from other h1 strains, particularly in swine and human populations, is an important issue to ascertain the magnitude of the disease caused by the pdmh1n1. in this study, we developed an elisa assay to discriminate pdmh1n1 strains from other swine and human h1 viruses. madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa) were maintained in modified eagle's medium (mem) containing 5% fbs. a ⁄ california ⁄ 04 ⁄ 09 ⁄ h1n1 virus (ca ⁄ 04) was kindly provided by the centers for disease control and prevention (cdc), atlanta, georgia. other viruses are listed in table 1 . viruses were propagated in mdck cells and stored at )70°c until use. viruses were titrated by the reed and muench method to determine the median tissue culture infectious dose (tcid 50 ). 15 three monoclonal antibodies (3b2, 5h7, and 12f3) against ha of 2009 pandemic h1n1 were prepared in our laboratory following previously described methods (shao and perez et al., unpublished). purification and labeling of mabs mab 3b2, 5h7 and 12f3 were purified on a protein g-sepharose affinity column (upstate biotechnology, lake placid, ny, usa). biotinylation of the detection antibody in the elisa was performed using sulfo-nhs-lc-biotin (sulfosuccinimidyl-6-(biotinamido)hexanoate; pierce, rockford, il, usa) according to the manufacturer's instructions. purified 5h7 and 12f3 were selected as the capture antibody, and biotin-conjugated 3b2 was selected as the detection antibody, and hrp-conjugated streptavidin (abcom, cambridge, ma, usa) was developed using the tmb substrate system (kpl, gaithersburg, md, usa). in brief, the mixture of the purified 5h7 and 12f3 (2ae0 and 2ae2 lg ⁄ ml respectively, in carbonate ⁄ bicarbonate buffer, ph 9ae6) was coated to 96-well plates (test well, t) for 12 h at 4°c. at the same time, a control antibody was coated to 96-well plates (control well, c). after blocking the plates with 5% (w ⁄ v) non-fat milk in pbs for 1 hour at 37°c, the samples were diluted in extract buffer (1%tween-20, 0ae5%bsa in pbs) and added to the wells (100 ll ⁄ well, each sample was table 1 . specificity assay of the sandwich elisa result (t ⁄ c) added to four wells-two for t wells and two for c wellsand the mixture was incubated at 37°c for 1 hour. after four washes, 100 ll biotin-conjugated 3b2 (0ae25 lg ⁄ ml) in dilution buffer (0ae5% bsa in pbs) was added to the wells and the mixture was incubated for 1 h at 37°c. following three washes, 100 ll diluted hrp-conjugated streptavidin (62ae5 ng ⁄ ml) in dilution buffer was added to the plates. after incubation for 1 h at 37°c, the plates were washed five times, and the binding developed using the tmb substrate system for 30 minutes. the ratio of the average od 650 value of the t wells to that of the c wells (t ⁄ c) of individual samples was calculated. t ⁄ c values >1ae5 were considered positive in the sandwich elisa. we developed three monoclonal antibodies, 3b2, 5h7, and 12f3, against a prototypical pdmh1n1 strain, a ⁄ california ⁄ 04 ⁄ 2009 (h1n1) (ca ⁄ 04). these monoclonals were used to develop a rapid sandwich elisa for specific diagnosis of pdmh1n1 strains. purified 5h7 and 12f3 were used as capture antibodies, whereas the biotin-conjugated 3b2 was used as detection antibody. the sandwich elisa showed strong reaction with different pdmh1n1 strains as described in in order to evaluate if the sandwich elisa could distinguish the pdmh1n1 from other swine h1 clusters (a, b, c, d), 14 swine influenza strains spanning these clusters were tested. these viruses were first diluted 1:10 in extract buffer, and then added to the coated plates. as shown in table 1 , the t ⁄ c ratios of these viruses were <1ae5, and therefore showed negative elisa result. likewise, testing of human seasonal virus strains a ⁄ brisbane ⁄ 59 ⁄ 2007 (h1n1), a ⁄ malaya ⁄ 302 ⁄ 1954(h1n1), a ⁄ wsn ⁄ 1933 (h1n1), and a ⁄ brisbane ⁄ 10 ⁄ 2007 (h3n2) also showed negative elisa results. furthermore, the sandwich elisa showed no cross reaction with avian influenza viruses, including strains of the h2, h3, h5, h6, h7, h8, h9, h10, h11, h12, and h13 subtypes. more recently, the mutation d222g in the ha of some pdmh1n1 strains has been associated with exacerbated disease and altered receptor binding. [16] [17] [18] [19] [20] to evaluate if such mutant could be detected in our sandwich elisa, we tested a mutant of a ⁄ netherland ⁄ 602 ⁄ 2009 (h1n1) carrying the d222g mutation (engineered by reverse genetics). as described in table 1 , our elisa could still capture the d222g mutant virus and showed a positive reaction, which highlights the specificity of our assay for pdmh1n1 strains, even those with mutations. to evaluate the sensitivity of the elisa, we used the serially diluted pdmh1n1 viruses to determine the limit of detection (lod). as shown in table 2 , in our elisa the highest positive dilutions of nl ⁄ 602 and ca ⁄ 04 were 1:320 and 1:160, respectively. the lod of the sandwich elisa by tcid 50 was 3ae2 · 10 3 and 1ae5 · 10 4 tcid 50 ⁄ ml, for nl ⁄ 602 and ca ⁄ 04, respectively. it is important to note that the t ⁄ c ratio from nl ⁄ 602 and ca ⁄ 04 viruses showed clearly a dose dependent effect, while the t ⁄ c ratio of a ⁄ swine ⁄ iowa ⁄ 30 (h1n1) did not show the same dependence and was always <1ae5, corroborating the high specificity of the sandwich elisa for pdmh1n1 strains. although we did not compare our elisa with other current commercial rapid influenza detection kits, the lod of our elisa assay is similar to other commercial kits that detect human seasonal influenza virus. 21 comparison of the sandwich elisa with the ''gold standard'' -virus isolation in order to further evaluate the feasibility of the application of the elisa to clinical samples, 70 nasal wash samples 1ae58 · 10^6 )(0ae8) )(0ae8) )(0ae9) )(1ae0) )(0ae9) )(0ae8) )(1ae1) )(0ae9) -from ferrets, 56 of those previously infected with ca ⁄ 04 and shown positive by virus isolation, were tested. the samples were diluted 1:1 in extract buffer and then tested using the sandwich elisa. result showed 51 out of 56 positive samples by virus isolation were positive also by the sandwich elisa (sensitivity 90ae1%). the 14 samples tested that were negative by virus isolation were also negative in the elisa, indicating 100% specificity for our assay. these results show not only that our elisa has high compatibility with the virus culture method, but also indicates this application can be used for clinical samples. although real time rt-pcr targeting the ha gene has been used for specific diagnosis of pdmh1n1 with high sensitivity, [22] [23] [24] [25] [26] [27] it is a method that requires manipulation of the sample to extract viral rna, and it is prone to crosscontamination during the pcr steps. in this study, we described a convenient sandwich elisa based on three mabs developed against the pdmh1n1 strain. the elisa not only shows high specificity for pdmh1n1 strain, but also shows great sensitivity. the elisa could distinguish pdmh1n1 strains from human seasonal h1 and h3 viruses and, more importantly, from other swine h1 viruses. we must note that current rapid diagnostic tests cannot be used to differentiate pdmh1n1 from swine or human h1 viruses. 28 it is also worth noting that the sensitivity of commercial rapid antigen-based diagnostic tests for detecting pdmh1n1 is lower than that for human seasonal influenza viruses. 28, 29 a study by kok et al. 32 showed that sensitivity of the current rapid antigenic tests for pdmh1n1 is only 53ae4%, whereas that for seasonal influenza a is 74ae2%. chen et al. 33 developed a dot-elisa and increased the sensitivity for influenza rapid antigen detection. however, the dot-elisa developed by chen cannot distinguish among subtypes. the lod of our elisa is between 3ae2 · 10 3 to 1ae5 · 10 4 tcid50 ⁄ ml, comparable to the lod of rapid diagnostic tests for human seasonal influenza viruses. 21 compared to the ''gold standard''-virus isolation-our sandwich elisa showed 90ae1% sensitivity using ferret nasal washes. our results highlight the potential application of our sandwich elisa for the specific diagnosis of pdmh1n1 viruses. 17 the timely and reliable laboratory evidences are vital factors for field epidemiologists trying to control outbreaks of infectious diseases and for the practicing clinicians to properly manage disease cases. therefore, analysis of new detection methods in comparison to the routine ''classical'' methods is essential to select new methods to be introduced into health service practices, especially in developing countries. in this study we have compared rt-rt-pcr detection of influenza viruses and direct fluorescent-antibody assay using r-mix hybrid cells (a549&mv1lu) with the ''classical'' cell culture methods in developing country settings. in this study, we analyzed 503 nasopharyngeal swabs col the detection of influenza h1, h3, b, and pandemic influenza (h1)pdm virus-specific nucleic acids was performed by rt-rt-pcr in abi7500 fast real time pcr system using primers recommended by cdc, usa, and super-scriptô iii one-step rt-pcr and platinum ò taq dna polymerase kits (invitrogen). the cycling protocol was: 30 minutes at 50°c, 2 minutes at 95°c, and 45 cycles of 15 seconds at 95°c, 30 seconds at 55°c. 1 rapid detection of influenza infected cells has been performed by dfa using the infected hybrid cells of r-mix within 48 hours after inoculation, according to the manufacturers instruction (diagnostic hybrids, inc., usa). 2 the isolation of influenza viruses was performed on mdck cell culture by the protocol recommended by cdc, usa. 3 we detected 1891(25ae2%) influenza virus-specific nucleic acid fragments from all tested samples by rt-rt-pcr. among the positive samples, there were 25ae6% a(h1n1), 0ae4% a(h3n2), 5ae6% influenza b, and 19ae5% a(h1n1)pdm with different distributions by time series in different age-groups. inoculation of the cell lines by rt-rt-pcr positive samples selected randomly has detected influenza virus in 52ae3% (521 ⁄ 995) on mdck cell culture and 51% (77 ⁄ 151) on r-mix hybrid cell culture with varying distribution for different strains. in other words, mdck cell culture technique was better for isolation for pandemic influenza viruses and dfa using r-mix hybrid cell culture technique for detection of seasonal influenza viruses (table 1) . average times needed for the final results for different methods were: 4 hours for rt-rt-pcr, 48 hours for dfa on r-mix and 10 days for mdck cell culture with two passages at least. the peak of the seasonal influenza a virus detection occurred in the 7-8th weeks of 2009, however the pandemic influenza detection peak was observed in the 43-44th weeks of 2009 ( figure 1 ). the outbreaks by seasonal influenza viruses was observed mostly among the children of 0-15 years of age, and pandemic influenza virus outbreak was observed mostly in the adults of 16-64 years of age. the results of this study indicate that rt-rt-pcr is the most suitable method for decision makers in epidemiological and clinical settings by sensitivity and timeliness. the final results show that r-mix dfa requires 12 times longer, and by mdck cell culturing, 60 times longer periods, than by rtrt-pcr. mdck cell culture technique has a higher isolation of pandemic influenza viruses, and r-mix dfa has a greater detection rate of seasonal influenza viruses by our results. according to our study, with rtrt-pcr, the isolation of positive samples by tissue culture of influenza a viruses was 24% and influenza b viruses was 66ae5%, which is lower than in similar spanish study. 4 however our study illustrates similar results with a canadian study 5 where the sensitivity of dfa method and tissue culture technique was shown to be lower than rtrt-pcr sensitivity. as recorded by a study of american researchers, 6 r-mix hybrid and conventional cell culture techniques have had similar sensitivity, which does not match the results of our study. however, the results of our study match with the results of italian and american scientists 7, 8 where the r-mix hybrid method for seasonal influenza viruses is higher than mdck cell culture technique. background: viral kinetics is increasingly used to study influenza infectiousness. the choice of the study design, i.e. when and how many times nasal samples are to be collected in individuals depending on the sample size, is crucial to efficiently estimate the viral kinetics (vk) parameters. material and methods: we performed a model based optimal design analysis in order to determine the minimal number of nasal samples needed to be collected per subject and when to collect them in order to correctly estimate the vk parameters. the model used was a non linear mixed effect model developed with data collected from 44 patients sampled nine times in 9 days (initial design -504 samples collected), and we used d-optimization for design identification. we also computed the minimal number of participants necessary. results: considering that 25% of the influenza-like illness cases are not due to volunteer challenge studies have been used since the 60's to provide data on virus shedding from the respiratory tract during influenza infection. 1 recently, vk was studied in naturally acquired influenza infection. 2, 3 these data are invaluable to describe the natural history of influenza-infection and to compute natural history parameters such as the latent period, generation time, or the duration of infectiousness. [1] [2] [3] [4] however, among the 56 studies used in a meta-analysis about viral shedding kinetics, 1 the designs varied greatly from one to another. these differences led to variable amount of available information concerning the vk. the lack of adequate sampling leads to imprecise estimates. on the other hand, intensive sampling or over-sampling, while associated with highly informative data, may lead to unnecessary discomfort for the patient and cost to the investigator. optimal design is increasingly used to conceive studies 5 and provides cost-efficient designs. here we propose an optimised design to model vk in the case of influenza infection. we defined the number of participants, the number of samples to collect and their allocations. this design allows, at a minimum cost and discomfort, accurate vk curves and allows the natural history parameters to be well described. model a vk population model was proposed for influenza infection. this model describes with eight parameters the relations between free virus, uninfected target epithelial cells, infected epithelial cells, and early immune response. this model was built on a dataset of 44 volunteers from which nasal samples were collected once a day over 9 days. we call this dataset the ''original dataset''. three parameters, the induction of the early immune response, the virus production rate, and the virus clearance, did not show inter-individual variability and were precisely estimated (relative standard error below 10%). we considered them as fixed in this research work. five parameters were hence considered here: b the infection rate, d the infected cell mortality rate, w the effect of early immune response on virus production rate and v init the initial value of virus titre. in order to correctly estimate these parameters it is crucial to determine a design to collect informative data. optimal designs maximise the amount of information provided by the study. it involves the determination of the number and allocation of sample times per subject as well as the number of participants. 6 d-optimization is based on the maximization of the determinant of the fisher information matrix and thus minimizes the variance of the parameters. 7 we used the fedorov-wynn algorithm implemented in pfim 3.0 to maximize this determinant, 8 which implies to pre-define a set of possible sample times. with the hypothesis that the inoculation occurred at 8:00 am, we chose three possible hours (8:00, 14:00, and 20:00) for each day with respect of the sleep-time. 8 to validate the design, we simulated 100 datasets of 44 volunteers with the optimised design obtained. we then estimated the population parameters using monolix 3.1 for each of the datasets. we compared the estimated parameters obtained with the simulated datasets to the parameters used to build the optimal design. we computed the relative bias as: with n: number of successful estimations among the 100 simulated datasets. h i : parameter value obtained with the ith dataset. h: parameter value obtained with the original dataset. we also compared the observed rse from these simulations with the rse predicted by pfim and the rse obtained with the original dataset. the rse is proportional to ffiffiffi n p , where n is the population size. we can hence deduce the smallest number of participants necessary to obtain rse below 50%. where rse predicted is the highest predicted rse (here rse for w) with 44 participants and n predicted = 44 and rse min is equal to 0ae5. considering that 25% of the influenza-like illness cases are not due to influenza virus, the total number of participants should be multiplied by 1ae25. we found that the best design was when all the participants are sampled five times: three times during the second day post-inoculation at 8:00, 14:00, and 20:00 hours and twice on the third day post-inoculation at 8:00 and 20:00 ( figure 1 ). the comparison of the relative bias and rse predicted by pfim and those obtained after simulation and re-estimation of the parameters are shown in figure 2 . v init and d in a lesser extent present bias. fixed effect parameters are precisely estimated and accordingly to pfim except for v init . we found that 16 participants shedding virus or 20 participants with ili symptoms are necessary if 25% of them are not infected with influenza virus. we propose an optimised design to accurately study the vk of influenza virus with the minimal number of samples. this design is well balanced between the amount of necessary information and the precision of estimation. we found that 100 samples are necessary to precisely fit the vk curves, which is five times less than the number of samples collected in the original study. 4??? the samples should be collected during the second and third days after inoculation. yet we showed in a previous work that the incubation period lasted 1ae9 days. 4??? hence, the optimised sample times correspond to the two-first days of symptoms and this design could be applied to naturally acquired infections studies in which the inoculation time is unknown. an advantage of this design is its practicality and convenience. all samples are collected during the daytime and after the onset of symptoms. it can thus be used for studies with naturally acquired infections. the design was validated with several criteria concerning the accuracy of the estimation with the optimised design. the parameters estimates were generally satisfactory. the parameter describing the effect of the early immune response on the virus production rate was, however, less precisely estimated (predicted rse = 62%), and the initial value of the viral titre was very different of the one obtained with the original dataset (bias v init on figure 2 ). this is probably due to the fact that it was measured at day 2 post inoculation, and that the inter-individual variability is much higher than at day 0. furthermore, d (the infected cell mortality rate) seems also to be biased. this may be due to the fact that three parameters were fixed. the model used was developed from experimentally inoculated healthy volunteers with low serum haemagglutinin antibody titre and with virus inoculation time at 8:00 am. the applicability of the design to naturally acquired infection would depend on the pathogenicity of the virus as well as pre-existing immunity and the relevance of challenge method to natural influenza acquisition. 1 our design could be directly used to accurately study vk during influenza infections and would reduce the discomfort of patients and the cost of the experimentation. usefulness of a self-blown nasal discharge specimen for use with immunochromatography based influenza rapid antigen test introduction influenza rapid antigen tests (irat) have become very popular and are widely used for confirming suspected clinical diagnosis of influenza in japan. 1 most of the currently used irat that are based on immunochromatography (ic), nasopharyngeal swab, nasopharyngeal aspiration, and throat swab have been approved as specimens for japanese national health insurance purposes. but the specimen collection by these methods gives patients considerable discomfort, and sometimes appropriate specimens cannot be obtained due to patient resistance, especially by children. in the present studies, self-blown nasal discharge was used as the specimen for an irat, and the results were compared with the results of viral isolation and an identical kit primed with nasopharyngeal swab specimens for seasonal influenza viruses and pandemic (h1n1)2009 virus. patients who visited any of the 10 clinics that belong to the influenza study group of the japan physicians association in the 2006-2007 and the 2009-2010 influenza seasons with influenza-like illnesses exhibiting findings were registered after providing informed consent. a square plastic sheet of 20 · 20 cm was handed to the patient. nasal discharge was collected by blowing the nose into the plastic sheet as a specimen for irat, i.e. self-blown specimen. two nasopharyngeal swab specimens were also obtained at the same time for irat and virus isolation. self-blown specimens were obtained successfully by 152 (82ae2%) of 185 consecutive outpatients in the 2006-2007 season, as seen in table 1 the sensitivity and specificity of various influenza rapid antigen tests have been reported in various settings. [1] [2] [3] [4] direct comparison of the results is difficult because of differences in patient or influenza virus, characteristics such as age, study designs, and other features. in this study of the 2006-2007 influenza season, the sensitivity, specificity, and accuracy of the ic kit primed with nasopharyngeal swab specimens were 87ae0%, 94ae0%, and 90ae8%, respectively. these results were quite comparable to our results of the 2005-2006 season, 3 in which the overall results of other ic kits were 82ae9%, 81ae0%, and 82ae3%, respectively, indicating that the ic kit used is quite reliable. the sensitivity, specificity, and accuracy of an ic kit will vary by the method of specimen collection. in general, virus titer is considered to be highest with nasopharyngeal aspiration, lower with nasopharyngeal swabs, and lowest with throat swabs. practically, nasopharyngeal swab is the most popular. the sensitivity, specificity, and accuracy of the ic kit with self-blown discharge specimens compared well with those of an identical ic kit primed with nasopharyngeal swab specimens. for self-blown specimens, sensitivity and specificity were 87ae0% and 94ae6% for influenza a, 85ae7% and 99ae1% for influenza b, 88% and 88ae9% for pandemic (h1n1) 2009. self-blown specimens display sensitivity, specificity, and accuracy comparable to that of conventional nasopharyngeal swab specimens. there was no significant difference in sensitivity, specificity, or accuracy between self-blown specimens and nasopharyngeal swab for influenza a, influenza b, and pandemic (h1n1) 2009. these results suggest that selfblown specimens are as useful as nasal cavity swab specimens for the diagnosis of influenza in the clinical settings. nasal discharge, obviously, cannot be collected from infants incapable of blowing their own nose or patients who do not develop a nasal discharge. in this study, self-blown specimens were obtained from 82ae2% of the patients. the rate of successful collection was over 70% in the age groups of 0-6 and 7-15 years. these rates would seem to be sufficient for clinical use. the procedure of self-blown specimen collection using a plastic sheet is easy and causes no pain or discomfort. it seems to be more acceptable and safe than the other methods, especially for children. furthermore, this procedure reduces the risk of influenza transmission from patients to the medical staff members involved in sample collection. self-blown sample collection may be superior to other sample collection methods in these respects. we previously reported an inverse correlation between the amount of virus in a specimen and the time to a positive reaction. 4 in this study, there was no significant difference in the mean time to a positive between self-blown self-blown specimens enough to be examined were obtained 152 from 185 consecutive outpatients, and specimens showed a tendency to be obtained large amount from children rather than the aged. there were no statistically significant differences between the ic kit results primed with self-blown discharge and nasopharyngeal swab specimens for influenza a, influenza b and pandemic (h1n1) 2009. and nasal swab specimens, suggesting that the self-blown specimens contained sufficient viral antigen for the ic kits. the influence of the presence or absence of nasal congestion on the results of the kit was assessed. the sensitivity of selfblown specimens from patients with nasal congestion was significantly lower than that from patients without nasal congestion. it is possible that insufficient capability to blow the nose due to nasal congestion might tend to lead to false negatives. the observation that the time to positive is longer for patients with nasal congestion than for patients without nasal congestion is concordant. application of self-blown specimen collection only to appropriate patients would increase the sensitivity, which would be important in a clinical setting. we tested only two commercial antigen detection kit, the quick vue rapid sp influ kit and quicknaviô-flu (denka-seiken co., ltd). the resulting sensitivity, specificity, and accuracy of the ic kit primed with self-blown specimens were considered adequate for clinical use. to confirm the usefulness of self-blown nasal discharge specimens, further investigation is necessary using other kits and in different settings. the usefulness of a self-blown nasal discharge specimen for an influenza rapid antigen test based on immunochroma-tography was evaluated in the 2006-2007 and 2009-2010 influenza season. results suggest that self-blown nasal discharge specimens are useful as specimens for influenza rapid antigen tests based on immunochromatography for not only seasonal influenza viruses, but also pandemic (h1n1) 2009 virus. the specimen collection by the patients themselves will reduce the burden of other collection methods and the risk of infection to the medical staff. in april 2009, a mixed-origin h1n1 influenza virus was recognized as a new causative agent of influenza-like illnesses (ili) in humans. since its emergence, the virus has spread rapidly throughout the world and caused a pandemic. most commercial rapid antigen tests (rat) can detect influenza a or b viruses, but cannot specifically distinguish pandemic (h1n1) 2009 virus with seasonal influenza. recent studies have indicated that the poor performance of the rat approach and nonspecific detec-tion of the pandemic (h1n1) 2009 virus was the main obstacle to their widespread use in private clinics. 1,2 with the need for a new rapid kit with reasonable sensitivity and specificity for pandemic (h1n1) 2009 virus, we developed a new rat kit in collaboration with company, standard diagnostics, inc., (yongin-si, gyonggi, korea). monoclonal antibody (mab) against haemagglutinin (ha) of the pandemic (h1n1) 2009 virus was developed using korean isolate and applied to the new kit with the mab to seasonal influenza virus. we examined the detection limit of the kit using the serial dilution of korean pandemic virus isolate (a ⁄ korea ⁄ 01 ⁄ 2009). during december 2009, 432 clinical specimens from patients with ili were collected at 11 sentinel clinics of six provinces in korea. the specimens were tested by the new rat, and the results were compared with those of real-time reverse transcription polymerase chain reaction (rrt-pcr) by us cdc and virus isolation in mdck cell culture to determine the sensitivity and specificity for the diagnosis of pandemic (h1n1) 2009. the detection limit of the new kit against ha of a ⁄ korea ⁄ 01 ⁄ 2009 virus was confirmed to be 10 4 pfu ⁄ ml. by contrast, the detection limit against the np protein was 10 3 pfu. however, when the kit was applied to clinical specimens, no difference between the two targets was found. using rrt-pcr and viral culture as the references, the performance of the ridt is shown in table 1 . among 432 specimens, 178 were tested positive by rrt-pcr and 186 were tested positive by viral culture. among the 178 rrt-pcr confirmed cases, 122 were positive, and among the 186 viral culture confirmed cases, 120 were positive with the new rat. using rrt-pcr as the reference standard, the overall sensitivity of rat was 68ae5% (95% confidence interval (ci): 61ae7-75ae3%) and specificity was 98ae4% (ci: 96ae9-99ae9%). with viral culture as the reference, the rat sensitivity and specificity was 64ae5% (ci: 57ae6-71ae4%) and 97ae6% (ci: 95ae7-99ae5%), respectively. when analyzed by the regions tested, the sensitivity ranged between 57ae1% and 95ae5% for rrt-pcr and between 53ae3% and 87ae0% for viral culture as a reference. among 340 patients who had a record of their symptom onset and sample collection date, 86 (25ae3%) visited the clinic on the day of symptom onset, and 158 (46ae5%) visited 1 day later. when the rat performance was evaluated by day of onset, the sensitivity was lower at three or more days after the onset of symptoms; however, the sensitivity was highest at 2 days after onset and reasonable on the day of onset or at 1 day after ( table 2 ). we found that this new rat had reasonable sensitivity and high specificity compared with rrt-pcr and viral culture for detecting the pandemic (h1n1) 2009 virus. in one recent study, the sensitivity and specificity of the new rat kit was 77% and 100%, respectively, and the ha protein for pandemic (h1n1) 2009 was detected more sensitively than the np protein for influenza a virus. 3 the sensitivity and specificity of our new rat were lower than those of that study. we found that the test performance varied depending on the clinics in which the tests were performed, and this might be attributable to the persons who collected the specimens. although the clinicians were trained well for *ci, confidence interval. **ppv, positive predictive value. ***npv, negative predictive value. collecting specimens, there might be some differences in performance. the new rat kit could detect pandemic (h1n1) 2009 virus specifically. although the sensitivity was lower than those of rrt-pcr and virus culture, and negative rat results should be confirmed with more sensitive methods, this kit could be useful in sentinel clinics if used with caution. determination of infectious virus titres is central to many experiments designed to study the biology of influenza virus. assays based on the measurements of viral components, whether viral protein or nucleic acid, does not differentiate infectious virus from non-infectious or defective viral particles, which may have no infectivity or biological *three hundred and forty samples with a known date of onset and sample collection were analyzed. ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 132-158 activity. therefore the ''gold standard'' of virus measurement requires bioassays that examine the ability of viral particles to replicate and further infect other cells. titration on madin-darby canine kidney (mdck) cells in a 96 well plate format is commonly used to measure influenza virus titre. this method is labour intensive, subjective in their read out of cytopathic effect, and takes several days to obtain a result. microneutralization tests that quantitate neutralizing antibody titres and assays of drugs for antiviral activity also require 96 well based assays of residual virus infectivity. therefore, technologies that improve on the titration of infectious virus will be of great benefit. this study utilized the xcelligence system (roche applied science), which adopts microelectronic biosensor technology to monitor dynamic, real-time label free and non-invasive analysis of cellular events. 1 the system measures electronic impedance using an array of microelectrodes located at the bottom of each culture well (e-plate 96). adherent cells are attached to the sensor surface of electrode arrays, and changes in impedance can be detected and recorded. the xcelligence system can monitor cell events induced by viral infection, such as changes in cell number, adhesion, viability, morphology, and motility. measured electrode impedance is expressed as dimensionless cell index and is graphically represented using software to show the phenotypic changes of a cell population over time. the aim of this study is to demonstrate that using this platform to measure real-time cell index has potential to circumvent many of the limitations of the currently established procedures of end point titration of virus infectivity and for microneutralization assays. madin-darby canine kidney cells were propagated in growth medium consisting of minimum eagle's medium (invitrogen) supplemented with 10% fetal bovine serum (invitrogen), 0ae6 mg ⁄ l penicillin (invitrogen), and 60 mg ⁄ l streptomycin (invitrogen), with incubation at 37°c in a 5% co 2 humidified atmosphere. influenza a ⁄ hong kong ⁄ 54 ⁄ 1998 (h1n1), a seasonal influenza virus from a patient who suffered from a mild febrile illness, was propagated in mdck cells maintained in virus medium consisting of minimum eagle's medium (invitrogen) supplemented with 0ae6 mg ⁄ l penicillin (invitrogen), 60 mg ⁄ l streptomycin (invitrogen), and 2 mg ⁄ l np-tosyl-l-phenylalaninechloromethyl ketone-treated trypsin (sigma, st louis, mo, usa), with incubation at 37°c in a 5% co 2 humidified atmosphere. virus stocks were aliquoted and stored at 70°c until use, and the 50% tissue culture infectious dose (tcid 50 ) of the virus stock was determined by titration in mdck cells according to standard procedures, 2 and the tcid 50 of the stock virus was calculated by the method of reed and muench. 3 to perform a microneutralization assay, mdck cells seeded at a density of 1000 cells ⁄ well in an e-plate 96 was removed from the xcelligence system after approximately 48 hour; growth medium was then removed, cells washed, and replaced with 100 ll virus-medium. a human serum, which is known to contain high titre antibody against the h1n1 virus was heat inactivated for 30 min at 56°c, and twofold serial dilutions were performed in virus medium. the diluted serum was mixed with an equal volume of virus medium containing influenza virus at 400 tcid 50 ⁄ 100 ll. after incubation for 2 h at 37°c in a 5% co 2 humidified atmosphere, 50 ll of virus-antibody mixture was added to the mdck cells to give each well an equivalent virus dose of 100 tcid 50 . a back titration of the virus challenge dose was performed, and a cell control (free of virus) was performed in quadruplicates. after incubation at room temperature for 30 minutes, the e-plate 96 was then placed back onto the xcelligence system in the incubator and maintain at 37°c with 5% co 2 , and the cell index values were measured every 15 minutes for at least a further 72 hour. the same procedures were performed with cells seeded in conventional 96 well cell culture plates for parallel comparison with the currently used standard method. in this case, cells were examined for cytopathic effect under an inverted microscope after 3 days of infection and the lowest virus dilution, which protected the cells from viral induced cytopathic effect taken as the neutralizing end point. after 48 hour of seeding mdck cells at 1000 cells ⁄ well, standard microneutralization assay for influenza virus was performed. integral to this assay, a serial titration of the input virus at 0ae5 log 10 increments was carried out. wells infected with the undiluted virus (100 tcid 50 ⁄ well), the cell index commenced dropping at a steeper gradient than the no-virus cell control after approximately 3 hour of infection ( figure 1 ). this drop in cell index continues at a consistent slope until it flattened out when approaching zero cell index. this steep decrease in cell index with constant gradient was also observed for virus dilutions up to and including 2 log 10 (100-folds), and the profile shifted with increased time in proportion to the dilution made to the virus. virus dilutions beyond 2 log 10 have cell index profiles similar to the no virus input control, and this corresponds to the absence of cytopathic effect as determined by microscopic observation at 72 hour after infection. hence, there was a correlation between the amount of virus used for infection, the onset of the influenza virus-mediated cytopathic effect, and the steep decline in cell index. a human serum with known microneutralization antibody titre to h1n1 virus was used in this study to investigate the real time cell index changes that occur during the assay ( figure 2 ). using influenza virus treated with serum dilutions up to and including a dilution of 1:160, the cell index profile remained essentially the same as the no virus cell control, which correlates with the lack of cytopathic effects under microscopic observation at 72 hour of infection. at a serum dilution of 1:320, the steep decrease in cell index, which is characteristic of cellular cytopathic effect induced by the virus, became evident at around 50 hour post infection, and this was reduced to 40 hour when serum dilution of 1:940 was used. in contrast, for the virus -no antibody control, the onset time for this steep decrease in cell index occurs at approximately 7 hour. for both serum dilutions of 1:320 and 1:940, full cytopathic effect was observed microscopically at 72 hour of infection. from microscopic observation of cytopathic effect, according to the current standard procedures, the neutralizing titre of the human serum used in this study is at 1:160 as it is the last dilution of the serum that prevented cytopathic effect from being detected. an essential part of the microneutralization assay is to confirm the titre of the input virus (normally 100 tcid 50 ⁄well) by performing a titration assay with decreasing serial dilutions of the virus. under normal procedures, cells are examined microscopically after 72 hour of infection for sign of cytopathic effects. in the case of mdck cells, the cytopathic effect is cell death, which is indicative of the presence of live influenza virus infecting and replicating in the cells. therefore, the titre of the virus is taken as the last dilution in which cytopathic effect is present. parallel realtime cell index measurements demonstrated that for wells with cytopathic effects, the profile exhibits a steep gradient linear decrease in cell index after infection with the virus, which can be termed the ''cpe plunge.'' the time in which the cpe plunge became evident appears to be inversely proportional to the amount of virus, therefore the opportunity exists to utilize this aspect to calculate or compare quantitatively different virus concentrations. for unequivocal assignment of cytopathic effect, it normally requires 2-3 days after infecting the cells, with 3 days after infection being the standard time to read virus titration and microneutralization assays. using the real-time cell index monitoring, it is found that apparent cytopathic effect can only be observed microscopically when the cell index has dropped to near zero. as the time of onset of the ''cpe plunge'' becomes evident many hours prior to observable cytopathic effect, it is possible that the time to results can be drastically reduced after some formulation of the method. we compared the current standard method in perfoming a microneutralization assay with one utilizing the real-time cell index measurement to investigate whether this approach is able to offer better performance over the existing one. the current standard neutralization assay is the microscopic observation of antibody mediated protection from virus cytopathic effect in mdck cells. this study showed that this may also be achieved by examining the profile generated from the real-time measurements of the cell index. using real-time cell index monitoring, it is possible to detect inhibitory activity at higher dilutions of the anti-serum than can be detected by the standard microscopic observation of cytopathic effect. therefore, the realtime cell index monitoring could potentially be developed to be a more sensitive method for measuring anti-viral activity. as drug resistant strains of influenza a viruses 4 including the 2009 pandemic h1n1 are being reported, the real-time cell based monitoring system may also have the potential to be developed for use as a diagnostic platform for drug resistance assays. this study suggests that real-time cell index monitoring has the potential to substantially reduce human resources in reading results, as well as reducing time-to-result of these assays from 3 days to two. the saving could be substantial for work involving bio-hazard level 3 ⁄ 4 pathogens such as h5n1 viruses as personnel working with these organisms are require to be highly trained and experienced. in addition, the reduction in transferring plates to and from the microscope in reading cytopathic effect will substantially reduce the possibility of accidents from occurring. furthermore, the system provides objective digital data to an otherwise subjective assay method, which can improve standardization, data exchange, and hence collaboration between different laboratories. with more detailed validation and development, real-time cell index monitoring could transform the way we study and diagnose infection with pathogens such as influenza viruses. the emergence of a novel h1n1 influenza a virus of swine origin, the pandemic a(h1n1) 2009, with transmissibility from human to human in april 2009 posed pandemic con-cern and required modifications to laboratory testing protocols. a new protocol for universal detection of influenza a and b viruses and simultaneous subtyping of influenza a (h1n1) 2009 virus, composed of two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h1n1v (relab, italy), was evaluated and compared to the reference protocol recommended by who. fast set infa ⁄ infb was able to detect influenza a and b viruses circulating between 1995 and 2008 belonging to different subtypes and lineages, and no cross reactions were observed by either fast set infa ⁄ infb or fast set h1n1v. the who assay was found to have a slightly lower end-point detection limit (10 )5 dilution) in comparison to the new protocol (10 )6 ). specificity of the assays was 100% as assessed on a panel of stored clinical samples including adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, s. pneumoniae, n. meningitidis, h. influenza, and human influenza viruses. the new assay panel allows the detection, typing, and subtyping of influenza viruses as requested for diagnostic and surveillance purposes. the high sensitivity of the protocol is coupled with capacity to detect viruses presenting significant heterogeneity by fast set infa ⁄ infb and with high discriminatory ability by fast set h1n1v. a rapid and sensitive assay for the detection of influenza virus in clinical samples from subjects with ili or low respiratory tract infections is a fundamental tool for epidemiological and virological surveillance, management of hospitalized patients, and control of virus nosocomial transmission. the emergence, in april 2009, of a novel h1n1 influenza a virus of swine origin, the pandemic (a(h1n1) 2009), with transmissibility from human to human poses pandemic concern and required modifications to the laboratory testing protocols. 1 molecular diagnosis of influenza is generally achieved through a twophase process: a screening phase for the detection of virus, and the subsequent strain characterization performed by either sub-type-specific rt-pcr or entire ⁄ partial genome sequencing. 2 during a pandemic, simultaneous implementation of both the detection of influenza a and b influenza viruses and identification of the new subtype is useful for clinical and epidemiological reasons. here, we describe a new protocol including two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h1n1v (relab, italy) that allows universal detection of all influenza a viruses and, simultaneously, all subtypes that are influenza a(h1n1) 2009. specificity and clinical sensitivity of the two-one-step rt-pcrs (fast set infa ⁄ infb and fast set h1n1v; relab, italy) were evaluated by testing 306 selected specimens, including: • fifty samples collected from nasopharyngeal swabs representative of influenza viruses, belonging to differ-ent subtypes and lineages, and other respiratory viruses and bacteria circulating in italy between 1995 and 2008. • six purified a(h5n1), a(h7n7), and a(h9n2) strains, kindly supplied by alan hay, who influenza centre, london, uk. • two hundred-fifty influenza positive samples selected according to type, subtype, clade and viral concentration from >2500 specimens received by the liguria influenza reference laboratory between january 1st and december 31st, 2009. since 1995, nasopharyngeal swabs sampled from patients suspected of having contracted the influenza virus have been collected in viral transport medium, and upon arrival into the laboratory, the samples were divided in ‡3 aliquots. those not immediately processed were stored frozen at )80°c. stored samples were used for this evaluation, and all specimens were re-extracted for the study. samples collected between 1995 and 2008 included specimens positive for: no seasonal a(h1n1) have been detected since january 1st, 2009. furthermore, 1 weak positive sample using fast set infa ⁄ infb, but negative at block pcr and typing ⁄ subtyping assays was tested. the analytical sensitivity of the test under investigation was determined testing ten-fold serial dilutions of seasonal influenza a(h1n1), seasonal influenza a(h3n2), new pandemic influenza a(h1n1) 2009, and b cell culture-grown viruses. the intra-assay reproducibility was measured by testing the same a(h1n1) 2009 positive sample 15 times in the same experiment, while the inter-assay reproducibility was confirmed by testing the same samples in 3 independent experiments. to evaluate the performance of the protocol, all samples were tested using a block pcr confirmation test (seeplex ò rv12 ace detection), 3 and all specimens collected between january 1st and december 31st, 2009 and dilutions were also assayed using the recommended who ⁄ cdc protocol of real-time rtpcr for influenza a(h1n1). typing and sub typing were performed using the who protocol and ⁄ or sequencing. 4 viral rna was extracted from swabs using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's protocol. fast set infa ⁄ infb and fast set h1n1v are two multiplex one-step real time pcr assays developed and evaluated by the liguria regional reference centre for diagnosis and surveillance of influenza in collaboration with relab diagnostics. both assays contain primers and a dual-labelled hydrolysis probe that targets two regions of the matrix gene (table 1) . amplification conditions were as follows: reverse-transcription 50°c for 15 minutes, denaturation 94°c for 2 minutes, then 40 cycles of 95°c for 15 seconds, 58°c for 30 seconds. the entire amplification process extended for 110 minutes. an internal control real-time assay was also incorporated in order to detect pcr inhibition, failed extraction ⁄ pcr and technical error. the cdc realtime rtpcr (rrtpcr) protocol for detection and characterization of swine influenza includes a panel of oligonucleotide primers and dual-labelled hydrolysis (taqman ò ) probes to be used in real-time rtpcr assays for the in vitro qualitative detection and characterization of swine influenza viruses in respiratory specimens and viral cultures. this protocol recommends three primer-and-probe sets: infa, amplifying a conserved region of the matrix gene from all influenza a viruses; sw infa, designed to specifically detect the nucleoprotein (np) gene segment from all swine influenza viruses and sw h1, designed to specifically detect the hemagglutinin gene segment from a(h1n1) 2009. 5 the seeplex ò rv12 ace detection for auto-capil-lary electrophoresis is a multiplex block rt-pcr that applies dpoô (dual priming oligonucleotide) technology and is designed to detect 12 major respiratory viruses, 11 respiratory rna (influenza a and b virus, parainfluenza virus type 1, 2 and 3, respiratory syncytial virus a and b, rhinovirus a ⁄ b, coronavirus oc43 and 229e ⁄ nl63) viruses and 1 dna (adenovirus) virus, from patients' samples including nasopharyngeal aspirates, nasopharyngeal swabs and bronchoalveolar lavage. 3 conventional viral culture was performed inoculating 0ae1 ml of each specimen into mdck-siat1 seeded into 24-well plates for influenza isolation. virus detection was performed by the hemagglutination test using 0ae75% guinea pig red blood cells (rbc). specificity and clinical sensitivity results of the new protocol are reported in table 2 . fast set infa ⁄ infb was able to detect influenza a and b virus circulating between 1995 and 2008 belonging to different subtypes and lineages, and no cross-reactions were observed by either fast set infa ⁄ infb or fast set h1n1v. among specimens collected between january 1st and december 31st, 2009, all 60 fast set infa ⁄ infb and fast set h1n1v high titre positive samples resulted positive using the who ⁄ cdc assay and showing reactivity using infa and sw infa primer-andprobe sets. among 90 low titre a(h1n1) 2009 positive samples at fast set infa ⁄ infb, 28 (31ae1%) were not detected by the who ⁄ cdc assay, but were positive using seeplex ò rv12. the who ⁄ cdc sw h1 primer-and-probe set works in 96ae7% (58 ⁄ 60) and 1ae1% (1 ⁄ 90) of high and low titre a(h1n1) 2009 positive samples, respectively. all a(h3n2) strains collected during 2009 and initially detected by fast set infa ⁄ infb were confirmed after rna re-extraction by seeplex ò rv12 and who ⁄ cdc assay showing reactivity using the infa primer-and-probe set. all infa ⁄ infb were confirmed after rna re-extraction by seeplex ò rv12. one influenza a case identified by the who ⁄ cdc kit (infa primer-and-probe set, ct values: 37ae5, sw infa primerand-probe set: negative) and new protocol (a primer-andprobe set, ct values: 34ae1, a(h1n1) 2009 primer-and-probe set, ct values: 34ae6) was not detected by either seeplex ò rv12 or by who subtyping protocol and ⁄ or sequencing, suggesting a very low viral load or unspecific results by real time assays. the analysis of serial dilutions of cell culturegrown a(h1n1) 2009 showed that the detection limit of fast set infa ⁄ infb, fast set h1n1v, and seeplex ò rv12 was identical (10 )6 ) and 1log 10 lower than that using the who ⁄ cdc protocol (10 )5 ). a similar analysis with respect to a(h1n1) and a(h3n2) strains indicated that fast set infa ⁄ infb sensitivity (10 )6 and 10 )7 , respectively) was 1log 10 lower than that showed by seeplex ò rv12 (10 )5 and 10 )6 , respectively). in comparison with the new protocol, the who ⁄ cdc assays, considering infa primer-and-probe set, was found to have a slightly lower end-point detection, detecting the 10 )5 a(h1n1) and a(h3n2) dilution. also in detecting influenza b virus, fast set infa ⁄ infb sensitivity (10 )6 and 10 )7 , respectively) was 1log 10 lower than that showed by seeplex ò rv12 and the who ⁄ cdc protocol. data on intra-assay and inter-assay precision, measured as cv% of ct showed that the dispersion indices observed had values of less than 3%. since 2009 samples were detected using the new protocol that resulted negative using the who ⁄ cdc assays. the unfortunately low quantity of low titre a(h3n2) samples collected during 2009 did not allow us to highlight differences between assays fast set infa ⁄ infb, and fast set h1n1v positivity was always confirmed by seeplex ò rv12, which demonstrated high sensitivity, showing a detection limit comparable or lower when compared with those observed using the who ⁄ cdc assays. the high analytical sensitivity of seeplex ò rv12 is reported by kim 3 who observed a detection limit of 10 copies per reaction for each type ⁄ subtype of influenza viruses. the high sensitivity of the new protocol is coupled with its capacity to detect viruses presenting a significant heterogeneity by fast set infa ⁄ infb and high discriminatory ability by fast set h1n1v. fast set infa ⁄ infb was able to identify representative influenza viruses of circulating strains during the last decade belonging to different subtypes, lineages, and clusters, and fast set h1n1v primerand-probe set reacted selectively with a(h1n1) 2009 target. a recent report demonstrated that the sw infa assay is not specific to a(h1n1) 2009 and is able to detect both human and avian (h5n1) influenza a viruses and so there is the potential for misidentification. 15 high titre (ct 8ae5 and 15ae6 at fast set infa ⁄ infb) a(h5n1) viruses did not react with fast set h1n1v primer-and-probe set (data not shown). available human a(h5n1) sequences are similar within the h1n1v primer-and-probe regions, but having 4-5 mismatches in the forward primer and, more notably, two of the mismatches occurred within 9 nucleotides of the 3 end, an important determinant for primer specificity. in conclusion, this protocol can be a powerful tool in the diagnostic laboratory setting for specific simultaneous analysis of several samples in minimal time, showing enhanced sensitivity in detecting influenza viruses, and high discriminatory ability in identifying the new pandemic a(h1n1) 2009. a university-corporate partnership to enhance vaccination rates among the elderly: an example of a corporate public health care delivery public health campaigns usually rely on governmental infrastructure and finance for vaccine implementation programs. however, there are many financial and physical barriers which preclude widespread and effective vaccine administration, especially among the elderly. on an international scale, both government agencies and citizen groups have a vested interest in searching for more resourceful methods of attaining significant immunization levels (>75% of the population). in fact, it seems to have become both a grassroots civic and governmental goal, especially among developing countries. we implemented the unique strategy of enlisting the assistance of a privately-owned food market chain to address the public health issue of mass vaccination for the elderly. in this context, publix pharmacy and the university of south florida (usf) recently developed both a handbook and a training program to facilitate the administration of vaccinations. between 2008 and 2009, the publix-usf partnership resulted in administration of over thirty thousand influenza a (h3n2) vaccinations, 76% of which were given to adults over 55 years of age. consequently, vaccine administration costs were decreased by using corporate resources and bypassing overly strained municipal resources. this unique university-corporate partnership successfully delivered h3n2 vaccine to a vulnerable cross-section of society at a lower cost and with minimal side effects and morbidity. it may be safely projected that university-corporate partnerships could result in an effective method for rendering a vital service to an aging and especially vulnerable segment of the population. government policy and funding are the foundation of immunization programs on an international scale. for example, in the united states, governmental programs account for over 50% of the monetary outlay used for immunization. 1 until 2006, the global alliance for vaccines and immunizations (gavi) acted as a catalyst for implementing vaccine and immunization programs in each targeted country. 2 under the auspices of gavi-collaborations between governments, charitable organizations, and multinational health agencies (such as uncief and the who)-many countries have increased their spending for vaccination programs. however, development of financially sustainable immunization programs geared toward reaching the majority of the population are still at a nascent level of evolution. 3 the development of more innovative and costeffective approaches has become imperative in order to reach a greater number of vaccination candidates. administering the influenza vaccine only to the subpopulation of over 65 year olds would save an estimated 220 000 quality-adjusted life years in a cohort of approximately half the world's population. 4 widespread public vaccination programs are made more complex by the continuing development of newer vaccines, concomitant specialized administration costs, and the logistical challenge of conveying recipients to vaccination points of service. 1, 5 in spite of the increasing complexity of mass vaccination, cost-benefit analyses clearly favor annual influenza vaccination in the elderly population on an international scale. 4, 6 recently, in 2008, influenza vaccine administration was reported to reach between 32% and 82% of the elderly population, which denotes varying degrees of success within each particular country. 7, 8 however, there was also a report of a uniform plateau effect at around 75% of the population, beyond which additional vaccination coverage was difficult to achieve. 9 physical limitations to vaccination seem to be more insurmountable for the elderly. unfortunately, this is the population segment which could experience the most significant vaccination-associated mortality reduction. 10 we employed the unique strategy of involving the resources of publix supermarkets, a corporate food market chain, to address the public health issue of widespread vaccination for the elderly. we took advantage of recent 2005 changes in the florida statutes, which expanded the scope of pharmacists' practice to include administration of vaccines. subsequently, publix pharmacy and the university of south florida (usf) developed a handbook and training program to facilitate and enhance vaccine administration by publix pharmacists. by using proprietary pharmacists and more practical supply storage, we were able to decrease the costs of vaccine administration. the consumer was charged $10 for administration costs plus the cost of the injection itself, regardless of insurance or eligibility for governmental subsidy. although patients were initially self-selected, they were ultimately excluded if they had demonstrated prior adverse effects to influenza vaccinations or to any of the components of such vaccinations. between 2008 and 2009, the publix-usf partnership vaccinated 30 063 people against influenza a (h3n2), of which 29 404 were florida residents. the age range was 1-105 years old with a median age of 65 years old. seventysix percent of the participants were over 50 years old (see figure 1 ). within the population surveyed, the reported side effects of the vaccine in this study were not serious, but included: vertigo, cold sweats, chills, vomiting, syncope, rash, nausea, stomach pain, elevated blood pressure, injection site reaction, inflamed bursa, and bilateral thigh discomfort. participants from all socioeconomic classes were vaccinated. an income-by-zip code analysis revealed 44% of those vaccinated resided in zip code areas where the average household income was <$50 000 per year. of those remaining, 25% had an average income of $50 000-$70 000 per year, and 31% had an income of >$70 000 per year. each person vaccinated was charged ten dollars for administration costs. this represents a decrease in the administration costs ranging from one dollar to ten dollars saved per vaccine. 11, 12 conclusion this unique university-corporate partnership successfully delivered h3n2 vaccine to a high-risk population with decreased vaccine administration costs. the influenza vaccine is well-tolerated, with minimal side effects when patients who have a history of adverse reactions are excluded. we can postulate that university-corporate partnerships may indeed be effective at reaching the aging population which is a challenge in most communities. this delivery model may prove to be another tool for improving the efficiency of mass immunization by facilitating accessibility, which results in wider coverage. this model also enhances delivery of healthcare by decreasing costs of immunization regardless of whether the payer is a government, insurance company, or self-pay consumer. the gavi initiative stressed three goals for accomplishing sustainability and independence in immunization programs. 3 the goals were to: (i) mobilize additional resources from governmental and non-governmental sources; (ii) improve program efficiency to minimize additional administration resources needed; and (iii) increase the reliability of funding. empowering privately owned corporations within the community, such as food markets or pharmacies, to administer vaccines mobilizes additional resources to readily achieve the first goal of gavi. mobilizing resources of non-healthcare, corporate vaccination locations enhances accessibility due to travel convenience. in our study, participants came from all socioeconomic classes, suggesting that ease of access is independently hindering mass vaccination, and that people of all incomes are more likely engaged when access issues are eliminated. the second and third goals were also accomplished by recruiting a corporation's resources for vaccine administration (refrigeration, storage, and employees). this minimizes the money spent from vaccine program funds to support the infrastructure of immunizations, thus improving financial efficiency and sustainability. financial efficiency implies that money is spent to safely reach as large a portion of the population as possible. by using corporate storage facilities instead of paying for independent facilities, money can be spent elsewhere. more vaccines can be purchased and more money can be spent on media communications to encourage vaccination. sustainability requires the ability to fund annual vaccination programs which reach 75% of the population or greater. key to the control of pandemic influenza are surveillance systems that raise alarms rapidly and sensitively. in addition, they must minimise false alarms during a normal influenza season. we develop a method that uses historical syndromic influenza data from the existing surveillance system 'servis' 1 monitoring seasonal ili activities in scotland. we develop an algorithm based on wcr of reported ili cases to generate an alarm for pandemic influenza. wcr is defined as the ratio of the number of reported cases in a week to the number of cases reported in the previous week. from the seasonal influenza data from 13 scottish health boards, we estimate the joint probability distribution ( figure 1) we compare our method, based on our simulation study, to the mov-avg cusum and ili rate threshold methods and find it to be more sensitive and rapid. the wcr method detects pandemics in larger fraction of total runs within the same early weeks of pandemic starting than does any of the other two methods ( figure 2 ). as shown in the table, for 1% pandemic case reporting rate and detection specificity of 95%, our method is 100% sensitive and has mdt of 4 weeks, while the mov-avg cusum and ili rate threshold methods are, respectively, 97% and 100% sensitive with mdt of 5 weeks. at 99% specificity, our method remains 100% sensitive with mdt of 5 weeks. although the threshold method maintains its sensitivity of 100% with mdt of 5 weeks, sensitivity of mov-avg cusum declines to 92% with increased mdt of 6 weeks. for a two-fold decrease in the case reporting rate (0ae5%) and 99% specificity, the wcr and threshold methods, respectively, have mdt of 5 and 6 weeks with both having sensitivity close to 100%, while the mov-avg cusum method can only manage sensitivity of 77% with mdt of 6 weeks. the first cases of the 2009 pandemic were reported in scotland in the 29th week of the season. the wcr algorithm as well as the mov-avg cusum method detects the pandemic 12 weeks later in week 41. the ili threshold method detects it 1 week later in week 42. both the wcr and mov-avg cusum methods therefore outperform the ili threshold method by 1 week in the retrospective detection of the 2009 pandemic in scotland. while computationally and statistically very simple to implement, the wcr method is capable of raising alarms rapidly and sensitively for influenza pandemics against a background of seasonal influenza. although the algorithm has been developed using the servis data, it has the capacity to be used at large scale and for different disease systems where buying some early extra time is critical. more generally, we suggest that a combination of different statistical methods should be employed in generating alarms for infectious disease outbreaks. different detection methods would provide cross-checks on one another, boosting confidence in the outputs of the surveillance system as a whole. real-time evidence being created worldwide will greatly contribute to the full understanding of influenza pandemics. here we report the real-time epidemiology and virology findings of the influenza a(h1n1)2009 pandemics in mongolia. the epidemiological and virological data collected through isss of nic, nccd, mongolia (real-time information on registered ili cases and virological laboratory results are available from the weekly updates in the nic, mongolia website: http://www.flu.mn/eng/index.php?option=com_ content&task=category&sectionid=5&id=36&itemid=51) were used for analysis in relation to the previous seasonal influenza activities in the country. influenza viruses were detected in naso-pharyngeal samples from ili patients by rt-rt-pcr with applied biosystems fast real time pcr system 7500, using primers and instructions supplied by cdc, usa. 1 influenza viruses were isolated by inoculation of rt-rt-pcr-positive samples of mdck cell culture according to the standard protocol. 2 ten representative strains of a(h1n1)pdm viruses were selected for sequencing of different gene segments, namely: a ⁄ ula, and a ⁄ dundgovi ⁄ 381 ⁄ 2010. sequencing of influenza virus gene segments was performed in applied biosystems 3130xl genetic analyzer using primers and instructions supplied by cdc, usa, 3 and bioinformatic analysis was performed with abi ⁄ seqscape v.2.5 and mega4 programs. the pandemic alert in mongolia was announced by the government on april 28, 2009, just after the who announcement of the pandemic alert phase, and planned containment measures were intensified. despite intensive surveillance, no a(h1n1)pdm virus was detected in mongolia until the beginning of october 2009. around 40 suspected cases, mostly arriving from the a(h1n1)pdm epidemic countries, tested zero by rt-rt-pcr for a(h1n1)pdm virus. the first a(h1n1)pdm case detected by the routine surveillance system in ulaanbaatar city, the capital of mongolia, was confirmed by rt-rt-pcr on october 12, 2009 (41st week of 2009). the reported ili cases escalated rapidly, reached the peak in the 43-44th week of 2009, and gradually decreased thereafter ( figure 1 ). week of 2010. however, the registered ili cases increased again from the 5th week of 2010, and peaked at the 8-9th weeks of 2010. the viruses isolated during this 2nd peak were influenza b strains ( figure 1 and table 1 ). for the genetic characterization of the mongolian pandemic isolates, 3 gene segments i (pb2), 2 gene segments ii (pb1), 3 gene segments iii (pa), 9 gene segments iv (ha), 3 gene segments v (np), 4 gene segments vi (na), 7 gene segments vii (m), and 3 gene segments viii (ns) of the representative a(h1n1)pdm mongolian strains were sequenced, and all sequences have been deposited in the genbank (accession numbers: cy050844, cy0533648, cy050846, cy050845, cy065987, cy065989, cy065990, cy 065988, cy065991, cy065985, cy065984, cy065986, cy052366 , cy053365, cy054546, cy054547, cy055169, cy 055170, cy055171, cy056363, cy055308, cy057191, cy057083, cy065995, cy065997, cy065998, cy065996, cy 065999, cy065993, cy065992, cy065994, cy054548, cy054549, cy073448). all 8 genes of mongolian strains were possessing 99ae4-99ae9% similarity with the genbank deposited gene sequences of the original pandemic strain a ⁄ california ⁄ 072009(h1n1). the who declared the pandemic alert phase (phase iv) on april 27, 2009, 4 and was prompted to announce the pandemic phase (phase v) two days later. 5 after 43 days, the who declared the beginning of the pandemic peak period (phase vi) on june 11, 2009. 6 however, in mongolia, the pandemic alert period continued for 168 days. mongolia was free of the pandemic virus during the whole first wave of the pandemics in the northern hemisphere. with the confirmation of the 1st influenza a(h1n1)pdm case on october 12, 2009 in ulaanbaatar, mongolia entered into the pandemic phase (phase v), and after just 2 weeks, the registered ili cases peaked, confirming mongolia shifted into the pandemic peak period (phase vi), which i i i v i i v i v v v i i i i iii iv v vi vii viii worldwide by who in mongolia coincided with the 2nd wave of pandemics in many countries of the northern hemisphere (see, picture 1 and table 1 ). despite the relatively milder clinical manifestations, the disease burden for the health service was enormous, while the morbidity per 10 000 population at the peak period was 5-6 times higher above the upper tolerant limit, and 3-4 times higher above the seasonal influenza outbreaks. in contrast to the seasonal influenza outbreaks where over 80% of the registered ili cases have been in the age group under 15, 2 it has been observed that over 50% of the registered cases in this pandemic peak period were in the age group of 16-60. on january 18, 2010, we regarded the pandemic had entered into the post-peak period (phase vii) when the registered ili cases became lower than the upper tolerant limit, during which time mongolia experienced an influenza b outbreak. on may 24, 2010 we determined that mongolia entered the post-pandemic period (phase viii) as the influenza virus isolations were almost stopped, and after no pandemic virus detected for 3 months. who announced pandemic vii and viii phases much later. 7, 8 this first ever real-time laboratory confirmed influenza pandemics in mongolia and confirmed some variations of pandemic spread in different parts of the world. the comparison of deduced amino-acid sequence changes have shown that the mongolian strains belong to the clade 7, according to the classification of a(h1n1)pdm influenza strains suggested by m. nelson, 9 which has circulated worldwide since july 2009. this is also evidence that the 1st wave of the pandemics did not hit mongolia. the who public health research agenda for influenza 1 is aimed to support the development of evidence needed to strengthen public health guidance and actions essential for limiting the impact of influenza on individuals and populations. each stream-specific group reviewed and discussed the proposed organization, content, rationale, and global health importance of their designated research stream. specific research recommendations were made for topics within each stream: background: a syndromic surveillance system using nonclassical data sources for detection and monitoring evolution of flu and flu-like illness (ili) in djibouti is reported here as part of the preliminary report of djibouti who-copanflu international study (wcis)**. methodology: clinical reports, over-the-counter drug sales, lab diagnosis report, and health communication trends were obtained for an integrated statistical analysis. results: transition to winter is concomitant with upsurge of ili cases and ili drug sales. in addition, more rural folks manage ili infections on self medicament than through clinical consultancy. inefficient and vague data collections were observed. a successful implementation of wcis will create a platform upon which challenges faced in djibouti health department in routine surveillance will be addressed to achieve a near-real time surveillance of flu pandemic. conclusion: innovations, prompt reporting, and instituting open source syndromic surveillance system software's in resource limited environment like djibouti will enhance early detection and evolution monitoring of pandemic flu. the spanish flu in 1918 ⁄ 1919 infected and killed millions of people, and threatened to wipe humanity off the face of planet. however, the recent scenarios of influenza h1n1 (2009) pandemics' worldwide occurrence fell short of most scientific prediction on its magnitude and intensity. this dampened their confidence; they cannot state precisely as to when, how, where, and which of the spanish flu-like pandemic will occur in the future. in support of scientific community and governments, the who hasn't gone to slumber, but is reminding its member states to up their post pandemic surveillance and monitoring of influenza virus in circulation for advance preparedness in case of an outbreak. 1 despite all uncertainty around the pandemic flu h1n1 2009, there remains a common knowledge and understanding that this flu has shown a great potential to evolve and cause huge morbidity and mortality. although its future magnitude may be unpredictable, its recurring events have severe consequences on human health and the economic well being of everyone. 1 and therefore, advance planning and preparedness is critical in protecting any population in the future, especially those located in resource limited environment without universal health cover and generous disaster emergency funds. 2 . two collapsed sets of a weekly and monthly mean data (of four years period) were clustered in five categories of ili cases, drug sales, lab results, vaccine consumption, and health promotion. this was followed by a descriptive statistics analysis of cumulative weekly and monthly data to establish presence or absence of trend. time series analysis was not done due to data limitation. copanflu program: as at the time of going to press, the cohort study is at the household recruitment and inclusion phase and the study covers the djibouti city. it is in our intention to use the cohort study findings to validate or improve the niph ministry of health djibouti ili surveillance effort for better preparedness. clinical service: 83% of all health facilities are in djibouti city. of the 39ae9% (326 445) of the population that seeks medical care on influenza and influenza like illness each year, 58ae2% (187 586) and 11ae9% (38 389) of them are attended to at the city's public and private clinics, respectively ( figure 1 ). the rest are attended from the regional health centers. the majority of ili incidence sharply rise with the onset of the winter season (october to april), affecting mostly the middle age group (1-24 years). pharmaco-surveillance: 2% (99 350) of total prescriptions were antipyretic and antiflu drugs, 91ae4% (90 765) of which were consumed by peripheral regions, the non djilab diagnostics: the annual ili lab diagnosis was negligible 0ae0072% (250), which can be attributed to less equipped virology laboratories to warrant routine service utility. documented cases were from previous bouts of avian influenza that had a human incidence from 2006 and 2009. 5 with support of egypt-based naval army medical research unit three (namru 3), clinicians were motivated to sample all ili patients and submit to collaborating international reference influenza lab in cairo, egypt. vaccination: influenza vaccinations were undocumented, but at least 0ae4% (3122) of population sought the service (for yellow fever and meningitis) as mandatory travel advisory or as childhood immunization need. at the time of going to the press, there were at least 80 000 vaccine doses of h1n1 (2009) virus donations yet to be administered. health promotion and hygiene: print and audiovisual risk communication remained favorite means of reaching out to urban dwellers (70ae6%). while to the rural and nomadic population, person to person communications was the preferable means. to increasing public awareness that will encourage reporting of ili cases and entrench risk aversion health behavior that limits flu spread, who-copanflu international study djibouti has incorporated basic training on ili infection and personal hygiene by interviewers during household inclusion. improving national epidemic surveillance capacity and response under new international health regulation 6 is important for any nation, including djibouti. our finding indicates the winter season predisposes one to ili infections; they therefore opt for medical services or self medication depending on their capability and ⁄ or understanding. in djibouti, almost no city dwellers favors self medication over clinical consultation, suggesting the presence of inhibitory factors like distance from the health centers and the cost of accessing consultancy. common in the absence of universal primary health care setting, it therefore calls for active innovativeness in outbreak detection, disease reporting, and preventive medicine on the part of health authority so as to achieve good population health. 7 in respond to these, niph has turned resource limitation to a motivation instead and is working towards institutionalizing a near-real-time syndromic surveillance system as a core functional unit. it capitalizes on three major aspects within its reach: prompt accurate data generation for analysis, ehesp wcic-study input, and information technology use. prompt accurate data generation for analysis: data used in our analysis suffered from un-timeliness (weekly instead of daily basis), incompleteness (vague over-counter drug sales records), entry errors (incidence case reports), and poor collection format (most of data collection forms). use of satellite handset phones for regional health centers and mobile phones for city sentinel clinics will reduce unnecessary data delivery delays. in addition, creating awareness to data entry personnel on the importance of careful and completeness of entries is important, as is the need to reformat data collection forms to capture exact aspects of surveillance needs for relevant executable analysis. 8 besides alerting for immediate impending epidemics, these data can also be adopted for projective predictive modeling of annual epidemics, including that for influenza. 9 ehesp wcic-study input: djibouti wcic-study is complementary to the existing syndromic surveillance system, but with emphasis on flu and flu-like illness. various innovations as suggested above are used in seeking to overcome the prevailing challenges. while every attempt is made to realize its (wcic-study) objective and for global comparison, lessons learned from successful implementation will form a platform for future refined syndromic surveillance protocol as equally reported elsewhere in asian countries. 7, 10 information technology: national institute of public health djibouti has an informatics department with sufficient working pcs and personnel to execute efficient data collection and management for epidemiological analysis. however, licensing cost of near-real time syndromic surveillance software is prohibitive, but the open access software with capacity to generate custom graphs, maps, plots, and temporal-spatial analysis output for specific syndromes should make implementation a lot easier. such output for conditions like flu (or gastroenteritis) will be essential to cause prompt response of the local public health office and international partners in saving lives and suffering of djibouti people. 11 pandemic flu surveillance and preparedness requires multifaceted, interdisciplinary, and international approach whose efficiency and efficacy can only be refined over time. building on the health care system's swot for preparedness, the ehesp wcic-study promises to refine surveillance system operation and knowledge on individual's risk determinants to swine flu (h1n1) 2009 virus infection at the household level in djibouti. these efforts are ultimately creating available control options at the time of need (pandemic occurrence), and at the same time exploring investment in quality data profiling and information technology, which will include syndrome surveillance software systems like essence, ewors, or other open sourced ones. the antibody efficacy -which compares the illness frequency between those with and those without a protective level of pre-epidemic hi antibodies ( ‡1:40) -has been proposed 1 ; however, this index has rarely been used due to practical difficulties in confirming the strain-spe-cific disease corresponding to each of the vaccine-induced antibodies. we followed 114 elderly individuals residing in a nursing home, whose serum specimens were obtained before and after undergoing trivalent influenza vaccination, in 2002 ⁄ 2003 influenza season (medium-scale mixed [a ⁄ h3n2 and b] epidemic in study area, and a ⁄ h3n2 was circulating at the nursing home). 2 the serum antibody titre to each strain of influenza virus was measured by the hi method, using the same antigens as those in the vaccine. all participants' body temperatures, respiratory symptoms, other general symptoms, hospitalization, discharge, and death were recorded daily from 1 november 2002 to 30 april 2003 in a prospective manner. when the participants suffered any influenzalike symptoms, such as sudden fever ‡37ae8°c, throat swabs were collected and tested using a rapid diagnosis kit for influenza, which utilizes an immunochromatographic method. the adjusted odds ratios (or adj ) for febrile illness and kit diagnosed influenza were evaluated using multiple logistic regression models adjusting for possible confounders (i.e., age, sex, coexisting conditions, and vaccine strains). after vaccination, the proportion of subjects achieving an hi antibody titre ‡1:40 (seroprotection level) were 61ae4% (51ae8-70ae4%) for a ⁄ h1n1, 79ae8% (71ae3-86ae8%) for a ⁄ h3n2, and 26ae3% (18ae5-35ae4%) for b. during the follow-up period, the a ⁄ h3n2 strain was isolated therein, and 44 subjects experienced sudden-onset fever ( ‡37ae8°c), and eight subjects were positive for rapid diagnosis kit. patients with a seroprotection level of the hi antibody titre ( ‡1:40) had lower incidences of febrile illness (or adj , 0ae35; 95% ci, 0ae09-1ae28) and rapid kit diagnosed influenza (or adj , 0ae35; 95% ci, 0ae03-4ae64) than those with a lower titre. thus antibody efficacy (1 ) or adj ) against fever related to a ⁄ h3n2 and kit diagnosed influenza were both estimated to be 65%. although statistical significance was not detected due to limited sample size, these results lend support for the usefulness of antibody efficacy. some data presented within this manuscript was also published in hara et al. asia via a regional network from which epidemics in the temperate regions were seeded. 1 the virus isolates obtained from nasopharyngeal swab specimens from outpatients were typed and subtyped by the hemagglutination (ha) inhibition assay. 2 the emergence of a ⁄ fujian ⁄ 411 ⁄ 2002 coincided with higher levels of influenza-like illness in korea than what is typically seen at the peak of a normal season. most of the intermediates and fujian-like strains were isolated from asian countries, and the mutational events associated with the fujian strains took place in asia. closely dated phylogeny from december 26, 2001 to august 11, 2002 showed that the antigenic evolution of the h3n2 fujian strains had periods of rapid antigenic changes, equivalent to 10 amino acid changes per year ( figure 1 ). the fujian-like influenza strains were disseminated with rapid sequence variation across the antigenic sites of the ha1 domain. the antigenic evolution of the fujian strains was initiated by exceptionally rapid antigenic change that occurred in asia, which was then followed by relatively modest changes. some of the data presented in this manuscript was previously published in kang et al. we compared reactivity to the novel virus strain using haemagglutination inhibition (hi) assays performed on discarded plasma specimens left over from routine testing. samples were taken from healthy adult blood donors (>16 years) before and after the ph1n1 influenza epidemic that occurred during the southern hemisphere winter of 2009, and again prior to onset of the 2010 southern hemisphere influenza season. reactivity to the novel h1n1 2009 strain of influenza was relatively uncommon among the healthy adult population during the first australian winter wave, rising from a baseline of 12% to 22%. a further increase in the seropositive proportion from 22% to 43% was observed over the summer months, most likely attributable to immunisation. this level of immunity appears to have been sufficient to constrain the 2010 winter epidemic. together with a final serum collection, planned for late 2010, these data will aid evaluation of the extent and severity of disease in this 'second wave' of ph1n1. assessment of the extent of disease due to novel influenza a(h1n1) virus (ph1n1) during the 2009 winter outbreaks in australia was made difficult by the generally mild nature of disease. the epidemic was experienced in a staggered fashion around the country, 1 reflecting the considerable geographical distances between state and territory capital cities ( figure 1 ). differences in the intensity of case-finding during the evolving pandemic response and between jurisdictions hindered comparisons of disease burden in distinct geographical regions. 2 rates of reported hospitalisations and deaths appeared fairly similar across states 1 but, without a consistent exposure denominator, assessment of relative severity was difficult. we conducted a national serosurvey of antibody to ph1n1 using residual plasma from healthy blood donors collected before and after the 2009 epidemic to estimate ph1n1 exposure. 3 here we report the findings of that first collection, together with new data on seroprevalence of ph1n1 antibody in specimens gathered in march-april 2010. these latter samples were collected prior to onset of seasonal influenza activity to assess the impact of a national ph1n1 vaccine program conducted in spring ⁄ summer 2009 ⁄ 10 on the proportion of individuals with antibody titres deemed protective. 4 findings informed estimates of population susceptibility to ph1n1 prior to the 2010 influenza season and provided a baseline for a subsequent serosurvey that will be collected at the end of 2010 to assess the extent of exposure during the 'second wave.' tralian red cross blood service (the blood service) for dengue fever surveillance studies. these samples were used to provide a baseline estimate of prevalence of cross-reactive antibody to ph1n1 in the australian population. discarded plasma specimens, taken for virologic testing from healthy adult blood service donors, were prospectively collected at two additional timepoints for measurement of antibody to ph1n1. collection periods were as follows: approximately 120 plasma samples were randomly selected from donors in each of brisbane, hobart, melbourne, newcastle, perth, sydney, and townsville on each occasion. up to 20 specimens were identified in each of the following age strata: 16-24, 25-34, 35-44, 45-54, 55-64, and >65 years. at the last collection timepoint, there was deliberate over-sampling of the oldest and youngest age strata in which approximately 40 specimens were collected (i.e., up to 160 specimens per site). in accordance with the provisions of the national health and medical research council's national statement on ethical conduct in human research, individual consent was not required for use of these specimens, given the granting of institutional approval by the blood service human research ethics committee. reactivity of plasma against ph1n1 was measured in haemagglutination inhibition (hi) assays using turkey red blood cells (rbc). 5 egg-grown a ⁄ california ⁄ 7 ⁄ 2009 virus was purified by sucrose gradient, concentrated and inactivated with b-propiolactone, to create an influenza zonal pool preparation (a gift from csl limited). plasma samples were pretreated with receptor destroying enzyme ii (denka seiken co. ltd), 1:5 (volume ⁄ volume) and tested as previously described. 6 following 1 hour incubation, 25 ll 1% (volume ⁄ volume) of rbc was added to each well. hi was read after 30 minutes. any samples that bound to the rbc in the absence of virus were adsorbed with rbc for 1 hour and reassayed. samples in which background activity could not be eliminated by these means were excluded from the analysis. titres were expressed as the reciprocal of the highest dilution of plasma where haemagglutination was prevented. a panel of control sera and plasma samples was included in all assays. it comprised paired ferret sera pre-and postinfection with the pandemic virus or seasonal influenza a(h1n1), a(h3n2), or influenza b viruses and paired human plasma and sera collected from donors before april 2009 or after known infection with the pandemic virus or after immunisation with the australian monovalent pandemic 2009 vaccine. all assays were performed by the who collaborating centre for reference and research on influenza. for each of the three study timepoints and within each age group, the proportion of seropositive individuals (hi titres ‡40) was calculated, with exact (clopper-pearson) confidence intervals. the contribution of individual variables (age, gender) and location to seropositive status was assessed in separate multivariate logistic regression models developed to assess the post-pandemic and pre-influenza season 2010 collections. all statistical analyses were conducted in stata 10. locations of specimen collection are shown in figure 1 , together with the number of samples tested from each centre. samples with high background hi titres or discrepancies between assays were excluded at each timepoint as follows: 5 at baseline, 27 from the post-pandemic collection, and 0 in early 2010. pared with baseline was 10% overall, rising from 12% to 22% (table 1 ). the only jurisdictions in which seropositive proportions were higher in october ⁄ november than in the baseline collection were hobart [31% (95% ci 22ae3, 39ae7)], perth [24% (16ae3, 31ae7)], and sydney [24% (16ae3, 31ae7)]. in the multivariate regression model, the only jurisdiction in which exposure appeared somewhat higher than the reference population of brisbane was hobart [or 1ae83 (95% ci 0ae99, 3ae4), p = 0ae06]. a marked age effect on antibody status was observed at this timepoint, with an increase in the proportion of seropositive individuals in relation to the baseline collection only noted for those aged between 16 and 34 years (table 1) . according to the multivariate model, the youngest and oldest cohorts had similar titres, with all other groups showing significantly lower seropositive proportions than the reference population of 16-24 years [e.g. 45-54 years or 0ae36 (95% ci 0ae21, 0ae64, p < 0ae0001)]. an overall increase in the seropositive proportion from 22% to 43% was observed between october 2009 and april 2010, distributed throughout all jurisdictions ( (17, 25) ]. antibody titres prior to the 2010 influenza season rose in all age groups, but remained significantly lower among [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] year olds than in the youngest age cohort (table 1) . adjusted ors for the seropositive proportion in the multivariate model in these age groups were: 35-44 years [or 0ae49 (95% ci 0ae31, 0ae77)]; 45-54 years [or 0ae48 (0ae31, 0ae75)]. the relatively low titres observed in these groups reflected small incremental increases in the seropositive proportion across each of the time points studied, suggestive of both low rates of infection and vaccination. the rise in immunity observed across the population was most likely attributable to immunisation in the majority, given the absence of observed outbreaks and very few notified cases of ph1n1 during the period between the two plasma collections. this study suggests that, while adult exposure to ph1n1 during the 2009 southern hemisphere winter was uncommon at around 10%, vaccine uptake in the australian population over the period november 2009-may 2010 was in the order of 20%. this latter estimate is in keeping with recently published figures for adult ph1n1 vaccine coverage from a national immunisation survey conducted by the australian institute of health and welfare. 4 in that survey, vaccine coverage was significantly higher in tasmania than in other states, but mostly in those over 65 years of age, possibly in a subgroup whose health status may have differed from that of the donor population. 4 no allowance has been made in this analysis for likely waning of natural or vaccine induced immunity, possibly resulting in lower estimates of natural and ⁄ or vaccine exposure than may have occurred over the period. regardless of such intervening processes, the seropositive proportion among australian adults at the start of the 2010 winter season appeared likely to be sufficient to constrain transmission of infection in the age groups tested. this assertion has been borne out in practice, with only modest levels of influenza reported during the late and protracted 2010 season. 7 a final serum collection is planned for the end of the 2010 influenza season in australia from which to assess the level of exposure in relation to the baseline observed here. the need for epidemiologic studies such as this has been highlighted by groups such as the european centre for disease control to aid evaluation of the extent and severity of the 'second wave,' 8 known to be variable from historical reports of past pandemics in disparate populations. 9 in 2009-2010, the first wave of the swine-origin novel h1n1 flu (h1n1) pandemic swept across the world, including japan. to examine the epidemiological nature of this novel infectious disease among school children within and among small regional communities, we have carried out a complete survey on the incidence of h1n1 among school children using absentee reports provided by school health teachers in two small administrative districts (population: about 140 000 in total) in japan. we then examined the epidemiological diversity on the inci-dence of h1n1 within and among small regional communities. we investigated seventeen elementary and ten junior high schools in moroyama-town and sakado-city located in the central part of saitama prefecture. populations are: all ages, 37 015 and 100 634; elementary schools, 1873 and 5389; junior high schools, 1131 and 2568, respectively. the number of school children in each school ranges from 137 to 876. the surveillance system was built on an apache-and mysql-based web server using html, php, and java-script. 1 school health teachers enter information on children absenteeism due to school infectious diseases via web browsers at each school infirmary on a daily basis. in addition to the trend graphs shown on the web browser, detailed analyses were reported to the schools and local educational boards weekly. the basic reproduction number (r 0 ) of h1n1 was estimated according to becker. 2 agentbased modeling and simulations were also performed using a multi-paradigm simulator anylogic version 6.5 (xj technologies, st. petersburg, russia). by the end of march 2010, cumulative incidence (ci) of h1n1 among school children in moroyama and sakado reached 30% and 34%, respectively. the overall r 0 among school children in this area was 1ae43. vaccination rate of children in this area during the surveillance period was reported to be very low (<10%). there was no considerable difference between the epidemic curves in this neighboring town and city. on the other hand, in the individual schools, the cis as of the end of march 2010 scattered from 16% to 51% ( figure 1 ) even though the schools are closely located. to examine the cause of this diversity, we built an agent-based community model 3 consisted of the same numbers of agents as those of children in the actual schools and people in moroyama and sakado to simulate the infection. the ratio of probability of infection in schools and the remaining places were assumed to be 1:1 or 10:1. using a heuristic optimization scheme, we estimated the parameters for the simulations to give the overall ci of 33% (the ci as of the end of march 2010). we then performed simulations repeatedly. the cis obtained with the repetitive simulations with the assumption of higher probability of infection in schools scattered from 23% to 44%, indicating that the cis of the small population communities may vary considerably, even though all the agents were assumed to have the same susceptibility to infection at the beginning, and the other conditions were the same. the policies for surveillance ⁄ analyses ⁄ prevention of communicable diseases in local communities have generally been decided on governmental-and ⁄ or each local administrative district-basis (populations: several hundred thousands to several millions) in japan. we found the considerable variations in the cis of h1n1 for children among much smaller areas, i.e., the school districts (populations: all ages, several thousands; school children, several hundreds). we thus conclude that the granularity of surveillance ⁄ analyses ⁄ prevention should be finer than in the past to achieve the most effective policies against influenza and similar communicable diseases in the local communities. the cause of this diversity can be explained in part by the stochastic nature of infection transmission processes in the small populations shown by the agent-based simulations. we have already conducted a complete questionnaire survey for the school children and their parents to clarify the relevance of the other issues including differences in environmental factors, preventive policies (e.g., vaccination, school closures), etc., in each school. the detailed analyses will be reported elsewhere. a www-based surveillance system for transmission of infectious diseases among school children within and among small regional communities. j epidemiol 2010; 20(s1):s151. this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. the first cases of the 2009 a ⁄ h1n1v pandemic influenza were reported in mexico and the united states in april 2009. given the context of this new influenza virus and considering the likelihood of its pandemic spread, the cohorts for pandemic influenza (copanflu) international consortium was created in order to study individual and collective determinants of pandemic a ⁄ h1n1v influenza across different countries by setting up prospective cohorts of households, followed during 2 years. this study relies on the first available data from the copanflu-france project, which is part of the copanflu international consortium. we studied factors associated with elevated haemagglutination antibody titers against a/h1n1v at entry in the copanflu-france cohort. we focused in this primary analysis on the association between the titers and influenza vaccination (seasonal or pandemic) across age groups. the copanflu-france cohort was set up in fall 2009. inclusions began on december 4, 2009 and ended on july 23, 2010. households were sampled using a random telephonic design (mitofsky-waksberg method) 1 in a stratified geographical sampling scheme, aimed at including a sample of subjects representative of french general population. all household members were eligible to the cohort, without any age limit. the inclusion of a household required the participation of all members: the refusal of one or more member(s) prevented the inclusion of other members. the protocol was approved by a research ethics committee and written informed consent was obtained for all subjects. this study requires several visits to the households by nurses who collect written data with questionnaires and biological samples. during the inclusion visits, nurses collected from all subjects detailed data regarding medical history, including vaccination and preventive measures against influenza. blood samples were collected at entry and centralized. a standard hai technique was adapted to the detection and quantification of antibodies to the 2009 a ⁄ h1n1v virus. 2 the titration endpoint was the highest dilution that exhibited complete inhibition of haemagglutination in two independent readings. the lowest read dilution was 1 ⁄ 40. geometric mean titers were calculated for hai assays with the use of generalized estimating equations for interval-censored data, 3, 4 taking into account a within-household correlation. multivariate models were derived from this method to identify factors associated with elevated gmts. we defined the ''gmt ratio'' (gmtr) as the multiplicative factor applied to the gmt in presence of an explanatory variable. for qualitative explanatory variables, a gmtr of n means a predicted n-fold higher gmt for subjects exposed to the considered factor compared to others. for continuous explanatory variable, the same interpretation applies to a unit difference. the following variables were included in the multivariate models: age, history of pandemic or seasonal influenza vaccination, and history of ili. age was categorized in three groups: 0-15 years (reference group), 15-50 years and over 50 years. the definition of ili was that used by the cdc 5 : fever ‡37ae8°c and cough and ⁄ or sore throat without another known cause. history of ili was defined as an ili reported by the subject between september 12, 2009 (beginning of the influenza epidemic in france) and the date of inclusion. this preliminary analysis included 1304 subjects belonging to 544 households. results reported hereafter do not account for missing data. participating households were sized 1-7 subjects, mean size = 2ae4. in comparison, the mean size of french households is 2ae3 according to the latest national census. 6 the median age of subjects at entry was 40ae6 years [iqr: 16ae5; 58ae6] versus 34ae4 [15ae2; 52ae5] for french population. 7 the proportion of subjects reporting a history of ili since the beginning of the epidemic varied from 2ae9% for subjects over 50 years to 9ae1% for subjects below 15 years (table 1) . vaccination with the pandemic strain was the highest in subjects below 15 (16%) whereas vaccination with the seasonal strain was the highest in subjects over 50 (44ae9%). detailed data regarding vaccination is given in table 1 . this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. [8] [9] [10] [11] [12] [13] among non-vaccinated subjects, elevated gmt in the elderly may be the result of exposure to similar viruses in early life, whereas children and young adults with elevated gmt are likely to have been infected by the 2009 a ⁄ h1n1v virus. [13] [14] [15] [16] interestingly, a significant drop in the hai titer is observed during the months following vaccination with the pandemic strain. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. the reason for this association is not obvious: although we cannot discard the hypothesis of a higher incidence of a ⁄ h1n1v infections in seasonal vaccine recipients, as described by several other studies, 17-19 the main explanation may be a cross-reaction between pandemic and seasonal strains. 16, 20, 21 further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. in april 2009, the cdc alerted about the appearance of a new strain of ia h1n1 with unknown virulence. infants under 5 years old had higher risks of hospitalization, complications, and rate of death for sari. materials and methods: a cross-sectional study was executed from may to december in 2009. the sources were: mandatory reporting form of the province surveillance system, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports. inclusion criteria: children under 5 years old with diagnostic of ili or sari and confirmed cases with epidemiological nexus or laboratory confirmation (rrt-pcr, ifi). the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. results: the ili rate in infants under 5 years old was 1295ae19 ⁄ 100 000 people (95% ci 1236-1356) being higher in infants of 4 years old (1569 ⁄ 100 000 people of 4 years (95% ci 1426-1722) ( table 1) . infants had less risk of getting sick in relation to the rest of the population (rr 0ae40 [95% ci 0ae33-0ae47]) (p < 0ae05). the chance of sari in infants was 2ae89 (95% ci 2ae46-3ae39) compared to the rest of the population. the lethality rate was higher in infants under 1 year old (14 ⁄ 100 000 people [4 ⁄ 28 364]). discussion: the evidence suggests that the infants under 5 years old had lower risk of getting sick than the rest of the population, but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under 1 year old. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. in april 2009, the cdc alerted about the appearance of a new strain of ia h1n1 with unknown dissemination and virulence. in june, the world health organization declared the pandemic. 1, 2 the ili often presents an unspecific clinical picture in infants under 5 years old, from mild symptoms to sari, especially in the newborn babies. infants under 5 years old have higher risks of hospitalization, complications, and rate of death for sari. 3, 4 on may 7th, argentina declared the first imported case of ia h1n1, and by the end of the month, it announced the viral circulation in the country. the epidemiological surveillance system of the province arranged that all the patients with influenza diagnosis made by a doctor must be reported. from april 24th to november 14th, 20 212 suspected cases of ili in the province of tucumán were reported. the ili rate was 1353 ⁄ 100 000 people, and ia h1n1 comprised 40 ⁄ 100 000 people. the lethal rate of sari ia h1n1 was 306ae7 ⁄ 100 000 people (62 ⁄ 20 212). 5 the objective of this research was to determine the epidemiological characteristics of the pandemic ia h1n1 in infants under 5 years old in the province of tucumán between may and december in 2009. the province of tucumán is placed in the center of the northwest of the republic of argentina. it has a population of 1 493 488 inhabitants of which 138 821 are infants under 5 years old. the crude birth rate for 2008 was 19ae9&. the infant mortality rate was 13ae8&. respiratory pathologies in infants under 5 years old were the third cause of death in the province (12%). the public health system of the province is composed by three sectors: public, private, and welfare. with 91 health facilities as a total, the average of available beds is 3& per inhabitants and 6& per neonates. a cross-sectional study was executed from may to december in 2009 in the province of tucumán, argentina. the following sources were used: mandatory reporting form of the surveillance system of the province filled by a doctor, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports (patients with sari). inclusion criteria: • suspected case of ili: sudden appearance of fever higher than 38°c, cough, or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nausea or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea. ) were used for the analysis. the odds rations, risk ratio and 95% confidence interval were calculated to compare ambulatory with hospitalized patients, confirmed and dismissed, <5 years old and the rest of the population. it was considered significant a rate of p < 0ae05. the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. the epidemiological surveillance system of the province received 21 012 ili reports, 11ae77% (2474 ⁄ 21 012) were infants under 5 years old. twenty seven percent were dismissed (676 ⁄ 2474), and 34% (623 ⁄ 1798) of suspected cases were confirmed. the first ia h1n1 case was a child of 2 years from the province of buenos aires, in 22th epidemiological week, and the last suspected case was reported in october 26, 2009 ( figure 1 ). the ili rate in infants under 5 years old was 1295ae19 ⁄ 100 000 people (95% ci 1236-1356), being higher in infants of 4 years old (1569 ⁄ 100 000 people of 4 years, [95%ci 1426-1722]). the higher ili rates in confirmed the pandemic of ia h1n1 (2009) was detected for the first time in the province of tucumán. the evidence suggests that infants under 5 years old had lower risk of getting sick than the rest of the population (protective factor), but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under 1 year old. towns with the highest demographic density had superior proportion of cases. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. outbreak of h1n1 influenza -2009: behavior of influenza h1n1 in school children in the province of tucumá n, argentina criteria: patients treated with antiviral medication for prophylaxis, respiratory pathologies which did not justify specific medication, and incomplete forms. results: from all notifications, 6342 were cases of ili in the group aged 5-17 years old; 53% were males. the incidence rate in this group was 17ae4 per thousands of inhabitants. the 13% of laboratory samples were influenza a h1n1, 34% were confirmed as unspecific influenza, and 49% were dismissed. the school aged children group had a high risks of getting sick (r.r. 1ae78 [95% c.i. 1ae55-2ae04]), especially males. it appeared that school aged children had a protective factor for presenting sari (or 0ae66 [95% c.i. 0ae66-0ae89], p < 0ae05). the lethality rate in this group was 9ae32 ⁄ 10 thousands. headaches, myalgia, coryza, and sore throat were very common and significantly different (p < 0ae05) than the rest of the population. it was reported a decrease in the ew 28 coinciding with winter holidays (ew 27). the epidemic curve was different in males compared to females during the winter holidays. discussion: school aged children got sick more than the rest of the population, although they presented less proportions of sari. however, comorbidities were decisive in order to present sari or death. the epidemic curve was different in males compared to females. through its analysis, the beneficial effect of school closure was observed, as long as children meet the recommendation to stay home. in april 2009, different countries reported cases of influenza a h1n1; mexico reported a high mortality rate associates with this disease. 1 the world health organization (who) declared the phase 6 influenza pandemic alert on june 11. 2 several reports from different countries describe the behavior of the pandemic in school aged children. this group plays an important role in the transmission of influenza. in germany, during the summer peak, pandemic hardly spread within this group. this might be explained by the timing of the summer school holidays, which started between ew 27 and 31. since mid october, after the autumn holidays, the school-aged children began to be more affected, and the proportion increased from 16% in the initiation period to 43ae8% in the acceleration period. 3 in australia, 55% of h1n1 cases were school aged children (5-17 years), with a median age of 16 years (29% of cases were aged 13-17 years and, and 26% between 5-12 years). 4 in canada, the infection rate was highest in this group. 5 in chile, the incidence rate was 4500 ⁄ 100 000 inhabitants, although in general they had mild desease. 6 school closure can operate as a proactive measure, aimed at reducing transmission in the school and spread into the wider community, or reactive, when the high levels of absenteeism among students and staff make it impractical to continue classes. the main health benefit of proactive school closure comes from slowing down the spread of an outbreak within a given area and, thus, flattening the peak of infections. this benefit becomes especially important when the number of people requiring medical care threatens to saturate health care capacity. it has its greatest benefits when schools are closed very early in an outbreak, before 1% of the population falls ill. school closure can reduce the demand for health care by an estimated 30-50% at the peak of the pandemic under ideal conditions, but too late in the course of a community-wide outbreak, the resulting reduction in transmission is likely to be very limited. policies for school closure need to include measures that limit contact among students when they are not in school. 7 tucumán is placed in northwest argentina and has a total area of 22 524 km 2 . the population (2001 census, projection 2009) was 1 493 488 inhabitants; of wich 421 638 were 5-19 years old. the health system of the province is composed of 3 sectors: public, private, and welfare. it has a total of 91 health facilities with internement available and an average of 3 & inhabitants. influenza-like illness (ili) has seasonal and endemic behavior in this province, as evidenced by past records from the national health surveillance system and influenza sentinel surveillance unit of the province. an increase of ili was reported in 2009, with a peak in the ew 28. the objectives were: general objective to describe the behavior of the influenza a h1n1 2009 epidemic in school aged children from the province of tucumán, argentina. specific objectives • to explore the response to preventive measures by school aged population. • to assess the effect of the suspension of classes in this group. • to estimate the magnitude and severity of the disease. • to observe the effect of co-morbidities in this group. a cross-sectional study was executed from may to december 2010. data were gathered through mandatory reporting forms, wich were collected from all public and private health centers. inclusion criteria: patients with compatible symptoms with influenza a; school aged children 5-17 years old. exclusion criteria: patients treated with antiviral medication as prophylaxis, respiratory pathologies which did not justify specific antiviral medication, and incomplete forms. • suspected case of ili: cases considered by clinical criteria (fever higher than 38°c, cough or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nauseas or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea). • confirmed case: person with positive laboratory results for influenza a h1n1 or unspecificed influenza a (by laboratory results through rrt-pcr or immunofluorescence techniques). • dismissed case: by negative or different laboratory results, or different clinical evolution. • comorbidities: chronic illnesses like arterial hypertension, diabetes, asthma, recurrent obstructive bronchial syndrome (robs), smoking, chronic obstructive pulmonary disease (copd), immunosuppression, hiv ⁄ aids, cancer, nephropathy, obesity; pregnancy was also considered. data were analyzed using epi2000 software (epi infoô cdc, atlanta, eeuu). rates were calculated and rr was estimated with their respective confidence interval (ci). population data were taken from 2001 national census projections. an estimation based on the same census was used for the group between 5 and 17 years old. to observe the effects of other co-variables, the or and their ci were calculated. logistic regression was used to evaluate the influence of the comorbidities. x 2 was used to compare proportions. respiratory samples (nasopharyngeal and faryngeal swabs) were obtained. they were analyzed at influenza sentinel surveillance unit of tucumán, and ⁄ or sent to national reference laboratory dr. c. malbrán (rt-pcr). from all notifications (20 212), 6342 were cases of ili in the group aged between 5 and 17 years old, 53% (3340 ⁄ 6342) of which were males. the incidence rate was 17ae4, and it differed according to the sexes: 18ae0 males and 16ae7 females per thousands of inhabitants (p < 0ae05). of all laboratory samples (370) 13% were confirmed as influenza h1n1, 34% were confirmed as unspecificied influenza, and 49% were dismissed. the remaining percentage corresponded to the isolation of other viruses (parainfluenza, respiratory syncytial virus, and adenovirus). the school aged group had higher risk of getting sick, in relation to the rest of the population (rr 1ae78 [95% ci 1ae55-2ae04]), especially males (rr 1ae97) compared with females (rr 1ae66). the highest attack rate was observed in the capital of tucumán (42 ⁄ 1000 inhabitants). according to the rest of the population, it looked like being school aged children meant a protective factor for presenting sari (severe acute respiratory infection) (or 0ae66 [95% ci 0ae66-0ae89], p < 0ae05). the lethality rate was 9ae32 ⁄ 10 thousand. the risk of dying was low compared to other ages. persons with comorbidities had significantly higher risk of presenting sari (or 1ae8 [95% ci 1ae35-2ae58], p < 0ae05) and of dying (or 8ae1 [95% ci 19ae6-3ae34], p < 0ae05). respiratory comorbidities were the most frequent: asthma 2ae5% (162 ⁄ 6342) and 2% rors (122 ⁄ 6342). the symptoms headaches, myalgia, coryza, and sore throat were very common and significantly different (p < 0ae05) than the rest of the population. if we compared the group aged 5-17 years with 18-39 years old, the epidemic curve of the first group showed a decrease in the ew 28, coinciding with winter holidays (ew 27) (figure 1 ). there was a slight increase in the tendency when classes began, but it showed a clear declination afterwards. the analysis of rates in school aged children by ew showed a reduction of 22ae2% in males and 26ae6% in females (p < 0ae05) at ew 28. however, after the first week of winter holidays, the curve in males had a significant increased to 65ae6% compared to ew 27, reaching the highest weekly rate of the epidemic (18 ⁄ 10 000 inhabitants). the reopening of classes coincided with a significant decrease of the rate (34ae9%), from 18 to 11ae7 ⁄ 10 000 inhabitants in ew 31 (p < 0ae05). in females, the school closure coincided with a plateau-shaped curve, and the reopening with a significant decrease of 43ae4% of the rate, from 12ae1 to 6ae9 in ew 32 ( figure 2 ). the school children got sick a lot more than the rest of the population, although they presented less proportions of sari. however, comorbidities were determined in order to present sari or death. symptoms like headache, myalgia, coryza, and sore throat were considered more conducting for the definition of cases in this population in tucumán. the epidemic curve was different in males compared to females during the winter holidays. the beneficial effect of school closure was observed as long as persons met the recommendations. the difference between males compared to females during winter holidays could mean that women would have carried out social distance recommendations much better, for example, remained at home. the significant reduction after the opening of classes is a factor to be considered as an effective intervention in the declining stage of the curve. here, we report pdmh1n1 infection attack rate (iar) during the first wave of the pandemic. we used our iar estimates to infer the severity of the pandemic strain, including the age-specific proportion of infections that led to laboratory confirmation, hospitalization, intensive care unit (icu) admission, and death. [1] [2] [3] [4] part of these results are now available in ref. 5 subjects of a community study, 5-14 years old between 1 november 2008 and 31 october 2009, we conducted a cohort study of pediatric seasonal influenza vaccination and household transmission of influenza. one hundred fifty-one children aged 5-14 were recruited and provided baseline sera in november and december 2008. between september and december 2009 a further 766 children aged 5-14 were recruited and provided baseline sera for the second phase of the study. for this serologic survey, we tested the 151 sera collected before the first wave and the 766 sera collected after the first pandemic wave. written informed consent was obtained from all participants. parental consent was obtained for participants aged 15 or younger, and children between the ages of 8 and 15 gave written assent. all study protocols were approved by the institutional review board of the university of hong kong ⁄ hospital authority hong kong west cluster. age-stratified data on virologically confirmed outpatient consultations, hospitalizations, icu admissions, and deaths associated with pdmh1n1 from 29 april 2009 to 15 november 2009 were provided by the hong kong hospital authority (the e-flu database). 6 since may 2009, patients admitted with acute respiratory illnesses routinely underwent laboratory testing for pdmh1n1 virus by molecular methods. sera were tested for antibody responses to a ⁄ california ⁄ 4 ⁄ 2009 by viral microneutralization (mn). 7 most individuals infected with influenza develop antibody titers ‡1:40 by viral microneutralization after recovery. 8 we defined the pdmh1n1 seroprevalence rate as the proportion of individuals who had antibody titers ‡1:40. while mn antibody titers of ‡40 are not by themselves conclusive evidence for pdmh1n1 infection, we have assumed that the increase in cross-sectional seroprevalence between the pre-and post-first wave time periods are evidence of recent pdmhn1 infection. the iar was defined as the proportion of individuals infected by pdmh1n1 during the first wave. the case-confirmation rate (ccr), case-hospitalization rate (chr), case-icu-admission rate (cir), and case-fatality rate (cfr) were defined as the proportion of pdmh1n1 infections that led to laboratory-confirmation, hospitalization, icu admission, and death. due to containment efforts until june 29, 2009 all laboratory-confirmed cases were required to be hospitalized for isolation regardless of disease severity. as such, only surveillance data from june 30 onwards were used to estimate severity measures. we estimated the iar as the difference between the prefirst-wave and post-first-wave seroprevalence rate. we used the estimated iar as the denominator for calculating the ccr, chr, cir, and cfr. we used an age-structured sir model with 5 age classes (0-12, 13-19, 20-29, 30-59, and ‡60) to describe the transmission dynamics of pdmh1n1 in hong kong between 10 june and 15 november 2009. we assumed that the mean generation time was 2ae3 days. using the age-structured transmission model, we estimated the following transmission parameters from the serial cross-sectional serologic and hospitalization data: (i) r o , the basic reproductive number; (ii) p 1 and p 2 , the reduction in within-age-group transmission for 0-12 and 13-19 years old during summer vacation (compared to school days during september-december 2009); (iii) d r , the average time for neutralization antibodies titer to reach ‡1:40 after recovering from infection; (iv) h a , the age-specific relative susceptibility with 20-29 years old adults as the reference group. we assumed non-informative priors for all parameters and used monte carlo markov chain methods to obtain posterior distributions of the parameters. sources of specimens: [1] pediatric cohort study (2-29 april 2009 virological surveillance data suggested that the first wave of pdmh1n1 in hong kong occurred from august to october 2009. most of the laboratory-confirmed infections in this first wave occurred in individuals aged below 25 years old accounting for >72% of the lab-confirmed cases and hospitalizations, 32% of icu admissions, and 6% of deaths. taking into account a delay of 2-3 weeks for antibody titers to appear during convalescence, 8 we found that these virological surveillance data were consistent with our serial cross-sectional seroprevalence data, which indicated a sharp rise in seroprevalence among the 5-25 years old from september to november and a plateau thereafter (data not shown). among individuals aged 5-14 years, the seroprevalence rates were similar across time between pediatric outpatient subjects and pediatric cohort study subjects (data not shown). similarly, for older age groups, the seroprevalence rates were largely similar between blood donor subjects and hospital outpatient subjects (except for the 20-29 years old in november-december). this provided some evidence that despite biases in our convenience sampling scheme, the resulting serologic data provided a reasonably representative description of seroprevalence in the community. the estimated pre-and post-first-wave seroprevalence rates and the corresponding iar estimates are shown in table 1 . the severity estimates (ccr, chr, cir, and cfr) are shown in table 2 . in summary, we estimated the iar was 43ae4% among 5-14 years old, 15ae8% among 15-19 years old, 11ae8% among 20-29 years old, 4ae3% among 30-39 years old, 4ae6% among 40-49 years old, and 4ae0% among 50-59 years old. overall, we estimated a population-weighted iar of 10ae7% (9-12%) among individuals aged 5-59 years through the first wave in hong kong. ccr were around 2ae8-5ae4% among the 5-59 years old. chr were around 0ae47-0ae87% among the 5-59 years old. cir increased from 7ae9 (5ae2-12ae6) per 100 000 infections in 5-14 years old to 75 (32ae7-281) per 100 000 infections in 50-59 years old. cfr followed a similar trend with 0ae4 (0ae1-2ae3) death per 100 000 infections in 5-14 years old to 26ae5 (10ae4-109) deaths per 100 000 infections in 50-59 years old. compared to children aged 5-14, adults aged 50-59 were 9ae5 and 66 times more likely to be admitted to icu and die if infected. the best-fit age-structured transmission model gave the following parameter estimates: 1. the basic reproductive number was 1ae38 (95%ci, 1ae36-1ae41). 2. it took an average of 14 (9-21) days for recovered individuals to develop neutralization antibody titer ‡1:40. table 2 . estimated age-specific proportions of individuals with pdmh1n1 infections that were laboratory-confirmed, were hospitalized, were admitted to icu, and died. case-icu and case-fatality rates are expressed as number of episodes per 100 000 infections 3. compared to 20-29 years old, 0-12 years old children and 13-19 teenagers were 3ae7 (3ae2-4ae5) and 1ae6 (1ae3-2) times more susceptible to pdmh1n1 infection, respectively. 4. compared to 20-29 years old, 30-59 years old older adults and 60-79 years old elderly were only 0ae42 (0ae3-0ae6) and 0ae33 (0ae18-1ae5) times as susceptible as the 20-29 years old, respectively. 5. compared to the school period during september-december 2009, summer vacation reduced within-agegroup transmission by 61% (53-72%) among 0-12 years old, but only 12% (3-18%) among 13-19 years old. using computer simulations, we estimated that if preexisting seroprevalence is zero, real-time serologic monitoring with about 1000 specimens per week would allow accurate estimates of iar and severity as soon as the true iar has reached 2% (data not shown). we estimated that during the first wave in hong kong, 43ae4% of school-age children and 10ae7% of individuals aged 5-59 were infected by pdmh1n1. a serologic survey in england found similar iars in london and the west midlands. 8 both studies highlight the importance of including serologic surveys in pandemic surveillance. the geographically compact and well-mixed population in the urban environment of hong kong permits some degree of confidence in the validity of our iar and severity estimates. the completeness of the pdmh1n1 surveillance system, welldefined population denominator, and our large-scale serologic survey provide accurate numerators and denominators for the severity measures. we based severity estimates for pdmh1n1 on the iar as the denominator. in most previous studies of pdmh1n1 severity, the denominator was clinical illness attack rate, which depends on the probability of symptoms as well as medical care seeking behavior of the population. 2, 9 our estimated cirs and cfrs are broadly consistent with presanis et al.'s 2 'approach 2' severity estimates, but around 7-9 times lower than their 'approach 1' estimates. our estimates of chr are 2-10 times higher than their approach 2 estimates of symptomatic chr. however, the hospitalization-death ratio was 4253 ⁄ 27 = 164 as of november 15 in hong kong, but 996 ⁄ 53 = 19 as of june 14 in new york, 2 suggesting that the clinical threshold for admission in terms of disease severity at presentation may have been lower in hong kong. our study has a number of limitations. first, we have used antibody titers of ‡1:40 by viral microneutralization as an indicator of recent infection, correcting for pre-existing seroprevalence levels, but this may lead to underestima-tion of the iar if some infections led to antibody titers <1:40, or if some individuals with baseline titers ‡1:40 were infected. second, our estimates of the iar would be biased upwards if infection with other circulating influenza viruses led to cross-reactive antibody responses resulting in antibody titers ‡1:40. however between august and october 2009, 83% of influenza a viruses detected in hong kong were pdmh1n1, and only 3% of isolated viruses were seasonal h1n1 viruses. 10 third, a minority of severe illnesses associated with pdmh1n1 infection might not be identified by molecular detection methods, for example if admission occurred after viral shedding from the primary infection has ceased, in which case we may have underestimated the disease burden of pdmh1n1. finally, our analyses are primarily based on seroprevalence among blood donors to the hong kong red cross, who may not be representative of the whole population. we do not have detailed data on donors to compare their risk of infection with the general population, but we did observe very similar seroprevalence rates across the three groups of subjects in our study, i.e., blood donors, hospital outpatients and participants in a community cohort (data not shown). in conclusion, around 10ae7% of the population aged 5-59 and half of all school-age children in hong kong were infected during the first wave of pandemic h1n1. compared to school-children aged 5-14, older adults aged 50-59, though less likely to acquire infection, had 9ae5 and 66 times higher risk of icu-admission and death if infected. thus, although the iar of pdmh1n1 is similar to that of a seasonal epidemic, the apparently low morbidity and mortality of 2009 pandemic influenza (h1n1) appears to be due to low infection rates in older adults who had a much greater risk of severe illness if infected. the reasons why older adults appear relatively resistant to pdmh1n1 infection even though they appear to lack neutralizing antibody remains unclear. if antigenic drift or other adaptation of the pdmh1n1 virus allows these older age groups to be infected more efficiently, the morbidity and mortality of subsequent waves of the pandemic could yet become substantial. and the national institute of allergy and infectious diseases, national institutes of health (contract no. hhsn266200700005c; adb no. n01-ai-70005). the funding bodies had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to publish. bjc reports receiving research funding from medimmune inc., a manufacturer of influenza vaccines. the authors report no other conflicts of interest. some data presented in this manuscript were previously published in wu et al. 5 it is well known that a primary goal of vaccination is to generate immunological memory against the targeted antigen to prevent disease in a vaccinated person. this ensures an accelerated immune response in the event of future contact with the pathogenic agent, such as a virus. therefore, it is very important to develop criteria for the assessment of vaccine immunogenicity by measuring both t and b memory cell levels from the vaccinated host. in contrast to inactivated influenza vaccines, live attenuated influenza vaccines (laivs) have been shown to provide primarily cellular and local immune responses. 1-3 to date, however, the hemagglutination-inhibition (hai) test (i.e. detection of serum antibodies) remains the method widely accepted for evaluation of an influenza vaccine's immunogenicity. improved understanding of the role of cellular and mucosal immunity and their contribution to protecting against severe illness caused by influenza infection has emphasized the need to reconsider methodologies used to evaluate the immunogenic impact of various influenza vaccines. such new assays need to include methods to measure local antibodies and virus-specific lymphocytes, especially in the case of live attenuated influenza vaccines, because of their potential to induce such broad-based immune responses. 4 the aim of this study was to assess the ability of new russian pandemic laivs a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) ('ultragrivak,' registered 25ae03ae2009) and a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) ('influvir,' registered 13ae10ae2009) to induce memory t-cells in naïve human subjects and to compare results to levels of hai antibodies from each subject. a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) laiv was generated by 7:1 genetic reassortment of low-pathogenic avian influenza virus a ⁄ duck ⁄ potsdam ⁄ 1406-86(h5n2) and master donor strain a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2). 5, 6 the vaccine strain contains ha gene from avian virus, as well as na and internal genes from the master donor virus. a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) laiv was generated by classical (6:2) reassortment of a ⁄ california ⁄ 07 ⁄ 2009 (h1n1) with the master donor virus. 7 the vaccine strain contains ha and na genes from a 'wild-type' h1n1 strain and internal genes from the master donor virus. participants were aged 18 to 20 years and were without contra-indication of laiv vaccination. immunogenicity of a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) laiv was assessed in ten vaccinated persons and ten volunteers inoculated with a placebo (sterile physiological saline solution). immunogenicity of a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) laiv was estimated in 16 vaccinated volunteers and nine volunteers inoculated with placebo. viruses or placebo were administered intranasally twice with an interval period of 21 days at a dosage of 0ae25 ml per nostril for each vaccination. physical examination, venous blood and nasal swab samples were collected at four time points during the study: (i) before vaccination (day 0); (ii) 21 days after first vaccination (day 21); (iii) 21 days after the second vaccination (day 42); and (iv) 6 weeks after the second vaccination (day 63). serum hai antibodies were measured by standard hai assay 8 using 1% human red blood cells. test antigens for the assay were a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) or a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) to match the appropriate vaccine antigen. local iga antibodies in nasal swabs were evaluated by elisa 9 using whole purified a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) or a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) viruses at 16 hau per 0ae05 ml for absorption to elisa plates. endpoint elisa titers were expressed as the highest dilution of sera that gave an optical density (od) greater than twice the mean od of six negative controls in the same assay. percentages of virus-specific cd3 + cd8 + ifn-c + and cd3 + cd4 + ifn-c + peripheral blood memory cells were determined using a flow cytometry iccs assay performed by the published method. 3 pbmcs were prepared with standard histopaque-1077 gradient centrifugation from heparinized whole blood. wilcoxon matched pair test, mann-whitney u test and the students t-test were used for statistical data analysis. prior to the first vaccination (day 0), gmts of hai antibodies to a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) and a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) laivs were 1 ⁄ 5ae4 and 1 ⁄ 5ae7, respectively. in addition, gmts of siga against these specific antigens from nasal swabs were 1 ⁄ 5ae7 and 1 ⁄ 9ae1, respectively. no hai antibody titers greater than 1:40 were observed prior to vaccination. background levels of virusspecific t-cells varied significantly within groups. mean levels of virus-specific cd8 + ifnc + cells were 0ae151% to a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) and 0ae173% to a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1). for cd4 + ifnc + cells, initial levels were 0ae240% and 0ae336%, respectively. thus, background levels of virus-specific antibodies were low, but prior vaccination or virus exposure in some volunteers produced some pre-existing levels of t cells, thus they were not absolutely immunologically naïve in this sense. preexistence of h5n1-crossreactive antibodies and t-cells has been observed previously. [10] [11] [12] effect of vaccination antibody immune responses both influenza a (h5n2) and influenza a (h1n1) laivs stimulated production of serum hai antibodies and local iga antibodies in nasal swabs. following the first vaccination with influenza a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) laiv, 20% percent of volunteers exhibited seroconversion of hai antibodies; after the second vaccination, 30% of volunteers exhibited seroconversion. after the first vaccination, a 10% conversion rate of siga was observed; after the second vaccination, 60% showed conversions in levels of siga. the first vaccination with a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) laiv showed 6ae3% of hai antibodies seroconversions vaccination, and 50% seroconversion after second vaccination. for local siga, those results were 18ae8% and 31ae3% following the first and second inoculation, respectively. figure 1 summarizes cellular immune responses observed in the vaccinated versus the placebo group. after the influenza a (h5n2) laiv inoculation, significant differences in both cd4 and cd8 ifnc-producing t-cells were observed at day 42 after the second vaccination (d63). these data indicate that healthy young people who never received such avian influenza vaccines and were not exposed to h5n1 wild-type viruses were able to respond to the live attenuated h5n2 influenza vaccine. after the first influenza a (h1n1) laiv vaccination, reliable increases were observed in cd8 + cells only. after the second vaccination, increases in both cd4 + and cd8 + fold changes were significantly higher in vaccinated volunteers compared to the placebo group. it is noteworthy that cellular immune responses (cd4 + and cd8 + cells) were more marked in the a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1). considering the long-term circulation of h1-subtype viruses among humans in contrast to the novelty of h5viruses, such a result would be expected. similar data were also observed following vaccination with the h5n2 laiv. after first vaccination, the percent of people with notable increases in virus-specific cd4 + and cd8 + t-cells was 20% and 10% to h5n2 and 38% and 75% to h1n1, respectively. after the second vaccination, these results were 40% and 30% to h5n2 and 69% and 69% to h1n1, respectively. importantly, a significant number of vaccinated volunteers without remarkable increases ( ‡4-fold) in hai antibodies had notable increases in cd4 + and ⁄ or cd8 + memory cells. the percent of people with notable increases in virus-specific t cells after the second vaccination among hai()) volunteers was 40% and 75% to h5n2 and h1n1, respectively. these results indicate that laivs were able to induce broadly responsive, key antiviral immune responses that would not have been detected by the hai assay alone. thus, it can be deduced that hai data alone fails to reveal important broad and specific immune responses to laiv. consequently, the hai test alone is not suitable for assessment of laiv immunogenicity. furthermore, vaccination with h5n2 laiv was able to induce cross-reactive memory t-cells to a seasonal vaccine strain, a ⁄ 17 ⁄ solomon islands ⁄ 06 ⁄ 9 (h1n1) ( table 1) . reliable increases to a (h1n1) were observed in up to 20% of volunteers. there was an inverse dependence between levels of memory t cells before and after vaccination. authors are thankful to path for the financial support of these studies. we are also thankful to jessica d'amico and dr. rick bright for their editorial review. options for the control of influenza vii background: increased susceptibility of older populations to secondary bacterial pneumonia-like infections following influenza infection has been well documented. 1 recent evidence in mouse models suggests that this increased risk from secondary bacterial infection occurs through a desensitization of the innate immune response. 2 this recent finding, however, does not account for potential differences in immune responsiveness due to age. materials and methods: to address this parameter, we used three age groups (aged, adult, and young mice) to evaluate the role of age in influenza-mediated vulnerability to secondary bacterial challenge with pseudomonas aeruginosa. all mice were evaluated for multiple parameters including: (i) survival; (ii) lung bacterial load; (iii) total lung protein content; (iv) immune cell infiltration; (v) cytokine ⁄ chemokine expression; and (vi) toll-like receptor (tlr) rna expression profiles. results: prior challenge with influenza contributed to aberrant cytokine ⁄ chemokine profiles and increased lung cellular infiltrate in response to secondary bacterial infection across all age groups, supporting a critical role for influenza infection in the alteration of immune responses to other pathogens. also similar to human influenza, these changes were exacerbated by age in mice as demonstrated by increased bacterial load, mortality, and total lung protein content (an indicator of lung damage) after p. aeruginosa challenge. conclusions: these data support a potential role for virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and the increased susceptibility of influenza virus infected mice to secondary bacterial infection. the understanding of the complex interaction of host and pathogen -and the role of age -in human influenza is critical in the development of novel therapeutics and improved vaccine approaches for influenza. our results support further examination of influenza-mediated alterations in innate immune responses in aged and non-aged animals to allow elucidation of the molecular mechanisms of influenza pathogenesis in humans. there is considerable evidence in the clinical literature to support the role of influenza infections with an enhanced risk for secondary bacterial pneumonias. [3] [4] [5] given the increased pneumonia-related morbidity and mortality in both the young and elderly populations, there is rationale for gaining a deeper understanding as to the systemic changes in the pulmonary microenvironment. although there are some recent reports that account for some of the molecular mechanisms at work in this disease process, 2 there is a paucity of experimental evidence that considers the potential effects of age. developmental changes in the immune system that occur in the aged environment have been well documented with regard to senescence of the adaptive immunity, global changes in myeloid cell function, and the establishment of a general pro-inflammatory state. 6, 7 the aim of this work was to provide evidence for the contribution of the aged immune environment to the pathology of influenza mediated secondary bacterial infections. animals used in this study were housed under conditions approved by tulane university's institutional animal use and care committee. female balb ⁄ c mice used in these studies were divided into three age groups: aged (18 months old), adult (6 months old), and young (2 months old). each age group was subdivided into two groups: influenza infected and naïve (control). mice were infected by the intranasal route with 4 · 10 5 pfu of mouse-adapted influenza a ⁄ pr ⁄ 8 ⁄ 34. clinical disease was measured by body weight changes over a 6 week period post influenza challenge, and recovery was determined as return to pre-infection weight. all mice were subsequently challenged intransally with1 · 10 7 cfu pseudomonas aeruginosa strain pao1. twenty-four hours post-pseudomonas challenge, bal with sterile pbs was performed on all mice in all groups. total rna from the cellular fraction was pooled from three experimental animals from each group. tlr mrna was detected by qrt-pcr, where expression levels were determined as relative to b-actin mrna levels. cdna was synthesized from total cellular rna from bal samples using iscript cdna synthesis kit (biorad). pcr reactions were composed of 0ae1 lg cdna forward and reverse primers according to optimized conditions and 12ae5 ll of 2 · syber green icycler supermix (biorad), in a total vol-ume of 25 ll and were run using a biorad icycler utilizing melting point determination. primers and concentrations used in this study included: mus_tlr2f: tgctttcct-gctggagattt-600 nm, mus_tlr2r: tgtaacgcaac agcttcagg-900 nm, mus_tlr3f: atatgcgcttcaa tccgttc-300 nm, mus_tlr3r: caggagcatactggt gctga-600 nm, mus_tlr4f: ggcagcaggtggaattg tat-600 nm, mus_tlr4r: aggccccagagttttgttc t-900 nm, mus_tlr5f: ctggggacccagtatgctaa-600 nm, mus_tlr5r: acagccgaagttccaagaga-900 nm, mus_tlr7f: ggagctctgtccttgagtgg-900 nm, mus_tlr7r: caaggcatgtcctaggtggt-600 nm, mus_ b-actinf: agccatgtacgtagccatcc-600 nm, mus_b-actinr: ctctcagctgtggtggtgaa-900 nm. as a measure of protein leakage into the alveolar space, total protein content in each bal was measured by bca assay of each supernatant fraction according to manufacturer's instructions (pierce). cytokine and chemokines levels were measured by multiplexed bead array (bioplex, biorad). immune cell characterization of bal was estimated by flow cytometry. lymphocyte populations were gated by forward versus side scatter and characterized as b cells (f4 ⁄ 80 ) , cd19 + ) or t cells (cd11b ) , cd4 + ). the myeloid population that is composed of macrophages, neutrophils, dendritic cells, and natural killer cells was enumerated by gating all but those found in the lymphocyte gate using forward versus side scatter plots. flow cytometry data was analyzed using flojo software (treestar). statistical analysis, where appropriate, was performed using a two-way analysis of variance (age versus influenza infection status) supported by bonferonni's correction for multiple comparisons. 8 a recent finding by didierlaurent, et al., 2 described an influenza mediated desensitization of tlr function as a primary contributor to an increase in bacterial burden when challenged after resolution of the primary influenza infection. this finding, however, was obtained using animals that were 6-8 weeks of age, where our study included two cohorts of older mice (6 months and 18 months). using whole protein content of the bal as an estimate of protein leakage into the lumen of the lung, we found elevated protein content in aged mice as compared to young and adult mice. in aged mice, a slightly lower total lung protein when comparing influenza infected to protein in the bal from influenza naïve mice challenged with p. aeruginosa (table 1) . supporting previously published studies showing a generalized pro-inflammatory cytokine environment in the aged immune system, we provide evidence for significantly (p = 0ae0465) and an increase in ifnc (p = 0ae0323) was detected. the decrease in gm-csf correlates well with a previous report that gm-csf is less prevalent in influenza resolved animals (table 1 ). 2 we also report a noticeable change in the immune cell populations with respect to b-cells, cd4 + t-cells, and the myeloid cell populations. there is a trend of increased prevalence in cd4 t-cells in the post-influenza environment across all ages. b-cell numbers also trend toward increase in influenza treated animals in young and adult animals; however, there is a noticeable decrease in the bcells in aged animals. across all age groups, there is a general decrease in frequency of cells that would normally make up the myeloid cellular fraction of the bal (macrophages, neutrophils, dendritic cells, and natural killer cells) ( table 1 ). our study also shows, as cited by others, that toll-like receptor (tlr) gene expression in the post-influenza environment is decreased in cells found in the bal 2 after both influenza and pseudomonas infection. our data support the previous finding of a reduced expression of tlr mrna in influenza-cleared mice when we measured tlr 2, 3, 5, and 7. only tlr4 showed differences with respect to age with young mice showing little or no detectable change in tlr4 mrna expression. our results show an increase in the expression across all tlrs examined in the aged mice group (table 1) irrespective of influenza infection status. these data support earlier studies performed with adult mice that showed reduced tlr mrna expression in the post-influenza environment. this study also expands the current understanding of the potential role of age in influenza mediated bacterial infection-induced mortality. the impact of these alterations in the immune microenvironment across age groups and infection status is highlighted by the ability of bacterially challenged animals to clear infection. assessment of bacterial load in the lungs of p. aeruginosa challenged mice indicated a difference in young and adult mice if previously infected with influenza virus. in aged mice, both influenza challenged and influenza-naïve mice had higher bacterial loads and less variability when comparing within the age group, supporting the risk of age alone in susceptibility to bacterial pneumonia (table 1, figure 1 ). taken together, these data support the potential role for both virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and increased the susceptibility to secondary bacterial infection that results from influenza infection in mice. these findings highlight distinct differences in the immune environment between age groups and thus reveal necessity for further examination as to the mechanisms of immunity across age with respect to current infection status. garnering a clearer understanding as to the complex interaction of host and pathogen with respect to age in influenza infections is central to the development of increased efficacy in vaccine and therapeutic strategies. prospective estimation of the effective reproduction background pandemic influenza a (h1n1) virus (ph1n1) emerged in early 2009 and rapidly spread to every continent. an urgent priority for international and national public health authorities was to estimate the transmissibility of the pandemic strain for situational awareness and to permit calibration of mitigation strategies. the basic reproductive number, r 0 , is defined as the average number of secondary cases that 1 index case generates in a completely susceptible population, and is a common measure of transmissibility. however, it is difficult to estimate r 0 without an understanding of the degree of any pre-existing immunity in the population. the effective reproductive number, r, is defined as the average number of secondary cases that 1 index case generates, and can be estimated over time (i.e. r t ). wallinga and teunis 1 described a method to estimate r t based on illness onset dates of the cases while assuming that all secondary cases would have been detected, and cauchemez et al. 2 extended the method to permit prospective estimation by adjusting for secondary cases that have not yet experienced illness onset at the time of analysis. we describe how the method can further be extended to account for reporting delays, allowing true real-time estimation of r t during an epidemic, and we illustrate the methodology on notifications of ph1n1 and associated hospitalizations in hong kong. we obtained data on all laboratory-confirmed ph1n1 infections ('cases') reported between may 1 and november 15, 2009 to the hospital authority and center for health protection in hong kong collated in the eflu database. a subset of the cases was hospitalised. the database also included information on age, sex, illness onset date, laboratory confirmation date, and contact history (for the early cases). laboratory-confirmed ph1n1 infection was a notifiable condition throughout our study period. we extended existing methods for estimating r t over time to allow for reporting delays between illness onset and notification, and between illness onset, notification, and hospitalisation for those cases that were hospitalised, where the reporting delay distribution were estimated empirically from the data. 3 we further extended the methodology to allow for imported cases (infected outside hong kong) contributing to the estimation of r t as infectors but not infectees. we used multiple imputation to allow for missing data on some symptom onset dates to make best use of all available data. 4 we used a serial interval with mean (standard deviation) of 3ae2 (1ae3) days, 5 and in sensitivity analyses, we used serial intervals with mean 2ae6 days 6 and 3ae6 days. 7 statistical analyses were performed in r version 2.9.2 (r development core team, vienna, austria). in late april 2009 following the who global alert, hong kong initiated containment protocols to attempt to delay local transmission of ph1n1 for as long as possible. these measures included screening at ports, airports, and border crossings, and enhanced surveillance for people with influenza-like illness, particularly for those who had recently returned from abroad. laboratory testing capacity was substantial due to heavy investment in local infrastructure following previous experiences with avian influenza a ⁄ h5n1 in 1997 and severe acute respiratory syndrome in 2003. laboratory-confirmed ph1n1 cases were isolated until recovery, and their close contacts were placed under quarantine for 7 days. imported cases were identified sporadically through may and early june 2009. the first case of ph1n1 not traceable to importation (i.e. a local case) was identified on june 11 and triggered a change to mitigation phase measures. some containment measures, including isolation of cases, were continued until the end of june to allow a soft transition between containment and mitigation phases. as an immediate measure to try to reduce community transmission of ph1n1, all childcare centres, kindergartens, and primary schools were proactively closed for 14 days (subsequently extended for another 7-14 days to summer vacation in early july). 8 any secondary schools in which one or more confirmed ph1n1 case was identified were reactively closed for 7 days. on june 13 the government opened eight designated flu clinics across the territory to provide free medical consultation for outpatients with influenza-like illness and free laboratory testing for ph1n1. these clinics resumed regular chronic disease services in mid-august, and laboratory testing and antiviral treatment was restricted to high risk groups in september. the various interventions are highlighted in figure 1 (a), superimposed on the epidemic curve of laboratory-confirmed ph1n1 cases and ph1n1-associated hospitalizations. around 15% of the cases were hospitalised, and this proportion increased somewhat towards the end of the epidemic. 3 figure 1(b) shows the estimates of r t based on laboratory-confirmed ph1n1 cases. the estimated r t peaked at 1ae5 on june 12, and fell below 1 between 20 june and 3 july (which was within the school closure period). r t fluctuated between 0ae8 and 1ae3 through the school summer vacations in july and august, it subsequently increased to around 1ae2-1ae3 after schools reopened in september until the epidemic peaked in late september, and then fluctuated below 1 as the epidemic declined. the trends in r t based on h1n1-associated hospitalizations were similar, although with wider confidence intervals due to the smaller number of events ( figure 1c ). the extension of the methods to allow for reporting delays avoided substantial bias in realtime estimates of r during the epidemic for the most recent 7 days, and closely tracked the final estimates of r t . 3 our results suggest that ph1n1 may have had slightly lower transmissibility in hong kong than elsewhere. for example, estimates of r t were around 1ae5-2ae0 in new zealand 9 and australia. 10 lower transmissibility in hong kong has been associated with school closures in june and july followed by summer vacations from july through august. 8 furthermore, in hong kong the influenza virus usually does not circulate after august, 11 and therefore seasonality could also be a cause for the lower r t . on the other hand, the interventions applied during the mitigation phase, such as the widespread use of antiviral treatment in hong kong and the pre-existing immunity in the ageing population in hong kong, may also be associated with lower transmissibility. there are some limitations to our work. first, we only used aggregated data, and we did not consider the heterogeneity among the cases in terms of sex and age or other factors. therefore our estimates can only provide a snapshot of the overall trend, but limited information for any specific subset of population. secondly, we did not consider the possibility that cases might be infected in hong kong and exported to other countries, which could lead to slight underestimation of the transmissibility. one has to be careful in translating the estimated r t to the effectiveness of any specific interventions, as interventions may not be the only factor influencing the transmissibility; for example, a depletion of the susceptible population during an epidemic can also be a factor for the decline in r t . 12 in conclusion, real-time monitoring of the effective reproduction number is feasible and can provide useful information to public health authorities for situational awareness and planning. in affected regions, laboratory capacity was typically focused on more severe cases, and changes in laboratory testing and notification rates meant that that case counts may not necessarily reflect the underlying epidemic. a useful alternative to case-based surveillance is surveillance of the subset of severe infections, for example hospital admissions, or icu admissions, 13 and our results show that it was feasible to monitor ph1n1-associated admissions in real-time to estimate transmissibility. influenza antigenic cartography projects influenza antigens into a two or three dimensional map based on immunological datasets, such as hemagglutination inhibition and microneutralization assays. a robust antigenic cartography can facilitate influenza vaccine strain selection since the antigenic map can simplify data interpretation through intuitive antigenic map. however, antigenic cartography construction is not trivial due to the challenging features embedded in the immunological data, such as data incom-pleteness, high noises, and low reactors. to overcome these challenges, we developed a computational method, temporal matrix completion-multidimensional scaling (mc-mds), by adapting the low rank mc concept from the movie recommendation system in netflix and the mds method from geographic cartography construction. the application on h3n2 and 2009 pandemic h1n1 influenza a viruses demonstrates that temporal mc-mds is effective and efficient in constructing influenza antigenic cartography. the web sever is available at http://sysbio.cvm. msstate.edu/antigenmap. as a segmented, negative stranded rna virus, influenza virus is notorious for rapid mutations and reassortments. the mutations on the surface glycoproteins (ha and na) of influenza viruses are called antigenic drifts, and these antigenic drift events allow the virus to evade the accumulating immunity from previous infection or vaccination and lead to seasonal influenza epidemics. a reassortment event with a novel influenza antigen may result in antigenic shift and cause influenza pandemic. for instance, the 2009 h1n1 pandemic virus is a reassortant with a swine origin ha antigen. vaccination is the primary option for reducing the effect of influenza, and identification of the right vaccine strains is the key to development of an effective vaccination program. the antigenicity of an optimal vaccine strain should match that of the epidemic strain. in influenza surveillance program, the influenza antigenic variants are generally identified by the immunological tests, such as hemagglutination inhibition (hi) assay, microneutralization (mn) assay, or elisa. these immunological assays measure the antigenic diversity between influenza viruses by comparing the reaction titers among the test antigens and reference antisera. however, data interpretation of the data from these assays is not trivial due to the embedded challenges such as data incompleteness, high noises, and low reactors. by mimicking geographic cartography, influenza antigenic cartography projects influenza antigens into a two or three dimensional map using immunological datasets. 1 antigenic cartography can simplify the data interpretation, and thus, facilitate influenza antigenic variant identification. recently, we developed a novel computational method, temporal matrix completion-multidimensional scaling (mc-mds), in antigenic cartography construction. 2 in this paper, we described the details of temporal mc-mds, especially the original concepts introduced in this method, and how they can achieve the robustness in antigenic cartography construction. our method included two integrative steps: it first reconstructs the hi matrices using low rank mc method, and then generates antigenic cartography using mds with a temporal regularization. the mc concept was adapted from the movie recommendation system in netflix and the cartography concept from geographic cartography. in 2006, netflix, an online dvd and blu-ray disc rentalby-mail and video streaming company, held a 3-year netflix prize contest (http://www.netflixprize.com/) on computational methods for improving its recommendation system. 3 in its recommendation system, netflix collected the rating data from the individuals. based on his or her renting history and the ratings in the systems (e.g., from evaluators and other renters), netflix recommendation system suggests certain movies to a renter. apparently, no individuals would be feasible to provide ratings for all of the movies, as it will take hundreds of years for a single person to rate over 50 000 movies available from netflix. thus, the resulting rating data is an incomplete matrix, and it can be as sparse as less as 1%. 4 the challenge in netflix recommendation system is a classic mc problem. [4] [5] [6] [7] [8] as the inspiration of netflix prize contest, many efficient low rank mc algorithms were developed, for instance, opt-space, 7 svt, 5 cf, 9 bellkor, 10 pf, 11 and fwls. 12 eventually, the team bellkor's pragmatic chaos won this contest. their methods combines nonlinear probe blending and linear quiz blending to come up with a predictor bigchaos. 13 matrix completion estimates the unobserved values based on the observed values. the users can refill the missing data without repeating the experiments. furthermore, mc will help reduce the noises in the data, for instance, those biases by different individuals performing experiments. in influenza antigenic characterization, hi assay is a commonly used assay for antigenic analysis, since hi assay is relatively economic and easy to perform. however, hi is labor intensive, and it is almost impossible for any individual lab to complete the hi assays for all pairs of antigens and antisera during influenza surveillance. in addition, both testing antigens and the reference antisera are dynamic. for instance, in seasonal influenza surveillance, generally only contemporary antisera are used in experiments. thus, we will have to integrate multiple hi tables in order to evaluate the overall antigenic changes for influenza vaccine strain selection. the resulting hi tables will be incomplete, and the observed entries in the integrated hi data can be as less as 3%. the completion of this matrix can be formulated as a typical mc. briefly, given the combination of hi matrix with m antigens and n antisera, the hi matrix can be represented as m m·n = (m ij ) m·n , where m ij denotes the hi values from the reaction between testing antigen i and antiserum j. the low rank mc assumes that both antigen and antiserum can be embedded into a low rank space. to be specific, the low rank mc method is to seek matrix u m·r , v n·r and a diagonal matrix r r·r , where m = u m·r r r·r (v n·r ) t . in order to achieve this goal, the optimization formulation has been employed, which can be represent as following, where e denotes the observed entries in hi matrix and g(x) is a regularization function. the eqn (1) is the standard format of a low rank mc formulation. the geographic cartography is a common technique to display the cities and their geographic distances in a map. this cartography can be generated using mds based on a geographic distance matrix. figure 1(a) shows the antigenic cartography generated using a distance matrix with seven cities, and figure 1 (b) is a map for comparison. as an analog of geographic cartography, the influenza antigenic cartography maps the influenza antigens into a two or three dimensional map based on the distance matrix generated using immunological data. this incomplete matrix can be filled through mc algorithm discussed in section mc and netflix. low reactors, non-random date incompleteness, and temporal model generally, three types of data are present in a combined hi matrix: high reactor, low reactor, and missing values. among these three data types, high reactors are the most reliable data points. the low reactors are those values present in the hi matrix as ''equal to or less than a threshold h'', where h can be 5, 10, 20, or 40. low reactors have similar values in the affinity dataset but could be from different binding settings. these low reactors are present due to the detection limits of biotechnology, and they are not reliable. both these missing values and low reactors make it very difficult to analyze and interpret antigenic correlations amongst tested antigens and reference antigens. to our best knowledge, none of the existing mc method can handle the threshold values. in addition, the non-random incompleteness of influenza immunological datasets generates an additional challenge in traditional mc methods, which are based on the assumption that the observed values are randomly distributed among the matrix. in a typical combined antigenic hi data, most of the off-diagonal entries are missing values or low reactor values. 1 in order to overcome the above issues, we incorporated a regularization function into the eqn (1), where this indicator function is only valid for those entries with low reactor values. an alternating gradient decent method is applied to solve the optimization problem in eqn (2) . in addition, a temporal mds method is proposed to project the antigens into a 2 or 3 dimensional map. x where d ij is the average distance between virus i and virus j, t i is the isolation year of virus i, d ij is the distance between virus i and virus j in cartography, d ac i is the distance between virus a and center of group i, and d c i c j is the distance between the centers of group i and group j. all the parameters are tuned by cross validation. we named this method as temporal mc-mds. by applying temporal mc-mds method in an h3n2 dataset, 2 low reactors. figure 2 (a) is a three-dimensional influenza antigenic map based on this data by using mc-mds method. the reported 11 clusters (hk68, en72, vi75, tx77, bk79, si87, be89, be92, wu95, sy97, and fu02) were displayed in the core of a spiral s-shape, and bk79 and be92 are located at the turning point of this s-shape. however, the antigenic distances between some viruses are incorrect. for example, the distance between hk68 and fu02 in the projection is 7ae1223 units, which is close to the distance between hk68 and bk79 (6ae5113 units). the main reason leading to those inaccurate distances is the unique distribution of hi datasets described in section 2.3. in comparison, with the temporal model, not only the viruses in 11 clusters have been clearly separated, but also the antigenic distances between each cluster are proportional to their isolation time interval. in this updated cartography ( figure 2b ), the antigenic distance between hk68 and fu02 is 15ae0633 units, where the distance between hk68 and fu02 is 6ae3984 units. this result suggested that the temporal information is critical for antigenic cartography construction for immunological datasets spanning a long time period. the hi data from seasonal influenza surveillance belong to this category. for seasonal influenza virus ⁄ pandemic influenza viruses within a short time span, the temporal model is probably not necessary, as there is lack of long-term immunological pressure present in the population. figure 2 (c) is an antigenic cartography generated using a hi dataset with 2009 h1n1 influenza viruses spanning from april of 2009 to june of 2009. this map demonstrates that there is lack of antigenic drifts during the first wave of this pandemic influenza as all of these viruses are mixed altogether. our limited studies on h5 and h7 avian influenza viruses suggested the temporal model is not needed for avian influenza viruses. however, extensive studies are required to investigate whether there is any special data structure present in this type of data. in this study, we described in details the concepts and applications of new computational method, temporal mc-mds for influenza antigenic cartography construction. we formulate the influenza cartography as two integrative steps: low rank mc problem from the concept of netflix movie recommendation system and mds from geographic cartography construction. in order to handle two additional challenges, including low reactor and non random distribution of antigenic data, a temporal model is incorporated into mc-mds as temporal mc-mds. our applications demonstrated that temporal mc-mds is effective in constructing influenza antigenic cartography. the three dimensional antigenic cartography for a ⁄ h3n2 seasonal influenza virus without temporal model, and the antigenic clusters were defined in ref. [2] ; (b) the three dimensional antigenic cartography for a ⁄ h3n2 seasonal influenza virus with temporal model; (c) the two dimensional antigenic cartography for 2009 a ⁄ h1n1 pandemic influenza without temporal model, and these viruses were labeled in shape by the corresponding month for them to be detected. one grid is corresponding to a twofold change in hemagglutination inhibition experiment. the mechanisms driving the three waves of infection and mortality in the uk in 1918-1919 are uncertain. although the circulation of three distinct viruses could have generated three waves of infection, 1 the virological evidence required to prove or disprove this hypothesis is lacking. social distancing, an alternate mechanism for generating fluctuations in the effective susceptible pool and therefore explaining multiple waves of infection, 2, 3 was not generally imposed in the uk as it was in the us and australia. we are therefore motivated to explore the possible role of continual population-level changes in the average protective response against the circulating virus in generating a multi-wave pandemic, within a biologically motivated deterministic model for influenza transmission. the nature and duration of protection against further infection following recovery from influenza is uncertain and depends on the mode and tempo of viral evolution, as well as the response of the cellular and humoral arms of the adaptive immune system. 4 for a given seasonal ⁄ pandemic strain, memory b-cells may generate a specific antibody response in a portion of the adult ⁄ elderly population, depending on the exposure to related antigenic sub-types. 5 however neutralising antibodies are unlikely to be a widespread immunological response to a novel (pandemic) strain. memory t-cells which recognise conserved internal viral proteins may be a more common mechanism for protection; the generation of very high levels of cytotoxic cd8 + t-cells potentially facilitates rapid viral clearance, 6, 7 and lower levels of cd8 + t-cells perhaps provide partial protection. 8 in this work we explore key drivers of multi-wave pandemics within phenomenological models that incorporate different immune response mechanisms building on existing models 9,10 incorporating the role of evolving population-level protection in multi-wave pandemics. we use weekly reports of influenza mortality rates 11 for five administrative units in the uk (blackburn, leicester, newcastle, manchester and wigan) where records from block censuses instigated by local medical officers to record the cumulative incidence of reported symptoms in each wave in a sample of 1000 or more households are also available. 10 the symptom reporting data allows us to estimate the case fatality rate and thus use the mortality time series to constrain our transmission model. furthermore, the incidence of individuals reporting symptoms in multiple waves provides information about the acquisition and loss of immunity. we extract the death rate and symptomatic (re)infection rates predicted by our model prevalence for a given set of parameters and estimate a likelihood-based on a comparison to all the death and cumulative reported incidence data assuming a negative binomial error distribution. we utilise monte carlo markov chain (mcmc) methods with parallel tempering algorithms to maximise this likelihood and obtain parameter estimates. parallel tempering -which concurrently searches for maximal likelihood parameter solutions on a set of scaled likelihood surfaces -allows for relatively rapid exploration of the parameter space. we use bayesian information criteria (combined with qualitative assessment of biological plausibility) to aid model selection. we have implemented a deterministic compartmental transmission model, which allows for a variety of phenomenological modes of protection against the pandemic virus. to facilitate this, we stratify the population into two groups; the 'experienced' population (stratum 1) who have had been exposed to an influenza virus and the 'naive' population (stratum 2) who have not. in each stratum, i hosts may be classified as either susceptible s i , exposed e1 i and e2 i , having (recovered from) a symptomatic i i (r i ), or asymptomatic a i (ra i ) infection. note that the states tq i , tq2 i , e2 i , t i , and t2 i are included so that the hosts move between the key epidemiological states with a peaked (rather than exponential) distribution of waiting times. hosts in the experienced stratum may exhibit reduced susceptibility, infectiousness, and symptomatic proportion compared to naive hosts, parameterised by e i , e s , and e a , respectively; however note that depending on the model parameters, there may be fully susceptible hosts within the experienced stratum. in addition, we assume homogeneous population mixing and a constant basic reproduction number r 0 with the force of infection: modulated by a sinusoidal seasonal term with amplitude b 1 with phase chosen to maximise transmission in the winter season. here n is the total population size, and x e is the initial fraction in the experienced strata. the proportion of symptomatic cases a and the case fatality rate l are permitted to vary from wave to wave (and given indices 1, 2 or 3 accordingly). the transmission dynamics is described by the following set of coupled ordinary differential equations. where s in,1 = p utq2 i and s in,2 = 0 in order to divert recovered infectious hosts from the naive stratum into the experienced stratum. the probabilities of gaining permanent protection are q 1 = q and q 2 = 0. the latent exposed period is fixed to be c = 1 ⁄ 1ae3 days, and the rate of recovery is parameterised by m = 1 ⁄ t inf , where t inf is the infectious period. hosts with prior sterilising protection begin in q 1 and move into s 1 at rate u q = 3 ⁄ t wq . recovered hosts (r i ) migrate back to s 1 at a rate u = 3 ⁄ t w . the state p 1 contains hosts with permanent protection. the modes of protection captured in this model are: i. permanent prior protection (beginning in state p 1 ), ii. waning prior protection (beginning in state q 1 ), iii. permanent acquired protection with probability q (moving into state p 1 ), iv. waning acquired protection with probability 1 ) q, and, v. partial prior protection (beginning in state s 1 ) resulting in reduced infectiousness (e i ), susceptibility (e s ), and symptomatic proportion (e a ). in the context of this model, 'permanent' protection refers to protection which lasts for the duration of the epidemic. here we explore the results of parameter fitting to two models which differ in the nature of the assumed pre-existing protection in the community at the beginning of the pandemic. protection hypothesis 1 assumes that the prior protection is sterilising but temporary, whilst protection hypothesis 2 assumes that the prior protection is partial but permanent and may act on susceptibility, infectiousness, and ⁄ or asymptomatic proportion. each model allows waning acquired protection and for a proportion q of the experienced population to gain permanent protection following infection. fitted parameters common to each model are t inf , b 1 , q, t w , a, l and the proportion beginning in p x i . prior protection hypothesis 1: sterilising, waning prior protection we fix x e = 1 and fit for q 1 (t = 0) ⁄ n and t wq so that protective modes i, ii, iii, and iv are enabled ( figure 1 ). it is important to note that due to the slow convergence of the mcmc chains, we cannot guarantee that our parameter estimates correspond to the global minimum. furthermore, parameter estimates can only be meaningfully interpreted for good fits to the data. due to the prediction of a fourth (unobserved) wave for the model fit to blackburn, we do not report these parameter estimates here. the fits to the leicester data are generated with the parameter set r 0 = 5ae7, a 1 = 0ae25, a 2 = 0ae65, a 3 = 0ae65, t w = 0ae28years, t wq = 0ae45 years, we fix q 1 (t = 0) ⁄ n = 0 and fit for x e , e a , e i , and e s so that protective modes i, iii, iv, and v are enabled (figure 2) . the parameters corresponding to the fit in figure 2 for leicester are r 0 = 7ae4, a 1 = 0ae09, a 2 = 0ae50, a 3 = 0ae61, t w = 0ae22 years, p 1 (t = 0) ⁄ n = 0ae01, s 2 (t = 0) ⁄ n = 0ae48, b 1 = 0ae021, t inf = 0ae94 days, q = 0ae561, e a = 0ae9966, e i = 0ae946, and e s = 0ae594. our model with protection hypothesis 1 -which, similarly to the model discussed in ref. [9] , assumes that a sub-population has waning sterilising prior protection -is able to generate multiple waves of infection via the continual replenishment of s 1 from an initially large proportion (over 40%) of hosts with prior protection in q combined with the waning of acquired immunity in around 23% of cases on a time-scale of 3 months. disease severity as measured by symptomatic proportion increases from 25% in the first wave to above 60% for the second and third waves. over a quarter of the population are initially permanently immune, and a large r 0 value of 5ae7 drives transmission in the remaining population. protection hypothesis 2 -which assumes that prior protection offers partial susceptibility and ⁄ or reduced infectiousness or symptomatic disease -performs slightly more poorly; the fit to the leicester data has an inferior likelihood (although the mortality data only likelihood is a little larger), despite the higher dimensionality of the model. nevertheless, the model fit still mirrors many characteristics of the data, particularly for leicester. we note that for this model, a 1 is very near the lower limit, corresponding to ubiquitous exposure in the first wave. in this scenario, refuelling of the susceptible pool to generate secondary and tertiary waves is still possible due to a shorter waning time of acquired protection (well within 3 months) and a lower probability of gaining permanent protection following infection, when compared with the parameter estimate for hypothesis 1. the parameter estimates suggest that approximately 50% of the population initially experiences reduced disease severity (e a $ 0ae66), but similar susceptibility and infectiousness. a larger value for r 0 $ 7ae4 is required to drive transmission despite low numbers beginning in p 1 , due to the large number of hosts who acquire temporary or permanent immunity early on in the pandemic. it is clear that, at least mathematically and perhaps biologically, there are multiple possibilities for the structure of population-level protection which are compatible with the generation of multiple pandemic waves. however, whilst the models considered here are able to explain the observed mortality and reinfection data for some patterns of infection and mortality (e.g. leicester), they are not consistently able to reproduce a pandemic which dies out after three waves across the connected populations we are studying (e.g. for blackburn). it is challenging to construct a deterministic model for the spread of disease within multiple locations in the uk in 1918, which assumes homogeneous mixing without modulation of the transmission rate by social distancing. an improved model working with these assumptions likely requires a richer structure for the host protection response than the structures we have explored thus far. we are currently seeking improved fits to the data by implementing a number of biologically defensible exten-sions to our model, including incremental immunity whereby t w increases by a factor v after each exposure to the pandemic flu, and incremental loss of prior protection whereby a increases as hosts lose their sterilising prior protection. it is important to note that the mechanism(s) generating differences in the pandemic experience recorded in geographically connected locations is an open question; true differences in demography, varying degrees of reactive social distancing, inhomogeneities in the circulation (or circulation history, i.e. prior immunity) of viral strains, stochastic variations, and ⁄ or unique socio-cultural ⁄ behavioural conditions may all contribute to this effect. the 2009 h1n1 experience in australia and elsewhere highlighted the difficulties faced by public health authorities in diagnosing infections and delivering antiviral agents (e.g. oseltamivir) as treatment for cases and prophylaxis for contacts in a timely manner. consequently, forecasts from mathematical models of the possible benefits of widespread antiviral interventions were largely unmet. we summarise results from a recently developed model that includes realworld constraints, such as finite diagnostic and antiviral distribution capacities. we find that use of antiviral agents might be capable of containing or substantially mitigating an epidemic in only a small proportion of epidemic scenarios given australia's existing public health capacities. we then introduce a statistical model that, based on just three characteristics of a hypothetical outbreak [(i) the basic reproduction number, (ii) the reduction in infectiousness of cases governments and public health agencies worldwide, spurred by outbreaks of sars and h5n1, have developed preparedness strategies to mitigate the impact of emerging infectious diseases, including pandemic influenza. pandemic response plans are presently being revised in light of the 2009 h1n1 experience. [1] [2] [3] many developed countries amassed large stockpiles of neuraminidase inhibitors (nais) with the expectation that they could be used to not only treat the most severely ill, but curb transmission in the community. without relevant field experience indicating how nais should be distributed, mathematical and computational modelling has been used to inform optimal deployment policy in a pandemic scenario. 4-10 models of population transmission were used to infer likely effects on epidemic dynamics, using data from human and animal studies of experimental infection and nai efficacy trials. in the australian (and wider) context, models indicated the potential for substantial benefit at the population level if nais were distributed in a liberal manner, targeting close contacts of indentified cases. 11 furthermore, results indicated that use of limited nai resources in this way may improve the impact of case treatment due to the effects on epidemic dynamics. 11 however, these models did not take into account logistic and other real-world constraints, such as finite diagnostic and antiviral distribution capacities, which were identified as limiting factors during the australian 2009 h1n1 pandemic response. [12] [13] [14] in particular, if using positive pcr diagnosis as a 'decision to treat' test, delays to confirmation of diagnosis, particularly once total laboratory capacity was exceeded, prevented timely delivery of nais to both cases and contacts of cases. 12 in previous work, 15 we have extended our existing models to examine how diagnostic strategies [e.g. using pcr confirmation versus syndromic influenza-like illness (ili) presentation as a decision to treat], diagnostic-capacity, and nai distribution capacity each impact on the ability to deliver an effective intervention. the model uses case severity (the proportion of infections deemed severe) to determine the overall presentation proportion, and so the ability to identify individuals eligible for nai treatment and contact prophylaxis. figure 1 (a) shows a key result from the model. for each curve shown, we simulated thousands of epidemics, sam-pling across plausible ranges of parameters describing virus, population, and intervention characteristics using a latin hypercube sampling (lhs) approach. without intervention, the proportion of the population infected either symptomatically or subclinically by the end of the epidemic is around 50%. if a syndromic strategy (ili presentation) is used to determine provision of nais as treatment and prophylaxis, excessive distribution of drug to individuals who are not infected with influenza occurs early in the epidemic. early stockpile expiry accounts for a marginal impact of the antiviral intervention on the final outbreak size, in the order of a few percent. the second strategy modelled (pcr ⁄ syndromic) is one where pcr confirmation of diagnosis is required early in the epidemic to make treatment decisions until such time as laboratory capacity is exceeded. from this point, individuals are treated on the basis of symptoms alone -during an epidemic phase in which a substantial proportion of ili presentations will be attributable to influenza. under this strategy, the intervention is able to control the outbreak in approximately 10% of the simulated epidemics given the 'base case' constraints on diagnosis and delivery assumed in the model. the results highlight that a successful antiviral intervention requires a highly sensitive diagnostic strategy in the initial stages of the epidemic and comprehensive distribution of post-exposure prophylaxis. a pcr ⁄ syndromic strategy for decision to treat and provide contacts with prophylaxis is thus optimal. the surface in figure 1(b) shows the percentage of simulation runs for the pcr ⁄ syndromic strategy that have a final population attack rate of <10% (a substantial reduction from the no intervention case of approximately 50%) as a function of pcr capacity and nai daily distribution capacity. as indicated by the arrow, the estimated australian pcr laboratory capacity appears to be sufficient, while significant benefits for the public health outcome may be achieved if logistical delivery constraints for nai distribution can be ameliorated. however, the probability that such an interventioneven with substantial increases in pcr and nai distribution capacity -would successfully mitigate an epidemic is low (12-25%), and consequently it is difficult to universally recommend an antiviral intervention. 15 in this study, we introduce a statistical model that predicts whether or not an nai distribution strategy based on a pcr ⁄ syndromic antiviral distribution policy will be successful in mitigating an epidemic. we thereby provide proof-of-principle for the design of a decision support tool that may be used by public health policy makers during an epidemic when faced with formulation of context specific nai distribution policy. synthetic data of hypothetical outbreaks and interventions were generated using the lhs simulations developed in ref. [15] . we selected a random sample of 100 outbreaks from a total of 2000 simulated epidemics (5% of model simulations). using these data, we identified independent model parameters that were most highly rank-correlated with the final attack rate. these parameters were included in a logistic regression model to assess their ability to predict whether an influenza epidemic would be successfully mitigated by an antiviral intervention (ar < 10%). model predictions were then validated against the full simulated dataset. full details of the simulation model, its structure, parameterisation and parameter distributions are available in ref. [15] . use of the lhs simulation approach, and the method of model analysis and evaluation was similar to that previously described. 16 matlab 2010a (mathworks, natick, ma, usa) was used for the analysis and statistical model fitting. table 1 shows results from our logistic regression model. key parameters sufficient to predict whether or not an outbreak may be controlled by the deployment of av agents are: 1. r 0 , the basic reproductive number of the outbreak (assigned values between 1ae35 and 1ae45 for this example). as the value of r 0 increases, the epidemic progresses more rapidly and is more difficult to control, explaining the negative correlation coefficient. 2. e t , the relative infectiousness of treated individuals (assigned values between 0ae8 and 1ae0). higher values for this parameter indicate only modest drug effects on transmission, explaining the negative correlation coefficient. 3. g, the proportion of infections that are severe (assigned values between 0ae001 and 0ae1), and which in turn determines the presenting proportion (derived values between 0ae11 and 0ae56). as the presenting proportion increases, the ability to identify and treat cases and deliver prophylaxis to contacts also rises, increasing the impact of the antiviral intervention. the roc curve (1-specificity versus sensitivity, not shown) for the logistic regression model specified in table 1 has an area under the curve of 0ae937, demonstrating that the model predicts the success of an antiviral intervention extremely well. for example, with a sensitivity of 95% we still have a specificity of approximately 80%. evaluation of the 2009 pandemic response has emphasised the need for early informed decision-making to implement proportionate disease control measures. our model identifies a low probability of successful epidemic mitigation using targeted antivirals alone (figure 1 and ref. 15 ), in distinction to results from models that fail to account for the diagnosis and delivery constraints inherent in any public health response. the decision support tool (table 1) highlights key epidemic characteristics that are predictive of a high likelihood of effective mitigation. the reproduction number was one of the earliest parameters estimated from early outbreak data during the 2009 h1n1 outbreak. 17, 18 our findings reinforce the importance of characterising epidemic severity as early and as accurately as possible, in order to inform a proportionate pandemic response. critically, a typically mild pandemic (low g), such as that experienced in 2009, is predictably difficult to contain using a targeted antiviral strategy due to the low proportion of infectious cases that present to health authorities. the relative infectiousness of treated individuals, e t , is strongly negatively correlated with successful mitigation, perhaps a surprising result given the model's underlying assumption (based on available epidemiological and human clinical trials data) that e t lies in the range [0ae8, 1]. that is, nais provided as treatment have a maximum impact of just a 20% reduction in infectiousness. however, our previous results 4 show a strong synergistic effect of treatment when overlayed on a contact prophylaxis strategy, explaining the observation here that e t is critical in determining likely success of an intervention. despite the limited impact of treatment at the individual-level, the model outcomes are highly sensitive to the value of the relative infectiousness of treated cases. it follows that determination of e t is important for predicting the population-level outcome of a control effort. a 'small' reduction (of the order approximately 10%) may be extremely valuable in terms of success of a public health control strategy, and so should not be discounted. using a mathematical model which takes into account some of the key logistic constraints that are inherent to healthcare responses, we have derived a logistic regression model for estimating the probability that an antiviral intervention based on liberal distribution of nais as treatment and prophylaxis could successfully mitigate an influenza epidemic. the model demonstrates an excellent degree of accuracy when applied to synthetic data. the choice of parameters for the regression model was restricted to those that were both highly correlated with the success of the intervention and hopefully feasible to measure during the early stages of an emerging epidemic. the model could therefore be a useful near real-time decision support tool for public health policy in the face of an influenza epidemic, although further validation on a range of synthetic data (and real-world data where available) is required. influenza to seasonal flu status to avoid overstretching the demands on healthcare services. a great deal of information has emerged as the result of the pandemic response exercises conducted by affected countries. however, uncertainties remain regarding the effectiveness of intervention measures, as well as the feasibility and the timing of their implementation. mathematical and computational models [2] [3] [4] have been used to project the outcomes of influenza outbreaks under various scenarios and epidemiological hypotheses. motivated by the events of 2009 and public health measures adopted by the taiwan cdc, we use a stochastic, individual-based simulation model 5 to study the spatio-temporal transmission characteristics of the h1n1 virus, so as to quantitatively assess the effects of early intervention strategies. our stochastic disease simulation model 5 builds upon a highly connected network of individuals interacting with each other via social contact groups. to represent the daily interactions of approximately 23 million people living in taiwan, we constructed a computer-generated mock population based on national demographic and employment statistics (to derive daily commute patterns) from the 2000 taiwan census (http://www.stat.gov.tw/). each individual is created with a set of attributes, including age, sex, residence, family structure, and social standing (employment status, etc.). based on their attributes and the time of day, each individual is assigned to miscellaneous contact groups, where the potential of interactions between any two individuals resulting in flu virus transmission occurs. such epidemiological properties are defined by empirically parameterized attributes such as basic reproduction number r 0 , transmission probability, contact probability and associated probability distributions outlining the disease's natural history. additionally, intervention measures are implemented as scheduled events that could alter control parameters during the course of a simulation run. the targeted basic reproduction number (r 0 ) in all our simulations is 1ae6, following the suggested range by who of 1ae2-1ae7. 6 as the latent ⁄ incubation and infectious periods for h1n1 have not yet been reliably ascertained, we adopt the natural history of the 1957 and 1968 pandemic influenza viruses. 2, 7 here, the latent period ranged from 1 to 3 days, with a median value of 1ae9 days. the infectious periods begin 1 day prior to symptom onset and can continue for 3-6 days, with a median value of 4ae1 days. twothirds of the infected individuals will develop clinical symptoms, and the asymptomatic cases will have half the infectious strength. the efficacy of antiviral drugs (oseltamivir) and vaccines are based on these studies. 8, 9 for the source region of the infected cases, we use the north american continent (canada, mexico and united states) with an estimated total population of 450 527 697 and an average 16 hours of flight time to taiwan. the average daily passenger number is 2489 based on the 2009 annual statistical report on tourism, tourism bureau, taiwan (http://admin.taiwan.net.tw/english/statistics/year.asp? relno=61). each simulation lasts 365 days and starts with a baseline simulation of r 0 % 1ae6 h1n1pdm outbreak at the source region. the outbreak was adjusted to approximate clinical attack rate (car) in the united states, april 2009-march 13, 2010. 10 we estimate the daily number of imported cases according to average daily passenger numbers and their probability of holding a disease status. we then apply airport exit ⁄ entry screening per corresponding success rates, by subtracting the number of identified symptomatic cases. we also consider latently infected passengers with inflight disease progression, by fitting a gamma distribution to the cumulative distribution of time to onset data with 16 hours average flight-time, as presented by pitman et al. 11 the daily imported cases are seeded according to the traveling patterns of foreign tourists and residents returning home. from the disease's natural history, we derive that roughly 50% of the infected travelers present no symptoms; the percentage increases if most symptomatic individuals elect not to travel in their condition, or are stopped by airport screening. we use the official epidemic data provided by the taiwan cdc to calibrate the simulation model and perform regression analysis on scenario parameters. this data is a close estimation of the weekly new clinical cases of h1n1pdm patients. it consists of weekly opd (outpatient department) icd-9 code 487 (influenza) tallies collected by the bureau of national health insurance, taiwanadjusted to exclude seasonal flu patients and to account for uninsured patients. we formulate our scenario settings according to 2009 events in taiwan, and establish settings to approximate the actual events. with domestic events and intervention schedules fixed in time, the start date determines the simulation outcomes and the data range for selected indicators, such as the mean car, the epidemic peak, and several significant dates for the incoming index case events. we plot the 2009 taiwan weekly h1n1 opd487 cases alongside the weekly new clinical cases from our simulation results in figure 1 . our simulations not only capture the epidemic trend, but also pick out the most likely date, may 20, for identifying the first symptomatic case at airport screening based on practical assumptions. we further analyze the effectiveness of various mitigation measures with february 6, 2009 as the empirical start date for h1n1pdm in north america. the simulation result confirms that by the time we identified the first symptomatic case at the border screening, infected cases had already made their way to the public. by our calculation, roughly four such cases had passed in each of our scenario settings, with the first case happening as early as 3 weeks before detection. figure 1 also highlights the importance of the timing for the implementation of mitigation measures; for example, a 6-day-delay of the identical intervention plan results in nearly an additional 1% of the population being infected. therefore, the rule of thumb for healthcare officials is to implement intervention measures as early as possible. in our study, we have ignored the possibility of inflight transmission and any false positive results by airport screening procedures. to assess the effectiveness of each mitigation strategy of interest and their combinations, we take the calibrated simulation model and perform 100 simulation realizations for groups of scenarios containing only those intended mitigation measures, and analyze the averaged results. for example, in the airport exit screening policy only scenario, the first imported symptomatic case can be delayed up to 2 months, and the epidemic peak can be delayed up to 13 days. as the data suggests, the exit screening policy alone has very little impact on car. combining various screening success rates for both exit and entry screening allows us to quantitatively assess their beneficial ramifications on the epidemic. for example, there is very little additional benefit between 100% and 80% suc-cess rates for entry screening policies when exit screening policies are adequate, as the enhanced border screening only delayed the epidemic peak by 1 day, and reduced car by <0ae01%. base on this result, the government should not attempt to exhaust all its resources in securing the border during a pandemic event, because the return of such a policy will be disappointing. instead, a response plan with a shifting focus on health resource allocation and the capacity of adjusting intervention strategies in line with the developing epidemic will be most effective. based on the same principle, we perform experiments with assorted scenarios, including relaxing entry screening policies after identifying the first imported symptomatic case, mass vaccination based on the actual vaccination schedule of h1n1pdm in taiwan, and altering the start dates of the vaccination schedule. our results show that with a reasonable reduction in the airport entry screening success rate, we conserve valuable healthcare resources, but loose a few days for the strategic planning and preparation of subsequent response measures. in other simulation scenarios, a national vaccination campaign has very little impact on the outcome, due to the late start of the vaccination schedule. we then explore the effect of a national vaccination campaign with various starting dates. the simulation results are illustrated in figure 2 , where the benefit of an early start date for mass vaccination is clearly demonstrated. considering a scenario with an 80% airport exit screening success rate, 80% airport entry screening success rate and 80% symptomatic case tracing success rate, the combined intervention strategy results in: a 2% reduction in car if the vaccination campaign starts in mid-november; 11% reduction if the campaign starts in mid-october; 26% reduction if the campaign starts in mid-september; and 37% reduction if the campaign starts in mid-august. in retrospect, the taiwanese government's response to h1n1pdm proved to be effective. first and foremost, it initiated enhanced border monitoring and on-board quarantine inspection as soon as the threat of a flu pandemic became clear. at the same time, the domestic preparations towards h1n1pdm were escalated, such as antiviral drug stockpiling and distribution, and vaccine acquisition. as the h1n1 cases increased worldwide, various revised plans were adopted and implemented; such as the shift from labor-extensive on-board quarantine inspection to the notifiable infectious disease reporting system and realtime outbreak and disease surveillance system in order to effectively track down symptomatic and exposed passengers, apply prophylaxis treatment and mandatory in-home quarantine. as a result, all h1n1pdm related statistics are well below the international average. in modern society, countries rely heavily on the global economy for their own prosperity. shutting down the border for any length of time is not only costly, but could have disastrous economic effects that linger long after the event is over. moreover, with nearly 50% of the infected passengers presenting no symptoms whatsoever, they are not detectable by any port authority's screening procedures, and the importation of the novel flu virus is therefore inevitable. many studies conclude that entry screening is unlikely to be effective in preventing or delaying the importation of influenza, and has negligible impact on the course of subsequent epidemic. however, these studies are based on the assumption that effective exit screening is in place. our study shows that as the exit screening success rate decreases, the sensitivity of the entry screening policy becomes more pronounced. with the same methodology, we can also study the effects of varying the length of flight time, or the disease's incubation time. lastly, the benefit of entry screening is even more crucial for a small island country such as taiwan, since all incoming traffic must go through the port authority where entry screening can be enforced. in england and wales, three waves of the pandemic struck in summer, autumn, and winter seasons of 1918-1919. although the proportion of people reporting symptoms was often greater in the first wave, 1-3 a puzzling feature was the much higher mortality in the second wave, in which 0.27% of the population died, compared with 0.03% in the out-of-season first wave and 0.10% in the third wave. 4 an obvious hypothesis to explain the changes in mortality from wave to wave would be that the 1918 virus mutated to higher virulence after the (lower mortality) first wave. although pandemic virus reconstituted from the high mortality waves has proven to have high virulence in animals, 5 it has not been possible to recover virus from the first wave in 1918 for comparative purposes. indeed it is questionable whether virulence mutation(s) occurring between wave 1 and wave 2 could have spread to so many different populations in the time-frames observed. furthermore, in all three pandemic waves, there was the same agedistribution of mortality, with more deaths occurring amongst younger adults than older adults. [1] [2] [3] this 'pandemic signature', arguably due to immune protection of older adults who were exposed to a similar virus in the years before 1890, 6, 7 suggests that the 1918-1919 viruses were at least immunologically similar in all three waves. a second hypothesis would be that the higher case fatality in the later waves was due to higher rates of complicating bacterial pneumonia, 8 to increased transmission of influenza virus in the cooler months of the year, or to other seasonal effects. 9 we have considered a third (immunological) hypothesis to explain the greatly increased mortality in waves 2 and 3. the underlying idea is that the mortality rate in the first wave was lower than in later waves because most persons were protected by prior immunity in the first wave, and that the mortality was higher in later waves because of waning of that short-lived immunity. this hypothesis builds on our earlier modelling papers suggesting that even before the first wave in 1918, military, 10 school, and urban 11 populations in england and wales apparently had (short-lived) immune protection, presumably induced by recent prior exposure to seasonal influenza. [10] [11] [12] we suggest that this short-lived strain-transcending protection was in addition to the longer-lasting immunity, presumably induced by exposures to a similar virus circulating prior to 1890, that arguably reduced pandemic mortality for older adults in 1918-9. 6,7 cumulative mortality rates attributed to pandemic influenza were available for each of the three waves in 1918-1919 for 330 populations in england and wales. 13 we have built immunological models to potentially explain the variation in mortality rates across waves and populations. to show proof of principle, we have fitted these models to mortality data from a randomly selected sub-set of twenty populations. our key assumption was that the risk of a fatal infection would be limited to persons with inadequate immunity who were being exposed to the pandemic virus for the first time. persons who were exposed and who survived an earlier wave were assumed to be protected against death in a later wave. model a and assumptions (see figure 1) before the first wave, we assumed that people could be fully susceptible (s 0 ), or partially protected (q 0 ), or fully protected (p) by prior immunity which was not necessarily specific for the new virus. we assumed that exposure to the new pandemic virus would be fatal (m) in a proportion h of fully susceptible persons who were actually exposed (e) in the relevant wave. for those surviving that first exposure, it was assumed that they would be permanently protected against death in later waves by an immune assumed that viral exposure and multiplication would induce an immune response specific for the pandemic virus that would protect them against death in that wave and in subsequent waves. in contrast, for persons with strong prior immune protection, p, the virus would not be able to multiply to induce pandemic-specific immune protection. between waves, it is assumed that due to the waning of non-specific prior immunity, persons in the p state can move to the q state, and persons in the q state can move to an s state before the next wave. the proportion (e) of susceptible persons exposed to productive infection in each population was estimated by applying the following version of the final size equation 11 to the proportion susceptible (s & q) in each wave, for each population: note: in both figures 1 and 2 , we have omitted the flows out of the q and e states that removed persons from the risk of death. parameters: s 0 = proportion fully susceptible to infection and death before wave 1; q 0 = proportion susceptible to immunising infection, but not to death from exposure in wave 1; p 0 = proportion temporarily protected against both immunising infection and death from exposure in wave 1; n 0 = proportion even more protected against both immunising infection and death from exposure in wave 1 (model b only); r 0 = basic reproduction number (the average number of secondary cases for each primary case) in a fully susceptible population; f = proportion moving from q to s between waves; g = proportion moving from p to q between waves; d = proportion moving from n to p between waves (model b); h = proportion of e that actually move to m and die. model a could provide a very good fit for the summer, autumn, and winter waves of the 1918-1919 pandemic (results not shown). however, because of the replenishment of the pool of susceptible persons over time, model a also predicted a fourth wave of influenza in the spring season of 1919. as no such wave was seen, and as we could not find parameters values for model a that did not predict a fourth wave, we must regard model a as inadequate. model b was similar to model a, but with an additional stage of prior immunity (n), which could wane to p. model b allowed us to not only fit the three observed waves, but also to fit the imputed data (zero cases) corresponding to the absent fourth wave. following earlier work, 10,11 we used a bayesian approach with markov chain monte carlo (mcmc) procedures to estimate model parameters, and we used hyper-parameters to allow for parameter variation between populations. 11 the initial conditions were specified by the parameters: p 0 , q 0 , s 0 and n 0 . from these and the other parameters, it was possible to simulate the behaviour of model a over three waves, and of model b over four waves, and to estimate the expected numbers dying in each wave in each population. we calculated the log likelihood of the observed numbers of deaths given the parameter estimates, and we used mcmc simulation to generate the posterior distributions of parameters. although we obtained an excellent fit between observed and expected numbers of deaths in each of the three waves for the 20 populations for model a, we could not find parameter values for model a that would fit the three observed waves without giving rise to a fourth wave in the spring of 1919. accordingly, in the modified model b, we allowed for an additional stage of prior immunity (figure 2) , and we fitted the model to the same data, plus imputed data corresponding to 'the absent fourth wave'. we obtained a very good fit to the three observed waves and the absent fourth wave in each population. the 95% credibility intervals for parameter estimates, derived from the posterior distributions of the hyper-parameters were: h = 0.05-0.08, s 0 = 0.015-0.032, q 0 = 0.40-0.47; n 0 = 0.2 (fixed); p 0 = 1 ) s 0 ) q 0 ) n 0 ; f = 0.33-0.44; g = 0.87-0.94; d = 0.94-0.97 and r 0 = 2.6-3.6. this analysis had allowed all parameters to vary from population to population under the constraints of the hyper-parameters. however, several of the biologically determined parameters might be expected to be more constant from population to population, whereas those dependent on mixing history and other social characteristics which vary more widely from population to population. to test this possibility, we fixed the mean values for the more biological parameters (f = 0.385; d = 0.955; g = 0.905) and estimated the 95% credibility intervals for the others as: h = 0.05-0.10; s 0 = 0.012-0.032, q 0 = 0.36-0.44; and as before n 0 = 0.2 (fixed); in a subsequent paper we will be able to provide more details of the method, the robustness of the assumptions, and the results from fitting to many more populations. this short report suggests that the observed patterns of mortality in england and wales over the three waves of the 1918-1919 influenza pandemic 4,13 can be explained by an immunological model. in particular, the lower mortality in wave one can be explained by the assumption of protective immunity antedating the first wave, arguably induced by prior exposure to seasonal influenza. 10, 11 the much greater mortality in wave two can be explained by the waning, between wave one and wave two, of that short-lived and less-specific immune protection. the somewhat lesser mortality in wave three and the 'absent fourth wave' can be explained in terms of the progressive acquisition of immunity specific to the pandemic virus. the credibility estimates for parameters are of potential interest. for example, r 0 estimates of 3.1-4.5 across different populations are consistent with our earlier findings. 10, 11 if all persons had been susceptible, such r 0 values imply that the virus would have infected most people in all populations. however, even in the first wave, the proportion susceptible, s 0 + q 0 , was <50% in all populations, so that a considerable number of persons escaped productive infection in that wave; as their immunity waned, they became susceptible to infection in the later waves. it is likely that the variation in r 0 between populations is due to different rates of population mixing. estimates for h indicate that between 5% and 10% of infections in the most susceptible persons were fatal; the higher values of h could reflect higher rates of secondary bacterial infection in the most socially disadvantaged and overcrowded populations. 4 although we have shown the plausibility of an immunological explanation for wave to wave changes in pandemic mortality, we cannot assume that our particular model is even approximately correct. nor can we exclude the possibility that the higher mortality in the later pandemic waves in 1918-1919 was because of genetic change in the virus in later waves, or because of changing rates of secondary bacterial infection 8 or seasonal effects. 9 nevertheless, there is growing evidence that the population spread of pandemic influenza, whether in 1918-1919 10, 11 or in 2009, 12, 14 can be constrained by significant prior immunity, even for viruses that are ostensibly novel. previous reports, reviewed in ref. [10, 11] , support the idea of strain-transcending immune protection, which can wane over periods of a few months. this form of protection, probably induced by recent exposure to seasonal influenza, may not be mediated by hi or neutralizing antibody. 11 in contrast, strain-specific immunity, most often mediated by hi or neutralizing antibodies can be so long-lasting that after several decades it will still provide significant protection against any closely-related virus that re-appears in the population. 14 it has not escaped our notice that although attack-rates in the h1n1 2009 pandemic were low in many countries, with generally mild symptoms, the virus did cause lifethreatening illness in a small proportion of younger affected persons. 15 it seems likely that those who were most severely affected in 2009 were doubly unlucky: they had missed out on seasonal influenza infection or vaccination in the preceding season(s), and they were born too late to have been protected by the closely-related viruses that are thought to have circulated before 1950. 14 during the early phases of the 2009 influenza pandemic in italy, real-time modeling analysis were conducted in order to estimate the impact of the pandemic. in order to evaluate the results obtained by the model we compared simulated epidemics to the estimated number of influenza-like illness (ili) collected by the italian sentinel surveillance system (influnet), showing a good agreement with the timing of the observed epidemic. by assuming in the model mitigation measures implemented in italy, the peak was expected on week 44 (95% ci: 44, 45). results were consistent with the influnet data showing that the peak in italy was reached in week 46. these predictions have proved to be a valuable support for public health policy makers for planning interventions for mitigating the spread of the pandemic. mathematical models have recently become a useful tool to analyse disease dynamics of pandemic influenza virus can-didates. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] as of april 2009, after the pandemic threat emerged worldwide, 12 it was crucial for policy makers to have early predictions on the possible spread of the 2009 pandemic influenza virus in order to support, with quantitative insight into epidemic, policy decisions. thus, after the first pandemic alert was announced by the world health organization (who) in late april 2009, a national crisis management committee headed by the minister of health was established in italy in order to provide weekly advice to the italian ministry of health. real-time analyses using an individual based model were undertaken. the transmission model was previously used for evaluating the effectiveness of the control measures adopted in the national pandemic preparedness plan 3 and for assessing the age-prioritized distribution of antiviral doses during an influenza pandemic. 2 to parameterize the transmission model, we used data derived from the national surveillance system until 17 june 2009 and estimates of key epidemiological parameters as available at that time. in order to provide a preliminary assessment of the model predictions performed during the early stages of the epidemic, we compare model predictions with surveillance data of influenza-like illness (ili) available since august 2009. after the first pandemic alert was announced by the who in late april 2009, a national active surveillance system for the 2009 pandemic influenza was set up from 28 april to july 2009. 13 however, over the period from april to october 2009, surveillance systems, laboratory testing, and diagnostic strategies have varied considerably in italy. since end of july 2009, following who recommendations, 14 the focus of surveillance activities has changed in reporting requirements, as active case-finding became unsustainable and unnecessary. for this reason, the ministry of health (ministry of health, available in italian at the website: http://www.normativasanitaria.it) requested regional health authorities to report the weekly aggregated ili cases according to a new case definition (sudden onset of acute respiratory symptoms and fever >38°c plus at least one of the following systemic symptoms: headache, malaise, chills, sweats, fatigue; plus at least one of the following respiratory symptoms: cough, sore throat, nasal obstruction). by october 2009, following the increasing number of cases, the sentinel influenza surveillance system (influ-net available at: http://www.flu.iss.it) became the official surveillance system for ili cases in italy (ministry of health, available in italian at the website: http://www. normativasanitaria.it). since 1999, influnet is routinely based on a nation-wide, voluntary sentinel network of sentinel community based physicians in the 21 regions and autonomous provinces of the country. incidence rates are, therefore, not based on consultations, but on the served population of each reporting physician each week. influ-net usually consists of an average of 830 (range 648-902) general practitioners (including physicians and pediatricians) per year, covering about 1ae5-2% of the general population, (representative for age, geographic distribution, and urbanization level) reporting ili cases (according with a specific case definition). italian influnet surveillance system is part of the european influenza surveillance scheme (eiss). a stochastic, spatially explicit, individual-based simulation model 3 was used. individuals are explicitly represented and can transmit the infection to household members, to school ⁄ work colleagues, and in the general population (where the force of infection is assumed to depend explicitly on the geographic distance). the national transmission model was coupled with a global homogeneous mixing susceptible-exposed-infectious-removed (seir) model accounting for the worldwide epidemic, which is used for determining the number of cases imported over time. regarding the epidemiological assumptions (e.g., length and shape of the infectivity period, which lead to an effective generation time of 3ae2 days), this study is consistent with refs [3, 4, 11, 15] , but for the proportion of symptomatic individuals, which is assumed to be 66ae7%. 16 the basic reproductive number of the national transmission model was set to 1ae4, according to the early estimates as obtained during the initial phase of the epidemic in mexico in a community setting. 17, 18 we initialized our simulations through the global homogeneous mixing model in such a way that 158 imported cases were generated until 7 june 2009. this gives a reliable way for fixing the time in the simulations and thus determining the timing of school closure and vaccination in the simulations. the model accounts for school closure for both summer and christmas holidays: we assumed that in these periods contacts among students decrease, while contacts in the general community increase, as in ref. [19] . we also considered scenarios accounting for partial immunity in the population. 20 in order to investigate the effects of recommendations of the ministry of health (confirmed cases coming from affected areas were isolated for 7-10 days, either in hospital or at home) established in the early phase of the pandemic (april-july 2009), we assumed that a fraction of the imported symptomatic cases were isolated on the first day after the symptoms onset. this recommendation was in place until 27 july 2009. we also assumed, according to the italian school calendar, that schools were closed from 10 june 2009 to 10 september 2009 for the summer holidays, and from 22 december 2009 to 6 january 2010 for christmas holidays. the effects of prolonged school closure were also investigated. when considering vaccination, we assumed 6 weeks for the logistical distribution of doses of pandemic vaccine. since at the time of simulation specific recommendations regarding the administration of a single dose of pandemic vaccine from ema were not available yet, we considered the administration of 2 vaccine doses 1 month apart). the pandemic vaccine was considered effective after the administration of the second dose with a vaccine efficacy of 70%. we assumed the vaccine to be administered by priority, vaccinating first the target population accounting for essential services workers (including health care workers and blood donors), pregnant women at the second or third trimester, and at risk patients (with chronic underlying conditions) younger than 65 years old. the vaccination coverage was assumed 90%. regarding antiviral treatment and prophylaxis, recommendations of the ministry of health in the initial phase of the epidemic were to administer antivirals to all confirmed cases and to their close contacts. we assumed that the surveillance system would be able to detect 90% of symptomatic cases. after 8 july 2009, recommendations changed and antiviral treatment was considered only for cases with severe complications and in case of local clusters. since it was difficult to establish the proportion of treated cases, we considered different scenarios: antiviral treatment from 0% to 30% of the symptomatic cases. consistently with ref. [3] , both treatment and prophylaxis were assumed to start 1 day after the clinical onset of symptoms in the index case. treatment was assumed to reduce infectiousness by 70%, whereas antiviral prophylaxis was assumed to reduce susceptibility to infection by 30%, infectiousness by 70%, and the occurrence of symptomatic disease by 60%. as of 26 july 2009, approximately 1238 confirmed cases have been reported to the italian surveillance system for pandemic influenza. during july, the sudden increase of ili confirmed cases suggests for sustained autochthonous transmission in italy. by analyzing the number of ili cases reported to the surveillance during the weeks from 39 to 44, we found that the exponential growth rate was 0ae832 ⁄ week and thus we estimated the national reproductive number to be r 0 = 1ae38. this estimate of the basic reproductive number supports the choice of the value adopted in the model simulations (r 0 = 1ae4). in the absence of intervention measures, the predicted cumulative attack rate was 30ae6% (95% ci: 30ae6, 30ae7), and the peak was expected on week 42 (95% ci: 41, 42) with a peak day incidence of 0ae35% (95% ci: 0ae329%, 0ae37%). by assuming case isolation, antiviral treatment, and prophylaxis to 90% of symptomatic cases until 8 july 2009, the peak was expected on week 43 (95% ci: 43, 44). when considering 33ae3% of natural immunity in the population aged more than 59 years, the peak was expected 1 week later than in the previous scenario, i.e., on week 44 (95% ci: 44, 45). to validate the model, we compared model predictions (which are based only on the available information on the early phases of the epidemic) with ili data (figure 1 ). based on model predictions, we estimated the underreporting factor of influnet ranging from 3ae3 to 3ae7, considering different scenarios. by aligning the simulations with the ili data adjusted by the underreporting factor, we can observe that almost all the points in the increasing phase of the epidemic lie within the 95% ci of the model results (both considering or not natural immunity). the decay phase of the simulated epidemics shows a small delay with respect to the ili data. when introducing single and combined mitigation measures, such as case isolation, antiviral treatment, prophylaxis, and vaccination in the model, results showed that even a low proportion of symptomatic cases treated with antiviral drugs could have led to a relevant reduction in the epidemic size (table 1) . we simulated the planned italian vaccination strategy (begun on 15 october 2009), obtaining a limited but not negligible reduction in the attack rate with respect to the scenarios accounting only for antiviral treatment. moreover, the effect of vaccination would be higher if coupled with antiviral treatment; vaccination would have no effect on delaying the peak incidence. model predictions produced in italy during the early phase of the 2009 pandemic influenza are in excellent agreement with italian surveillance data on the beginning of the epidemic (when case isolation, antiviral treatment of index cases, and antiviral prophylaxis to close contacts were implemented by the italian regional public health authorities) and are basically consistent with the influnet data during the course of the epidemic. the model has been useful for predicting the timing of the epidemic, while it has overestimated the impact of the 2009 influenza pandemic for adult and elderly individuals. however, the disalignment is probably due to the model parameterization. based on literature values, 4,5 we assumed a similar fraction of cases in the different social contexts considered in the model (namely 1 ⁄ 3 in households, 1 ⁄ 3 in schools ⁄ workplaces, and 1 ⁄ 3 in the general community), since analysis on the relative transmissibility of the virus was not carried out for any country yet. we were also able to estimate an underreporting factor for the influnet data in the range 3ae3-3ae7. if we focus our attention on the reporting factor computed by considering the total number of cases (instead of symptomatic cases), the resulting value lies in the range 18-20ae2%, which is in excellent agreement with the range estimated in ref. [21] on previous a ⁄ h1n1 influenza seasons, namely 16ae2%-21ae6%. moreover, based on our results showing that vaccinating 40% of the italian population was more than adequate to mitigate the pandemic, the ministry of health decided to stockpile a limited number of vaccines. we have also shown that starting the vaccination program in october (or later) could have had only a limited effect on reducing the impact of the epidemic, although it may have been useful to prevent a possible second wave and to protect essential workers and at-risk patients. finally, our results have shown that antiviral treatment would have been the most efficient strategy to reduce the impact of the influenza pandemic, even with a limited antiviral stockpile. a population-wide passive immunotherapy program in this paper, we assume that convalescent plasma (cp) is efficacious in treating severe cases of pandemic influenza. under this premise, we test the hypothesis that a population-wide passive immunotherapy program that collects plasma from a small percentage of convalescent individuals can harvest sufficient cp to treat a substantial percentage of severe cases during the first wave of the pandemic. the proposed program involves recruiting adults (individuals age 20-55 years) to donate blood if they have experienced influenza-like symptoms more than 2 weeks ago (to account for the time needed for neutralizing antibodies to build up). the blood samples would be screened for infectious diseases (including hiv, hbv, hcv, htlv, and syphilis, etc., as in routine blood donation screening) and neutralizing antibodies against the pandemic virus. donors whose blood samples are free of known infectious agents and contain a sufficiently high titer of neutralizing antibodies would then be invited to donate plasma by plasmapheresis or routine whole blood donation. qualified donors with higher titers may be given higher priority for plasma donation. in this paper, we use the demographic and logistical parameters of hong kong as a case study. see figure 1 for a schematic of the proposed passive immunotherapy program. we examine the following questions regarding the logistical feasibility and potential benefits of the proposed passive immunotherapy program: (i) what percentage of convalescent individuals (donor percentage) is needed in order for the program to significantly reduce pandemic mortality? (ii) how many severe cases can be offered passive immunotherapy? (iii) what are the ratelimiting factors in the supply of passive immunotherapy? (iv) what are the epidemiologic and logistical factors that determine the demand-supply balance of passive immunotherapy? a more detailed presentation of our results is now available in ref. [ 5 ] . transmission and natural history model for pandemic influenza we use an age-structured disease transmission model to simulate the spread of pandemic influenza. the natural history model is similar to that used by basta et al. 6, 7 the most important parameter in characterizing the growth of an epidemic is the basic reproductive number r 0 , which is defined as the average number of secondary cases generated by a typically infectious individual in a completely susceptible population. we consider values of r 0 between 1ae2 and 2, which is consistent with recent estimates. 6, [8] [9] [10] logistical model for the passive immunotherapy program we assume that q d (%) of 20 to 55 year-old individuals who have recovered from symptomatic infections of pandemic influenza donate their blood for screening t r = 14 days after cessation of symptoms. follow-ups of convalescent individuals infected with h1n1pdm in an ongoing clinical trial of passive immunotherapy suggested that neutralizing antibodies level reaches maximal level around 14-21 days after recovery and stays at that level for months after. 11 we assume that q s (%) of these donors are qualified for plasma donation of which q r (%) are recurrent donors who return to donate plasma every t w = 14 days. screening involves both detection of infectious agents and neutralizing antibodies against the pandemic virus. the latter is the rate-limiting step because neutralization tests of pandemic viruses can only be done in a bsl3 setting. we assume that five bsl3-trained technicians are available to test the blood specimens, each running 150 viral neutralization tests in 3 days. therefore, the capacity and turnaround time of blood screening are u s = 750 and t s = 3 days, respectively. hong kong currently has nine plasmapheresis machines which allow a maximal throughput of 162 plasma donations per day (assuming 12-hour daily operation with each donation taking 40 minutes). therefore, the capacity and turnaround time of plasmapheresis are u p = 9 and t p = 1 ⁄ 18 days, respectively. collected cp are ready for use in transfusion after final quality check, which takes t q = 2 days. we assume that r t plasma donations are required to treat one severe case on average. the expert panel of the abovementioned study of passive immunotherapy for h1n1pdm in hong kong suggested that r t < 10. we assume that p h (%) of symptomatic cases will be severe cases for whom passive immunotherapy is suitable. although p h will be smaller than the case-hospitalization rate (passive immunotherapy may not be suitable for some hospitalized cases), we assume that the two have similar ranges and consider p h ranging from 0ae1% to 1%. because each severe case requires r t plasma donations on average, demand for cp is simply r t p h times the number of symptomatic cases. therefore, r t p h can be regarded as a single parameter, which we refer to as the lumped demand parameter. we define the outcome as the percentage of severe cases that can be offered passive immunotherapy by the proposed program during the first wave of the local epidemic. we refer to this outcome as treatment coverage and denote it by q. we consider the base case scenarios assuming q r = 20% and q s = 80%. in general, the treatment coverage q increases sharply as the basic reproductive number r 0 and the lumped demand parameter r t p h decrease (figure 2a ). in particular, when r 0 is large and r t p h is small, q is very sensitive to r t p h , but insensitive to r 0 . similarly, when r 0 and r t p h are small, q is very sensitive to both. with a donor percentage of q d = 15%, the proposed program can supply passive immunotherapy to more than 82% of severe cases (q > 82%) if r 0 < 1ae4 and r t p h < 1ae5%, but <35% if r 0 > 1ae8 and r t p h > 1ae5%. in general, the treatment coverage q increases sharply as the donor percentage q d rises from 0%, but with rapidly decreasing marginal increase ( figure 2b ). when r 0 < 1ae4 and r t p h < 1ae5%, q > 67% even if q d is as low as 5%, which is comparable to the current average blood donation rate of 38ae1 donations per 1000 population in developed countries. 12 when q d is >15%, q becomes largely insensitive to further increase in q d in most scenarios. the treatment coverage q for q d = 15% is more than 81% that for q d = 50% across all values of r 0 and r t p h considered in the base case. therefore, increasing the donor percentage q d beyond 15% has a relatively small impact on cp supply. this is because increasing q d can boost supply only when plasmapheresis is not yet the supply bottleneck. for the same reason, once the donor percentage q d has reached 15%, the treatment coverage q is insensitive to further increase in q d even when the plasmapheresis and screening capacity are doubled ( figure 2b , lower panel). we conduct an extensive multivariate sensitivity analysis to test the robustness of our base case observations against uncertainties in parameter values. we generate 15 000 epidemic scenarios by randomly selecting parameter values from their plausible ranges using latin-hypercube sampling. although there are numerous model parameters, the treatment coverage q is mainly determined by three lumped parameters: (i) r t p h , which indicates the magnitude of demand; (ii) q s q d , which indicates the magnitude of supply; (iii) the initial growth rate of the epidemic r (results not shown). while the dependence of q on r t p h and q s q d is readily comprehensible, it is not obvious a priori that q depends on the natural history and transmission dynamics of the disease via only the initial epidemic growth rate. when the plasmapheresis and screening capacity are very large, the supply-demand dynamics is further simplified: the treatment coverage q depends on lumped demand parameter r t p h and the lumped supply parameter q s q d only via their ratio. finally, q becomes insensitive to q s q d when the latter increases beyond 15-20%, which is consistent with our base case observations. our results suggest that with plasmapheresis capacity similar to that in hong kong, the proposed passive immunotherapy program can supply cp transfusion to treat 67-82% of severe cases in a moderate pandemic (basic reproductive number r 0 < 1ae4, lumped demand parameter r t p h < 1ae5%) when the donor percentage is 5-15%. increasing the donor percentage beyond 15% has little additional benefit because cp supply is constrained by the capacity of plasmapheresis during most stages of the epidemic. increasing plasmapheresis capacity could significantly boost cp supply, especially when there is a substantial pool of recurrent donors to alleviate the dependence of cp supply on donor percentage. in an ongoing clinical trial of passive immunotherapy for h1n1pdm virus infection in hong kong, 20% of convalescent individuals agreed to donate their plasma for the study. therefore, the donor percentage required by the proposed passive immunotherapy program (5-15%) is likely to be feasible. in view of the logistical feasibility of such program, we recommend that further clinical studies are conducted to evaluate the safety and efficacy of passive immunotherapy as a treatment for severe cases of pandemic influenza virus infection. our study is based on the premise that cp will be efficacious in reducing morbidity and mortality associated with pandemic influenza. in theory, the polyclonal nature of neutralizing antibodies in cp would lower the probability of an escape mutant emerging in treated patients. further, besides providing neutralizing antibodies against the pandemic virus, cp also might carry antibodies to other bacterial pathogens, which might decrease the severity of coexisting bacterial infections. 4 as such, cp not only might reduce the case fatality rate but might also increase the recovery rate and shorten duration of hospitalization of severe cases. the proposed passive immunotherapy program can thus significantly reduce the burden on the healthcare system, especially the intensive care unit, which will likely be stressed, if not overloaded, at the peak of an influenza pandemic wave, hence benefiting the general public and not only those receiving passive immunotherapy. although the hypothesized efficacy of cp has yet to be proven in clinical trials, our modeling results show that a public health system similar to that in hong kong has the capacity to support a population-wide passive immunotherapy program that can supply cp treatment to a substantial percentage of the severe cases in a moderately severe pandemic. we estimate that compared to other developed countries, hong kong has a relatively low plasmapheresis capacity. our conclusions regarding donor percentage needed and rate-limiting factors remain valid for plasmapheresis capacity ranging from 50% to 400% of what we have assumed in the base case (results not shown). our conclusions are robust against uncertainties in the natural history and transmission dynamics of pandemic influenza. our sensitivity analysis shows that the outcome depends on these epidemiological characteristics only via the initial growth rate of the epidemic. as such, our results are applicable not only to pandemic influenza, but also to other emerging infectious diseases for which the time-scales of disease transmission and antibody response are similar to that for influenza virus. the three determinants of treatment coverage (the initial epidemic growth rate, the lumped demand parameter r t p h , and the lumped supply parameter q d q s ) are all readily measurable in real-time during an epidemic. therefore, our methods and results can be used as a general reference for estimating the treatment coverage of the proposed passive immunotherapy program for a given plasmapheresis capacity. background highly pathogenic h5n1 virus continues to pose a serious threat to human health and appears to have the capacity to cause severe disease in previously healthy young children and adults. at present, antiviral therapy by oseltamivir remains the mainstay for managing h5n1 patients. while early treatment improves survival, approximately 50% of patients treated within 4 days of illness still succumb to the disease. in addition to the role of viral replication, there is good evidence that the host proinflammatory responses contributes to h5n1 pathogenesis. this suggests that both antiviral and immune-modulatory drugs may have a role in therapy. we previously demonstrated that cyclooxygenase 2 (cox-2) plays a regulatory role in h5n1 hyperinduced pro-inflammatory responses, and its inhibitor has potent effects at modulating this host response. now we demonstrate that, in addition to its immune-modulatory effect, a selective cox-2 inhibitor, ns-398 has a direct antiviral effect against h5n1 infection. materials and methods human primary monocytederived macrophages or alveolar epithelial cells (a549) were pre-treated with ns-398 or drug-vehicle for 1 hour before h5n1 virus infection. h5n1 viruses at multipicity of infection (moi) of 2 was used to infect the cells. following virus adsorption for 30 mins, the virus inoculum was removed, and the cells were washed and incubated in corresponding medium with ns-398 or drug-vehicle as controls for 3, 6, 24, 48, and 72 hours post-infection. cells were harvested for rna isolation at 6 hours post-infection to study viral matrix (m) gene expression. supernatants were collected for 50% tissue culture infection dose (tcid 50 ) assay to determine the virus titers at 3, 24, 48, and 72 hours after h5n1 infection. results ns-398 was found to suppress virus gene transcription and infectious virus yield in h5n1-infected human cells. conclusion we demonstrate that a selective cox-2 inhibitor, ns-398, shows an inhibitory effect on h5n1 viral replication in addition to its immune-modulatory effect that could counter the detrimental effects of excessive proinflammatory cytokine production. the findings suggest that selective cox-2 inhibitors may be a therapeutic target for treating h5n1 disease in combination with appropriate antiviral therapy. the emergence and spread of the highly pathogenic avain influenza viruses (h5n1) in poultry and wild birds with repeated zoonotic transmission to humans has raised pandemic concern. at the time of writing, 507 human cases have been reported with 302 fatalities, an overall case fatality rate of around 60% (cumulative number of confirmed human cases of avian influenza a ⁄ (h5n1) reported to world health organization updated to 18 october 2010). our previous data demonstrated that cox-2 was markedly up-regulated in h5n1-infected primary human macrophages, and that it played a regulatory role in the h5n1hyperinduced host pro-inflammatory responses. 1 such cytokine dysregulation is proposed to be a major contributor to the pathogenesis of h5n1 disease in humans. 2 with the use of selective cox-2 inhibitors, we found that the h5n1-hyperinduced cytokine response was significantly suppressed by the drug in a dose-dependent manner. 1 selective cox-2 inhibitor is a form of a non-steroidal anti-inflammatory drug that selectively targets cox-2, and it is an inducible enzyme responsible for inflammatory process and immune response. here, we report a novel finding of a direct antiviral effect of a selective cox-2 inhibitor, ns-398, against h5n1 infection in human primary macrophages and alveolar epithelial cells. taken together with our previous findings that suggest an immuno-modulatory effect that can modulate virus driven cytokine dysregula-tion, these findings highlight a role for cox-2 and its downstream signaling as potential novel targets for adjunctive therapy of severe viral pneumonia, such as that caused by h5n1. such therapy may be combined with conventional antiviral drugs. the h5n1 virus used was a ⁄ vietnam ⁄ 3212 ⁄ 04 (3212 ⁄ 04) (h5n1), a virus from a patient with h5n1 disease in vietnam during 2004. the viruses were grown and titrated in madin-darby canine kidney cells cells as described elsewhere. 3 virus infectivity was expressed as tcid 50 . all experiments were performed in a biosafety level 3 facility. monocyte-derived macrophages: peripheral-blood leucocytes were separated from buffy coats of healthy blood donors (provided by the hong kong red cross blood transfusion service) by centrifugation on a ficoll-paque density gradient (pharmacia biotech) and purified by adherence as reported previously. 3 the research protocol was approved by the ethics committee of the university of hong kong. macrophages were seeded onto tissue culture plates in rpmi 1640 medium supplemented with 5% heat-inactivated autologous plasma. the cells were allowed to differentiate for 14 days in vitro before use in the infectious experiments. alveolar epithelial cells: a549 cells were obtained from atcc and maintained in culture using dulbecco's modified eagle medium supplemented with 10% fetal calf serum, 0.6 mg ⁄ l penicillin, and 60 mg ⁄ l streptomycin. differentiated macrophages or a549 cells were pre-treated with a selective cox-2 inhibitor, ns-398 (cayman), at concentrations as indicated or drug-vehicle for 1 hour before infection. cells were infected with h5n1 viruses at moi of 2. following virus adsorption for 30 min, the virus inoculum was removed, the cells were washed and incubated in corresponding medium with ns-398 or drug-vehicle as controls throughout the experiments. cells were harvested for rna isolation at 6 hours post-infection to study viral m gene expression. supernatants were collected for tcid 50 assay to determine the virus titers at 3, 24, 48, and 72 hours after h5n1 infection. total rna was isolated using the rneasy mini kit (qiagen) according to the manufacturer's instructions. the cdna was synthesized from mrna with poly(dt) primers and superscript iii reverse transcriptase (invitrogen). transcript expression was monitored by real-time pcr using power sybr ò green pcr master mix kit (applied biosystems) with specific primers. the fluorescence signals were measured using the 7500 real-time pcr system (applied biosystems). the specificity of the sybr ò green pcr signal was confirmed by melting curve analysis. the threshold cycle (ct) was defined as the fractional cycle number at which the fluorescence reached 10 times the standard deviation of the base-line (from cycle 2 to 10). the ratio change in target gene relative to the b-actin control gene was determined by the 2 )ddct method as described elsewhere. 4 ns-398 reduced the viral m gene expression in h5n1infected human macrophages in a dose-dependent manner ( figure 1) . similarly, production of infectious virus yield in h5n1 infected macrophages was found to be suppressed in the presence of ns-398 at 100 lm compared to vehicletreated cells (figure 2a) . a comparable effect of ns-398 was observed in h5n1-infected human alveolar epithelial cells ( figure 2b ). we have previously demonstrated that cox-2 expression was dramatically upregulated following h5n1 infection in human macrophages in vitro and in epithelial cells of lung tissue samples obtained from autopsy of patients who died of h5n1 disease. 1 this suggests that cox-2 may be an important host factor involved in h5n1 pathogenesis and also provide a possible explanation on why h5n1 virus replication is susceptible to a selective cox-2 inhibitor. cox-2 was previously reported to play an important role in the pathogenesis of other influenza a viruses. 5 an in vivo study has highlighted the importance of cox-2 in h3n2infected mice. findings showed that infection induced less severe illness and reduced mortality in cox-2 knock-out mice than in wild-type mice. on the other hand, cox-1 knock-out mice had enhanced inflammation and earlier appearance of proinflammatory cytokines in the bal fluid, whereas the inflammatory and cytokine responses were dampened in cox-2 knock-out mice. these data suggests that cox-1 and cox-2 may lead to opposite totally contrasting effects on influenza h3n2 infected mice. cox-1 deficiency is detrimental, whereas cox-2 deficiency is beneficial to the host during influenza viral infection. therefore in the present study, instead of blocking cox enzymes in general as reported by others, 5 we have chosen ns-398 that selectively block cox-2 but preserve cox-1 activity and showed that this drug significantly reduced h5n1 virus replication in a dose-dependent manner. taken together with our previous report suggesting its immuno-modulatory effects, 1 we believe that selective cox-2 inhibitors and cox-2 signaling pathways deserve investigation as a promising approach for targeting therapy in h5n1 diseases. however, a few reports have suggested the importance of cox-2 in the late stage of inflammation for the resulution of inflammation, [6] [7] [8] and this raises concern whether inhibition of cox-2 may be harmful in treating diseases related to dysregulation of host inflammatory response such as acute lung injury, 9 which is a leading cause of death in h5n1 patients. we previously looked at the autopsy samples of lung tissues from h5n1 patients and found that cox-2 expression was markedly up-regulated compared with that from persons who died of non-respiratory causes. 1 moreover, data also demonstrated that pro-inflammatory cytokines, such as tnf-a, was markedly elevated in the h5n1 infected lung autopsies. 10 taken together, with the histo-pathological findings, which showed predominant features of exudative inflammatory phase in autopsy lung samples from h5n1 patients, 11, 12 we may therefore speculate that people who had fatal h5n1 infection died during acute inflammation phase, and before the resolution could occur, especially for the cases with a short disease duration (<10-12 days). 13 in conclusion, the roles of cox-2 in both pro-inflammation and pro-resolution phases deserves detailed investi-gation. the timing of selective cox-2 inhibitor therapy in h5n1 infected patients may be extremely critical. therefore a time-dependent study using selective cox-2 inhibitors on h5n1-infected animal models will be particularly important in order to address the effectiveness of this drug in treating h5n1 disease. avian antibodies to combat potential h5n1 pandemic and seasonal influenza highly pathogenic avian influenza a virus (hpaiv) strain a ⁄ h5n1 with unprecedented spread through much of asia and parts of europe in poultry remains a serious threat to human health. passive immunization (transfer of protective immunoglobulins) offers an alternative and ⁄ or additional strategy to prevent and cure influenza. here, we report that virus-specific immunoglobulin y (igy) isolated from eggs of immunized hens provide protection in mice against lethal h5n1 virus infection by neutralization of the viruses in the lungs upon intranasal administration. importantly, chicken eggs obtained from randomly selected supermarkets and farms in vietnam, where mass poultry vaccination against a ⁄ h5n1 is mandatory, contain high levels of igy specific for a ⁄ h5n1 virus. when administered before or after the infection, igy prevented and significantly reduced replication and spread of hpaiv h5n1 and related h5n2 strains. thus, the consumable eggs readily available in markets of countries that impose poultry vaccination against a ⁄ h5n1 could offer an enormous source of valuable biological material that provides protection against a ⁄ h5n1 virus with pandemic potential. the approach could be used to control seasonal influenza. since 2004, hpaiv of the h5n1 subtype has resulted in more than 430 cases of laboratory-confirmed human infection in 15 countries with a death rate of more than 50% (http://www.who.int/csr/disease/avian_influenza/). h5n1 influenza virus remains a global threat because of its continued transmission among domestic poultry and wild birds. passive immunization (the transfer of antigen-specific antibodies (abs) to a previously non-immune recipient host) offers an alternative and ⁄ or additional countermeasure against influenza. 1 development of human monoclonal antibodies (mabs) against h5n1 influenza haemagglutinin (ha) using epstein-barr virus (ebv) immortalization of b cells isolated from patients infected with h5n1, 2 phage display, 3 humanized mabs, 4 and human recombinant abs 5 has been attempted. chickens produce a unique immunoglobulin molecule called igy that is functionally equivalent to mammalian igg. 6 igy is found in the sera of chickens and is passed from hens to the embryo via the egg yolk. 7 egg igy has been used to prevent bacterial and viral infections (see review 8 ) of the gastrointestinal tract and recently for protection against pseudomonas aeruginosa infection of the respiratory tract of patients with cystic fibrosis (cf). 9 the epidemic of hpaiv h5n1 virus has resulted in serious economic losses to the poultry industry, mostly in southeast asia. therefore, many countries including china, indonesia, thailand, and vietnam have introduced mass vaccination of poultry with h5n1 virus vaccines that controls the h5n1 epidemic to some extent. 10 chickens immunized with recombinant h5 and ⁄ or inactivated h5n1 reassortant vaccines produced a high level of virus-specific serum antibodies (abs) and were protected from h5n1 virus challenge. 11 theoretically, these abs could be found in egg yolk and separated for use in humans to prevent and cure h5n1 hpaiv infection and disease, respectively. here, we examined the possibility that igy isolated from consumable eggs available in supermarkets in vietnam, where mandatory h5n1 vaccination has been implemented, provide prophylaxis and therapy of hpaiv h5n1 infection in mice. six-to 8-week-old female balb ⁄ canncrl (h-2d) mice (charles river and jackson laboratory) and hy-line . igy abs were extracted from egg yolks as previously described. 12 the 50% egg infectious dose (eid 50 ) was determined by serial titration of virus stock in eggs, and eid 50 ⁄ ml values were calculated according to the method of reed and muench. 13 human virus stocks were grown in mdck cells as described previously ,14 with viral titers determined by standard plaque assay. the 50% tissue culture infectious dose (tcid 50 ) of virus was determined by titration in mdck cells. 15 the standard elisa was performed for detection of anti-igy in the sera of igy-immunized mice. fifty percent lethal dose (ld 50 ) titers were determined by inoculating groups of eight mice i.n. with serial 10-fold dilutions of virus as previously described. 16 for infection, ketamine-anesthetized mice were inoculated intranasally with a lethal dose with 250 pfu (5 · ld 50 ) of a ⁄ pr ⁄ 8 ⁄ 34 (h1n1) virus as previously described, 17 10 · ld 50 of vn ⁄ 1203 (h5n1) or 5 · ld 50 a ⁄ aquatic bird ⁄ korea ⁄ w81 ⁄ 2005 (h5n2) resuspended in 50 ll pbs per animal. ketamine-anesthetized mice were treated intranasally with 50 ll of igy before or after infection. mice were observed for weight loss and mortality. subsets of animals were scarified for virus titre. we found comparable hai titers in the sera and egg yolks obtained from a farm in vietnam that was participating in a national mass vaccination program. furthermore we found 90% of eggs purchased in randomly selected supermarkets in hanoi, vietnam containing h5-specific igy. the hai and vn titers of pooled egg yolk igy are comparable with those of sera obtained from hens selected randomly from the farm that underwent supervised h5n1 vaccination. in contrast, igy separated from eggs purchased in korean markets where poultry are not vaccinated against avian influenza h5n1 has no detectable h5-specific hai or vn activity. we first treated naïve mice intranasally with h5n1-specific igy before infection with hpaiv h5n1 strain, a ⁄ vietnam ⁄ 1203 ⁄ 2004, isolated from a fatal case. such treated mice displayed mild weight loss and recovered completely by the end of the first week after inoculation ( figure 1a ). when animals were treated once with h5n1specific igy after h5n1 inoculation they exhibited minimal weight loss during the first week after inoculation, and virus titers in the lungs were substantial reduced at day 3 after infection; however, 50% of treated mice succumbed to infection during the second week after inoculation ( figure 1b) . it is possible that not all the hpaiv a ⁄ h5n1 viruses were neutralized upon the single treatment with igy, and escaping viruses can spread systemically to organs outside of the lungs. these viruses may reappear in lung tissue later when specific igy is absent. indeed, vn ⁄ 1203 virus injected intravenously or into the brain can spread to the lungs. 18 to circumvent the virus escape, we administered multiple treatments with h5n1specific igy after the infection. as a result, all infected mice recovered completely by the second week post-infection ( figure 1c) , and virus titers in the lungs were substantially reduced to the level that seen in protected mice that received single prior-infection treatment ( figure 1d) . similarly, the protective efficacy of h5n1-specific igy was observed in mice infected with lethal dose of mouseadapted avian influenza virus strain a ⁄ aquatic bird ⁄ korea ⁄ w81 ⁄ 2005 (h5n2). this virus shares 94.4% nucleotide sequence homology with ha (h5) but has different na (n2) from the one used for mass immunization in vietnam (reassortant avian h5n1 influenza virus a ⁄ goose ⁄ gd ⁄ 96-derived, strain re-1). the results indicate that h5n1-specific igy isolated from eggs purchased in markets have preventive and therapeutic effects against infection with hpaiv h5n1 and the related strain h5n2. the findings suggest that while a single treatment with igy prior to lethal infection was sufficient to protect the animals from the infection, multiple treatment is required for complete therapeutic effect after infection with hpaiv such as vn ⁄ 1203 strain. we further examined the protective efficacy of igy isolated from eggs laid by hens immunized in the laboratory with heat-inactivated human influenza a ⁄ h1n1 virus, a ⁄ pr ⁄ 8 ⁄ 34. we found substantial levels of hai and vn abs in the sera and yolks derived from immunized hens. when naïve mice were administered intranasally with such anti-pr ⁄ 8 igy at 6-8 hours before or after infection with lethal dose of pr ⁄ 8 virus, they were protected from the infection or lethal disease, respectively. the virus titers in the lungs of a ⁄ pr8 specific igy-treated mice at day 3 after infection were also significantly lower than those seen in untreated mice or mice receiving normal igy. intranasal administration is the most effective route as compared to oral or peritoneal or intravenous administration for protection against lethal challenge, and the presence of virus-specific igy in bronchoalveolar lavage (bal) is required for the protection. the results provide a proof-of-concept that intranasal administration of virus-specific igy prevents influenza virus infection and cures the disease. the concept could be applied to control influenza outbreaks including seasonal and pandemic influenza. the protection was correlated with hai and vn activities of the igy and reduced virus titers in the lungs after treatments, suggesting that the protection is mediated by vn. we asked if administration of igy in the respiratory tract induces anti-igy ab response in mice. if this is the case, the next question is whether pre-existing anti-igy abs block igy-mediated protection. indeed, significant levels of anti-igy were observed in animals that received single or multiple administration of igy. when igy-immune mice were treated with virus-specific igy before or after lethal challenge, the results were identical to those obtained from treated naive mice, indicating that pre-existing anti-igy abs do not interfere with the protection mediated by virus-specific igy. consistently, incubation with anti-igy serum did not interfere with hai and vn activity of the virus-specific igy, indicating that anti-igy abs do not block virus binding by virus-specific igy (figure 2 ). the finding suggests that the igy treatment could be applied to persons who have developed anti-igy during the individuals' life, and such treatment strategy could be repeated if multiple treatment is required and ⁄ or necessary later on to protect infections with other pathogens. the approach using specific igy for prevention and therapy of hpaiv h5n1 infection offers a practical alternative to immunotherapy using convalescent plasma 19 and an additional therapeutic option to antiviral drugs since widespread drug resistance has been recently reported among influenza virus strains. igy is relatively stable. we found no change in protective activity after at least 12 months storage at 4°c, and lyophilization does not affect the activity, making production of igy practical. the use of igy immunotherapy has many advantages, since igy does not activate the human complement system or human fc-receptors, which all are well-known cell activators and mediators of inflammation. 20 we chose the water dilution method for preparation of igy. the method is simple, efficient and does not require any toxic compounds or any additives. such igy preparations by this method have been used in other human study. 9, 21 eggs are normal dietary components, so there is minimal risk of toxic side effects, except for those with egg allergy. thus, our study demonstrated that influenza virus-specific igy can be used in passive immunization that provides great help for immunocompromised patients and elderly who have weaken immune response to influenza vaccines. importantly, the consumable eggs readily available in the markets of countries that impose mandatory h5n1 vaccination offer an enormous source of valuable, affordable, and safe biological material for prevention and protection against potential h5n1 pandemic influenza. parts of the information and data presented in this manuscript were previously published in http://www.plosone.org/ article/info:doi%2f10.1371%2fjournal.pone.0010152. the polyphenol rich plant extract cystus052 is highly introduction the 2009 ⁄ 2010 h1n1influenza a virus pandemic clearly demonstrates that influenza is still a major risk for the public health. although the pandemic swine origin influenza a virus (soiv) caused only mild symptoms, the control of the outbreak still remains difficult. even as vaccine is available against this virus, the possibility of reassortment between the pandemic and a seasonal or avian a ⁄ h5n1 influenza virus strain is indeed a frightening, but a likely event. this reassortant strain might be able to transmit easily between humans causing fatal infections, and the current soiv vaccine might no longer be sufficient to protect against the reassorted virus. in such a case, we can only rely on effective antiviral drugs. today, neuraminidaseinhibitors, such as oseltamivir, represent the most common clinically approved medication against influenza a viruses. unfortunately, the frequency of reports describing the appearance of drug-resistant seasonal h1n1 and also h5n1 influenza a viruses dramatically increased in the recent past. [1] [2] [3] [4] drug resistance to the known antivirals highlights the urgent need for alternative antiviral compounds with novel defense mechanisms. recently, we have reported that a polyphenol rich plant extract, cystus052, which showed antiviral activity against influenza a viruses in cell culture and in mice. 5, 6 moreover, the antiviral activity of cy-stus052 against seasonal influenza virus and common colds was also demonstrated in humans. 7 however, the efficiency of cystus052 against soiv and a ⁄ h5n1 isolates was unknown so far. therefore, we investigated cy-stus052 effectiveness against the pandemic strain and seven natural influenza a ⁄ h5n1 isolates detected in several avian species during 2006 ⁄ 2007 avian influenza outbreak. additionally, the potency of the most common neuraminidase inhibitor oseltamivir was also investigated against these isolates. here, we show that cystus052 treatment was effective in in vitro studies against soiv and a ⁄ h5n1 influenza virus. viruses avian h5n1 isolates were originally obtained from the bavarian health and food safety authority, oberschleissheim, germany. the soiv a ⁄ hamburg ⁄ 4 ⁄ 2009 was obtained from the robert-koch-institut, berlin, germany. all h5n1 viruses were further propagated in embryonated chicken eggs or mdck ii (h1n1v) cells at the friedrich-loeffler-institut, tübingen, germany. for the cytopathological effect (cpe) inhibition screening, in accordance with sidwell, 8 mdck ii cells were infected with different viruses at moi of 0ae005. virus-infected cells were then treated with antiviral compounds cystus052 from 0ae1 to 1000 lg ⁄ ml or oseltamivir from 0ae01 nm to 1 mm. after incubation for 48 hours at 37°c and 5% co 2 , cells were fixed, and viable cells were stained with crystal violet. after extraction of crystal violet from viable cells with 100% methanol, the extinction was measured with an elisa reader. immediately before infection, mdck ii cells (8 · 10 4 cells ⁄ well) were washed with pbs and subsequently incubated with virus diluted in pbs ⁄ ba (0ae2% ba) 1 mm mgcl 2 , 0ae9 mm cacl 2 , penicillin and streptomycin to a multiplicity of infection (moi) of 0ae001 for 30 minutes at 37°c. cystus052 was added in a concentration of 50 lg ⁄ ml directly to the virus-stock and on the cell monolayer simultaneously with the infection. after 30 minutes incubation period, the inoculums were aspirated and cells were incubated with either mem or mem containing 1 lm oseltamivir. at indicated time points, supernatants were collected. infectious particles (plaque titers) in the supernatants were assessed by a plaque assay under avicel as described previously. 9 in order to investigate the antiviral potential of cy-stus052, ec 50 values based on the inhibition of the cpe on mdck ii cells were determined for cystus052 and in addition for oseltamivir. the ec 50 values for cystus052 ranged from 1ae53 to 18ae88 lg ⁄ ml. cystus052 demonstrated the highest sensitivity against the soiv, sn1 and mb1 isolates with ec 50 values below 5 lg ⁄ ml. compared to these virus strains, cystus052 showed a slightly increased ec 50 value for gsb1 (18ae88 lg ⁄ ml). in contrast the ec 50 values for bb1 and bb2 were notably elevated (65ae68 and 76ae22 lg ⁄ ml). thus, the weakest antiviral effect of cystus052 was observed against these two isolates. the ec 50 values evaluated for oseltamivir ranged from 0ae07 to 512ae76 lm ( table 1 ), indicating that bb2 (512ae76) and gsb1 (356ae92 lm) can be considered resistant against oseltamivir. to confirm these results we investigated the ability of cystus052 to block virus replication as published before. 1 as a control, virus infected cells were treated with oseltamivir as described earlier. 6 in the absence of the drugs all influenza strains showed similar growth properties (figure 1, black squares) . first progeny viruses were detectable between 8 and 20 hours post infection (figure 1, black squares) . treatment with cystus052 resulted in reduction of virus titers of all influenza virus strains (fig. 1a-h, open triangles) . surprisingly, oseltamivir failed to inhibit the replication of two h5n1 influenza virus strains (gsb1 and bb2), supporting the data of ec 50 values ( figure 1d+h , grey rhombes). we assessed the antiviral activity of cystus052 against the newly emerged soiv and seven avian h5n1 influenza viruses. cystus052 showed efficient antiviral activity against the pandemic h1n1v strain and was effective to a wide range of h5n1 viruses. furthermore, cystus052 demonstrated a broader and more efficient antiviral potential than oseltamivir. cystus052 treatment leads to a stronger reduction of progeny virus titers, and more importantly, cystus052 was effective against all tested viruses, while oseltamivir was unresponsive against two of seven a ⁄ h5n1 viruses. even though the pandemic strain in general is still sensitive to oseltamivir treatment, there are increasing numbers of reports of emerging resistant variants. the treatment with cystus052 does not result in the emergence of viral drug resistance since the mode of action is an unspecific physical binding of the virus particle that is also beneficial to reduce opportunistic bacterial infections. 5,7,10 cystus052 is an extract from a special variety of the plant cistus incanus, and it is very rich in polymeric polyphenols. 11 it is well known that polyphenols exhibit protein-binding capacity. 12 however, cystus052 exhibited no neuraminidase inhibiting activity. therefore, ingredients of cystus052 may act in a rather unspecific physical manner by interfering with the viral hemagglutinin at the surface of the virus particle as demonstrated before. 5 while this prevents binding of the virion to cellular receptors, it does not block accessibility and action of the viral neuraminidase. since, infections with influenza a viruses are still a major health burden and the options for control and treatment of the disease are limited, plant extracts such as cystus052 should be considered as a new candidate drug for a save prophylactic and therapeutic use against influenza viruses. attenuation of respiratory immune responses by antiviral neuraminidase inhibitor treatment and boost of mucosal immunoglobulin a response by co-administration of immuno-modulator clarithromycin in paediatric influenza the antiviral neuraminidase inhibitor osv and zanamivir are widely used treatment options for influenza infection and are being stockpiled in many countries. although mucosal immunity is the frontline of defense against pathogens, the effects of neuraminidase inhibitor treatment on airway mucosal immunity have not been reported. the suppression of viral rna replication and viral antigenic production by these drugs may result in a limited immune response against influenza virus. 1 macrolides, such as cam and azithromycin, have anti-inflammatory and immunomodulatory properties that are separate from their antibacterial effects. [2] [3] [4] this study examined the impact of osv treatment on immune responses in the airway mucosa and plasma in mice infected with iav and pediatric influenza patients. we also assessed the immuno-modulatory effects of cam in influenza patients who were treated with or without osv. female 3ae5-week-old weanling balb ⁄ c mice were nasally inoculated with 25 pfu of iav ⁄ pr8 ⁄ 34 h1n1 at day 0. immediately after infection, mice were given 50 lg of osv orally or vehicle at 12-hours intervals for 11 days. the levels of virus-specific siga in nws and bronchoalveolar fluids (balf) and igg in plasma were measured by elisa as reported previously. 5 a retrospective clinical study was conducted. for the study, 40 children with acute influenza were recruited and grouped according to the treatment received: 5 days treatment with osv (n = 14), cam (n = 8), osv + cam (n = 12), and untreated (n = 6). since parents in japan are well aware of the adverse effects of osv especially the neuropsychiatric complications, 6 the decision on whether to administer osv or not and to prescribe cam was made by the parents and the attending paediatricians, based on their anti-viral and immuno-modulatory activity. 3, 6 comparisons were made of the levels of siga against iav ⁄ h3n2 and iav ⁄ h1n1, total siga, in nws and disease symptoms before and after treatment. anti-ha siga and total siga in nws of patients were determined from the standard regression curves with human iga of known concentration in a human iga quantitation kit (bethy laboratories). because an affinity purified human anti-ha-specific siga standard of each influenza a subtype is not available, the relative value of anti-haspecific siga amount was expressed as unit (u). one unit was defined as the amount of one lg of human iga detected in the assay system as reported previously. 6 the concentrations of siga in individual nws were normalized by the levels of total siga (lg ⁄ ml). oseltamivir suppresses viral rna replication and viral antigenic protein production. to investigate the influence of daily treatment with osv on ha-specific mucosal and systemic immune responses, we analyzed ha-specific siga levels in nws and balf as well as igg levels in plasma at days 8 and 12 post-infection in mice treated orally with osv or methylcellulose (mc) as vehicle. the osv treated mice showed lower antibody responses in nws and balf than control mice treated with mc solution (table 1) . significantly reduced ha-specific siga responses were particularly noted in the osv group at day 12, the period of maximal mucosal siga induction. the airway secretions and plasma from mice at day 0 did not contain detectable levels of ha-specific antibodies. these findings were supported by other data whereby mice treated with osv displayed significantly lower numbers of ha-specific iga antibody-forming cells (afcs) in the nasal lamina propria, mediastinal lymph nodes, and lungs compared with mc-treated mice. these results clearly indicate that oral administration of osv downregulates ha-specific siga responses in mucosa. on the other hand, there were no significant differences in the elevated levels of ha-specific plasma iga and igg antibodies or the increased numbers of ha-specific iga and igg afcs in the spleen between osv-and mc-treated mice. taken together, these results implicated the oral administration of osv in a suppressed induction of haspecific siga responses in respiratory lymphoid tissues, although systemic ha-specific antibody responses were not significantly affected by osv. since cam up-regulates il-12, a mucosal adjuvant cytokine in the airways, and promotes the induction of siga and igg in the airway fluids of mice infected with iav, 4, 7 we assessed the impact of treatment with osv and ⁄ or cam on the levels of anti-influenza siga in nws and clinical status of influenza patients. the concentration ratio of table 1 . anti-ha-specific siga to total siga in nws was expressed as titer: anti-ha-specific siga (u ⁄ mg) ⁄ total siga (lg ⁄ mg) · 100. figure 1 shows changes in the anti-ha(h3n2) siga ratio (titer) and fold of increase in siga titer in each patient during the 5-days' treatment for the four different treatment groups. it is noteworthy that, upon admission to the hospital, the siga titers were <3 in 93% of patients. during the 5 days of treatment, rapid increases in the titers were observed in almost all patients in cam, osv + cam, and no treatment groups. in contrast, in the osv group, the anti-ha-specific siga titers remained unchanged or decreased in the majority of patients. the finding of significant low induction of anti-viral siga in the osv group was supported by the results of animal experiments. however, the addition of cam to osv augmented siga production and restored mucosal siga levels; 75% of patients treated with osv + cam showed >5-fold increase in the titers during treatment. these observations suggest that cam stimulated the local mucosal immunoresponse in the nasopharyngeal region of patients treated with osv. the prevalence of disease manifestations was also analyzed. 6 among the symptoms listed, a significant decrease in the prevalence of cough was recorded between the no treatment group and the osv + cam group and between the osv group and the osv + cam group (**p < 0ae01), despite the limited number of patients in each group. the duration of the febrile period was significantly shorter in the osv and osv + cam groups than the no treatment group. however, no significant difference was observed between the osv group and osv + cam group. it has been reported that osv does not affect the cellular immune responses, such as cytotoxic t lymphocytes and natural killer cells. 8 however, the effects of osv on mucosal immunity have not been studied so far. the present study showed that osv treatment of mice infected with iav induced insufficient protective mucosal siga responses in the respiratory tract, although treated mice showed the similar levels of systemic igg and iga antibody responses in plasma to those in mice treated with vehicle (table 1) . 1 the observed effect of osv on mucosal immunity was probably due to a suppression of viral replication and viral antigen production in the mucosal layer. these observations in mice are further supported by our clinical reports of siga in nws and balf of osv treated influenza patients. 6 the 14 membered-and 15 membered-ring macrolides have been found to possess a wide range of anti-inflammatory and immuno-modulatory properties, 2,3 and to be effective in the treatment of respiratory syncytia and iav infection. 9,10 the efficacy of low doses administered on the long term against pathogens that are insensitive to macrolides indicates a mode of action that is separate from their antibacterial activity. 2, 3, 9, 11 in the present study, we evaluated the immunomodulatory effects of cam on mucosal immune responses in pediatric influenza. a decrease in the proportion of total siga that was anti-ha-specific siga during treatment was observed in 21.4% of patients in the osv group (those represented by the dotted lines and closed diamonds in figure 1 ), whereas an increase in the proportion was observed in most patients of the other groups (except for one patient of the untreated group). despite the low or unchanged induction of anti-ha-specific siga in the majority of osv-treated patients, the additional use of cam with osv boosted the mucosal immune response and restored local mucosal siga levels. we are currently engaged in detailed immunological studies of the effects of cam and osv on the levels of mediators controlling iga class switching in nws of influenza patients and airway secretion of mice infected with iav. further studies should clarify the boost mechanisms of cam and the suppression mechanisms of osv in iga class switching. our findings suggest the risk of re-infection in patients showing a low mucosal response following osv treatment and cam effectively boosts the siga production for protection of re-infection. to date there is an urgent need to develop new antivirals against influenza. most of the molecules reported target influenza proteins that acquire rapid mutations of resistance. the development of new molecules that have a broad antiviral activity and are not subjected to influenza mutation is of particular interest. our laboratory and others recently showed that proteases can participate to the innate immune response in the airways through the activation of a family of receptors called par. in particular, through the release of interferon, par2 agonists curbed viral replication significantly in infected cells. in this study, since erk activation is crucial for virus replication, we investigated whether par2 could inhibit virus replication through inhibition of the erk pathway. results showed that while influenza a infection alone or par2 stimulation alone induced erk activation, par2 stimulation does not inhibit erk activation in influenza infected cells. thus, par2 agonists may be a potential new drug against influenza viruses that could be used in combination with other anti flu therapy such as the inhibition of the erk pathway. respiratory tract-resident proteases are key players during influenza virus type a infection. 1, 2 in addition to their direct activating effect on surface viral proteins, lung mucosal proteases can regulate cellular processes by their ability to signal through protease-activated receptors (pars). 3 after cleavage of the receptor by proteases, the new aminoterminal sequence of par binds and activates the receptor internally. these receptors are highly expressed at epithelial surfaces, in particular in the lung, where human influenza virus replicate in vivo. pars are thus directly exposed to proteases present in the airways. among the four different pars, par2 acts as an antiviral through an interferondependent pathway. 4, 5 thus, agonists of par2 are potential new drugs against a broad range of influenza viruses, which is in accordance with the broad antiviral action of interferon. however, the signalling pathway induced by par2 agonists in influenza a infected cells has still to be investigated. in this manuscript, we showed that influenza infection or activation of par2 induced erk activation, a crucial step for efficient virus replication. 6, 7 however, par2 agonists do not impaired erk activation in influenza a virus infected cells. since the pathway of par2 protection is likely to be erk-independent, the use of anti erk molecules in combination with par2 agonists maybe of potential interest in future anti-influenza therapy. influenza viruses a ⁄ wsn ⁄ 33 (h1n1) (a kind gift from nadia naffakh) was used in the present study. mdck (madin-darby canine kidney) and the human alveolar type ii a549 cell were obtained from atcc and grown as previously described. 8 for western blot analysis, the following antibodies were used: monoclonal antibody for phospho-erk1 ⁄ 2 (t202 ⁄ y204) and for erk1 ⁄ 2 antibodies from cell signaling technology (beverly, ma), horseradish peroxydase (hrp)-coupled rabbit polyclonal antibodies against mouse or rabbit igg from paris (compiègne, france). a549 cells were infected with iav at an moi of 1 in emem medium, as previously described. 9,10 at various time points post infection, cells were collected and proteins were analysed as previously described. 6,11 par2 stimulation was performed at 37°c in emem medium as previously described. 4 after infection and ⁄ or stimulation, cells were lysed in ice-cold lysis buffer. lysates were centrifuged at 12 000 g for 20 min, and total proteins of the supernatants were analyzed by western blot analysis as previously described. 12, 13 results since activation of the erk pathway is essential for efficient influenza replication, 7 we first investigated the kinetics of erk activation after influenza infection in human a549 alveolar epithelial cells. for this purpose, a549 cells were infected with influenza viruses at a moi of 1 at different time point post-infection, and activation of erk1 ⁄ 2 pathway was assessed by western blot analysis using an anti-erk antibody. results showed that erk was phosphorylated after influenza infection in a time course depen-dent manner when compared to uninfected cells. in contrast, erk phosphorylation was not observed with heatinactivated viruses, suggesting that productive infection is needed for erk activation ( figure 1a ). antibodies against erk1 ⁄ 2 were used as controls. since erk is activated after influenza infection, we then tested whether activation of par2 in uninfected cells also leads to activation of this pathway. for this purpose, a549 cells were stimulated with the selective human (h) or mouse (m) par2 agonist or a control peptide for the indicated time ( figure 1b ). when exposed to the par2 agonists and compared to controltreated cells, erk phosphorylation increased over the time course of stimulation. thus, influenza infection or stimulation of par2 without infection in a549 cells induced activation of the erk pathway at different time point post-infection. since influenza infection and par2 stimulation induced erk activation, we then investigated whether par2 could inhibit erk activation in influenza infected a549 cells. results in figure 2 showed that in influenza infected cells, par2 activation for ten minutes does not inhibit erk activation after influenza infection. thus, erk activation is not inhibited by par2 activation in influenza stimulated cells. in this manuscript, we studied the activation of the erk pathway after par2 stimulation and or influenza infection. particularly interesting is the fact that either influenza infection or par2 stimulation alone induce erk phosphorylation in a549 epithelial cells, while erk activation is not inhibited in a549 infected cells compared to uninfected ones after par2 stimulation. proteases are key factor in the pathogenicity of influenza viruses. in addition to the cleavage of ha, necessary for iav replication, extracellular proteases also play a role in the modulation of the immune system against influenza viruses through the activation of pars. particularly par2, activated by extracellular trypsin-like proteases, could inhibit virus replication through the release of interferon, 4,5 thus, strengthening the immune system via agonist peptides and providing new therapeutic potential against a broad range of influenza strains. in addition, targeting the host instead of the virus could provide a way to escape from virus resistance. 14 thus, a better understanding of how virus escapes from immune surveillance may provide new therapeutic strategies to block iav. in addition, combinations of drugs that block virus replication via different pathways are of interest. the non classical molecules hla-g maybe an interesting new target as we recently showed that it is upregulated after influenza infection, 13 and it is a well known immunotolerant molecule. 15 indeed, it inhibits the innate immune response 16 as well as the adaptive immune response. 17, 18 also, as previously suggested, the erk signal transduction cascade is also of potential interest since it is crucial for virus replication and particularly influenza replication. 6, 7 as shown here, it is unlikely that par2 protection occurs through an erkdependent pathway. thus strengthening the immune response with par2 agonists and blocking nuclear retention of the viral ribonucleoprotein complexes with inhibitors of the mek ⁄ erk pathway may be alternative combinatory approaches for influenza therapy. in addition, since those potential drugs target the host instead of the virus, this could help in the design of new antivirals molecules more resilient to iav mutations and thus to virus resistance. the initial waves of the first influenza pandemic of the 21st century have passed. in june 2009, vaccine companies estimated they could produce in 6 months almost 2.5 billion doses of pandemic vaccine. 1 instead, they actually produced only 534 million doses, of which 64% were non adjuvanted preparations. had these doses been produced with adjuvants (i.e., 3.75 lg instead of 15 lg ha per dose), an additional 1 billion doses could have been made available. yet there was public opposition to adjuvants in many countries, especially by regulatory officials in the united states. misperceptions about the safety of both adjuvanted and nonadjuvanted vaccines were widespread. added to this, shortfalls in vaccine production, delays in vaccine delivery, and the ''mildness'' of the pandemic itself meant that only a few countries achieved reasonable levels of vaccine coverage. millions of doses went unused and had to be destroyed. supplies of antiviral agents were even more limited. thus, despite the best efforts of influenza scientists, health officials, and companies, more than 90% of the world's people did not have timely access to affordable supplies of vaccines and antiviral agents. instead, they had to rely on 19th century public health ''technologies.'' given current understanding of biology in the early 21st century, they should have had -and probably could have had -something better. this report reviews evidence for an alternative approach to serious and pandemic influenza that could be used in all countries with basic health care systems. instead of confronting the influenza virus with vaccines and antiviral agents, it suggests that we might be able to modify the host response to influenza virus infection by using anti-inflammatory and immunomodulatory agents. this idea was introduced several years ago 2 and has been reviewed in several publications. [3] [4] [5] [6] [7] [8] the central importance of the host response in the 1918 pandemic, young adults had high mortality rates. ever since, influenza virologists have sought to answer the question ''why did young adults die?'' by defining the molecular characteristics of the 1918 virus that were responsible for its virulence. 8 in doing so, they have overlooked a crucial piece of clinical evidence from the 1918 pandemic: compared with young adults, children were infected more frequently with the same virus, yet they seldom died. consequently, the more important question is ''why did children live?'' this can only be explained by recognizing that children must have had a different host response to the 1918 influenza virus than adults. physicians have long recognized that for several other medical conditions, both infectious (e.g., pneumococcal bacteremia) and non-infectious (e.g., multiple trauma), children have a more benign clinical course than adults. 6, 8 a corollary of this observation is that secondary bacterial pneumonia, although commonly found in young adults in 1918, could not have been the primary cause of death. children must have had the same or higher rates of nasopharyngeal colonization with the same bacteria that were associated with pneumonia deaths in adults, yet children seldom died of secondary bacterial pneumonia. 6 if young adults died with secondary bacterial pneumonia, underlying host factors must have made them more susceptible. few people who die of influenza do so during the first few days of illness when pro-inflammatory cytokine levels are high. instead, like patients with sepsis, they usually die in the second week, when anti-inflammatory cytokines and immunosuppression dominate. 6, 8, 9 influenza deaths occur more frequently in older persons with cardiopulmonary conditions, diabetes, and renal disease, but as seen in the 2009 h1n1 pandemic, they also occur in younger adults with obesity, asthma, and in women who are pregnant. regardless of age, people with all of these conditions share one characteristic in common: they have chronic low-grade inflammation. in effect, their ''innate immune rheostats'' have been set at different, and perhaps more precarious, levels that make them more vulnerable to influenza-related complications. 10 laboratory studies of influenza virus infection confirm the importance of the host response. in several studies in mice in which the host response has been modified (e.g., cytokine knockout), survival has been improved without increasing virus replication in the lung. 5 in fact, severe disease can be induced without any influenza virus replication. for example, fatal acute lung injury has been induced in mice by inactivated (not live) h5n1 virus. 11 in this model, antiviral agents would be useless; only the host response could be responsible for disease. these observations raise the following question: could the host response be modified so patients with severe seasonal and pandemic influenza might have a better chance of surviving? influenza is associated with acute coronary syndromes, and influenza vaccination and statins reduce their occurrence. these associations led to the suggestion in 2004 that statins might be used to treat pandemic influenza. 2 other agents that might also be effective include ppara and pparc agonists (fibrates and glitazones, respectively) and ampk agonists (e.g., metformin). 5, 8 these agents have been studied in laboratory models of inflammation, sepsis, acute lung injury, ischemia ⁄ reperfusion injury, energy metabolism, mitochondrial function, and programmed cell death. the results of these studies cannot be reviewed in detail here, but the major findings for cell signaling are summarized in the table 1 . unfortunately, the results of experimental studies are not always clear cut. for example, in one study of influenza virus infected mice, il-10 was necessary for containing infection, 12 but in another study il-10 appeared to be harmful. 13 nonetheless, overall understand-ing of cell signaling pathways in influenza virus infections and the actions of statins, glitazones, fibrates, and ampk agonists strongly suggest that these agents could benefit patients with severe influenza. laboratory studies in mice infected with pr8 (h1n1) h2n2 and pandemic h1n1 viruses show that resveratrol, fibrates, glitazones, and ampk agonists reduce mortality by 30-50%, often when treatment is started 2-4 days following infection. 14-17 (resveratrol is a polyphenol found in red wine. it shares with these other agents many of the same cell signaling effects.) in h5n1-infected mice, treatment with celecoxib and mesalazine, together with zanamivir, showed better protection than zanamivir alone. 18 remarkably, these immunomodulatory agents have not increased virus replication. even more remarkable, in another model of a highly inflammatory and frequently fatal conditionhepatic ischemia ⁄ reperfusion injury -glitazone treatment ''rolled back'' the host response of ''young adult'' mice (8-10 weeks old) to that of ''children'' (3-4 weeks old). 19 this unique study suggests that immunomodulatory treatment might roll back the damaging and sometimes fatal host response of young adults with influenza to the more benign and rarely fatal response of children. several, but not all, observational studies have shown that outpatient statins decrease hospital admissions and mortality due to community-acquired pneumonia. 8 for influenza itself, preliminary evidence presented in october 2009 suggests that immunomodulatory treatment of influtable 1 . cell signaling targets that might be affected by immunomodulatory treatment of severe seasonal and pandemic influenza* down regulate pro-inflammatory cytokines (e.g., nf-kappab, tnfa, il-1, il-6) up regulate anti-inflammatory cytokines (il-10, tgfb) up regulate pro-resolution factors (lipoxin a4, resolvin e1) up regulate ho-1 and decrease tlr signaling by pamps and damps up regulate enos, downregulate inos, restore inos ⁄ enos balance and stabilize cardiovascular function decrease formation of reactive oxygen species and decrease oxidative stress improve mitochondrial function and restore mitochondrial biogenesis decrease tissue factor and its associated pro-thrombotic state stabilize the actin cytoskeleton in endothelial cells and intracellular adherins junctions, and thereby increase pulmonary barrier integrity and decrease vascular leak differentially modify caspase activation and apoptosis in epithelial and endothelial cells, macrophages, neutrophils and lymphocytes in the lung and other organs increase the bcl-2 ⁄ bax ratio in influenza virus-infected cells and prevent the apoptosis necessary for virus replication. *see references 2,5,7,8 for details. nf-kappab, nuclear factor kappab; tnfa, tumor necrosis factor alpha; tgfb, transforming growth factor beta; ho-1, heme oxygenase -1; tlr, toll-like receptor; pamp, pathogen-associated molecular pattern; damp, damage associated molecular pattern; enos, endothelial nitric oxide synthase; inos, inducible nitric oxide synthase. enza patients with severe illness could be beneficial. in a study of almost 4000 patients hospitalized with laboratoryconfirmed seasonal influenza, inpatient statin treatment reduced hospital mortality by 66%. 20 in these patients, the cell signaling effects of statin treatment, summarized in the table 1 , probably acted to reduce pulmonary infiltrates, maintain oxygenation, stabilize myocardial contractility and the peripheral circulation, reverse immunosuppression, restore mitochondrial biogenesis, and prevent multi-organ failure. achieving these clinical effects led to a decrease in mortality. because of the molecular cross-talk between statins, fibrates, glitazones, and ampk agonists, 5,8 similar clinical benefits might be expected from other members of this ''family'' of immunomodulatory agents. simvastatin, pioglitazone, and metformin are produced as inexpensive generics in developing countries. they are used throughout the world in the daily treatment of millions of patients with cardiovascular diseases and diabetes. global supplies are huge. because most people with influenza recover without specific treatment (this was true in 1918), not all patients would require immunomodulatory agents. instead, only those at risk of ards, multi-organ failure, and death would need to be treated. importantly, the cost of treatment for an individual patient would be less than $1.00 (d.s. fedson, unpublished observations). moreover, unlike vaccines they could be used on the first pandemic day. thus far, influenza scientists and the institutions that support their work (e.g., nih and cdc, national health agencies in many countries, the bill and melinda gates foundation, the welcome trust, and the world health organization) have shown little interest in immunomodulatory treatment. nonetheless, when more than 90% of the world's people have no access to influenza vaccines and antiviral agents, their physicians must have access to an effective ''option,'' especially one that might be lifesaving. research on immunomodulatory agents for influenza must involve investigators in many fields outside influenza science -those with expertise in the molecular and cell biology of inflammation, immunity, sepsis, cardiopulmonary diseases, endocrinology and metabolism, ischemia ⁄ reperfusion injury, mitochondrial function, and cell death. laboratory studies needed to identify promising treatment agents would probably cost $5-15 million (d.s. the results of these studies would inform clinical trials that critical care physicians are already eager to undertake. 21, 22 this work will be especially important for people in developing countries where critical care capacity is extremely limited and not likely to improve. 23 like critical care physicians, influenza scientists too must recognize that they cannot afford not to undertake research to determine whether generic immunomodulatory agents might be useful in managing severe seasonal and pandemic influenza. the nf-kappab-inhibitor sc75741 efficiently blocks h5n1 influenza virus propagation in vitro and in vivo without the tendency to induce resistant virus variants introduction influenza is still one of the major plagues worldwide. the appearance of highly pathogenic avian influenza (hpai) h5n1 viruses in humans and the emergence of resistant h5n1 variants against neuraminidase inhibitors highlight the need for new and amply available antiviral drugs. we and others have demonstrated that influenza virus misuses the cellular ikk ⁄ nf-kappab signalling pathway for efficient replication, suggesting that this module may be a suitable target for antiviral intervention. 1 here, we show that the novel nf-kappab inhibitor sc75741 efficiently blocks replication of influenza a viruses, including avian and human a ⁄ h5n1 isolates in vitro in concentrations that do not affect cell viability or metabolism. in a mouse infection model with hpai a ⁄ h5n1 and a ⁄ h7n7 viruses, we were able to demonstrate reduced clinical symptoms and survival of sc75741 treated mice. moreover, influenza virus was reduced in the lung of drug-treated animals. besides this direct antiviral effect, the drug also suppresses h5n1-induced overproduction of cytokines and chemokines in the lung, suggesting that it might prevent hypercytokinemia we hypothesise to be associated with pathogenesis after infections with highly pathogenic influenza viruses, such as the a ⁄ h5n1 strains. thus, a sc75741-based drug may serve as a broadly active nontoxic anti-influenza agent. to assess the number of infectious particles (plaque titers) in organs a plaque assay using avicel ò was performed in 96-well plates as described by mastrosovich and colleagues. 2 virus-infected cells were immunostained by incubating for 1 hour with a monoclonal antibody specific for the influenza a virus nucleoprotein (serotec) followed by 30 minutes incubation with peroxidase-labeled anti-mouse antibody (dianova) and 10 minutes incubation with true blueô peroxidase substrate (kpl). stained plates were scanned on a flat bed scanner and the data were acquired using microsoft ò paint software. the virus titer is given as the logarithm to the basis 10 of the mean value. the detection limit for this test was <1ae7 log 10 pfu ⁄ ml. organs of infected and control mice were homogenized and incubated over night in 1 ml trizol ò reagent (invitrogen) at 4°c. total rna isolation was performed as specified by the manufacturer (invitrogen). rna was solubilised in 50 ll rnase free water and diluted to a working concentration of 50 ng rna ⁄ ll. reverse transcription real-time pcr was performed using quantifastô sybr ò green rt-pcr kit and quantitect primer assays (qiagen) . all samples were normalized to gapdh and fold expression analyzed relative to uninfected controls. 3 ct values were obtained with the smartcycler ò (cepheid). to answer the question whether the nf-kappab inhibitor sc75741 shows antiviral properties against influenza virus, h5n1 infected mdck cells were treated with different concentrations of the inhibitor (figure 1 ). already treatment with 1 nm of sc75741 led to a reduction of viral cpe of more than 70%. almost 100% protection of cells was achieved when cells were treated with 50 lm sc75741. the results indicated that sc75741 has antiviral properties at concentrations ranging from 1 to 5 nm. we next tested whether sc75741 would also be effective in the mouse model of influenza virus infection. when h7n7 mice were treated i.v. once daily for 5 days with 5 mg ⁄ kg sc75741, survival rate of the animals increased significantly (p < 0ae05). the same results were found when h7n7 influenza virus infected mice were treated i.p. with 15 mg ⁄ kg sc75741 (data not shown). moreover, sc75741 treatment was not only effective when the inhibitor was given prior to h5n1 influenza virus infection, but also in a therapeutic setup when sc75741 was applied to the animals 4 days after infection (data not shown). since influenza virus infected mice showed increased survival after lethal infection, we next questioned whether the amount of influenza virus was reduced in the lung. therefore, we performed quantitative real-time (qrt) pcr to detect viral mrna. mice were treated with either sc75741 or the solvent, and 48 hour later the lungs were prepared to perform qrt-pcr. as shown in figure 2a the amount of viral mrna was reduced by 90% in sc75741 treated mice compared to solvent treated controls, indicating that sc75741 leads to a reduced expression of h5n1 specific mrna in the lung of infected mice. since infection of mice with h5n1 leads to hypercytekinemia, 4 we also investigated the expression of cytokines in sc75741 treated mice. as shown in figure 2b the amount of il-6 specific mrna was drastically reduced in sc75741 treated mice compared to solvent treated controls. moreover, also the expression of ip-10 was altered in sc75741 treated h5n1 influenza virus infected mice. here, roughly 90% reduction of specific mrna was detectable ( figure 2c ). thus, sc75741 leads to a reduced transcription of il-6 and ip-10 in h5n1 infected mice. there is an urgent need for new concepts to develop antiviral drugs against influenza virus. targeting cellular factors is a promising but challenging approach, and the concerns about side effects are obvious. however, it should be considered that drugs targeting viral factors, such as amantadine or oseltamivir, also exhibit a wide range of side effects in patients. thus, drug safety has to be rigorously tested in clinical trials regardless whether a drug targets a cellular or a viral factor. moreover, resistance against human h1n1 influenza viruses and highly pathogenic avian h5n1 virus strains to oseltamivir and amantadine have been reported. 5 in that respect, the strategy to target cellular factors 6,7 might be one way to ensure that new drugs against influenza virus will be useful and effective for a long time without causing the development of resistant virus variants. we were able to demonstrate that the nfkappab inhibitor sc75741 is able to reduce influenza virus activity in cell culture. moreover, the compound was also effective against highly pathogenic avian influenza viruses of the h5n1 and h7n7 subtypes in the mouse model. next to the reduction of virus sc75741 was also able to reduce h5n1-induced overproduction of cytokines and chemokines in the lung in the lung of mice after infection with h5n1. most importantly, the drug did not show any tendency to induce resistant virus variants (data not shown). thus, a sc75741based drug may serve as a broadly active non-toxic antiinfluenza agent. [1] [2] [3] [4] [5] in hong kong, the first confirmed case was a tourist from mexico reported on may 1, 2009. the local government made its first attempt to contain the spread of h1n1 in the local community by closing the metropark hotel where that tourist was staying, and quarantining 350 guests and staff for 7 days. following identification of the first local case around 6 weeks later on june 11, 2009, the government closed all kindergartens and primary schools from june 12 until early july. fever clinics were also opened, the alarm levels in hospitals were raised to the highest, and a public education campaign was implemented. previous studies of the community responses to severe acute respiratory syndrome (sars) and human-to-human h5n1 avian flu identified the importance of understanding the background perceptions of risk and psychological impact on the community. [6] [7] [8] [9] [10] in this study we investigated the psychological and behavioral responses of the general local community throughout the first wave of ph1n1, and we also examined the factors associated with greater use of preventive measures. 11 a total of 13 surveys were conducted between april and november 2009, covering the entire first wave of the ph1n1 pandemic. computer generated random-household telephone numbers from all land-based local telephone numbers covering over 98% of hong kong households were used to recruit a total of 12 965 local adults. one cantonese-speaking adult (age ‡18) was invited for interview in each selected household on the basis of a kish grid. the survey instrument was based on previous experience in sars and avian influenza projects. information, including knowledge on modes of transmission, psychological responses to pandemic influenza, preventive behaviors, attitudes towards the new vaccines and socio-demographics, was collected. informed consent was obtained prior to the interview. ethics approval was obtained from the institutional review board of the university of hong kong. descriptive statistics were weighted by sex and age based on the reference population data provided by the hong kong government census and statistics department. 12 multivariable logistic regression analyses were used to examine the association between the use of preventive measures and knowledge, perceptions and behaviors, sociodemographic characteristics, and psychological responses to pandemic influenza. multiple imputation was used to cope with a small proportion of missing data and make the best use of all available data. 13 statistical analyses were conducted in r version 2.9.1 (r development core team, vienna, austria). twelve thousand and nine hundred and sixty-five local adults were recruited throughout the study period, with a total of 127 715 telephone calls being made; the response rate among eligible participants was 69.9%. 11 hong kong entered the containment phase after the world health organization (who) announced a global alert, and policies including border screening, tracing, and quarantine of doi:10.1111/j.1750-2659.2011.00219.x www.influenzajournal.com suspected cases were implemented. hong kong transitioned to the mitigation phase on june 10, 2009 when the first local case was reported. the chronology of these and other events plus the epidemic curve of laboratory-confirmed ph1n1 cases are shown in figure 1(a) . the anxiety scores and risk perception of the respondents are shown in figure 1(b,c) . anxiety, measured by the state trait anxiety inventory, remained steady throughout the study period. in response to the announcement made by who and the unknown nature of the new virus, a higher proportion of the respondents expressed worry (more, much more, or extremely more worried than normal) if developed ili and perceived ph1n1 severity (same, more, or much more serious than sars) initially in early may 2009. fewer respondents reported worry if they developed ili as the pandemic proceeded, with a slight perturbation around the first deaths in july 2009 and a steady decline to 40.0%, while perceived severity of ph1n1 declined more dramatically after an early high. perceived risks of infection of respondents (absolute susceptibility) and risk relative to others (relative susceptibility) were also investigated and found to remain relatively stable throughout the first wave, with no indication of an increase during the period of peak ph1n1 activity in september (figure 1c) . as the first wave of ph1n1 progressed, knowledge on modes of transmission did not improve. on the contrary, later in the epidemic increasing proportions of respondents reported oral-fecal and cold weather as modes of transmission of ph1n1. around 35-40% of the respondents did not recognize direct and indirect contact or touching infected persons and contaminated objects as transmission routes for ph1n1 throughout the first wave ( figure 1d ). higher proportions of respondents avoided crowded places and rescheduled travel plans in the second half of june 2009 when local kindergartens and primary schools were closed and the first ph1n1-associated deaths were announced. social distancing measures such as avoiding crowded places and rescheduling travel plans remained stable with slightly decreasing trends thereafter. the use of hygiene measures and other social distancing strategies was relatively stable with slightly decreasing trends during the study period ( figure 2 ). female sex and older age were generally associated with greater reported use of hand hygiene measures, home disinfection, avoidance of crowded places, and rescheduling of travel plans. 11 female sex was also positively correlated with use of face masks and cough etiquette. we found a negative correlation between anxiety and use of all hand hygiene measures and cough etiquette, but a positive correlation between anxiety and use of home disinfection and (c) proportion of the respondents reporting higher worry if developed flu-like symptoms (more, much more, or extremely worried), higher perceived seriousness of h1n1 compared to sars (much more or more severe), higher probability to contract h1n1 over the next 1 month (certain, much more, or more likely), higher probability to contract h1n1 over the next 1 month compared to others outside family (certain, much more, or more likely). (d) proportion of the respondents identifying 5 possible modes of transmission as the actual modes of transmission of h1n1. social distancing measures. 11 other significant factors contributing to greater use of preventive measures were worry and knowledge. 11 greater worry was associated with higher probability of home disinfection, social distancing measures, and use of face masks. knowledge that h1n1 could be spread by indirect contact was associated all the investigated preventive measures, and knowledge that h1n1 could be spread by droplets was associated with cough etiquette, but not face masks. there were no consistent trends between all the investigated preventive measures and absolute and relative susceptibility. 11 community transmission emerged in hong kong in mid-june 2009, and prior to emergence of community transmission, perceived risk and perceived severity were high. as ph1n1 spread in hong kong, risk perception declined, even at the same time as incidence was increasing. anxiety was low throughout, at around 1.8 on the 4-point scale, compared to a maximum of 2.5 during sars on the same scale. 9 anxiety has been showed to be positively correlated to personal hygiene measures and social distancing in previous studies; 9,14 however, we found a negative correlation between anxiety and use of all hand hygiene measures, cough etiquette, and face masks, and a positive correlation between anxiety and home disinfection. 11 the differences in findings may be due to the fact that our anxiety measure was not specific to h1n1, and the score could be affected by other factors including economics. unlike hygiene measures, higher anxiety level, greater worry, and higher risk of perception were all associated with more social distancing. 6, 7, 9, 14 social distancing is the most direct strategy in avoiding infection from other people, and it is commonly observed in an outbreak that the general public avoids crowded places, travelling to other countries, and social gatherings, 9, 14 but the economic impact could be substantial. 15 as community incidence of h1n1 peaked, we did not observe any increase in use of preventive measures (figure 2) . we found that face mask use peaked at the early stage of the pandemic, while hand hygiene remained fairly constant, and the knowledge on the modes of transmission of ph1n1 did not improve over time. the lack of substantial change in preventive measures or knowledge about the modes of ph1n1 transmission in the general population suggests that community mitigation measures played little role in mitigating the impact of ph1n1 in hong kong. on the other hand, knowledge that ph1n1 could be spread by indirect contact was associated with all of the preventive measures studied. consistent with reports during the sars period, 9, 16 this study also showed that females and those of older age were more likely than others to use hygiene measures, avoid crowded places, and reschedule travel plans. this study has some limitations. first, this was a crosssectional study that was carried out at different time points, rather than a longitudinal study following the same individuals over time, and so the inferences on changes in behavior may need to be interpreted more cautiously. second, we recruited samples from all land-based local telephone numbers that cover 98% of hong kong households, but the response rate was not high enough to guarantee a representative sample, and this could be a source of selection bias. third, the responses were self-reported, and this may lead to social desirability bias in estimating knowledge, attitudes, and preventive behaviors. fourth, since the hong kong population has previously gone through unique experiences from sars in 2003 and avian flu in 1997, our results may not be comparable to other countries or settings. in conclusion, this study revealed that the ph1n1 pandemic failed to generate an increase use of preventive measures in the local community. there was no association between anxiety level and the events of the pandemic. with a relatively low mortality and morbidity rates compared to sars, ph1n1 was not a matter of concern in the hong kong community. the lack of substantial change in the use of preventive measures and improvement in knowledge on the modes of transmission of ph1n1 suggested that public health campaigns during the pandemic may not have had substantial effects on the general public. london is a major tourist destination, the seat of government and finance in the uk, and in 2012 will host much of the olympic and paralympic games. along with the rest of the global community, in 2009 and early 2010 london faced the challenges of responding to the first pandemic of the 21st century. at the time, nhs in london was composed of 72 organisations, including the london ambulance service, acute hospitals, mental health and primary care trusts, and the strategic health authority. while london's nhs is well practiced at responding to large, big bang incidents, the influenza a ⁄ h1n1v pandemic was a rising tide event that lasted many months. significant preparatory work had been undertaken prior to april 2009, which meant that the nhs in london was ready to respond. nhs london (the strategic health authority for london) led the response in partnership with local managers in all nhs organisations. the first uk cases of influenza a ⁄ h1n1v were reported in scotland on 27 april, with the first in london on 30 april. cases continued to increase, and the first wave peaked in london in july. cases reduced over the school summer holidays, but increased again when children returned to school at the start of september, and a second, smaller wave occurred. it is essential that the nhs learns from the 2009 ⁄ 10 influenza a ⁄ h1n1v pandemic to ensure it is prepared for future challenges. nhs london provided a standardised debriefing pack to all nhs organisations in the region to identify, capture, and learn lessons. each debrief event involved health and inter-agency partners to ensure all viewpoints were considered and brought together in a single local report. all local reports were compiled in an over-arching document, which brings together common themes to inform ongoing preparedness in the region. 1 the debrief process identified a number of common themes, such as the need for clear and appropriate communication, the importance of working with partners, and the benefits of strong and early leadership. however, differences between and within organisations were also highlighted; for example, some wanted more freedom for local decision making, whereas others would have preferred more stringently applied central direction. the following paragraphs considers individual areas assessed in the debrief process. command and control was in the main effective, with clear direction delivered from the national centre through nhs london to local nhs organisations. effective leadership is essential; the identification of senior local individuals to lead the response with teams of people to support them was critical. appropriate use of technology to communicate messages and coordinate command and control processes greatly aided the response. this included the development of the nhs london noon brief, a daily digest and associated web portal, and regular teleconferencing. key points are: • operational management at all levels must be considered in pandemic planning. • appointing an executive lead in each organisation was invaluable in the response. • pandemic flu planning for london must continue to be regionally led. communication is an essential component of the response to any incident. it must be clear, timely, and accurate. in the main, communication was excellent and met these criteria. one of the most challenging aspects was when messages from partner organisations differed, which occasionally led to confusion, unnecessary work, or frustration. the use of technology greatly aided communication across the region and supported the response; this included secure web sites, bluetooth, and text messaging etc. key points are: • regular internal communications and staff briefings are critical in the response to emergencies. • regular teleconferencing should be incorporated into future plans. • organisations should consider proactive and innovative methods for communicating during emergencies. robust partnership working was an essential component of pandemic preparedness work; however in the event, the a ⁄ h1n1v pandemic had little impact on sectors in london other than health. resilient communication networks between organisations, a common understanding, and the ability to make decisions were essential to the response at local level. ipcs proved an excellent mechanism to maintain local working relationships and resolve problems. clarity on the seniority of those attending these meetings and whether multi-site organisations such as mental health trusts should attend every ipc should be considered on a local and regional basis. key points are: • pandemic planning must remain part of inter-agency working. • social care resilience and planning must be embedded and integrated in health planning. 'vulnerable groups' is a universal term that covers a large and fluid group of individuals with different needs. ensuring access to healthcare during the pandemic for those who became vulnerable due to the situation, or those identified as such prior to the event, was the role of the pct in partnership with the local authorities. work continues to ensure that communication with vulnerable people is appropriate and timely in all incidents, and that organisations work together to achieve this. key points are: • planning to support the breadth of vulnerable people must continue. • pandemic preparedness for the prison sector should be further developed. • red ⁄ amber ⁄ green ratings for assessing vulnerabilities of mental health service users in an emergency should be further developed across the region. correct and appropriate usage of ppe is an essential component of reducing influenza spread, particularly in healthcare settings. london's nhs had been working towards developing local stockpiles of ppe when the pandemic commenced; however, there was little in place. the unanticipated national stockpile, while providing ppe to all organisations, was accompanied with some challenges in that it was often unfamiliar stock. key points are: • work around local stockpiling of non-standard consumables should continue. • regular training and fit testing of respirators should be embedded in all organisations. antiviral treatment was a core component of the response to influenza a ⁄ h1n1v, and was provided free of charge from a national stockpile. npfs reduced pressure on frontline nhs services once it was activated; however, there were concerns that patients could 'cheat' the system and obtain the drugs prior their clinical need. information about storage requirements of countermeasures must be clearly explained when they are delivered to frontline services, and the potential for recall into national stockpiles should be planned for. key points are: • regular exercising of local mass countermeasures centres and antiviral collection points (acps) should continue. • the use of community pharmacies as acps should be further considered in the capital. pandemic influenza vaccine uptake by healthcare workers was better than usual seasonal influenza uptake in the majority of nhs organisations, but could have been even better. this was largely due to the second pandemic wave not being as significant as expected, lack of clarity around when the vaccine would be delivered, and limited amounts being available initially. • gp-led and mass vaccination models for pandemic vaccination should be considered in local plans. • local lessons from the pandemic vaccination campaign should be applied to seasonal flu vaccination. the ability to maintain or increase capacity in response to a surge in demand, no matter what the cause, must be planned for. any of a number of situations could result in reduced staff or more patients, such as industrial action, transport disruption, disease outbreak, major incident, or poor weather. the work undertaken during planning for and responding to the pandemic will stand organisations in good stead for future disruptions. the importance of robust business continuity planning locally cannot be overlooked, as this is a key component of maintaining and increasing capacity. key points are: • local gp 'buddy schemes' should be encouraged for response to extreme pressure events. • organisations should regularly run staff skills audits so as to be aware of their overall capability for managing emergencies. • less emphasis should be placed on the use of retired staff when planning service continuity. reporting is a necessary but onerous task, and is often one of the most time-demanding parts of any incident response. it is also the aspect least likely to be tested through exercising. nhs london worked with organisations to endeavour to reduce reporting pressures, but much of this was dictated by central government. it is essential that future reporting requirements are proportional, informative, and realistic. while recognising it is not possible to predict the detail of information that may be requested, some broad assumptions can be made. key points are: • organisations should consider how they would collect and collate data from disparate parts of their organisation, rather than focussing on the detail of what that might be. • national and regional planning should consider the need for information and how this is balanced with the demand this places on organisations. • the introduction of the concept of a daily dashboard to identify areas of pressure should be incorporated into pandemic flu planning. the winter and pandemic influenza resilience assurance process undertaken in autumn 2009 was a useful process to inform planning for the first winter when the pandemic virus would be circulating in the uk. this consisted of a regional inter-agency exercise and a comprehensive review of the winter and pandemic plans of all nhs organisations in london. • regular assurance of pandemic flu preparedness should be maintained. • future resilience assurance processes should be undertaken in a timely and measured manner. • local organisations should continue to undertake regular pandemic flu exercises. the recovery period is as important as the response, but often receives minimal attention and has the potential to suffer as staff return to their normal jobs. one of the aspects that was not anticipated during the pandemic was the amount of stock (ppe, antivirals, and vaccine consumables) that would be recalled into national stockpiles. this proved particularly challenging for pcts who had to coordinate the process across their local areas. key points are: • the recovery period of an emergency must be given the same status and importance as the response. • future pandemic flu planning must include the recovery of national stockpiles of equipment and medicines. it is essential the lessons from the 2009 ⁄ 10 influenza a ⁄ h1n1v pandemic are learnt and embedded into business-as-usual and emergency response processes in preparation for the next pandemic and other incidents. even though the a ⁄ h1n1v pandemic was generally milder than previous pandemics, it still presented challenges to the nhs in london. the biggest challenge that remains is to ensure that the public and nhs staff are aware that a more virulent virus could cause significantly more illness, death, and disruption, and that we must maintain our preparedness should this happen. the influenza a ⁄ h1n1v pandemic has been a major stimulus to business continuity planning and emergency preparedness across health in london, and many of the experiences during the pandemic proved invaluable in the unusually severe weather in early 2010. it is important that this impetus and focus is maintained. changes to the nhs landscape in london will be considered in ongoing pandemic and emergency preparedness to ensure we remain as well prepared as possible for future events, particularly as london approaches the 2012 olympic and paralympic games. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. the way national health systems operate during inter-pandemic and the pandemic alert periods and the methods they use to address potential threats posed by zoonotic viruses with pandemic potential, as well as sea-sonal influenza epidemics, can clearly indicate whether the countries have enough capacities to respond adequately to unexpected influenza outbreaks. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during a pandemic scenario, the group prioritized topics and questions relating to rapid action and response. five to 10 key public health needs associated with a pandemic scenario have been identified for each of the research agenda streams: five priority public health topics were identified for a pandemic scenario as follows: • examination of host range and transmission dynamics of animal influenza viruses to guide surveillance, control strategies, and risk communication. • enhanced surveillance in animals and humans to monitor virus evolution: o early detection of novel reassortants or changes in genotype and ⁄ or phenotype related to virulence. o development of epidemiological and laboratory diagnostic tools and capacity building to optimize case finding. o develop a framework for surveillance in animals that address ethical, legal, and social barriers to intra-pandemic surveillance and reporting. • deconstruct the origins of the pandemic virus to identify factors that permitted efficient human transmission. • develop strategies to limit economic, social, and cultural disincentives of animal-based interventions to reduce intra-and inter-species transmission. • operational research to optimize risk communication in the early phases of the pandemic linked to animal husbandry and food safety. stream 2: limiting the spread of pandemic, zoonotic and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: transmissibility of influenza across the progression of infection and spectrum of disease: • relative contributions of the different modes of transmission for influenza. five priority public health topics were identified for a pandemic scenario as follows: • identification of groups at higher risk of infection and severe disease outcome through enhanced surveillance. • understanding disease severity and identification of predictors of severe outcomes. • investigation of vaccine effectiveness, especially in high risk groups in diverse geographic areas. • establishment ⁄ enhancement of pharmacovigilance, particularly for adverse events among at-risk groups. • optimization of strategies for rapid and targeted vaccine deployment. • rapid assessment to optimize acceptance of pandemic vaccine. six priority public health topics were identified for a pandemic scenario as follows: • collaboration and coordinated sharing of data, protocols, regulatory, and other implementation strategies and databases from different countries on all aspects of patient management and outcome to accelerate improvements in patient care. • development of best practices in patient management in different settings, including checklists and algorithms for clinical care and treatment, prognostic parameters, and tests to predict potential for the development of severe disease. • rapid, reliable, simple, low-cost point-of-care diagnostic tools for influenza. • best use of current antiviral drugs and optimal formulations in different target populations, such as parenteral and other routes of administration for severe infections. • use of combination therapies, including use of adjunctive therapies (e.g., use of convalescent serum and immunomodulators). • role of ongoing viral replication, host responses, and the effect of co-infections in the pathogenesis of severe disease. modern tools for early detection and monitoring of disease the group on surveillance tools concluded that the agreed topics of interest were equally applicable during a pandemic or inter-pandemic period: • studies to appraise and adapt modern technologies for early detection of influenza outbreaks in surveillance at the human-animal interface. • develop, integrate, and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems. • study efficient mechanisms on sharing data, clinical specimens, and viruses with consideration for local, ethical, legal, and research perspectives. • examine the timeliness and quality of data required for early detection from local to national and global levels for the respective stakeholders. five priority public health topics were identified for a pandemic scenario as follows: • identify environmental determinants of seasonal variation in influenza transmissibility in tropical and temperate regions. • estimate the transmission risk associated with types of contacts by comparing measured contact patterns with outbreak data. • incorporation of validated models of behavioral responses to risk and control measures in virus transmission. • development and implementation of novel technology for real-time sero-surveillance during a pandemic. • develop experimental and theoretical framework to assess host adaptation to study host receptor, antigenicity, and virulence. modern tools for strategic communication three priority public health topics were identified for a pandemic scenario as follows: • evaluate tools to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups to guide future communication efforts; develop tools and methods to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups, and thereby, guide future communication efforts. for communicating in different cultural settings, which engage and empower individuals and communities to practice and promote appropriate risk reduction measures. implementation of the identified research priorities is expected to underpin public health decision making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs and economic loss, and mitigate potential social disruption. complemented by an analogous research roadmap for a pandemic influenza scenario, the research recommendations for an interpandemic period represent a framework to provide evidence to guide public health policies on influenza control. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza 1 developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting public health needs for inter-pandemic periods according to its five key research streams: • stream 1. reducing the risk of emergence of pandemic influenza. • stream 2. limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza. • stream 3. minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza. • stream 4. optimizing the treatment of patients. • stream 5. promoting the development and application of modern public health tools. stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza inter-pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during an inter-pandemic phase, a more comprehensive approach was applied to establish research topics and prioritizing a range of questions that will build a solid foundation to guide research activities to support public health decision making. five to ten key public health needs associated with an inter-pandemic scenario have been identified for each of the research agenda streams: stream 2: limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: 1. transmissibility of influenza across the progression of infection and spectrum of disease 2. relative contributions of the different modes of transmission for influenza 3. biological, behavioral, and social host factors that influence the risk of transmission and infection 4. patterns, drivers, and mechanisms affecting the seasonality of transmission 5. viral and population factors that influence transmission and spread of different influenza types, subtypes, and strains 6. strategies to reduce the transmission of influenza in community, household, and health care settings, especially in less-resourced areas 7. impact and cost effectiveness of social measures, such as school closures, and the role of surveillance in assessing timing of these interventions 8. impact, effectiveness, and cost effectiveness of individual measures, such as isolation and quarantine 9. role of vaccination in limiting the spread of influenza and strategies for its use 10. impact of antiviral treatment and prophylaxis in reducing transmission of influenza stream 3: minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza 1. identify higher risk groups and severe disease through surveillance; disease severity and identification of predictors of severe outcomes 2. evaluate vaccination preventable disease burden and the potential impact of immunization programs through vaccine demonstration projects 3. enhancement of the properties of existing vaccines, including duration and breadth of protection, safety, immunogenicity, and dosesparing 4. development of new vaccines and vaccine platforms, especially suitable for under-resourced country settings 5. study the effectiveness of vaccine strategies to reduce disease burden in children and other high risk groups in a wide range of settings 6. improved uptake and acceptability of vaccines for both seasonal and pandemic influenza seven priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: inter-pandemic seasonal influenza scenario 1. research on the burden of severe disease with a focus on regionalspecific factors, such as the burden of tb and hiv and optimization of pandemic and management 2. development of new antiviral strategies and validation of surrogate endpoints which may aid in advancing understanding of disease progression 3. further clinical evaluation of current antiviral drugs, particularly in populations at risk 4. integration of seasonal influenza with pandemic preparedness; strengthen surveillance, health care systems, capacity, and preparedness planning 5. improving diagnostics (e.g., multiplex assays for viruses and bacteria), including antiviral resistance testing at point-of-care 6. dissemination of best practices, situation analysis, preparation for next epidemic (e.g., establish protocols for rotating stockpiles of antiviral drugs) 7. increased attention to basic science research such as studying immunomodulatory drugs five priority public health topics were identified for an inter-pandemic zoonotic influenza scenario as follows: inter-pandemic zoonotic influenza 1. antiviral susceptibility of circulating zoonotic viruses (e.g., h5, h9, h7 influenza viruses) 2. reassortment between zoonotic and human influenza viruses and the potential for inter sub-type spread of antiviral resistance and virulence modern tools for early detection and monitoring of disease the group focusing on surveillance tools concluded that the agreed topics of interest were equally applicable during both pandemic and inter-pandemic period: 1. identify modern technologies for early detection of influenza outbreaks as well as their application in surveillance at the human-animal interface 2. develop and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems 3. studies to address challenges on data, clinical specimens, and viruses sharing with consideration for local, ethical, legal, and research perspectives 4. examine the timeliness and quality of data required for early detection from local to regional, national, and global levels role of modeling in public health decision making five priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: 1. integration of genetic and epidemiological data to understand spatiotemporal spread to forecasts evolution for vaccine strain selection and to anticipate likely burden of disease 2. quantifying the relative contributions of different modes of transmission of human influenza and developing mechanistic modeling of transmission processes 3. research using data-capture technologies to characterize human contact and mobility patterns at local, regional, and global scales, and their correlation with transmission risk 4. integration of genetic, antigenic, and epidemiological analyses to optimize surveillance for newly emerging pathogens at the animal ⁄ human interface 5. identifying and quantifying human and environmental ecological, behavioral, and demographic determinants of the risk of cross-species transmission and pandemic emergence modern tools for strategic communication four priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: 1. review of evidence and experience related to health crisis communication from fields to organize knowledge and support evidencebased practice in strategic communication 2. identify and develop tools to rapidly and accurately monitor knowledge, attitudes, and practices in different population groups and guide future communication efforts 3. identify and develop communication tools and approaches for cultural settings and communities to practice and promote appropriate risk reduction measures 4. understand the potential ethical, social, economic, and political communication in crisis and develop strategies to work within constraints while maximizing opportunities complemented by an analogous research roadmap for a pandemic influenza scenario, the research topic recommendations for an inter-pandemic period represent an important outcome of joint international efforts by who, academicians, and public health experts. implementation of the identified research priorities is expected to underpin public health decision-making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs, and economic loss and mitigate potential social disruption over a medium-tolong term period. the impacts of school resumption on the incidence of pandemic (h1n1) 2009 in school students introduction school closure is one non-pharmaceutical intervention that is often suggested in pandemic preparedness plans, and it was widely implemented in pandemic (h1n1) 2009 to reduce transmission amongst school students. however, from past epidemiological studies, the effect of school closure in reducing respiratory disease transmission was inconclusive. 1 given this public health intervention causes major disruption to the education system and potentially raises childcare issues to working parents, evaluating its effect in the recent pandemic is necessary to improve future pandemic planning. in hong kong, since school closure was implemented early in the pandemic and closure was effectively continued with the commencement of summer holiday, the lack of incidence data in the absence of school closure makes it difficult to analyse its effect directly. this has prompted us to analyse the situation indirectly from the angle of school resumption after summer holiday. in hong kong, public health surveillance on pandemic (h1n1) 2009 was effective from 25th april-30th september 2009: healthcare professionals were advised to report suspected cases of infection to centre for health protection, department of health, hksar, for further laboratorial confirmation. demographics of reported cases were subsequently recorded into a computerised system (the ''e-flu'' database). following institutional approval, a dataset of all confirmed cases diagnosed from may to september 2009 was obtained, which included the age, gender, confirmation date, and notification date of each report. all cases were classified into four defined socio-economic classes by age: pre-schoolers (0-5), school students (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) , adults (20-60), and retirees ( ‡61). assuming cases had contracted infection on the earlier date between confirmation and notification, daily incidence in each age class was counted for epidemic curve construction. upon observing an unusual rise in the epidemic curve of school students when school season resumed in september, interrupted time series analysis (also known as intervention analysis) 2 was applied to obtain the statistical significance of this observation. the analysis was applied to the incidence in school students from 12th july to 26th september 2009, which covered the period from the start of summer holiday to the end of the 4th week of new school season. incidence in school students before summer holiday was deliberately dropped since not all schools were closed when the school closure policy was effective: all primary schools were closed proactively, whereas secondary schools were individually closed on a reactive basis if students were identified to have contracted the infection. school activity was formulated as a step function, which takes value from 1st september 2009 onwards (st = 0: t < 1st september, st = 1 otherwise). a range of times series models were fitted by the maximum likelihood method and aic (akakine information criterion) was used to select the one with best fit. all computations were performed in sas version 9.2. a total of 3905 (14ae8%) pre-schoolers, 13758 (52ae1%) school students, 8383 (31ae8%) adults, and 338 (1ae3%) retirees were diagnosed with the infection in the surveillance period. the epidemic curves of preschoolers, school students, and adults showed a steady rise from 11th june onwards when local transmission of pandemic influenza was identified. an upsurge in the epidemic curve of school students can be observed in early september, coinciding with the commencement of the new school year (figure 1) . interrupted time series analysis on the epidemic curve of school students returned an arima(0,1,0) model with equations: where st, yt, yt denote school activity, predicted and actual incidence in school students on day t, respectively. standard error and significance for model constants were: 60ae27 (se = 33ae5, p = 0ae08), 2ae73 (se = 3ae84, p = 0ae48). in short, the model can be interpreted as: the number of infected school students rose by 2ae73 per day on average during the entire study period, with a sharp increase by 60ae27 coming into effect when the new school year began. time series analysis showed, at the marginally significance level, that daily incidence in school students had a major increase when school season resumed. on the assumption that the increase was not caused by any change in health seeking behaviour, this result suggests that school resumption had facilitated transmission amongst school students. on the basis that school activity significantly increases incidence of pandemic influenza in school students, this study suggests closures of schools in the early phase of pandemic (h1n1) 2009 and subsequently in the summer holiday probably had a major effect in mitigating transmission amongst school students. youngsters were postulated to be major vector for transmission in pandemic (h1n1) 2009. if this were true, it would be reasonable to expect the epidemic curves of the other age classes to show a similar upsurge when one is observed in school students. the absence of such observation in the epidemic curve of hong kong suggests school students were mostly disseminating the virus amongst themselves, but not to the other age groups. in november 2009, gip convened the first global consultation on a public health research agenda for influenza to identify key research topics in each of the five main streams of public health research. during this meeting, the scientific working group (swg) of the sub-stream in ''modern tools for risk communication'' identified the requirements in research during influenza pandemics and inter-pandemic periods to provide clear, credible, and appropriate messages which meet the needs of diverse communities. the swg suggested that who hold a follow-up workshop to assess the use of modern tools related to strategic and risk communication and to further promote research in these areas. communication'' in may 2010. one of the main objectives of the meeting was to generate a roadmap of public health research priorities related to strategic and risk communication. the research roadmap was developed by the group of invited experts on the basis of an analysis of available evidence and experience on public health and health crisis communication from relevant disciplines across global regions, as well as critical assessment of existing communication methods related to influenza control in different cultural, social, and ethnic settings. the workshop consisted of a series of presentations by experts in relation to experiences and lessons learned about communication during the sars, h5n1 epidemic, and h1n1 pandemic. there were also a series of group discussions on identifying research needs for pandemic and interpandemic periods in order to strengthen the research agenda. the expert group identified important public health needs in relation to communication during pandemics as well as in the inter-pandemic times. the main topics of discussion centered on communicating issues of influenza virus transmission, the use of influenza vaccines safety and efficacy, and use of antivirals as well as definition of the severity of the pandemic and the phase changes. in this context a number of research areas were identified, which can be broadly classified into four areas: understanding of communication principles and mechanisms is associated with an array of research topics covering different subject areas. one of the key questions here relates to the link between communication and ''behaviour change'' models and their application and appropriateness for different settings. the expert group defined the term ''behaviour change'' in this context as the modification of behaviour towards better health practices that are supported by clinical and scientific evidence for personal protection against infectious diseases and other adverse health risks. research topics related to these models require understanding and differentiating information and ''behaviour change'' needs of different audience segments, such as stakeholder mapping, target audience analysis, research into behaviour motivation, social norms, and the cultural, religious, social, legal, and political barriers and enablers of particular behaviors that are beneficial in influenza control. this research area also includes the analysis of media consumption among different audiences, role models, including ways to analyse how rumours and misinformation are spread, and ways to provide evidence-based information correctly. other important areas of investigation embrace methods to communicate uncertainty, learning how to build trust while communicating about a pandemic, and understanding what needs to be done before, during, and after a pandemic in order to create the best environment for influenza pandemic communication. critical key audiences identified for more intensive analysis were health workers, religious, public health, and societal (political and community) leaders. • investigation of the role of different communication channels and communication formats for different target audiences in a pandemic, particularly for groups that are ''hard-to-reach.'' • determining effects of perceptions related to pandemic influenza (severity, susceptibility, response efficacy, self efficacy, perceived social norms) on protective behaviours in different groups. • understanding audience in terms of their knowledge, preventive activities, and reasons why engaged ⁄ not engaged. • developing mechanisms to synergies between risk communication and behavior oriented approaches in the pandemic and inter-pandemic phases. • determining social, economic, cultural, and religious factors which support behaviours to limit spread and minimize impact in different settings. • identification of the key predictors ⁄ factors that influence people's behavior among different groups and populations vis-à -vis pandemic flu behaviors. • identification of elements that contribute to trust among populations and in different settings (country, public, professional, community), particularly where trust was previously compromised. • understanding psychology of different groups regarding their response to uncertainty, and finding the best way to communicate uncertainty. the research questions in this section relate to the planning, development, and evaluation of tools that can be quickly accessed and used in a pandemic situation. these may include communication materials and channels; the setting up of key stakeholder and champion communication networks; research protocols that are ready for rapid assessment during a pandemic or new communication tools. the use and understanding of terminology and language by both lay and professional groups and communities in planning for and ⁄ or reacting to a pandemic are important areas of research. acute examples, such as the naming of the viruses or the use of the word ''pandemic,'' illustrate this need well. the research focus of this area is to look at lessons learned from the a(h1n1) 2009 pandemic and to document and evaluate case studies, both looking at best practices, challenges, and barriers that were experienced. different communication strategies need to be evaluated and models to be built not only in terms of reach, but also in terms of impact on thinking, emotional response, and behavioural modification. a key question was how to prepare communication for a pandemic and how can the pandemic communication contribute to longer term ''behavioural change.'' mathematical modelling on gauging outcomes of such ''behaviour change'' would provide strategic approaches in risk communication. this section aims to answer the question whether the modeling, mapping, and scenario planning are actually useful in the pandemic situation. the expert group agreed that the research on the above issues should use a variety of methods and engage a number of disciplines. this would include literature reviews, case studies, trials, ethnographic studies, modelling, surveys, network analysis, as well as any other useful methodology. in an inter-pandemic situation for actual behaviour under pandemic conditions. • study the synergies and develop priority research topics on strategic ⁄ risk communication for influenza under inter-pandemic situations that includes zoonotic and seasonal infections. the who public health research agenda for influenza initiated and facilitated a multi-disciplinary discussion for communication during pandemic and inter-pandemic situations. it focused on both theoretical and practical issues to improve practice and ensure the health of the public for influenza. critical areas for research were identified to build evidence in this field. it was recognized that there are extensive bodies of knowledge in a number of disciplines, 2,3 such as health promotion, behavioural psychology, social sciences, social and behaviour change communication, social marketing, and communication for development relating to these questions, and that these should be explored. outcomes of these research activities are expected to widen the evidence base which will support developing communication strategies for influenza by countries, institutions, and individuals and will, consequently, help to improve public health world-wide. abstract background: cytokine dysregulation contributes to the unusual severity of h5n1 (reviewed in 1 ). previously, we demonstrated that interferon regulatory factor 3 (irf3) and p38 map kinase (p38) signaling pathways separately contribute to the induction of pro-inflammatory cytokines and chemokines in h5n1-infected cells. 2 here we investigate the role of innate sensing receptors in the induction of these cytokines and chemokines in response to h5n1 and seasonal h1n1 infection. materials and methods: human macrophages derived from peripheral blood monocytes were infected with h5n1 (483 ⁄ 97) or seasonal h1n1 (54 ⁄ 98) viruses. the role of innate sensing receptors in cytokine and chemokine induction by h5n1 virus was investigated using transient knock-down of these receptors with sirnas. the expression of innate sensing receptors in infected cells, and as a result of paracrine activation (by virus free supernatants of infected cells) of adjacent uninfected cells were also monitored by real-time pcr and ⁄ or western blotting. the involvement of janus kinase (jak) signaling pathways in these autocrine ⁄ paracrine cascades was investigated using a jak inhibitor. results: we previously showed that tnf-alpha, ifn-beta, and ifn-lambda 1 are the key mediators directly induced by the h5n1 virus in primary human macrophages with other cytokines and chemokines being induced as part of a secondary autocrine and paracrine cascade. here we demonstrated that retinoicacid-inducible gene i (rig-i) rather than toll-like receptor 3 (tlr3) plays the predominant role in h5n1-induced cytokines and chemokines in human macrophages via the regulation of irf3 and nf-kb nuclear translocation. in addition to the effects on virus infected cells, paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the upregulation of sensing receptors. conclusions: h5n1 directly induced tnf-alpha and ifnbeta mainly via rig-i signaling, and the subsequent activa-tion and nuclear translocation of irf3 and nf-kb in human macrophages. in addition to the effects on cytokine signaling, the innate immune sensing regulators themselves were also up-regulated by h5n1 infection, much more so than by seasonal influenza infection, via jak signaling. the up-regulation of innate sensing receptors was not limited to the infected cells, but was also found in adjacent uninfected cells through paracrine feedback mechanisms. this may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. a more precise understanding of the signaling pathways triggered by h5n1 virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h5n1 influenza and also for treating other causes of acute respiratory disease syndrome. human h5n1 infection is associated with a mortality rate of more than 60%. the basis for the unusual severity of h5n1 disease has not been fully explained. cytokine dysregulation has been suggested to contribute to the disease severity of h5n1 (reviewed in 1 ). however, signaling pathways involved in the cytokine induction by h5n1 virus are not fully understood. previously, we demonstrated that irf3 and p38 map kinase (p38) are separate signaling pathways which contribute to the induction of pro-inflammatory cytokines and chemokines in h5n1-infected cells. 2 rig-i and melanoma differentiation-associated gene 5 (mda5) are important cytosolic sensors of nucleic acid of pathogens, while tlr3 and tlr8 also recognize nucleic acid species of pathogens, but they are localized at the endosomal membrane. 3 rig-i was found to be responsible for the recognition of influenza a virus infection, 4 and the transfection of vrnps induces ifn-beta expression. 5 while many studies have shown the role of rig-i in the induction of ifn-beta by influenza virus infection, the majority of these studies used either immortalized cell lines or mouse embryonic fibroblasts. there is a lack of data on the role of these innate sensing receptors in highly pathogenic avian influenza h5n1 infection in primary human cells in vitro, which are more physiologically relevant. furthermore, there is little data on the autocrine and paracrine up-regulation of these innate immune sensors following virus infection. human macrophages were obtained from peripheral blood monocytes by adhesion and differentiation in vitro for 14 days in rpmi medium supplemented with 10% autologous plasma. the cells were infected with h5n1 (483 ⁄ 97) or seasonal h1n1 (54 ⁄ 98) viruses at a moi of 2ae0. a549 cells were obtained from atcc and cultured in mem medium supplemented with 10% fcs and 1% penicillin and streptomycin. the role of innate sensing receptors in cytokine induction by h5n1 and h1n1 viruses was investigated using transient knock down of these receptors with sirnas in human macrophages as previously described 2 using specific sirnas purchased from qiagen. immunofluorescence staining assay of irf3 and nf-jb was employed to detect the nuclear translocation of these transcription factors after h5n1 infection. rabbit polyclonal antibodies against human irf3 and and nf-kb were obtained from santa cruz biotechnology. goat anti-rabbit igg antibody conjugated with alexa fluor 488 was a product of molecular probes. for investigation of paracrine effects on rig-i and tlr3 expression, culture supernatants collected from mock, 54 ⁄ 98 or 483 ⁄ 97 infected human macrophages were used to treat uninfected cells. the supernatants were first passed through a filter with 100-kda cut-off. virus particles as well as molecules with a molecular weight higher than 100 kda were retained and removed, while the filtrate was collected for treatment of uninfected cells. the expression of innate sensing receptors in infected cells and in adjacent uninfected cells following paracrine activation by virus free supernatants of infected cells was monitored by real-time pcr. the involvement of jak signaling pathways in these paracrine cascades was investigated using a jak inhibitor (calbiochem). we previously showed that tnf-alpha, ifn-beta, and ifnlambda 1 are the key mediators directly induced by the h5n1 virus in primary human macrophages with others being induced as part of a secondary autocrine and paracrine cascade. 2 in this study, we demonstrate that knockdown of rig-i or tlr3 led to the reduction of ifn-beta and tnf-alpha in human macrophages by both 54 ⁄ 98 (h1n1) and 483 ⁄ 97 (h5n1) infection. as shown in figure 1a , 483 ⁄ 97 virus induced higher level of ifn-beta mrna expression than 54 ⁄ 98 infection. cells transfected with rig-i or tlr3 sirna significantly reduced the expression of ifn-beta after 483 ⁄ 97 infection, by 63% and 29%, respectively. rig-i silencing also significantly reduced the ifn-beta expression in 54 ⁄ 98 infected cells by 52%. in contrast, silencing of mda5 or tlr8 did not suppress the induction of ifn-beta by either 54 ⁄ 98 or 483 ⁄ 97 infection; in fact, there was a slight (15%) increase of ifn-beta in cells transfected with mda5 sirna. based on these results we conclude that while both rig-i and tlr3 contribute to h5n1-induced interferon-beta induction in human macrophages, rig-i plays the dominant role. in order to investigate the relationship between these innate sensing receptors and the activation of transcription factors irf3 and nf-jb, we next measured the nuclear translocation of irf3 and nf-jb in cells with rig-i or tlr3 silencing after h5n1 infection. immunofluorescence staining assay on irf3 and nf-jb was performed and the number of cells with nuclear translocation was quantitated. the percentages of cells with nuclear translocation were plotted in figure 1b . we demonstrated that rig-i knockdown led to a significant reduction of irf3 nuclear translocation after 483 ⁄ 97 infection, whereas the nuclear translocation of nf-jb after 483 ⁄ 97 infection was significantly suppressed by rig-i or tlr3 silencing. these results suggest that the involvement of rig-i and tlr3 in the cytokine induction by 483 ⁄ 97 was via the regulation of irf3 and nf-jb nuclear translocation. since rig-i and tlr3 are important in influenza a virus-induced cytokine expression, we next explored the expression of these innate receptors in neighboring uninfected human macrophages by treating the uninfected macrophages with the filtered culture supernatants collected from mock, 54 ⁄ 98, or 483 ⁄ 97 infected macrophages. as shown in figure 2a , 483 ⁄ 97 supernatant differentially induced the mrna expression of rig-i, mda5, and tlr3 compared to 54 ⁄ 98 supernatant treated human macrophages. the induction of rig-i was higher than the induction of mda5 and tlr3. in the presence of 1 lm of jak inhibitor, the up-regulation of all three innate sensing receptors was significantly reduced showing their induction was dependent on jak activity. human lung epithelial a549 cells were also treated with the supernatants collected from macrophages infected with mock, 54 ⁄ 98, or 483 ⁄ 97 virus. differential induction of rig-i, mda5 and tlr3 by 483 ⁄ 97 supernatant compared to 54 ⁄ 98 supernatant treated cells was observed (figure 2b) . 483 ⁄ 97 supernatant dramatically induced all three innate sensing receptors, while 54 ⁄ 98 supernatant only marginally induced rig-i and mda5, but not tlr3. as in human macrophages, treatment with 1 lm of jak inhibitor caused a significant suppression of 483 ⁄ 97 supernatantinduced rig-i, mda5, and tlr3 expression in a549 cells. these results, taken together with the direct effects on virus infected cells, suggest that paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the up-regulation of innate sensing receptors. h5n1 directly induced ifn-beta ( figure 1 ) and tnf-alpha (data not shown) mainly via rig-i signaling and the consequent activation and nuclear translocation of irf3 and nf-kb in human macrophages. these results were consistent with a previous study using beas-2b cells showing the essential role of rig-i in ifn-beta reporter activity by h3n2 influenza virus infection. 4 while tlr3 also played a role in induction of ifn-beta and the activation of irf3 and nf-kb, it plays a less important role compared to rig-i. the reduction of irf3 and nf-kb activation was also confirmed with the study by le goffic 4 showing differential regulation of irf3 and nf-kb by rig-i and nf-kb can also be regulated by tlr3. in addition to the direct role of rig-i and tlr3 in sensing and signaling the presence of influenza virus, the innate immune sensing regulators were themselves also highly upregulated in both infected (data not shown) and adjacent uninfected cells by influenza virus infection. compared with seasonal h1n1 virus, the h5n1 viruses had a much more dramatic effect on inducing innate sensing receptors via jak signaling pathways activated by autocrine and paracrine mediators. the up-regulation of rig-i, mda5, and tlr3 was markedly induced by virus free culture supernatants from h5n1-infected macrophages, while supernatant from 54 ⁄ 98-infected cells induced the expression of these receptors only to a lesser degree. the soluble mediators in the virus infected cell supernatant caused paracrine upregulation of rig-i, mda5, and tlr3 in uninfected macrophages as well as human lung epithelial cells. these effects may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. taken together these results provide, at least, part of the explanation on the hyper-induction of cytokines in h5n1 infection. a more precise identification of the signaling pathways triggered by h5n1 virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h5n1 influenza and also for treating other causes of acute respiratory disease syndrome. we generated mutants of y55 (h9n2) and a ⁄ duck ⁄ hokkaido ⁄ vac generation and characterization of mutant viruses rgy55sub (h9n2), rgvac1sub (h5n1), and rgvac1ins (h5n1), which have a serial basic amino acid residues at their ha cleavage sites were generated by site-directedmutagenesis and reverse genetics. rgy55sub (h9n2) and rgvac1ins (h5n1) required trypsin to replicate in mdck cells, and showed similar levels of growth to their parental viruses (table 1) . chickens intravenously inoculated with rgy55sub (h9n2) or rgvac1ins (h5n1) did not show any signs of disease. rgvac1sub (h5n1) replicated in mdck cells without exogenous trypsin, and one of the eight chickens inoculated with the virus showed slight depression at 1 day post-infection. the h9 and h5 mutant viruses were serially passaged in the air sacs of chicks to assess their ability to acquire pathogenicity. plaque formation in mdck cells and pathogenicity in 3-day-old chicks and 4-week-old chickens are shown in table 1 . rgy55sub (h9n2) replicated in mdck cells in the absence of trypsin and killed all of the chicks after six consecutive passages. two of the eight-four-weekold chickens inoculated intravenously with rgy55sub-p8 (h9n2) died within 5 days. eventually, over 75% of the chickens intravenously infected with rgy55sub-p10 (h9n2) died by 2 days post inoculation, and its pathogenicity was comparable to that of hpaivs. 10 rgvac1sub-p1 (h5n1) was pathogenic to both chicks and 4-week-old chickens, and mortality increased after one more passage. rgvac1ins-p1 (h5n1) replicated in mdck cells in the absence of trypsin, killed all of the chicks, and caused 75% mortality among 4-week-old chickens. the lethal effect of rgvac1ins-p1 (h5n1) on chickens increased with one additional passage in the air sacs of chicks, as in the case of rgvac1sub (h5n1). to examine whether the pathogenicity of each virus via the natural route of infection correlated with that by intravenous infection or not, three 4-week-old chickens were challenged intranasally with the viruses at an eid 50 of 10 6ae5 and observed for clinical signs until day 14 post-infection (data not shown). all chickens inoculated with rgy55sub-p10 (h9n2) or its parental viruses survived without showing any clinical signs, and serum antibody responses were detected in the hi test. on the other hand, rgvac1sub-p2 (h5n1) and rgvac1ins-p2 (h5n1) were pathogenic as in the intravenous experiment, killing two of three chickens by day 11 post-inoculation. one of three chickens were not infected with rgvac1sub-p2 (h5n1) or rgvac1ins-p2 (h5n1) via intranasal route (data not shown), indicating these p2 viruses had not been completely adapted to the host. to investigate the possibility of these p2 viruses to acquire further pathogenicity for chicken, rgvac1sub-p3 (h5n1) and rgvac1ins-p3 (h5n1) were obtained from the brain homogenates of the chickens that died on 11 days post intranasal inoculation with the p2 viruses. although mortality rate of chickens inoculated with the p3 viruses was equal to that with p2 viruses, enhancement of pathogenicity was observed in intranasal inoculation study; all of the chickens inoculated with rgvac1sub-p3 (h5n1) were infected, and time to death was shortened to 4-6 days post inoculation in chickens with rgvac1ins-p3 (h5n1) (data not shown). to investigate whether tissue tropism of the viruses was involved in their pathogenicity, we determined viral titers in the tissue and blood samples from 4-week-old chickens intranasally inoculated with each virus on 3 days post infection ( table 2) . rgy55 (h9n2) and rgvac1 (h5n1) were scarcely recovered from the samples, and the mutant strains before passage showed broader tissue tropism than the parental viruses. none of the chickens inoculated with rgy55sub-p10 (h9n2) showed any signs of disease, and viruses were recovered from each of the samples except the brain and the blood. one chicken inoculated with rgvac1sub-p2 (h5n1) showed clinical signs such as depression, and viruses were recovered from virtually all of its organs and blood samples. two of three chickens inoculated with rgvac1ins-p2 (h5n1) showed disease signs, and one died 2 days post inoculation. the viruses were recovered from almost all samples of the two chickens showing signs of disease. p3 viruses were efficiently replicated in systemic organs of the chickens as compared with p2 viruses. throughout the study, the viruses were recovered from the brains of all of the chickens showing clinical signs. here, we demonstrated that the h9 influenza virus acquired intravenous pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the ha and passaged in chicks. rgy55sub-p10 (h9n2) killed 75% of chickens infected intravenously, and its pathogenicity was comparable to that of hpaivs (table 1) . however, chickens intranasally inoculated with rgy55sub-p10 (h9n2) did not show any clinical signs of disease (data not shown). these results are consistent with a previous study in chickens that found some h10 influenza viruses did not show intranasal pathogenicity although their intravenous pathogenicity index was over 1ae2, classified as hpaiv according to the definition by european union. 11 ohuchi et al. 12 reported that the insertion of additional basic amino acids into the h3 ha cleavage site resulted in intracellular proteolytic cleavage. other groups reported that h3 and h6 has tolerated amino acid mutations into their cleavage sites, and the viruses with the mutated has replicated in mdck and ⁄ or qt6 cells in the absence of trypsin. 13, 14 the results in the present study is in agreement with these, namely, cleavage-based activation by a ubiquitous protease is not restricted to the h5 and h7 has. the intranasal pathogenicity of the h9 and h5 mutants were different (data not shown), although these viruses similarly replicated in mdck cells in the absence of trypsin and killed chickens by intravenous inoculation ( table 1) . the viruses were recovered from the brain and the blood of some chickens infected with rgvac1 mutants (h5n1), and morbidity was closely associated with viral titers in the brain (table 2) . on the other hand, no viruses were recovered from the brain of chickens infected with rgy55 mutants (h9n2), explaining why rgy55sub-p10 (h9n2) did not show intranasal pathogenicity. all the viruses passaged in the air sacs of chicks killed chicken embryos by 48 hours post allantoic inoculation (data not shown). rgvac1sub-p3 (h5n1) and rgvac1ins-p3 (h5n1) were more pathogenic to chicken embryos than rgy55sub-p10 (h9n2); the allantoic fluid obtained from the embryonated eggs inoculated with the h5 viruses passaged in air sacs was turbid. it has been reported that infection of a highly pathogenic h7 virus were strictly confined to endotherial cells in chicken embryos or chickens. 15, 16 therefore, it is suggested that endotheliotropism differed between the h9 and h5 viruses passaged in air sacs and affected their intranasal pathogenicity. taken together, it is assumed that rgvac1sub-p3 (h5n1) and rgvac1ins-p3 (h5n1) showed marked intranasal pathogenicity with high levels of viremia caused by replication in vascular endothelial cells, leading to invasion of the brain. in the intravenous experiment, rgy55sub-p10 (h9n2) easily reached systemic organs, including the brain hematogenously, replicated through the cleavage of ha by a ubiquitous protease, and then exerted its pathogenicity. further study including a pathological analysis is currently underway to test this hypothesis. for all hpai viruses of subtypes h5 and h7 known to date, the cleavage of ha occurs at the c-terminal r residue in the consensus multibasic motifs, such as r-x-k ⁄ r-r with r at position p4 and k-k ⁄ r-k ⁄ t-r with k at p4, and leads to a systemic infection. early studies demonstrated that the ubiquitously expressed furin and pcs are activating proteases of hpai viruses. 1 furin and pcs cleave the consensus multi-basic motif r-x-k ⁄ r ⁄ x-r with r at position p4. 2 however, replacement of p4 r by k and a nonbasic amino acid significantly suppresses the processing activities of furin and pcs. 2 most of the type ii transmembrane serine protease identified so far recognize a single r at position p1, but the newly isolated mspl and its transcript variant tmprss13 preferentially recognize paired basic residue, particularly r and k at position p4, at the cleavage site. [3] [4] [5] thus, mspl and tmprss13 can activate various bioactive polypeptides with multibasic residue motifs, including fusogenic viral envelope glycoproteins. the present study was designed to characterize the proteolytic processing of the hpai virus ha by mspl and tmprss13 in comparison with furin. hpai virus a ⁄ crow ⁄ kyoto ⁄ 53 ⁄ 2004 (h5n1) 6 was isolated from embryonated eggs inoculated with tracheal homogenates from dead crows. then, the mutant ha sequence was constructed by changing r residue to k residue (n'-rkkr-c' to n'-kkkr-c') at the ha cleavage site by sitedirected mutagenic pcr as described. 7 we used human cell line ecv304, which expresses mspl and tmprss13 at levels below detection, and established the cells stably expressing mspl and tmprss13, such as ecv304-mspl and ecv304-tmprss13. 7 to determine the cleavage specificities of mspl ⁄ tmprss13 and furin, peptides (20 lg each) were incubated with 0ae25 mu mspl ⁄ tmprss13 for 1 hour and furin for 8 hours at 37°c, respectively. after incubation, the samples were separated by reverse-phasehigh-performance liquid chromatography (rp-hplc) with the use of a c 18 column. the elution samples were then identified by amino acid sequence analysis and by maldi-tof-ms. we analyzed the cleavability of 14-residue synthetic peptides derived from ha cleavage sites of hpai strains, such as a ⁄ chick ⁄ penn ⁄ 1370 ⁄ 83 (h5n2) 8 and a ⁄ fpv ⁄ rostock ⁄ 34 (h7n1), 9 and low pathogenic strain a ⁄ aich ⁄ 2 ⁄ 58 (h3n2). after incubation with human mspl or human furin, the digested samples were separated by rp-hplc, and peptide fragments were characterized by mass-spectrometry and protein sequencing. in contrast to the low cleavage efficiencies of the h3 ha peptide with a single r at the cleavage site ( figure 1a) , both the h5 ha peptide with the k-k-k-r motif ( figure 1b ) and the h7 ha peptide with the r-k-k-r motif ( figure 1c) were fully processed at the correct positions by mspl within 1 hour. in the case of h7 ha peptide with multiple basic residues, mspl cleaved two carboxyl-terminal sides of r in the cleavage site sequence of n'-k-k-rfl-k-k-rfl-g-c', while furin cleaved only at a single site of r with r at position p4, n'-k-k-r-k-k-rfl-g-c' in the presence of 1 mm cacl 2 . these cleavage site specificities of furin were consistent with that reported for the h5 ha peptide of hpai virus a ⁄ hong kong ⁄ 156 ⁄ 97 (h5n1) with r-k-k-r motif. 1 however, the h5 ha peptide with k at position p4 ( figure 1b) was hardly cleaved by furin under the same experimental conditions. tmprss13 showed similar results (data not shown). these findings suggest that mspl and tmprss13 cover diverse cleavage specificities, including non-susceptible specificity to furin. full length recombinant ha of hpai virus with kkkr cleavage motif was converted to mature ha subunits with membrane-fused giant cell formation in mspl or tmprss13 transfectant cells. 7 in addition, this conversion was suppressed by bowman-birk trypsin inhibitor, a membrane non-permeable highmolecular mass inhibitor against mspl ⁄ tmprss13. to test for the generation of infective virus, the conditioned media of 1-day culture of ecv304-wt and ecv304-mspl cells infected with wt and mutant hpai h5n1 viruses were inoculated into newly prepared cells and cultured for 24 hours. although spreading of wt virus infection with ha cleavage motif of r-k-k-r was detected from the conditioned medium of both ecv304-wt and ecv304-mspl cells, that of mutant virus with ha cleavage motif of k-k-k-r was only detected from the condition medium of ecv304-mspl cells. these results strongly suggest that the expression of mspl, but not furin, potentiates multicycles of hpai virus with k-k-k-r ha cleavage motif. seasonal human influenza a virus has have consensus monobasic cleavage site sequence, n'-q ⁄ e-x-rfl-g-c', and all hpai virus has have two types of cleavage site sequences with multiple basic amino acids, n'-r-k ⁄ r-k ⁄ r ⁄ x-rfl-g-c' with r at position p4 in a large number of hpai viruses and n'-k-k ⁄ r-k ⁄ t-rfl-g-c' with k at position p4 in a small number of hpai viruses. figure 1 shows furin efficiently cleaved synthetic hpai a ⁄ hong kong ⁄ 156 ⁄ 97 (h5n1) ha cleavage site peptide with the r-k-k-r motif, but hardly cleaved the hpai virus a ⁄ chick ⁄ penn ⁄ 1370 ⁄ 83 ha cleavage site peptide with the k-k-k-r motif. furthermore, cleavage of the full-length ha of hpai virus with r-k-k-r motif was detected, but cleavage of hpai virus ha with k-k-k-r motif was hardly detected in ecv304-wt cells containing furin ( figure 2 ). these substrate specificities of furin suggest that proteases other than furin and pc5 ⁄ 6 play a role in the processing of has of hpai virus with k-k ⁄ r-k ⁄ t-r cleavage motif. mspl and tmprss13 show unique cleavage site specificities of the double basic residues at the cleavage site, and r or k at position p4 greatly enhanced the efficiency, which none of the other ttsps have shown similar substrate specificities so far. furthermore, infectious and multicycle viral replication along with ha processing was also noted in genetically modified mutant recombinant live hpai virus a ⁄ crow ⁄ kyoto ⁄ 53 ⁄ 2004 (h5n1) with k-k-k-r cleavage motif in ecv304-mspl cells (figure 2) . these results were supported by the data of two cleaved peptides by mspl in figure 1c . these findings suggest that mspl has diverse cleavage specificities and may cleave ha at least two sites, although multiplicity of the mutant hpai virus was observed under the conditions. these results also suggest that mspl and tmprss13 in the membrane might potently activate the ha membrane fusion activity of hpai viruses and promote their spread. highly pathogenic avian influenza viruses replicate in various organs in birds, and the ha processing proteases might be widely distributed in these organs. indeed, tmprss13 and mspl are ubiquitously expressed in almost all human organs tested and are highly expressed in lungs, leukocytes, pancreas, spleen, and placenta. 4, 5 in addition, mspl and tmprss13 are strictly localized in the plasma membranes, suggesting that proteolytic activation of hpai virus ha occurs not only through the trans-golgi network by furin and pc5 ⁄ 6, but also on the cell surface by mspl and tmprss13. the pb1-f2 protein, which is translated from the +1 reading frame of the pb1 gene segment, has been linked to the pathogenesis of both primary viral and secondary bacterial infections in a mouse model. 1-3 a mitochondrial targeting sequence is located in the c-terminal portion of the pb1-f2 open reading frame, and expression of full length pb1-f2 has been associated with mitochondrial targeting and apoptosis in a monocyte dependent manner. 1, 4 it has been theorized that enhanced virulence could result from mitochondrial disruption with subsequent cell death mediated by pb1-f2. 4, 5 a suggested second function of the pb1-f2 protein is that it enhances immunopathology by triggering the inflammatory response. 3, 6 in earlier studies from our group, the pro-inflammatory phenotype was markedly upregulated when the pb1-f2 from the 1918 pandemic strain was expressed, arguing that this protein may be an important virulence factor for highly pathogenic pandemic viruses. 3, 6 in this report we analyze the pb1-f2 protein's contribution to pathogenesis in a mouse model, examining both inflammation and cell death. pb1-f2 proteins from a variety of epidemiologically important iav strains including all pandemic strains from the 20th century, a highly pathogenic avian influenza virus of the h5n1 subtype, and representative seasonal strains were utilized to determine the relevance to pandemic disease. we demonstrate that macrophage mediated immunopathology, but not apoptosis, are relevant functions of pb1-f2 proteins from past or potential pandemic influenza viruses. using the predicted amino acid sequences of the pb1-f2 proteins from pr8, a ⁄ brevig mission ⁄ 1 ⁄ 1918, 1 ⁄ singapore ⁄ 1 ⁄ 1957, a ⁄ hong kong ⁄ 1 ⁄ 1968, a ⁄ wuhan ⁄ 359 ⁄ 1995, and a ⁄ vietnam ⁄ 1203 ⁄ 2004, peptides from the c-terminal end were synthesized as described. 3 an additional n-terminal peptide was synthesized from the pr8 sequence as a positive control (mgqeqdtpwilstghistqk) as described. 3 a panel of viruses were reverse engineered as described 7, 8 and included laboratory strain pr8, a virus unable to express pb1-f2 (dpb1-f2 ⁄ pr8), or expressing the pb1-f2 of the 1918 pandemic strain (1918 pb1-f2 ⁄ pr8) or the truncated 1956 h1n1 strain (beij pb1-f2 ⁄ pr8). 3, 7 in addition, 7:1 reassortants encoding pb1 gene segments from a *current address: department of immunology and microbiology, university of melbourne, melbourne, vic., australia. 2004 highly pathogenic avian influenza of the h5n1 subtype (h5n1 pb1 ⁄ pr8), or from a 1995 human h3n2 strain (h3n2 pb1 ⁄ pr8) were utilized along with their isogenic deletion mutants for pb1-f2 (h5n1 dpb1-f2 ⁄ pr8 and h3n2 dpb1-f2 ⁄ pr8). cell lines and cell death assays raw264.7 cells were grown under conditions as described. 9 cells were infected with one multiplicity of infection (moi) of virus for 2-12 hours, or exposed to 50 lm (final concentration) of peptides derived from the c-terminal portion of pb1-f2 for 1 hour. cells from the supernatant and monolayers were harvested, washed, and stained with annexin (apc) and propidium iodide (pi) (becton dickinson, san jose, ca, usa), then analysed for cell death as described. 3 six-to eight week old female balb ⁄ cj mice (jackson laboratory, bar harbor, me, usa) were maintained in a biosafety level 2 facility in the animal resource center and procedures approved by the animal care and use committee at sjcrh. infectious agents and peptides were diluted in sterile pbs and administered intranasally to anesthetized mice (n = 6-10) in a volume of 100 ll (50 ll per nare) and monitored for overt signs of illness and weight loss daily. following euthanasia by co 2 inhalation, the trachea was exposed and cannulated with a 21 gauge plastic catheter (bd insyte; becton dickinson, sandy, ut, usa). bronchoalveolar lavage fluid (balf) was collected, red blood cell depleted, and cellular content analyzed via flow cytometry as described. 3 one way analysis of variance (anova) was used for multiple comparisons of cell death and cellularity of balf. a p-value of <0ae05 was considered significant for these comparisons. graphpad prism version 5.00 for windows (graphpad software, san diego, ca, usa) was utilized for all statistical analyses. to assess the contribution of pb1-f2 to inflammation, we utilized a panel of previously described reverse engineered viruses in the mouse infection model. 3, 7 the effect of pb1-f2 expression was observed clearly in the inflammatory infiltrate in response to infection in the lungs. deleting pb1-f2 from pr8 or expression of the c-terminally truncated beij pb1-f2 had a significantly reduced influx of macrophages ( figure 1a) . expression of the 1918 pb1-f2 caused similar inflammatory effects as the pr8 virus. disruption of pb1-f2 expression the virus containing the h5n1 pb1 gene segment in a pr8 background also significantly decreased the inflammatory response compared to the virus maintaining the ability to express full length pb1-f2 ( figure 1a) . however, no differences were seen that could be attributed to the 1995 h3n2 derived pb1-f2. the lungs of mice infected with the panel of pb1-f2 variant viruses were examined at 72 hours. pathologic changes typical of pr8 viral infection were observed in all lungs. these typical findings included perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris (figure 2 ). in the lungs of mice infected with pr8 or 1918 pb1-f2 ⁄ pr8, however, significantly more perivascular cuffing was noted, with a prominent increase in numbers of macrophages (figure 2a, c) . the overall number of inflammatory cells throughout the lungs, including both airways and alveoli, was quantitatively greater in these mice than in mice infected with dpb1-f2 ⁄ pr8 or beij pb1-f2 ⁄ pr8 ( figure 2b, d) . as the function and influence of pb1-f2 protein on normal viral function is not currently understood, and given the abrogation of enhanced inflammation induced by the truncated pb1-f2 beij ⁄ pr8 virus, we sought to elucidate whether the c-terminal domain of pb1-f2 could alone induce this inflammatory response. mice were exposed to a panel of peptides and were euthanized 24 hours later for collection of balf. significant influxes of macrophages into the balf were seen following exposure to c-terminal pb1-f2 peptides derived from pr8, the pandemic strains from 1918 (h1n1), 1957 (h2n2), and 1968 (h3n2), and the 2004 h5n1 virus compared to controls ( figure 1b) . similar effects were not seen with the peptide derived from a more recent h3n2 strain, a ⁄ wuhan ⁄ 359 ⁄ 1995. when peptide exposed mice were followed for morbidity for 7 days, peptides proven to induce a heightened inflammatory response correlated strongly with overt clinical signs of illness (data not shown). thus, the ability to cause lung inflammation appears to be a property of pb1-f2 proteins of viruses containing pb1 gene segments reassorted directly from the avian reservoir. the pb1-f2 protein may contribute to virulence by rendering the host cellular immune response ineffective through inducing apoptosis. 5 we sought to determine whether this was an epidemiologically important function for combating the host immune response to infection by testing the ability of pb1-f2 proteins from several different iav strains to cause cell death. we therefore infected raw264.7 cells with the panel of recombinant viruses at an moi of 1 for 2-12 hours. as has been demonstrated previously, 1,4,5 pr8 virus induces significant cell death compared to uninfected controls ( figure 1c ). when raw264.7 cells were infected with pr8 virus, necrotic death peaked 8 hours after infection. viruses lacking the c-terminal portion of pb1-f2, including the dpb1-f2 ⁄ pr8 and the beij pb1-f2 ⁄ pr8 were unable to cause cell death ( figure 1c ). in addition, expression of the 1918 pb1-f2 also did not cause significant increases in cell death over controls. expression of pb1-f2 or deletion of pb1-f2 in either an h3n2 or h5n1 pb1 gene segment background similarly did not alter the cell death phenotype. to examine additional strains for which we did not have isogenic virus pairs, we next exposed the balbcj mouse derived macrophage cell line raw264.7 to the panel of pb1-f2 peptides derived from pr8, the pandemic strains from 1918 (h1n1), 1957 (h2n2) and 1968 (h3n2), and the 2004 h5n1 for 1 hours. cell death in raw264.7 cells was caused only by the peptides derived from the laboratory strain pr8 and the peptide derived from the 1918 pandemic strain ( figure 1d ). viability was not affected by exposure of raw264.7 to peptides derived from other virus strains. we conclude from these data that the mechanism by which pb1-f2 contributes to the pathogenicity of pandemic influenza is unlikely to be through its reported ability to cause cell death. these data presented here demonstrate that the lung inflammatory response is enhanced by the influenza a virus pb1-f2 protein in a mouse model. this inflammatory response was characterized by increased cellular infiltration of macrophages into the interstitial and alveolar spaces of the lungs, as well as enhanced perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris. this augmentation was shown to be induced by pb1-f2 proteins only from those strains contributing to the formation of all pandemic strains of the 20th century and from the currently circulating, highly virulent h5n1 strains that constitute an imminent pandemic threat. the iav h1n1 strains circulating in humans since around 1950 code for a truncated pb1-f2. these viruses may lack the cterminal residues responsible for the inflammatory effects demonstrated in this publication. additionally, recently circulating h3n2 strains, in contrast to their pandemic forbear from 1968, have lost the capacity to cause pb1-f2 mediated inflammation through mutation of the c-terminus of this protein. in 2009 a novel h1n1 iav emerged from an animal reservoir and caused a human pandemic. disease burden from this strain has been considered mild 10 in contrast to the three pandemics of the 20th century. 11 the reasons for this disparity in pathogenesis are unclear. an examination of the origins of the three 20th century pandemics shows that only 2 the hemagglutinin (ha) and pb1 gene segments were reassorted directly from the avian reservoir in every case, suggesting gene products of one or both of these may be important. 12 the ha surface glycoprotein provided the antigenic novelty required for the each virus to achieve pandemic status. however, the significance of inclusion of a novel pb1 gene segment in each of the 20th century pandemics is not yet understood. we show here that the pb1-f2 of these pandemic strains contributes to virulence through induction of inflammatory responses. thus pb1-f2 may serve as a marker of the pathogenicity of pandemic strains. since the 2009 h1n1 strain codes a truncated pb1-f2 of only 11 predicted amino acids, the lack of pb1-f2 mediated inflammation may account in part for its relatively lower virulence. 13, 14 of the panel of pb1-f2 proteins studied, only that from the laboratory strain pr8 was capable of rendering responding host-immune cells ineffective by induction of cell death. we therefore hypothesize that molecular signatures specific to induction of apoptosis may have been lost through genetic mutation of the pb1-f2 gene throughout the evolution of the iavs. our findings suggest that this apoptotic function is unlikely to be important for the virulence of any of the known pandemics. rather, the inflammatory phenotype appears to be the dominant contribution of pb1-f2 to pandemic disease. influenza virus-cytokine-protease cycles are principal mechanisms of multi-organ failure in severe influenza and therapeutic approaches introduction influenza a virus is the most common infectious pathogen in humans, causing significant morbidity and mortality, particularly in infants and the elderly. mof with severe edema is observed in the advanced stage of influenza pneumonia. 1 however, the relationships amongst factors that induce vascular hyper-permeability in severe influenza remain unclear. it is reported that significant increases in levels of pro-inflammatory cytokine levels, such as tnf-a, il-6, and il-1b, affect host survival both positively and negatively. 2 the inflammatory response affects cell adhesion, permeability, apoptosis, and mitochondrial reactive oxygen species, potentially resulting in vascular dysfunction and mof. 3 in addition, iav infection up-regulates several cellular proteases including ectopic trypsin 4 and mmp-9. 5 up-regulated ectopic trypsin mediates the post-translational proteolytic cleavage of viral envelope hemagglutinin (ha), 6 which is crucial for viral entry and replication 7 and the subsequent tissue damage in various organs. 8 the aim of the this study was to define the pathogenic impact of cytokine storm in iav infection and the molecular mechanisms by which pro-inflammatory cytokines and proteases cause vascular dysfunction in animal model. weanling female mice aged 3 weeks (c57bl ⁄ 6crslc) were infected with iav ⁄ wsn ⁄ 33 (250 pfu) with and without treatment of pdtc (2.5 mg ⁄ kg), nac (10 mg ⁄ kg), and ndga (10 mg ⁄ kg). these inhibitors were administrated once daily for 4 days after infection. the levels of cytokines in tissue homogenates were measured by elisa kits. the effect of inhibitors on viral replications was determined by real-time pcr. gelatin zymography and western blotting were conducted as reported previously. 9 host cellular responses in the airway after iav infection figure 1 shows schematic view of typical biological responses in the airway of mice after iav infection. an initial response before viral proliferation is significant increases in pro-inflammatory cytokine levels. immediately after cytokine inductions, there is a marked up-regulation of ectopic trypsin along with an increase in virus titer in the airway, lung, and brain. 4 ectopic trypsin mediates the post-translational proteolytic cleavage of iav ha, which is crucial for viral entry and replication and the subsequent tissue damage in various organs. we also found that iav infection markedly induces mmp-9 and matrix degradation. 5 just after the peak of viral proliferation, the innate and adaptive immune responses of protective immunity are induced for defense and recovery, or oppositely on rare occasions, mof with vascular hyper-permeability is started into the advanced stage of influenza. the levels of tnf-a and il-6 in the lungs were increased persistently for 6 days after iav wsn infection, and that of il-1b peaked at days 4-6 post-infection (figure 2a ). since these cytokine responses are associated with activation of nf-jb and ap-1, we treated mice once daily for 4 days with anti-oxidant inhibitors: pdtc and nac against nf-jb activation, and ndga against ap-1 activation. pdtc and ndga significantly suppressed the up-regulation of tnf-a and il-1b (p < 0.001), and nac suppressed tnf-a (p < 0.001), and il-6 (p < 0.01) at day 4 post-infection. gelatin zymography showed up-regulation of ectopic trypsin and mmp-9 in mice lung, brain, and heart during infection for 4 days ( figure 2b ). trypsin and mmp-9 induction was inhibited by treatment with pdtc, nac, and ndga, probably via blockade of nf-jb and ap-1 binding in the promoter region of the genes. viral rna replication in various organs at day 4 post-infection was suppressed by more than one order of magnitude by pdtc, nac, and ndga ( figure 2c ). suppression of viral multiplication and induction of cellular factors by pdtc, nac, and ndga, significantly improved the survival of mice at day 14 post-infection, the late stage of infection ( figure 2d ). to elucidate the mechanisms underlying brain vascular dysfunction of influenza-associated encephalopathy, changes in the levels of tight-junction proteins, intracellular zonula occludens-1 (zo-1) and transmembrane occludin, and the matrix protein laminin, were analyzed by western blotting. marked reductions in the expression levels of tight-junction constituents were detected at day 4 post-infection, which were partly rescued by pdtc, nac, or ndga (figure 2e ). 9 no other tight-junction protein, claudin-5 or matrix fibronectin and type iv collagen, were affected. the present study reports several new observations: (i) proinflammatory cytokines, tnf-a, il-1b, and il-6, when up-regulated by iav infection, induce trypsin and mmp-9 expression in various organs in mice; (ii) inhibitors of nf-jb and ap-1 effectively suppress the up-regulation of proinflammatory cytokines, trypsin, and mmp-9 and improve survival rates of infected mice. based on these results, we propose the 'influenza virus-cytokine-protease cycle' hypothesis as one of the mechanisms of vascular dysfunction in mof with cytokine storm in severe influenza and influenza-associated encephalopathy. 9 the significance of pro-inflammatory hyper-cytokinemia, or 'cytokine storm,' in the pathogenesis of iav infection remains unclear. on the positive effects, cytokines promote lymphocyte activation and infiltration at the sites of infection and exert direct antiviral effects. however, on the negative effects of excess cytokines, the hyper inflammatory process evoked by viral infection may become harmful through intracellular activation of nf-jb, ap-1, and the janus kinase-signal transducers and activators of transcription signaling pathways. 3, [10] [11] [12] the in vivo experiments presented here showed that nf-jb and ap-1 inhibitors markedly suppress the expression of cytokines, trypsin, mmp-9, and viral replication, resulting in a significant increase in the survival of infected mice. furthermore, cytokines interact with mitochondria to increase the production of reactive oxygen species, resulting in the production ⁄ activation of vasodilatory mediators such as nitric oxide and bradykinin, and subsequent endothelial dysfunction and edema in various organs. 3 the molecular mechanisms underlying tight-junction disruption in endothelial cells and vascular hyper-permeability following the 'cytokine storm' remain unclear. tnfa up-regulation alters the cellular redox state, reduces the expression of four complex i subunits by increasing mitochondrial o 2 ) production and depleting atp synthesis, decreases oxygen consumption thereby resulting in mitochondrial damage, 3, 13 and increases [ca 2+ ] i 14 atp depletion dissociates zo-1 from the actin cytoskeleton and thereby increases junctional permeability. 15 endothelial dysfunction induced by 'influenza virus-cytokine-protease cycle' in the early stage of severe influenza may further affect various circulating factors, coagulation factors and complement systems, and vascular interacting cells, such as neutrophils, macrophages and lymphocytes. mof is the final outcome of metabolic and mitochondrial fuel disorder, immunosuppression, endocrine disorder, and tissue injury followed by endothelial dysfunction in many organs. another key pathway of acute lung injury in the highly pathogenic avian influenza virus h5n1and acute respiratory syndrome-corona virus infection reported recently involves oxidative stress and formation of oxidized phospholipids, which induce lung injury via toll-like receptor 4 signaling pathway. 16 in addition to these data, up-regulated trypsin and pro-inflammatory cytokines may also affect tissue destruction and immunosuppression in the late stage of iav infection. further studies are required on the role of the 'influenza virus-cytokine-protease cycle' in the pathogenesis of mof, particularly in the late stage of viral infection. though influenza a virus replication kinetics and host responses have been previously studied in umbilical vein endothelial cell or transformed endothelial cell lines, the tropism of influenza a virus including h5n1 and pandemic h1n1pdm for primary human lung microvascular endothelial cell has not been well defined. 1 in this study we employed primary human lung microvascular endothelial cells, which are more physiologically relevant for understanding pathogenesis of influenza in the lung as to obtain a better understanding of the links of endothelial cell infection to systematic virus dissemination and multiple organ involvement in severe human influenza. supernatants of cells infected at moi of two were collected for cytokine protein assays, and total rna was extracted for gene expression analysis using qpcr. we found that seasonal influenza h1n1 and h3n2 viruses initiated viral gene transcription and viral protein expression, but did not produce infectious progeny, while the highly pathogenic avian influenza h5n1 and the pandemic influenza h1n1pdm virus could replicate even with the absence of exogenous protease (figure 1) . furthermore, when compared to seasonal h1n1 and h3n2, the h5n1 virus was a more potent inducer of cytokine and chemokine including ifn-b, mcp-1, rantes, ip-10 (figure 2) , and il-6, in virus infected endothelial cells, whereas h1n1pdm induced intermediate levels of cytokine and chemokine. avian influenza h5n1 and pandemic h1n1pdm virus (but not the seasonal h1n1 and h3n2 virus) can productively replicate in human lung microvascular endothelial cells. this is likely to be of relevant to pathogenesis and provides a possible explanation for the extra-pulmonary infection seen in animal infection models. this extra-pulmonary spread may support the previous speculation and anecdotal evidence that h5n1 and h1n1pdm virus can infect the gastrointestinal tract through the virus dissemination from the infected respiratory tract as the first target cells for influenza infection. [2] [3] [4] in addition, the release of proinflammatory cytokine and chemokine induced by influenza h5n1 and h1n1pdm virus infection in lung microvascular endothelial cells may be important contributors to the pathogenesis of severe human influenza disease leading to endothelial cell dysfunction that contributes to severe pulmonary disease symptoms. during its replication, influenza virus utilizes the host cellular machinery for many aspects of its life cycle. characterization of such virus-host protein-protein interactions is a must to identify determinants of pathogenesis. the m2 ion channel protein plays a crucial role during the entry and late stages of the viral life cycle where its c-terminal domain, well conserved among influenza a viruses, is accessible to cellular machinery after fusion with endosomal membrane and during its trafficking along the secretory pathway prior to assembly and budding. 1 the aim of the study is to identify cellular interactants of m2 that play important regulatory roles during influenza infection. to identify cellular partners of m2 we performed a genome-wide yeast-two-hybrid (y2h) screening approach 2 using the cytosolic domain of m2 as bait and a human placenta random primed cdna library as prey and tested more than 60 million interactions. from the y2h screening, an interesting interaction with the human annexin a6 (anxa6) protein, 3 a member of annexin family proteins that binds to phospholipds in a ca 2+ -dependent manner, was identified. co-immunopre-cipitation of myc-tagged anxa6 and viral m2 proteins coexpressed in hek293t cells after transfection and infection confirmed the direct interaction between anxa6 and m2. we further investigated whether this interaction had any functional significance with regards to influenza life cycle. using a rna interference strategy to silence the anxa6 gene in human lung epithelial a549 cells, we observed increased progeny virus titers either in a single or multiple viral growth kinetics study, suggesting a negative regulatory role for anax6 during viral infection (figure 1 ). a novel interaction between m2 and anxa6 was identified. more functional studies are in progress to define precisely the potential negative regulatory role of this interaction during viral infection. a systematic dissection of the viral life cycle will be performed to identify the step(s) affected by the anxa6 cellular factor using specific assays such as real-time quantitative rt-pcr in a single or multiple viral growth kinetics study, cell transduction with ha-and m2-pseudotyped lentiviral particles, virion attachment and internalization assay, immunofluorescence staining of np protein as a marker of viral ribonucleoproteins localization, viral polymerase activity measurement, and viral budding observation by electron microscopy. rna extraction was achieved by qiagen biorobot ez1 prior to respiratory multiplex pcr analysis. what remained of the extracted material of each specimen was stored by refrigeration at 4°c. electronic patient records were searched for parameters, such as c-reactive protein (crp), white cell count (wcc), length of admission in days, and patient co-morbidities. patients were divided into three groups according to clinical severity: mild, moderate, and severe. the 'mild' group comprised of those admitted for three days or fewer, or not admitted at all. the 'moderate' group comprised those who required admission to hospital for more than 3 days as a result of swine flu, but who did not require admission to an intensive care unit (itu). the 'severe' group comprised those who had required itu admission. invitrogen '2· reaction mix': 0ae4 mm of each dntp + 6 mm magnesium sulphate. primer ⁄ probe mix recipe applied biosystems 7500 fast real-time pcr system, 'respiratory multiplex' program. well content 25 ll; thermocycler initial stage 50ae0°c for 15 minutes, then 95°c for 2 minutes. subsequent cycles of 95ae0°c for 15 seconds followed by 60°c for 33 seconds for 45 cycles. sequence detection software version 1.4 (applied biosystems). of 126 clinical isolates analyzed, all samples produced amplification of pdh material; 100 produced amplification of both swine flu and pdh material. human male dna (lot no. 36048611039 at 10 ng ⁄ l, applied biosystems) at concentration calculated at 16 666ae6 cells ⁄ ll was diluted from 10 )1 to 10 )4 , yielding mean average ct values of respectively 30ae10, 33ae60, 37ae78, and 37ae22. plotting log of cell number versus ct gave a y = mx + c line from which ct could be interpolated into cell numbers. for swine flu quantification, a sample of swine flu ct 19ae28 was diluted through 10 )1 to 10 )10 . it must be noted that due to variability in resultant swine flu ct values, repetitions at these dilutions were done using an rna carrier (1350 lg ⁄ l, qiagen; cat no. 1017794) in place of rnasefree water. the 10 )4 concentration was positive in nine out of 15 assays; this fraction was used in the calculation described by simmonds 2 to obtain a copy number of targets per reaction by the equation copy value = )ln(f), where f is decimal fraction of failure rate. here, f = 6 ⁄ 15 = 0ae4; )ln0ae4 = 0ae916 copies. a control curve was generated with ct values of 26ae73, 30ae02, 33ae67, and 37ae23 giving copy values of 916, 91ae6, 9ae16, and 0ae916, respectively. using excel (microsoft office, 2010), these control series were adapted into formulae to convert swine flu and pdh ct values into copy numbers of these elements per reaction. simple division derived a value for swine flu copy per pdh copy, but this was chosen to be expressed as swine flu copy number per 100 human cells. this will be referred to as the 'c' value. forty-two patients had known clinical details; average age was 29ae74, female to male ratio 63:37, and average admission length of 7 days. of the mild group (n = 26), nine cases were not admitted to hospital. of the remainder, the mean average admission length was 2ae5 days. mean average c value for all samples was 1ae49 · 10 6 , with a standard deviation of 1ae49 · 10 7 ; geometric mean was 4ae28, and median average was 6ae45. log(mean average c value) is shown for each severity group and for identified risk factors in the 'mild' severity group (figures 2a, b respectively) . in each case variation was too great to yield statistical significance. figure 1 shows the range of c values observed in the 'moderate' severity group. > -4 · 0 ; < -4 · 5 > -3 · 5 ; < -4 · 0 > -3 · 0 ; < -3 · 5 > -2 · 5 ; < -3 · 0 > -2 · 0 ; < -2 · 5 > -1 · 5 ; < -2 · 0 > -1 · 0 ; < -1 · 5 > -0 · 5 ; < -1 · 0 > -0 · 0 ; < -0 · 5 > 0 · 0 ; < 0 · 5 > 0 · 5 ; < 1 · 0 > 1 · 0 ; < 1 · 5 > 1 · 5 ; < 2 · 0 > 2 · 0 ; < 2 · 5 > 2 · 5 ; < 3 · 0 in a study by duchamp et al., 3 no significant correlation was observed between viral ct value and presence or absence of cardiaorespiratory disease, myalgia, digestive symptoms, or upper or lower respiratory tract infection (although a trend was observed towards patients presenting with signs of upper respiratory tract infection). to our knowledge, no other study has used a dual pcr for analysis of respiratory virus concentrations, and no study has attempted to correlate biochemical markers with respiratory virus concentration. the data exhibited a spectrum of c values, from values <5 · 10 )5 to over 1 · 10 8 . the three severity group standard deviations all overlapped with each other, preventing statistical significance. analysis of co-morbidities showed a high mean average c value when asthma was present (6ae05 · 10 7 ), but again this was associated with an excessive standard deviation. whereas the median average c value in the presence of asthma was higher than the overall average c value (9ae09 versus 5ae85), it was significantly lower than the median c value when no co-morbidity was documented (15ae55). there are multiple caveats that may be the cause of such variety of c values obtained. the duration between initial rna extraction and study pcr had a range of 21 to 285 days, with mean average delay of 211 days. the degradation of viral rna is an important contributor to assay variance and failure; rna degradation in clinical samples has been studied. [4] [5] [6] degradation of human dna in clinical samples may have occurred. several studies have chartered degradation of stored human dna. 2, 7 with regards to sampling, the clinical collection of throat swabs is naturally variable according to the method of the collector. a small number of bronchoalveolar lavage samples were analyzed, yet did not amplify, presumably due to rna degradation. the upper respiratory tract may be only a physical stepping stone for the virus, and take no further role in pathogenesis of severe disease (although undoubtedly is crucial for transmission). interestingly, a ferret study of pathogenesis observed that swine flu yields from the upper respiratory tract were greater than those given by ordinary seasonal h1n1, with consequently increased shedding. 8 the review by mansfield 9 cites significant findings regarding influenza pathogenesis, including the predilection of h5n1 strains for type ii pneumocyte cells and alveolar macrophages. it also highlights the limitation of knowledge through dearth of human autopsy studies; an exception is the recognition of haematophagocytic syndrome in severe cases. it is known that specific immunoglobulin is effective against establishment of infection in the upper respiratory tract, whereas specific cytotoxic t lymphocytes (ctls) are necessary for clearance of the virus from the lower respiratory tract. 10 it is also suggestive that a gap of two whole days transpires between initial infection and instigation of a specific immune response. 11 it is plausible that in the healthy individual, virus progression is confounded by efficient natural mucosal immunity, in part through good secretory immunoglobulin levels. airway inflammation associated with asthma exacerbation is known to increase both risk of respiratory viral infection and poorer outcome. it is unproven but likely that the local inflammatory processes give rise to increased virion burdens in the upper airways; however, the same effect is conceivable for epithelial cell turnover. there will likely be variance within each clinical category due to patient circumstances and clinicians' judgment of required admission. unfortunately, the duration of symptoms prior to swab collection was often omitted in the clinical notes. finally, stratification of patient group by receipt of antiviral treatment was not studied. no correlations were observed with c values and crp, wcc or admission length. trends were observed towards higher c values in 'mild' cases, but without statistical significance. the relative small study size, coupled with the intrinsic variability of the parameters studied, warrants larger, better controlled, prospective studies to elucidate clinical use of the c value for influenza illness prediction and management. in mid-april 2009 a novel variant of a(h1n1) influenza virus began to spread rapidly throughout the world, causing the first pandemic of the 21st century. the majority of the cases associated with this new virus show to be mild, but severe and fatal cases have been reported. molecular markers associated with severity have already been identified, as is the case of the mutation d222g. 1 resistant viruses to antiviral drugs have also been identified, highlighting the importance of rapid determination of the antiviral drug profile. global a(h1n1) 2009 genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. the objective of this ongoing research study was, primarily, to thoroughly characterize the genetic profile and evolution of the emergent influenza a(h1n1) 2009 virus circulating in portugal and its phenotypic expression on antiviral drugs susceptibility. the cases considered in this study were obtained from the community and from two collaborating hospitals in lisbon -a reference hospital for adults (hospital de curry cabral) and a reference hospital for children (hospital dona estefânia). the cdc real-time pcr protocol, recommended by world health organization (who), was the method used to confirmed all influenza a(h1n1) 2009 cases. from a total of 577 a(h1n1) 2009 positive cases diagnosed and confirmed, 163 were selected for this study, taking in consideration that they should cover the period of epidemic activity in portugal and include cases from persons belonging to risk groups and cases associated with more severe clinical features. ninety-six a(h1n1) 2009 strains were isolated in mdck-siat1 cells, from combined naso-oropharyngeal swabs. for the evaluation of the genetic profile of a(h1n1) 2009 virus circulating in portugal, 37 of the 96 isolates were characterized by genetic analysis of the ha, na, and mp genes. the remaining five gene segments (pb1, pb2, pa, ns, and np) were also sequenced for six of this 37 isolates. briefly, sequencing was performed according to the protocol developed by cdc and recommended by who, 2 using bigdye terminator v.1.1 technology. nucleotide sequences were determined in a dna automatic sequencer abi prism 3130xl genetic analyzer. for each genomic segment, genetic analysis was performed with lasergene v.4.05 software (dnastar inc, usa) using an average of 4-6 overlapping readings, including sense and antisense, for precise nucleotide and amino acid sequence determination. genetic mutation and phylogenetic analysis were performed by neighbor-joining method, using mega4.0 software, against published sequences from the vaccine strain (a ⁄ california ⁄ 7 ⁄ 2009) and from selected a(h1n1) 2009 strains available on gisaid epiflu database. all mutations were identified with reference to the vaccine strain genome sequence. antiviral drug susceptibility profile of a(h1n1) 2009 influenza virus circulating in portugal was evaluated both phenotypically and genotypically for nais and genotypically for amantadine. phenotypic evaluation to nais, oseltamivir and zanamivir, was performed for all 96 isolates by ic 50 determination through munana fluorescence assays. 3 genotypic evaluation was performed by searching for mutations associated with resistance to nais in all 37 na gene sequences. amantadine susceptibility profile was performed for all 96 isolates by searching on m2 sequence for the 5 molecular markers associated with resistance to this antiviral drug (l26f ⁄ i; v27a ⁄ d; a30t; s31n; g34e). genetic characterisation of the ha1 subunit of ha reveals point mutations in different strains. all 37 analysed strains present p83s and i321v mutations, which distinguish them from the vaccine strain ( figure 1a ). thirty-three of the 37 sequenced strains group in the s203t branch. this mutation is referred in the literature as being associated with the putative antigenic site ca. 4 most of these strains (19) further subgroup in the d222e branch, this mutation being associated with one loop of the receptor-binding site. 1 from the early to the late epidemic period, an increased circulation of virus carrying the mutation s203t was observed. this is in agreement with the association between this mutation and an enhanced viral fitness that is described in the literature. 5 additional mutations were also observed in a small number of virus, of which we highlight: regarding the genetic characterisation of na, the majority of strains analysed (34 of 37) presents the mutations n248d and v106i ( figure 1b) . as mutation s203t in ha gene, these two na mutations are described in the literature as associated with enhanced viral fitness. 5 the few strains not carrying these mutations have circulated in the beginning of the epidemic period. fifteen of the 37 analysed strains further subgroup in y155h branch. additionally, mutation i223v was identified in two strains. for the remaining gene segments available for the six analysed strains, the observations include: (i) no previously described virulence markers in pb2, pb1-f2, and ns1 were detected; (ii) pb1-f2 protein is present in the truncated form of 11 amino acids; (iii) the presence of mutations i123v and l122q in ns1 and v100i in np; (iv) the described association of mutation i123v in ns1 and v100i in np genes with viral fitness. phenotypic evaluation of nais susceptibility revealed the existence of three minor and two major outliers to oseltamivir ( figure 2 ). the two minor outliers exhibited a reduction of approximately twofold in the susceptibility to this antiviral drug, comparing to the baseline level, while the reduction exhibited by the two major outliers was of approximately three-and fourfold. regarding zanamivir, two minor outliers were identified with a reduction of approximately twofold in the susceptibility, compared to the baseline level. these two minor outliers (a ⁄ portugal ⁄ 17 ⁄ 2009 and a ⁄ portugal ⁄ 82 ⁄ 2009) correspond to the two major outliers identified for oseltamivir. genetic analysis revealed the presence of the mutation i223v in the na sequence of these two strains. the contribution of this mutation for the profile of reduced susceptibility identified for both nais is not known, but a mutation in the same na position (i223r) has been referred to as being associated with a reduction in nais susceptibility. 6 full genome sequence analysis of these strains shows that both strains also present the v480i mutation in pb2 gene. however, no association of this mutation with antiviral drug susceptibility is referred in the literature. concerning genetic evaluation of susceptibility to amantadine, all 96 analysed strains present a serine in position 31, which is a molecular marker of resistance to m2 inhibitors. these preliminary results allow us to discuss several points. however, the additional data that is being obtained through this ongoing study will be essential for a more complete analysis. for example, more information is needed to determine if the mutations found alter the biology and the fitness of the virus or if there are associated with an increased prevalence of the virus. the majority of the mutations identified in ha1 subunit have been detected in a(h1n1)2009 strains distributed throughout the epidemic curve, not evidencing a specific evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h1n1)2009 virus. 7 the occurrence of mutations in the position 222 of the ha1 subunit of a(h1n1)2009 virus have been described. however, more studies are needed to clarify the outcome of these mutations, as for example in patients with severe complications. it could also be relevant to investigate the presence of single and mixed variants in viruses and in clinical specimens and the possibility of these mutations affecting the binding specificity. regarding the susceptibility of a(h1n1)2009 pandemic viruses to antiviral drugs, all analysed strains were found to be resistant to amantadine. this resistant profile was not unexpected since the mp gene from this new variant had originated in the eurasian swine lineage, which is characterised by being resistant to this antiviral drug. 8 the majority of the a(h1n1)2009 strains analysed revealed to be susceptible to both nais, with only five strains exhibiting a profile of reduced susceptibility, three to oseltamivir and two to both nais. for these last two, the presence of the i223v mutation in the na sequence could explain the reduction observed, but a more complete analysis is needed to confirm this. the french national pandemic plan includes an early containment phase followed by a limitation phase. the efficacy of such a plan depends on pre-existing surveillance and laboratory networks. the grog community surveillance network and the hospital lab networks organized by the two french nics carried out the virological monitorthe efficacy of such plan depends on pre-existing influenza surveillance and laboratory networks. in france, the community surveillance is carried through the grog surveillance network. in addition, surveillance is also carried out in hospitals by the renal network. this renal network is divided in two sub-networks: the so-called h5-labs network, activated during the containment phase and the extended renal lab network activated in the limitation phase. the h5-labs have bsl-3 facilities that can be used for diagnosis purposes. as part of the national influenza surveillance system led by the french institute for public health surveillance (invs), the grog community surveillance network and the lab networks linked to the two french nics carried out the virological monitoring of the a(h1n1)2009 pandemic from the early containment phase up until the end of the pandemic phase. during the containment phase, all suspected cases were hospitalized and declared to invs. each patient was tested on the same day by specific virological diagnosis. hospital admission was not mandatory during the limitation phase, (i) the clustered cases were monitored to study transmission chains, and (ii) the circulation of the virus in the community was monitored through grog swabs collected by practitioners. the nics organized the influenza surveillance to fulfill several objectives according to the epidemiological situation. first, rt-pcr tools (influenza a m gene rt-pcr and a(h1n1)2009 specific h1 and n1 genes rt-pcrs) were developped and distributed to the lab networks on the 14th of may 2009. 1 from the early phase, the nics and the h5-lab network analyzed all the samples collected from hospitalized and community patients. during the early phase of the limitation phase, an increasing number of labs were performing the specific assays. when the pandemic wave started, all hospital labs could do the testing. results were centralised by nic and reported on a weekly basis. in addition, nics carried out the monitoring of antiviral resistance emergence (na pyrosequencing, specific h275y rt-pcr, and phenotypic assays), and real-time surveillance of genetic changes involved in virus adaptation (pb2) virulence factors or antigenic variations (ha). this sequencing was carried out by the pf8 sequencing platform of the institut pasteur. the first imported a(h1n1)2009 influenza cases were observed from the 28th of april 2009. a limited number of cases have been reported in may. local transmission could be detected end of may. clusters were observed in schools in june and in summer camps during summer. as opposed to the epidemiology of the a(h1n1)2009 virus in other european countries, no summer wave was observed in france. only a limited number of sporadic cases were reported up until october. early september, a significant number of cases presenting with influenza-like illness was reported (figure 1 ). the virological investigation of these cases showed high prevalence of rhinovirus infection. this circulation of rhinovirus was a counfounding factor of the pandemic. the pandemic wave lasted 11 weeks between mid-october and the end of december (week 43 to week 53, figure 1 ). the pandemic wave started week 42-43 in the ile-de-france area, and only week 44-45 in the rest of france. the peak was recorded week 48 ( figure 1 ). the impact of the pandemic was mainly observed in the 5-15 years group of age. overall, 1334 severe cases have been admitted to the hospital, and 308 deaths have been recorded by the end of the pandemic wave. the major impact was observed in the 15-65 years group of age (66% of deaths recorded). amongst the severe cases and the deceased cases, 20% and 16% of cases had no risk factor, respectively. these specimens, 24 279 were positives for h1n1, representing 99ae7% of total influenza virus detections. only nine brisbane-like h1n1, 62 brisbane-like h3n2, and eight b viruses have been detected in the same period of time. the weekly positive rate ranged from 0% to 48%. phylogenetic and antigenic analyses of the viruses collected during the pandemic wave did not show any emerging genetic or antigenic variants (figure 2a,b) . eight patients, all among cases presenting with severe illness, were infected by a virus harbouring the d222g mutation in the ha. 2 amongst the virus tested for antiviral susceptibility or screened for the h275y mutation by or specific rt-pcr, only 11 oseltamivir-resistant viruses related to the na h275y mutation have been detected. one of these cases also had an i223r mutation associated to a reduced sensitivity to zanamivir. all but one resistant virus were detected in treated immunocompromised patients. overall, eight patients presented a virus with the d222g mutation in the ha. all these patients had a severe infection; one of these had also a h275y mutation in the na asociated to oseltamivir resistance. the pandemic started by the end of april 2009. although the first cases recorded were as early as the 28th of april, the epidemic wave associated with a widespread spread of the virus was only recorded in october. the french population did not have to face a summer wave, as observed in north america and in numerous european countries. 3, 4 it is difficult to speculate the reasons for the lack of summer wave; the specimens collected were negative for influenza. moreover, during september, it was anticipated that school openings would be the trigger for the beginning of the pandemic wave. as a matter of fact, a significant increase of influenza-like syndromes were observed at that time, but the virological investigation carried out by the laboratories showed thta is was related to a very large epidemic of rhinovirus. 5 the epidemic circulation of other respiratory viruses can be counfounding factors for the surveillance of the influenza epidemic clinical when the survellance is only based on collection of clinical information. the starting of the pandemic wave was heterogeneous in france. the ilede-france region (paris and its suburbean area), where the population is dense, experienced an early start as compared to the rest of france. however, once the pandemic started in the rest of the county, the epidemic curves were quite similar. the peak was reached at identical times, although it may have been delayed in some remote places in france. overall, we estimate that 10% of the french population consulted for an ili presentation. the impact was mainly observed in the 5-15 years groupe of. however, this age groupe represented only a limited number of severe cases and deaths. on the other hand, the 15-65 years groupe of age, where the prevalence was not high, was the age group where the majority of severe cases and deaths was recorded (74% and 66%, respectively). 6 this data is consistent with the observational data reported by numerous other countries. 7 according to the profile of hospitalized cases, a(h1n1)2009 was more aggressive than seasonal viruses. the number of admission to the hospital was ten-fold that observed during a normal influenza epidemic. even if the mortality was limited (312 cases), the age distribution of the deceased patients was different as compared to seasonal influenza (75% mortality in <65 years of age). the lack of recordeable excess mortality has been interpreted to be the consequence of a very mild pandemic, milder than some seasonal epidemics. however, the median age of the fatal cases was much younger than those observed during the seasonal flu, leading to a mis-interpretation of the real impact of the pandemic. when the impact is measurered in loss of years of life, the impact of this pandemic is larger than seen with seasonal influenza, and is quite comparable to these of the two last pandemics. 8 the pandemic preparadness of numerous countries, the develoment of new intensive care techniques and equipment, and the large use of antivirals have reduced the overall impact of this pandemic. these are new factors that should be taken into account when evaluating the real impact of the 2009 h1n1 virus. 8 the virological monitoring of the pandemic was achieved by the community-based and hospital-based seasonal influenza networks, reminding the importance of maintining such networks. the diagnosis of influenza in most of the patients was carried out by molecular techniques. it has been clearly stated from the beginning of the pandemic that near-patient tests were lacking of susceptibility and could not be used for patient management. the distribution of a set of validated and comprehensive techniques by the two nic was very helpfull for the monitoring of the pandemic and the patients. however, this diagnostic procedure change should not preclude maintaining virus isolation that is necessary for whole genome analysis, monitoring of antigenic changes, and phenotypic testing for antiviral testing. some of the mutants that have been recorded, including viruses with antiviral resistance phenotype or genotype, could be analysed from grown virus strains. it is striking that despite a large antiviral usage, only a limited number of isolates had mutations associated to resistance. however, the frequent isolation of such resistant virus was observed in immunocompromised patients that presented severe infections and long virus shedding. 9 the impact of the pandemic is still under evaluation. sero-epidemiological analysis will be performed to asses for the real attack rate of the 2009 pandemic virus. as in other countries, it has been recorded that asymptomatic infections could be observed frequently. 10 it is quite unlikely that the impact of the pandemic was reduced by the vaccination campaign, although this vaccination started on the 12th of november, just when the pandemic started in france. it is estimated that 6 millions received the vaccination. pandemic strains of the influenza virus sporadically emerge, deviating from the regular endemic strains of seasonal influenza. in april 2009, a novel pandemic influenza virus a ⁄ h1n1 emerged, swiftly spreading across the world. immediately, domestic and international public health agencies were forced to develop containment and mitiga-tion strategies in response to the pandemic. however, the dynamics and transmission patterns of this novel virus are yet to be fully understood. simultaneously, seasonal strains of influenza (a ⁄ h1n1, a ⁄ h3n2, and b) continued to circulate in many nations. both pandemic and seasonal variants of influenza are responsible for significant morbidity and mortality. 1 to characterize the dynamics of this disease and the variation within strains, a more detailed understanding of the patterns in viral shedding during natural infection is required. the majority of data on the patterns of viral shedding during influenza infection are a result of volunteer challenge studies. 2 in these studies, volunteers are commonly screened for pre-existing immunity against the challenge strain and are of a certain demographic and age. information on the patterns of viral shedding in natural influenza infections, pandemic or seasonal, is limited but should provide greater generalizability. we describe the trends of viral shedding and clinical illness in community acquired cases of pandemic and seasonal strains of influenza. in 2008, a community-based study was conducted to analyse the effectiveness of non-pharmaceutical interventions to prevent the spread of influenza in households. 3 in 2009, a similar community-based study was initiated to collect comparative data from individuals infected with seasonal and pandemic influenza. 4 both studies were conducted with very similar protocols, involving 617 households in total. the specimens and symptom data required for this study all arise from secondary infections ascertained in these two community-based studies. the recruitment process in both studies was essentially identical. index cases were first recruited from their healthcare provider if they presented with influenza-like illness (ili). this individual would be included in the follow-up if he ⁄ she tested positive for influenza virus infection by rapid antigen test (quickvue) and was the first person in his ⁄ her household that showed signs of ili in the previous 2 weeks. follow-up consisted of three home visits that spanned approximately 7-10 days. at each home visit, nasal and throat swab (nts) specimens were collected from all household members, regardless of the presence or absence of symptoms. symptoms were recorded in daily symptom diaries provided for every household member, and digital thermometers were provided to record daily tympanic temperature. the symptoms recorded were fever ‡37ae8°c, headache, myalgia, cough, sore throat, runny nose, and phlegm. influenza virus infection and subtype was identified by reverse transcription polymerase chain reaction (rt-pcr) on the nts specimens. viral shedding was quantified from the same specimens by rt-pcr to determine viral loads, as well as by quantitative viral dilutions to determine median tissue culture infectious dose (tcid 50 ). the details concerning laboratory methods have been described in a previous study. 5 all analyses in this study focus exclusively on secondary cases; these are household contacts of recruited index cases who acquire influenza virus infection following the initial home visit. index cases generally presented with a certain threshold of illness severity requiring medical attention, whereas infections among household contacts can vary from asymptomatic to severe representing naturally acquired influenza infections. these secondary cases must be negative for influenza for their first nts specimen, and subsequently tested positive. we analysed mean viral loads measured by rt-pcr and quantitative culture by plotting by day since acute respiratory illness (ari) onset according to strain of influenza (pandemic a ⁄ h1n1, seasonal a ⁄ h1n1, seasonal a ⁄ h3n2, and seasonal b). ari is the reference time point, because the day of infection is unknown and is defined as the presence of ‡2 of the symptoms mentioned above. average symptom scores were also plotted according to ari onset and grouped into upper respiratory symptoms (sore throat and runny nose), lower respiratory symptoms (cough and phlegm), and systemic signs and symptoms (fever ‡ 37ae8°c, headache, and myalgia). mean daily tympanic temperatures were also plotted since date of ari onset and according to strain of influenza virus. all analyses were conducted using r software (version 2.10.1; r development core team). 6 a total of 617 households and 2499 individuals were followed-up in the two studies. of 1887 household con-tacts tested by rt-pcr, 153 were found to be influenza positive. among these influenza infections, 13 (8ae5%) were asymptomatic (rt-pcr positive plus 0 symptoms recorded), 20 were subclinical (rt-pcr positive plus 1 symptom recorded), and 88 presented with an onset of ari during the follow-up period. from the cases with ari onset, seven pandemic a ⁄ h1n1, 40 seasonal a ⁄ h1n1, 22 seasonal a ⁄ h3n2, and 19 seasonal b influenza virus infections were identified. the age distribution among secondary cases was observed to be largely comparable across the four strains of interest (table 1 ). there were a lower proportion of males who acquired pandemic a ⁄ h1n1 compared to the seasonal strains of the virus. cough was the most commonly reported symptoms during follow-up in cases of pandemic a ⁄ h1n1 and seasonal b, whereas runny nose was most common in seasonal a ⁄ h1n1 and a ⁄ h3n2 cases. cumulatively, fever ( ‡37ae8°c) was reported in approximately half (51%) of the secondary cases. patterns of viral shedding were analysed in a subset of 88 influenza positive individuals who recorded an onset of ari in their symptoms diaries (figure 1 ). household contacts that were asymptomatic, subclinical, or did not have an ari onset were excluded from the analysis. viral shedding in all three influenza a strains were recorded to occur on the day of ari onset or 1 day post-ari onset. following the peak, measured levels of viral shedding declined steadily to undetectable levels over 5-6 days. the trend of viral shedding in influenza b infected individuals rose 2 days before ari onset, fluctuated for around 6 days before eventually resolving. the patterns of viral shedding over time measured by quantitative viral culture were generally similar to the patterns measured by rt-pcr. the patterns of symptoms and signs were comparable in the four strains of influenza included in this study, peaking on the day or 1 day post-ari onset, and gradually declining over a period of 5-7 days. in all strains, systemic symptoms and signs were observed to resolved faster than upper and lower respiratory symptoms. the trend of tympanic temperature in each influenza strain was comparable to the respective symptom pattern. patterns of viral shedding observed in influenza a strain infections (pandemic a ⁄ h1n1, seasonal a ⁄ h1n1, and seasonal a ⁄ h3n2) were broadly similar. the pattern differed from the observed pattern of viral shedding in seasonal influenza b infections. the majority of viral shedding in influenza a strains occurred at and near ari onset, whereas there were variable amounts of viral shedding preand post-ari onset for those with influenza b. the biological reason for this difference is yet to be clarified. these differences are consistently observed regardless of laboratory method used to quantify the viral loads. it was observed that viral shedding measured by tcid 50 resolved more quickly than when measured by rt-pcr, suggesting that rt-pcr is more sensitive, but it could be detecting inactivated fragments of rna instead of active virus. the trends observed for the seasonal strains of influenza in this study were similar to those reported in literature. 2 the patterns of symptoms and signs as well as tympanic temperature in the four different strains of interest in this study were found to be comparable. these patterns closely resemble the patterns of viral shedding observed in the influenza a virus strains, but not in the influenza b virus strain. the trends of viral shedding, symptom scores, and tympanic temperature for pandemic a ⁄ h1n1 were similar to trends observed for seasonal a ⁄ h1n1 and seasonal a ⁄ h3n2 infections, suggesting that the dynamics of these viruses are largely the same. the clinical course of infection with pandemic a ⁄ h1n1 influenza virus appeared to be similar to the seasonal b influenza virus, but the patterns of viral shedding over time diverges. in general, our results suggest that the dynamics of the pandemic a ⁄ h1n1 virus were similar to the seasonal a ⁄ h1n1 and a ⁄ h3n2 viruses, and clinically similar to the seasonal b virus. this study faced sample size limitations; very few cases of pandemic a ⁄ h1n1 were detected and the secondary attack rate in general was low, though a total of 617 households were followed up. this lack of power led to the inability to analyse the differences between adult and children and other characteristics that could be correlated with amount of viral shedding. there are also biases that must be factored in during recruitment. the eligibility criteria of only healthy households could select for households with higher innate immunity. on the other hand, recruitment at health care providers can be biased towards index cases that had more severe illness that required medical attention. the strength of the study is the broad generalizability of the results due to the strict classification of secondary cases. the infections reported in this study were all community-based and should represent true natural infections. pandemic potency of the influenza virus is largely determined by its transmissibility. the first objective of this study was to model the transmission of influenza h1n1 and h5n1 viruses. at present, vaccination with laiv has been used as a widespread, effective public health measure for influenza prophylaxis. some unsubstantiated concerns have been raised about a potential possibility of reassortment of circulating influenza viruses with laiv viruses following vaccination with laiv. thus, another objective of this study was to assess the probability of pig-to-pig transmission of cold-adapted viruses and their potential reassortment with wt influenza strains. female albino guinea pigs weighing 300-350 g were inoculated intranasally with 10 5 eid 50 of virus without anaesthesia. transmission studies were then performed 24 hours after inoculation. inoculated animals were housed at 25% relative humidity and 22°c in the same cage with noninfected guinea pigs or in cages placed 3 m away from non-infected pigs. virus replication was determined by virus isolation in hen eggs and by pcr. sera were collected at 0 and 28 days post inoculation. seroconversions were assessed by routine hai test. genome composition of reassortants was monitored by rflp analysis. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated, and virus growth properties were observed following virus titration in hen eggs. when infected pigs were co-caged with non-infected (naïve) individuals, vn1203, indo ⁄ 5, a ⁄ california ⁄ 7 ⁄ 2009, and nibrg-23 were isolated in 0%, 25% 83ae3%, and 100% of contact animals, respectively. serological confirmation of virus transmission was higher than virological data (25%, 100%, 100%, and 100%, respectively). in addition, it was shown that when pigs inoculated with a ⁄ california ⁄ 7 ⁄ 2009 were co-caged with animals inoculated with nibrg-23, they got infected with both viruses ( table 1) . the ability of direct transmission of cold-adapted viruses was also investigated. data show that the a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 laiv candidate was detected in the upper respiratory tract of 87ae5% vaccinated pigs. the mdv was identified in 100% of infected animals. however, neither group of contact pigs, co-housed with the vaccinate pigs, had evidence of infection with cold-adapted viruses. in addition, none of the contact pigs had any evidence of seroconversion to the coldadapted viruses as determined by hai assay. it was also most interesting to note that pig-to-pig transmission of the highly transmittable nibrg-23 reassortant virus was not seen when pigs, vaccinated with mdv, were co-caged with animals infected with nibrg-23 virus (table 1) . this strongly implies a form of interference or protection from transmissibility that was provided by the cold-adapted virus. the results show that nibrg-23 and indo ⁄ 5 viruses were able to spread between cages over the 3 m distance (100% and 50% naïve animals were successfully infected, respectively). a ⁄ california ⁄ 7 ⁄ 2009 influenza and vn1203 viruses did not transmit between infected and non-infected guinea pigs housed in separated cages (table 1) . pigs with confirmed a ⁄ california ⁄ 7 ⁄ 2009 virus replication were also infected with nibrg-23 virus if h1n1-and h5n1-infected animals were separated by a space. thus, influenza virus transmission from h5n1-to h1n1-infected pigs has been shown, but the reverse pattern did not occur. transmission of nibrg-23 or a ⁄ california ⁄ 7 ⁄ 2009 viruses was not observed when contact pigs were first vaccinated with the mdv and housed at a 3 m distance ( table 2) . it was also shown that efficiency of transmission of nibrg-23 was much higher than of other studied h5n1 viruses; it can be transmitted between naïve guinea pigs separated from infected animals at a distance of 4-5 m (data not shown). five reassortants were isolated from animals which were infected with a ⁄ california ⁄ 7 ⁄ 2009 virus and co-caged with pigs inoculated with nibrg-23. two reassortants possessed different combinations of pr8, nibrg-23, and a ⁄ california ⁄ 7 ⁄ 2009 genes and demonstrated the non-ca ⁄ non-ts phenotype typical of wt viruses. unexpectedly, two other reassortants inherited ha gene from nibrg-23, na gene from a ⁄ california ⁄ 7 ⁄ 2009, and other genes from pr8 became ca and ts. 7:1 non-ts reassortant inherited pa gene from pr8 and seven other genes from a ⁄ california ⁄ 7 ⁄ 2009, gained ca properties. in spite of aforesaid experimental data, we cannot exclude the theoretical possibility of simultaneous infection of human host with cold-adapted and wt influenza viruses. to better understand possible consequences of such a reassortment event, we co-infected guinea pigs with a mixture of mdv and nibrg-23 viruses. nasal washes were collected and cloned by limited dilutions in hen eggs in the presence or absence of immune serum to the mdv. cloning of nasal washes without antiserum led to isolation of over 100 clones, which were all identical to the mdv (data not shown). when nasal washes were cloned in the presence of antiserum, only nine clones were isolated. genome composition analysis showed that all isolates were triple reassortants, which had inherited pb2 and na genes from mdv, pa gene from pr8, and ha gene from nibrg-23. the origin of the other gene segments (pb1, np, m, ns) in the genome of guinea pig-derived reassortants varied. reassuringly, all reassortants generated in vivo had the phenotype typical of the mdv. the severity of influenza outbreaks is partly determined by efficient spreading of the causative virus strain between human hosts. however, little is known about mechanisms underlying influenza virus transmission in humans. guinea pigs have been shown to be a suitable model for influenza transmission studies. 1 our in vivo study showed that influenza a viruses vary in their transmissibility. nib-rg-23 and indo ⁄ 5 viruses were able to transmit to naïve animals caged distantly from infected animals. in contrast, cold-adapted viruses, the same as those used for licensed laivs, showed no signs of transmission from one guinea pig to another. our study also provided evidence of a lower level of transmissibility of the novel pandemic h1n1 virus compared to the nibrg-23 and indo ⁄ 5 h5n1 strains evaluated. benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. 2 in our study, the mdv inoculated into guinea pigs appeared to interfere with and even offer protection from transmission of the highly transmissible nibrg-23 virus. the ability to immunize with the laiv and subsequently block the spread of a homologous h3n2 subtype and a heterologous h3n2 subtype influenza virus between guinea pigs has been shown. 3 interference between cold-adapted and wildtype influenza virus infection was the most likely explanation for the data observed in our study. the mdv inoculated into guinea pigs might in some way interfere with transmission of highly transmissible influenza viruses. it is believed by some that widespread use of laiv could increase the potential risk of reassortment of the vaccine strain with circulating influenza viruses immediately following vaccination. however, it was shown that any such potential reassortments would most likely lead to yet attenuated viruses. 4 our in vivo studies have shown that introduction of mdv genes into the genome of nib-rg-23 virus led to the generation of triple reassortants inherited pb2 and na genes of mdv and ha gene of h5n1 virus. all isolates possessed phenotypical markers associated with attenuation of mdv. our data suggest that even if a reassortment event of such rare occurrence between a laiv strain and a circulating virus were to occur, it would most likely lead to a reassortant that would retain highly attenuated phenotypic properties of the vaccine strain. our data strongly support the safety of laivs, especially those developed against highly transmissible h5n1 and h1n1 pandemic influenza viruses. this information builds upon databases that have clearly shown the low likelihood of transmitting an laiv, as well as the high likelihood of any field reassortment of laiv with a circulating influenza virus to retain important properties of the cold-adapted, temperature-sensitive vaccine master composition. very interestingly, we also present data that show the potential of a laiv to prevent the transmission of highly infectious influenza viruses, perhaps identifying a broader role for laiv in the overall scheme of influenza virus prophylactic use. background: schlieren imaging is a non-invasive, real-time airflow visualization technique that relies on differences in air temperatures (and the resulting changes in the refractive index) to allow exhaled human airflows to be seen clearly against the background of more-stationary, ambient air. recently, this technique, well-known to engineers, has been applied to better understand and characterize airflow behaviors associated with everyday, as well as healthcarerelated, human respiratory activities. materials and methods: as a surrogate marker for the behavior of airborne infectious agents, schlieren imaging was used to visualize the airflow patterns produced by adult human volunteers of different ages while coughing with and without the wearing of standard surgical and n95 masks. results: the cough plumes were generally similar in shape and range for all the adult volunteers used in this study. although both the surgical and n95 masks decelerated and blocked some of the forward momentum of the coughed airflows, much of the cough plume was redirected and escaped around the top, bottom, and side edges of the masks to merge with the volunteer's natural, verticallymoving thermal plume. conclusions: schlieren imaging is a safe technique for visualizing exhaled airflows from human volunteers without the need for potentially-irritant or toxic particle tracers. findings from these schlieren imaging experiments will assist the development of more effective aerosol infection control guidelines in healthcare premises where patients infected with potentially airborne infectious agents (e.g., influenza and tuberculosis) are present. these infectious agents may be transmitted to healthcare workers, other patients, and their visitors by way of exhaled airflows. with the recent influenza pandemic 1,2 and the ongoing concerns about human cases of avian influenza h5n1 infections, 3 there is now a very real concern about the potential for the aerosol transmission of respiratory pathogens. such concerns amongst staff and patients in healthcare environments have led to a greater emphasis on the understanding and control of infectious airflows. 4,5 previous visualization techniques have used potentially-toxic or irritant gas or particulate tracers with hazardous laser light sources that have precluded the use of human volunteers as subjects. instead, various forms of lung models that simulate human respiratory patterns with such particulate tracers have been used. 6, 7 schlieren imaging is a technique familiar to engineers and offers a non-invasive (i.e., no tracer required) airflow visualization method that depends only on differences in the refractive index of the warmer, human-exhaled air and the cooler ambient air. 8 the use of a simple incandescent or light-emitting diode (i.e., non-laser) light source is safe and allows human volunteers to be used as experimental subjects, where their exhaled airflows are then observed using a large, precise spherical or parabolic telescopic mirror and a camera, and are recorded for later analysis and presentation. [9] [10] [11] the analysis of these patterns of 'real-life' human airflows will be useful in optimizing aerosol infection control guidelines, which aim to reduce the transmission of airborne infectious agents to other healthcare personnel, patients, or their visitors. the images and analysis presented here have all been obtained from the large 1 m diameter parabolic mirror (figure 1 ) situated at the gas dynamics laboratory of penn state (directed by gary s. settles). this large schlie-ren imaging system has been in use for over 30 years to obtain high quality schlieren images for various engineering applications. it has only recently been applied to clinically-relevant imaging. the objective of this paper is to augment and expand upon the details of the methods and results presented in an earlier study using this same schlieren imaging system. 10 the aim of this series of studies is to visualize and capture a series of airflow images produced by coughing from adult human volunteers of different ages (24-80 years old). these included males (three of 26 years, one of 80 years of age) and females (one of 24 years, one of 30-40 years, and one of 40-50 years of age). each volunteer was tested with and without wearing either a standard surgical mask or n95 mask. more specifically, the aim was to visualize the extent and direction of leakage around the mask whilst each subject was coughing. penn state institutional approval for experiments involving human subjects was also obtained. each volunteer was asked to stand approximately 1 m in front of the schlieren mirror, facing across the surface of the mirror on one side, and to cough several times as the real-time, color image and video footage was recorded by the operator (using a nikon d90 camera; nikon inc. melville, ny, usa). this process was repeated whilst each volunteer was wearing a standard surgical mask then an n95 mask (supplied by 3mô, st paul, mn, usa). some of the schlieren images obtained from some of these volunteers have been published previously: for a 26-year old male, 9 the 24 year-old female and a 26-year old male, 10 and the 40-50 year-old female. 11 this article completes this series of schlieren images obtained from these experiments by including the images recorded for the older, 80 year-old man. generally, it was found that the shape of the cough plumes (shown in the figure as darker shadows emanating from the subject's mouth) produced by adult humans of different ages was relatively similar. cough plumes are roughly conical in shape and very turbulent, usually passing beyond the extent of the 1 m mirror (figure 2a) . a previous detailed study of one of these images measured a maximum airflow velocity of 8 m ⁄ second for an adult cough. 9 similarly, the effects of wearing surgical and n95 masks can be generalized across different ages. wearing a surgical mask allows leakage of the coughed air from the sides, top, and bottom of the mask ( figure 2b ). there is also some leakage through the mask, as indicated by the darker patches of air directly in front of the mask ( figure 2b, c) . the useful effect of the mask appears to be a deceleration and redirection of this coughed (and potentially infectious) air into the natural, upward-rising human thermal plume, which captures it and carries it upwards where it is diluted and less likely to transmit infection to others. the effects of the n95 mask are similar (i.e., deceleration and redirection), yet due to its tighter (mask-fitted) face seal, more of the coughed air appears to penetrate the front of the mask ( figure 2c ). this penetrating air is, however, also decelerated sufficiently to allow the wearer's natural thermal plume to carry it upwards. 10, 11 discussion from these series of schlieren images presented in this and other related studies, [9] [10] [11] it is clear that schlieren imaging offers a safe, non-invasive, real-time technique to visualize human exhaled airflows for all age groups. it is apparent that, at least where airflow patterns are an acceptable surrogate marker for airborne transmission risks, there are beneficial effects of wearing either type of mask, even when the mask fit is relatively poor. this is often the case when n95-style masks are purchased and used by the general public -in contrast to the situation with healthcare workers, who are often accurately fit-tested for this type of mask. the immediate significance of this can be seen when masks are bought by parents for their children. often, these will not be of pediatric size and the mask-fit will be loose. children are well-known to be major sources of infection in the community because of their relatively poor immunity to many types of infectious agents due to their young age and, therefore, limited past-exposure history. 12 these images allow infection control teams to literally see how far and how fast potentially-infectious human exhaled airflows can travel from an individual. this may have significant implications for guidance on the wearing of masks for infected staff and patients, on ward bed-spacing, as well as for the types of masks to be used in different situations. the important practical potential lies in the non-intrusive visualization of airflows associated with human volunteers, to assist in heightening the awareness amongst healthcare workers of the risks and potential for the airborne transmission of infectious agents, as well as the development of more effective aerosol infection control policies. schlieren images can be analysed more quantitatively, e.g., with the 'schlieren-piv' technique, 9, 13 though this additional quantitative data is probably more of research interest than being of immediate practical use to everyday hospital infection control teams. these are the subtypes that we have studied. clearly, the question arises as to whether the changes in antigenicity are coupled with changes in germicide susceptibility. we have employed a modified log-reduction method 3 in a cell culture system employing mdck cells 4 in serum-free ex-cellô 5 medium supplemented with trypsin. microscopic examination of cpe was the marker for infectivity together with plaque assay. we confirmed antiviral potency by using specific subtype influenza identification subtype technology, quidel quickvue ò influenza a + b test. the log inactivation and percent inactivation by bac after a 60 second contact time for the h1, h2, and h3 pandemic strains are as follows: a ⁄ swine ⁄ iowa ⁄ 12 ⁄ 30 h1n1, 3ae5 log ⁄ 99ae97%; a ⁄ swine ⁄ cal ⁄ 2009 h1n1, 4ae8 logs ⁄ 99ae998%; a ⁄ j305 ⁄ 57 ⁄ h2n2, 5 logs ⁄ 99ae999%; and a ⁄ hong kong 8 ⁄ 68 h3n2, 5ae0 logs ⁄ 99ae999% (table 1 ). comparable results of antiviral efficacy are obtained with the tcid 50 and plaque assays against all subtypes studied. when performing the plaque assay the sensitivity of virus recovery was better in the vessel with a larger surface area and overall recovery was in agreement with the potency determined by tcid 50 assay. in our plaque assay, we inoculated a ⁄ hong kong ⁄ 8 ⁄ 68 virus dilutions into two different vessels with 2 hours adsorption time: 6-well plate and t-25 flask, 9 ml inoculum per replicate. virus titers obtained were: 1ae4 · 10 6 pfu ⁄ ml from 6-well plate and 2ae2 · 10 6 pfu ⁄ ml from t-25 flask ( table 2 ). the discrepancy on virus potency can possibly be explained as: the binding of virus to host cell occurs only when virus gets a chance to interact with the cell on the monolayer during adsorption time. the percentage of virus population in the inoculum that has the opportunity to bind to the cell mainly depends on the surface area where this interaction takes place. therefore, in our experiment the plaque assay in the t-25 flask gave higher virus recovery 2ae2 10 6 versus 1ae4 · 10 6 pfu ⁄ ml. the increased virus recovery can translate into better sensitivity of the test system for disinfectant and antiviral agents. the potency of the virus used in this study was determined by tcid 50 was 5 · 10 6 tcid 50 ⁄ ml. rapid diagnostic testing for influenza (quickvue ò influenza a + b test, quidel) for aj305 versus bac was studied. the presence of influenza viral nucleoprotein a determined by quickvue kit correlated 100% with the viral infection based on by cpe in viral culture. interestingly, the inactivation of viral nucleoprotein was able to be revealed with diagnostic kit in the dilutions of virus ⁄ bac reaction mixture, which possessed prominent cytotoxic effect for the host cells in viral culture system. this type of molecular testing method is useful for interpreting antiviral efficacy against a background of cytotoxicity. these experiments are intended for the sponsor to substantiate to us fda that their antiviral substances are safe and effective. the data shows that the three hemagglutinin subtypes were highly susceptible to the quaternary ammonium compound in the short term in vitro experiment. the appearance of novel subtypes in the future can be met with the assurance that disinfectant and ⁄ or antiseptic resistance will be unlikely. certainly, from the above data, although genetic reassortment of human and swine viruses may modulate influenza pathogenesis and limit existing vaccine benefit, it is not likely be a factor in control of viruses on environmental surfaces by benzalkonium-type disinfectant ⁄ cleaning agents in community or health care environments. table 2 . comparison of viral titer obtained in different vessels using quantal tcid 50 and plaque assay methods plaque assay tcid 50 assay t-25 (25 cm 2 ) 6-well plate (9 cm 2 ) tcid 50 ⁄ ml tcid ⁄ ml 2ae2 · 10 6 pfu ⁄ ml 1ae4 · 10 6 pfu ⁄ ml 5 · 10 6 2ae5 · 10 6 options for the control of influenza vii outbreak influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. in this paper we share our experience of managing an outbreak of viral respiratory infection in an acf very early in the 2009 influenza pandemic and also describe some of the emerging issues relating to crossreacting antibodies to the pandemic (h1n1) 2009 influenza virus in the very elderly. the outbreak investigation was conducted as part of an urgent public health intervention initiated by the new south wales (nsw) department of health during the early stages of the first southern hemisphere wave of the 2009 pandemic. nose and throat swabs for nucleic acid testing (nat) plus acute and convalescent serum samples (6 weeks apart) were collected from all the residents of an acf where an influenza-like illness (ili) outbreak occurred. the investigation revealed dual outbreaks of pandemic (h1n1) 2009 influenza and rhinovirus infection. out of 28 residents, three had laboratory confirmed influenza [two with pandemic (h1n1) 2009], and 10 had rhinovirus infection on nat. testing of acute sera collected from every subject found elevated ( ‡1:40) pandemic (h1n1) 2009 hai antibody in 60% (9 ⁄ 15) subjects aged 85 years or more (born before 1925 and median age 88 years; geometric mean titre-gmt 48ae1) compared with none of the 13 residents aged under 85 years (born after 1924 and median age 79 years; gmt 10ae1, p = 0ae01). the acf was closed to visi-tors for 7 days. the symptomatic residents received treatment-dose oseltamivir, and all other residents were given oseltamivir prophylaxis. more than one virus may be circulating in an acf with an ili outbreak at any one time in winter. a significant proportion of elderly residents had pre-existing cross reacting antibody to the pandemic (h1n1) 2009, which may explain the minimal clinical impact of pandemic (h1n1) 2009 in this elderly population. influenza is one of the leading causes of infectious death in elderly people, principally due to co-morbidities and declining immune competence with age. it is the most important agent in outbreaks of respiratory illness. 1 influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. 2 the clinical presentation of influenza in residents of acfs can be subtle, with a blunted febrile response and a non-specific decline in mental and functional status. 3 residents commonly have underlying diseases that can be exacerbated by influenza infection, and in addition, they are at higher risk of serious influenza-related complications than community dwelling elderly people. 4 people aged over 65 years are also at higher risk of influenza-related death, and more than 90% of annual influenza-related mortality is usually confined to this high risk group. 5 in australia, influenza and pneumonia have sub-stantial health impacts; recorded as being the underlying causes of death for 2623 persons in 2007. 6 since the world health organization declared an influenza pandemic in june 2009, australia has suffered one of the highest rates of confirmed infection during the first southern hemisphere wave. by late october 2009 there were 187 reported deaths due to pandemic influenza in australia, 7 and to date there have been about 18 449 deaths reported worldwide. 8 although disproportionately far fewer elderly people developed clinical influenza during the current pandemic than occurs with seasonal influenza, their case-fatality rate remained substantial. 9 early in the pandemic (june 2009), we investigated a suspected pandemic influenza outbreak in a rural acf in the state of nsw, australia. the epidemiology (including virulence and clinical outcome in the elderly) of the pandemic (h1n1) 2009 virus was mostly unknown at the time of investigation, and as time passed, this investigation provided clarity on some important issues of the influenza epidemiology in the elderly population. in this paper we share our experience of managing a dual outbreak of viral respiratory infections early in the pandemic, and also describe some of the emerging issues relating to the cross-reacting antibodies to pandemic influenza in the very elderly. the outbreak investigation was conducted as part of urgent public health intervention initiated by the nsw department of heath in conjunction with the local public health unit, the national centre for immunisation research and surveillance (ncirs), and the institute of clinical pathology and medical research (a who national influenza centre). to determine the extent and cause of the outbreak, a public health research doctor (gk) was dispatched from sydney over a weekend to assist with outbreak investigation and control. on june 12th 2009, the greater southern public health unit surveillance officer (bd) received a report of a possible pandemic (h1n1) 2009 outbreak in a local acf. on investigation, it was discovered that 3 days earlier a 77 year old female resident had become generally unwell, but without specific symptoms of influenza like illness (ili). soon after, nine of the 27 co-residents (but no staff) had developed symptoms suggestive of influenza. one other resident had returned from a melbourne (victoria) hospital (where pandemic (h1n1) 2009 was known to be circulating) the previous week after surgery, but did not have ili symptoms. on june 10th, the 10 symptomatic residents had nasal swabs taken by the local doctor for influenza [including pandemic (h1n1) 2009] nucleic acid testing (nat). there was rising concern due to reports of widespread pandemic (h1n1) 2009 influenza in a local army camp just over the border in nearby victoria, where pandemic (h1n1) 2009 influenza was known to be circulating widely. on june 12th, the 77 year old lady proved nat positive for pandemic (h1n1) 2009, but none of the other samples were pandemic (h1n1) 2009 nat positive. concern arose that there might be an outbreak of pandemic (h1n1) 2009 in the facility, and that some of the swabs from other residents might be false negatives. between 14 and 15 june, after consent was obtained, directly or through next of kin in 13 demented residents, all 28 submitted to venipuncture for serology, 27 successfully, and the other 18 as yet un-swabbed residents were swabbed. basic demographic data were collected from every resident with clinical information on co-morbidities and current medication use. convalescent blood samples were collected after 4 weeks on 16th july 2009 from 23 of the 28 residents. swabs were sent to icpmr where nat for influenza a [including pandemic (h1n1) 2009] and b was performed. the acute and convalescent serum samples were tested later (in december 2009), using haemagglutination inhibition assay (hai) to detect pandemic (h1n1) 2009 antibody. 10, 11 interventions the acf was closed to visitors from 12th until 18th june. treatment of the positive case and the nine symptomatic residents, with twice daily oseltamivir, was begun on saturday june 13th, and all other residents were started on once daily oseltamivir prophylaxis. the facility manager and local general practitioner (gp) monitored patient health on a daily basis, and none had to stop oseltamivir due to adverse events. one resident with ili who was known to have moderately impaired renal function was given once daily rather than twice daily oseltamivir treatment. the age range of the residents was 58-97 years with a median of 85 years. all residents had underlying medical conditions, e.g., chronic cardiac and respiratory diseases ( table 1) testing of acute sera collected from every subject found elevated ( ‡1:40) cross-reacting hai antibody to the pandemic (h1n1) 2009 in 60% (9 ⁄ 15) of subjects aged 85 years or more (born before 1925 and median age 88 years; geometric mean titre-gmt 48ae1). however, the hai titre was consistently <1:40 and significantly lower (gmt 10ae1, p = 0ae01) in the 13 residents aged under 85 years (range 58-83 years, median 79 years) (figure 1 ). the index case (nat positive) did not show a significant raise in hai level in convalescence (going from 20 to 40). the pandemic (h1n1) 2009 case that was determined by serology was pandemic (h1n1) 2009 nat negative. to our surprise, seven of the other 18 asymptomatic residents had rhinovirus detected on extended nat (reported on june 25th), despite being asymptomatic at time of swabbing and remaining so. the original nine influenza nat negative samples were then tested and three of these were also nat positive for rhinovirus; in total, ten proved nat positive for rhinovirus (35ae7%). the serologically confirmed pandemic (h1n1) 2009 case was also positive for rhinovirus infection. of interest was that only one resident had a documented fever. this investigation illustrates some of the difficulties in managing and investigating possible influenza outbreaks in real time in the context of an influenza pandemic. 12 finding a nat positive case of pandemic (h1n1) 2009 influenza among many other symptomatic cases raised the possibility (although not the probability) that pandemic (h1n1) 2009 was the cause of the outbreak. rhinovirus infection, however, was confirmed by nat in ten residents. this outbreak illustrates that more than one virus (in this case 2 and perhaps 3) may be circulating in an acf at any one time in winter. in ili outbreaks in acfs, broad laboratory testing is recommended; nat is the most sensitive method of detecting influenza or other viruses in respiratory tract samples. 13 studies have found that the pandemic (h1n1) 2009 haemagglutinin (ha) gene is more closely related phylogenetically to the 1918 h1n1 virus and classical swine influenza a ⁄ h1n1 viruses than more recent seasonal human influenza a ⁄ h1n1 viruses. 14 it is antigenically similar to the 1918 h1n1 pandemic virus in terms of the immunodominant antibody response to haemagglutinin. [15] [16] [17] it is likely that individuals alive during the emergence and initial persistence of the 1918 pandemic virus would have higher levels of cross-reacting hai antibodies to the pandemic (h1n1) 2009, which would contribute towards better clinical protection. 18 in our investigation, 60% of the residents born before 1925 (aged 85 years or above in 2009) had pre-existing cross-reacting hai antibody to the pandemic (h1n1) 2009. in elderly populations, severe illness may be associated with organisms typically considered to be mild, such as rhinovirus. however, studies have shown that nursing home residents may be susceptible to outbreaks of rhinovirus that may cause mild to severe respiratory illness, particularly in those with a history of lung disease. one rhinovirus outbreak in a nursing home in the usa caused 12 fatalities. 19 another outbreak showed residents with underlying lung disease are more likely to have longer infection, require antibiotics, develop bronchospasm, and have difficulty breathing; two residents with underlying lung disease required emergency treatment and one died. 20 a previous influenza outbreak in a nsw aged care facility in 2006 caused significant mortality and morbidity. that outbreak resulted in 14 hospital admissions and six deaths. 21 in our investigation we have found that 17% of the residents had chronic lung disease and 64% had chronic cardiac conditions both considered as high risk for severe complications of both rhinovirus and influenza infection. however, there were no hospitalisations or deaths in our outbreak investigation. indeed only one resident developed fever, indicating that non-specific signs of illness (such as in our index case) may be the only, or early, indication of an ili. our own experience with managing other ili outbreaks has also taught us that staff of acfs may not be vigilant enough to detect fevers. in this outbreak, the nursing home staff, local gp, public health unit and the outbreak investigation team and supporting laboratory staff acted quickly and in a coordinated way. pre-existing cross-reacting antibody in the very elderly (aged ‡85 years) probably helped to limit the spread of the pandemic virus (compared to the circulation of rhinovirus) within the acf. exposure to the 1918 pandemic (or a close variant occurring before 1925) appears to be responsible for a high hai titre in the very elderly, which contributed towards better clinical protection. however, wider testing early on would have alerted us more quickly to the main cause of the outbreak. treatment and prophylactic use of oseltamivir may also have contributed to halting the spread of pandemic (h1n1) 2009 and also to symptom relief. pandemic (h1n1) 2009 influenza virus (ah1pdm) has spread worldwide since march 2009. in a paper of ah1pdm, 25% of infected individuals have experienced gastrointestinal symptoms such as diarrhea and vomiting, which is higher than that of seasonal influenza. however, little is known whether viable virus shed from stool and replication of viruses are ongoing in the gastrointestinal tract. 1,2 viral load and isolation of ah1pdm in cell culture in stool samples has been reported. 3 stool specimens were collected from 35 patients suspected to have pandemic (h1n1) 2009 infection from november 2009 through may 2010. virus isolation was conducted in cell culture by using madin-darby canine kidney (mdck) cells and taqman based rt-pcr from 10% (w ⁄ v) stool suspension in phosphate-buffered saline. taqman based rt-pcr was conducted by using primers, probes, and positive controls provided by niid (national institute of infectious diseases of japan). to confirm presence of ah1pdm viral rna, lamp (loop-mediated isothermal amplification) was used as supplemental testing. of patients, one child (case 1) submitted one nasal swab and four stool samples, another one nasal swab and two stool samples, and the other one stool sample. informed consent was obtained. strand specific rt-nested pcr was performed for only case 1 by using only one primer at the rt reaction and also assayed neu5aca2-3gal and neu5aca2-6gal binding specificity about isolated strain derived from nasal swab and stool. receptor binding specificity was performed using a solid-phase binding assay with the sialylglycopolymers (poly a-l-glutamic acid backbones containing neu5aca2-3galb1-4glcnacb-pap or neu5aca2-6galb1-4glcnacb-pap bond as described. 4 ) nucleotide sequences of the ha gene of ah1pdm viruses isolated from stool sample and nasal swab were analysed. in order to exclude the possibility of contamination, the stool samples and nasal swabs were subjected to virus isolation separately. after getting the results on the nucleotide sequence, we also confirmed no strain harboring identical sequence was isolated in our laboratory before and after the day of sample collection. ah1pdm viral rna was detected in nine (25%) of the subjects from stool samples. among nine subjects, one case (case no. 1) was positive for viral isolation. case1, a healthy 9-year-old girl, experienced fever and abdominal pain, and the others had gastrointestinal symptoms without upper respiratory symptoms. in case 1, influenza a virus was diagnosed by rapid antigen test on the day of symptom onset. viable ah1pdm virus was isolated from the stool sample and nasal swab on the second day from onset using mdck cells (table 1 ). viral load decreased gradually after symptom onset. however, viral shedding was still present 8 days after symptom onset. positive stranded rna was detected 6 days after symptom onset from the stool specimen ( figure 1 ). above two ah1pdm strains (isolated from nasal swab and stool specimen) bound exclusively to human type receptor, neu5aca2-6gal. sequence analysis demonstrated that isolated virus from stool samples was identical with that from nasal swabs in comparison of ha gene (990 bp). ah1pdm influenza virus was isolated from the stool and nasal swab samples in the same patient simultaneously by using mdck cells. our results suggests the detection of viral rna and viable ah1pdm influenza virus from stool samples may serve as a potential mode of transmission and has important implications in understanding the context of ah1pdm influenza virus. strategies to prevent transmission of influenza include use of respirators. ffp2 and n95 respirators are certified to fil-ter at least 95% of particles (0ae3 lm in diameter), and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. [1] [2] [3] we have developed a proprietary acid-polymer formulation to coat a standard ffp2 respirator with an antiviral layer. we aimed to test this coated respirator for antiviral efficacy against a range of influenza viruses. a series of tests compared the antiviral efficacy of coated and uncoated respirators in conditions designed to simulate real-life exposure to influenza by varying the route of inoculation, contact time, temperature, humidity, moisture, and contaminating substances. we also investigated whether infectious viruses could be transferred from contaminated respirator surfaces to gloves. we tested human, swine, and avian influenza viruses, including influenza a and b viruses. influenza a subtypes were the a ⁄ h1n1 2009 pandemic strain, seasonal h1n1, h5n1, h3n2, h5n9, and h2n2. in each test, suspensions of influenza viruses were prepared to 4-8 log 10 tcid 50 ⁄ ml in mem. in some tests, organic contaminants (yeast, bsa, and mucin) were added. one set of respirators was maintained at 40°c and 75% relative humidity for 24 hours before the viral challenge, and repeatedly sprayed with he-pes buffer to simulate respiratory secretions. for each test, three coated (glaxosmithkline actiprotect) and three uncoated (sperian willson easy fit) ffp2 respirator samples were inoculated with 0ae2 ml of a viral suspension, which was applied with a pipette, sprayed, or aerosolised to create airborne droplets. after 1 minute at room temperature (on a shaker), the respirator samples were assayed for the presence of infectious viruses using standard methods. 4 in one test, after a 1 minute contact time of the respirator with the virus, nitrile gloves were applied with light pressure to the outer surface of inoculated respirator samples and then assayed after 1 minute. samples were put into test medium (mem, supplemented with antibiotics [penicillin, gentamycin, or streptomycin] and amphotericin b or l-glutamine). the supernatants were vortexed, extracted, and used to prepare serial 10-fold dilutions in mem. each dilution was used to inoculate four wells of rmk cells in a multi-well plate, and these cultures were incubated and scored over 7 days for cytopathic effects, cytotoxicity, and viability. (some tests substituted mdck cells; others used inoculated embryonated chick eggs.) all tests included negative cell controls, cytotoxicity controls, and neutralisation controls. the spearman-karber formula was used to calculate viral loads as tcid 50 or eid 50 . 4 antiviral efficacy was calculated from the difference between the geometric mean loads of influenza virus on the coated and uncoated respirators after 1 minute of exposure. the viral loads applied to respirators in these experiments ranged from 5ae5 to 8ae1 log 10 tcid 50 , and were therefore high in comparison with respiratory secretions from infected patients at the peak of influenza symptoms (range 3-7 log 10 tcid 50 ). 1 tables 1-2 show that the average viral loads detected on uncoated ffp2 respirator samples remained high in all conditions tested, ranging from 3ae2 to 6ae9 log 10 tcid 50 (or 4ae5-5ae0 log 10 eid 50 ). in contrast, the average viral load on coated respirators after 1 minute of exposure ranged from below the limits of detection to £1ae5 log 10 tcid 50 (1ae0 log 10 eid 50 ). therefore, the relative antiviral efficacy of the coating ranged from ‡2ae7 to 6ae4 log 10 . table 1 shows that the relative antiviral efficacy of the coated mask remained high in simulated-use conditions such as organic contaminants and repeated saturation at high temperature and humidity. in the experiment to test transfer of viruses from respirators, the gloves applied to regular uncoated inoculated respirators had a viral load of 3ae5 log 10 eid 50 (table 1) . by contrast, no viruses were detected on either the coated respirators or the gloves applied to them. the relative reduction in contamination was therefore ‡2ae5 log 10 . ‡4ae8 log 10 viral load with organic contaminants* 6ae1 0 ae7 5 ae3 log 10 viral load after heat, moisture, and simulated secretions** 5ae1 0 ae8 4 ae3 log 10 viral load transferred to glove** 3ae5 £1ae0 ‡2ae5 log 10 eid 50 *influenza subtype was a ⁄ h5n1, and strain was vnh5n1-pr8 ⁄ cdc-rg. **influenza subtype was a ⁄ h3n2, and the strain was hong kong ⁄ 8 ⁄ 68. results are mean log 10 tcid 50 , unless specified otherwise. results are mean log 10 tcid 50 , unless specified otherwise, based on an infectivity assay in triplicate. limits of detection varied. *2009 pandemic strains. **results are mean log 10 eid 50 , based on a haemagglutinin assay in duplicate. options for the control of influenza vii ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 301-327 strategies to prevent transmission of influenza include use of respirators, and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings for protection against pandemic influenza. 1-3 ffp3 respirators are certified in europe to filter at least 99% of nacl particles (0ae3 lm in diameter), and ffp2 and ffp1 respirators must filter at least 95% and 80% of these particles, respectively. influenza a viruses are typically 0ae1 lm, and can be carried in aerosolised droplets smaller than 1 lm in diameter, which can disperse widely, remain airborne for 8 hours, and be inhaled deeply into the respiratory tract. 4 we have developed an acid-polymer formulation to coat the outer layer of a standard ffp2 respirator, in order to provide antiviral activity on the outer surface. we compared this coated respirator against standard ffp1, ffp2, and ffp3 respirators for filtration of aerosolised influenza viruses. the aim was to simulate protection against infectious viruses in droplets released when infected people cough and sneeze, and during aerosol-generating procedures in healthcare settings. the first assay compared three samples of coated ffp2 respirators (glaxosmithkline actiprotect) with three ffp2 controls (sperian willson easy fit). for each test, suspensions of influenza a (h3n2) at 7ae1 log 10 tcid 50 ⁄ ml in 0ae1· minimum essential medium (mem) were aerosolised with a nebulizer. the airborne droplets were introduced into a sterile chamber upstream of a respirator sample for 2 minutes, at a flow rate of 28ae3 l ⁄ minute. constant airflow was maintained for another 2 minutes after exposure to the virus. then the collection dish in the downstream sieve sampler (anderson) was assayed for infectious viruses using standard techniques. 5 briefly, serial dilutions of the collection medium (mem with 1% fbs, 5% gelatine, and 2% hepes, supplemented with antibiotics and amphotericin b) in mem + trypsin were used to inoculate madin-darby canine kidney epithelial (mdck) cells in quadruplicate in a multi-well plate. these cultures were then incubated and scored over 4-6 days for cytopathic effects, cytotoxicity, and viability. negative cell controls and cytotoxicity and neutralisation controls were also performed. the spearman-karber formula was used to calculate tcid 50 . the second assay compared five samples of coated respirators with five ffp1 controls (3m 9310) and five ffp3 controls (3m 1863). a suspension of influenza a (h1n1), at 8ae3 tcid 50 ⁄ ml, was nebulized for 1 minute and 40 seconds into the aerosol chamber, at a flow rate of 28ae3 l ⁄ minute, followed by constant airflow for 5 minutes after exposure to the virus. then the collection medium in the downstream chamber (as before, with 1% nahco 3 ) was assayed as described above. initial viral loads in the first and second assays were 8ae1 and 7ae9 log 10 tcid 50 , respectively, and were therefore high in comparison with respiratory secretions from infected patients at the peak of their influenza symptoms (range 3-7 log 10 t-cid 50 ). table 1 shows that the average viral load that passed through the uncoated ffp2 respirators in the first assay was 3ae6 log 10 tcid 50 . the average viral load that passed through the coated respirators was 1ae4 log 10 tcid 50 . therefore, for active filtration of viruses, the relative efficacy of the respirator with antiviral coating was 2ae2 log 10 greater than the uncoated respirator. for surface inactivation, the relative antiviral efficacy of the coated respirator was 3ae9 log 10. in the second study, table 2 shows that the average viral load that passed through the uncoated ffp1 respirators was 5ae2 log 10 tcid 50 . in contrast, 3ae1 log 10 tcid 50 passed through the coated ffp2 respirators. by comparison with the viral load when no respirator was present (8ae9 log 10 tcid 50 ), the ffp1 respirators reduced the viral load by 3ae7 log 10 , and the coated ffp2 by 5ae8 log 10 . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load by 2ae1 log 10 more than the ffp1 respirators. in this second study, the average viral load that passed through the uncoated ffp3 respirators was also 5ae2 log 10 tcid 50 . by comparison with the viral load when no respirator was present (8ae9 log 10 tcid 50 ), the ffp3 respirators reduced the viral load by 3ae7 log 10 . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load passing through the mask by 2ae1 log 10 more than the ffp3 respirators. table 2 also shows that the coated respirators reduced the infectious viruses remaining on the mask surfaces by 3ae8 log 10 more than the ffp1 respirators, and 3ae6 log 10 more than the ffp3 respirators. even with a very high viral challenge, the coated respirators prevented passage of at least an additional 2ae1 log 10 infectious viruses, compared with uncoated respirators. large numbers of infectious virions passed through all uncoated respirators tested. ffp3 respirators were no more effective than ffp1 respirators at blocking airborne influenza viruses. based on these in-vitro results, respirators with the antiviral coating could be expected to provide more protection than standard respirators from the risk of inhaling influenza viruses. strategies to prevent transmission of influenza include use of respiratory protection. ffp2 and n95 respirators are certified to filter at least 95% of nacl particles (0ae3 lm in diameter), and many guidelines have recommended that healthcare workers wear these respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. 1,2 we have developed a proprietary acid-polymer formulation, designed to coat a standard respirator and inactivate influenza viruses on contact. we tested this coated respirator for cytotoxicity, skin irritation, and sensitisation potential. the antiviral coating was also tested for stability and leaching under extreme environmental conditions, such as physical abrasion and simulated breathing at different temperatures, levels of humidity and co 2 , and saturation with contaminants. eight coated respirators were tested at standard relative humidity (60% rh) for 8 hours, and one at elevated humidity (80% rh) for 2 hours. four coated masks were treated with synthetic blood or oral secretions, and then tested at 30% rh for 1 hour. the sample respirators were sealed onto a mannequin head inside an airtight chamber, and air at 30°c and 5000 ppm co 2 was pumped through the masks by a cyclic breathing machine at 24 l ⁄ minute. a 37 mm glass-fibre filter was placed behind the respirator, over the mannequin's mouth opening. at the end of all tests, these filters were eluted and analysed using high-performance liquid chromatography (hplc). standard in vitro methods were used to assess the cytotoxicity of the coated polyester and uncoated polypropylene layers of the respirator (glaxosmithkline actiprotect). 3 samples were extracted in minimum essential medium (mem), supplemented with serum, penicillin, streptomycin, amphotericin b, and l-glutamine, at 37°c for 24 hours. triplicate monolayers of mouse fibroblast cells (l-929) were dosed with each extract (including a reagent control and negative and positive controls), and incubated at 37°c in 5% co 2 for 48 hours. after 48 hours of incubation with samples or controls, the monolayers of mouse fibroblast cells were examined microscopically for abnormal cell morphology or cellular degeneration. samples of the coated respirator (comprising four polypropylene layers bonded to the coated polyester outer layer) were applied under occlusive patch conditions to the skin of 51 adults. controls, including individual layers, were applied in the same way. in a separate patch test, samples of the coated polyester outer layer and controls were applied under the same conditions to 219 adults. after 24 hours, test patches and controls were removed. sites were then scored for itching, erythema, oedema, epidermal damage, and papular response after 48 and 72 hours. the patches were applied three times a week for 3 weeks. to evaluate sensitisation, test patches were applied 10-15 days later for 24 hours at different sites to the original samples. after this challenge, skin was assessed and graded for sensitisation potential after 48 and 72 hours. table 1 shows that no residues of the antiviral coating or degradation products were detected in the air that had passed through any of the eight respirators. cytotoxicity tests showed that the coated respirator material caused 10% cell lysis or toxicity, classified as slight reactivity (grade 1), and that uncoated material caused no cell lysis or toxicity (grade 0) ( table 2) . results for positive and negative controls were severe reactivity and no reaction, respectively. from the results of the two human repeat-insult patch tests, neither the coated or uncoated layers nor the fullthickness respirator fabric caused irritation (including itching, erythema, edema, vesiculation, epidermal damage, papules, or reactions beyond the patch site) or sensitisation in any of the adult volunteers at any of the time points. based on these results, in conjunction with published data on acute and repeat-dose toxicity, mutagenicity, local irritation, dermal sensitisation, and inhalation safety for all components of the antiviral coating, the potential topical or inhalation exposure to the coated antiviral respirator does not pose a safety risk. the antiviral coating is durable and stable, and stays on the outer surface of the respirator, even in extreme environmental conditions. the coated respirator is non-irritating and non-sensitising. therefore, this respirator is considered to be well-tolerated and safe for its intended use. ies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. knowing how influenza virus is transmitted at home and in school is the key to preventing its spread. at the previous two meetings of this conference, 1,3 we introduced our study of household transmission of seasonal influenza and reported our conclusion that protracted survival of the virus even after treatment increases household transmission, and is a major factor in the transmission of the virus to infants. on the other hand, during the recent pandemic, many schoolchildren developed serious respiratory tract disorders, which again highlights the significance of schoolbased transmission of the disease. in this study, we compared transmission of a new influenza strain at home and in school with that of seasonal influenza and proposed countermeasures. the for the analysis of school-based transmission, the epidemic status of seasonal influenza in 4237 children at six elementary schools over the past two seasons (2002-2003 and 2003-2004 seasons) was compared with that of pdmh1 in 1913 children at two primary schools. using observational data of school-based transmission, we also constructed a model for influenza transmission 3, 4 and evaluated the effects of factors that could affect influenza transmission (e.g., antibody prevalence, transmission rate, non-infectious latent period, infectious latent period, school closure) through the use of 1000 simulations. in this study, a diagnosis of influenza was confirmed by rapid influenza antigen detection kit. we previously reported the high sensitivity of the kits, 5-7 not only for seasonal influenza, but also for h1n1 pandemic 2009 compared to virus isolation and pcr. serum antibody was not investigated. most of the index patients were treated with oseltamivir or zanamivir, and 30 patients were treated with amatadine. no treatment was done for 16 patients. no nai therapy was done as prophylaxis within the family. the incidence of households with an initial case patient who subsequently infected another member of the household was 21ae6% (275 of 1271 households) for seasonal influenza or 18ae3% (112 of 612 households) for pdmh1. thus, the household incidence of pdmh1 was lower than that of seasonal influenza. in addition, the percentage of family members in households who were infected by initial case patients (household transmission rate) was 10ae5% (375 of 3573 individuals) for seasonal influenza or 7ae9% (141 of 1792 individuals) for pdmh1. thus, the household transmission rate was also lower for pdmh1 than that for seasonal influenza. effect of family size on household incidence and household transmission rate an analysis of the effect of family size on household incidence showed that, in families consisting of 2-7 individuals, the incidence of seasonal influenza in order of increasing family size was 3ae5%, 17ae4%, 24ae5%, 24ae2%, 32ae4%, and 42ae9%, respectively, and the incidence of pdmh1 was 0ae0%, 13ae5%, 17ae8%, 24ae7%, 42ae9%, and 33ae3%, respectively, indicating that household incidence tends to increase with increasing family size. in contrast, no definite relationship was noted between household transmission rate and family size. transmission rates for seasonal influenza in order of increasing family size were 3ae5%, 10ae6%, 11ae8%, 8ae0%, 10ae3%, and 7ae1%, respectively, or 0ae0%, 6ae7%, 7ae7%, 8ae0%, 15ae2%, and 5ae6%, respectively, for pdmh1 (shown in table 1 ). effect of age cohort of initial case patient in household on household incidence and household transmission rate an analysis of the effect of the age cohort of the initial case patient in the household on household incidence and transmission rate showed that the household incidence of seasonal influenza in c1, c2, c3, and c4 was 26ae7% (112 of 419 households), 21ae4% (66 of 308 households), 16ae3% (21 of 129 households), 9ae9% (9 of 91 households), and for m and f was 19ae6% (37 of 189 households) and 22ae5% (29 of 129 households), respectively. therefore, household incidence was the highest in c1, followed by the parents. when the initial case patient was a child, the household incidence increased with decreasing patient age. in contrast, the household incidence of pdmh1 in c1, c2, c3, and c4 was 23ae1% (36 of 156 households), 17ae3% (42 of 243 households), 8ae1% (9 of 111 households), 10ae0% (4 of 40 households), and for m and f was 30ae8% (12 of 39 households) and 39ae1% (9 of 23 households), respectively. therefore, household incidence was higher when the initial case patient was a parent, rather than a child. the household transmission rates for seasonal influenza from c1 to f were 13ae4%, 10ae1%, 6ae0%, 3ae9%, 10ae3%, and 12ae6%, respectively. therefore, as for household incidence, the highest rate (13ae4%) was observed in c1. the corresponding household transmission rates for pdmh1 were 9ae9%, 6ae9%, 4ae0%, 4ae3%, 12ae7%, and 22ae1%, respectively, with the highest transmission rates observed for infections from parents (shown in table 1 ). if the rate of individuals with a secondary infection transmitted from the initial case patient in a household is presented as a percentage of the total number of affected individuals, the rates for seasonal influenza and pdmh1 were 22ae8% (375 of 1646 individuals) and 18ae9% (142 of 753 individuals), respectively. therefore, the rate of individuals with a secondary infection was lower for pdmh1 than that for seasonal influenza. by age cohort, the corresponding rates of individuals for seasonal influenza in c1, c2, c3, and c4 were 22ae3% (120 of 539 individuals), 11ae5% (40 of 348 individuals), 12ae2% (18 of 147 individuals), 7ae1% (7 of 98 individuals), and for m and f was 42ae7% (141 of 330 individuals) and 27ae1% (48 of 177 individuals), respectively. for pdmh1, the corresponding rates in c1, c2, c3, and c4 were 24ae3% (50 of 206 individuals), 9ae3% (25 of 268 individuals), 9ae0% (11 of 122 individuals), 4ae8% (2 of 42 individuals), and for m and f was 51ae9% (42 of 81 individuals) and 35ae3% (12 of 34 individuals), respectively. these findings indicate that, especially in the case of pdmh1, most secondary infections in parents tend to be transmitted from another household member. the mean annual prevalence of seasonal influenza and the new influenza strain at the elementary schools for the two seasons was 19ae6% and 10ae3%, respectively, whereas the prevalence determined 7 days after appearance of the first case in school was 3ae9% and 4ae5%, respectively. in the recent season at the same elementary schools, however, the prevalence was a high 43ae5%. since the prevalence at 7 days after the appearance of the first case in school was already 17ae5%, these data show that the influenza virus spread quickly throughout the schools. at the schools with high transmission rates in the early period of the pandemic, new infections were confirmed even 4 days after the school closure action was taken. these findings indicate that pdmh1, the current influenza virus, has a long latent period during which it becomes infectious and spreads from infected individuals to numerous others in their vicinity. we constructed a model for influenza transmission in schools and estimated the time course of changes in the number of expected cases and the expected prevalence during the season. in this model, school children were divided into six groups depending on the stage of infection: uninfected period with no immunity, non-infectious latent period, infectious latent period, onset, post-onset infectious period, and immune period. it was assumed that schoolbased transmission occurred during the infectious latent period prior to onset and that no infections occurred during the post-onset infectious period because children were absent from school. due to the long latent period of pdmh1, the distribution of the non-infectious latent period of pdmh1 was established as (day 1, day 2, day 3, day 4) = (10%, 40%, 40%, 10%) and the distribution of the infectious period as (day 0, day 1, day 2) = (10%, 80%, 10%). when simulations were performed under these conditions using the model for school-based transmission of influenza in which children from classes with an outbreak were kept at home for 3 days, the time course of changes in the number of affected individuals actually observed and the time course of changes in the number of expected cases were determined. the expected prevalence under these conditions was 35%. to evaluate the effect of school closure, simulations were performed based on the assumption that children from affected classes were not kept at home for 3 days. it was shown that there was an increase in the expected number of cases during the 3 days corresponding to the period of actual school closure and that the expected prevalence increased to 52%. based on these findings, it was concluded that keeping children home from classes with an outbreak is an effective means of controlling the transmission of influenza in schools (shown in figure 1 ). if the transmissibility of pdmh1 virus at home is estimated based on the speed of transmission and the degree to which pdmh1 is prevalent in schools, it would be expected that the household transmission of pdmh1 is also higher than that of seasonal influenza. in fact, the opposite is the case. this paradox can be explained in two ways. 1. the number of children aged 19 or more and parents with pdmh1 influenza as a percentage of the total number of affected individuals is lower than those with seasonal influenza (20ae8% versus 37ae2%). further, although the number of parents with a secondary infection was high at home, the percentage of the total number of individuals with pdmh1 was a low 3ae8% (29 of 754 individuals), compared to that for seasonal influenza (5ae5% [91 of 1640 individuals]). in other words, adults are less susceptible to pdmh1 infections and there was a correspondingly small number of affected individuals. therefore, it was considered that the transmission rate at home was lower than that at school for this reason. 2. the percentage of households with more than one affected individual within the same family was higher for pdmh1 at 28ae5% (157 of 550 households) than for seasonal influenza at 24ae4% (309 of 1265 households). in the patients secondarily infected with pdmh1, 26ae8% of them showed symptoms of infection 10 days or more after the onset in the first patient, suggesting that they were not infected at home, and the actual household transmission was 20ae9% (115 of 550 households). therefore, although the prevalence was higher for pdmh1, it seems that household transmission was lower because households with an affected individual implemented satisfactory control measures against infection. seasonal influenza differs greatly from pdmh1 influenza in its transmissibility at home and in school. in the household transmission of pdmh1 influenza, both the household incidence and household transmission rate of pdmh1 were low compared to those for seasonal influenza. although transmission of seasonal influenza from infants to parents was marked, in the case of pdmh1, the reverse was true with transmission from parents to children being predomi-nant. it should be noted that household transmission in mothers was common in all eight seasons, suggesting the need to reconsider control measures against infection when nursing unwell family members. in the case of school-based transmission, pdmh1 was more prevalent than seasonal influenza, indicating that the virus spread quickly throughout the schools. this difference was attributed to the long infectious latent period when pdmh1 rapidly became rampant in the schools. an analysis of school-based transmission using a model for influenza transmission showed that, when 10% of the student population is infected, schools should be closed for five consecutive days in order to minimize the spread of the disease. the effectiveness of seasonal influenza vaccine in preventing pandemic and seasonal influenza infection: a randomized controlled trial introduction household transmission has been estimated to account for one-third of all influenza transmission, 1,2 and children are at high risk of spreading the disease. with reference to previous evidence, 3-15 some vaccine deployment strategies target children to prevent them from infection and transmitting influenza. 16 nevertheless, few studies evaluated the effectiveness of vaccinating children in reducing household transmission. 10, 11 during 2008-09, a pilot randomized controlled trial was conducted to investigate such effect by studying households with school age children randomized to receive trivalent inactivated seasonal influenza vaccine (tiv). 17 the monovalent vaccine against pandemic influenza a (h1n1) (ph1n1) had yet been available until the end of the first wave. various conclusions have been made as to whether seasonal influenza vaccine might possibly protect against ph1n1. [18] [19] [20] [21] [22] [23] [24] [25] we report findings on the effectiveness of tiv against ph1n1 observed in our cohort. households were screened if they expressed interest after receiving invitation letters distributed via their children's school or an existing pediatric cohort study. 26 to be eligible, the household had to include at least one child aged 6-15 years who was not allergic or hypersensitive to any of the tiv components. children known to have immunosuppressive conditions or other contraindications against tiv were also excluded. written consent and assent were obtained from participants aged above 18 years and those aged 8-17 years, respectively. proxy written consent was obtained from legal guardians or parents for participants younger than 18 years. ethical approval was obtained from the institutional review board of the university of hong kong. consented households were allocated to the tiv and placebo group (in ratio 3:2) according a code generated by block randomization with random block sizes of 5, 10, and 15. an independent nurse prepared 0. one child (study subjects) from each household in the tiv group received a single dose of tiv with one child from each household in the placebo group receiving a single dose of saline placebo. parents and legal guardians were asked to report any adverse reactions 4 days following vaccination. all participants, study nurses, and other research staff were blinded to the allocation and administration of vaccine or placebo. the vaccine allocation sequence was only disclosed to the investigators at completion of the study. serum specimens were collected from subjects shortly before (november-december 2008), one month after vaccination (december 2008-january 2009), and after the winter (april 2009) and summer influenza seasons (august-october 2009). serum specimens were obtained from household contacts at baseline and after the winter and summer influenza seasons. all household members recorded any fever ‡37.8°c, chills, headache, sore throat, cough, presence of phlegm, coryza, or myalgia daily on a symptom diary. they were also invited to report to the study hotline immediately if they experienced at least 2 of the above signs or symptoms. as a response, the study nurse would visit the households with any sick members and collect nose and throat swab from all household members. the households were also telephoned monthly or increased to fortnightly during influenza seasons to monitor for signs and symptoms and remind them to report to the hotline. supermarket or book vouchers (for children) were given to the households including us$13 for each serum specimen collected, us$6.5 for each home visit, and us$65 for completion of the study. serologically-indicated influenza infection was the primary outcome of this study. it was define as a ‡4 fold rise in antibody titer within each influenza season. other study outcomes included rt-pcr confirmed influenza virus infection, acute respiratory illness (ari) (two of any of the above listed signs or symptoms), and influenza-like illness (ili) (fever ‡37.8°c with cough or sore throat). antibody titers against the vaccine strains were obtained by testing each serum specimens by haemagluttination inhibition (hai). viral microneutralization (vn) using standard methods was found to be more sensitive than hai in detecting antibody response against a ⁄ california ⁄ 04 ⁄ 2009(h1n1) in another study conducted by our group 27 and was, therefore, used in this study. the sera was initially diluted at 1 ⁄ 10 and further tested in serial doubling dilutions. nose and throat swabs were tested by reverse transcription polymerase chain reaction (rt-pcr) for influenza a and b viruses. technical details of the laboratory methods have been reported elsewhere. 27, 28 fisher's exact test and chi-squared tests were used to compare count data including occurrence of side effects, laboratory confirmed, and clinically defined influenza infections. wilcoxon signed-rank test were used to compare the serum antibody titers between groups. exact binomial method or the wald approximation was used to estimate 95% confidence intervals where appropriate. all analyses were carried out in r version 2.8.1 (r development core team, vienna, austria). twenty-five primary and secondary schools in the district of the study clinic were invited to participate. to parents of three schools that agreed to take part and another study cohort, 3690 invitation letters were sent and 105 households were enrolled. personal referrals were made from these parents to enroll 14 additional households. among 119 enrolled households, 1 subject with history of epileptic seizure was assessed to be contra-indicated against receiving the vaccine. blood taking failed in another subject, and both of them withdrew from the study. eleven households did not complete the study. table 1 shows subject and household contacts of the tiv and placebo group were similar in demographics and prior influenza vaccination history. antibody titers before vaccination were comparable between groups (data not shown). most study subjects who received tiv showed antibody titer ‡40 against the vaccine strains 1 month after receiving tiv, and the proportion was significantly higher than those who received placebo (a ⁄ h1n1 93% in tiv versus 68% in placebo group, p < 0.01; a ⁄ h3n2 97% versus 61%, p < 0.01; b 99% versus 91%, p = 0.15). none of the study subjects had antibody titer ‡40 against ph1n1 following receipt of seasonal tiv. no serious adverse reactions were reported, and only pain at injection sites was slightly higher in tiv group (data not shown). subjects who received tiv had lower rates of serologically confirmed seasonal influenza a(h1n1) (8% versus 21%, p = 0.10), a(h3n2) (7% versus 12%, p = 0.49) and b infection (3% versus 8%, p = 0.36, although the differences were not statistically significant (table 2) . study subjects had higher rate of serologically confirmed ph1n1 infection (32% versus 17%, p = 0.09), yet it was not statistically significant. after adjusting for potential cross reactive antibody response, 31% of subjects in tiv versus 12% in placebo groups showed ph1n1 infection confirmed by either serology or rt-pcr (p = 0.04). little differences were observed for rt-pcr confirmed infection, ari, and ili in results combining the winter and summer influenza seasons. during winter season when seasonal influenza predominated, study subjects who had received tiv showed a lower tendency to develop ili (15% versus 23%, p = 0.43) or ari (46% versus 54%, p = 0.52). an opposite tendency was seen (ili 27% versus 19%, p = 0.43; ari 49% versus 44%, p = 0.68) during summer when ph1n1 predominated. however, these differences were not statistically significant. rates of ili in subjects infected with ph1n1 did not differ statistical significantly between subject who received tiv and placebo (40% versus 25%, p = 0.30). the study was not powered to detect indirect benefits to household contacts of vaccines resulting from reduced household transmission. attack rates were found to be similar between household contacts of subjects received tiv and placebo (data not shown). to examine potential factors that might affect risk of laboratory confirmed ph1n1 infection, a multivariable logistic regression model was fitted to study all subjects and their household contacts. younger participants aged below 16 years were found to have a higher risk (<16 years or = 6.60, 95% ci 2.17, 20.13; 16-45 years or = 2.53, 95% ci 0.80, 7.99, >45 or = 1.00). after adjusting for age, sex, and date of study completion, receipt of tiv for the 2008-9 influenza season was not found to affect risk of ph1n1 infection. however, participants who had laboratory confirmed seasonal influenza infection during the study period had 65% lower risk of ph1n1 infection (infected with seasonal influenza or = 0.35, 95% ci 0.14, 0.87; not infected with seasonal influenza or = 1.00). as (see table s1 for winter and summer results separately). influenza-like illness (ili) defined as temperature ‡37.8°c plus cough or sore throat; acute respiratory illness (ari) defined at least any two of fever ‡37.8°c, chills, headache, sore throat, cough, presence of phlegm, nasal congestion, runny nose, muscle or joint pain. limited by the sample size, we were not able to differentiate between the protective effect of seasonal a(h1n1) and a(h3n2) infection against ph1n1. other details of the results from the study were published elsewhere. 17 discussion a non-significantly higher rate of ph1n1 infection was observed in study subjects who received tiv compared to placebo. results from a multivariable logistic regression suggested that such a pattern might be explained by more common seasonal influenza infection in placebo group prior to the pandemic, protecting the placebo group against ph1n1. seasonal influenza infection within 3-6 months observed in our study might have conferred better cross protection than tiv against ph1n1. this resembles similar previous findings on cross protection between influenza infections in human and animal studies. [29] [30] [31] [32] [33] [34] [35] [36] however, the same phenomenon has not been observed in some studies on seasonal influenza vaccine against ph1n1. 18, 21, [23] [24] [25] apart from differences in study design and vaccine used, we speculate that a short time interval between ph1n1 and most recent seasonal influenza peak activities might be crucial for the phenomenon. hong kong is a subtropical area where the 2009 pandemic was preceded immediately by summer seasonal influenza circulation and a few months apart from the winter 2008-9 influenza peak. if cross protection from seasonal influenza lasts for only a short period, it might have waned below partial cross protection from tiv over time from last seasonal influenza infection. the current study is limited by a small sample size, and further studies are required to confirm our hypothesis. while tiv is only effective against matching strains, a universal influenza vaccine could provide better protection against the ever evolving influenza viruses. introduction immunisation of healthy, as well as high risk, children has been the focus of much recent attention both in prevention of seasonal influenza and during the 2009 h1n1 pandemic. detailed information on reactogenicity, particularly for newer vaccine formulations that include adjuvants, is limited. we recently reported results of a head-to-head comparison of two 2009 h1n1 pandemic influenza vaccines in children in the uk. 1 here we present new, detailed analyses of reactogenicity data from that study, which has important potential implications for future paediatric influenza vaccine development and use. we compared the safety, reactogenicity, and immunogenicity of two h1n1 influenza vaccines, one as03 b (tocopherol based oil in water emulsion) adjuvanted egg culture derived split virion, the other non-adjuvanted cell culture derived whole virion, given as two dose schedules 21 days apart, in a randomised, open label trial as previously reported. the study was age stratified (6 months to under 3 years & 3-12 years) to ensure adequate data in young children. age appropriate safety data (simplified for under 5 year olds) were collected for 7 days after each vaccine dose and serum was collected at enrolment & 21 days after the second dose. nine hundred-thirty seven children received vaccines as per-protocol. when comparing the two vaccines, grade 3 ( ‡50 mm) local reactions were seen more frequently following the adjuvanted than the non-adjuvanted vaccine in both age groups, after both vaccine doses. in children over 5 years old, 7ae2% versus 1ae1%, p < 0ae001, after dose one; 8ae5% versus 1ae1%, p = 0ae002, after dose two, in children under 5 years old, 1ae5% versus 0ae0%, p = 0ae06, after dose one (non significant, ns); 5ae9% versus 0ae0%, p < 0ae001 after dose two. fever ‡38°c (axillary measurement) was seen more frequently following the second dose of the adjuvanted vaccine compared to the non-adjuvanted vaccine in <5 year olds (22ae4% versus 12ae5%; p < 0ae05). looking specifically at the adjuvanted vaccine in under 5 year olds, comparing the second dose with the first, there were significantly higher rates of fever ‡38°c (axillary measurement) (22ae4% versus 8ae9%, p < 0ae001), local grade 3 ( ‡50 mm) reactions (5ae9% versus 1ae5%, p = 0ae02), pain (39ae4% versus 31ae5, p = 0ae02), use of analgesia or antipyretic medication (43ae7% versus 31ae5%, p < 0ae001), and decreased activity (31ae9% versus 20ae4%, p < 0ae001). the adjuvanted vaccine was significantly more immunogenic, most notably in the younger children. in <3 year olds, haemagglutination inhibition (hi) seroconversion rates were 98ae2% versus 80ae1%, p < 0ae001. among all general and local reactions measured, only the maximum temperature measured during the 7 days after the second dose of the adjuvanted vaccine showed a significant (positive) association with post vaccination hi titres. for each 1°c rise in temperature there was a 27% increase in titre (p < 0ae001). these reactogenicity data demonstrate a step towards the future possibility of one-dose influenza immunisation programmes for young children associated with low rates of fever and other reactions. the occurrence of fever following adjuvanted vaccine, seen particularly after a second dose in younger children, was quantitatively associated with enhanced antibody titres. this association was not seen with unadjuvanted vaccine. this apparent difference between the relatedness of the pyrogenic and immunogenic effects of the two vaccines merits further investigation. novel adjuvants appear to have the potential to overcome the relatively poor immunogenicity previously experienced with inactivated influenza vaccines in infants and young children. however, careful adjustment may be needed to optimise the balance between high protection and acceptable reaction rates. tries causing sporadic human infections. vaccination has been used as an effective public health tool for influenza prophylaxis. the goal of this study was to evaluate live attenuated influenza vaccine (laiv) vaccine candidates for subtypes h1 and h5. the attenuated phenotype of h1 and h5 laiv candidates has been proven in experiments in ovo and in vivo. in randomized clinical trials among adult volunteers, no significant adverse reactions attributable to the live vaccine occurred. our results indicate that pandemic laiv candidates were well tolerated and elicited serum, local, and cellular immune responses. the emergence and spread of highly pathogenic avian influenza h5n1 viruses in avian populations and concurrent infections in humans since 1997 has prompted efforts to develop vaccines for use in the event of an influenza pandemic. in 2009, the world faced a new h1n1 pandemic. immunization with inactivated or live vaccines is the primary measure for preventing influenza. laivs appear to be safe and efficacious, and might possibly provide broader immune responses than inactivated vaccines. our study evaluated laiv pandemic candidates as part of the global influenza pandemic preparation project outlined by the who. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated by routine technique in embryonated hen eggs. laiv and placebo were supplied by microgen (irkutsk, russia). the monovalent laiv was produced from the pandemic vaccine candidates and formulated to contain 10 7 and 10 6ae9 eid 50 per dose (0ae5 ml) of a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 and a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92, respectively. the vaccine or placebo was administered intranasally with a single-use dosing nasal sprayer. two doses were given at an interval of 21 days. one hundred-ninety healthy adults aged 18-60 years were randomly divided into groups to receive either pandemic vaccine candidates (204) or placebo (28) . subjects were informed about purposes and methods of the study and potential risks associated with participation. all participants had an hai antibody titer of £1:10 to a ⁄ california ⁄ 7 ⁄ 2009 (h1n1) pandemic virus. in all there were 47 and 42 vaccines and 19 and 8 participants who received placebo, and were further tested for immune responses to h1n1 or h5n2 pandemic vaccine, respectively. another 29 participants vaccinated with h1n1 laiv were children between 12 to18 years old. before the children were vaccinated, their parents were advised about study and their consent was required before any child was enrolled. on the advice of the national ethics committee, we did not include a placebo group in this study. individuals were not enrolled if they had an acute illness or fever at the beginning of the study or a history of egg allergy. immune responses of subjects were assessed by routine hai test (evaluation of serum igg antibodies), elisa (evaluation of iga antibodies eluted from the nasal swabs into steril pbs), and cytokine flow cytometry assay (evaluation of virus-specific cd3 + cd4 + ifnc + and cd3 + cd8 + ifnc + peripheral blood mononuclear cells). the results of phenotypic analysis in ovo showed that pandemic vaccine candidates retained the cold adapted-temperature sensitive (ca ⁄ ts) phenotype, typical of the coldadapted parental mdv. in contrast and as expected, a ⁄ california ⁄ 07 ⁄ 2009 and a ⁄ duck ⁄ potsdam ⁄ 1406-86 parental strains had the non-ts ⁄ non-ca phenotype typical of wt viruses. the h5n2 pandemic vaccine candidate demonstrated an attenuated phenotype in mice and in java macaques and did not infect chickens. the vaccine attenuation study confirmed the attenuated phenotype of a a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 pandemic laiv candidate in mouse, ferret, and guinea pig models. the phase i ⁄ ii randomized, controlled, double-blind clinical study safety evaluation of pandemic vaccine candidates in adults clinical examination of subjects who received two doses of pandemic vaccine candidates indicated that both vaccines were well tolerated. no fever reactions were observed after the first or second vaccination. after the first vaccination, 33ae0% and 40ae0% of reactogenicity events consisting of catarrhal symptoms, such as pharyngeal irritation or hyperemia, were observed for h1n1 and h5n2 vaccine candidates, respectively. after revaccination, subjects did not report local or systemic reactions. to determine whether a serological response occurred in the cohort of immunologically naïve subjects vaccinated with pandemic vaccine candidates, hai and elisa tests were used (table 1) . post-vaccination geometrical mean titers (gmt) among 20 subjects who received two doses of h5n2 vaccine were significantly higher than pre-vaccination titers. the frequency of ‡4 fold antibody rises was significantly higher (47ae1%) after revaccination than after one dose (5ae9%). the percentage of subjects with post-vaccination serum hai titers to h5n2 ‡ 1:20 was 47ae1% and for titers ‡1:40, it was 29ae4%. no seroconversions in the placebo group were detected. the virus-specific nasal iga antibody response to vaccination after two doses of the h5n2 vaccine candidate demonstrated significant increases of ‡4 fold rise iga antibodies (65%) compared to one dose. cumulative data of h5n2 vaccination (all applied tests) showed 35% and 80% of conversions after the first and the second vaccination, respectively. increasing h5n2 vaccine virus infectivity from 10 6ae9 to 10 8ae3 eid 50 ⁄ dose lead to an enhancement of post-vaccination hai titers in vaccinees after the first vaccination to homologous h5n2 antigen from 5ae9% to 31ae0% of ‡4 fold antibody rises. values of post-vaccination serum hai antibody titers in subjects vaccinated with another pandemic vaccine candidate, a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38, also proved to be rather low. after the primary vaccination, the percentage of subjects with hai protective antibody titers ‡1:40 were 2ae1%. after revaccination, this parameter increased to 17ae0%. four-fold increases in serum hai antibody titres were four-fold conversions after the first and the second vaccination was 23ae4% and 34ae4%, respectively. elisa antibodies in nasal swabs showed had an advantage in detecting induction of local iga as compared to serum hai antibodies. after revaccination four-fold serum hai antibody conversions were 34ae4% vs. 63ae8% of iga conversions in nasal swabs, respectively. taking into account cumulative data of h1n1 vaccination (hai and elisa data), the obtained results were here and in the 42ae5% and 70ae2% of conversions after the first and the second vaccination, respectively. fourty-seven subjects were vaccinated with h1n1 laiv, and 19 who received a placebo were chosen for evaluation of cellular immune response by cytokine assays. after revaccination, the mean increases of both cd4 + and cd8 + memory cells were significantly higher in vaccinated subjects compared to the placebo group. interestingly, the same effect of vaccination was observed in vaccinees without detectable conversions of hai antibody titers. even after a single vaccination, the rate of subjects with significant increases of these cells in the blood was 37ae5% (cd8 + ) and 75% (cd4 + ). after the revaccination, the percentage of subjects with significant increases in cd8 + and in cd4 + cells was 68ae8%. immunogenicity of h1n1 pandemic vaccine candidate in children hai antibody results among children aged 12 to 18 years proved to be significantly higher when compared to adult subjects: after the first vaccination, 41ae4% of the children seroconverted; after revaccination, seroconversions reached 83ae3% ( table 2 ). the gmt rise to h1n1 vaccine with primary vaccination was 3:1; after revaccination it increased to 6:7. benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. 1 many years of laiv seasonal trials have shown excellent tolerability and low reactogenicity. [2] [3] [4] indeed, data showed that live influenza vaccines cause minimal systemic, local, and thermal reactions, generally from 0 to 3%. a different situation was observed in the cohort of immunologically naïve volunteers vaccinated with pandemic vaccines. the rate of local reactions to a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 and a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 9 vaccine candidates increased to 33ae0% and 40ae0%, respectively. after revaccination no significant local and systemic reactions were observed. this confirms, indirectly, the development of a sufficiently high level of protection after the first vaccination with pandemic laiv. the most important criterion for assessing the quality of vaccines is their estimated safety, epidemic effectiveness, and immunogenicity. however, current regulatory documentation 5 mandates that induction of serum antibodies, measured by hai, as the only criterion for a laiv immunogenicity evaluation. in addition to the standard hai assay, we determined serum (igg) and local (iga) antibodies in adult subjects vaccinated with an h1n1 pandemic vaccine candidate. evaluation of overall results obtained in these additional serological tests, as well as those from the hai assay, showed an immune response to the vaccine in the majority of subjects (42ae5% of ab seroconversions after the single vaccination and 70ae2% after revaccination, respectively). these data show that methods used to routinely measure laiv immunogenicity should be revised to include a number of additional immunological methods such as igg and iga elisa, and cytokine assays consistent with the recently updated who recommendations on laiv monitoring. these clinical studies clearly demonstrated that pandemic laiv candidates are effective at generating pandemic specific influenza immunity. a key finding from this study is that it may be practical to give the vaccine as a single dose to both children and adults. evaluation of our laiv pandemic vaccine candidates was performed as part of the global influenza pandemic preparation project outlined by the who. 6 it was considered that laiv could be produced in greater quantities and more rapidly than inactivated vaccines. together with the generation of herd immunity by laiv, this suggests that laiv implementation during the first wave of a pandemic may provide significant social, economic, and health benefits to the community. authors are thankful to path for the financial support of h1n1 pandemic vaccine study. we are grateful for the the main evolutionary mechanism of influenza viruses during inter-pandemic period is the antigenic drift, but the epidemiological picture of circulating viruses is complicated by a high level of heterogeneity of strains, even though drift does not occur, due to co-circulation of drifted and old strains or to co-circulation of viruses belonging to the same type ⁄ subtype but with different antigenic patterns. [1] [2] [3] [4] [5] [6] lack of data exists on the impact of the wide heterogeneity of circulating strains on the seroprotection and on-field effectiveness of influenza vaccine: in particular, little is known about the ability of influenza vaccine to elicit an effective immune response against isolates with few amino acid mutations with respect to vaccine strains that represent the majority of circulating viruses. mf59-adjuvanted vaccines, which are currently used for the prevention of seasonal influenza epidemics in elderly, are showed to confer higher seroprotection against homologous and drifted a(h3n2) strains than non-adjuvanted vaccines. [7] [8] [9] the broader immune response showed by mf59-adjuvanted vaccine was measured using hi and nt assays against egg-grown drifted strains representing vaccine composition changes during the following seasons, but its ability to elicit a broader immune response against circulating viruses belonging to vaccine cluster and presenting amino acid mutations onto antigenic sites or against on-field isolates not-antigenically distant from vaccine strains has not yet been investigated. showing amino acid changes onto antigenic sites in position 145 (n145k), 189 (n189k), and 227 (p227s) with respect to a ⁄ california ⁄ 7 ⁄ 04. in particular, a ⁄ genoa ⁄ 13 ⁄ 04 and a ⁄ genoa ⁄ 27 ⁄ 04 presents n126d amino acid mutation detected in clade 2 a ⁄ wyoming ⁄ 3 ⁄ 03-like viruses. 1 the ha sequences of a ⁄ genoa ⁄ 59 ⁄ 04, a ⁄ genoa ⁄ 2 ⁄ 05, genoa ⁄ 11 ⁄ 05, a ⁄ genoa ⁄ 47 ⁄ 05, and a ⁄ genoa ⁄ 62 ⁄ 05 fell within the clade represented by the ha of a ⁄ califor-nia ⁄ 7 ⁄ 04; among these isolates, a ⁄ genoa ⁄ 2 ⁄ 05 and a ⁄ genoa ⁄ 11 ⁄ 05 showed antigenic site sequences very close to that of the 2005 ⁄ 06 vaccine strain, whereas ha sequences of a ⁄ genoa ⁄ 62 ⁄ 05, a ⁄ genoa ⁄ ⁄ 47 ⁄ 05 and a ⁄ genoa ⁄ 59 ⁄ 05 posses amino changes onto antigenic site a(r142k), c(g50e) and d(r208k), respectively. the ha sequences of more recent isolates fell within the clade represented by the ha of a ⁄ brisbane ⁄ 10 ⁄ 07 and characterized by the amino acid changes, relative to the ha of a a ⁄ california ⁄ 7 ⁄ 04, g50e and k140i, with the exception of a ⁄ genoa ⁄ 3 ⁄ 07, showing r142g and l157s amino acid changes present in viruses belonging to a ⁄ nepal ⁄ 921 ⁄ 06 clade. measure of genetic distance between vaccine and circulating strains was calculated as previously described by gupta. 10 two blood samples were collected from each subject, just before and 22 ± 2 day post-vaccination. all sera were stored at )20°c. all samples were tested at the laboratory of health sciences department, university of genoa, by haemagglutination-inhibition (hi) and neutralization (nt) assays, performed following the who criteria and standardised method in our laboratory, respectively. [11] [12] [13] guinea pig red blood cells were used for hi assay. all samples were assayed twice for hi and for nt. the obtained antibody titre was expressed as the reciprocal of the last sera haemagglutinating or inhibiting virus dilution. immunogenicity was determined by: geometric mean titre (gmt); mean-fold increase (mfi; ratio of post-to pre-vaccination titre); seroprotection rate (the percentage of subjects achieving an hi and nt titre ‡40 iu); and seroconversion rate (percentage of subjects with a fourfold increase in hi or nt antibody titers, providing a minimal post vaccination titer of 1:40). post-vaccination gmt was reported as ratio, with the corresponding 95% confidence interval, of gmts after vaccination with mf59-adjuvanted vaccine and with non-adjuvanted subunit vaccine. seroprotection and seroconversion rate 95% confidence interval was calculated using modified wald method. comparisons of seroconversion and seroprotection rates between subunit and mf59-adjuvanted vaccine groups have been analyzed by fischer's exact test. the results were evaluated against the committee for medicinal products for human use (chmp) criteria for approval of influenza vaccines in the elderly, which require that at least one of the following criteria be met: mfi >2; seroprotection rate >60%, or seroconversion rate >30%. furthermore, hi titres were also transformed into binary logarithms, corrected for pre-vaccination status, as described by beyer et al. 14 and were expressed as median titres, with the corresponding 25°-75°i nter-quantile range. comparisons of corrected post-vaccination titers between subunit and mf59-adjuvanted vaccine groups were analyzed by wilcoxon test. difference in immunogenicity profile between vaccine groups, expressed by ratio of different parameters, was correlated with genetic and antigenic distance between vaccine and viruses used in the study using spearman test. pre-vaccination titres were not significantly different between vaccine groups, for all 15 strains (data not shown). post-vaccination gmt ratios between mf59-adjuvanted and non-adjuvanted vaccine groups determined using hi and nt assays, with the corresponding 95% confidence interval, according to viral strain are shown in figure 1 . both vaccines met chmp requirements for mfi (>2), seroconversion (>30%), and seroprotection rate (>60%) against a ⁄ wyoming ⁄ 3 ⁄ 03-like, with the exception of a ⁄ genoa ⁄ 13 ⁄ 04 and a ⁄ california ⁄ 7 ⁄ 04-like circulating viruses and against egg-grown a ⁄ wyoming ⁄ 3 ⁄ 03, a ⁄ california ⁄ 7 ⁄ 04, and a ⁄ wisconsin ⁄ 67 ⁄ 05 strains; the immune response against a ⁄ genoa ⁄ 13 ⁄ 04 met the requirements for mfi and seroprotection rate only in mf59-adjuvanted vaccine group. requirements for mfi, seroconversion, and seroprotection rate against the a ⁄ brisbane ⁄ 10 ⁄ 07-like virus a ⁄ genoa ⁄ 2 ⁄ 07 and the a ⁄ nepal ⁄ 921 ⁄ 06-like genoa ⁄ 3 ⁄ 07 viruses and against egg-grown a ⁄ brisbane ⁄ 10 ⁄ 07 strain were reached only in subjects vaccinated with the mf59adjuvanted vaccine. a similar pattern emerged from the analysis of mfi, seroconversion and seroprotection rates using nt assays. subjects vaccinated with the mf59-adjuvanted vaccine showed significantly higher post-vaccination hi gmts against a ⁄ wyoming ⁄ 3 ⁄ 03-like, a ⁄ california ⁄ 7 ⁄ 04-like, a ⁄ nepal ⁄ 921 ⁄ 06-like and a ⁄ brisbane ⁄ 10 ⁄ 07like viruses, with the exception of a ⁄ genoa ⁄ 3 ⁄ 06, and against egg-grown a ⁄ california ⁄ 7 ⁄ 04, a ⁄ wisconsin ⁄ 67 ⁄ 05, and a ⁄ brisbane ⁄ 10 ⁄ 07 strains, compared with individuals immunized with the non-adjuvanted vaccine ( figure 1 ). the mf59-adjuvanted vaccine also induced significantly higher seroconversion and seroprotection rates against following correction for pre-vaccination status, hi titres were significantly higher for the mf59-adjuvanted vaccine group when evaluated against a ⁄ wyoming ⁄ 3 ⁄ 03-like viruses, a ⁄ brisbane ⁄ 10 ⁄ 07-like a ⁄ genoa ⁄ 2 ⁄ 07, and a ⁄ nepal ⁄ 921 ⁄ 06-like a ⁄ genoa ⁄ 3 ⁄ 07 strain ( figure 2 ). pre-vaccination titre corrected response was higher in subjects vaccinated with mf59 adjuvanted vaccine also against egg-grown a ⁄ wyoming ⁄ 3 ⁄ 03, a ⁄ california ⁄ ⁄ 7 ⁄ 04, a ⁄ wisconsin ⁄ 67 ⁄ 05, and a ⁄ brisbane ⁄ 10 ⁄ 07. among viruses more closely related to a ⁄ california ⁄ 7 ⁄ 04, subjects immunized with mf59-adjuvanted vaccine showed a significantly higher corrected titres against a ⁄ genoa ⁄ 59 ⁄ 04, a ⁄ genoa ⁄ 47 ⁄ 05, and a ⁄ genoa ⁄ 62 ⁄ 05 strains compared with the non-adjuvanted vaccine ( figure 2) . spearman test showed a clear correlation between the distances and the advantage offered by mf59 expressed by ratio between mfi, post-vaccination gmts, corrected post-vaccination median, seroconversion, and seroprotection rates calculated using hi test in the two vaccine groups. similarly, ratio between mfi, seroconversion, and seroprotection rates calculated with nt test correlated with the genetic and antigenic distance between vaccine and viruses used for the study. the ability of mf59 to enhance the immunogenicity and to elicit a broader immune response against drifted strains than non-adjuvanted vaccine is consistent with other findings reported during the last decade. [7] [8] [9] 15 in subjects vaccinated with the mf59-adjuvanted vaccine containing a ⁄ california ⁄ 7 ⁄ 04, the immune response, expressed by a number of parameters, such as crude and corrected postvaccination titers, seroconversion, and seroprotection rates calculated using hi and nt assays, is higher than that observed in individuals immunized with subunit vaccine when it is evaluated against a drifted strains, such as a ⁄ brisbane ⁄ 10 ⁄ 07-like and a ⁄ nepal ⁄ 921 ⁄ 06-like strains, and against egg-grown a ⁄ brisbane ⁄ 10 ⁄ 07 virus. for the first time in this study, the impact of heterogeneity of circulating strains antigenically close to the vaccine on the antibody response elicited by mf59-and non-adiuvanted vaccines is evaluated. immune response against viruses isolated during the 2004 ⁄ 05 season, that appear more phylogenetically close to 2004 ⁄ 05 vaccine strain a ⁄ wyoming ⁄ 3 ⁄ 03, was higher in subjects vaccinated with mf59-adiuvanted vaccine as demonstrated by higher crude and corrected post-vaccination hi titres and higher postvaccination nt titres, with the exception of a ⁄ genoa ⁄ 27 ⁄ 04, against whom the nt post-vaccination gmt is identical in mf59 and subunit vaccine groups. furthermore, hi seroconversion and seroprotection rates were higher in mf59 vaccine group when evaluated against a ⁄ genoa ⁄ 13 ⁄ 04 and a ⁄ genoa ⁄ 27 ⁄ 04. as far as the immune response against a ⁄ california ⁄ 7 ⁄ 04-like viruses, the small number of enrolled subjects did not allow appreciating differences using qualitative response indicators, but crude post-vaccination hi titres were higher in mf59 vaccine group for all the strains. interestingly, a ⁄ california ⁄ 7 ⁄ 04-like viruses with at least one amino acid change onto antigenic sites, i.e. a ⁄ genoa ⁄ 59 ⁄ 04, a ⁄ genoa ⁄ 47 ⁄ 05, and a ⁄ genoa ⁄ 62 ⁄ 05, showed a more marked difference in terms of response between the two vaccine groups. individuals immunized with mf59-adiuvanted vaccine showed higher corrected post-vaccination hi titres and post-vaccination nt titres in comparison with subjects vaccinated with plain vaccine. these response indicators were similar in the two vaccine groups when the response was evaluated against a ⁄ genoa ⁄ 2 ⁄ 05 and a ⁄ genoa ⁄ 11 ⁄ 04, which present no amino acid changes onto antigenic sites and identical hi titers respect with a ⁄ california ⁄ 7 ⁄ 04 at molecular and antigenic characterization, respectively. thus, the advantage offered by mf59 in terms of higher immunogenicity expressed by higher post-vaccination hi titres is observable also against viruses showing antigenic and molecular pattern undistinguishable from vaccine strain, but it became even more evident as the antigenic and molecular distance between vaccine and circulating strains grew. as emerged for a ⁄ genoa ⁄ 59 ⁄ 04, a ⁄ genoa ⁄ 47 ⁄ 05, and a ⁄ genoa ⁄ 62 ⁄ 05, one amino acid was a sufficient change in antigenic sites for 2-fold decrease of hi titre against homologous vaccine strain to observe 2-fold higher post-vaccination nt titers (mf59 ⁄ subunit postvaccination gmt ratio range between 1ae79 and 2ae45, figure 1) and one-dilution higher corrected post-vaccination hi titers in mf59 vaccine group ( figure 2) . finally, the correlation between the distance and the improvement offered by mf59 in terms of higher immunogenicity clearly emerged by spearman correlation analysis: it remains wellfounded both using a number of different response parameters obtained from hi and nt assays and calculating the distance by serological and genetic methods. outbreaks of h1n1pdm in pigs in commercial swine operations have been reported in several countries. in all incidents, epidemiological investigations have linked humans as the possible source of the infection to pigs. experimentally, it was established that the virus is pathogenic and transmits readily in pigs. 1 the natural outbreaks of h1n1pdm and laboratory studies underscore the threat that the virus poses to the swine industry and highlight the need for developing effective control strategies. in the united states, a trivalent live attenuated influenza vaccine (flumistò) has been licensed for use in humans since 2003. 2 in swine medicine, however, temperature-sensitive laivs are not available. currently, only inactivated vaccines are available for pigs, but they provide limited protection against antigenically diverse influenza viruses. additionally, the use of inactivated vaccines has been associated with enhanced pneumonia when immunized pigs were challenged with divergent viruses. 3 thus, the development of laivs has the potential to circumvent the drawbacks associated with commercial vaccines. with the aim of developing laiv temperature-sensitive influenza vaccines against the h1n1pdm virus, we have used reverse genetics to introduce attenuation markers in the polymerase genes of a swine-like tr h3n2 influenza virus, a ⁄ turkey ⁄ ohio ⁄ 313053 ⁄ 04 (h3n2) (ty ⁄ 04). 4 we chose this isolate because it grows well in both eggs and cell culturebased substrates, displays a broad host range, and has internal genes similar to the h1n1pdm virus. safety and efficacy studies of the ty ⁄ 04 att vaccine candidates in pigs demonstrated that this vaccine backbone is attenuated in swine and conferred sterilizing immunity upon an aggressive intratracheal challenge of pigs with the 2009 h1n1 pandemic virus. thus, introduction of genetic signatures for att in the backbone of a swine-like tr influenza virus resulted in highly attenuated and efficacious live influenza vaccines with promising applications veterinary medicine. 293-t cells and mdck cells were maintained as previously described. 5 a ⁄ turkey ⁄ ohio ⁄ 313053 ⁄ 04 (h3n2) (ty ⁄ 04) has options for the control of influenza vii ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 328-394 been previously described and it was kindly provided by yehia saif, ohio state university. 4 a ⁄ california ⁄ 04 ⁄ 09 (h1n1) (ca ⁄ 04) was kindly provided by the centers for disease control and prevention (cdc). generation of recombinant viruses by reverse genetics (rg) was done using a previously described method. 6 the genetic signatures for attenuation 5 were introduced into the pb2 and pb1 genes of ty ⁄ 04. ny 2:6 ty ⁄ 04 att is a 2:6 reassortant with the surface genes from the a ⁄ new york ⁄ 18 ⁄ 2009 (h1n1) virus and the ty ⁄ 04 att internal genes. all viruses were amplified in mdck cells to produce viral stocks. twenty-five pigs were divided into five groups (n = 5) and intranasally inoculated with 10 5 tcid 50 ⁄ animal of either h3n2:6ty ⁄ 04 att or with ny(h1n1)2:6ty ⁄ 04 att vaccines diluted in 2 ml of mem. two other groups were similarly inoculated with h3n2:6ty ⁄ 04 wt and h3n2:6ty ⁄ 04 rg and served as controls, whereas a fifth group was mockvaccinated with pbs alone. clinical observations were performed as previously described. 1, 7 efficacy of h1n1 ty ⁄ 04 att vaccine in pigs fourty pigs were divided in four groups (n = 10)( table 1) . group 1 was vaccinated with 10 5 tcid 50 ⁄ animal of ny(h1n1)2:6ty ⁄ 04 att through intranasal route, whereas group 2 was vaccinated intramuscularly with 2 ml of an adjuvanted uv-inactivated ca ⁄ 04 vaccine (uvadj-ca ⁄ 04). 7 group 3, non-vaccinated and challenged (nv+ca ⁄ 04), and group 4, non-vaccinated, mock-challenged (nv+mock), were also included. pigs were boosted two weeks later. fourteen days post boost (dpb), pigs from groups 1-3 were challenged intratracheally with 2 ml of 1 · 10 5 tcid 50 of ca ⁄ 04. following challenge, pigs were monitored using methods as previously described. 7 all statistical analyses were performed using graphpad prism software version 5ae00 (graphpad software inc., san diego, ca). the differences were considered statistically significant at p < 0ae05. the ty ⁄ 04 att-based vaccines are attenuated in swine pigs inoculated with wt ty ⁄ 04 viruses developed fever (>40°c) that peaked 24 hpi ( figure 1a) and shed large amounts of in nasal secretions ( figure 1b) . similarly, viral titers in bronchoalveolar lavage fluid (balf) collected at 3 dpi ranged from 10 5 to 10 6 tcid 50 ⁄ ml ( figure 1c ). at necropsy, the lungs from animals inoculated with these viruses had severe pneumonia ( figure 1d ). in contrast, none of the animals inoculated with h3n2 or h1n1 ty ⁄ 04 att viruses developed clinical signs following vaccination, indicating that the ty ⁄ 04 att viruses were safe for administration to pigs ( figure 1a) . correspondingly, there was 100-1000 fold less virus shedding from the nose of pigs vaccinated with ty ⁄ 04 att viruses as compared to unmodified ty ⁄ 04 viruses. in general, ny(h1n1)2:6ty ⁄ 04 att -vaccinated pigs shed less virus than h3n2:6 ty ⁄ 04 att inoculated pigs ( figure 1b ). in addition, viral titers in balf were significantly reduced (p < 0ae01) in ty ⁄ 04 attvaccinated pigs as compared to ty ⁄ 04 wt-infected pigs ( figure 1c ). although both vaccines caused mild gross and microscopic lesions in the lungs, the percentage of lung 0ae2 ± 0ae1* 0 ± 0* 0 ± 0* 0 ± 0* balf, bronchoalveolar lavage fluid, uvadj-ca ⁄ 04, uv-inactivated ca ⁄ 04 vaccine; nv+ca ⁄ 04, non-vaccinated, challenged positive control group; nv+mock, non-vaccinated, non-challenged negative control group. *significantly different from nv+ca ⁄ 04 control group at p < 0ae05. geometric mean hi titer against ca ⁄ 04 at the day of challenge. à percentage of macroscopic lung lesions given as mean score ± sem. § average viral titer (log 10 ) measure as tcid 50 per ml. -average viral titer (log 10 ) in balf at 5 dpc. involvement was not significantly different from mock-vaccinated pigs, corroborating the clinical findings that these vaccines are sufficiently attenuated in pigs ( figure 1d, e) . histopathologically, nasal turbinates and trachea obtained from pigs immunized with either vaccine were similar to control animals, as opposed to the wt-inoculated pigs ( figure 1e ). vaccination with h1n1 ty ⁄ 04 att-based vaccines provides sterilizing immunity against h1n1pdm in pigs the clinical performance in pigs of the h1n1 vaccines is summarized in table 1 . nv+ca ⁄ 04 animals had macroscopic pneumonia, viral replication in balf and shedding in the nose. uvadj-ca ⁄ 04 vaccine provided satisfactory protection, but this protection was not sterilizing. remarkably, animals vaccinated with ny(h1n1)2:6ty ⁄ 04 att had sterilizing immunity. in both vaccine groups there was a significant reduction (p < 0ae001) in the percentage of macroscopic lung pathology compared to the nv+ca ⁄ 04 group. control pigs had neither significant macroscopic nor microscopic lesions in the lungs. hi antibody titers measured at the day of challenge in both vaccine groups were approximately the same (table 1 ). in the present study, we developed for the first time, temperature-sensitive laiv for use in pigs. data from our safety studies showed that both the h3n2 and h1n1 ty ⁄ 04 att vaccines were attenuated in pigs. although the ty ⁄ 04 att vaccines were detected in balf samples, the level of viral replication was significantly reduced in comparison to unmodified virus and, more importantly, caused no overt clinical signs. a minimal amount of replication is likely beneficial for eliciting t-cell responses to internal genes that may provide heterologous cross-protection. one of the most challenging tasks in producing effective live attenuated vaccines is to achieve an adequate balance between safety and efficacy. by introducing the att modifications into the polymerase genes of a swine-like tr strain, this desirable balance was achieved. the vaccines were histopathologic scores of nasal turbinates, trachea and lungs at 3 dpi. ny(h1n1)2:6ty ⁄ 04 att (a virus that carries the surface genes of a ⁄ new york ⁄ 18 ⁄ 09 (h1n1) and ty ⁄ 04 att internal genes). all h3n2 viruses have their surface genes derived from ty ⁄ 04. values are shown as the mean ± sem. * p < 0ae05; **p < 0ae01; *** p < 0ae001. options for the control of influenza vii ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 328-394 attenuated in pigs and, more importantly, provided sterilizing immunity upon an aggressive challenge with pandemic h1n1 as opposed to an experimental ca ⁄ 04 inactivated vaccine, which elicited protective but not sterilizing immunity in all animals. in the face of influenza pandemics that have the ability to overcome the species barriers such as the 2009 h1n1, the supply of vaccines for use in agriculture could be jeopardized. our cell culture-based live att h1n1 vaccines could be an attractive alternative for this possible pandemic vaccine shortage. because the ty ⁄ 04 att live vaccines developed here are efficacious in swine, are easier to manufacture than inactivated vaccines, and do not require adjuvants, our study represents a major advance in vaccine development for the 2009 h1n1 pandemic. in conclusion, our second generation of live att influenza vaccines based on modifications of the pb2 and pb1 genes of ty ⁄ 04 retains its safety properties in vivo and can induce excellent protection against aggressive h1n1 challenges in the swine host. influenza virus is one of the most important respiratory pathogens worldwide. 1, 2 type a influenza causes an acute disease of the upper airways, and affects 20-40 million persons yearly. moreover, the threat of human influenza epidemic and pandemic has dramatically increased in recent years. 3 vaccination is one of the crucial interventions for reducing the spread and impact of influenza. the generally used parenteral inactivated influenza vaccines induce mainly systemic antibody responses and only weak cell-mediated immunity and low levels if any mucosal immunity. on the other hand, intranasal immunization with live virus can induces a broad spectrum of both systemic and mucosal antibodies, and the immune response localized in the mucosa blocks the virus even during the first phase of infection. unfortunately, the use of live vaccines is always associated with a certain risk. the development of a crossprotective vaccine against potentially pandemic strains is an essential part of the strategy to control and prevent a pandemic outbreak. we induced intrasubtypic and intersubtypic cross-protection in balb ⁄ c mice by intratracheal (it) immunization with inactivated influenza viruses together with dead delipidated bacillus firmus (dbf) as an adjuvant. ten days after the 2nd immunization dose, the mice were infected with live influenza virus b ⁄ lee ⁄ 40 lethal for mice (total infection dose corresponded to 5 · ld 50 ) or a⁄ pr 8 ⁄ 34 (total infection dose corresponded to 0ae5-5 ld 50 ). dbf adjuvant markedly increased both systemic and mucosal anti-viral antibody formation when applied together with inactivated influenza a or b viruses. 4 protective significance was tested in vivo. mice were preimmunized with 1) pbs (controls), 2) dbf alone, 3) virus alone, and 4) vir-us+dbf. influenza b virus strains b ⁄ lee and b ⁄ yamanashi 166 ⁄ 98 (58 years phylogenetically distant and antigenically substantially different, especially in terms of the main protective antigen -surface haemagglutinin) or two different influenza a subtypes -a ⁄ pr 8 ⁄ 34 (h 1 n 1 ) and a ⁄ california 7 ⁄ 04 (h 3 n 2 ) -were used (figures 1 and 2) . the mice were challenged with 5 · ld 50 of either b ⁄ lee ⁄ 40 or a ⁄ pr 8 ⁄ 34 as appropriate. all controls died. the mice treated with dbf alone died with a delay or survived, which could be explained by stimulation of innate immunity. the animals immunized with virus alone were protected against homologous strains. adjuvant immunization was cross-protective: the mice immunized with a heterologous b strain (figure 2 ) fell ill (pronounced body mass loss), but almost all survived and recovered. 5 the mice immunized with a heterologous a subtype were excellently protected (negligible weight loss and zero mortality). 6 intratracheal dbf (500 lg per mouse) given to non-immunized mice 24 hour before influenza infection eliminated the lethal effect in 40-100% of infected animals depending on infection dose (0ae5-5 ld50); in mice infected with lower than lethal doses (0ae5 ld50), weight loss was minimized or did not occur. the current mode of vaccination-induced immunity is mostly effective against a homologous strain of the virus used for vaccination. the attention is therefore focused on vaccines that are able to induce cross-protection and could be effective also in case of sudden appearance of a new virus variant. inactivated influenza viruses are known to be often insufficiently effective when used for mucosal immunization and for induction of cross-protection against drifted influenza viruses or novel subtypes. the drawback of vaccination with dead virus can be overcome by using a suitable adjuvant. mouse models were successfully immunized with vaccine containing inactivated virus in combination with cholera toxin or the escheria coli heat-labile toxin (lt). [7] [8] [9] the use of cholera toxin in humans is precluded because of its high toxicity; a number of lt mutants that retain their adjuvant activity have been prepared; these mutants were likewise tested on the mouse model and should not cause any serious side effect in humans. for this reason, current studies aim at finding a suitable and safe mucosal and systemic immune response. dbf has been shown to be a very efficient adjuvant for mucosal immunization stimulating both innate and adaptive immunity. intratracheal immunization with inactivated influenza viruses and dbf as adjuvant induced efficient and even heterosubtypic cross-protection. dbf given 24 hour before infection provided partial protection probably because of its strong stimulatory effect on the innate immunity. temperature-sensitive and cold-adapted candidates for live attenuated influenza vaccine with genomic composition of 7:1 based on highly pathogenic influenza a ⁄ h5n1 viruses with pandemic potential were generated by the replacement of six internal genes from the influenza a ⁄ puerto rico ⁄ 8 ⁄ 34 (pr8) virus from pr8-based rg-candidates for inactivated vaccine with appropriate internal genes of influenza a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) master donor virus (mdv) for russian laiv by methods of classical reassortment. all attempts to capture avian n1 neuraminidase into the genome of the mdv laiv production were ineffective. 6:2 reassortants were not generated. step by step co-infection of triple reassortants (h5n1-h1n1-h2n2) with h2n2 mdv in some cases was the only possibility to generate influenza a ⁄ h5n2 cold-adapted vaccine reassortants. difficulties in generating 6:2 reassortants could be explained by a substantial gene constellation in the genome of pr8based h5n1 reassortant viruses. strong coupling of pb2 ⁄ pr8 and avian n1 genes in a ⁄ h5n1-pr8-rg reassortants was revealed. annually updated laiv strains are generated by classical reassortment of circulating influenza viruses with well characterized, attenuated, ts ⁄ ca mdvs. resulting attenuated reassortants inherit the relevant ha and na of wild type parental virus and six internal genes of the mdv. 1 candidates for inactivated influenza vaccines based upon avian influenza viruses with pandemic potential are generally generated by reverse genetics methods. 2 in these cases, like with laiv, vaccine strains are 6:2 reassortants which possess the modified ha and na from potentially pandemic virus and six internal genes from the pr8 virus. the pr8 virus is considered to be of low virulence, i.e. attenuated, for humans, yet offers properties of high seed virus growth for influenza vaccine production. the ha of avian h5 influenza viruses with pandemic potential is engineered to remove four basic amino acid codons from the cleavage site of ha, resulting in a virus that is considered attenuated for natural hosts and safe for people. the objective of this study was to safely generate vaccine candidates for a laiv using highly pathogenic avian influenza viruses by the replacement of six internal pr8 genes in the genome of candidates for inactivated vaccine subtype h5n1 (a ⁄ h5n1-pr8-rg) with internal genes of the laiv mdv by methods of classical reassortment. len17-mdv and a ⁄ h5n1-pr8-rg virus were co-infected in embryonated chicken eggs. five rounds of selective propagation were performed, three of which were at low temperature (25°c). the production and selection of reassortants were carried out in the presence of rabbit antiserum to len17-mdv. cloning by endpoint dilution was performed in each of the last three passages. a virus sample in an open petri dish was rocked gently for 20 sec while being irradiated with a ge 15 watt germicidal lamp at a distance of 20 cm from the dish. the residual infection titer was measured by titration in embryonated chicken eggs. genome composition of reassortant viruses was monitored by rflp analysis. 3 in addition, capacity of reassortant viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) for influenza viruses was determined by virus titration in chicken eggs. reassortment of the mdv with the vn-pr or indo-pr viruses either resulted in reassortants that contained six internal genes from len17-mdv. however, all generated clones contained the na from the mdv. of ten such 7:1 reassortants based on vn-pr three reassortants had the pa gene from pr8 and one had ns gene from pr8. 6:2 reassortants from the targeted h5n1 composition were not generated. after repeated attempts, 7:1 temperature sensitive and cold adapted reassortants based on vn-pr and indo-pr viruses were obtained, but again, none had inherited the avian n1 neuraminidase (table 1) . in contrast, nibrg-23 didn't reassort with the mdv at all. twelve unsucsessful attempts to develop 6:2 or 7:1 reassortants of nibrg-23 with mdv showed that the classical reassortment procedure (cloning by limited dilutions in the presence of anti-mdv serum, followed by co-infection of equal doses of two parental viruses in eggs and two selective passages at 25°c) did not work for this virus pair. to disharmonize the incredibly strong gene constellation of nibrg-23, various modifications of the co-infection step were studied, such as: altering the nibrg-23 to mdv ratio (from 1:1 to 1: tions of anti-mdv serum alone or together with anti-pr8 serum. it was noted that even if the h5n1 to mdv ratio was 1:10 6 , the clones obtained were presumably parental h5n1 viruses without the transfer of any mdv-genes into genome of nibrg-23. in all, 234 clones were isolated, and 209 of them were identical to nibrg-23 parental virus. in nine clones, only the pa gene from mdv was included, whereas in three clones only the 'cold' ns gene was included (data not shown). using uv inactivation of nibrg-23 prior co-infection was more encouraging. after the first round of co-infection of partially uv-inactivated nibrg-23 with mdv (at ratio 1:10 2 ), reassortants that inherited several internal genes of mdv were obtained in the context of the nibrg-23 background (b3, c2, c4, d1) ( table 2 ). some of them (c2, c4, d1) were chosen for the next round of co-infection. after the second round of co-infection, c2, c4, and d1 'intermediate' reassortants with mdv (at ratio 1:1 or 1:10) 7:1 vaccine reassortants finally were obtained. live attenuated influenza vaccine is considered as one of the most promising pandemic vaccines. according to the who there is evidence that laiv might be more effective than inactivated vaccines. 4 this study attempted the safe development of laiv for potential pandemic highly pathogenic avian a ⁄ h5n1 viruses on the base of rg-reassortants for inactivated vaccine with modified h5 hemagglutinin and mdv for laiv. replacement of pr8based internal genes into genome of vn-pr and indo-pr reassortants with appropriate genes of mdv was realized by the classical reassortment procedure. difficulties were encountered in obtaining 6:2 reassortants that contained both the ha and na from the wild type avian h5n1 parental virus. in attempts to reassort the nibrg-23 with mdv, the classical reassortment procedure was unsuccessful. the challenge faced was to break an incredibly strong gene constellation of the nibrg-23 virus. partial uv-inactivation of nibrg-23 was encouraged in replacement of some pr8 internal genes with mdv genes 5 in some cases avian-human reassortant viruses with gull h13n6 and human influenza h1n1 genes were difficult to generate, and reassortants with the desired genotype of six gull virus genes with human influenza a h1 and n1 genes were not isolated despite repeated attempts. the gull pb2, np, and ns genes were not present in any of the gull-human h1n1 reassortants generated. 6 it is difficult to fully understand potential reasons for observed difficulties to reassort some avian viruses with human strains. unsuccessful attempts to develop 6:2 vaccine reassortants may be caused by an observed strong connection of pb2 and na genes in the genome of a ⁄ h5n1-pr8-rg viruses. in our attempts, each reassortant that possessed avian n1 neuraminidase inheritied pb2 gene of pr8 as well. and vice versa, the 'cold' pb2 gene always appeared to be coupled with the n2 neuraminidase of the mdv. in some cases, step by step co-infection of triple reassortants (h5n1-h1n1-h2n2) with h2n2 mdv may be the only possibility to generate a cold-adapted vaccine reassortant. our studies demonstrate unique and significant challenges that are faced in the development of influenza vaccines for avian influenza viruses with pandemic potential. such challenges must be further studied to identify methodologies to allow for rapid development and response to emerging viruses in a crisis. it is imperative that these studies be continued and expanded to identify either mechanisms of such tight gene constellations in influenza viruses produced by rg-derived vaccine strains or inability some genes of human h2n2 and avian h5n1 viruses to cross. in addition, further studies to improve the efficiency of classical reassortment processes will be conducted. during the period from 1997 to 2009, avian influenza outbreaks among humans have been registered in 15 countries of asia, europe, and africa. morbidity and mortality of humans followed the global spread of avian influenza h5n1 among wild and domestic birds, which caused great economic loss to the poultry industry in many regions including some highly developed countries. the global threat from avian influenza forced scientists to develop technologies for the production of a ⁄ h5n1 human vaccine. the development of ai a ⁄ h5n1 vaccines using strains isolated in kazakhstan and the organization of local production and creation of strategic stockpiles of effective vaccines is the an important issue for public health protection in the republic of kazakhstan. to address this, a scientific program 'influenza a ⁄ h5n1 vaccine development for public health protection in kazakhstan' was approved and financed from 2008 to 2010. in this article we give basic results of the development of a recombinant ai a ⁄ h5n1 inactivated whole virion vaccine with aluminium hydroxide as adjuvant for public health protection in kazakhstan. [1] [2] [3] the development of vaccine technology was conducted with the use of a ⁄ astanarg ⁄ 6:2 ⁄ 2009(a ⁄ h5n1) recombinant strain made of a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05(h5n1) and a ⁄ pr ⁄ 8 ⁄ 34(h1n1) strains by the reverse genetics. inactivation of virus containing allantoic fluid was carried out with the use of formalin in different concentrations. complete-ness of the virus inactivation was tested by 3-fold virus passaging in embryos. 4, 5 purification and concentration of the inactivated viruscontaining allantoic fluid was conducted with the use of ultra filtration in tangential flow, which was followed by gel filtration. then we evaluated the content of total protein, hemagglutinin, and ovalbumin in purified and concentrated material. 6 vaccine was composed of clarified and inactivated virus concentrate with the known ha dose containment, and 0ae4% aluminum hydroxide was added in 1:1 proportions. composition components and quality control of finished vaccine was determined in the stages of semi-finished product and finished biopreparation. determination of quantitative ovalbumin content was conducted by elisa applying a strip test-system chicken egg ovalbumin elisa kit cat. n 6050 (alpha diagnostic international, usa). vaccine immunogenicity was evaluated by hai micro test in u-bottom 96-well plates produced by 'costar' (usa). 7 vaccine apyrogenicity was evaluated after intravenous injection of the studied preparation to rabbits. 8, 9 for confirmation of the results vaccine series were tested for bacterial endotoxins with the use of limulus amebocyte lysate produced by charles river laboratories, inc. usa. 8 the vaccine toxicity was evaluated in white mice with body weight 18-20 gm and in rats with body weight 180-210 g both males and females according to glp principles. 9 allergenic characteristics of the inactivated vaccine was determined in white outbred mice and guinea-pigs both males and females according to 'methodic guideline for evaluation of allergenic characteristics of pharmacological substances'. 10 in the first series of experiments, we conducted work for obtaining influenza a(h5n1) recombinant strain. bidirectional expression plasmid phw_b754 with full-length sequences of ha and na gene segments of the strain a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05 (h5n1) isolated in kazakhstan were synthesized in geneart ag, (regensburg, germany). ha gene was modified by deleting the region encoding multiple basic amino acid rrrk motif in ha cleavage site. moreover, to prevent recovery of repeating basic amino acids motif due to polymerase slide, we inserted replacements g fi t and k fi t. thus the ha cleavage site consists of the following sequence ntpqgerrrkkrglfgai ntpqtetrglfgai. the basic amino acid motif of highly pathogenic strain a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05 (h5n1) was replaced by the sequence tetr ⁄ glf, which is characteristic of low pathogenic strains of influenza h5n1. sequence of gene coding na in the strain a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05 (h5n1) was cloned without modifications. the other segments pb1, pb2, pa, np, m and ns were obtained from influenza virus ivr-116 and synthesized and cloned in two-forked expression plasmid phw_b754 in geneart ag company, germany. the origin of genetic segments of vaccine strain a ⁄ astanarg ⁄ 6:2 ⁄ 2009 (h5n1) is presented in table 1 . vero cell culture (134 passage) (who) was received from european cell culture collection (salisbury, wiltshire sp4 0jg, great britain). the cell culture was grown in dmem ⁄ f12 medium with the addition of 10% of fetal bovine serum and 2 mm l-glutamine. to obtain reassortant virus a ⁄ astana ⁄ 6 ⁄ 05r-6:2, vero cells were infected with correlative plasmids by way of electroporation using nucleofector ii (amaxa) equipment. infected cells were placed in 6-well plates. after 6 hour, dmem ⁄ f12 medium was changed into 4 ml of opti-pro sfm (gibco) medium adding 2 mm l-glutamine and 1lg ⁄ ml trypsin. two days after cytopathic effect appearance supernatant was collected and used for infection of spf-eggs. the virus a ⁄ astanarg ⁄ 6 ⁄ 05-6:2 was grown in chicken embryos, and then virus titer was determined in chicken embryos and madine-darby canine kidney (mdck) cell culture. the titer of two final a ⁄ astanarg6 ⁄ 05-6:2 virus stocks was 9ae1 log 10 eid 50 ⁄ ml (chicken embryos); 1ae8 log 10 tcid 50 ⁄ ml (mdck cells); ha titer 1:512. a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05 (h5n1) virus contains motif of repeating basic amino acids in ha cleavage site. it is known that this sequence is the main determinant of ai virus pathogenicity. that is why this site was deleted in vaccine candidate strain. sequence results confirmed that influenza virus a ⁄ astanarg ⁄ 6 ⁄ 05r-6:2 strain ha gene sequence contains modified ha cleavage site and keeps mutations inserted for prevention of return to virus wild type. to confirm stability of modified ha gene sequence, five additional passages of recombinant strain a ⁄ astana rg ⁄ 6 ⁄ 05-6:2 were conducted in chicken embryos. sequencing and following phylogenetic analysis of the recombinant strain a ⁄ astana rg ⁄ 6 ⁄ 05-6:2 ha gene sequence proved the presence of modification in ha cleavage site. deletion of pathogenicity site of the obtained virus was confirmed by lethality test for chicken embryos, intravenous pathogenicity test in chicken, and in plaque-forming test with trypsin. pathogenicity test in chicken embryos showed that recombinant strain a ⁄ astanarg6 ⁄ 05-6:2 is capable of growing up to high titers without causing embryos' death. a ⁄ astanarg6 ⁄ 05-6:2 strain pathogenicity evaluation was conducted in 5-6 week-age white leghorns chicken, and this study proved that the strain a ⁄ astanarg6 ⁄ 05-6:2 (h5n1) is not virus pathogenicity inductor in chickens, which got intravenous injections of this virus (pathogenicity index is equal to 0). h5n1 strain ha cleavage site modification provides its cleavage capability only with tripsin-like proteases, which shows low level of pathogenicity. aiming at confirmation of ha cleavage site modification, we experimentally studied virus replication ability both with trypsin and without this enzyme. and we got the following results. in the plaque-forming test, a ⁄ astanarg6 ⁄ 05-6:2 strain produced plaques in mdck cells only with trypsin, proving the trypsin-dependent phenotype characteristic of low pathogenic avian influenza viruses. to prove the ha subtype antigenic analyses of a ⁄ astana ⁄ 6 ⁄ 05r-6:2 strain was conducted by means of serological methods in hemagglutinin inghibition test with the use of postinfection antisera of rabbits and rats (influenza research institute swd rams), standard serum received from cdc, atlanta, usa. hai test proved that a ⁄ astana ⁄ 6 ⁄ 05r-6:2 strain belongs to h5 subtype. furthermore, toxicity of vaccine candidate strain was evaluated by way of subcutaneous injection of viral material to balb mice. the strain appeared to be non-toxic for white mice getting subcutaneous injection of 0ae5 ml of the preparation. the conducted research showed that according to all tested characteristics, a ⁄ astana ⁄ 6 ⁄ 05r-6:2 strain can be used for influenza a ⁄ h5n1 inactivated vaccine production. according to its genetic characteristics, this strain belongs to the group of vaccine strains recommended by who for the development of influenza pre-pandemic inactivated vaccines. we determined basic cultivation parameters of the recombinant strain a ⁄ astanarg6 ⁄ 05-6:2 in 10-11 day chicken embryos. the determined parameters are the following: infection dose, 1000-10 000 eid 50 ; cultivation period, 72 hour; incubation temperature, 33°c. these cultivation parameters allow obtaining virus containing material with biological eid and hemagglutinating activity of 8ae5-9ae0 log 10 eid 50 ⁄ cm 3 and 1:512 ha titre and even higher. in the next series of experiments, we conducted research on the determination of optimal sequence of technological stages of virus clarification, concentration, and inactivation in the order of vaccine production. samples of viral material were subjected to inactivation before and after clarification and concentration. the regimen of virus inactivation by formaldehyde with final concentration of 0ae05%, period of inactivation of 3 days, temperature of inactivation medium of 4-6°c, ph of inactivation medium of 7-7ae5. on the basis of the conducted experiments we determined that the selected regimen of inactivation provides complete and irreversible inactivation of viral suspensions of the hpai strain irrespective of the kind of inactivated material. we did not observe reduction of ha activity in non-clarified viral suspensions. however, when we inactivated clarified and concentrated material, ha activity reduced by an order of magnitude. comparison of forms and sizes of virion structural elements in native (non-clarified) and formalin inactivated preparations did not reveal any significant differences. concentration of virus particles in the studied preparations was similar. the selected inactivation regimen provides obtaining completely avirulent viral suspension of the strain a ⁄ astanarg6 ⁄ 05-6:2, and it does not influence the structure of the virus. on the basis of the experiments results, we selected method of viral allantoic fluid inactivation without preliminary clarification. during further research, we tried to get highly clarified viral concentrate. this study resulted in the combined scheme, which includes clarification of inactivated viral allantoic fluid by low speed centrifugation at 4000 circulations per min for 30 minutes, filtration through membrane filters with pore diameter of 0ae45 lm, ultrafilatration ⁄ diafiltration, gel filtration in 6b sepharose, and sterilization of viral suspension through membrane filters with pore diameter of 0ae22 lm. the experiments resulted in the development of production technology of embryonic inactivated vaccine based on recombinant strain a ⁄ astanarg ⁄ 6:2 ⁄ 2009 (h5n1) contain-ing aluminium hydroxide as adjuvant. the developed influenza a ⁄ h5n1 human vaccine has the trade name kazfluvacò. its composition components are presented in table 2 . preclinical testing of the vaccine kazfluvacò was conducted according to the following parameters: general health condition of animals, change of body weight and temperature of immunised animals (for ferrets), presence of post vaccination antibodies response in sera, forming protective immune response against reassortant viruses of h5 subtype, study of acute and chronic toxicity of three experimental vaccine series in different doses and semi-finished vaccine product applying different ways of injection, study of allergic and immunotoxic characteristics of the vaccine, as well as study of pyrogenic reaction and analysis for bacterial endotoxins presence. [11] [12] [13] [14] preclinical tests of kazfluvacò vaccine safety showed that this vaccine does not have toxic effect on organisms of warm-blooded laboratory animals. double intramuscular injection of kazfluvacò vaccine in inoculative dose does not effect appearance, general health condition, behaviour of animals, their muscular strength and physical activity, does not have negative effect on biochemical parameters of blood and basic physical functions of animals organism, and does not cause pathomorphological changes. this shows the safety of the vaccine. local irritation action was not observed. the results of the vaccine allergic action study showed that the vaccine does not have allergic effect at the intravenous injection. the research also showed that the vaccine does not have negative effect on immune system of laboratory animals. research conducted on mice and ferrets showed high immunogenic activity of the vaccine at one-and two-dose regimen of injection. the research showed 100% of protective effect of kazfluvacò vaccine at two-dose injection regimen in ferrets infected by homological strain of influenza virus. the devised inactivated influenza a ⁄ h5n1 vaccine kaz-fluvacò is a safe and immunogenic biopreparation that is not worse than the overseas analogues in its immunobiological characteristics. [15] [16] [17] [18] to date the whole-virion inactivated influenza a ⁄ h5n1 vaccines of the producers such as omnivest (hungary), biken, denka seiken, kitasato institute, kaketsuken (japan), gsk biologicals (belgium), sinovac biotech (china) are registered. all of them are produced on the basis of chicken embryos and aluminum is used as an adjuvant. kazfluvacò differs from its analogues in the flowchart of the virus purification and concentration that makes possible to produce a safer preparation. 19, 20 the results of the conducted research and preclinical testing allow starting work towards implementation of phase i preclinical tests on volunteers. it is planned to conduct a randomized blind placebo-controlled phase i study on double application of kazfluvacò vaccine in increasing doses. the preparation will be administered to volunteers aged 18-60 years for assessment of its safety and immunogenicity in doses of 7ae5 and 15ae0 lg of ha. when the world health organization (who) announced the sixth phase of a ⁄ h1n1v influenza pandemic, scientists all over the world started investigation to develop technology for production of prophylactic means against the disease. having taken into consideration the threat of a pandemic for kazakhstan, the ministry of education and science of the republic of kazakhstan launched the program ''monitoring, study, and development of diagnostic, prophylactic, and therapeutic means for influenza a ⁄ h1n1.'' this paper presents the experimental data obtained at the ribsp in the course of the studies towards the development of technology for production of an inactivated a ⁄ h1n1 influenza vaccine, as well as the results of pre-clinical testing of the developed vaccine. the development of vaccine production technology was conducted with the use of who recommended vaccine strain nibrg-121xp constructed by the method of reverse genetics in the national institute for biological standards and control (nibsc, great britain). the virus was inactivated with formalin at different final concentrations, and the extent of inactivation was evaluated via threefold virus passages in developing chicken embryos. 1 the inactivated virus was purified and concentrated by the method of ultrafiltration in tangential flow followed by gel filtration. the purified and concentrated material was evaluated judging on the total protein, hemagglutinin (ha), and ovalbumin. the vaccine was prepared by pooling the purified and concentrated virus material with the certain weight content of ha and the work solution of aluminum hydroxide (0ae4%) in the ratio 1:1. the ovalbumin content was quantified in elisa with the use of the strip test system chicken egg ovalbumin elisa kit (cat. no. 6050 alpha diagnostic international, san antonio, texas, usa). weight content of the virus ha was determined according to sominina, burtseva. 2 the content of the residual formaldehyde, aluminum (al +3 ) ions, and thiomersal in the vaccine was measured according to the operating instructions. 3 the vaccine immunogenicity was assessed in the hemagglutination inhibition test, which was carried out as a microassay in 96-welled u-bottomed plates (''costar'', new york, usa). 3, 4 apyrogenicity of the vaccine was assessed post intravenous administration of the tested preparation to rabbits. 5, 6 to confirm the obtained results the vaccine batches were tested for bacterial endotoxins with use of the limulus amebocyte lysate (charles river laboratories, inc., wilmington, ma, usa). 7 the toxicity of the vaccine was assayed in white mice weighing 18-20 g and in rats weighing 180-210 g (male and female) in compliance with the principles of good laboratory practice. 8 allergenic properties of the inactivated vaccine were determined according to the ''operating instructions on assessment of allergenic properties of pharmaceutical substances'' 9 in white outbred laboratory mice and guinea-pigs of both sexes. the first step in the course of developing technology for vaccine production was to determine the major conditions for influenza virus cultivation: usage of 10-days embryonated chicken eggs at the infectious dose within 1000-10 000 eid 50 , incubation temperature (34 ± 0ae5)°c, and duration of the incubation period 72 hours. the established parameters for virus cultivation made it possible to produce virus-containing materials of infectious activity within 8ae5-9ae0 log eid 50 ⁄ cm 3 and hemagglutinating activity 1:256 and higher. in the subsequent experiments, an optimal method for virus inactivation was selected. on the basis of the experimental findings, the following conditions for inactivation of the native virus-containing material were elected: formalin of 0ae05% final concentration as an inactivating agent; inactivation period of 72 hours at temperature (4 ± 2)°c. these conditions provide the complete inactivation of the virus (nibrg-121xp strain) material, did not impact distinctly the structural organization of the virus, and did not reduce the antigenic activity. as it is well known, virus purification and concentration means very much in the development of technology for production of an inactivated whole-virion influenza vaccine. the investigation into optimization of the technological step of purification and concentration of the recombinant influenza virus nibrg-121xp strain resulted in selection of an optimal pattern including such steps as clarification of the virus suspension by filtration through membranes with pore size 0ae45 lm, virus concentration by ultrafiltration in a tangential flow, dialysis filtration in a tangential flow, gel filtration on sepharose 6b, and sterilization of the viral suspension through membrane filters with pore size 0ae22 lm. the studies conducted by the ribsp specialists resulted in the development of technology for production of the first domestic whole-virion inactivated a ⁄ h1n1 influenza vaccine with aluminum hydroxide as adjuvant and with the brand name refluvac ò . the key processing characteristics of the whole-virion inactivated a ⁄ h1n1 influenza vaccine vaccine refluvac ò are shown in table 1 . simultaneous with the performance of all process operations, the parameters such as sterility, inactivation extent, ph, vaccine specificity, total protein content, weight content of has, aluminum and formalin contents, content of thiomersal, and ovalbumin, pyrogenicity of the vaccine and its immunogenicity for mice, were optimized. the key qualitative characteristics of the designed influenza a ⁄ h1n1 vaccine refluvac ò are shown in table 2 . before implementation of phase i clinical trials on volunteers, preclinical testing of three experimental batches of refluvac for immunogenic activity and safety was carried out. it was conducted in three laboratory bases of research institutions: the toxicology institute ⁄ federal medicobiological agency, russia (st petersburg), the research institute for biological safety problems (republic of kazakhstan), and the influenza research institute ⁄ north-western branch of the russian academy of medical sciences (st petersburg), with use of different animal models (mice, rats, chinchilla rabbits, guinea-pigs, ferrets). the results of the preclinical testing are as follows: • electron microscopy of the preparation has shown that the viral particles are well dispersed and do not aggregate. the portion of whole (intact) particles is over 95%, which is evidence of virion integrity; • assessment of polypeptide composition of the vaccine refluvac by electrophoresis in 10% polyacrylamide gel with sodium dodecyl sulfate has shown the vaccine to contain both surface antigens (ha, na) and highly purified inner virion proteins (np, m1) that are typespecific antigens, so the vaccine is a preparation of full immunological value; • judging on the parameters of acute and chronic toxicity for white mice and rats of both sexes, the vaccine is a non-toxic and safe preparation; • under conditions of a chronic experiment on white mice and rats, it was found that refluvac does not produce changes in behavior, somatic, or vegetative responses; • assay of hematological and biochemical blood characteristics of white mice and rats following vaccine administration did not reveal any significant differences as compared to the animals of the control group; • refluvac does not cause allergenic and immunotoxic impact; • the vaccine refluvac does not cause local irritative effect; • refluvac is apyrogenic for laboratory animals; • the pathomorphological and hystopathological analysis did not reveal any changes due to immunization in animal organs; • testing of immunogenic characteristics of the vaccine on mice and ferrets has shown formation of hemagglutinating antibodies in animals after single administration; • refluvac induces 100% protection in immunized ferrets at their challenge with the wild-type influenza virus a ⁄ california ⁄ 07 ⁄ 2009 (h1n1v). the results of the performed preclinical testing have allowed concluding that refluvac, an inactivated whole-virion vaccine with aluminum hydroxide as adjuvant, is a safe and highly effective preparation against influenza a ⁄ h1n1v. the implemented study resulted in development of technology for production of the first domestic inactivated allantoic whole-virion influenza a ⁄ h1n1 vaccine with aluminum hydroxide as an adjuvant under the brand name refluvac ò based on the recombinant strain nibrg-121xp. the devised pandemic vaccine meets who requirements as well as requirements concerning safety and immunogenicity of the national pharmacopeias of the republic of kazakhstan and russian federation. [9] [10] [11] [12] the devised technology for vaccine production differs from the previous technologies for production of allantoic whole-virion influenza a ⁄ h1n1 vaccines in its processdependent parameters. presence of an adjuvant (aluminum hydroxide) increases significantly the vaccine immunogenicity and allows maximal reduction of the dose of the administered antigen that, in turn, results in diminished reactogenicity of the vaccine. aluminum hydroxide is an adjuvant that is most frequently used in clinical practice. 13 to date the results of the double-centered randomized study of the europe-licensed vaccine fluval p [monovalent inactivated whole-virion influenza vaccine with aluminum phosphate based on strain a ⁄ california ⁄ 07 ⁄ 2009 (h1n1) nymc x-179a (omninvest, pilisborosjeno, hungary)] that is similar to the refluvac preparation are published. the data of this research are an evidence of safety and high immunological effectiveness of the vaccine in dose 6 lg ha at single administration both in adults and elderly persons. 14 the results of the pre-clinical tests allow recommending carrying out phase 1 clinical testing of the refluvac ò vaccine for safety and immunogenicity. single immunization of volunteers with refluvac ò in doses 3ae75, 7ae50, and 15ae00 lg of ha are planned. mid 50, respectively. the study results confirm that new h1n1 laiv and h7n3 laiv candidates are safe and immunogenic and confer protection from homologues influenza virus infection in mice. the recent emergence of a new pandemic h1n1 virus and the threat of transmission of avian viruses to humans had stimulated research and development of live attenuated cold-adapted influenza vaccines against newly appeared influenza viruses. formulations of live attenuated influenza a vaccine (laiv) against pandemic influenza strains, including h1n1, h5n1, h9n2, and h7n3 are currently being tested in preclinical and phase i clinical studies. 1 the following paper describes the preclinical study of new h1n1 and h7n3 laiv candidates in mice. the study addressed the following three objectives: (i) to demonstrate that cold-adapted (ca) reassortant influenza a(h1n1) and a(h7n3) vaccine candidates are indistinguishable from the parental a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) master donor strain (mds) virus with regard to replication efficiency in upper and lower respiratory tract of mice; (ii) to demonstrate the immunogenicity of different doses of cold-adapted (ca) reassortant influenza a(h1n1) and a(h7n3) vaccine candidates in mice; and (iii) to demonstrate the protective efficacy of cold-adapted (ca) reassortant influenza a(h5n1) and a(h7n3) vaccine candidates in mice against a homologous wild-type virus challenge. the a ⁄ 17 ⁄ mallard ⁄ netherlands ⁄ 00 ⁄ 95 (h7n3) reassortant containing the ha and na genes from a ⁄ mallard ⁄ netherlands ⁄ 00 (h7n3) and six other genes from mds, the a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) reassortant containing the ha and na genes from a ⁄ california ⁄ 7 ⁄ 2009 (h1n1) and six other genes from a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) were generated by classical genetic reassortment in embryonated chicken eggs (ec). viruses were propagated in 10days old eggs (34°c, 48 hours). fifty percent egg infectious dose (eid 50 ) titers were determined by serial titration of viruses in eggs. titers were calculated by the method of reed and muench. 2 female balb ⁄ c mice, 6-8 weeks of age were used in all experiments. mice were lightly anesthetized with ether and then inoculated intranasally (i.n.) with 50 ll of infectious virus diluted in phosphate-buffered saline (pbs). mice were inoculated with 100 mid 50 (50% mouse infectious dose) of a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1), a ⁄ 17 ⁄ mallard ⁄ netherlands ⁄ 00 ⁄ 95 (h7n3), and a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) mds. viral loads were measured in respiratory and brain tissues collected at 3 and 6 days post-infection (dpi). tissue homogenates prepared using a disruptor and clarified supernatants were titrated on eggs at permissive temperature to determine infectious concentrations. groups of animals were inoculated with 1000 mid 50 or 100 mid 50 of either h1n1 laiv or h7n3 laiv intranasally after collecting a pre-immunization blood sample. a second blood sample was collected at 28 dpi. on the same day, the animals received a second intranasal inoculation with the same virus that was used for priming at 0 dpi. to assess protection, all animals were infected 42 dpi with either 100 mid 50 of a ⁄ california ⁄ 7 ⁄ 2009 (h1n1) or 100 mid 50 a ⁄ mallard ⁄ netherlands ⁄ 00 (h7n3) virus by the intranasal route. four animals from each group were euthanized at 45 dpi, and the respiratory and systemic organs were harvested for virus titration. a forth blood sample was collected at 56 dpi from the remaining animals. hi antibody titers were determined for individual serum samples collected on days 0, 28, 42, and 56. body weights were taken daily following challenge through day 14 postchallenge. sera were tested for hi against homologous h1n1 and h7n3 viruses. the h1n1 laiv, h7n3 laiv and h2n2 mds influenza viruses replicate in mice lungs at level 2ae1-2ae3 lgeid 50 ⁄ ml at 3 dpi (figure 1 ). at 6 dpi, replication of the viruses in the lungs decreased to 1ae6-2ae0 lgeid 50 ⁄ ml (data not shown). in contrast, the wild-type virus a ⁄ mallard ⁄ netherlands ⁄ 00 (h7n3) demonstrated high level replication in lungs -6ae4 lgeid 50 ⁄ ml. the levels of replication of studied viruses in nasal turbinates were 2ae5-3ae7 lg eid 50 ⁄ ml at 3 dpi (figure 1) , and 2ae0-2ae2 lgeid 50 ⁄ ml at 6 dpi (data not shown). there were no significant differences between the viruses in regard to replication in upper respiratory tract of mice. thus, it was shown that a ⁄ 17 ⁄ mallard ⁄ netherlands ⁄ 00 ⁄ 95 (h7n3) and a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) vaccine candidates was indistinguishable from parental a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) in terms of replication in the lungs and noses of mice at 3 and 6 dpi. no virus was found in the brain tissue of immunized mice at 3 and 6 dpi (in undiluted samples tested). thus, it was shown that a ⁄ 17 ⁄ mallard ⁄ netherlands ⁄ 00 ⁄ 95 (h7n3), a ⁄ 17 ⁄ cali-fornia ⁄ 2009 ⁄ 38 (h1n1) vaccine candidates are identical to a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) in lacking neuroivasive capacity, and all three viruses similarly fail to replicate in the brain. it was shown that all immunized animals survived after challenge with wild-type a ⁄ mallard ⁄ netherlands ⁄ 12 ⁄ 00 (h7n3) virus. the mice in vaccine groups showed no signs of morbidity. average weight changes were tracked from day 2 to day 6 in all study groups, but the changes did not exceed 5%. as shown in figure 2 , the challenge virus actively replicated in respiratory tissue taken from mock immunized animals (5ae7 lgeid 50 in the lung and 4ae2 lgeid 50 in the nose), but failed to infect the brain and spleen. on the other hand, in both h7n3 laiv vaccinated groups, all tested organs were free from presence of challenge virus. thus, immunization of mice with either 1000 mid 50 or 100 mid 50 h7n3 laiv protected the animals from the subsequent challenge infection with a homologous with wild-type h7n3 virus. both h1n1 and h7n3 laiv candidates were found to be immunogenic. after one dose of 100 mid 50 of h1n1 laiv, gmt of hi antibodies were 17ae6. one dose of 1000 mid 50 or 100 mid 50 h7n3 laiv elicited hi antibody level with gmt of 8ae7 and 6ae6, respectively. the second dose of h7n3 laiv further stimulated serum hi antibody levels to gmt 40ae0 and 23ae3, for 1000 mid 50 or 100 mid 50, respectively (data not shown). the mouse model is widely used to better understand the pathogenicity of avian influenza viruses for mammalian species, to be able to predict the pandemic potential of such viruses, and to develop improved methods for the prevention and control of the virus in a potential pandemic. 3 a subset of the h7 viruses was evaluated for the ability to replicate and cause disease in balb ⁄ c mice following intranasal administration. h7 subtype viruses were able to infect mice without adaptation and manifested different levels of lethality and kinetics of replication. 4 there is limited preclinical information available for laiv. thus, live monovalent vaccine against pandemic influenza virus h1n1 (influvir) was tested for acute toxicity and its effect on the systems and organs of laboratory animals. according to toxicology and necroscopy results, the live monovalent influenza vaccine influvir, when applied intranasally, was safe and was well tolerated. 5 in our current study we demonstrate that a(h1n1) and a(h7n3) laiv are indistinguishable from the parental mds virus with regards to replication kinetics in the upper and lower respiratory tract of mice. both h1n1 and h7n3 laiv candidates were immunogenic and protect mice against subsequent a challenge with the wild-type virus. live attenuated cold-adapted (ca) influenza vaccines are an effective means for the control of influenza, most likely due to their ability to induce both humoral and cellular immune responses. in our study we confirm that new h1n1 laiv and h7n3 laiv candidates are safe, immunogenic, and confer protection from influenza infection in mice. health organization (who) declared a pandemic by raising the worldwide pandemic alert level to phase 6. therefore, h1n1 inactivated monovalent vaccine formulated with our proprietary oil-in-water emulsion based adjuvant was evaluated in ferrets for its potential to induce with low antigen dose efficient, robust, and rapid protective immunity against a wild type challenge virus (a ⁄ netherlands ⁄ 602 ⁄ 2009). this adjuvant was also tested in ferrets in a h5n1 avian influenza model for its ability to induce a cross-clade immunity and cross-protection. two independent studies (a&b) were carried out with male and female outbred ferrets (musleta putorius furo) in compliance with ''guide for the care and use of laboratory animals,'' ilar recommendations and aaalac standards. ferrets used in both studies were influenza seronegative by anti-nucleoprotein elisa and by hi assay against the pandemic and seasonal strains. in study a, four groups of seven ferrets aged approximately of 6 months received one or two im vaccinations 3 weeks apart of either af03-adjuvanted (3ae8 lg of ha with af03) or unadjuvanted ( body weight loss was monitored as an indicator of disease and a mean body weight loss of 20% was recorded in the control group at day of necropsy. body weight loss was reduced to £10% and £8% in animals that had received 1 and 2 doses of either unadjuvanted or af03-adjuvanted vaccine, respectively. viral lung titration showed high levels of virus replication ( ‡4ae7 tcid50 ⁄ g tissue) in the lungs of all control ferrets 4 days after challenge. one or two administrations of unadjuvanted vaccine reduced lung viral load by 2 and 3 log 10 , respectively. interestingly, ferrets that received either one or two doses of af03-adjuvanted h1n1 2009 vaccine, showed significantly greater reduction of lung viral loads (>4 log 10 ). no virus was detected in the lungs of 6 ⁄ 7 (86%) animals immunized with a single injection of the af03-adjuvanted vaccine and in 100% of ferrets vaccinated twice. assessment of viral shedding from the upper respiratory tract showed that the af03-adjuvanted a ⁄ h1n1 monovalent vaccine was able to reduce the viral load in the nose and in the throat by 3ae8 and 3ae1 log 10 , respectively, as compared to the control group. conversely, viral loads were only slightly reduced in the nose and mostly unchanged in the throat in ferrets immunized with either one or two doses of unadjuvanted a ⁄ h1n1 monovalent vaccine. gross pathology and histology examinations revealed lung lesions consistent with influenza a ⁄ h1n1 virus infechowever, a second dose of af03-adjuvanted vaccine strongly increased hi and mn titers, which persisted for 3 months (table 2 ). antibody responses cross-reactive to heterologous clade 2.2 strain were elicited ferrets vaccinated with the af03-adjuvanted clade 2.1 vaccine. hi antibody titers ‡40 crossreactive to clade 2.2 and persistent up to d110 were observed in vaccinated animals. an inter-clade low crossreactive hi response to a clade 1 strain was only detected in a few ferrets that had been vaccinated with the af03-adjuvanted clade 2.1. all af03-adjuvanted clade 2.1 antigen vaccinated animals survived challenge either with the homologous or heterologous virus until euthanized day 5. after challenge, mean body temperature and mean body weights were monitored as indicators of disease. in the control ferrets, mean body temperature increased by 2-3°c (depending on the challenge virus strain) 24 h post challenge, with an accompanying mean body weight loss ranging from 15ae4% to 20ae7%. ferrets vaccinated with the af03-adjuvanted clade 2.1 vaccine showed a lower and delayed fever compared to control ferrets that received the same viral challenge, whereas no significant differences were observed between vaccinated animals and their respective controls upon challenge with clade 2.2 or clade 0 viruses. body weight loss was reduced in all vaccinated animals when compared to controls after challenge with either the homologous clade 2.1 strain or with one of the heterologous strains. lung virus titration showed high levels of virus replication in all control animals 5 days after homologous challenge with the clade 2.1 virus. lung viral loads of all ferrets immunized with the af03-adjuvanted clade 2.1 vaccine were reduced more than 4 log 10 . vaccination resulted in complete viral clearance from the lungs of 80% of animals assessed 5 days after challenge. as compared to controls, a reduction of the mean viral load of about 2 log 10 was observed in ferrets vaccinated with the af03-adjuvanted clade 2.1 vaccine after heterologous challenge with either the clade 0 or clade 1 virus. conversely, vaccination with af03-adjuvanted clade 2.1 vaccine did not result in reduction of lung viral loads after challenge with the clade 2.2 heterologous virus strain. titration of pharyngeal swabs showed high levels of viral shedding in all control ferrets after challenge with clade 2.1 strain, whereas virus was not detected in any vaccinated animal. similarly, 5 log 10 reduction of viral shedding was seen in vaccinated versus control ferrets following clade 1 heterologous challenge. lower reductions in viral shedding were observed after clade 2.2 challenge (2ae6 log 10 ) and clade 0 challenge (1ae4 log 10 ). gross pathology and histology revealed lung lesions consistent with influenza a ⁄ h5n1 virus infection all control animals challenged with the clade 2.1, clade 2.2 or clade 0 strains. mild to moderate lung lesions were observed in control animals following challenge with clade 1 virus. macroscopic evaluation (percentage of affected lung parenchyma) and histopathological analysis (extent and severity of alveolitis, alveolar oedema and hemorrhage) showed that lung lesions were significantly reduced in af03-adjuvanted clade 2.1 vaccinated animals after challenge with the homologous clade 2.1 virus strain as compared to controls. similarly, a reduction of the macroscopic and microscopic lung lesions was observed in vaccinated animals upon heterologous challenge with clade 2.2 and clade 0 virus strains, whereas no differences were observed between control and vaccinated animals after challenge with clade 1 virus. the results of these ferret challenge studies demonstrated that low doses of pandemic influenza vaccines formulated with an oil-in-water emulsion adjuvant, af03, elicited strong antibody responses specific to the immunizing strain. importantly, these vaccines provided protection after homologous challenge with complete virus clearance in ferret lungs and reduced viral shedding from the upper respiratory tract suggesting an ability to reduce virus transmission. moreover, af03-adjuvanted h5n1 vaccine can provide cross-protection upon challenge with different h5n1clades by preventing mortality and reducing the viral burden in the lower and the upper respiratory tract. in conclusion, the results of these studies highlighted the ability of af03-adjuvanted influenza vaccines to induce potent immune responses and full protection in ferrets against homologous challenge and suggested that protection may be mediated, at least in part, by antigenspecific humoral immunity. since 1993, outbreaks of h9n2 influenza virus infection in poultry have occurred in eurasian countries. phylogenetic and antigenic analysis of h9n2 isolates revealed that there are three sublineages, consisting of g1, g9, and korean, among ha genes of the eurasian h9n2 viruses. h9n2 viruses do not cause severe disease in poultry, but co-infection of h9n2 viruses with bacteria such as staphylococcus aureus, haemophilus paragallinarum, or attenuated coronavirus vaccine may exacerbate the disease. 1,2 h9n2 viruses were isolated from domestic pigs in china and korea and from humans with febrile respiratory illness in hong kong in 1998 kong in , 1999 kong in , and 2003 it is, thus, postulated that in the present study, h9 virus strains were analyzed antigenically and phylogenetically to select a proper h9n2 vaccine strain. inactivated whole virus particle vaccine was prepared, and its potency against h9 virus challenge was assessed in mice. viral rnas were extracted from the allantoic fluid of chicken embryos infected with viruses by using a commercial kit (trizol ls reagent; invitrogen, california, usa) and reverse-transcribed with the uni12 primer 6 and m-mlv reverse transcriptase (invitrogen). the primers used for the ha gene amplification were h9-101f 7 and h9-1341r. for phylogenetic analysis, sequence data of the genes together with those from public database were analyzed by the neighbor-joining method. 8 h9 influenza viruses were analyzed by hemagglutinationinhibition (hi) test. 9 chicken hyperimmunized antisera against seven h9 viruses were prepared according to previous report. 10 virus replication and pathogenicity against embryonated chicken eggs viruses were inoculated into 10-day-old embryonated chicken eggs and incubated for 48 hours at 35°c. ha titers and 50% egg infectious dose (eid 50 ) were measured every 12 hours post-inoculation. pathogenicity of dk ⁄ hok ⁄ 49 ⁄ 98 against embryonated chicken eggs was evaluated by mean death time (mdt) as described previously. 11 dk ⁄ hok ⁄ 49 ⁄ 98 was injected into the allantoic cavities of 10-day-old embryonated chicken eggs and propagated at 35°c for 48 hours. the virus in the allantoic fluids (512ha) was purified by differential centrifugation and sedimentation through a sucrose gradient according to previous report. 12 the concentration of protein was measured by od using ultrospec 3100 pro (amersham biosciences, tokyo, japan). the purified virus was inactivated with 0ae1% formalin at 4°c for 7 days. immunization of mice and challenge of immunized mice with hk ⁄ 1073 ⁄ 99 four-week-old female balb ⁄ c mice were purchased from japan slc, inc. (shizuoka, japan). the mice were injected subcutaneously with 10, 2, 0ae4, or 0ae08 lg proteins of inactivated dk ⁄ hok ⁄ 49 ⁄ 98 whole virus vaccine. two weeks later, the mice were boosted by subcutaneous injection with the same dose of the vaccine. control mice were injected with pbs. serum samples were tested by enzyme-linked immunosorbent assay (elisa) according to previous report. 10 one week after the second vaccination, 10 mice in each group were challenged intranasally with 30 ll of 10 6ae5 eid 50 of hk ⁄ 1073 ⁄ 99 under anesthesia. on 3 days postinfection, five mice in each group were sacrificed, and the lungs were separately homogenized to make a 10% (w ⁄ v) suspension with minimal essential medium (nissui, tokyo, japan). the virus titers of the supernatants of lung tissue homogenates were calculated in 10-day-old embryonated chicken eggs and expressed as the eid 50 ⁄ gram of tissue. the other five mice in each group were monitored for body weight for 14 days after challenge. the ha genes of 22 h9 viruses were sequenced and analyzed by the neighbor-joining method. all of the 22 h9 viruses were classified into the eurasian lineage ( figure 1) . eleven, seven, and four strains were classified in the korean, g9, and g1 sublineages, respectively. the h9 viruses of the korean and g9 sublineages were isolated from waterfowl, poultry, pigs, and humans in the east asian countries, and those of the g1 sublineage were isolated from poultry in the west asian countries. the cross-reactivity between these antisera and h9n2 viruses were analyzed by hi test. the antisera against h9 viruses belonging to the korean sublineage were broadly cross-reacted to h9 viruses belonging to the g9 and g1 sublineages. h9 viruses belonging to the korean lineage were reacted to the antisera against h9 viruses belonging to the g9 and g1 sublineage compared with h9 viruses belonging to the other sublineage (data not shown). thus, it was suggested that h9 vaccine strain should be selected from the viruses of korean sublineage to prepare for the vaccine strain of h9 viruses. dk ⁄ hok ⁄ 49 ⁄ 98 replicated efficiently in 10-day-old embryonated chicken eggs (data not shown). pathogenicity of dk ⁄ hok ⁄ 49 ⁄ 98 against embryonated chicken eggs was determined by mdt. dk ⁄ hok ⁄ 49 ⁄ 98 was low pathogenic against embryonated chicken eggs (data not shown) and was selected as an h9 vaccine strain. to assess the potency of the vaccine against h9 virus infection, mice vaccinated subcutaneously with inactivated dk ⁄ hok ⁄ 49 ⁄ 98 were challenged intra-nasally with hk ⁄ 1073 ⁄ 99. immunogenicity of the inactivated vaccine was assessed by measuring the igg antibodies in mouse sera by elisa. antibody was detected in the group of mice injected 10 lg protein after the first immunization and detected in the group of mice injected 2 lg protein after the second immunization. thus, potency of the present inactivated whole virus vaccine was demonstrated in mice. next, to assess the protective immunity of the inactivated vaccine in mice, viral titers in the lungs was determined. the virus titers in the lungs were 10 1ae5 -10 3ae7 eid 50 ⁄ g in the groups of mice injected 10, and 2 lg protein, and 10 5ae3 -10 6ae0 eid 50 ⁄ g in the other vaccinated groups. body weight reduction of mice were observed in the group of mice injected 0ae4, 0ae08 lg protein, and control groups from 3 dpi, and reached to 10% body weight loss from 4-to 6-day post-infection ( figure 2 ). this result correlates with antibody titer in mouse sera and viral titers in the lungs. these results suggest that the test h9 inactivated whole vaccine confers prevent of weight loss and reduction of virus replication against h9 influenza virus infection in mice. recently, h9n2 viruses of all of three sublineage have been isolated from wild birds and poultry in worldwide. h9n2 viruses were isolated from pigs and humans in china 3 and korea, suggesting that h9n2 virus would be a potential for a pandemic influenza virus in human population. h9n2 viruses were isolated from pigs in china and korea and were classified into the g9 and korean sublineage. in human cases, all h9n2 virus isolated from humans in china was classified into the g1 sublineage. it was suggested that h9n2 viruses isolated from pigs and humans vary in antigenicity of isolates between the korean, g9, and g1 sublineages. therefore, it is important for the preparedness of influenza pandemic to develop h9 influenza virus vaccine, which could broadly cross-react to antisera of all sublineage viruses. so, we selected the vaccine candidate strain, dk ⁄ hok ⁄ 49 ⁄ 98, which could broadly cross-react to antisera of all sublineage viruses, and which could replicate in this study, it was suggested that the test vaccine has potency to protect against challenge with h9 virus using mice for mammalian model. the challenge virus, hk ⁄ 1073 ⁄ 99, was isolated from human, replicates efficiently in mice, and shows pathogenicity in mice. the test vaccine inhibited viral replication and body weight loss in mice. whole inactivated vaccine produced protective immunity, supporting our approach of using whole virus particles for vaccine development. furthermore, whole particle virus vaccine could induce igg and mucosal iga levels after intranasal vaccination with whole particle vaccine. 13 the present results may facilitate the studies of the vaccine for future pandemic caused by h9 influenza virus in humans. tants to attempt to improve growth. to determine whether wild type h1n1pdm grew better in the novartis mdck suspension cell line (mdck33016pf) than in eggs, isolations from h1n1pdm positive clinical samples were attempted in both substrates. the isolation rate of h1n1pdm viruses was higher in mdck33016pf cells (89%) (31 ⁄ 35) compared to allantoically inoculated eggs (66%) (23 ⁄ 35) . however the yields were lower than observed with seasonal viruses. little improvement in virus yield was seen with extra passaging or dilutions of h1n1pdm viruses isolated in mdck33016pf cells. with the emergence of the swine-origin pandemic h1n1 (h1n1pdm) influenza in april 2009, 1 the need for efficient production of a suitable vaccine was a high priority. 2 virus isolates were distributed by the who for the urgent development of suitable vaccine strains early in the pandemic. vaccine viruses can be grown in embryonated chicken eggs 2 or in certified mammalian cells. 3, 4 unfortunately wildtype h1n1pdm virus strains distributed by the who grew poorly in cell lines and eggs, requiring the generation of a series of 3 conventional and 8 reverse genetics 5 derived reassortants to attempt to improve growth. from these reassortants, only the conventional egg derived reassortants nymc-x-179a and nymc-x-181 (both based on one of the earliest known viruses a ⁄ california ⁄ 7 ⁄ 2009) showed high enough growth and yield in eggs and cell culture to make them suitable for vaccine manufacture. these reassortants, while acceptable, still only gave haemagglutinin (ha) yields of approximately 60% that of seasonal h1n1 reassortants. to determine if more recent wild type h1n1pdm viruses grew better in the novartis mdck suspension cell line (mdck33016pf), h1n1pdm positive clinical samples were cultured in mdck33016pf cells and also in embryonated hen's eggs. in addition, to improve virus yields from mdck33016pf isolates, extended passaging of three wild type h1n1pdm influenza viruses was performed using various virus dilutions at each passage level. the results were assessed using various serological and molecular biology techniques and compared to viruses isolated in eggs and conventional mdck cells. h1n1pdm viruses were received at the centre from who national influenza centres, who influenza collaborating centres and other regional laboratories and hospitals in australia, new zealand, and the asia ⁄ pacific region. viruses were received as original clinical specimens consisting of nasal swabs, throat swabs, nasopharyngeal aspirates, or nasal washes that had previously been shown to be h1n1pdm positive by real time rt-pcr. these specimens were then cultured in mdck33016pf cells with serum free medium containing trypzean (optaflu) 3 and also independently inoculated into the allantoic cavity of 11 day-old embryonated hen's eggs. virus cultures in mdck33016pf cells were sampled at 48 and 72 hour and evaluated by various means including ha titres. at 72 hour, virus cultures were further passaged at varying dilutions ranging from 10 )5 to 10 )8 up to a total of 10 passages. embryonated hen's eggs were incubated at 35°c for 3 days and allantoic fluid was harvested and ha titres performed to determine whether a further passage was required in order to improve growth. the conventional reassortants were produced by a mixed infection of eggs or mdck 33016pf cells with the wild type virus and a donor virus carrying the internal genes of the a ⁄ puerto rico ⁄ 8 ⁄ 34 virus. the reassortants were obtained by sequential passages using immuno-selective antisera against the surface antigen of the donor virus to remove virus populations carrying the ha and na protein of the donor strain. 6 the reverse genetics viruses were rescued in vero cells using the 12 plasmid system. 7 both types of reassortants were generated and supplied by who collaborating centres and essential regulatory laboratories except the nvd-c-07 strain, which was produced by novartis. in this small study with recent h1n1pdm viruses, the isolation rate was higher in mdck33016pf cells (89%) (31 ⁄ 35) compared to allantoically inoculated eggs (66%) (23 ⁄ 35) . assessment of ha titres, however, showed higher ha titres in egg-isolated viruses compared to viruses isolated in mdck33016pf cells after two passages. egg generated or cell generated reassortant viruses gave higher ha titres compared to the homologous wild type viruses (table 1) . no amino acid changes were observed in mdck33016pf isolated influenza viruses compared to original specimens or viruses isolated in conventional atcc derived mdck cells, unlike egg isolated viruses which showed a number of amino acid changes, many consistent with egg adaptation mutations (table 1) . 10 viruses isolated in mdck33016pf cells grouped phylogenetically with viruses isolated in conventional atcc derived mdck cells or viruses sequenced from original clinical samples, while egg isolated viruses grouped slightly differently (data not shown). as a result of the poor growth of h1n1pdm viruses in mdck33016pf cells, serial dilutions were performed over a number of passages ( figure 1 ). based on the results obtained from the virus isolates, a ⁄ victoria ⁄ 2081 ⁄ 2009, a ⁄ wellington ⁄ 188 ⁄ 2009, and a ⁄ darwin ⁄ 2131 ⁄ 2009, a supplemental protocol was developed and used in the isolation of a ⁄ brisbane ⁄ 10 ⁄ 2010 (figure 1 ). only small differences in ha titer were seen between different dilutions, and copy number showed a similar trend to ha titer at each passage ( figure 1 ). following the supplemental protocol for the isolation of a ⁄ brisbane ⁄ 10 ⁄ 2010 results showed slightly higher ha titres with little variation between passages. the egg derived reassortants nymc-x-179a and nymc-x-181 were also assessed for growth in mdck33016pf cells and were found to be superior by ha titer to other conventional reassortants (egg or cell derived), reverse genetics derived reassortants, or wild type viruses (table 1) . two methods were used to determine the ratio of ha to other viral proteins: densiometric analysis using sds-page and reversed-phase hplc using a subtype specific standard. 11 ha content in different vaccine seeds of influenza a subtypes demonstrated that the ha content per total virus protein from the nymc h1n1pdm reassortants was significantly different to the seasonal influenza a subtypes. for the seasonal h1n1 the ratio of ha to p4 p5 p6 p7 p8 p9 p10 p3 p4 p5 p6 p7 p8 p9 p10 p3 n and m1 was ‡30%, for the h3n2 the ratio of ha to n and m1 was £30%, while for the pandemic a ⁄ h1n1, the ratio of ha to n and m1 was much lower at £20% (data not shown). the results of this study has observed the growth of a series of 2009-2010 h1n1pdm viruses in vaccine suitable mdck33016pf cells to be generally lower than what has been seen with other seasonal influenza viruses. 11 little improvement in virus yield was seen with extra passaging of h1n1pdm viruses isolated and passaged in mdck33016pf cells. passaging up to 10 times in mdck33016pf cells using dilutions ranging from 10 )5 to 10 )8 resulted in supernatants with viral ha titres ranging from 8 ha ⁄ 25 ll to 512 ha ⁄ 25 ll. the isolation rate of h1n1pdm viruses was higher in mdck33016pf cells (89%) compared to allantoically inoculated (and passaged) eggs (66%), a trend also seen in previous work with seasonal influenza viruses. 12 in contrast a study by hussain and colleagues 13 found similar rates of isolation and replication of seasonal influenza viruses in mdck cells and eggs. the virus load as determined by matrix gene copy number showed a similar trend to ha titers. two of the isolates exhibited small rises and falls in ha titer during passaging, while a third, a ⁄ victoria ⁄ 2081 ⁄ 2009 gave consistently higher titers. interestingly this virus was unable to be isolated in eggs. the ha sequences of all strains were assessed at p1, p2, p3, p4, p10 and when available compared to the original clinical sample ha sequence. mdck33016pf-isolated viruses had few if any changes in their ha amino acid sequence, while the majority of egg isolates showed 1-2 amino acid changes compared to the clinical sample, with an egg adaption change (l191i) evident in a number of them. the ha sequence of one of the better growing viruses, a ⁄ victoria ⁄ 2081 ⁄ 2009, was found to have a g155e change compared to the a ⁄ california ⁄ 7 ⁄ 2009 reference virus. this change was also seen in the virus isolated in conventional, adherent mdck cells. these viruses with g155e change when tested by hai have shown reduced reactivity with ferret antisera to a ⁄ california ⁄ 7 ⁄ 2009-like viruses, but normal reactivity with ferret antisera to h1n1pdm a ⁄ bayern ⁄ 69 ⁄ 2009-like viruses. despite this mutation all mdck33016pf derived viruses appeared to be a ⁄ california ⁄ 7 ⁄ 2009-like by hai. the h1n1pdm egg-derived reassortants (nymc x-179a and nymc x-181) when grown in mdck33016pf cells were superior to wild type h1n1pdm viruses, reverse genetics derived reassortants, and other egg-derived reassortants. the yields of haemagglutinin from the nymc h1n1pdm reassortants were still below those seen with sea-sonal h1n1 reassortants as was also seen in eggs. this trend has also been noted in other studies. 10 in summary, attempts to improve growth and yield of the h1n1pdm wild types for mdck 331016pf cells by extended passaging were not successful, and reassortants did not perform as well as seasonal h1n1 reassortants have in the past. however, using higher dilutions for the passaging of h1n1pdm viruses in mdck33016pf cells did result in higher ha titres (a ⁄ brisbane ⁄ 10 ⁄ 2010). further work is therefore required to generate pandemic h1n1 seed viruses that grow well in a variety of cell culture and egg based vaccine production systems. the aim of this study is to evaluate antibody response to influenza virus neuraminidase (na) following immunization with live attenuated influenza vaccine (laiv). we adjusted the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. (1990) to assay neuraminidase inhibition (ni) antibody in sera taken from immunized mice and from human subjects in a clinical trial. for the assay, we prepared the a(h7n1) reassortant virus containing the na of a ⁄ california ⁄ 07 ⁄ 2009 (h1n1) and the hemagglutinin (ha) of a ⁄ equine ⁄ prague ⁄ 1 ⁄ 56(h7n7). in addition, we used an na-specific igg elisa assay to test sera from immunized mice and volunteers. in mice, one dose of laiv induced ni antibody of a geometric mean titer (gmt) of 31ae7, compared to 10ae6 in the control group. gmt of ni from human subjects who received two doses of pandemic a(h1n1) were significantly higher than pre-vaccination titers. in unvaccinated human subjects, na-specific cross-reactive antibodies to pandemic a(h1n1) were detected more often than cross-reactive antibodies to ha. antibody response to influenza virus na contributes to the overall immune response to influenza and may provide partial protection against influenza infection and reduce severity of disease in the host. 1 a number of preclinical studies using purified or recombinant na have shown that various two-dose vaccine regimens in mice may significantly reduce pulmonary virus titers following viral challenge. [2] [3] [4] a plasmid dna-vaccine model demonstrated cross-reactive antibodies to human n1 in mice could provide partial protection against a lethal challenge against h5n1 or recombinant pr8 bearing the avian n1. 4 immunogenicity of current influenza vaccines, including laivs, is measured primarily as a level of strain-specific hemagglutination inhibition (hi) antibodies. 5 however, the who meeting on the role of na in inducing protective immunity against influenza infection (2008) specified a need to develop suitable assays for anti-na antibody detection to enhance influenza vaccine evaluation in preclinical and clinical studies. 6 the aim of the current study was to evaluate anti-na antibodies to pandemic a(h1n1) 2009 influenza virus following laiv immunization. the rn1 ⁄ 09-swine a(h7n1) reassortant influenza virus containing the na of a ⁄ california ⁄ 07 ⁄ 2009(h1n1) and the ha of a ⁄ equine ⁄ prague ⁄ 1 ⁄ 56(h7n7) generated by classical genetic reassortment in embryonated chicken eggs (ce). parental a ⁄ equine ⁄ prague ⁄ 1 ⁄ 56(h7n7) influenza virus was obtained from the center for disease control and prevention, atlanta, ga, usa. viruses were propagated in 10 day old ce and purified by sedimentation out of the allantoic fluid, followed by ultracentrifugation on 30-60% sucrose step gradient. for the mouse studies, 10 week old cba mice were inoculated intranasally with one dose 10 7 eid 50 ⁄ 0ae05 ml of a ⁄ 17 ⁄ california ⁄ 09 ⁄ 38(h1n1) vaccine strain or received 0ae05 ml pbs. blood samples were collected on day 15 post inoculation. healthy young adults were immunized twice, 10 or 21 days apart in the fall 2009 with a ⁄ 17 ⁄ california ⁄ 09 ⁄ 38(h1n1) laiv manufactured by microgen, irkutsk, russia. for the human studies, peripheral blood specimens were collected from volunteers before vaccination, 21 days after the first vaccination, and 21 days after the second dose of vaccine. sera from five subjects diagnosed with influenza a(h1n1) were collected in december 2009, 3 to 4 weeks post infection and kindly provided by e. vo ıtsekhovskaia from biotechnology laboratory, institute of influenza, rams. also, sera obtained in 2005 from unvaccinated vol-unteers were tested for presence of cross-reactive antibodies to a ⁄ california ⁄ 07 ⁄ 2009 (h1n1). sera were treated with a receptor-destroying enzyme from vibrio cholera (denka-seiken, tokyo, japan) and then were tested in duplicates for hemagglutination-inhibition (hi) h1 specific antibodies by standard procedures 7 using a ⁄ 17 ⁄ california ⁄ 09 ⁄ 38(h1n1) test antigen. the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. 8 was adjusted to assay ni antibody. briefly, 96-well plates (sarstedt, inc., nümbrecht, germany) were coated overnight with 150 ll of 50 lg ⁄ ml fetuin. the purified a(h7n1) reassortant virus was diluted in pbs with 1% bsa and 10 mm ca 2+ to give a four times higher optical density at 450 nm (od 450 ) compared to control wells not containing virus. fifty-microliter volumes of serially diluted serum samples were incubated with an equal volume of prediluted virus for 1 hour at 37°c. after incubation, the plates were washed and neuraminidase activity was measured by subsequently adding peroxidase-labeled lectin (2 lg ⁄ ml; sigma, st. louis, mo, usa), incubating for 1 hour at room temperature, washing the plates, and adding 100 ll of peroxidase substrate (tmb). the reaction was stopped after 5 minute by adding 100 ll of 1n sulfuric acid. od values were measured at 450 nm using the universal microplate reader (el x 800; bio-tek instruments, inc., winooski, vt, usa). the ni titers were expressed as the reciprocal dilution that gave 50% od of positive control (virus, no serum control). in addition we used an igg elisa assay 9 with 0ae5 lg ⁄ ml of purified na from a ⁄ california ⁄ 07 ⁄ 09(h1n1) to test sera from immunized mice and volunteers. data were analyzed with statistica software (version 6ae0) (statsoft, inc. tulsa, oklahoma, usa). geometric mean titers (gmt) were calculated and used to represent the antibody response. the comparisons were made within groups between pre-and postvaccinated titers (expressed as log 2 ) after first and second vaccination using wilcoxon matched pairs test. to compare multiple independent groups we used a kruskal-wallis anova with subsequent multiple pairwise comparison based on kruskal-wallis' sums of ranks. a p-value of <0ae05 was considered to be statistically significant. in mice, one dose of laiv induced antibody responses to both ha and na components of the a ⁄ california ⁄ 07 ⁄ 2009(h1n1) influenza virus vaccine (table 1) . geometric mean titers of ni antibody levels from vaccinated mice were 31ae7 and were significantly higher compared to those in unvaccinated control animals (p < 0ae02). elisa igg titers expressed as log 10 were 1ae7 compared to 1ae0 in control group. there was good correlation between antibody rises obtained using ni or elisa tests (r = 0ae7). in a study during the fall of 2009, 85% of 60 examined unvaccinated subjects were negative to pandemic a(h1n1) (hi titers £1:10). serum hi antibody titers to pandemic a(h1n1) ‡1:40 were considered to be protective against *the postvaccination gmts of hi antibodies after revaccination were higher than respective prevaccination titers (p = 0ae04) **the postvaccination gmts of ni antibodies after revaccination were higher than respective prevaccination titers (p = 0ae02) serum hi and ni antibodies to a ⁄ california ⁄ 07 ⁄ 09(h1n1) after one or two doses of pandemic laiv were evaluated in subjects who had pre-vaccination hi titers £1:10 ( table 2) . post-vaccination gmts of a(h1n1)-specific antibodies were significantly higher than pre-vaccination titers only among subjects who received two doses of laiv ( table 2 ). the frequency of subjects with ‡ fourfold rises in hi antibody titers was higher after two doses (32ae3%) compared to responses after one dose (7ae1%) although the differences were not statistically significant ( table 2 ). the highest antibody titers of hi and ni antibodies were achieved after natural infection (p < 0ae01 compared to all post-vaccination groups). all five subjects with confirmed influenza also had high levels of n1-specific igg measured by elisa using purified na as the coating antigen (data not shown). influenza ha and na surface proteins are primary targets of neutralizing antibodies that provide protection against influenza infection. the correlation of strain-specific hi antibody titers ‡1:40 to protection of 50% of the subjects against influenza infection is based on a number of reports published in 1980s. 10 serum antibodies against viral na as result of influenza infection or vaccination also can neutralize the virus from infecting cells; however, little is known about protective levels of such antibodies. to evaluate ni antibodies directed against pandemic a(h1n1) we used the reassortant a(h7n1) influenza virus with mismatched ha to avoid non-specific inhibition. we demonstrated laiv immunization effectively increased levels of ni antibody, although in smaller amounts compared to influenza infection. our data suggest that an antibody to neuraminidase, resulting from an earlier infection of the circulating seasonal influenza a(h1n1), evidently cross-reacted with the n1 of pandemic influenza virus, perhaps due to the previously reported 17% of conserved na epitopes in pandemic a(h1n1). 11 the peroxidase-linked lectin test using the reassortant a(h7n1) influenza virus was shown to be a sensitive and time effective means of revealing homologous and cross-reactive anti-na antibodies after laiv immunization or influenza infection. this could be a useful method for influenza vaccine evaluation. significant levels of anti-na antibodies detected in peripheral serum from subjects infected with wildtype h1n1 virus or with h1n1 laiv. and the cross-antibody response to ph1n1. for calculation of geometric mean titer (gmt), a titer of <10 was assigned a value of 5. statistical significance was determined by paired t-test. cross-reactive antibody response to ph1n1 in vaccinated populations of seasonal influenza virus table 1 shows the antibody response to seasonal influenza viruses and ph1n1 of participants. before vaccination, no or little antibody response to ph1n1 had been detected in all age groups. vaccination with seasonal influenza vaccines resulted in seroresponse in over 70% of subjects, except children aged 0-7 years (68%) and subjects aged of 18-59 years (69%) vaccinated with 2007-2008 season influenza vaccine and adults aged ‡60 years (59%) vaccinated with 2008-2009 season influenza vaccine. seroconversion was detected in over 40% of subjects of all ages. postvaccination to prevaccination gmt ratios for response to seasonal influenza viruses was more than 2ae5-fold. in contrast, seroresponse to a ⁄ california ⁄ 07 ⁄ 2009 after vaccination with 2007-2008 and 2008-2009 seasonal influenza vaccines were detected in only 3% and 4% of those aged 0-7 years, 3% of those aged 8-17 years, 7% and 3% of those aged 18-59 years, 7% and 5% of those aged ‡60 years, respectively. seroconversion in all participants ranged from 2% to 4%, and postvaccination to prevaccination gmt ratios were <2ae5-fold. preexisting antibody response to ph1n1 among subjects born before 1920s in china according to a recent report, people who were born from 1910 to 1929 had a preexisting immunity to ph1n1. 7 although only a very low level of cross-reactive antibody response to ph1n1 had been observed among older subjects aged more than 60 years old in china, we further analyzed these data by different age distribution of subjects, which can trace back to the previous infection that is genetically and antigenically more closely related to this new ph1n1 influenza virus. the proportion of seroresponse to ph1n1 with the titer of 40, 80, and 160 (highest titer detected from participants of all ages in this study) and the value of gmt were analyzed according to the birth decade of subjects from 1915. similarly, a peak of antibody response and the value of gmt occurred both in subjects born from 1915 to 1925 and sharply decreased afterward ( figure 1 ). the seroresponse of subjects born in and before 1925 is significantly higher than subjects born afterward (p < 0ae05). similar to recent studies in some asia countries (guangxi province of china and singapore), limited antibody response to ph1n1 had been detected in children and adults. 8, 9 but, some other studies from european countries (finland, germany, the united kingdom) and the united states reported a high proportion of older individuals aged >60 years with pre-existing cross-reactive antibodies to ph1n1, which may possibly ba a result of previous exposure to antigenically related h1n1 influenza viruses circulating in earlier decades or a lifetime of exposure to influenza a, which has resulted in broad heterosubtypic immunity among older individuals in those countries. previous infection and vaccination with a ⁄ new jersey ⁄ 76 may also contribute to the high level of cross-reactive antibody response to ph1n1 among adults older than 60 years in the us. 10, 11 the peak of the antibody response to ph1n1 in subjects born between 1915 and 1925, which is consistent with recent reports, may suggest the previous viral infections of 1918 spanish flu or closely related influenza viruses, which is before and little after the year of 1918. recent antigenic report of new ph1n1 viruses indicated that they are antigenically homogeneous among historical viruses, which are most similar to classical swine a(h1n1) viruses. a number of reviews [12] [13] [14] [15] confirmed that the 1918 virus is the likely ancestor of all four of the human and swine h1n1 and h3n2 lineages, as well as the 'extinct' h2n2 lineage. in 1930, a(h1n1) influenza viruses were first isolated from swine. they have been shown to be antigenically highly similar to the recently reconstructed human 1918 a(h1n1) virus. 16 the cellular responses may contribute to the sustaining and long term antibody response. probably, boosting by persisting antigenically related viruses in the early decades of the 20th century, may have contributed to the ability of these subjects to sustain memory b cells, 17 and it is well established that a subset of plasma cells is long-lived, and these cells contribute to durable humoral immune responses, 18 such as that observed after childhood smallpox vaccination. furthermore, t cells that recognize cross-reactive epitopes are preserved and might be enriched in the memory population; the course of each infection is influenced by the t-cell memory pool that has been laid down by a host's history of previous successive infections. 19 our study indicated that wide transmission of this new virus or any antigenically close related influenza a(h1n1) viruses may not have circulated among populations in china before the outbreak of ph1n1. our data also suggests the need for vaccination with ph1n1 vaccine in all age groups. hypo-and agammaglobulinemia patients have an impaired immune system and are particularly susceptible to bacterial infections that are normally defended against by antibodies. therefore, patients routinely receive replacement therapy with immunoglobulins isolated from healthy blood donors. 1 these patients are also prone to get viral infections, possibly due to defects in toll-like receptors 8 and 9. 2 because these patients lack an antigen specific humoral immune response, they are rarely vaccinated. the ability of hypogammaglobulinemic patients to produce a specific cell-mediated immune response upon vaccination has only been sparsely investigated. in contrast to local mucosal antibodies, vaccine-induced cell-mediated immunity is not believed to protect against pathogen entry per se, but may be sufficient to provide protection against severe disease and death following transmission of some microbes. 3, 4 the aim of this pilot study was to investigate if influenza vaccination of hypogammaglobulinemic patients can induce an influenza-specific cell-mediated immune response. we therefore vaccinated hypogammaglobulinemic patients and healthy controls with pandemic h1n1 virus vaccine and subsequently investigated the bcell and t-cell responses. the percentages of ifn-c, il-2, and tnf-a cytokine producing cd4+ th1-cells were determined, as these cytokines are important indicators of cell-mediated immunity. five a-or hypogammaglobulinemic patients were classified based on the freiburg classification 5 : patient #1 is diagnosed with x-linked agammaglobulinemia, patient #2 and #4 are in group ia, patient #3 is in group ib and patient #5 is in group ii. the monovalent egg grown split virus vaccine adjuvanted with as03 was manufactured by glaxosmithkline (gsk), belgium. the vaccine strain was produced by reassortment between influenza a ⁄ california ⁄ 7 ⁄ 2009 (h1n1) and a ⁄ pr ⁄ 8 ⁄ 34 (h1n1) to produce a ⁄ california ⁄ 7 ⁄ 2009-like virus (x179a). the vaccine was mixed with adjuvant to contain 7ae5 lg haemagglutinin (ha) of a ⁄ california ⁄ 7 ⁄ 2009-like virus (h1n1), squalene (21ae38 mg), dl-atocopherol (23ae72 mg), and polysorbate 8 (9ae72 mg) per ml. healthy controls and hypogammaglobulinemia patients were vaccinated by intramuscular (im) injection. hypogammaglobulinemia patients received one or two vaccine doses 21 days apart. the intention was to vaccinate the hypogammaglobulinemic patients with two doses of 3ae75 lg ha, but 7ae5 lg ha was inadvertently administered to the patients as the first dose. for patient #4 this was the second dose as he had received an initial dose of 3ae75 lg ha 3 months prior to the study. patient #1, #2, and #5 received a second dose of 3ae75 lg ha. four healthy controls were immunised with one dose of 3ae75 lg ha according to norwegian national guidelines. peripheral blood mononuclear cells (pbmcs) were harvested and washed in pbs with 10% fbs. the pbmcs were resuspended in lymphocyte medium (rpmi 1640 with l-glutamine, 0ae1 mm non-essential amino acids, 10 mm hepes ph 7ae4, 1 mm sodium pyruvate, 100 iu ⁄ ml penicillin, 100 lg ⁄ ml streptomycin, 0ae25 lg ⁄ ml fungizone and 10% fbs) prior to use in the enzyme-linked immunospot (elispot) and influenza-specific cd4+ t-cell assays. serum haemagglutination inhibition antibodies were tested by a standard method using 8 ha units and 0ae7% turkey erythrocytes. all samples were tested in duplicate and the test was repeated at least two times. titres <10 were assigned a value of 5 for calculation purposes. for numeration of antibody-secreting cells (asc), an eli-spot assay was conducted as previously described 6 with the following modifications. ninety-six well elispot plates were coated with 2 lg ⁄ ml of a ⁄ california ⁄ 7 ⁄ 2009like (x179a) h1n1 virus diluted in pbs overnight at 4°c. after blocking with rpmi (10% fbs), 10 5 pbmcs were added and incubated (37°c, 5% co 2 ) for 16 hour. secreted antibodies were detected with biotinylated goat anti-human igg, iga and igm specific antibody (southern biotech, birmingham, alabama, usa), incubated for 2 hour at room temperature and developed with extravidin peroxidase and aec substrate. the numbers of spots were counted using an elispot reader (immunoscanô) and immunospot ò software. the influenza-specific cd4+ th1-cell response was measured by intracellular cytokine production of ifn-c, il-2, and tnf-a. peripheral blood mononuclear cells (10 6 per well) were incubated for 16 hour (37°c, 5% co 2 ) in 200 ll lymphocyte medium containing 1 lg ⁄ ml anti-cd28, 1 lg ⁄ ml anti-cd49d, 0ae7 lg ⁄ ml monensin, 1 lg ⁄ ml brefeldin a, (bd biosciences, franklin lakes, new jersey, usa), and the h1n1 influenza split virus vaccine x179a (either 2ae5 lg ⁄ ml or 10 lg ⁄ ml ha). basal cytokine production was determined by incubating pbmcs in lymphocyte medium without influenza virus, and the percentage of cytokine positive cells without influenza stimulation were subtracted from influenza-stimulated cells. cells were stained for cd3, cd4, cd8, ifn-c, il-2, and tnf-a (bd biosciences) as previously described. 7 finally, cells were resuspended in pbs containing 5% fbs and 0ae1% sodium azide and analysed by bd facscanto flow cytometer (300 000-500 000 cells acquired). flowjo v8ae8ae6 (tree star, ashland, oregon, usa) was used for data analysis. five to six fold lower gmts were found in the patient group as compared to the healthy controls throughout the study ( figure 1a) . the lowest hi titres were obtained in patients #1, #2, and #3, whilst patients #4 and #5 and all healthy controls fulfilled two of three european medicines agency committee for medicinal products for human use (chmp) 8 seasonal influenza vaccine licensing criteria, by obtaining an hi titre >40 and a mean geometric increase of 2ae5 between pre-and post-vaccination. thus, the hi data indicate that two vaccine doses was sufficient to induce a protective hi antibody response in two out of five of the hypogammaglobulinemia patients tested in this study. the numbers of influenza-specific iga, igg, and igm asc were tested pre-vaccination and 7 days post-vaccination with the h x179a virus. few or no ascs were detected pre-vaccination (data not shown). at 7 days post-vaccination the patient's iga, igg, and igm asc levels were significantly lower (p < 0ae01) compared to the healthy controls ( figure 1b) . but, the post-vaccination asc numbers in the patients were generally higher than at pre-vaccination stage (0-2 ascs). patient #3 had the highest iga and igg asc numbers, followed by patients #2 and #5, whilst patient #4 and #1 had few or no asc's. these results confirm that the patients are indeed hypogammaglobulinemic and that some of the patients (#1 and #4) could be agammaglobulinemic in the context of producing influenza-specific antibodies. the asc levels of patients #2, #3, and #5 were lower than those of the healthy controls, but could possibly be adequate for reducing the severity of influenza disease. the influenza-specific th1-cell response was evaluated by stimulating pbmcs with the influenza x179a virus 21, 42, and 90 days post-vaccination. stimulation of healthy control pbmcs with x179a 21 days after vaccination, induced ifn-c, il-2, and tnf-a production by an average of 0ae10%, 0ae09%, and 0ae03% cd4+ t-cells, respectively. patient #1 and #2 had higher responses than the healthy controls and stimulation with x179a induced 0ae45%, 0ae34%, and 0ae05% of t-cells from patient #2 to produce ifn-c, il-2, and tnf-a, respectively (figure 2a) . the response of patient #2 was further boosted by a second vaccine dose, which resulted in 0ae73%, 0ae54%, and 0ae25% cd4 t-cells producing ifn-c, il-2, and tnf-a, respectively at day 42 ( figure 2b ). these results show that the hypogammaglobulinemia patients studied here did not have a common impaired influenza-specific cd4+ th1 cytokine response. rather, there was a tendency towards increased responses, suggesting that the diminished antigen specific b-cell responses could induce a compensatory antigen specific th1-cell response. the results from this pilot study suggest that some hypogammaglobulinemia patients may benefit from influenza vaccination. we found very different patient responses to influenza vaccination, but some of the patients (patient #2 and #3) did mount low influenza-specific asc responses. in addition, the vaccine-induced hi antibody titres above the protective level in patient #4 and #5. these results are in accordance with previous publications, which described that polypeptide vaccines induce humoral responses in subgroups of common variable immunodeficiency patients. [9] [10] [11] in this study, we also investigated cell-mediated immunity and found the percentages of homologous and cross-reactive influenza-specific cd4+ th1-cells to be in the same range (for patient #3, #4, and #5) or higher (for patient #1 and #2) in the a-or hypogammaglobulinemic patients compared to the healthy controls. the higher response is probably due to the patients having received a vaccine dose of 7ae5 lg ha, whilst the controls received 3ae75 lg ha. in addition, the patients received a second booster dose, which influences the day 42 and 3 months responses. nonetheless, these results are the first to demonstrate that proliferation of pandemic influenza antigen specific th1cells can be induced in hypogammaglobulinemic patients. in addition, vaccination induced influenza-specific asc's in some patients. the findings are promising and provide hope that hypogammaglobulineamic patients could be vaccinated against influenza and other diseases preventable by figure 2 . peripheral blood mononuclear cells s from patients and healthy controls were isolated at day 21 (a), 42 (b), and day 90 (c) and stimulated for 17 hour with x179a virus before staining and flow cytometric analysis. the figure shows the mean ± sd frequency of influenza-specific cd4+ cytokine producing cells (%) where the basal cytokine production from unstimulated cells has been subtracted. data for the hypogammaglobulinemia patients are additionally shown as a number for each patient. **significantly higher frequency of il-2 producing cd4+ t-cells in the patients compared to the healthy controls (students t-test p < 0ae05). titres are presented as the geometric mean titre ± 95% confidence interval. elispot data (b) are presented as the mean number of influenza-specific iga, igg, and igm ascs per 100 000 peripheral blood mononuclear cells ± sem. data for the hypogammaglobulinemia patients are additionally presented by a number for each patient. *significantly higher numbers of ascs were detected in the healthy controls as compared with the hypogammaglobulinemia group (students t-test, p < 0ae01). vaccination. however, this hypothesis should be tested in larger clinical studies. the influenza virus undergoes antigenic evolution under intense immune selection pressure from herd immunity in humans through the process called antigenic drift and shift. 1,2 because of antigenic drift, yearly updating of vaccine strain is needed. a mismatch between the circulating strains and the vaccine strain in the subsequent season is often encountered, resulting in reduction of vaccine effectiveness and lack of protection from the circulating strain. 3 in order to address this, a universal influenza vaccine based on a more conserved part of the influenza virus, which is not affected by antigenic change and that is conserved across all strains, remains the ultimate goal to afford cross-protection to drifted strains as well as to other subtypes of influenza which may arise from antigenic shift. 4, 5 previous studies have investigated the potential of the m2e. 6, 7 m2e has remained highly conserved since it was first isolated in 1933. 6 several studies have examined the use of m2e as a vaccine component, using various approaches including proteins, peptides, dna vectors, and attenuated viral vectors. 6, [8] [9] [10] [11] [12] [13] although m2e is a weak antigen, by linking the protein to a carrier hepatitis b virus core particle, protection against influenza has been achieved in mice particularly when administered with an adjuvant. 3 some articles found that vaccination with m2e coupled to hbc induces protective antibodies, whereas the contribution of t cells to protection was negligible. protection induced by vaccination with m2e-hbc was weak overall and failed to prevent weight loss in vaccinated infected animals, and mice succumbed to high dose infection. 14 we aimed to address the poor immunogenicity of m2e-hbc by using igv as adjuvant. igv domain is common and conserved in the tim family. ligand binding sites of t cell immunoglobulin mucin (tim) located at igv domain. [15] [16] [17] tim function is done by anti tim antibody which recognized the ligand binding sites of igv domain. 18 tim family members share a common motif, including an igv domain. they are differentially expressed on th1 cells and th2 cells with the ability to regulate the immune system. 19, 20 the igv domain of human b7-2 is sufficient to co-stimulate t lymphocytes and induce cytokine secretion. 21 soo hoo et al. vaccinated with tim-1 antibody and inactivated influenza and found enhanced vaccine-specific immune response. 22 we report here for the first time the use of igv recombinant protein as adjuvant to immunize mice with influenza m2e-hbc. results indicated that igv can induce the strong cellular immune response and cross reaction with different subtype influenza virus antigen. target igv may be used to develop the new method for vaccination strategies. expression and purification of recombinant igv protein rna was extracted from healthy human pbmc. one-step rt-pcr (qiagen, valencia, ca, usa) was done for the amplification igv gene. the pcr product was purified and cloned into pet32a (novagen, madison, germany). the resultant construct pet32a-igv has a histidine (his) tag (6his) at the n terminus. dna sequence of the insert was determined by sequencing. igv. recombinant protein was expressed in escherichia coli and was purified on a ni column (novagen). the purified protein was examined by sds-page and western blotting. six-eight weeks female balb ⁄ c mice (institute of zoology chinese academy of sciences, china) was used for the study. mice were immunized twice intradermally with 10 ug m2e-hbc (provided by cnic, china) combined with different doses of recombinant igv protein 5, 10, 50 ug, respectively, or without igv as control. the area proximal to the tibialis anterior muscle was sterilized with 70% ethanol and different groups of mice were injected bilaterally with 5, 10, 50 ug igv plus 10 ug m2e-hbc in 50 ul phosphate buffer saline per mouse using a 1 ml syringe with attached 1 ⁄ 2¢¢ 30g needle. the immunization was given at 3 weeks intervals. four blood samples were obtained from every mouse: before immunization, after the first and second immunization, and after virus challenge by retro-orbital plexus puncture. after clotting and centrifugation, serum samples were collected and stored at )80°c prior to use for assays. mouse-adapted a ⁄ pr ⁄ 8 ⁄ 34 (h1n1), a ⁄ brisbane ⁄ 1 ⁄ 10 (h3n2), a ⁄ xinjiang ⁄ 1 ⁄ 2006 (h5n1), and a ⁄ guangzhou ⁄ 333 ⁄ 1999 (h9n2) were provided by chinese national influenza centre. nine to eleven days old embroynated specific pathogen free (spf) chicken eggs were inoculated with virus, and the eggs were incubated at 35°c for 2-3 days. the allantoic fluid was collected and purified by sucrose density gradient centrifugation, and the virus was inactivated by formaldehyde at 4°c overnight. to identify igg, igg1, igg2a against m2e, elisa assays were used. in brief, 96-well (nunc, brunei, denmark) were coated with 100 ul ⁄ well of m2e recombinant protein (provided by gene lab of ivdc, xuanwu district, beijing, china) in carbonate buffer (ph 9ae6) overnight at 4°c. immediately before use, the coated plates were incubated with blocking solution (2% bsa in pbs) for 2 h at 37°c and washed four times with pbs containing 0ae05% tween 20 (pbs-t). the serum samples were serially diluted and added in the plates. the detection color was developed by adding hrp-labeled goat anti-mouse igg, igg1, or igg2a ( figure 1) . no cross-strain response was observed in the control group. the igv adjuvented groups show splenocytes stimulation with seasonal h1n1, h3n2, h5n1, and h9n2 antigens. m2e-hbc immunization without igv showed splenocyte stimulation, but the extent was lower than animals immunized in the presence of the igv adjuvent. these data suggested that igv had enhanced effect on priming against the conserved viral antigen matrix protein and generation cross-strain immune response. influenza is a respiratory disease causing epidemics every year. h5n1 viruses and swine-origin h1n1 have also infected humans in recent years. seasonal influenza vaccine cannot cope with significant antigenic drift or with the emergence of pandemic viruses of different subtypes not contained in the vaccine. the high extent of conservation of the m2e makes it a promising immunogen. a vaccine based on coupling of the m2e peptide to an appropriate carrier may provide a universal vaccine with effectiveness and safety. m2e based vaccination induces protective antibodies not only in mice, but also in ferrets and monkeys. the carrier hepatitis b core as carrier with m2e forms a virus like particle (vlp). vaccination with m2e coupled to hbc induces protective antibody, 11 whereas the contribution of t cell protection was negligible. protection induced by vaccination with m2 coupled to hbc was weak overall. in order to improve the vaccination effect of m2e-hbc, new adjuvant igv was evaluated in combination with the m2e-hbc. the tim molecules are a recently discovered class of proteins with the ability to regulate the immune system. crystal structures of the tim molecules has revealed a unique, conserved structure with ligand-binding sites in the igv domain. to determine the potential immunostimulatory molecular properties of igv, we have evaluated immune response of the igv in combination with m2e-hbc vlp. previous papers reported that vlp immunized mice can induce the th1 and th2 immune response. 23 different adjuvant combined vlp can produce biased immune response th1 ⁄ th2 mixed immune response, or th1-preferred th1 ⁄ th2 profile. 24 thus, the response following the use of igv as a new adjuvant combined with m2e-hbc vlp needs to be evaluated. results indicated that igv combined groups showed th1 biased immune response and enhanced cross reactive t cell immune responses. this may show that igv immunized the mice and antiigv antibody can cross link the igv on t cells and enhance the cell figure 1 . t cell proliferation assay. mice were immunized twice with 50, 10, 5, 0 ug ⁄ ml igv plus m2e-hbc, respectively, and naive group was immunized with pbs. three weeks after a boosting immunization, spleens were harvested from immunized and naive mice. different subtypes of inactivated virus antigen (a) h1n1, (b) h3n2, (c) h5n1, (d) h9n2 were added and cocultured with different group splenocytes for 96 h. quick cell proliferation assay kit was used to detect the cell proliferation. the 420-480 nm absorbance was read on a plate reader. data were showed were shown as mean values. the difference between naive group and different doses igv plus m2e groups was determined using the student's t-test. all significance level is p < 0ae05. response. we also evaluated the cross-protection produced by igv combined m2e-hbc. we challenge with mouseadapted strain pr8 and prove the cross protection via reaction between the cells from the immunized animal and different subtypes of virus antigen. some subtypes of virus cannot infect the mice naturally, and therefore, virus challenge cannot be used to evaluate the effect. we co-cultured the t cells with inactivated antigen h1, h3, h5, and h9, and t cell proliferation was measured. results indicated that after immunization with igv plus m2e-hbc, the t cells show cross-protection with other subtypes. this provides evidence that igv can enhance the cross protection across subtypes. the results of this study demonstrated that recombinant igv can be useful as an adjuvant and polarize the m2e-hbc vlp immune response to a th1 profile. igv induced the m2e-hbc vlp to induce t cell proliferation and cross-reactive responses to different influenza virus subtypes. this finding represents a new direction for the promotion of cell mediated immunity in m2e based vaccine against influenza. a core european protocol, i-move, describing the methods to estimate influenza vaccine effectiveness (ive) was proposed by the european centre for disease prevention and control (ecdc) and epiconcept for the 2009-2010 season. it includes a case control method for pooled analysis based on a randomized ''systematic'' sample of swabs. 1, 2 collection of swabs using a non randomized, i.e., ''ad hoc,'' sampling strategy, left at the appreciation of sentinel practitioners, provides a greater number of cases and con-trols for ive estimation more easily than using a systematic randomized sampling strategy. the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than 5000 specimens yearly from cases of acute respiratory illness (ari), using both sampling methods. 3, 4 during the circulation of pandemic influenza 2009 viruses in france, it gave an opportunity to compare ive estimates using systematic randomized versus non systematic ''ad hoc'' sampling. influenza vaccine effectiveness was estimated by a casecontrol methodology according to ecdc i-move protocol, using on the one hand a systematic random sampling, on the other hand ''ad hoc'' non random sampling. the study was proposed to 608 primary care practitioners of the grog network (493 general practitioners and 115 pediatricians) trained to collect data and swabs. the study population was patients from the community of all ages consulting a grog practitioner for an influenza like illness (ili) and having a nasal or throat swab taken within an interval of <5 days after symptom onset. ili was defined according to the european union (eu) case definition as sudden onset of symptoms with at least one of the following four systemic symptoms: fever or feverishness, malaise, headache, myalgia; and at least one of the following three respiratory symptoms: cough, sore throat, shortness of breath. swabs were performed through usual surveillance. no ethical approval was needed, but an oral informed consent was requested. cases were excluded if they refused to participate in the study or if they were unable to give informed consent or to follow the interview in native language because of aphasia, reduced consciousness, or other reasons. an individual was considered as vaccinated against pandemic influenza if he or she reported having received a pandemic influenza vaccination during the current season, and if at least one vaccine dose occurred more than 14 days before ili onset. the study period started with the initiation of active influenza surveillance by the grog network, i.e., 15 days after the beginning of the influenza vaccination campaign, and finished at the end of the influenza period defined as the last week with at least one swab positive for influenza within the grog network. ''ad hoc'' sampling patients from which swabs were taken were selected by the grog practitioners during the study period. systematic random sampling during the same period, patients were selected at random as follows. an age-group 0-4 years (gps and pediatricians); 5-14 years (gps and pediatricians); 15-64 years (gps); 65 years or more (gps) was assigned to each practitioner, who was requested to swab the first patient of the week presenting with an ili within the pre-assigned age-group. swabs were collected in appropriate transport medium (virocult ò , viralpack ò , utm copan ò ) and sent by post to the laboratory in triple packaging following the international guidelines for the transport of infectious substances (category b, classification un 3373). laboratory confirmation of influenza was by rt-pcr to detect currently circulating influenza a (subtypes h3, seasonal and pandemic h1) and b viruses. an influenza case was defined as an ili case with a respiratory sample positive for influenza during the study period. controls were cases of ili having a swab negative for influenza during the study period. the outcome of interest is laboratory confirmed influenza. confounding factors and effects modifiers identified during the i-move preliminary study 5 were registered: risk factors, chronic diseases, severity of underlying conditions, smoking history, former vaccinations, and functional status. data on cases and controls were collected by the practitioners using a standardized questionnaire adapted from the i-move study. questionnaires were sent by the practitioners with the swab to the virology laboratory, and sent to the grog national coordination. data entry and validation were ensured by open rome through the vircases computing tool. validation steps included control of exhaustiveness of centralization of questionnaires, comparison of data entered by the labs and the national grog coordination, coherence control, and identification of missing data. analysis was done for the two sampling groups (systematic and ad hoc) on cases ⁄ controls following the european method proposed by epiconcept, using excel 2007 ª (microsoft corp. redmond, washington, usa) and stata ª . baseline characteristics of cases and controls in unmatched studies were compared using the chi-square test, fisher's exact test, or the mann-whitney test (depending on the nature of the variable and the sample size). the association between vaccination status and baseline characteristics was assessed for both case and control groups. the vaccine effectiveness was computed as ive = 1)or (odds ratio). an exact 95% confidence interval (ci) was computed around the point estimate. analysis was stratified according to age groups, time (month of onset), presence or absence of chronic disease, and previous influenza vaccination. effect modification was assessed comparing the or across the strata of the baseline characteristics. confounding factors were assessed by comparing crude and adjusted or for each baseline characteristic. a multivariable logistic regression analysis was conducted to control for negative and positive confounding factors using a complete case analysis (with records with missing data dropped) and using multiple imputation with chained equations. the complete model included age group, number of gp visits, onset week, seasonal vaccination, previous seasonal influenza vaccination, presence of chronic disease and associated hospitalizations in the previous 12 months, gender, and smoking status. variables were tested for multi-colinearity. interactions were tested using the likelihood ratio test (or wald test) and included in the model if significant at 5% level. a model with fewer variables (age group, number of gp visit, onset week, and seasonal vaccination) was also tested. several models were applied to both the ''ad hoc'' and systematic sampling groups of cases and controls. as shown in table 1 , whatever the analysis method used, the ''ad hoc'' sampling strategy led to a slightly lower estimate of ive. the ci were extended when data were missing and reduced when using multiple imputations with chained equations. however, from a statistical point of view, comparison of ''ad hoc'' versus systematic strategies is not straightforward, because ''ad hoc'' sampling is not randomized and does not allow comparisons with statistical tests using statistical distribution laws. there are more missing data with the ad hoc sampling method. this is mainly due to our validation procedure: in the case of missing data in the systematic sampling group, as required by the i-move study protocol, queries were sent to sentinel practitioners using mail and phone calls. this specific heavy workload is not usually performed during routine surveillance and has not been achieved for the ''ad hoc'' sampling group given the great number of cases and controls (2690). within the framework of the i-move study, several items were added to the grog's usual clinical form accompanying swabs (hospitalizations, number of gp visits, smoking status, help needed for bathing or walking). in 2009-2010, gps explained that this added workload was not compatible with their daily additional workload due to the pandemic situation. therefore, many of them refused to fill these new items systematically and threatened to leave the network. we thus obtained that the ''i-move items'' would be filled in for the clinical forms linked to systematic sampling, but were not in a position to obtain that for ''ad hoc'' sampling. the weekly distribution of systematic swabbing is not similar to that of ad hoc swabbing. the percentage of ad hoc swabs was higher than systematic swabs during the pandemic wave (mid-november to end of december) during which time the percentage of swabs positive for influenza was also higher ( figure 1 ). this could explain the higher rate of positive swabs within the ''ad hoc'' samples. the vaccination campaign was launched by the ministry of health on october 20, 2009, 6 and vaccination coverage increased during the surveillance period. in february, the vaccination coverage was 10ae2% in patients swabbed in the systematic group (9ae6% on imputed data) and 11ae5% [4ae4-23ae4] in the ad hoc group (11ae9% on imputed data). at the national level, vaccine coverage is estimated at 7ae95%. 7 due to the over-mediatisation of pandemic vaccination and to rumors about its poor effectiveness, overconsultation of vaccinated patients and over-swabbing of vaccinated patients in the ad hoc group are not surprising. age distribution is significantly different between our two samples (p < 0ae0001): the rate of 5-14 years old is lower in the systematic sampling group (25ae5%) than in the ad hoc sampling group (35ae7%). this can be explained by the fact that for the systematic sampling procedure, each grog practitioner had to swab the first ili patient in his assigned age group, whereas for ''ad hoc'' sampling, every grog practitioner could swab any ili patient irrespective of age. given the emphasis by health authorities and media on the burden of pandemic influenza among children and teenagers, one can hypothesize that when they were able to, sentinel practitioners focused on these age groups. gps in the ad hoc sampling scheme seem to have been more likely to select cases and further, to select vaccinated cases. those patients may have consulted earlier with specific symptoms (strong headache being more prevalent among cases). over-swabbing of patients having these symptoms in the ad hoc group is likely. the 2009-2010 pandemic influenza season was markedly different from previous ones: vaccination rate increased during and mainly after the pandemic peak; behaviors were strongly modified by unusual media hype; clinical features and risk factors might be different. it will be necessary to see if similar results are observed during a regular influenza season during which the vaccination rate increases before the epidemic peak with usual messages about vaccination and usual clinical influenza features. influenza early warning networks can estimate ive, taking into account many covariates. from a stakeholders and patients point of view, during the 2009-2010 influenza pandemic wave, there were no major discrepancies between ive estimated with an ad hoc sampling strategy, based on sentinel practitioners instinct, and ive estimated with a systematic random sampling strategy whatever the multivariable analysis methodology. although from a statistical point of view, comparison of the two strategies is not readily feasible because of the non random nature of ad hoc sampling. this latter strategy seems to result in slightly lower ive estimates, which could potentially be attributed to sentinel practitioners swabbing behavior. the ability to avoid missing data is a key point to decide which sampling method must be adopted, because ci extent depends greatly on the proportion of missing data among covariates. to match ive evaluation to surveillance networks practicality, selection of only those data essential for the study endpoint and easily collected by sentinel practitioners is paramount. it will be necessary to determine if results similar to those observed during the 2009-2010 pandemic season are found during a regular influenza season. influenza a viruses are important pathogens which remain a major cause of morbidity and mortality worldwide, and large numbers of the human population are affected every year. the first influenza pandemic in this century broke out in humans in march 2009, and it was declared to be pandemic by mid-june. as of 1 august jul 2010, the pandemic virus had caused more than 18 449 deaths worldwide, according to the world health organization (http:// www.who.int/csr/don/2010_08_06/en/index.html). the infection and spread of the pandemic influenza was reduced in part due to the use of vaccines. however, the lack of h1n1pdm vaccine early in the pandemic illustrates the need to improve vaccine production and to generate vaccines that induce stronger cross-protection. inactivated split vaccines or live attenuated influenza virus vaccines (laivs) against h1n1pdm viruses were approved for human use by the united states food and drug administration. both the inactivated vaccines and laivs are produced by creating reassortant viruses that generally contain six vrnas (pb2, pb1, pa, np, m, and ns) from a master donor strain, plus the two glycoprotein vrnas (ha and na) from a virus that antigenically matches the strain predicted to circulate in upcoming influenza season (e.g. a ⁄ ca ⁄ 07 ⁄ 2009). the reference viruses containing inactivated split virus vaccines are produced in embryonated chicken eggs, and primarily result in the production of antibodies that recognize the viral glycoproteins. both of these vaccine approaches require significant lead time for vaccine production, and modern approaches to speed preparation of vaccines and improve their efficacy is a global priority. 1, 2 the ns1 protein of influenza a virus is a multifunctional protein that plays important roles in virus replication and as potent type i ifn antagonist. 3, 4 mutations and ⁄ or deletions in ns1 typically induce stronger ifn responses by the host; those in turn suppress the replication of influenza virus 3-7 and can enhance immune recognition. [8] [9] [10] [11] in this study, we created a panel of experimental h1n1pdm ns-laiv candidates that have different deletions in the ns vrna and analyzed the vaccine potential of each ns-laiv in mice and ferrets to identify the best candidate(s). wt h1n1pdm influenza a virus a ⁄ new york ⁄ 1682 ⁄ 2009 (ny1682) was created by reverse-genetics directly from a human swab specimen collected in new york state in april 2009. 12 deletions were introduced into the ny1682 ns plasmid to create three mutant ns segments: ns1-73, ns1-126, and nsd5. nucleotides 246-482 (cdna of ns segment) and 405-482 were replaced by stop codons to generate ns1-73 and ns1-126; nucleotides 612-626 were deleted to generate nsd5, whose open reading frames for ns1 and nep were maintained. recombinant viruses were generated by co-transfection of eight reverse-genetics plasmids carrying the cdna of each gene segment into 293t ⁄ mdck cocultured monolayer adapted from hoffmann et al. 12, 13 mouse studies experiments were performed in a biosafety level 3 laboratories approved by the u.s. centers for disease control and prevention and the u.s. department of agriculture, and were conducted under approved animal care and use protocols. groups of 6-week-old female balb ⁄ cj (jackson laboratory, bar harbor, me, usa) were anesthetized with isoflurane and inoculated intranasally with 10 5 tcid 50 of each recombinant virus in 20 ll of pbs diluent, or pbs as controls. body weights and clinical symptoms of the mice were monitored daily for 10 days. nine mice in each group were euthanized on 1, 2, and 5 days post inoculation (dpi), and nasal washes and lungs were collected for virus titration by tcid 50 assay in mdck cells. at 30 dpi, 12 mice per group were challenged intranasally with 5 · 10 4 tcid 50 (100 ld 50 in 6-week-old mice) of a mouse-adapted variant of ny1682 (a ⁄ ny ⁄ 1682 ⁄ 2009-ma7) (accepted, journal of virology). disease symptoms and weights of the vaccinated mice were monitored for 10 days, and four mice from each virus group were euthanized at 3 and 6 days post challenge. lungs were removed and homogenized for virus titration by tcid 50 assay. the mice that became moribund or lost >25% of their starting body weight were euthanized for humane reasons. male fitch ferrets (triple f farms, sayre, pa, usa), 7-10 months of age and serologically negative by hemagglutination inhibition (hi) assay for currently circulating influenza viruses were used in this study. groups of 3 or 4 ferrets were inoculated intranasally with 10 6ae5 tcid 50 of one of the viruses: ny1682 wt (n = 4), ns1-73 (n = 3), ns1-126 (n = 4), or nsd5 (n = 4). ferrets were monitored for clinical signs through 14 dpi as previously described. 14 nasal washes were collected on 1, 3, 5, and 7 dpi and were titrated in mdck cells by tcid 50 assay. serum was isolated from blood collected 6ae5 weeks after immunization and used for neutralization assays. the ferrets were challenged with 10 6 pfu of a ⁄ mexico ⁄ 4482 ⁄ 2009 15 6ae5 weeks postimmunization and monitored for clinical signs of disease through 14 dpi. nasal washes were collected on 1, 3, 5, and 7 dpi, and were titrated in mdck cells by plaque assay. using reverse genetics, we created three laiv candidates weight loss of wt virus inoculated mice became evident at 3 dpi, and the mice did not recover until 8 dpi (figure 1a) . in contrast, mice inoculated with any one of the vaccine candidates had no clinical signs of disease and continued to gain weight at the same rate as did the mockinoculated mice ( figure 1a ). viral titers in the lungs of ns1-73, and ns1-126 infected mice were $100-fold lower than titers from wt virus-infected mice at all the time points analyzed (1, 2, and 5 dpi) ( figure 1b) . notably, the nsd5 laiv was cleared from the mouse lungs very rapidly, and the mean titers were $100-fold and 50 000-fold lower than the titers of the wt virus at 1 and 2 dpi, respectively ( figure 1b) . the vaccinated mice were challenged with a mouseadapted variant of ny1682 (accepted, journal of virology) on 30 dpi. no disease symptoms were observed in the mice immunized by any of the ns-laiv candidates or the wt control. in contrast, disease symptoms including ruffled fur, hunched posture, and weight loss were observed in the mock-immunized mice as early as 2 days post challenge (dpc); the symptoms progressed to severe disease, and the animals showed dramatic weight loss, became moribund, and succumbed to infection by 5 dpc (figure 1c ). high titers of virus ($10 7 tcid 50 ⁄ ml) were present in the mock-immunized mice at 3 dpc and at 6 dpc ( figure 1d ). in contrast, virus was not detected in the lungs of immunized mice ( figure 1d ). this challenge data demonstrates that all of the ns-laiv candidates, including the highly attenuated nsd5, induced sterilizing immunity that protected mice from a lethal ny1682 h1n1pdm variant. groups of ferrets were intranasally immunized with 10 6ae5 tcid 50 of each vaccine candidate or the wt virus. the titer of viruses recovered from nasal washes ranged from 10 6ae8 to 10 4ae5 tcid 50 ⁄ ml through day 5 in the wt virusinfected group, while the ns-laivs showed various degrees of attenuation (figure 2a) . the viral titer of all of the ns-laivs is at least 100-fold lower than that of wt in the nasal wash collected at 1 dpi. the ns1-73 laiv was the least attenuated in ferrets, and its replication was similar to that observed in mice. relative to the wt virus, the ns1-126 laiv showed 100-fold reduction in titer, and the nsd5 laiv was below the limit of detection (at least 1000fold reduction) at 3 dpi. sera from blood collected 6ae5 weeks after immunization was analyzed for the presence of neutralizing antibodies by micro-neutralization assays. the ns-laiv candidates all induced very strong neutralizing antibody responses (1280-5120) that were similar to the titer elicited by wt virus infection ( figure 2b ). the ferrets were challenged with 10 6 pfu of a ⁄ mexico ⁄ 4482 ⁄ 2009 (h1n1pdm) 6ae5 weeks post immunization. little disease or weight loss were observed in the naïve ferrets, and the ferrets immunized by infection with wt virus or the ns-laiv candidates didn't show any disease symptoms or weight loss. in contrast to the high titer of virus detected in the naïve ferrets through 5 dpc, the ns-laiv immunized ferrets had very low levels of a ⁄ mexico ⁄ 4482 ⁄ 2009 in their nasal washes at 1 dpc ( figure 2c ). the ferrets immunized with the ny1682 ns-laivs had $10 000-to 100 000-fold lower viral titers than did the naïve animals ( figure 2d ). in summary, the ns-laiv candidates dramatically inhibited initial replication of the h1n1pdm virus under stringent challenge conditions (10 6 pfu), and that the vaccinated animals rapidly cleared the infection (to below the limit of detection, by 3 dpc). our results demonstrate that all of the ns-laiv candidates are attenuated compared to the wt h1n1pdm virus, and the degree of attenuation is dependent on the specific ns mutation. ns1-73 was the least attenuated and does not represent a good vaccine candidate; whereas, nsd5 and ns1-126 were highly attenuated in both the mouse and ferret models. although they were markedly attenuated, they elicited strong neutralizing antibody responses and protected mice and ferrets from subsequent challenge. nsd5 has a subtle in-frame deletion (15 nt) that affects both the ns1 (residues 196-200) and nep (residues 39-43), and is analogous to a naturally attenuated variant of a normally highly pathogenic h5n1 virus (a ⁄ sw ⁄ fj ⁄ 03). 16 the analogous ns1 deletion in a ⁄ sw ⁄ fj ⁄ 03 (residues 191-195) was shown to reduce binding to host cleavage and polyadenylation specificity factor (cpsf), reduce ns1 protein stability, and enhance the type i ifn response of this h5n1 virus. 16 our study indicates that deletion of these 15nt in the ns vrna of the h1n1pdm also stimulates the host ifn response, specifically, ifn-ß, ifn-k1, ip10, and mxa (data not shown). the role of the deletion of residues 39-43 from nep has not been elucidated, but the induction of ifn and isgs by nsd5 was similar to, or slightly lower than, their induction by ns-126, suggesting that the nep mutation also has an attenuating effect that warrants future investigation. in summary, we have generated a panel of laivs directly from a swab specimen containing a new pandemic virus and analyzed their attenuation and immunogenicity in two animal models. our study demonstrates that nsd5 is a novel ns-laiv that could be used to create laivs for diverse influenza a viruses. this study also validates the use of ns-laiv candidates, which are not only highly attenuated, but they also elicit strong innate and adaptive immune responses, resulting in protection of mice from subsequent challenge with a lethal mouse-adapted variant of ny1682, and ferrets from challenge with a ⁄ mexico ⁄ 4482 ⁄ 2009 (h1n1pdm). currently, a total of approximately 300 million doses of inactivated influenza vaccine are being produced worldwide each year. one of the limitations in vaccine production is poor growth of human isolates in embryonated chicken eggs. this is essential to develop high yield seed viruses for large scale production of influenza vaccines. influenza a vaccine production utilizes high yield reassortants carrying ha and na genes from a wild type (wt) strain with generally 5-6 internal genes from the a ⁄ pr ⁄ 8 ⁄ 34 (pr8) strain, an highly egg adapted high growth donor strain. 1 influenza b vaccines, however, have been produced directly from wt strains, partly because no high yield donor analogous to pr8 has been identified. in recent years, reverse genetics has been used as an alternative means of developing high growth vaccine viruses. 2, 3 since in this plasmid-based technology, a 6:2 reassortant (six internal genes from a donor strain and two surface antigen genes from wild type strain) can be directly rescued, reverse genetics-derived reassortant viruses were expected to grow as efficiently as those derived from classical reassortment. however, reverse genetics reassortants have not produced the expected high growth for several reasons: (i) the 6:2 configuration is not always the best for virus yield, (ii) there is no process included for positive selection of adaptive mutants from quasispecies, and (iii) cell-derived viruses are not readily adapted to grow efficiently in eggs. our laboratory at new york medical college has been preparing b reassortants for several years by classical reassortment using b ⁄ lee ⁄ 40 as a donor. it has been possible to develop b reassortants, which produce higher virus yields than wt strains in eggs, and it was found that the np gene of b ⁄ lee ⁄ 40 was important in producing high yield b reassortants. 4 however, b ⁄ lee ⁄ 40 is inconsistent in providing high yield properties to b reassortants. in this study, in an attempt to find an alternative donor, we investigated the usefulness of b ⁄ panama ⁄ 45 ⁄ 90 for developing high yield b reassortants. as a wt strain, b ⁄ brisbane ⁄ 60 ⁄ 2008 was used, which is one of the recommended influenza b virus vaccine strains for the 2009 ⁄ 10 and 2010 ⁄ 11 seasons. we found that b ⁄ panama ⁄ 45 ⁄ 90 is a useful donor, and some of the resultant reassortants were considered as vaccine candidates. 5 b reassortant viruses were prepared by the classical reassortment method described by kilbourne. 1 the antiserum to b ⁄ panama ⁄ 45 ⁄ 90 hemagglutinin and neuraminidase (hana) was raised in this study by immunizing rabbits with hana isolated from b ⁄ panama ⁄ 45 ⁄ 90; purified igg was used for antibody selection. the yields of the reassortants and their corresponding parent viruses were assessed by hemagglutination assay. viral rna was extracted directly from the allantoic fluid and amplified by rt-pcr to produce cdna for analyzing the gene composition. restriction fragment length polymorphism (rflp) analyses were performed to determine the origin of each gene segment of the high yield reassortants. restriction enzyme sets for each gene segment are available upon request. in this study we investigated the usefulness of b ⁄ panama ⁄ 45 ⁄ 90 as a donor for transferring high yield phenotype. b ⁄ panama ⁄ 45 is a yamagata lineage strain with high growth phenotype (ha titer: 1024-2048). b ⁄ panama ⁄ 45 ⁄ 90 itself was a recommended b virus vaccine strain for 1989 ⁄ 90-1994 ⁄ 95 seasons. as a wt virus, a victoria lineage strain, b ⁄ brisbane ⁄ 60 ⁄ 2008, was used, which is a recommended b virus vaccine strain for use in the 2009 ⁄ 10 and 2010 ⁄ 11 seasons. reassortants were prepared according to classical reassortment protocol. after co-infection of b ⁄ panama ⁄ 45 ⁄ 90 and b ⁄ brisbane ⁄ 60 ⁄ 2008, progeny viruses carrying surface antigens (ha and na) of the vaccine strain were negatively selected by anti-b ⁄ panama hana antibodies, followed by passages without antibodies for positive selection of eggadapted viruses and finally limited dilution cloning. nymc bx-33, bx-33b, bx-33d, and r-3a are representative of resultant reassortants, which have significantly higher ha titers than the wt strain. the complete gene compositions of these reassortants were determined by rt-pcr ⁄ rflp analyses. as shown in table 1 , all of these reassortants contained the pb2 of b ⁄ panama ⁄ 45 ⁄ 90. other genes of b ⁄ panama ⁄ 45 ⁄ 90 (np of bx-33, m of bx-33b, and pb1 of bx-33d) may not be involved in the high virus yield, since no significant growth difference among these reassortants in eggs was found as assayed by hemagglutination test. accordingly, the pb2 of b ⁄ panama ⁄ 45 ⁄ 90 is considered to be the sole factor involved in the high yield phenotype donated to the vaccine strain. we previously found that the b ⁄ lee ⁄ 40 np gene was important in producing high yield b reassortants. it was of interest to examine whether b ⁄ lee ⁄ 40 np and b ⁄ panama pb2 could work together to produce even higher yields. to test this possibility, bx-33b (2:6 reassortant: pb2 and m genes from b ⁄ panama and the rest of the genes from b ⁄ brisbane) was selected and further reassorted with b ⁄ lee ⁄ 40. despite some difficulty in removing the na gene of b ⁄ lee ⁄ 40 (r-4c, 7b, 10b in table 2 ), by monitoring ha and na genes of resultant viruses after each antibody selection passage with anti b ⁄ lee ⁄ 40 hana antibodies, we were able to isolate and clone a triple reassortant, nymc bx-35, which contains the np gene from b ⁄ lee ⁄ 40 and pb2 and m genes from b ⁄ panama; the remaining genes are from b ⁄ brisbane ⁄ 60 ⁄ 2008 (table 2 ). in comparison with bx-33b, no significant growth enhancement (nor reduction) in eggs was found for bx-35 over that seen for bx-33b. nevertheless, bx-35 stably produces high virus yield and has been utilized as a seed virus for influenza b vaccine production for the 2010-2011 season by one or more vaccine manufacturers. there are contradictory reports 6-8 about the usefulness of reassortment for high yield influenza b viruses. however, we have been preparing b reassortants for several years by classical reassortment 1 using b ⁄ lee ⁄ 40 as a donor, and have been able to generate higher virus yield than wt strains. 4 in this study, we found that b ⁄ panama ⁄ 45 ⁄ 90 serves as an efficient donor in providing the high growth capacity to b ⁄ brisbane ⁄ 60 ⁄ 2008 (a recommended vaccine virus of victoria lineage for 2009 ⁄ 10-2010 ⁄ 11 seasons), and that the pb2 of b ⁄ panama ⁄ 45 ⁄ 90 is associated with the high yield phenotype. this particular strain from yamagata lineage might be useful to prepare high yield reassortants for other victoria lineage vaccine viruses. we noticed in this study that there may be segment incompatibilities between b ⁄ panama ⁄ 45 ⁄ 90 and b ⁄ brisbane ⁄ 60 ⁄ 2008. as shown in table 1 , the pa and ns genes of all the high yield reassortants examined are derived from wt, b ⁄ brisbane ⁄ 60 ⁄ 2008, not from the donor, b ⁄ panama ⁄ 45 ⁄ 90. this indicates that in this reassortment, the pa and ns genes are not replaceable with that of the donor to obtain high yield viruses. this degree of incompatibility might be common in b reassortment, resulting in low donor ⁄ wt reassortants, such as 2:6 and even 1:7 reassortants that we obtained in this study. if this is the case, reverse genetics based on 6:2 configuration may not result in generating high yield b reassortants unless a variety of donor ⁄ wt combinations are designed. one can speculate that in influenza b viruses, the surface glycoproteins (ha and na) and some of the internal proteins are functionally more closely related than in influenza a virus, as was seen in that pa and ns genes of b ⁄ brisbane ⁄ 60 ⁄ 2008 reassort together with the ha and na genes of the same parent (table 1 ). in our recent study on a reassortment between b ⁄ lee ⁄ 40 and b ⁄ panama ⁄ 45 ⁄ 90, it appeared that ha shapes overall gene constellations of the resultant reassortants, namely the reassortants tend to have more internal genes from the same parent of ha, no matter which parent's ha is selected by antibodies against the surface antigens of the other parent (data not shown). because of success in influenza a virus reassortment with pr8, it is generally believed that reassortant with 6:2 or 5:3 configuration is optimal for virus yield. this may be the case in most instances of influenza a reassortment, but is not necessarily so in b reassortment. as shown in this study, only a single donor gene is capable of improving the yield of vaccine strain by reassortment. influenza a ⁄ h1n1v has spread rapidly in all parts of the world in 2009 as a true pandemic. 1 epidemic events in russia occurred during the last week of september 2009 starting from far east region (yuzhno-sakhalinsk). kaliningrad (the western most russian city) was the second starting point of the epidemic. during october 2009 the epidemic spread over the whole russian territory. in a short period the new virus started to change genetically as it began to adapt to human populations during this pandemic (http://www.who.int; http://www.euroflu.org). in the period from may to december 2009, 1558 clinical samples (nasopharyngeal swabs and postmortem materials) of patients with influenza-like illness from different regions of russian federation were analyzed to confirm the diagnosis using real-time reverse transcription pcr (rrt-pcr). clinical nasopharyngeal swabs and bronchoalveolar lavage and post mortal fragments of trachea, lungs, bronchi, spleen from saint petersburg hospitals and 49 basic laboratories of federal influenza center were included in this study. all specimens were taken from patients with influenza-like illness or viral pneumonia. specimens were tested by rrt-pcr according to cdc protocols, i.e. using superscript iii platinum one-step qrt-pcr system (invitrogen) with primers and probes for infa, h1 seasonal, and h1sw (biosearch technologies). in addition, the test-systems 'amplisense influenza virus a ⁄ b-fl' and 'influenza virus a ⁄ h1-swine-fl' for pcr-detection, typing and subtyping of influenza viruses were also used. these test-systems are produced by central institute of epidemiology, moscow, russia and recommended by russian ministry of health as tests for influenza diagnosis. sequencing was carried out on an abi prism 3100-avant genetic analyzer (applied biosystems, usa) with bigdye terminator cycle sequencing kit. phylogenetic analysis was performed using programs vector nti 10.0 (invitrogen) and mega 4.1 (psu, usa) by maximum likelihood with the tim+i+g model for ha, and -hky+i+g model for na. evolutionary model was selected by akaike information criterion (aic) in model-test (posada, crandall, 1998). statistical reliability of tree branches was evaluated by bootstrap test (100 replications). immunohistochemical study was performed using novalink antibodies to ha and np with novocastra visualization system. influenza virus a ⁄ h1n1v rna was detected in 409 patients with severe form of influenza-like illness and 163 fatal cases. out of pcr-confirmed flu recovered cases 58% were patients under 14 years of age, 41% were aged 15-64 years, and 1% were older than 65 years. mean age of recovered patients was 15ae7 years (from 1 month to 77 years). viral rna in postmortem materials was detected mostly in lung tissue (86% of specimens) and trachea fragments (64%), and less commonly in spleen (17%). mean age of the deceased with confirmed flu (h1n1v) infection was 39ae3 years with age ranging from 7 months to 75 years. in 84% of fatal cases, influenza was complicated by viral or secondary bacterial pneumonia. median time from the onset of illness until death was 10 days. according to our data, 4% of patients died had diabetes, 4ae5% were obese, and 4% were pregnant women in the 2nd or 3rd trimester. ha and np were detected by immunohistochemical assay in lung tissue of dead patients with confirmed influenza virus a ⁄ h1n1v infection. ha and np was revealed in the endothelium of different sized blood vessels (capillaries and arterioles). these influenza virus proteins were also detected in some tissue macrophages apart from epithelium and endothelium. the localization of the two proteins was different: ha is mostly localized in cell membrane and cytoplasm, and np -mostly in the nucleus. here we present data on molecular genetic characteristics of 51 strains of pandemic virus, 40 strains obtained from clinical specimens, and 11 from post mortal ones isolated in the research institute of influenza. all the strains studied contain the s31n substitution in m2 protein, which indicates resistance to the adamantane antivirals, and have no h275y substitution in the neuraminidase, which indicates resistance to oseltamivir. the phylogenetic analysis showed that russian viruses were similar to influenza viruses a ⁄ texas ⁄ 05 ⁄ 2009 and a ⁄ california ⁄ 07 ⁄ 2009 (ha similarity 98ae9%). all russian viruses could be divided in two clusters: the first one includes viruses similar to the reference strain a ⁄ california ⁄ 07 ⁄ 2009, and the second one, which is the majority of viruses analyzed includes strains with substitutions ha s203t, na n248d, v106i, and ns i23v (figure 1 ). bootstrap support was 59. the isolates with ha s203t substitution can be classified in one of the five minor genome variants of a ⁄ h1n1v viruses found in the united states and mexico in 2009. 2 several viruses had strain-specific substitutions in antigenic sites sb and ca and the mutation d222g in ha receptor-binding site. the substitution of amino acid residue asp to gly at position 222 of ha was found in eight of eleven isolates (73%) from postmortem lung and trachea samples and two of forty isolates (5%) from nasopharyngeal swabs of patients with severe course of the disease. appearance of amino acid substitutions in the ha receptor-binding site (d187e and d222g ⁄ e) could be associated with influenza virus passaging on eggs. 3 five strains that contained g at position 222 of ha were isolated from post mortal specimens on mdck cells in this study, thereby excluding the possibility of substitution appearance hence to virus adaptation on eggs. in order to reveal genome changes in a(h1n1)v, strains isolated on the territory of russian federation during the 2009 pandemic, 67 full genome sequences from genbank, and research institute of influenza database were analyzed comparing two groups of viruses (isolated before and after 22 sept 2009). nine amino acid changes observed predominantly in late pandemic strains were found. five of them (s128p, s162n, d222g, v234i, v321i) reside in ha, two in na (i263v, n386k), two in pb2 (k340n, t588i), and one in pa (f35l). towards the end of the epidemic the viral population had demonstrated statistically certain rise in number of strains containing mutations in four genes. difference between groups was statistically significant (chisquare test, p = 0ae05). if v 2 > 3ae84, than difference between early and late strains is statistically significant. additionally fisher's test determined whether 'early strains' and 'late strains' differ significantly in the proportion of 'no mutation event' and 'mutation event' attributed to them in each particular position. all calculations were performed in fisher_tk freeware by vladimir belyaev similar to calcfisher (haseeb, 2003) fully described here (http://www.jstatsoft.org/v08/i21/paper). we have selected positions with statistically significant amino acid changes in late strains (p-value 0ae05). according to full genome analysis of influenza virus a ⁄ h1n1v 2009 strains, seven clades were distinguished, but the divergence between representatives of different clades remained small. 4 (figure 2 ). besides the strain a ⁄ perth ⁄ 12 ⁄ 2010 also contains substitution s181f in the same ha antigenic site. according to data obtained, the 2009 epidemic in russia was caused only by influenza virus a ⁄ h1n1v. unlike the previous epidemic periods when most severe influenza cases were registered among the children under 5 years and among elderly people aged over 65 years, the first wave of pandemic due to influenza virus a (h1n1)v resulted in increased level of mortality mainly among the people aged 18-53 years. though all pandemic viruses showed comparative genetic homogeneity, some evolutionary trends could be outlined. for clarification of the exact pathogenic role of mutation d222g in ha receptor binding site, further studies are necessary. full-genome analysis of influenza virus a ⁄ h1n1v strains circulating in the southern hemisphere in the new epidemic season 2010 revealed the phylogenetic subgroup distinguished by seven substitutions in inner proteins (pb2, pb1, np, ns1) and sa antigenic site of ha (n142d). the changes revealed could be caused by adaptation of the virus to an immunized human population. nasal and throat swabs (placed in 3 ml mem and frozen at )80°c until use for viral rna extraction and tissue culture inoculation) were collected from patients with febrile illness, i.e., >38ae0°c. samples were received from clinics in us embassies and us military laboratories located throughout the world since the initial who declaration of 2009 novel h1n1 outbreaks as a global pandemic on june 11, 2009. viral isolates were obtained from inoculating cultures of mdck cells with 0ae1-0ae2 ml viral suspensions collected in mem originated from patients after 2-7 days incubation. [10] [11] [12] [13] due to low viral titers in normal clinical samples, most of full viral genome sequences were derived from viral stocks obtained by tissue culturing passages (mdck, 1-2 times). viral rna was extracted from clarified supernatant fluid of nasal ⁄ throat swabs or mdck cultures using the 'charge-switch' rna extraction system based on the user manual protocol from the manufacturer (invitrogen inc., ca, usa). total rna was eluted into volume equal to original sample volume, i.e., 100 ll starting viral supernatant used to yield final 100 ll rna in molecular grade water (invotrogen inc.) and stored at )80°c until tested. generating ⁄ preparing overlapped cdnas for full genome coverage of 2009 novel h1n1 viruses by multiple rt-pcr amplifications the first step in the high-throughput sequencing pipeline for full influenza genome sequences was to establish a robust rt-pcr amplification scheme consisting 67 different rt-pcr primer pairs covering all 8 rna segments to ensure 100% amplification coverage of full viral genomes of all the incoming targeted viruses (houng, hs. 2011, submitted for publication). extracted viral rna (5 ll), derived from mostly mdck culturing stock or clinical sample containing sufficient viral load (>100 infectious units per ml) was added to primer-free rt-pcr total master mixture (10ae5 ll) for each virus followed by adding primer pair (2 ll, 3 pmole ⁄ ll per primer). rt-pcr was then performed: rt reaction through two hold-steps (50°c, 15 minutes and 94°c, 2 minutes); 35 cycling amplifications (94°c for 15 seconds, 53°c for 15 seconds, 72°c for 2ae5 minutes). specific cdna amplicons corresponding to each individual primer pair were routinely monitored and visualized by agarose gel electrophoresis. pooled 67 cdna products (2-5 lg) from each viral rt-pcr amplification run were used as sequencing substrates according to the roche 454 flx user manual and bulletins by incorporating adaptors containing individually multiplex identifier [mid]-key assigned to each individually pooled viral cdna. up to 12 different mid-keyed viral cdna were further pooled together to be clonally amplified on capture beads in water-in-oil emulsion micro-reactors (em amplifications), and pyrosequenced using one of two regions of a 40 · 75 mm picotiterplate. for each individual viral genome containing multiple assemblies (8 rna segments), we obtained sff file(s) containing raw sequencing reads from which nucleotide sequence data and phredlike quality scores were extracted. on average, 80ae0-90ae0% of 3-9 million mid-key specific nucleotides were extracted and mapped for consensus genome sequences. roche 454 gsmapper (v. 2.0 and 2.3) software was used to assemble all sequencing raw data and sff files into consensus sequences. new reference mapping projects were created to assemble each individually mid-keyed viral cdna into consensus viral sequences. one of the earliest 2009 h1n1 genomes of california origin, a ⁄ california ⁄ 04 ⁄ 2009(h1n1), deposited in genbank, was routinely employed as a reference genome sequence for most of gsmapper projects. the resultant consensus sequences obtained were further verified and validated through the ncbi annotation utility check and ultimately deposited to the ncbi influenza database, genbank. nucleotide sequences specific to each individual rna gene were aligned by the geneious pro 4.7.5 software (http://www.geneious.com). 14 trees were built based on the tamura-nei genetic distance model using the neighbor-joining method with no outgroup used via geneious pro 4.7.5. phylogenetic trees of the 2009 h1n1 genomes were constructed by importing fasta files containing specific concatenated target sequences of pb1, pb2, pa, ha, np, na, mp, and ns from each individual virus into the geneous pro software and going through the sequence assembling and tree building steps. high-throughput pyrosequencing of pooled 2009 novel h1n1 cdnas by roche 454 flx system up to 24 viral cdnas could be routinely sequenced to completion for 24 different full viral genomes from a single roche 454 flx picotiter plate by utilizing the combination of pico-titer plate's two distinct regions as well as 12 different mid-keyed adaptors. the 'shotgun' sequencing approach employed in this study is a feasible method to viral isolates (n) sequence multiple pooled 2009 h1n1 viral genomes. for each pyrosequencing experiment, approximately 800 000-1 000 000 passed key reads (single fragment per bead) were obtained that yielded readable nucleic acid sequences. among those close to a million passed key reads, only 500 000-600 000 passed key reads had an average sequencing read length of >220 bps, defined as 'long reads' (220 bps · 500 000 reads = total of 110 million bases of nucleic acid sequences) that were used to assemble into influenza genome sequences. mathematically, 110-130 million bases of raw sequencing data from each single roche 454 flx experiment would provide sufficient sequencing bases to cover 24 full genome sequences with approximate 3-400 · of sequencing depth coverage of influenza a with average genome size of 13 500 bps for the total of eight segmented rnas. so far, more than 100 full 2009 h1n1 genomes sampled worldwide have been successfully sequenced and deposited in the ncbi database by division of viral diseases, walter reed army institute of research (wrair). the bioinformatics derived from unique viral genome sequences generated from this study based on constant rt-pcr amplification scheme and identical roche 454 pyrosequencing protocols provide a reliable data set in predicting the evolutionary patterns of pandemic viruses. wrair received clinical samples from us embassies and military personnel throughout the world since the initial who announcement of 2009 novel h1n1 outbreaks. nearly equal distributions of sequenced viruses derived from three broadly categorized geographic regions, north america, central ⁄ south america, and asia ⁄ europe ⁄ africa (data not shown). besides the geographic distribution pattern of viral isolates, figure 1 displays the viral isolation time lines of all the sequenced viruses reflecting two peaks that coincided with two waves of 2009 pandemic infections, early-mid summer and fall of 2009. 15 phylogenetic trees of the eight influenza a segments of all sequenced viruses were tentatively generated. it was found that the substitution frequencies per site for the ha, na, and ns genes are at much higher rate than the other five genes, pb2, pb1, pa, np, and mp genes (data not shown). the observed higher genetic variations for ha and na genes of 2009 h1n1 are consistent with the historical genomic and epidemiological dynamics data of human influenza a revealing higher temporal fluctuations in ha and na genes. [16] [17] [18] [19] analysis of full influenza genomes containing concatenated eight complete rna segments revealed the existence of two distinctive genetic clades in circulation since the beginning of 2009 pandemic, as shown in fig-ure 2. it is noteworthy that all viruses of mexico and california origins (clade 1 shown at the top of figure 2 ) were isolated at the beginning of 2009 pandemic prior to the isolation of all other viruses belonging to the second genetic clade 2. 18, 20 discussion during the past decade, the advance of dna sequencing technology, such as development of ngs, in making full viral genome sequences readily available have enabled study of far broader and more detailed aspects of evolutionary change for any new emergent infectious pathogen. the massive sequencing capacity of roche 454 flx system allows simultaneously process and sequence millions of individual cdna molecules, in contrast to processing and sequencing individual cdna fragments by conventional sanger sequencing method. within a short period of few months since the beginning of the 2009 pandemics, wrair accomplished large number of representative 2009 h1n1 full genomes of worldwide origins via roche 454 flx system. sequencing data derived from this study illustrates a much higher genetic variation rate for ha and na genes of 2009 h1n1 that is compatible to the higher temporal fluctuation rate for ha and na genes of seasonal influenza a derived from decades of intensive monitoring and comparison studies and analyses. [16] [17] [18] [19] following the mexican and us reported cases, confirmed outbreaks of 2009 swine h1n1 rapidly proliferated and spread throughout europe, asia, africa, and south america, most probably via global airline travel. 5, 6 it seemed that new cases in the us and most cases throughout the world had been clinically mild relative to the initial reported cases in mexico. [21] [22] [23] [24] here we demonstrate through the phylogenetic relationship of sequenced 2009 h1n1 full genomes that the clinical isolates could be divided into two different clades of viruses, i.e., the clade 1 genetic group contains only viruses isolated at the beginning (march ⁄ april 2009, mexico and california) of 2009 pandemics and the rest of other viruses all belong to the 2nd genetic group, clade 2. thus, it's likely that the currently circulating 2009 h1n1 of clade 2 causing worldwide infections is genetically different from the initial 2009 h1n1 isolates that caused the early infections in mexico and california. 18, 20 introduction a pandemic influenza virus (2009 h1n1) was recently introduced into the human population. the hemagglutinin (ha) gene of 2009 h1n1 is derived from 'classical swine h1n1' virus, which likely shares a common progenitor strain with the human h1n1 virus that caused the pandemic in 1918. 1 since antigenic changes of influenza virus ha occur more slowly in swine than in humans, 2 we hypothesized that 2009 h1n1 might still retain an antigenic structure similar to that of 1918 h1n1 or the early isolates of its descendants. in this study, we compared ha antigenic structures of 2009 h1n1 and human h1n1 viruses by a molecular modeling approach to demonstrate the existence of shared epi-topes for neutralizing antibodies. we found that has of 2009 h1n1 and the 1918 h1n1 virus shared a significant number of amino acid residues in known antigenic regions. from this observation, we hypothesize that the 2009 h1n1 ha antigenic sites will be targeted by antibody-mediated selection pressure in humans in the near future. we further discuss possible directions of antigenic changes in the evolutionary process of 2009 h1n1. sequence data of ha genes modeller 9v6 was used for homology modeling of ha structures. after 100 models of the ha trimer were generated, the model was chosen by a combination of the mod-eller objective function value and the discrete optimized protein energy statistical potential score. after addition of hydrogen atoms, the model was refined by energy minimization with the minimization protocols in the accelrys discovery studio 2.1 software package using a charmm force field. steepest descent followed by conjugate gradient minimizations was carried out until the root mean square gradient was less than or equal to 0ae01 kcal ⁄ mol ⁄ a. the generalized born implicit solvent model was used to model the effects of solvation. the ha model was finally evaluated by using procheck, whatcheck, and verify-3d. custom-made programs were developed with the ruby language and used for investigating the numbers of potential n-glycosylation sites and candidate codons (cand1) in ha sequences. it is known that the h1 ha molecules have four distinct antigenic sites: sa, sb, ca, and cb. 3, 4 as a result, these sites consist of the most variable amino acids in the ha molecule of the seasonal human h1n1 viruses that have been subjected to antibody-mediated immune pressure since its emergence in 1918, although it was absent in humans from 1957 to 1976. to investigate the structures of these antigenic sites of 2009 h1n1, 3d structures of the ha molecules of sc1918, the recent seasonal human h1n1 virus (br2007), and 2009 h1n1 (ca2009) were constructed by a homology modeling approach, and compared by mapping all the amino acid residues that were distinct from those of sc1918 ha (data not shown). we found that most of these antigenic sites of br2007 ha predominantly contained altered amino acid residues if compared with sc1918. by contrast, amino acid residues at these positions were relatively conserved in ca2009 ha when compared with sc1918 ha. notably, the sa and sb sites, which contain many amino acids involved in neutralizing epitopes near the receptor binding pockets, remain almost intact ( table 1 ), suggesting that antibodies raised by natural infection with sc1918 or its antigenically related descendant viruses play a role in specific immunity against ca2009. these observations lead us to hypothesize that such antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in the human population. based on this hypothesis, we speculated that 2009 h1n1 would undergo patterns of amino acid substitutions in ha similar to those seen in seasonal human h1n1 viruses during its epidemic period (i.e. those that have been substituted since 1934) (figure 1) . we then predicted possible amino acid substitutions of 2009 h1n1 from the sequence similarity of the antigenic sites. for example, both sc1918 and ca2009 had an asn residue at position 142 in the sa site. for sc1918, the residue at this position has altered from asn to lys since 1942. combining these two facts, it seems reasonable to hypothesize that ca2009 will also undergo an amino acid substitution from asn to lys at position 142 in the future. interestingly, we found that some of the recent variants of the 2009 h1n1 virus have indeed undergone substitutions identical to those predicted in figure 1 . it is important to monitor whether such variants will be selected and survive in sustained circulation in humans. next, we analyzed the acquisition of potential n-glycosylation sites associated with antigenic changes. previously, we reported that cand1 sites, a set of three codons that require single nucleotide substitution to produce n-glycosylation sequons, were important motifs to rapidly acquire n-glycosylation sequons. 5 therefore, we investigated the number and location of potential n-glycosylation sites and cand1 sites in 2009 h1n1 ha. we found that ca2009 also had a single n-glycosylation sequon at the same position in the globular head region of ha, and lacked the multiple n-glycosylations that have been observed in the antigenic changes of the human h1n1 virus during the early epidemic of this virus. we also found that ca2009 ha possessed three cand1 sites that were present at the same position in sc1918 ha (positions of the first asn residue, 177, 179, and 184). of these, the cand1 sites with positions at 177 and 179 had actually become potential n-glycosylation sites in human h1n1 viruses. this result suggests the likelihood of additional n-glycosylation at these sites during future antigenic changes of 2009 h1n1 ha. notably, some of the recent 2009 h1n1 variants (as of march 24, 2010) have an additional n-glycosylation sequon at position 179, where the 1918 h1n1 virus readily acquired an n-glycosylation site during its circulation. the present study suggests that the antigenic structure of 2009 h1n1 ha is similar, at least in part, to that of the 1918 h1n1 ha. the 2009 and 1918 h1n1 has share unique three-codon motifs that are important to readily acquire n-glycosylation sequons in their globular head region. based on these similarities, we predicted possible amino acid substitutions that might be associated with future antigenic changes of 2009 h1n1, and confirmed that such substitutions occurred in some of the recent variants of this virus. the present study provides an insight into likely future antigenic changes in the evolutionary process of 2009 h1n1 in the human population. influenza viruses are classified into three types, a, b, and c, based upon the antigenic properties of nucleoproteins and matrix proteins. influenza a virus infects a wide range of hosts, including human, bird, swine, equine, and marine mammal species, while influenza b and c are less pathogenic than influenza a and are mainly found in humans, although there is evidence that they can also infect other species. influenza a has evolved in association with its various hosts on different continents for extended periods of time. 1 to survive as a successful pathogen, the influenza viruses have developed a number of mechanisms, including antigenic mutation and genome reassortment, to continuously evolve and evade the surveillance of the host immune systems. antigenic and genetic analyses have provided important insights into the molecular dynamics of influenza virus evolution. 2 however, a comprehensive understanding of influenza viral genetic divergence and diversity remains lacking. neuraminidase (na) is a major surface glycoprotein of influenza a and b, but is absent in influenza c. it plays a key role in virus replication through removing sialic acids from the surface of the host cell and releasing newly formed virions. influenza a viral na genes are classified into nine subtypes (na1-na9) based upon their antigenic properties, while na genes of influenza b are not classified into subtypes. furthermore, most na subtypes of influenza a have evolved into distinct lineages and sub-lineages, which correspond to specific hosts or geographical locations. in this study, we conducted large-scale analyses of influenza na sequences in order to infer their evolution and to identify lineages (or sub-lineages) of influenza a viruses. a total of 11 454 na sequences that excluded laboratory recombinant sequences were downloaded from genbank. sequences were aligned with muscle and mafft. the alignments were adjusted manually using translatorx, 3 based upon corresponding protein sequences. phylogenetic analyses were conducted using the maximum-likelihood (ml) method in raxml. 4 a set of perl scripts were written by us to facilitate this computational analysis. lineages and sub-lineages were determined based on the topology of the ml trees. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, 5 but with the following modifications: a single digit is used to represent one of the nine subtypes and a letter is used to represent a lineage; a sub-lineage is also represented using a digit; a dot is used to separate a lineage and a sublineage. for example, 2a.2 means na2 subtype, lineage a, and sub-lineage 2. the time of most recent common ancestor (tmrca) was estimated using the bayesian mcmc method in beast. 6 in all cases, we employed the gtr + u4 nucleotide substitution model, in which the first and second codon positions are allowed different rates relative to the third codon position. all data sets were analyzed under a relaxed molecular clock and the bayesian skyline population coalescent prior. the maximum clade credibility (mcc) tree across all plausible trees was computed from the beast trees using the treeannotator program, with the first 10% trees removed as burn-in. phylogenetic analysis based upon na sequences revealed two large groups corresponding to influenza a and b, respectively ( figure 1a ). within influenza a, two subgroups were found, one consisting of na1, na4, na5, and na8 and the other consisting of the remaining five subtypes. subtype na9 was found to be a sister subtype of na6, na1 being a sister subtype of na4, and na8 a sister subtype of na5. finally, each na subtype forms a distinct cluster, indicating its genetic uniqueness. influenza a and b viral na were estimated to have diverged around 2641 years ago ( figure 1b ). however, it had large 95% hpd values which ranged from 1259 years to 4299 years ago. the na subtypes of influenza a diverged from more than 1000 to several hundred years ago. the time of most recent common ancestor (tmrca) of each subtype of influenza a virus was generally recent and ranged from the calendar years 1767 to 1928 (figure 1b ). in addition, the tmrca for influenza b viral na was dated back to 1936. a total of 23 lineages were identified in influenza a (table 1) . three lineages, 1a, 1b, and 1c, were identified for na1 based upon the tree topology. linage 1a originated from avian viruses and was further divided into 5 sub-lineages: 1a.0, 1a.1, 1a.2, 1a.3, and 1a.4. linage 1b consists of north american swine influenza viruses whereas 1c is a human lineage. two large lineages, 2a and 2b, were identified in na2. lineage 2a is a human-specific lineage. interestingly, five major swine clades were observed within this lineage. lineage 2b is an avian-specific lineage, and consists of 3 sub-lineages, 2b.0, 2b.1, and 2b.2. three lineages were found in na3. lineage 3a was found in north american avian, 3b in eurasian ⁄ oceanian avian, and 3c also in avian, but it does not show any geographical pattern. for na4, na5, and na6, each was classified into 2 lineages, one found in north american avian (4a, 5a, 6a) and the other in eurasian ⁄ oceanian avian (4b, 5b, 6b). three lineages identified respectively in na7 and na8 are north american avian (7a, 8a), equine (7b, 8b), and eurasian avian (7c, 8c). na9 was also found to have 3 lineages: north american avian (9a), eurasian ⁄ oceanian avian i (9b), and eurassian ⁄ oceanian avian ii (9c), respectively. in this study, we conducted large-scale phylogeny and evolutionary analyses using influenza viral na sequences. the results showed that divergence between influenza a and b viruses occurred earlier than between any influenza a subtypes. this observation was consistent with previous findings based upon phylogenetic analysis of the ha gene, one of the most important genes related to host infection. 7 within influenza a, two sub-groups were found, one consisting of na1, 4, 5, and 8 and the other consisting of the rest of five subtypes (na2, 3, 6, 7, 9) . this observation does not agree with the result described by liu et al., 8 where na subtypes 1, 3, 4, 5, and 8 formed one group and the remaining four subtypes (na2, 6, 7, and 9) formed the other group. this difference is apparently caused by the fact that an outgroup was not used in their phylogenetic analyses. in the present study, both influenza a and b viral na sequences were included in the analysis. high bootstrap values were obtained for major groups, indicating that the inferred evolutionary relationship should be highly reliable. classification and designation of the lineages and sublineages within the influenza a virus are essential for studies of viral evolution, ecology, and epidemiology. a total of 23 lineages were identified within nine influenza a viral na subtypes and with the majority of the identified lineages found to be host or geographic specific or both. our results demonstrated a comprehensive view for the evolution of na genes and provided a framework for the inference of evolutionary history of pandemic viruses and for further exploring of viral circulations in multiple hosts. for example, the global pandemics of human h1n1 in 1918, h2n2 in 1957, the pandemic of human h3n2 virus in 1968, 9 the crisis of h5n1 hpai in hong kong in 1997, 10 and swine-origin h1n1 influenza in 2009, 11 all can be mapped onto the lineages and sub-lineages identified in this study. such information will facilitate not only identification of known genetic origins but also early detection of novel influenza a viruses. influenza viruses constantly evolve to avoid the human immune pressure in the process of antigenic drift. 1 through sequencing of viral genomes, the rates and direction of virus evolution can be observed. moreover, comparison of protein sequences allows us to determine amino acid substitutions that are related to immune pressure and antigenic drift. the creation of global influenza genetic databases, along with concurrent development of analytical tools, allows the comparison of multiple influenza virus strains. 2 the main aim of this study was to perform antigenic and genetic comparison of pandemic influenza viruses (h1n1) isolated during the 2009-2010 pandemic in ukraine and in other countries. nasopharyngeal swabs and autopsy materials collected from infected patients were received from the areas of ukraine. in addition, field isolates of influenza viruses from the 2009 ⁄ 10 season and strain specific serum were used for identification by hemagglutinin inhibition assay. influenza viruses were identified and subtyped using real-time rt-pcr analyses using cdc primers and adopted protocols. 3 sequencing was performed in two world health organization (who) influenza collaboration centers (centers for disease control and prevention, atlanta and national institute for medical research, london). hemagglutinin inhibition assay was conducted using chicken and guinea pig red blood cells following standard who protocols. 4 the all 6 ukrainian isolates of influenza viruses, which were isolated in ukraine during august-november 2009, were identified as a ⁄ california the phylogenetic analyses confirmed the evolutionary relationship between ukrainian isolates and viruses from other countries, which were isolated during the first wave of the pandemic. high genetic and antigenic conservation of pandemic influenza viruses from ukraine and other countries also were demonstrated. considering that the emergence of the novel pandemic influenza strain occurred in countries of northern hemisphere during summer, it was very interesting and significant tracking the dynamics of genetic changes in influenza viruses, which were isolated at the beginning of epidemic and those isolated during the rise of the epidemic in ukraine. influenza a virus causes moderate to severe epidemics annually and catastrophic pandemics sporadically. due to the evasiveness of the influenza virus and the nature of its genome (eight single-stranded and negative-sense rna segments), it is essential to understand the evolution of this important pathogen. 1 influenza virus evolves by two major mechanisms: mutation and reassortment. antigenic and genetic analyses have revealed partially the molecular dynamics of influenza virus evolution. 2,3 however, important questions, such as how many genotypes in the influenza a virus, remain unanswered. one of the major issues pertaining to this genotyping problem is how many lineages or sub-lineages can be determined for a subtype and according to what criteria. because of the unique structure of the influenza a viral genome, the computational genotyping methods developed for other viruses cannot be applied to the influenza virus. constructing phylogenetic trees is a powerful technique for the identification of evolutionary groupings (i.e., lineages ⁄ clades). however, for large trees, it is hard to determine how many lineages and the boundaries for each lineage. in this regard, multivariate analysis methods, such as multidimensional scaling (mds) and model-based hierarchical clustering, both taking advantage of dimension reduction and visualization, can complement conventional phylogenetic methods. 4 hemagglutinin (ha), the fastest evolving segment, is recognized as the most important gene in the influenza virus that plays a key role in viral pathogenesis. however, we have only limited knowledge of lineages and sub-lineages occurring in the hemagglutinin (ha) gene of influenza a virus, 5 although much effort has been made in assigning clades or sub-clades in highly pathogenic avian influenza (hpai) virus ha. 6 in this study, both model-based hierarchical clustering and phylogenetic methods were used for sequence analysis. one objective for this study is to explore and develop a more accurate lineage approach for further comprehensive influenza lineage and genotype analyses. a total of 11 821 hemagglutinin (ha) sequences (approximately 1778 nucleotides long), excluding laboratory recombinant sequences, were downloaded from genbank as of march, 2010. 7 sequences were aligned with muscle 8 and mafft. 9 the genetic distance matrix of all pairwise sequences was computed using the k2p model under mega 5.0. we then used the distance matrix as input to the cmdscale module in r 2.5.1 for the mds analysis. the principle coordinates resulting from mds were used for the model-based hierarchical clustering analysis, again in r 2.5.1 (the r foundation. available at: http://www.r-project.org/). the bayesian information criterion (bic) values were computed based upon ten different statistical data models -eii, vii, eei, evi, vei, vvi, eee, eev, vev, and vvv. the highest bic value was used to determine the number of clusters in the given sequence data. phylogenetic analysis was conducted using maximumlikelihood (ml) in raxml. 10 raxml uses rapid algorithms for bootstrap and maximum likelihood searches and is considered one of the fastest and most accurate phylogeny programs for large-scale sequence analysis. all the analyses were conducted on the supercomputer cluster (holland computing center, http://hcc.unl.edu/main/index.php). the trees were visualized in figtree (version 1.3.1) . lineages and sub-lineages were determined based on both the topology of the ml trees and model-based clustering results. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, 11 with the following modifications: lineage analysis was conducted for each ha subtype, which agrees with the convention of influenza virologists that 16 ha subtypes were identified in influenza a virus; ha lineages are represented with digits and letters, where the digit(s) represent one of the 16 subtypes and a letter represents a lineage; here, we present sub-lineages or sub-sub-lineages also in digits, with smaller numbers representing earlier lineages or sub-lineages within the same subtype (e.g., lineage 1 occurs earlier than lineage 2); the digit 0 is used to indicate inclusion of ancestral viruses in a lineage (or sub-lineage); a dot is used to separate lineages, sub-lineages, and sub-sub-lineages. for example, 1a.1ae2 means ha1 subtype, lineage a, and sub-lineage 1, and sub-sub-lineage 2. the sub-lineage level can be extended as necessary. the model-based clustering method corroborates commonly used phylogenetic methods in lineage and sub-lineage assignment. here we use the h12 subtype as an example to show the lineage and sub-lineage assignment. the bayesian information criterion (bic) reaches its maximum when the number of clusters for h12 equals 3, regardless of which mode we choose ( figure 1a ). therefore, based on bic, the optimal number of clusters for the h12 subtype is 3. as a result, a total of 3 clusters based upon the vvv model were identified ( figure 1b) . a significant correlation was found in lineage assignments by the phylogenetic method and the model-based hierarchical clustering method ( figure 1b,c) . lineages 12a and 12b were identified for h12, which correspond to north american avian and eurasian avian, respectively. lineage 12a was further divided into 2 sub-lineages, 12a.0, 12a.1, where 12a.0 is the ancestral sub-lineage in 12a. based on both model-based hierarchical clustering and phylogenetic analyses, a total of 41 distinct lineages were identified among 16 subtypes, averaging out to be 2ae56 lineages per subtype ( table 1 ). the majority of the identified lineages were found to be host or geographic specific or both. for example, three lineages, 1a, 1b, and 1c, were identified for ha1. lineage 1a was further divided into two sub-lineages, 1a.0 and 1a.1. the 1a.0 is swine-specific, whereas 1a.1 is a human pandemic 2009 h1n1 sub-line how to accurately identify an evolutionary lineage of influenza a viruses is challenging. one commonly used approach is molecular phylogeny, where phylogenetic trees are constructed, and the tree topology is used for lineage determination. here, we used a bayesian model-based clustering method, along with phylogenetic methods, to decide lineages and sub-lineages of influenza a viruses based upon sequence data. the results demonstrated that the modelbased clustering method corroborates phylogenetic methods and increases the accuracy of lineage assignment. one salient feature of this study is its large-scale analysis of all available influenza a hemagglutinin sequences. a total of 41 distinct lineages and 81 sub-lineages were classified; the majority of them were found to be host or geographic specific. this observation agrees largely with previous findings. 5 we are conducting further analyses of other influenza a segments 12 and expect to identify their lineages and create a comprehensive genotypes database for all influenza a viruses. such information will allow us to detect the genetic origin of newly found viruses, track their genetic changes, and identify potential genome reassortments. a hierarchical nomenclature system has been proposed and adopted for hpai ha clades and sub-clades by who influenza surveillance centers. 6 wan et al. also proposed a hierarchical approach for influenza a viral genotypes system. 13 the work presented here is one of the first steps towards the development of a nomenclature system for influenza a virus lineages (at the segment level) and genotypes (at the genome level). whether the naming system will be accepted and used by the influenza research community is more challenging than the lineage analysis itself. identification of the genetic origins of influenza a viruses will enhance our understanding the evolution and adaptation mechanisms of influenza viruses. the phylogenetic analysis is the traditional approach to identify the influenza progenitor. first, the nucleotide sequences are aligned using multiple sequence alignment methods, such as clustalw, 3 muscle, 4 and t-coffee. 5 second, phylogenetic analysis is performed on these aligned sequences to infer their evolutionary relationship using neighbor-joining (nj), 6 likelihood, or bayesian inference. 8 bootstrap analyses or computation of posterior probability are usually applied to estimate the phylogenetic uncertainty. however, this phylogenetic analysis is time consuming due to intensive computations in multiple sequence alignments and phylogenetic inferences. it is difficult to perform an analysis using this method on a large dataset, for instance, with more than 1000 taxa, as is the common case for influenza studies. alternatively, blast 9 is applied to identify the prototype genes in the database. blast determines a similarity by identifying initial short matches and starting local alignments. since influenza viral sequences have very high similarities, especially for most conserved regions, blast usually generates a large number of outputs, which will not be helpful for progenitor identification. since blast is a local sequence alignment, the results from blast may not reflect the global evolutionary information between the sequences. the blast scores cannot be used to define the evolutionary relations between viruses, especially in the context of the entire genetic pool. recently, we have developed a distance measurement method, complete composition vector (ccv), that can calculate genetic distance between influenza a viruses without performing multiple sequence alignments. 10, 11 we also adapted the minimum spanning tree (mst) clustering algorithm for influenza reassortment identification. 12 the application of this approach in the analyses of pb2 genes of influenza a virus showed that the integration of ccv and mst allows us to identify the potential progenitor genes rapidly and effectively. based on these results, here we develop a webserver called ipminer for influenza progenitor identification. ipminer can identify potential progenitors for a query sequence against all public influenza datasets within a few minutes. in order to improve the computing efficiency, 31 distance matrices were pre-computed by ccv, and they include 16 for ha (h1 to 16), 9 for na (n1 to n9), and one for each of the internal gene segments (pb2, pb1, pa, np, ns, and mp). these 31 pre-computed matrices will be updated weekly. ipminer just needs to compute the query matrices for a query sequence and sequences in the database. the standalone ccv program is also available at http://sysbio.cvm.msstate.edu/ipminer. in order to identify the influenza progenitor genes, ipminer first integrates the query matrix and a corresponding pre-computed matrix into a full distance matrix, which is then clustered by mst clustering algorithm. we adapted the threshold we measured previously in mst, u + nr, where u is the average distance and r is the standard deviation of a cluster. 12 as a result, mst will generate a hierarchical structure for the clusters. in each cluster, we will randomly select 20 viruses or 10% of the cluster size if this cluster has more than 200 viruses. ipminer will return the viruses with the smallest distances when the search reaches to the lowest level (the largest n) in this hierarchical structure. our analyses have shown that the level 5 has generally yielded good results for influenza a viruses. to visualize the overall mst structure, ipminer applies multi-dimensional scaling (mds) method to project all the viruses in the genetic pool onto a two dimensional graph, and the precursor viruses are marked in different shapes ( figure 1 ). the users can select other prototype viruses from the graph for further phylogenetic analyses. a single job with one query sequence takes <2 min. the genbank identifiers and associated genetic distances and sequence identities are displayed. the users can download the sequences for the identified precursor viruses as well as those from the prototypes viruses. in addition, for the users' convenience, ipminer generates a phylogenetic tree using nj method implemented in phylip 13 to illustrate the phylogenetic relationship among the query sequence(s), the identified progenitors, and the selected prototypes viruses. the programs in this solution package are written in java. the shell scripts are written in korn shell script in order to achieve high performance. cascading style sheets (css) are used for a consistent look across the pages. this also enables to change the overall design just by replacing the css definition file. php has been used as server side scripting and is written in java. in order to achieve high performance for computing in a genomic scale, we apply hash function or a binary tree, which enables that the precursor identification has a time complexity of o(n). for single queries, the users can visualize the results online. for batch queries of multiple sequences, the results will be sent to the users by e-mail. ipminer has been tested on microsoft internet explorer, mozilla firefox, and safari. the users need javascript to obtain full function of ipminer server. the webserver is available at http://sysbio.cvm.msstate.edu/ipminer. in summary, ipminer webserver has three major computational features for influenza progenitor identification: (i) it calculates the genetic distances through ccv and identifies the viruses with the shortest ccv distances against the query virus to be the progenitor genes; (ii) it projects influenza viruses onto a two dimensional map, which illustrates the global relationship between the progenitor genes and other viruses in the genetic pool; and (iii) it performs phylogenetic analyses between the query virus, the identified progenitor genes, and other selected prototype viruses. ipminer provides a user friendly web service for influenza progenitor identification in real time. the gisaid initiative offers an alternative to current public-domain database models in response to growing needs of the global influenza community for the sharing of genetic sequence and associated epidemiological and clinical data of all influenza strains. gisaid's publicly accessible epifluô database is governed by a unique sharing mechanism that protects the rights of the submitter, while permitting ongoing research as well as the development of medical interventions, such as drugs and vaccines. for the gisaid initiative, the max planck institute for informatics (mpii) saarbrücken, germany, has developed a web portal that is accessible at http://www.gisaid.org featuring the gisaid epifluô database that offers a unique collection of nucleotide sequence and other relevant data on influenza viruses. the database is based on software by oracle and the dante ò system by a3systems gmbh, germany. extensive metadata are also collected for most isolates. the database provides features for searching, filtering specific datasets for download, and user friendly upload functionality. to uphold gisaid's unique sharing mechanism, all users must positively identify themselves. while access is free of charge, all users agree that they will not attach any restrictions on the data, but will acknowledge both the originator of the specimen and the submitter of the data, and seek to undertake to collaborate with the submitter. all uploaded sequence data are submitted to rigorous curation by the friedrich-loeffler-institute for animal health (fli), germany. the database has been live since september 14, 2009. among its contributors are all five who collaborating centers for influenza who routinely contribute data in addition to using the epifluô database for their semiannual vaccine strain selection. to provide a complete picture of data, all data available in the public domain is routinely imported. as of october 8, 2010, the rapidly growing gisaid dataset comprises 166 989 nucleotide sequences (from 48 779 isolates) with 28 949 (from 11 857 isolates) uniquely submitted to this database. software development is underway to continually extend the spectrum of available data analysis tools. the intergovernmental process of the 62nd world health assembly specifically mentions gisaid as a publicly available database for depositing virus sequence data. starting in 2011, germany's federal ministry of food, agriculture and consumer protection will be the long-term host of the gisaid platform. the mpii will continue to develop the portal and database software and enable gisaid to act as a catalyst for the development of advanced bioinformatics software connected directly to the database. gisaid has become an indispensible resource for the international scientific community on influenza. the consortium will expand its activities and offers to catalyze research and development on a wide variety of issues pertaining to risk analysis, drug development, and therapy of influenza. options for the control of influenza vii ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 416-424 the pandemic h1n1 virus emerged in 2009 and spread rapidly throughout the world, principally affecting children and young adults. as this virus is new to the human population, it is important to determine if these influenza infections are more commonly associated with other respiratory pathogens compared to previously circulating influenza strains. co-infecting respiratory viruses may cause increased morbidity in individuals with pandemic h1n1, and may also be unwanted contaminants in influenza vaccines if original clinical samples containing these adventitious viruses are used to directly inoculate certified cell lines for vaccine production. to examine this issue, stored rna from 300 original clinical samples (nasal swabs, nasal aspirates, throat swabs) from australian and new zealand subjects that were collected in 2009 that were positive for pandemic h1n1 and 300 samples collected in 2008 that were positive for seasonal influenza by real time pcr assay (using the cdc, usa kits), were subjected to a resplex ii -panel version 2.0 (qiagen) pathogen screen. the resplex ii assay detects 18 common respiratory viruses, such as respiratory syncytial viruses (rsv a, b), influenza a and b viruses, parainfluenza viruses (piv1-4), human metapneumo-viruses (hmpv), coxsackieviruses ⁄ echovirus (cvev), rhinoviruses (rhv), adenoviruses (adv b, e), coronaviruses (nl63, hku1, 229e, oc43), and bocaviruses. resplex ii uses a combination of multiplex rt-pcr, hybridization of pcr onto target specific beads followed by detection using luminex-xmap technology. original clinical samples were received at the center from who national influenza centers, who influenza collaborating centers, and other regional laboratories and hospitals from australia, new zealand, and the asia ⁄ pacific region. most samples were from australia and new zealand. these samples consisted of nasal swabs, nasopharyngeal swabs, nasal washes, throat washes, and throat swabs. all samples were stored at )80°c until rna was extracted. rna was extracted from 200 ll of clinical sample using either the magnapure extraction system (roche, australia) or the qiaxtractor system (qiagen, australia) according to the manufacturer's recommendations with an elution volume of 90 ll and stored at )80°c until used. a 5 ll aliquot of rna was used to amplify the selected influenza virus gene using specific primers and probes as supplied by cdc (atlanta, usa) 1 along with super-script iii platinum one-step rt-pcr reagents (invitrogen, australia). real time pcr detection was performed on a 7500 fast system with sds software (applied biosystems, ca, usa). a cut off of a cycle threshold (c t ) of 35 or below was considered positive. resplex ii panel ver2.0 detection the qiagen molecular differential detection (mdd) system was used, which combines qiaplex amplification (multiplex rt-pcr) with detection on the liquichip 200 workstation (luminex's xmap microsphere based multiplexing system) and qiaplex mdd software according to the manufacturer's instructions. a low level cutoff was used (150) to obtain maximum sensitivity. from the 300 clinical specimens that were positive for influenza from 2008 by real time pcr, there were 18 (6%) a(h1n1) seasonal influenza viruses, 106 (35ae3%) a(h3n2) viruses, 174 (58%) b viruses, and 2 (0ae7%) viruses which were influenza a positive, but could not be typed. clinical samples from 2009 selected to study were all influenza a(h1n1) pandemic 2009 positive by real time pcr. detection of influenza virus in respiratory samples was much lower with the resplex ii assay (using a low cut off of 150 units) for pandemic influenza a virus (160 ⁄ 320; sensitivity 53ae3%) and to a lesser extent for seasonal influenza a (116 ⁄ 126; sensitivity of 92ae1%) and b viruses (161 ⁄ 174; sensitivity of 92ae5%) when compared to real time pcr. there were relatively few co-infecting respiratory viruses with either pandemic h1 infections in 2009 (5ae7%) or seasonal influenza infections in 2008 (6ae3%) ( table 1 ). the most common dual infection seen with pandemic h1n1 2009 viruses and 2008 seasonal b viruses was with cvev (7 ⁄ 18; 2009 and 5 ⁄ 14; 2008, respectively) while for 2008 a(h3) viruses there were no dominant co-infecting viruses ( table 2 ). in 2009 one case was detected with three respiratory pathogens in the same sample, a 25 year old female who had pandemic h1n1, cvev, and rhv, and in a 2008 seasonal influenza sample, one case with a triple infection was detected (bocavirus, piv2 and influenza b). the median age of subjects with co-infections was younger for both pandemic h1n1 with a median age of 10 years (range: 1 months to 27 years), compared to the full 2009 sample set which had a median age of 29 years (range: 1 months to 85 years), while for the patients from 2008 with seasonal influenza viruses with co-infections they had a median age of 6ae5 years (range: 1 months to 26 years) compared to all 2008 samples which had a median age of 16 years (range: 1 months to 84 years). there was good concordance in detecting influenza a and b in respiratory samples collected in 2008 between real time rt-pcr and the resplex ii system (100% versus >92ae1% for seasonal influenza a and b respectively). this data compares well with other studies such as li et al. 2 who found that resplex ii had 82ae8% sensitivity and 100% specificity for seasonal influenza a viruses and 90ae0% sensitivity and 100% specificity for influenza b viruses. in contrast, the present study found only 53ae3% sensitivity for the resplex ii detection of influenza a with the 2009 samples that were positive for pandemic h1n1 by real time rt-pcr. a recent study by rebbapragada et al. 3 also showed lower sensitivity for pandemic h1n1 viruses in nasopharyngeal samples with the resplex ii system (55% sensitivity and 100% specificity) compared to other commercial platforms seeplex rvp (95% sensitivity and 100% specificity) and luminex rvp (100% sensitivity and 97% specificity). interestingly the latest version of the resplex system offered by qiagen the resplex ii plus panel ruo now has a separate target for the pandemic h1n1 virus (mexico 2009). 4 in terms of detection of other respiratory viruses such as piv-1, piv-3, rsv and hmpv, high sensitivities (87ae5%, 72ae2%, 73ae2%, and 80%, respectively) and specificities (99ae7-100%) compared to taqman rt-pcr have been reported from testing of nasal wash and nasopharyngeal clinical samples. 5 in both the seasonal influenza positive 2008 and the pandemic h1n1 positive (by real time rt-pcr) clinical specimens, few other respiratory viruses were detected. only 18 of the 2008 samples had another virus detectable and one had two other viruses, while in 2009 16 out had another virus and one had two other viruses detected from a total of 300 influenza virus positive samples collected in each year. enteroviruses, coronaviruses, and parainfluenza viruses were most often found with both seasonal and pandemic infections. younger age appeared to be associated with co-infections with those subjects in 2008 with dual infections having a median age of only 10 years compared to the study groups 29 years; and similarly for 2009, the median age for subjects with dual infections was only 6ae5 years compared to the study groups' median age of 16 years. a study by chong et al. 6 on 298 nasopharyngeal swabs collected during 2007-2009 using resplex ii and luminex xtag rvp fast, they found dual respiratory virus infections in 27 ⁄ 179 (15ae1%) of samples and only 2 (0ae7%) with triple respiratory viral infections; however, these were from cases with any combination of multiple respiratory viruses not necessarily influenza, although influenza positive cases were the most common respiratory virus detected (37ae4% of all positive samples). given the low level and variety of viral co-infections along with both seasonal and pandemic influenza seen in this study, it is unlikely that influenza infections predispose subjects to particular respiratory viruses, but may still allow bacterial colonization, such as has been seen with severe and fatal cases with pandemic h1n1 with various bacteria including streptococcus pneumoniae, streptococcus pyogenes, staphylococcus aureus, or haemophilus influenzae. 7, 8 low levels of other respiratory viruses along with the finding that certain cell lines (like the mdck 33016-cells used in this study) do not propagate a number of these viruses (e.g. rsv a and b, rhinoviruses, coronaviruses), but do propagate others (e.g. parainfluenza 3) 9 should make testing for unwanted viruses that might be co-isolated with influenza viruses more focused and hence easier to detect and eliminate this isolate for future vaccine production. global influenza surveillance is one of the most important approaches to combat spread of disease. current laboratory methods for characterizing influenza are time-consuming and labor-intensive, and few viral strains undergo full characterization. 1 even fewer strains from domestic poultry and swine or from wild aquatic birds are wellcharacterized. these strains are important for global surveillance since they are thought to be the precursors to pandemic influenza strains. 2 we have designed a highthroughput global bio laboratory to address these surveillance needs. the goal of this project was to develop highspeed and high-volume laboratory capabilities for extensive surveillance and rapid and accurate detection and analysis of influenza. the workflow consists of surveillance, sample transportation, laboratory testing, data management and analysis. five robotic systems have been designed for this laboratory: sample accessioning, biobanking, screening, viral culture, and sequencing. sample accessioning logs barcodes, centrifuges, and aliquots samples are then sent to biobanking. the robotic biobank stores samples at )80°c and reformats tubes for screening. the screening system extracts rna and confirms the presence and subtype of influenza. aliquots of positive samples are sent to the viral culturing system for scale-up. finally, cultured samples are extracted and sent to the sequencing system for full genome sequencing. the sample accessioning, sequencing, and biobanking systems have been built, delivered, and validation processes are currently being completed. robotic screening and culturing systems have been fully designed and are ready to be built. a biosafety level 3-enhanced containment laboratory was built to enable the flow of samples containing highly pathogenic avian influenza viruses. in full operation, this approach to surveillance is designed to enable the sequencing of up to10 000 full virus genomes per year, more than the total of all full influenza genomes sequenced to date. the design of a robotic laboratory for influenza surveillance presents unique challenges and opportunities. before a robotic system is built, each assay is worked out on the bench top, each movement of the plates and reagents is defined, and the laboratory information management system (lims) must be able to address each step of the process. alternate assays are conceived for processes that are not automation-friendly. waste streams, worker safety, and space constraints are considered. each possibility is taken to reduce processes that have the potential to aerosolize or cross-contaminate influenza samples. instruments must be found that fit the capabilities needed. detailed specifications for each of the robotic systems were written including all the parameters listed above. once the systems are built, a long validation process takes place where the processes and instruments in each system are adjusted to function together properly. finally, a validation study is performed to ensure that the system is able to produce useful data for influenza research. the entire process takes months from start to finish for each robotic system and requires complete cooperation from a diverse team of researchers. the accessioning system logs initial sample information with the lims system. samples arrive in barcoded cryotubes. the liquid handler brings all samples up to a common volume and clarifies samples by centrifugation. samples are then transferred from screw-cap sample vials into storage plates containing 96 individually punchable storage tubes. each tube (0ae3 ml) is individually identifiable with a 2d barcode on the bottom. six archive aliquots are made, and tubes are individually weld-sealed for storage. tips for aspiration are fixed and undergo a high-pressure plasma process between each use to sterilize tips and destroy nucleic acids. samples are stored at )80°c. each module has a capacity of 600 remp plates or $60 000 samples. the automated freezer system can assemble requested samples as 96-well plates while samples remain frozen. the screening system uses magnetic bead extraction chemistry, real-time pcr, and a liquid handling system to extract samples, confirm and quantify the presence of influenza, and reformat extracted samples for input into the sequencing system. serotype of human influenza samples will be performed by real-time pcr. many samples will not have enough material for further analysis and will need to be scaled up. the culturing system combines incubators, a liquid handling platform, plate reader, and real-time pcr to culture, monitor growth, harvest, and quantify influenza. when the system is not being used for culture and scale-up, it can be used to assay previously cultured influenza samples for drug resistance. a challenge to sequencing large numbers of influenza samples is the manpower required for sample preparation. the sequencing system has the capacity to prepare up to 10 000 samples for sequencing per year for sanger sequencing. sanger sequencing was chosen because it is well-established for influenza surveillance, and automation-friendly. the system is designed to work with multiple primer sets (48, 96, 192) . robotic systems all report to the lims. each process completion, plate movement, and data point are entered and checked by an online, web-based lims. status updates, notification, reporting, and data analysis can be achieved without entering the bsl3 containment facility. routine data analysis such as determining whether a cultured sample is ready to be harvested will be performed by the lims. complex data analysis, while still requiring significant human input, will be made easier by the data-acquisition functions of the lims. the implementation of a high-throughput influenza surveillance laboratory will provide an influenza research and response capacity that far exceeds what is available today. with the addition of each new system, we add a new capability to the influenza community and new opportunities to foster partnerships and collaborations with government, foundations, businesses, and academic institutions. this laboratory will not only enable cutting edge research, but will also enable a more effective response of near real-time surveillance during a pandemic outbreak. pandemics of 1957 and 1968 were believed to arise from avian influenza viruses. 1 the tropism of avian and human seasonal influenza viruses for the human lower respiratory tract deserves investigation. the target cell types that support replication of avian influenza a viruses in the human respiratory tract in the early stages of clinical infection have not well defined. in a previous autopsy studies of human h5n1 disease, influenza a virus were found to infect alveolar epithelial cells 2 and macrophages. 3 in this study, viral infectivity and replication competence of human and high and low pathogenic avian influenza viruses were systematically investigated in the human conducting and lower respiratory tract using ex vivo organ cultures. we compared the replication kinetics of human seasonal influenza viruses (h1n1 and h3n2), low pathogenic avian influenza viruses (h9n2, h5n8) with that of the highly pathogenic h5n1 viruses isolated from human h5n1 disease. a range of human seasonal influenza a viruses of subtypes h1n1 and h3n2 viruses were included in this study from 1975 to 2009. two isolates of low pathogenic avian influenza a (lpai) (h9n2) viruses from different virus lineages isolated from poultry in hong kong in 1997, a low pathogenic influenza a (h5n8) virus isolate from wild ducks in hong kong in 2005, and two virus isolates of highly pathogenic avian influenza (hpai) a subtype h5n1 were included. fragments of human bronchi and lung were cut into multiple 2-3 mm fragments within 2 hours of collection and infected in parallel with influenza a viruses at a titer of 10 6 tcid 50 ⁄ ml and as control cultures were infected with ultraviolet light inactivated virus. these tissues fragments were infected for 1 hours and washed twice with pbs and incubated for 1, 24, and 48h at 37°c. the bronchial tissue was cultured in an air-liquid interface using sponge. viral yield was assessed by titration in mdck cells. one part of the infected tissue were fixed in formalin and processed for immunohistochemistry for influenza antigen. other part of infected tissue was homogenized and underwent rna extraction, and the expression of influenza virus matrix gene was measured by quantitative rt-pcr. human bronchus ex vivo cultures supported human seasonal influenza virus to replicate efficiently. avian influenza h9n2 virus replicated, although less efficiently than that of seasonal influenza viruses, whereas hpai h5n1 did not productively replicate in ex vivo cultures of human bronchus. this is in agreement with our previous finding in the well-differentiated bronchial epithelial cells in vitro. 4 on the other hand, human lung ex vivo cultures supported prominent productive replication of human seasonal influenza h1n1 ( figure 1a ) and hpai h5n1 ( figure 1f ) viruses. lpai, such as h9n2 ( figure 1c -d) and h5n8 ( figure 1e ), also replicated productively, but with a lower viral yield. surprisingly, the replication of human influenza h3n2 viruses ( figure 1b ) across the last three decades was greatly inhibited. there are clear differences in viral tropism of human seasonal and avian influenza viruses for replication in the human bronchus and lung. hpai h5n1 virus can infect and productively replicate in the lower lung, which may account for the severity of human h5n1 disease, but not in the conducting airways. surprisingly, there are marked differences in the replication competence of seasonal influenza viruses in ex vivo lung tissues, with influenza h1n1 viruses being able to replicate efficiently while h3n2 viruses do not. this may be related to the more strict siaa2-6gal binding preference of h3n2 viruses. on the other hand, the efficient replication of influenza h1n1 viruses in the alveolar spaces indicates factors other than tissues tropism alone play a role in the differences in disease severity between human seasonal h1n1 and avian h5n1 virus infections. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells, suggesting that viral protein(s) other than m gene-translational products facilitates the splicing of viral mrnas. the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns1 (c ⁄ ns1) and ns2 (c ⁄ ns2), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns1 and c ⁄ ns2 proteins, drastically reduced the splicing of ns mrna, raising the possibility that c ⁄ ns1 or c ⁄ ns2 enhances the splicing of viral mrnas. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns1, whereas it was reduced by co-expression of influenza a virus ns1 protein (a ⁄ ns1). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns1, whereas it was inhibited by that of a ⁄ ns1. these results suggest that influenza c virus ns1, but not a ⁄ ns1, can up-regulate the splicing of viral mrnas. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. 1, 2 the inefficient splicing of viral pre-mrnas can be understood partly by the fact that influenza a virus ns1 protein is associated with spliceosomes and inhibits pre-mrna splicing. 3, 4 cis-acting sequences in the ns1 transcript also negatively regulate splicing. 5 by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. 6 the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. 7 the yamagata ⁄ 1 ⁄ 88 strain of influenza c virus was grown in the amniotic cavity of 9-day-old embryonated hen's eggs. cos-1 and 293t cells were cultured in dulbecco's modified eagle's medium containing 10% fetal calf serum. subconfluent monolayers of cos-1 cells were transfected with pme18s containing influenza c virus m gene cdna using the lipofectamine procedure and then incubated at 37°c. total rna was extracted from both the transfected cells and cells infected with c ⁄ yamagata ⁄ 1 ⁄ 88 virus using the rneasy mini kit (qiagen). ribonuclease protection assay was performed using a ribonuclease protection assay kit rpa iii (ambion). 6 briefly, a [ 33 p]-labeled influenza c virus rna 6-specific rna probe (vrna sense) was synthesized by in vitro transcription and hybridized with the total rna at 42°c overnight. hybrids were digested with rnase a (0ae08 u) and rnase t1 (3 u) at 37°c for 30 minutes and then analyzed on a 4% polyacrylamide gel containing 4 m urea. hmv-ii cells infected with c ⁄ yamagata ⁄ 1 ⁄ 88 and cos-1 cells transfected with pme18s expressing influenza c virus ns1 were fixed with carbon tetrachloride at various times after infection and transfection, respectively. the cells were then stained by an indirect method using anti-gst ⁄ ns1 serum as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit igg (seikagaku kogyo) as the secondary antibody. the splicing efficiency of influenza c virus m gene-specific mrna (m mrna) in infected cells was higher than that in m gene-transfected cells the ratio of m1 encoded by a spliced m mrna to cm2 encoded by an unspliced m mrna in influenza c virusinfected cells was about 10 times larger than that in m gene-transfected cells. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells (figure 1 ). these data suggest that viral protein(s) other than m gene-translational products facilitates viral mrna splicing. the influenza c virus ns gene translational product may up-regulate the splicing of viral mrnas the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns1 (c ⁄ ns1) and ns2 (c ⁄ ns2), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns1 and c ⁄ ns2 proteins, drastically reduced the splicing of ns mrna, suggesting that c ⁄ ns1 or c ⁄ ns2 enhances viral mrna splicing. immunofluorescent staining showed that ns1 localized in the nucleus in the early phase of infection, and was distributed in both the nucleus and cytoplasm in the late phase of infection, raising the possibility that influenza c virus ns1 protein plays a role in viral mrna splicing that occurs in the nucleus. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns1, whereas it was reduced by co-expression of influenza a virus ns1 protein (a ⁄ ns1) (figure 2a ). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns1, though it was inhibited by that of a ⁄ ns1 ( figure 2b ). these results suggest that influenza c virus ns1, but not a ⁄ ns1, can up-regulate the splicing of viral mrnas. 7 in influenza a virus-infected cells, splicing is controlled so that the steady-state amount of spliced mrnas is only 5-10% of that of unspliced mrnas. 1, 2 the mechanisms by which influenza a virus ns pre-mrnas are poorly spliced have been investigated and the following confirmed. influenza a virus ns1 protein associates with spliceosomes and inhibits pre-mrna splicing. 3, 4 two cis-acting sequences in the ns1 transcript (positions 153-465 in the intron and positions 775-860 in the 3¢ exon region) inhibit splicing. 5 by contrast, influenza c virus m gene-specific mrna (m mrna) is efficiently spliced in influenza c virus-infected cells. 6 in this study, we examined the mechanism by which influenza c virus m mrna is efficiently spliced and the regulatory mechanism of the splicing of ns gene-specific mrna (ns mrna). the introduction of a translational pre-mature termination into the influenza c virus ns gene, thereby blocking the synthesis of influenza c virus ns1 (c ⁄ ns1) and ns2 (c ⁄ ns2) proteins, drastically reduced the splicing rate of ns mrna. we further examined whether c ⁄ ns1 potentially facilitates viral mrna splicing. the splicing rate of m mrna of influenza c virus was increased by co-expression with c ⁄ ns1, whereas it was reduced by co-expression with influenza a virus ns1 protein (a ⁄ ns1) (figure 2a ). the splicing of influenza a virus m gene-specific mrna was also increased by co-expression with c ⁄ ns1, though it was inhibited by co-expression with a ⁄ ns1 ( figure 2b ). these results suggest that influenza c virus ns1 can facilitate viral mrna splicing, but in no way inhibit it, which is in striking contrast to the inhibitory effect of influenza a virus ns1 on pre-mrna splicing. 3, 4 the mechanism for splicing enhancement by c ⁄ ns1 also remains to be determined. we speculate that c ⁄ ns1 may interact with some host proteins involved in splicing, thereby leading to an up-regulation in splicing, or that c ⁄ ns1 may bind to pre-mrna, increasing its accessibility to the spliceosome. the spliced mrna of the influenza c virus m gene encodes the m1 protein, which plays an important role in virus formation and determines virion morphology. 8, 9 therefore, it is speculated that the mechanism for efficient splicing of m mrna, which provides the m1 protein necessary for virus assembly in a redundant amount, has been maintained in the influenza c virus. by contrast, unspliced mrna from the influenza c virus m gene encodes the cm2 ion channel, which is permeable to chloride ions, 10 and also has ph-modulating activity. 11 although the role of the influenza c virus cm2 ion channel in virus replication remains to be determined, it is conceivable that the over-expression of the cm2 protein has a deleterious effect on virus replication since the fact that a high level of influenza a virus m2 protein expression inhibits the rate of intracellular transport of the influenza a virus ha protein and other integral membrane glycoproteins has been demonstrated. 12 if this is the case, efficient splicing of m mrna may control the amount of cm2 synthesized to optimize virus replication. therefore, we speculate that efficient splicing of m mrna leads to a high level of m1 expression and the reduced expression of cm2, thereby creating conditions that are optimal for virus replication. in this study, we provided evidence that c ⁄ ns1 facilitates the splicing of m mrna. furthermore, c ⁄ ns1 may regulate the splicing efficiency of its own ns mrna during infection, controlling the amount of c ⁄ ns1 and c ⁄ ns2 proteins in infected cells. c ⁄ ns2 plays an important role in the nuclear export of vrnp, and is also associated with vrnp in the later stages of infection in virus-infected cells and is incorporated into virions, 13 suggesting that c ⁄ ns2 is involved, not only in the sorting of vrnp into the assembly site, but also in virus assembly. therefore, it is likely that there is a mechanism by which an appropriate amount of c ⁄ ns2 is provided during infection to accomplish these functions. in conclusion, c ⁄ ns1, which enhances the splicing of viral mrna, may regulate both the expression level of m gene-derived m1 and cm2 proteins, and that of ns gene-derived ns1 and ns2 proteins, thereby leading to optimal virus replication. propagation of the human influenza viruses in embryonated hen's eggs always results in a selection of variants with amino acid substitutions in the hemagglutinin (ha) that affect viral receptor-binding characteristics (reviewed 1 ). brookes et al. 2 recently studied infection in pigs using the egg-grown virus that contained a mixture of the original a ⁄ california ⁄ 07 ⁄ 09 (h1n1pdm) and its two egg-adaptation mutants with single amino acid substitutions d222g and q223r (225 and 226 in h3 numbering system). only the original virus and the variant with 222g were detected in the directly inoculated animals, indicating that the variant with 223r failed to infect. only the original virus was detected in nasal secretions of contact infected pigs, suggesting that the d222g mutant failed to transmit. in contrast, there was an apparent selection of the d222g mutant in the lower respiratory tract samples from directly inoculated pigs. the d222g substitution is of a special interest as it can emerge during virus replication in humans and was associated with severe and fatal cases of pandemic influenza in 2009-2010 3-7 and 1918. 8 here we compared phenotypic properties of the original clinical isolate of h1n1pdm virus a ⁄ hamburg ⁄ 5 ⁄ 09 and its d222g and d223r mutants to explain observed effects of these mutations on virus replication in swine 2 and to predict their potential effects on virus replication in humans. a ⁄ hamburg ⁄ 5 ⁄ 09 (ham) was isolated from clinical material by two passages in mdck cells. the virus was passaged twice in 11-day-old embryonated hen's eggs and plaqued in mdck cells. the plaques were amplified in mdck cells and the sequences of the viral ha were determined. the variants with single mutation d222g and q223r were aliquoted and designated ham-e and ham-e1, respectively. the receptor-binding specificity of the viruses was assessed by assaying their binding to desialylated-resialylated peroxidase-labeled fetuin containing either a2-3-linked sialic acid (2-3-fet) or a2-6-linked sialic acid (2-6-fet). 9 in brief, viruses adsorbed in the wells of 96-well eia micro plates were incubated with serial dilutions of 2-3-fet or 2-6-fet, and the amount of bound fetuin probe was quantified by peroxidase activity. the binding data were converted to scatchard plots (a490 ⁄ c versus a490), and the association constants of the virus-fetuin complexes were determined from the slopes of these plots. viral cell tropism and replication efficiency in human airway epithelium were studied using fully differentiated cultures of human tracheo-bronchial epithelial cells (htbe). 10, 11 to determine cell tropism, cultures were infected at a moi 1, fixed 8 hours after infection, and double immuno-stained for virus antigen and cilia of ciliated cells. infected cells were counted under the microscope (100· objective with oil immersion) in the epithelial segment that included 15-30 consecutive microscopic fields containing between 5% and 20% ciliated cells relative to the total number of superficial cells. percentages of infected ciliated cells and infected non-ciliated cells relative to the total number of infected cells were calculated. ten segments per culture were analyzed and the results were averaged. to compare growth kinetics of ham and ham-e, replicate htbe cultures were infected with 200 plaque-forming units of the viruses followed by incubation at 35°c under airliquid interface conditions. at 24, 48, and 72 hours postinfection, we added dmem to the apical compartments of the cultures and incubated for 30 minutes at 35°c. the apical washes were harvested, stored at )80°c, and analyzed simultaneously for the presence of infectious virus by titration in mdck cells as described previously. 11 the non-egg-adapted h1n1pdm virus ham, similarly to the seasonal human virus a ⁄ memphis ⁄ 14 ⁄ 96 (h1n1), bound to 2-6-fet ( figure 1a ) and did not show any significant binding to 2-3-fet. this result contrasted with the binding of h1n1pdm viruses to several 2-3-specific probes in carbohydrate microarray analysis. 12 reduced avidity of virus interactions with soluble glycoprotein in solution as compared to its binding to the probe clustered on the microarray surface could account for these differences in the assay results. the d222g mutant ham-e differed from the parent virus by its ability to bind to 3-fet and by its reduced binding to 6-fet. the q223r mutant only bound to 2-3-fet, although less strongly than did the avian virus a ⁄ duck ⁄ alberta ⁄ 119 ⁄ 98 (h1n1). the viral cell tropism in htbe cultures ( figure 1b ) correlated with receptor specificity. ham and mem ⁄ 96 showed a typical human-virus-like tropism 10, 11 with preferential infection of non-ciliated cells (<5% of infected cells were ciliated). the mutant with 223r and control duck virus displayed a typical avian-virus-like tropism (preferential infection of ciliated cells). the d222g mutant displayed a cell tropism that was intermediate between those of human and avian viruses; in particular, this mutant infected significantly higher proportion of ciliated cells than ham and mem ⁄ 96. observed alteration of receptor specificity and cell tropism ( figure 1 ) suggested that egg-derived mutations can affect replication of the h1n1pdm virus in human airway epithelium. to test this, we first compared the capacity of the viruses to initiate infection in htbe cultures. replicate cultures were infected with identical doses of the viruses, fixed 8 hours post-infection, and immuno-stained for viral antigen. under these conditions, ham and ham-e infected comparable numbers of cells, whereas ham-e1 infected at least 10 times less cells (data not shown). this result indicated that the mutation q223r markedly impaired the ability of ham-e1 to infect human airway epithelial cultures. we next compared two other viruses ham and ham-e for their multi-cycle replication in htbe cultures and found that the original virus reached threefold higher peak titers 48 hours post infection than did the d222g mutant ( figure 2 ). the d222g mutation in h1n1pdm virus facilitates virus binding to 2-3-linked receptors and alters viral cell tropism in human airway epithelium. these changes could account for increased replication of the d222g mutant in the lower respiratory tract in humans 5-7 and pigs 2 and correlation of this mutation with severe pulmonary disease. [3] [4] [5] [6] [7] the d222g mutant replicates less efficiently in human airway cultures than the original virus. this finding correlates with an apparent lack of transmission of variants with 222g in humans 3 and pigs. 2 egg-derived mutation q223r abolishes virus binding to 2-6-linked receptors and strongly decreases infection in cultures of human airway epithelium. this result agrees with poor infectivity of the q223r mutant in pigs 2 and highlights potential pitfalls of using egg-adapted viruses with this mutation for the preparation of live influenza vaccines. nin-esterase-fusion (hef), nucleoprotein (np), matrix (m1) protein, cm2, and the non-structural proteins ns1 and ns2. 1,2 cm2 is the second membrane protein of the virus and is encoded by rna segment 6 (m gene). [3] [4] [5] [6] [7] [8] it is composed of three distinct domains: a 23-residue n-terminal extracellular domain, a 23-residue transmembrane domain, and a 69-residue cytoplasmic domain. 3, 9, 10 it is abundantly expressed at the plasma membranes of infected cells and is incorporated in a small amount into virions. 10,11 cm2 forms disulphide-linked dimers and tetramers, and is posttranslationally modified by n-glycosylation, palmitoylation, and phosphorylation. [10] [11] [12] analyses of a number of cm2 mutants revealed the positions of the amino acids involved in the posttranslational modifications. 10, 13 evidence was obtained that the n-glycosylation was not required for either the formation of disulfide-linked multimers or transport to the cell surface, 10 and that none of dimer-or tetramer-formation, palmitoylation or phosphorylation was essential to the transport of cm2 to the cell surface. 13 in the present study, in order to investigate the effect of cm2 palmitoylation on influenza c virus replication, we generated a cm2 palmitoylation-deficient influenza c virus, in which a cysteine at residue 65 of cm2 was mutated to alanine, and examined the viral growth and viral protein synthesis in infected cells. 293t and hmv-ii cells were maintained as described previously. 14,15 llc-mk 2 cells were maintained at 37°c in minimal essential medium with 5% foetal bovine serum and 5% calf serum. monoclonal antibodies (mabs) against the hef, np, and m1 proteins of c ⁄ ann arbor ⁄ 1 ⁄ 50 (aa ⁄ 50), and antisera against the aa ⁄ 50 virion and the cm2 protein were prepared as described previously. 3, [16] [17] [18] the seven pol i plasmids for the expression of viral rnas of aa ⁄ 50, and the nine plasmid dnas for the expression of the influenza c viral proteins were reported previously. 6, 14 plasmid dna, ppoli ⁄ cm2-acy(-), in which 995-tgt-997 of the m gene was replaced with 995-gct-997, was constructed based on ppoli ⁄ m. to generate a recombinant wild-type (rwt) virus, the above-mentioned 16 plasmids were transfected into 293t cells as described previously. 6 to rescue a mutant virus, rcm2-c65a, a recombinant influenza c virus lacking a cm2 palmitoylation site, the plasmid ppoli ⁄ cm2-acy(-), instead of ppoli ⁄ m, was transfected together with the other 15 plas-mids. at 90 hours posttransfection (p.t.), the respective culture medium of the transfected-293t cells was inoculated into the amniotic cavity of 9-day-old embryonated chicken eggs, and a stock of the recombinant virus was prepared. the infectious titres of the stocked recombinant viruses and the supernatants of recombinant-infected hmv-ii cells were determined according to the procedure reported previously. 19 radioimmunoprecipitation hmv-ii cells infected with recombinants were labeled with [ 35 s]methionine or [ 3 h]palmitic acid. cells were then disrupted and subjected to immunoprecipitation with the indicated antibodies. the immunoprecipitates obtained were then analysed by sds-page on 17ae5% gels containing 4 m urea, and processed for fluorography. 18 flotation analysis was performed according to the procedure described previously. 6 to examine whether the cm2 protein without palmitoylation is synthesized in rcm2-c65a-infected cells, hmv-ii cells infected with the recombinants were subjected to , and the lysates of the cells were immunoprecipitated with anti-cm2 serum and analysed by sds-page. as shown in figure 1 , the cm2 protein was synthesized both in the rwt-and rcm2-c65a-infected cells, but no incorporation of [ 3 h]palmitic acid into the cm2 proteins synthesized in the rcm2-c65ainfected cells was observed, indicating that cm2 in the rcm2-c65a-infected cells was not palmitoylated. the rwt or rcm2-c65a viruses were infected to hmv-ii cells at an m.o.i. of 5 and incubated at 33°c for up to 96 hours. the infectious titres (p.f.u. ⁄ ml) of rwt were approximately 5-to 10-fold higher than those of rcm2-c65a at 24-96 hours p.i. (data not shown), indicating that rwt grew more efficiently than did rcm2-c65a. thus palmitoylation of cm2 appears to have some effect on the generation of infectious virions in cultured cells. to investigate the reason(s) for the difference in growth kinetics between the two recombinants, we analysed viral proteins synthesized in the infected hmv-ii cells. pulsechase experiments of hmv-ii cells revealed no significant differences in the synthesis and maturation of the hef, np, m1, and cm2 proteins between the rwt-and rcm2-c65a-infected cells (data not shown). the infected cells pulse-labeled and chased were respectively immunoprecipitated with anti-cm2 serum in the presence of 50 mm iodoacetamide and analysed by sds-page in non-reducing condition. in both populations of infected cells, several bands corresponding to cm2a-monomer, -dimer, and -tetramer, as well as cm2b-dimer and -tetramer were detected (data not shown). these results demonstrate an absence of any significant differences between palmitoylation-deficient cm2 and authentic cm2 in terms of conformational maturation and transport in infected cells. membrane flotation analysis revealed that no significant differences in the kinetics of the hef, m1, and cm2 proteins were observed between rwt-and rcm2-c65ainfected cells (data not shown). in contrast, a slight difference in np kinetics was observed. the pulse-labeled np proteins were recovered in the bottom fractions in both rwt-and rcm2-c65a-infected cells. in the chase experiment, the amount of membrane-associated np proteins in fractions 3 and 4 was 30 % of the total np in the rwt-infected cells, which was higher than that (19%) in the rcm2-c65a-infected cells (data not shown). this finding may suggest that the affinity of the np protein, presumably representing the viral ribonucleoprotein (vrnp) complex, to the plasma membrane in the rcm2-c65ainfected cells is lower than that in rwt-infected cells, leading to the less efficient generation of infectious virions. since cm2 is structurally similar to m2, an influenza a virus membrane protein known to be involved in infectious virus production, [20] [21] [22] [23] [24] it is possible that the cytoplasmic tail of cm2 participates in the genome packaging through interaction with vrnp. in the present study, we showed that the affinity of np to the plasma membrane of rcm2-c65a-infected cells was slightly lower than that to the plasma membrane of rwt-infected cells. this observation may suggest that palmitoylation of cm2 is involved in the viral ribonucleoprotein (vrnp) incorporation, leading to efficient infectious virion generation. we hypothesize that palmitoylation contributes to proper regional structure formation in the cm2 cytoplasmic tail, which is competent to recruit vrnp efficiently into virions. alternatively, the cm2 cytoplasmic tail without palmitoylation is not likely to reach the proper conformation, resulting in reduced interaction with vrnp and less efficient generation of infectious progeny virions. the questions of if and how the m1 protein is involved in the interaction between the cm2 cytoplasmic tail and np remains to be clarified. we showed that cm2 synthesized in rcm2-c65ainfected cells was oligomerized and transported to the cell surface. this finding is consistent with the previous observation that palmitoylation is not required for the transport of cm2 to the cell surface in cm2-expressing cos-1 cells. 13 however, the use of reverse-genetics system has enabled us to conclude that the palmitoylation of cm2 is required for efficient infectious virus production. this suggests that the significance of the other posttranslational modifications of cm2 during virus replication can be clarified using recombinant viruses lacking the respective modification sites. sialic acid (sia) linked glycoproteins are the classical influenza receptors for influenza virus haemagglutinin to bind. the distribution of sia on cell surfaces is one of the determinants of host tropism, and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. previous research has shown the differences in apical versus basolateral infection and release of different influenza virus from polarized epithelial cells 1 and correlated this with sialic acid distribution in the human respiratory tract. moreover, mass spectrometric analysis was recently employed to elucidate the glycans present in the tissue in a higher resolution in human lung. 2 the objective of this study was to examine in detail the distribution of these sia-linked glycans at the cellular level by the use of confocal microscopy. human primary type i-like and type ii pneumocytes were isolated from human non-tumor lung tissue by tissue fragmentation, percoll density gradient centrifugation, and magnetic cell sorting. 1 the cells were seeded on coverslips and maintained in small airway growth medium. when confluence was reached, cell monolayers were fixed with 4% paraformaldehyde. we used the plant lectins, sambucus nigra glutinin (sna) from roche which binds to siaa2-6gal, maackia amurensis agglutinin (maa)i and maaii from vector lab, which bind the siaa2-3gal linked glycans using vector red as fluorescent chromogen. the cells were counter-stained with dapi or with fitc-conjugated antibody against endoplasmic recticulum (protein disulfideisomerase, pdi). the cells were imaged with multi-photon excitation laser scanning microscopy using zeiss 510 lsm. the optical cross-section pictures were reconstructed by zeiss lsm510 meta. we found that there was more binding of maai and ma-aii to type ii pneumocytes than type i-like pneumocytes and more overall binding of these lectins than binding of sna ( figure 1 ). in keeping with results from other polarized cells there was more binding to the apical than basolateral aspect, thus, explaining the previously published data on apical versus basolateral infection. 1 as sialic acid has been implicated in the targeting of proteins to the surface, the relative lack of sialic acid on the basolateral aspect can explain why there is little seasonal influenza virus dissemination to the systemic circulation in human infections. furthermore, though there was little binding of sna to the figure 1 . primary human type i-like and type ii pneumocytes stained with lecins (red), pdi (green), and dapi (blue) and imaged captured with confocal microscope. apical or basolateral aspects of the pneumocytes, the experimental findings of infection by influenza h3n2 virus that has a strict siaa2-6gal tropism 3 suggests that there are siaa2-6gal glycans present, which are not readily bound by the lectin sna. the in vitro model of primary human type i-like and type ii pneumocytes system formed a polarized epithelium that has a similar lectin distribution to human alveoli in vivo which demonstrated that it is a physiologically relevant model to study the tropism and pathogenesis of influenza a virus. human disease caused by highly pathogenic avian influenza (hpai) h5n1 virus is associated with fulminant viral pneumonia and mortality rates in excess of 60%. 1 cytokine dysregulation is thought to contribute to its pathogenesis. 2, 3 we previously found delayed onset of apoptosis in h5n1 infected human macrophages and, therefore, a longer survival time of the target cells for prolonged virus replication and cytokine and chemokine secretion, which may contribute to the pathogenesis of h5n1 disease in humans. 4 as bronchial and alveolar epithelial cells are target cells of influenza virus because of their proximal physiological location and interaction with macrophages, we further investigated if the differential onset of apoptosis could be found in influenza h5n1 and seasonal influenza h1n1 infected human respiratory epithelia. we dissected the apoptotic pathways triggered by influenza virus infection. seasonal influenza h1n1 virus (a ⁄ hk ⁄ 54 ⁄ 98), a low pathogenic avian influenza h9n2 lineage isolated from poultry (a ⁄ quail ⁄ hk ⁄ g1 ⁄ 97), and two virus isolates of hpai a subtype (a ⁄ hk ⁄ 483 ⁄ 97 and a ⁄ vn ⁄ 1203 ⁄ 04) were included. primary human bronchial and alveolar epithelial cells were infected with influenza viruses at moi of 2 and the cell monolayer was collected at 24, 30, and 48 hours post infection for tunel assay, and supernatant were collected for ldh assay. fragments of human lung tissues were cut into multiple 2-3 mm fragments within 2 hours of collection and infected with influenza a viruses at a titer of 10 6 tcid 50 ⁄ ml. these tissues fragments were infected for 1 hours and incubated for 48 hours at 37°c. one part of the infected tissue was fixed in formalin and processed for immunohistochemistry for influenza antigen, and the other part was homogenized and underwent rna extraction. apoptosis cdna superarray platform (sabioscience) was employed to conduct apoptosis pathway analysis. in bronchial epithelial cells, seasonal influenza h1n1 virus induced a high percentage of apoptotic cells by tunel assay at 24, 30, and 48 hours post infection with a peak of (figure 1) . a similar observation of delayed onset of apoptosis was found in influenza h5n1 and h9n2 infected alveolar epithelial cells. besides, cdna array data of ex vivo infected human lung showed that both influenza h5n1 and h1n1 virus induced trail expression compared with mock-infected tissue (approximately 15 folds) at 48 hours post infection, but influenza h3n2 virus infected lung induced significantly more trail (27 folds compared to mock infected cells), albeit with a limited viral replication ( figure 2 ). influenza h3n2 virus infected lung also elicited more tnf-alpha and fasr transcription than either h5n1 or h1n1. these observations can account for the greater apoptotic response in influenza h3n2 virus infected lung. as little impact on the expression of intrinsic pathway components was observed, it seems that the apoptotic response to influenza virus infection in lung was mainly through the extrinsic pathways. no significant changes in the expression of anti-apoptotic protein gene was found, except for a moderate induction of birc3 by influenza h3n2 virus, which may act to modulate the apoptotic response. the delayed onset of apoptosis by hpai h5n1 and low pathogenic avian influenza h9n2 virus infected respiratory epithelial cells may be a mechanism for the influenza viruses to have more prolonged replication within the human respiratory tract, and this may contribute to the pathogenesis of human disease. hemagglutination (ha) assay 10% crbc suspension was treated by 625 mu a2, 3-specific sialidase at 37°c for 15 minutes. complete elimination of a2, 3-receptor on sialidase-treated crbcs was confirmed by receptor staining and flow cytometry. ha assay of live viruses with 1% crbc or 1% sialidase-treated crbc were performed in bsl-3 facility. synthetic 3¢sln-paa-biotin(pa191), 6¢sln-paa-biotin(pa190), 6¢sln-ln-paa-biotin(pa343) was provided by the scripps research institute (tsri). as described elsewhere with some modifications, 3 generally, serial dilutions of sialyglycopolymers were coated in 96-well-flat-bottom polystryrene plates, and 32 hau live virus ⁄ well were added. alternatively, the plates were precoated with 5 lg ⁄ ml sialyglycopolymers, and then 8, 16, 32, 64, 128 hau live virus ⁄ well influenza viruses were added. rabbit antisera against a ⁄ ah ⁄ 1 ⁄ 05 diluted in pbs containing 1% bsa was added into the wells. bound antibody was detected by use of hrp-conjugated anti-rabbit igg antibody and tetramethylbenzidine substrate solution. each sample was determined in duplicates and the absorbance read at 450 nm. a total of 31 h5n1 virus strains were obtained from 2003 to 2009. the name and passage history of influenza viruses used in the study are listed in table 1 . as the same sequences of eight rna segments were detected in a ⁄ js ⁄ 1 ⁄ 07 and a ⁄ js ⁄ 2 ⁄ 07, only a ⁄ js ⁄ 2 ⁄ 07 was tested here. three amantadine-resistant variants with m2 mutation of screening of receptor-binding preference by ha assay representative results from three sets of independent experiments are shown in table 1 . complete ha with sialidasetreated crbcs, which were only with a2,6-receptors, was detected in human influenza virus (a ⁄ brisbane ⁄ 59 ⁄ 2007, h1n1) and two human h5n1 virus strains, a ⁄ gd ⁄ 1 ⁄ 06 and a ⁄ gx ⁄ 1 ⁄ 08. high binding of a2,3 oligosaccharides to h5n1 viruses was detected ( figure 1a -c). and enhanced a2, 6-binding preference was also detected in a ⁄ gd ⁄ 1 ⁄ 06 and a ⁄ gx ⁄ 1 ⁄ 08. the a2, 6-binding was dose dependent for sialyglycopolymers and virus titer. notably, as compared with a ⁄ gd ⁄ 1 ⁄ 06 of both short-and long-a2, 6 recognition, a ⁄ gx ⁄ 1 ⁄ 08 prefers to bind to long-a2, six oligosaccharides at low viral titer ( figure 1b,c) . however, both of them showed strong affinity to short-and long-a2, 6 oligosaccharides at high viral loads ( figure 1d ). sialoside-, galactoside-, mannoside-and sulfo-os-binding are the four types of carbohydrate-binding properties of influenza virus. 12 binding of influenza virus to the a2, 3-or a2, 6-linked sialylated glycans on cell surface is important for host range restriction, and the preference to a2, 3 of h5n1 virus limited its efficient infection in human. here, dual receptor-binding preferences were detected in a ⁄ gd ⁄ 1 ⁄ 06 and a ⁄ gx ⁄ 1 ⁄ 08, which are of clade 2ae3ae4. although there is no direct evidence supporting the occurrence of human-to-human transmission in these infection events or the association between viral virulence and receptor-binding switching, viral systemic disseminations are found in the both fatal cases (data not shown). furthermore, with the introduction of clade 2ae3ae4 into the adjacent countries of china, 4 the finding of h5n1 virus with 2-6binding in human should be of concern. though h5n1 virus with human-type receptor-binding was isolated from one patient treated by oseltamivir and those viruses were with ha and ⁄ or na substitutions, 13 whether the substitutions responsible for receptor specificity switching is pre-existed or selected in human host remains unknown. our finding that three mutant viruses bearing m2 mutations of a30s, a30t, and s31n cloned from one isolate a ⁄ hb ⁄ 1 ⁄ 06 suggested it is likely that the resistant viruses emerged in the host environment. no variation was found in their ha and na sequence, and all of them show high affinity to a2-3-binding. our data suggest that the binding-specificity was not affected by the mutations on viral envelope protein m2. with the adaptation from wild aquatic birds to domestic poultry or even in human host environment, influenza virus may possess broader carbohydrate-binding spectrum or topology conformation. 11, 14 we demonstrated differential a2, 6-binding property of two human h5n1 viruses, a ⁄ gd ⁄ 1 ⁄ 06 and a ⁄ gx ⁄ 1 ⁄ 08. though minor effect of short-a2, 6-binding was detected in viruses a ⁄ gx ⁄ 1 ⁄ 08 at low virus titer, both were of high affinity to long-a2, 6 glycans, even at the low titer which are rich on apical side of human upper respiratory epithelia. 11 notably, no evident binding preference switching was detected in the viruses isolated from the sporadic human infection cases at the early of 2009 in china (table 1) . however, higher affinity to the long-a2, 6 glycans was observed in bj ⁄ 1 ⁄ 09, gz ⁄ 1 ⁄ 09, and xj ⁄ 1 ⁄ 09 (data not shown). the discrepancy from the findings obtained by sialidase-treated crbc maybe associated with a limited abundance of n-linked a2-6 with long branches on crbc, as demonstrated in a recent study. 11 therefore, glycan dose-dependent binding assay is valuable and should be applied in flu surveillance. the underlying cause of the tendency is unknown, and further research on receptor-binding specificity of h5n1 viruses is required. influenza a viruses of migrating wild aquatic birds in north america towards improved influenza a virus surveillance in migrating birds european union council directive 92 ⁄ 40 ⁄ eec the neighbor-joining method: a new method for reconstructing phylogenetic trees confidence limits on phylogenies: an approach using the bootstrap prospects for inferring very large phylogenies by using the neighbor-joining method mega4: molecular evolutionary genetics analysis (mega) software version 4.0 the influenza virus resource at the national center for biotechnology information characterization of low-pathogenic h5 subtype influenza viruses from eurasia: implications for the origin of highly pathogenic h5n1 viruses h5n1 virus outbreak in migratory waterfowl a ⁄ h13 and a ⁄ h16 influenza viruses: different lines of one precursor evolution and ecology of influenza a viruses evolutionary processes in influenza viruses: divergence, rapid evolution, and stasis antigenic and genetic conservation of h3 influenza virus in wild ducks biologic characterization of chicken-derived h6n2 low pathogenic avian influenza viruses in chickens and ducks genetic and pathogenic characterization of h6n1 avian influenza viruses isolated in taiwan between 1972 and experimental selection of virus derivatives with variations in virulence from a single low-pathogenicity h6n1 avian influenza virus field isolate evolution and ecology of influenza a viruses is china an influenza epicenter genesis of a highly pathogenic and potentially pandemic h5n1 influenza virus in eastern asia evolution and molecular epidemiology of h9n2 influenza a viruses from quail in southern china establishment of influenza a virus (h6n1) in minor poultry species in southern china h5n1 influenza viruses: outbreaks and biological properties seroprevalance and identification of influenza a virus infection from migratory wild waterfowl in china avian flu: h5n1 virus outbreak in migratory waterfowl migration of waterfowl in the east asian flyway and spatial relationship to hpai h5n1 outbreaks avian influenza monitoring in migrating birds in taiwan during establishment of an h6n2 influenza virus lineage in domestic ducks in southern china evolution and molecular epidemiology of h9n2 influenza a viruses from quail in southern china the genesis and evolution of h9n2 influenza viruses in poultry from southern china human infection with influenza h9n2 human infection with an avian h9n2 influenza a virus in hong kong in 2003 antigenic and genetic characterization of h9n2 swine influenza in china cocirculation of avian h9n2 and contemporary ''human'' h3n2 influenza a viruses in pigs in southeastern china: potential for genetic reassortment? h9n2 influenza a viruses from poultry in asia have human virus-like receptor specificity characterization of h9 subtype influenza viruses from the ducks of southern china: a candidate for the next influenza pandemic in humans? bioedit: a user-friendly biological sequence alignment editor and analysis program for window 95 ⁄ 98 ⁄ nt genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion phylogenetic analysis using parsimony (and other methods) 4.0 beta a novel genotype h9n2 influenza virus possessing human h5n1 internal genomes has been circulating in poultry in eastern china since 1998 characterization of h9n2 influenza viruses isolated from vaccinated flocks in an integrated broiler chicken operation in eastern china during a 5 year period characterization of avian h9n2 influenza viruses from united arab emirates phylogenetic analysis of influenza a viruses of h9 haemagglutinin subtype h9n2 subtype influenza a viruses in poultry in pakistan are closely related to the h9n2 viruses responsible for human infection in hong kong diversified reassortants h9n2 avian influenza viruses in chicken flocks in northern and eastern china genotypic evolution and antigenic drift of h9n2 influenza viruses in china from the nucleoprotein as a possible major factor in determining host specificity of influenza h3n2 viruses pigs as the ''mixing vessel'' for the creation of new pandemic influenza a viruses origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic pandemic (h1n1) 2009 outbreak on pig farm reassortment of pandemic h1n1 ⁄ 2009 influenza a virus in swine from where did the 2009 'swine-origin' influenza a virus (h1n1) emerge? substitution of lysine at 627 position in pb2 protein does not change virulence of the 2009 pandemic h1n1 virus in mice evolution and ecology of influenza a viruses the origins of pandemic influenza -lessons from the 1918 virus characterization of the 1918 influenza virus polymerase genes origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic emergence of a novel swine-origin influenza a (h1n1) virus in humans antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans genetic reassortment of avian, swine, and human influenza a viruses in american pigs detection of two antigenic subpopulations of a(h1n1) influenza viruses from pigs: antigenic drift or interspecies transmission genetic relatedness of hemagglutinins of the h1 subtype of influenza a viruses isolated from swine and birds cases of swine influenza in humans: a review of the literature triple-reassortant swine influenza a (h1) in humans in the united states reassortment of pandemic h1n1 ⁄ 2009 influenza a virus in swine ferrets as a transmission model for influenza: sequence changes in ha1 of type a (h3n2) virus the ferret: an animal model to study influenza virus human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals medical management of influenza infection incidence of adamantane resistance among influenza a (h3n2) viruses isolated worldwide from 1994 to 2005: a cause for concern surveillance of resistance to adamantanes among influenza a(h3n2) and a(h1n1) viruses isolated worldwide peramivir emergency use authorization peramivir and its use in h1n1 influenza cs-8958, a prodrug of the new neuraminidase inhibitor r-125489, shows long-acting antiinfluenza virus activity detection of influenza viruses resistant to neuraminidase inhibitors in global surveillance during the first 3 years of their use surveillance of influenza isolates for susceptibility to neuraminidase inhibitors during the 2000-2002 influenza seasons surveillance for neuraminidase inhibitor resistance among human influenza a and b viruses circulating worldwide from oseltamivir-resistant influenza viruses a (h1n1), norway, 2007-08 influenza activity -united states and worldwide, 2007-08 season emergence of resistance to oseltamivir among influenza a(h1n1) viruses in europe oseltamivir-resistant influenza virus a (h1n1), europe, 2007-08 season widespread oseltamivir resistance in influenza a viruses (h1n1), south africa and composition of the 2009-10 influenza vaccine emergence of h274y oseltamivir-resistant a(h1n1) influenza viruses in japan during the 2008-2009 season pyrosequencing as a tool to detect molecular markers of resistance to neuraminidase inhibitors in seasonal influenza a viruses neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir host cell selection of influenza neuraminidase variants: implications for drug resistance monitoring in a(h1n1) viruses neuraminidase receptor binding variants of human influenza a(h3n2) viruses due to substitution of aspartic acid 151 in the catalytic site -role in virus attachment? neuraminidase inhibitor susceptibility testing in human influenza viruses: a laboratory surveillance perspective update: drug susceptibility of swine-origin influenza a (h1n1) viruses comprehensive assessment of 2009 pandemic influenza a (h1n1) virus drug susceptibility in vitro detection of molecular markers of drug resistance in 2009 pandemic influenza a (h1n1) viruses by pyrosequencing pandemic (h1n1) 2009 and oseltamivir resistance in hematology/oncology patients fluview: a weekly influenza surveillance report prepared by the influenza division development of a sensitive chemiluminescent neuraminidase assay for the determination of influenza virus susceptibility to zanamivir evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor susceptibility network surveillance for neuraminidase inhibitor resistance among human influenza a and b viruses circulating worldwide from different neuraminidase inhibitor susceptibilities of human h1n1, h1n2, and h3n2 influenza viruses isolated in germany from oseltamivir-and amantadine-resistant influenza viruses a (h1n1) infections with oseltamivirresistant influenza a (h1n1) virus in the united states isolation of oseltamivirresistant influenza a ⁄ h1n1 virus of different origins in yokohama city, japan, during the 2007-2008 influenza season a two-year survey of the oseltamivir-resistant influenza a(h1n1) virus in yamagata, japan and clinical effectiveness of oseltamivir and zanamivir oseltamivir-resistant influenza a pandemic (h1n1) 2009 virus detection of an oseltamivir-resistant pandemic influenza a ⁄ h1n1 virus in hong kong a novel small-molecule inhibitor of the avian influenza h5n1 virus determined through computational screening against the neuraminidase aurintricarboxylic acid is a potent inhibitor of influenza a and b virus neuraminidases oseltamivir-resistant influenza a viruses are transmitted efficiently among guinea pigs by direct contact but not by aerosol oseltamivir is adequately absorbed following nasogastric administration to adult patients with severe h5n1 influenza prevention and control of influenza influenza-associated hospitalizations in the united states mortality associated with influenza and respiratory syncytial virus in the united states severe respiratory disease concurrent with the circulation of h1n1 influenza staphlococcus aureus pneumonia studies on influenza in the pandemic of 1957-1958. ii. pulmonary complications of influenza bacterial pneumonia during the hong kong influenza epidemic of 1968-1969 streptococcus pneumonia diagnostic value of microscopic examination of gram-stained sputum and sputum cultures in patients with bacteremic pneumococcal pneumonia pneumonitis-associated hyperprocalcitoninemia clinical review 167: procalcitonin and the calcitonin gene family of peptides in inflammation, infection, and sepsis: a journey from calcitonin back to its precursors procalcitonin in sepsis and systemic inflammation: a harmful biomarker and a therapeutic target procalcitonin in children with suspected novel influenza a (h1n1) infection in vitro and in vivo calcitonin i gene expression in parenchymal cells: a novel product of human adipose tissue serum procalcitonin and c-reactive protein levels as markers of bacterial infection: a systematic review and meta-analysis procalcitonin as a diagnostic test for sepsis in critically ill adults and after surgery or trauma: a systematic review and meta-analysis respiratory syncytial virus infection in elderly and high-risk adults procalcitonin assay in systemic inflammation, infection, and sepsis: clinical utility and limitations simultaneous detection of chlamydophila pneumoniae and mycoplasma pneumoniae by use of molecular beacons in a duplex real-time pcr 10 years influenza therapy with antivirals (zanamivir and oseltamivir), 2nd world summit of antivirals the art of viewing and influenza -its importance for diagnosis, 3rd european influenza conference 9 emergence of a novel swine origin influenza a (h1n1) virus in humans origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic molecular detection of a novel human influenza (h1n1) of pandemic potential by conventional and real-time quantitative rt-pcr assays pandemic influenza a(h1n1)v: human to pig transmission in norway? an investigation into human pandemic influenza virus (h1n1) 2009 on an alberta swine farm pandemic (h1n1) 2009 infection in swine herds implications of the emergence of a novel h1 influenza virus emergence of a novel swine-origin influenza a virus (s-oiv) h1n1 virus in humans rapid detection of reassortment of pandemic h1n1 ⁄ 2009 influenza virus rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) universal primer set for the fulllength amplification of all influenza a viruses cocirculation of avian h9n2 and contemporary ''human'' h3n2 influenza a viruses in pigs in southeastern china: potential for genetic reassortment? hog in the limelight: swine flu's got new genes on reassortment of pandemic h1n1 ⁄ 2009 influenza a virus in swine pediatric hospitalizations associated with 2009 pandemic influenza a (h1n1) in argentina factors associated with death or hospitalization due to pandemic 2009 influenza a(h1n1) infection in california mortality from pandemic a ⁄ h1n1 2009 influenza in england: public health surveillance study antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans characterization of a newly emerged genetic cluster of h1n1 and h1n2 swine influenza virus in the united states experimental inoculation of pigs with pandemic h1n1 2009 virus and hi cross-reactivity with contemporary swine influenza virus antisera triple-reassortant swine influenza a (h1) in humans in the united states identification of human h1n2 and human-swine reassortant h1n2 and h1n1 influenza a viruses among pigs in ontario genetic and pathobiologic characterization of pandemic h1n1 2009 influenza viruses from a naturally infected swine herd archive number pandemic (h1n1) 2009 outbreak on pig farm reassortment of pandemic h1n1 ⁄ 2009 influenza a virus in swine a simple method for estimating 50 percent endpoints occurrence of haemagglutinin mutation d222g in pandemic influenza a(h1n1) infected patients in the west of scotland rrt-pcr) protocol for detection and characterization of swine influenza (version diagnostic hybrids, inc. readycells respiratory training r-mixô&d3ô dfa respiratory virus screening & id kit handbook centers for disease and control and prevention. influenza laboratory course comparison study of a real-time reverse transcription polymerase chain reaction assay with an enzyme immunoassay and shell vial culture for influenza a and b virus detection in adult patients comparison of a commercial qualitative real-time rt-pcr kit with direct immunofluorescence assay (dfa) and cell culture for detection of influenza a and b in children prospective comparison of r-mix shell vial system with direct antigen tests and conventional cell culture for respiratory virus detection optimized detection of respiratory viruses in nasopharyngeal secretions evaluation of r-mix freshcells in shell vials for detection of respiratory viruses time lines of infection and disease in human influenza: a review of volunteer challenge studies comparative epidemiology of pandemic and seasonal influenza a in households viral shedding and clinical illness in naturally acquired influenza virus infections population modeling of influenza a/h1n1 virus kinetics and symptom dynamics sensitivity of influenza rapid diagnostic tests to h5n1 and 2009 pandemic h1n1 viruses clinical evaluation of an immunochromatography test kit, capilia flua,b, for rapid diagnosis of influenza time to positive reaction of influenza rapid diagnosis kit and its clinical significance the speed and reliability of the antigen detection kit capilia flu a, b when used in a clinical setting options for the control of influenza vi role of rapid immunochromatographic antigen testing in diagnosis of influenza a virus 2009 h1n1 infection poor clinical sensitivity of rapid antigen test for influenza a pandemic (h1n1) 2009 virus evaluation of new rapid antigen test for the detection of pandemic influenza a ⁄ h1n1 2009 virus application of real-time cell electronic sensing (rt-ces) technology to cell-based assays world health organization. pandemic (h1n1) role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide systembased multiplex pcr assay world health organization. influenza world health organization. cdc protocol of realtime rtpcr for influenza a (h1n1) development of a reverse transcription-loop-mediated isothermal amplification assay for detection of pandemic (h1n1) 2009 virus as a novel molecular method for diagnosis of pandemic influenza in resource-limited settings one-step real-time rt-pcr for pandemic influenza a virus (h1n1) 2009 matrix gene detection in swine samples detection of pandemic influenza a ⁄ h1n1 ⁄ 2009 virus by real-time reverse transcription polymerase chain reaction detection of influenza a(h1n1)v virus by real-time rt-pcr development of a real-time rt-pcr for the detection of swine-lineage influenza a (h1n1) virus infections evaluation of multiple test methods for the detection of the novel 2009 influenza a (h1n1) during the new york city outbreak design and clinical application of a molecular method for detection and typing of the influenza a ⁄ h1n1pdm virus confirmation of the first hong kong case of human infection by novel swine origin influenza a (h1n1) virus diagnosed using ultrarapid, real-time reverse transcriptase pcr evaluation of four real-time pcr assays for detection of influenza a(h1n1)v viruses diagnostic assay recommended by the world health organization for swine origin influenza a (h1n1) virus cross-reacts with h5n1 influenza virus increasing immunization coverage the cost-effectiveness of influenza vaccination for people aged 50 to 64 years: an international model national seasonal influenza vaccination survey in europe seasonal influenza and vaccination coverage influenza vaccination coverage rates in five european countries during season 2006 ⁄ 07 and trends over six consecutive seasons influenza in the elderly -a mini-review cost of vaccine administration among pediatric practices cost of universal influenza vaccination of children in pediatric practices surveillance report: respiratory infections strategies for mitigating an influenza pandemic methods for monitoring influenza surveillance data approaches to syndromic surveillance when data consist of small regional counts rrt-pcr) protocol for detection and characterization of swine influenza (version influenza virus strains circulating in mongolia in 2003-2007 swine influenza who (2009) influenza a(h1n1) world now at the start of 2009 influenza pandemic general statement following the ninth meeting of the emergency committee who (2010) influenza updates us dept of defense global emerging infections surveillance è me recensement general de la population et l'habitat (rgph 2009), ministè re de l'economie des finances et de la planification ministry of health djibouti a survey of human cases of h5n1 avian influenza report by who before international health regulation (ihr) electronic public health surveillance in developing settings statistical analyses in disease surveillance systems potential for a global dynamic of influenza a (h1n1) infectious disease informatics: syndromic surveillance for public health and biodefense visualization techniques and graphical user interfaces in syndromic surveillance systems statistical inference for infectious diseases. risk-specific household and community transmission parameters evaluation of seasonal influenza vaccination effectiveness based on antibody efficacy among the institutionalized elderly in japan australian influenza surveillance summary report. canberra: australian government department of health and ageing australia's winter with the 2009 pandemic influenza a (h1n1) virus seroprevalence of 2009 pandemic influenza a(h1n1) virus in australian blood donors pandemic vaccination survey: summary results. cat. no. phe 128 concepts and procedures for laboratory-based influenza surveillance. who collaborating centres for reference and research on influenza influenza a(h1n1) seroconversion rates and risk factors among distinct adult cohorts in singapore ecdc. forward look risk assessment: likely scenarios for influenza in 2010 and the 2010 ⁄ 2011 influenza season in europe and the consequent work priorities. stockholm: european centre for disease prevention and control epidemiology of influenza -summary of influenza workshop iv sampling of populations: methods and applications comparison of influenza serological techniques by international collaborative study eliminating bias in the estimation of the geometric mean of hi titres reply to ''nauta jjp, eliminating bias in the estimation of the geometric mean of hi titers novel influenza a virus infections. 2010 case definition institut national de la statistique et des é tudes é conomiques. taille des mé nages dans l'union europé enne estimation de la population au 1er janvier par ré gion, dé partement, sexe et â ge sero-immunity and serologic response to pandemic influenza a (h1n1) 2009 virus in hong kong pandemic influenza a(h1n1) virus in scotland: geographically variable immunity in spring 2010, following the winter outbreak high prevalence of antibodies to the 2009 pandemic influenza a(h1n1) virus in the norwegian population following a major epidemic and a large vaccination campaign in autumn incidence of 2009 pandemic influenza a h1n1 infection in england: a cross-sectional serological study prevalence of seroprotection against the pandemic (h1n1) virus after the 2009 pandemic antibodies in residents of new south wales, australia, after the first pandemic wave in the 2009 southern hemisphere winter older age and a reduced likelihood of 2009 h1n1 virus infection pre-existing antibody response against 2009 pandemic influenza h1n1 viruses in taiwanese population cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus seasonal influenza vaccine and increased risk of pandemic a ⁄ h1n1-related illness: first detection of the association in british columbia association between the 2008-09 seasonal influenza vaccine and pandemic h1n1 illness during spring-summer 2009: four observational studies from canada does seasonal influenza vaccination increase the risk of illness with the 2009 a ⁄ h1n1 pandemic virus seasonal h1n1 2007 influenza virus infection is associated with elevated pre-exposure antibody titers to the serum cross-reactive antibody response to a novel influenza a (h1n1) virus after vaccination with seasonal influenza vaccine world now at the start of 2009 influenza pandemic rki working group pandemic. the first wave of pandemic influenza (h1n1) 2009 in germany: from initiation to acceleration department of health and ageing. australian influenza surveillance canadian surveillance studies help to understand h1n1 patterns influenza pandé mica a un añ o de la primera ola. ¿qué podemos decir ahora? revista chilena de infectología pandemic (h1n1) 2009 briefing note 10 options for the control of influenza vii managing and reducing uncertainty in an emerging influenza pandemic the severity of pandemic h1n1 influenza in the united states assessing the severity of the novel influenza a ⁄ h1n1 pandemic tecumseh study of illness. xiii. influenza infection and disease, 1976-1981 the infection attack rate and severity of 2009 pandemic influenza (h1n1) in hong kong school closure and mitigation of pandemic (h1n1) 2009, hong kong comparative epidemiology of pandemic and seasonal influenza a in households incidence of 2009 pandemic influenza a h1n1 infection in england: a cross-sectional serological study mortality from pandemic a ⁄ h1n1 2009 influenza in england: public health surveillance study monthly summary tables of influenza virus isolation different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures estimating in real time the efficacy of measures to control emerging communicable diseases the effective reproduction number of pandemic influenza in hong kong: prospective estimation multiple imputation: a primer comparative epidemiology of pandemic and seasonal influenza a in households strategies for containing an emerging influenza pandemic in southeast asia estimation of the serial interval of influenza school closure and mitigation of pandemic (h1n1) 2009, hong kong estimating the reproduction number of the novel influenza a virus (h1n1) in a southern hemisphere setting: preliminary estimate in new zealand early transmission characteristics of influenza a(h1n1)v in australia: victorian state methods for monitoring influenza surveillance data comparative estimation of the reproduction number for pandemic influenza from daily case notification data how to maintain surveillance for novel influenza a h1n1 when there are too many cases to count a computational framework for influenza antigenic cartography mapping the antigenic and genetic evolution of influenza virus proc of kdd cup and workshop matrix factorization techniques for recommender systems exact matrix completion via convex optimization a singular value thresholding algorithm for matrix completion matrix completion from a few entries matrices with prescribed entries and eigenvalues combining predictions for accurate recommender systems the bellkor solution to the netflix grand. prize the pragmatic theory solution to the netix grand prize feature-weighted linear stacking technical report. of health and ageing (australia). the australian health management plan for pandemic influenza influenza pandemic contingency plan, version 8.0 world health organisation. pandemic influenza preparedness and response prophylaxis or treatment? optimal use of an antiviral stockpile during an influenza pandemic the response to novel influenza a (h1n1) epidemic in taiwan and analysis of the initial 61 confirmed cases mitigation strategies for pandemic influenza in the united states strategies for mitigating an influenza pandemic containing pandemic influenza at the source efficient simulation of the spatial transmission dynamics of influenza world health organization. mathematical modelling of the pandemic h1n1 a mathematical model for the global spread of influenza pandemic potential of a strain of influenza a (h1n1): early findings effectiveness of the 2003-2004 influenza vaccine among children 6 months to 8 years of age, with 1 vs 2 doses cdc estimates of 2009 h1n1 influenza cases, hospitalizations and deaths in the united states accessed 1 entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation pandemic of influenza 1918-1919 america's forgotten pandemic. the influenza of 1918 influenza: a survey of the last 50 years in the light of modern work on the virus of epidemic influenza understanding mortality in the 1918-19 influenza pandemic in england and wales genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus the age pattern of mortality in the 1918-1919 influenza pandemic: an attempted explanation based on data for england and wales protective immunity and susceptibility to infectious diseases: lessons from the 1918 influenza pandemic deaths from bacterial pneumonia during 1918-1919 influenza pandemic understanding influenza transmission, immunity and pandemic threats a biological model for influenza transmission: pandemic planning implications of asymptomatic infection and immunity prior immunity helps to explain wave-like behaviour of pandemic influenza in 1918-1919 influenza: accounting for prior immunity 1918-1919 influenza pandemic mortality in england and wales. colchester, essex: uk data archive [distributor] cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus australia's winter with the 2009 pandemic influenza a (h1n1) virus model predictions and evaluation of possible control strategies for the 2009 a ⁄ h1n1v influenza pandemic in italy the role of population heterogeneity and human mobility in the spread of pandemic influenza age-prioritized use of antivirals during an influenza pandemic mitigation measures for pandemic influenza in italy: an individual based model considering different scenarios strategies for containing an emerging influenza pandemic in southeast asia modeling targeted layered containment of an influenza pandemic in the united states reducing the impact of the next influenza pandemic using household-based public health interventions mitigation strategies for pandemic influenza in the united states effective, robust design of community mitigation for pandemic influenza: a sytematic examination of proposed us guidance containing pandemic influenza at the source strategies for mitigating an influenza pandemic new influenza a (h1n1) virus: global epidemiological situation velluci l. enhanced epidemiological surveillance of influenza a(h1n1)v in italy changes in reporting requirements for pandemic (h1n1) 2009 virus infection use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza guidance document: pandemic influenza preparedness and response situation updates -pandemic (h1n1) pandemic potential of a strain of influenza a (h1n1): early findings estimating the impact of school closure on influenza transmission from sentinel data preexisting immunity to pandemic (h1n1) understanding the dynamics of seasonal influenza in italy: an analysis of disease incidence and population susceptibility what is the optimal therapy for patients with h5n1 influenza meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? treatment with convalescent plasma for influenza a (h5n1) infection prophylactic and therapeutic efficacy of human monoclonal antibodies against h5n1 influenza logistical feasibility and potential benefits of a population-wide passive-immunotherapy program during an influenza pandemic strategies for pandemic and seasonal influenza vaccination of schoolchildren in the united states mitigation strategies for pandemic influenza in the united states pandemic potential of a strain of influenza a (h1n1): early findings transmission potential of the new influenza a(h1n1) virus and its age-specificity in japan the transmissibility and control of pandemic influenza a (h1n1) virus effect of clinical and virological parameters on the level of neutralizing antibody against pandemic influenza a virus h1n1 world health organization. 10 facts on blood transfusion hyperinduction of cyclooxygenase-2-mediated proinflammatory cascade: a mechanism for the pathogenesis of avian influenza h5n1 infection the role of influenza virus gene constellation and viral morphology on cytokine induction, pathogenesis, and viral virulence induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? analysis of relative gene expression data using real-time quantitative pcr and the 2 acetylsalicylic acid (asa) blocks influenza virus propagation via its nf-kappab-inhibiting activity cyclooxygenase 2 plays a pivotal role in the resolution of acute lung injury role of 15-deoxy delta(12,14) prostaglandin j2 and nrf2 pathways in protection against acute lung injury resolution of inflammation in murine autoimmune arthritis is disrupted by cyclooxygenase-2 inhibition and restored by prostaglandin e2-mediated lipoxin a4 production inhibition of cox-2 aggravates neutrophil migration and pneumocyte apoptosis in surfactantdepleted rat lungs re-emergence of fatal human influenza a subtype h5n1 disease apoptosis and pathogenesis of avian influenza a (h5n1) virus in humans h5n1 infection of the respiratory tract and beyond: a molecular pathology study molecular biology, and pathogenesis of avian influenza a (h5n1) infection in humans passive immunity in prevention and treatment of infectious diseases prophylactic and therapeutic efficacy of human monoclonal antibodies against h5n1 influenza neutralizing human monoclonal antibody against h5n1 influenza ha selected from a fab-phage display library passive immunoprophylaxis and therapy with humanized monoclonal antibody specific for influenza a h5 hemagglutinin in mice generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza a h5n1 viruses igy: clues to the origins of modern antibodies chicken antibodies: a clinical chemistry perspective oral immunotherapy with yolk antibodies to prevent infections in humans and animals good effect of igy against pseudomonas aeruginosa infections in cystic fibrosis patients avian influenza vaccines: a practical review in relation to their application in the field with a focus on the asian experience vaccination: a tool for the control of avian influenza immunoglobulins from egg yolk: isolation and purification a simple method of estimating fifty per cent endpoints characterization of the reconstructed 1918 spanish influenza pandemic virus detection of antibody to avian influenza a (h5n1) virus in human serum by using a combination of serologic assays avian influenza (h5n1) viruses isolated from humans in asia in 2004 exhibit increased virulence in mammals heterosubtypic immunity to influenza a virus infection requires a properly diversified antibody repertoire early control of h5n1 influenza virus replication by the type i interferon response in mice successful treatment of avian influenza with convalescent plasma chicken antibodies: taking advantage of evolution -a review oral administration of specific yolk antibodies (igy) may prevent pseudomonas aeruginosa infections in patients with cystic fibrosis: a phase i feasibility study avian flu: isolation of drug-resistant h5n1 virus reduced sensitivity of influenza a (h5n1) to oseltamivir enzymatic properties of the neuraminidase of seasonal h1n1 influenza viruses provide insights for the emergence of natural resistance to oseltamivir surveillance for neuraminidase inhibitor resistance among human influenza a and b viruses circulating worldwide from a polyphenol rich plant extract, cystus052, exerts anti influenza virus activity in cell culture without toxic side effects or the tendency to induce viral resistance cystus052, a polyphenol-rich plant extract, exerts anti-influenza virus activity in mice cistus incanus (cystus052) for treating patients with infection of the upper respiratory tract. a prospective, randomised, placebocontrolled clinical study in vitro and in vivo assay systems for study of influenza virus inhibitors new low-viscosity overlay medium for viral plaque assays antibacterial and antifungal activities of cistus incanus and c. monspeliensis leaf extracts flavan-3-ols and proanthocyanidins from cistus incanus inhibition of the infectivity of influenza virus by tea polyphenols attenuation of inducible respiratory immune responses by oseltamivir treatment in mice infected with influenza a virus anti-inflammatory activity of macrolides: a new therapeutic potential? macrolide antibiotics as immunomodulatory medications: proposed mechanisms of action proteases essential for human influenza virus entry into cells and their inhibitors as potential therapeutic agents modified pulmonary surfactant is a potent adjuvant that stimulates the mucosal iga production in response to the influenza virus antigen boost of mucosal sectretory immunoglobulin a response by clarithromycin in paediatric influenza intranasal interleukin-12 is a powerful adjuvant for protective mucosal immunity immunological effects of the orally administered neuraminidase inhibitor oseltamivir in influenza virus-infected and uninfected mice clarithromycin in the treatment of rsv bronchitis: a double-blind, randomized placebo-controlled trial bird flu''), inflammation and anti-inflammatory ⁄ analgesic drugs clinical effects of low-dose long-term erythromycin chemotherapy on diffuse panbronchiolitis proteolytic activation of influenza viruses by serine proteases tmprss2 and hat from human airway epithelium proteolytic activation of the 1918 influenza virus hemagglutinin protease-activated receptors: new concepts in regulation of g protein-coupled receptor signaling and trafficking protective role for protease-activated receptor-2 against influenza virus pathogenesis via an ifn-gamma-dependent pathway agonists of proteinaseactivated receptor-2 enhance ifn-gamma-inducible effects on human monocytes: role in influenza a infection plasmacytoid dendritic cells migrate in afferent skin lymph annexin ii incorporated into influenza virus particles supports virus replication by converting plasminogen into plasmin plasminogen promotes influenza a virus replication through an annexin ii-dependent pathway in absence of neuraminidase costimulatory receptors in a teleost fish: typical cd28, elusive ctla4 trypsin increases pseudorabies virus production through activation of the erk signalling pathway characterization of hla-g1, -g2, -g3, and -g4 isoforms transfected in a human melanoma cell line immunosuppressive hla-g molecule is upregulated in alveolar epithelial cells after influenza a virus infection influenza virus propagation is impaired by inhibition of the raf ⁄ mek ⁄ erk signalling cascade influenza viruses and the nf-kappab signaling pathway -towards a novel concept of antiviral therapy the immunotolerance role of hla-g hla-g1 co-expression boosts the hla class i-mediated nk lysis inhibition hla-g inhibits the allogeneic proliferative response hla-g-mediated inhibition of antigen-specific cytotoxic t lymphocytes the world health organization. h1n1 influenza vaccine task force pandemic influenza: a potential role for statins in treatment and prophylaxis commentary: from scarcity to abundance: pandemic vaccines and other agents for ''have not'' countries new technologies for meeting the global demand for pandemic vaccines confronting the next influenza pandemic with inexpensive generic agents: can it be done? was bacterial pneumonia the predominant cause of death in the 1918-1919 influenza pandemic? meeting the challenge of influenza pandemic preparedness in developing countries confronting the next influenza pandemic with antiinflammatory and immunomodulatory agents: why they are needed and how they might work molecular and cellular aspects of sepsis-induced immunosuppression immune homeostasis in the respiratory tract and its impact on heterologous infection identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury effector t cells control lung inflammation during acute influenza virus infection by producing il-10 a detrimental effect of interleukin-10 on protective pulmonary humoral immunity during primary influenza a virus infection inhibition of influenza a virus replication by resveratrol increased survival after gemfibrozil treatment of severe mouse influenza tnf ⁄ inos-producing dendritic cells are the necessary evil of lethal influenza virus infection peroxisome proliferator-activated receptor and amp-activated protein kinase agonists protect against lethal influenza virus challenge in mice acetylsalicylic acid (asa) blocks influenza virus propagation via its nf-kappab-inhibiting activity new low-viscosity overlay medium for viral plaque assays cyproquant-pcr: a real time rt-pcr technique for profiling human cytokines, based on external rna standards, readily automatable for clinical use role of hypercytokinemia in nf-kap-pab p50 deficient mice after h5n1 influenza a virus infection developing new antiviral agents for influenza treatment: what does the future hold? influenza viruses and the nf-kappab signaling pathway -towards a novel concept of antiviral therapy rna viruses and the mitogenic raf ⁄ mek ⁄ erk signal transduction cascade world health organization writing group. non-pharmaceutical interventions for pandemic influenza, international measures effects of school closures, 2008 winter influenza season, hong kong entry screening to delay local transmission of 2009 pandemic influenza a (h1n1) simple physical interventions such as hand washing and wearing masks can reduce spread of respiratory viruses hand hygiene and virus transmission demographic and attitudinal determinants of protective behaviours during a pandemic: a review perceptions related to human avian influenza and their associations with anticipated psychological and behavioral responses at the onset of outbreak in the hong kong chinese general population perceptions about status and modes of h5n1 transmission and associations with immediate behavioral responses in the hong kong general population longitudinal assessment of community psychobehavioral responses during and after the 2003 outbreak of severe acute respiratory syndrome in hong kong public perceptions, anxiety, and behaviour change in relation to the swine flu outbreak: cross sectional telephone survey community psychological and behavioral responses through the first wave of the 2009 influenza a(h1n1) pandemic in hong kong main tables of the 2006 population census. hong kong: government of the statistical analysis with missing data the impact of community psychological responses on outbreak control for severe acute respiratory syndrome in hong kong economic impact of sars: the case of hong kong sars-related perceptions in hong kong group members: stream 1 -ilaria oie -world organisation for animal health, france; malik peiris us department of health and human services, usa; masato tashiro, national institute of infectious diseases, japan; marie-paule kieny 1 , david wood 1 . stream 4 -tawee chotpitayasunondh world influenza centre at national institute for medical research philippe veltsos 1 , cathy roth 1 , gregory härtl 1 options for the control of influenza vii who public health research agenda for influenza outbreak communication best practices for communicating with the public during an outbreak, doc who ⁄ cds ⁄ 2005.32. geneva: world health organization who outbreak communications guidelines, geneva: world health organization characterization of h9 subtype influenza viruses from the ducks of southern china: a candidate for the next influenza pandemic in humans? genetic conservation of hemagglutinin gene of h9 influenza virus in chicken population in mainland china continuing evolution of h9 influenza viruses in korean poultry evolution and molecular epidemiology of h9n2 influenza a viruses from quail in southern china characterization of the pathogenicity of members of the newly established h9n2 influenza virus lineages in asia sequence analysis of the hemagglutinin gene of h9n2 korean avian influenza viruses and assessment of the pathogenic potential of isolate ms96 molecular characterization of h9n2 influenza viruses: were they the donors of the ''internal'' genes of h5n1 viruses in hong kong? h9n2 influenza viruses possessing h5n1-like internal genomes continue to circulate in poultry in southeastern china role of infectious bronchitis live vaccine on pathogenicity of h9n2 avian influenza virus manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees) an avian influenza virus of h10 subtype that is highly pathogenic for chickens, but lacks multiple basic amino acids at the haemagglutinin cleavage site human influenza virus hemagglutinin with high sensitivity to proteolytic activation insertion of a multibasic cleavage motif into the hemagglutinin of a low-pathogenic avian influenza h6n1 virus induces a highly pathogenic phenotype acquisition of a polybasic hemagglutinin cleavage site by a low-pathogenic avian influenza virus is not sufficient for immediate transformation into a highly pathogenic strain pathology and virus distribution in chickens naturally infected with highly pathogenic avian influenza a virus (h7n7) during the 2003 outbreak in the netherlands implication of the proprotein convertases furin, pc5 and pc7 in the cleavage of surface glycoproteins of hong kong, ebola and respiratory syncytial viruses: a comparative analysis with fluorogenic peptides substrate cleavage analysis of furin and related proprotein convertases. a comparative study cloning and expression of novel mosaic serine proteases with and without a transmembrane domain from human lung host envelope glycoprotein processing proteases are indispensable for entry into human cells by seasonal and highly pathogenic avian influenza viruses world health organization ⁄ oie ⁄ fao h5n1 evolution working group. toward a unified nomenclature system for highly pathogenic avian influenza virus (h5n1) novel type ii transmembrane serine proteases, mspl and tmprss13, proteolytically activate membrane fusion activity of hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication is virulence of h5n2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? targeted infection of endothelial cells by avian influenza virus a ⁄ fpv ⁄ rostock ⁄ 34 (h7n1) in chicken embryos detection of circulating asian h5n1 viruses by a newly established monoclonal antibody a novel influenza a virus mitochondrial protein that induces cell death influenza a virus pb1-f2 protein contributes to viral pathogenesis in mice expression of the 1918 influenza a virus pb1-f2 enhances the pathogenesis of viral and secondary bacterial pneumonia the influenza a virus pb1-f2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function influenza virus pb1-f2 protein induces cell death through mitochondrial ant3 and vdac1 a single mutation in the pb1-f2 of h5n1 (hk ⁄ 97) and 1918 influenza a viruses contributes to increased virulence the effects of influenza a virus pb1-f2 protein on polymerase activity are strain specific and do not impact pathogenesis eight-plasmid system for rapid generation of influenza virus vaccines pb1-f2 proteins from h5n1 and 20 century pandemic influenza viruses cause immunopathology pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico emergence of a novel swine-origin influenza a (h1n1) virus in humans dating the emergence of pandemic influenza viruses in vitro and in vivo characterization of new swine-origin h1n1 influenza viruses emergence of a novel swine-origin influenza a virus (s-oiv) h1n1 virus in humans influenza: emergence and control systemic cytokine responses in patients with influenza-associated encephalopathy inflammatory cytokines in vascular dysfunction and vascular disease identification of trypsin i as a candidate for influenza a virus and sendai virus envelope glycoprotein processing protease in rat brain mechanisms of matrix metalloprotease-9 upregulation and tissue destruction in various organs in influenza a virus infection activation of influenza a viruses by trypsin treatment host envelope glycoprotein processing proteases are indispensable for entry into human cells by seasonal and highly pathogenic avian influenza viruses dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation influenza virus-cytokine-protease cycle in the pathogenesis of vascular hyperpermeability in severe influenza molecular pathogenesis of influenza a virus infection and virus-induced regulation of cytokine gene expression active nf-jb signaling is a prerequisite for influenza virus infection nf-jb and virus infection: who controls whom tnf-induced mitochondrial damage: a link between mitochondrial complex i activity and left ventricular dysfunction acute encephalopathy associated with influenza and other viral infections molecular structure and assembly of the tight junction identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury human influenza virus infection and apoptosis induction in human vascular endothelial cells fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia h5n1 infection of the respiratory tract and beyond: a molecular pathology study fatal avian influenza a (h5n1) in a child presenting with diarrhea followed by coma influenza virus m2 protein mediates escrt-independent membrane scission the protein-protein interaction map of helicobacter pylori development of a real-time rt-pcr for the detection of swine-lineage influenza a (h1n1) virus infections long-term storage and recovery of buccal cell dna from treated cards pandemic a(h1n1)2009 influenza virus detection by real time rt-pcr: is viral quantification useful? the effect of storage at different temperatures on the stability of hepatitis c virus rna in plasma samples impact of various handling and storage conditions on quantitative detection of hepatitis c virus rna blood storage at 4 degrees c-factors involved in dna yield and quality effects of prolonged storage of whole plasma or isolated plasma dna on the results of circulating dna quantification assays pathogenesis and transmission of swine-origin 2009 a(h1n1) influenza virus in ferrets viral tropism and the pathogenesis of influenza in the mammalian host influenza: pathogenesis and host defense cutting edge: stealth influenza virus replication precedes the initiation of adaptive immunity genetic and antiviral drug susceptibility profiles of pandemic a(h1n1)v influenza virus circulating in portugal observed association between the ha1 mutation d222g in the 2009 pandemic influenza a(h1n1) virus and severe clinical outcome world health organization. sequencing primers and protocol antiviral drug profile of seasonal influenza viruses circulating in portugal from predicting the antigenic structure of the pandemic (h1n1) 2009 influenza virus hemagglutinin genomic signature and mutation trend analysis of pandemic (h1n1) 2009 influenza a virus weekly update on oseltamivir resistance to influenza a (h1n1) 2009 viruses pandemic a(h1n1)2009 influenza virus detection by real-time rt-pcr: is viral quantification useful? virulence associated substitution d222g in hemagglutinin of 2009 pandemic influenza a(h1n1) virus affects receptor binding the first wave of pandemic influenza (h1n1) 2009 in germany: from initiation to acceleration new york city swine flu investigation team, et al. the severity of pandemic h1n1 influenza in the united states from rhinovirus delayed the circulation of the pandemic influenza a(h1n1)2009 virus in france dynamics and impact of the a(h1n1)2009 epidemic in metropolitan france clinical aspects of pandemic 2009 influenza a(h1n1) virus infection preliminary estimates of mortality and years of life lost associated with the 2009 a ⁄ h1n1 pandemic in the us and comparison with past influenza seasons oseltamivir resistance in adult oncology and hematology patients infected with pandemic (h1n1)2009 virus household transmission of the 2009 pandemic a ⁄ h1n1 influenza virus: elevated laboratoryconfirmed secondary attack nrates and evidence of assymptomatic infections time lines of infection and disease in human influenza: a review of volunteer challenge studies facemasks and hand hygiene to prevent influenza transmission in households comparative epidemiology of pandemic and seasonal influenza a in households viral shedding and clinical illness in naturally acquired influenza virus infections r: a language and environment for statistical computing the guinea pig as a transmission model for human influenza viruses in the control of epidemic and pandemic influenza 12-13 blocking interhost transmission of influenza virus by vaccination in the guinea pig model phenotypic properties resulting from directed gene segment reassortment between wild-type a ⁄ sydney ⁄ 5 ⁄ 97 influenza virus and the live attenuated vaccine strain emergence of a novel swine-origin influenza a (h1n1) virus in humans pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico cumulative number of confirmed human cases of avian influenza a ⁄ (h5n1) reported to who factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises airborne transmission of disease in hospitals noninvasive positive-pressure ventilation: an experimental model to assess air and particle dispersion airflow and droplet spreading around oxygen masks: a simulation model for infection control research coughing and aerosols. images in clinical medicine antimicrobial products registered for use against influenza a virus on hard surfaces. office of pesticide programs u.s. environmental protection agency antimicrobials division the genesis of a pandemic influenza virus influenza in the elderly -a mini review control of influenza in the long term care facility: a review of established approaches and newer options a study of the impact of influenza on the functional status of frail older people influenza in long-term care facilities: preventable, detectable, treatable mortality associated with influenza and respiratory syncytial virus in the united states causes of death australia 2007: leading causes of death australian government department of heath and ageing. pandemic (h1n1) 2009 update bulletins for pandemic (h1n1) 2009 update bulletins for 26 world health organization. pandemic (h1n1) 2009-update 112 factors associated with death or hospitalization due to pandemic 2009 influenza a (h1n1) infection in california influenza a (h1n1) 2009 antibodies in residents of new south wales, australia, after the first pandemic wave in the 2009 southern hemisphere winter qualification of the hemagglutination inhibition assay in support of pandemic influenza vaccine licensure managing outbreaks of viral respiratory infection in aged care facilities -challenges and difficulties during the first pandemic wave laboratory diagnosis of influenza virus infection antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans incidence of 2009 pandemic influenza a h1n1 infection in england: a cross-sectional serological study cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus in vitro and in vivo characterization of new swine-origin h1n1 influenza viruses cross-reacting antibodies against pandemic influenza a (h1n1) 2009 virus in elderly australians rhinovirus outbreak in a long term care facility for elderly persons associated with unusually high mortality a rhinovirus outbreak among residents of a long term care facility lessons from a respiratory illness outbreak in an aged-care facility detection and isolation of pandemic (h1n1) influenza virus infection among pediatric patients reporting diarrhea and influenza-like illness significance of seasonal influenza viruses in the stool of pediatric patients viral load in patients infected with pandemic h1n1 2009 influenza a virus haemagglutinin mutations responsible for the binding of h5n1 influenza a viruses to human-type receptors biedermann e references 1 world health organization writing group. nonpharmaceutical interventions for pandemic influenza, international measures avian influenza, including influenza a (h5n1), in humans: who interim infection control guideline for health care facilities inactivation of influenza a viruses in the environment and modes of transmission: a critical review quantitative method for evaluating virucidal activity of biocides used on hard surfaces nonpharmaceutical interventions for pandemic influenza, international measures avian influenza, including influenza a (h5n1), in humans: who interim infection control guideline for health care facilities inactivation of influenza a viruses in the environment and modes of transmission: a critical review distribution of airborne influenza virus and respiratory syncytial virus in an urgent care medical clinic characterisation of stability, cytotoxicity, and skin sensitisation and irritation for respirators with antiviral coating nonpharmaceutical interventions for pandemic influenza, international measures avian influenza, including influenza a (h5n1), in humans: who interim infection control guideline for health care facilities c600cf/0/infectioncontrol.pdf international organization for standardization. 10993: biological evaluation of medical devices, part 5: tests for cytotoxicity: in vitro methods effects of antiviral drugs on viral detection in influenza patients and on the sequential infection to their family members. serial examination by quick diagnosis (capiria) and virus culture intrafamilial transmission of influenza a and b analysis of infectious diseases data stochastic models for analysis of household transmission data: examining human-made transmission experiments factors influencing the effectiveness of oseltamivir and amantadine for the treatment of influenza. a japanese study of the multi-center study of the 2002 ⁄ 2003 influenza season clinical evaluation of an immunochromatography test kit, capilia flua, b for rapid diagnosis of influenza usefulness of a self-blown nasal discharge specimen for use with immunochromatography based influenza rapid antigen test. options for control of influenza vii 2010; 184 abstract book. options for the control of influenza vii strategies for containing an emerging influenza pandemic in southeast asia flute, a publicly available stochastic influenza epidemic simulation model the japanese experience with vaccinating schoolchildren against influenza mass vaccination of schoolchildren against influenza and its impact on the influenza-associated mortality rate among children in japan effect of vaccination of a school-age population upon the course of an a2-hong kong influenza epidemic herd immunity in adults against influenza-related illnesses with use of the trivalent-live attenuated influenza vaccine (caiv-t) in children the effect of mass influenza immunization in children on the morbidity of the unvaccinated elderly effect of influenza vaccination of children on infection rates in hutterite communities: a randomized trial efficacy of live attenuated and inactivated influenza vaccines in schoolchildren and their unvaccinated contacts in novgorod, russia effectiveness of influenza vaccination of day care children in reducing influenza-related morbidity among household contacts effectiveness of schoolbased influenza vaccination strategies for pandemic and seasonal influenza vaccination of schoolchildren in the united states strategy for distribution of influenza vaccine to high-risk groups and children estimating the impact of childhood influenza vaccination programmes in england and wales population-wide benefits of routine vaccination of children against influenza prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) protective efficacy of seasonal influenza vaccination against seasonal and pandemic influenza virus infection during 2009 in hong kong partial protection of seasonal trivalent inactivated vaccine against novel pandemic influenza a ⁄ h1n1 2009: case-control study in mexico city association between the 2008-09 seasonal influenza vaccine and pandemic h1n1 illness during spring-summer 2009: four observational studies from canada cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus effectiveness of 2008-09 trivalent influenza vaccine against 2009 pandemic influenza a (h1n1) -united states seasonal influenza vaccine and protection against pandemic (h1n1) 2009-associated illness among us military personnel interim analysis of pandemic influenza (h1n1) 2009 in australia: surveillance trends, age of infection and effectiveness of seasonal vaccination pandemic (h1n1) 2009 risk for nurses after trivalent vaccination infection and death from influenza a h1n1 virus in mexico: a retrospective analysis birth weight, infant growth, and childhood body mass index: hong kong's children of 1997 birth cohort comparative epidemiology of pandemic and seasonal influenza a in households facemasks and hand hygiene to prevent influenza transmission in households: a cluster randomized trial partial protection against challenge with the highly pathogenic h5n1 influenza virus isolated in japan in chickens infected with the h9n2 influenza virus prior h1n1 influenza infection and susceptibility of cleveland family study participants during the h2n2 pandemic of 1957: an experiment of nature transmission of pandemic h1n1 influenza virus and impact of prior exposure to seasonal strains or interferon treatment prior infection with an h1n1 swine influenza virus partially protects pigs against a low pathogenic h5n1 avian influenza virus protection against a european h1n2 swine influenza virus in pigs previously infected with h1n1 and ⁄ or h3n2 subtypes infection of mice with a human influenza a ⁄ h3n2 virus induces protective immunity against lethal infection with influenza a ⁄ h5n1 virus evidence of a cross-protective immune response to influenza a in the cotton rat model heterosubtypic immunity to influenza a virus: where do we stand? safety and immunogenicity of as03b adjuvanted split virion versus non-adjuvanted whole virion h1n1 influenza vaccine in uk children aged 6 months-12 years: open label, randomised, parallel group, multicentre study options for live attenuated influenza vaccines (laiv) in the control of epidemic and pandemic influenza 12-13 pilot studies on recombinant cold-adapted live type a and b influenza virus vaccines development of cold-adapted recombinant live, attenuated influenza vaccines in usa and ussr clinical and epidemiological evaluation of a live, cold-adapted influenza vaccine for 3-14-year-olds revised requirements for influenza vaccine (live) global pandemic influenza action plan to increase vaccine supply characteristics of human influenza a ⁄ h1n1, a ⁄ h3n2 and b viruses isolated characteristics of human influenza a ⁄ h1n1, a ⁄ h3n2 and b viruses isolated characteristics of human influenza a ⁄ h1n1, a ⁄ h3n2 and b viruses isolated characteristics of human influenza a ⁄ h1n1, a ⁄ h3n2 and b viruses isolated dynamics of antigenic and genetic changes in the hemagglutinins of influenza a ⁄ h3n2 viruses of three consecutive seasons molecular epidemiology of a ⁄ h3n2 and a ⁄ h1n1 influenza virus during a single epidemic season in the united states an mf59-adjuvanted inactivated influenza vaccine containing a ⁄ panama ⁄ 1999 (h3n2) induced broader serological protection against heterovariant influenza virus strain a ⁄ fujian ⁄ 2002 than a subunit and a split influenza vaccine response of influenza vaccines against heterovariant influenza virus strains in adults with chronic diseases cross-protection by mf59 tmadjuvanted influenza vaccine: neutralizing and hemagglutinationinhibiting antibody activity against a (h3n2) drifted influenza viruses quantifying influenza vaccine efficacy and antigenic distance detection of antibody to avian influenza a (h5n1) virus in human serum by using a combination of serologic assays world health organization global influenza programme. who manual on animal influenza diagnosis and surveillance antigenic characterization of influenza b virus with a new microneutralisation assay: comparison to haemagglutination and sequence analysis seroprotection rate, meanfold increase, seroconversion rate: which parameter adequately expresses seroresponse to influenza vaccination? the adjuvanted influenza vaccines with novel adjuvants: experience with the mf59-adjuvanted vaccine experimental inoculation of pigs with pandemic h1n1 2009 virus and hi cross-reactivity with contemporary swine influenza virus antisera current status of live attenuated influenza vaccine in the united states for seasonal and pandemic influenza failure of protection and enhanced pneumonia with a us h1n2 swine influenza virus in pigs vaccinated with an inactivated classical swine h1n1 vaccine isolation and characterization of h3n2 influenza a virus from turkeys a new generation of modified live-attenuated avian influenza viruses using a two-strategy combination as potential vaccine candidates a dna transfection system for generation of influenza a virus from eight plasmids efficacy of inactivated swine influenza virus vaccines against the 2009 a ⁄ h1n1 influenza virus in pigs mortality associated with influenza and respiratory syncytial virus on the united states realities and enigma of human viral influenza: pathogenesis, epidemiology and control immune response after adjuvant mucosal immunization of mice with inactivated influenza virus stimulation of protective and cross-protective imunity against influenza b virus after adjuvant mucosal immunization of mice protective and cross-protective mucosal immunization of mice by influenza virus type a with bacterial adjuvant synergistic action of cholera toxin b subunit (and escherichia coli heat-labile toxin b subunit) and a trace amount of cholera whole toxin as an adjuvant for nasal influenza vaccine effects of intanasal administration of chlera toxine (or escherichia coli heat-labila enterotoxin b subunits)supplemented with a trace amount of the holotoxin on the brain the adjuvants mf59 and lt-k63 enhance the mucosal and systemic immunogenicity of subunit influenza vaccine administrated intranasaly in mice generation of influenza vaccine strains by genetic reassortment a description of the process of seasonal and h5n1 influenza vaccine virus selection and development pcr restriction analysis if genome composition and stability of cold-adapted reassortants live influenza vaccines options for live attenuated influenza vaccines (laiv) in the control of epidemic and pandemic influenza 12-13 characterization of an influenza a h5n2 reassortant as a candidate for live-attenuated and inactivated vaccines against highly pathogenic h5n1 viruses with pandemic potential are there alternative avian influenza viruses for generation of stable attenuated avian-human influenza a reassortant viruses? organization of production, quality control and provision of the republic of kazakhstan with the a ⁄ h5n1 vaccine development of diagnostics and vaccines against influenza a ⁄ h5n1 and a ⁄ h1n1 is the basis for biological safety and protection of population in kazakhstan maior results of developing a technology for production of a pandemic influenza a ⁄ h5n1 vaccine» abstract book ''options for the control of influenza vii abstract book international scientific conference of students, graduates and young scientists ''lomonosov isolation of influenza viruses in cell cultures and chicken embryos and their identification development of quality and safety control methods of the inactivated vaccine based on the strain a ⁄ astanarg ⁄ 6:2 ⁄ 2009 (h5n1 control methods of medical immunobiological preparations injected to humans. guidelines, muk 4.1/4.2.588-96 (adopted by goskomsanepidnadzor rf ob 31 scientific center for expertise of medical products, moscow. russian national pharmacopeia. ist edn the guidance for pre-clinical testing of pharmaceutical substances for safety and effectiveness (regulations of good laboratory practice in the russian federation -glp). manual on experimental (pre-clinical) assay of new pharmaceutical substances. moscow: ministry of public health ⁄ department of quality, effectiveness and safety control of medicines ⁄ scientific center of expert examination and national control of medicines ⁄ pharmacological national committee manual on experimental (pre-clinical) assay of new pharmaceutical substances. moscow: medline status of the development of influenza a ⁄ h5n1 vaccines in the world and in russia approval of instruction for preclinical testing of pharmacological and medical substances in the repubilic of kazakhstan preclinical testing of new medical immunobiological substances. basic principles. moscow: ussr ministry of health order and methods of control of vaccine immunobiological safety. general methodic principles. moscow: ussr ministry of health planning and performance of clinical study of medicines federal pharmaceutical substances law. #122-fz russian federation national standard. good clinical practice. # 232-st order of the minister of health of russian federation # 266 signed 19.06.2003. rules for clinical practice in russian federation a single-dose influenza a (h5n1) vaccine safe and immunogenic in adult and elderly patients: an approach to pandemic vaccine development cross-reactive immunity to clade 2 strains of influenza virus a subtype h5n1 induced in adults and elderly patients by fluval, a prototype pandemic influenza virus vaccine derived by reverse genetics, formulated with a phosphate adjuvant, and directed to clade 1 strains develop of domestic vaccinal and diagnostic preparations for influenza a ⁄ h5n1 and a ⁄ h1n1 as a basic principle of biological safety and security for the population of kazakhstan guidelines: ''isolation of influenza viruses in cell cultures and chicken embryos and their identification''. moscow: a-print validation of control methods for chemical and physicochemical characteristics of mibp quality: organization the guidance for pre-clinical testing of pharmaceutical substances for safety and effectiveness (regulations of good laboratory practice in the russian federation -glp). manual on experimental (pre-clinical) assay of new pharmaceutical substances. moscow: ministry of public health ⁄ department of quality, effectiveness and safety control of guidance on assessment of allergenic characteristics of pharmaceutical substances on adoption of the instructions for performance of pre-clinical assays and tests of pharmaceutical substances and medicines in the republic of kazakhstan national pharmacopoeia of the republic of kazakhstan planning and performance of clinical study of medicines federal law on medicines no.122-aii dated national standard of the russian federation ''proper clinical practice'' no. 232-st guidance on clinical practice in the russian federation'' (in russian) aluminum hydroxide adjuvants activate caspase-1 and induce il-1beta and il-18 release safety and immunogenicity of a 2009 pandemic influenza a h1n1 vaccine when administered alone or simultaneously with the seasonal influenza vaccine for the 2009-10 influenza season: a multicentre, randomised controlled trial current status of live attenuated influenza vaccine in the united states for seasonal and pandemic influenza a simple method of estimation fifty per cent endpoints use of animal models to understand the pandemic potential of highly pathogenic avian influenza viruses evaluation of replication and pathogenicity of avian influenza a h7 subtype viruses in a mouse model results of preclinical studies of live monovalent influenza vaccine influvir swine influenza a (h1n1) infection in two children -southern california comparison of rna hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro concepts and procedures for laboratory-based influenza surveillance. us department of health and human services and pan-american health organization detection of antibody to avian influenza a (h5n1) virus in human serum by using a combination of serologic assays fda ⁄ nih ⁄ who public workshop on immune correlates of protection against influenza a viruses in support of pandemic vaccine development coinfection of avian influenza virus (h9n2 subtype) with infectious bronchitis live vaccine co-infection of staphylococcus aureus or haemophilus paragallinarum exacerbates h9n2 influenza a virus infection in chickens human infection with an avian h9n2 influenza a virus in hong kong in 2003 avian-to-human transmission of h9n2 subtype influenza a viruses: relationship between h9n2 and h5n1 human isolates human infection with influenza h9n2 universal primer set for the full-length amplification of all influenza a viruses phylogenetic analysis of hemagglutinin and neuraminidase genes of h9n2 viruses isolated from migratory ducks the neighbor-joining method: a new method for reconstructing phylogenetic trees application of a microtechnique to viral serological investigations biological activity of monoclonal antibodies to operationally defined antigenic regions on the hemagglutinin molecule of a ⁄ seal ⁄ massachusetts ⁄ 1 ⁄ 80 (h7n7) influenza virus development of vaccine strains of h5 and h7 influenza viruses isolation of ortho-and paramyxoviruses from feral birds in hokkaido head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines: effect of comparison, spatial organization and immunization route on the immunogenicity in a murine challenge model evaluation of growth, genetic, and antigenic characteristics of pandemic h1n1 viruses isolated and passaged in qualified mdck suspension cells a (h1n1) influenza virus pandemic: a review influenza vaccine: from surveillance through production to protection trivalent mdck cell culture-derived influenza vaccine optaflu (novartis vaccines) validation of the safety of mdck cells as a substrate for the production of a cell-derived influenza vaccine genetic composition of a high-yielding influenza a virus recombinant: a vaccine strain against ''swine'' influenza rescue of influenza a virus from recombinant dna of health, public health service. concepts and procedures for laboratory based influenza surveillance a rapid method for immunotitration of influenza viruses using flow cytometry generation of live attenuated novel influenza a ⁄ california ⁄ 7 ⁄ 09 (h1n1) vaccines with high yield in embryonated chicken eggs haemagglutinin quantification and identification of influenza a&b strains propagated in per.c6 cells: a novel rp-hplc method comparison of egg and high yielding mdck cell-derived live attenuated influenza virus for commercial production of trivalent influenza vaccine: in vitro cell susceptibility and influenza virus replication kinetics in permissive and semi-permissive cells effect of neuraminidase antibody on hong kong influenza infection-permissive immunization with influenza virus neuraminidase prevents weight loss in infected mice protection of mice with recombinant influenza virus neuraminidase cross-reactive neuraminidase antibodies afford partial protection against h5n1 in mice and are present in unexposed humans note for guidance on harmonization of requirements for influenza vaccines who meeting on the role of neuraminidase in inducing protective immunity against influenza infection who manual on animal influenza diagnosis and surveillance [electronic resource] ⁄ world health organization, department of communicable disease surveillance and response measurement of anti-influenza neuraminidase antibody using a peroxidase-linked lectin and microtitre plates coated with natural substrates detection of antibody to avian influenza a(h5n1) virus in human serum by using a combination of serologic assays large trials confirm immunogenicity of h1n1 vaccines pre-existing immunity against swine-origin h1n1 influenza viruses in the general human population yuelong shu references 1 novel swine-origin influenza a (h1n1) virus investigation team. emergence of a novel swine-origin influenza a (h1n1) virus in humans serum crossreactive antibody response to a novel influenza a (h1n1) virus after vaccination with seasonal influenza vaccine detection of antibody to avian influenza a (h5n1) virus in human serum by using a combination of serologic assays concepts and procedures for laboratory-based influenza surveillance manual on animal influenza diagnosis note for guidance on harmonization of requirements for influenza vaccines (cpmp ⁄ bwp ⁄ 214 ⁄ 96). european agency for the evaluation of medicinal products cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus serologic survey of pandemic (h1n1) 2009 virus influenza a(h1n1) seroconversion rates and risk factors among distinct adult cohorts in singapore swine influenza a outbreak reflections on the 1976 swine flu vaccination program the 1918 flu and other influenza pandemics: ''over there'' and back again evidence of an absence: the genetic origins of the 1918 pandemic influenza virus the 1918 influenza virus: a killer comes into view antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans pathogenesis and immunogenicity of influenza viruses with genes from the 1918 pandemic virus lifetime of plasma cells in the bone marrow humoral immunity due to long-lived plasma cells no one is naive: the significance of heterologous t-cell immunity infection outcomes in patients with common variable immunodeficiency disorders: relationship to immunoglobulin therapy over 22 years signaling by toll-like receptors 8 and 9 requires bruton's tyrosine kinase antiviral cytotoxic t-cell memory by vaccination with recombinant listeria monocytogenes cell-mediated immune response of human lymphocytes to influenza a ⁄ ussr (h1n1) virus infection severe deficiency of switched memory b cells (cd27(+)igm(-)igd(-)) in subgroups of patients with common variable immunodeficiency: a new approach to classify a heterogeneous disease an early humoral immune response in peripheral blood following parenteral inactivated influenza vaccination multifunctional th1 cells define a correlate of vaccine-mediated protection against leishmania major note for guidance on harmonization of requirements for influenza vaccines (cpmp ⁄ bwp ⁄ 214 ⁄ 96) active vaccination in patients with common variable immunodeficiency (cvid) serum bactericidal antibody response to serogroup c polysaccharide meningococcal vaccination in children with primary antibody deficiencies specific anti-influenza virus antibody production in vitro by lymphocytes from a subset of patients with hypogammaglobulinemia shu a a state key laboratory for infectious disease control and prevention, national institute for viral disease control and prevention, chinese center for disease control and prevention, beijing, china. b state key laboratory for molecular virology and genetic engineering, national institute for viral disease control and prevention roles of adjuvant igv combined with influenza m2e based virus like particle in the immune response and cross protection usa) 1ae5 h at 37°c. tetramethylbenzidine (tmb) was applied, and the optical den epidemiology and pathogenesis of influenza epidemiology and virology of influenza illness influenza vaccine: the challenge of antigenic drift evolving strategies for the prevention of influenza infection: potential for multistrains targeting a ''universal human influenza'' a vaccine universal influenza a vaccine: optimization of m2-based constructs the epitope recognized by a monoclonal antibody in influenza a virus m2 protein is immunogenic and confers immune protection high-epitope density in a single recombinant protein molecule of the extracellular domain of influenza a virus m2 protein significantly enhances protective immunity pre-clinical study of influenza virus a m2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys induction of influenza type a virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2 a universal influenza a vaccine based on the extracellular domain of the m2 protein protective immunity against influenza a virus induced by immunization with dna plasmid containing influenza m gene protection against influenza virus challenged by topical application of influenza dna vaccine influenza a vaccine based on the extracellular domain of m2: weak protection mediated via antibody-dependent nk cell activity t cell immunoglobulin mucin-3 crystal structure reveals a novel ligand binding surface structures of t cell immunoglobulin mucin protein 4 show a metal-ion-dependent ligand binding site where phosphatidylserine binds structures of t cell immunoglobulin mucin receptors 1 and 2 reveal mechanisms for regulation of immune responses by the tim receptor family the tim gene family: emerging roles in immunity and disease th1-specific cell surface protein tim-3 regulates macrophage activation and severity of and autoimmune disease identification of a surface glycoprotein on african green monkey kidney cells as a receptor for hepatitis a virus the igv domain of human b7-2 (cd86) is sufficient to co-stimulate t lymphocytes and induce cytokine secretion vaccination with cell immunoglobulin mucin-1 antibodies and inactivated influenza enhances vaccine-specific lymphocyte proliferation, interferon-r production and cross-strain reactivity incorporation of membrane-anchored flagellin into influenza virus-like particles enhances the breadth of immune response virus-like particle induces protective immunity against homologous and heterologous strains of influenza virus site accessed on 1st study designs for timely estimation of influenza vaccine effectiveness using european sentinel practitioner networks a new influenza surveillance system in france: the ile-de-france ''grog''. 1. principles and methodology site accessed on 1st i-move'' towards monitoring seasonal and pandemic influenza vaccine effectiveness: lessons learnt from a pilot multi-centric case-control study in europe population and risk group uptake of h1n1 influenza vaccine in mainland france 2009-2010: results of a national vaccination campaign of global health and emerging pathogens ns deletions convert the 2009-h1n1 pandemic virus into a live attenuated vaccine. influenza and other respiratory viruses the influenza virus enigma vaccine production capacity for seasonal and pandemic (h1n1) 2009 influenza interferon-induced isg15 conjugation inhibits influenza a virus gene expression and replication in human cells the multifunctional ns1 protein of influenza a viruses influenza a and b viruses expressing altered ns1 proteins: a vaccine approach multiple anti-interferon actions of the influenza a virus ns1 protein the ns1 protein of a human influenza virus inhibits type i interferon production and the induction of antiviral responses in primary human dendritic and respiratory epithelial cells immunization with live attenuated influenza viruses that express altered ns1 proteins results in potent and protective memory cd8+ t-cell responses plasmacytoid dendritic cells delineate immunogenicity of influenza vaccine subtypes regulation of adaptive immunity by the innate immune system the influenza virus ns1 protein: inhibitor of innate and adaptive immunity single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza a viruses unidirectional rna polymerase i-polymerase ii transcription system for the generation of influenza a virus from eight plasmids avian influenza (h5n1) viruses isolated from humans in asia in 2004 exhibit increased virulence in mammals transmission and pathogenesis of swine-origin 2009 a(h1n1) influenza viruses in ferrets and mice a naturally occurring deletion in its ns gene contributes to the attenuation of an h5n1 swine influenza virus in chickens future influenza vaccines and use of genetic recombinants eight-plasmid system for rapid generation of influenza virus vaccines rescue of influenza b virus from eight plasmid high yield reassortants of influenza type b viruses np gene obtained from high yield donor. options for control of influenza vi. abstract #330 summary of status of development and availability of b ⁄ brisbane ⁄ 60 ⁄ 2008-like candidate vaccine viruses and potency testing reagent reassortants of influenza b viruses for use in vaccines: an evaluation preparation of influenza b virus recombinant strains genetic and phenotypic analysis of reassortants of high growth and low growth strains of influenza b virues pandemic influenza in russia: detection and molecular references evolutionary pattern of pandemic influenza (h1n1) 2009 virus in the late phases of the 2009 pandemic antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans effects of egg adaptation on the receptor-binding properties of human influenza a and b viruses the early diversification of influenza a ⁄ h1n1pdm update on influenza a (h1n1) 2009 monovalent vaccines clinical and epidemiologic characteristics of 3 early cases of influenza a pandemic options for the control of influenza vii virus infection, people's republic of china genome evolution of novel influenza a (h1n1) viruses in humans introduction and transmission of 2009 pandemic influenza a (h1n1) virus -kenya emergence of a novel swine-origin influenza a (h1n1) virus in humans first season of 2009 h1n1 influenza origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic modeling gene sequences over time in 2009 h1n1 influenza a virus populations differentiation of two distinct clusters among currently circulating influenza a(h1n1)v viruses conversion of mdck cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production trivalent mdck cell culture-derived influenza vaccine optaflu (novartis vaccines) use of mdck cells for production of live attenuated influenza vaccine flow cytometric monitoring of influenza a virus infection in mdck cells during vaccine production cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences seroprevalence following the second wave of pandemic molecular evolution of human influenza a viruses in a local area during eight influenza epidemics from evaluation of hemagglutinin subtype 1 swine influenza viruses from the united states the early molecular epidemiology of the swine-origin a ⁄ h1n1 human influenza pandemic the genomic and epidemiological dynamics of human influenza a virus reconstructing the initial global spread of a human influenza pandemic: a bayesian spatial-temporal model for the global spread of h1n1pdm mild form of 2009 h1n1 influenza infection detected by active surveillance: implications for infection control the pandemic 2009 (h1n1) swine influenza virus is mild compared to the pandemic 1918 (h1n1) virus because of a prolineto-serine substitution in the receptor-binding site of its hemagglutinin -a hypothesis pathogenesis and transmission of swine-origin 2009 a(h1n1) influenza virus in ferrets investigation of the first cases of human-to-human infection with the new swine-origin influenza a (h1n1) virus in canada antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans molecular evolution of hemagglutinin genes of h1n1 swine and human influenza a viruses antigenic structure of influenza virus haemagglutinin defined by hybridoma antibodies the predicted antigenicity of the haemagglutinin of the 1918 spanish influenza pandemic suggests an avian origin genetically destined potentials for nlinked glycosylation of influenza virus hemagglutinin global patterns of influenza a virus in wild birds antigenic and genetic characteristics of swine-origin 2009 a (h1n1) influenza viruses circulating in humans translatorx: multiple alignment of nucleotide sequences guided by amino acid translations raxml-iii: a fast program for maximum likelihood-based inference of large phylogenetic trees flugenome: a web tool for genotyping influenza a virus beast: bayesian evolutionary analysis by sampling trees origin and evolution of influenza virus hemagglutinin genes panorama phylogenetic diversity and distribution of type a influenza virus early alterations of the receptor-binding properties of h1, h2, and h3 avian influenza virus hemagglutinins after their introduction into mammals human influenza a h5n1 virus related to a highly pathogenic avian influenza virus origin and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic keywords novel influenza viruses, oseltamivir resistance phylogenetic analyses of pandemic influenza viruses textbook of influenza mapping the antigenic and genetic evolution of influenza virus cdc protocol of realtime rt-pcr for swine influenza a(h1n1) revision 1 the hemagglutination and hemagglutination ingibitions test. who collaborating center for surveillance molecular evolutionary genetics analysis (mega) software version 4.0 pandemic influenza: an inconvenient mutation large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution origin and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic antigenic and genetic characteristics of swine-origin 2009 a (h1n1) influenza viruses circulating in humans genetic subtyping using cluster analysis panorama phylogenetic diversity and distribution of type a influenza virus continuing progress towards a unified nomenclature for the highly pathogenic h5n1 avian influenza viruses: divergence of clade 2.2 viruses the influenza virus resource at the national center for biotechnology information muscle: multiple sequence alignment with high accuracy and high throughput multiple alignment of dna sequences with mafft raxml-iii: a fast program for maximum likelihood-based inference of large phylogenetic trees flugenome: a web tool for genotyping influenza a virus evolution of influenza viral neuraminidase (na) genes revealed by large-scale sequence analysis. proceedings of the options for the control of influenza vii a quantitative genotype algorithm reflecting h5n1 avian influenza niches xiu-feng wan a a department of basic sciences complete composition vector, influenza a virus, minimum spanning tree, phylogeny, progenitor ipminer: a progenitor gene identifier for influenza a virus avian-to-human transmission of the pb1 gene of influenza a viruses in the 1957 and 1968 pandemics triple-reassortant swine influenza a (h1) in humans in the united states clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighing, positionspecific gap penalties and weight matrix choice muscle: multiple sequence alignment with high accuracy and high throughput coffee: an objective function for multiple sequence alignments the neighbour-joining method: a new method for reconstructing phylogenetic trees mrbayes: bayesian inference of phylogenetic trees basic local alignment search tool a quantitative genotype algorithm reflecting h5n1 avian influenza niches computational identification of reassortments in avian influenza viruses ubiquitous reassortments in influenza a viruses phylip -phylogeny inference package (version 3.2) pandemic influenza 2009 in russia: isolation, antigenic produced in our laboratory. normal equine serum was purchased from sigma (usa) comparative data on the development of influenza pandemic in russia and in the other european countries (in russian). materials of the ii annual all-russian congress on infectious diseases isolation on 24.05.2009 and deposition in the state virus collection of the first strain a ⁄ moscow ⁄ 01 ⁄ 2009 (h1n1)swl, similar to the swine virus a(h1n1) from the first patient diagnosed in moscow isolation of influenza viruses in cell cultures and in chicken embryos and their identification. methods guidance (in russian) molecular genetic characterization of the strains of pandemic influenza a(h1n1)v isolated on the territory of russian federation in 2009 (in russian). materials of the ii annual all-russian congress on infectious diseases genetic and antigenic analyses of influenza a (h1n1) viruses, 1986-1991 sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses pandemic influenza a(h1n1) virus mutations reported to be associated with severe disease antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans barr b,c a novartis vaccines and diagnostics gmbh, marburg, germany. b who collaborating centre for reference and research on influenza co-infecting viruses in pandemic h1n1 and seasonal influenza positive human respiratory samples emergence of a novel swine-origin influenza a (h1n1) virus in humans simultaneous detection and high-throughput identification of a panel of rna viruses causing respiratory tract infections comparison of multiple commercial assays for the detection of swine-origin influenza a (h1n1) virus (soiv) evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections performance comparison of res-plex ii and xtag rvp fast for detecting respiratory viruses clinical virology symposium communityacquired respiratory co-infection (carc) in critically ill patients infected with pandemic 2009 influenza a (h1n1) virus infection bacterial co-infections in lung tissue specimens from fatal cases of 2009 pandemic influenza a (h1n1) -united states a quantitative risk assessment of exposure to adventitious agents in a cell culture-derived subunit influenza vaccine john's hopkins bloomberg school of public health design of an automated laboratory for high-throughput influenza surveillance human influenza surveillance: the demand to expand influenza: an emerging disease avian-to-human transmission of the pb1 gene of influenza a viruses in the 1957 and 1968 pandemics proinflammatory cytokine responses induced by influenza a (h5n1) viruses in primary human alveolar and bronchial epithelial cells induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? influenza h5n1 and h1n1 virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation mapping of the two overlapping genes for polypepetides ns1 and ns1 on rna segment 8 of influenza virus genome sequences of mrnas derived from genome rna segment 7 of influenza virus: collinear and interrupted mrnas code for overlapping proteins influenza virus ns1 protein inhibits pre-mrna splicing and blocks mrna nucleocytoplasmic transport the influenza virus ns1 protein: a novel inhibitor of pre-mrna splicing identification of cis-acting intron and exon regions in influenza virus ns1 mrna that inhibit splicing and cause the formation of aberrantly sedimenting presplicing complexes identification of a second protein encoded by influenza c virus rna segment 6 influenza c virus ns1 protein upregulates the splicing of viral mrnas identification of an amino acid residue on influenza c virus m1 protein responsible for formation of the cord-like structures of the virus a mutation on influenza c virus m1 protein affects virion morphology by altering the membrane affinity of the protein detection of ion channel activity in xenopus laevis oocytes expressing influenza c virus cm2 protein evidence that the cm2 protein of influenza c virus can modify the ph of the exocytic pathway of transfected cells the ion channel activity of the influenza virus m2 protein affects transport through the golgi apparatus intracellular localization of influenza c virus ns2 protein (nep) in infected cells and its incorporation into virions receptor specificity, host range and pathogenicity of influenza viruses replication, pathogenesis and transmission of pandemic (h1n1) 2009 virus in non-immune pigs world health organization. preliminary review of d222g amino acid substitution in the haemagglutinin of pandemic influenza a (h1n1) 2009 viruses observed association between the ha1 mutation d222g in the 2009 pandemic influenza a(h1n1) virus and severe clinical outcome quasispecies of the d225g substitution in the hemagglutinin of pandemic influenza a(h1n1) virus from patients with severe disease in hong kong a possible association of fatal pneumonia with mutations of pandemic influenza a ⁄ h1n1 sw1 virus in the receptor-binding site of the ha1 subunit severe outcome of influenza a ⁄ h1n1 ⁄ 09v infection associated with 222g ⁄ n polymorphisms in the haemagglutinin: a multicenter study influenza pandemic caused by highly conserved viruses with two receptor-binding variants overexpression of the alpha-2,6-sialyltransferase in mdck cells increases influenza virus sensitivity to neuraminidase inhibitors human and avian influenza viruses target different cell types in cultures of human airway epithelium avian-virus-like receptor specificity of the hemagglutinin impedes influenza virus replication in cultures of human airway epithelium receptor-binding specificity of pandemic influenza a (h1n1) 2009 virus determined by carbohydrate microarray fields virology. philadelphia, pa: lippincott williams & wilkins the molecular virology and reverse genetics of influenza c virus identification of a second protein encoded by influenza c virus rna segment 6 identification of a 374 amino acid protein encoded by rna segment 6 of influenza c virus influenza c virus cm2 protein is produced from a 374 amino acid protein (p42) by signal peptidase cleavage a mutation on influenza c virus m1 protein affects virion morphology by altering the membrane affinity of the protein influenza c virus cm2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence evidence that the matrix protein of influenza c virus is coded for by a spliced mrna functional properties of the virus ion channels the cm2 protein of influenza c virus is an oligomeric integral membrane glycoprotein structurally analogous to influenza a virus m2 and influenza b virus nb proteins characterization of a second protein (cm2) encoded by rna segment 6 of influenza c virus phosphorylation of influenza c virus cm2 protein the sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza c virus cm2 protein identification of an amino acid residue on influenza c virus m1 protein responsible for formation of the cord-like structures of the virus a human melanoma cell line highly susceptible to influenza c virus antigenic characterization of the nucleoprotein and matrix protein of influenza c virus with monoclonal antibodies construction of an antigenic map of the haemagglutinin-esterase protein of influenza c virus the synthesis of polypeptides in influenza c virus-infected cells new low-viscosity overlay medium for viral plaque assays the influenza virus m2 protein cytoplasmic tail interacts with the m1 protein and influences virus assembly at the site of virus budding the cytoplasmic tail of the influenza a virus m2 protein plays a role in viral assembly the influenza a virus m2 cytoplasmic tail is required for infectious virus production and efficient genome packaging distinct domains of the influenza a virus m2 protein cytoplasmic tail mediate binding to the m1 protein and facilitate infectious virus production influenza virus m2 ion channel protein is necessary for filamentous virion formation influenza h5n1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells das181 inhibits h5n1 influenza virus infection of human lung tissues receptor binding specificity of recent human h3n2 influenza viruses differential onset of apoptosis in avian influenza h5n1 and seasonal h1n1 virus infected human bronchial and alveolar epithelial cells: an in vitro and ex vivo study human influenza virus a ⁄ hongkong ⁄ 156 ⁄ 97 (h5n1) infection induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? proinflammatory cytokine responses induced by influenza a (h5n1) viruses in primary human alveolar and bronchial epithelial cells differential onset of apoptosis in influenza a virus h5n1-and h1n1-infected human blood macrophages avian flu: influenza virus receptors in the human airway haemagglutinin mutations responsible for the binding of h5n1 influenza a viruses to humantype receptors an avian influenza h5n1 virus that binds to a human-type receptor evolution of highly pathogenic h5n1 avian influenza viruses in vietnam between evolutionary dynamics and emergence of panzootic h5n1 influenza viruses writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza a (h5n1) virus recent avian h5n1 viruses exhibit increased propensity for acquiring human receptor specificity a simple screening assay for receptor switching of avian influenza viruses glycan topology determines human adaptation of avian h5n1 virus hemagglutinin h5n1 chicken influenza viruses display a high binding affinity for neu5acalpha2-3galbeta1-4(6-hso3)glcnac-containing receptors a strain of human influenza a virus binds to extended but not short gangliosides as assayed by thin-layer chromatography overlay search for additional influenza virus to cell interactions avian flu: isolation of drug-resistant h5n1 virus the surface glycoproteins of h5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties this study was supported by the li ka shing foundation, the national institutes of health (niaid contract hhsn266200700005c), and the area of excellence scheme of the university grants committee (grant aoe ⁄ m-12 ⁄ 06) of the hong kong sar government. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn266200700005c, the li ka shing foundation, and we thank all french and vietnamese field staff involved in the data collection in viet nam for their enthusiasm and support and we are grateful to the pig farmers participating in the study for their cooperation and patience. this study was a part of the gripavi project and was funded by the french ministry of foreign affairs. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn26600700005c and the area of excellence scheme of the university grants commission (grant aoe ⁄ m-12 ⁄ 06) of the hong kong sar government. we acknowledge the food and environmental hygiene department of hong kong for facilitating the study. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn266200700005c, the li ka shing foundation, and the area of excellence scheme of the university grants committee (grant aoe ⁄ m-12 ⁄ 06) of the hong kong sar government. we gratefully acknowledge our colleagues from iiii, shantou university and skleid, hku for their excellent technical assistance. the study was supported by the rfcid commissioned study (lab#16) from research fund secretariat, food and health bureau, hong kong sar; area of excellence scheme of the university grants committee (grant aoe ⁄ m-12 ⁄ 06), hong kong sar; and by niaid contract (sjceirs, hhsn26620070005c), nih, usa.ferrets in all groups inoculated with a ⁄ turkey ⁄ 15 ⁄ 06 virus survived the infection and were observed once daily for 14 days. below lower limit of detection (<0ae75 log 10 eid 50 ⁄ ml).statistical cutoff of ic 50 values for nai susceptibility, determined by x 0ae75 + 3iqr. outliers with ic 50 above this cutoff and >10 times the mean ic 50 for each drug were characterized as extreme outliers; those with known drug-resistance mutations such as h275y were classified as resistant and analyzed separately. h275 wildtype, oseltamivir-susceptible isolates. h275y variants, oseltamivir-resistant virus isolates. iqr, interquartile ranges; nai, neuraminidase inhibitors. we wish to thank our collaborators in the who global influenza surveillance network and united states public health laboratories for the submission of virus isolates and clinical specimens. we also thank our colleagues from the virus reference team and the influenza sequence activity, influenza division, cdc, for their valuable technical assis-the findings and conclusions of this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention (cdc). we are indebted to yonas araya, theresa wolter, and ivan gomez-osorio for their excellent laboratory techniques and animal handling assistance. we would like to thank andrea ferrero for her laboratory managerial skills. this research was possible through funding by the cdc-hhs grant (1u01ci000355), niaid-nih grant, (r01ai052155), csrees-usda grant (1865-05523), and niaid-nih contract (hhsn266186700010c). we thank c bazzoli for advice. this work was supported by a grant from the european union fp7 project flu-modcont (no. 20160). we thank staff at seoul, incheon, daejeon, gwangju, gangwon, and jeonbuk provincial research institute of health and environments for their laboratory testing. additionally, we would like to acknowledge the contributions of participating sentinel doctors for evaluating the new rat kit. this study was supported by a grant from the korea cdc. we thank roche applied science for providing the materials and equipment for this evaluation. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn26600700005c and the area of excellence scheme of the university grants committee (grant aoe ⁄ m-12 ⁄ 06) of the hong kong sar government. the authors would like express their sincere thanks to cdc, usa for supporting the routine surveillance of ili in we would like to acknowledge the australian red cross blood service (the blood service) and the australian government, which fully fund the blood service for the provision of blood products and services to the australian community. we also wish to thank the donors and staff of the blood service, who have assisted in provision of specimens for testing in this protocol, as well as the staff at the who we are grateful to liping long for her assistance in map generation. this project was supported by nih niaid 1rc1ai086830. cz is supported partially by canadian nserc postdoc fellowship. the authors thank the national investigation team based at the national institute of health (istituto superiore di sanita'), italy (in particular antonino bella, maria cristina rota, stefania salmaso) for providing their support in data collection, and the european union this study was supported in part by a grant-in-aid (21249061) and the special coordination funds for promoting science and technology of ministry of education, science, sports and culture of japan. this study was supported in part by a grant-in-aid from the ministry of education, science, and culture of japan (21249061) and the special coordination funds for promoting science and technology of mext of japan. the work described here was supported by phs grant ai-66349 (jam) and alsac. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. py, po, dw and kb are employees of glaxosmithkline. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the stud authors are thankful to path for the financial support of this research. we would like to acknowledge jessica d'amico and dr. rick bright of path for their editorial review. this study was supported by path. the authors would like to thank rick bright, jessica d'amico, and vadim tsvetnitsky for editing assistance. the we thank dr. m. enami (kanazawa university) for generously providing plasmids containing cdnas to influenza a virus m and ns genes. we also gratefully thank dr. r. sho (department of public health, yamagata university faculty of medicine) for statistical analysis. some data shown in this study have also been presented in the reference paper. 7 this work was supported in part by a grant-in-aid for scientific research from the ministry of education, culture, sports, science, and technology, japan, takeda science foundation, terumo life science foundation, and a grant-in-aid from the global coe program of the japan society for the promotion of science. we thank markus eickmann for his help in isolation and initial characterization of a ⁄ hamburg ⁄ 5 ⁄ 09 and for providing antisera against h1n1pdm. this study was supported by the european union fp6 global a(h1n1)2009 genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. in this study we analysed the genetic and antiviral drug susceptibility profiles of pandemic a(h1n1)2009 influenza virus circulating in portugal. genetic profile analysis was performed in 37 isolates to the hemagglutinin (ha), neuraminidase (na) and mp genes, and in six of these isolates the pb1, pb2, pa, np and ns genes were also analysed. antiviral drug susceptibility profile was analysed for 96 isolates, phenotypically and genotypically to neuraminidase inhibitors (nai) and genotypically to amantadine. the point mutations identified in ha, na, and mp genes of different strains do not seem to evidence an evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h1n1) 2009 virus. all analysed strains were found to be resistant to amantadine, and five of these strains exhibited a reduced susceptibility profile to nai, three only for oseltamivir and two for both inhibitors. introduction: the dynamics of pandemic influenza a ⁄ h1n1 compared to seasonal strains of influenza is not clearly understood. it is important to understand the patterns of viral shedding and symptoms over time in community-based infections.materials and methods: household infections were followed-up in two large community-based studies. patterns of viral shedding, symptoms and signs, and tympanic temperature were plotted over time and grouped according to strain for analysis.results: the patterns of viral shedding, symptoms and signs, and tympanic temperature in three influenza a strains (pandemic a ⁄ h1n1, seasonal a ⁄ h1n1, and seasonal a ⁄ h3n2) were comparable. peak viral shedding occurred close to the onset of symptoms and resolved after 6-7 days. patterns of viral shedding in influenza b virus infections differed.discussion: the patterns of viral shedding and clinical course of pandemic influenza a ⁄ h1n1 infections were broadly similar to seasonal influenza a ⁄ h1n1 and a ⁄ h3n2. only the clinical course of seasonal influenza b infections was similar to pandemic influenza a ⁄ h1n1. the dynamics of pandemic influenza a ⁄ h1n1 were observed to be largely alike to the dynamics of seasonal influenza a ⁄ h1n1 and a ⁄ h3n2. the coated respirators inactivated a broad range of influenza strains within 1 minute, including the 2009 pandemic strain and human, swine, and avian influenza viruses. antiviral effectiveness was not reduced by hot, humid conditions or repeated saturation, which might occur during prolonged use of respirators. in contrast, infectious virions were detected on the surfaces of all uncoated ffp2 respirators, and could be transferred to glove surfaces during handling of contaminated masks. growth of the viruses was monitored by ha titer using turkey red blood cells, by quantitative real time rt-pcr (qrt-pcr) to detect the influenza a matrix gene, and also by flow cytometry to detect virus positive cells using monoclonal antibodies (imagen influenza virus a and b). 8, 9 matrix gene copy number was determined using qrt-pcr and analysed using the sequence detection software on a 7500 fast system sds (applied biosystems, california, usa). further characterisation was performed through sequence analysis and the ha inhibition (hai) assay. 8 sequence analysis was performed using dnastar 8 and all sequences obtained were compared with the sequence of either the original clinical specimen if available or the conventional atcc derived mdck cell isolate. the hai assay was used to characterize the viruses against a panel of known standard reference viruses and their homologous ferret antiserum.options for the control of influenza vii abstract background: we measured the cross-reactive antibody response to pandemic h1n1 2009 in children and adults before and after vaccination with [2007] [2008] [2008] [2009] influenza season vaccines as part of the rapid public health response to the emergence of ph1n1 and to provide evidence for ph1n1 vaccination policy development in mainland china. materials and methods: archived serum specimens from previous vaccine studies were detected by hemagglutination inhibition assay. results: limited crossreactive antibody response to ph1n1 had been detected among participants of all age groups before and after they had been vaccinated with 2007-2008, 2008-2009 influenza seasonal vaccines. vaccination with seasonal influenza viruses resulted in limited seroconversion to ph1n1 in all age groups, compared with 59-94% of seroconversion to seasonal influenza viruses. but similar to recent studies, a peak of cross-reactive antibody response to ph1n1 was observed in 23% and 39% of participants born from 1915 to 1925 before and after vaccination. conclusions: in order to protect our populations in china, our study strongly suggests vaccination with ph1n1 is required in all age groups and that older populations born before 1925 may be associated with a lower infection rate of ph1n1. on april 15 and april 17, 2009, cases of ph1n1 were identified in specimens obtained from two epidemiologically unlinked patients in the united states and soon thereafter in texas and mexico. 1 since that time, the virus has spread across the globe. assessment of cross-reactive antibody response to the ph1n1 after vaccination with sea-sonal influenza vaccine was first reported from us centers of disease control and prevention (us cdc). according to their results, the seasonal influenza vaccines provided little or no protection against the ph1n1, but some degree of preexisting immunity to the virus existed, especially among adults aged ‡60 years. 2 in this study, using archived serum samples from previous vaccine studies, we measure the level of cross-reactive antibody response to ph1n1 in children and adults vaccinated intramuscularly with trivalent inactivated vaccine developed for the northern hemi serum specimens were collected and provided by provincial centers for disease control and prevention of china as a public health response to the emergence of ph1n1 exempt from human-subjects review. a total of 1308 serum samples were collected from xinjiang uygur autonomous region, yunnan, and shandong provinces. all the serum specimens were grouped by the age of subjects (0-7, 8-17, 18-59, ‡60 years) and by different influenza seasons.hemagglutination inhibition assay was performed according to standard procedures in this study. [3] [4] [5] as with h1n1 components of the vaccine, the seasonal influenza viruses used in this study were a ⁄ solomon islands ⁄ 3 ⁄ 2006 and a ⁄ brisbane ⁄ 59 ⁄ 2007. the ph1n1 influenza virus used in this study was a ⁄ california ⁄ 07 ⁄ 2009 provided by us cdc. all the viruses were propagated in specific pathogenfree embryonated chicken eggs and inactivated by 1& paraformaldehyde. the criteria 6 recommended by the european agency for the evaluation of medical product was applied for the assessment of seasonal influenza vaccine gmt, geometric mean titer; hi, hemagglutination inhibition. three weeks after boosting immunization, spleens were harvested from immunized and control mice. splenocytes were prepared by lymphocyte separation media (ez-sepô, shen zhen, china). the cells were washed and resuspended in complete rpmi-1640 containing fetal bovine serum (hyclone, logan, ut, usa), glutamax, 5 um b-me. splenocytes were cultured in vitro in the presence of inactivated h1n1, h3n2, h5n1, and h9n2 influenza virus antigen for 96 h. quick cell proliferation assay kit (biovision, san francisco, ca, usa) was used to detect the cell proliferation. the 420-480 nm absorbance was read on a plate reader. all experiments have been repeated at least three times.results are presented as mean standard error of the mean (sem). comparison of the data was performed using the student's t-test. significance was defined as a p value of <0ae05. to evaluate the adjuvant effect of recombinant igv, the anti m2e antibody subclasses was measured. igg1 and igg2a were detected after the first and second immunization ( table 1 ). the ratio of igg2a ⁄ igg1 was calculated. immunization with only m2e-hbc showed a lower igg2a ⁄ igg1 ratio <0ae5. igv combined with m2e-hbc led to a high igg2a ⁄ igg1 ratio of up to 5-15 after first and second immunization. these igg subclass distributions indicated that igv can induce a th1 immune response. to determine whether the splenocytes were stimulated in vitro with different subtypes of inactivated influenza antigen after the igv plus m2e-hbc antigen immunization, h1n1, h3n2, h5n1, h9n2 inactivated antigen was used table 1 . the serum igg, igg1, igg2a, and igg2a ⁄ igg1 ratio were measured by elisa after first and second immunization. m2e were coated on the 96 wells plate overnight, and serial dilution sera of day 7, 14, 21 after first and second immunization were added 0, 2ae5, 5, 10, 20, 40 ug ⁄ ml of igg, igg1, igg2a purified antibody were also added for obtaining the standard curve. hrp-labeled goat anti-mouse igg, igg1, or igg2a was then added, washed, and the optical density was read at 492 nm. the results were showed at mean ± sem. day after first immunization days after second immunizationoptions for the control of influenza vii the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than 5000 specimens yearly from cases of acute respiratory illness (ari), using two sampling methods: systematic randomized and non systematic ''ad hoc'' sampling. although vaccines against influenza a virus are the most effective method by which to combat infection, it is clear that their production needs to be accelerated and their efficacy improved. a panel of recombinant live attenuated human influenza a vaccines (laivs), including ns1-73, ns1-126, nsd5, were generated by rationally engineering mutations directly into the genome of a pandemic-h1n1 virus. the vaccine potential of each laiv was determined through analysis of attenuation, immunogenicity, and their ability to protect mice and ferrets. the data indicate that the novel nsd5-laiv was ideally attenuated and elicited strong protective immunity. this study also shows that attenuating mutations can be rapidly engineered into the genomes of emerging ⁄ circulating influenza a viruses in order to produce laivs. the influenza virus exhibits complicated evolutionary dynamics due to multiple reasons, such as diverse hosts, high mutation rates, and rapid replications. in this study, large-scale analyses of 11 454 influenza neuraminidase (na) sequences revealed influenza a and b na genes diverged first around 2641 years ago, and subsequently the na subtypes of influenza a emerged around 1125 years ago. all nine na subtypes of influenza a were genetically distinct from each other, with a total of 23 lineages identified. in addition, five and three sub-lineages were further identified in lineage 1a of na1 and lineage 2b of na2, respectively. the majority of lineages and sub-lineages were found to be host or geographic specific. this study provides not only a better understanding of influenza na evolution, but also a database of lineages and sub-lineages that can be used for early detection of novel genetic changes for improved influenza surveillance. although phylogenetic approaches are commonly used and often found to be powerful, how to accurately identify lineages or sub-lineages of a gene segment of the influenza a virus remains a challenging issue. in this study, we address this issue by analyzing hemagglutinin (ha) sequences using a combination of statistical and phylogenetic methods. following a hierarchical nomenclature system that uses a letter to represent a lineage and a digit for a sub-lineage, we identified 41 distinct lineages and 81 sub-lineages in all 16 ha subtypes through large-scale analyses of 11 821 influenza a hemagglutinin sequences. the majority of the lineages or sub-lineages were host or geographic specific or both. further analysis of other segments will allow us to construct a comprehensive database for influenza a lineages and genotypes, facilitating early detection of new viral strains and genome reassortments and hence improve influenza surveillance. identification of the genetic origin of influenza a viruses will facilitate understanding of the genomic dynamics, evolutionary pathway, and viral fitness of influenza a viruses. the exponential increases of influenza sequences have expanded the coverage of influenza genetic pool, thus potentially reducing the biases for influenza progenitor identification. however, these large amounts of data generate a great challenge in progenitor identification. clinical (nasopharyngeal swabs) and post-mortem materials (fragments of trachea, bronchi, lungs, spleen) were obtained from clinics and ⁄ or out-patients from st. petersburg and from 19 base virological laboratories (bvls) of the research institute of influenza in different regions of the country, which cover approximately 3 ⁄ 4 of the territory of russia. the informed consent for the bio-materials collection and studies was obtained from research subjects or from their relatives in cases of post-mortem materials. isolation of viruses was carried out in the mdck cell culture (cdc, atlanta, ga, usa) and in 10-day-old chicken embryos (e). isolation was done according the standard internationally accepted methods. 3 the reaction of hemagglutination (ha) and the inhibition of hemagglutination (hai) were performed according the who recommended standard method. 3 for the identification of epidemic isolates, we used the hyperimmune diagnostic bovine or ovine antisera annually obtained from the who reference center (cdc). for a detailed antigenic analysis we used the hyperimmune rat antisera against epidemic and reference influenza strains during the period from july 20, 2009 up to april 30, 2010, we have obtained 772 swabs from clinics and out-patients in st. petersburg and 374 swabs from the bvls. in this period, rather high incidence of lethality from pneumonia was observed, which developed on the background of the pandemic flu h1n1v. thus, we received from bvls 173 postmortem materials from 91 deceased patients which manifested pcr+ influenza h1n1v-specific rna. all materials were tested for a possibility of isolation of influenza virus h1n1v both in eggs and in mdck cells. pcr-negative materials were discarded. we isolated 229 strains of pandemic influenza from the materials collected in st. petersburg and region, which comprised 29ae7% of the total number of analyzed samples. at the same time, we did not isolate any other sub-types of influenza in the season 2009-2010 except the pandemic flu. from the swabs purchased from bvls, 47 strains were isolated, which compose 37ae9% of the pcr+ samples, and 35 strains from the post-mortem materials (43ae2% of the pcr+ samples).altogether in the season 2009-2010, we isolated, retrieved, and analyzed in hai 453 influenza strains. 91ae2% of them were pandemic strains a(h1n1)v, and only 7ae9% influenza b viruses. these data together with the epidemiologic data and the results of pcr-diagnostic provide evidence in favor of nearly mono-etiological character of epidemic season 2009-2010 in russia for pandemic influenza a(h1n1)v.though the isolation of pandemic viruses was fulfilled in two traditional model systems, in the case of pandemic 2009 virus, we could observe the tendency of preferential multiplication in embryos compared to mdck, especially in cases of post-mortem material for which chicken embryos are the preferential system of isolation.h1n1v viruses, which were isolated and passaged in mdck, even with significant ha titers, quickly lost their ha activity provided they were kept at +4°c. moreover, some other tested cell lines proved to be practically nonsensitive to the pandemic viruses h1n1v. we used hai reaction for the typing and antigenic characterization of isolated viruses. in the course of isolation of viruses in the reported period, we produced rat polyclonal antisera to the strains a ⁄ california ⁄ 07 ⁄ 09 and a ⁄ st. petersburg ⁄ 56 ⁄ 09 (h1n1)v and the antisera to the strains a ⁄ new jersey ⁄ 8 ⁄ 76 -the virus isolated during the epidemic 1976 in the united states and also of the swine origin -and to the 'swine' strains a ⁄ sw ⁄ 1976 ⁄ 31 and a ⁄ iowa ⁄ 15 ⁄ 30. the hai results of representative strains are given in figure 1 . table 1 shows that the isolated strains were homogenous in their antigenic properties and interacted with the diagnostic antiserum cdc for a(h1n1)v and also with the antisera to the strains a ⁄ california ⁄ 07 ⁄ 09 and a ⁄ st. petersburg ⁄ 56 ⁄ 09 up to 1-1 ⁄ 4 homologous titer. viruses that were isolated from post-mortem materials did not differ by their antigenic characteristics from those isolated from swabs of live patients. only two strains could be attributed to the drift-variants of the strain a ⁄ california ⁄ 07 ⁄ 09 because they reacted with the appropriate antiserum up to 1 ⁄ 8 homologous titer; these strains were a ⁄ pskov ⁄ 1 ⁄ 09 and a ⁄ belgorod ⁄ 6 ⁄ 09. it is interesting that the isolated strains reacted with the antisera to the strains a ⁄ new jersey ⁄ 8 ⁄ 76 and a ⁄ sw ⁄ 1976 ⁄ 31 to 1 ⁄ 4-1 ⁄ 8, and some particular strains even to 1 ⁄ 2 homologous titer. it is even more interesting that some pandemic isolates reacted with the antiserum to the strain iowa isolated in 1930 up to 1 ⁄ 4-1 ⁄ 8 homologous titer. despite of the fact that since the outbreak of 'swine flu' in the usa in new jersey 30 years had gone (and for the strain iowa this period is nearly 70 years) the ha of these viruses and of the pandemic influenza 2009 share some common antigenic determinants as was shown in hai.one more interesting feature of a considerable part of isolated strains is their capability to react with high titers with normal equine serum heated to 56 and to 80°c, while all the strains of swine origin isolated earlier were inhibitor-resistant ( figure 1 ). russian isolates of 2009 divided, in this respect, in two clear and approximately equal in number groups: one of them is similar to the reference strain amino acid substitutions, among them more than 30 were disclosed in antigenic sites, so the degree of similarity to this strain is 78%. a new site of glycosylation was also discovered in the position 276 of ha. 4 essential distinctions of the aminoacid sequence of ha and antigenic properties of the h1n1v strains as compared with actual circulating and vaccine strains is one of the factors that determine the pandemic potential of this new influenza virus.according to the literature, the mutation in the ha gene d222g could cause a broadening of the spectrum of receptor specificity of influenza virus by the acquisition of the capacity to bind both the residues a(2 fi 6) and a(2 fi 3) of the sialic acid of cellular receptors. 5 both types of receptors are present at the human respiratory tract, but in different parts of it, and they exist in different proportions. 6 according to the data of the european center of disease control and prevention (ecdc), the varieties g222 of the h1n1v virus were isolated in 20 countries from subjects deceased of influenza or who suffered a severe form of illness, as well as from those who sustained only a light course of influenza. 7 concerning the strains isolated in rii, this mutation was discovered in nine cases: four were isolated from live patients and five from post-mortem materials. thus, there are no convincing data at present that could prove a causal relationship of the given substitution and the aggravation of a disease course. this is in accordance with previous observations. 7 concerning the resistance of studied strains to the widely used antiviral preparations, it was shown that all tested strains possessed the substitution s31n in the m2 protein that determine the resistance to adamantanes. there was no substitution in the position 275 of neuraminidase (na), which determines the resistance to oseltamivir (h275y). these substitutions are the characteristic indices of the eurasian lineage of swine influenza viruses. 8 thus all studied russian h1n1v isolates were resistant to adamantanes (rimantadine) and sensitive to oseltamivir. respiratory clinical samples taken in 2008 and 2009 that tested positive by real time reverse transcription (rt)-pcr for seasonal influenza viruses (a and b) and pandemic h1n1 2009 respectively were assessed for other respiratory viruses using the resplex ii panel ver2.0 system distributed by qiagen. results showed that co-infections with another 16 respiratory viruses were relatively rare, with a small number of samples having another co-infecting virus present, very few samples having two other viruses detectable in their samples, and none with further viruses. this low number of co-infecting viruses and the ability of certain cell lines not to support infection with particular viruses may make primary isolation of influenza viruses in cell lines easier than might have been thought previously. cm2 is the second membrane protein of influenza c virus and is posttranslationally modified by phosphorylation, palmitoylation, n-glycosylation, and dimer ⁄ tetramer formation. in the present study, we generated rcm2-c65a, a recombinant influenza c virus lacking cm2 palmitoylation site, and examined viral growth and viral protein synthesis in the recombinant-infected cells. the rcm2-c65a virus grew less efficiently than did the wild-type virus. membrane flotation analysis of the infected cells revealed that less np was recovered in the plasma membrane fractions of the rcm2-c65a-infected cells than that in the wild-type virus-infected cells, suggesting that palmitoylation of cm2 is involved in the affinity of the ribonucleoprotein complex to the plasma membrane, leading to the efficient generation of infectious viruses. influenza c virus has seven single-stranded rna segments of negative polarity, encoding pb2, pb1, p3, haemagglutiboth the a2, 6 linkage and its topology on target cells were critical for human adaptation of influenza a viruses. the binding preference of avian flu virus h5n1 ha to the a2, 3-linked sialylated glycans is considered the major factor that limited its efficient infection in human. currently, the switch in binding-specificity of human h5n1 viruses from a2, 3 to a2, 6-glycans did naturally occur, and limited humanto-human transmission was found. to monitor their potential adaptation in the human population, receptor-binding specificity surveillance was made in china. here, the binding specificity of 32 human h5n1 virus strains isolated from 2003 to 2009 was demonstrated. dual binding preference to a2, 3 and a2, 6-glycans were found in a ⁄ guangdong ⁄ 1 ⁄ 06 and a ⁄ guangxi ⁄ 1 ⁄ 08. furthermore, both of them showed a high affinity to the long-branched a2, 6-glycans, which predominate on the upper respiratory epithelial in human. our data suggests that the existence of h5n1 virus with binding specificity to humans should be of concern.introduction via envelope glycoprotein hemagglutinin (ha), influenza viruses bind to cell-surface glycosylated oligosaccharides terminated by sialic acids (sa) where their linkage is celland species-specific. differential receptor binding preference is a host barrier for influenza virus transmission. although most h5n1 viruses have low affinity to neu5aca2, 6gal (human-type) receptor, recent findings suggested that the adaptation of h5n1 virus to human by mutations in the receptor-binding site (rbs) do indeed happen and resulted in enhanced affinity to human-type receptor. [1] [2] [3] in contrast to its putative precursor, a ⁄ gs ⁄ gd ⁄ 1 ⁄ 96, diverse genotypes were presented in currently circulating h5n1 virus, accelerating evolution and widespread occurrence. 4, 5 to date, 10 distinct phylogenetic clades (0-9) were identified based on h5n1 ha, and the confirmed human infections were caused by clade 0, 1, 2ae1, 2ae2, 2ae3, and 7. 6 in china, human h5n1 disease was mostly caused by clade2ae3ae4, which was identified in 28 isolates from 39 confirmed patients from 17 provinces since 2003. clade 7 and clade 2ae2 are responsible for the case in 2003 and 2006, respectively. two current cases of 2009 and 2010 were due to clade 2ae3ae2. now information on receptor property has been documented in some h5n1 viruses of clade 1, 2ae1, and 2ae2. [1] [2] [3] 7 little is known about h5n1 virus of clade 2ae3ae4, particularly from human.recently, a2, 3-specific sialidase-treated red blood cell (rbc) agglutination assay was developed and used for receptor specificity screening of h5n1 virus. 3, 8 the a2, 6 or a2, 3-binding preference can be distinguished by the change of hemagglutination titer reacted with rbcs and enzymatic rbcs. since fine receptor specificity existed in h5n1 viruses, 9,10 the glycan array including sulfated-, fucosylated-, linear sialosides, di-sialosides, or direct binding assay with synthetic polyacrylamide (paa)-based sialylglycopolymers was also recommended for the receptor-specificity surveillance on h5n1 viruses. furthermore, the long-branched a2, 6 sialylated glycans were currently identified to predominate on the upper respiratory epithelial in human and the recognition of this topology, 6¢sln-ln is the key determinant for the human-adaptation of influenza a virus. 11 here, we analyzed the receptor-binding specificity of human h5n1 viruses isolated in china from 2003 to 2009. since 2003, a total of 39 h5n1 infection cases were confirmed in china from 17 provinces. the pharyngeal swabs and lower airway aspirations from the patients were collected within 12 days after disease onset, maintained in viral-transport medium, and tested within 24 hours.options for the control of influenza vii 