key: cord-256303-bpa571ys authors: hotez, peter j.; bottazzi, maria e.; singh, sunit k.; brindley, paul j.; kamhawi, shaden title: will covid-19 become the next neglected tropical disease? date: 2020-04-10 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008271 sha: doc_id: 256303 cord_uid: bpa571ys nan the daily world health organization (who) coronavirus situation reports highlight the rapid spread of covid-19 across europe, the united states, and many of the advanced nations in east asia [1] . currently, the major low-and middle-income nations such as bangladesh, brazil, democratic republic of congo, india, indonesia, mexico, nigeria, pakistan, philippines, and south africa, as well as central american and other nations are beginning to report an increase in covid-19 cases, but the numbers are still relatively small. for example, these highly populated nations now only account for about 1% of the confirmed cases, even though they represent approximately one-third of the global population. almost certainly, this situation will shift in the coming weeks and months. we believe there is a high probability the current numbers represent underestimates due to inadequate testing. surely, lack of access to diagnostic kits comprises one of the many components of weak health systems in resource-poor nations. beyond testing, it may turn out that the seasonal nature of some respiratory virus pathogens might also extend to the sars cov2. this means that cases would decline with warming temperatures, independent of control efforts. however, this would also indicate that though covid-19 might be peaking in the northern hemisphere this winter and spring, there is a real possibility that it will advance into tropical countries and the southern hemisphere later this year. in such case, some of the nations highlighted above might be vulnerable to the next wave of sars cov2 dissemination. sars cov2 is a new virus pathogen, and we need to appropriately assess its behavior over the span of a full year. if sars cov2 becomes a major respiratory virus pathogen in resource-poor countries of the tropics and subtropics, we might envision unprecedented levels of global morbidity and mortality. we have already seen how even strong health systems in new york and northern italy quickly became overwhelmed, and one can only imagine the terrible consequences of this virus in the poorest reaches of africa, asia, and latin america. based on the levels of illness we have seen to date in the northern hemisphere, we are especially worried about the fate of thousands of dedicated doctors, nurses, and other health care providers. accordingly, plos neglected tropical diseases will consider articles from the community of scientists and public health experts in asia, africa, and latin america now shifting their efforts to combat the covid-19 pandemic. we therefore welcome original high quality research papers and front matter articles, including viewpoints and editorials. our plos neglected tropical disease editors have just completed an exhaustive assessment of what constitutes a neglected tropical disease in order to keep our scope both relevant and timely [2] . however, as john lennon once said, "life is what happens to you while you're busy making other plans," and on that basis we now invite our community of ntd scientists to submit covid-19 papers on what may become a global health terror on a scale that rivals or even exceeds some of the world's major neglected tropical diseases. coronavirus disease 2019 (covid-19) situation report-68 what constitutes a neglected tropical disease? key: cord-306952-cpltrsa7 authors: de souza, pedro mansueto melo; gerson, gunter; dias, josebson silva; de melo, deborah nunes; de souza, sarlene gomes; ruiz, erasmo miessa; fernandes tavora, fabio rocha; cavalcanti, luciano pamplona de góes title: validation of verbal autopsy and nasopharyngeal swab collection for the investigation of deaths at home during the covid-19 pandemics in brazil date: 2020-11-04 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008830 sha: doc_id: 306952 cord_uid: cpltrsa7 nan brazil currently holds the second highest record of cases and deaths due to the coronavirus disease 2019 (covid-19) in the world [1] . the state of ceará, with >9 million inhabitants, in northeastern brazil, was the epicenter of the epidemic and one of the first states to present community transmission of the covid-19. forty-five days after the first confirmed case, there was a collapse of health services, contributing to the increase in the number of people dying at home [2] . in the scenario of a pandemic associated with an increase in the number of deaths outside a hospital environment, a specialized service in the investigation of fatalities was necessary to increase the capacity of the health system and to detect and report the death burden from the disease. "dr. rocha furtado" death verification service (svo-rf) is located in the capital fortaleza and, in partnership with the epidemiological surveillance office and the local public health laboratory, it acts in the investigation of deaths in the state, which was already reported in recent arboviruses epidemics [3, 4] . to fulfill its mission, svo-rf performs complete diagnostic autopsies (cda) for the investigation of deaths at home without medical assistance, as well as deaths without bona fide diagnosis prior to death [5] . moreover, unlike most other death verification services in brazil, the svo-rf also implemented a medical team that drives to the houses where the death occurred, to investigate cases where a clinical necropsy was not indicated, such as with patients with advanced cancer diagnoses or other chronic terminal illnesses that die at home, without any home care program. this team, called svo-mobile, is composed of a physician, a social worker, and a driver and usually acts in a complementary way to clinical necropsies that take place at the headquarters. in the covid-19 pandemic, however, the svo-mobile played a significant role since clinical necropsies were suspended following the ministry of health's guidelines [6] . due to the lack of biosafety at the headquarters, including an air treatment system at the facility, added to the community transmission of the virus and the risk of transmission by asymptomatic and presymptomatic patients, all autopsies were suspended. the svo-rf leadership saw then the need to expand the number of svo-mobile teams from 1 to 3, while still maintaining its regular team at the headquarters. all deaths from this period were investigated through physician-certified verbal autopsies (pcva), external body examination, and collection of nasopharyngeal swab samples in cases of suspected covid-19, with the 3 svo-mobile teams moving to their homes while continuing to receive bodies at headquarters. from the 10th to the 31st epidemiological week (ew) of 2020, including march 1 to august 1, the number of household deaths in the city of fortaleza attended by svo-rf was 2,215, representing an increase of 69.00% to the same period of 2018 and 2019. the weekly percentage variation between the number of consultations in 2020 with the average of 2018 to 2019 varied from −14.56% in the 30th ew to +355.65% in the 19th ew (table 1) . among the 2,115 household deaths in this period, 353 (16.69%) cases had clinical-epidemiological criteria for suspected covid-19, with the weekly variation starting from 0.00% in the first 2 weeks studied, up to 37.71% in the 20th ew (table 1) . for any suspected case, nasopharyngeal swab samples were collected and sent to the central laboratory of public health of ceará for the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus polymerase chain reaction technique in real-time (rt-pcr) testing. among the suspected cases, the rate of positivity in the sars-cov-2 survey ranged from 0.00% at the beginning and end of the period analyzed to 100.00% positivity in the 18th ew (table 1) . it is a noteworthy fact that there were no confirmed cases of sars-cov-2 among home deaths in the last 5 weeks, which may represent both a decrease in the circulation of the validation of verbal autopsy for the investigation of deaths at home during the covid-19 virus as well as the reestablishment of the capacity of the health services of providing medical assistance to possible new cases of the disease. therefore, in the light of an increase of up to 355.65% in the weekly number of home deaths, it was the ability to rapidly expand the investigation teams that made it possible to meet the excessive demand of these 2,115 deaths that occurred outside the hospital environment and confirm 280 deaths from covid-19 that would hardly have been identified without this service (table 1) . nevertheless, the use of the verbal autopsy did not allow the medical team to answer some questions about other home deaths that did not meet the criteria for suspicion for covid-19. for example, would it be possible that all the excess of unsuspected deaths from covid-19 resulted only from the difficulty of accessing health services? or could some of these deaths have been caused by covid-19 with an atypical clinical presentation without criteria for suspicion? in view of these limitations, we believe it is necessary to invest also in other forms of investigation, such as minimally invasive necropsies, whose applicability and safety have already been demonstrated in other developing countries [7] , with infectious diseases in general [8] and with covid-19 specifically [9] . we also advocate the expansion of biosafety measures for the entire network of death verification services, so that complete clinical necropsies can be performed even during pandemics, following the recommended good laboratory practices [10] and contributing to the understanding of the pathophysiological mechanisms that lead to death by covid-19 [11] worldwide. despite these and other limitations of the verbal autopsy certified by a physician [12] , we consider that, in a pandemic scenario, the investigation of home deaths by this method associated with the collection of samples for laboratory research constitutes a safe, financially viable, and secure method. it probably contributes to an increase in the detection of the disease and a consequent decrease in underreporting, especially in places where the collapse of the health system can lead to a rise in home deaths without medical assistance. health system collapse 45 days after the detection of covid-19 in ceará , northeast brazil: a preliminary analysis postmortem diagnosis of dengue as an epidemiological surveillance tool experience of the arbovirus death investigation committee in ceará , brazil institui a rede nacional de serviços de verificação de ó bito e esclarecimento da causa mortis (svo). brasília: ministério da saúde secretaria de vigilâ ncia em saúde. departamento de análise em saú de e vigilâ ncia de doenças não transmissíveis pathological methods applied to the investigation of causes of death in developing countries: minimally invasive autopsy approach infectious cause of death determination using minimally invasive autopsies in developing countries ultrasound-guided minimally invasive autopsies: a protocol for the study of pulmonary and systemic involvement of covid-19 autopsy in suspected covid-19 cases postmortem examination of patients with covid-19 performance of physician-certified verbal autopsies: multisite validation study using clinical diagnostic gold standards key: cord-260693-8mfuwx8l authors: seelig, frederik; bezerra, haroldo; cameron, mary; hii, jeffrey; hiscox, alexandra; irish, seth; jones, robert t.; lang, trudie; lindsay, steven w.; lowe, rachel; nyoni, tanaka manikidza; power, grace m.; quintero, juliana; stewart-ibarra, anna m.; tusting, lucy s.; tytheridge, scott; logan, james g. title: the covid-19 pandemic should not derail global vector control efforts date: 2020-08-31 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008606 sha: doc_id: 260693 cord_uid: 8mfuwx8l nan the covid-19 pandemic is placing immense pressure on health systems worldwide. this is particularly apparent in resource-poor settings with limited capacity to treat and contain new disease outbreaks. the world health organization (who) has emphasised the crucial need to sustain efforts to prevent, detect, and treat malaria during this pandemic [1] . however, a similar approach should also be adopted for the control of arboviral diseases of global importance, including dengue, zika, chikungunya, and yellow fever, as recommended by the pan-american health organization (paho) in their interim guidance on control of aedes aegypti mosquitos during the covid-19 pandemic [2] . for example, an unprecedented dengue epidemic continues to affect latin america and the caribbean, with a surge of cases experienced in the first half of 2020 [3] . even before the covid-19 pandemic began, many countries were struggling to control dengue. the public health interventions initiated to halt the covid-19 spread have already severely affected routine vector surveillance and control activities (such as regular household surveys) [4, 5] . the combined impact of both covid-19 and epidemics of dengue or other vector-borne diseases (vbds) could have potentially devastating consequences [6] . in view of these combined challenges, we reiterate our solidarity with the global partners who are dealing with the covid-19 pandemic, while strongly urging them to consider these recommendations for the control of vbds: • continue the implementation of the who's global vector control response 2017-2030 (gvcr) strategy and regional policies for vector control [7, 8] , with respect to inter-and intrasectoral collaboration, engagement and mobilisation of communities, and scaling up of vector control if required, according to the implementation plan of vector control activities, while adapting activities as necessary to prevent further spread of covid-19, in particular vector surveillance, which may need to be scaled down [9, 10] . and, more specifically with respect to vbd control, we present a list of recommended actions grouped into three categories: 1. maintain vector control operations in the context of covid-19 (adapting activities to improve worker safety and to prevent further spread): a. continue and enhance protection of vulnerable populations against vbds, e.g., through providing topical repellents and distributing bed nets, and deliver vector control safely while practising physical distancing. b. ensure the safety of community workers and volunteers through training on safe methods to conduct their work in the face of covid-19 and provide personal protective equipment (see section 2.5 in [2] for further details). c. perform intensive risk assessment and contingency planning for continuity of vector control by dedicated health personnel with appropriate training. this may involve moving from community-based control to household-, clinic-, hospital-, or school-level control measures. a. encourage global funding bodies and dedicated donors to provide long-term funding for capacity building in vector surveillance and control, including training and retention of local staff, and for contingencies during any anticipated lockdown. it is imperative not to reduce existing funding on vector control programmes or to lose sight of the value of vector control, which would offset any previous gains. b. improve the validity, availability, and proper use of rapid testing, both for covid-19 and vbds, to prevent misdiagnosis and to improve case management for potential coinfections and comorbidities [11] . c. identify gaps in practices and knowledge related to vector-borne disease control and encourage local and national stakeholders to address these deficiencies. d. improve preparedness for outbreaks (vector-borne or not) by devising a comprehensive preparedness plan that will identify all activities and materials required for each stage (from no risk to epidemic) and by providing strategic reserves of pharmaceuticals, personal protective equipment, public health insecticides, and vector control equipment. e. despite significant progress to date, many research activities are currently on hold. the search for new and effective tools for both malaria and dengue control must continue, including the use of artificial intelligence and novel technological solutions, such as drones, to assist in vector surveillance and control. f. integrate modelling to predict the combined impact of vbds and covid-19. 3. covid-19 disease monitoring and control innovations that could be applied to vector control: a. one of the encouraging aspects of the response to the covid-19 pandemic has been the rapid and open global dissemination of data, such as virus sequence data. this has allowed development of the first vaccine candidates in record time. we strongly recommend a similar effort to promote relevant data sharing on vectors, arboviruses, and other vbds to support public health interventions. the global vector hub (gvh) online platform was designed to facilitate easy access and exchange of information, data, and resources among relevant stakeholders, and an early version was launched in june 2020 to support global vector control efforts in the context of the covid-19 pandemic. b. provide accurate and up-to-date information and data from trusted sources to fight the spread of misinformation. c. support studies that map and evaluate the impact of distancing and isolation measures on vector control interventions and vbds, especially in large urban centres. it is expected that the most vulnerable and poorest populations will suffer most from covid-19, due to lack of access to appropriate care and the impacts of isolation measures on fragile livelihoods. it is vital that the covid-19 response does not increase vbd threats in these communities by derailing global vector control efforts. who urges countries to ensure the continuity of malaria services in the context of the covid-19 pandemic control of aedes aegypti in the scenario of simultaneous transmission of covid-19 pan american health organization/world health organization covid-19 in latin america: the implications of the first confirmed case in brazil dengue kills too'-latin america faces two epidemics at once. reuters world news preventing dengue epidemics during the covid-19 pandemic action plan on entomology and vector control 2018-2023. 56th directing council of paho, 70th session of the who regional committee for the americas. washington, d.c.: paho, 2018 (resolution cd56.11) world health organization. tailoring malaria interventions in the covid-19 response. geneva: who, 2020 community-based health care, including outreach and campaigns, in the context of the covid-19 pandemic. geneva: who, 2020 covert covid-19 and false-positive dengue serology in singapore the opinions expressed in this letter are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention and pan-american health organization/world health organization. key: cord-268329-apl6n6jl authors: antunes, douglas eulálio; goulart, isabela maria bernardes; goulart, luiz ricardo title: will cases of leprosy reaction increase with covid-19 infection? date: 2020-07-17 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008460 sha: doc_id: 268329 cord_uid: apl6n6jl nan the coronavirus disease 2019 (covid-19)-caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a betacoronavirus (betacov)-emerged for the first time as an outbreak of pneumonia in wuhan, china, and it is now spreading to several countries around the world [1] . the clinical spectrum of covid-19 ranges from asymptomatic presentation to sars [2] . the main symptoms of the disease are fever and cough, occurring in 94% and 79% of symptomatic individuals, respectively [3, 4] . elderly individuals are at a higher risk of complications and death associated with covid-19 because a less vigorous immune response is fatal, especially in those who have comorbidities, such as hypertension, diabetes, coronary heart disease, and chronic obstructive pulmonary disease [4] . the disease has an incubation period that ranges from 5.1 to 11.5 days. the spread of the virus is favored during the asymptomatic period of infected individuals. in addition, sars-cov-2 remains viable for 3 hours in the form of aerosols and 72 hours on plastic and stainless steel surfaces [5, 6] . the numbers related to disease incidence are alarming every day, as the total number of deaths increases in proportion to increases in the number of disease cases. the impact of the pandemic on public health in these countries has been disturbing because the percentage that has advanced to severe forms of the disease will require care in intensive care units. this type of care might cause a collapse in the overall health system. the brazilian society of hansen's disease issued an alert on leprosy and covid-19 coinfection that warned of the risk of infection in those individuals being treated for leprosy reactions. however, further clarification on epidemiological predictions for the covid-19 infection and its possible role as an immune stimulus triggering leprosy reactions are necessary [7] . leprosy reactions are immunological events that affect 8% to 33% of leprosy patients, reaching 64.5% in some regions, manifesting before, during, and/or after treatment with multidrug therapy (mdt) [8] . these phenomena are subdivided into type 1 reactions (t1r) and erythema nodosum leprosum (enl), which is part of the type 2 reaction (t2r). in the t1r, the type 1 helper (th1) response predominates in lesions and the serum of patients with high levels of cytokines tumor necrosis factor-alpha (tnf-α), interferon-gamma (ifn-γ), interleukin-17 (il-17), and c-x-c motif chemokine 10 (cxcl10) [9] . some studies of sars-cov-2 infection have reported the presence of a cytokine storm syndrome and a subgroup of patients who progressed to severe forms of the disease, expressing a pro-inflammatory profile in plasma with il-2, il-7, tnf-α, and others as significant complications, such as occurs in t1r [10, 11] . furthermore, the elevation of ferritin and il-6 were characterized as predictors of fatality for the disease, suggesting that mortality may be caused by hyperinflammation induced by the virus [11] . in relation to enl, the formation of immune complexes occurs in the blood and deposits in the tissues, especially the skin, kidneys, and joints and is, therefore, a type iii hypersensitivity reaction [12] . an extensive neutrophil infiltration in pulmonary capillaries may be induced by covid-19, evidenced in an autopsy specimen from lungs of severe patients [13] . neutrophils are associated with enl, since skin lesions present an intense perivascular infiltrate of neutrophils throughout the dermis, and these cells are able to trigger enl, allowing tnf-α and il-8 release after stimulation with lipopolysaccharide (lps) [14, 15] . we support that neutrophils, influenced by covid-19, may propitiate enl in those infected patients. in both reactions, we warn of the possible effect that covid-19 infection may have on the number of cases of these immunological events because the presence of infection is an important risk factor for triggering leprosy reactions [8] . another disturbing factor, which may contribute to the susceptibility of those affected by leprosy reactions, are the treatments implemented during these events that interfere with the inflammatory response of these patients. in the treatment of t1r, prednisone is used at a dose of 1 mg/kg and sometimes even higher (1.5 mg/kg) [16] . prednisone is a drug similar to the endogenous hormone cortisol that allows several effects, such as anti-inflammatory, immunosuppressive, anti-proliferative, and vasoconstrictive actions [17] . the genetic mechanism of this drug is related to interference in the transcription of nuclear factor kappa b (nf-κb), which may cause suppression of the synthesis of pro-inflammatory cytokines, such as il-1, il-2, il-6, il-8, tnf-α, ifn-γ, and vascular endothelial growth factor (vegf) [17] . the anti-inflammatory and immunosuppressive effects of glucocorticoids are dose dependent. therefore, at high doses, their effects are immunosuppressive, causing emerging infections, such as covid-19, in these susceptible patients [17] . in enl, thalidomide is prescribed, an immunomodulatory drug that inhibits the expression of tnf-α and ifn-γ, affecting the pro-inflammatory activity of these patients. it also interferes in response to other microorganisms, such as covid-19, which also may favor the manifestation of severe forms referring to the clinical spectrum of this disease [18] . despite the fact that some t2r patients may express pro-inflammatory cytokines in several tissues, the expression of il-10 may be elevated in those individuals without the use of thalidomide, indicating mycobacterium leprae viability, which can contribute to a low immune response to covid-19 in these individuals [19] . even though some clinical trials are underway to investigate the use of thalidomide-alone or combined with systemic glucocorticoids, such as methylprednisolone-to treat moderate and severe covid-19 pneumonia, there are no final conclusions ensuring that they are an effective treatment [20] . thus, we believe that, as the number of new cases of covid-19 infection increases, the incidence of leprosy reactions may also increase considerably. in addition, it is worth remembering that reactive patients are treated with drugs that affect the stability of the immune system, which, in turn, can contribute to the manifestation of sars, especially in those who are elderly with comorbidities. it is important to remember that, as early as possible, social isolation measures should be started to flatten the curve and reduce the number of new covid-19 cases, consequently preventing an unprecedented collapse of all levels of care and guaranteeing the monitoring of leprosy reactions. finally, for leprosy patients on treatment or after discharge from mdt, we recommend what medical doctors and health teams advise with the goal to avoid exposure to sars-cov-2, such as the following: the use of masks in the community; stay home and self-isolate from others; wash hands regularly for 20 seconds, with soap and water or alcohol-based hand rub; avoid close contact (staying 2 meters away from other people); and cover your nose and mouth with a disposable tissue or flexed elbow when you cough or sneeze. these precautions will aid prevention of the spread of covid-19 infection-and its severe manifestations, including leprosy reactions. evaluation and treatment coronavirus (covid-19) early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia. the new england journal of medicine clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china. jama internal medicine the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application. annals of internal medicine aerosol and surface stability of sars-cov-2 as compared with sars-cov-1. the new england journal of medicine guidelines to doctors of the brazilian society of hansen's disease (sbh) on the possibility of coinfection leprosy and covid-19 identification of clinical, epidemiological and laboratory risk factors for leprosy reactions during and after multidrug therapy increased serum circulatory levels of interleukin 17f in type 1 reactions of leprosy clinical features of patients infected with 2019 novel coronavirus in wuhan covid-19: consider cytokine storm syndromes and immunosuppression reactions in leprosy: an epidemiological study of 386 patients in west nepal targeting potential drivers of covid-19: neutrophil extracellular traps. the journal of experimental medicine a systematic review of immunological studies of erythema nodosum leprosum neutrophils isolated from leprosy patients release tnf-alpha and exhibit accelerated apoptosis in vitro understanding the type 1 reactional state for early diagnosis and treatment: a way to avoid disability in leprosy. anais brasileiros de dermatologia a practical guide to the monitoring and management of the complications of systemic corticosteroid therapy tnf alpha signaling beholds thalidomide saga: a review of mechanistic role of tnf-alpha signaling under thalidomide. current topics in medicinal chemistry differential expression of ifn-gamma, il-10, tlr1, and tlr2 and their potential effects on downgrading leprosy reaction and erythema nodosum leprosum a review of sars-cov-2 and the ongoing clinical trials. international journal of molecular sciences the authors are grateful for the entire assistance and laboratory team of the center for sanitary dermatology and leprosy (credesh). key: cord-260412-yjr83ef6 authors: hotez, peter j.; bottazzi, maria elena title: developing a low-cost and accessible covid-19 vaccine for global health date: 2020-07-29 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008548 sha: doc_id: 260412 cord_uid: yjr83ef6 nan there is an urgent need to advance safe and affordable covid-19 vaccines for low-and middle-income countries of asia, africa, and latin america. such vaccines rely on proven technologies such as recombinant protein-based vaccines to facilitate its transfer for emerging market vaccine manufacturers. our group is developing a two-pronged approach to advance recombinant protein-based vaccines to prevent covid-19 caused by sars-cov-2 and other coronavirus infections. one vaccine is based on a yeast-derived (pichia pastoris) recombinant protein comprised of the receptor-binding domain (rbd) of the sars-cov formulated on alum and referred to as the cov rbd219-n1 vaccine. potentially, this vaccine could be used as a heterologous vaccine against covid-19. a second vaccine specific for covid-19 is also being advanced using the corresponding rbd of sars-cov-2. the first antigen has already undergone current good manufacturing practices (cgmp) manufacture and is therefore "shovel ready" for advancing into clinical trials, following vialing and required good laboratory practice (glp) toxicology testing. evidence for its potential efficacy to cross-protect against sars-cov-2 includes cross-neutralization and binding studies using polyclonal and monoclonal antibodies. evidence in support of its safety profile include our internal assessments in a mouse challenge model using a lethal mouse-adapted sars strain, which shows that sars-cov rbd219-n1 (when adsorbed to aluminum hydroxide) does not elicit eosinophilic lung pathology. together, these findings suggest that recombinant protein-based vaccines based on the rbd warrant further development to prevent sars, covid-19, or other coronaviruses of pandemic potential. "the thing we have to think about now that's different is, how do we produce vaccines specifically for the developing world if this is a truly global epidemic."-seth berkley, ceo, gavi as of june 2020, covid-19 caused by the sars-cov-2 coronavirus has infected more than 7 million people globally (confirmed cases) and caused almost 400,000 deaths [1] . although the epidemic began in china, europe, and the united states, there are significant concerns about the risks of disease emergence in low-and middle-income nations. there are now more almost 750,000 cases in brazil, 300,000 cases in india, and 50,000 cases in south africa, such that covid-19 will become widespread among the poor living in the group of 20 nations [1, 2] . moreover, sars-cov-2 infection is expected to emerge in the global south [3] . in the african region of the world health organization (who), covid-19 is now spreading in the populated areas of ghana, nigeria, and democratic republic of congo, and presumably across the region [1] . in nations such as india, for example, the feasibility of enforcing social distancing in large and crowded urban centers will be particularly daunting [3] , so that ensuring access to a safe and affordable covid-19 vaccine will become a global priority. dr. seth berkley, the ceo of gavi, the vaccine alliance, has highlighted the importance of prioritizing a covid-19 vaccine specifically for these countries [4] . at least a dozen covid-19 candidate vaccines are under development using different technology platforms [5] , with an emphasis on speed, maximizing safety, and avoiding vaccineinduced immunopathology [6] . many of these will enlist cutting-edge nucleic acid delivery technologies and other innovative approaches. in the meantime, there is urgency to address and rapidly respond to gavi's charge and pursue safe, low-cost, easily administered, and rapidly scalable approaches. for instance, texas children's center for vaccine development (cvd) at baylor college of medicine, in collaboration with its nonprofit product development partners-seattle-based path and infectious disease research institute (idri)-have been spearheading a coronavirus vaccine program focusing on recombinant subunit protein vaccines produced in a globally available microbial fermentation platform, and optimized to maximize yield following expression and protein purification [7, 8] . towards this goal, we are now also developing the sars-cov-2 rbd recombinant protein as a potential vaccine candidate, in parallel with the existing cov rbd219-n1 candidate vaccine, which was previously developed and manufactured under cgmp in 2016 [7] [8] [9] [10] . the bulk drug substance has been stored frozen (−70˚c to 80˚c) and remains stable through ongoing testing. furthermore, an independent quality assessment confirmed the suitability of the material through phase 2 clinical trials. both rbd vaccine candidates have potential as vaccine antigens to prevent sars-cov-2 infection and/or covid-19. overall, our initial approach relies on advancing the already manufactured cov rbd219-n1 as a heterologous recombinant subunit vaccine to protect against both sars and covid-19 [9] , and in parallel accelerate the advancement of the sars-cov-2 rbd candidate as a homologous covid-19 vaccine (fig 1) . our preliminary studies now show that the sars-cov-2 rbd candidate, which is specific for the sequence of the sars-cov-2, can also be highly produced in the yeast p. pastoris. both approaches reinforce each other, as the processes developed for the cov rbd219-n1 candidate also apply to the sars-cov-2 candidate, and both antigens downstream could be further developed as potentially a bivalent or a universal coronavirus vaccine. the sars-cov protein known as cov rbd219-n1 was selected on its ability to elicit high titers of neutralizing antibodies against both sars-cov pseudotype virus and live sars-cov virus [7, 8] , prior to confirmatory testing against sars-cov challenge in animal models. it also induced high-level neutralizing antibodies and protective immunity with minimal immunopathology in mice after a homologous virus challenge with sars-cov (ma15 strain) [9, 10] . there are several advantages of the cov rbd candidate antigens and vaccines for purposes of global health: 1. high yield and low cost. the antigens are expressed in p. pastoris, a low-cost expression platform, which can be produced and scaled at high yields [7, 8] . by deleting an n-linked glycosylated asparagine at the n-1 position of rbd219, both the yield and antigenicity improved. at a 10-liter scale production process, the cov rbd219-n1 antigen was produced through fermentation at 400 mg/l fermentation supernatant (fs) with purification recovery >50% [7, 8] . a panel of characterization tests indicates that the process is reproducible and robust and that the purified, tag-free rbd219-n1 protein has high purity and a well-defined structure. it is therefore suitable for both pilot scale manufacturing and for transition into process improvements leading to industrial scale manufacturing. 2. technology transfer. the process is suitable for technology transfer to emerging market vaccine manufacturers (aka dcvms, developing country vaccine manufacturers) having expertise in fermentation technology (https://www.dcvmn.org/) [11] . the p. pastorisderived recombinant protein is currently produced by several dcvms, including those in bangladesh, brazil, cuba, india, and indonesia. 3. shovel ready. the cov rbd219-n1 antigen was manufactured under cgmp and can be vialed to produce between 20,000 and 200,000 doses, with the possibility of transferring production processes and cell banks to dcvms for large-scale production sufficient to meet global needs. beyond low cost and ease of potential technology transfer to dcvms, an advantage of employing a recombinant protein subunit vaccine is the long-standing safety record of this class of vaccines, and the fact that this technology has been used for the licensure of two other antiviral vaccines-hepatitis b and human papillomavirus, as well as biologics (e.g., insulin) [11] . in addition to their low cost and suitability for use in public immunization programs in lowand middle-income countries, we pursued rbd recombinant protein-based vaccines as a technology to maximize safety relative to other platforms, such as virus vectors that have previously been found to induce immune enhancement. for instance, immune enhancement in children following a formalin-inactivated respiratory syncytial virus (rsv) vaccine was first reported in the 1960s and later shown to occur in laboratory animals with early prototype sars-cov vaccines using virus-vectored platforms or inactivated virus constructs [12] . we have recently summarized the major safety concerns of some prototype coronavirus vaccines based on studies conducted in laboratory animals (rodents, ferrets, and nonhuman primates) [12] . they include the following points. some of the earliest sars-cov vaccine candidates used vectored-based platforms, and these were associated with immune enhancement or activation. in 2004-2005, scientists at the public health agency of canada's national microbiology laboratory in winnipeg, manitoba (who helped to develop the first successful ebola vaccine), found that a recombinant modified vaccinia ankara (rmva) expressing the s-spike protein resulted in severe liver pathology upon sars-cov virus challenge. similarly, rmva expressing the s-spike also resulted in lung immunopathology in rhesus macaques, as did other virus-vectored constructs. lung immunopathology is also linked to whole inactivated viral vaccines. however, it was determined that in many cases eosinophilic pathology is driven by the sars nucleocapsid (n) protein, although a recent trial in nonhuman primates found that an alum-adjuvanted inactivated sars-cov-2 vaccine did not induce immunopathology [13] . among the major conclusions of these studies was that they may be driven by t helper-17 (th17) responses linked to interleukin-6 [12, 14] , and that aluminum formulations exhibit greatly reduced immunopathology [15] . given the history of virus-vector platforms and inactivated vaccines in eliciting eosinophilic immunopathology, our emphasis has been on the evaluation of inexpensive recombinant proteins produced in microbial systems. these are comprised of the cov rbd219-n1 antigen, encoding amino acids 319-536 (219 aa) of the sars-cov s-spike protein [7] [8] [9] [10] , and now a second, cov2 rbd antigen, which is also expressed without the n-terminal amino acid. the rationale for selecting the rbd domain of the s protein includes focusing on the key component that binds to the human angiotensin converting enzyme 2 (ace2) receptor, and removing the known elements of the s protein involved in immune enhancement. supporting studies summarized elsewhere emphasize how s protein peptides outside of the rbd can induce immune enhancement in non-human primates [12] . moreover, cov rbd219-n1 induce high titers of neutralizing antibodies in mice and 100% infection against sars cov virus challenge [10] . alum formulations of cov rbd 219-n1 do not induce immunopathology [10] , a finding consistent with other published studies [13] [14] [15] . there is evidence to justify advancing the cov rbd219-n1 antigen as either a homologous vaccine against sars [7] [8] [9] [10] or as a heterologous vaccine against covid-19 [9] . in parallel, a cov2 rbd protein candidate is being advanced. regarding the former, against sars cov homologous virus challenge the vaccine formulated on alum exhibits high levels of protective immunity and with evidence of minimal or no immune enhancement [10] . with regards to cross-protection against sars cov2, the rbd of the sars-cov-2 and cov rbd219-n1 share significant similarity of amino acid sequence (> 75% identity, >80% similarity) and there is evidence that both viruses use the human ace2 receptor for cell entry [9] . further published studies indicate strong antigenic similarities between the sars-cov and sars-cov-2 rbds, and the potential for cross protection. for example, serum from a convalescent sars-cov patient was shown to neutralize sars-cov-2 driven entry [16] . moreover, new studies by tai and colleagues find that using pseudotyped sars-cov-2, the sars-cov rbd blocks the entry of both sars-cov and sars-cov-2 pseudovirus into human ace2-expressing 293t cells [17] . through pseudovirus neutralization activity, it was found that sars-cov rbd-specific antisera could neutralize sars-cov-2 pseudovirus infection, suggesting that sars-cov rbd-specific antibodies can cross-react with sars-cov-2 rbd and cross neutralize sars-cov-2 pseudovirus infection [17] . additional studies find that multiple (but not all) neutralizing monoclonal antibodies bind to both rbds [9, 18, 19 ]. an international priority is the scale-up and global access of an affordable and safe recombinant vaccine to prevent emerging coronavirus infections, including covid-19. our aspirational goal is to protect global populations at risk for this important emerging virus infection. a low-cost recombinant protein antigen expressed in p. pastoris and formulated on aluminum or other accessible adjuvants represents a highly accessible technology to transfer to low-and middle-income countries. it represents one of several key mechanisms for ensuring that populations across the major affected nations of africa, asia, and the americas will benefit from covid-19 vaccinations. world health organization. covid-19 situation reports poverty and the impact of covid-19: the blue-marble health approach millions in india under coronavirus lockdown as major cities restrict daily life will vaccines reach low-income countries during a global pandemic the outbreak of sars-cov-2 pneumonia calls for viral vaccines don't rush to deploy covid-19 vaccines and drugs without sufficient safety guarantees optimization of the production process and characterization of the yeast-expressed sars-cov recombinant receptor-binding domain (rbd219-n1), a sars vaccine candidate yeast-expressed recombinant protein of the receptor-binding domain in sars-cov spike protein with deglycosylated forms as a sars vaccine candidate potential for developing a sars-cov receptor-binding domain (rbd) recombinant protein as a heterologous human vaccine against coronavirus infectious disease (covid)-19 yeast-expressed sars-cov recombinant receptor-binding domain (rbd219-n1) formulated with alum induces protective immunity and reduces immune enhancement the yeast stands alone: the future of protein biologic production covid-19 vaccine design: the janus face of immune enhancement rapid development of an inactivated vaccine candidate for sars-cov-2 the potential role of th17 immune responses in coronavirus immunopathology and vaccine-induced immune enhancement. microbes and infection covid-19 vaccines: neutralizing antibodies and the alum advantage pö hlmann s. the novel coronavirus 2019 (2019-ncov) uses the sars coronavirus receptor ace2 and the cellular protease tmprss2 for entry into target cells characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine cryo-em structure of the 2019-ncov spike in the prefusion conformation identification of sars-cov rbd-targeting monoclonal antibodies with cross-reactive or neutralizing activity against sars-cov-2 key: cord-001484-va0teako authors: ahmed, sarah a.; van den ende, bert h. g. gerrits; fahal, ahmed h.; van de sande, wendy w. j.; de hoog, g. s. title: rapid identification of black grain eumycetoma causative agents using rolling circle amplification date: 2014-12-04 journal: plos negl trop dis doi: 10.1371/journal.pntd.0003368 sha: doc_id: 1484 cord_uid: va0teako accurate identification of mycetoma causative agent is a priority for treatment. however, current identification tools are far from being satisfactory for both reliable diagnosis and epidemiological investigations. a rapid, simple, and highly efficient molecular based method for identification of agents of black grain eumycetoma is introduced, aiming to improve diagnostic in endemic areas. rolling circle amplification (rca) uses species-specific padlock probes and isothermal dna amplification. the tests were based on its sequences and developed for falciformispora senegalensis, f. tompkinsii, madurella fahalii, m. mycetomatis, m. pseudomycetomatis, m. tropicana, medicopsis romeroi, and trematosphaeria grisea. with the isothermal rca assay, 62 isolates were successfully identified with 100% specificity and no cross reactivity or false results. the main advantage of this technique is the low-cost, high specificity, and simplicity. in addition, it is highly reproducible and can be performed within a single day. black grain eumycetoma represents the most common fungal mycetoma worldwide. this chronic, erosive infection of subcutaneous tissues particularly affects the lower extremities and leads to severe disability [1] . the disease is considered a major health problem in tropical areas and is prevalent among people of low socio-economic status [2] . mycetoma presents as a subcutaneous mass with multiple sinuses that discharge pus, serous fluid and grains, i.e. the characteristic compact grains of the causative agent formed inside the lesion [3] . a wide range of microorganisms has been reported to cause mycetoma. for treatment, not only differentiation between (fungal) eumycetoma and (bacterial) actinomycetoma is important, but also the identity of the causative agent, since species differ in their response to antimicrobial drugs [4] . in endemic countries, clinical diagnosis may be the only diagnostic method. a fully developed mycetoma lesion is easily identified clinically, whereas in early stages with the absence of grains, the infection may be confused with phaeomycosis or soft tissue tumors [1] . in such cases fine needle aspiration cytology or deep surgical biopsy for histological examination are useful [1, 5] . some fungal and bacterial grains have a characteristic histological appearance which helps in provisional identification, but recognition of the causative species remains impossible [6] . isolation of the pathogen from discharged grains or from biopsies allows identification of agents that sporulate, but most of the species lack phenotypic characteristics [3] . molecular techniques have been introduced to facilitate the identification of nondescript organisms [7, 8, 9] , but are of high cost and time-consuming. thus, there is a need for a fast, simple and reliable method for identification. rolling circle amplification (rca) is a powerful diagnostic method based on detection of specific nucleic-acid sequences and enzymatic amplification of circularized oligonucleotide probes under isothermal conditions [10] . the probes are linear oligonucleotides that contain two target-complementary sequences at their ends joined by linkers [11] . the ends of the probe hybridize to the complementary target in juxtaposition and then ligate which allows the circularization of the probe [11] . the circular structured molecule then amplifies with dna polymerase that has strand displacement and progressive dna synthesis activity resulting in series of repeats of the original circular template [11, 12] . the technique has been proven to be rapid, specific and low-cost for molecular identification of viruses, bacteria, and fungi [13, 14, 15, 16] . it has been applied for identification of a rare black grain mycetoma species exophiala jeanselmei [17] . in addition, rca has been used successfully for identification of white grain mycetoma species scedosporium boydii [18] . the aim of the present study is to develop rca-based diagnostics for the most common agents of black-grain eumycetoma. the study included 62 isolates belonging to eight species causing black grain mycetoma: madurella mycetomatis (n = 32), m. fahalii (n = 1), m. pseudomycetomatis (n = 3), m. tropicana (n = 2), trematosphaeria grisea (n = 10), falciformispora senegalensis (n = 6), f. tompkinsii (n = 2), and medicopsis romeroi (n = 6). strains were obtained from the reference collections of cbs-knaw fungal biodiversity centre (utrecht, the netherlands) and the mycetoma research centre (mrc, khartoum, sudan) and are listed with metadata in table 1 . type strains of all tested species were included. all strains were identified down to species level by sequencing of the rdna its region [19, 20] . dna was extracted using cetyltrimethyl ammonium bromide (ctab) method as described by möller et al. [21] . amplification of the its region was performed using primers v9g and ls266 [22] in a 25 ml reaction mixture containing: 10 ng of template dna, 0.1 mm each dntp, 0.6 u taq polymerase (gc biotech, alphen aan den rijn, the netherlands), 1 ml of each primer (10 pmol) and 2.5 ml reaction buffer (0.1 m tris-hcl, 0.5 m kcl, 25 mm mgcl 2 , 0.1% gelatin, 1% triton x-100). pcr reactions consisted of a 5 min predenaturation step at 95uc, followed by 30 cycles of 95uc for 30 s, 52uc for 30 s and 72uc for 1 min, with final post elongation step at 72uc for 7 min. pcr products were detected by electrophoresis using 1% agarose gels. sequences of the its region were used to design 8 probes specific for each species used in this study. two alignments were generated since the analyzed species were known to belong to two different fungal orders [19, 20] . its derived from madurella (sordariales) were aligned with 200 isolates of chaetomiaceae including chaetomium, thielavia, and achaetomium. for the remaining species (pleosporales) an alignment was constructed to include representative isolates of the family trematosphaeriaceae and of coelomycetes in the suborder pleosporineae. sequences were aligned using bionumerics v4.61 (applied maths, sint-martens-latem, belgium). probes were designed with minimum secondary structure and were checked using primerselect (dnastar lasergene, wi, u.s.a.). to insure specificity of the probes, target-specific sites of each padlock probe was submitted to blast in ncbi sequence database for homologous sequences. the melting temperature of the 59 end of the probe binding arm was designed to be.63uc while for the 39 end binding arm it was designed to be at least 15uc below the annealing temperature. the probes were phosphorylated at the 59 end and are listed in table 2 . probe linkers were taken from zhou et al. [23] . padlock probe ligation was performed in a mixture consisting of 1 ml purified its amplicons, 2 u pfu dna ligase (epicentre biotechnologies, madison, wi, u.s.a.), and 0.1 mmol/l padlock probe in buffer (20 mm tris-hcl ph 7.5, 10 mm mgcl 2 , 20 mm kcl, 0.1% igepal, 0.01 mm ratp, 1 mm dtt), with a total reaction volume of 10 ml. ligation conditions were: 5 min denaturation at 94uc, followed by 7 cycles of 94uc for 30 sec, 63uc for 4 min, and final cooling at 10uc. prior to rca amplification reaction and in order to reduce the ligation-independent amplification, ligation products were treated by addition of 10 u exonucleases i and 10 u exonucleases iii (new england biolabs, hitchin, u.k.) with a final volume of 20 ml. the mixture was then incubated for 30 min at 37uc, followed by 3 min at 94uc to deactivate the endonuclease enzymes. rca amplification reaction was performed in a 50 ml mixture containing; 2 ml ligation product, 8 u bst dna polymerase (new england biolabs), 10 pmol of each rca primer (table 2) , and 400 mm dntp mix. the mixture was incubated at 65uc for 60 min and cooled at 10uc. electrophoresis on a 1% agarose gel was used to visualize rca products. a positive reaction is indicated by the presence of ladder-like pattern. the reaction was also visualized by adding 1.0 ml of a 10-fold diluted sybr green i (cambrex bioscience, workingham, u.k.) to 10 ml of the amplification product. accumulated double stranded dna was detected with uv transilluminator (vilber lourmat, marne-la-vallée, france). the specificity of the 8 rca probes was tested using strains of black-grain mycetoma causative species listed in table 1. analytical sensitivity was determined using 10-fold serial dilution of m. mycetomatis (cbs 109801) and m. fahalii (cbs 129176) dna and the test was performed as mentioned above. in addition, rca was performed directly using dna samples without amplification of the target gene. to evaluate the detection limit from direct dna samples two-fold serial dilutions of target dna were tested. the sensitivity of the rca probes was also determined by 10fold serial dilution of myc and mfah probes tested with amplified its of m. mycetomatis and m. fahalii respectively. rca was used to identify 62 strains belonging to eight species causing human eumycetoma. since black grain eumycetoma species are known to be phylogenetically distant, it is easy to find unique sites for their identification. the ribosomal its region was sufficient for identification of all species and showed no intraspecific variability within a set of 100 m. mycetomatis strains in our collection. for m. mycetomatis, m. tropicana, m. pseudomycetomatis, and f. senegalensis the its1 region was treatment of eumycetoma largely depends on the causative pathogen. identification of mycetoma agent with phenotypic features is too limited, and physiological and biochemical techniques are laborious, time-consuming and nonspecific, whereas the currently available molecular methods based on dna sequencing are specific but extremely expensive. we describe rolling circle amplification method for identification of black grain eumycetoma using species-specific padlock probes. eight probes were designed and successfully used for species identification and the results were easily visualized in 1% agarose gel. rca provides a simple, reproducible, and costeffective method for rapid identification of mycetoma agent that can be used in low-resource clinical settings. rca results for the tested strains were easily visualized in 1% agarose gel. positive reactions demonstrated ladder like patterns while negative reactions resulted in a clear background (fig. 1) . with sybr green, positive results showed green fluorescence when exposed to uv light, while negatives did not. when exonucleolysis was performed some inhibition was observed with low rca positive signals on gel or with fluorescence. faint nonspecific bands were observed when this step was omitted. rca reactions were performed successfully without digestion with exonucleases, as the non-specific bands did not interfere with rca results. all m. mycetomatis strains were correctly identified with rca, irrespective of their geographical origin (sudan, india, mali) (fig. 2) . for the other agents, each individual species-specific probes yielded positive results with their corresponding species and with 100% agreement with its sequencing (fig. 2, table 3 ). no cross reactivity or false positive and negative results were observed. the sensitivity of rca when using amplified product of the target gene was less than 32610 23 ng of dna. a higher concentration of 100 ng is needed when the test is carried out directly from the dna samples without amplification of the its. the probes were very sensitive and a concentration of 6.6610 25 ng was successfully ligated and then amplified with rca. the turnaround time required for conducting the entire experiment including pcr amplification of target dna, rca processing and analysis was found to be 6 hours. dna sequencing of the its region took more than 8 hours to be performed (fig. 3 ). mycetoma is a unique tropical disease, endemic in many tropical and subtropical regions that has been recently added to the who list of neglected tropical diseases. [24] . it is mainly prevalent in what is known as ''mycetoma belt'' which includes mexico, senegal, sudan, india and other countries between tropic of cancer [1] . in 2014, a mycetoma consortium of scientists and physicians published research gaps on mycetoma which need to be addressed in the coming years [2] . one of the research priorities identified was the need to develop a reliable and cost-effective method for species identification to improve diagnosis [2] . mycetoma agents have been extensively studied in recent years [8, 9, 20] . the large phylogenetic distance between a number of these agents provides the possibility to use a moderately variable marker like rdna its for species identity. ahmed et al. [25] developed pcr-restriction fragment length polymorphism (rflp) for identification of m. mycetomatis targeting the its region. however, with the description of the molecular siblings m. fahalii, m. pseudomycetomatis, and m. tropicana [26] the method might be insufficiently accurate. moreover, there is a need for identification these siblings species; madurella grisea appeared to be distantly related and was re-named as t. grisea [20] . in the present study we developed a simple, fast and highly specific molecular method for the identification of agents of black grain mycetoma. in this method, the its region is easily amplified using one set of primers, which simplifies the use. in a second, isothermal amplification reaction padlock probes are used to identify the species by rca. the only equipment necessary is a thermocycler for the pcr reaction and a water bath or heating block for the rca reaction. this relative simplicity enhances possible use in routine laboratories in endemic areas. due to its robustness, high potential, and reproducibility, rca is increasingly used as a diagnostic tool in pathogenic fungi, e.g. agents of chromoblastomycosis, dermatophytes, aspergillus, candida, and talaromyces marneffei [16, 23, 27, 28] . the method does not require dna sequencing and is therefore considered as a rapid and cost-effective. applications are being expanded to nano-and biotechnology [29] . in the present study eight species-specific probes were designed and used for identification of 62 isolates. for the rca reaction species probe hybridization to the 39 and 59 ends of target dna and joining of adjacent ends by dna ligase when both show perfect complementarity. the ligation appears to be highly specific and thus the method can detect single nucleotide polymorphism [30] . the amplification reaction is driven by an isothermal dna polymerase to amplify the circularized probes with high efficiency and an estimated capacity to synsthesize more than 70,000 bp per hour [31] . rca products can be detected with different methods including gel electrophoresis, radiolabeling, uv absorbance, fluorescence, and single molecule detection [32] . it was known that the positive signals can be detected within 15 min after starting the rca reaction by real time pcr [23] . in the present study the rca positive signal was easily visualized using both gel electrophoresis and fluorescent dye. the duration of our rca protocol was 2 h, but additional time is required for dna extraction and its amplification. compared to the dna sequencing the turnaround time for rca is 2 hours less than sequencing and this even more if there is no in-house sequencer available. our results with eight padlock probes showed that rca accurately identified all species with no cross reactivity (fig. 1) . it may be concluded that rca is extremely useful for specific identification of agents of mycetoma. performance and rapid turnaround time features make the rca suitable for quick and reliable diagnosis, which is an enormous improvement compared to the current phenotypic identification of mostly non-sporulating cultures. future application of rca could be the detection of agents dna directly from clinical samples without requirement of culturing. the mycetoma knowledge gap: identification of research priorities management of mycetoma a new technique for the diagnosis of mycetoma using fixed blocks of aspirated material histological diagnosis of madura foot (mycetoma): a must for definitive treatment diagnostic problem with imported cases of mycetoma in the netherland molecular identification of black-grain mycetoma agents molecular detection and identification of agents of eumycetoma: detailed report of two cases dna replication: the rolling circle model padlock probes: circularizing oligonucleotides for localized dna detection improvements of rolling circle amplification (rca) efficiency and accuracy using thermus thermophilus ssb mutant protein rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification a novel rolling circle amplification assay to detect members of the family anelloviridae in pigs and humans rolling circle amplification for direct detection of rpob gene mutations in mycobacterium tuberculosis isolates from clinical specimens rapid identification of fungal pathogens by rolling circle amplification using fonsecaea as a model detection and identification of opportunistic exophiala species using the rolling circle amplification of ribosomal internal transcribed spacers rapid identification of pseudallescheria and scedosporium strains by using rolling circle amplification phylogenetic findings suggest possible new habitat and routes of infection of human eumyctoma revision of agents of black-grain eumycetoma in the order a simple and efficient protocol for isolation of high molecular weight dna from filamentous fungi, fruit bodies, and infected plant tissues variability and molecular diagnostics of the neurotropic species cladophialophora bantiana practical method for detection and identification of candida, aspergillus, and scedosporium spp. by use of rollingcircle amplification the 17 neglected tropical diseases. geneva: world health organization development of a species-specific pcr-restriction fragment length polymorphism analysis procedure for identification of madurella mycetomatis new species of madurella, causative agents of black-grain mycetoma use of rolling circle amplification to rapidly identify species of cladophialophora potentially causing human infection rapid identification and differentiation of trichophyton species, based on sequence polymorphisms of the ribosomal internal transcribed spacer regions, by rolling-circle amplification rolling circle amplification: a versatile tool for chemical biology, materials science and medicine padlock probes reveal single-nucleotide differences, parent of origin and in situ distribution of centromeric sequences in human chromosomes 13 and 21 highly efficient dna synthesis by the phage phi f 29 dna polymerase. symmetrical mode of dna replication signal amplification of padlock probes by rolling circle replication key: cord-000680-vsgd9v1w authors: lee, linda k.; gan, victor c.; lee, vernon j.; tan, adriana s.; leo, yee sin; lye, david c. title: clinical relevance and discriminatory value of elevated liver aminotransferase levels for dengue severity date: 2012-06-05 journal: plos negl trop dis doi: 10.1371/journal.pntd.0001676 sha: doc_id: 680 cord_uid: vsgd9v1w background: elevation of aspartate aminotransferase (ast) and alanine aminotransferase (alt) is prominent in acute dengue illness. the world health organization (who) 2009 dengue guidelines defined ast or alt≥1000 units/liter (u/l) as a criterion for severe dengue. we aimed to assess the clinical relevance and discriminatory value of ast or alt for dengue hemorrhagic fever (dhf) and severe dengue. methodology/principal findings: we retrospectively studied and classified polymerase chain reaction positive dengue patients from 2006 to 2008 treated at tan tock seng hospital, singapore according to who 1997 and 2009 criteria for dengue severity. of 690 dengue patients, 31% had dhf and 24% severe dengue. elevated ast and alt occurred in 86% and 46%, respectively. seven had ast or alt≥1000 u/l. none had acute liver failure but one patient died. median ast and alt values were significantly higher with increasing dengue severity by both who 1997 and 2009 criteria. however, they were poorly discriminatory between non-severe and severe dengue (e.g., ast area under the receiver operating characteristic [roc] curve = 0.62; 95% confidence interval [ci]: 0.57–0.67) and between dengue fever (df) and dhf (ast area under the roc curve = 0.56; 95% ci: 0.52–0.61). there was significant overlap in ast and alt values among patients with dengue with or without warning signs and severe dengue, and between those with df and dhf. conclusions: although aminotransferase levels increased in conjunction with dengue severity, ast or alt values did not discriminate between df and dhf or non-severe and severe dengue. dengue is a mosquito-borne arboviral infection endemic to most tropical and subtropical countries [1] . elevation of the liver enzymes aspartate aminotransferase (ast) and alanine aminotransferase (alt) is common in acute dengue illness, occurring in 65-97% [2, 3, 4, 5] of dengue patients, peaking during the convalescent period of illness (days 7-10) [2, 4, 6] . in dengue-endemic countries, dengue is an important cause of acute viral hepatitis [7] . elevated ast and alt levels have been associated with bleeding [2, 4, 6] and dengue hemorrhagic fever (dhf) [3, 8] . liver failure has been recognized as a complication and unusual manifestation of dengue [9, 10] but occurred infrequently in 3 of 270 patients in taiwan [6] and 5 of 644 patients in vietnam [4] . in malaysia, 8 of 20 pediatric dhf patients developed liver failure, 1 died, and the rest recovered completely [11] . in singapore, ast or alt levels were not independent predictors of dhf in 1973 adult dengue patients [12] . in 2009, the world health organization (who) revised its dengue guidelines and proposed severe organ impairment as one category of severe dengue in addition to severe plasma leakage and severe bleeding [1] . severe liver involvement was defined as ast or alt$1000 units/liter (u/l). in taiwan, ast.10 times the upper limit of normal (uln) occurred in 11% of dengue patients [6] , while in brazil this occurred in 4% of their cohort [3] . in this study, we aimed to evaluate the clinical relevance of elevated ast and alt levels and correlate liver aminotransferase levels with dengue severity according to who 1997 and 2009 classifications. all laboratory-confirmed dengue patients identified from our hospital microbiology database and treated using a standardized dengue clinical care path at the department of infectious diseases, tan tock seng hospital (ttsh), singapore from 2006 to 2008 were retrospectively reviewed for demographic, serial clinical and laboratory, radiological, treatment, and outcome data. these cases were positive by real-time polymerase chain reaction (pcr) [13] . we included patients with only positive dengue serology in only subgroup analyses, as we did not have paired sera, and other etiologies for elevated ast and alt could not be excluded without more extensive evaluation. cases were categorized using serial clinical and laboratory data from the entire clinical course as dengue fever (df), dhf, or dengue shock syndrome (dss) using who 1997 classifications [9] . dengue fever classification requires fever and at least two of the following: headache, eye pain, myalgia, arthralgia, rash, bleeding, and leukopenia. dengue hemorrhagic fever requires all of the following: fever, platelet count #100610 9 /liter, bleeding, and plasma leakage [9] . dengue shock syndrome is a case of dhf with either tachycardia and pulse pressure ,20 mmhg or systolic blood pressure ,90 mmhg [9] . cases were also categorized as dengue without warning signs (ws), dengue with ws, or severe dengue using who 2009 classifications [1] . dengue (who 2009) requires fever and two of the following: nausea, vomiting, rash, aches and pains, leukopenia, or any warning sign [1] . warning signs include abdominal pain or tenderness, persistent vomiting, clinical fluid accumulation, mucosal bleeding, lethargy or restlessness, hepatomegaly, or hematocrit rise ($20%) with rapid drop in platelet count (,50,000/liter) [1, 14] . we modified the who 2009 warning sign of rise in hematocrit concurrent with rapid drop in platelet count by quantifying it as hematocrit $20% concurrent with platelet count ,50,000/liter, as this was shown to correlate significantly with dengue death in our adult dengue death study [14] . severe dengue includes severe plasma leakage, severe bleeding, and severe organ impairment [1] . we performed a subgroup analysis for median maximum ast and alt values stratified by febrile (days 1-3 of illness), critical (days 4-6), and convalescent (days 7-10) phases as defined by who 2009 [1] and compared across dengue severity classification according to who 1997 [9] and 2009 [1] . we excluded severe dengue due to isolated elevation of ast or alt$1000 u/l from our definition of severe dengue outcome, as this would be a confounder in assessing the relevance of ast or alt levels in defining dengue severity. patients had ast/alt taken at presentation and then throughout hospitalization at the physician's discretion. maximum ast and alt values recorded at a median of 4 days of illness (interquartile range [iqr]: 3-5 days) were used in this analysis. those with pre-existing liver diseases were excluded. at ttsh, the uln for ast is 41 u/l; for alt, it is 63 u/l for males and 54 u/l for females. we assessed the clinical relevance of elevated ast or alt levels using four liver failure criteria-two for acute liver failure, and two that determine prognosis from chronic liver disease. the american association for the study of liver diseases (aasld) recommends defining acute liver failure in a patient as: international normalized ratio (inr)$1.5, any degree of altered mental status, and illness ,26 weeks in duration without preexisting cirrhosis [15] . the king's college criteria assess prognoses in those with acute liver failure; the criteria are: prothrombin time .100 seconds or 3 of the following: age .40 years, prothrombin time .50 seconds, serum bilirubin .18 mg/dl, time from jaundice to encephalopathy .7 days [16] . the model for endstage liver disease (meld) determines three-month mortality based on the following formula: 3.86(log serum bilirubin [mg/ dl])+11.26(log inr)+9.66(log serum creatinine [mg/dl])+6.4 [17] . the child-pugh criteria include assessment of degree of ascites, serum bilirubin and albumin, prothrombin time, and encephalopathy to determine one-and two-year survival [18] . the mann-whitney u and kruskal-wallis tests were used to determine statistical significance for continuous variables, and chisquare or fisher's exact test for categorical variables. statistical tests were conducted at the 5% level of significance. receiver operating characteristic (roc) curves showing the area under the curve (auc) were generated to determine the discriminatory performance of aminotransferase values. all statistical analyses were performed using stata 10 (stata corp., college station, tx). this was a retrospective study involving data collection from medical records. all patient data were anonymized during analysis. this study was approved by the institutional review board, national healthcare group, singapore [dsrb e/08/ 567]. from 2006 to 2008, 690 dengue pcr positive cases were reviewed. males comprised 493 (71%) of the cases, and the median age of the cohort was 35 years (iqr: 27-43 years). a charlson comorbidity index $3, which predicts increased one-year mortality [19] , was noted in 5 (0.7%) patients. with who 1997 classification, 62% had df, 31% dhf, and 7% dss. with who 2009 classification, 14% had dengue, 62% had dengue with warning signs, and 24% had severe dengue. hence, by who 1997 classification, 38% of patients with dhf/dss needed close monitoring, while by who 2009 classification, 86% of patients with warning signs or severe dengue required close monitoring. median length of illness from onset to hospital presentation was 4 days (iqr: 3-5 days), while median length of hospital stay was 5 days (iqr: 4-6 days). intravenous fluid was administered to 641 (93%) and platelet transfusion to 86 (12%). intensive care unit (icu) admission was required in 3 patients, and death occurred in 1 patient due to pneumonia. (1) elevation of ast/alt and risk of liver failure overall, 595 (86%) had ast above the uln, and 316 (46%) had alt above the uln. seven patients (1.0%) had severe dengue according to who 2009 criteria concurrent with ast or alt$1000 u/l while three additional patients had severe dengue due to ast or alt$1000 u/l only. of the former seven patients, 86% had severe plasma leakage, 29% had severe bleeding, and none had severe organ impairment other than isolated ast or alt$1000 u/l. among the 3 patients admitted to the icu, ast or alt values were above the uln but below 1000 u/l. no patients in our cohort developed acute liver failure under aasld or king's college criteria. with child-pugh scoring, 2 (0.3%) belonged to child-pugh class c. with meld scoring, predicted three-month mortality of 6% were identified in 68 (10%) dengue is a global public health problem, as the incidence of the disease has reached hyperendemic proportions in recent decades. infection with dengue can cause acute, febrile illness or severe disease, which can lead to plasma leakage, bleeding, and organ impairment. one of the most prominent clinical characteristics of dengue patients is increased aspartate and alanine aminotransferase liver enzyme levels. the significance of this is uncertain, as it is transient in the majority of cases, and most patients recover uneventfully without liver damage. in this study, we characterized this phenomenon in the context of dengue severity and found that, although liver enzyme levels increased concurrently with dengue severity, they could not sufficiently discriminate between dengue fever and dengue hemorrhagic fever or between non-severe and severe dengue. therefore clinicians may need to use other parameters to distinguish dengue severity in patients during early illness. association between transaminase levels and dengue www.plosntds.org patients in our cohort and 19.6% in 2 (0.3%) patients. the same two patients who were child-pugh class c also had a predicted 19.6% three-month mortality using meld scoring; they both had dss and severe dengue. (2) dengue severity and ast/alt values median ast and alt values for dengue without warning signs, dengue with warning signs, and severe dengue (table 1) in other hemorrhagic fevers, higher ast:alt ratios correlated with disease fatality [20] . in our pcr-positive cohort, median ast:alt ratios for df, dhf, and dss were 1.68, 1.68, and 1.88 (p = 0.29) and for dengue without ws, dengue with ws, and severe dengue, they were 1.60, 1.68, and 1.78 (p = 0.10), respectively. the majority of our patients' maximum ast and alt values were recorded during febrile (n = 258) and critical (n = 377) phases of acute dengue illness. by who 2009 dengue severity classification, the median ast and alt values were significantly higher for severe dengue compared to dengue with and without warning signs during both the febrile and critical phases but not the convalescent phase (table 3) . by who 1997 classification, the median ast and alt values were significantly higher for dhf versus df and dss in the febrile phase only but not critical and convalescent phases (table 4 ). (4) does a threshold ast or alt value defining severe dengue exist? in order to determine the reliability of ast and alt values in defining dengue severity, roc curves for ast and alt against severe dengue excluding isolated transaminitis were determined (figure 1 ). the auc for ast was 0.62 (95% confidence interval [ci]: 0.57-0.67) and for alt, 0.60 (95% ci: 0.54-0.64). this demonstrates that ast or alt levels are insufficient to differentiate among the who 2009 dengue classifications. they were also poorly discriminatory between df and dhf, as the areas under the curve (auc) for ast and alt were 0.56 (95% ci: 0.52-0.61) and 0.55 (95% ci: 0.51-0.59), respectively ( figure 2 ). in our serology-positive cohort, the auc values for ast and alt were 0.56 and 0.54 for differentiating between df and dhf. the auc values for severe and non-severe dengue were 0.64 and 0.60 for ast and alt, respectively. the box plots in figure 3 for the distributions of ast values show considerable overlap among the liver enzyme values for those with dengue with and without warning signs, and severe dengue. because there were extreme outliers in our cohort, only those with ast below 1000 u/l were included in these plots. figure 4 shows overlapping ast values among those with df and dhf. similarly, considerable overlap was observed in alt values for patients with dengue with and without warning signs, and severe dengue, as well as for df versus dhf (data not shown). our analysis showed that liver aminotransferase levels were associated with but did not adequately differentiate between dengue severity. although median ast and alt values were significantly higher in those with dhf/dss versus df, and severe dengue versus non-severe dengue, very few (1.0%) had ast or alt$1000 u/l. notably, none developed liver failure, and death occurred in only 1 patient (0.1%). the majority of patients recovered uneventfully. the lack of acute liver failure in our study was not unusual, as the incidence of acute liver failure in dengue patients was 1.1% in studies by trung and kuo [4, 6] . the largest study to date reported no acute fulminant hepatitis [3] . in contrast to these adult studies, it is noteworthy that in dengue-endemic countries, dengue may be an important cause of acute liver failure in children [21, 22] . while some studies have shown that ast and alt values differ between df and dhf [3, 4, 8] , few studies support ast or alt as an independent predictor of dhf [23] . two studies in singapore found liver aminotransferase levels to be significantly elevated among df and dhf patients [12] and survivors and nonsurvivors of dengue [24] on univariate analysis, but this association was lost after adjusting for confounders on multivariate analysis. trung et al. showed significant differences comparing other febrile illness, dengue without plasma leakage, and dengue with plasma leakage with and without shock during critical and convalescent phases for ast but during critical phase for alt only [4] . we made the novel finding that liver aminotransferase association between transaminase levels and dengue www.plosntds.org levels may significantly vary according to dengue severity during the febrile phase. for dhf by who 1997 classification, both ast and alt were significantly higher during the febrile phase compared to df or dss, and for severe dengue by who 2009, ast and alt were significantly higher during the febrile and critical phases. the impact of co-infection with hepatitis viruses or concomitant hepatotoxic drugs was not assessed in our retrospective study, although we did exclude those with known liver comorbidities. kuo et al. found that hepatitis b or c did not increase the extent of liver aminotransferase elevation in a retrospective adult dengue study in taiwan [6] . in contrast, trung et al. found that hepatitis b co-infection modestly increased alt levels without significant clinical impact in a prospective adult dengue study in vietnam [4] . tang et al. showed that dengue and hepatitis b co-infected patients showed an aberrant cytokine secretion profile compared with those with dengue alone. [25] . in singapore, seroprevalence for hepatitis b was 2.8% [26] and hepatitis c 0.37% [27] . the etiology of elevated aminotransferase levels during acute dengue illness is unclear since ast is expressed in the heart, skeletal muscle, red blood cells, kidneys, brain, and liver, while alt is secreted primarily by the liver [28, 29] . because dengue infection can cause acute damage to these non-hepatic tissue types that express ast, raised aminotransferase levels may not be entirely due to severe liver involvement. it is therefore possible that the patients with high ast levels were also more likely to be classified as severe dengue under the 2009 criteria due to the common pathways to non-hepatic tissue damage, even though there is no association with poorer outcome. our retrospective study has some limitations. aspartate and alanine aminotransferase values were tracked according to clinical judgment rather than at regular intervals during illness. we did not have dengue serotype data for each patient, but in 2006, denv-1 was predominant in singapore with a switch to denv-2 in 2007-2008 [30] . serology-positive cases were not included in primary analyses because our clinical laboratory used a rapid diagnostic test with potential for false positive results [31] , we did not have paired sera to confirm dengue diagnosis [9] , and not every patient with elevated ast or alt was comprehensively evaluated for other etiologies of viral and non-viral hepatitis. although serology-positive cases presented later during illness, we saw no difference in outcome. five serology-positive patients (0.34%) required icu admission versus 0.43% of pcr-positive cases, while four patients (0.27%) died in the serology-positive cohort, versus 1 patient (0.14%) among pcr-positive cases. however, relative data accuracy in our retrospective study was made possible by using a standardized dengue clinical care path. another limitation of this study is the relatively few cases with substantially elevated liver aminotransferase levels. at the same time, since our cohort comprised primarily adults, additional studies in pediatric populations will be useful to confirm our findings. in patients with dhf/dss or severe dengue, early diagnosis and proper management may improve outcome in most patients without comorbidities. however, in resource-limited countries, patients with severe disease may present late to the hospital with shock, with or without organ impairment at the time of admission. our study highlights that early diagnosis and proper management of dengue patients may lead to excellent prognosis without organ injury. in conclusion, elevated aminotransferase levels were associated with dhf/dss and severe dengue in our cohort of adult patients with confirmed dengue. however, no threshold values discriminated between df and dhf or between severe dengue and nonsevere dengue. dengue: guidelines for diagnosis, treatment, prevention and control severity of acute hepatitis and its outcome in patients with dengue fever in a tertiary care hospital karachi aminotransferase changes and acute hepatitis in patients with dengue fever: analysis of 1,585 cases liver involvement associated with dengue infection in adults in vietnam use of simple laboratory features to distinguish the early stage of severe acute respiratory syndrome from dengue fever liver biochemical tests and dengue fever the infective causes of hepatitis and jaundice amongst hospitalised patients in vientiane early clinical and laboratory indicators of acute dengue illness dengue hemorrhagic fever: diagnosis, treatment, prevention and control world health organization regional office for southeast asia (2011) comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever fulminant hepatitis in dengue infection predictive value of simple clinical and laboratory variables for dengue hemorrhagic fever in adults the performance of rt-pcr compared with a rapid serological assay for acute dengue fever in a diagnostic laboratory confirmed adult dengue deaths in singapore: 5-year multi-center retrospective study the management of fulminant hepatic failure early indicators of prognosis in fulminant hepatic failure a model to predict survival in patients with end-stage liver disease transection of the oesophagus for bleeding oesophageal varices a new method of classifying prognostic comorbidity in longitudinal studies: development and validation blood chemistry measurements and d-dimer levels associated with fatal and nonfatal outcomes in humans infected with sudan ebola virus prevalence of dengue infection in north indian children with acute hepatic failure dengue virus infection: a major cause of acute hepatic failure in thai children clinical characteristics of dengue and dengue hemorrhagic fever in a medical center of southern taiwan during the 2002 epidemic fatal dengue hemorrhagic fever in adults during a dengue epidemic in singapore unique impacts of hbv co-infection on clinical and laboratory findings in a recent dengue outbreak in china changing seroprevalence of hepatitis b virus markers of adults in singapore a study on the epidemiology of hepatitis c infection among blood donors in singapore aga technical review on the evaluation of liver chemistry tests biochemical investigations in the management of liver disease, in textbook of hepatology: from basic clinical science to clinical practice dengue virus surveillance for early warning a systematic review and meta-analysis of the diagnostic accuracy of rapid immunochromatographic assays for the detection of dengue virus igm antibodies during acute infection we would like to acknowledge dr. wah wah lin for data entry. key: cord-000625-cpjlzutk authors: ablordey, anthony; amissah, diana ackon; aboagye, isaac frimpong; hatano, ben; yamazaki, toshio; sata, tetsutaro; ishikawa, koichi; katano, harutaka title: detection of mycobacterium ulcerans by the loop mediated isothermal amplification method date: 2012-04-03 journal: plos negl trop dis doi: 10.1371/journal.pntd.0001590 sha: doc_id: 625 cord_uid: cpjlzutk background: buruli ulcer (bu) caused by mycobacterium ulcerans (m. ulcerans) has emerged as an important public health problem in several rural communities in sub-saharan africa. early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where m. ulcerans is most prevalent. methodology: we compared conventional and pocket warmer loop mediated isothermal amplification (lamp) methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction) for the detection of m. ulcerans in clinical specimens. the effect of purified and crude dna preparations on the detection rate of the lamp assays were also investigated and compared with that of is2404 pcr, a reference assay for the detection of m. ulcerans. thirty clinical specimens from suspected bu cases were examined by lamp and is2404 pcr. principal findings: the lower detection limit of both lamp methods at 60°c was 300 copies of is2404 and 30 copies of is2404 for the conventional lamp at 65°c. when purified dna extracts were used, both the conventional lamp and is2404 pcr concordantly detected 21 positive cases, while the pocket warmer lamp detected 19 cases. nine of 30 samples were positive by both the lamp assays as well as is2404 pcr when crude extracts of clinical specimens were used. conclusion/significance: the lamp method can be used as a simple and rapid test for the detection of m. ulcerans in clinical specimens. however, obtaining purified dna, as well as generating isothermal conditions, remains a major challenge for the use of the lamp method under field conditions. with further improvement in dna extraction and amplification conditions, the pwlamp could be used as a point of care diagnostic test for bu buruli ulcer (bu) caused by mycobacterium ulcerans (m. ulcerans) is a necrotizing skin disease endemic mostly in rural wetland of tropical countries of africa, america, asia and australia. the disease also occurs in non-tropical areas of australia, china and japan. globally, bu has been reported in over 30 countries [1] [2] [3] . the burden of bu is however most severe in sub saharan africa where the true incidence of the disease is difficult to determine as a result of poor surveillance measures and case confirmation [2] . available data however reveals an increase in bu incidence over the last several years in the west african countries of ivory coast, ghana and benin. in these countries bu has replaced leprosy as the second most prevalent mycobacterial disease [1] , [3] [4] [5] . bu begins as painless nodule, papule, plaque or edema that evolves into characteristic ulcers with undermined edges. if untreated, extensive ulceration (that can cover 15% of the body), scarring and contractures may cause serious functional disabilities in patients [5] [6] [7] . unfortunately most patients seek treatment late and present with large ulcers [8] [9] [10] . previously treatment of such lesions involved surgical removal of all the affected tissue and part of the surrounding tissues, eventually followed by skin grafting [9] [10] [11] [12] . in 2004 antimycobacterial treatment alone (if necessary in combination with surgery) was introduced and has since been considered as the treatment of choice for bu [6] , [13] [14] [15] [16] . laboratory confirmation of clinically suspected bu cases has therefore become crucial for the clinical management of bu [17] . four laboratory tests are recommended for the diagnosis of bu. these include microscopic examination, culture, is2404 pcr and histopathological analysis. microscopic examination detects 29%-78% of clinically suspected bu cases and is currently the only rapid and affordable test available for bu diagnosis in many endemic areas. the detection rate of culture is between 34%-79% and takes an average of 9-12 weeks to yield positive results. culture therefore cannot be used for rapid laboratory confirmation of bu. histopathological analysis is reported to detect 30% additional cases than other confirmatory tests, however this technique is restricted to external reference laboratories and are unavailable in peripheral health centres or district or regional hospitals. is2404 pcr has close to 96% sensitivity and is considered the method of choice for laboratory confirmation of bu [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . the who recommends that at least 50% of cases must be confirmed by is2404 pcr before commencement of antibiotic therapy [22] [23] [24] [25] . however technical difficulties (eg, cold chain requirement, stable power supply and qualified laboratory staff) limit the use of this diagnostic test in bu endemic areas. a dry reagent pcr consisting of lyophilized pcr mix which is reconstituted with water for testing dna was developed to simplify bu diagnosis by pcr [26] but this method also requires the use of a thermocycler, electrophoresis and gel imaging equipment and therefore similarly makes the use of this diagnostic test for bu diagnosis in endemic areas unlikely. the loop mediated isothermal amplification (lamp) is a novel nucleic acid amplification method for molecular detection and identification [27] . the principle of lamp is autocycling strand displacement dna synthesis in the presence of bst dna polymerase with high strand displacement activity under isothermal conditions between 60-65uc within 60 minutes [28] . the assay is highly specific due to the recognition of target dna by 4 to 6 independent sequences and the amplification efficiency of lamp is equivalent to that of pcr based methods ( [27] , [29] , [30] ). the lamp reaction enables easy identification of positive tests due to the accumulation of high amounts of amplification products in the reaction tubes. further improvement in visual identification can be realized through the addition of intercalating dyes such as sybr green or hydroxynapthtol blue to reaction tubes [31] . this therefore precludes the need for post amplification analysis and hence reduces cost and labour. lamp has also been shown to be less affected by a number of inhibitors of conventional pcr [32] . additionally the closed tube format of this assay reduces problem of carry over contamination which is likely in less controlled environments [33] . with all of these characteristics lamp of dna has emerged as a powerful tool to facilitate point of care diagnostic test [31] . in order to develop a field applicable technique that offers high detection sensitivity and specificity for the diagnosis of bu, we explored the use of the pocket warmer lamp (pwlamp) technique, a dna amplification method using isothermal conditions (60uc) provided by a disposable pocket warmer [34] . ethical approval for analysing patients' specimens was obtained from the ethical review board of the noguchi memorial institute for medical research. specimens used were anonymously taken from an already existing collection of patients' specimens processed for diagnosis of bu from agogo presbyterian hospital in ghana. thirty clinical specimens consisting of 20 swabs and 10 fine needle aspirates taken respectively from ulcers and pre-ulcerative lesions of suspected bu patients were used in this study. the fine needle aspirate specimens were kept in 1 ml phosphate buffered saline (pbs) and swabs were stored dry in sterile tubes. each swab was transferred into a tube containing 2 ml milli-q purified water (millipore corporation, billerica, ma) and gently vortexed for 5 sec and then removed. portions 250 ml of the sample suspensions were transferred to separate new sterile eppendorf tubes containing 250 ml of lysis buffer (1.6 m guhcl, 60 mm tris ph 7.4, 1% triton x-100, 60 mm edta, tween-20 10%), 50 ml proteinase-k and 250 ml glass beads. the mixtures were incubated horizontally in a shaker (200 rpm) at 60uc overnight. to capture the dna, 40 ml of diatomaceous earth solution (10 g diatomaceous earth obtained from sigma aldrich chemi gmbh in 50 ml of h 2 o containing 500 ml of 37% (wt/vol) hcl) was added to the suspensions and incubated at 37uc with shaking (200 rpm) for 60 min. the mixtures were centrifuged at 14,000 rpm for 10 sec and the resulting pellets were washed twice with 900 ml of 70% ethanol (2-8uc) followed by 900 ml of acetone. the pellets were dried at 50uc for 20 min and resuspended in 100 ml milli q purified water and centrifuged at 14,000 rpm for 10 sec. the purified dna was used as templates for both is2404 pcr and lamp assays to detect m. ulcerans. to investigate the performance of the lamp assay on crude dna preparations, we obtained 2 types of dna extracts for each clinical specimen. one crude extract consisted of 250 ml suspensions of the specimen boiled for 10 min followed by centrifugation at 14,000 rpm for 5 min (boiled extract). the other crude extract used was a 250 ml suspension of the unboiled specimen. ten m. ulcerans strains grown on lj slants were harvested and dna was extracted as previously described [35] . serial dilutions of purified m. ulcerans dna containing 300,000, 30,000, 300, 30 and 3 copies of is2404 element per 5 ml were prepared. the number of copies of the insertion sequence element was determined based on the genome size of 5,806 kb and presence of an average number of 207 copies of is2404. this was used to determine the detection limit of the lamp assays. in order to develop a simple and rapid test that can be used to diagnose buruli ulcer under field conditions, we modified the conventional lamp assay by using a disposable pocket warmer as a heating device for generating a constant temperature for the test reaction and employed the use of crude sample preparations consisting of boiled and unboiled extracts of the clinical specimen instead of using purified dna as the diagnostic specimen. thirty clinical specimens from suspected buruli ulcer patients were investigated by the modified lamp (or pocket warmer lamp) and the conventional lamp, as well as is2404 pcr, a reference method for the detection of mycobacterium ulcerans. there was no significant difference in the detection rate (63-70%) in all of the methods when purified samples were used for the tests. on the other hand the use of crude specimen preparation resulted in a drop in detection rate (30-40%) . this study demonstrates that the lamp test can be used for rapid detection of m. ulcerans when purified dna preparations are used. with further improvements in the sample reaction, as well as in specimen purification, the pocket warmer lamp may provide a simple and rapid diagnostic test for buruli ulcer. lamp for detection of mycobacterium ulcerans www.plosntds.org pcr for is2404 pcr targeting is2404 was performed as described previously [21] . the first and second round pcrs used primers pgp1: 59-agggcagcgcggtgatacgg-39and pgp2: 59-cagtg-gattggtgccgatcgag-39 and pgp3: 59-ggcgcagat-caacttcgcggt-39 and pgp4: 59-ctgcgtggtgcttt-acgcgc-39, respectively. for the first round, the 30 ml reaction volume contained 3 ml dna, 25 pmol/ml of each primer (pgp1 and pgp2), 3 ml of 106 pcr buffer (containing 1.5 mm magnesium chloride), 6.0 ml qsolution, 0.2 mm deoxynucleotide triphosphates (dntps) and 1.0 u hotstar taq polymerase (qiagen). for the second run, 1 ml of the first run product was added to 24 ml reaction volume containing, 25 pmol/ml of each primer (pgp3 and pgp4), 2. pocket warmer lamp (pwlamp) was performed using a loopamp dna amplification kit (eiken chemical) described previously [32] . each 25 ml reaction mixture contained 1.6 mm each of fip and bip, 0.2 mm each of f3 and b3, 0.8 mm each of lf and lb, 26 reaction mixture (12.5 ml), 1 ml of bst dna polymerase, 1 ml of fluorescence detection reagent (eiken chemical), 3.5 ml distilled water and 1 ml sample. reaction tubes were incubated at 60uc for 60 min in the heat block (geneamp 9700, applied biosystems, foster city, ca) while with the pwlamp, the tubes were sandwiched in a twofold pocket warmer (hokaron haru-type, lotte health products, tokyo, japan) surrounded by a paper towel, and put in a styrofoam box for 120 min (60 min reaction incubation). the reaction was terminated at 85uc for 5 min and the results were read by eye in ambient light and also using uv illumination. a chi-squared test was performed to reveal the statistical difference using spss (version 16.0; spss inc., chicago, il) software. lamp reaction requires a constant temperature of about 60u-65uc for 60 min for amplification of dna [27] [28] [29] [30] [31] [32] [33] [34] . in a previous study a pocket warmer reached 58uc in 30 min and stayed around 60uc for more than 60 min in a styrofoam box [34] . the 3 pocket warmers (of a pack of 30 hand warmers) tested in this study achieved a temperature of 60uc after 60 min and maintained this temperature for about 90 min. the pocket warmer thus provided a suitable temperature (60uc) and time range (60 min) for amplification. both pwlamp and the conventional lamp assays were able to detect to the limit of 300 copies of the target sequence after 60 min of amplification. this limit improved to 30 copies when the conventional lamp was carried out at 65uc (the pocket warmer was not able to attain this temperature and was therefore not investigated). the sensitivity and specificity of the lamp assays for the detection of m. ulcerans is shown in tables 1 and 2. under ambient illumination, positive specimens in the lamp assay produced greenish colouration (figure 2 ). when purified dna extracts were used, 21 (16 swabs, 5 fine needle aspirates) (70%) of 30 clinical specimens were positive by is2404 pcr as well as by the conventional lamp. none of the pcr positive specimens were negative by conventional lamp. however 19 samples of purified dna extracts were positive with the pwlamp, but the 90.5% sensitivity (19/21) of the pwlamp compared to the results by is2404 pcr was not statistically significant (p = 0.58, chi-square test). all negative specimens in is2404 pcr were negative in both lamp assays, indicating specificities of both lamp assays to the reference method were 100%. twelve unboiled (9 swabs and 3 fine needle aspirates) and 9 boiled (6 swabs and 3 fine needle aspirates) extracts were positive by all 3 detection assays with sensitivities of 57.1% (unboiled, 12/21) and 42.9% (boiled, 9/21) compared to results using purified dna extracts respectively for both lamp and is2404 pcr assays. the positivity of swabs was found to be in the range of 30% to 80% compared to 30% to 50% for fine needle aspirates. when the positivities in crude dna specimens were compared with those in purified dna, the differences were statistically significant by chi-square test (unboiled vs purified dna, p = 0.0195, and boiled vs purified dna, p = 0.0019). none of the is2404 pcr negatives was positive in the lamp assays irrespective of the dna extracts type used. these data suggest that sensitivity of lamp and pcr assays for the detection of m. ulcerans in clinical specimens is enhanced when purified dna extracts are used. bu is a neglected tropical disease that mostly affects the poor in resource limited communities in sub-saharan africa [1] [2] [3] . is2404pcr, the method of choice for confirmation of bu diagnosis cannot be operational in bu endemic areas [16] , [17] , [24] [25] . the development of rapid and reliable point of care diagnostic assays is of high priority to bu management and prevention. this study explored the potential use of the lamp method for field diagnosis of bu. some important limitations to the use of this assay in the field include specimen purification and difficulty in maintaining isothermal condition for the reaction. a study suggested that omission of dna extraction or the use of crude dna extracts have no effect on the lamp test [32] . hatano et al used a disposable pocket warmer to provide isothermal condition for lamp reaction [34] . based on this knowledge, we applied the pwlamp to crude and purified dna extracts in order to determine whether this method will be suitable for use as a point of care diagnostic test for bu. although the pocket warmers used in this study reached 60uc after 1 hr (instead of 30 min in previous studies [34] ), both devices achieved the requisite temperature and holding time for executing lamp reaction, a major advantage in the use of amplification based assay for the detection of an infectious agent under field condition. the pwlamp did not cross-react with other mycobacteria ( figure 1) . moreover, the experiment on clinical specimens demonstrated that the pwlamp had a 100% specificity in clinical specimens of bu. the pwlamp was found to have comparable sensitivity as the conventional lamp at 60uc as both assays were able to detect 300 copies of is2404 element (equivalent of 1.5 genomes of m. ulcerans). the detection limit of the conventional lamp at 65uc improved to 30 copies of is2404 and this level of sensitivity may probably be achieved with a pocket warmer that can attain a temperature of 65uc and maintain a holding time of 60 min. when applied to purified dna extracts of clinical specimens, the pwlamp, conventional lamp and is2404 pcr yielded concordant results (tables 1 and 2) . however, 2 of the samples that were positive by is2404 pcr/ conventional lamp were negative by the pw lamp. none of the is2404 pcr negative samples were positive in both types of lamp assays. on the other hand we observed a drop in detection rate from 63-70% to 30-40% when crude extracts of clinical specimens were used (tables 1 and 2) . this indicates that the use of crude dna extracts as template may not be appropriate for the detection of m. ulcerans by the lamp method. this observation contradicts a previous study that suggested omission of dna extraction had no effect on sensitivity of the lamp assay [32] . for the crude preparations however, it is noteworthy that the detection rate of the lamp assay was significantly higher for the unboiled extracts than for the boiled extracts. explanations for these results were not explored. the observation that the lamp assay was not inhibited especially for the unboiled specimens is quite consistent with previous work that have shown lamp to be tolerant to culture medium and to certain biological substances including phosphate buffered saline, serum, plasma, urine and vitreous [32] . in conclusion, the study demonstrates that the lamp assay yields comparable results as is2404 pcr when it is performed at 60u-65uc for 60 min on purified dna extracts and further supports the use of the pocket warmer as a device for providing isothermal amplification condition for the lamp assay. this therefore is a potential boost to the application of pwlamp in resource poor settings. challenges of obtaining pure dna extracts of clinical specimen as well as the use of a pocket warmer capable of maintaining 65uc for 1 hr, however needs to be addressed in order to improve the performance of the pwlamp assay. further development and testing in larger numbers of specimens is therefore necessary to access the potential use of pwlamp as a simple and rapid point of care diagnostic test for bu. buruli ulcer. mycobacterium ulcerans infection distribution of mycobacterium ulcerans in buruli ulcer endemic and non-endemic aquatic sites in ghana mycobacterium ulcerans infection: an overview of reported cases globally mycobacterium ulcerans infection: control, diagnosis, and treatment buruli ulcer in ghana: results of a national case search buruli ulcer: management of mycobacterium ulcerans disease: a manual for health care providers mycolactone: a polyketide toxin from mycobacterium ulcerans required for virulence mycobacterium ulcerans disease: role of age and gender in incidence and morbidity mycobacterium ulcerans infection (buruli ulcer): first reported patients in togo socio-economic implications of buruli ulcer in ghana: a three-year review buruli ulcer. mycobacerium ulcerans infection. who/cds/cpe/gbui mycobacterium ulcerans infection and buruli ulcer disease: emergence of a public health dilemma provisional guidance on the role of specific antibiotics in the management of mycobacterium ulcerans disease (buruli ulcer). world health organization efficacy of the combination rifampin-streptomycin in preventing growth of mycobacterium ulcerans in early lesions of buruli ulcer in humans promising clinical efficacy of streptomycin-rifampin combination for treatment of buruli ulcer (mycobacterium ulcerans disease) mycobacterium ulcerans infection (buruli or bairnsdale ulcer): challenges in developing management strategies laboratory confirmation of buruli ulcer disease in togo consensus recommendations for the diagnosis, treatment and control of mycobacterium ulcerans infection (bairnsdale or buruli ulcer buruli ulcer. diagnosis of mycobacterium ulcerans disease: a manual for health providers. world health organization (who) development of a pcr assay for rapid diagnosis of mycobacterium ulcerans infection identification and characterization of is2404 and is2606: two distinct repeated sequences for detection of mycobacterium ulcerans by pcr buruli ulcer: progress report buruli ulcer: advances in understanding mycobacterium ulcerans infection comparative study of the sensitivity of different diagnostic methods for the laboratory diagnosis of buruli ulcer disease laboratory diagnosis of buruli ulcer disease dry-reagentbased pcr as a novel tool for laboratory confirmation of clinically diagnosed mycobacterium ulcerans-associated disease in areas in the tropics where m. ulcerans is endemic loop-mediated isothermal amplification of dna loop-mediated isothermal amplificationmethod for rapid detection of the toxic dinoflagellate alexandrium, which causes algal blooms and poisoning of shellfish accelerated reaction by loop-mediated isothermal amplification using loop primers rapid detection of the severe acute respiratory syndrome (sars) coronavirus by a loop-mediated isothermal amplification assay loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium and m. intracellulare in sputum samples tolerance of loop-mediated isothermal amplification to a culture medium and biological substances poon loop-mediated isothermal amplification for influenza a (h5n1) virus. emerging infectious diseases n www lamp using a disposable pocket warmer for anthrax detection, a highly mobile and reliable method for anti-bioterrorism a comparison of dna extraction procedures for the detection of mycobacterium ulcerans, the causative agent of buruli ulcer, in clinical and environmental specimens key: cord-276271-3nz3169p authors: deborggraeve, stijn; coronado, ximena; solari, aldo; zulantay, ines; apt, werner; mertens, pascal; laurent, thierry; leclipteux, thierry; stessens, tim; dujardin, jean-claude; herdewijn, piet; büscher, philippe title: t. cruzi oligoc-test: a simplified and standardized polymerase chain reaction format for diagnosis of chagas disease date: 2009-06-02 journal: plos negl trop dis doi: 10.1371/journal.pntd.0000450 sha: doc_id: 276271 cord_uid: 3nz3169p background: pcr has evolved into one of the most promising tools for t. cruzi detection in the diagnosis and control of chagas disease. however, general use of the technique is hampered by its complexity and the lack of standardization. methodology: we here present the development and phase i evaluation of the t. cruzi oligoc-test, a simple and standardized dipstick format for detection of pcr amplified t. cruzi dna. the specificity and sensitivity of the assay were evaluated on blood samples from 60 chagas non-endemic and 48 endemic control persons and on biological samples from 33 patients, 7 reservoir animals, and 14 vectors collected in chile. principal findings: the lower detection limits of the t. cruzi oligoc-test were 1 pg and 1 to 10 fg of dna from t. cruzi lineage i and ii, respectively. the test showed a specificity of 100% (95% confidence interval [ci]: 96.6%–100%) on the control samples and a sensitivity of 93.9% (95% ci: 80.4%–98.3%), 100% (95% ci: 64.6%–100%), and 100% (95% ci: 78.5%–100%) on the human, rodent, and vector samples, respectively. conclusions: the t. cruzi oligoc-test showed high sensitivity and specificity on a diverse panel of biological samples. the new tool is an important step towards simplified and standardized molecular diagnosis of chagas disease. accurate diagnosis of chagas disease, the most important parasitic disease in the americas [1] , is challenging due to the latent character of the infection. following a brief acute phase, infected individuals can progress to a lifelong chronic phase and about 30% will eventually develop the typical cardiac and intestinal complications that can be fatal [2] . trypanosoma cruzi, the protozoan pathogenic agent, is naturally transmitted by blood sucking triatominae bugs. additional transmission routes include vertical transmission from mother to child, transfusion with infected blood and organ transplantation. oral t. cruzi transmission by the consumption of contaminated food is linked to localized outbreaks of acute chagas disease [3] . the t. cruzi species is genetically heterogeneous and has been classified in two major phylogenetic lineages, t. cruzi i and t. cruzi ii, of which the latter is subdivided in 5 sublineages (iia to iie). t. cruzi i predominates in endemic countries north of the amazon, whereas t. cruzi ii is predominant throughout the southern cone countries of south america. t. cruzi i is associated with domestic and sylvatic transmission cycles, while iia and iic seem to be restricted to sylvatic cycles and iib, iid and iie to domestic cycles [4] . both lineages are associated with cardiac lesions in human infection, but it seems that digestive tract lesions only occur in infection with t. cruzi ii [5] . t. cruzi is closely related to t. rangeli that is apparently harmless to man but shares the same reservoir animals and vectors [6] . acute infections, although rarely recognized because of the nonspecific clinical manifestations, can be diagnosed by parasitological tests since the parasite load in the blood at that stage is generally high enough. chronic infections, on the contrary, are associated with a very low parasite load in the blood and are therefore mainly diagnosed by means of antibody detection tests. however, antibody detection tests are liable to specificity problems due to cross-reaction with antibodies induced by other parasites, especially leishmania and t. rangeli [7] . hence, it is generally recommended that specimens are tested in two or three different immunodiagnostic tests, such as indirect immunofluorescence (iif), indirect hemagglutination (iha) and enzyme-linked immuno sorbent assays (elisa). furthermore, immunodiagnostic tests are less useful in testing the effectiveness of treatment and congenital infections. xenodiagnosis is regularly used for parasite detection but results of this invasive test are only available after 1 to 3 months and the method shows generally low sensitivity with chronic infections [8] . accurate tools to rapidly diagnose active infections, to assess treatment efficacy and to determine the impact of transmission control measures are needed. the introduction of the polymerase chain reaction (pcr) to amplify specific dna sequences opened promising diagnostic perspectives. pcr has been used to detect t. cruzi in the blood of chronic chagasic patients [9] , in congenital transmission [10] , in patient follow-up after treatment [11] and in disease reactivation after heart transplantation [12] . despite the reported high sensitivity and specificity, the pcr technique is restricted to high-level equipped laboratories. this is partly due to the laborious and timeconsuming detection of the pcr products. amplicons are generally detected by electrophoresis in agarose gel followed by u.v. illumination in the presence of the carcinogenic ethidium bromide. quantitative real-time detection of pcr amplified t. cruzi dna has been developed [13] and offers major advantages such as increased sensitivity, high-throughput analysis and single tube formats. however, the technique is complex and expensive. besides the need for simplification of the dna amplification assay, standardization is a crucial requirement for moving molecular tools towards patient diagnosis and disease control. oligochromatography (oligoc) provides a simple and rapid one-step dipstick format for detection of pcr products (coris bioconcept, gembloux, belgium; patent nu wo 2004/ 099438a1) [14] . after the pcr step, the detection step takes only 5 minutes and no other equipment than a water bath and pipette are needed. controls for the pcr reaction and the chromatographic migration are incorporated in the assay. the technique has already been developed for diagnosis of human african trypanosomiasis [15] , leishmaniasis [16] , toxoplasmosis [17] , schistosomiasis [18] and severe acute respiratory syndrome (sars) [19] , and high sensitivity and specificity were reported. we here describe the development and phase i evaluation of a t. cruzi pcr-oligochromatography test (t. cruzi oligoc-test), targeting a highly conserved satellite dna sequence [20] . we assessed the diagnostic accuracy of the assay on a broad spectrum of biological samples from chagas non-endemic and endemic control persons, infected children, adults, reservoir animals and vectors. t. cruzi y (lineage iib) epimastigotes were grown in glucoselactalbumine-serum-hemoglobine (glsh) medium [21] with 10% fetal calf serum at 26uc. at day 3 post-inoculation, parasites were counted in a bürker counting chamber (marienfeld, germany). tenfold dilution series of parasites ranging from 10,000 parasites/ 180 ml to 1 parasite/180 ml blood were made in human blood freshly taken on edta from a healthy volunteer. non-spiked blood was used as negative control. purified dna from t. cruzi ops21 cl11, stc35r, ivv cl3, x110/8, rn pcr 0 and vmv4, belonging to lineage i, iia, iib, iic, iid and iie, respectively, was obtained from the dna reference bank at the institute of tropical medicine antwerp (itma), belgium. dna from 25 different trypanosoma rangeli isolates (table 1) and from leishmania donovani, trypanosoma brucei gambiense, mycobacterium tuberculosis, schistosoma mansoni and plasmodium falciparum was obtained from other research groups. dna concentrations were measured in the nanodrop nd-1000 uv-vis spectrophotometer (nanodrop technologies, wilmington, usa) and the dna was stored at 220uc. ethical clearance for the study was obtained from the ethics committee of the faculty of medicine, university of chile, santiago, chile. written informed consent was given by the patients or guardians and from non-diseased persons. research on biological samples from animals was conducted adhering to the institution's guidelines for animal husbandry. blood samples from t. cruzi infected persons. 2 ml whole blood from 6 children (0-10 years old) and 27 adults (.18 years old) infected with t. cruzi were collected in chile (region iv), between the years 2000 and 2004. the blood was mixed with an equal volume of 6 m guanidine-hcl and 0.2 m edta, and 200 ml was subjected to dna extraction. persons were considered as infected with t. cruzi if positive in kdna pcr amplifying the minicircle variable region followed by southern blot hybridization with a t. cruzi specific probe, as described by zulantay et al. [22] . for 20 of the 27 adult chagas disease patients, clinical, serological and xenodiagnosis data were available. six patients showed normal electrocardiogram (ecg) results, 11 showed prolonged qtc syndrome and one showed sinus tachycardia, while for 2 patients no ecg was done. all 20 patients chagas disease (american trypanosomiasis) is caused by the protozoan parasite trypanosoma cruzi and represents a major public health problem in latin america. furthermore, growing human population movements extend the disease distribution to regions outside the south american continent. accurate diagnosis is crucial in patient care and in preventing transmission through blood transfusion, organ transplantation, or vertical transmission from mother to child. routine diagnosis of trypanosoma cruzi infection generally is based on detection of the host's antibodies against the parasite. however, antibody detection tests are liable to specificity problems and are of limited use in assessing treatment outcome and congenital infections. the introduction of the polymerase chain reaction (pcr) to amplify specific dna sequences opened promising diagnostic perspectives. despite its reported high sensitivity and specificity, broad use of the pcr technique in diagnosis of chagas disease is hampered by its complexity and the lack of any standardization. we here present the development and evaluation of the t. cruzi oligoc-test, a simple and standardized dipstick format for detection of pcr amplified t. cruzi dna. the new tool is an important step towards simplified and standardized molecular diagnosis of chagas disease. were positive in two independent immunodiagnostic tests (iif and elisa). all 20 patients were negative in xenodiagnosis but 5 showed a positive result when the kdna pcr was performed on the xenodiagnosis specimens. biological samples from reservoir animals and vectors. 200 ml blood from 2 octodon degus and 5 abrothrix olivaceus rodents and the intestinal content from 14 mepraia spinolai bugs were collected. all animals originated from las chinchillas national reserve (chile, region iv) and were classified as infected with t. cruzi based on positive results in kdna pcr followed by southern blot hybridization [22] . dna from t. cruzi y culture was extracted with the qiaamp dna mini kit (qiagen, hilden, germany). spiked blood and blood from the endemic control persons were extracted using the qiaamp dna blood mini kit (qiagen, hilden, germany). dna from blood of patients and animals was extracted with the ezna blood dna kit (omega bioteck, inc., doraville, ga). intestinal content of the vectors was mixed with 200 ml pbs, boiled for 10 minutes and centrifuged at 10,000 g. the supernatant was used as dna template. an alignment of the internal control and target dna, primers and probes sequences is presented in figure 1 . primers, probes and internal control dna were synthesized by biomers.net (ulm, germany). primers. 103 sequences of the 195 bp t. cruzi satellite dna (genbank accession numbers ay519985 to ay520098) were aligned using the ''dna man'' software (lynnon corporation, quebec, canada). the sense primer tc-sat-f 59-cactctc-tgtcaatgtctgtttgcgtg-39 and anti-sense primer tc-sat-r 59-gaaattcctccaagcagcggata-39 were designed targeting a conserved 81 bp sequence within the t. cruzi satellite dna. the anti-sense primer tc-sat-r was biotinylated at the 59 end. internal control dna. the internal control (ic) dna sequence (81 bp) is identical to the t. cruzi target sequence except for a 20 bp central part. the ic central sequence was designed with the same length and the same gc content as the t. cruzi central sequence. probes. the t. cruzi detection probe (59-tggaca-ccaaacaaccc-39) and the ic detection probe (59-agggtctactgggttacctg-39) were designed to hybridize the t. cruzi and ic central sequences respectively. the detection probes were conjugated with gold particles using the procedure described in patent wo 2004/099438a1 [14] . the t. cruzi and ic migration control probes were synthesized with the reverse complement sequence of the respective detection probes. the 50 ml pcr reaction mixture was prepared by adding 2.5 ml sample dna and 1 unit of hot star taq polymerase (qiagen, hilden, germany) to 47.3 ml of t. cruzi ampli-mix (coris table 1 . code, origin, host, and amount of dna detected by the t. cruzi oligoc-test of 25 t. rangeli isolates. principle. the t. cruzi oligo-strip construction and principle is the same as the hat-pcr-oc dipstick described by deborggraeve et al. [15] but a migration control line was added to the test side. a schematic overview of the t. cruzi oligoc-test principle is presented in figure 2 . the ic line and the migration control lines determine whether the t. cruzi oligoc test is valid or invalid. an invisible migration control line indicates an invalid detection step, while an invalid pcr reaction is indicated by a negative ic line in combination with a negative test line. the latter may happen due to inhibitory factors in the extracted dna. the assay is repeated when the control lines indicate failure of migration or of the pcr reaction. when a sample contains high concentrations of t. cruzi dna, competition between the t. cruzi and the ic template dna can result in an invisible ic control line but now combined with a strongly visible t. cruzi test line. in this case the test result is considered as a valid positive result. assay procedure. after pcr amplification the pcr product is denaturated at 94uc for 30 seconds and transferred immediately to ice. forty microliters are mixed with an equal volume of oligostrip running buffer preheated at 55uc, followed by dipping the t. cruzi oligo-strip into the solution. test results are read after 5 minutes qualitatively. whenever a test line appears that is visible by naked eye, the test is considered positive. once test results are read the oligo-strips should not be stored for future reading as background may appear after drying of the strip. the detection limit of the t. cruzi oligoc-test was evaluated on three independent tenfold serial dilutions of t. cruzi strain y dna in water containing 0.1 mg/ml acetylated bovine serum albumin (bsa). the lower detection limit was consistently 1 fg dna per assay ( figure 3a ) which is about 1/200 of the dna content of one parasite. the analytical sensitivity was also evaluated on dna extracted from three independent blood sample series spiked with decreasing numbers of living t. cruzi epimastigotes. the assay was able to detect 1 parasite in a 180 ml blood sample ( figure 3b ), while non-spiked control blood samples always remained negative. the analytical specificity of the assay was evaluated on tenfold serial dilutions of dna from t. cruzi strains representative for the 6 different lineages and on dna from relevant non target pathogens. the t. cruzi oligoc-test was able to detect 1 fg dna of the strains x110/8 (lineage iic), rn pcr o (iid) and vmv4 (iie), 10 fg dna of the strains stc 35r (iia) and ivv cl3 (iib), and 1 pg dna of the strain ops21 (i). negative test results were observed with 5 ng purified dna per test of l. donovani, t. brucei gambiense, m. tuberculosis, s. mansoni and p. falciparum. to assess the level of t. rangeli detection by the t. cruzi oligoc-test, tenfold serial dilutions of purified dna from 25 different t. rangeli isolates were tested (table 1 ). four t. rangeli isolates could be detected at a dna concentration of 10 pg per assay, 13 at a dna concentration of 100 pg or 1 ng, while 8 isolates remained negative when tested at 1 ng dna per assay. negative results were observed when testing dna extracts from blood of 20 t. brucei gambiense infected persons from drc, 20 l. donovani infected persons from nepal, 20 p. falciparum infected persons from zambia, and 48 chagas endemic control blood samples from chile (table 2) . hence, the specificity of the assay on 60 chagas non-endemic and 48 endemic control blood samples was 100% with a 95% confidence interval [ci] scored by wilson's method of 96.6% to 100% [23] . the diagnostic accuracy of the t. cruzi oligoc-test was tested in 2007 on biological samples from 6 t. cruzi infected children and 27 infected adults, 2 infected o. degus rodents, 5 infected a. alivaceus rodents and 14 infected m. spinolai bugs ( table 2) . all samples showed a positive test result except for 2 blood samples from children. hence, the test showed a sensitivity of 66.7% (95% ci: 30%-90.3%), 100% (87.5%-100%), 100% (95% ci: 64.6%100%) and 100% (95% ci: 78.5%-100%) on the specimens from infected children, adults, rodents and vectors, respectively. the two oligoc-test negative blood samples showed invalid results in two independent repetitions. therefore, dna was ethanol precipitated and washed as described elsewhere [15] . the purified dna samples were retested in t. cruzi oligoc-test and valid negative test results were observed. antibody detection is routinely used for diagnosis of chagas disease but suffers from specificity problems because of crossreactivity with antibodies against other infectious agents such as leishmania spp. and t. rangeli. furthermore, immunodiagnosis is less useful in cure assessment after treatment and in diagnosis of congenital transmission. xenodiagnosis is cumbersome, time consuming and invasive. molecular detection of the parasite by the pcr technique is able to overcome most of these drawbacks since high sensitivity and specificity is combined with detection of dna as surrogate for parasite detection. despite the major advantages, pcr is still restricted to research applications and did not enter many diagnostic laboratories yet, which is mainly due to the complexity of the technique. furthermore, the numerous in house diagnostic pcr assays and the neglect of any standardization hamper this promising technique to occupy a major position in clinical diagnosis. next to patient management, pcr can play an important role as an accurate surrogate for parasite detection in epidemiological studies on reservoir animals and vectors. in this study a molecular dipstick assay for simple, rapid and standardized detection of t. cruzi dna was developed and evaluated. the t. cruzi oligoc-test consists of pcr amplification of a short sequence within the t. cruzi satellite dna followed (1) is performed using a pcr mix containing single-stranded internal control (ic) template (2) . the ic sequence is the same as the target sequence within the t. cruzi satellite dna but with a specific internal sequence (dotted line). both templates are amplified with the same primers of which the reverse primer is biotinylated (n). when the pcr and subsequent denaturation is completed, the pcr product solution contains single-stranded t. cruzi and ic dna (3), and is mixed with an equal volume of migration buffer preheated at 55uc. the t. cruzi oligo-strip is dipped into the mixture and test results are read after 5 minutes migration at 55uc (4). during migration, the solution takes up the gold-labelled (#) detection probes. the t. cruzi detection probes (5) at the test side and the ic detection probes (6) at the control side hybridise with their respective amplicons. the biotinylated pcr products accumulate on the neutralite avidin lines at both sides of the dipstick and the t. cruzi and ic amplicons are visualized by their respective detection probes (7) . in case of a negative sample, only the ic amplicon is present and visualized at the control side of the dipstick (8) . the excess of detection probes migrate further and hybridise on the complementary probes coated at both sides of the dipstick as a control for migration (9) . doi:10.1371/journal.pntd.0000450.g002 molecular dipstick for chagas disease www.plosntds.org by a single step pcr product detection in dipstick format. the detection step is performed in 5 minutes and the only equipment needed is a pipette and water bath. the lower detection limits of the assay are 1 fg of purified t. cruzi strain y dna and 1 parasite in a 180 ml blood sample. this is similar to the detection limits of the primers tcz1/tcz2 and diaz1/diaz2 targeting the same 195 bp satellite dna as reported by moser et al. [24] and diaz et al. [25] , respectively. however, when we tested serial dilutions of dna from t. cruzi strains representative for the 6 lineages we observed a 100 to 1000 times lower sensitivity on the t. cruzi strain of lineage i compared to the strains of lineage ii. the lower sensitivity on t. cruzi lineage i is probably due to the lower copy number of the satellite repeats in lineage i as reported by elias et al. [26] . this is confirmed by the fact that diaz et al. also observed a lower sensitivity of their primers on dna from t. cruzi i strains [25] . the test showed a positive signal when tested on purified dna from t. rangeli. to assess the level of cross-reaction, serial dilution series of dna of 25 different t. rangeli isolates were tested. four of the 25 isolates could be detected at 10 pg dna per test which is 10 times more than the required dna of t. cruzi lineage i and 100 to 1000 times more than t. cruzi lineage ii. fourteen t. rangeli isolates required a higher amount of dna to be detectable with the t. cruzi oligc-test and 8 remained negative at 1 ng dna per assay. this is in contrast with ochs et al. [27] and virreira et al. [10] who reported no amplification of t. rangeli dna by the tcz1/tcz2 primers. however, these authors used only one t. rangeli isolate and one dna concentration. the observed cross-reaction with t. rangeli is not surprising since the table 2 . diagnostic accuracy of the t. cruzi oligoc-test on biological samples from chagas non-endemic controls and endemic controls and from t. cruzi infected persons, reservoir animals and vectors. satellite dna has been described in t. rangeli. breniere et al. [28] reported that the number of repeats of the 195 bp satellite dna is far lower in t. rangeli than in t. cruzi, which might explain the higher detection threshold of the assay for t. rangeli. the variable t. rangeli detection levels are probably due to variable copy numbers of the satellite repeats within the t. rangeli population, as described for t. cruzi [26] . however, a parasitaemia of 10,000 t. rangeli parasites per ml of blood in human is unlikely since several studies reported no or very low t. rangeli multiplication in mammalian host cells [29, 30] . the specificity of the t. cruzi oligoc-test was estimated at 100% on blood samples from 60 chagas non-endemic and 48 endemic control persons. sensitivities of 93.9% on 33 infected patients and of 100% on 7 infected rodents and 14 infected vectors were observed. the high sensitivity on blood samples from adults is encouraging given the general low parasitaemia in the blood of such patients. however, we used a kdna pcr followed by southern blot as the main reference test instead of immunodiagnosis, that is currently the standard diagnostic technique for chronic chagas disease. all 20 adult patients for which additional diagnostic test results were available showed positive immunodiagnostic test results but were negative in conventional xenodiagnosis, indicating the high sensitivity of pcr based parasite detection. further evaluations are needed to estimate the diagnostic accuracy of the test on chronic chagasic patients diagnosed on the basis of immunodiagnostic tools alone. the negative results on the two blood samples from children are unexpected since parasite loads are generally higher in children than in adults. the two samples showed initially invalid t. cruzi oligoc-test results, indicating inhibition of the pcr reaction. therefore, the dna was precipitated, washed and retested with the t. cruzi oligoc-test where after the tests became valid but negative. the dna might have been lost during the multiple purification steps. the test showed high sensitivity on reservoir animals and vectors, which is promising regarding the coexistence of different t. cruzi lineages, including lineage i, at the collection site as reported by rozas et al. [31] . in conclusion, the diagnostic accuracy on this diverse panel of biological samples indicates the potential of the t. cruzi oligoc-test. further phase ii and iii evaluation studies are needed to assess the diagnostic sensitivity and specificity of the test on human target populations in different endemic regions. persons with potentially cross-reacting diseases such as leishmaniasis should be included in the control group during future phase ii evaluations. this new tool is a first step towards low-tech and user-friendly molecular diagnostics for t. cruzi detection. besides the simplification, presentation of the t. cruzi oligoc-test as a selfcontaining kit with ready-to-use pcr mix and dipsticks offers excellent prospects for pcr standardization. standardization of the pcr technique is crucial for its implementation as a reference parasite detection test for diagnosis and clinical studies. chagas disease is routinely diagnosed by antibody detection but a simple and standardized pcr format can play an important role in situations were the use of immunodiagnosis is limited, such as congenital chagas disease, patient follow-up after treatment and disease re-activation after organ transplantation. an additional potential niche for the t. cruzi oligoc-test may be patients with acute chagas disease but negative test results in direct microscopy and patients with chronic chagas disease but inconsistent or borderline immunodiagnostic test results. isothermal amplification of the parasite's dna through novel methods such as the loop mediated isothermal amplification (lamp) [32] and the nucleic acid sequence based amplification (nasba) [33] might further simplify the assay. furthermore, standardized pcr formats to discriminate the 6 lineages of t. cruzi would be most welcome, since accurate lineage discrimination is important for disease and vector control programmes [34] . additional standardization of the sampling procedures and dna extraction is needed as well. in 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sars-cov by oligochromatography of rt-pcr amplicons characterization of satellite dna in trypanosoma brucei and trypanosoma cruzi structures antigéniques de trypanosoma brucei (protozoa, kinetoplastida) use of polymerase chain reaction (pcr) and hybridization assays to detect trypanosoma cruzi in chronic patients treated with itraconazole or allopurinol probable inference, the law of succession, and statistical inference detection of trypanosoma cruzi by dna amplification using the polymerase chain reaction an improved polymerase chain reaction assay to detect trypanosoma cruzi in blood organization of satellite dna in the genome of trypanosoma cruzi postmortem diagnosis of autochthonous acute chagasic myocarditis by polymerase chain reaction amplification of a species specific dna sequence of trypanosoma cruzi copy number differences in the 195 bp repeated satellite dna from trypanosoma cruzi and trypanosoma rangeli: potential use for epidemiologic surveys infectivity of trypanosoma rangeli in a promoncytic mammalian cell line interaction of trypanosoma rangeli tejera, 1920 with different cell lines in vitro coexistence of trypanosoma cruzi genotypes in wild and peridomestic mammals in chile loop-mediated isothermal amplification of dna nasba and other transcription based amplification methods for research and diagnostic microbiology american trypanosomiasis (chagas' disease) and the role of molecular epidemiology in guiding control strategies key: cord-269004-hj03n13h authors: krähling, verena; dolnik, olga; kolesnikova, larissa; schmidt-chanasit, jonas; jordan, ingo; sandig, volker; günther, stephan; becker, stephan title: establishment of fruit bat cells (rousettus aegyptiacus) as a model system for the investigation of filoviral infection date: 2010-08-24 journal: plos negl trop dis doi: 10.1371/journal.pntd.0000802 sha: doc_id: 269004 cord_uid: hj03n13h background: the fruit bat species rousettus aegyptiacus was identified as a potential reservoir for the highly pathogenic filovirus marburg virus. to establish a basis for a molecular understanding of the biology of filoviruses in the reservoir host, we have adapted a set of molecular tools for investigation of filovirus replication in a recently developed cell line, r06e, derived from the species rousettus aegyptiacus. methodology/principal findings: upon infection with ebola or marburg viruses, r06e cells produced viral titers comparable to veroe6 cells, as shown by tcid(50) analysis. electron microscopic analysis of infected cells revealed morphological signs of filovirus infection as described for humanand monkey-derived cell lines. using r06e cells, we detected an unusually high amount of intracellular viral proteins, which correlated with the accumulation of high numbers of filoviral nucleocapsids in the cytoplasm. we established protocols to produce marburg infectious virus-like particles from r06e cells, which were then used to infect naïve target cells to investigate primary transcription. this was not possible with other cell lines previously tested. moreover, we established protocols to reliably rescue recombinant marburg viruses from r06e cells. conclusion/significance: these data indicated that r06e cells are highly suitable to investigate the biology of filoviruses in cells derived from their presumed reservoir. bats have been shown to be hosts for many pathogens, including those causing tropical diseases, such as leptospira, hendra virus, nipah virus and sars-like coronavirus [1] [2] [3] [4] [5] [6] . in some cases the development of disease in humans has been directly linked to contact with infected bats. recently, several species of fruit bats were identified as probable reservoirs for the filoviruses marburg virus (marv) and ebola virus (ebov) [7] [8] [9] [10] [11] . filoviruses cause a severe hemorrhagic fever with case fatality rates of up to 90%, for which there is neither an approved vaccine nor specific treatment currently available [12] . as a result of this, as well as the fact that filoviruses represent a serious hazard for laboratory workers, they are classified as biosafety level 4 (bsl4) agents. the filovirus outbreaks in central africa occur sporadically and unpredictably, the latter contributes considerably to the public awareness of filovirus outbreaks. for more than 40 years the search for the natural reservoir of filoviruses was one of the most interesting endeavours in the field of highly pathogenic agents, and was fuelled by the dramatic outbreaks, cases of filovirus infected tourists and concerns that filoviruses might be abused as biological weapons. in the case of ebov, outbreaks could frequently be traced back to the preparation of bush meat, often from sick monkeys, for consumption [13, 14] . since filovirus infection of monkeys results in a rapid and fatal hemorrhagic fever, it was considered that monkeys do not represent the natural reservoir of ebov or marv. right from the beginning of the recorded history of filovirus outbreaks, the marv outbreak in 1967, it was suspected that bats might also be connected to the spread of infection. this was emphasized by the observation that, in those cases where the consumption of contaminated bush meat could be ruled out as the source of infection, often a close contact between index cases and bats was observed [15] . in 1996, swanepoel et al. were able to show that certain species of bats could be productively infected with ebov without showing signs of disease, which was considered a prerequisite for serving as natural hosts [16] . supporting this hypothesis, filoviral genomic rna and antibodies could be detected in bats of different species from the region where outbreaks had occurred, providing the first evidence that bats are infected in a natural context [8, 11] . finally, while marv was isolated from samples of the megachiropteran rousettus aegyptiacus that were trapped in regions where outbreaks took place [15] the assumption that this fruit bat species can also serve as a reservoir for ebov is based on serologic data [9] . the filoviruses ebov and marv are enveloped rna viruses with a filamentous shape and constitute the family filoviridae within the order mononegavirales. the family of filoviridae contains the genera marburgvirus and ebolavirus [12] . filoviruses contain a nonsegmented negative-strand 19 kb rna genome, which encodes seven structural proteins and an additional nonstructural protein in the case of ebola virus. the genome is associated with four nucleocapsid proteins: np, vp30, vp35 and l [17] . np encapsidates the viral genome and is, together with the polymerase l and the polymerase cofactor vp35, necessary and sufficient for viral replication. vp30, the fourth nucleocapsid protein, represents an essential transcription factor for ebola virus [18] [19] [20] . the filoviral nucleocapsid is enclosed by two matrix proteins, vp40 and vp24 that connect the nucleocapsid with the lipid envelope [17] . the transmembrane glycoprotein gp is inserted in the envelope, where it recognizes target cells and induces fusion between cellular and viral membranes [21] [22] [23] . so far, little is known about the filoviral life cycle in the presumed reservoir. one study describes persistent infection of a mexican free-tailed bat cell line tb1.lu with ebov. the authors showed that ebov replication in these cells was low but could be stimulated by inducing the ras/mapk pathway [24] . the mexican free-tailed bat belongs to the order microchiroptera and is abundant in north america but is only very distantly related to megachiroptera such as rousettus aegyptiacus, the presumed natural reservoir of filoviruses. the unavailability of a cell line from a bat species that is relevant for filovirus transmission to humans constrains research of filovirus biology in the natural reservoir. recently, jordan et al. presented a newly established cell line derived from rousettus aegyptiacus (r06e), which could presumably close this gap by allowing in vitro studies to understand the replication of filoviruses in bats [25] . so far, filoviruses have been propagated in human or monkey cell lines and it was now of interest to determine whether cells from the natural host replicate filoviruses, and if so to characterize the infection. here we have examined whether the r06e cell line is suitable for investigations of filovirus infection. veroe6 (african green monkey kidney cells), hek293 (human embryonic kidney cells), huh7 (human hepatoma cells) and r06e cells (derived from rousettus aegyptiacus) [25] were cultured in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal calf serum (fcs), penicillin (50 units/ml), and streptomycin (50 mg/ml). the leiden strain of marv, which was isolated in 2008 from a tourist visiting a bat-infested cave in uganda [26] , or the mayinga strain of zaire ebolavirus (zebov) (genbank accession number nc002549) were propagated in veroe6 cells. virus titers were determined by 50% tissue culture infectious dose (tcid 50 ) assays. cells were infected with marv or zebov with 0.1, 0.5 or 5 tcid 50 /ml per cell, as indicated. all work with filoviruses was performed in the biosafety level 4 (bsl4) facility of the philipps university, marburg. veroe6 cells were cultured in 96-well plates to 50% confluence and infected with 10-fold serial dilutions of supernatants from infected cells. at 10 to 14 days post infection (p.i.), when the cytopathic effect had stabilized, cells were analyzed by light microscopy. the tcid 50 /ml was calculated using the spearman-kä rber method [27] . infected cells were fixed 2 or 3 days p.i. in paraformaldehyde (pfa) and glutaraldehyde (4% pfa, 0.1% glutaraldehyde in 60 mm pipes, 25 mm hepes, 2 mm mgcl 2 , 10 mm egta, ph 7.0), scraped off after 30 minutes and centrifuged at 20,0006g for 20-30 minutes. the fixative solution was replaced with 4% pfa in dmem to completely inactivate the sample overnight before removal from the bsl4 laboratory. dehydration of infected cells and embedding in epon were performed as described previously [28] . ultrathin sections were stained with uranyl acetate and lead citrate and observed with a zeiss 109 electron microscope. supernatants of the infected cells were centrifuged through a 20% sucrose cushion at 77,0006g at 4uc to concentrate the viral particles. the pellet was inactivated and fixed with 4% pfa in dmem overnight. negative staining of viral suspensions was done with 2% phosphotungstic acid. cells were transfected with plasmids encoding a marv minigenome (3m-5m-luc, 1 mg) [20, 29, 30] and the viral proteins necessary for replication and transcription: np (0.1 mg), vp35 (0.5 mg), vp30 (0.1 mg), and the polymerase l (1 mg) using transit-lt1 (mirus, madison, wi, usa) according to the manufacturer's instructions. the minigenome contained a renilla luciferase reporter gene instead of the cat gene [30, 31] . in addition, a plasmid coding for the t7 polymerase (0.5 mg) and a construct constitutively expressing a firefly luciferase reporter (pgl4, promega, 0.1 mg), suitable for normalization of transfection efficiency, were co-transfected. at 48 h post transfection (p.t.), cells were lysed in passive lysis buffer (promega, madison, wi, usa). luciferase assays were performed using the promega dual-luciferase reporter assay system. relative firefly luciferase signals were used to normalize for transfection efficiency. the marv ivlp assay was performed as described by wenigenrath et al. [30] . at 48 h p.t., cells and supernatants were harvested. ivlps from the supernatant were used to infect cells that were either untreated or pretransfected with plasmids marburg virus and several species of ebola virus are endemic in central africa and cause sporadic outbreaks in this region with mortality rates of up to 90%. so far, there is no vaccination or therapy available to protect people at risk in these regions. recently, different fruit bats have been identified as potential reservoirs. one of them is rousettus aegyptiacus. it seems that within huge bat populations only relatively small numbers are positive for filovirus-specific antibodies or filoviral rna, a phenomenon that is currently not understood. as a first step towards understanding the biology of filoviruses in bats, we sought to establish a model system to investigate filovirus replication in cells derived from their natural reservoir. here, we provide the first insights into this topic by monitoring filovirus infection of a rousettus aegyptiacus derived cell line, r06e. we were able to show that filoviruses propagate well in r06e cells, which can, therefore, be used to investigate replication and transcription of filovirus rna and to very efficiently perform rescue of recombinant marburg virus using reverse genetics. these results emphasize the suitability of the newly established bat cell line for filovirus research. student's t test was performed to analyze statistical significances within the different in vitro assays. whole-cell extracts were prepared by lysing cells in sodium docecyl sulfate (sds) sample buffer (25% glycerol, 2.5% sds, 125 mm tris [ph 6.8], 125 mm dithiothreitol, 0.25% bromophenol blue). samples were boiled for 10 minutes at 99uc and transferred into a fresh tube before removal from the bsl4 facility. proteins were separated on 12% sds polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. immunostaining was performed with dilutions of primary antibody in phosphate-buffered saline containing 1% (w/v) skim milk and 0.1% tween-20, as indicated below. vp40-specific monoclonal antibodies (marv: anti-vp40 40-2-2 (1:2000); zebov: anti-vp40 2c4 (1:200)) were used to detect the vp40 proteins of the respective virus. a mouse monoclonal antibody was used to detect a-tubulin (clone dm 1a, 1:5000; sigma-aldrich, st.louis, mo, usa) as a cellular control protein. western blot detection was performed with alexa 680-conjugated anti mouse immunoglobulin g secondary antibody using the odyssey infrared imaging system (li-cor, lincoln, ne, usa). the total amount of protein in the cell lysates was determined by separating cell lysates on 12% sds polyacrylamide gels which were stained with coomassie brilliant blue. gels were then quantified by using the odyssey infrared imaging system application software (version 2.1.12). r06e cells were grown in 6-well plates to 50% confluence. transfection of plasmids coding for the supporting nucleocapsid proteins np (0.5 mg), vp35 (0.1 mg), vp30 (0.1 mg) and l (2 mg) as well as the t7 polymerase (0.5 mg) and the full-length cdna construct of marv (2 mg) was performed with transit-lt1 (mirus, madison, wi, usa) according to the manufacturer's instructions. five hours p.t. the medium was replaced with 4 ml dmem with 2.5% fetal calf serum. cells and supernatants were harvested between day 6 and 12 after cpe formation. supernatant was used to infect fresh r06e cells (passage 1). when passage 1 cells displayed a cpe, viral rna was isolated from the supernatant using the qiaamp viral rna mini kit (qiagen, venlo, netherlands). one-twelfth of the eluted rna was used for reverse transcription-pcr (rt-pcr) using the transkriptor onestep rt-pcr kit (roche, basel, switzerland) and glycoproteinspecific primers designed to amplify nt 5889 to 7370 of the marv genome (forward: 59-caggtcgactcagtgaatatattct-catat-39; reverse 59-gaggcaccagaactagagga). amplified fragments were restricted with kpni to distinguish recombinant and wild type virus. furthermore, integrity of recombinant viral rna was verified by sequencing of the complete genome. cells and supernatants of passage 1 were to determine whether r06e cells are susceptible to filovirus infections, we infected r06e and veroe6 cells, which are commonly used to prepare stock virus [32] [33] [34] , with marv, strain leiden [26] , or zebov, strain mayinga using 0.5 tcid 50 per cell. both cell lines exhibited a strong cytopathic effect (cpe) at day 7 p.i.. supernatants of infected cells collected at day 1, 2, 3 and 7 p.i. were subjected to tcid 50 analysis, which revealed that marv showed similar growth kinetics and end titers in r06e cells and veroe6 cells. zebov grew faster in veroe6 cells than in r06e cells but reached the same maximum titer at 7 d p.i. (fig. 1a) . when the two cell lines were infected with 5 tcid 50 per cell of ebov or marv no differences between r06e and veroe6 cells were observed (not shown). investigations to analyze the reason of the differences between high and low infectious doses are underway. the protein composition of viral particles from the supernatants of both cell lines was analyzed by coomassie brilliant blue staining. this analysis did not show significant differences in the viral protein composition between virus produced during infection of r06e cells and veroe6 cells (fig. 1b) . next we investigated the structure of filovirus-infected r06e cells and viral particles, by electron microscopy (em). viral inclusions in perinuclear regions were composed of hexagonally arranged nucleocapsids with characteristic electron-transparent internucleocapsid zones for zebov (fig. 1c 1) and a higher density of nucleocapsid packaging for marv (fig. 1c 2) . at the plasma membrane, mature viral particles in the process of budding or completely formed were readily observed (fig. 1c 3 and 4) . em analysis of negatively stained samples of the supernatants of r06e cells infected with marv or zebov showed typical filamentous particles (fig. 1c, 5 and 6) . notably, the number of intracellular marv and ebov nucleocapsids in r06e cells was considerably higher than in veroe6 cells. this is reflected by the larger size of viral inclusions, which represent accumulations of viral nucleocapsids in r06e (fig. 1d 3-6 ) compared to veroe6 cells (fig. 1d 1,2) . we were, therefore, interested to further investigate whether the amount of intracellular viral proteins was different in veroe6, huh7 and r06e cells. the three cell lines were infected with 0.1 tcid 50 per cell and western blot analysis was performed with lysed cells at 48 and 72 h p.i. using monoclonal antibodies specific for marv or zebov vp40 or the cellular cytoskeleton protein tubulin. these analyses revealed that the fruit bat cells accumulated considerably more viral protein than the other cell lines tested ( fig. 2a) . to further support this result, we quantified total protein levels of the different cell lines by coomassie staining of cell lysates separated figure 1 . infection of r06e cells with marv and zebov. (a) veroe6 or r06e cells were infected with marv or zebov with 0.5 tcid 50 /cell. supernatants were collected at 1, 2, 3 and 7 days p.i. and used for tcid 50 assays. (b) supernatant from day 7 was concentrated via ultracentrifugation and viral particles were analyzed by coomassie staining for protein composition. (c) r06e cells were infected with marv and zebov at a high moi, fixed and inactivated at day 3 p.i.. cells were dehydrated and embedded in epon prior to ultrathin sectioning. analysis by transmission electron microscopy showed viral inclusions in the perinuclear region (1, 2) and mature viral particles (3, 4) . marv and zebov particles were purified via ultracentrifugation through a 20% sucrose cushion, fixed with 4% paraformaldehyde, negatively stained and analyzed by electron microscopy (5, 6). (d) veroe6 and r06e cells were infected with marv and zebov at a high moi, harvested at 48 h p.i. and treated as described under b. analysis by transmission electron microscopy showed viral inclusions (broken lines) in the perinuclear region of veroe6 (1, 2) and r06e (3, 4) fig. 2a) the amount of vp40 was then normalized against the total cell protein (fig. 2b) . quantification clearly shows that at 48 as well as 72 h p.i. the highest amount of viral protein can be found in r06e cells when compared to huh7 or veroe6 cells (fig. 2b) . however, our data also indicate that r06e cells support filoviral propagation to an extent comparable with veroe6 or huh7 cell lines. furthermore, em analysis of ultrathin sections of r06e cells revealed morphological signs of filoviral infection filovirus infection of fruit bat cells www.plosntds.org similar to those detected previously in cells originating from monkeys or humans [35, 36] (fig. 1c) . taken together, these observations suggest that r06e cells may not release viral particles with a comparable efficiency to that with which they produce structural proteins. further experiments to examine the viral budding efficiency and interaction of viral proteins with proteins of the endosomal sorting complex required for transport (escrt) in fruit bat-derived cells will be very important, as it has been shown that interaction of marv vp40 with tsg101 or interaction of zebov vp40 with tsg101 and nedd4 plays an important role during the budding process [37] [38] [39] . to further investigate the suitability of r06e cells for the analysis of filovirus replication and transcription we used artificial filovirus minigenome systems [20, 29] . four different cell lines, r06e, hek293, huh7 and veroe6 were transfected with plasmids encoding a marv minigenome and the viral proteins necessary for replication and transcription (np, vp35, vp30 and l). cell lysates were analyzed 48 h p.t. for luciferase reporter activity. all analyzed cell lines showed nearly the same reporter activity, except for the fruit bat cell line where activity was slightly decreased (fig. 3a, upper panel) . using a plasmid encoding the green fluorescence protein (gfp) for transfection of cells we found that the transfection efficiency was 15% for r06e cells, 50% for hek293, 38% for huh7 and 27% for veroe6 cells (data not shown). when the transfection efficiency was taken into account, reporter signal in all tested cell lines showed no significant differences (fig. 3a, lower panel) . furthermore we have tested the ebola virus minigenome system with r06e, huh7, veroe6 and 293 cells and got comparable reporter gene activity (data not shown). it was then investigated whether it was possible to produce marv infectious virus-like particles (ivlps) in r06e cells [30] and to establish a naïve ivlp assay for marv to investigate primary transcription, as has already been described for zebov [31] . r06e or hek293 cells (producer cells, pc) were transfected with plasmids encoding all marv structural proteins, the minigenome and the t7 support plasmid [30] . at 48 h p.t., cells and supernatants were harvested and ivlps from the supernatant were used to infect huh7 or r06e cells (indicator cells, ic), which were either untreated or pretransfected with plasmids encoding the nucleocapsid proteins. we did not detect a significant difference in the luciferase reporter signal in ic, when these were pretransfected (fig. 3b) . interestingly, when non-transfected ic (naïve) were infected with ivlps, only r06e cells showed reporter gene signals (fig. 3c) . further experiments with the naïve ivlp assay revealed that using hek293 cells to produce the ivlps (pc) and r06e cells as ic produced the highest reporter gene signals in the indicator cells (data not shown). this combination took advantage of the very high transfection efficiency of hek293 cells for ivlp production and the high capacity for efficient production of viral proteins in r06e cells. it was then investigated whether recombinant marv could be rescued from r06e cells. previous attempts using the experimental set-up described by enterlein et al. had not worked in our hands [40] . finally, after having unsuccessfully tested several combinations of cell lines for transfection and passage of potentially rescued virus, we used r06e cells for transfection of plasmids encoding the marv nucleocapsid proteins as well as the t7 polymerase together with the plasmid containing the full length genome of marv under the control of the t7 promoter. after 6-12 days cells and supernatant were harvested. the supernatant was used to infect fresh r06e cells (passage 1). this method allowed us to rescue three different recombinant viruses of which rescue of clone #16 is shown in fig. 4 . in the transfected cells, cpe formation was visible at day 7 p.t. and at day 8 after passaging the supernatant to fresh cells. at this time, rna was purified from supernatant and rt-pcr was performed with gp-specific primers. the amplified fragments were restricted with kpni, the restriction site for which was mutated in the recombinant full-length genome. agarose gel electrophoresis of the fragments showed that, while control pcr fragments derived from the wild-type marv genome were restricted by kpni, fragments from clone #16 were not, indicating that clone #16 was a recombinant virus. in addition, western blot analyses with vp40-and np-specific monoclonal antibodies were performed to confirm the presence of recombinant virus in the supernatant. taken together, our experiments suggest that the efficient rescue in r06e cells is the result of an exceptional support of marv replication in the fruit bat cell line. this observation is consistent with the highly efficient expression of marv proteins in r06e cells and their ability to release high titers of marv in the supernatant. at first glance the high expression levels of viral proteins and high viral titers found in the supernatants of the fruit bat-derived cell line r06e are puzzling in light of the presumption that rousettus aegyptiacus is a reservoir for filoviruses. however, experimental infection of fruit bats with zebov has been shown to result in productive infection and high titers of virus without clear signs of illness in the infected animals [16] . a more recent study described a similar observation in the reservoir fruit bat of nipah virus. in this study nipah virus-infected pteropus bats developed a subclinical infection, neutralizing antibodies were filovirus infection of fruit bat cells www.plosntds.org raised in the serum of all animals and virus was excreted periodically [41] . while the mechanisms that result in persistence of filoviruses in the natural host are not understood, it is possible that the huge amounts of viral protein produced by primary target cells induce a rapid immune response leading to clearance of the virus from circulation, with the exception of tissues that are less accessible to the immune response. for filoviruses it has been shown that in the course of human infection, infectious virus continues to be detected in the semen for more than 80 days after infection and can lead to sexually transmitted disease as was the case for marburg virus [42, 43] . it is therefore of interest to investigate whether hidden repositories of virus are present within infected rousettus aegyptiacus that are then reactivated under certain unknown conditions. in addition, using the r06e cells, it will be of great interest to investigate how the bats interferon system responds to filovirus infection. studies are underway to explore whether filoviruses are able to induce a persistent infection in r06e cells and to further analyze the innate immune response of the cells to infection with filoviruses. in summary, we were able to show that r06e cells can be infected productively with filoviruses, giving rise to infectious viral particles. infected cells show morphological signs of filoviral infection similar to those detected previously in cells originating from monkeys or humans. further, r06e cells seem to be more effective in producing recombinant marv than other cell lines tested and allowed for the establishment of a naïve infectious vlp assay for marv. these results emphasize the suitability of the newly established bat cell line for further research in numerous areas of filovirus biology. the contribution of bats to leptospirosis transmission in sao paulo city, brazil bats: important reservoir hosts of emerging viruses henipavirus rna in african bats isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats transmission of human infection with nipah virus the ecology of ebola virus fruit bats as reservoirs of ebola virus large serological survey showing cocirculation of ebola and marburg viruses in gabonese bat populations, and a high seroprevalence of both viruses in rousettus aegyptiacus studies of reservoir hosts for marburg virus marburg virus infection detected in a common african bat therapy and prophylaxis of ebola virus infections multiple ebola virus haemorrhagic fever outbreaks in gabon ebola hemorrhagic fever -gabon: gorilla-to-human? promedmail post isolation of genetically diverse marburg viruses from egyptian fruit bats experimental inoculation of plants and animals with ebola virus interactions of marburg virus nucleocapsid proteins oligomerization of ebola virus vp30 is essential for viral transcription and can be inhibited by a synthetic peptide crystal structure of the c-terminal domain of ebola virus vp30 reveals a role in transcription and nucleocapsid association comparison of the transcription and replication strategies of marburg virus and ebola virus by using artificial replication systems computer simulations of proteolysis of marburg and ebola-zaire filovirus coded proteins to generate nonapeptides with motifs of known hla class i haplotypes and detection of antigenic domains in the viral glycoproteins characterization of marburg virus glycoprotein in viral entry proteolytic processing of marburg virus glycoprotein stimulation of ebola virus production from persistent infection through activation of the ras/ mapk pathway cell lines from the egyptian fruit bat are permissive for modified vaccinia ankara response to imported case of marburg hemorrhagic fever, the netherland virus isolation and quantitation vp40, the matrix protein of marburg virus, is associated with membranes of the late endosomal compartment three of the four nucleocapsid proteins of marburg virus, np, vp35, and l, are sufficient to mediate replication and transcription of marburg virus-specific monocistronic minigenomes establishment and application of an infectious virus-like particle system for marburg virus infection of naive target cells with virus-like particles: implications for the function of ebola virus vp24 vp35 knockdown inhibits ebola virus amplification and protects against lethal infection in mice inhibition of filovirus replication by the zinc finger antiviral protein protective efficacy of neutralizing antibodies against ebola virus infection differentiation of filoviruses by electron microscopy ultrastructural organization of recombinant marburg virus nucleoprotein: comparison with marburg virus inclusions overlapping motifs (ptap and ppey) within the ebola virus vp40 protein function independently as late budding domains: involvement of host proteins tsg101 and vps-4 ebola virus matrix protein vp40 interaction with human cellular factors tsg101 and nedd4 interaction of tsg101 with marburg virus vp40 depends on the pppy motif, but not the pt/sap motif as in the case of ebola virus, and tsg101 plays a critical role in the budding of marburg virus-like particles induced by vp40, np, and gp rescue of recombinant marburg virus from cdna is dependent on nucleocapsid protein vp30 experimental nipah virus infection in pteropid bats (pteropus poliocephalus) spermatogenic transmission of the ''marburg virus''. (causes of persistence and genetic stability of ebola virus during the outbreak in kikwit, democratic republic of the congo all work with wild-type or recombinant marv and zebov was performed in the bsl4 facility of the philipps university marburg. we thank g. ludwig and m. schmidt for technical support with the bsl4 procedures, a. herwig for technical assistance with the cell culture and k. kowalski for excellent support with the bsl4 work. we also thank a. groseth for carefully editing the manuscript. key: cord-272250-asuxx1ln authors: robertson, kis; lumlertdacha, boonlert; franka, richard; petersen, brett; bhengsri, saithip; henchaichon, sununta; peruski, leonard f.; baggett, henry c.; maloney, susan a.; rupprecht, charles e. title: rabies-related knowledge and practices among persons at risk of bat exposures in thailand date: 2011-06-28 journal: plos negl trop dis doi: 10.1371/journal.pntd.0001054 sha: doc_id: 272250 cord_uid: asuxx1ln background: rabies is a fatal encephalitis caused by lyssaviruses. evidence of lyssavirus circulation has recently emerged in southeast asian bats. a cross-sectional study was conducted in thailand to assess rabies-related knowledge and practices among persons regularly exposed to bats and bat habitats. the objectives were to identify deficiencies in rabies awareness, describe the occurrence of bat exposures, and explore factors associated with transdermal bat exposures. methods: a survey was administered to a convenience sample of adult guano miners, bat hunters, game wardens, and residents/personnel at buddhist temples where mass bat roosting occurs. the questionnaire elicited information on demographics, experience with bat exposures, and rabies knowledge. participants were also asked to describe actions they would take in response to a bat bite as well as actions for a bite from a potentially rabid animal. bivariate analysis was used to compare responses between groups and multivariable logistic regression was used to explore factors independently associated with being bitten or scratched by a bat. findings: of 106 people interviewed, 11 (10%) identified bats as a potential source of rabies. a history of a bat bite or scratch was reported by 29 (27%), and 38 (36%) stated either that they would do nothing or that they did not know what they would do in response to a bat bite. guano miners were less likely than other groups to indicate animal bites as a mechanism of rabies transmission (68% vs. 90%, p = 0.03) and were less likely to say they would respond appropriately to a bat bite or scratch (61% vs. 27%, p = 0.003). guano mining, bat hunting, and being in a bat cave or roost area more than 5 times a year were associated with history of a bat bite or scratch. conclusions: these findings indicate the need for educational outreach to raise awareness of bat rabies, promote exposure prevention, and ensure appropriate health-seeking behaviors for bat-inflicted wounds, particularly among at-risk groups in thailand. rabies is an exceptionally fatal encephalitis caused by rhabdoviruses in the lyssavirus genus. transmission typically occurs when broken skin is contaminated with saliva from an infected mammal-usually in association with a bite but in rare instances by scratches. the most well-known and ubiquitous lyssavirus is the rabies virus (rabv), which circulates in new world bats and both old and new world terrestrial mammals. the vast majority of human rabies cases worldwide are transmitted by dogs infected with rabv. a lesser known member of the genus is the mokola virus, which has been isolated from a number of terrestrial mammals in africa (most notably shrews) and has caused at least two human cases [1] , [2] . reservoirs for the remaining nine members of the lyssavirus genus appear to be exclusively old world bats [3] . rabies is a major public health problem in asia. of the estimated 55,000 human cases that occur annually worldwide, more than half occur in asian countries [4] . in recent decades, initiatives aimed at raising rabies awareness (e.g. the world rabies day campaign) and lowering human exposure risk through mass vaccination of leading reservoir species have been implemented globally, coinciding with the development of highly potent human rabies vaccines [4] , [5] , [6] . notable trends have subsequently followed. of all asian countries, thailand has experienced the steadiest decline in human rabies cases, with a near 10-fold decrease in reported cases during the last 20 years [7] . much of this decline is attributable to the country's very extensive use of rabies vaccine in the treatment of persons bitten by dogs. in 2003, for instance, more than 400,000 people in thailand were vaccinated against rabies following potential rabies exposures [8] . historically in southeast (se) asia, animal-based prevention efforts for rabies have almost exclusively been centered on dogs. most reported human cases in the region are traced to these animals either through an exposure history or through molecular or antigenic subtyping of variants from rabid human patients [9] , [10] . the canine-associated rabies variant has been the only one linked to terrestrial wildlife and domestic animals in thailand [8] , further evidence that dogs are the main lyssavirus reservoir in the region. to date, no human cases of rabies linked to lyssaviruses other than canine-associated rabv have been reported in thailand or the rest of se asia. like other dog-rabies endemic countries, the majority of human rabies victims in thailand are children under the age of 15 years [11] . within the last ten years, however, interest in bats and their role in lyssavirus transmission has increased in the region. the discovery of the australian bat lyssavirus (ablv) in australian flying fox bats (pteropus spp.) in the mid-1990's and the isolation of new bat lyssaviruses in the former soviet union [12] , [13] were pivotal in turning scientific interest towards the study of potential asian bat reservoirs. in the last 10 years, evidence of lyssavirus maintenance in se asian chiropterans has emerged from surveillance in cambodia, thailand, bangladesh, and the philippines [14] , [15] , [16] , [17] . although lyssaviruses have not yet been isolated from these mammals, neutralizing antibodies associated with lyssaviruses have been detected in sera from both regional mega and microbats. these findings strongly suggest that asian bats maintain lyssaviruses like their counterparts in europe, africa, australia, and the americas. human deaths due to bat-borne rabies infection have been well documented in these continents [12] , [18] , [19] , [20] . in particular, rabid vampire bats are a major cause of human mortality in south america's amazon region [21] . routine surveillance for bat rabies is lacking in asia and as a consequence, understanding is limited regarding the extent of lyssavirus circulation among se asian bats and the impact on animal and public health. however, evidence thus far raises pressing questions about human health risks. the potential implications of bat rabies are particularly salient in se asia because human-bat interaction occurs routinely in many locales. bat guano is regularly mined from caves for use as a fertilizer. hunting of bats for sale and personal consumption occurs as well, despite laws to stop this practice. the presence of large numbers of bats at many buddhist temples also promotes exposures, as these sites are focal points for commerce, tourism, and religious expression. because the severity of skin trauma inflicted by bats is usually minor and unlikely to prompt a medical visit on the basis of physical injury alone, public knowledge of appropriate healthseeking behaviors following a bat exposure is especially important in preventing cases of bat-borne rabies. rabies postexposure prophylaxis (pep) for bat bites and scratches, as recommended by both the world health organization (who) and the u.s. advisory committee on immunization practices (acip), includes thorough wound washing and the administration of rabies immune globulin (in non-immunized individuals) and rabies vaccine administered in a series of doses [4] , [22] . when administered promptly and properly, rabies pep is highly effective in preventing the disease. to date, however, no intervention has proven effectiveness in stopping the clinical course after symptom onset, a fact which further underscores the importance of early care following a possible lyssavirus exposure. little is known about the extent of bat-specific rabies awareness in se asia. to assess the knowledge and practices of individuals who are most at-risk of bat exposures in thailand, we surveyed persons who regularly come in contact with bats or bat dwellings through occupational activities and other practices. we sought to elucidate gaps in knowledge that potentially have bearing on rabies prevention, describe the occurrence of bat-associated exposures in this population, and explore factors associated with bat exposures that are of potential consequence to lyssavirus transmission. the desired sample size was 200 individuals. surveyed individuals were a convenience sample of adults who collect guano from caves, engage in bat hunting, work or reside at temples that serve as sites for mass bat roosting, or work as game wardens responsible for monitoring and protecting bat caves. engagement in at least one of these activities within the last 5 years and being 18 years or older were inclusion criteria for participation. individuals were recruited from eight provinces as shown by figure 1 . recruitment areas were selected based on proximity to bat caves and/or mass bat roosting sites and certain community characteristics known to promote batassociated activities (e.g., an agrarian-economy that benefits from guano fertilizer use). participants were classified based on the activity in which they most frequently engaged, and were primarily recruited from rural and semi-rural localities. in farming communities near bat caves, village leaders and other local contacts provided assistance in locating individuals known to engage in guano mining. such individuals were also found through referral from existing participants. a similar method was employed to locate bat hunters/trappers in communities where fruit farms are known to attract flying fox bats. to recruit temple workers/residents and game wardens, permission from supervisory officials at buddhist temples and national parks was obtained before individuals under their management were approached for participation. recruits were not offered or given incentives for participating. the study design and consent process was approved by the institutional review board (irb) at cdc (protocol# 5709). all participants were verbally informed of the study's purpose and assured that their responses would be kept anonymous, even if they engaged in illegal activities. oral consent was obtained to ensure rabies is a fatal encephalitis caused by lyssaviruses. evidence of lyssavirus circulation has recently emerged in southeast asian bats. we surveyed persons regularly exposed to bats and bat habitats in thailand to assess rabies-related knowledge and practices. targeted groups included guano miners, bat hunters, game wardens, and residents/personnel at buddhist temples where mass bat roosting occurs. of the 106 people interviewed, 11 (10%) identified bats as a source of rabies. history of a bat bite/ scratch was reported by 29 (27%), and 38 (36%) expressed either that they would do nothing or that they did not know what they would do in response to a bat bite. guano miners were less likely than other groups to indicate animal bites as a mechanism of transmission (68% vs. 90%, p = 0.03) and were less likely to say they would respond appropriately to a bat bite or scratch (61% vs. 27%, p = 0.003). these findings indicate a need for educational outreach in thailand to raise awareness of bat rabies, promote exposure prevention, and ensure health-seeking behaviors for bat-inflicted wounds, particularly among atrisk groups. anonymity and accommodate illiterate participants, and was documented by the interviewer electronically via personal digital assistants (pda) prior to administering the survey. this method of obtaining informed consent was approved by cdc's irb. a 41-item structured questionnaire was developed in english and translated and reviewed by native thai speakers employed by the office of the u.s. cdc, international emerging infections program in bangkok. the questionnaire was designed to be administered in thai via face-to-face interviews, with responses entered in pdas using geoage fast software. not all questions provided data used in this study. the questionnaire was developed based on socio-ecological reasoning about gaps in rabies knowledge that potentially translate into failed prevention on the individual level. data were collected on demographics; primary bat-associated activity and years of experience; history of rabies vaccination; and type and frequency of bat exposures such as cave entry, direct contact with bats, bites and scratches from bats, and bat consumption. individuals who reported receiving rabies vaccination were asked to indicate whether it was in direct response to an animal exposure (i.e. pep) or for pre-exposure immunization (prep), which is a vaccination series most often administered to people who have a relatively high likelihood of rabies virus exposure due to occupational risks or other factors. those who reported receiving prep were asked to describe its administration and only those who indicated receiving a series of injections spaced over multiple days were counted as having prep. to assess rabies-related knowledge, participants were asked to rate their understanding of the disease as either ''little or none'', ''basic'', or ''extensive''; explain how humans acquire the disease, and identify animal sources of the disease. each knowledge question was evaluated independently, and the validity of a participant's self-reported knowledge level was not verified using other responses. participants were also asked to describe the severity of rabies. only responses that emphasized death or profound suffering with no suggestion that recovery was likely were considered evidence that the participant recognized rabies as being severe. awareness of other diseases that humans can get from bats was also elicited. to assess health-seeking practices following transdermal bat exposures, participants were asked about actions they would take if they were bitten or scratched by a bat. responses to this openended question were compared to a similar question later asked about actions a person should take following a bite from a potentially rabid animal, based on the participant's own understanding of what constitutes a potentially rabid animal. questions that were specifically asked about bats preceded all questions asked about rabies to minimize reporting bias, and whenever feasible, participants were asked open-ended questions to minimize the interviewer's influence on responses. participants were also interviewed away from other people. interviewers were instructed to not ask questions in a leading manner and to allow as much time as necessary for participants to answer. survey responses were transferred to a computer, exported into microsoft excel, and then imported into sas version 9.2 for analysis. data were summarized using descriptive statistics and comparisons by bat-associated activity group were made using chi-square or fisher's exact test. multivariable logistic regression analysis was used to explore factors independently associated with being bitten or scratched by a bat. variables related to the bats & rabies kp www.plosntds.org outcome at p-values#0. 25 were included in the model. crude and adjusted odds ratios (or) with 95% confidence intervals (ci) were calculated. associations were statistically significant at p-values less than 0.05. the study was conducted during august 3-18, 2009. a total of 106 people were interviewed. interviews lasted an average of about 10 minutes. demographic characteristics, history of rabies vaccination, and primary bat-associated activity of the participants are described in table 1 . all temple workers/residents and game wardens were involved in their activity at the time of interview; 71% of guano miners and 53% of bat hunters reported that their most recent engagement had occurred within the previous 12 months. of all groups, guano miners had fewer years of schooling, with 89% educated at the primary level or less versus 55% of nonguano miners (p = 0.001). temple workers/residents were more likely to have greater than 15 years of experience in their activity than other groups (59% vs. 26%, p = 0.001). thirty-one percent of participants reported a history of receiving either rabies prep (7.5%) or pep (23.5%) within their lifetimes. of those who had received rabies pep, 96% reported that they had been vaccinated in response to a dog exposure and 4% for a cat exposure. no participants reported receiving pep for a bat exposure and none reported receiving both prep and pep. there were no statistically significant differences between activity groups with respect to rabies vaccination. table 2 describes participant's responses to rabies-related knowledge questions by activity group. a majority of participants (54%) reported having little or no knowledge of rabies. proportionately more temple workers/residents reported basic or extensive knowledge than non-temple workers/residents (p = 0.03). self-assessed rabies knowledge appeared to be lowest among guano miners, but not to a statistically significant degree (p = 0.06). although most (85%) participants seemed to be aware that animal bites cause rabies, significant differences were observed between activity groups. only 68% of guano miners indicated animal bites as a mechanism of transmission compared to 90% of non-guano miners (p = 0.03). when asked to identify which animals are sources of rabies, only 11 (10%) participants named bats. in contrast, dogs were named by 80 (76%), cats were named by 41 (39%), and other mammals (including rodents and large domestic animals) were named by 24 (23%). fourteen participants (13%) were unable to name any animals as rabies sources. differences between activity groups with respect to bat attribution were not statistically significant, and individuals who attributed rabies to dogs were no more likely to also attribute rabies in bats than those who did not attribute rabies to dogs (11% vs 8%, p = 1.0) (not shown in table 2 ). when asked whether they were aware of any other diseases (besides rabies) that humans can get from bats, 18 (17%) answered yes; all were temple workers/residents (not shown in table 2 ). table 3 shows how participants responded when asked ''what actions would you take if you were bitten or scratched by a bat?'' and ''if someone has been bitten by an animal potentially infected with rabies what should that person do?'' twenty-eight (26%) participants expressed that they would seek medical care or rabies pep for a bat bite or scratch, while a significantly higher proportion (95%) advocated these actions if the bite came from a potentially rabid animal (p = 0.0001). the proportion of participants who either said they would do nothing or that they didn't know what they would do if bitten or scratched by a bat was significantly higher than the proportion answering similarly when asked about an exposure to a potentially rabid animal (36% vs. 2%, p = 0.0001). guano miners were more likely than non-guano miners to give this response for bat exposures (61% vs. 27%, p = 0.003). an incidental finding (not shown in the table) was that previous recipients of prep or pep were more likely than nonrecipients to advocate a health-seeking behavior for bat bites and scratches but not to a stasticially significant extent (82% vs 56%; p = 0.15). table 4 describes bat-related exposures by the number and proportion of participants who reported experiencing it at least once in their lifetimes, along with those who reported experiencing it more than five times a year. a history of transdermal bat exposure (bite or scratch) was reported by 29 (27%) participants. table 5 shows factors independently associated with a bat bite or scratch history. variables considered for inclusion in the multivariable model on the basis of biological plausibility included age, sex, years of experience, education, knowledge self-assessment, frequency in bat caves/roost areas, and bat-associated activity. all except the latter three variables were removed from the final model due to unadjusted p-values.0.25. no two variables were so strongly associated with one another or the outcome as to suggest the presence of colinearity. in the final model, self-assessed rabies knowledge of ''little or none'' was not significantly predictive of a bat bite or scratch history, although a strong association was observed prior to adjustment for other variables. individuals who engaged in guano mining, bat hunting, or visiting a bat cave or roost area more than 5 times a year were more likely to report a history of bat bites or scratches. in this survey among persons at risk for bat exposure in thailand, we found that although general awareness of rabies transmission and severity were relatively high, awareness of bat rabies in particular was low, with only 10% of participants identifying bats as a potential source of rabies and 36% failing to say they would take any specific action if bitten or scratched by a bat. bat exposures conducive to potential lyssavirus transmission were also common in this population and were reported by members of all four activity groups, supporting more than just a theoretical risk for these types of incidents. we found that guano miners reported the highest frequency of transdermal bat exposures, were the least knowledgeable about rabies, and were the least likely to say they would respond to bat exposures in a manner that would ensure rabies prevention. based on these findings we conclude that of the groups we surveyed, bat rabies has the greatest potential impact on guano miners. the potential risk associated with guano mining is even more stark given that moribund bats (i.e., those most likely to be rabid) normally fall to the floor of caves, where they can readily come in contact with someone collecting bat droppings by hand. the effectiveness of any bat-borne rabies prevention strategy may hinge upon how well it diffuses into communities where guano mining regularly occurs. education at the community level is an important strategy in the prevention of human rabies [4] . although the decreasing incidence of human rabies in thailand points to the effectiveness of past and present rabies education efforts, our findings demonstrate a need to raise public awareness of the potential risk of rabies associated with bat exposures. special attention should be placed on communities where bats or bat guano are commonly utilized, and if school-based, programs should include primary level students to ensure that they reach those who do not progress past this level of schooling. in addition to emphasizing the importance of exposure avoidance and countering attitudes that inappropriately lower risk perception towards bats, wound washing and healthcare utilization following bat bites and scratches are practices that should be promoted. similarly, if the awareness we observed in the public is indicative of awareness in the medical community, outreach to healthcare professionals might also be needed to ensure that patients presenting with bat exposures are treated in accordance with who guidelines [4] . studies aimed at assessing knowledge and practices in the thai medical community should be explored to ensure that such outreach is well-informed. education at temples and national parks is also recommended to ensure personnel at these sites know to avoid unnecessary bat contact and respond appropriately to bat-inflicted injury. a a strategy that integrates community outreach with law enforcement should be considered as well. comparing the proportion of participants who advocated a given action for a bat exposure with the proportion who advocated the same action for an exposure to a potential rabid animal. b includes cleaning the wound with water, soap, and/or a common antiseptic agent (e.g. betadine). c includes prayer or visiting a traditional healer. d includes bleeding the wound, bandaging, or using topical home remedies (e.g. local herbs). doi:pntd.0001054.t003 table 4 . bat-associated exposure histories reported by participants. to date, there have been no reported cases of human rabies cases associated with bats in thailand. one plausible explanation is that the prevalence of bat lyssaviruses in se asia is so low that humans are rarely if ever exposed to these pathogens. it is also possible that the prevailing assumption about dogs as the usual source of rabies leads patients and their family members to overlook relevant bat encounters when recounting animal exposures, resulting in misdiagnosis or misattribution. additionally, the rate of human rabies vaccination in the population may be high enough to protect many people against bat rabies. in our study, we found that almost a third of all participants reported a history of rabies vaccination, mostly as a result of dog-associated pep. this suggests that the percentage of individuals in our study population with at least some lyssavirus immunity is relatively high and may help account for why bats have yet to be linked to any human rabies cases in the country. however, immunity levels could change if pep use becomes more conservative in the future. currently, funds annually spent on the purchase of human rabies biologics by the thai government are quite substantial [8] , and this financial burden may be difficult to sustain indefinitely. there are some limitations to our study that should be noted. first, it is unlikely that our relatively small convenience sample is representative of all persons engaged in bat-related activities in thailand. our findings may have also been subject to reporting bias, since guano miners and bat hunters may have been less willing than others to answer questions truthfully due to the illegal nature of their work. this potential bias may have led participants to understate their years of experience, which could explain why this variable was not found to be associated with a history of transdermal bat exposures. estimated participation rates for these two groups were also much lower than the other two groups (participation rates were hard to definitively ascertain because participation was ultimately premised on self-identification). additionally, we classified individuals based on their self-reported primary batassociated activity; however, a few participants indicated involvement with other activities (e.g,. guano miners that also hunt bats) either presently or in the past. having such a history was not accounted for this study, although it potentially could be associated with an increased lifetime risk of transdermal bat exposures. the desired sample size of 200 persons was somewhat arbitrarily determined given the lack of reliable estimates for the study population size. failure to meet this number was largely due to the difficulty in finding willing participants who engaged in bat hunting and guano mining, and the limited availability of personnel and funds that could be used to extend the study period. as a consequence of our small sample size and low statistical power, truly significant associations may have gone undetected in this study. however, by recruiting from several provinces, we minimized the influence that geography might have imparted on the associations we observed. another limitation is that the validity and reliability of the questionnaire may have been suboptimal because the survey instrument was not subject to very rigorous in-field testing. our findings have relevance to zoonotic diseases other than rabies. se asian bats have been linked to the encephalitis-causing nipah virus and hendra virus [23] , [24] , [25] , and the corona virus associated with severe acute respiratory syndrome (sars) [26] , [27] . less novel diseases associated with bats also include histoplasmosis, an invasive fungal respiratory disease linked to bat guano exposure [28] . additionally, evidence suggests that bat ectoparasites may transmit pathogens such as bartonella and rickettsia [29] , [30] . in this study, we found that exposures that could potentially facilitate transmission of these diseases appear to occur relatively frequently, with 36% of surveyed participants reporting that they experience direct contact with bats at least twice a year. bat consumption-an activity that in and of itself may be low risk (assuming the bat is well cooked) but could be associated with increased disease risk through contact with bat carcasses-was reported by more than half the participants. exposure to toxic or infectious aerosols is another potential hazard for this population as well, since almost all participants reported regularly being in bat caves and roosting areas. more epidemiological studies are needed to better assess the risks associated with bat-related exposures, particularly in regions of the world where outbreaks of severe zoonoses have occurred and questions remain regarding animal reservoirs for such diseases. parnkaew rattanasilpkancharn from the thailand ministry of health; dr. sumalee boonmar, benchawan pongurgsorn, pongpun sawatwong, and manoon hirunsalee from cdc-thailand ieip; and dr. amy turmelle, felix jackson, and dustyn palmer, of the poxvirus and rabies branch, cdc. disclaimer statement: the findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention. isolation of a rabies related virus from the cerebrospinal fluid of a child with aseptic meningitis a fatal human infection with mokola virus shimoni bat virus, a new representative of the lyssavirus genus world rabies day: focusing attention on a neglected disease oral vaccination of wildlife against rabies: opportunities and challenges in prevention and control the rabies situation in far east asia transmission dynamics of rabies virus in thailand: implications for disease control complex genetic structure of the rabies virus in bangkok and its surrounding provinces, thailand: implications for canine rabies control rabies situation in cambodia moving towards the elimination of rabies in thailand australian bat lyssavirus: a recently discovered new rhabdovirus rabies surveillance in the former soviet union serologic evidence of lyssavirus infection in bats survey for bat lyssaviruses lyssavirus surveillance in bats serologic evidence of lyssavirus infections among bats, the philippines rabies surveillance in the united states during epidemiology and pathogenicity of african bat lyssaviruses first encounter of european bat lyssavirus type 2 (eblv-2) in a bat in finland bat-transmitted human rabies outbreaks, brazilian amazon human rabies prevention-2008: recommendations of the advisory committee for immunization practices nipah virus infection in bats (order chiroptera) in peninsular malaysia duplex nested rt-pcr for detection of nipah virus rna from urine specimens of bats bat nipah virus review of bats and sars identification of a novel coronavirus in bats fungal infections among returning travelers association of baronella with the fleas (siphonaptera) of rodents and bats using molecular techniques dectection of rickettsia, borrelia, and bartonella in carios kelleyi (acari: argasidae) the authors thank dr. robert gibbons and the us department of defense's armed forces research institute of medical sciences (afrims); dr. rattiya naksuwan from the thailand ministry of agriculture; key: cord-012911-d6ct94d9 authors: jalloh, mohamed f.; kaiser, reinhard; diop, mariam; jambai, amara; redd, john t.; bunnell, rebecca e.; castle, evelyn; alpren, charles; hersey, sara; ekström, anna mia; nordenstedt, helena title: national reporting of deaths after enhanced ebola surveillance in sierra leone date: 2020-08-18 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008624 sha: doc_id: 12911 cord_uid: d6ct94d9 background: sierra leone experienced the largest documented epidemic of ebola virus disease in 2014–2015. the government implemented a national tollfree telephone line (1-1-7) for public reporting of illness and deaths to improve the detection of ebola cases. reporting of deaths declined substantially after the epidemic ended. to inform routine mortality surveillance, we aimed to describe the trends in deaths reported to the 1-1-7 system and to quantify people’s motivations to continue reporting deaths after the epidemic. methods: first, we described the monthly trends in the number of deaths reported to the 1-1-7 system between september 2014 and september 2019. second, we conducted a telephone survey in april 2017 with a national sample of individuals who reported a death to the 1-1-7 system between december 2016 and april 2017. we described the reported deaths and used ordered logistic regression modeling to examine the potential drivers of reporting motivations. findings: analysis of the number of deaths reported to the 1-1-7 system showed that 12% of the expected deaths were captured in 2017 compared to approximately 34% in 2016 and over 100% in 2015. we interviewed 1,291 death reporters in the survey. family members reported 56% of the deaths. nearly every respondent (94%) expressed that they wanted the 1-1-7 system to continue. the most common motivation to report was to obey the government’s mandate (82%). respondents felt more motivated to report if the decedent exhibited ebola-like symptoms (adjusted odds ratio 2.3; 95% confidence interval 1.8–2.9). conclusions: motivation to report deaths that resembled ebola in the post-outbreak setting may have been influenced by knowledge and experiences from the prolonged epidemic. transitioning the system to a routine mortality surveillance tool may require a robust social mobilization component to match the high reporting levels during the epidemic, which exceeded more than 100% of expected deaths in 2015. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 from may 2014 to november 2015, sierra leone experienced the largest epidemic of ebola to date, which also affected neighboring liberia and guinea. the epidemic in sierra leone resulted in more than 14,000 cases and nearly 4,000 deaths of ebola [1] . traditional practices involving physical contact with corpses and sick people [2] contributed to ebola transmission [3] [4] [5] . it has been estimated that an average of approximately 2.5 new ebola cases resulted from every unsafe traditional burial during the epidemic in west africa [3] . in one extreme situation, 28 confirmed cases were epidemiologically linked to a single traditional burial of a prominent pharmacist in moyamba district, sierra leone in september 2014 [4] . out of these 28 cases, approximately 75% of them had direct physical contact with the pharmacists' corpse. epidemic control efforts heavily focused on halting risky behaviors, such as washing and touching of corpses as part of traditional burial rites, and providing alternatives for safe burials by specially trained teams [6] [7] [8] . there was a need to promptly identify all deaths occurring in communities to test them for ebola and ensure safe burial [9] [10] [11] . community-based reporting of deaths consequently constituted an important component of responding to the epidemic [5, 12] . in august 2014, the government of sierra leone repurposed an existing national, toll-free telephone line (1-1-7 system) for communities to report all deaths and suspected ebola patients as part of the epidemic response [13] . the 1-1-7 system's design, implementation and adaptations have been described elsewhere [14] . although the government of sierra leone required communities to report deaths of all-causes to the 1-1-7 system during the 2014-2015 ebola epidemic [14] , burial teams were not always successful in responding to all death alerts within 24 hours due to the high community demand and call volumes. this resulted in dissatisfaction among communities where families had to wait longer than 24 hours for safe burial services. furthermore, communities were at times dissatisfied with how corpses were handled by burial teams [15, 16] . as the epidemic progressed, safe alternatives to traditional burials were made available to families such as observing the burial from a safe distance and allowing a religious leader to pray on the corpse. religious leaders played a key role in advocating for incorporating safe alternatives that show respect for the deceased and their family [17] . social mobilization efforts were implemented nationwide to promote ebola protective behaviors including community acceptance of safe burial measures and reporting of deaths to the 1-1-7 line during the epidemic [9, 10, 18, 19] . after the outbreak ended, social mobilization and risk communication interventions that promoted the use of the 1-1-7 line were scaled down. analysis of calling trends indicated that the numbers of deaths reported to the 1-1-7 system sharply declined after the epidemic ended even though the official government policy mandated that all deaths occurring in communities must still be reported. all reported deaths that were suspected to be ebola or otherwise 'suspicious' were supposed to be forwarded to district-based surveillance officers by the 1-1-7 call center operators for screening and further investigations depending on the circumstances of the death. strategies for continuing death reporting in sierra leone or other post-ebola-outbreak settings are scarce. factors contributing to the decline in death reporting after the epidemic and enhanced surveillance ended are not well understood, and neither are motivating factors for those who continued to report. moreover, the potential influence of ebola experiences on death reporting motivations in post-ebola-outbreak settings has not been examined. to inform routine mortality surveillance, we aimed to describe deaths reported to the 1-1-7 system, perceptions of the reporting system, and motivations to report the deaths after the epidemic and enhanced ebola surveillance had ended. previous analysis of the number of sick people and deaths reported to the 1-1-7 system has been published for the period of september 2014 to december 2016 [14] . to establish more comprehensive trends in death reporting during a five-year period spanning september 2014 -september 2019, we obtained monthly unadjusted aggregated data of death alerts placed to the 1-1-7 call center managed by ehealth africa on behalf of the sierra leone ministry of health and sanitation [20] . to inform strategies for improving routine surveillance of deaths, in april 2017, we then conducted a cross-sectional, telephone-based survey with individuals aged 18 years and above who reported a death to the 1-1-7 system after the end of enhanced ebola surveillance in sierra leone. the methods and materials of the survey have been described in this paper in accordance with guidelines for strengthening the reporting of observational studies in epidemiology [21] . in april 2017 we obtained a sampling frame of 7,025 callers who reported a death to the 1-1-7 system. survey respondents were randomly selected from a stratified sampling frame of all callers who reported at least one death between december 2016 and april 2017. fewer than 5% of the records were duplicates and were removed from the sampling frame. for callers who reported multiple but non-duplicate deaths (< 10% of sample), the most recent death was kept while all others were removed to mitigate recall bias. we then stratified the sampling frame by geographic region of residence (west, north, east, south). in our sample size calculations, we assumed 70% call-success rate, 50% overall-response rate agreeing to consent, and 90% item-response rate. we aimed to obtain a final sample of approximately 1,355 callers, to allow for a 2.5% margin of error for national estimates and 5.0% margin of error for regional estimates of death-reporting motivations. significance level was set to α = 0.05. this resulted in a random list of 4,300 people to contact by telephone. trained interviewers made up to three attempts at different times of the day to contact potential participants. a telephone number was marked as unreachable and removed from the telephone database after three unsuccessful attempts. the questionnaire development was informed by a focus group discussion with a convenience sample of 12 respondents to assess appropriateness of item formats, respondents' understanding and interpretation of questions, appropriate sequencing to mitigate bias, and categorization of expected responses to open-ended questions (for example, motivations to report a death). the questionnaire was subsequently revised and piloted with a convenience sample of 25 eligible respondents. participants in the pilot were excluded from the final sample selection to avoid repeat-interview bias. a team of ten interviewers and two supervisors were trained on the proper administration of the survey including informed consent, oral translations of items from english to common local languages (krio, mende, temne, and fullah), use of the open data kit (odk) digital data collection tool [22] , and interviewing techniques. the interviews were conducted in respondent's preferred local language. calls were placed by interviewers using a telephone system setup within the 1-1-7 call center in freetown. on average, interviews lasted approximately 15-20 minutes. the interviews were administered using odk (www.opendatakit.org) installed on computer tablets pre-programmed with a digital copy of the questionnaire. supervisors oversaw the data collection process including monitoring phone interviews, verifying data entered in odk, and reviewing final submission of processed data to a secured, webbased hosting server. verbal informed consent was obtained from all participants before initiating the telephone interview. sociodemographic variables included region of residence, sex, age, education, religion, and occupation. in addition, we collected data on circumstances surrounding the death including the nature of death (accident-related, possible stillbirth, possible maternal death), signs and symptoms, place of death, and treatment seeking history within the month prior to dying. call history to 1-1-7 during the ebola epidemic was defined as anyone who responded "yes" to the question "did you ever call 1-1-7 during the ebola crisis in sierra leone (may 2014 to november 2015)?" it is worth noting that by 2015 the epidemic response capacity had generally improved compared to 2014 when response capacity was severely challenged as new ebola cases peaked nationwide in november of that year [23] . past ebola experience was dichotomized into "yes" and "no" such that anyone who responded by saying "yes" to one or more of the following three questions was categorized as having some past ebola experience: (a) "do you personally know anyone who died from ebola?" (b) "do you personally know anyone who survived ebola?" and (c) "do you personally know anyone who was quarantined due to ebola?" item wording and grouping for past ebola experience was directly informed by a prior assessment in sierra leone [24] . motivations to report a death were captured by asking an open-ended question: "what made you call 1-1-7 to report the death?" without prompting for any specific responses, interviewers recorded the reason(s) for calling provided by respondents into the following six categories: (a) "find out the cause of death;" (b) "protect self or others from possible infection;" (c) "obey government policy/law;" (d) "obtain burial permit (to allow traditional burial);" (e) "obtain death certificate;" and (f) "other." selection of multiple reasons for calling was allowed, and data collectors probed to get an exhaustive list of motivations. first, we described trends in reporting by plotting a bar graph (fig 1) the survey data were analyzed using stata version 15 se (statacorp llc, college station, tx). frequencies, proportions and other descriptive statistics were generated for all variables. responses indicating "don't know", "don't remember", and "declined to respond" were treated as missing values. a composite outcome variable was created for scoring motivations expressed by respondents. the score could range from 0 to 6 depending on the number of motivations that respondents cited. two composite binary exposure variables were then generated. first, a binary variable was generated to indicate if ebola-like symptoms (fever, diarrhea, vomiting) were present in the decedent (coded 0 if none and 1 if one or more symptoms). second, a binary variable was generated for knowing someone who died from ebola, survived ebola or quarantined due to ebola exposure during the 2014-2015 epidemic (coded 0 if none and 1 if one or more such experiences). given the ordered outcome for motivations using a count variable, we used ordered logistic regression modeling in our multivariate analyses to estimate odds ratios (ors) and their 95% confidence intervals (cis). we fitted a model to examine the possible associations between motivations to report the death and (a) experiencing ebola-like symptoms before dying, (b) previously calling the 1-1-7 line during the epidemic, and (c) knowing someone who died from ebola, survived ebola, or was quarantined due to ebola during the epidemic in sierra leone. the model was adjusted for sociodemographic characteristics of the person who reported the death (region, sex, age, education, religion, health worker status) and of the deceased person (sex, age, religion). educational attainment and occupation of the deceased persons were excluded in the models due to high frequency of missing values. the covariates in the model were assessed for collinearity. subsequently, region of residence of the deceased persons was excluded because it was collinear with region of residence of the person who reported the death. in all models, significance level was set to α = 0.05 for a two tailed wald test. the assessment was approved as non-research by the sierra leone ministry of health and sanitation. participation of u.s. centers for disease control and prevention (cdc) staff was approved as a non-research activity by cdc's center for global health (cgh-hsr# 2016-276). the monthly aggregated data of deaths reported to the 1-1-7 system showed a sharp decline after the 2014-2015 ebola epidemic ended in sierra leone compared to the post-outbreak enhanced mortality surveillance period. for instance, in the final month before the ebola epidemic was declared over in sierra leone (october 2015), a total of 8,164 deaths were reported through the system compared to 5,090 in november 2015 when enhanced surveillance began. moreover, in the last month of enhanced surveillance (june 2016), 3,851 deaths were reported through the system compared to 2,456 in the beginning of post-outbreak routine surveillance of deaths. the number of deaths reported to the 1-1-7 system continued to plummet to as low as 1,550 in january 2017, 673 in january 2018, and 586 in january 2019. (fig 1) . in the year when we conducted the survey (2017), a total of 11,642 deaths were reported to the 1-1-7 system compared to 32,469 in 2016 and 117,036 in 2015. sierra leone has a crude death rate of 11.9 per 1000 population according to the 2015-2020 estimates by the united nations [25] . therefore, approximately 95,000 deaths could be expected yearly in the total estimated population of 8 million. thus, the number of deaths reported to the 1-1-7 system in 2017 was approximately 12% of expected total deaths in the country compared to approximately 34% in 2016 and over 100% in 2015. table 1) . in the sample obtained, deceased persons were more frequently male (54.9%), had no education (53.5%), affiliated as muslim (78.5%), and were 50 years old or above (41.6%) ( table 2) . overall, 376 (29.1%) deaths were women of reproductive age, 127 (9.8%) were infants, and 59 (4.9%) were accident-related deaths. among deaths of women of reproductive age, 24 (6.4%) were pregnant at the time of the death. thirty-three of the infant deaths (30.3%) were stillbirths. overall, 83.3% of deceased persons reportedly received some form of treatment from one or more sources within the past month of dying, and non-exclusively cited the place of treatment as health facility (82.4%), home (18.5%), pharmacy or drug store (10.9%), traditional healer (6.4%), and other sites (<1%). the most frequently cited symptoms that the deceased persons had purportedly experienced within the past month of dying were fever (32.1%), joint pain (21.0%), headache (19.6%), and abdominal pain (15.7%). ebola-like symptoms (fever, diarrhea, or vomiting) were reportedly experienced by 35.5% of the decedents (table 3) . missing values were higher for the variables on occupation of the deceased person (n = 474; 36.7%), education level of the deceased person (n = 392; 30.4%), and treatment received before dying (n = 222; 17.2%) when compared to other variables (less than 10%). missing values were mostly due to reporting of deaths by health workers who did not know certain details about the deceased person. nearly all respondents (94.1%) wanted the government to continue using the 1-1-7 system in sierra leone, and to keep the current '1-1-7' number (89.7%). of those who wanted continuation of the reporting system (n = 1174), reporting of all deaths was the most commonly reported preference (79.7%) (fig 2) . the most frequently cited motivations were to obey government policy (81.6%), find out the cause of death (36.5%), obtain burial permit (28.7%), and protect self or others from infection (25.5%) (fig 3) . compared to deaths that did not exhibit ebola-like symptoms, exhibiting one or more ebola-like symptoms was associated with a two-fold increase in the odds of being motivated to report the death (adjusted or [aor] 2.26 ci 1.78-2.87). motivations to report were not associated with previously calling the 1-1-7 line during the ebola epidemic (aor 1.0 ci 0.79-1.27) or knowing someone who died, survived or was quarantined due to ebola during the epidemic (aor 0.94 ci 0.73-1.21) ( table 4 ). our descriptive analysis of the five-year trends in the number of deaths reported to the 1-1-7 system identified a substantial decline in reporting after the period of enhanced ebola surveillance ended. in 2017, the system maximally captured about 12% of the total expected deaths in the country compared to approximately 34% in 2016 and more than 100% (sic) in 2015. in the telephone survey we identified motivations related to death reporting that have practical implications for improving routine mortality surveillance in a post-ebola-outbreak setting. nearly all respondents wanted the death reporting system to continue. the leading motivation for reporting was the desire to obey the government's reporting mandate of all deaths. reasons for this desire to comply with reporting mandate were not directly evaluated in our assessment but may be linked to altruistic intentions to help prevent potential ebola as documented in a prior qualitative assessment near the end of the epidemic in sierra leone [26] . in fact, we found that people who reported deaths that had experienced ebola-like symptoms were more motivated to report, which may have also been influenced by knowledge and experiences from the prolonged ebola epidemic. effective mortality surveillance is an important pillar of promptly identifying and responding to deaths from notifiable diseases such as ebola, especially in outbreak-prone areas. recent epidemics of ebola in sub-saharan africa have reinforced the need for effective surveillance systems including mechanisms to promptly detect cluster-deaths that may be tied to a possible outbreak of ebola or other infectious diseases [4, 23, 27] . it should be noted that prior to the ebola outbreak deaths were usually reported in-person to local city councils mainly to obtain a burial plot [14] . therefore, telephone reporting of deaths to the national government was a new behavior for people in sierra leone before the ebola outbreak. the context in which a death reporting system is implemented poses ethical considerations. deaths tended to become widely publicized in communities during the epidemic in sierra leone. anyone in the community could report a death to the 1-1-7 line even without having full information about the details of the death or the approval of close relatives. given the limited capacity to respond to all incoming death notifications, multiple reports of the same death with incomplete or contradictory information may have made it difficult to prioritize the dispatch of safe burial teams. however, during the post-outbreak period, our survey revealed that mostly family members and health workers reported deaths to the 1-1-7 system, which likely improved the completeness and accuracy of the information provided about the death. despite confidentiality guidelines, in both the outbreak and post-outbreak contexts it is unclear if callers to the 1-1-7 line were assured confidentiality when they reported a death. in addition, the training guidelines of call operators stated that callers were supposed to be informed that they may receive a follow-up call to get more information about the reported death. we cannot verify whether operators informed all callers about potential follow-up calls. it is possible that concerns about confidentiality may have influenced death reporting behaviors over time, including in our assessment. continued implementation of the 1-1-7 death reporting system is just one example of how sierra leone leveraged its ebola response infrastructure for routine surveillance [23] . another example is seen in how the government of sierra leone, with support from partners, transitioned from a paper-based to a web-based electronic integrated disease surveillance and response (eidsr) system after the ebola epidemic. sierra leone became the first country to have a fully functional eidsr system in sub-saharan africa in 2019 [28] . the eidsr system is used to track 28 priority notifiable diseases in all 1,300 health facilities in the country [29] . the platform was developed using the country's existing district health information system. however, the 1-1-7 system is presently not integrated into eidsr. going forward, if the government maintains the 1-1-7 system, it is critical to ensure its interoperability and integration with the eidsr system to open the opportunity to connect case-based reporting of notifiable diseases with event-based mortality surveillance to rapidly detect outbreaks and initiate public health response. the expectation that the 1-1-7 system was going to be successfully converted to a routine mortality surveillance system does not seem to have been fulfilled given that the system only captured about 12% of the expected deaths in the aftermath of ebola. the low level of death reporting to the 1-1-7 system after the epidemic ended was likely due to several factors including the lack of continued social mobilization to promote reporting and the lack of a clearly communicated government policy for routine use of the system. sierra leone's implementation of the 1-1-7 system holds important lessons for the development and sustainability of similar telephone-based surveillance systems in sub-saharan africa. as reminded by the 2018-2020 ebola outbreaks in the democratic republic of congo and the ongoing covid-19 pandemic, surveillance tools such as the 1-1-7 system when coupled with adequate public engagement can facilitate early detection of cases and deaths to curb disease spread. our assessment has limitations. duplicate reporting may have been more frequent in the early stages of the epidemic in 2014 when the capacity to respond to deaths was at times unable to meet the demand for safe burial services. improvements in response capacity in 2015 may have reduced the likelihood of families placing repeated calls for the same death in trying to ensure a safe burial. the number of duplicate reports likely stabilized starting in 2015 when response capacity improved. duplicate records only accounted for about 5% of the total records in the database of deaths reported between december 2016 and april 2017. assuming duplicate reporting level was similar between 2015 and 2019, it is unlikely that duplicate reporting substantially influenced the overall reporting trends. the descriptive trends provided in the paper are meant to help lay a foundation to examine and discuss the reporting motivations in a post-ebola-outbreak context. additional research could consider leveraging the recently launched sierra leone ebola database [30] , which contains deduplicated, anonymized, linked data on 1-1-7 alerts, ebola cases, laboratory results, ebola treatment unit, ebola treatment unit clinical records, and burial records, to ascertain death reporting trends using case-based data that account for duplicates. the generalizability of the results of our telephone survey with death reporters also have limitations. for instance, the sample of respondents were mostly men. we do not know if this was because proportionally more men reported deaths to the 1-1-7 call center or whether it was due to a systematic bias of higher unsuccessful call rates to women who reported deaths. lastly, our assessment only targeted individuals who reported deaths to the 1-1-7 system in order to understand their motivations for reporting. people who had deaths in their households but failed to report may be demographically different from our sample and held concerns that prohibited them from reporting. future research should consider using qualitative approaches to better understand barriers to death reporting among household heads who fail to report deaths in their households. support for compliance with government death reporting policy motivated users of the 1-1-7 system in sierra leone after the ebola epidemic ended. increased motivation to report deaths that resembled ebola in the post-outbreak setting may have been influenced by knowledge and experiences from the prolonged epidemic. post-ebola-outbreak periods offer an opportunity for instituting routine mortality reporting, as people have been sensitized about the importance of reporting through the experiences of the outbreak. transitioning the system to a routine mortality surveillance tool may require a robust social mobilization component [31] to match the high reporting levels during the outbreak, which exceeded more than 100% of the expected deaths in 2015. as global health security efforts try to strengthen surveillance systems [32], routine use of death reporting systems like 1-1-7 could play an important role in early detection of clusters of deaths linked to potential infectious disease outbreaks including ebola. world health organization. ebola situation report-30 social pathways for ebola virus disease in rural sierra leone, and some 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3-day house-to-house campaign-sierra leone, september 2014 the 117 call alert system in sierra leone: from rapid ebola notification to routine death reporting improving burial practices and cemetery management during an ebola virus disease epidemic-sierra leone facilitators and barriers to community acceptance of safe, dignified medical burials in the context of an ebola epidemic keeping the faith: the role of faith leaders in the ebola response. freetown: cafod christian aid, tear fund, islamic relief worldwide lessons of risk communication and health promotion-west africa and united states social mobilization and community engagement central to the ebola response in west africa: lessons for future public health emergencies 117 and district call log analysis monthly reports. freetown: ehealth africa strengthening the reporting of observational studies in epidemiology (strobe): explanation and elaboration. international journal of surgery odk: tools for the common case ebola response impact on public health programs impact of ebola experiences and risk perceptions on mental health in sierra leone population dynamics trust, fear, stigma and disruptions: community perceptions and experiences during periods of low but ongoing transmission of ebola virus disease in sierra leone ebola in west africa-cdc's role in epidemic detection, control, and prevention leone leads the way in africa with fully functional electronic disease surveillance system centers for disease control and prevention. ebola outbreak sparks disease surveillance transformation in sierra leone centers for disease control and prevention. data hosting: sierra leone ebola database (sled) the development of standard operating procedures for social mobilization and community engagement in sierra leone during the west africa ebola outbreak of 2014-2015 we thank the 1,291 sierra leoneans who took the time to share their experiences with reporting the deaths of their loved ones to the 1-1-7 system. we acknowledge various contributions made by dayo spencer-walters, alex taylor, mohamed kabia, and maseray sesay from ehealth africa; laura shelby, mitsuaki hirai, wenshu li, and erika myer from cdc. key: cord-270481-rrpqz0uy authors: hays, russell; pierce, doris; giacomin, paul; loukas, alex; bourke, peter; mcdermott, robyn title: helminth coinfection and covid-19: an alternate hypothesis date: 2020-08-17 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008628 sha: doc_id: 270481 cord_uid: rrpqz0uy nan in their recently published commentary, bradbury and colleagues [1] drew attention to the possible negative interactions between helminth infection and covid-19 severity in helminth-endemic regions. helminth infections are known to be powerful modulators of the human immune response, and numerous studies now highlight the effects this may have on human infectious, inflammatory, and metabolic diseases. we believe, however, that any interaction between pre-existing helminth infection and the subsequent severity of covid-19 need not necessarily be a negative one, and theoretical and empirical evidence suggests that helminths may indeed have a mitigating effect. one of the clear predictors of severe covid-19 that has emerged during the pandemic has been the presence of obesity, metabolic syndrome, or type 2 diabetes mellitus (t2dm) in patients contracting the virus. [2] these conditions are associated with the second week "cytokine storm" phenomenon [3] that often results in the need for ventilatory support and increased mortality, including in younger patients. these metabolic diseases are characterised by an inflammatory milieu, with increased levels of proinflammatory cytokines, many of which are also implicated in the severe form of covid-19. elevated levels of acute phase reactants and proinflammatory cytokines including ifn-γ, il-1β, il-6, il-12, il-17, il-18, il-27, and tnf have been shown to be predictors of clinical deterioration and the onset of severe disease. lymphopaenia (and, in particular, a reduction in regulatory t cells [treg] numbers) and eosinopaenia are also closely associated with disease severity. [4] epidemiological studies over the past decade have consistently reported an inverse relationship between a variety of chronic helminth infections and the presence of metabolic syndrome and t2dm, particularly in transitional societies in which both conditions are prevalent. [5] recent reports have shown reduced levels of proinflammatory cytokines such as il-1α, il-1β, il-6, il-12, il-18, il-23, il-27, g-csf, and gm-csf in subjects with coexisting helminth infection and t2dm and a partial reversal of this effect following treatment of the worm infection. [6] in addition, chronic helminth infections are associated with increased numbers of treg cells, m2 macrophages, and eosinophils. it is therefore feasible to propose that a reduced capacity for the production of proinflammatory cytokines and increased numbers of regulatory immune cells due to the immunomodulatory effects of pre-existing helminth infection could result in a reduced risk of severe covid-19. although the interaction between helminth infection and viral pneumonia is poorly defined, there is some evidence that helminth infection may moderate the process of pulmonary inflammation in viral infections. some studies have suggested that helminth infection may impair responses to viral immunization [7] and viral infection [8] , but no clear clinical evidence exists that it acts to worsen outcomes. [9] the increased levels of il-4 and il-10 (anti-inflammatory cytokines associated with chronic helminth infection) found in some studies [10] need not represent a virus induced pathological response but could instead reflect the normal regulatory and tissue-repair response to inflammation. epidemiological studies of the prevalence of severe covid-19 in societies in which helminth infection is common would clearly be of great interest, but currently, no reliable data exists. the pandemic has been most active in developed countries where helminth infection is rare, and the data coming from less-developed societies may be difficult to interpret given the early phase of the pandemic, the lack of extensive testing, unreliable information regarding case fatality rates and cause of death, and their generally younger populations with lower prevalence of metabolic disease and obesity. the numbers currently emerging from the who do not indicate a widespread increase in case fatality rates in the developing world, with the number of reported deaths being generally low. [11] we believe that, as the understanding of the mechanisms of severe covid-19 evolves, there may be a case for exploring the possible effects of experimental helminth infection (ehi) on covid-19 severity in a study setting. this is particularly so should the mechanism of severe disease rest principally in endothelial invasion and vascular injury secondary to unchecked inflammatory responses, rather than ongoing viral replication. [12] although the outcomes of trials have been mixed [13] , experimental inoculation with the hookworm necator americanus has been established as both practical and safe for use in study settings and has been successfully deployed in trials involving atopic and autoimmune disorders. [14] importantly, no data exists studying the effect on metabolic outcomes, but one study is currently underway examining the effect of ehi on individuals with obesity and metabolic syndrome. [15] in the present crisis, a prospective study examining the effect of ehi on subsequent severe covid-19 could produce valuable insights into the immunology of this condition. clearly, the design of such a study would pose considerable ethical and practical challenges. experimental coronavirus infection would seem impossible, particularly given that the trial would logically target those at most risk of severe disease. a case-matched cohort study conducted at multiple locations around the world would require large numbers of subjects and would be dependent on the unpredictable future course of the pandemic. nevertheless, such a trial could demonstrate a potential mitigation of severe disease in susceptible individuals and give some evidence-based guidance on how to best manage the helminth elimination programs currently operating in many countries as the pandemic unfolds over coming years. will helminth co-infection modulate covid-19 severity in endemic regions? coronavirus infections and type 2 diabetes-shared pathways with therapeutic implications diabetes is a risk factor for the progression and prognosis of covid-19 immune response to sars-cov-2 and mechanisms of immunopathological changes in covid-19 do worms protect against the metabolic syndrome? a systematic review and meta-analysis helminth infection modulates systemic pro-inflammatory cytokines and chemokines implicated in type 2 diabetes mellitus pathogenesis helminth infections suppress the efficacy of vaccination against seasonal influenza virus-helminth coinfection reveals a microbiota-independent mechanism of immunomodulation. science helminth modulation of lung inflammation clinical features of patients infected with 2019 novel coronavirus in wuhan who coronavirus disease (covid-19 cytokine storms: understanding covid-19 harnessing helminth-driven immunoregulation in the search for novel therapeutic modalities a proof of concept study establishing necator americanus in crohn's patients and reservoir donors safety and tolerability of experimental hookworm infection in humans with metabolic disease: study protocol for a phase 1b randomised controlled clinical trial key: cord-002581-r7mskri0 authors: magnani, diogo m.; silveira, cassia g. t.; rosen, brandon c.; ricciardi, michael j.; pedreño-lopez, núria; gutman, martin j.; bailey, varian k.; maxwell, helen s.; domingues, aline; gonzalez-nieto, lucas; avelino-silva, vivian i.; trindade, mateus; nogueira, juliana; oliveira, consuelo s.; maestri, alvino; felix, alvina clara; levi, josé eduardo; nogueira, mauricio l.; martins, mauricio a.; martinez-navio, josé m.; fuchs, sebastian p.; whitehead, stephen s.; burton, dennis r.; desrosiers, ronald c.; kallas, esper g.; watkins, david i. title: a human inferred germline antibody binds to an immunodominant epitope and neutralizes zika virus date: 2017-06-12 journal: plos negl trop dis doi: 10.1371/journal.pntd.0005655 sha: doc_id: 2581 cord_uid: r7mskri0 the isolation of neutralizing monoclonal antibodies (nmabs) against the zika virus (zikv) might lead to novel preventative strategies for infections in at-risk individuals, primarily pregnant women. here we describe the characterization of human mabs from the plasmablasts of an acutely infected patient. one of the 18 mabs had the unusual feature of binding to and neutralizing zikv despite not appearing to have been diversified by affinity maturation. this mab neutralized zikv (neut(50) ~ 2 μg/ml) but did not react with any of the four dengue virus serotypes. except for the expected junctional diversity created by the joining of the v-(d)-j genes, there was no deviation from immunoglobulin germline genes. this is a rare example of a human mab with neutralizing activity in the absence of detectable somatic hypermutation. importantly, binding of this mab to zikv was specifically inhibited by human plasma from zikv-exposed individuals, suggesting that it may be of value in a diagnostic setting. zika virus (zikv) belongs to the genus flavivirus of the flaviviridae family and is related to dengue virus (denv), yellow fever virus (yfv), japanese encephalitis virus (jev), and west nile virus (wnv) [1] . the globally distributed mosquito species of the aedes genus, vectors for many flavivirus, can also transmit zikv [2, 3] . however, zikv remained a relatively minor and obscure cause of human disease for most of the second half of the 20 th century and was featured in a limited number of scientific reports. in fact, it was not until 2007 that autochthonous human infection was described outside africa and continental asia-in the federated states of micronesia [4] [5] [6] . since then, reports from brazil have chronicled a rapidly spreading epidemic that co-exists with denv and chikungunya virus (chikv). the epidemic has spread north with mosquito-borne transmission being reported in many nations of the americas as far north as mexico and southern florida [7] [8] [9] . more ominously, zikv has been implicated as the causative agent in fetal developmental problems [10, 11] . there are reports of zikv-associated birth defects, primarily brain abnormalities and microcephaly in infants born to mothers infected with zikv [12] . virus has been recovered from amniotic fluid, placental, and brain tissues [13] [14] [15] [16] [17] [18] [19] [20] [21] . zikv infection has been classified as an ongoing threat by the world health organization. in the united states, the centers for disease control and prevention has issued guidance for the management of the infection in the general population, pregnant women, and infants [22] [23] [24] . due to recent reports of sexually transmitted zikv infection, the cdc has also developed guidelines for prevention of this mode of transmission [22] [23] [24] [25] [26] [27] . more recently, zikv transmission has also been described in miami, florida [28] , suggesting that autochthonous spread could occur in any region of the u.s. inhabited by aedes spp. treatment of a variety of human ailments using mabs is revolutionizing our ability to ameliorate human suffering. for infectious disease, the ebola epidemic highlighted the potential utility of a cocktail of three neutralizing (n)mabs that block infection by the ebola virus [29] . most convincingly, the administration of a single nmab up to five days post infectious virus exposure prevents the development of disease in ebola-infected macaques [30] . because mabs can be engineered to prevent antibody-dependent enhancement by incorporating the l234a and l235a (lala) mutations which reduce fcγr binding [31] , they are a promising intervention in flaviviral therapies. our long-term goal is to use a cocktail of lala-mutated nmabs to prevent zikv infection in at-risk individuals, primarily pregnant women. therapeutic nmabs must be potent in order to be clinically viable, and most nmab isolation strategies are based on the identification of high-titer, antigen-selected repertoires. somatic hypermutation (shm) in germinal center (gc) b cells provides the basis for selection of b cells producing abs with increased affinity-a hallmark of the adaptive humoral response. this feature is conserved among mammals, highlighting the importance of ab affinity enhancement for evolutionary fitness [32] . thus, it is unsurprising that the vast majority of human abs in the memory immunoglobulin (ig)g pool have undergone affinity maturation and have, on average, 10-26 nucleotide substitutions from precursor genes [33] . the contribution of shm to ab-mediated viral neutralization is particularly clear for the chronicallyinduced broadly neutralizing antibodies to hiv [34] [35] [36] [37] . reversion of these anti-hiv nmabs to precursor germline antibodies results in a drastic reduction or complete loss of viral neutralization [38] [39] [40] [41] . although mutated mabs are found after secondary denv infection, the role of these mutations in acute virus-neutralization and clearance is less clear [42] [43] [44] [45] . still, the prevalent thought is that antiviral ab response involves the engagement of poor-or non-neutralizing germline clones generated by v(d)j rearrangement, followed by shm-mediated refinement in germinal centers to enhance neutralization potency. here we describe the isolation of 18 plasmablast-derived human mabs, sorted 12 days post onset of symptoms from a zikv-patient in são paulo, brazil. the patient reported a previous history of dengue infection and yellow fever vaccination (table 1) . a few of the isolated abs neutralized zikv, most of them at relatively high concentrations. interestingly, one of these mabs (p1f12) exhibited no nucleotide mutations when compared to its corresponding germline sequences, but still recognized a zikv immunodominant epitope and neutralized the virus. these results suggest unforeseen roles for gc-independent responses against zikv and possibly other viruses. blood samples were collected from volunteer 533, a 56-year-old woman who reported a pruriginous skin rash that started six days prior to the beginning of acute neurological deficits suggestive of gbs. zikv infection was confirmed by a positive real-time reverse-transcriptase pcr assay for zikv rna in urine samples collected at days 11 and 12 after the onset of the first rash symptoms. blood and cerebrospinal fluid were negative for zikv rna. previous history of a single dengue infection and yellow fever immunization were also reported. peripheral blood mononuclear cells (pbmcs) were obtained from blood samples collected 12 days post onset of symptoms. blood samples from patient 533 were obtained after signing a written consent form approved by the university of são paulo's institutional review board (cappesq 0652/09). anonymized plasma samples from volunteers in brazil and us were obtained from naïve and convalescent subjects with rt-pcr-confirmed zikv or denv infection (s2 table) . four volunteers donated samples post yellow fever vaccination. we conducted reverse transcription followed by a nested pcr to amplify the variable region of the immunoglobulin (ig) chains using described protocols with minor modifications [46] . briefly, cdna was synthesized in a 25 μl reaction using the original sort plates. each reaction contained 1 μl of 150 ng random hexamer (idt), 2 μl of 10 mm dntp (life technologies), 1 μl of superscript iii reverse transcriptase (life technologies), 1 μl molecular biology grade water, and 20 μl of single sorted cell sample in lysis buffer (described above). the reverse transcription reaction was performed at 42˚c for 10 min, 25˚c for 10 min, 50˚c for 60 min, 94 c for 10 min. after the reaction was completed, cdna was stored at -20˚c. heavy and light chains were amplified in three different nested pcr reactions, using a mix of 5' v-specific primers with matching 3' primers to the constant regions of igg, igl, and igk. pcr reactions were conducted using hotstartaq plus dna polymerase (qiagen). the second set of pcr reactions was carried out with primers redesigned to incorporate restriction sites compatible with subcloning into rhesus igg1 expression vectors, instead of the original human vectors [46] . we sequenced the amplified and cloned products using primers complementary to the ig constant regions. sequences were analyzed using igblast and imgt/v-quest to identify v (d) j gene rearrangements, as well as shm levels [47, 48] . we expressed mabs in expi293f (thermofisher) human cell lines. the plasmids encoding heavy and light chains were co-transfected using the expifectamine 293 transfection kit (a14525, thermofisher). after 5-6 days, we harvested the secreted mab in the supernatant. ig concentration in the supernatant was determined by an anti-rhesus igg elisa, before we proceeded with the functional assays. for the experiments with purified mabs, we used protein a plus (pierce)-containing columns to remove the impurities. the concentration of purified protein was determined by measurement of absorbance at 280 nm (nanodrop, thermo scientific). virus capture assay and recombinant e protein elisa p1f12 binding was determined by both virus capture assay (vca) and recombinant (r)e elisas. the vca plates were coated overnight with the mouse-anti-flavivirus monoclonal antibody 4g2 (clone d1-4g2-4-15, emd millipore) followed by incubation with viral stocks (zikv or denv). the re elisa plates were coated with zikv e protein (mybiosource, mbs596001) diluted to 5 μg / ml in pbs. after the coating step, the plates were washed with pbs and mab samples diluted to 1 μg / ml were added to designated wells and incubated for 1 h at 37˚c. subsequently, the plates were washed and detection was carried out using a goat anti-human igg hrp secondary ab (southern biotech), which was added to all wells at a dilution of 1:10,000. following a 1 h incubation at 37c, the plates were washed and developed with tmb substrate at room temperature for 3-4 min. the plates were developed with tmb substrate at room temperature for 3-4 min. the reaction was stopped with tmb solution and absorbance was read at 450 nm. the neutralizing potency of the mabs was measured using a flow cytometry-based assay [49, 50] . in brief, recombinant mabs (transfection supernatant or purified) were diluted and preincubated with zikv (paraiba) or the reference denv serotypes in a final volume of 220 μl for 1 h at 37˚c. the virus and mab mixture (100 μl) was added onto wells of a 24-well plate of 100% confluent vero cell monolayers in duplicate. a new seed of vero cells (ccl-81tm) was obtained from the american type culture collection (atcc) repository for this study. the inoculum was incubated in a 37˚c incubator at 5% co 2 for one hour with agitation of the plates every 15 min. after one hour, the virus and mab-containing supernatants were aspirated and the wells were washed with media. fresh media was then added and the plates were incubated for a total of 24 hours. cells were trypsinized with 0.5% trypsin (life technologies), fixed (bd cytofix), and permeabilized (bd cytoperm). viral infection was detected with the 4g2 antibody (millipore) recognizing zikv or denv, followed by staining with an anti-mouse igg2a apc fluorophore-conjugated secondary reagent (biolegend). the concentration to achieve half-maximal neutralization (neut 50 ) was calculated using a nonlinear regression analysis with prnts were conducted as previously described [51] . briefly, purified p1f12 was serially diluted in optimem supplemented with 2% human serum albumin (vwr), 2% fetal bovine serum, and gentamicin. zikv paraiba 2015 was diluted to a final concentration of~500-1000 pfu / ml in the same diluent added to equal volumes of the diluted ab. the virus/mab mixture was incubated at 37˚c for 30 min. cell culture medium was removed from 90% confluent monolayer cultures of vero cells on 24-well plates and 100 μl of the virus/ab mixture was transferred onto duplicate cell monolayers. cell monolayers were incubated for 60 min at 37˚c and overlaid with 1% methylcellulose in optimem supplemented with 2% fbs 2mm glutamine + 50 μg / ml gentamicin. samples were incubated at 37˚c for four days after which plaques were visualized by immunoperoxidase staining, and a 50% plaque-reduction neutralization titer was calculated. inhibition of p1f12 mab binding was determined by elisa. to begin, the elisa plate was coated with mouse anti-flavivirus monoclonal antibody 4g2 (emd millipore) diluted 1:1,000 in carbonate binding buffer and incubated overnight at 4˚c. the next day, the plate was washed five times with pbs-tween20 and wells were blocked with 5% skim milk in pbs for 1h at 37˚c. after the block step, the plate was washed and virus samples were added to designated wells for 1h incubation at room temperature. subsequently, the plate was washed with pbs only and corresponding blocking plasma samples were added for 1h at 37˚c. following the plasma block, the plate was washed and p1f12 was added to corresponding wells for 1 h at 37 c. p1f12 was detected using a rhesus igg-specific antibody (mouse anti-monkey igg-hrp clone sb108a; southern biotech). thereafter, the plate was washed and wells were developed with tmb substrate at room temperature for 3-5 min before the reaction was stopped with tmb stop solution. absorbance was determined at 450 nm. we isolated plasmablasts from patient 533 who presented with suspected guillain-barré syndrome (gbs) ( table 1 ) (first day of symptoms = d0). the patient had a previous history of dengue infection and yellow fever vaccination ( table 1 ). the previously healthy 56-year-old woman presented to the emergency room (d6) reporting a progressive paresthesia mainly in the extremities of her hands, along with acute, intermittent pain in her left forearm during the previous four days. at physical examination, the patient presented with a grade iv asymmetric muscular weakness and hypoesthesia in her left limbs, with abolished deep tendon reflexes in the lower limbs. a mild weakness of her left facial muscles was also noted. the patient reported no respiratory disorders and no hoarseness, and no signs of dysautonomia were detected at the clinical evaluation. fever, conjunctivitis, and myalgia or joint pain were absent during the illness. afterwards, the patient was hospitalized with a clinical diagnosis of gbs, for which an intravenous human ig (ivig) treatment was initiated at a dosage of 0.4 g / kg / day for 5 days. cerebrospinal fluid analysis and an electroneuromyogram were performed on fourth (d10) and fifth (d11) days after neurological symptom onset, respectively; the results were within normal limits. the electroneuromyogram was repeated on the 15 th day of neurological symptoms, but no significant abnormalities were noted despite the persisting weakness in the patient's left leg and arm. during the treatment with ivig, the patient presented with transient worsening of her hemiparesis, but progressively recovered over the course of weeks after discharge from the hospital. at 32 days post-neurological symptom onset (d38), a physical exam revealed significant improvement of muscular strength and abolished deep tendon reflexes in the lower limbs. the remittent skin rash cleared completely 10 days after its initial emergence. blood, cerebrospinal fluid and urine samples were collected on the 5 th day of neurological symptoms (d11) for detection of zikv by rt-pcr. the urine sample was zikv-positive by pcr, while blood and cerebrospinal fluid were negative. a saliva sample collected on d15 was negative for zikv. we isolated plasmablasts from peripheral blood mononuclear cells (pbmcs) collected on day 12 (table 1) . from wells containing single-sorted cells, we amplified, cloned, and sequenced heavy and light ab chains using 5' primers complementary to the v gene segments and a 3' primer annealing to the constant igg region [46] . this resulted in 18 paired heavy and light chains (s1 table) . eight of the 18 mabs bound to zikv (fig 1) . seven of these mabs exhibited cross-reactivity to one or more of the denv serotypes, and a single mab-p1f12-bound exclusively to zikv. interestingly, two mabs bound to denv but not zikv. we tested the neutralization potency of the zikv-specific p1f12 mab in a flow-based neutralization assay and a plaque reduction neutralization test (prnt) and found that it neutralized zikv at approximately 2 μg / ml (prnt 50 ) (fig 2) . analysis of the isolated antibody variable (v) domain sequences revealed five mabs with average gene mutation levels (10-26 nucleotide modifications), two mabs with over 30 nucleotide substitutions, and 11 mabs with unusually low levels of shm for isotype-switched mabs (lower than 10 changes) (s1 table) . the most highly mutated mabs (p1b08 and p1c03) were not zikv-specific by binding (s1 table) . in fact, the eight zikv-binding mabs had the lowest shm levels, including four mabs lacking clearly recognizable mutations when compared with putative heavy and light chain germline precursors (s1 table, fig 3) . except for junctional diversity, the zikv-neutralizer p1f12 mab heavy chain did not exhibit signs of antigenselected ig diversification. p1f12 had an identical sequence to the ig heavy chain variable (ighv) genes segment ighv3-7 ã 01 up to the amino acid c105, prior to the cdr-h3 (international immunogenetics information system [imgt]) [52] . however, position g106-the site of the junction between ighv and the igh diversity (ighd) genes-differed from the germline reference. interestingly, this region is part of a segment (n1) with non-germline nucleotides corresponding to six amino acids identified between the ighv and ighd genes (fig 3c) . this segment is likely the result of n nucleotide additions generated during b cell ig gene rearrangement, prior to antigen selection. because of the lack of mutations elsewhere in the sequences, it is likely that the r106g substitution was also generated during this developmental step. the downstream sequence corresponding to the junction between ighd3-22 ã 01 and the igh joining (ighj) ighj6 ã 02 genes also revealed similar nucleotide insertions. likewise, the kappa (k) chain junction between the igkv1-8 ã 01 and igkj4 ã 01 genes also contained one insertion. although we cannot rule out the possibility of shm-mediated nucleotide changes in the n insertion regions, no mutation was identified in the remainder of the regions of the heavy and light chains. thus, the p1f12 mab is likely very close or identical to the original v-(d)-j gene rearrangement in the naïve b cell before antigen contact. to investigate whether p1f12 recognizes an immunodominant zikv epitope, we used a serological blocking assay. in brief, this assay detects the presence of competing abs that can inhibit the p1f12 mab binding to its epitope. because p1f12 did not bind to recombinant e protein (fig 4) we used whole virus in our binding assays. we captured zikv on the plate using the 4g2 mab (pan-flavivirus), and incubated zikv with plasma from patients with diverse histories of denv and zikv exposure (s2 table) . we added unlabeled p1f12 (engineered with rhesus igg1 constant regions) and detected binding of the mab using a hrplabeled mouse anti-rhesus mab (fig 5) . nine of ten plasma samples from individuals that had been infected with zikv blocked the binding of p1f12 in a blinded test (fig 5, s2 table) . similar blocking activity was observed regardless of whether individuals had been previously infected with denv or had been vaccinated for yellow fever. in contrast, little or no blocking activity was observed by denv+ plasma in the absence of prior zikv exposure (fig 5) . furthermore, this recognition was specific in that it was not observed in 14 of 14 denv-only infected individuals. thus, the p1f12 serological blocking assay accurately predicted previous zikv exposure, as confirmed by rt-pcr, in all but one of the patient plasma samples tested. although this patient, donor 1302, had a positive urine rt-pcr result for zikv, plasma from 1302 did not block p1f12 binding to zikv (s2 table) . interestingly, the plasma did not exhibit detectable zikv-neutralizing activity, suggesting that this patient did not mount a measurable antibody response against zikv. in conclusion, only the plasma that inhibited zikv infection of vero cells contained p1f12-blocking antibodies. here we show that a igg mab with no detectable shm was generated against zikv early in infection. remarkably, despite being germline-encoded, this mab is zikv-specific and does not bind to any of the four denv serotypes. furthermore, this mab not only neutralizes zikv, but it also binds to an immunodominant epitope on the virus. remarkably, despite being germline-encoded, p1f12 binds specifically to zikv and does not cross-react with any of the four denv serotypes. our results also suggest that p1f12 recognizes a unique epitope on zikv. it is unclear how this ab developed such specificity without shm. finally, these findings suggest that affinity maturation is not necessary for the generation of isotype switched virus-neutralizing abs. low levels of shm in abs possessing neutralizing activity have been previously reported in mice and humans [53] [54] [55] , supporting the idea that germline-encoded mabs can indeed neutralize. abs with low levels of shm have also been reported during the acute phase of human denv infection, but it was not clear that these abs contributed to the antiviral neutralization activity [56] . in studies in mice, vsv-specific mabs lacking shm have been isolated previously [53] . interestingly, secondary, but not primary, mouse abs against vsv had mutations [57] . furthermore, the reversion of these mutated abs to non-mutated precursors reduced, but did not abrogate, vsv binding and neutralizing activity. the binding differences between the mutated and germline abs were much less pronounced than might be expected [57] . additionally, mice that cannot conduct shm due to aid knockout still mounted neutralizing ab responses against friend virus, a strain of murine leukemia virus [55] . it has been suggested that these abs lacking extensive shm undergo a gc-independent developmental pathway [58] , although the mechanistic basis for this phenomenon remains to be elucidated. rapid, gc-independent responses might be particularly relevant in the control of acute cytopathic viruses [55, 58, 59] . the gc-independent abs would arise quickly after infection and then curtail viral replication, preventing virus-mediated damage [60] . even more provocatively, hangartner et al. have argued that cytopathic viruses specifically evolved to retain binding to these germline sequences to decrease host lethality and increase fitness. on the other hand, chronic viruses may have evolved to avoid germline-binding and development of neutralizing responses to persist [60] . so far, these hypotheses remain unsubstantiated by the lack of evidence for strictly germline neutralizing ab responses in humans. while our experiments were not specifically designed to detect gc-independent responses, it seems likely that the isotype-switched p1f12 originated directly from a germline precursor. we isolated p1f12 from a zikv-infected individual that developed neurological complications compatible with gbs and was treated with ivig. underlying factors that influence the potential association of gbs and zikv infection might involve an autoimmune process, which could influence the development of immune responses [61] . additionally, ivig may have had a role in the selection of the ab responses mounted by peripheral b cell repertoires [62] . this is unlikely, however, since the patient initiated ivig treatment on the same day that the plasmablasts were isolated. it is possible, then, that gbs or ivig-treatment influenced the development of p1f12. these potential associations are difficult to determine and were outside the scope of this study. it is clear, however, that these responses were not exclusive to volunteer 533, as p1f12 binding can be blocked by the serum of most zikv-infected individuals (fig 5) . recently [45] . these mabs were isolated from memory cells sorted with soluble and monomeric zikv e proteins and, in contrast to p1f12, bind to the recombinant protein [45] . in contrast, we isolated zikv-specific mabs from circulating plasmablasts at d12. the peak recall of memory b-cell derived plasmablasts is thought to occur within the first week post-secondary infection [63, 64] . thus, it is probable that most of the isolated mabs did not have a memory-b cell origin, and it remains possible that some of the plasmablasts were sorted from the basal population that circulate in low frequencies in the blood. in conclusion, the isolation of mabs using different b cell methods suggest that anti-zikv mabs with germline characteristics are not limited to specific b cell subtypes [42, 45] . notably, the anti-zikv mabs isolated to date are less mutated than the mabs isolated after related denv infections [42] [43] [44] [45] . together, these findings suggest possible differences in the development of ab responses against zikv. unfortunately, despite our efforts, we were unable to map p1f12's binding site. we first employed an in vitro escape assay [65] , which did not result in a single mutated consensus sequence. also, p1f12 did not bind to the prm/e proteins expressed in cells, precluding our ability to map this interaction using an ala-mutated envelope panel [66, 67] . characterizing this interaction will, thus, require a significant effort that is beyond the scope of the current manuscript. because the p1f12 mab retains the ability to bind virions, our conclusion is that it binds to a conformational epitope. based on the cohort of human plasma samples tested in this study, it appears that most zikv-infected individuals mount ab responses against the epitope recognized by p1f12. this epitope is recognized by abs in individuals previously infected by zikv, thereby preventing the binding of p1f12. by contrast, abs in the plasma from individuals previously infected by any of the denv serotypes, do not prevent binding of p1f12. p1f12 may, therefore have potential as a diagnostic. several diagnostic options for testing for zikv exposure exist, including rt-pcr, igm elisa, and prnt methods [22, 68] . while it is relatively straightforward to detect zikv nucleic acid during the acute phase in blood, urine, saliva, and semen, it has proven more difficult to design rapid and effective diagnostics for zikv exposure in the chronic phase. for samples collected after the first week of symptoms, the initial test is an anti-zikv, anti-denv, anti-chikv virus igm elisa [68] . however, in patients who have received a flaviviral vaccine (denv, yfv, or jev) and/or have been infected with any flaviviruses in the past, these assays may be difficult to interpret due to the cross-reactivity of the abs [68] [69] [70] [71] [72] [73] . thus, a positive igm test needs to be confirmed with a laborious prnt assay. igm antibodies persist for 2-12 weeks in serum, and sera from individuals previously infected for more than 12 weeks would also have to be confirmed with a virus neutralization-based method [68] . our plasma inhibition assay may, perhaps, provide an alternative to these other techniques. in this study, we isolated plasmablast-derived abs from a zikv-infected individual with unusual characteristics. the human igg p1f12 has no or limited shm yet binds to an immunodominant zikv epitope that is not present on any of the four denv serotypes. furthermore, this mab can neutralize the virus with a neut 50 of approximately 2 μg / ml. our results suggest that shm-independent pathways may generate neutralizing abs in the responses against zikv. molecular evolution of zika virus during its emergence in the 20(th) century a simple technique for 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we thank the patients for their volunteer donations. we also thank the laboratory and clinical personnel responsible for collecting the plasma samples used in this study. conceptualization: dmm diw egk. key: cord-007383-5yb3dxse authors: kang, jun-gu; jeon, kyeongseok; choi, hooncheol; kim, yuri; kim, hong-il; ro, hyo-jin; seo, yong bok; shin, jua; chung, junho; jeon, yoon kyung; kim, yang soo; lee, keun hwa; cho, nam-hyuk title: vaccination with single plasmid dna encoding il-12 and antigens of severe fever with thrombocytopenia syndrome virus elicits complete protection in ifnar knockout mice date: 2020-03-20 journal: plos negl trop dis doi: 10.1371/journal.pntd.0007813 sha: doc_id: 7383 cord_uid: 5yb3dxse severe fever with thrombocytopenia syndrome (sfts) is an emerging tick-borne disease caused by sfts virus (sftsv) infection. despite a gradual increase of sfts cases and high mortality in endemic regions, no specific viral therapy nor vaccine is available. here, we developed a single recombinant plasmid dna encoding sftsv genes, gn and gc together with np-ns fusion antigen, as a vaccine candidate. the viral antigens were fused with fms-like tyrosine kinase-3 ligand (flt3l) and il-12 gene was incorporated into the plasmid to enhance cell-mediated immunity. vaccination with the dna provides complete protection of ifnar ko mice upon lethal sftsv challenge, whereas immunization with a plasmid without il-12 gene resulted in partial protection. since we failed to detect antibodies against surface glycoproteins, gn and gc, in the immunized mice, antigen-specific cellular immunity, as confirmed by enhanced antigen-specific t cell responses, might play major role in protection. finally, we evaluated the degree of protective immunity provided by protein immunization of the individual glycoprotein, gn or gc. although both protein antigens induced a significant level of neutralizing activity against sftsv, gn vaccination resulted in relatively higher neutralizing activity and better protection than gc vaccination. however, both antigens failed to provide complete protection. given that dna vaccines have failed to induce sufficient immunogenicity in human trials when compared to protein vaccines, optimal combinations of dna and protein elements, proper selection of target antigens, and incorporation of efficient adjuvant, need to be further investigated for sftsv vaccine development. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 severe fever with thrombocytopenia syndrome (sfts) is an emerging tick-borne infectious disease caused by sfts virus (sftsv), belonging to the phenuiviridae family of bunyavirales [1, 2] . the genome of sftsv is composed of three segmented rnas: large (l) segment encoding rna-dependent rna polymerase (rdrp), medium (m) encoding the envelope glycoproteins, gn/gc, and small (s) encoding the nucleocapsid and nonstructural proteins (np and ns) [1] . clinical manifestations include fever, gastrointestinal symptoms, leukocytopenia, and thrombocytopenia [3, 4] . disease mortality of sfts patients have been estimated to be 52 0% [3] . even though the majority of sfts cases has been reported from china [3] , korea [4] , and japan [5] , sftsv infections in southern asia, including vietnam, have been recently reported in a retrospective survey [6] . currently, no specific viral therapy nor vaccine is available. an effective vaccine is needed to combat its relatively high mortality, especially in elderly patients, and spread of sftsv between humans [7, 8] . vaccine development for sfts is at an early discovery phase and there have only been a few studies on vaccine candidates using animal infection models [8] [9] [10] [11] . immunization of ns antigen with freund's adjuvant in c57bl/6 mice, which are naturally resistant to sftsv but partially mimic human infections [12] , failed to enhance viral clearance, although it induced high titer of anti-ns antibodies and significantly elevated ifn-γ levels in sera upon viral challenge [9] . vaccination of plasmid dnas encoding np and ns peptides also enhanced antigen-specific cellular immunity of t cells, such as ifn-γ and tnf-α secreting cd4 + and cd8 + t cells, in balb/c mice when applied by nano-patterned microneedles [10] . however, the protective effect of cellular immunity induced by dna vaccination was not confirmed by in vivo infection with sftsv. recently, dong f. et al. reported that a single dose of live attenuated recombinant vesicular stomatitis virus (rvsv) vaccine expressing the sftsv gn/gc glycoproteins elicited high titers of protective neutralizing antibodies in both wild type and interferon α/β receptor knockout (ifnar ko) mice [11] . they clearly showed that a single dose rvsv carrying sftsv gn/gc could provide complete protection against lethal challenge with sftsv in young and old ifnar ko mice, a promising infection model for severe human infection of sftsv [11, 13, 14] . nevertheless, the potential role of cellular immunity against the viral antigens in complete protection was not examined in this study, although adoptive transfer of immune sera from mice immunized with rvsv-gn/gc to naïve ifnar ko mice provide partial (~60%) protection against lethal sftsv challenge [11] . in this study, we developed a recombinant plasmid dna (psftsv) encoding extracellular domains of gn and gc, and np-ns fusion antigen as a dna vaccine candidate. we examined whether it could provide protective immunity against lethal sftsv infection in ifnar ko mice. in order to facilitate the processing and presentation of the sftsv antigens by dendritic cells (dcs) and enhance antigen-specific t cell responses, these recombinant antigens were fused with fms-like tyrosine kinase-3 ligand (flt3l) [15, 16] . moreover, we generated a recombinant dna encoding il-12α and β in addition to the recombinant viral antigens (psftsv-il12) to further enhance cell-mediated immunity [17] . vaccination of psftsv-il12 provided complete protection of ifnar ko mice upon lethal sftsv challenge, whereas immunization with psftsv elicits only partial protection, indicating that antigen-specific cellular immune responses enhanced by co-expression of il-12 could play a significant role in protection against lethal sftsv infection. we confirmed significantly higher levels of gn and np-specific cd4 + and cd8 + t cell responses in mice vaccinated with psftsv-il12 when compared to those in mock vector-immunized mice. therefore, our results indicate that enhanced antigenspecific t cell immunity against multiple sftvs antigens by dna vaccination could be a promising direction for developing an effective vaccine against sftsv infection. animal experiments were conducted in an animal biosafety level 3 facility at seoul national university hospital and international vaccine institute. this study was approved by the seoul national university hospital and international vaccine institute institutional animal care and use committee (snuh iacuc no. 15-0095-c1a0 and ivi iacuc no. 2018-018) and conducted in strict accordance with the recommendations in the national guideline for the care and use of laboratory animals. in order to generate plasmids for dna vaccination, genes encoding ectodomains of gn, gc (from genebank accession no. ajo16082.1), and np/ns fusion protein (from genebank accession no. ajo16088.1 and aki34298.1, respectively) were synthesized (genscript, piscataway, nj, usa) and cloned into pgx27 vector (genexine, seongnam, republic of korea). all these genes were fused with signal peptide of tissue plasminogen activator (tpa, uniport no. p00750) and flt3l (uniport no. p49771) in their n-terminus [15] (psftsv, fig 1) . in addition, murine il-12α and β genes (uniport no. p43432) [17] were also synthesized (genscript) and cloned into psftsv (psftsv-il-12, fig 1) . to confirm the expression of cloned genes in psftsv and psftsv-il12, hek 293t cells (atcc crl-1573, manassas, va, usa) were transfected with pgx27, psftsv, or psftsv-il12 plasmid using the polyethylenimine (pei) transfection method [18] . briefly, plasmids and pei were diluted with opti-mem media (gibco, gaithersburg, md, usa) and incubated with hek 293t cells. after 4 h of incubation, media were changed with dulbecco's modified eagle medium (dmem) (gibco) containing 10% fetal bovine serum (fbs) and incubated in humidified co 2 atmosphere at 37˚c. after 48 h of culture, cells and culture supernatants were harvested. cells were washed with phosphate-buffered saline (pbs) and lysed with np-40 lysis buffer (1% np-40 in 50 mm tris-hcl, ph 8.0, 150 mm nacl) containing protease inhibitor cocktail (sigma-aldrich, st. louis, mo, usa). lysates and supernatants were stored at -80˚c until use. for quantification of flt3l-fused proteins and mouse il-12 in the supernatants of transfected hek293t cells, human flt3l and mouse il-12p70 quantikine elisa kits (r&d systems, minneapolis, mn, usa) were used according to manufacturer's instructions. expression of gc, gn, and np/ns fusion protein in the culture supernatants and cell lysates were examined by immunoblotting using rabbit anti-gn, gc (nbp2-41153 and nbp2-41156, novus biologicals, centennial, colorado, usa), and np (produced by custom polyclonal antibody production service via abclon, seoul, republic of korea) antibodies, respectively. each sample was separated on 8% polyacrylamide gels and transferred onto 0.45 μm pvdf membranes (merck, darmstadt, germany). pvdf membranes were blocked with pbs containing 0.05% tween 20 (pbst) and 5% skim milk at room temperature for 2 h. the membranes were sequentially incubated with primary and secondary antibodies conjugated with horse radish peroxidase (hrp, invitrogen, waltham, ma, usa). the membranes were then visualized using a las-4000 system (fujifilm, tokyo, japan). the gene encoding the np of the kasjh strain (genbank accession no. kp663733) was cloned into the pet28a (+) vector (novagen, gibbstown, nj, usa). np protein was then purified from e. coli strain bl21 (de3) harboring the recombinant plasmid. following induction with 0.1 mm isopropyl β-d-thiogalactoside (iptg) for 18 h at 16˚c, the protein was purified using histrap hp histidine-tagged protein columns (ge healthcare, chicago, il, usa) according to the manufacturer's instruction. recombinant gn and gc glycoproteins fused to fc region of human immunoglobulin heavy chain were purified as previously described [19] . briefly, vectors cloned with gene encoding gn or gc were transfected into hek293f cells (thermo fisher scientific, waltham, ma, usa) using polyethylenimine. the transfected cells were cultured in freestyle 293 expression medium (gibco) for 6 days. overexpressed recombinant proteins in supernatants were purified by akta start affinity chromatography system hitrap mabselect (ge healthcare) according to the manufacturer's instructions (s1 fig). to determine the antibody titers specific to gc, gn, and np in sera of immunized mice, immunoassay plates (96-well plates; nunc, rochester, ny, usa) were coated with 100 ng/well of purified antigens at 4˚c overnight. his-tagged gn and gc recombinant proteins were purchased from immune technology co. (new york, ny, usa). his-tagged np recombinant protein was purified as mentioned above. after antigen coating, the immunoassay plates were blocked for 2 h at room temperature with pbst containing 5% skim milk. 100 μl of seriallydiluted serum samples were incubated for 1 h at room temperature and subsequently detected using hrp-conjugated goat anti-mouse igg (santa cruz biotechnology, santa cruz, ca, usa). 3,3',5,5'-tetramethylbenzidine peroxidase substrate solution (kpl, gaithersburg, md, usa) was then added to develop color for 7 min, and the reaction was stopped by the addition of 1 m h 3 po 4 solution. absorbance was measured at 450 nm using a microplate reader (tecan, mannedorf, switzerland). spleen cells were released into rpmi 1640 media (gibco) by mincing the spleen through a 70 μm cell strainer (bd biosciences, san jose, ca, usa). after lysis of red blood cell with a red blood cell lysing buffer hybri-max (sigma, st. louis, mo, usa), splenocytes were cultured for 18 h in rpmi media containing 10% fbs (gibco) and 1% penicillin/streptomycin (gibco) in the presence of 10 μg of purified gn or np antigens in 96 well v-bottomed culture plates (nunc, roskilde, denmark). for intracellular detection of ifn-γ, splenocytes (2 × 10 6 cells/well) were treated with 1 μg of golgiplug (bd bioscience) for the final 6 h. cells were then blocked with ultra-block solution (10% rat sera, 10% hamster sera, 10% mouse sera (sigma), and 10 μg/ml of anti-cd16/32 (2.4g2) (bd pharmingen, franklin lakes, nj, usa), followed by staining with anti-cd3 (145-2c11) (bd biosciences), cd4 (rm4-59) (bd biosciences) and cd8 (53-6.7) (biolegend, san diego, ca, usa) antibodies conjugated to different fluorescent dyes. after surface staining, splenocytes were fixed and permeabilized with cytofix/cytoperm kit (bd bioscience) and incubated with anti-ifn-γ antibody (xmg1.2) (bd pharmingen). the stained cells were analyzed on a cytoflex s flow cytometer (berkman coulter inc, brea, ca, usa). flow cytometry data were analyzed using flowjo software version 10.6 (tree star, ashland, or, usa). gating strategies for the flow cytometric analyses are summarized in s2 fig. sftsv (genbank accession no. mn329148-mn329150) isolated from a korean sfts patient was propagated in vero e6 cells (atcc crl-1586). the supernatant of infected cells was harvested at 5 d after infection and stored at-80˚c after filtering with 0.45 μm syringe. focusforming unit (ffu) of sftsv was determined by plaque assay using methylcellulose media [20] . briefly, the filtered supernatants were serially diluted and added to a monolayer of vero e6 cells and incubated for 1 h at 37˚c. viral supernatants were removed and cells were incubated under an overlay media (dmem supplemented with 5% fbs and 1% methylcellulose) at 37˚c for 7 days. cells were fixed with 4% paraformaldehyde (intron, seongnam, republic of korea) and 100% methanol (merck, darmstadt, germany). the sftsv foci were detected using rabbit anti-sfts np antibody (abclon) and goat anti-rabbit igg secondary antibody conjugated with alkaline phosphatase (thermo fisher scientific). viral plaques were visualized by incubation with nbt/bcip solution (roche, mannheim, germany). to evaluate the neutralizing activity of immune sera, a focus reduction neutralization titer (frnt) assay was performed using immunized mice sera [21] . sftsv (0.0001 multiplicity of infection) was pre-incubated with serially diluted sera from mice at 4˚c for 1 h. the mixture of virus and sera was added onto a monolayer of vero e6 cells in 24-well plate. after incubation for 2 h, supernatant of cells were removed and cells were cultured under an overlay media at 37˚c for 7 days. viral foci were visualized as described above. the percentage of focus reduction was calculated as [(no. of plaques without antibody)-(no. of plaques with antibody)] / (no. of plaques without antibody) x 100. 50% focus reduction neutralization titers (frnt 50 ) were calculated by a nonlinear regression analysis (log[inhibitor] versus normalized response method) embedded in graphpad prism software v5.01 (graphpad software; https:// www.graphpad.com). six to eight-week-old female interferon α/β receptor knockout (ifnar ko, b57bl/6) mice [22] were used for immunization and challenge tests. they were housed and maintained in the specific pathogen-free facility at seoul national university college of medicine. mice were intramuscularly immunized by electroporation using orbijector ep-i model (sl vaxigen inc., seongnam, republic korea) in the hind leg three times at two-week intervals. 4 μg of purified pgx27, psftsv, or psftsv-il12 in 100 μl of pbs was used for each immunization. mice were also subcutaneously immunized with 20 μg of gn-fc or gc-fc protein absorbed in aluminum hydroxychloride (alhydrogel adjuvant 2%, invivogen, hong kong). mice sera were collected from immunized mice at one week after the third immunization to determine the levels of specific antibody titers. two weeks after the final immunization, mice were subcutaneously challenged with 1 x 10 5 ffu of sftsv. body weight and mice survival were monitored until surviving mice fully recovered. blood and tissues of mice were collected at the indicated time after viral challenge and applied for viral quantitation using qrt-pcr, or hematological analysis. total rna was extracted from the plasma of sftsv-infected mice using trizol ls reagent (life technologies, carlsbad, ca, usa) according to the manufacturer's instruction. total rna was reverse transcribed into cdna using hisenscript rh (-) rt premix kit (intron, seongnam, republic of korea). cdna was quantified using taqman universal master mix 2 (applied biosystems, waltham, ma, usa). qrt-pcr was performed on biorad cfx connect real-time system (bio-rad, hercules, ca, usa) under following conditions: uracil-n-glycosylase incubation at 50˚c for 2 min, polymerase activation at 95˚c for 10 min, denaturation at 95˚c for 15 s, annealing and extension at 53˚c for 1 min. amplification was performed for 45 cycles and the fluorogenic signal was measured during annealing/extension step. primer set and detecting probe for qrt-pcr was derived from the np gene of sftsv: np forward (5'-ccttcaggtcatgacagctgg-3'), np reverse (5'-accaggctctcaatcactcctg t-3') and detecting probe (5'-6fam-agcacatgtccaagtgggaaggctctg-bhq1-3'). copy numbers were calculated as a ratio with respect to the standard control. to collect hematological data, animals were euthanized and bled by cardiac puncture. blood was prepared in tubes coated with 0.5 m edta (enzynomics, daejeon, republic of korea). prior to evaluation of platelet counts, 4% paraformaldehyde was added to edta-anticoagulated whole blood samples at a 1:1 ratio to inactivate virus [23] . the platelet counts were analyzed using advia 2012i hematology system (siemens healthimeers, erlangen, germany). data was analyzed using the graph pad prism 5.01 software (graphpad software, la jolla, ca, usa). statistical analysis was performed using two-tailed student's t-test with 95% confidence interval or one-way analysis of variance (anova) followed by newman-keuls t-test for comparisons of values among different groups. data are expressed as the mean ± standard deviation (s.d.). statistical analysis on survival rates were performed using the mantel-cox log rank test. a p-value of < 0.05 was considered statistically significant. four viral genes of sftsv were cloned into pgx27 vector to be expressed in mammalian cells (fig 1) . extracellular domains of viral glycoproteins, gn and gc, were separately cloned into the plasmid vector under control of cmv promoter and ires sequence. full lengths of viral np and ns genes fused with a linker peptide (gsgsgsgsgsgra) were also cloned into the vector under control of rsv promoter. all the proteins were fused with the extracellular domain of flt3l and the signal sequence of tissue plasminogen activator (tpa) in their n-terminus to promote antigen presentation and trafficking of the fusion proteins as previously described [15] . in addition, psftsv-il12 plasmid also encodes murine il-12α and β to enhance antigen-specific t cell responses [17] . expression and secretion of the viral antigens and il-12 in hek293 cells transfected with the plasmids were confirmed by elisa and immunoblot analysis (fig 2) . all the viral antigens were detected in cell culture supernatants, as well as in cellular lysates (fig 2b) . next, we assessed antigen-specific adaptive immunity against the viral antigens after vaccination in ifnar ko mice. antibody responses to np antigen was significantly elevated (mean titer ± s.d.: 255 ± 63, n = 3) only in mice immunized with psftsv-il12, and not in mice vaccinated with mock vector or psftsv at two weeks after third immunization (fig 3a) . specific antibodies against gn and gc were barely detectable in all the vaccinated groups, suggesting that these dna vaccines may not efficiently induce specific antibodies against gn or gc, but produce np antibody responses in the presence of il-12 expression. in order to characterize the potential difference in quality of cell-mediated immunity in ifnar ko mice immunized with the dna vaccines, we analyzed antigen-specific t cell responses by assessing ifn-γ secreting t cells in an antigen-dependent manner (fig 3b and 3c ). splenocytes collected from immunized mice were stimulated with gn or np antigens and cytokine-positive t cell subsets were analyzed by flow cytometry. the frequencies of cytokinepositive cd4 and cd8 t cells induced by splenocytes of psftsv-il12-immunized mice were significantly higher than those of vector-immunized mice. even though the cellular responses of immune splenocytes from psftsv-vaccinated mice were generally increased, the levels were not statistically significant, suggesting a potent role of il-12 co-expression in enhancing cell-mediated immunity against the viral antigens. since we observed significant elevation of t cell responses specific to the viral antigens in ifnar ko mice immunized with psftsv-il12 dna vaccine, we tested whether it could provide protective immunity against lethal sftsv infection. the susceptibility of ifnar ko mice to a korean sftsv isolate was examined by inoculating the mice with 1 x 10 1 to 1 x 10 5 ffu (s3a fig). upon infection, all the mice gradually lost body weight and became moribund from the third to fifth day after infection, depending on the infection dose. all the infected mice died at 5~9 d after infection (s3a fig). similar survival kinetics were previously reported in ifnar ko mice infected with other sftsv strains [11, 14] , indicating that our korean sftsv isolate possesses an equivalent degree of virulence to prior chinese isolates. the platelet counts in mice infected with 1 x 10 5 ffu of sftsv significantly declined up to 4 d after infection, and the mean platelet volumes of the infected mice were significantly higher than those of control animals (s3b fig), suggesting that platelet destruction and the activation of platelet production simultaneously occur during lethal infection [14] . to assess the protective efficacy of the dna vaccine, groups of mice were immunized with mock vector, psftsv, or psftsv-il12 three times and then subcutaneously challenged with a lethal dose (10 5 ffu/mouse) of sftsv (fig 4) . all the mock-immunized mice expired by 5 d after sftsv infection. in contrast, all the mice immunized with psftsv-il12 were protected from lethal viral challenge, and 40% (2 of 5) of mice vaccinated with psftsv survived ( fig 4a) . consistently, psftsv-il12-vaccinated animals lost weight until 4 d after the viral challenge and recovered thereafter, whereas decrease in body weight of psftsv-immunized mice was observed up to 8 d after the challenge and the surviving mice gradually recovered. when we examined the progression of thrombocytopenia during the acute phase of infection in the mice, platelet counts in vector-immunized mice rapidly declined until expiration (fig 4b, left panel) . however, platelet counts only marginally decreased in mice vaccinated with psftsv or psftsv-il12 and counts rebounded in psftsv-il12-immunized mice at 4 d after viral infection. in addition, viral loads were significantly reduced in the plasma of psftsv or psftsv-il12-vaccinated mice when compared to those of vector-immunized control at 4 d after infection. notably, despite similar initial viral loads (~10 6 copies/ml of plasma) at 2 d after viral challenge among the mice groups, psftsv-il12-vaccinated mice (mean ± s.d. = 4.2 × 10 6 ± 4.6 × 10 6 ) were approximately five and fifty times lower than those of psftsvvaccinated mice (mean ± s.d. = 2.1 × 10 7 ± 1.7 × 10 7 ) and vector-immunized mice (mean ± s.d. = 2.0 × 10 8 ± 1.9 × 10 8 ) at 4 d, respectively. since antibody responses against the viral glycoproteins, gn and gc, were poorly induced in ifnar ko mice immunized with the dna vaccines, we further investigate the potential protective role of neutralizing antibodies against gn or gc protein by immunization with the protein antigens fused with human fc fragment (fig 5) . vaccination with gn-fc or gc-fc protein with alum adjuvant efficiently induced specific antibodies against the immunized antigens in ifnar ko mice; levels of antibody titers against both antigens were fairly similar (mean titer ± s.d. = 3,584 ± 1,024, n = 4). in addition, levels of neutralizing antibodies against sftsv in immune sera from mice immunized with either protein antigen were significantly higher than those from fc-immunized controls, when assessed by focus reduction neutralization test (frnt, fig 5b) . frnt 50 titers of immune sera from mice immunized with gc-fc (mean titer ± s.d. = 209 ± 140, n = 4) and gn-fc (mean titer ± s.d. = 929 ± 1,134, n = 4) were approximately five and twenty folds higher than those of fc-immunized mice (mean titer ± s.d. = 42 ± 29, n = 4). it is also notable that frnt 50 titers of gn-fc immune sera were generally higher than those of gc-fc sera, although the difference was not statistically significant. finally, we examined the protective efficacy of the protein vaccines against lethal infection with sftsv (fig 5c) . all the control mice immunized with fc antigen died at five days after infection, whereas 50% of gn-fc immunized mice were protected from lethal challenge and regained their lost body weight. interestingly, weight loss of gc-fc vaccinated mice was delayed and observed from 4 d after infection, whereas the control mice lost weight from 2 d after infection. even though all the mice immunized with gc-fc succumbed to death, they survived three days more than control mice and the extension of median survival time was statistically significant (p = 0.0046). dna vaccination has been widely investigated for various infectious diseases, especially targeting viral infections, during the last two decades [24, 25] . although, human clinical trials of dna vaccines have yielded poor immunogenicity and less than optimal results, the approval of a few veterinary vaccines is a testimony of proof-of-concept and the hope that licensed dna vaccines for human use may not be too far away [24] . while we were preparing this manuscript, kwak j.e. et al. reported a promising demonstration of dna vaccination against lethal sftsv infection in old-aged (> 4-years-old) ferret model [26] . they used five plasmid dnas encoding individual viral gene, gn, gc, np, ns, or rdrp, and found that vaccination of old ferrets with a mixture of the five plasmids completely protected ferrets from lethal sftsv challenge without developing any clinical symptoms and systemic viremia [26] . in addition, adoptive transfer of immune sera from ferrets immunized with two plasmids encoding gn and gc could not perfectly prevent the systemic viremia after sftsv infection, but provided complete protection against lethal challenge, suggesting that gn/gc may be the most effective antigens for inducing protective immunity. they also found that non-envelop (np, ns, and rdrp)-specific t cell responses also contribute to protection against sftsv infection although it failed to induce neutralizing activity [26] . here we also observed that vaccination with a plasmid encoding gn, gc, np, and ns, together with murine il-12, could provide protective immunity in ifnar ko mice against lethal sftsv challenge. however, our current dna vaccine failed to suppress initial systemic viremia and weight loss (fig 4) . this might be due to the lack of type i interferon signaling and/or poor antibody responses against gn and gc glycoproteins, which are required for viral neutralization during the early stage of viral infection. inefficient generation of antibodies against gn and gc might be due to the absence of type i interferon signaling in ifnar ko mice or fusion of flt3l with the glycoproteins, resulting in conformation defect of the antigens and/or dysregulation of b cell responses. given that immunization with gn-fc and gc-fc proteins can induce specific antibodies in ifnar ko mice (fig 5) , the absence of type i ifn signaling might not be the cause of inefficient antibody generation. instead, flt3l may suppress specific antibody responses as observed in other studies [16, [27] [28] [29] . flt3l may exert tolerogenic effect on cd4 t cells via dendritic cells, thereby promoting b cell hypo-responsiveness in vivo; however, the underlying mechanisms of cd4 t cell dysregulation are still unclear [28] . nevertheless, flt3l can significantly enhance cd8 t cell responses when fused with a target antigen and promote persistent maintenance of antigen-specific cd8 t cells in vivo [16, 29] . in this study, we also observed significant enhancement of gn and np-specific cd8 t cell responses, as well as cd4 t cells, in the presence of il-12 expression (fig 3b and 3c) , which correlated well with vaccine protective efficacy in mice infected with lethal doses of sftsv. the potent effect of antigen-specific t cell responses in protection against sftsv infection is consistent with the results of the aforementioned ferret infection model [26] . in this study, we did not include a negative control using a plasmid encoding il-12 alone, since it has been shown that il-12 alone failed to boost antigen-specific cellular immunity [30] . finally, we evaluated the degree of protective immunity provided by the individual glycoprotein, gn or gc, after protein antigen immunization for the first time (fig 5) , since previous studies showed that vaccination with both antigens retained in a live viral vector [11] or dna vaccine [26] can provide complete protection against lethal sftsv challenge in ifnar ko mice or old ferrets, respectively. both protein antigens are immunogenic and induce significant levels of neutralizing activity against sftsv, although anti-gn antibodies retain relatively higher neutralizing capacity than anti-gc antibodies. consistently, the protective efficacy of gn protein vaccination is relatively higher than gc protein immunization. while vaccination with either protein antigen failed to provide complete protection against lethal sftsv challenge, gc vaccine prolonged the survival time of infected mice and gn antigen can provide partial protection. considering that gn and gc glycoproteins function as viral ligands for cellular receptor binding and viral membrane fusion in host endosome, respectively [31] [32] [33] , neutralization of both antigens might be required to completely block viral attachment and entry into host cells. taken together, we confirmed that antigen-specific t cell responses against sftsv induced by dna vaccination can provide complete protection against lethal viral challenge and immunization with each individual glycoprotein of sftsv can also confer partial protective immunity. given that the achilles heel of dna vaccines remains their poor immunogenicity in human trials when compared to protein vaccines [25] , optimal combinations of dna and/or protein vaccines, proper selection of target antigens, and incorporation of efficient vaccine adjuvant, need to be further investigated for development of the best sftsv vaccine formulation for human application. the emergence of severe fever with thrombocytopenia syndrome virus taxonomy of the order bunyavirales: update 2019 epidemiological and clinical features of laboratorydiagnosed severe fever with thrombocytopenia syndrome in china, 2011-17: a prospective observational study severe fever with thrombocytopenia syndrome: comparison with scrub typhus and clinical diagnostic prediction viral load and inflammatory cytokine dynamics associated with the prognosis of severe fever with thrombocytopenia syndrome virus infection: an autopsy case endemic severe fever with thrombocytopenia syndrome nosocomial person-to-person transmission of severe fever with thrombocytopenia syndrome current status of severe fever with thrombocytopenia syndrome vaccine development. current opinion in virology immunization with recombinant sftsv/ nss protein does not promote virus clearance in sftsv-infected c57bl/6j mice nano-patterning of a stainless steel microneedle surface to improve the dip-coating efficiency of a dna vaccine and its immune response single dose of a rvsv-based vaccine elicits complete protection against severe fever with thrombocytopenia syndrome virus pathogenesis of emerging severe fever with thrombocytopenia syndrome virus in c57/bl6 mouse model the pathogenesis of severe fever with thrombocytopenia syndrome virus infection in alpha/beta interferon knockout mice: insights into the pathologic mechanisms of a new viral hemorrhagic fever animal models of emerging tick-borne phleboviruses: determining target cells in a lethal model of sftsv infection. front microbiol clearance of persistent hpv infection and cervical lesion by therapeutic dna vaccine in cin3 patients long-term maintenance of gp120-specific immune responses by genetic vaccination with the hiv-1 envelope genes linked to the gene encoding flt-3 ligand engineering n-glycosylation mutations in il-12 enhances sustained cytotoxic t lymphocyte responses for dna immunization transient mammalian cell transfection with polyethylenimine (pei) an anti-gn glycoprotein antibody from a convalescent patient potently inhibits the infection of severe fever with thrombocytopenia syndrome virus methylcellulose media for plaque assay of murine leukemia virus sequential emergence and wide spread of neutralization escape middle east respiratory syndrome coronavirus mutants, south korea a type i interferon and il-10 induced by orientia tsutsugamushi infection suppresses antigen-specific t cells and their memory responses modeling severe fever with thrombocytopenia syndrome virus infection in golden syrian hamsters: importance of stat2 in preventing disease and effective treatment with favipiravir dna vaccine delivery and improved immunogenicity the future of human dna vaccines development of a sftsv dna vaccine that confers complete protection against lethal infection in ferrets intramuscular co-injection of naked dna encoding hbv core antigen and flt3 ligand suppresses anti-hbc antibody response co-expression of flt-3 ligand gene ablates tumor immunity elicited by her-2/neu dna vaccine in transgenic mice enhancement of dna vaccine potency by linkage of antigen gene to a gene encoding the extracellular domain of fms-like tyrosine kinase 3-ligand. cancer research enhanced protection against viral infection by co-administration of plasmid dna coding for viral antigen and cytokines in mice the role of phlebovirus glycoproteins in viral entry, assembly and release structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion structures of phlebovirus glycoprotein gn and identification of a neutralizing antibody epitope key: cord-310870-w8wu8vno authors: shorten, robert j.; wilson-davies, eleri title: the risk of transmission of a viral haemorrhagic fever infection in a united kingdom laboratory date: 2017-05-18 journal: plos negl trop dis doi: 10.1371/journal.pntd.0005358 sha: doc_id: 310870 cord_uid: w8wu8vno nan contaminated with splashes or droplets of blood or body fluids, and inoculation with sharps. experts from the uk advisory committee on dangerous pathogens (acdp) agree that there is no circumstantial or epidemiological evidence of an aerosol transmission risk from vhf patients. a review of previous cases of vhfs imported into the uk shows that biochemistry, haematology, microbiology, and virology assays were performed using routine analysers in standard cl2 pathology laboratories. often, these assays were performed prior to the diagnosis being made, yet no transmissions to laboratory workers were recorded (table 1) . [9] [10] [11] [12] in addition, over 9,000 cases of crimean congo haemorrhagic fever (cchf) were reported in turkey between 2002 and 2014, with an estimated minimum 180,000 blood samples processed in routine laboratories with no additional precautions. a review was performed of 51 healthcare exposures that occurred in 9 centres where 4,869 of these patients were managed. of these, only 2 cases in laboratory staff were identified. one may have been associated with a needle-stick injury and the other with handling samples while not wearing appropriate ppe (gloves). [13] there is no evidence of any risk of transmission when good laboratory practice is followed within a cl2 laboratory. although it is reassuring that large numbers of samples from patients with cchf infection have been processed safely in routine laboratories in turkey, it should be noted that this bunyavirus is rarely transmitted person to person, so the parallels between this and other vhf viruses need to be carefully considered. the potential routes of transmission within a cl2 laboratory setting are, however, the same for all vhf agents. laboratory-acquired cases of evd were reported in the west african outbreak; however, it should be remembered that initial laboratory work in this outbreak was performed with extremely limited facilities and resources. we are not aware of any imported cases of evd to resource-rich settings that have resulted in laboratory transmission, even when samples were analysed with no additional precautions prior to diagnosis. in vitro diagnostic systems (ivds) include analysers that automate the diagnostic process in clinical laboratories. these in vitro diagnostic medical devices (ivdmd) therefore process blood or blood in suspension of testing fluids. consequently, all ivdmd which perform assessments on patients' blood will regularly be challenged by exposure to blood-borne viruses (bbv) which circulate within the population. therefore, they should be designed to eliminate or reduce as far as reasonably practicable the risk of infection to users and other persons, which includes the staff who service the devices. fundamental to this is the manufacturer's design to minimise the leakage of fluid and contamination, which may lead to microbial exposure during normal use or servicing. [14] viruses are either naked or enveloped in structure (fig 1) . the envelope (where present) is derived from the surrogate host cell, which, along with the associated viral receptors, is essential for attachment and host cell entry. removal of the viral envelope inactivates the virus, preventing replication and subsequent host infection (fig 2) . all of the high hazard viral diseases cited are caused by enveloped viruses. ivds should be decontaminated prior to inspection, maintenance, repair, or disposal, either on site or at the manufacturer's or agent's premises. [15] decontamination must be carried out in line with the manufacturer's instructions using methods validated to inactivate enveloped viruses. the enveloped viruses are among the most susceptible pathogens to disinfectants. [16] this is due to the presence of the lipid envelope, which is compromised by most biocides. compounds with validated efficacy against enveloped viruses include alcohols, aldehydes, biguanides (e.g., chlorhexidine), halogens, peroxygen compounds (e.g., hydrogen peroxide), peracetic acid, some phenols, and some quaternary ammonium compounds. manufacturers must validate their decontamination method against appropriate surrogate model enveloped viruses. laboratory viruses vary between strains and wild-type viruses. studies, by necessity, use laboratory strains which may be grown to high titre and efficiently assayed. any virus used in a validation study is, therefore, a model virus. viral inactivation validation studies have successfully used surrogate model viruses with properties similar to wildtype viruses to provide evidence for the safety of human blood plasma products for over a decade. [17] studies using model viruses are also used for pharmaceuticals, derived from human and/or animal sources, including recombinant proteins produced in eukaryotic cell lines, vaccines, and some class iii medical devices. [18] viral inactivation validation studies must inactivate a range of enveloped viruses, using at least 3 viruses to represent different properties that ivdmds are expected to be challenged against. a suitable model virus for hiv, hbv, and hcv must be included. if an enveloped virus has been shown to have a higher resistance to the manufacturer's disinfectant class, then a model for that virus should also be included. once dried on inanimate surfaces, viruses are less susceptible to decontamination than when hydrated in suspension. [19] [20] validation studies should therefore include a quantitative suspension test for the assessment of internal decontamination procedures [15] and quantitative carrier tests [21] or appropriate alternative methods. an effective and reliable decontamination method will show a reduction of 4 log 10 . [22] despite some pathogens causing a very high titre viraemia, the design of ivdmds reduces their contamination as far as reasonably practicable. the design of ivdmds means they only require a small volume of blood to perform an analysis. consequently, when an effective and reliable decontamination method is in place that provides a reduction of 4 log 10 , this will inactivate any residual enveloped virus within the ivdmd. decontamination therefore renders the ivdmd safe to use and service after challenge with any enveloped virus. once assessed by appropriately designed viral inactivation validation studies on relevant surrogate model enveloped viruses, the decontamination process has been shown to be effective against all known and future emerging enveloped viruses, which includes ebola, cchf, lassa fever, and marburg virus. guidance issued by the acdp therefore states that samples taken from these patients may be safely processed using standard precautions, good laboratory practice, and ppe, as the risk of infection from these samples is low. [23] it also states that routine decontamination procedures are adequate in these situations. the guidance further explains that autoanalysers are the preferred method for processing such specimens. sealed buckets should be used for any centrifugation procedures that are not undertaken within an autoanalyser. in certain circumstances, the use of discrete analysers may need to be considered, but these are not a safer option. point of care (poc) blood gas analysers present a high risk of splashing and should only be used in exceptional circumstances with suitable barrier protection ppe for staff and in a controlled environment. when preparing blood film slides for malaria testing, consideration should be given to the potential for splash and therefore should be carried out in a microbiological safety cabinet or, alternatively, facial protection should be used. likewise, blood cultures and blood cross-matching may be performed at cl2 following appropriate risk assessment. we also know from health & safety executive reporting of injuries, diseases and dangerous occurrence regulations (riddor) data that rates of infection with bbvs in healthcare workers are low, and the majority of these are needle-stick associated and not in laboratory staff. [24] this is also supported by data from the uk significant occupational exposures surveillance system. [24] tens of thousands of samples are processed daily in routine pathology laboratories that are, often unknown to us, positive for bbvs. the important consideration here is about the clinical well-being and appropriate investigation and management of the patient. a delay in diagnosis of other traveller-associated infections, such as malaria or typhoid, as well as lack of access to supportive pathology assays, can be fatal. laboratory staff welfare is equally important, and the application of good laboratory practice in a risk-assessment-led setting indicates that such samples may be analysed safely in routine cl2 laboratories. ebola virus disease cluster in the united states the first case of ebola virus disease acquired outside africa transmission of ebola hemorrhagic fever: a study of risk factors in family members lessons from nosocomial viral haemorrhagic fever outbreaks the management, design and operation of microbiological containment laboratories advisory committee on dangerous pathogens. the approved list of biological agents the public health response to a case of lassa fever in london in 2000 the first case of lassa fever imported from mali to the united kingdom a fatal case of lassa fever in london first confirmed case of crimean-congo haemorrhagic fever in the uk healthcare-associated crimean-congo haemorrhagic fever in turkey, 2002-2014: a multicentre retrospective cross-sectional study global harmonization task force. essencial principles of safety and performance of medical devices chemical disinfectants and antiseptics. quantitative suspension test for the evaluation of virucidal activity in the medical area. test method and requirements similarities and differences in the responses of microorganisms to biocides is hepatitis b-virucidal validation of biocides possible with the use of surrogates? guideline on virus safety evaluation of biotechnological investigational medicinal products. doc ref emea/chmp/bwp/398498/2005-corr resistance of surface-dried virus to common disinfection procedures susceptibility of hepatitis b virus to disinfectants or heat inactivation of hepatitis b virus by intermediate-to-highlevel disinfectant chemicals note for guidance on virus validation studies: the design, contribution and interpretation of studies validating the inactivation and removal of viruses cpmp/bwp/268/95. comm propr med prod management of hazard group 4 viral haemorrhagic fevers and similar human infectious diseases of high consequence bloodborne viruses: eye of the needle microbiology and biotechnology unit, health and safety executive, united kingdom. key: cord-002394-n85ptr5p authors: reddy, vijayalakshmi; desai, anita; krishna, shankar susarla; vasanthapuram, ravi title: molecular mimicry between chikungunya virus and host components: a possible mechanism for the arthritic manifestations date: 2017-01-26 journal: plos negl trop dis doi: 10.1371/journal.pntd.0005238 sha: doc_id: 2394 cord_uid: n85ptr5p background: chikungunya virus (chikv), a reemerging pathogen causes a self limited illness characterized by fever, headache, myalgia and arthralgia. however, 10–20% affected individuals develop persistent arthralgia which contributes to considerable morbidity. the exact molecular mechanisms underlying these manifestations are not well understood. the present study investigated the possible occurrence of molecular mimicry between chikv e1 glycoprotein and host human components. methodology: bioinformatic tools were used to identify peptides of chikv e1 exhibiting similarity to host components. two peptides (a&b) were identified using several bioinformatic tools, synthesised and used to validate the results obtained in silico. an elisa was designed to assess the immunoreactivity of serum samples from chikv patients to these peptides. further, experiments were conducted in a c57bl/6j experimental mouse model to investigate if peptide a and peptide b were indeed capable of inducing pathology. findings: the serum samples showed reactivity of varying degrees, indicating that these peptides are indeed being recognized by the host immune system during chikv infection. further, these peptides when injected into c57bl/6j mice were able to induce significant inflammation in the muscles of c57bl/6j mice, similar to that observed in animals that were injected with chikv alone. additionally, animals that were primed initially with chikv followed by a subsequent injection of the chikv peptides exhibited enhanced inflammatory pathology in the skeletal muscles as compared to animals that were injected with peptides or virus alone. collectively these observations validate the hypothesis that molecular mimicry between chikv e1 protein and host proteins does contribute to pathology in chikv infection. introduction chikungunya fever is caused by a arbovirus belonging to family togaviridae and genus alphavirus. chikv is positive sense rna virus with about 11.8 kb long genome. the prevalence of chikv has increased globally. it caused massive outbreaks when it re-emerged in 2005 in french reunion islands where it affected about 33% of the total population. chikv outbreaks also occurred in india during the same period, southern states in india recorded a total of 1.3 million cases [1, 2] . chikungunya fever is characterized by fever, headache, myalgia and arthralgia. though chikungunya is a self limiting illness [3] , a small proportion of 10-20% of affected individuals develop persistent arthralgia. the risk factors associated with the development of persistent arthralgia include older age of patients (>40 years) and pre existing rheumatic problems. however, the precise molecular mechanisms of pathogenesis that lead to the development of these complications are poorly understood. experimental evidence of chikv persistence in macrophages of macaca species has been demonstrated [4] and it has been suggested as one of the factors contributing to residual arthralgia. although, molecular mimicry as the cause of prolonged joint manifestations had not been proved conclusively in chikungunya infection, there are reports which suggest that such a phenomenon might be operational. therefore, in this study we investigated the possible occurrence of molecular mimicry between chikv e1 and host components using a three pronged strategy: (i) identification of homologous regions between chikv proteins and host tissue components using bioinformatics tools, (ii) establishing cross reactivity between serum samples obtained from chikv infected patients and peptides exhibiting molecular mimicry and (iii) validating the ability of the cross reactive peptides in inducing joint and muscle pathology in a mouse model. we demonstrate the occurrence of molecular mimicry between chikv envelope glycoprotein (e1) and the host components. a clinical isolate of chikv (chikungunya virus strain drde-06; genbank accession number: ef210157.2) was used for all the in vivo experiments in this study. the bioinformatics related work was carried out using the chikv e1 protein sequence from the prototype strain chikv s27 available in the swiss prot (id:q8jux5). further, a multiple sequence alignment of the e1 glycoprotein of drde-06 sequences and chikv s27 revealed a 98% homology between the two strains. peptides chikv peptides were custom synthesised from commercial sources (hysel pvt ltd., india) and obtained as a lyophilised powder. the non-specific peptide was a gift from xcyton diagnostics private ltd, bangalore, india. rabbit anti-human polyclonal-hrp conjugate was procured from dako, denmark, while goat anti-mouse igg-hrp was obtained from genei, bangalore. all work related to animals was conducted with good animal practice defined by committee for the purpose of control and supervision of experiments of animals. the use of animals was approved by the institutional animal ethics committee (iaec) of nimhans (approval reference no: aec/41/222(b)/nv dated 05. 10 .2010). the animals were housed in cages maintained in hygienic conditions with good ventilation, in a room maintaining the usual day and night cycle. the animals used for the experiments were euthanized by cervical dislocation and animal ethics were strictly adhered to at all times, while bleeding and sacrificing the animals. the use of human samples for the study was approved by was approved by institute ethics committee at nimhans (approval reference no: nimhans/68th iec/2010) which adheres to the ethical guidelines for biomedical research on human participants developed by the indian council for medical research (icmr). written informed consent was obtained from all the subjects themselves in the study. c57bl/6j strain of mice were obtained from nimhans central animal research facility and used in the study. eight day old mouse pups were procured from the animal facility along with the mother and the mouse pups were used for the experiments. the human samples used in this study were received at the department of neurovirology, national institute of mental health and neurosciences (nimhans), which is one of the twelve designated national apex laboratories for the diagnosis of chikungunya in india. all the subjects enrolled in the study presented to the hospital/clinics with fever, joint pain, rash, myalgia, conjunctival redness, and headache. additionally, the prevalence and local outbreaks in the region aided in making a clinical diagnosis of chikungunya fever. blood samples (3-5 ml clotted blood) were collected from thirty six subjects, serum separated and stored in aliquots at -70˚c until all the tests were performed. the chikv infection was confirmed by detection of chikv specific igm antibodies using an elisa (national institute virology, pune) and/or chikv rna by taqman real time pcr targeting the nsp4 region [5] . serum samples collected from 31 healthy individuals served as controls. chikv was grown in c6/36 cell line and infectious fluid was harvested. chikv infected c6/36 fluid was centrifuged at 10,000 rpm for 20minutes to remove debris and nacl was added to the supernatant to obtain a final concentration of 0.5 molar. subsequently, polyethylene glycol was added to the mixture to obtain a final concentration of 10% (w/v) and the suspension stirred on ice bath for 20 minutes. the mixture was incubated overnight at 4˚c, and centrifuged at 3000 rpm for 30 minutes to obtain the virus rich precipitate. the precipitate was dissolved in 1/100th of original infected cell culture fluid volume using gtne buffer. chikv was purified using a discontinuous sucrose gradient method. briefly, 5ml of 20% sucrose (w/v) in gtne buffer was carefully overlaid onto 2.5ml of 50% (w/v) sucrose. subsequently, 2.5ml of chikv obtained after peg concentration was overlaid onto the discontinuous sucrose gradient and centrifuged at 28,000 rpm for 2 hours at 4˚c using a ultracentrifuge (beckman coulter, usa). the band at the inter-phase was collected and resuspended in 10-12 volumes of pbs (ph7.2) and centrifuged at 28,000 rpm for 2 hours to obtain a purified virus pellet. the pellet was dissolved in 1ml of fresh pbs and frozen in small aliquots at -70˚c. the complete genome sequence of a prototype chikv s27 belonging to african genotype was obtained from the gen bank. the sequence of chikv e1 glycoprotein was obtained from swissprot (q8jux5). this sequence was uploaded into immune epitope database and analysis resource (iedb) server available at http://www.immuneepitope.org/. the server predicts the antigenic determinants using five different algorithms-chou and fasman beta turn prediction, emini surface accessibility prediction, karplus and schulz flexibility prediction, kolaskar and tongoankar antigenicity prediction, parker hydrophilicity prediction. the antigenic peptides from e1 region were deduced after considering hydrophilicity, surface probability, chain flexibility and secondary structure antigenic index both as text and graphs. the results obtained from the server were combined to construct a graph using ms-excel, which in turn yielded putative epitopic regions of chikv e1 glycoprotein. the peaks with antigenic propensity, surface accessibility, flexibility, hydrophilicity and beta turns were considered. the results obtained through iedb were further confirmed using additional server-european molecular biology open software suite (emboss) available at http://liv.bmc.uu.se/cgi-bin/emboss/antigenic. the results obtained using the chou and fasman criteria for beta turn prediction was also verified using coudes server. the existence of sequence similarity between chikv e1 glycoprotein and human hla-b27 was investigated using blast. the existence of structural similarity between the chikv e1 glycoprotein and human host components were determined by using bioxgem server and number of hits obtained in the non-redundant protein database (nrpdb) were limited to first 100 in the output. multiple sequence alignment of e1 glycoprotein sequences of alphaviruses-chikv, onnv, rrv, sfv, and eeev was done using clustalw available at http://www.ebi.ac.uk/tools/clustalw2/index.html. the optimal concentration of the peptide to be coated onto the elisa plate was predetermined in an initial experiment and was found to be 25μg/well. the peptides were coated onto the elisa microwells using carbonate buffer and incubated overnight at 4˚c. the plate was washed three times with phosphate buffered saline with tween (pbst) and quenched using 1% skimmed milk powder in pbst for one hour at 37˚c. the plates were washed with pbst and reacted with 100μl of serum samples (1:100 dilution in pbs containing 0.25% triton-x 100) obtained from patients infected with chikv (n = 36) as well as serum samples obtained from control subjects (n = 31). the samples were incubated for 90 minutes at 37˚c, followed by five washes with 1x pbst. subsequently, rabbit polyclonal anti-human antibodies tagged with hrp (dako, denmark) was diluted 1:1000 and 100μl was added to each well and the plate was incubated at room temperature for 90 minutes. the plate was washed five times with pbst and 100μl of the tmb solution was added and incubated in the dark for 10 minutes. the reaction was stopped by the addition 4n sulphuric acid and the od values were read at 492nm using elisa reader (thermo scientific, usa). mouse experiments to evaluate the role of chikv peptides in causing tissue damage by molecular mimicry eight day old c57bl/6j pups were procured along with the mother and the pups were used for the in vivo experiments. they were assigned to nine different groups with each group comprising of 6 pups as shown in table 1 . prior to determining the role of peptides in the possible enhancement of pathology related to chikv infection, the pathological changes induced by the chikv in c57bl/6j mice were studied (group 1). for this purpose, chikv (10 5 pfu/50μl) was inoculated into the foot pad of 8 day old mice. the control group of mice (group 2) received equal volume (50 μl) of eagles minimum essential medium (emem). the mice were kept under observation for 12 days only post infection. at the end of the observation period, blood was collected from the mice through retro orbital plexus bleeding, and the mice euthanized to obtain the following organsbrain, thymus, heart, lungs, spleen, liver, kidneys, upper limbs and lower limbs. for histopathological studies the tissues were fixed in 4% paraformaldehyde, while for pcr the tissues were collected in emem. chikv infection was confirmed by two methods-presence of chikv specific igg antibodies in the serum and detection of chikv nucleic acid in the serum and harvested tissues using taqman real-time pcr [5] . eight day old pups in group 3, 4 and 5 were injected with two doses (50μg /dose) of chikv peptide a, chikv peptide b and non-specific peptide respectively on day 0 and day 5. the peptides were reconstituted in sterile 1x pbs (ph 7.2). equal volumes of peptide solution and freund's incomplete adjuvant were mixed and emulsified to obtain water in oil emulsion. the emulsion was stored at -70˚c until use. in all these three groups blood was collected 12 th day post inoculation by retro orbital bleeding and fresh tissues were harvested and processed for pcr and paraformaldehyde fixed tissues for histopathology. mice in group 6 were injected with emem alone followed by pbs emulsified in freund's incomplete adjuvant 5 days post infection with chikv. c57bl/6j mice in group 7, 8 and 9 were injected with chikv (10 5 pfu/50 μl) followed by 50μg of the chikv peptide a, chikv peptide b and non-specific peptide respectively, on 5 th day post chikv inoculation through the same route at the same site. in these groups of mice the blood was collected and tissues harvested on 12 th day post infection, and processed for pcr and histopathological examination. detection of anti chikv igg antibodies in the serum of c57bl/6j mice serum was separated from the blood and stored at -70˚c. polystyrene elisa microwells (nunc, denmark) were coated with purified chikv in carbonate buffer at a concentration of 1μg/well diluted in carbonate buffer (appendix i). the plate was incubated overnight at 4˚c, washed thrice with pbst and quenched using 1x pbst containing 1% skimmed milk powder. serum samples were diluted 1:100 in pbst and 100μl added to the wells and incubated at 37˚c for 1 hour followed by washing for five times with pbst. subsequently, 100 μl of a 1:5000 dilution of goat anti-mouse igg conjugated with hrp (genei, bangalore) was added to the wells, incubated at 37˚c for 1 hour followed by washing for 5 times with pbst. finally, 100μl of the substrate solution (tmb) was added to all the wells and incubated in the dark for 10 minutes. the reaction was stopped by the addition 4n sulphuric acid. the od values were read at 450nm using an elisa reader (thermo scientific, china). the tissues collected in emem were homogenised using a motorised hand held homogeniser. the homogenates were spun at 8,000 rpm for 10 minutes at 4˚c. the supernatants were collected, 200 μl of the supernatant was used for rna extraction using qiamp viral rna extraction kit (qiagen, germany). the eluted rna was converted to cdna using high capacity reverse transcription kit (abi, usa). the cdna was stored at -20˚c until tested. similarly whole blood rna extraction kit was used to extract rna from blood and converted to cdna which was used in the taqman real time pcr as described earlier [5] . all tissues were fixed in paraformaldehyde and embedded in paraffin for processing and 4 μm thick sections obtained were mounted on selin coated glass slides. subsequently, the tissue sections were de-paraffinised with two changes of xylene and rehydrated in absolute alcohol followed by washing briefly in tap water. staining of the sections was carried out using harris haemotoxylin for 5-8 minutes, followed by washing under running tap water for 5 minutes and differentiated in 1% acid alcohol for 30 seconds. the sections were subsequently washed under running tap water for 1 minute, treated with bluing saturated lithium carbonate solution for 30-60 seconds and washed under tap water for 1 minute. counter staining of sections was carried out using eosin-phloxine solution for 1 minute followed by dehydration in 95% alcohol and absolute alcohol for 5 minutes each. the sections were finally immersed in xylene twice for 2 minutes each for clearing and then mounted with dpx. bioinformatics approach to deducing molecular mimicry as described in the materials and methods section, the sequence of the african prototype of chikv s27 ei glycoprotein (q8jux5) was obtained from the swissprot database and subjected to immune epitope analysis using five algorithms in iedb and emboss programs. the results are depicted in fig 1. the scores obtained for the five algorithms were uploaded into an ms excel sheet to generate a combined graph which yielded the putative epitopic regions of e1 glycoprotein of chikv. analysis of the peaks in the graph with respect to antigenic propensity, surface accessibility, flexibility, hydrophilicity and b turns enabled prediction of the following epitopic regions: these regions of chikv e1 glycoprotein satisfy all the criteria necessary for a given peptide to be considered immunogenic and capable of eliciting an immune response in the host system. subsequently multiple sequence alignment of e1 glycoprotein of alphaviruses-chikv, onyong onyong virus (onnv), ross river virus (rrv), semiliki forest virus (sfv), and eastern equine encephalitis virus (eeev) was done through clustalw. the results of the alignment are depicted in fig 2. as evident from the figure, the alignment revealed two motifs skd and kca present only in arthritogenic alphaviruses (chikv,onnv, rrv) and not in encephalitogenic alphaviruses (sfv, eeev). furthermore, the amino acid sequences skd and kca were present in the immunodominant peptides 1 and 2 deduced from e1 glycoprotein respectively. all further experiments using human serum samples and mouse models were therefore restricted only to these two peptides. the sequence similarity between the chikv e1 glycoprotein and hla-b27 was determined using blast. the alpha chain of hla-b27 molecule shared a partial homology from the stretch ranging from 216-220 of chikv e1 glycoprotein as well as the immunodominant region of peptide a (fig 3) . the output obtained through the bioxgem 3d blast performed on the chikv e1 was limited to first 100 hits. the output of the blast was further analyzed and limited only to human proteins ( table 2 ). further analysis of these human proteins was limited to those that are known to contribute to the inflammation and arthritic pathology. amongst these, six human proteins-human complement component 3, complement component 5 [6] , fibronectin [7] , kelch like protein [8] , mast/stem cell growth receptor [9] and beta arrestin 1 [10] which exhibited maximum similarity to chikv ei protein were only considered for further analysis. prominent among these six were complement proteins c3 and c5. when the structural similarity between the e1 glycoprotein and complement component c3 was analyzed, the sequence of amino acids in the region of chikv e1 glycoprotein which shared homology with complement component c3 were also present in peptide a and peptide b (fig 4) . all the patients in the study were from the state of karnataka, south india. among the 36 patients with confirmed chikv infection, 17 were females and 19 were males. the mean age of the patients was 40.64 years. all the patients whose samples were used in the study presented with fever, while 28(78%) had arthralgia, 30(83%) had myalgia, 20(56%) had complained of headache. rash and gastrointestinal symptoms were seen in 14(39%) and 10(28%) of the patients each, and conjunctival redness was seen in 1(3%) of the patients. amongst 3/36 patients (8%) persistent arthralgia was reported at 12 weeks after onset of initial symptoms. hence follow up samples blood samples could be collected from these three patients at 12 weeks. in all other patients no follow up samples could be collected. a sample was considered to be positive in the peptide elisa if it had an od value equal to or greater than that of the cut-off value. the cut off value for each of the peptides was calculated by using od values obtained with sera of healthy control subjects (n = 31) using the formula mean od of control samples + 2sd. the cut off od value for peptide a was 0.373 and for peptide b it was 0.408. amongst the 36 samples obtained from confirmed chikv patients, 24 (66.66%) showed reactivity to the peptide a and 27 (75%) to peptide b (fig 5) . the od values of chikv positive samples towards these peptides ranged from 1.501 to 0.11 for peptide a, while it varied from 1.378 to 0.203 for peptide b. these experiments were carried out to determine the possible synergistic role of peptides in the enhancement of pathology in chikv infection: all the mice were confirmed to have chikv infection by the detection of anti chikv antibodies in the serum by elisa. the cutoff in the elisa was determined using the mean + 2sd od values of serum samples obtained from uninfected control mice and it was 0.253. the od values of serum samples from all the infected animals were found to be > 0.253 and hence considered positive for chikv-specific antibodies. in addition, the presence of chikv nucleic acids was demonstrable in the muscle tissue by taqman real time pcr while, it was not detected in the blood and other tissues of the infected group of mice. all the control group of animals were negative for chikv nucleic acids. in order to have an objective assessment of the pathological features noted in all groups of animals, a semi quantitative scale for scoring the degree of inflammation centred mainly around the muscles was evolved and the slides were evaluated by a pathologist blinded to the groups and the same is depicted in fig 6. the inflammation was graded as minimal (1+), mild (2+), moderate (3+) and severe (4+). the salient histopathological features noted in chikv infected mice were as follows: (i) the soft tissue around the elbow and knee joints were relatively normal with no evidence of synovitis or arthritis, while tissues close to the shoulder and the hip joint revealed moderate degree of lymphohistiocytic infiltration virus, (ii) the major muscles of the hip and the shoulder had multifocal lymphocytic infiltrate in the endomysium with myonecrosis, (iii) random and occasion muscle fibres close to inflammation revealed cytoplasmic basophilia and central nucleation with prominent nucleolus indicative of regenerative activity, similar to polymyositis noted in human subjects (iv) the synovium and periarticular soft tissue had sparse inflammation while the articular cartilage and the articular cavity were free of inflammation, (v) the epimysium around the muscle and tendinous portion close to the insertion had variable lympho-histiocytic inflammation indicating tenosynovitis, (vi) the striking pathology was mineralization of the necrosed muscle belly especially the lateral group of muscles close to the hip and shoulder joints similar to some cases of chronic polymyositis in human subjects. other than these features noted in the limbs and joints all the other organs did not reveal any significant pathological changes. some of the salient features noted in chikv infected mice (group 1) are depicted in fig 7. the degree of pathological damage centred on the muscles in the various groups of mice was graded and a comparative chart was prepared ( table 3) . as evident from the table, the group of mice that were mock infected (group 2) and subsequently did not receive any peptides revealed sparse (1+) inflammation in the muscles probably related to the procedure. similar findings were also noted in the mock infected animals that received a single dose of freund's incomplete adjuvant (group 6). in mice that received two doses of non-specific peptide but no virus (group 5) hyperplasia of the bone marrow was noted with sparse inflammation (1+). on the other hand, mice that received two doses of chikv specific peptides but no virus (groups 3 & 4) exhibited myositis, muscle necrosis, vasculitis and hyperplasia of the marrow (immune mediated inflammatory muscle and marrow reactive changes) and an overall inflammation score of 3+ (fig 8) . in the three groups of mice that received an initial inoculum of virus followed by a single dose of either chikv specific peptides (groups 7&8) or non-specific peptide (group 9) the pathological features were more florid (fig 9) . amongst these three groups of animals, the mice that received virus followed by non-specific peptide exhibited features similar to those observed in mice that received virus alone (group 1). the animals in groups 7 and 8 had the highest overall inflammatory score (4+) and showed multiple features including myositis, muscle necrosis, focal regeneration, mineralization and calcification of the necrotic muscles, hyperplasia of marrow in the long bones. molecular mimicry represents shared immunologic epitopes between a microbe and the host. in a viral system, viruses have been shown to have cross reactive epitopes with host self proteins [11] . molecular mimicry can be either in the form of sequence homology wherein the host and the infectious agent share the sequence of similar or identical amino acids or it might be due to the conformational similarity between the host and the infectious agent in question [11] . molecular mimicry is one of the major mechanisms for the induction of autoimmune diseases through the activation of autoreactive t cells in the host immune system. several elegant examples of molecular mimicry leading to autoimmune manifestations have been described following bacterial and viral infections [11] . chikungunya fever is a self limiting illness, however in 20-30% of the patients arthralgia persists for a period of two years and above [12] . more than half of all chikv infected patients in la reunion island during the 2005-2006 epidemics had complaints of persistent joint pain / recurring stiffness [13] . the arthritis attributed to chikv infection indeed mimics rheumatoid arthritis, as discussed by bouquillard et al [14] wherein 21 patients infected with chikv in reunion islands developed ra. further, malvy et al [15] suggested that molecular mimicry may be responsible for chronic manifestations as symptoms continue to persist despite chikv not being detectable in the synovial tissue. we investigated molecular mimicry in this study by using a combined approach of identifying homologous regions between chikv glycoprotein e1 protein and host tissue components using bioinformatics tools, the ability of these designed peptides to cross react with serum samples from chikv infected patients and inducing immune mediated joint and muscle pathology in a mouse model. in order to determine if there are any "arthritogenic" motifs within the e 1 protein, a multiple sequence alignment of amino acid sequences of e1 glycoprotein of chikv was carried out with other alphaviruses such as onnv, rrv, sfv and eeev using clustalw (fig 2) . the alignment revealed that two common motif(s) skd and kca were present only in arthritogenic alphaviruses such as chikv, rrv and onnv but not in the 'encephalitogenic' alphaviruses such as sfv and veev (fig 2) . the presence of these two motifs seen only in arthritogenic alphaviruses lead us to postulate that these two motifs may have a role in the development of arthralgia which is a hallmark in the disease produced by these group of viruses. simultaneously, the immunodominant epitopes in the chikv e1 glycoprotein were deduced using iedb and emboss programs which use criteria like presence of beta turn, surface accessibility, flexibility, antigenicity and hydrophilicity of the regions to be studied. on combining the common results obtained with these two programs, four immunodominant regions were obtained in chikv e1 glycoprotein (peptide a, b, c and d). interestingly, it was observed that the "arthritogenic" motifs skd and kca were also present in the immunodominant peptides a and b respectively. sequence homology comparisons between chikv e1 glycoprotein and various human proteins using blast revealed that a homology of four consecutive amino acids tqlv/telv exist between the chikv e1 glycoprotein and hla-b27 molecule (fig 3) . it is a well established that hla b27 has been implicated in the pathogenesis of autoimmune arthritis and ankylosing spondylitis [16] . interestingly this consecutive sequence of amino acids was also present in one of the immunodominant peptides (peptide a) of chikv e1 glycoprotein ( fig 3) . having ascertained that there is amino acid homology between chikv e1 glycoprotein and hla b27 molecule, the occurrence of structural homology was also explored using the bioxgem program. this program searches for the longest common substructures existing between the query structure and every structure in the database. the output of the query while executing the program was however limited to the first 100 hits. amongst these 100 proteins, further analysis was restricted only to human proteins present in the output. amongst the 20 human proteins in the list (table 2) , six proteins were shortlisted as they are known to contribute to either inflammation or arthritic pathology. the most prominent amongst them was complement components c3and c5. indeed, c3 complement component has been implicated in inflammation and tissue injury in other related alphaviral infections [17] and therefore further analysis was confined to the homology between the chikv e1 glycoprotein and the c3 complement component. the analysis showed that the homology occurred between von willebrand factor (vwf) domain of c3 and chikv e1 glycoprotein (panel a, fig 5) . interestingly, the amino acid sequences in the region of chikv e1 glycoprotein exhibiting homology were also present in the immunodominant peptides a and b (panel b, fig 5) . in summary, combining the data from the various bioinformatics approaches and employing a logical algorithm relevant to the pathogenesis of arthritis the choice narrowed down to two immunodominant peptides of chikv e protein (peptides a & b) .therefore all further experiments were carried out using only these two peptides.the peptides a and b were used in an elisa to assess the immunoreactivity of serum samples obtained from chikv confirmed patients as well as healthy control subjects. the serum samples obtained from chikv confirmed patients (n = 36) showed reactivity to both the peptides to varying degrees. antibodies to peptide a was noted in 24/36 (66.66%) of samples while 27/36 (75%) of serum samples showed reactivity to peptide b (fig 5) . these results indicate that the two peptides are indeed being recognized by the host immune system during chikv infection. as evident form fig 5, it was interesting to note that the sera from three patients who had persistent arthralgia 12 weeks after the onset of initial symptoms indeed exhibited much higher od values (0.9 to 1.3 for peptide a and 1.2 for peptide b) to the two peptides a & b as compared to other patients (od values were close to cut off and between 0.4 and 0.6). although, a quantitative elisa would have delineated these differences better our elisa was designed only as qualitative assay. further, experiments were conducted to investigate if peptide a and peptide b were capable of inducing pathology in an experimental mouse model. the results indicated that these two peptides on their own were able to induce significant inflammation in the muscles of c57bl/ 6j mice (fig 8 and table 3 ) similar to that observed in animals (3+) that were injected with chikv. further, animals that were primed initially with chikv followed by a subsequent injection of the two chikv peptides exhibited enhanced inflammatory pathology (4+) as compared to animals that were injected with peptides or virus alone (fig 9 and table 3 ). on the contrary, animals that received an unrelated peptide (i.e. not containing the arthritogenic motifs or exhibiting homology to host proteins) either before or after priming with chikv exhibited minimal muscle inflammation (1+). collectively these observations validate the hypothesis that molecular mimicry between chikv e1 protein and host proteins does contribute to pathology in chikv infection. such observations have not been reported hitherto in chikv infection although molecular mimicry as a mechanism leading to autoimmune phenomena has been demonstrated in several microbial infections including viruses and bacteria [18] . among the viruses, molecular mimicry has been noted in theiler murine encephalitis virus (tmev), hepatitis b virus (hbv), and sfv and coxsackie viral infections [11] . in sfv infection of c57bl/6j mice a similar approach using bioinformatic tools derived peptides and validation of these peptides invivo for their ability to induce autoimmune demyelination was undertaken [19] . an algorithmic approach was used to demonstrate amino acid homology between immunogenic epitopes of sfv and various myelin proteins. the criterion used for the occurrence of molecular mimicry was the presence of three similar consecutive amino acids. it was observed that myelin oligodendrocyte protein (mog) shared homology with sfv e2 glycoprotein. the injection of a peptide containing the sequence of shared amino acids into mice caused demyelination with presence of multifocal vacuolation in the cns white matter [19] . the phenomenon of molecular mimicry has also been studied in sars infection [20] . eleven peptides derived from sars spike(s) protein which shared homology with various human proteins were synthesised and their reactivity was assessed using serum samples obtained from sars patients. serum samples recognised only 2/11 peptides and the authors concluded that these two peptides may contribute to viral pathogenesis through the phenomena of molecular mimicry. the limitations of the present study are twofold. firstly, hla-b27 typing was not performed in the chikv confirmed patients and controls of this study. however, serum reactivity of peptide a which shared homology with hla b27 molecule suggests that this molecule may have an important role to play in persistent arthralgia as 3/36 patients exhibited high reactivity to it. the geographical and ethnic variation in the prevalence of hla-b27 globally is well documented [21] and further its occurrence in chikv related complications is also reported to be very low [15, 22] . therefore it may be argued that the observance of molecular mimicry between chiv e1 glycoprotein and hla-b27 may not be the major factor contributing to complications that ensue following acute infection. secondly, the time interval between the injection of chikv and the peptides into animal the model (groups 7& 8) is rather short rendering it difficult to conclude whether the inflammatory response noted was due to the immunopathologic process of molecular mimicry or a direct inflammatory effect of both the virus and the peptide. however, the muscle fibrosis and calcification in the muscles noted in the experimental animals recapitulates immune mediated polymyositis in humans during the evolution and progression. the other limitation with all studies conducted using animal models to demonstrate molecular mimicry as a mechanism of pathogenesis is that an adjuvant such as cfa/fica is invariably required to elicit immune mediated damage. this suggests that in addition to having cross reacting disease inducing epitopes, sufficient activation of antigen presenting cells is required. consequently, the most difficult part in the studies investigating molecular mimicry is to correlate/extrapolate the findings obtained in vitro and/or animal studies to the disease occurring in the natural host. despite this limitation, the concept of molecular mimicry remains a viable hypothesis for framing questions and approaches to understanding the pathogenetic mechanisms involved during the disease process. therefore, it would be definitely worthwhile to pursue future studies with the two chikv peptides described in this study using transgenic animal models and human t and b cell responses to the peptides. for their help with histopathology and mr. kumar from department of neurovirology for help with the animal experiments. we would like to thank late. dr. v. kumaraswami for being inspiration for the study. re-emergence of chikungunya virus in india chikungunya virus infection-a resurgent scourge imported chikungunya infection chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages utility of igm elisa, taqman real-time pcr, reverse transcription pcr, and rt-lamp assay for the diagnosis of chikungunya fever the role of complement in inflammatory diseases from behind the scenes into the spotlight citrullination of fibronectin in rheumatoid arthritis synovial tissue identification of specific autoantigens in sjögren's syndrome by serex the critical role of mast cells in allergy and inflammation increased expression of beta-arrestin 1 and 2 in murine models of rheumatoid arthritis: isoform specific regulation of inflammation molecular mimicry and immune-mediated diseases chikungunya virus infection. a retrospective study of 107 cases post-epidemic chikungunya disease on reunion island: course of rheumatic manifestations and associated factors over a 15-month period rheumatoid arthritis after chikungunya fever: a prospective follow-up study of 21 cases destructive arthritis in a patient with chikungunya virus infection with persistent specific igm antibodies hla-b27: natural function and pathogenic role in spondyloarthritis complement contributes to inflammatory tissue destruction in a mouse model of ross river virus-induced disease molecular mimicry as a mechanism of autoimmune disease molecular mimicry between a viral peptide and a myelin oligodendrocyte glycoprotein peptide induces autoimmune demyelinating disease in mice peptide mimicrying between sars coronavirus spike protein and human proteins reacts with sars patient serum what's new? specific management of post chikungunya rheumatic disorders: a retrospective study of 159 cases in reunion island from we thank ms. priyanka s and ms. pavithra s for help with bio-informatics analysis. we thank mr. prasanna and ms. rajashakti from human brain tissue repository (hbtr) from nimhans conceived and designed the experiments: vr rv ad ssk. analyzed the data: vr rv ssk ad. wrote the paper: rv vr ssk ad. key: cord-263044-o8aosx2q authors: lipsitch, marc; donnelly, christl a.; fraser, christophe; blake, isobel m.; cori, anne; dorigatti, ilaria; ferguson, neil m.; garske, tini; mills, harriet l.; riley, steven; van kerkhove, maria d.; hernán, miguel a. title: potential biases in estimating absolute and relative case-fatality risks during outbreaks date: 2015-07-16 journal: plos negl trop dis doi: 10.1371/journal.pntd.0003846 sha: doc_id: 263044 cord_uid: o8aosx2q estimating the case-fatality risk (cfr)—the probability that a person dies from an infection given that they are a case—is a high priority in epidemiologic investigation of newly emerging infectious diseases and sometimes in new outbreaks of known infectious diseases. the data available to estimate the overall cfr are often gathered for other purposes (e.g., surveillance) in challenging circumstances. we describe two forms of bias that may affect the estimation of the overall cfr—preferential ascertainment of severe cases and bias from reporting delays—and review solutions that have been proposed and implemented in past epidemics. also of interest is the estimation of the causal impact of specific interventions (e.g., hospitalization, or hospitalization at a particular hospital) on survival, which can be estimated as a relative cfr for two or more groups. when observational data are used for this purpose, three more sources of bias may arise: confounding, survivorship bias, and selection due to preferential inclusion in surveillance datasets of those who are hospitalized and/or die. we illustrate these biases and caution against causal interpretation of differential cfr among those receiving different interventions in observational datasets. again, we discuss ways to reduce these biases, particularly by estimating outcomes in smaller but more systematically defined cohorts ascertained before the onset of symptoms, such as those identified by forward contact tracing. finally, we discuss the circumstances in which these biases may affect non-causal interpretation of risk factors for death among cases. the case-fatality risk (cfr) is a key quantity in characterizing new infectious agents and new outbreaks of known agents. the cfr can be defined as the probability that a case dies from the infection. several variations of the definition of "case" are used for different infections, as discussed in box 1. under all these definitions, the cfr characterizes the severity of an infection and is useful for planning and determining the intensity of a response to an outbreak [1, 2] . moreover, the cfr may be compared between cases who do and do not receive particular treatments as a way of trying to estimate the causal impact of these treatments on survival. such causal inference might ideally be done in a randomized trial in which individuals are randomly assigned to treatments, but this is often not possible during an outbreak for logistical, ethical, and other reasons [3] . therefore, observational estimates of cfr under different treatment conditions may be the only available means to assess the impact of various treatments. however, observational studies conducted in the early phases of an outbreak, when public health authorities are appropriately concentrating on crisis response and not on rigorous study design, are challenging. a common problem is that disease severity of the cases recorded in a surveillance database will differ, perhaps substantially, from that of all cases in the population. this issue has arisen in the present epidemic of ebola virus disease in west africa and in many previous outbreaks and epidemics [4] [5] [6] [7] [8] [9] and will continue to arise in future ones. here we outline two biases that may occur when estimating the cfr in a population from a surveillance database, and three more biases that may occur when comparing the cfr between subgroups to estimate the causal effect of medical interventions. we also briefly consider the applicability of these biases to a different application: comparing the cfr across different groups of people, for example, by geography, sex, age, comorbidities, and other "unchangeable" risk factors. such factors are "unchangeable" in the sense that they are not candidates for intervention in the setting of the outbreak, though some could, of course, change over longer timescales. the goal of estimating the cfr in groups defined by such unchangeable factors is not to understand the causal role of these factors in mortality, but to develop a predictive model for mortality that might be used to improve prognostic accuracy or identify disparities. such box 1. definition of the cfr. the cfr itself is an ambiguous term, as its definition and value depend on what qualifies an individual to be a "case." several different precise definitions of cfr have been used in practice, as have several imprecise ones. the infection-fatality risk (sometimes written ifr) defines a case as a person who has shown evidence of infection, either by clinical detection of the pathogen or by seroconversion or other immune response. such individuals may or may not be symptomatic, though asymptomatic ones may go undetected. the symptomatic case-fatality risk (scfr) defines a case as someone who is infected and shows certain symptoms. infection in many outbreaks is given several gradations, including confirmed (definitive laboratory confirmation), probable (high degree of suspicion, by various clinical and epidemiologic criteria, without laboratory confirmation), and possible or suspected (lower degree of suspicion). this paper describes issues in estimating any of these risks or comparing them across groups, but does not go into the details of each possible definition. furthermore, unlike risks commonly used in epidemiologic research (e.g., the 5-year mortality risk), the length of the period during which deaths are counted for the cfr is rarely explicit, probably because it is considered to be short enough to avoid ambiguity in the definition of cfr. however, a precise definition of the cfr would need to include the risk period, e.g., the 1-month cfr of ebola. clearly, the definition of cfr for a particular investigation should be specified as precisely as possible. predictions may be affected by survivorship bias and selection bias, but not by confounding, as we discuss. two biases that may affect the estimation of an overall cfr are presented in table 1 : for diseases that have a spectrum of clinical presentation, those cases that come to the attention of public health authorities and are entered into surveillance databases will typically be people with the most severe symptoms, who seek medical care, are admitted to hospital, or die. therefore, the cfr will typically be higher among detected cases than among the entire population of cases, given that the latter may include individuals with mild, subclinical, and (under some definitions of "case") asymptomatic presentations. laboratory confirmation as an inclusion criterion may reduce this bias if it is able to detect a wider spectrum of presentations, or may exacerbate it if the probability of receiving a laboratory test is higher for more severe cases and/or if test sensitivity is higher for more severe cases. the magnitude of this bias may be uncertain for a long period because the spectrum of clinical presentations is itself uncertain at the start of an outbreak of a new disease [12, 26] . all proposed approaches to estimate and correct for this bias (table 1 ) require auxiliary data sources to estimate how the reported subset of cases compares with the overall population of cases. the availability of such auxiliary data sources will depend on the context of the outbreak. during an ongoing epidemic, there is a delay between the time someone dies and the time their death is reported. therefore, at any moment in time, the list of cases includes people who will die and whose death has not yet occurred, or has occurred but not yet been reported. thus dividing the cumulative number of reported deaths by the cumulative number of reported cases at any moment will underestimate the true cfr. the key determinants of the magnitude of the bias are the epidemic growth rate and the distribution of delays from case-reporting to death-reporting; the longer the delays and the faster the growth rate, the greater the bias. heuristically, the underestimate will be proportionate to the expansion of the epidemic during the delay between the time a case enters the database to the time the death of that case enters the database (if it occurs). fig 1 illustrates an example where the delay is 3 weeks, the epidemic doubling time is 2 weeks, and the underestimate is by a factor of 2 3/2 2.8. this bias may be corrected for in various ways, and to varying degrees, using information on the growth rate of the epidemic, the distribution of times from case-report to death-report, and the distribution of times from case-report to recovery-report (i.e., report that the case is no longer at risk of dying of the infection). a simple approach is to limit analysis to those cases with sufficiently long follow-up for a death to have been recorded had a death occurred, but this approach may result in an exceedingly small sample size if applied early in the epidemic. several such strategies are described in table 1 . here, and in table 2 , we discuss the sources of three biases that threaten the validity of a causal interpretation of a difference in cfr between groups who have received different interventions. such a difference might be measured as a risk ratio (rr), the ratio of cfr in group a to that in group b, or as an odds ratio (or), the ratio of the odds of dying in group a and group b, or as table 1 . potential biases that can affect the estimation of cfr (and thereby also the comparison of cfr across groups). direction outbreaks in which analysts have noted this bias may be operating preferential ascertainment of severe cases: in an infection with a range of manifestations from relatively mild to highly severe, milder cases are less likely to appear in surveillance databases than more severe ones; therefore, the cfr among ascertained cases will be higher than that among all cases. spuriously increases estimate of cfr influenza h1n1pdm [10] [11] [12] , influenza h7n9 [6] , influenza h5n1 [7] (though this hypothesis has been refuted [8] ), middle east respiratory syndrome [4] , ebola (this article) [13] note: these solutions are listed in approximately the temporal order in which they may be practical, from early in the outbreak to later on; details will depend on the epidemiology of the outbreak. use sentinel surveillance sites to estimate multipliers between various levels of severity and extrapolate to a larger population [6] . survey-or health-facility-based surveillance for symptomatic infection [14] in a defined population, combined with enhanced surveillance for severe outcomes (particularly death) in the same population. use travelers from high-burden areas with low ascertainment to low-burden areas with higher ascertainment to estimate incidence of infection in source population [15, 16] , thereby providing a more accurate denominator for comparison to deaths in source population. surveillance pyramid approaches: reconstruct conditional probabilities of appearing at one severity level conditional on reaching a lower severity level; combine data sources that have relatively complete ascertainment of higher severity levels (e.g., hospitalization, icu, death) with those having relatively complete ascertainment of lower levels (e.g., seeking medical attention, hospitalization) [10, 11] . cfr can then be estimated as a product of conditional probabilities with associated uncertainties [17] . serologic ascertainment of infection [18, 19] to provide a population denominator for infections regardless of symptoms, combined with active surveillance for more severe outcomes. individuals ascertained by a different mechanism, e.g., named healthy contacts of cases who subsequently test positive, could be a more representative group in whom to assess severity [20] . bias due to delayed reporting. during an ongoing epidemic, at any week w the persons who have died up to time w will not be the only ones to die of the infection among those who became cases by w. the denominator of the cfr (cases) includes persons who have not yet died of the infection, but will do so in the future. thus the cfr by w will be less than the true cfr. this bias will be particularly severe for infections that are increasing rapidly in incidence and for which the infection-death time interval is long. spuriously decreases estimate of cfr sars [9] , influenza h1n1pdm [21] , ebola [22, 23] limit analysis to those cases with sufficiently long follow-up for a death to have been recorded had a death occurred. while this may lead to extremely small sample sizes near the beginning of an epidemic, this strategy is more feasible after a local epidemic wave, including reporting delays, has passed or nearly passed [10, 11] . limit analysis to those cases known either to have died or recovered, but exclude those with unknown outcome (biased if severity affects outcome ascertainment) [22] [23] [24] . apply a competing-risk kaplan-meier-like method or a parametric mixture model to the full dataset (biased if the times to death and time to recovery have different distributions) [24, 25] . fit the distribution of times to death and to recovery to estimate the true cfr [10, 11] , or inverse-probability weight deaths using the conditional probability of having survived by w, given that one dies [21] (biased if the probability distribution is incorrect). a risk difference (rd), the difference between the cfr in group a and group b. we use the term relative cfr to refer to any of these measures, and call a relative cfr non-null when it differs from 1 (ratio) or 0 (difference). when these biases are present, a relative cfr, different from the null value in group b compared with a does not imply a causal effect of group. for example, if group a is non-hospitalized patients and group b is hospitalized patients, an odds ratio of death less than 1 may not imply a beneficial effect of hospitalization on the odds of death. similarly, a relative cfr greater than 1 may not imply that hospitalization is harmful. we use the estimation of the causal impact of hospitalization on mortality as our example throughout this section. note that exactly the same reasoning applies to assessment of another intervention or to a comparison of two interventions, for example, a comparison of treatment at center a versus treatment at center b. the first bias arises in a naïve comparison of mortality between those who have and those who have not been hospitalized. if some individuals die before they can be admitted to a hospital, they will by definition not become hospitalized. therefore, even in the absence of any effect of hospitalization on the risk of death, there will be fewer deaths among those hospitalized than among those not hospitalized. we will refer to this bias as "survivorship bias." in an ongoing epidemic, there will typically be a delay between the reporting of a case and the reporting of the death of that case, if the infected person dies. thus, at any moment, there will be some cases reported who will die of the infection but who have not yet died, or whose deaths have not yet been reported. simple division of the number of deaths reported by week w (green), by the number of cases reported by week w (blue) will underestimate the cfr because the numerator does not include all those cases in the denominator who will eventually die. with a reporting delay of 3 weeks for deaths compared to cases, the reported deaths curve will be shifted 3 weeks to the right, relative to the curve of the total number of cases reported by week w who will die (red). if the epidemic doubling time is 2 weeks, as shown here, the underestimate of cfr will be by a factor of about 2 3/2 2.8, with the exponent being the number of epidemic doubling times that pass between case reporting and death reporting. in reality, there will be a distribution of reporting delays rather than a fixed delay, making this a heuristic rather than exact approach. the problem is ameliorated in an epidemic that grows more slowly or less than exponentially. for more details, see references in table 1 . this bias can be eliminated using data on the time d since the person became a case. the analysis would then compare the risk of death between those individuals who became table 2 . potential biases that can affect the comparison of cfr across groups (relative cfr), using the example of comparing the cfr among hospitalized and non-hospitalized persons to assess the relative cfr for hospitalization. direction outbreaks in which analysts have noted this bias may be operating possible solutions/means of detecting the bias survivorship bias: those who die before being hospitalized cannot, by definition, be hospitalized; a crude comparison of deaths among hospitalized and non-hospitalized cases will therefore reflect the "protective" effect of death against hospitalization. this is an example of reverse causality because for these individuals, death prevented hospitalization, rather than hospitalization preventing death. spurious protective effect of hospitalization on risk of death ebola (this article) conditioning analysis on survival up to day d of symptoms, and analyzing hospitalization on day d as the intervention, will avoid this bias, as individuals who die before hospitalization will not be included in the analysis. this analysis can be repeated for different values of d and potentially combined in a parametric model. individuals identified before becoming cases (e.g., as healthy contacts of infected persons) and actively followed regardless of clinical severity could be analyzed separately as a prospective cohort for whom the course of disease could be observed and this restriction readily made. confounding: if individuals are hospitalized in response to predictors of poor prognosis, hospitalization will be noncausally associated with poor outcome. this problem is common in the pharmacoepidemiology literature [27] . alternatively, in situations of triage, when beds or other resources are limited, individuals with better prognosis may receive hospitalization (or another intervention), creating a spurious beneficial effect of hospitalization. may be in either direction, depending on whether those receiving the intervention have better or worse prognosis. ebola (this article), h1n1pdm (effect of antiviral treatment on death) [28] , influenza h5n1 (effect of antiviral treatment on death) [29, 30, 31] in principle, analysis can adjust for prognostic factors that also predict hospitalization via matching, stratification, or multivariable analysis. in practice, such information may be unavailable [27] . such adjustments will be more readily made if data are obtained prospectively from a cohort of cases identified before becoming cases. selection bias occurring because mortality and hospitalization both affect the probability a case will appear in the database [32] : when inclusion in a database can occur as a result of either of two (or more) factors, the association between these two factors within the database will be biased relative to that in the source population. for example, if death and hospital admission are both means by which cases are ascertained and enter a database, as may be the case for ebola datasets, hospitalization will be spuriously associated with death in the dataset even if hospitalization has no causal effect in preventing death. direction of bias depends on the probabilities of inclusion in the dataset depending on exposure and outcome. ebola (this article) without knowledge of how cases came to enter a dataset, the magnitude of this bias cannot be evaluated. under assumptions about the proportion of cases entering the dataset for various reasons, a sensitivity analysis could be performed to assess the plausibility of assigning any observed protective effect to this bias [33] . this bias too may be avoided by prospectively following a cohort of individuals who are identified before becoming cases. the second source of bias is confounding. severity of disease will likely affect the probability of hospitalization and the probability of death. as a common cause of the exposure of interest (hospitalization) and the outcome (death), disease severity is a confounder of the causal effect of hospitalization on death. if hospitalization is offered to especially severe cases or-in the setting of extreme triage-to especially mild cases, then hospitalization would spuriously appear harmful (if hospitalization went to especially severe cases) or beneficial (if it went to especially mild cases). there may be other confounders of this effect besides disease severity. individuals living in rural areas may be at greater risk of mortality (e.g., due to malnutrition) and also less likely to be hospitalized (due to longer travel time to hospital). place of residence (or travel time to hospital) in this setting would be a confounder of the effect of hospitalization on death. the standard approach to reducing confounding is to stratify, restrict, or adjust for prognostic factors that affect the propensity to receive the treatment (in this case to be hospitalized) [27] . however, such information may frequently be limited or unavailable in databases compiled during outbreaks, especially in resource-limited settings. the third source of bias is selection occurring because mortality and hospitalization both affect the probability a case will appear in the database. during an outbreak, many cases may not appear in the database because they are not ascertained or because information about them is not obtainable. in particular, cases who are not hospitalized, and cases who do not die, may be less likely than other cases to appear in the database because they are less likely to come to medical or public health attention. if appearance in a database is the common effect of hospitalization and death, then the association between hospitalization and death among cases in the database may be non-null even if hospitalization and death were independent in the population of all cases. the direction and magnitude of the association between hospitalization and death among cases in the database will then be the result of combining the association due to this selection bias, the association due to a potential effect of hospitalization on mortality, and the association due to confounding. hypothetical examples are shown in tables 3-5 . in these tables, the association in the population between hospitalization on day 8 (an arbitrarily chosen day) and death is negative; individuals hospitalized on day 8 (an arbitrarily chosen day) of symptoms have a lower probability of death than those who are not hospitalized on day 8 of symptoms. if we assume that this analysis has avoided survivorship bias by limiting analysis to cases still alive on day 8, then the population-level association would reflect a combination of the causal effect of hospitalization on day 8 on risk of death, and confounding by severity or other factors. this population-level association is the same in tables 3, 4, and 5, but different probabilities are assumed for inclusion in the database, depending on whether an individual is hospitalized on day 8, dies, or both. relative cfrs on the rr, or, and rd scales for hospitalization on day 8 are calculated for each hypothetical example. the hypothetical data in these tables show that selection bias in such a circumstance may be either positive or negative on each of the three scales, depending on the specific probabilities of selection in each of the four states. table 3 shows an example of negative bias on the rr, or, and rd scales (overestimating the protective effect of hospitalization on day 8 expressed as a lower value of each relative risk measure). table 4 shows an example of a positive bias on the rr and rd scales and a negative bias on the or scale. table 5 shows an example of positive bias (underestimating the protective effect of hospitalization on day 8 expressed as a higher value of each measure) on all three scales. from experience, it seems that when databases are assembled in this way, it is rarely possible to tell why an individual case has come into the database. in the absence of such information, it is difficult to imagine how adjustments could be performed. however, sensitivity analyses could be performed to assess how strong such biases are likely to be [33] . we have stated already that survivorship bias can be avoided by limiting analyses of the intervention to those who remain alive on a certain day after becoming a case. one strategy that would help to resolve the other two sources of bias is to limit analysis to a cohort of cases who were identified before they became cases; for example those who were identified as healthy contacts of known cases, and were followed prospectively. confounding occurs because individual factors like severity of infection or place of residence (which could affect both the probability of exposure-receiving the intervention-and the probability of the outcome-mortality) are not accounted for in the analysis through stratification, restriction, or adjustment. selection bias in this setting occurs because the exposure and the outcome both affect the probability of inclusion in the database. follow-up of a cohort of contacts ascertained before becoming cases could eliminate hospitalization and mortality as predictors of inclusion in the database, thus eliminating the form of selection bias we have discussed. it would provide an opportunity for subscript p represents the population values, while subscript d represents the values measured for those cases included in the data base; selection bias produces the discrepancy. the extent of selection bias may be measured as or s ¼ s 00 s 11 s 01 s 10 , where s ij is the probability a case with exposure (hospitalization at day 8) i and outcome (mortality) j appears in the database. in this example, selection bias spuriously enhances the negative association between hospitalization on day 8 and death, on all scales: rr, or, and rd. gathering data on severity and other predictors of exposure and outcome, which would facilitate control of confounding, though not guarantee to eliminate it. such a cohort would also provide a natural setting for analyses that avoid survivorship bias. the cost of such improvements in inference would be the need to ascertain such contacts and maintain surveillance of those individuals, following them to obtain data on relevant covariates. such a strategy-which has been followed in cases of exposed health-care workers in settings with high resources and few cases-would likely have benefits for the individuals followed (e.g., increasing the probability they receive care if infected) and for reducing transmission (if such individuals were promptly isolated upon evidence of infection). however, it has not been possible so far in the large ebola outbreaks in west africa to do this routinely. it is often of interest to predict the probability of mortality for an individual case of an infectious disease based on that individual's demographic and clinical data, without placing any causal interpretation on the factors used to predict outcome. for example, in 2009, there was much interest in whether morbid obesity (or obesity in general) was predictive of worse outcome in infection with the novel pandemic strain of influenza a/h1n1 [34] .the primary goal was to improve estimates of clinical prognosis, although observations about prognosis could later be used to generate causal hypotheses for further testing. similarly, observations of disparate rates of severe outcomes by geography within new york city did not initially involve causal judgments about why certain areas had worse outcomes, although they could be used to guide enhancement of services in areas with worse outcomes [35] . even for a well-understood disease like polio, it may be necessary to identify unusual demographic patterns of mortality in order to understand and respond effectively to an outbreak [36] . prognostic exercises such as these cannot suffer from confounding bias because no causal interpretation is attached to the conclusions. they can, however, suffer from selection bias. returning to the ebola context, one might wish to know whether pregnant women infected with ebola are at greater risk of death from ebola infection than other cases [37] , for example, in order to give them greater supportive care. if the probability of entering the database depends on whether an ebola patient is pregnant and on whether she ultimately dies of the infection, then the probability of death given pregnancy will likely differ in the database from the value in the population of direct interest for a clinical or public health decision maker. if the goal of analysis is to inform public health decision makers on the value of efforts to prevent infection in pregnant women, then the population-wide cfr among pregnant women is the value of direct interest. if, on the other hand, the goal of analysis is to inform health care providers at a treatment center to make a better clinical decision based on an accurate prognosis of the patient presenting to them, the quantity of direct interest is the probability of death among pregnant women in the population they encounter-those admitted to the treatment center. table 5 . effect of selection bias on estimates of relative cfr on the risk ratio (rr) odds ratio (or) and risk difference (rd) scales. joint frequencies of hospitalization and death in the whole population among those alive at day 8 of symptoms this value, again, will differ from that in the database, which may (in our running example) have been enriched for individuals entered in the database because they died of the infection. it will also differ from that in the overall population. the general point is that selection bias can be operative if the population on which analysis is performed is not a representative sample of the population for which the value of the cfr is sought, and selection bias of this form can lead to spurious conclusions in prognostic estimates as well as in causal ones. as in the case of causal inference, prognostic estimates will avoid selection bias to the extent they can be performed on a randomly chosen cohort of cases, identified via tracing of healthy contacts, for example. to determine the appropriate scope and magnitude of public health response to an infectious disease outbreak, it is important to estimate the cfr and the determinants of its variation [1, 5] . for example, in the 2009 influenza pandemic, early point estimates of the cfr ranged over orders of magnitude, from a value below that of seasonal influenza, which would have justified a modest response, to values around 1%, approximately half that of the 1918 pandemic, which would have indicated the need for massive interventions to protect public health [12, [15] [16] [17] 21] . to a large degree, this variation reflected judgments that one or the other of the biases in table 1 was more important, judgments that were difficult to make accurately and confidently on the rapid timescale required for decision making [26] . in other situations, accurate assessment of the cfr is not as crucial for decisions about the scale of response required; for example, in the ongoing 2014 ebola epidemic in west africa, the uncertainty about the cfr is limited to a range between high values and very high values, and it is not clear that any greater response would be indicated by a 90% cfr than a 60% cfr [22] . either way, a rapid and massive response is warranted. even when the overall cfr is not a key input to decision making, there is obvious value to inferences about which conditions lead to a lower cfr, whether these be specific treatment, particular types of supportive care, or hospitalization in general. moreover, treatment facilities might be evaluated by the proportion of their patients who survive; here the relative cfr calculated would be for treatment in one facility versus treatment in another. there will be a temptation to conclude that treatment facilities with higher cfr are doing a worse job-that is, to apply a causal interpretation to observed differences in the cfr. even in settings with more resources to measure covariates, methods of risk-adjustment of comparative outcomes to account for the mix of patients seen are complex and controversial [38] . in an emergency setting, with few covariates available to characterize the "case mix" of a health care provider, causal interpretation of differences in cfr would be particularly prone to error, potentially producing conclusions that mislead and thereby damage control efforts. for instance, if through confounding, larger referral treatment centers primarily receive patients who have survived infection for some time and are therefore less likely to die, independently of treatment, this may be erroneously interpreted as more effective treatment in these centers. similarly, if certain treatment centers preferentially admit the most symptomatic patients, they may falsely appear to be less effective or even harmful to patient outcome. with at least five separate sources of bias in cfr or relative cfr estimates, and only imperfect solutions typically available for most due to lack of data, separating causal from non-causal factors in relative cfr estimation seems extremely risky. this is not to deny that data should be gathered or analyzed; on the contrary, the biases here suggest that more thorough data gathering is necessary before analyses of such quantities as relative cfr are relied upon for any decision. there has been much debate, particularly in the area of ebola treatments, about whether randomized studies comparing a treatment to a placebo are ethical [3, 39] . whatever one's view on this debate, it seems likely that some observational (non-randomized) studies of the effectiveness of particular therapies, or the comparative effectiveness of two or more therapeutic approaches will occur, whether for ethical reasons, logistical reasons, or both. such studies-in which a key endpoint will be mortality-will be vulnerable to the sorts of biases described in this article, particularly in cases in which the true effect size of the treatment is limited. the biases described here should be kept in mind when evaluating the conclusions of such studies, and wherever possible, studies should be designed to minimize them. small studies conducted using systematic approaches to enrollment and follow-up of patients may be more precise and less biased than studies with larger sample sizes that use databases collected for other reasons. similarly, there may be situations in which efforts are made to administer scarce therapeutic agents to those most likely to benefit from them. such efforts rely on estimates, formal or informal, of the prognosis of patients with and without the treatment, depending on variables such as the time since they became symptomatic. these estimates, too, may be affected by the biases discussed. in the current ebola outbreak in west africa, such data gathering has not routinely occurred, for a number of reasons, including lack of health system infrastructure [40] and prioritization of crisis response and other directly lifesaving activities. in future outbreaks of other diseases, as in the past with pandemic influenza, setting up systematic approaches to gather data useful for such assessments should be a priority [1, 5] . meanwhile, emphasis on recording for each patient in a database the time, place, and circumstances (e.g., hospital, clinic, funeral, contact tracing) under which the information is being gathered can substantially improve our ability to account for biases induced by a database with unplanned entry criteria. to reduce the impact of the biases identified on causal and (where applicable) prognostic inference, it appears desirable when possible to limit analysis to a subset of cases who have been followed prospectively since they became cases. these individuals might most likely be identified by forward contact tracing, in which cases are asked to name healthy individuals with whom they have had contact, and those individuals are followed to identify further infections. it has previously been noted that cases identified by contact tracing are more representative of cases in the general infected population than those identified because of symptoms, medical need, or death [20, 41] . use of such a sample does not guarantee to eliminate biases, as there may be residual confounding not adequately controlled in the analysis or subtler forms of selection bias (e.g., differential loss to follow up within the sample) [32] , but should significantly reduce them. we have emphasized the relevance of several biases to interpretation of datasets gathered in an emergency, such as the early phases of an emerging infection. while the downward bias in estimation of the cfr due to delayed reporting of deaths is most acute in rapidly growing epidemics, the other biases described may apply regardless of the overall trajectory of an epidemic, and thus may apply to endemic diseases as well as emerging ones. nonetheless, due to the sense of urgency to gather data and scale-up a response simultaneously, datasets assembled during infectious disease outbreak or emergency settings are especially prone to include unplanned mixes of cases who enter the dataset for various reasons. biases of the sorts described here should be systematically considered whenever one attempts to extract causal inferences from such observational data, and alternative, more systematic data collection should be considered when possible. • datasets available at the onset of new epidemics of infectious diseases are often collected for reasons other than epidemiologic analysis of absolute and comparative casefatality risks (cfr), and estimates of such quantities based on these data may be subject to biases, the relative magnitudes of which are difficult to ascertain and vary by situation. • major sources of bias affecting the estimation of absolute cfr are differences in severity between all cases and the subset of cases who enter the dataset, typically leading to inflated estimates of cfr, and more rapid reporting (less delay) in reporting cases than in reporting the deaths of those cases, typically leading to underestimates of cfr. • biases affecting the causal interpretation of relative cfr (causal attribution of different cfr in different groups to a particular intervention in one group, e.g., hospitalization) may arise from survivorship bias, in which individuals who survive longer may be more likely to receive the intervention; from confounding, in which a common factor (e.g., disease severity) affects the probability of both the intervention and mortality; and from selection bias, in which individuals are more or less likely to enter the dataset as a function of whether they receive the intervention and whether they have the outcome. • these biases may be severe enough to lead to qualitatively mistaken inferences about the severity of the infection or about the impact of interventions (such as hospitalization) on mortality, and may be particularly misleading when comparing, for example, the effect of hospitalization at different centers, given that cases hospitalized at different centers may enter the dataset for different reasons. • methods exist to identify and reduce these biases. in particular, the use of small but carefully defined cohorts of individuals who are followed from the time of infection or symptom onset (perhaps those identified via contact tracing) may ameliorate many of these biases. epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong basic methods for sensitivity analysis of biases assessing the severity of the novel influenza a/h1n1 pandemic a structural approach to selection bias h1n1 surveillance group. improving the evidence base for decision making during a pandemic: the example of 2009 influenza a/h1n1 studies needed to address public health challenges of the 2009 h1n1 influenza pandemic: insights from modeling interim pre-pandemic planning guidance: community strategy for pandemic influenza mitigation in the united states-early targeted layered use of nonpharmaceutical interventions. department of health and human services randomised controlled trials for ebola: practical and ethical issues middle east respiratory 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to severe acute respiratory syndrome (sars) managing and reducing uncertainty in an emerging influenza pandemic assessment and control for confounding by indication in observational studies 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 effectiveness of antiviral treatment in human influenza a(h5n1) infections: analysis of a global patient registry strengthening observational evidence for antiviral effectiveness in influenza a (h5n1) determinants of antiviral effectiveness in influenza virus a subtype h5n1 a structural approach to selection bias basic methods for sensitivity analysis of biases risk factors for severe outcomes following 2009 influenza a (h1n1) infection: a global pooled analysis pandemic (h1n1) 2009 surveillance for severe illness and response investigation of elevated case-fatality rate in poliomyelitis outbreak in pointe noire ebola hemorrhagic fever and pregnancy use of risk adjustment in setting budgets and measuring performance in primary care ii: advantages, disadvantages, and practicalities evaluating novel therapies during the ebola epidemic ebola: the teaching and learning moment household transmission of 2009 pandemic influenza a (h1n1) virus in the united states we thank lina nerlander for helpful suggestions on an earlier draft. key: cord-292157-hrm69640 authors: stull-lane, annica r.; lokken-toyli, kristen l.; diaz-ochoa, vladimir e.; walker, gregory t.; cevallos, stephanie a.; winter, andromeda l. n.; muñoz, ariel del hoyo; yang, guiyan g.; velazquez, eric m.; wu, chun-yi; tsolis, renée m. title: vitamin a supplementation boosts control of antibiotic-resistant salmonella infection in malnourished mice date: 2020-10-02 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008737 sha: doc_id: 292157 cord_uid: hrm69640 disseminated disease from non-typhoidal salmonella enterica strains results in >20% mortality globally. barriers to effective treatment include emerging multidrug resistance, antibiotic treatment failure, and risk factors such as malnutrition and related micronutrient deficiencies. individuals in sub-saharan africa are disproportionately affected by non-typhoidal s. enterica bloodstream infections. to inform a clinical trial in people, we investigated vitamin a as a treatment in the context of antibiotic treatment failure in a mouse model of vitamin a deficiency. vitamin a-deficient (vad) mice exhibited higher systemic bacterial levels with a multidrug-resistant clinical isolate in comparison to mice on a control diet. sex-specific differences in vitamin a deficiency and disseminated infection with s. enterica serotype typhimurium (s. typhimurium) were observed. vad male mice had decreased weight gain compared to control male mice. further, infected vad male mice had significant weight loss and decreased survival during the course of infection. these differences were not apparent in female mice. in a model of disseminated s. typhimurium infection and antibiotic treatment failure, we assessed the potential of two consecutive doses of vitamin a in alleviating infection in male and female mice on a vad or control diet. we found that subtherapeutic antibiotic treatment synergized with vitamin a treatment in infected vad male mice, significantly decreasing systemic bacterial levels, mitigating weight loss and improving survival. these results suggest that assessing vitamin a as a therapy during bacteremia in malnourished patients may lead to improved health outcomes in a subset of patients, especially in the context of antibiotic treatment failure. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in susceptible populations globally, there is a vicious cycle of infection and malnutrition [1] . malnutrition overlaps with deficiencies in micronutrients, such as iron, iodine, zinc and fatsoluble vitamins like vitamin a [2] . severe or recurrent infections can lead to vitamin a deficiency due to impaired nutrient absorption and decreased food intake, as well as direct nutrient loss through urine [3, 4] . in turn, vitamin a deficiency compromises the gastrointestinal mucosal epithelial barrier, increasing susceptibility to enteroinvasive bacteria and sepsis [4] . one of the most common enteric pathogens is salmonella enterica [5] . non-typhoidal serotypes of s. enterica cause significant morbidity and mortality worldwide, resulting in 93.8 million cases of gastroenteritis and 155,000 associated deaths annually [6] . invasive non-typhoidal salmonella (ints) infection, a severe febrile illness with bacteremia, disproportionately affects infants, older adults and immunosuppressed individuals. an estimated 3.4 million cases of invasive disease occur globally per year, resulting in 680,000 deaths [7] . malnutrition and concurrent malaria are key risk factors for systemic disease, and populations in sub-saharan africa are disproportionately affected [8] . barriers to effective treatment of ints illness include appropriate diagnostic tools, antibiotic treatment failure and antimicrobial resistance [9] . notably, the mortality rate due to ints in children reaches 20-25% [10] , indicating that more effective treatment is vital. vitamin a administration has been studied as treatment of infectious disease, such as for measles in children [11] . in addition, the who recommends vitamin a supplementation in the treatment of children that present with severe acute malnutrition [12] . given the importance of vitamin a in immune function [4, 13, 14] , these treatment recommendations suggest a role for vitamin a as a host-directed therapy [15] . however, vitamin a has not been studied as a treatment for nonspecific febrile illnesses like ints disease, especially in the context of antibiotic treatment failure. this study aimed to explore the therapeutic potential of vitamin a administered after onset of infection, simulating care of a sick patient presenting to clinic. our hypothesis was that vitamin a therapy could boost the effect of subtherapeutic antibiotic treatment and improve treatment outcomes for a drug-resistant ints infection. to address this question, we used mice to model antibiotic treatment failure of s. enterica serotype typhimurium in the setting of vitamin a deficiency. mouse experiments were carried out in compliance with the national institutes of health policies on animal welfare, the animal welfare act, and all other applicable federal, state and local laws. mice were cared for by research staff and university of california (uc) davis teaching and research animal care services (tracs) under a program accredited by the association for assessment and accreditation of laboratory animal care international (aaalac). tracs maintains a health monitoring program administered through the uc davis comparative pathology laboratory. when needed, campus veterinary services is available to provide interventional and supportive care. all mouse experiments were approved by the uc davis institutional animal care and use committee (iacuc) under protocol 19900. inbred c57bl/6j-slc11a1 +/+ mice are a c57bl/6j congenic strain containing a genomic segment with slc11a1 from s29s1 mice that was introgressed into chromosome 1 [16] . this mouse line was maintained at uc davis under specific pathogen-free conditions in cages with sterile alpha-dri bedding manufactured by shepherd specialty papers (watertown, tn). to generate vad and control mice, c57bl/6j-slc11a1 +/+ dams were given a vad diet at 2 weeks of gestation. pups were placed on either vad or control diets at weaning and were maintained as groups throughout each experiment. mouse weights were monitored weekly during growth. custom vad and control diets were obtained from envigo teklad diets (madison, wi). when the model was established, liver retinol concentration was confirmed by hplc. liver retinol concentration of control mice ranged from 120-300 nmol/g, whereas liver retinol concentration of vad mice ranged from 0-7 nmol/g. inbred c57bl/6j mice were purchased from the jackson laboratory (bar harbor, me) and were maintained on standard chow. d23580, a multidrug-resistant salmonella enterica serotype typhimurium clinical bloodstream isolate, was received from robert heyderman at the malawi-liverpool-wellcome trust clinical research centre [17] . inocula were cultured aerobically in luria-bertani (lb) media with 30 μg/ml chloramphenicol shaking for 16-18 hours at 37˚c, subcultured to a calculated optical density (od) of 0.001, and grown 16-18 hours rocking at 37˚c in a 96-well plate containing lb with serial dilutions of enrofloxacin ranging from 0 μg/ml to 2.56 μg/ml. enrofloxacin (baytril 100 by bayer) was obtained from the pharmaceutical service at the uc davis veterinary medical teaching hospital. the od was read at 595 nm on a biorad model 680 microplate reader (hercules, ca). the experiment was repeated six times, and the average minimum inhibitory concentration (mic) was recorded. a determination of susceptible (s), intermediate (i) or resistant (r) was made according to clsi guidelines [18] . inocula of s. typhimurium strains d23580, a multilocus sequence type (st) 313 strain isolated from blood, sl1344, an st19 isolate from a calf with salmonellosis [19] and sara16, an st19 human isolate and reference strain [20, 21] , were cultured in lb with shaking for 16-18 hours at 37˚c. the identity of each strain was confirmed by antibiotic resistance profiling. mice received 100 μl of sterile pbs or 100 μl of 1000 colony-forming units (cfu) diluted in sterile pbs by intraperitoneal (i.p.) injection. mouse weights were monitored daily during infection. mice were euthanized if they showed signs of lethal morbidity. systemic bacterial levels were characterized at day 1, 4 or 5 post-infection by determining tissue loads of s. typhimurium (cfu). liver and spleen were collected in pbs, weighed and homogenized using an ultra turrax t25 basic mixer from ika (staufen, germany). blood was collected in k2 edta microtainer tubes (becton, dickinson and company, franklin lakes, nj), kept on ice for one hour, and then incubated for 10 minutes with 100-200 μl of 1% triton x-100. liver, spleen and blood samples were serially diluted and plated on lb agar plates containing appropriate antibiotic selection. cfu/gram tissue or cfu/ml was calculated after overnight growth at 37˚c. mice were infected with s. typhimurium d23580 as previously described. for preliminary antibiotic treatment experiments, s. typhimurium systemic bacterial levels (cfu) were assessed for male (n = 5) and female (n = 5) mice on a standard diet 5 days post-infection for the following treatment groups: 0 mg/ml, 0.01 mg/ml, 0.05 mg/ml, and 0.10 mg/ml enrofloxacin delivered in the drinking water. water and mouse weights were collected daily, and the average antibiotic consumed (μg/kg/day) was determined. for co-treatment experiments with mice on special diets, 4-6 mice were assessed for control and vad male and female mice in the following groups: mock-treated, mock-treated and enrofloxacin, vitamin a only, and vitamin a and enrofloxacin co-treatment. mock-treated mice received 100 μl of sterile pbs administered by oral gavage on day 1 and day 2 post-infection. vitamin a-treated mice received 600 iu of retinyl palmitate (interplexus, inc., kent, wa) in 100 μl of sterile pbs administered by oral gavage on day 1 and day 2 post-infection and were changed to control diet at day 1 post-infection. enrofloxacin (0.05 mg/ml or 0.01 mg/ml) was administered at day 2 post-infection in the drinking water. enrofloxacin (baytril 100 by bayer) was obtained from the pharmaceutical service at the uc davis veterinary medical teaching hospital. water weights were monitored daily to determine average antibiotic consumption per mouse. cfu/ gram tissue or cfu/ml was calculated for liver, spleen and blood. for measurement of plasma enrofloxacin concentrations, blood was first centrifuged for 10 minutes at 1000 x g. plasma was stored at -80˚c for further analysis by liquid chromatography with tandem mass spectrometry (lc-ms/ms). the remaining blood sample was incubated at room temperature for 10 minutes with 100-200 μl of 1% triton x-100 and plated for cfu as previously described. lc-ms/ms was used to quantify enrofloxacin and ciprofloxacin concentrations in the mouse plasma. first, a mixed master stock solution of 10 μg/ml of enrofloxacin (milliporesigma, st. louis, mo) and ciprofloxacin (milliporesigma, st. louis, mo) was made in control cd-1 mouse plasma (bioreclamation ivt, westbury, ny) with k2 edta. this stock solution was then further diluted with the same control mouse plasma to establish a mouse plasma calibration curve. the following calibrator concentrations were used: 0, 1, 2.5, 5, 10, 50, 100, 500, 1000 and 5000 ng/ml. calibrators 10, 100 and 1000 ng/ml also served as quality control (qc) samples. moxifloxacin (milliporesigma, st. louis, mo) in acetonitrile (acn) (fisher scientific, hampton, nh) was used as an internal standard such that 160 μl of 200 ng/ml moxifloxacin in acn was added to 40 μl of the mouse plasma calibrators, qc samples and the experimental mouse plasma samples collected as previously described. all samples were vortexed for 10 seconds. samples were then centrifuged at room temperature for 5 minutes at 17,000 x g, and 25 μl of resulting supernatant was diluted with 175 μl of water. next, 2 μl of the final solution was injected into the acquity ultra performance liquid chromatography (uplc) system with a beh c18 column, 1.7 μm, 2.1 mm x 50 mm (waters corporation, milford, ma). the following mobile phases were used: a (h 2 o with 0.1% formic acid), and b (acn with 0.1% formic acid) (fisher scientific, hampton, nh). mobile phase a was also used for purging, and acn/h 2 o at 50/50 (v/v) was used for the needle washing between each injection. the flow rate was 0.2 ml/min, the column temperature was set at 50˚c and the autosampler temperature was set at 10˚c. the following uplc gradient program was used for the separation: 0-1 min, 10% b; 2.5 min, 72.5% b; 2.51-4.75 min, 95% b; 4.76-7 min, 10% b. the output of the uplc was fed to a xevo tq-s triple quadrupole mass spectrometry (ms/ms) system (waters corporation, milford, ma), which was used to ionize target molecules with electrospray ionization (esi+) and monitor the ion m/z fragmentation transitions from 360.1-245.2 for enrofloxacin quantification, 332.2-245.2 for ciprofloxacin quantification and 402.2-261.1 for moxifloxacin quantification in multiple reaction monitoring (mrm) mode. the retention times were 2.45, 2.38 and 2.58 minutes for enrofloxacin, ciprofloxacin and moxifloxacin, respectively. the calibration curve was fitted with a weighted (1/x 2 ) least-squares linear regression algorithm. the detection range was from 1 ng/ml to 5000 ng/ml for enrofloxacin and from 2.5 ng/ml to 5000 ng/ml for ciprofloxacin. the extraction yield was about 80-100% for all three antibiotics and the matrix effect enhanced both enrofloxacin and ciprofloxacin ms signals by 10% and 4%, respectively. the matrix effect enhanced moxifloxacin ms signals by 48%. both inter-and intra-batch accuracy were lower than 10% (% deviation) except that ciprofloxacin had 11% of inter-batch deviation at 2.5 ng/ml. both intra-and inter-batch precision were also lower than 10% coefficient of variation (cv), except that enrofloxacin and ciprofloxacin had 20-40% cv at 1 ng/ml and 2.5 ng/ml, respectively. the lower limit of quantification (lloq) for enrofloxacin was determined to be 1 ng/ml and the lloq for ciprofloxacin was determined to be 2.5 ng/ml. to assess survival in untreated mice, control and vad male and female mice were infected with s. typhimurium d23580 as previously described, and weights were monitored daily for up to 4 days post-infection. percent survival for each group (n = 8-9) was reported with a kaplan-meier survival curve. to assess male-specific survival with treatment conditions, control and vad male mice were infected with s. typhimurium as previously described. control mice and one group of vad mice were mock-treated with pbs. other vad groups received either enrofloxacin treatment alone (0.01 mg/ml) or co-treatment of enrofloxacin and vitamin a as previously described. percent survival of 6-16 mice/group at 8 days post-infection was assessed with a kaplan-meier survival curve. to assess treatment of vitamin a alone, control and vad male and female mice were infected with s. typhimurium as previously described. control mice were mock-treated with pbs, one group of vad mice was treated with vitamin a as previously described and a second group of vad mice received mock-treatment. percent survival of 12 mice/group at 13 days post-infection was assessed with a kaplan-meier survival curve. for all survival curve experiments, mice were weighed daily and humanely euthanized when they approached 20% weight loss or displayed signs of lethal morbidity, according to our institution's humane endpoints policy. statistical analyses were performed with graphpad prism 8 (graphpad, la jolla, ca). since data were found not to have a normal distribution, nonparametric analyses were utilized. significance of differences between two groups was determined with a mann-whitney test. significance of differences between multiple groups from a control group was determined with a kruskal-wallis test and a post-hoc dunn's multiple comparisons test. for the survival curves, statistical significance was determined using a log-rank (mantel-cox) test. data for weights, systemic bacterial levels, antibiotic consumed and plasma enrofloxacin concentration were reported as mean ± sem. a p<0.05 was considered significant. in order to model how malnutrition and associated vitamin a deficiency are risk factors for developing systemic s. typhimurium infection in people, we established a mouse model of vitamin a deficiency and disseminated disease (fig 1a) . mice were maintained on either a vad diet or a control diet containing adequate vitamin a. to model systemic infection, mice were infected with s. typhimurium d23580 via the intraperitoneal (i.p.) route. at 4d postinfection, systemic levels of s. typhimurium in liver, spleen and blood were significantly higher in vad mice compared to control (fig 1b) , and there was no sex difference observed in bacterial colonization (s1a fig). in a similar experiment with a necropsy at 5d post-infection, no sex differences in systemic bacterial burden was observed in control mice (s1b to generate vitamin a-deficient (vad) mice, c57bl/6 slc11a1 +/+ pregnant mice were put on a vad diet 2 weeks into gestation (a). at 3 weeks, pups were weaned onto either a vad or control diet. male (n = 7) and female (n = 7-8) mice on each diet were infected with s. typhimurium (stm) d23580 via the intraperitoneal (i.p.) route at 9 weeks of age and systemic bacterial burden was characterized by colony-forming units (cfu) in liver, spleen and blood collected at necropsy (nx) 4 days after infection (b). data represent mean ± sem. significance between control and vad mice was determined with a mann-whitney test of log-transformed values. a p<0.05 was considered significant. data demonstrated that our model of vitamin a deficiency could be utilized to study its effect on susceptibility to systemic s. typhimurium infection. it has been known for decades that vitamin a deficiency affects males and females differently [22] . to assess if this held true for our mouse model, both male and female mice were included. during maintenance on vad and control diets, mice were weighed weekly. while diet had no effect on weight gain of female mice, vad male mice gained significantly less weight than control mice starting at postnatal week 5 (fig 2a) . during the course of infection with s. typhimurium d23580, vad male mice had significant weight loss from day 0 to day 4 of infection, and 50% of mice in the vad male group exhibited lethal morbidity by day 4 ( fig 2b and 2c ). in contrast, vad females and control mice of both sexes maintained their weight over the course of the experiment (fig 2c) . while vitamin a deficiency had sex-specific effects on infection-induced morbidity ( fig 2b and 2c ), both male and female mice were similar in exhibiting significantly higher systemic burdens of s. typhimurium in vad compared to control mice (s1c fig). these results indicated that while vitamin a deficiency compromised control of systemic s. typhimurium infection in both sexes, male mice were affected more strongly, both by reduced growth while on the vad diet and by increased morbidity during infection. since there was no sex difference in systemic bacterial burden by day 4 post-infection (fig 1b) but vad males lost weight and had decreased survival compared to female mice (fig 2) , we investigated whether an earlier time point would yield sex differences in salmonella pathogenesis. further, we assessed if this trend would hold true for multiple strains of s. typhimurium, including d23580 (an st313 strain), and the st19 strains sl1344 and sara16. there was no sex difference in systemic bacterial burden for any of the s. typhimurium strains in mice fed a control diet (fig 3a) . however, at day 1 post-infection, male mice on a vad diet had significantly more or a trend towards a higher systemic burden of all three s. typhimurium strains than vad female mice (fig 3b) . these results suggest that vitamin a deficiency has a more marked effect in male mice on responses that control systemic replication of s. typhimurium in the early stages of infection. emerging multidrug-resistant s. typhimurium strains like d23580 are resistant to the firstline drugs for ints: ampicillin, chloramphenicol and trimethoprim/sulfamethoxazole. for this reason, the fluoroquinolone ciprofloxacin is one of the currently recommended treatments for ints [23] . however, emergence of fluoroquinolone resistance in salmonella strains and host factors like malnutrition can contribute to treatment failure [24, 25] . we chose to model antibiotic treatment failure with the fluoroquinolone enrofloxacin since it can be administered orally through the drinking water to mice and because we could measure it in the bloodstream. we first confirmed susceptibility of d23580 to enrofloxacin in vitro. the average mic was 0.04 μg/ml, which by clsi guidelines is susceptible [18] . we next sought to determine an in vivo mic. in order to assess the subtherapeutic concentration of enrofloxacin administered in the drinking water, systemic bacterial levels were assessed for male and female mice at a range of concentrations at day 5 post-infection. to model antibiotic treatment failure, enrofloxacin administration was delayed to day 2 post-infection. we used c57bl/6j mice fed a standard diet for titration experiments. no sex difference was observed in systemic bacterial levels (s2 fig) . a significant decrease in bacteria in liver, spleen and blood was seen at an enrofloxacin concentration of 0.10 mg/ml (fig 4a) . the concentration 0.01 mg/ml was subtherapeutic, as it failed to significantly decrease systemic s. typhimurium levels. the concentration 0.05 mg/ml was partly subtherapeutic, as it did not significantly decrease s. typhimurium levels in the liver. water weights and mouse weights were monitored daily during infection, and the vitamin a and antibiotics synergize against antibiotic resistant salmonella average amount of enrofloxacin consumed was calculated in μg/kg/day for days 2-3, 3-4 and 4-5. enrofloxacin consumption tracked with dosage, although 0.01 mg/ml and 0.05 mg/ml were not statistically significant from the untreated group (fig 4b) . taken together, these data demonstrated that 0.01 mg/ml and 0.05 mg/ml could model antibiotic treatment failure and thus were used for subsequent experiments with mice on special diets. to assess the therapeutic potential of vitamin a in the context of antibiotic treatment failure, systemic levels of s. typhimurium d23580 at day 4 post-infection were assessed for male and female mice on either control or vad diets with the following treatment groups: mocktreated, vitamin a only, enrofloxacin (0.05 mg/ml) only, and vitamin a and enrofloxacin cotreatment (fig 5a) . in vad male mice, co-treatment significantly decreased bacterial levels at all three systemic sites in comparison to mock treatment; however, individual treatments did not (fig 5b) . systemic colonization of vad male mice in all groups combined was significantly higher if the mouse had >10% weight loss than if the mouse had <10% weight loss (s3 fig). mock-treated vad male mice lost significant weight from day 0 to day 4, but none of the vitamin a and antibiotics synergize against antibiotic resistant salmonella treatment groups were significant (s4 fig). in contrast, vad female mice either showed no significant improvement with any treatment (spleen), or equal effectiveness with enrofloxacin alone and co-treatment (liver, blood) (fig 5c) . in male and female mice on a control diet, both enrofloxacin (0.05 mg/ml) and co-treatment yielded significant or trending decreases in systemic s. typhimurium (s5a and s5b fig). since 0.05 mg/ml enrofloxacin was successful, independent of vitamin a, at decreasing s. typhimurium in liver and blood in vad female mice and control mice for both sexes, a concentration of 0.01 mg/ml was also assessed for these groups. however, co-treatment of 0.01 mg/ml enrofloxacin and vitamin a yielded no benefit in vad female mice compared to mock-treated animals (s6 fig). for males on a control diet, co-treatment of 0.01 mg/ml enrofloxacin and vitamin a showed a slight decrease in splenic bacterial levels, but there was no difference in the liver and blood (s7a fig). there was no benefit of any therapy for females on a control diet (s7b fig). enrofloxacin is a common antibiotic used in veterinary medicine, and it is converted to ciprofloxacin by the liver [26] . no significant differences in plasma enrofloxacin or plasma ciprofloxacin levels were detected between enrofloxacin only and co-treated male (s8a fig) and as 0.01 mg/ml was subtherapeutic such that there was still lethal morbidity in vad male mice, these conditions were used to assess survival of vad male mice with treatments. mice were monitored for 8 days post-infection and euthanized upon showing signs of lethal morbidity, and a kaplan-meier survival curve was generated (fig 5d) . of the infected mice fed control diets, 100% of males survived. the percent survival of mock-treated vad males was only 25%, but this was improved with enrofloxacin only (41.7%) or co-treatment of vitamin a and enrofloxacin (66.7%). significance between each treatment group and survival of mice on a control diet was assessed with a log-rank (mantel-cox) test. further, for the 44 male mice 6-16 ). an enrofloxacin concentration of 0.01 mg/ml was used in drinking water treatment, and oral gavage treatments were given as previously described. significance was determined with pairwise comparisons between the control group and each other treatment group using a log-rank (mantel-cox) test ( � , p<0.05; ns, not significant). data from untreated controls are also included in fig 1. https://doi.org/10.1371/journal.pntd.0008737.g005 represented in the survival curve (fig 5d) , weight loss correlated with survival. over the course of the experiment, if a mouse lost less than 10% of its weight, it was more likely to have survived (16/21; 76% survival) than if a mouse had lost more than 10% of its weight (7/23; 30% survival). in a separate experiment with a longer time course grouping sexes, vitamin a treatment alone improved survival by 66.7% in vad mice (s9 fig). taken together, these data indicated that vitamin a synergized with subtherapeutic (0.05 mg/ml) enrofloxacin treatment to decrease systemic s. typhimurium levels and improve survival in vad male mice. epidemiologic studies have shown that vitamin a deficiency and disseminated non-typhoidal salmonella infections are co-endemic, disproportionately affecting sub-saharan africa [10, 27] . the high mortality rate of disseminated infection has been attributed to antimicrobial resistance of st313 strains that circulate regionally in sub-saharan africa, to genomic features of these strains associated with increased systemic dissemination, and to the high prevalence of co-morbidities that predispose to systemic disease [17, 23, 24] . the current study was designed as a preclinical trial in mice to assess the potential utility of vitamin a as an adjunct to antibiotic therapy. our results suggest a benefit of combined antibiotic treatment and vitamin a supplementation, with the effect being most apparent in male mice. it is well-appreciated that vitamin a supplementation can be a useful tool for disease prevention and treatment. in populations with a �1% prevalence of night blindness or a �20% prevalence of vitamin a deficiency, the who recommends vitamin a supplementation for children 12-59 months of age [28] . vitamin a supplementation was found to significantly reduce the risk of all-cause morbidity and mortality due to diarrhea [29] . in children 2 and under presenting with measles, the who-recommended treatment of two consecutive daily megadoses of vitamin a [30] reduces overall and pneumonia-specific mortality [11] and decreases duration of diarrhea and fever. however, the utility of vitamin a for bacterial bloodstream infections has not been assessed. s. typhimurium bloodstream infections typically present as a febrile illness; diarrhea is often absent or not a prominent symptom [10, 31, 32] . our results suggest that co-treatment with vitamin a and antibiotics may improve outcomes during bloodstream infections with drug-resistant s. typhimurium. despite who's recommendation for vitamin a supplementation, full coverage is still lacking in many countries. therefore, supplementing with vitamin a during episodes of febrile illness is a potential strategy to increase coverage and improve infection outcomes. a notable finding of our study was the sex-specific effect of vitamin a supplementation with antibiotics for treatment of s. typhimurium infection in mice. the picture emerging from recent studies of innate immunity is that multiple sex-specific differences impact resistance of males and females to infection. human males are more susceptible to many infectious diseases, whereas females are more at risk of developing an autoimmune condition [33] . in current events, males are at a higher risk of severe covid-19 infection [34] . males also have a higher risk of developing sepsis [35, 36] . a study on sepsis in children found that vitamin a deficiency was more common in the sepsis group as compared to control, and vitamin a deficiency rates were~80% in the subgroups of severe sepsis and septic shock [37] , indicating how a vad status can predispose to severe disease. the majority of patients with sepsis in this study were male. in addition, some genes encoding for innate immune molecules are located on the x chromosome [33] , suggesting a genetic basis for some of the differences observed. it is therefore possible that malnutrition and vitamin a deficiency may compromise the immune response to systemic salmonella infection in a sex-specific manner. we speculate that vitamin a may be playing the role of a host-directed therapy by boosting the immune response of vad male mice, synergizing with an initially subtherapeutic dose of antibiotic, and leading to better immunological control of infection. while the current study did not investigate the mechanisms underlying sex differences in the response to vitamin a supplementation, possible explanations include sex differences in innate immune cell function [33] , intestinal microbiota composition [38] , and hormone levels [39] . antibiotic treatment failure is the persistence of symptoms despite initiation of antibiotics. common causes include wrong diagnosis, infection with a resistant organism, or host failure such as malnutrition [25] . we modeled antibiotic treatment failure with a subtherapeutic enrofloxacin concentration that yielded no difference in systemic bacterial burden, but circulating antibiotic was still present in the mouse. it can be the joint action of antibiotics and host defense mechanisms that leads to clearance of bacteria and resolution of disease. while antibiotics are typically studied for their bactericidal or bacteriostatic activity against susceptible bacteria, they can also exist at subtherapeutic or subinhibitory concentrations, i.e. at concentrations below the mic. subinhibitory antibiotic concentrations can still affect both the host and the bacterium [40] . it has been proposed that some antibiotics may influence antiphagocytic cell-surface structures and lead to enhanced phagocytosis, modifying immune cell function [41] . for example, neutrophil phagocytosis and intracellular killing activity have been enhanced by pre-incubation with subinhibitory antimicrobial concentrations in some studies, although it depends on the antibiotic used and organism studied [42] . one study reported that subinhibitory levels of fluoroquinolone antibiotics like ciprofloxacin lead to enhanced binding of anti-lps antibodies to enterobacteriaceae like escherichia coli [43] . taken together, these findings suggest that vitamin a treatment may synergize with subtherapeutic antibiotics to enhance immune function in male mice. our study has limitations. we studied the effect of vitamin a co-treatment with only a single antibiotic, enrofloxacin, and a single clinical isolate, d23580. common treatments for ints have included first-line antibiotics of ampicillin, chloramphenicol and trimethoprim/sulfamethoxazole [23] . d23580 is resistant to all of these first-line drugs. it is sensitive to fluoroquinolones at certain concentrations in vitro and in vivo. however, future studies should incorporate s. typhimurium strains also resistant to fluoroquinolones, as this is an emerging issue [9] . in addition, it would be important to determine whether vitamin a supplementation can prevent treatment failure with other commonly used antibiotics. there are also limitations to using a mouse model of infection, including the low genetic variation of inbred mice used and the short infection time course permitted by the vad male mouse model. future studies could incorporate additional mouse lines, routes of infection and challenge doses. however, given the track record of using vitamin a as prevention and treatment for other infections, this information on whether vitamin a supplementation could be effective against disseminated infections with antibiotic-resistant salmonella might be best obtained in a clinical study. 1-7) . the same data is represented in two different ways. initially there were more vad male mice in the study but they reached lethal endpoint prior to day 5 and were likely to have a higher cfu than in the figure. c. levels of s. typhimurium in liver, spleen and blood at day 4 and 5 post-infection combined (n = 8-15). data shown in this figure are a re-analysis of data presented in s1a and s1b. data represent mean ± sem. significance between groups was determined with a mann-whitney test of logadditionally, vad mice treated with retinyl palmitate were placed on a control diet replete with vitamin a one-day after s. typhimurium infection. data represent percent survival of 12 mice per group from three independent experiments. pairwise comparisons between the control diet group and each other group was determined with a survival analysis log-rank (mantel-cox) test ( � , p<0.05). (tif) burden of infection on growth failure malnutrition and health in developing countries increased urinary retinol loss in children with severe infections vitamin a, infection, and immune function antimicrobial susceptibility testing of bacteria that cause gastroenteritis the global burden of nontyphoidal salmonella gastroenteritis global burden of invasive nontyphoidal salmonella disease the global burden and epidemiology of invasive non-typhoidal salmonella infections fluoroquinolone-resistant enteric bacteria in sub-saharan africa: clones, implications and research needs invasive non-typhoidal salmonella disease: an emerging and neglected tropical disease in africa vitamin a for treating measles in children guideline: updates on the management of severe acute malnutrition in infants and children. who guidelines approved by the guidelines review committee. geneva: world health organization vitamin a supplementation in infants and children vitamin a deficiency alters rat neutrophil function host-directed therapies for bacterial and viral infections tlr signaling is required for salmonella typhimurium virulence loss of multicellular behavior in epidemic african nontyphoidal salmonella enterica serovar typhimurium st313 strain d23580 m100: performance standards for antimicrobial susceptibility testing the estimation of doses of salmonella typhimurium suitable for the experimental production of disease in calves reference collection of strains of the salmonella typhimurium complex from natural populations defining the core genome of salmonella enterica serovar typhimurium for genomic surveillance and epidemiological typing the sex difference in vitamin-a metabolism fluoroquinolone resistance in salmonella: insights by whole-genome sequencing epidemic multiple drug resistant salmonella typhimurium causing invasive disease in sub-saharan africa have a distinct genotype recommendations for treatment of childhood non-severe pneumonia pharmacology of the fluoroquinolones: a perspective for the use in domestic animals global prevalence of vitamin a deficiency in populations at risk 1995-2005: who global database on vitamin a deficiency. geneva: world health organization guideline: vitamin a supplementation in infants and children 6-59 months of age. who guidelines approved by the guidelines review committee. geneva: world health organization vitamin a supplementation for preventing morbidity and mortality in children from six months to five years of age treating measles in children. department of immunization, vaccines and biologicals. geneva: world health organization non-typhoidal salmonella bacteraemia among hiv-infected malawian adults: high mortality and frequent recrudescence clinical presentation of non-typhoidal salmonella bacteraemia in malawian children sexual dimorphism in innate immunity sex differences in mortality from covid-19 pandemic: are men vulnerable and women protected? jacc case rep predictors of sepsis in moderately severely injured patients: an analysis of the national trauma data bank gender differences in human sepsis vitamin a deficiency in critically ill children with sepsis neonatal vitamin a supplementation and vitamin a status are associated with gut microbiome composition in bangladeshi infants in early infancy and at 2 years of age the early postnatal period, mini-puberty, provides a window on the role of testosterone in human neurobehavioural development role of antibodies and antibiotics in aerobic gram-negative septicemia-possible synergism between antimicrobial treatment and immunotherapy. reviews of infectious diseases subinhibitory antimicrobial concentrations: a review of in vitro and in vivo data influence of subinhibitory levels of antibiotics on expression of escherichia-coli lipopolysaccharide and binding of antilipopolysaccharide monoclonal-antibodies the authors thank greg barton for providing inbred c57bl/6-slc11a1 +/+ mice, robert hey key: cord-003006-lk2ny1wd authors: cantoni, diego; rossman, jeremy s. title: ebolaviruses: new roles for old proteins date: 2018-05-03 journal: plos negl trop dis doi: 10.1371/journal.pntd.0006349 sha: doc_id: 3006 cord_uid: lk2ny1wd in 2014, the world witnessed the largest ebolavirus outbreak in recorded history. the subsequent humanitarian effort spurred extensive research, significantly enhancing our understanding of ebolavirus replication and pathogenicity. the main functions of each ebolavirus protein have been studied extensively since the discovery of the virus in 1976; however, the recent expansion of ebolavirus research has led to the discovery of new protein functions. these newly discovered roles are revealing new mechanisms of virus replication and pathogenicity, whilst enhancing our understanding of the broad functions of each ebolavirus viral protein (vp). many of these new functions appear to be unrelated to the protein’s primary function during virus replication. such new functions range from bystander t-lymphocyte death caused by vp40-secreted exosomes to new roles for vp24 in viral particle formation. this review highlights the newly discovered roles of ebolavirus proteins in order to provide a more encompassing view of ebolavirus replication and pathogenicity. ebolaviruses are negative-sense single-stranded rna (ssrna) viruses capable of causing acute haemorrhagic fever. the prototypical ebola virus (ebov; zaire ebolavirus) was responsible for the recent west africa outbreak that resulted in 28,000 cases and 11,000 deaths between 2014 and 2016 [1] . the reservoir of this lethal pathogen has not been conclusively proven, though there is good evidence suggesting fruit bats as a primary source of exposure [2] . furthermore, survivors of ebolavirus disease (evd) can carry the virus in immunologically privileged sites for over one year, post-recovery from the acute infection [3, 4] . resultantly, there is an a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 increased awareness of the risk of alternate forms of transmission in evd as survivors may carry ebov in semen for over 12 months, causing sexual transmission, or in breast milk, which can lead to infection of newborns [4, 5] . in 2004, funding from project bioshield (united states department of health and human services) spurred research that has led to the development of novel therapeutics to prevent and treat evd, resulting in a very promising vaccine that was evaluated during the 2014 outbreak and showed 100% efficacy in disease prevention [6, 7] . following project bioshield and the 2014 west africa outbreak, there has been extensive research into the ebolaviruses that has greatly expanded our understanding of viral replication and pathogenesis. ebolavirus is a genus within the family filoviridae, which also includes the genus marburgvirus (e.g., marburg virus: marv) and cuevavirus (e.g., lloviu virus) [8] . the ebolavirus genus contains five species: zaire ebolavirus, sudan ebolavirus, taï forest ebolavirus, bundibugyo ebolavirus, and lastly, reston ebolavirus, the only member that is nonpathogenic in humans for reasons that are still unclear [9] . the majority of research has focused on zaire ebolavirus, as this species has been associated with the greatest number of outbreaks and has the highest case-fatality rates of all the ebolaviruses (30%-90%, depending on the specific outbreak), though as a consequence, many novel or species-specific functions of ebolavirus proteins may be undiscovered. the 19 kb viral genome encodes for seven main proteins: nucleoprotein (np), glycoprotein (gp), l-polymerase (l) protein, viral protein (vp) 24, vp30, vp35, and vp40 (fig 1) [10] . l is an rna-dependent rna polymerase (rdrp) and forms the rdrp complex with vp30 that is responsible for viral genome transcription and replication. vp24 and vp35 inhibit interferon (ifn) signalling and facilitate evasion of the host immune response. np encapsidates the viral genome into the nucleocapsid, whilst vp40 drives viral assembly and budding. gp is the only protein on the surface of the virion and is essential for binding to target cells and subsequently mediating membrane fusion and the release of the viral genome. however, recent research has uncovered a multitude of new, often overlapping roles for ebolavirus proteins, and it is not possible to view viral replication as a one-protein-one-function process (table 1 ). in this review, we examine the collection of recently identified secondary functions of ebolavirus proteins in order to provide a more comprehensive understanding of roles of each protein in viral replication and pathogenicity. vp24 is one of the most studied filovirus proteins, with the majority of studies focusing on its primary role in inhibiting the host ifn response. vp24 is known to inhibit ifn-α/β and ifn-γ activation by binding to key host proteins from the karyopherin α family (karyopherin α1, α5, and α6) preventing their binding to and the subsequent nuclear import of signal transducer and activator of transcription 1 (stat1) [11] . in addition, vp24 blocks ifn signalling by directly binding to stat1, preventing its phosphorylation, nuclear import, and transcription of ifn-stimulated genes (isg) [11] [12] [13] . recent data suggest vp24 is also able to block ifn induction by supressing nuclear factor-kappa b (nf-kb) activation following tumour necrosis factor alpha (tnf-α) stimulation [14] and by supressing retinoic acid-inducible gene i (rig-i)-dependent activation of ifn-γ1 gene expression [15] . furthermore, the innate response antagonist domains (irad) in ebov vp24 and vp35 have been implicated in preventing the maturation of ebov-infected dendritic cells by modulating global gene expression [16, 17] . this effect required both vp24 and vp35 irad domains, though the vp24 irad domain alone was sufficient to down-regulate cytokine signalling pathways [17] . these results highlight the importance of cooperative action of multiple viral proteins for evasion of the host immune response. vp24 has also recently been seen to function in capsid assembly [18] . np and vp35 are known to be the major components of the viral nucleocapsid, though vp24 is weakly associated and may act as a catalyst for particle formation [18] . further evidence for a structural role of vp24 comes from the observation that n-or c-terminal deletions in vp24 inhibited the formation of nucleocapsid-like structures mediated by vp24, vp35, and np coexpression [19] . it was suggested that the n-terminal domain of vp24 facilitates capsid formation by mediating protein-protein interactions. this is supported by the observation that mutation of the vp24 n-terminal domain results in protein aggregation [19] . a recent study confirmed the interaction between vp24 and np, showing that vp24 residues v170 and n171 are located on a highly conserved exposed loop that interacts with np during nucleocapsid assembly [20] . coexpression of vp24 and vp40 results in a greater production of virus-like particles (vlps) than when vp40 is expressed alone [21] . similarly, in live ebov infection, vp24 small interfering rna (sirna) knockdown decreases viral budding and increases the retention of viral proteins within the cell [22] . further evidence suggests that vp24 binds to vp35 on the outer surface of the nucleocapsid where it organises the adjacent np layer, promoting nucleocapsid stability and explaining the observed interactions between np, vp24, and vp35 during nucleocapsid formation [23]. rna editing by addition of us occurs on the gp transcript in order to produce gp and the ssgp. post-translational modification of the gp protein results in two gp subunits after cleavage by host furin. slight differences in gene overlaps and the presence of intergenic regions are evident between the different ebolavirus species, though the functional consequence of these differences is not clear at present. ebov in addition to its role in nucleocapsid stability, vp24 may be necessary for incorporation of the viral rna (vrna) genome into the nucleocapsid. several studies have shown that vp24, together with vp35, induce conformational changes in np that are necessary for rna encapsidation [18, 24, 25] . it was shown that vp24 may be directly involved in length-dependent vrna interactions and packaging [26] . in the study, transcription-and replication-competent virus-like particles (trvlps) were analysed for rna content, and a significant reduction of packaged rna was observed when vp24 was knocked down with an interfering micro-rna (mirna). the trvlps that were produced showed a 2-fold reduction in rna content and a 10-fold reduction of infectivity, suggesting that vp24 may play an essential role in rna packaging. furthermore, disruption of the vp24-np interaction reduced rna packaging and resulted in a significant reduction in reporter activity, highlighting the importance of vp24 in rna packaging [20] . in addition, trvlp reporter gene activity was significantly affected by the presence of vp24 in a genome-length-dependent manner. using the trvlp tetracistronic genome system, the presence of vp24 during vlp production resulted in a 25-fold increase in reporter gene activity upon subsequent infection, whereas the presence of vp24 had no effect when monocistronic minigenome systems were used in the trvlp, suggesting a genomelength-dependent role for vp24 in rna packaging and vlp infectivity. surprisingly, vp24 may also have a length-dependent role in transcriptional regulation. it was observed that vp24 moderately inhibited the expression of reported genes from monocistronic minigenome plasmids [27] . however, vp24 expression had no effect on protein expression from the trvlp tetracistronic genomes [26] . whilst the impact of vp24 on protein expression is not clear, vp24 itself may be subject to length-dependent transcriptional regulation. recent work has implicated the length of the intergenic region (ir) between vp30 and vp24 as having a significant as with vp24, vp35 is primarily known for its multifaceted ability to suppress the host cell immune response. vp35 is a type-i ifn antagonist, inhibiting the activation of interferon regulatory factor (irf)-3 via double-stranded rna (dsrna) binding and reducing ifn-α/β production by inhibiting rig-i signalling [30] [31] [32] . vp35 also blocks ifn production by increasing protein inhibitor of activated stat1 (pias1)-mediated sumoylation of irf-7, thus inhibiting ifn production following toll-like receptor (tlr) and rig-i activation [33] . lastly, vp35 is a suppressor of rna silencing, functionally equivalent to the human immunodeficiency virus (hiv-1) trans-activator of transcription (tat) protein and important for viral evasion of the innate immune response [34] . together, there is significant evidence demonstrating vp35's intricate ability to inhibit innate immune signalling and the host antiviral response (fig 2) . recent work suggests that vp35 may have more diverse functions during virus replication, as vp35 was shown to interact with l and facilitate genome transcription through the formation of the rdrp complex and genome packaging through association with np [35-37]. the first 450 residues of vp35 appear to be essential for binding to l and thus rdrp function, whereas the c-terminus associates with np, thus linking np and l during nucleocapsid assembly [36, 38] . n-terminal deletions in vp35 block these interactions and were sufficient to inhibit the replication and transcription of an ebov minigenome system [38] . the role of the vp35 c-terminus in capsid assembly is perhaps surprising, as this region contains the ifn inhibitory domain responsible for its main role in immune evasion. however, this domain contains several conserved stretches of basic residues involved in dsrna binding and ifn inhibition, whereas a preceding stretch mediates interaction with np [36]. further research found the vp35-np interaction controls the switch between rna-bound np and free np, thus switching between genome replication and genome packaging in the nucleocapsid [39] . an n-terminal peptide derived from the vp35 np-binding protein region (npbp) binds np with high affinity, causing the release of rna from np and resulting in the activation of genome transcription and the inhibition of np oligomerisation (fig 2) [39]. additional investigation of vp35-np binding showed two further interaction sites. hydrophobic vp35-np binding at these sites inhibited np oligomerisation and prevented np-rna binding by blocking access to the rna binding domain [40] . work by leung and colleagues suggests that during nucleocapsid formation, the npbp peptide first disassociates from np, then rna binds to np, followed by np oligomerisation. in contrast, kirchdoerfer and colleagues show that monomeric np has no significant affinity for rna, suggesting that the npbp peptide would be displaced by an additional np molecule, causing np oligomerisation that would then allow for rna binding. however, in either process, vp35-np interactions are crucial for virus replication and are being explored as targets for future therapeutics [41, 42] . vp35 also undergoes further protein-protein interactions that may affect viral genome transcription through the interaction with the cytoplasmic dynein light chain (lc8) [43] . lc8 is a highly conserved 8 kda subunit of the cytoplasmic dynein motor complex but can also exist as a dimer in soluble form, which can affect viral transcription and assembly [44, 45] . lc8 was seen to stabilise vp35 n-terminal oligomerisation in a dose-dependent manner and enhance viral genome synthesis [46] . it was noted that lc8 functions mostly in the early stages of infection, enhancing early viral gene expression before the host cells are able to establish the antiviral state. thus, vp35 modulation of vrna transcription can facilitate virus replication while simultaneously enhancing immune evasion. the minor nucleoprotein vp30 has the primary role of initiating ebov transcription [47] . it is dynamically phosphorylated, whereby upon phosphorylation, transcription is negatively regulated, enabling binding to np [48, 49] . in turn, this permits interactions that regulate vrna synthesis [37] . vp30 binds zinc ions due to the presence of an unconventional zinc-binding motif, facilitating rna binding and increasing viral genome transcription [50-53]. in addition, vp35 blockade of irf3 and irf7 phosphorylation inhibits the production of ifn-β. recent studies have also highlighted the importance of vp35 in regulating np-rna association. during viral genome replication, the vp35 n-terminal peptide binds to np, enabling the vrna to associate with the rdrp complex for replication. during virus assembly, vp35 disassociates, enabling np to oligomerise, bind rna, and form the nucleocapsid. 5'ppp, 5' triphosphate; dsrna, double-stranded rna; ifn, interferon; ikk, inhibitor of nuclear factor kappa b kinase subunit epsilon; irf, interferon regulatory factor; mavs, mitochondrial antiviral-signalling protein; mda5, melanoma differentiation-associated protein 5; np, nucleoprotein; pact, protein activator of the interferon-induced protein kinase; rdrp, rna-dependent rna polymerase; rig-i, retinoic acid-inducible gene i; tank, tumour necrosis factor-receptor-associated factor family member-associated nuclear factor kappa b activator; tbk1, tumour necrosis factor-receptor-associated factor family member-associated nuclear factor kappa b activator binding kinase 1; traf3, tumour necrosis factor-receptor-associated factor 3; vp, viral protein; vrna, viral rna. https://doi.org/10.1371/journal.pntd.0006349.g002 in addition to its rna-binding role in transcription, vp30 also interferes with cellular rna silencing [54] . in the presence of sirna, vp30 was seen to interact with the essential rna interference (rnai) protein dicer, though the vp35 n-terminus rna binding domain was not required for the interaction or for the suppression of rnai (fig 3) . as with the rna silencing suppressor activity of vp35, the exact role of rnai in antiviral immunity is not clear, nor is the consequence on ebov replication of blocking mirna/sirna processing as mediated by vp30 [55]. however, despite the fact that rna binding is not required for rna silencing suppression, vp30 was seen to bind to a variety of nonviral rnas. vp30-rna binding required specific base composition and structure of the target rna molecule [53], though it is not clear if there is a function of vp30 binding to nonviral rna or if this is a consequence of its necessary binding to vrna during transcription. the matrix protein vp40 has roles predominantly in virus assembly and budding [56, 57] . vp40 can assemble either as a hexamer, which appears to be involved in budding, or as an octamer that functions in genome replication and rna binding [58, 59] . most research has focused on the role of vp40 in assembly and budding; however, recent studies have begun to elucidate novel roles. vp40 is known to be sufficient to mediate the formation and budding of vlps; however, recent results have demonstrated that vp40 induces the formation for exosomes that are capable of inducing bystander cell death [60, 61] . exosome release has been seen in virally infected cells, and vp40 expression was seen to increase the expression of several endosomal sorting complex required for trafficking (escrt) proteins involved in exosome biogenesis, including tumour susceptibility gene 101 (tsg101), vacuolar protein-sorting-associated protein 25 (vps25), and vps36 [61, 62] . this is consistent with previous reports showing vp40 utilising escrt proteins to aid viral budding, though how vp40 switches between budding and exosome release is not clear [63] [64] [65] . transfer of vp40-induced exosomes to naïve t lymphocytes and monocytes induced apoptosis and significantly reduced cell viability similarly to that seen when exosomes from virally infected cells were used [61, 62] . however, pleet and colleagues noted that the presence of vp40 in the exosomes caused a down-regulation of mirna machinery in both the donor and recipient cells, including a reduction in the expression of dicer, argonaute-1, and drosha ( fig 3) . it was previously noted that vp35, vp30, and vp40 are capable of interacting with the mirna/rnai pathway [54]; however, this was the first demonstration that the suppression of rna silencing can be transferred to naïve cells in the absence of virus. currently, the precise mechanism in which vp40 interacts with mirna machinery has yet to be characterised, and it is not yet known if the action of vp40 on the mirna machinery directly causes apoptosis or if the exosomes contain other proteins or rnas that may be responsible for causing the induction of apoptosis. however, the repression of key proteins in the mirna pathway has been previously linked to the induction of apoptosis; thus, the suppression of rna silencing may serve both to directly counteract the innate cellular immune response and to induce apoptosis of bystander immune cells, blocking the activation of adaptive immunity [57, 61, 66] . due to the essential role of vp40 in viral assembly and budding, several studies have looked at inhibiting vp40 for the creation of new antiviral therapeutics [67, 68] . as vp40 also plays a role in immune evasion (via rnai suppression and exosome-bystander cell death), there is increased motivation for developing therapeutics that target one or multiple functions of vp40. it is known that viral replication requires vp40 phosphorylation at tyrosine 13 by the cellular tyrosine kinase abelson murine leukaemia viral oncogene homologue 1 (c-abl1) [69] . in addition, recent results showed that cyclin-dependent kinase 2 (cdk2) in complex with cyclin a or cyclin e phosphorylated exosomal vp40 at serine-233 [61] . thus, the inhibition or modulation of vp40 phosphorylation may be a target for new therapeutics. lastly, pleet and colleagues showed that treatment with the fda-approved drug oxytetracycline reduced vp40 exosome release and significantly increased donor cell viability upon treatment with vp40 exosomes, further suggesting that targeting the secondary functions of vp40 may be a new approach for developing antivirals for evd. the gp gene has been shown to encode for three different products due to transcriptional editing by l: full length gp, which consist of gp1 (receptor binding) and gp2 (viral fusion) subunits; soluble gp (sgp), which lacks the transmembrane domain; and small soluble gp (ssgp) [70, 71] . due to furin cleavage of sgp, a smaller cleaved fragment is also produced, called δpeptide [72] . gp is the only viral protein located on the surface of the virion and has a critical role in attachment and fusion [73] [74] [75] . ebolaviruses are thought to predominantly enter cells via gp-dependent macropinocytosis, though other mechanisms have been reported, depending on factors such as host cell type [76, 77] . after entry, gp directs fusion between the viral membrane and endolysosomes that contain the viral receptor niemann-pick c1 (npc1) and two-pore segment channel 2 (tpc2), enabling release of the viral genome [78, 79] . during the 2014-2016 outbreak, a mutation in gp at a82v was detected with high frequency [80] [81] [82] . this mutation increased gp membrane fusion activity and increased infectivity in a variety of cell types, including chimpanzee fibroblasts (s008842), rhesus epithelial (frhk4), african green monkey epithelial (vero), and human dendritic cells. the authors propose that this mutation is likely a result of ebov adaptation to the human host, as several viral variants have been seen to increase human cell infectivity while decreasing virus entry in nonhuman primates [83] [84] [85] . thus, the specific nature of protein function needs to be considered in the context of the given host. gp has been shown to have a multitude of secondary roles beyond attachment and fusion that affect both virus replication and pathogenicity. several studies have shown that gp contributes to ebov virulence; however, it is not sufficient on its own to be defined as a virulence marker, despite having marked effects beyond entry and fusion [86] . ebov gp expression has been well established as having a cytotoxic effect on host cells [87] [88] [89] . gp cytotoxicity is mediated through the mucin-like domain and its effect on the extracellular signal-regulated kinase (erk) mitogen-activated protein kinase (mapk) pathway [90] . ebov gp reduces the phosphorylation and catalytic activity of erk2, resulting in the loss of cell adherence, cell rounding, and the induction of non-apoptotic cell death. the decrease in erk2 activity was also necessary for gp-induced down-regulation of αv integrin expression, further impairing cell adherence and tight junction formation. in addition, the sgp cleavage product, δ-peptide, may play a role in ebov pathogenicity by acting as a viroporin [91] . δ-peptide is able to form pores in the plasma membrane of mammalian cells, increasing ion permeability and causing cytotoxicity [92] . however, it is not known if the δ-peptide can be released from cells or if its induction of cytotoxicity is limited to within the infected cell. in order to regulate the toxicity caused by gp and its cleavage products at early stages of infection, gp expression is dynamically regulated [93] . the balance and timing of ebov gp/sgp/ssgp/shed-gp/δ-peptide expression has been found to be pivotal in virus replication, affecting not only cell death but viral assembly and budding [94] . whilst vp40 expression is sufficient to produce vlps, gp expression enhances vlp generation, suggesting a possible secondary role for gp in viral egress [21] . recent work suggests that gp does not directly affect viral assembly or budding, but rather counteracts the cellular budding restriction factor tetherin [95] . in the absence of gp, vlps assemble and bud but are retained on the cell surface through the antiviral tethering actions of the tetherin protein. gp expression enabled vlp release but did not affect tetherin cell-surface localisation, nor was a specific gp-tetherin interaction found [96] . it was shown that the gp glycosylation and its receptor-binding domain (rbd) were critical for anti-tetherin activity, though mutation of the rbd did not affect interactions with tetherin, and inhibition of gp-npc1 binding did not affect the anti-tetherin activity [97] . instead, it is thought that gp is specifically able to block the association between vp40 and tetherin, though the nature of tetherin-vp40 interaction and the mechanism of gp inhibition is not known [98] . ebov infection causes significant impairment of the endothelial barrier function. gp repression of erk2 activity reduces integrin expression and cell adherence; however, gp also induces endothelial cell activation, further decreasing endothelial barrier functions [99] . during ebov infection of endothelial cells, cell adhesion molecules (cam) icam-1 and vcam-1 were found to be transcriptionally upregulated, with increased cell surface expression of cams. the activity occurs with cellular gp expression as well as following the transfer of gpcontaining vlps; viral replication does not appear to be required. endothelial activation was not observed in the absence of gp, nor with the gp transcriptional variant sgp or the gp cleavage product δ-peptide [99] . gp-induced endothelial cell activation may facilitate decreased barrier function, whilst the up-regulation of cams may facilitate adhesion and subsequent infection of immune cells, such as macrophages. a model was proposed whereby activated endothelial cells result in increased leukocyte recruitment, resulting in thrombomodulin release, resulting in an activated, leukocyte-rich endothelium in a procoagulant state [100] . whether this is an unintended consequence of gp or has a role in increased viral spread and infectivity is yet to be investigated. ebov gp has also been implicated in modulation of the host immune system. gp on the cell plasma membrane has been shown to be cleaved by tnf-α-converting enzyme (tace), resulting in the release of a soluble cleaved product called shed gp that is missing the transmembrane domain [101] . shed gp was seen to activate uninfected macrophages and dendritic cells, resulting in the production of multiple pro-and anti-inflammatory cytokines, including tnf-α, with subsequent effects on vascular permeability [102] . it is thought that shed gp activates macrophages by binding to and activating toll-like receptor 4 (tlr4) in a manner requiring gp glycosylation. recently it was also shown that full-length gp on vlps can also trigger the activation of tlr4 in macrophages, resulting in a similar activation phenotype [103] . in contrast, gp binding to tlr4 on t lymphocytes directly triggers cell death through an up-regulation of caspase 9, even in the absence of infection [104] . in dendritic cells, gp was found to interact with the liver and lymph node sinusoidal endothelial cell c-type lectin (lsectin), a ctype lectin that contains two amino acids, residues n256 and n274, that bind gp in a ca 2 + -dependant manner, triggering the activation of spleen tyrosine kinase (syk) signalling and the production of inflammatory cytokines tnf-α and interleukin-6 (il-6) [105] . as shed gp retains most of the structure of full-length gp, this soluble molecule is capable of binding to and neutralising circulating anti-gp antibodies, facilitating viral immune evasion [101] . similarly, sgp has been implicated in evading the immune system via antigenic subversion [106] . it was found that boosting gp-immunised mice with sgp biased b-cell response towards epitopes that were shared between sgp and gp, reducing gp-specific antibody production and possibly impeding the immune-mediated clearance of ebov infection. the structure of sgp in complex with antibodies was recently solved and highlights differences in antibody reactivity between gp and sgp [107] . whilst gp is trimeric, sgp oligomerises into a parallel homodimer. cross-reactive c13c6 antibody epitopes are presented similarly on gp and sgp, though the authors report that one c13c6 antibody binds to one gp trimer, whereas multiple different immune-complexes were formed with sgp, ranging from rectangular complexes with a 2:2 ratio of c13c6 antibody to sgp dimer up to pentagonal 5:5 antibody:sgp complexes [107] . this further supports the hypothesis that sgp enhances viral immune evasion by biasing the antibody response towards sgp binding. whilst the multitude of gp secondary effects have a significant impact on ebov infection, pathogenesis, and immune evasion, gp remains the immunodominant protein on the ebov virion, and vaccination with the gp protein on pseudotyped viruses, recombinant vesicular stomatitis virus with zaire ebolavirus gp (rvsv-zebov), has been highly effective in preventing evd [7] . np has a distinct function in the replication cycle as it is a key component of the viral ribonucleoprotein complex and has critical roles in protecting vrna from degradation and in mediating genome encapsidation during virus assembly [10] . at present, all research has focused on these primary activities of np, and any secondary roles remain to be determined [18, 25, 40] . similarly, the rna-dependent l-polymerase is an essential component of the rdrp complex and required for viral genome transcription and replication [10] . it has been observed that l can also edit mrna, as seen with the gp gene, where l-editing results in the production of the gp transcript instead of sgp [108] . l-editing may also regulate the different expression levels of gp, sgp and ssgp. during serial passage in tissue culture cells, l was found to add a single uridine (u) residue to a site consisting of 7 us in the gp gene, changing the expression ratio of gp:sgp to 80:20. a single passage in guinea pigs caused reversion of the genome back to 7 us and changed the gp:sgp expression ratio back to 20:80, which may facilitate immune evasion during in vivo replication [109] . in contrast, during viral replication in the human hepatocarcinoma cell line (huh7), a 9u variant was seen that retained the high level expression of sgp but had enhanced expression of ssgp [71] . it is speculated that these rapid alterations in the gp gene may act as a regulatory mechanism, enabling efficient virus replication in different host environments. at present, no other roles for the l protein have been postulated. for many years now, the fundamental principles of ebolavirus replication have been known, with each viral protein playing a specific role: l and vp30 form the rdrp and mediate viral genome transcription and replication, np packages the vrna genome and forms the nucleocapsid, vp40 mediates virion assembly and budding, gp mediates virion attachment to and fusion with the host cell, and vp24 and vp35 enable evasion of the host immune system. however, in recent years, our understanding of ebolavirus replication and pathogenesis has significantly increased, and we now know that each viral protein plays a multitude of overlapping roles during virus replication. vp24 and vp35 were thought to primarily mediate immune evasion but have now been seen to function in viral replication and in the formation of the nucleocapsid. similarly, many of the viral proteins that were thought to play functional roles in replication (i.e., vp30, vp40, gp) have now been discovered to have significant roles in immune evasion. this broadens our understanding of the ways in which ebolaviruses can evade and subvert the host immune system. of particular interest is the observation that vp30, vp35, and vp40 all appear to supress the host rna silencing system, though the relevance of this to viral replication is not yet clear (fig 3) . in addition, many ebolavirus proteins are seen to interact with immune cells, causing cell activation and/or cell death and facilitating both viral replication and spread (e.g., by recruiting monocytes to infected cells or by increasing vascular leakage) as well as enabling immune evasion (e.g., antibody neutralisation by sgp and by cleaved gp), roles that were previously solely attributed to vp24 and vp35. since project bioshield was initiated in 2004, there has been a substantial increase in our understanding of ebolavirus replication and pathogenesis. several vaccines and antiviral therapeutics have been developed and were used during the 2014-2016 west africa ebola virus outbreak. however, the outbreak was difficult to contain and highlighted the fact that there are many significant aspects of ebov that we do not yet understand (e.g., ebov persistence in immunologically privileged tissue) and that there is a pressing need for continued research to better understand and combat this deadly pathogen. • viral structural proteins, such as vp40 and gp, have been found to cause cell death in a wide range of cell types, including bystander t lymphocytes. • viral immune modulating proteins, such as vp24 and vp35, now have significantly expanded functions during virus replication, including the regulation of viral particle formation. 14. guito after ebola in west africa-unpredictable risks, preventable epidemics fruit bats as reservoirs of ebola virus persistence and genetic stability of ebola virus during the outbreak in kikwit, democratic republic of the congo ebola rna persistence in semen of ebola virus disease survivors-final report ebola virus in breast milk efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola {ç }a suffit!) proposal for a revised taxonomy of the family filoviridae: classification, names of taxa and viruses, and virus abbreviations risks posed by reston, the forgotten ebolavirus the pathogenesis of ebola virus disease structural characterization and membrane binding properties of the matrix protein vp40 of ebola virus the role of exosomal vp40 in ebola virus disease oligomerization and polymerization of the filovirus matrix protein vp40 structural rearrangement of ebola virus vp40 begets multiple functions in the virus life cycle a ppxy motif within the vp40 protein of ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding ebola vp40 in exosomes can cause immune cell dysfunction human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis overlapping motifs (ptap and ppey) within the ebola virus vp40 protein function independently as late budding domains: involvement of host proteins tsg101 and vps-4 ebola virus matrix protein vp40 interaction with human cellular factors tsg101 and nedd4 alix rescues budding of a double ptap/ppey l-domain deletion mutant of ebola vp40: a role for alix in ebola virus egress inducing cell proliferation inhibition and apoptosis via silencing dicer, drosha, and exportin 5 in urothelial carcinoma of the bladder could the ebola virus matrix protein vp40 be a drug target the natural compound silvestrol is a potent inhibitor of ebola virus replication productive replication of ebola virus is regulated by the c-abl1 tyrosine kinase the nonstructural small glycoprotein sgp of ebola virus is secreted as an antiparallel-orientated homodimer a new ebola virus nonstructural glycoprotein expressed through rna editing delta-peptide is the carboxy-terminal cleavage fragment of the nonstructural small glycoprotein sgp of ebola virus tyro3 family-mediated cell entry of ebola and marburg viruses ebolavirus glycoprotein structure and mechanism of entry ebola virus entry: a curious and complex series of events ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to npc1+ endolysosomes is a rate-defining step ebolavirus glycoprotein directs fusion through npc1+ endolysosomes ebola virus glycoprotein with increased infectivity dominated the 2013-2016 epidemic human adaptation of ebola virus during the west african outbreak functional characterization of adaptive mutations during the west african ebola virus outbreak spontaneous mutation at amino acid 544 of the ebola glycoprotein potentiates virus entry and selection in tissue culture biochemical basis for increased activity of ebola glycoprotein in the 2013-2016 epidemic a polymorphism within the internal fusion loop of the ebola virus glycoprotein modulates host cell entry the ebola virus glycoprotein contributes to but is not sufficient for virulence in vivo identification of the ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury ebola virus glycoprotein-mediated anoikis of primary human cardiac microvascular endothelial cells ebola virus glycoprotein toxicity is mediated by a dynamin-dependent protein-trafficking pathway the erk mitogen-activated protein kinase pathway contributes to ebola virus glycoprotein-induced cytotoxicity modeling of the ebola virus delta peptide reveals a potential lytic sequence motif ebola virus delta peptide is a viroporin ebola virus glycoprotein gp is not cytotoxic when expressed constitutively at a moderate level less is more: ebola virus surface glycoprotein expression levels regulate virus production and infectivity tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein ebola virus glycoprotein counteracts bst-2/tetherin restriction in a sequence-independent manner that does not require tetherin surface removal the tetherin antagonism of the ebola virus glycoprotein requires an intact receptor-binding domain and can be blocked by gp1-specific antibodies ebola virus glycoprotein promotes enhanced viral egress by preventing ebola vp40 from associating with the host restriction factor bst2/tetherin effects of ebola virus glycoproteins on endothelial cell activation and barrier function ebola hemorrhagic fever: novel biomarker correlates of clinical outcome ectodomain shedding of the glycoprotein gp of ebola virus shed gp of ebola virus triggers immune activation and increased vascular permeability ebolaviruses associated with differential pathogenicity induce distinct host responses in human macrophages ebola virus glycoprotein directly triggers t lymphocyte death despite of the lack of infection the myeloid lsectin is a dap12-coupled receptor that is crucial for inflammatory response induced by ebola virus glycoprotein antigenic subversion: a novel mechanism of host immune evasion by ebola virus structures of ebola virus gp and sgp in complex with therapeutic antibodies gp mrna of ebola virus is edited by the ebola virus polymerase and by t7 and vaccinia virus polymerases genomic rna editing and its impact on ebola virus adaptation during serial passages in cell culture and infection of guinea pigs key: cord-000113-d0eur1hq authors: fooks, anthony r.; johnson, nicholas; freuling, conrad m.; wakeley, philip r.; banyard, ashley c.; mcelhinney, lorraine m.; marston, denise a.; dastjerdi, akbar; wright, edward; weiss, robin a.; müller, thomas title: emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century date: 2009-09-29 journal: plos negl trop dis doi: 10.1371/journal.pntd.0000530 sha: doc_id: 113 cord_uid: d0eur1hq the diagnosis of rabies is routinely based on clinical and epidemiological information, especially when exposures are reported in rabies-endemic countries. diagnostic tests using conventional assays that appear to be negative, even when undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. these tests are rarely optimal and entirely dependent on the nature and quality of the sample supplied. in the course of the past three decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection of rabies virus. these tests enable viral strain identification from clinical specimens. currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. indeed the challenges in the 21st century for the development of rabies diagnostics are not of a technical nature; these tests are available now. the challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted validation of a test that will then lead to its acceptance by organisations globally. secondly, the areas of the world where such tests are needed are mainly in developing regions where financial and logistical barriers prevent their implementation. although developing countries with a poor healthcare infrastructure recognise that molecular-based diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and transferring technology through laboratory twinning between the developed and the developing countries. importantly for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human diseases that present with similar clinical symptoms. antemortem testing for human rabies is now possible using molecular techniques. these barriers are not insurmountable and it is our expectation that if such tests are accepted and implemented where they are most needed, they will provide substantial improvements for rabies diagnosis and surveillance. the advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of techniques capable of high throughput testing with a low turnaround time for rabies diagnosis. clinical and epidemiological information, especially when exposures are reported in rabies-endemic countries. diagnostic tests using conventional assays that appear to be negative, even when undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. these tests are rarely optimal and entirely dependent on the nature and quality of the sample supplied. in the course of the past three decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection of rabies virus. these tests enable viral strain identification from clinical specimens. currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. indeed the challenges in the 21st century for the development of rabies diagnostics are not of a technical nature; these tests are available now. the challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted validation of a test that will then lead to its acceptance by organisations globally. secondly, the areas of the world where such tests are needed are mainly in developing regions where financial and logistical barriers prevent their implementation. although developing countries with a poor healthcare infrastructure recognise that molecularbased diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and transferring technology through laboratory twinning between the developed and the developing countries. importantly for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human diseases that present with similar clinical symptoms. antemortem testing for human rabies is now possible using molecular techniques. these barriers are not insurmountable and it is our expectation that if such tests are accepted and implemented where they are most needed, they will provide substantial improvements for rabies diagnosis and surveillance. the advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of techniques capable of high throughput testing with a low turnaround time for rabies diagnosis. validated diagnostic tests that confirm the presence of rabies virus or a lyssavirus variant have been the foundation of rabies control strategies in many countries. historically, histopathological techniques such as the sellers stain technique [1] were used to determine the presence of negri bodies as rabies virus-specific antigen, however due to poor sensitivity and specificity this technique is no longer recommended by the world health organization (who). the fluorescent antibody test (fat) [2] relies on the ability of a detector molecule (usually fluorescein isothiocyanate) coupled with a rabies specific antibody forming a conjugate to bind to and allow the visualisation of rabies antigen using fluorescent microscopy techniques. microscopic analysis of samples is the only direct method that allows for the identification of rabies virus-specific antigen in a short time and at a reduced cost, irrespective of geographical origin and status of the host. it has to be regarded as the first step in diagnostic procedures for all laboratories. autolysed samples can, however, reduce the sensitivity and specificity of the fat. the rabies tissue culture infection test (rtcit) [3] and the mouse inoculation test (mit) [4] are based on the propagation and isolation of the virus. these diagnostic tests are used to detect virus particles either directly in tissue samples (fat) or indirectly in animals and in tissue culture (mit and rtcit, respectively). the rationale for the use of virus isolation (rtcit/mit) from a sample where there is a suspicion of infection with rabies virus is always recommended, especially when koch's postulates are likely to be met. such amplification of the viral pathogen facilitates additional molecular analysis to be undertaken, including sequencing of the viral isolate and subsequent phylogenetic analysis. conventional diagnostic tests for rabies (fat, rtcit, mit) are not labour intensive and rely upon low throughput. the fat can be completed in less than two hours. in contrast, both the rtcit and mit require longer turnaround times (4-days and 28-days, respectively). the fluorescent antibody virus neutralisation (favn) test [5] and the rapid fluorescent focus inhibition test (rffit) [6] utilise a similar principle, to measure the level of virus neutralising antibody in vaccinated individuals. 'indirect' serological methods, including the favn and rffit measure the host response to infection/vaccination only and do not detect the presence of infectious virus/antigen directly. however, host antibody detection (favn/rffit) is an indirect tool to measure the presence of rabies virus in a non-immunised individual by evaluating the host response to infection. the test may lack sensitivity and specificity, and the interpretation of the test results may be difficult as the host response to infection varies substantially between individuals. as such, the negative predictive value of serological tests for rabies diagnosis is considered poor. therefore, serological assays are not suitable as diagnostic tools for routine rabies testing. these internationally approved methods have provided accurate and timely information of animal and human rabies cases thereby supporting surveillance for rabies and providing a greater understanding of the epidemiology of this disease (box 1). for numerous laboratories in rabies-endemic regions in the developing world, cost and simplicity are critical factors in the delivery of disease diagnosis and cannot be neglected, even when the principal consideration is for rapid diagnosis. therefore, cost and simplicity need to be considered if new technologies are to be adopted in the regions of the world where they are most needed. molecular tools based on the detection and manipulation of the genetic information of the virus are becoming more widely accepted and accessible for the diagnosis of rabies. the advent of molecular biology is changing the face of diagnostic virology generally enabling high throughput and short turnaround-time analysis of samples. in the 21 st century, it is expected that diagnostic virology techniques for high throughput rabies virus detection will progress rapidly towards the use of molecular diagnostics replacing more conventional testing techniques such as virus isolation and histopathology. it is also possible that immunological tests, measuring 'surrogate' markers such as cytokines and electrolytes, will augment the standard diagnostic approach; nevertheless they will continue to remain oddities outside the realms of the routine diagnostic laboratory and be confined to a few reference laboratories. semi-automated or automated instruments and robotics-based techniques are useful when large numbers of the same test are undertaken and these tests will continue to increase in popularity and use, especially in central reference laboratories rather than in each local or regional facility. new technological advances will undoubtedly be faster, more accurate and may, in time, offer a cost-effective alternative to traditional rabies diagnostic tests. these paradigm shifts including modern advances in technology will lead to the effective control of rabies in animals and wildlife [7] (box 2). this review provides information on some of the latest developments and diagnostic techniques for determining the presence of rabies virus or nucleic acid in diagnostic samples. the principal focus of this review is to highlight the new developments in virology related to techniques for the diagnosis and surveillance of rabies. literature reviews were identified through web of science, pubmed and scopus using various search phrases. this review also drew on information provided to international organisations, mainly who and oie, funded by the uk department for environment, food and rural affairs (defra) in an advisory context on diagnostic and surveillance strategies for rabies. this review however, does not reflect the views of defra, who or oie. this review provides information on some of the latest developments and diagnostic techniques for determining the presence of rabies virus in diagnostic samples. our aim is to provide a viewpoint on the current thinking in diagnostic virology for rabies, reflecting the 'neglected' nature of this tropical disease and the contrasting needs of diagnostic laboratories in developed and developing countries. development of rapid immunohistochemical test (drit) in the evaluation of suspect rabies tissue samples a direct rapid immunohistochemical test (drit) for the postmortem detection of rabies virus antigen in brain smears has been developed [8] . using a cocktail of highly concentrated and purified biotinylated monoclonal antibodies, rabies antigen can be detected by direct staining of fresh brain impressions within 1 hour. this test employs anti-rabies monoclonal antibodies specific for the nucleoprotein, a viral protein produced in abundance during productive infection. the fat is based on antibodies specific for the same protein but, being conjugated to fluorescein isothiocyanate, requires a fluorescent microscope to visualise any specific antibody bound to viral protein within the test sample [2] . in contrast, the new box 1. key learning points drit antibody cocktail is biotinylated such that following a short incubation with a streptavidin-peroxidase complex, antibody-antigen binding complexes can be visualised through the addition of the substrate, 3-amino-9-ethylcarbazol. performed on brain tissues, the drit has proven as sensitive as the fat for fresh specimens [9, 10] . brain impressions stained using the drit technique can be read within one hour and the antibody cocktail used has been shown to detect classical rabies virus strains (genotype 1) that have been assessed [11] . currently, the fat is routinely used to detect virus antigen in badly decomposed sample material. for the purpose of testing samples in the developing world where suitable cold storage for samples is often unavailable, this factor is important in the development of new tests. this obstacle has been overcome through evaluating sample preservation in phosphate buffered 50% glycerol at a range of temperatures for different time periods prior to testing for virus antigen. glycerol saline solutions have been previously recognised as suitable storage media for tissue samples in the absence of cold storage [12] (box 2). using the drit in field studies in tanzania, viral antigen could be detected in samples after considerable time periods post collection regardless of the regimen of glycerol preservative used [11] . applications of the drit to analyse field samples in other rabies endemic regions have also proven highly successful. field trials in chad sought to study the drit in direct comparison to the fat to attempt to confirm previous studies as to the incidence of rabies within a district known to be endemic. in this study, results between the two tests were 100% in agreement [9] and the only issue regarding use of the drit over the fat was the need for the drit kit to be stored refrigerated prior to use. the drit will enable developing countries to perform routine rabies surveillance at greatly reduced cost and without the need for prohibitively expensive microscopic equipment along with the expertise and financial input needed to maintain them. the cost effectiveness of the drit will allow knowledge and technology transfer to areas of the developing world that currently are unable to diagnose rabies cases. another method for the detection of rabies virus antigen from postmortem samples is a recently developed rapid immunodiagwww.plosntds.org nostic test (ridt) based on the principles of immunochromatography [13] . the immunochromatographic lateral flow strip test is a one-step test that facilitates low-cost, rapid identification of various analytes including viruses [14] . briefly, suspect material is subjected to a test device similar to a lateral flow device. conjugated detector antibodies attached to two different zones on a membrane indicate the presence of viral antigen. preliminary validation studies with a limited number of samples showed that the ridt might have great potential as a useful method for rabies diagnosis without the need for laboratory equipment [13] . however, thorough validation including various circulating variants of rabv and other lyssaviruses is still needed before this test could be relied upon and be used as an alternative for the gold standard fat. reverse-transcriptase polymerase chain reaction (rt-pcr) various conventional rt-pcr protocols for the diagnostic amplification of lyssavirus genome fragments have been published (tables 1-3 ). since primers were selected from conserved regions of the genome, most assays amplify parts of the nucleoprotein (n-) gene as earlier proposed [15] . in generic approaches intended to detect all lyssaviruses either hemi-nested or fully nested amplifications are used and have applications for both antemortem (saliva, csf, brain) and postmortem samples (principally brain tissue) ( table 2 ). some of these diagnostic procedures are also applied for further virus characterization, including sequencing reactions [16] or restriction fragment length polymorphism (rflp) [17] . also, strain-specific rt-pcrs have been developed to distinguish various rabies virus (rabv) strains in a particular region [18] . classical rt-pcr assays proved to be a sensitive and specific tool for routine diagnostic purposes [19, 15] , particularly in decomposed samples [20, 21, 22] or archival specimens [23, 24] . the sole detection of amplified rt-pcr products by gel-based systems however, especially when using hemi-nested rt-pcrs generates the risk of cross-contamination, does not allow an exact quantification of genome copies and does not include tests for specificity [25] . hybridisation methods [26] and pcr-elisa methods were established to overcome these difficulties [27] , although these techniques have not become universally accepted. additionally, many laboratories now use partial sequencing to confirm the detection of a lyssavirus and obtain data that can be used in a phylogenetic analysis of viruses circulating in a specific region. the importance of sequencing the pcr products was highlighted in an experimental study [28] . this study demonstrated that although the nested rt-pcr was shown to be the most sensitive of the diagnostic techniques employed, host genomic amplicons of the same size as the target amplicons were observed on the agarose gels, which were subsequently confirmed as false positives following direct sequencing [28] . with the introduction of fluorogenic probes, detection of sequence specific templates can be achieved in real-time. specificity is ensured by an inherent hybridization reaction, and cross-contamination is avoided due to the closed tube nature of the test [29, 30] . consequently, for rabv and other lyssaviruses, various pcr assays using taqman technology have been described (tables 4-5) . a generic real-time taqman-pcr for the detection and differentiation of lyssavirus genotypes 1, 5, and 6 has also been developed [31] . this assay utilises a pan-lyssavirus primer set, which has been shown to amplify a large panel of representative lyssaviruses, with probes specifically designed to discriminate between classical rabies virus and the european bat lyssaviruses type-1 and -2 (eblv-1 and eblv-2). pcr assays using taqman technology have applications for antemortem and postmortem samples. the pan-lyssavirus primer www.plosntds.org set can also be used in conjunction with a specific dye such as sybr green to allow for rapid detection of the amplicons. validation of probe based assays relies on the availability of representative viruses or nucleic acid. however, for some lyssavirus genotypes only a limited number of viruses or sequences are available for primer/probe design, and they may not represent the genetic diversity of all current variants that are circulating. single mutations for the north american rabv strains [32] in the region of the primers or the probe can alter the sensitivity of the pcr. thus the genetic diversity among lyssaviruses may hamper the use of a single assay for all lyssaviruses. as such scanning surveillance may benefit more from the use of a pan-lyssavirus primer sybr green assay rather than a strain or specific based assay. the use of a rapid automated nasba technique was successfully applied to the ante-mortem saliva and cerebrospinal fluid (csf) of four rabies patients in thailand and shown to have a ten-fold increase in sensitivity compared to rt-pcr [33] . the assay detected rabies viral rna as early as two days after onset of symptoms. the nasba technique involves the use of three enzymes (reverse transcriptase, rnase h and t7 rna polymerase) to synthesise multiple copies of target rna under isothermal conditions. briefly, a large number of rna copies are generated using a pair of specific primers, one of which contains the t7 rna polymerase binding site, and the other which has an electrochemiluminescence detection region attached to the 59 end. the table 3 . conventional, gel-based pcr-assays for the detection of lyssavirus species other than rabv. www.plosntds.org amplified rna is detected using an automated reader, enabling rapid throughput testing. it is relatively easy to use and the whole process from extraction to detection can take as little as four hours. this technology has already been applied for point of care testing of bacterial pathogens [34] and viral pathogens [35, 36] . the nasba technique has also been adapted to investigate rabies virus replication in situ, whereby the relatively lower isothermal temperatures of nasba compared to in-situ rt-pcr ensure that cell integrity is maintained [37] . lamp offers an alternative dna amplification method to the polymerase chain reaction for applications to the ante-mortem saliva and csf testing. the originators of the technique suggest that it amplifies with high specificity, efficiency and without the need for thermal cycling [38] . amplification is achieved through the specific binding of two inner and two outer primers to the target sequence. the inner primers initiate strand synthesis whilst the outer primers displace the inner primers, allowing them to self-anneal to the nascent strand. this creates hairpin structures that trigger further strand synthesis that in turn lead to concatenation of the target sequence [38] . polymerisation and strand displacement are achieved using a single enzyme, bst 1 dna polymerase. the technique is rapid, generating large quantities of target sequence within minutes. for the amplification of rna viruses, a reverse transcription step is undertaken prior to the lamp reaction. primer sets have been successfully developed to detect a range of pathogenic viruses including west nile virus [39] , japanese encephalitis virus [40] , foot and mouth disease virus [41] and chikungunya virus [42] . to assess the applicability of lamp to the detection of rabies virus we designed a primer set using primerexplorer v4 software (eiken chemical company ltd., japan) that can detect the challenge virus standard (cvs) fixed strain of rabies virus (table 6 ). in addition to the standard set of four primers, two further loop-binding primers have been added to increase the rate of strand displacement and synthesis [43] . the reverse transcription and lamp reactions were undertaken simultaneously (rt-lamp) in a single tube at 65uc using a www.plosntds.org thermostable reverse transcriptase, hence avoiding the step process inherent in an rt-pcr. target amplification was monitored by the incorporation of the double stranded dna binding fluorophore picogreen (figure 1a) or by separation on a 1% agarose gel (figure 1b) . at a constant temperature of 65uc, cvs rna could be detected within 30 minutes. development of rt-lamp assays for use in diagnosis and surveillance is challenged by the considerable sequence variation observed within the rabies virus genome [44] that can frustrate specific primer design. preliminary attempts at this suggest that multiple combinations of primers (up to 12 different primers) can lead to sensitive, rapid amplification of rabv genomes from a wide range of geographical locations. the use of isothermal amplification has the benefit of reducing the technological requirements of thermal cycling used in rt-pcr. this in turn offers the opportunity, when linked with lateral flow devices, to develop surveillance protocols where testing can take place in the field or in less sophisticated laboratories. microarray linked to sequence independent pcr amplification offers the ability to rapidly identify pathogenic viruses from postmortem samples [45, 46, 47] . we have undertaken a study that has demonstrated the ability of a microarray to detect each of the seven lyssavirus genotypes (vla weybridge, unpublished data). the microarray is composed of oligonucleotide probes 70 nucleotides in length and includes probe sets for each of the seven classified genotypes and sets for the unclassified lyssaviruses. the www.plosntds.org diagnostic capability of the array was illustrated showing the ability of the array to detect rabv in a human case of rabies as the amplified rna bound specifically to the classical rabies virus (genotype 1) probe set ( figure 2 ). development of a novel ultrasensitive and stable potentiometric immunosensor a stable potentiometric immunosensor for the detection of various analytes of interest from complex real world samples such as blood, serum and milk has been described [48] . the biosensor detects enzyme labelled immunocomplexes formed at the surface of polypyrrole coated, screen-printed gold or silver electrodes. detection is through a secondary reaction that produces charged products with a shift in potential, being measured by local changes in redox state, ph and/or ionic strength. the magnitude of the change in potential is directly related to the level of target in the matrix such that the assays are quantitative and the numerical output is rapidly transmissible. the bioassays produce very rapid results (5-15 minutes), are highly reproducible (%cv,5%) and are ultra-sensitive (,50 fm). thus this sort of biosensor offers the rapidity of production of signal of a lateral flow device with the sensitivity of third generation elisas. immunoassays can be developed in a sandwich or competitive format for small (e.g. digoxin; mw 780 da) or large (e.g. hepatitis b surface antigen; .300 kda) molecules [49] . in addition to immunoassays, it is also possible to detect specific nucleic acids and whole cells using the technology. due to the method of production of the electrode strips, the base assays are inexpensive (,us$1 per strip) as is the detection hardware (us$2,000). in addition, most immunoassays can be readily adapted to this format with minimal additional optimisation. potentiometric immunosensors for detecting the rabies virus nucleoprotein are in progress and offer the ability to rapidly screen complex non-clarified matrices, possibly at pen-side, in a cost-effective manner. serological assays are not suitable as diagnostic tools for routine rabies testing as the host response to infection varies substantially between individuals. however, serology is still useful, particularly to monitor the development of the immune response. we would suggest that detection of rabies antibodies in serum and csf, early after presentation and in the absence of a history of vaccination may be a positive indicator for a therapeutic intervention. viral pseudotypes, the core of one virus coated with envelope protein derived from a second virus, offers a safe alternative to the use of pathogenic viruses in neutralisation assays. using pseudotypes expressing genotype 1 cvs-11 glycoprotein, high titre stocks (1.3-3.2610 5 infectious units/ml) were produced that proved 100% specific and highly sensitive compared with neutralisation titres achieved using the favn [50] . a high correlation was also observed (r = 0.89). using pseudotypes expressing eblv-1 (genotype 5) and eblv-2 (genotype 6) g-proteins, neutralising antibody titres broadly correlated with the degree of g-protein diversity. a vaccine study in tanzania compared the two assays with pseudotypes showing 100% specificity and 94.4% sensitivity to the favn with a high correlation of antibody titres (r = 0.92). incorporation of lagos bat virus (genotype 2), mokola virus (genotype 3) and duvenhage virus (genotype 4) g-proteins, as well as lacz as a reporter gene, makes the pseudotype assay an attractive option for serosurveillance in africa and other resource limited countries. in addition, as the pseudotype assay uses substantially less input serum (10 ml) compared to favn and rffit, multiple tests can be undertaken on samples where collection volumes are limited or valuable e.g. bat sera. due to the neurotropic nature of rabies virus, infection results in enormous viral replication in the cns in the final stage of the disease that leads to massive antigen and viral genome concentrations. this makes detection of viral antigen in brain tissue by tests such as the fat or the drit [7] very robust and relatively simple to perform, and these have become rapid gold standard tests. as for detection of viral genome, approaches are now available which process multiple specimens from nucleic acid extraction through to genetic typing, with significantly reduced risks of contamination. in addition, the use of taqman rt-pcr or similar technologies on robotics platforms, allow for rapid largescale rabies detection, typing and quantification in real time [32, 31, 51] . the development of pcr-based methods (box 2) provided an alternative method for post mortem rabies diagnosis [26] , and the possibility of ante mortem diagnosis of human rabies [52] . rt-pcr methods invariably involve multiple transfers of nucleic acids between different tubes. coupled with the high sensitivity of pcr methodologies, any small amount of contamfigure 1 . amplification of rabies virus rna by reverse transcription loop-mediated isothermal amplification (rt-lamp). for each reaction 1 mg rna, prepared using trizol (invitrogen) from normal mouse brain (1a, circles; 1b 2) or infected mouse brain (1a, triangles; 1b, +) was added to a reaction mixture containing all six primers at concentrations indicated in table 1 ination will undoubtedly produce false-positive results. attempts have been made to adapt rt-pcr to reduce manipulations thereby reducing contamination risks. the visualisation of pcr products by gel electrophoresis exposes facilities and operators to large quantities of amplified material and thus many adaptations have been directed at replacing this step. new and improved rapid diagnostic tools for rabies using taqman technology have been developed that avoid cross-contamination due to the closed tube nature of the test [29, 30] . a further benefit of rt-pcr has been to enable practical molecular characterization of rabies viruses [26] that has added significantly to the understanding of virus evolution and epidemiology. this approach has superseded the use of monoclonal antibodies for typing and characterising new strains of rabies virus. this has provided the evidence to support the delineation of lyssaviruses into genotypes [53] (box 2) and was used for the classification of another four putative members of the genus [54, 55] . also, this technique was a prerequisite for the understanding of the molecular biology of lyssaviruses [56] and underpinned future developments in rabies diagnosis and prevention. it is likely that in the future microarray techniques in combination with sophisticated bioinformatics and arrays with a hierarchical set of probes will provide an alternative to rapid virus discovery and characterisation. the development of 'real-time' rt-pcr techniques allows the quantification of this rna in 'real-time,' giving a relatively quick and reliable method for the measuring levels of viral rna. pcr based techniques are not currently recommended by the who for routine post-mortem diagnosis of rabies. however, in laboratories with strict quality control procedures in place and demonstrable experience and expertise, these molecular techniques have been successfully applied for confirmatory diagnosis and epidemiological surveys. for these reasons, it is likely that international bodies will accept their use in the future for routine rabies diagnosis. reverse transcription pcr has been reported to confirm rabies diagnosis intra-vitam in suspect human cases, when conventional diagnostic methods have failed and post-mortem material is not available (box 1) [57] . rabies virus rna can be detected in a range of biological fluids and samples (e.g. saliva, csf, tears, skin biopsy sample and urine). owing to the intermittent shedding of virus, serial samples of fluids such as saliva and urine should be tested but negative results should not be used to exclude a diagnosis of rabies. all positive pcr results should be sequenced to confirm the origin of the virus and rule out possible contamination. in terms of the rna concentrations in the brain, the sensitivity especially of nested or real-time pcrs may be beyond the threshold needed for routine post-mortem testing. also, contamination of negative samples could lead to an unjustifiable administration of a high number of costly post exposure prophylaxis and would produce false data for the rabies surveillance. however, with the introduction of accreditation for laboratories, quality control measures are being implemented in a growing number of laboratories worldwide. such quality controls for diagnostic rabies pcrs should encompass several measures, including the inclusion of appropriate positive, negative, and inhibition controls in assay runs. the consistency and the interassay reproducibility should also be ensured over time by monitoring performance. only if laboratories meet the required standard [58] , can pcr fulfil its full potential. the use of pcr should not be restricted only as a confirmatory diagnostic test for decomposed samples but also as a powerful tool for virus typing figure 2 . microarray identification of rabies virus rna prepared from a brain sample from a confirmed case of human rabies. total rna was extracted using trizol (invitrogen) and treated with dnase i prior to conversion to double stranded dna [45] . non-specific amplification was achieved using a dna polymerase (klen taq, sigma) and the products were labelled through binding of alexa fluor 555 reactive dye (invitrogen) to amplicons. labelled target dna was denatured at 95uc and chilled on ice before dilution in hybridization buffer and addition to the microarray slide. hybridization occurred at 50uc overnight. slides were washed and the target-probe binding was captured using genepix pro 6.1 software (molecular devices). statistical analysis of the data was conducted using detectiv software [64] . doi:10.1371/journal.pntd.0000530.g002 and molecular epidemiology studies. the lack of standardization is a major obstacle to the general use of pcr for rabies diagnosis, especially in developing countries. it is evident that the rt-pcr dominates genetic detection of rabies virus and it seems probable that this technique will dominate rabies diagnosis in the 21 st century. however, we should not discount alternatives that have the benefit of isothermal amplification that will enable implementation in laboratories where access to thermal cyclers is an obstacle. a nasba technique was successfully applied to the saliva and csf of four living patients with rabies and detected rabies viral rna 2-days after the onset of symptoms. this technique has also been adapted to investigate rabies virus replication in-situ. lamp also falls into this category and can be adapted for use with lateral flow devices thus making its application very simple. existing assays for rabies virus antibody prevalence studies either require high containment facilities or do not distinguish between neutralising and non-neutralising antibodies [59] [60] . recently however, a neutralisation assay using retroviral pseudotypes was described [50] , not bound by the restrictions listed above and also allowing a choice of endpoint reporter proteins (bgalactosidase, green fluorescent protein or luciferase) [61] . a further benefit of this technique is its adaption to using small volumes of sera thus making them useful for surveillance. currently, high-throughput rabies virus molecular detection methods augment standard diagnostic tests or are in the process of development and refinement for use alone. as we progress through the 21 st century, it is possible that these techniques will replace conventional tests (box 1). obstacles to adoption include cost, complexity and local acceptance of their use. it is also possible that immunological tests by measuring 'indirect' markers such as cytokines and electrolytes will increase in use. these tests however, will probably remain in the realm of large reference laboratories where resources allow the development of novel assays. as far as semi-automated or automated instruments and robotics-based techniques are concerned, they are useful when large numbers of the same test are undertaken such as surveillance and companion animal testing and these tests will continue to increase in popularity and use, especially in central reference facilities. there is a clear need to simplify molecular diagnostic techniques so these tests can be applied universally in developing and developed countries. it is likely that new developments will focus on generating low volume and yet affordable diagnostic tests for rabies. more use will be made of point-of-care (poc) diagnostic testing using portable extraction techniques linked to pcr machines with the use of lyophilised reagents to overcome coldchain dependencies in tropical countries. in the 21 st century, these technologies will have a demonstrable impact on people living in developing countries, especially where rabies is still considered a 'neglected' disease. by contrast in the developed world, these new technological advances will undoubtedly be faster, more accurate and cost-effective leading to a 'theragnostics approach' that combines therapeutics with diagnostics for the human treatment of rabies. interest in treating human rabies aggressively is gaining momentum, largely due to the reported success in treating a 15year-old girl, in whom clinical rabies developed one month after she was bitten by a bat, using a combination of therapeutic coma with antiviral drugs whilst allowing for the host immune system to confer immunity -the 'milwaukee protocol' (box 2) [62, 63] . bioinformatics, genomics, proteomics, and functional genomics are the molecular biology tools that are essential for the progress of molecular 'theragnostics', where both early diagnosis and monitoring of serology are critical factors for the successful treatment of a rabies patient. in addition, theragnostics could eliminate the unnecessary treatment of patients for whom rabies immunotherapy is not appropriate i.e. immunosuppressed patients, resulting in substantial drug cost savings for the healthcare system. rapid microscopic examination for negri bodies and preparation of specimens for biological tests the fluorescent antibody test. in laboratory techniques in rabies les techniques rapides de diagnostic de laboratoire de la rage early diagnosis of rabies by mouse inoculation. measurement of humoral immunity to rabies by mouse protection test development of a fluorescent antibody virus neutralisation test (favn test) for the quantitation of rabies-neutralising antibody a rapid reproducible test for determining rabies neutralizing antibody slate d (2006) control and prevention of rabies in animals: paradigm shifts standard operating procedure for the direct rapid immunohistochemistry test for the detection of rabies virus antigen. national laboratory training network course. atlanta: us department of health and human services rabies diagnosis for developing countries the primary application of direct rapid immunohistochemical test to rabies diagnosis in china evaluation of a direct, rapid immunohistochemical test for rabies diagnosis simple technique for the collection and shipment of brain specimens for rabies diagnosis evaluation of a rapid immunodiagnostic test kit for rabies virus immunochromatographic lateral flow strip tests the polymerase chain reaction (pcr) technique for diagnosis, typing and epidemiological studies phylogenetic comparison of the genus lyssavirus using distal coding sequences of the glycoprotein and nucleoprotein genes identification of regional variants of the rabies virus within the canadian province of ontario polymerase chain reaction protocols for rabies virus discrimination pcr technology for lyssavirus diagnosis rabies virus detection by rt-pcr in decomposed naturally infected brains heminested reversetranscriptase polymerase chain reaction (hnrt-pcr) as a tool for rabies virus detection in stored and decomposed samples rabies virus in the decomposed brain of an ethiopian wolf detected by nested reverse transcription-polymerase chain reaction an evaluation of immunofluorescence and pcr methods for detection of rabies in archival carnoy-fixed, paraffin-embedded brain tissue usefulness of reverse transcriptasepolymerase chain reaction for detection of rabies rna in archival samples molecular diagnosis of animal diseases: some experiences over the past decade pcr technique as an alternative method for diagnosis and molecular epidemiology of rabies virus molecular methods to distinguish between classical rabies and the rabiesrelated european bat lyssaviruses experimental infection of big brown bats (eptesicus fuscus) with eurasian bat lyssaviruses aravan, khujand, and irkut virus a novel method for real time quantitative rt-pcr real time quantitative pcr development of a real-time, taqman reverse transcription-pcr assay for detection and differentiation of lyssavirus genotypes 1, 5, and 6 evaluation of a taqman pcr assay to detect rabies virus rna: influence of sequence variation and application to quantification of viral loads nucleic-acid sequence based amplification in the rapid diagnosis of rabies integrated microfluidic tmrna purification and real-time nasba device for molecular diagnostics multi-analyte single-membrane biosensor for the serotype-specific detection of dengue virus parallel nanoliter detection of cancer markers using polymer microchips isothermal amplification of rabies virus gene loop-mediated isothermal amplification of dna real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus rapid detection and quantification of japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplification novel reverse transcription loopmediated isothermal amplification for rapid detection of foot-and-mouth disease virus rapid and real-time detection of chikungunya virus by reverse transcription loop-mediated isothermal amplification assay accelerated reaction by loop-mediated isothermal amplification using loop primers host switching in lyssavirus history from the chiroptera to the carnivora orders microarray-based detection and genotyping of viral pathogens microarrays for rapid identification of plant viruses microassay-based detection of viruses causing vesicular or vesicular-like lesions in livestock animals an ultrasensitive and stable potentiometric immunosensor a novel hepatitis b virus surface antigen immunoassay as sensitive as hepatitis b virus nucleic acid testing in detecting early infection a novel serological assay for the detection of rabies virus neutralising antibodies development of a taqman real-time rt-pcr assay for the detection of rabies virus intravitam diagnosis of human rabies by pcr using saliva and cerebrospinal fluid phylogenetic relationships of irkut and west caucasian bat viruses within the lyssavirus genus and suggested quantitative criteria based on the n gene sequence for lyssavirus genotype definition bat lyssaviruses (aravan and khujand) from central asia: phylogenetic relationships according to n, p and g gene sequences primary structure of leader rna and nucleoprotein genes of the rabies genome: segmented homology with vsv case report: rapid ante-mortem diagnosis of a human case of rabies imported into the uk from the philippines expert consultation on rabies longitudinally profiling neutralizing antibody response to sars coronavirus with pseudotypes a sensitive retroviral pseudotype assay for influenza h5n1-neutralizing antibodies retroviral pseudotypes survival after treatment of rabies with induction of coma applying the milwaukee protocol to treat canine rabies in equatorial guinea. scand detectiv: visualisation, normalisation and significance testing for pathogen-detection microarray data rabies in a captive colony of big brown bats (eptesicus fuscus) a heminested polymerase chain reaction for the detection of brazilian rabies isolates from vampire bats and herbivores regular exposure to rabies virus and lack of symptomatic disease in serengeti spotted hyenas preliminary report on a single-tube, non-interrupted reverse transcription-polymerase chain reaction for the detection of rabies virus in brain tissue heminested pcr assay for detection of six genotypes of rabies and rabiesrelated viruses molecular diagnosis of lyssaviruses and sequence comparison of australian bat lyssavirus samples screening of active lyssavirus infection in wild bat populations by viral rna detection on oropharyngeal swabs rt-pcr for detection of all seven genotypes of lyssavirus genus susceptibility of north american big brown bats (eptesicus fuscus) to infection with european bat lyssavirus type 1 susceptibility of ferrets (mustela putorius furo) to experimentally induced rabies with european bat lyssaviruses (eblv) possible emergence of west caucasian bat virus in africa encephalitis caused by a lyssavirus in fruit bats in australia ante mortem diagnosis of human rabies using saliva samples: comparison of real time and conventional rt-pcr techniques comparison of realtime pcr and heminested rt-pcr methods in the detection of rabies virus infection in bats and terrestrial animals we wish to acknowledge the following for constructive comments and reviewing the manuscript: drs. charles trimarchi (usa), jim powell (usa), david schnurr (usa) and daniel horton (uk). the opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the authors or the institutions with which the authors are affiliated. key: cord-004247-lagv3tp7 authors: hooft van huijsduijnen, rob; kojima, somei; carter, dee; okabe, hisafumi; sato, akihide; akahata, wataru; wells, timothy n. c.; katsuno, kei title: reassessing therapeutic antibodies for neglected and tropical diseases date: 2020-01-30 journal: plos negl trop dis doi: 10.1371/journal.pntd.0007860 sha: doc_id: 4247 cord_uid: lagv3tp7 in the past two decades there has been a significant expansion in the number of new therapeutic monoclonal antibodies (mabs) that are approved by regulators. the discovery of these new medicines has been driven primarily by new approaches in inflammatory diseases and oncology, especially in immuno-oncology. other recent successes have included new antibodies for use in viral diseases, including hiv. the perception of very high costs associated with mabs has led to the assumption that they play no role in prophylaxis for diseases of poverty. however, improvements in antibody-expression yields and manufacturing processes indicate this is a cost-effective option for providing protection from many types of infection that should be revisited. recent technology developments also indicate that several months of protection could be achieved with a single dose. moreover, new methods in b cell sorting now enable the systematic identification of high-quality antibodies from humanized mice, or patients. this review discusses the potential for passive immunization against schistosomiasis, fungal infections, dengue, and other neglected diseases. the last three decades have seen a dramatic rise in the use of monoclonal antibodies (mabs) as therapeutics. by 2017, a total of 78 antibodies had been approved by the us food and drug administration (fda) or the european medicines agency (ema) [1] , with a further 11 approved by the fda in 2018 [2, 3]. beyond this, over 570 antibodies are in clinical development [4] . the global mab market reached us$100 billion annually in 2017 [5], underscoring the considerable economic importance of these medicines. the success of mabs starts with the general applicability of the technology used to make them. antibodies can be developed that have not only high affinity for their targets but also high selectivity, meaning they are less likely to have unwanted side effects and unexpected safety problems. mabs are particularly good at targeting cell-surface proteins and circulating protein factors; this is in contrast to small molecules, in which cell surface protein-protein a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in infectious disease, the biggest success story has been the use of antibodies to provide prophylaxis against infection by respiratory syncytial virus (rsv). rsv is responsible for over 30 million episodes of new lower respiratory tract infections, particularly targeting children 5 years and younger, resulting in an estimated 48,000 to 74,500 deaths globally (2015 estimates) [14] . palivizumab, dosed at 15 mg/kg, is given as a monthly intramuscular injection throughout the rsv season, which is suitable for small babies but would not be appealing for adults. a follow-on antibody-medi8897, now in phase ii clinical studies [15]-is 100 times more potent than palivizumab in vitro. it is being developed as a single 50 mg injection to cover the typical 5-month season. taken together, these cases illustrate, that after initial proof of concept, it is possible to find second-generation antibodies with more simple routes of delivery and sufficient potency to support relatively infrequent dosing in the field. a major objection to the use of mabs in infectious diseases as a therapeutic class is their high price, which is presumed to be a consequence of a high cost of goods. manufacturing costs are a particularly important attribute for any medication that is developed for diseases of poverty in low-and middle-income countries (lmics). however, production efficiency of mabs has increased dramatically over recent decades, and cell-culture expression levels around 4 g/l or even higher are common. a published analysis from a decade ago already noted that the costs of mab production were dropping from us$300/g to us$20/g when being produced at the 10-tonne-per-year scale, potentially using large, 100,000 l reactors [16] . a more recent analysis has similar estimates [17] , which-depending on process and volume-range from us$20/g to us$80/g [18] . for a public health application against an infectious disease in an lmic, an intramuscular or subcutaneous delivery would be preferable (rather than the intravenous route). the intramuscular route effectively limits the dose; an injection volume of 1.0 ml is probably close to the maximum acceptable in small children, and antibodies are typically only soluble to around 100 mg/ml, which gives a limitation on the total dose of around 100 mg, corresponding to a range of 5 to 20 mg/kg [19] . taken together, the cost of goods of an injection based on current numbers would be in the range of us$1 to us$8. benchmarking such costs is difficult, but as a comparison, the recently launched malaria vaccine provides less than 50% protection for 3 injections at around us$5 each. the programmatic cost of protecting a child from malaria for a year in the sahel using existing low-cost oral medicines has been estimated at us$3.40 by unitaid [20] . an injectable therapeutic that could protect a child for a season for this price provides a cost-effective option. the other important factor to manage is the reduction of the frequency of administration, to make the prophylaxis useful in resource-poor settings. mabs used in therapy are generally iggs (immunoglobulin gs), which have plasma half-lives of 20 to 25 days in humans [21, 22] . such antibodies can be used to provide several months of protection, as in the case of the once-per-season anti-rsv antibody medi8897 (nirsevimab) discussed earlier or the anti-calcitonin gene-related peptide (anti-cgrp) fremanezumab, recently approved for prevention of migraines with a dose of 675 mg every 3 months. an alternative to simply increasing the dose would be increasing the half-life of the circulating mab. several strategies have been proposed here [23] : mutations can be made in the igg fc (fragment crystallizable) region, which interacts with the receptors responsible for the uptake of antibodies. such mutations, as for medi8897, can significantly extend an mab's plasma half-life. mutations derived from the neonatal fc receptor (fcrn) increase the strength of the interaction between the fc region and the fc receptors under the acidic ph conditions in lysosomes, and as a result, the receptor-fc complex is recycled back to the cell surface, escaping degradation [24, 25] . this approach was followed for bevacizumab and cetuximab, by mutating met 428 to leu and asn 434 to ser. discussion about even longer periods of protection has been stimulated by recent results from virally mediated expression of antibodies targeting hiv [26] . these technologies hold out the promise of sustained serum titers of antibodies for more than a year from a single injection, although the technological development is more complicated than for the direct injection of antibodies. one final objection to the goal of administering monoclonals is that vaccination would instead provide lifelong coverage. however, for some diseases, the development of long-lasting potent vaccines still remains elusive. many pathogens will not raise an adequate immune response, due to immunosuppression. finally, antibodies give immediate protection, important during rapidly evolving epidemics or pandemics and for individuals being deployed into zones of high transmission. antibodies would thus form part of the overall armamentarium, along with drugs, bed nets and insecticides, and vaccines. first and foremost for the development of mabs is the question of suitable antigen targets. viruses are the simplest pathogens, and their genomes allow limited means to evade host immunity; therefore, passive and active vaccinations have been most successful in this area. therapeutic antibodies have been developed and marketed for respiratory syncytial, varicella zoster, vaccinia, and hepatitis b viruses (hbvs) [27, 28] . historically, immune responses against viruses were considered purely cellular and those against other types of pathogens mostly humoral (antibody based) and less effective. however, nowadays the interplay between both systems is better understood, for instance, with therapeutic opportunities against viruses such as rabies that rely on passive immunization. mabs that target the initial stages of an infection may provide prophylaxis, and the repertoire of potential antigens for such approaches is greatly helped by the increased availability of genomic and transcriptional information. passive immunization is especially effective in infections, such as those with the rabies virus, which avoids apoptosis of infected neurons while killing protective t cells so that carriers do not mount an immune response themselves [29] . a recent review [30] lists 21 mabs (and a nanobody, a single-chain type of antibody produced by camelids) that are currently in clinical trials in the united states. in addition, polyclonal antibodies have been approved in the european union or the us for cytomegalovirus, hepatitis viruses a and c, and postexposure prophylaxis against measles, rabies, rubella, and tetanus [31]. these approaches appear most suitable for virus infections and for protection against the consequences of snake venom but might also be applied to other types of disease. there are fewer marketed antibodies that target bacteria, and these target toxins, namely anthrax and clostridium cytotoxin [27] . this paucity may be due to a lack of discovery efforts, a consequence of the historic availability of cheap and effective antibiotics. however, with the worrying rise of antibiotic resistance, the tide is turning [32] . immunization against protist parasites and fungi is generally ineffective. one problem is that organisms such as trypanosoma brucei (which causes sleeping sickness) and plasmodium (malarial parasites) encode dozens or even hundreds of different surface antigens that are sequentially exposed at their most abundant stage, outpacing the host's immune system. for some pathogens, clinical trials in infected patients are not possible or desirable. in the case of inhalational anthrax, raxibacumab has been approved using the fda "animal rule" [33]. this provides a route forward in disease areas in which trials with infected human patients are not possible or not ethical. recently, the case for discovering mabs for neglected tropical and other infectious diseases was reviewed [34] . this work took a broad, category-wide approach (viruses, bacteria, parasites) and also included venoms. snake bites, and the challenge of envenoming, represent a serious health problem, resulting in 81,000 to 138,000 deaths yearly [35] , and were recently included in the world health organization (who) list of neglected tropical diseases. the discovery of mabs in this area logically follows the long-standing therapeutic successes with passive immunization [36] , and the costs and feasibility of developing mabs were recently reviewed [34, 37] [38], including specific clinical development and regulatory challenges [39] . in this review, we selected diseases prioritized by the global health innovative technology (ghit) fund, an international collaboration between the public and private sectors, supporting collaborations between japanese and non-japanese entities to advance global health research and development [40, 41] . the present vaccine discovery statuses for these diseases are listed in table 1 . human schistosomiasis is caused by infection, mainly with one of 4 species of the blood fluke belonging to schistosomatoidea. it is estimated that there are over 200 million cases annually in africa, with two-thirds being schistosoma haematobium and the remainder s. mansoni. in southeast asia, other species such as s. japonicum and s. mekongi cause smaller numbers of infections [64] . the mortality associated with schistosomal infections is difficult to calculate. comorbidities linked to anemia are common, and infection with s. haematobium can lead to female genital schistosomiasis, which results in increased susceptibility to hiv infection [65] . the life cycle of the parasite is complex, involving both a snail and a human host. in the infective stage, cercariae migrate and penetrate the human host skin to become schistosomula, which then move with the venous circulation, initially to the lungs. after further maturation, the parasite migrates via the heart to the portal circulation and the intestines; from there, eggs are excreted with the feces or, passing through the bladder wall, in urine. prophylaxis could therefore either be causal-and prevent entry and survival of the cercariae-or suppressive, by killing the juvenile and adult worms. clearly, as with the malarial parasites, the more stages of the parasite life cycle that can be inhibited, the more effective the protection will be. the molecular targets best addressed by antibodies are still not clear. some life cycle differences can be characterized: schistosomula recovered from the lungs are elongated and more resistant to antibody-dependent, cell-mediated cytotoxicity than newly transformed schistosomula. praziquantel, the standard treatment for schistosomiasis, acts only against adult worms and has no effects on the juveniles, thus repeat treatment is required to eliminate all the worms in a patient. an ideal treatment would therefore kill both adult and juvenile worms at a submicromolar concentration. this is effectively the target candidate profile for a small molecule inhibitor [66] . no target-product profile (tpp) for prophylaxis against schistosomiasis has been published, but it is possible to start to build one, with the recent characterization of praziquantel for such use in murine models and in pediatrics. clinically, this treatment reduces infection by 70% to 80% using doses of 40 to 60 mg/kg [67] . in a murine model with infection by s. mansoni cercariae, the maximum dose of 400 mg/kg killed 96% of all adult worms [68]. however, this murine dose produced a peak exposure of 6 ng/ml, some 10-fold higher than the exposure observed in children at the recommended regimen of 40 to 60 mg/kg [69, 70] . it seems pragmatic to project that an antibody that can reduce the worm burden by 80% in the mouse model, at a dose that is clinically achievable in humans, could be acceptable. the maximally practicable injection of an antibody in children is 2 to 5 mg/kg, and given the relatively consistent allometry between humans and mice for mabs, this corresponds to a dose of 80 to 200 μg in mice [19] . target identification for schistosomiasis is not straightforward. unlike malaria, the vaccine field offers no clues as to useful antibody targets. two vaccine candidates are currently being tested in humans. the first is based on the fatty acid-binding protein (fabp) sm-14 (schistosoma mansoni-14), which is being tested in 2 small, open-label pilot studies in senegal (nct03041766 and nct03799510). a larger 300-participant trial in uganda, nct03910972, is being planned for a vaccine based on sm-tsp-2 (schistosoma mansoni-tetraspanin-2) but is not expected to report before 2023. clues about other possible ways forward, in terms of antigen selection, come from a variety of areas.first, schistosoma are helminths (or flatworms), and the initial immune response is a characteristically strong t helper 2 (th2) cytokine reaction, which is not seen in viral or bacterial infections and is less pronounced in protozoan infections. the cytokines that are released include interleukin (il)-5, which drives the production of eosinophils to attack the parasite, and il-4 and il-13, which drive the production of immunoglobulin e (ige). epidemiological studies in endemic areas suggest that an age-dependent immunity may develop against infection, or against reinfection after treatment [71-73]. there is a good correlation between this protection and the development of ige antibodies, resulting from th2 responses [73, 74] . a hybridoma that produces a monoclonal ige antibody to s. japonicum, sj18ε.1, was identified [57]. this mab was protective in an in vitro, antigen-dependent, cellular cytotoxicity assay with rat macrophages or eosinophils and also in vivo during the early phase of infection second, beyond the cell-surface proteins, schistosomes also express a large number of glycans as part of their glycoprotein and glycolipid repertoire, and an antibody response against those glycans is mounted by the infected host [80] . in 2 cases, the specific antibodies produced could be identified using b cell cloning [81] or after infection of mice genetically modified to express human antibody repertoires [82] . interestingly, there has been no systematic analysis of the antibodies produced that target the various stages of the parasite life cycle, although early work on s. mekongi suggests schistosomula and adult worm extracts would induce a better response [83] . in addition to stimulating a th2 response, schistosome infection can suppress t-cell activation [84-86]; s. mansoni uses 2 distinct mechanisms to suppress t-cell activation, resulting in the selective up-regulation of pd-l1 on the surface of splenic f4/80 + macrophages. the presence of fatigued or anergized t cells opens up the possibility for cotherapy of low doses of mabs against programmed cell death protein-1 (pd-1) or pd-l1 with antibodies that reverse the anergy. the treatment of invasive fungal infections (ifis) remains a major challenge worldwide. there are few broad-spectrum antifungal drugs; the most effective have substantial toxicity concerns, and well-tolerated drugs used prophylactically frequently induce resistance. even with the best current treatment, the risk for mortality due to an ifi can be higher than 40%. for low-income countries, this figure is substantially worse, as many invasive infections are uniformly fatal without treatment, and estimates of global mortality involving fungal infections are as high as 1.5 million annually [87-89]. in addition, fungal infections are the cause of significant comorbidity and mortality in hiv patients. modeling studies suggest that optimal therapy could save the lives of 1.6 million hiv patients over a 5-year period [90]. the discussion here will focus on mabs developed for the following significant fungal pathogens: cryptococcus, pneumocystis and paracoccidioides, and candida. in addition to antibodies that directly target and inhibit the fungal pathogen, mabs can be directed to checkpoints that control the host immune response. this approach may be particularly useful against fungal pathogens for which sustained infection is characterized by a shift from a protective th1 or th17 response to a noninflammatory th2 response. the clinical use of anti-il17 in rheumatoid arthritis clearly exacerbates fungal infection [91], although it remains to be seen whether the reverse-stimulating this pathway-has a clinically useful effect. cryptococcus neoformans predominantly causes opportunistic infection in patients with hiv/aids and is responsible for a large burden of aids-related disease and death in sub-saharan africa [92] . although the rate of infection has decreased in recent years through greatly improved access to antiretroviral therapy (art), mortality in infected patients has not declined, demonstrating the failure of antifungal development to keep pace with improved antiviral treatment. cryptococcosis first manifests as a pulmonary disease but spreads hematogenously to the cerebrospinal fluid and brain to cause meningitis and meningoencephalitis, adding the complexity of crossing the blood-brain barrier to drug development. the ability of mabs to protect against a lethal cryptococcal infection in mice was first demonstrated over three decades ago [93], with subsequent work demonstrating the efficacy of various mabs that target the polysaccharide capsule, an essential virulence factor and the primary host-pathogen interface of cryptococcus infection [94] [95] [96] . among these, mab 18b7 was evaluated in a phase i clinical trial [50] and was found to produce a modest reduction in circulating cryptococcal antigen. further development has been hampered, however, by difficulties in securing funding for a disease for which financial returns are likely to be low-a common problem for neglected infectious diseases. subsequent studies have examined mabs that target cryptococcal melanin [97] , β-glucan [98] , or glucosylceramide ( [99] [100] [101] ; see [102] for additional in vivo and in vitro examples). host cd40 has also been targeted to stimulate the immune response [103] . in addition to highlighting the potential of mabs as therapeutics, these studies have demonstrated the diversity of inhibitory actions that mabs can perform on cryptococcal cells, which can include opsonization and increased phagocytosis, inhibition of fungal growth, capsular polysaccharide release and biofilm formation, antibody-mediated target cleavage, and augmentation of the host response [104] [105] [106] [107] . pneumocystis, like cryptococcus, is an important opportunistic pathogen in hiv/aids and other immunosuppressed patient populations, with estimates of up to 500,000 cases per year [87]. however, unlike cryptococcus, which is acquired from the environment, pneumocystis spp. are commensals, with different species occurring in the lungs of many mammals. the capacity for endogenous infection and for acquisition from asymptomatic carriers makes pneumocystis an attractive target for immunoprophylaxis. to this end, a variety of programs are aimed at developing a pneumocystis vaccine [108, 109] , and passive immunization studies have been initiated using mabs raised to pneumocystis epitopes. intranasal administration of 4f11, an mab that recognizes the pneumocystis kexin-like protein kex1, was able to prevent transmission of pneumocystis pneumonia from infected to susceptible cohoused mice, demonstrating the feasibility of this approach [110] . emerging evidence of the importance of b cellmediated immunity to pneumocystis infection strongly supports further research in this area [111] . paracoccidioides brasiliensis, while less globally prevalent than cryptococcus or pneumocystis, is the most important cause of ifis in latin america. mabs have been raised against the major p. brasiliensis antigens glycoprotein 43 (gp43) and gp70. in an infected mouse model, anti-gp43 activity, mediated by mab e3-enhanced phagocytosis of p. brasiliensis cells, increased interferon-γ production and led to a reduction in the fungal burden [112] , while anti-gp70 mabs significantly reduced fungal colony-forming units and almost completely abolished granuloma formation in the lungs [113] . antibodies raised against mouse cd25 + regulatory t (t reg ) cells, which control immunity and excessive inflammation, depleted cd25 + cells, resulting in less severe tissue inflammation, with reduced mortality in susceptible mice [112] . again, this demonstrates the potential for mabs to exert control of fungal infection, either directly by incapacitating the fungal cells or indirectly via modulation of the host response. candida albicans is the most commonly isolated fungal pathogen globally and is associated with significant morbidity and mortality, particularly in patients with hiv or tuberculosis (tb) infection [114] . a recent paper demonstrated the cloning of antibody genes from b cell cultures derived from patients infected with c. albicans [115] . these antibodies were capable of stimulating opsonophagocytic macrophage activity and provided protection in a murine model of disseminated candidiasis. in summary, mabs offer tremendous potential to augment the antifungal arsenal: animal models have provided promising results, there is a wide range of potential targets, they have the capacity to both inhibit the pathogen and augment the host response, and it may be possible to target diverse fungal pathogens with an appropriate mab cocktail or by targeting panfungal antigens. use of mabs as adjuvants to existing antifungal drugs also shows promise [116] . however, it should be noted that not all mabs are inhibitory; indeed, some may worsen infection, species and strain specificity can be an issue, and the mechanistic basis of inhibition by different mabs can vary dramatically [117] . to date, only 2 mab treatments have advanced to clinical trials: the previously mentioned 18b7 in cryptococcus, and mycograb, a heat shock protein 90 (hsp90)-specific antibody fragment, which showed promise for treating candida infections but failed to make it to market due to production difficulties and unresolved safety issues. antifungal immunomodulation is a complex area, and the field is still emerging. these preliminary studies highlight the potential for exciting new advances in mab research and application, both for understanding fungal immunity and for manipulating it to tackle lifethreatening fungal infections. dengue fever is a mosquito-borne viral infection found in tropical and subtropical regions around the world. the dengue virus (denv) is transmitted by female mosquitoes, mainly of the species aedes aegypti and, to a lesser extent, a. albopictus. there are 4 distinct serotypes-denv-1 to denv-4-of the virus, and all serotypes are presently circulating in endemic areas. denv infects cells of the human immune system and other cell types, leading to symptoms that include high fever, severe headache, severe pain behind the eyes, joint pain, muscle and bone pain, rash, and mild bleeding. in severe cases, plasma leaks out of the circulatory system, which can be fatal. the global incidence of dengue has grown dramatically in recent decades. one recent study estimated that approximately 390 million people are infected, of which 96 million manifest clinically each year [118] . who estimates that, globally, 500,000 people with severe dengue require hospitalization each year and that 2.5% of these infections are lethal. antibody-dependent enhancement (ade) is problematic in dengue infection. the presence of subneutralizing levels of flavivirus cross-reactive serum antibodies (acting against one member of the virus family) may result in an increase in infectivity via ade of another virus member or serotype, which is observed particularly after secondary dengue infection [119, 120] . despite decades of effort, there is no effective treatment against dengue. currently, dengvaxia is approved by the fda and is the only licensed dengue vaccine in the world. this is also a live attenuated tetravalent dengue vaccine developed by sanofi pasteur [121] that has been approved in several countries. however, interim results from long-term safety follow-up studies demonstrated an increased risk for hospitalization of vaccine-sensitized individuals [122] , suggesting that ade-related concerns are relevant. it has been reported that non-neutralizing levels of anti-denv antibody can enhance viral entry into host cells by forming a denvantibody complex [123, 124] . there is concern that an incomplete antibody against denvs may cause ade-mediated severe dengue disease. hence, there is a need for a safe and highly efficacious dengue therapy or vaccine that provides immunity against all 4 serotypes simultaneously. mab therapy is an alternative to vaccines and other therapies against dengue. many mabs against dengue from mice and humans have been characterized, and the use of mabs has also been explored as a therapeutic option. antibody sign-3c, identified by the singapore immunology network, neutralized all 4 dengue serotypes and decreased viremia of all serotypes in mice when given 2 days after infection [43] . a humanized mab visterra 513 (vis513), a panserotype anti-denv developed by visterra [44, 45] , which binds e protein domain iii (ediii) and neutralizes all 4 serotypes of denv, also showed useful antiviral utility. vis513 (25 mg/kg or 50 mg/kg) was administered at 5 days post infection in nonhuman primates, and no infectious virus could be detected by either plaque assay or virus isolation after treatment, a finding that was, however, not mirrored by reverse transcription pcr (rt-pcr) findings. a human challenge model is now available for clinical research in dengue [125] . in this model, the efficacy and safety profile of therapeutic antibodies can be evaluated rapidly in small-scale clinical settings, prior to traditional large-scale field studies with naturally infected patients. to develop mabs for dengue therapy, it is important to consider an approach that prevents or reduces ade, and several studies that address this link have been undertaken. a neutralizing human mab, d23-1b3b9, that targets the fusion loop in domain ii showed strong neutralizing activity against all 4 denv serotypes [46] . however, at subneutralizing concentrations, it also elicited ade activity in vitro [47] . to reduce the ade, injampa and coworkers [48] modified the d23-1b3b9 antibody fc domain at position n297q. the modified antibody kept the same cross-neutralizing activity to all 4 serotypes as those of wild-type antibody but lacked ade activity against all 4 serotypes at subneutralizing concentrations. in another neutralizing mab, sign-3c, 2 leucine to alanine mutations were engineered in the fc part, abrogating binding to fc gamma receptors [43] . this mutant fc version (sign-3c-lala) protected mice, while ade was completely abrogated. similarly, mab11/mutated fragment crystallizable region (mutfc) (an mab that is unable to bind to cells with fcγ receptors [fcγr]) and potentiate ade have been used as a prophylactic therapy [49] . passive immunization with this mab (at 25 mg/kg) reduced viral load and disease progression in nonhuman primates. here again, therapeutic antibodies can also be used for prophylaxis, affording immediate and reliable protection. tb tb is a good example of the gap between a pathogen's prevalence and burden. mycobacterium tuberculosis spreads easily among human populations; presently, about one-third of all humans are infected, and new infections occur in 1% of the population each year. among these billions of carriers, there are, however, "only" 10 million active tb infections, with 1.3 million deaths in 2016. thus, the vast majority of carriers keep the pathogen in check. tb exerts 2 levels of immune evasion: one in which it is maintained in a latent state and one in which it breaks free and causes active disease [126] . several studies have tested antibody therapy in tb, with varying success (reviewed in [127] ). even if an mab treatment would not be curative, shortening the standard treatment of patients infected with multidrug-resistant (mdr) and extensively drug-resistant (xdr) strains would represent a major advance. in addition, "checkpoint blockade during chronic tb infection requires further consideration" [128] . the role of protective antibodies in malaria was demonstrated over 40 years ago with the finding that the passive transfer of sera from mice with radiation-attenuated sporozoites delayed the development of infection in other mice [129] . the tpps for malaria are well described [130, 131] . these include a profile for seasonal malaria chemoprevention, a treatment successfully launched in sub-saharan africa in the last 5 years, consisting of a full treatment course of 3 days of amodiaquine and 1 dose of sulfadoxine/pyrimethamine. it is given monthly to children during the rainy season. antibody therapeutics for malaria could (1) prevent the entry (initial infection) of the parasite, (2) block entry of the sporozoite into the liver cells, (3) block entry of the merozoites into the erythrocytes, or (4) block the uptake of the gametocytes into the mosquito (breaking the transmission cycle). one difficulty in targeting the merozoites in symptomatic malaria is that the extracellular phase of the pathogen is relatively short-lived and is only a small part of the life cycle. additionally, the number of merozoites invading erythrocytes is very large (up to 10 12 ), compared with the number of sporozoites invading the liver stages (dozens). as such, the most interesting place to intervene with an mab is at the initial infection of the liver. currently there are 3 mabs published with potent activity against the circumsporozoite protein, csp, and these can reduce the parasitaemia in sporozoite-infected frg (triple mutant fah/raγ2/il2rγ) mice that carry a human liver implant [132] . these mabs include mab317, cloned from b cells obtained from a patient in the recent rts,s vaccine trial [133] , cis43 (circumsporozoite protein 43 [56]), and a set that included mgu12 [134] , cloned from patients vaccinated with irradiated sporozoites. antibodies that block the invasion process of the red blood cells by merozoites represent another possible approach. in studies of the merozoite protein rh5 (reticulocytebinding protein homologue 5) as a potential vaccine, some antibodies were described that could block the cycle of erythrocyte infection [135] . the specific merozoite epitopes are now being characterized, and this could form the basis of a second-generation antibody [136, 137] . a general observation with plasmodium infections is that the human host is unable to mount a sterilizing immune response. one theory is that the parasite can control the t-cell response and induce a state of fatigue or anergy similar to that seen in tumor-invading lymphocytes in immuno-oncology. several studies in mice have demonstrated that blocking pd-l1 or ctla-4 (cytotoxic t-lymphocyte-associated protein 4) improves clearance in mice infected with plasmodium bergheii [138] , and from this and similar experiments, a strong argument can be made that reversal of this fatigue by mild checkpoint inhibitor blockade may be a way of facilitating the host response [11]. hiv mab therapies have also been proposed for hiv. the case for hiv was recently reviewed [139, 140] . two such mabs are in phase iii trials: pro 140 and cenicriviroc. pro 140 targets ccr5 (cysteine-cysteine chemokine receptor type 5) and recently entered phase iib/iii trials for weekly subcutaneous dosing for monotherapy maintenance [141] . cenicriviroc is a dual ccr2 and ccr5 antagonist investigated for a number of indications, including hiv infection [142] . as noted earlier, ibalizumab was recently approved as a second-line treatment for hiv treatment [13]. for hbv infection, the hypothesis is that high circulating hbsag levels prevent a proper immune response. a novel mab, e6f6, is being evaluated for reducing hbsag levels in patients [52] . in addition, the hbv s protein is being targeted for the discovery of therapeutic mabs [143] . in the case of visceral leishmaniasis (vl), il-10 and glucocorticoid-induced tnf-receptorrelated protein have been considered as mab targets [144] , but there has been no systematic analysis of antigens or reported cloning of b cells from infected patients. although mabs have made a massive impact in controlling autoimmune disease, inflammation, and cancer, the relative impact in the world of infectious disease has largely been confined to viral diseases. the use of mabs to protect against rsv infections and the profile of secondgeneration antibodies shows that it is possible to obtain mabs that are sufficiently potent to provide long-term protection with a single intramuscular or subcutaneous injection. with the development of new technologies for cloning antibodies from b cells or plasma cells taken from patients infected with bacteria, viruses, fungi, or even protozoal pathogens, it is possible to quickly obtain fully human antibody collections with potential activity against pathogens in vitro and in vivo. technologies for expressing antibodies are now at the stage in which it is not uncommon to see extremely high levels of expression in cell culture, and taken together with the progress in reducing costs of production, the cost of goods for an antibody injection is starting to enter the range of us$1 to us$10, reaching the edge of what is affordable for infectious diseases of neglected populations. studies of mutations in the fc region confirm that mab half-lives can be extended, and the goal of a single injection to cover an entire season for those infections with seasonality is now a possibility. new technologies with viral delivery offer the promise that a single injection could give protection for even longer periods. for many infectious diseases, we are now seeing the buildup of a portfolio of potential antibodies. in cases in which little progress has been made, a systematic attempt is needed to identify the antibodies resulting from successful control of an infection in patients. beyond these basic antibodies, the availability of new monoclonals with anti-infective activity in vivo would open up the door to even more creative options: bispecific antibodies could be used in key immune cells and could effectively support the natural response to infection. studies in animal models of chronic malaria infection led to the observation that this results in a reduction in the impact of cytotoxic t cells and modulates the t reg capacity, leading to an "exhausted" or ineffective t-cell response [145] . clinical trials are already underway that use immune checkpoint blockers for chronic hiv and hbv infection [11] . this has important implications for any infectious disease in which a single infection does not drive a sterilizing immune response. for such diseases, which include malaria, one question for the longer term is whether immune checkpoint inhibition, or interfering with interferon-α signaling, could be used [128] . two decades ago, one of the biggest challenges of working in anti-infective drug discovery was the need to have new medicines with activities against the widest range of pathogens. in more recent times, the tide has changed, and clinical diagnosis now is such that medicines with a high degree of selectivity and specificity are often preferred-as long as they show good clinical activity. this shift to highly specific medicines favors the use of mabs. given the overall rise in interest for new treatments in infectious diseases caused by concerns about antimicrobial resistance, there is a real opportunity now to progress the newly emerging families of mabs to target infectious diseases of neglected populations. because of outdated preconceptions about this class of therapeutics, few research funds are being allocated to their discovery, resulting in an egg-and-chicken problem (the absence of conspicuous success driving additional efforts). one of the goals of the analysis that we present here is to promote making additional funds available to pursue the initial discovery of mabs for neglected diseases. monoclonal antibodies approved by the ema and fda for therapeutic use protective murine monoclonal antibodies to cryptococcus neoformans antibody-mediated protection in mice with lethal intracerebral cryptococcus neoformans infection monoclonal antibodies to cryptococcus neoformans capsular polysaccharide modify the course of intravenous infection in mice passive immunization with melanin-binding monoclonal antibodies prolongs survival of mice with lethal cryptococcus neoformans infection protection by anti-betaglucan antibodies is associated with restricted beta-1,3 glucan binding specificity and inhibition of fungal growth and adherence human antibodies against a purified glucosylceramide from cryptococcus neoformans inhibit cell budding and fungal growth monoclonal antibody to fungal glucosylceramide protects mice against lethal cryptococcus neoformans infection fungal glucosylceramides: from structural components to biologically active targets of new 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developments in anti-malarial target candidate and product profiles assessing drug efficacy against plasmodium falciparum liver stages in vivo structural basis for antibody recognition of the nanp repeats in plasmodium falciparum circumsporozoite protein a public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein human vaccination against rh5 induces neutralizing antimalarial antibodies that inhibit rh5 invasion complex interactions kilchip v1.0: a novel plasmodium falciparum merozoite protein microarray to facilitate malaria vaccine candidate prioritization. frontiers in immunology human antibodies that slow erythrocyte invasion potentiate malaria-neutralizing antibodies programmed death-1 ligand 2-mediated regulation of the pd-l1 to pd-1 axis is essential for establishing cd4(+) t cell immunity antibody-mediated therapy against hiv/aids: where are we standing now? broadly neutralizing anti-hiv-1 monoclonal antibodies in the clinic the return of pro 140, a ccr5-directed mab apobec3g/3a expression in human immunodeficiency virus type 1-infected individuals following initiation of antiretroviral therapy containing cenicriviroc or efavirenz a human monoclonal antibody against hepatitis b surface antigen with potent neutralizing activity combined immune therapy for the treatment of visceral leishmaniasis immune checkpoints and their inhibition in cancer and infectious diseases we thank dr. kiyoshi kita, dean and professor at the school of tropical medicine and global health, nagasaki university; dr. yoshimasa tanaka, assistant professor at the center for therapeutic innovation, nagasaki university; and dr. simon croft, professor at the london school of hygiene and tropical medicine, all of whom provided insight and expertise that greatly assisted the research. key: cord-001690-cn21fgug authors: franceschi, valentina; parker, scott; jacca, sarah; crump, ryan w.; doronin, konstantin; hembrador, edguardo; pompilio, daniela; tebaldi, giulia; estep, ryan d.; wong, scott w.; buller, mark r.; donofrio, gaetano title: bohv-4-based vector single heterologous antigen delivery protects stat1((-/-)) mice from monkeypoxvirus lethal challenge date: 2015-06-18 journal: plos negl trop dis doi: 10.1371/journal.pntd.0003850 sha: doc_id: 1690 cord_uid: cn21fgug monkeypox virus (mpxv) is the etiological agent of human (mpx). it is an emerging orthopoxvirus zoonosis in the tropical rain forest of africa and is endemic in the congo-basin and sporadic in west africa; it remains a tropical neglected disease of persons in impoverished rural areas. interaction of the human population with wildlife increases human infection with mpx virus (mpxv), and infection from human to human is possible. smallpox vaccination provides good cross-protection against mpx; however, the vaccination campaign ended in africa in 1980, meaning that a large proportion of the population is currently unprotected against mpxv infection. disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. however, there are no fda-approved therapies against mpx, and current vaccines are limited due to safety concerns. for this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that mpxv could be used as a bioterror agent. in the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (bohv-4) vectors, each expressing different mpxv glycoproteins, a29l, m1r and b6r were investigated in terms of protection from a lethal mpxv challenge in stat1 knockout mice. bohv-4-a-cmv-a29lgd(106)δtk, bohv-4-a-ef1α-m1rgd(106)δtk and bohv-4-a-ef1α-b6rgd(106)δtk were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. a small challenge study was performed, and all three recombinant bohv-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. further, bohv-4-a-ef1α-m1rgd(106)δtk alone or in combination with bohv-4-a-cmv-a29lgd(106)δtk and bohv-4-a-ef1α-b6rgd(106)δtk, was shown to be able to protect, 100% alone and 80% in combination, stat1((-/-)) mice against mortality and morbidity. this work demonstrated the efficacy of bohv-4 based vectors and the use of bohv-4 as a vaccine-vector platform. monkeypox virus (mpxv) is an orthopoxvirus with a broad-host range capable of infecting many animal species [1] . in humans, mpxv causes a disease very similar to the closely related variola virus, the etiological agent of smallpox: a rash (a 2-4 week period where macules develop and form papules, vesicles and pustules) which is preceded by a 10-14 day incubation, followed by a 1-3 day interval characterized by a prodromal fever, malaise and severe lymphadenopathy of the neck, inguinal and axillary regions [2] [3] [4] . the more virulent strains of mpxv (from the congo basin region) can induce mortality rates of~10% [5] [6] [7] [8] . the main clinical difference between human mpx and smallpox is the presentation of lymphadenopathy in the former [9, 10] . mpxv is endemic to central and west africa with increasing numbers of human infections being reported. in 2003, the transmissibility of mpxv became acutely obvious when an outbreak occurred in the united states and mpxv was transmitted to humans from prairie dogs [11, 12] . the global vaccination campaign that eradicated smallpox utilized vaccines that are also efficacious against mpxv and was successful because there is no animal reservoir for variola [13] . indeed, prior to the eradication of smallpox, the existence of mpxv was unknown and it is likely that many cases of human mpx were reported as smallpox. cases of human mpx are increasing in africa, this may be due to several factors: 1) increasing interactions between humans and infected animals through environmental degradation; 2) the cessation of routine vaccination against smallpox; 3) an increase in human susceptibility to the virus; 4) an increase in animal-human and human-human transmissibility of the virus; and 5) adaption of the virus to new host species that co-exist in, or close to, human geographical regions [2, [14] [15] [16] [17] [18] . the high mortality rate and morbidity rate of mpxv in humans makes it an important emerging disease for study. to date, no antiviral has been fda-approved for the treatment of human mpx. vaccines against smallpox have demonstrated good protection against mpxv in animal models and anecdotally in humans [1] ; however, most first-and second-generation smallpox vaccines are associated with at least some level of morbidity and a large proportion of the population are contraindicated to vaccination [19] . third-generation vaccines, such as mva, have been shown to be safer in non-human primates but require 2 the a29l, m1r and b6r orfs, were amplified from monkeypox dna (strain v79-i-005; accession: hq857562.1) using primer pairs listed in table 1 . the sense primer contains at its 5 0 -end the nhei restriction site along with the kozak's sequence preceding the atg, for a better translation initiation, whereas the antisense primer contains at its 5 0 -end a sali restriction site for the in-frame cloning of the orf with a gd106 tag peptide present in pcmv-igk-vp2-gd 106 plasmid vector. pcmv-igk-vp2-gd 106 has the human cytomegalovirus immediate early gene (cmv) promoter, the immunoglobulin kappa light chain leader sequence (igk), the bluetongue virus vp2 orf, the gd 106 epitope of bovine herpesvirus 1 glycoprotein d, successfully used as a tag during the cloning, and the bovine growth hormone polyadenylation site [37] . the 3 orfs were cut with nhei/sali and cloned into pcmv-igk-vp2-gd 106 cut with the same enzymes. this strategy allowed the substitution of the igk-vp2 sequence with those of the 3 orfs to generate pcmv-a29lgd 106 , pcmv-m1rgd 106 and pcmv-b6rgd 106 . to generate ptk-cmv-a29lgd 106 -tk, pcmv-a29lgd 106 was cut with bamhi, treated with t4 dna polymerase for blunt ending and cut with nhei; a29lgd 106 (442 bp) was excised from the pcmv-a29lgd 106 and inserted into the shuttle vector pint2egfp [25] cut with nhei/smai to replace gfp orf with the chimeric protein. pint2egfp contains two bohv-4 thymidine kinase gene homology regions flanking the green fluorescent protein (gfp) expression cassette driven by the cmv promoter, cutting with nhei/smai the gfp orf was replaced with that of a29lgd 106 . to generate pef1α-m1rgd 106 and pef1α-b6rgd 106 , the cmv promoter of pcmv-m1rgd 106 and pcmv-b6rgd 106 was substituted with that of the human elongation factor 1 alpha (ef1α). ef1α was amplified by pcr from pwpi plasmid vector (addgene plasmid #12254). pwpi was first linearized with pmei, then the pcr reaction was carried out with 0.25 μm of a couple of primers (ef1α-ndei-sense and ef1α-nhei-antisense; table 1 ) in a final volume of 50 μl containing 10 mm tris-hydrochloride ph 8.3, 0.2 mm deoxynucleotide triphosphates, 3 mm mgcl2, 50 mm kcl and 5% dmso. each cycle of 35, consisted of denaturation at 94°c for 1 min, primer annealing at 50°c for 1 min and elongation for 90 sec with 1u of pfu dna polymerase at 72°c. the 1237 bp ef1α amplification product was checked in 1% agarose gel and visualized after ethidium bromide staining in 1× tae buffer (40 mm tris-acetate, 1 mm edta). ptk-ef1α-m1rgd 106 -tk and ptk-ef1α-b6rgd 106 -tk constructs were obtained by subcloning ef1α-m1rgd 106 and ef1α-b6rgd 106 expression cassettes, cut with ndei/mlui and blunted-end with t4 dna polymerase, from pef1α/m1rgd 106 and ef1α/b6rgd 106 respectively in smai cut pint2 [38] . all enzymes were purchased from thermo scientific. confluent hek293t cells were seeded into 6 well plates (3x10 5 cells/well) and incubated at 37°c with 5% co 2 ; when the cells were sub-confluent the culture medium was removed and the cells were transfected with ptk-cmv-a29lgd 106 -tk, ptk-ef1α-m1rgd 106 -tk or ptk-ef1α-b6rgd 106 -tk using polyethylenimine (pei) transfection reagent (polysciences, inc.). briefly, 3 μg of dna were mixed with 7.5 μg pei (1mg/ml) (ratio 1:2.5 dna-pei) in 200 μl of dulbecco's modified essential medium (dmem) at high glucose percentage (euroclone) without serum. after 15 min at rt, 800 μl of medium without serum was added and the transfection solution was transferred to the well and left on the cells for 6 h at 37°c with 5% co 2 in air, in a humidified incubator. the transfection mixture was then replaced with fresh medium (emem, with 10% fbs, 50 iu/ml of penicillin, 50 μg/ml of streptomycin and 2.5 μg/ml of amphotericin b) and incubated for 24 h at 37°c with 5% co 2 . bohv-4-a, bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk were propagated by infecting confluent monolayers of bek and vero cells at a multiplicity of infection (moi) of 0.5 50% tissue culture infectious doses (tcid 50 ) per cell and maintained in medium with only 2% fbs for 2 h. the medium was then removed and replaced with fresh emem containing 10% fbs. when the cytopathic effect (cpe) occurred in the majority of the cell monolayer (*72 h post infection), the virus was prepared by freezing and thawing cells three times and pelleting the virions through a 30% sucrose cushion, as described previously [39] . virus pellets were then resuspended in cold emem without fbs. tcid 50 were determined with bek cells by limiting dilution. a plaque-purified isolate of the mpxv strain zai-79 [40] was purified through a sucrose cushion [41] and propagated in bs-c-1 cells [42] . virus infectivity was estimated as described previously [43] . briefly, virus suspensions were serially diluted in pbs +1% fcs (fetal clone ii, hyclone), absorbed to monolayers for 1 hour at 37°c, and overlaid with a suspension of 1% carboxyl methyl cellulose in dmem +5% fcs. after 2 days at 37°c, virus plaques were visualized and virus inactivated by the addition to each well of a 0.3% crystal violet/10% formalin solution. protein cell extracts were obtained from a 6-well confluent plate of hek293t transfected with pint2/cmva29lgd 106 , pint2/ef1αm1rgd 106 or pint2/ef1αb6rgd 106 and from 25-cm 2 confluent flasks of bek cells or vero cells infected with bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk by adding 100 μl of cell extraction buffer (50 mm tris-hcl, 150 mm nacl, and 1% np-40; ph 8). a 10% sds-page gel electrophoresis was used to analyze cell extracts containing 50 μg of total protein, after protein transfer in nylon membranes by electroblotting, the membranes were incubated with bovine anti gd 106 monoclonal antibody (clone 1b8-f11; vrmd, inc., pullman, wa) antibody, probed with horseradish peroxidase-labelled anti-mouse immunoglobulin antibody (sigma) and visualized by enhanced chemiluminescence (ecl kit; pierce). recombineering was performed as previously described [44] with some modifications. five hundred microliters of a 32°c overnight culture of sw102 containing bac-bohv-4-a-kanagalkδtk, were diluted in 25 ml luria-bertani (lb) medium with or without chloramphenicol (sigma) selection (12.5 mg/ml) in a 50 ml baffled conical flask and grown at 32°c in a shaking water bath to an od 600 of 0.6. then, 10 ml were transferred to another baffled 50 ml conical flask and heat-shocked at 42°c for exactly 15 min in a shaking water bath. the remaining culture was left at 32°c as the uninduced control. after 15 min the two samples, induced and uninduced, were briefly cooled in ice/water bath slurry and then transferred to two 15ml falcon tubes and pelleted using 5000 r.p.m. (eppendorf centrifuge) at 0°c for 5 min. the supernatant was poured off and the pellet was resuspended in 1ml ice-cold ddh 2 o by gently swirling the tubes in ice/water bath slurry. subsequently, 9 ml ice-cold ddh 2 o were added and the samples pelleted again. this step was repeated once more, the supernatant was removed and the pellet (50 μl each) was kept on ice until electroporated with gel-purified fragments (*3.3, *4.4 and *4.6 kb respectively for tk-cmv-a29lgd 106 -tk, tk-ef1α-m1rgd 106 -tk and tk-ef1α-b6rgd 106 -tk) obtained by cutting ptk-cmv-a29lgd 106 -tk, ptk-ef1α-m1rgd 106 -tk and ptk-ef1α-b6rgd 106 -tk with clai/pvuii (thermo scientific). an aliquot of 25 μl (~200 ng) was used for each electroporation in a 0.1 cm cuvette at 25 μf, 2.5 kv and 201o. after electroporation, for the counter selection step, the bacteria were recovered in 10 ml lb in a 50 ml baffled conical flask and incubated for 4.5h in a 32°c shaking water bath. bacteria serial dilutions were plated on m63 minimal medium plates containing 15 g/l agar, 0.2% glycerol, 1mg/l d-biotin, 45 mg/l l-leucine, 0.2% 2-deoxy-galactose and 12.5 mg/ml chloramphenicol. all the complements for m63 medium were purchased from sigma. plates were incubated 3-5 days at 32°c; then several selected colonies were picked up, streaked on mcconkey agar indicator plates (difco, bd biosciences) containing 12.5 g/ml of chloramphenicol and incubated at 32°c for 3 days until white colonies appeared. white colonies were grown in duplicate for 5-8h in 1ml of lb containing 50 mg/ml of kanamycin (sigma) or lb containing 12.5 mg/ml of chloramphenicol. only those colonies that were kanamycin negative and chloramphenicol positive were kept and grown overnight in 5ml of lb containing 12.5 mg/ml of chloramphenicol. bac dna was purified and analyzed through hindiii restriction enzyme digestion for tk-cmv-a29lgd 106 -tk, tk-ef1α-m1rgd 106 -tk and tk-ef1α-b6rgd 106 -tk fragment targeted integration, was separated by electrophoresis overnight in a 1% agarose gel, stained with ethidium bromide, capillary transferred to a positively charged nylon membrane (roche), and cross-linked by uv irradiation by standard procedures [28] . hybridization with digoxigenin-labeled probes confirmed the identity of specific restriction fragments. the 353, 573, 977 bp amplicons for a29l, m1r and b6r probes were generated by pcr with the primers: a29l sense/antisense, m1r sense/antisense, and b6r sense/antisense listed in table 1 , as previously described [29] . original detailed protocols for recombineering can also be found at the recombineering website (http://recombineering.ncifcrf.gov). bek or bekcre cells were maintained as a monolayer with complete dmem growth medium with 10% fbs, 2 mm l-glutamine, 100 iu/ml penicillin and 10 mg/ml streptomycin. when cells were sub-confluent (70-90%) they were split to a fresh culture vessel (i.e., every 3-5 days) and were incubated at 37°c in a humidified atmosphere of 95% air-5% co2. bac dna (5 μg) was electroporated in 600 μl dmem without serum (equibio apparatus, 270 v, 960 mf, 4-mm gap cuvettes) into bek and bekcre cells from a confluent 25-cm 2 flask. electroporated cells were returned to the flask, after 24h the medium was replaced with fresh medium, and cells were split 1:2 when they reached confluence at 2 days post-electroporation. cells were left to grow until the appearance of cpe. bek cells were infected with bohv-4-a, bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk at a m.o.i. of 0.1 tcid50/cell and incubated at 37°c for 4 h. infected cells were washed with serum-free emem and then overlaid with emem containing 10% fbs, 2 mm lglutamine, 100 iu/ml penicillin, 100 mg/ ml streptomycin and 2.5-mg/ml amphotericin b. the supernatants of infected cultures were harvested after 24, 48, 72 and 96 h, and the amount of infectious virus was determined by limiting dilution on bek cells by the tcid50 method. eight-week old female 129 stat1 -/mice were bred in-house and housed in filter-top microisolator cages and fed commercial mouse chow and water ad libitum. the randomized mice were housed in an animal biosafety level 3 containment area, with 5 mice/group. animal husbandry and experimental procedures were approved by the institutional animal care and use committee. mice were monitored every day until the termination of the experiment. bohv-4s vectors were injected intraperitoneally (ip) in a total volume of 300 μl with dmem used as a vehicle. for vaccinations with one vector, injections were comprised of 100 #x03bc;l of vector + 200 μl of vehicle. for the combination injections, 100 #x03bc;l of each vector was included for a total of 300 #x03bc;l injections were given as a primary vaccination at t = 0 days and as a booster vaccination at t = 23 days (see table 2 ). each vector was injected at a modified vaccinia ankara (mva) (a gift from the niaid-nih, bethesda, md) was provided at a dose of 2x10 8 plaques forming units (pfu)/ml and was injected in 0.1 ml between the skin and underlying layers of tissue in the scapular region on the backs of mice. mice were anesthetized with 0.1 ml/10 g body weight of ketamine hcl (6 mg/ml) and xylazine (0.5 mg/ml) by intraperitoneal injections. anesthetized mice were laid on their dorsal side with their bodies angled so that the anterior end was raised 45°from the surface; a plastic mouse holder was used to ensure conformity [45] . mpxv was diluted in pbs without ca 2+ and mg 2+ to the required concentration and slowly loaded into each nare (5 #x03bc;l/nare). mice were subsequently left in situ for 2-3 minutes before being returned to their cages. paired t-tests (tailed) were used to compare means between groups of mice. mortality rates were compared using the fisher's exact test. blinded lesion pictures were measured qualitatively using a scoring system ranging 0-4 in severity. p values <0.05 were considered statistically significant. among the approximately 200 genes that comprise the monkeypox virus genome, only few genes encoding antigenic proteins-that are known-are able to elicit a neutralizing antibody response in vaccinated animals. among these antigens, a29l, m1r and b6r orthologs were selected as candidate antigens to be delivered by bohv-4 based-vector. a29l, m1r and b6r orfs were amplified by pcr from a cosmid library and sub-cloned in frame with a tag peptide, gd106 [46] (fig 1a, 1e and 1h) , which was contained in a mammalian expression vector plasmid construct. m1r and b6rgd 106 tagged orfs were placed under the transcriptional control of the ef1α promoter (fig 1f and 1i ), whereas a29lgd 106 tagged orf under the transcriptional control of the cmv promoter ( fig 1b) . so generated expression cassettes (cmv-a29lgd 106 , ef1α-m1rgd 106 and ef1α-b6rgd 106 ) were validated, in terms of protein expression, by transient transfection in 293t cells and western-immunoblotting with a monoclonal antibody directed against the gd 106 tag peptide. a29l, m1r and b6rgd 106 tagged antigens were all well expressed in the whole cell extracts of the transiently transfected cells (fig 1c, 1g and 1j), further a29lgd 106 was also secreted (fig 1d) in the supernatant of the transiently transfected cells. construction of bohv-4s-based vector expressing a29l, m1r and b6rgd106 tagged antigens an apathogenic strain of bohv-4 cloned as a bac was used to create a bohv-4-a-based vector [28] . the tk bohv-4-a genome locus was utilized as the integration site for the cmv-a29lgd 106 , ef1α-m1rgd 106 and ef1α-b6rgd 106 expression cassettes. the bohv-4 tk genomic region is strongly conserved among bohv-4 isolates [47] , ensuring the stability of the genomic locus from potential recombination when foreign dna sequences are inserted in. indeed, the bohv-4 tk genomic locus has been interrupted by the insertion of foreign dna sequences of different size, without interfering with viral replication in vitro and loss of heterologous protein expression [25,27-29,31,32,34]. cmv-a29lgd 106 , ef1α-m1rgd 106 and ef1α-b6rgd 106 expression cassettes were first sub-cloned into pint2, a shuttle vector plasmid containing 2 bohv-4 tk sequences [25] , to generate ptk-cmv-a29lgd 106 -tk, ptk-ef1α-m1rgd 106 -tk and ptk-ef1α-b6rgd 106 -tk targeting vectors. restriction enzyme linearized targeting vectors were used for heat-inducible homologous recombination sw102 e. coli containing pbac-bohv-4-a-kanagalkδtk [28, 39, 48] (fig 2a) to generate pbac-bohv-4-a-cmv-a29lgd 106 δtk, pbac-bohv-4-a-ef1α-m1rgd 106 δtk and pbac-bohv-4-a-ef1α-b6rgd 106 δtk. selected clones were first analyzed by hindiii restriction enzyme digestion and then by southern blotting (fig 2b) . because heat-inducible recombination in sw102 e. coli and repeated passages could establish altered bacterial phenotypes due to an aberrant recombenases transcription, sw102 e. coli carrying pbac-bohv-4-a-cmv-a29lgd 106 δtk, pbac-bohv-4-a-ef1α-m1rgd 106 δtk and pbac-bohv-4-a-ef1α-b6rgd 106 δtk were serially cultured over for 20 passages and checked by hindiii restriction enzyme digestion. no differences among restriction patterns at various passages were detected (s1 fig), thus ensuring the stability of the clones. infectious viable bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk were obtained by electroporating pbac-bohv-4-a-cmv-a29lgd 106 δtk, pbac-bohv-4-a-ef1α-m1rgd 106 δtk and pbac-bohv-4-a-ef1α-b6rgd 106 δtk dna into bek and bekcre cells. the only difference was that the recombinant viruses reconstituted from electroporated bekcre resulted in depletion of the bac plasmid backbone containing the gfp expression cassette, as shown by the loss of gfp expression (fig 3a, 3e and 3h ). because the time necessary to reconstitute the viable recombinant bohv-4s was different among them, it was of interest to know if the foreign antigens, encoded by the expression cassette integrated into the viral genome, could have a detrimental effect on the viral replication. therefore, the growth characteristics of bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk were compared with that of the parental virus, bohv-4-a. although bohv-4-a-cmv-a29lgd 106 δtk and bohv-4-a-ef1α-m1rgd 106 δtk demonstrated a slower replication kinetics respect to bohv-4-a, they reached the same viral titer at the end-point (~10 6 ) (fig 3b and 3f) . whereas bohv-4-a-ef1α-b6rgd 106 δtk replication was drastically impaired, a 2 log reduction of the viral titer end-point (~10 4 ) respect to bohv-4-a was observed ( fig 3i) ; however, transgene expression was well detected in the whole cell extract of bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk infected cells (fig 3c, 3g and 3j). further, a29lgd 106 glycoprotein was found to be expressed as a secreted protein in the supernatant of bohv-4-a-cmv-a29lgd 106 δtk infected cells (fig 3d) . in vivo efficacy testing of bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk to test the efficacy of the vectors in vivo, we sought to determine if they could protect mice against a lethal challenge with mpxv. several murine strains have been developed as models of mpxv, in this study we utilized the 129 stat1 -/strain. thirteen cages were prepared with 5 mice/cage ( table 2) . cages 1 and 2 were un-vaccinated. cage 3 was vaccinated with vehicle (dmem without fbs). cages 4 and 5 were vaccinated with mva where cage 4 received a primary injection of vaccine at t = 0 days and a vehicle booster at t = 23 days, and cage 5 cages 12 and 13 were also vaccinated following the above regimen; however, these mice received a combination (combo) of the 3 bohv-4 vectors. bohv-4 vectors were injected ip in a total volume of 0.3 ml. there was no apparent morbidity-as measured visually-or weightloss recorded in any of the mice (s2 fig). at t = 50 days, mice in cages 2-13 were challenged with 2x10 5 pfu/mouse of mpxv. mortality rates are shown in fig 4. as expected, the mva vaccinated mice in cages 4 (mva/ veh) and 5 (mva/mva) were 100% protected against challenge. mice in cage 9 (m1r/m1r) were also 100% protected (p = 0.004); and although mice in cage 13 (combo/combo) experienced 1 death, they were still 80% protected against the mpxv challenge (p = 0.02). when comparing weight-change (fig 5) , we found that mice in cage 5 (mva/mva) did not lose weight compared to the pbs control (cage 1) and that mice in cage 4 (mva/veh) only lost weight (5%) on days 6, 7 and 8 post challenge. we also found that mice in cages 10 (b6r/ veh), 11 (b6r/b6r), 12 (combo/veh), and 13 (combo/combo) had significantly reduced weight-loss from day 8 post challenge (15%, 15%, 11%, and 15% on day 8, respectively), even though mice in cage 13 were 80% protected against the challenge. some protection from weight-loss was also afforded to mice in cages 8 (m1r/veh) and 9 (m1r/m1r) on days 13, 14 and 15 post challenge (15%). these data indicate that the presence of m1r is required to protect the mice against challenge, and that a booster vaccination is required. it also indicates that a combination of the 3 vectors improves protection against weight-loss. although m1r could provide protection, it was inferior to that provided by vaccination with mva. the data also indicate that although b6r does not provide protection against mortality, it does provide protection against weightloss in surviving mice (see above). the aim of the current study was to ascertain the potential utility of bovine herpesvirus 4 (bohv-4)-based vectors as safe, potent, large-capacity vaccine vectors for category a agents-mpxv for this specific case. this study demonstrated protection in the stat1 -/model and consideration should be given to also evaluating the vectors in cast/eij mice which have also been established as a murine model of monkeypox [49, 50] . ultimately, this study could pave the way for further studies in other animal models such as prairie dogs (cynomys ludovicianus) [51] [52] [53] , the 13-lined ground squirrel (spermophilus tridecemlineatus) [54, 55] and non-human primates (reviewed in [1] )-all of which have been extensively used as models of human monkeypox-and ultimately in human protection studies. in fact, since mpxv also infects humans and causes clinical-signs very similar to smallpox, it is classified as a category a select agent [56] . primarily for ethical but also for cost reasons, c57bl/6 stat1 knockout mice (stat1 (-/-) ) were chosen as an in vivo model before progressing to gene delivery and immunogenicity studies in non-human primates. stat1 (-/-) miceare highly sensitive to mpxv and the disease course in mpxv infected stat1 (-/-) mice, characterized by weight loss and death by day 10 post infection-is similar to that observed in wild-type mice infected with mousepox/ectromelia virus (ectv), the etiology agent of mousepox-and probably the best small animal model of smallpox [35, 57] . moreover it was revealed that antiviral therapy could protect mice to a degree similar to that of vaccination with dryvax or mva, supporting the use of stat1 (-/-) mice as a reliable model to evaluate orthopoxvirus prophylactics and therapeutics [35] . stat1 (-/-) mice were found to be sensitive to a wide number of pathogens due to the loss of stat1, a key factor responsible for type 1 and 2 interferon (ifn) signaling [58] [59] [60] [61] [62] [63] . bohv-4-based vectors in wild type and immunocompromised mice behave as replicating incompetent viral vectors, showing absence of pathogenicity [31,32, [64] [65] [66] and bohv-4 replication is strongly impaired in vitro after the treatment of bohv-4 infected cells with ifnγ [67] . considering that ifn-γ is an activator of stat1, the potential pathogenicity of bohv-4-based vectors in stat1 (-/-) mice was a concern. however, intra peritoneal bohv-4 inoculated stat1 (-/-) mice did not show any overt clinical sign, detrimental effect or pathology correlated to challenge. a29l, m1r, and b6r mpxv antigens were selected, as candidate antigens to be delivered by bohv-4-based vectors, as they are orthologous to a27l, l1r and b5r vaccinia virus (vv) antigens respectively. a27l is a 14 kda protein thought to be involved in vv entry events [68] . l1r is a 23-29 kda myristoylated surface protein involved in a yet-to-be-identified post viral attachment and pre-fusion events [69] . whereas b5r is a 42 kda glycoprotein found on the surface of the virus [70, 71] and is involved in cell surface glycosaminoglycan-mediated disruption of the viral outer membrane [72] . further, they were shown to be able to elicit a protective immune response in mice and non-human primates when formulated in combination as a subunit vaccine consisted of purified proteins, plasmid dna vaccines, recombinant adenovirus and alphavirus replicons [73] [74] [75] [76] [77] [78] [79] . since the purpose of this study was to determine the capability of bohv-4-based viral vectors to protect stat1 (-/-) mice against a lethal mpxv infection, the first concern was the generation of optimized expression cassettes to be integrated into the bac-bohv-4-a genome that were able to efficiently express a29l, m1r and b6r antigens. because no antibodies are available for a29l, m1r and b6r proteins, a short in-frame sequence coding for a tag peptide (gd 106 ) was provided at the 3 0 end of their orfs and this allowed their expression to be monitored by western immunoblotting. initially a29l, m1r and b6r tagged orfs were customized under the transcriptional control of the cmv promoter; however, the only orf to be efficiently expressed was a29l. for this reason, the cmv promoter of the m1r and b6r expression cassette was substituted with the human ef1α promoter which induced expression. the reason why the cmv promoter did not work with the orfs of m1r and b6r has not been determined. another interesting observation was the presence of a29l protein into the supernatant of the transfected cells despite the absence of a canonical signal peptide within the primary sequence of the protein as deduced by different signal peptide prediction software (http://www.csbio.sjtu.edu.cn/bioinf/signal-3l/; http:// www.cbs.dtu.dk/services/signalp/; http://phobius.sbc.su.se/) [80, 81] . in fact, the a29l protein analysis by secretomep (http://www.cbs.dtu.dk/services/secretomep), a sequence based method for the prediction of mammalian secretory proteins targeted to the non-classical secretory pathway, included a29l within the group of non-classical secreted proteins like fibroblast growth factors, some interleukins and galectins. bohv-4 is considered a virus without a clear disease association, the existence of a bohv-4 potentially pathogenic biotype cannot be absolutely excluded when a virus is going to be exploited as a gene delivery vector. bovine herpesvirus 4 (bohv-4) has been most consistently associated with uterine disease in postpartum cattle and bohv-4 infection is often identified concurrently with bacteria that cause uterine diseases [82, 83] . the association between bohv-4 infection and uterine disease has been difficult to establish. it was suggested that there may be an association with bacterial endometritis which leads to secretion of prostaglandin e2 (pge2) and then stimulation of viral replication by pge2, tnf-α and lipopolysaccharide (lps)-which causes further endometrial tissue damage and inflammation [84] [85] [86] . for this reason, a putative non-pathogenic strain of bohv-4 (bohv-4-a) isolated from the cell milk fraction of a healthy cow whose genome was cloned as a bacterial artificial chromosome (pbac-bohv-4-a) [28] was used. cmv-a29lgd 106 , ef1α-m1rgd 106 and ef1α-b6rgd 106 were integrated into the tk locus of pbac-bohv-4-a, and proved to be stable through passages in e. coli sw102 and recombinant viable bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk, bohv-4-a-ef1α-b6rgd 106 δtk were successfully reconstituted in bekcre cells. when bohv-4-a-cmv-a29lgd 106 δtk, bohv-4-a-ef1α-m1rgd 106 δtk and bohv-4-a-ef1α-b6rgd 106 δtk were characterized in terms of replication kinetics, a reduction of replication was observed for bohv-4-a-ef1α-b6rgd 106 δtk and this was attributed to a partial toxic effect induced by the abundant expression of b6rgd 106 -which was also observed in transfections of cells with ef1α-b6rgd 106 expression cassette. this latter observation excluded a potential detrimental effect induced by the topological location of the foreign dna in the bohv-4 genome. despite their replication characteristics, all three recombinant bohv-4s abundantly expressed their transgene in infected cells. in vivo protection studies determined that m1r protected against a lethal mpxv challenge. one hundred % protection was achieved when the vectors were administered twice (prime followed by booster), although the m1r expression vector was not superior to vaccination with mva as measured by weight-loss. protection was also afforded to mice when the vectors were administered in combination (combo) as a prime and booster. these mice experienced less weight-loss than mice vaccinated with m1r alone. this finding is surprising as the other vectors included in the combo were not protective when administered individually, although we did find protection against weight-loss when mice were treated with b6r alone. nevertheless, our studies reveal that protection can be afforded even when a small number of mice are used. further studies should be considered that increase the dose of vector administered to the mice. also, since the combination of all 3 vectors gave 80% protection against mortality and morbidity, various vector permutations should be considered to elucidate the most efficacious combination and ratio of vectors. no overt clinical-signs were observed following vaccination with the prime or booster injection, suggesting low immuno-reactivity and therefore possibly low-levels of adverse events in nhps and humans. although second generation live vaccines, such as acam2000, provide the most robust immune response, they are quite reactogenic and induce some level of morbidity in all vaccines. furthermore, a significant portion of the human population are contraindicated to vaccination with first-and second-generation vaccines [19] . mva is a non-replicating vaccine that has demonstrated efficacy in many animal trials. the main draw-back to mva is its relatively low immunogenicity, meaning that booster administrations are usually required for 100% protection against morbidity and mortality [87, 88] . future studies could reveal that vectors' studied here could be used as alternatives to mva. in summary, our findings have demonstrated that bohv-4 based vectors can be used as vaccines to protect against a lethal mpxv challenge in mice. our studies utilized stat1 (-/-) mice; however, other strains have demonstrated sensitivity to mpxv, namely the caste/eij strain [49, 50, 89] . future studies should consider evaluating the protection of these vectors in this strain also. this work provides a "proof-of-concept" for the bohv-4-based vector as a potential vaccine for category a agents. future and ongoing studies are focused on the design of bohv-4-based vectors expressing antigens from other category a pathogens, as well as an assessment of protection in non-human primate. a review of experimental and natural infections of animals with monkeypox virus between 1958 and 2012 human monkeypox: an emerging zoonotic disease diagnosis and management of smallpox status of human monkeypox: clinical disease, epidemiology and research reemergence of monkeypox: prevalence, diagnostics, and countermeasures outbreaks of disease suspected of being due to human monkeypox virus infection in the democratic republic of congo in 2001 a tale of two clades: monkeypox viruses virulence differences between monkeypox virus isolates from west africa and the congo basin clinico-epidemiological features of monkeypox patients with an animal or human source of infection multistate outbreak 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recombinant outer membrane proteins of intracellular and extracellular virions neutralizing and protective antibodies directed against vaccinia virus envelope antigens subunit recombinant vaccine protects against monkeypox dna vaccination with vaccinia virus l1r and a33r genes protects mice against a lethal poxvirus challenge differential antigen requirements for protection against systemic and intranasal vaccinia virus challenges in mice vaccination with venezuelan equine encephalitis replicons encoding cowpox virus structural proteins protects mice from intranasal cowpox virus challenge molecular smallpox vaccine delivered by alphavirus replicons elicits protective immunity in mice and non-human primates improved prediction of signal peptides: sig-nalp 3.0 feature-based prediction of nonclassical and leaderless protein secretion bovine herpesvirus 4-associated postpartum metritis in a spanish dairy herd mice against monkeypox virus plos neglected tropical diseases seroprevalence and comparison of isolates of endometriotropic bovine herpesvirus-4 bacterial infection of endometrial stromal cells influences bovine herpesvirus 4 immediate early gene activation: a new insight into bacterial and viral interaction for uterine disease the chemokine il8 is up-regulated in bovine endometrial stromal cells by the bohv-4 ie2 gene product, orf50/rta: a step ahead toward a mechanism for bohv-4 induced endometritis bovine endometrial stromal cells support tumor necrosis factor alpha-induced bovine herpesvirus type 4 enhanced replication clinical and immunologic responses to multiple doses of imvamune (modified vaccinia ankara) followed by dryvax challenge safety, immunogenicity and efficacy of modified vaccinia ankara (mva) against dryvax challenge in vaccinia-naive and vacciniaimmune individuals susceptibility of the wild-derived inbred cast/ei mouse to infection by orthopoxviruses analyzed by live bioluminescence imaging we would like to thank alfonso rosamilia for technical assistance. key: cord-000283-7s6283y5 authors: nazmi, arshed; dutta, kallol; basu, anirban title: antiviral and neuroprotective role of octaguanidinium dendrimer-conjugated morpholino oligomers in japanese encephalitis date: 2010-11-23 journal: plos negl trop dis doi: 10.1371/journal.pntd.0000892 sha: doc_id: 283 cord_uid: 7s6283y5 background: japanese encephalitis (je), caused by a mosquito-borne flavivirus, is endemic to the entire south-east asian and adjoining regions. currently no therapeutic interventions are available for je, thereby making it one of the most dreaded encephalitides in the world. an effective way to counter the virus would be to inhibit viral replication by using anti-sense molecules directed against the viral genome. octaguanidinium dendrimer-conjugated morpholino (or vivo-morpholino) are uncharged anti-sense oligomers that can enter cells of living organisms by endocytosis and subsequently escape from endosomes into the cytosol/nuclear compartment of cells. we hypothesize that vivo-morpholinos generated against specific regions of 3′ or 5′ untranslated regions of jev genome, when administered in an experimental model of je, will have significant antiviral and neuroprotective effect. methodology/principal findings: mice were infected with jev (gp78 strain) followed by intraperitoneal administration of morpholinos (5 mg/kg body weight) daily for up to five treatments. survivability of the animals was monitored for 15 days (or until death) following which they were sacrificed and their brains were processed either for immunohistochemical staining or protein extraction. plaque assay and immunoblot analysis performed from brain homogenates showed reduced viral load and viral protein expression, resulting in greater survival of infected animals. neuroprotective effect was observed by thionin staining of brain sections. cytokine bead array showed reduction in the levels of proinflammatory cytokines in brain following morpholino treatment, which were elevated after infection. this corresponded to reduced microglial activation in brain. oxidative stress was reduced and certain stress-related signaling molecules were found to be positively modulated following morpholino treatment. in vitro studies also showed that there was decrease in infective viral particle production following morpholino treatment. conclusions/significance: administration of vivo-morpholino effectively resulted in increased survival of animals and neuroprotection in a murine model of je. hence, these oligomers represent a potential antiviral agent that merits further evaluation. the genus flavivirus is composed of more than 70 different closely related species [1] . many flaviviruses are arthropod-borne and causes significant human diseases. among these, the four serotypes of dengue virus (denv), yellow fever virus (yfv), west nile virus (wnv) and japanese encephalitis virus (jev) are categorized as emerging global pathogens [2] . jev is a mosquitoborne, positive sense, single stranded rna virus, responsible for frequent epidemics of encephalitis, predominantly in children, in most parts of southeast asia and adjoining regions. it is the causal factor for 30,000-50,000 cases of encephalitis occurring every year and accounts for about 10,000 deaths annually with serious neurological squeal in the survivors [3] . jev has been expanding its 'geographical footprint' into previously non-endemic regions and with several billion people at risk, japanese encephalitis (je) represents an internationally emerging concern in tropical and sub-tropical countries. currently three types of je vaccine are in use-the inactivated mouse-brain derived, the inactivated cellculture derived and the live attenuated cell-culture derived. however, there are limitations for their usage in terms of availability, cost and safety [4] . at present, chemotherapy against jev is largely supportive and not targeted towards the virus. a lot of avenues has been explored in the past and are also being currently tried, so as to develop a safe and effective molecule that would be able to prevent the virus from replicating within the host. the jev genome is approximately 11 kb in length that carries a single long open reading frame (orf) flanked by a 95-neucleotide 59 untranslated region (59 utr) and a 585-neucleotide 39 utr. the orf encodes a polyprotein which is processed by viral and cellular proteases into three structural and seven non structural proteins [5, 6] . the 59 and 39 utrs of the jev genome contain conserved sequence elements and can form conserved stem loop structure. 59 utr contain secondary structures which are required for the formation of translation pre-initiation complex [7] . jev requires long range rna-rna interaction between 59 and 39 regions of its genome for efficient replication; one such interaction occurs between a pair of 10 complementary nucleotides, located in coding sequence for the capsid protein at 136-146 nucleotides from 59 end of the genome, and 39 cyclization sequence, commonly denoted as 39csi (39 conserved sequence i) located at 104-114 nucleotides from 39 end of the genome [8, 9] . the 39csi is highly conserved across members of jev serocomplex, indicating the possibility that rna elements within the 59 and 39 utrs in jev genome are essential for its replication. anti-sense oligonucleotides have been shown to be effectively used as therapeutic agents against viral infection. in one such study sirna generated against the cd loop-coding sequence in domain ii of the viral envelope protein (which is highly conserved among all flaviviruses because of its essential role in membrane fusion) has been found to protect against lethal encephalitis [10] . similarly sirnas has also been generated against various nonstructural proteins of jev and were found to be effective in inhibiting viral replication [11, 12] . anti-sense approach has also been employed to inhibit flaviviral replication by generating anti-sense molecules against rna elements within the 59 and 39 utrs in flaviviral genome. in one such approach, dnazyme against 39 utr of jev genome has be found to be effective in controlling virus infection in a murine model [13] . under the same approach but with different kind of anti-sense oligonuleuotide called morpholino, flaviviral replication has been inhibited in cultured cells as well as in animal models [14, 15] . morpholino oligomers are single stranded dna analogues containing same nitrogenous bases as dna but joined by backbone consisting of morpholine rings and phosphorodiamidate linkages [16] . for efficient delivery into cells these morpholino are often conjugated with arginine rich peptide [17] . however, in the current study we have used a different type of morpholino oligomer called vivo-morpholino against 39csi and one of the secondary structures present in 59 utr of the jev genome. vivo-morpholino are specialized type of non-peptide morpholino oligomers, conjugated with a new transport structure that provides effective delivery into a wide variety of tissues in living animals, thereby raising the possibilities of their use as therapeutic agents. the transporter comprises of a dendritic structure assembled around a triazine core which serves to position eight guanidinium head groups in a conformation effective to penetrate cell membranes. vivo-morpholinos have also been shown to effectively enter and function within cultured cells [18] . vivo-morpholinos are also cost effective, non immunogenic, and stable under physiological conditions as compared to other types of morpholinos. this study was designed to evaluate whether the use of vivo-morpholinos as therapeutic agents, is possible in an experimental model of je. we intend to show that these specifically designed vivo-morpholinos are effective in countering the viral load in the body, thereby imparting significant protection to the animals that were infected with a lethal dose of jev. all animal experiments were approved by the institutional animal ethical review board named ''institutional animal and ethics committee of national brain research centre''. the animal experiment protocol approval no. is nbrc/iaec/2007/ 36. animals were handled in strict accordance with good animal practice as defined by committee for the purpose of control and supervision of experiments on animals (cpcsea), ministry of environment and forestry, government of india. vero cells (a kind gift from dr. guruprasad medigeshi, translational health science and technology institute, gurgaon, india) and neuro2a (obtained from national centre for cell science, pune, india) cells were grown in dmem (dulbecco's modified eagles medium, supplemented with 10% fetal bovine serum (fbs) and antibiotics. the gp78 strain of jev was propagated in suckling balb/c mice and their brains were harvested when symptoms of sickness were observed. a 10% tissue suspension was made in mem (minimum essential medium), followed by centrifugation at 10,0006g and finally filtered through a 0.22 m sterile filter [19] . jev was titrated by plaque formation on vero cell monolayer. vero cells were seeded in six-well plates to form semi-confluent monolayer in about 18 h. cell monolayer were inoculated with 10-fold serial dilutions of virus samples made in mem containing 1% fbs and incubated for 1 h at 37uc with occasional shaking. the inoculum was removed by aspiration and the monolayers were overlaid with mem containing 4% fbs, 1% low-melting-point agarose and a cocktail of antibiotic-antimycotic solution (gibco, invitrogen corporation, grassland, ny, usa) containing penicillin, streptomycin, and amphotericin b. plates were incubated at 37uc for 72-96 h until plaques became visible. to allow counting of the plaques, the cell monolayer was stained with crystal violet after fixing the cells with 10% formaldehyde. all vivo-morpholino (mo) oligos were commercially procured from gene tools llc, (philomath, or, usa). mos were japanese encephalitis (je) is caused by a flavivirus that is transmitted to humans by mosquitoes belonging to the culex sp. the threat of je looms over a vast geographical realm, encompassing approximately 10 billion people. the disease is feared because currently there are no specific antiviral drugs available. there have been reports where other investigators have shown that agents that block viral replication can be used as effective therapeutic countermeasures. vivo-morpholinos (mos) are synthetically produced analogs of dna or rna that can be modified to bind with specific targeted regions in a genome. in this study the authors propose that in an animal model of je, mos specifically designed to bind with specific region of je virus (jev) genome, blocks virus production in cells of living organisms. this results in reduced mortality of infected animals. as the major target of jev is the nerve cells, analysis of brain of experimental animals, post treatment with mos, showed neuroprotection. studies in cultured cells were also supportive of the antiviral role of the mos. the potent anti-sense effect in animals and lack of obvious toxicity at the effective dosage make these mos good research reagents with future therapeutic applications in je. vivo-morpholino in japanese encephalitis www.plosntds.org designed to be complementary to sequences in the jev (gp78 strain) genome, as shown in table 1 . these oligonucleotides targeted specific regions in the 39 and 59 utrs of the jev genomic rna (figure 1) . a 21 base scrambled mo of random sequence (sc-mo) was used as a negative control in all the experiments. all mo sequences were screened with blast (http://www.ncbi.nlm.nih.gov/blast) against primate and murine mrna sequences and the sc-mo was additionally screened against all flaviviral sequences. all mos were procured in 300 nanomole quantities as a liquid of 0.5 mm stock (approximately 4 mg/ml) in buffered saline. they were diluted with sterile 16 pbs to achieve desired concentrations, and stored at 4uc as aliquots. five to six weeks old balb/c mice of either sex were randomly distributed into 5 groups-sham, jev-infected, jev-infected and treated with scrambled morpholino (jev+sc-mo), jev-infected and treated with morpholino against viral 39 conserved region (jev+39 mo) and jev-infected and treated with morpholino against secondary structure in the 59utr of viral rna (jev+59 mo). initially each group contained 8 animals. animals belonging to all groups except sham were infected with 3610 5 plaque forming units (pfu) of jev (gp78 strain) and that day was considered as day zero [20] . animals of sham group received equal volume of filtered mem. starting from 3 h post infection on day zero, 100 mg of sc-mo, 39 mo and 59 mo, diluted in 0.1 ml of sterile 16pbs (corresponding to 5 mg/kg body weight), were administered to animals belonging to jev+sc-mo, jev+39 mo and jev+59 mo groups respectively, once per day, for 5 consecutive days. animals belonging to the sham-treatment group received equal volumes of sterile 16 pbs only. survivality of animals in each group following jev infection and morpholino treatment were monitored daily upto 15 days post jev infection (or till their death, whichever was earlier). toxicity of the morpholinos in mice was evaluated by weight loss and abnormal behavioral & clinical observations (including tremors, ruffled fur, hunching, ataxia, gait abnormalities), in a masked manner to minimize bias [14, 20] . mouse cytokine bead array (cba) kits were used to quantitatively measure cytokine levels in mouse whole-brain lysates. 50 ml of bead mix containing a population of beads with distinct fluorescence intensities that have been coated with capture antibodies for different cytokines, and 50 ml of whole-brain lysates were incubated together, along with equal volume of phycoerythrin (pe)-conjugated detection antibodies, for 2 h at room temperature, in dark. the beads were then washed and resuspended in 300 ml of supplied 16 wash buffer. the beads were acquired using cell quest pro software in facs calibur and analyzed using bd cba software (becton dickinson, san diego, ca). standard curve was prepared by incubating 50 ml of supplied mouse inflammation standards with 50 ml of bead mix and pe-conjugated detection antibodies [21] . protein concentrations of whole brain lysates were estimated by bradford method. sample volumes containing 20 mg of protein were electrophoresed on polyacrylamide gel and transferred onto nitrocellulose membrane. after blocking with 7% skimmed milk, the blots were incubated overnight at 4uc with primary antibodies against jev e-glycoprotein (abcam, usa), and jev ns5 (a kind gift from dr. chun-jung chen, taichung veterans general hospital, taichung, taiwan), inos (upstate-chemicon, usa), hsp-70, sod-1 (santa cruz biotechnology, ca, usa), trx (ab frontiers, korea; a kind gift from dr. ellora sen, nbrc), phospho nfkb, phospho erk1/2, total erk1/2 and phos-phop38 map kinase (cell signaling, usa) at 1:1000 dilutions. after extensive washes with pbs-tween, blots were incubated with appropriate secondary antibodies conjugated with peroxidase (vector laboratories, ca, usa). the blots were again washed with pbs-tween and processed for development using chemiluminescence reagent (millipore, usa). the images were captured and analyzed using chemigenius, bioimaging system (syngene, cambridge, uk). the blots were stripped and reprobed with antib-tubulin (santa cruz biotechnology, usa) to determine equivalent loading of samples [22] . for immunohistochemical staining, brains from scarified animals were excised following repeated transcardial perfusion with ice-cold saline and fixed with 4% paraformaldehyde. twenty micron thick cryosections were made with the help of leica cm3050s cryostat and processed for immunohistochemical staining to detect presence of jev antigen in the brain and to label activated microglia. sections were incubated overnight at 4uc with mouse anti-jev antigen (nakayama, 1:250) (chemicon, ca, usa) and rabbit anti-iba-1 (1: 500; wako, osaka, japan), respectively. after washes, slides were incubated with fitcconjugated anti-mouse or anti-rabbit secondary antibodies (vector laboratories inc. burlingame, usa) and following final washes, sections were sections were cover slipped after mounting with 49-6diamidino-2-phenylindole (dapi, vector laboratories inc.). the slides were observed under zeiss axioplan 2 fluorescence microscope and zeiss apotome microscope (zeiss, gottingen, germany), respectively [21] . cryosections of brain from sham-treated, jev-infected and jev-infected and mo treated animals were rinsed in de-ionized water followed by incubation with the thionin dye. the excess dye the level of ros produced within brain tissue of each treatment groups were measured by the cell permeable, nonpolar, h 2 o 2 -sensitive probe 5(and 6)-chlromethyl-20,70-dichlorodihydrofluoresceindiacetate (cm-h2dcfda; sigma, usa). cm-h2dcfda diffuses into cells, where its acetate groups are cleaved by intracellular esterases, releasing the corresponding dichlorodihydrofluorescein derivative. subsequent oxidations of cm-h2dcfda yields a fluorescent adduct dichlorofluorescein that is trapped inside the cell. brain homogenates were treated with 5 mm solution of cm-h2dcfda followed by incubation in dark at room temperature for 45 min and then the relative fluorescence intensity were measured with the help of varioskan flash multimode reader (thermo electron, finland) at excitation 500 nm and emission 530 nm. the fluorescence intensity of intracellular cm-h2dcfda is a linear indicator of the amount of h 2 o 2 in the cells. the measured mean fluorescence intensity was then normalized to equal concentrations of protein in each sample [23] . nitric oxide released from brain homogenates following mo treatment was assessed using griess reagent as described previously. briefly, 100 ml of griess reagent (sigma, st. louis, usa) was added to 100 ml of brain homogenate and incubated in dark for 15 min. the intensity of the color developed was estimated at 540 nm with the help of a benchmark plus 96-well elisa plate reader (biorad, ca, usa). the amount of nitrite accumulated was calculated (in mm) from a standard curve constructed with different concentrations of sodium nitrite [21] . mouse neuroblastoma cells (n2a) were plated in five 60 mm plates at a density of 5610 5 cells/plate, and were cultured for 18 h. after 6 h in serum free dmem, cells were either mockvivo-morpholino in japanese encephalitis www.plosntds.org infected with sterile 16pbs or infected with jev at multiplicity of infection (moi) of 5. after 1k h, cells were washed twice with sterile 16pbs to remove non-internalized virus. three of the four plates that were infected with jev, were treated with sc-mo, 39 mo and 59 mo at 10 mm concentrations and all plates were incubated for 24 h in serum free media. after two washes with 16 pbs, cells were first fixed with bd cytofix solution (bd biosciences) for 15 min and permeabilized by resuspending in permeabilization buffer (bd cytoperm plus; bd biosciences) and incubated at 25uc for at least 10 min. cells were then washed twice in wash buffer (pbs containing 1% bovine serum albumin) then resuspended in wash buffer at 1610 6 cells per 100 ml. primary antibody (jev nakayama strain; chemicon, usa) were added in 1:100 dilutions and incubated for 30 min at 25uc. the cells were washed with wash buffer and pelleted by centrifugation followed by incubation with fitc conjugated secondary antibody for 30 min. after final wash with wash buffer, cells were resuspended in 400 ml facs buffer and analyzed on a facs calibur. the percentage of population of jev-positive cells was calculated after gating the populations on a dot plot using cell quest pro software (bd biosciences). statistical analysis was performed using sigmastat software (spss inc., chicago, il, usa). data were compared between groups using one-way analysis of variance followed by post hoc test. differences upto p,0.05 were considered significant. mos confer protection to animal from japanese encephalitis mo treatment conferred significant protection to mice following jev infection. the survival of mice following jev infection was dramatically increased with treatments of both 39 and 59 mo. approximately 90% of all the animals that were treated with 39 mo survived as compared to 75% survival of those animals that were treated with 59mo, post infection with jev ( figure 2a ). infection with jev was accompanied with distinct symptoms and weight loss whereas treatments with both 39 and 59 mo post jev infection, prevented animals from suffering. not much considerable changes in the average body weights of jevinfected animals treated with both 39 and 59 mo were observed when compared to animals belonging to jev and jev+ sc-mo groups showing significant reductions in their body weights 6 days post infection ( figure 2b ). the symptoms associated with je in murine model were observed on daily basis and scores were attributed accordingly. the animals that had most symptoms received the highest scores. it was observed that 39 and 59 mo treated animals scored lesser than those belonging to the jevinfected or jev+sc-mo groups ( figure 2c ). to assess whether the mos has any effect on reduction of viral load in brain, homogenized brain samples from all the treatment groups were subjected to plaque assay as described in materials and methods section. number of pfu/ml of the brain homogenate was found to be significantly higher in both jev and jev+sc-mo groups when compared to sham (p,0.001). viral pfus were found to be significantly reduced following 39 mo and 59 mo treatment when compared to only jev-infected or jev+sc-mo group (p,0.001) ( figure 3a) . to further validate the results obtained from the plaque assay, immunoblot for some of the jev-specific proteins were performed. the expression of ns5, a non structural protein of jev, was significantly increased in jev and jev+sc-mo groups when compared to sham (p,0.01), but its level were found to be significantly reduced after both 39 and 59 mo treatments when compared to jev-infected group (p,0.01). similarly, e glycoprotein level showed significant increase in jev-infected and jev+sc-mo groups when compared to sham (p,0.01) which were then drastically reduced following 39 and 59 mo treatments (p,0.01) (figure 3b-d) . immunostaining of brain sections showed greater presence of jev antigen in jev-infected and jev+sc-mo groups, whereas 39 and 59 mo treatments resulted in lesser presence ( figure 3e ). to further characterize the inhibitory effects of mo on jevinduced neuronal death, brain sections from all the treatment groups were subjected to thionin staining. numerous healthy cells were seen in sections obtained from sham, jev+39 mo and jev+59 mo groups when compared to sections belonging to only jev-infected or jev+sc-mo groups which contained numerous unhealthy/dying neurons with altered morphology ( figure 4a ). microglial activation and increased proinflammatory cytokine production are the hallmarks of jev infection [24] . to see whether mo treatment helps in vitiation of these effects, immunostaining for microglial specific marker iba-1 was performed in brain sections of all treatment groups. in brain sections of jev and jev+sc-mo groups the number of activated microglia with characteristic morphology, appeared to be more frequent when compared to sections belonging to sham, jev+39 mo and jev+59 mo groups ( figure 4b ). cba performed to check the proinflammatory cytokines levels in the brain homogenates obtained from different treatments showed that levels of mcp-1, ifn-c, tnf-a, and il-6 were found to be significantly increased in both jev and jev+sc-mo groups when compared to sham infected groups (p,0.01). the elevated levels of these proinflammatory cytokines were drastically reduced with 39 and 59 mo treatments (p,0.01) ( figure 4c -f). increased oxidative stress in cns is a major outcome of jev infection [20] . to evaluate whether mo treatment of mice resulted in abrogation of oxidative stress following jev infection, we measured ros and no levels in brain homogenate obtained from all treatment groups. two fold increases were observed in the ros levels in the brain samples of jev and jev+sc-mo groups when compared to sham (p,0.01), significant reduction in the ros levels were observed in jev+39 mo and jev+59 mo groups when compared to only jev-infected groups (p,0.01). although ros levels has decreased in jev+39 mo group when compared to jev group, it remained significantly higher than that of sham (p,0.01) ( figure 5a ). superoxide dismutase 1 (sod-1) and thioredoxin (trx-1) are the proteins associated with oxidative stress. sod-1 levels were found to be elevated approximately 2-and 3-fold in jev-infected and jev+sc-mo groups respectively when compared to sham (p,0.01). its levels in jev+39 mo and jev+59 mo groups were reduced significantly when compared to jev-infected group (p,0.01). trx-1 levels were also found to be increased significantly in jev-infected and jev+sc-mo groups when compared to sham (p,0.01) but it were significantly reduced in brain samples obtained from jev+39 mo and jev+59 mo groups when compared to only jevinfected groups (p,0.01) (figure 5b, d&e) . hsp-70 is a heat shock vivo-morpholino in japanese encephalitis www.plosntds.org protein that has been associated with intracellular stress. significant twelve fold increases in the levels of hsp-70 were observed in jev and jev+sc-mo groups when compared to sham (p,0.01), this drastic increases in the levels of hsp-70 in jev and jev+sc-mo groups were reduced in jev+39 mo and jev+59 mo groups (p,0.01) ( figure 5b&c ). jev infection leads to increased nitric oxide (no) production in cns [25] . significant two fold increases were seen in the no levels in brain samples obtained from jev-infected and jev+sc-mo groups when compared to those obtained from sham (p,0.01). no levels subsequently got down to significantly lower levels following 39 and 59mo treatments (p,0.01) ( figure 5f ). immunoblot analysis showed nearly 8-fold increases in levels of inos in jev-infected and jev+sc-mo groups when compared to sham (p,0.01). inos levels showed significant decreases in jev+39 mo and jev+59 mo groups when compared to only jev-infected groups (p,0.01) ( figure 5g&h ). western blot analysis demonstrated a significant inhibition in the expression of different stress related proteins whose levels were elevated following jev infection. upon mo treatments there were approximately 4-fold increases in the levels of pnfkb in jev and figure 2 . mice are protected from jev following mo treatment. the survival of mice following jev infection was dramatically increased with treatments of both 39 and 59 mo, though the survival in 39 mo treated mice was greater (,90%) than those treated with 59mo (75%) (a). considerable changes in the average body weights of jev-infected animals treated with both 39 and 59 mo were not observed when compared to animals belonging to jev and jev+ sc-mo groups that showed significant reductions in their body weights from 6 th day post infection till their death. black arrows points to the days by which all the animals died. (b). infection with jev was accompanied with distinct symptoms that were alleviated following treatments with both 39 and 59 mo. animals were assigned scores according to the symptoms, in a blinded manner. the graph was plotted by taking the scores of one animal that was considered as the representative of that group (c). n = 8 for all experiments; data shown are representative of duplicate sets of experiments. doi:10.1371/journal.pntd.0000892.g002 vivo-morpholino in japanese encephalitis www.plosntds.org jev+sc-mo groups when compared to sham (p,0.01). the levels of pnfkb were found to be significantly reduced in jev+39 mo and jev+59 mo groups when compared to only jev-infected groups (p,0.01) ( figure 6a&b ). phospho p38 mapk levels also showed significant 3-fold increases in jev and jev+sc-mo groups when compared to sham (p,0.01), its levels were also found to be reduced significantly following treatment with 39 and 59 mo when compared to only jev-infected groups (p,0.01) ( figure 6a&c ). both phospho erk1 and erk2 levels were found to be significantly increased in jev and jev+sc-mo groups when compared to sham (p,0.01). the levels of phospho erk1 and erk2 showed considerable decreases in jev+39 mo and jev+59 mo groups when compared to only jev-infected groups (p,0.01) ( figure 6a&d ). to assess whether mo has any effect on viral load in vitro n2a cell lysates from all the treatment groups were subjected to plaque assay. pfu/ml of the cell lysates was found to be significantly higher in both jev and jev+sc-mo groups when compared to mock-infected cells (p,0.01). viral loads were found to significantly reduced in both jev+39 mo and jev+59 mo groups when compared to only jev-infected group (p,0.01) ( figure s1a ). to further ascertain the results obtained from plaque assay, intracellular staining of jev antigen in n2a was performed and number of jev-positive n2a cells was then counted by flow cytometry. only 16% and 9% of the total gated cells were found to be positive for jev antigen in jev+39 mo and jev+59 mo groups respectively as compared to 30% in jev-infected group, and 34% in jev+sc-mo group ( figure s1b ). use of anti-sense molecules for targeted inhibition of viral replication has been under investigation for quite sometime. though the application of these molecules has raised the possibilities of their future use as novel therapeutic agents, there vivo-morpholino in japanese encephalitis www.plosntds.org are many issues regarding their effectiveness in terms of their stability and delivery to targeted cells. recent studies are involved in developing techniques to minimize or eliminate these issues so that anti-sense therapy can be employed to a wide variety of intractable diseases such as splice-modifying genetic defects and viral diseases. the role of various anti-sense molecules in the inhibition of replication of jev has been reported with positive outcomes [10, 11, 12, 13, 26] . morpholino oligomers are single stranded anti-sense molecules that exert their action by steric blocking of complementary rna. unlike other types of anti-sense oligonucleotides, morpholinos provide all the desired properties of stability, nuclease resistance, high efficacy, long-term activity, water solubility, low toxicity, and exquisite specificity. morpholino oligomers has been used previously for the inhibition of flaviviral replication [14, 27] including jev [15] though all of them has utilized peptide based morpholinos. the peptide based morpholinos contain delivery moiety evolved from natural peptides whose active components are 6-9 arginine residues in a bio-available 6-aminohexanoicspaced structure [28] . however, these arginine-based peptides are not commercially available for research purposes and their greatest efficacies have been in delivering morpholinos to the cytosol of tissues like liver [29] or leaky muscle [30] , which would be considered as easily deliverable. as a result, the reach of peptide based morpholinos into a wide spectrum of tissues remains questionable [18] . also, owing to the peptidic nature, degradation of the peptide portion of the conjugates was found to be time and tissue dependent [31] . furthermore, the applications of the arginine-rich peptide transporters are limited due to their high cost, scalability and stability. added to that are the risks of immune responses against the peptides which limits repeated administrations for diseases requiring long-term treatment [32] . increases were observed in the ros levels in the brain samples of jevinfected and jev+sc-mo groups in comparison to sham, that were reduced following 39 and 59 mo treatments. although ros levels were decreased in jev+39 mo group when compared to jev-infected group, it remained significantly higher than that in sham (a). approximately 13-fold increases were observed in the levels of hsp-70 in jev-infected and jev+sc-mo groups as compared to sham. these drastic increases were significantly reduced in jev+39 mo and jev+59 mo groups, the levels remained significantly higher than sham (b&c). sod-1 levels were found to be elevated 2and nearly 3-fold in jev-infected and jev+sc-mo groups respectively compared to sham. 39 and 59 mo treatment caused significant reduction of sod-1 levels compared to jev-infected group (b&d). alterations in trx-1 levels were similar to that observed in sod-1 except that its level in jev+ sc-mo was not significantly different than only jev-infected group (b&e). nearly 2-fold increases were observed in no levels of jev-infected and jev+sc-mo groups when compared to those obtained from sham. no levels were subsequently reduced to significantly lower levels following 39 and 59mo treatments (f). inos expression was found to increase 8-fold in jev-infected and jev+sc-mo groups when compared to sham. following 39 and 59mo treatments inos levels decreased significantly as compared to jev-infected group (g&h). ( * p,0.01 for jev when compared to sham; ** p,0.01 for jev+sc-mo when compared to jev-infected only; # p,0.01 for jev+ 39mo to minimize the problems encountered by the peptideconjugated morpholinos, octaguanidinium dendrimer-conjugated morpholino oligomers have been developed that are commonly referred to as vivo-morpholino (mo). these custom-sequence anti-sense molecules have been reported to enable morpholino applications in adult animals. mo was our choice of anti-sense molecule as this enabled us to test the specifically designed oligonucleotides in both animal as well as cell culture models. though 'outstanding' results have been reported to be achieved by intravenous (i.v.) administration of the mo, we preferred the intraperitoneal route via which modest systemic delivery can be achieved. this was so done because brain has been reported to be an ineffective tissue when mos are administered i.v. [33] , though there is no direct evidence showing that mos can cross blood brain barrier, when administered via other routes. according to the manufacturer's (gene tools llc) instructions the maximum suggested dosage in mammals is 12.5 mg/kg in a 24 hour period. our aim was to determine the minimum dose at which our desired effects could be achieved. initially we had chosen two doses of 5 mg and 10 mg per kg body weight (b.w.) of the animals. we found that there was no significant difference between the survival rate of jev-infected and mo treated mice in either dose (data not shown for 10 mg/kg b.w.). the survival rate was approximately 90% in those jev-infected animals that were treated with 39 mo and 75% in animals treated with 59 mo. thus we decided to proceed with the 5 mg/kg b.w. dose for all subsequent experiments. plaque assay from the brain homogenates of animals of all groups revealed that the number of infective viral particle production was dramatically reduced following 39 and 59 mo treatment. the 39 mo was generated against the 39 csi region of the jev genome that interacts with 59 cs region located in coding sequence for capsid protein at 136-146 nucleotides from 59 terminal of the genome. this interaction results in cyclization of jev genome that is necessary for its efficient replication. the 59 mo was targeted towards one of the secondary structures of the 59 utr that are required for the formation of translation preinitiation complex. blocking of these two sites in the jev genome leads to the most likely effect, i.e. inhibition of replication and translation of viral genome. this was further corroborated by the decrease in the expressions of viral proteins (ns5, e glycoprotein vivo-morpholino in japanese encephalitis www.plosntds.org and general flaviviral envelop protein) in the brain. flaviviral ns5 is known to possess guanylyltransferase activity that helps in the synthesis of methylated cap structure at the 59 end of the viral genome that plays a crucial role in the translation and stability of mrnas [34] . the jev e glycoprotein is believed to be involved in viral adhesion and entry into host cells, hemagglutination, cellular tropism, viral virulence, and the induction of protective immune responses [35] . decreased expression of these proteins indicates that viral replication and production of new infective viral particles are inhibited due to the mos. immunohistochemical staining for viral antigen also provided visual confirmation of the fact that jev antigen was detected at much lower amounts in the brain following mo treatment. however, these data does not prove that mos directly inhibit infective viral particle production in the brain itself, as it cannot be conclusively stated whether the mos can reach brain. these data merely suggests that the number of replication-competent infective jev in the brain was significantly reduced, which subsequently leads to neuroprotection. it is well known that jev infection causes microglial activation. activated microglia releases an array of chemical mediators that are detrimental for the neurons in brain [24] . since there was reduction in the production of infective viral particles following 39 and 59 mo treatments, we studied the effect on microglial pathophysiology in mouse brain. our results show that there was significantly reduced number of activated microglia in the brain sections of both 39 and 59 mo-treated animals as compared to only jev-infected or jev-infected and sc-mo treated animals. since there were little or no activation of microglia, proinflammatory cytokine levels in the brain were found to be significantly downregulated. histochemical staining also revealed that neuronal population and morphology remained largely unaffected in 39 and 59 mo-treated animals' brains as compared to only jev-infected or jev-infected and sc-mo treated animals. generation of ros with the generation of oxidative damage has been implicated in neurodegenerative diseases and in the degradation of nervous system functions and are also reported to increase following jev infection [22] . increase in ros levels initiates various responses within the cell, including damage to proteins, dna and lipid [36] . in this study, ros levels were found to be many-fold increased in jev-infected or jev-infected and sc-mo treated animals that were then found to be counteracted by the treatment of 39 and 59 mo. the levels of stress related proteins such as sod-1, hsp-70 and trx where also found to be positively modulated following 39 and 59 mo treatment. no is a known antagonist of jev. it has been shown that no inhibits jev infection by preventing viral replication [37] . in our study no levels were increased in the brain in response to jev infection possibly due to the upregulation of inducible nitric oxide synthase (inos). treatments with 39 and 59 mo caused a decrease of no to basal levels as observed in sham-treated animals. activation of pnfkb regulates apoptotic genes, especially the traf1 and traf2, and thereby checks the activities of the caspases, which are central to most apoptotic processes. jev is known to activate pnfkb via a pi3k-dependent pathway in the brain of infected animals, which is associated with apoptosis [38] . jev infection has also been shown to activate stress kinases, which in turn results in activation of erk1/2, and p38 mapk pathway leading to apoptotic death of neurons [39] . in accordance with the established results, here also we found that there was similar activation pattern of these molecules in jev-infected and jevinfected and sc-mo treated animal brain samples. treatment with the mos resulted in abrogation of those changes that led to greater survivality of brain neurons as observed by histochemical staining. activation of p38mapk is also related to the transcriptional activation of proinflammatory genes in the brain [40] . thus the decrease in phophop38 mapk levels correlates with the decreased levels of proinflammatory cytokine levels in obtained from the brain. to confirm the anti-viral and neuroprotective property of the mos observed in in vivo models, cultured neuroblastoma cells were infected with jev, followed by mo treatment. though the mos are specifically developed for in vivo studies, they are also known to be taken up by cells in culture conditions [18] . there was a significant decrease in viral titer in samples obtained from the cells that were treated with 39 and 59 mos as compared to either jevinfected or jev-infected and sc-mo treated cells, as revealed by plaque assay. this data was supported by facs analysis following intracellular staining for jev antigen. this study was undertaken to determine the antiviral and neuroprotective efficacy of vivo-morpholinos in an experimental model of je so that it can be considered as a therapeutic agent in the near future. there have been studies regarding the anti-jev effects of other types of morpholino oligomers though none of them are yet to be considered for therapeutic purposes. this is the first study that investigates the role of morpholino oligomers specially designed for effective delivery into live animal models. generally, the i.p route of administration of any drug is preferred in animal studies over any other routes. however, the efficacy of these antisense molecules needs to be checked by administering through other applicable routes, as i.p. administration in humans is uncommon, though not unheard of. the amounts of oligomers required and the route of administration in this study marks these molecules as practicable therapeutic agents in je, though further studies are required before these can be recommended for clinical trials. figure s1 mo treatments decrease viral load in vitro. mouse neuroblastoma cell (n2a) lysates from all the treatment groups were subjected to plaque assay in order to determine viral loads. pfu/ml was found to be significantly higher in both jev and jev+sc-mo groups when compared to sham. viral loads were found to be significantly reduced in both jev+39 mo and jev+59 mo groups when compared to only jev-infected group (* p , 0.01 for jev and jev+sc-mo when compared to sham; # p , 0.01 for jev+39 mo and jev+59mo when compared to only jev-infected group) (a). intracellular staining for jev antigen in n2a was performed and number of jev-positive n2a cells was then sorted by flow cytometry. 30% of the total gated cells were found to be positive for jev antigen in jev-infected group as compared to 34% in jev+sc-mo group. only 16% and 9% of the total gated cells were found to be positive for jev antigen in jev+39 mo and jev+59 mo groups respectively (b). found at: doi:10.1371/journal.pntd.0000892.s001 (0.25 mb tif) present perspectives on flaviviral chemotherapy origin and evolution of japanese encephalitis virus in southeast asia japanese encephalitis-a pathological and clinical perspective preventive strategies for frequent outbreaks of japanese 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proinflammatory mediators released by activated microglia induces neuronal death in japanese encephalitis induction of nitric oxide synthase during japanese encephalitis virus infection: evidence of protective role inhibitory effect of rnai on japanese encephalitis virus replication in vitro and in vivo inhibition of dengue virus serotypes 1 to 4 in vero cell cultures with morpholino oligomers the design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters pharmacokinetics, biodistribution, stability and toxicity of a cell-penetrating peptide-morpholino oligomer conjugate cell-penetrating peptide-morpholino conjugates alter pre-mrna splicing of dmd (duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo cell penetrating peptide conjugates of steric block oligonucleotides design and synthesis of dendritic molecular transporter that achieves efficient in vivo delivery of morpholino antisense oligo gene knockdowns in adult animals: ppmos and vivo-morpholinos the flavivirus ns5 protein is a true rna guanylyltransferase that catalyzes a two-step reaction to form the rna cap structure recent advances in japanese encephalitis role of free radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment inhibition of japanese encephalitis virus infection by nitric oxide: antiviral effect of nitric oxide on rna virus replication antiviral and antiinflammatory effects of rosmarinic acid in an experimental murine model of japanese encephalitis japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response a novel p38 alpha mapk inhibitor suppresses brain proinflammatory cytokine up-regulation and attenuates synaptic dysfunction and behavioral deficits in an alzheimer's disease mouse model mfold web server for nucleic acid folding and hybridization prediction the authors would like to thank sulagna das and prosenjit pal for their help; kanhaiya lal kumawat and manish kumar dogra for their technical assistance. key: cord-001642-bom9fk1y authors: wang, junhua; vuitton, dominique a.; müller, norbert; hemphill, andrew; spiliotis, markus; blagosklonov, oleg; grandgirard, denis; leib, stephen l.; shalev, itay; levy, gary; lu, xiaomei; lin, renyong; wen, hao; gottstein, bruno title: deletion of fibrinogen-like protein 2 (fgl-2), a novel cd4(+) cd25(+) treg effector molecule, leads to improved control of echinococcus multilocularis infection in mice date: 2015-05-08 journal: plos negl trop dis doi: 10.1371/journal.pntd.0003755 sha: doc_id: 1642 cord_uid: bom9fk1y background: the growth potential of the tumor-like echinococcus multilocularis metacestode (causing alveolar echinococcosis, ae) is directly linked to the nature/function of the periparasitic host immune-mediated processes. we previously showed that fibrinogen-like-protein 2 (fgl2), a novel cd4(+)cd25(+) treg effector molecule, was over-expressed in the liver of mice experimentally infected with e. multilocularis. however, little is known about its contribution to the control of this chronic helminth infection. methods/findings: key parameters for infection outcome in e. multilocularis-infected fgl2(-/-) (ae-fgl2(-/-)) and wild type (ae-wt) mice at 1 and 4 month(s) post-infection were (i) parasite load (i. e. wet weight of parasitic metacestode tissue), and (ii) parasite cell proliferation as assessed by determining e. multilocularis 14-3-3 gene expression levels. serum fgl2 levels were measured by elisa. spleen cells cultured with cona for 48h or with e. multilocularis vesicle fluid (vf) for 96h were analyzed ex-vivo and in-vitro. in addition, spleen cells from non-infected wt mice were cultured with rfgl2/anti-fgl2 or ril-17a/anti-il-17a for further functional studies. for treg-immune-suppression-assays, purified cd4(+)cd25(+) treg suspensions were incubated with cd4(+) effector t cells in the presence of cona and irradiated spleen cells as apcs. flow cytometry and qrt-pcr were used to assess treg, th17-, th1-, th2-type immune responses and maturation of dendritic cells. we showed that ae-fgl2(-/-) mice exhibited (as compared to ae-wt-animals) (a) a significantly lower parasite load with reduced proliferation activity, (b) an increased t cell proliferative response to cona, (c) reduced treg numbers and function, and (d) a persistent capacity of th1 polarization and dc maturation. conclusions: fgl2 appears as one of the key players in immune regulatory processes favoring metacestode survival by promoting treg cell activity and il-17a production that contributes to fgl2-regulation. prospectively, targeting fgl2 could be an option to develop an immunotherapy against ae and other chronic parasitic diseases. alveolar echinococcosis (ae) is a very severe zoonotic helminthic disease in humans, exhibiting a fatal outcome if remaining untreated [1] . ae is characterized by chronic and progressive hepatic damage caused by the continuous proliferation of the larval stage (metacestode) of echinococcus multilocularis [2] , that behaves like a slowly growing liver cancer, progressively invading host tissues and organs [3] . during e. multilocularis infections in humans, a th2-oriented immunity is basically associated with increased susceptibility to disease leading to chronic ae, while th1 cell activation has been linked to protectivity, which may even yield aborted ("died-out") forms of infection [2, 3] . experimental murine ae is characterized, as studied in spleen or lymph node cells, by an initial th1 response during the early stage of infection (till 1 month p.i.) that gradually switches to a more dominant th2-biased response during the chronic phase of ae (2-4 months p.i.). nevertheless, this mostly mixed th1/th2 profile, characterized by the concomitant presence of il-12α, ifn-γ and il-4 at the very early stage of e. multilocularis infection [4] , is associated with the expression of pro-inflammatory cytokines in the periparasitic granuloma and partial/ relative protective immunity (restriction of parasite growth) through fibrosis and necrosis [5] . it has been previously reported that cd4 + cd25 + t regulatory cells (tregs) play a critical role in human ae by blunting immune responses to specific antigens, or by suppressing the secretion of proinflammatory cytokines, especially through interleukin (il)-10 and transforming growth factor beta1 (tgf-β1) [6] . moreover, increased cd4 + cd25 + tregs were also observed in peritoneal cells of mice intraperitoneally (i.p.) infected with e. multilocularis, a finding that concurred with other findings demonstrating that e. multilocularis antigens promote t cell differentiation into treg cells [7] . previous microarray analyses showed that expression of mrna coding for the fibrinogenlike protein 2 (fgl2) were significantly up-regulated in the liver of mice perorally infected with e. multilocularis eggs [8] . fgl2, a member of the fibrinogen-related superfamily of proteins secreted by t cells, has recently been reported by a number of groups to be highly expressed in tregs. its role was associated to treg effector functions [9, 10] . it was shown that fgl2 could inhibit dendritic cell (dc) maturation through binding to the low-affinity fcgammariib receptor, and thus contribute to treg activity [11] . there is evidence that fgl2 exerts an immunosuppressive effect on t cell proliferation. thus, fgl2 seems to play an important role both in innate and adaptive immunity, by the fact to be expressed by activated cd4 + and cd8 + t cells, and reticulo-endothelial cells as well [12] [13] [14] [15] [16] [17] . fgl2 has been propagated as a novel cancer biomarker, and was shown to be involved in the pathogenesis of inflammatory disorders such as allo-and xenograft rejection [12, [18] [19] [20] [21] [22] and cytokine-induced fetal loss [23] , as well as in the pathogenesis of infectious diseases, such as viral hepatitis [14, 17] . however, nothing is known about fgl2 and its potential role in parasite-induced immunotolerance. the major aims of this work were thus: 1) to study the role of fgl2 in t cell reactivity as well as its effect on the maturation of dcs in an early time-point and a late stage of e. multilocularis infection in fgl2 knock-out (fgl2 -/-) mice; 2) to elucidate how parasite components, i.e. metabolites represented by those expressed in the vf of the e. multilocularis metacestode, affect the immune response in fgl2 -/mice; 3) to explore how fgl2 is secreted during the course of e. multilocularis infection; and 4) to provide a comprehensive picture of the various cell and molecular components involved in the regulation of the peritoneal periparasitic immune cell infiltrate, and likewise in the spleen as a key immune organ. to achieve these goals, th1/ th2-related and treg/th17 related cytokines, the maturation of dcs, and the generation of tregs and their functions were studied at the different disease stages in an experimental model with active or knocked-out fgl2-expression. the animal study was performed in strict accordance with the recommendations of the swiss guidelines for the care and use of laboratory animals. the protocol was approved by the commission for animal experimentation of the canton of bern (approval no. be_103/11). 8-week-old female c57/bl6 (wild type [wt]) and c57/bl6 fgl2 -/mice [24] were bred and housed in specific-pathogen-free (spf) facilities according to recommendations of felasa, and monitored by daily assessment of health status, putative weight loss or gain during the experiments. e. multilocularis metacestodes (clone kf5) were maintained by serial passages (vegetative transfer) in c57bl/6 mice [25] and injected intraperitoneally as previously described [26, 27] . each experimental group included 6 animals unless otherwise stated. control mice (mock-infection) received 100 μl of rpmi-1640 only. mice were sacrificed at 1 or 4 month(s) post-infection, corresponding to early and late stage of disease, respectively. parasite tissues were surgically recovered and, if present, fat and connective tissues were carefully removed for subsequent wet-weight determination of the parasite mass. e. multilocularis 14-3-3 gene-expression analyses by quantitative reverse transcriptase real time pcr (qrt-pcr) total rna was extracted from parasite tissue previously put into trizol (invitrogen) according to the manufacturer's instructions. cdna was synthesized using the omniscript reverse transcription kit (qiagen, hilden, germany). sybr-green mix-based qrt-pcr was carried out on a rotor-gene 6000 qpcr detection system (corbett) with the faststart essential dna green master (roche, basel, switzerland) following the manufacturer's instructions. pcr cycling was performed in triplicates in final volumes of 20 μl containing 2 μl cdna and 10 pm of each primer (cycle scheme: initial denaturation at 95°c-15 min, 45 cycles of 95°c-15 s, 55°c-30 s and 72°c-30 s). fluorescence was measured in every cycle, and a melting curve was analyzed after the pcr by increasing the temperature from 55 to 95°c in 0.5°c increments. the primers used were described earlier [28] , and em14-3-3 mrna levels were quantified relative to the mrna level of a parasite housekeeping gene, the β-actin homologue e. multilocularis. respective mean values from triplicate determinations from 6 individual mice in each group were taken for the calculation of relative emii/3 and em14-3-3 mrna levels in relation to em-β-actin mrna levels). peritoneal exudate cells (pec) and spleen cells were collected by peritoneal rinsing or grinding in 5 ml rpmi-1640 (gibco, basel, switzerland) and incubation of pec or spleen cell suspensions in 15 ml rpmi-1640 +20%fcs in a petridish for 2 h at 37°c 5%co 2 , as described earlier [25] . the non-adherent cells were collected, and highly (n99%) enriched itreg cells were obtained by macs (magnetic cell-separation) using the mouse cd4 + cd25 + t cell isolation-kit (miltenyi biotec, germany) followed by facs. in vitro suppression assays were carried out with cultures of 2×10 4 cd4 + cd25 -t effector (teff) cells from wt-mice as responder cells, together with 8×10 4 irradiated spleen cells as apcs and titrated numbers of cd4 + cd25 + treg cells from either e. multilocularis-infected ae-fgl2 -/or ae-wt mice as suppressor-cells, compared with non-infected controls. for rfgl2/antibody blockade studies, 1 μg/ml mouse rfgl2 or anti-fgl2-(monoclonal igg2a; abnova, luzern, switzerland) were added to the cell cultures at a 1:1 treg:teff ratio in the presence of apcs and cona (2μg/ml). cell proliferation was assayed using the colorimetric brdu cell proliferation elisa kit (calbiochem, merck, switzerland) according to manufacturer's instructions. spleen cells were cultured at a density of 2×10 6 cells/ml in rpmi-1640 +10%fcs. for assessment of the effects of stimulation by recombinant fgl2 (rfgl2) or anti-fgl2 monoclonal antibodies (anti-fgl2-mab), (both from sigma-aldrich, basel, switzerland) they were incubated with 1 and 5 μg/ml rfgl2 or 1 μg/ml anti-fgl2-mab for 48h in the presence of a protein transport inhibitor cocktail (ebioscience, san diego, ca, usa) for cytokine staining. negative control reactions were performed without rfgl2 or without anti-fgl2-mab. the effects of recombinant il-17a (ril-17a) anti-il-17a antibodies (both from sigma), were assessed by stimulation of cells with 0.5, 1, 2 and 4 μg/ml ril-17a or 1 μg/ml anti-il-17a for 48h, while negative control reactions were performed without ril-17a or anti-il-17a antibodies. fgl2-levels in the serum and supernatant from cell cultures were measured by sandwich elisa (biolegend, san diego, ca) according to manufacturer's instructions. spleen cell cultures were also stimulated with 2 μg/ml cona for 48h, or with 10μg/ml of vf for 96 h, in the presence of protein transport inhibitor cocktail for cytokine staining. the same cell reactions performed without vf were used as negative controls. pec or spleen cells were incubated with 1 μg of purified anti-cd16/cd32 for 20 min in the dark to block non-specific binding of antibodies to the fcγiii/ii receptors, cells were then stained with surface markers separately for 15 min with 1 μg of primary antibodies: fitc-labeled anti-cd80, anti-cd86, anti-cd25; pe-labeled anti-cd11b, anti-cd11c, pecy 5.5-labeled anti-cd4. for intracellular staining, the cells were first incubated with inside-fix (miltenyi, bergisch gladbach, germany) for 20 min at room temperature then stained with pe-labeled anti-ifn-r, anti-il-4, anti-il-17a, anti-il-2, anti-il-10 and anti-foxp3 in inside-perm (miltenyi, bergisch gladbach, germany) for 15 min in the dark. corresponding fluorochrome-labeled isotype control antibodies were used for staining controls. for each sample, a minimum of 500,000 cells were acquired using a facs lsrii flow cytometer and analyzed using flowjo software (tree star, or, usa), employing the gating strategy shown in s1 all data were analyzed by spss 17.0. the results are presented as means ± sd. normality of data was assessed by d'agostino & pesrson and shapiro-willk test. for normally distributed groups of data, one-way-anova followed by bonferroni's post-test or unpaired two-tail student's t-test were used to compare the differences between groups, and two-tail spearman's rho was used to analyze the correlation coefficient. significance was defined as p<0.05 for all tests, except those subsequently corrected by bonferroni. the levels of fgl2 expression in sera of e. multilocularis infected (ae-wt) mice were significantly higher, both at 1 and at 4 month(s) post-infection, when compared to non-infected wt-controls ( fig 1a) . spearman correlation coefficients indicated a positive correlation between serum il-17a level and fgl2 (r = 0.435, p = 0.045) in ae-wt-mice. to examine whether il-17a contributes to fgl2-secretion, spleen cells from non-infected wt-mice were co-cultured either with recombinant il-17a (ril-17a) as an external stimulus, or with anti-il-17a antibodies, and fgl2 levels were quantitatively analyzed in respective culture supernatants by elisa. as shown in fig 1b, addition of ril-17a lead to increased fgl2-secretion in a dose-dependent manner, while the addition of anti-il-17a had no effect ( fig 1b) . to characterize the role of fgl2 in the control of parasite growth, e. multilocularis-infected fgl2 -/mice and control wt littermates were analyzed after 1 and 4 months p.i. with respect to parasite weight and the expression of em14-3-3 as a marker for cellular proliferation activity [24] . at the late stage of infection (4 months-p.i.), fgl2 -/mice exhibited a significantly lower parasite load compared to wt mice ( fig 1c and 1d ), and 14-3-3 expression levels in ae-fgl2 -/mice were significantly lower those in ae-wt mice ( fig 1e) . moreover, the parasite invaded i, upon comparison to the constitutively expressed house-keeping em ii/3-10 gene. data represent mean ± sd of three independent experiments of a total of 15-18 mice in each group (5-6 mice per group in each independent experiment). comparison between groups was performed using a one-way anova for statistical analysis. *p<0.05, ** p<0.01. 'wt', wild type mice; 'fgl2 -/-', fgl2 knock-out mice; 'ae-wt', e. multilocularis-infected wild type mice; 'ae-fgl2 -/-', e. multilocularis-infected fgl2 knock-out mice. 'control', non-infected mice; '1m', 1 month p.i.; '4m', 4 months p.i. au: arbitrary units. note: em 14-3-3 is used as a molecular marker to assess viability and growth activity of the e. multilocularis metacestode tissue, whereas em ii/3-10, so as actin, serve as constitutionally expressed house-keeping gene. the liver (a marker of pathogenicity) in only 33.3% of the ae-fgl2 -/mice compared to 94.4% of ae-wt mice. to explore the cellular source of secreted fgl2, cd4 + effector t cells (teffs), cd8 + t cells, cd4 + cd25 + tregs, antigen presenting cells (apcs) were facs sorted from spleen cell suspensions of ae-wt mice, and non-infected control mice. quantitative rt-pcr showed that fgl2 mrna-levels were significantly increased in cd4 + cd25 + tregs derived from ae-wt mice, when compared to non-infected controls, while no significant changes were evident with respect to cd4 + teffs, cd8 + t cells. a slight decrease in fgl2 mrna-levels were noted in apcs from ae-wt mice compared to those in non-infected mice (fig 2a) . the contribution of fgl2 in the generation and maintenance of tregs in ae-wt and ae-fgl2 -/mice was analyzed ex vivo, and in vitro by either addition of rfgl2 to spleen cells or treating cultures with anti-fgl2 antibodies. at 4 months p.i., an increased frequency of foxp3 + /cd4 + cd25 + cells could be observed in pecs as well as spleen cells from ae-wt mice, compared to respective preparations in ae-fgl2 -/mice (p<0.05) (fig 2b and 2c ). in addition, expression levels of foxp3 and il-10-transcripts were significantly increased in pecs from ae-wt mice ( fig 2d) . moreover, when in vitro cultured pecs from ae-wt mice were exposed to anti-fgl2-mabs and stimulated with vf, the frequency of cd4 + cd25 + foxp3 + cells was decreased compared to pecs cultured in the absence of anti-fgl2-mabs (fig 2e) . this indicated that in the absence of fgl2 e. multilocularis metabolic components might exert immune-modulatory activities. we then assessed the effect of the targeted deletion of fgl2 on the ability of treg cells to suppress the proliferation of teffs. treg cells from either non-infected or ae-fgl2 -/mice, were less efficient in suppressing normal cd4 + teff cell proliferation when compared to treg cells from wt-mice (s2 fig). to further study the role of fgl2 regarding treg functions, spleen cells from ae-wt mice were exposed to either anti-fgl2-mab or rfgl2. in response to rfgl2, tregs from ae-wt mice inhibited cona-induced cd4 + teff proliferation; conversely, the same tregs were not able to inhibit cd4 + teff proliferation in the presence of anti-fgl2-mabs ( fig 2f) . t cell functions are less affected in fgl2 -/mice at the late stage of infection to further explore the effects of fgl2 on the immune response during e. multilocularis infection, t cell functions, in ae-wt and ae-fgl2 -/mice were comparatively assessed. purified splenic cd4 + t cells from ae-fgl2 -/mice exhibited an increased proliferation in response to cona, as compared to splenic cd4 + t cells from ae-wt mice (p<0.01) (s3a and s3b fig). to further study the role of fgl2 in tcell proliferation, splenic cd4 + t cells from wt mice were cultured in the presence and absence of rfgl2. cd4 + teffs showed a pronounced proliferation in response to cona stimulation, which was inhibited by the addition of rfgl2 (s3c fig) . furthermore, t helper (th) cells from ae-fgl2 -/mice appeared oriented towards a lower th2 response at early stage of infection (1 months p.i.), and a stronger th1-response at late stage of infection (4 months p.i.) (fig 3a-3c and 3e ). this dichotomic polarization was, however, not confirmed by in vitro cultivation of the cells in the presence of cona stimulation (fig 3d) . respective flow cytometric analyses of cd4 + t cells in spleen from ae-fgl2 -/mice and ae-wt mice showed that there was no difference in expression of ifn-γ and il-4 at 48 h after of exposure to cona (fig 3d) . however, expression levels of il-17a and il-2 were significantly higher in cd4 + t cells in spleen cell cultures obtained from ae-fgl2 -/mice at 4 months p.i. when compared to ae-wt mice. the role of fgl2 in the maturation of different subsets of dcs, i.e. cd11b + and cd11c + dcs, was investigated. first, the maturation levels in both pecs and spleen cells from infected ae-fgl2 -/mice, ae-wt mice, and non-infected controls were studied. among cd11b + dcs, the frequency of the maturation marker cd80 in pecs and spleen cells was higher at 4 months p.i. in ae-fgl2 -/mice than in ae-wt mice (fig 4a and 4b) . the same was found for cd11c + dcs (fig 4g and 4h) . however, there was no difference in cd86 frequency in both subpopulations of dcs (fig 4d and 4e ). subsequently, we assessed the same parameters, but upon in vitro cultivation and stimulation with cona for 48 h. findings revealed that cd80 frequency, not cd86, in both dc subpopulations from ae-fgl2 -/mice at 4 months p.i. was significantly higher than in those from ae-wt mice (fig 4c,4f and 4i ). flow cytometry of spleen cells exposed to vf for 96 h showed that the expression of il-2 was significantly higher in cd4 + cells from ae-fgl2 -/mice obtained after 4 months p.i. than in the corresponding cell population isolated from ae-wt mice. however, there was no difference in expression of ifn-γ, il-4 or il-17a between cd4 + cells from ae-fgl2 -/mice and ae-wt mice (fig 5a) . to further explore the role of vf on tregs and fgl2 secretion, respectively, spleen cells from ae-wt mice and non-infected wt controls were each cultured in the presence of vf (10 μg/ml). fgl2 levels in the supernatants were determined by elisa. an identical experiment was performed with cd4 + cd25 + tregs and cd4 + teffs instead of spleen cells, in the presence of apcs. no differences in fgl2 levels in supernatants of sorted ae-wt tregs, nor in cd4 + teffs, could be detected in response to vf, when compared to cultures from non-infected animals (s4 fig) . for dcs at 96 h after exposure to vf, the cd80 frequency, both in cd11b + and cd11c + dcs from ae-fgl2 -/mice at 4 months p.i., was significantly higher than in dcs from ae-wt mice. however, there was no difference in cd86 frequency in both subpopulations of dcs from ae-fgl2 -/mice after exposure to vf, compared to dcs from ae-wt mice (fig 5b) . to further confirm the role of fgl2 on t cell functions and dc maturation, spleen cells from non-infected wt mice were cultured in the presence or absence of rfgl2 or anti-fgl2-mabs. flow cytometry showed that ifn-γ expression was specifically increased in response to vf in the presence of anti-fgl2 mabs, and il-17a expression was decreased in the presence of high concentration of rfgl2 (5 μg/ml) and there was no influence on il-17 expression upon exposure to anti-fgl2-mab (fig 6a) . the expression of cd62l, a cell adhesion molecule that is abundant on naïve lymphocytes, but not expressed on effector memory t-lymphocytes [29] , was found to be increased on cd4 + t cells in the presence of rfgl2 (1 μg/ml), but decreased in the presence of anti-fgl2 antibodies (fig 6a) . however, this was not the case upon analysis of total spleen cell populations (s5 fig), which indicated that fgl2 might play an important role in down-regulating lymphocyte co-stimulation and effector t cell production. for dcs, the expression of cd86 on cd11c + dcs was decreased in the presence of rfgl2, but increased in the presence of anti-fgl2 mabs. however, expression of cd86 on cd11b + dcs showed the opposite (fig 6b) . during infection with e. multilocularis metacestodes, immune tolerance and/or down-regulation of immunity is a marked characteristic that becomes more pronounced at the later stage of chronic disease in both humans [30] and in experimentally infected mice [5] . in the present study, we identified fgl2 as an important mediator of susceptibility to e. multilocularis infection in mice, and demonstrated for the first time that fgl2 a) partially contributes to treg functions; b) has the capacity to down-regulate the maturation of dcs; c) suppresses th1-and th17-type immune responses; d) promotes th2-biased and treg immune responses; and finally that e) il-17a contributes to fgl2-secretion. earlier microarray-based data obtained from e. multilocularis-infected liver tissue have shown that the peri-parasitic area is characterized by an increased fgl2 expression [7] . since -/mice, co-cultured with vf (10 μg/ml), in the presence of a protein transport inhibitor cocktail (ebioscience, san diego, ca, usa), normalized using cells from non-infected controls as baseline (e.g. 1.0). (b) relative expression level of dc maturation markers in spleen cells from infected ae-wt and ae-fgl2 -/mice, co-cultured with vf (10 μg/ml), normalized using cells from non-infected controls. data represent mean±sd of three independent experiments of a total of 15-18 mice in each group (5-6 mice per group in each independent experiment). comparison between groups was performed using a one-way anova with bonferroni's multiple comparison post-test for statistical analysis. *p<0.006. 'wt', wild type mice; 'fgl2 -/-', fgl2 knock-out mice; 'ae-wt', e. multilocularis-infected wild type mice; 'ae-fgl2 -/-', e. multilocularis-infected fgl2 knock-out mice. doi:10.1371/journal.pntd.0003755.g005 immunomodulation is a hallmark of ae, and treg cells are one of the key immune subsets to mediate this effect, our objective was to study the role of treg-expressed fgl2 in the outcome of e. multilocularis infection. we originally hypothesized that upregulated fgl2-expression at the host-parasite interface promotes parasite proliferation. comparative analysis of e. multilocularis growth in fgl2 -/mice versus wt mice at the late stage of infection showed clearly that ae-wt mice exhibited a significantly higher parasite load as compared to ae-fgl2 -/mice. in parallel, the parasite proliferation potential, assessed by determination of the e. multilocularis em14-3-3 gene-expression-level, was significantly higher in ae-wt mice, and respectively lower in ae-fgl2 -/animals. in previous experiments we had already documented that em14-3-3-expression significantly increased upon reduced protective immunity such as encountered in athymic nude mice, or decreased when the parasite growth was hindered as e.g. by albendazole therapy [28, 31] . ae-wt-mice also exhibited higher fgl2-levels in the serum as compared to non-infected mice, elevated fgl2 mrna expression levels in respective pec and spleen cells, and a concomitantly increased number of tregs, which represent one of the major treg producers [9] . taken together, these results support the hypothesis that treg-expressed fgl2 contributes to the pathogenesis of e. multilocularis infection. in other infection models, such as viral mhv-3 infection [10] , similar results have been demonstrated, while no such effects were found during protozoan (toxoplasma gondii), bacterial (yersinia enterocolitica, listeria monocytogenes, and mycobacterium tuberculosis), and other types of viral infections such as murine gamma-herpesvirus-68 and sendai infections [32] . the successful survival of pathogens depends mainly on evading the host immune response by, for example, varying their surface antigens, eliminating their protein coat, and/or modulating the host immune response. immunosuppression is sometimes directly caused by pathogen-derived metabolic products, and sometimes involves antigenic mimicry. one of the most sophisticated mechanisms of immune evasion is the selective activation of a directly targeted subset of t helper cells. different pathogens may target different regulative pathways of the host immune response, such as th2, tregs or th17. it is known that the regulation of such pathways is not dependent on single genes or molecules. rather, a complex immuno-network is usually involved, which requires a sophisticated modulatory process via specific modulatory molecules from e. multilocularis to become effective, some may even act in combination or exert together a synergistic effect. already know e. multilocularis bioactive molecules include e.g. alkaline phosphatase [33] , em492 [34] , emtip [7] , among others; if these are involved in e.g. fgl2-induction needs to be further explored. how fgl2 influences and/or modulates the outcome of experimental ae is still largely unclear, although some of our study data provide first indications. in experimental ae [5, 35, 36] , the control of metacestode proliferation appears to be predominantly t cell-dependent, as assessed upon use of different immune-compromised mouse models [27, 31, 37] , and confirmed by observations in human ae-patients with immune suppression-associated conditions [38] [39] [40] [41] . it is therefore conceivable that the proliferating metacestode itself specifically activates and concurrently modulates the immune response to its own advantage. the metacestode appears to secrete immuno-active metabolites to suppress the protective th1-type response, and to promote a secondarily reinforced th2-type response through functional induction and maintenance of treg cells [2, 25] . tregs, which over-express a subset of regulatory cytokine genes including those coding for il-10 and tgf-β, play an important role in promoting immune tolerance in a number of parasitic disease models [42] . in ae, the expression of both cytokines appears up-regulated in mice as shown by different studies [39] , and other studies strongly suggested that they are also up-regulated in human ae-patients [43] . various molecular and cellular mechanisms have been proposed to explain how tregs suppress immune responses. these include cell-to-cell contact-dependent suppression, cytotoxicity, and immunoregulatory cytokine secretion such as il-10 and tgf-β [44] . however, the importance of these cytokines remains controversial, as several reports have demonstrated that antibodies against il-10 and tgf-β failed to block treg suppressive function, and tregs from tgf-β-deficient mice retained normal suppressive activity in vitro [44] . in addition, the ambiguous role of tgf-β, which is both, a strong inducer of immune tolerance and an activator of the pro-inflammatory il-17 cytokine system, remains puzzling [45, 46] . undoubtedly, fgl2 represents an alternative candidate that could, at least partially, support regulatory, and thus immunosuppressive, functions of tregs. in tregs of wt balb/c mice, fgl2 encoding mrna is constitutively expressed at a high level, and expression even increased after mhv-3 infection, and adoptive transfer of wt tregs into mhv-3 resistant fgl2 -/mice suggested that fgl2 might be an important treg effector molecule [10] . previous studies on fgl2 had shown that rfgl2 suppressed t cell proliferation induced by anti-cd3/28 mabs and cona [47, 48] . in this study, we now demonstrated that rfgl2 suppressed cd4 + effector t cell proliferation in response to e. multilocularis antigenic metabolites present in the metacestode vf. we also showed that fgl2 inhibited the maturation of dcs, suppressed th1 and th17 immune responses, and fgl2 polarized an allogeneic immune response towards a th2-oriented cytokine profile, both in vivo and in vitro. conversely, in fgl2 -/mice, th1 cytokine levels and activity of dcs and t cells were all increased when compared to wt-animals, and fgl2-serum-levels correlated with il-4 expression in wt-mice before and after e. multilocularis infection. the development of a th2-oriented immune response in wt-mice during the course of e. multilocularis infection corroborated the generally known effect of fgl2 to promote a th2 cytokine production, with a concomitant inhibition of th1-and th17-oriented immunity [47] . furthermore, increased serum levels of il-17a correlated with high fgl2 serum levels, suggesting for the first time that il-17a could contribute to fgl2-secretion. this was confirmed in vitro by the finding that recombinant il-17a promoted the production of fgl2 in spleen cells. we also investigated whether fgl2 would affect the functional activities of treg cells, by directly assessing the effects of rfgl2 and of an anti-fgl2-mab on treg activities in vitro. the presence of recombinant fgl2 promoted treg function, while addition of anti-fgl2-mab completely abrogated treg activity. further evidence for the role of fgl2 as a molecule that promotes treg function, and thus immunosuppression, was given by the observation that fgl2 -/mice exhibited both decreased treg numbers and impaired treg function. the mechanism by which fgl2 mediates its immunosuppressive activity is currently under investigation. previous studies have indicated that fgl2 binds to the inhibitory fcγriib receptor (cd32) expressed primarily on apcs. both cd80 and cd86 ligands are found on apcs and are known to provide efficient costimulation, however, in our experiments, distinct functions may be attributed to cd80 and cd86. the differential functions of these molecules have already been the subject of considerable studies, with most data suggesting that the two ligands share substantially overlapping functions. there is, however, also an increasing evidence that supports the view that cd80 may be a more effective ligand for ctla-4 than cd86 [49] . this fgl2-fcγriib interaction through cd80 was shown to induce apoptosis in b cells, and to inhibit dc-maturation [11] . in e. multilocularis infection, several immune subsets may express the fcγriib receptor, such as macrophages (including the 'epithelioid cells' that line the 'immuno-modulating' laminated layer), and also the numerous cd8 + t cells present in the periparasitic infiltrate [50] ; cd8 + t cells have actually been shown to express the fcγriib receptor in a murine model of trypanosoma cruzi [51] . taken our recent data on the course of cytokine expression by the periparasitic immune infiltrate in e. multilocularis infection [4] combined with the results from this study we propose that in e. multilocularis infected mice the following events occur: (a) tnf-α, ifn-γ and il-17a are released by the host at early stage of infection; (b) these cytokines, and especially ifn-γ as demonstrated previously [32] but also il-17a as shown in the present study, contribute to fgl2-secretion by tregs and other cells; and (c) secreted fgl2 can bind to fcγriib receptor, leads to a late-stage immune suppression by down-regulating the maturation of dcs, decreases co-stimulation of effector t cells, suppresses th1 and th17 immune responses, and accelerates th2 immune responses. some parasite's specific molecules may be involved in the modulation of fgl2, as discussed earlier. overall, this will lead to a periparasitic immune suppressed (anergic) status that favors the continuous "tumor-like" progression of the parasite (fig 7) . direct inhibition of macrophage and/ or mast cell functions could also be induced by this interaction [24] . these findings on the role of fgl2 in e. multilocularis infection now open the door for more applied approaches. for instance, the experimental treatment of e. multilocularis infected mice with anti-fgl2-mab could provide a means of converting the immunological anergy during chronic disease into a more pro-active, th1-oriented immunity, with potentially fatal consequences for the metacestode. this is already under investigation. furthermore, our findings may provide a rationale for studying fgl2 as a target for an immunomodulatory ifn-γ as demonstrated previously, but also il-17a as presently demonstrated, contributes to fgl2 secretion by tregs and other cells; once fgl2 is released, it binds to fcγriib receptors, down-regulates the maturation of dcs, decreases co-stimulation of effector t cells, suppresses th1 and th17 immune response, accelerates th2 immune responses, and overall this leads to an immune suppressed status that favors the continuous "tumor-like" progression of the parasitic metacestode. doi:10.1371/journal.pntd.0003755.g007 fgl2 in murine alveolar echinococcosis treatment option in patients with progressive ae. in addition, fgl2 may be proposed also as a serum marker indicative for progression of e. multilocularis infection, may be useful to assess the clinical status of ae-patients and the course and outcome and/or parasite activity in human ae. and ae-fgl2 -/mice were cultured with cona (2 μg/ml) for 48h. the presented data were normalized with their own non-infected controls for statistical analyses (we considered non-infected control as baseline, e.g. as 1.0). (c) different concentrations of recombinant fgl2 (0, 1, 5 μg/ml), cona or vesicle fluid (vf), and anti-fgl2-mab (1 μg/ml) were added to primary spleen cells isolated from ae-wt mice, compared to cultures from non-infected animals. cd4 + t cell proliferation was determined by brdu elisa. comparison between groups was performed using a one-way anova. ã p<0.05. experiments include wt and fgl2 -/mice at 4 months post e. multilocularis infection. tregs and cd4 + t cells (teffs) in the presence of apcs from ae-wt and control-wt mice were cultured with vf (10 μg/ml) for 96h. fgl2-levels (culture supernatants) were determined by elisa. data represent mean±sd of three independent experiments of a total of 15-18 mice for each group (5-6 mice per group in each independent experiment). comparison between groups was performed using a one-way anova. ã p<0.05. (tif) s5 fig. cd62l -expression in response to rfgl2 and anti-fgl2. different concentrations of recombinant fgl2 (0, 1, 2 μg/ml) and anti-fgl2-mab (1 μg/ml), and pma as a positive control, were added to primary spleen cells isolated from non-infected wt mice. cd62l-percentage in total spleen cells was determined by flow cytometry. data represent mean±sd of three independent experiments of a total of 15-18 mice for each group (5-6 mice per group in each independent experiment). comparison between groups was performed using a one-way anova ã p<0.05. 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larval development experimental infection with trypanosoma cruzi increases the population of cd8(+), but not cd4(+), immunoglobulin g fc receptor-positive t lymphocytes we would like to thank cristina huber (institute of parasitology, university of bern) and franziska simon (institute for infectious diseases, university of bern) for their excellent technical assistance, prof. christoph müller (institute of pathology, university of bern) for his suggestions concerning some experimental approaches carried out and his pre-reviewing of the manuscript and respective improving suggestions, dr. britta stadelmann for providing additional infected mice needed to carry out additional experiments, the facs sorting facility of the department of clinical research, university of bern, for excellent assistance with cell sorting. key: cord-003482-f1uvohf0 authors: malmlov, ashley; bantle, collin; aboellail, tawfik; wagner, kaitlyn; campbell, corey l.; eckley, miles; chotiwan, nunya; gullberg, rebekah c.; perera, rushika; tjalkens, ronald; schountz, tony title: experimental zika virus infection of jamaican fruit bats (artibeus jamaicensis) and possible entry of virus into brain via activated microglial cells date: 2019-02-04 journal: plos negl trop dis doi: 10.1371/journal.pntd.0007071 sha: doc_id: 3482 cord_uid: f1uvohf0 the emergence of zika virus (zikv) in the new world has led to more than 200,000 human infections. perinatal infection can cause severe neurological complications, including fetal and neonatal microcephaly, and in adults there is an association with guillain-barré syndrome (gbs). zikv is transmitted to humans by aedes sp. mosquitoes, yet little is known about its enzootic cycle in which transmission is thought to occur between arboreal aedes sp. mosquitos and non-human primates. in the 1950s and ‘60s, several bat species were shown to be naturally and experimentally susceptible to zikv with acute viremia and seroconversion, and some developed neurological disease with viral antigen detected in the brain. because of zikv emergence in the americas, we sought to determine susceptibility of jamaican fruit bats (artibeus jamaicensis), one of the most common bats in the new world. bats were inoculated with zikv prvabc59 but did not show signs of disease. bats held to 28 days post-inoculation (pi) had detectable antibody by elisa and viral rna was detected by qrt-pcr in the brain, saliva and urine in some of the bats. immunoreactivity using polyclonal anti-zikv antibody was detected in testes, brain, lung and salivary glands plus scrotal skin. tropism for mononuclear cells, including macrophages/microglia and fibroblasts, was seen in the aforementioned organs in addition to testicular leydig cells. the virus likely localized to the brain via infection of iba1(+) macrophage/microglial cells. jamaican fruit bats, therefore, may be a useful animal model for the study of zikv infection. this work also raises the possibility that bats may have a role in zika virus ecology in endemic regions, and that zikv may pose a wildlife disease threat to bat populations. introduction zika virus (zikv) was first isolated from a sentinel rhesus macaque in uganda in 1947 and subsequently from aedes africanus mosquitoes in the same location [1] . the first human cases were identified in 1954 in nigeria and serosurveys found evidence of a broad geographic distribution for zikv throughout africa and asia with sporadic cases in humans [2, 3] . the first recognized zikv epidemic occurred in yap state, federated state of micronesia in 2007. an estimated 73% of residents were infected, and of those 18% presented with clinical disease [4] . in 2013, a second epidemic occurred in french polynesia with 28,000 cases reported. during the latter outbreak, the incidence rate of guillain-barré syndrome (gbs) increased 20-fold and first indication of a connection between zikv infection and gbs was established [5] . the virus spread to brazil in 2015 [6, 7] and has since disseminated throughout much of tropical south america, central america, the caribbean, and the southern united states, with more than 200,000 confirmed cases [8] . zikv can also cause congenital zika syndrome (czs) in naïve populations and is therefore a virus of high concern [3] . zika virus is maintained in an urban cycle, transmitted between an aedes mosquito vector and humans thereby maintaining endemicity [9] . it is generally accepted that the virus transmits between non-human primates and vectors in a sylvatic cycle; however, the sylvatic cycle has not been well characterized in the old world and little is known about a new world sylvatic cycle [9, 10] . molecular analysis of zikv to better understand viral phylogenetics suggests that animal hosts affected viral evolution and therefore may play an important role in viral ecology [11] . in the 1950s and '60s, the susceptibility of bats to zikv was investigated. shepherd and williams [12] screened 172 wild bats from 12 different species in uganda for antibodies against zikv and found 16/44 little free-tail bats (tadarida pumila) and 26/36 angolan freetail bats (t. condylura) were seropositive by hemagglutination inhibition assay. additionally, two angolan free-tail bats were experimentally inoculated with zikv and serially bled to test for viremia. both animals were viremic on days 2, 4 and 6 as determined by paralysis in mice inoculated with the sera from those two bats [12] . simpson and o'sullivan [13] experimentally inoculated three straw-colored fruit bats (eidolon helvum), three egyptian fruit bats (rousettus aegyptiacusi), and five angolan free-tail bats. two of the straw-colored fruit bats were viremic and had seroconverted. one of the egyptian fruit bats was viremic and two had seroconverted. the angolan free-tail bats were euthanized on days 1, 3, 5, 7 and 10 days post inoculation and screened for viral tropism. at one day post infection, a kidney was trace positive [13] . finally, reagan et al. [14] inoculated 20 new world little brown bats (myotis lucifigus) by 5 different routes: intracranial, intraperitoneal, intradermal, intrarectal and intranasal. bats in all groups, with the exception of the intranasal group, developed fatal neurological disease 4-7 days post inoculation. brain tissue was virus-positive in all animals with clinical disease, determined by inoculation of mice with brain homogenate suspension [14] . considering the evidence that african bats are naturally susceptible to zikv and that little brown bats develop disease, the question emerged: could bats serve as a natural reservoir host for zikv in the new world? to test this hypothesis, we inoculated jamaican fruit bats (artibeus jamaicensis), among the most abundant bats in the caribbean, central america and mexico, with zikv to examine virology, immunology and pathology of the infection. although virus was detected in several organs, including the testes and brains, no overt clinical signs were detected, and substantial viremia or viruria was not evident. these results suggest that jamaican fruit bats are unlikely to serve as amplification hosts but that zikv infection may constitute a wildlife disease threat to bats. bats for this project were obtained from the colorado state university breeding colony approved by the institutional animal care and use committee (protocol 16-6512a). two experimental infections were conducted; a pilot study and a time course study. in the pilotstudy, three male bats (aj-z7, aj-z8, aj-z9) were intradermally inoculated with 7.5x10 5 plaque forming units (pfu) zikv, strain prvabc59; a high dose to assess susceptibility. no signs of disease were apparent during this 28 day experiment; however, all three bats had antibody titers of 3200 on day 28 (table 1) . after demonstration of susceptibility in the pilot study, a time course study was conducted. six male bats (aj-z1 through aj-z6) were identically inoculated and two were euthanized at 2, 5 and 10 days post inoculation (dpi). no conspicuous signs of disease were observed in any of the inoculated bats. necropsies immediately followed euthanasia and no significant gross pathology was evident. quantitative probe-based reverse transcription pcr (qrt-pcr) was performed on seruminoculated vero cell supernatants, serum, brain, lung, liver, spleen, kidney, urinary bladder, prostate and testes from bats from both studies. in addition, urine collected during the time course study was similarly assayed. urine from bats aj-z6 at 3 dpi and aj-z7 at 5 dpi had low levels of vrna whereas bat aj-z1, euthanized at 2 dpi, had low levels of vrna in its brain ( fig 1) . all other samples were negative. sera from aj-z2 at 2 dpi, and aj-z3 and aj-z4 at 5 dpi were negative by elisa. sera were blind passaged on vero e6 cells in an attempt to isolate zikv and all were negative for cpe and pcr. hematoxylin and eosin stain (h&e). heart, lung, liver, kidney, testes, prostate, urinary bladder, and brain were collected from all 9 animals as well as salivary glands from 3/9 bats. all samples were blindly read by one pathologist. a summary of the consistent histopathology findings is listed in table 2 . for the time course study, aj-z1 at 2 dpi showed mild pulmonary congestion with multifocal areas of interstitial pneumonia, mild intra-alveolar hemorrhage and mild atelectasis. terminal airways had slightly increased amounts of mucus. kidneys had multifocal interstitial infiltrates of small numbers of lymphocytes. all other tissues were within normal limits. in ajz2 at 2 dpi, lungs showed milder pathology than aj-z1 with minimal interstitial to perivascular infiltrates predominately lymphocytes and macrophages with a band of collapsed air spaces subjacent to the pleural surface. there were focal lesions in the left ventricle of the heart where there was individual cell loss or else fragmentation of the sarcoplasm of scattered cardiomyocytes. degenerate/necrotic cardiomyocytes were accompanied by infiltrations of small numbers of macrophages, lymphocytes and satellite cells. all other tissues were within normal limits. lungs from aj-z3 at 5 dpi had minimal focal interstitial histiocytic pneumonia with atelectasis. kidneys showed multifocal chronic lymphohistiocytic pyelitis with a few degenerate and detached epithelial cells accumulating in the renal pelvis and infiltration of pelvic stroma by small numbers of mixed inflammatory cells. mandibular salivary gland showed focal moderate cellular infiltrates of periductular lymphocytes and macrophages. affected salivary ducts contained detached and degenerate epithelial cells and leukocytes. occasional ducts were encircled by granulation tissue and a few heterophils. rare apoptosis was evident in the lining epithelium of such ducts. all other tissues were within normal limits. aj-z4 at 5 dpi had lungs with minimal alveolar septal infiltrates scattered within collapsed lung parenchyma along with multifocal microscopic hemorrhages. kidneys had multifocal areas of mineralization. in the outer medulla and at the cortico-medullary junction were rare perivascular infiltrates of lymphoplasmacytes. esophagus and lymphoid tissue associated with palatine salivary gland showed focal mild lymphoplasmacytic inflammation. moderate numbers of lymphocytes and plasma cells were arranged in columns parallel to the respiratory mucosal epithelium of the nasophayrnx. the lumen contained increased amounts of mucus and a few inflammatory cells, mainly heterophils and lymphocytes. in the testicles, there was focal testicular degeneration manifested by presence of giant spermatids in the lumina of affected seminiferous tubules and accumulation of a small numbers of interstitial lymphocytes and macrophages. all other tissues were within normal limits. lungs from aj-z5 at 10 dpi had minimal interstitial to perivascular infiltrates with multifocal atelectasis and microscopic hemorrhages. the left papillary muscle of the heart showed rare multifocal cardiomyocyte necrosis characterized by rounding up of individual cardiomyocytes. necrotic cardiomyocytes appeared with hypereosinophilic cytoplasm, devoid of cross striations or fragmented and rarely vacuolated. minimal interstitial hypercellularity due to increased activity of satellite cells and infiltration of small numbers of lymphocytes was observed in the vicinity of degenerate/necrotic cardiac muscle fibers. kidneys had an area of focal lymphoplasmactyic pyelitis. additionally, there was a focal area of mineralization and inflammation in the inner medulla. all other tissues were within normal limits. aj-z6 at 10 dpi had occasional focal inflammation and cardiomyocyte degeneration in the left ventricle and interventricular septum. area ca3 of the hippocampus in the brain showed focal pyrimidal neuronal necrosis with a focal area of mineralization around a vessel in the cerebral cortex along with focal gliosis and individual neuronal necrosis (fig 2) . all other tissues were within normal limits. testicular, neural and salivary glands' lesions are believed to be associated with zikv infection as they were not seen with other viral infections. in the pilot study bats, aj-z7 at 28 dpi had more prominent interstitial pneumonia with congestion of the lungs compared to earlier time points. the heart had minimal cardiomyocyte degeneration and necrosis with hypercellular interstitium and increased amounts of mature fibrous connective tissue. the kidney had focal interstitial infiltrates of the cortical and outer medullary interstitium. the brain showed degenerate neurons in area a3 of the hippocampus. all other tissues were within normal limits. aj-z8 had minimal focal testicular degeneration (fig 3) . all other tissues were normal. aj-z9 had perivascular lymphocyte pulmonary infiltrates and atelectasis. heart demonstrated locally extensive lymphocytic and histiocytic pericarditis. kidneys showed multifocal interstitial lymphocytic infiltrates. brain had focal, perivascular infiltrates of small numbers of lymphocytes at the subfornical commissure. the reticular formation showed multifocal neuronal degeneration/necrosis. immunohistochemistry and immunofluorescence. tissues were stained with a polyclonal antibody for zikv (cdc, fort collins). ajz-3 at 5 dpi with inflammation of the mandibular salivary gland had moderate immunoreactivity in the lumen of affected ducts (fig 4) . aj-z5 at 10 dpi had immunoreactive cells in the brain and mononuclear cell immunoreactivity in the testes ( fig 5) . additionally, aj-z5 demonstrated immunoreactivity in purkinje cells of the cerebellum (fig 6a) . aj-z8 at 28 dpi had immunoreactive cells around the pulmonary arteries in the lungs ( fig 7a) . aj-z8 also had immunoreactivity perivascullarly in the tunica albuginea of the testes (fig 8a) . scrotal skin had focal lymphocytic dermatitis with immunoreactive mononuclear cells ( fig 8d) . cell morphology consistently identified mononuclear cells compatible with macrophages and fibroblasts as the primary cell types showing immunoreactivity against zikv antigen. brain and testicular tissues stained with both goat polyclonal goat anti-iba1 (green) and monoclonal 4g-2 flavivirus e specific antibodies (red) showed co-localization (yellow) of zikv antigen in cytoplasm of activated microglial cells with their characteristic morphology in the cerebral cortex of infected bats 10 dpi in the time course study and 28 day dpi in the pilot study (fig 9) . increased microgliosis was noted in the vicinity of co-localization sites. the gliosis was also prominent in the cerebellum and hippocampus especially around dead neurons. in the testicles, occasional macrophages showed similar co-localization similar to that noted in the brain in the testicular interstitium, inner layer of tunica albuginea and scrotum. cells consistent in morphology with leydig cells were similarly highlighted by zika viral antigen only showing strong immunoreactivity using polyclonal anti-zikv antibody. two bat infection experiments were conducted in this investigation; 1) a pilot study to determine susceptibility of jamaican fruit bats to zikv infection, and 2) a time course study to better understand pathophysiology and chronology of events pertaining to the dynamics of viremia, viral tropism, replication and shedding of the virus in a new world bat species. the goal was to determine whether bats can be used as an animal model for zikv pathogenesis and to assess the possible role of bats in zikv ecology in the new world. in the pilot experiment, no signs of disease were apparent during the 28-day study. sera collected at euthanasia indicated modest antibody titers of 3200 for each bat by elisa (table 1) , whereas the human -convalescent control serum titer was �12,800. bats typically have low to modest antibody titers, perhaps due to limited somatic hypermutation and affinity maturation [15] [16] [17] [18] [19] [20] [21] [22] . concerning viremia, cell-serum supernatants, blind passage supernatants, and neat serum results were all negative. although serum is routinely used for zikv diagnostics in humans, it may not be the most suitable sample [23] [24] [25] [26] [27] . in one investigation zikv patient had negative serum sample for the duration of the study, whereas whole blood yielded positive qrt-pcr results from days 9 to 101 [27] . one possible explanation for the phenomenon of negative serum in human patients is that the virus during acute infection disseminates via a cell-associated viremia or as novel findings suggest that the virus gets phagocytized in neutrophils and therefore whole blood is a more sensitive diagnostic sample than serum. viruria is commonly detected in zikv-infected humans [26] ; therefore, urine may be an equally important diagnostic sample with higher viral load in early infection when compared to blood in humans and other primates [23] [24] [25] [26] . although urine collection from bats was challenging, we collected urine from some of the inoculated bats in the time course study. aj-z6 exhibited viruria only at 3 dpi, and aj-z7 was equivocal only at 5 dpi, corroborating the findings in other mammals that urine may be a route of viral shedding early in infection. urine from one human patient was positive from the first time point (6 dpi) through 14 dpi and again on day 56. similarly, saliva from that same patient was positive from day nine through day 14 and again on day 49 [27] . another investigation compared diagnostic samples of 80 infected patients and showed that urine was positive in 50 of them, whereas serum was only positive in 19 patients by qrt-pcr. the study concluded that viral loads in urine were tenfold higher compared to serum and that uremia lasted longer [25] . these data corroborated the first study that identified zikv shed in urine in which there was a higher viral load in urine for longer duration compared to serum [26] . zikv rna in plasma was detected in the bats by qrt-pcr between 2 and 6 dpi, but between 2 to 17 dpi in urine [26] . the lack of detectable viremia in the serum of bats is congruent with some of the human and nhp investigations in that viremia is low and short-lived. detached renal pelvic urothelial cells and degenerate salivary gland ductular epithelium as seen in the current study will make urine and saliva equally important fluids to collect in order to maximize detection of zikv in the acute and established stages of infections. for this experiment, all male bats were used because female bats are prioritized for colony expansion. zikv exhibited tropism for the testes with strong immunoreactivity in reproductive organs (figs 4 & 7) . histologically, minimal focal testicular degeneration in two bats ( fig 2) suggests viral related pathology may be minimal. in humans it has yet to be completely elucidated what reproductive organs harbor zikv, it has been determined that semen contains zikv both in both vasectomized and unvasectomized men [27, 28] . this suggests that zikv is sequestered in the testes and/or accessory sex glands. mouse models have demonstrated zikv infection and associated pathology in the testes [29] [30] [31] of humanized blt mouse model with infection primarily targeting macrophages and leydig cells [32] . limited investigation has been done relating to infection of accessory sex glands in mouse models, but one study that assessed the prostate found no virus, possibly due to differential expression of the receptor candidate in the testes but not in the prostate [29] . for this experiment the finding of viral antigen and viral rna in the testes but not in the prostate is consistent with published animal models and may suggest the potential for bats to serve as another animal model. three bats had histopathological alterations in the hippocampus at later time points and one bat had viral nucleic acid present in the brain as determined by qrt-pcr demonstrating tropism for the cns, a tissue predilection also documented in humans and animal models. zikv has a predilection for nervous tissue in animal studies and disease manifestation in humans. as a neurological teratogen, zikv has been detected in the brain mononuclear cells in human newborns with fatal microcephaly and fetal miscarriages. histological lesions are varied but may include parenchymal calcification, microglial nodules, gliosis, cell degeneration, mononuclear infiltration and necrosis [33] [34] [35] . in non-human animal models, evidence for viral tropism has been found in brain and/or peripheral nervous tissue [36] [37] [38] [39] . in immunocompromised mouse models, the virus has a predilection for the brain but with the mice engineered for specific immune traits it is difficult to know to what extent this recapitulates natural zikv pathophysiology [40] . in the bats used in this experiment, evidence of zikvinduced pathology in the brain is consistent with what has been seen in human newborns and fetuses. the novel finding of co-localizing zikv antigen in bat iba1 + microglial/macrophage cells lends support to the earlier evidence of microglial cell infection via axl ligand bridging zikv particles to glial cells [41] . iba1 (aka, allograft inflammatory factor 1, aif1) is a microglia/macrophage-specific calcium-binding protein, which has actin-bundling activity that participates in membrane ruffling and phagocytic activity of activated microglia. activated microglial cells appeared with increased ability of cell migration and phagocytosis, which is controlled by remodeling of membrane cytoskeleton [42] . the morphology of cells with co-localization in the brain of infected bats is consistent with activated microglia depicting prominent branched processes. recent primate models in rhesus and cynomolgus macaques demonstrated similar viral distribution of zikv antigen to that in bats, described herein. high-level of zikv was evident in cerebellar neurons and the same studies documented involvement of iba1 positive microglial cells in cns infections. in primate models there is increasing evidence that zikv antigen was detected in individuals with the highest peak plasma viremia, which in part implies that zikv may initially seed the cns by a passive spillover from circulating monocytes to resident microglial cells. this is further substantiated in all of human and animal studies, which did not show any evidence of disruption to bbb or viral distribution reminiscent of circumventricular distribution seen in alphavirus animal models [43] . in addition to brain and testes immunoreactivity, scrotal skin and mandibular salivary gland also harbored viral antigen. distribution of viral antigen in bat tissues suggests that infection in this species recapitulates human infection, which is thought to start with infection of epidermal and dermal cells with subsequent dissemination to multiple organs including salivary glands as viral rna can be detected in human saliva [44, 45] . the histopathology for ajzika virus infects new world bats z5, 5 dpi showed sialoadenitis and the presence zikv antigen by ihc (fig 3) . this suggests zikv may be shed in the saliva, although additional animal experiments need to be performed to confirm such a route of shedding. the results presented here suggest that jamaican fruit bats may be a suitable animal model for examining zikv infection to elucidate its pathogenesis. jamaican fruit bats may also serve as a model to ascertain sexual transmission, in utero transmission, teratogenesis and neurological pathophysiology. it may be that zikv is a wildlife disease threat for bats that could lead to infertility in some males, which could impact bat populations. zikv is thought to be maintained in two different distinct cycles: sylvatic-cycling between non-human primates (nhp) and mosquito species, and urban-cycling between humans and mosquito species [3] . while there are limited data on what mosquito species feed on jamaican fruit bats, evidence for natural flavivirus infection has been identified in wild new world bats. dengue virus (denv) rna and antibodies to denv were detected in multiple species of bats, including jamaican fruit bats, in mexico [46] . additionally, antibodies to denv were detected in multiple bat species including those of the artibeus genus in costa rica and ecuador [47, 48] . these data indirectly provide evidence for mosquito-bat interactions in the wild; either through consumption of bat-blood meals taken by mosquitoes or bat consumption of infected mosquitoes. as it pertains to a wildlife reservoir, wild nhps have antibody to zikv including several monkey species trapped near ziika forest [10] , and wild and semi-captive orangutans in borneo [49] . not only have nhp been found to be seropositive, but also many other mammals, including rodents, horses, cows, and goats [50, 51] . furthermore, experimental inoculation of various north american species resulted in seroconversion (cottontail rabbits, boar goats, pigs, and leopard frogs) and demonstrated viremia (nine-banded armadillo and leopard frogs) [52] . molecular epidemiology suggests animals play an important role in an enzootic cycle [11] . much about the enzootic cycle of zikv has yet to be understood but it stands to reason that bats may be capable of maintaining the virus in nature. jamaican fruit bats are found in northern south america, central america, and the caribbean-areas that now have zikv potentially exposing bat populations to the virus [8, 53] . however, the data presented here suggest it is unlikely that jamaican fruit bats can serve as amplification hosts of zikv, unless virus sequesters in some as-yet unidentified way that could lead to periodic shedding of virus. it may also be that some bats become persistently infected and can transmit sexually to maintain virus within populations of bats. further experimental and field studies will be necessary to fully understand the ecological role of bats in zikv maintenance. all animal procedures were approved by the colorado state university (csu) institutional animal care and use committee (protocol 16-6512a) and were in compliance with u.s. animal welfare act. bats csu has a captive colony of jamaican fruit bats (artibeus jamaicensis), a neotropical fruit bat indigenous to much of south america, central america and the caribbean [53] . colony bats are kept in a free flight room measuring 19'w x 10'l x 9'h. roosting baskets are hung from the ceiling throughout the room and drapes of different cloth material are positioned for hanging and roosting. ambient temperature is maintained between 20˚c and 25˚c, with humidity between 50% and 70%, and a 12 hour light/12 hour dark light cycle via a computer-controlled system. diets consist of a combination of fruits (shamrock foods, fort collins, co), tekald primate diet (envigo, huntington, uk), molasses, nonfat dry milk and cherry gelatin that are placed in multiple feeding trays around the room once a day. fresh water is provided. in addition, fruit is hung around the room to stimulate foraging behavior and serve as enrichment. for infection experiments, bats were trapped using a butterfly net and placed in an 20"d x 12"w x 18"h cage for 24 hours prior to inoculations to allow for acclimation. hanging clothes were provided for roosting and coverage. food and water are placed in open trays in the bottom of the cage and changed daily. tray liners were changed every two days, and cages and hanging clothes are changed every two weeks. due to the social nature of these bats, minimums of two bats were kept in cages at all times to mitigate potential stress. two sets of experiments were performed; a pilot study and a time course study. zika virus strain prvabc59. prvabc59 was isolated in 2015 by centers for disease control and prevention (fort collins, co) from an infected individual who traveled to puerto rico (genbank accession no. hq234499). the virus stock titer is 3x10 7 plaque forming units (pfu) per ml of media, and the fourth passage was used for both studies. for the pilot study, three male bats were anesthetized with 1% to 3% isoflurane to effect with an oxygen flow rate of 1.5 l/min, administered with a gas mask. animals were placed on a heating pad to maintain body temperature and respirations continuously monitored. the dorsum of each animal was disinfected with 70% ethanol and 25ul containing 7.5x10 5 p.f.u of virus was administered subcutaneously (sc) at the level of the scapula with a sterile hypodermic 25 gauge needle in a biosafety cabinet. when procedures were finished, bats were removed from isoflurane and placed back in the cage in ventral recumbency. respirations were monitored until animal was fully awake and ambulated normally. bats were identified as aj-z7, aj-z8 and aj-z9. animals were euthanized at 28 days post-inoculation (dpi). for the time course study, six male bats were anesthetized under the same protocol as the pilot study. animals were placed in ventral recumbency. after disinfecting the dorsum of each animal with 70% ethanol, 0.15mls of 1% lidocaine was administered sc at the level of the last rib with a 25 gauge sterile hypodermic needle as a local anesthetic. iptt300 transponders (biomedic data systems, inc., seaford, de) were inserted sc at the level of the caudal edge of the scapula. twenty-five microliters containing 7.5x10 5 p.f.u of virus was administered sc at the level of the cranial edge of the scapula. recovery followed the same protocol as for the pilot study bats. animals were identified as aj-z1 through aj-z6. aj-z1 and aj-z2 were euthanized at two dpi. aj-z3 and aj-z4 were euthanized at 5 dpi. aj-z5 and aj-z6 were euthanized at 10 dpi. female bats were excluded from the study because they are prioritized for breeding to sustain and expand upon the colony. for the pilot study, bats were visually monitored twice daily for fourteen days, and then monitored once a day for an additional fourteen days. for the time course study, bats were monitored twice a day throughout the experiment. for both studies, energy levels, behavior, ability to ambulate, respirations, presence of oral or nasal discharge, and fecal consistency were all assessed. during the time course study urine was collected at 2, 3, 5 and 10 dpi from as many bats as possible. urine was collected by allowing bats to grasp screen cloth with their feet and then the bat was placed in a clear solo cup (dart container, lake forest, il) with the screen covering the top of the cup as a lid, and kept in place with a rubber band. this allowed the bats to hang in a clear container. bats were monitored for 45 minutes. if they urinated, bats were removed from the collection contraption and placed back in the cage without disrupting the urine. urine collection was attempted on all remaining bats at each time point, but not all bats would urinate at each collection attempt. urine was successfully collected as follows: two dpi from aj-z3 and aj-z4; three dpi from aj-z3, aj-z5 and aj-z6; five dpi from aj-z3, aj-z4, aj-z5 and aj-z6; and ten dpi from aj-z5 and aj-z6. urine was pipetted off the surface of the cup with a sterile pipette tip and put in a 1.5 ml microcentrifuge tube and stored at -80˚c for future use. urine volume ranged between 5 ul and 15 ul. bats were deeply anesthetized and maintained with 3% isoflurane and an oxygen flow rate of 1.5 l/min. deep pain was assessed by firmly pinching skin and toes with forceps and assessed for any response. a thoracotomy was then performed with sterile standard scissors to puncture through the skin, muscle and diaphragm just caudal to the sternum and cut through the wall of the chest cavity caudally to cranially-removing and preventing negative pressure from building in the thorax. cardiac blood was collected with a 21 gauge sterile needle inserted into the apex of the heart. a maximum blood volume of between 1 and 1.5mls is collected in a syringe and transferred to a red top tube (rtt). rtts sat at room temperature for one hour to allow a clot to form and then centrifuged at 1000 x g for 10 min at room temperature. serum was removed from the clot, placed in a new microcentrifuge tube and stored at -20˚c. serum from bats at 2 and 5 dpi were used to assess for viremia. serum from 10 dpi and the 28 dpi pilot study bats were used to determine antibody titers. because blood draws yield a small volume of blood (50 μl whole blood for a non-terminal blood draw, 500 μl whole blood for terminal blood draw) it was necessary to prioritize samples to optimize data retrieved. in order to assay the serum for viral rna and perform serology, earlier time points were used to assess for viremia and later time points for seroconversion. along with sample partitioning for data maximization, the small blood volume led to concerns that there would be an undetectably small viral load. to circumvent this issue, neat serum and 1:10 diluted serum were inoculated onto vero cells to amplify any virus that may have been present at low levels. one blind passage on vero cells was done and cell supernatants assayed by qrt-pcr. the remaining serum from three of the four bats was assayed directly for zikv rna. necropsies were performed immediately after euthanasia. bats were assessed for gross pathology. the following tissues were collected for both experiments: heart, lung, liver, spleen, kidney, urinary bladder, prostate, testes, and brain. a portion of tissues were collected and kept at -80˚c for rna extraction, and a portion placed in 10% buffered formalin for histology at a 1:10 weight to volume ratio for histology. for a negative control animal a male bat was trapped from the colony and euthanized under the same protocol as the experimental infection bats. vero e6 cells (atcc) were propagated to 60% confluency in a 96-well tissue culture plate and infected with zikv strain prvabc at an m.o.i. of 0.1. after a one hour incubation period, unbound virus was removed and replaced with 2% fbs-dmem and incubated for a maximum of three days. media was then replaced with 85% acetone for 20 minutes at -20˚c to fix virusinfected cells to plate and serve as an antigen for enzyme-linked-immunosorbent assay (elisa). plates were stored at 4˚c until use and used within two weeks. plates were washed 5x with 0.05% tween 20-pbs and blocked with superblock t20 (tbs) blocking buffer (thermo fisher scientific, waltham, ma) for one hour at room temperature. serum from an uninfected bat was used for a negative control. a convalescent human serum sample (kindly provided by b. foy, csu) was used as a positive control. a two-fold serial dilution was used starting at 1:100 to 1:12800. diluted serum was placed in wells and incubated for two hours at room temperature. serum was removed and plates washed. hrp-conjugated protein a/g (thermo fisher scientific, waltham, ma) was added at a concentration of 2 μg/ml to each well, and incubated for 30 minutes at room temperature. hrp-conjugated protein a/g was used in place of a secondary antibody as it targets the fc portion of an antibody, which is highly conserved and therefore can be used for multiple animal species [54] . plates were washed and 150 μl of abts peroxidase substrate (2 component) (kpl, gaithersburg, md) added according to manufacturers' instructions, incubated at room temperature for 30 minutes, and then 150 μl of abts peroxidase stop solution (kpl, gaithersburg, md) added. plates were read on an emax plus microplate reader (cambridge scientific, watertown, ma). absorbance was measured at 405 nm and the limit of detectable response was set at three standard deviation values above mean negative control serum. trizol reagent was used for rna extraction from serum-cell supernatants, serum, urine and tissues according to ambion, life technologies protocol. for tissues, approximately 50 mg of tissue was homogenized with one ml of trizol reagent. a 5mm stainless steel bead (qiagen, valencia, ca) was used with a tissuelyser lt (qiagen, valencia, ca) at 50 hz for 5 minutes. one ml of trizol was added to urine to 5 to 15 μl of urine. one ml of trizol was added to 160 μl of serum from aj-z2, aj-z3, and aj-z4. two-hundred microliters of serum-cell supernatants were added to one ml of trizol. samples were then incubated at room temperature for 5 minutes. chloroform (thermo fisher scientific, waltham, ma) was added, samples were mixed, incubated for 3 minutes at room temperature and centrifuged at 12,000 x g for 15 minutes at 4˚c. the aqueous phase was removed, 4 μg of glycogen (thermo fisher scientific, waltham, ma) and 100% molecular grade isopropanol added (thermo fisher scientific, waltham, ma). samples were incubated at room temperature for 10 minutes and then centrifuged at 12,000 x g for 10 minutes at 4˚c. supernatant was removed and 75% molecular grade ethanol (thermo fisher scientific, waltham, ma) was added to rna pellet. samples were vortexed and centrifuged at 7500 x g for 5 minutes at 4˚c. wash was removed and air-dried. rna was resuspended in rnase-free water and stored at -80˚c for future use. vero cells were grown to 70 to 80% confluency in a 48-well tissue culture plate with 10% fbs-dmem. media was removed and 100 ul of bat serum from 2 dpi bats and 5 dpi bats was inoculated onto cells. additionally, serum from each bat was diluted 10-fold in 2% fbs (millipore sigma) pbs supplemented with 1% calcium and magnesium, and inoculated onto cells. samples were incubated for one hour at 37˚c. inoculum was removed and cells washed twice in sterile pbs. two-percent fbs-dmem was added to wells and plates were incubated at 37˚c, 5% co 2 . cells were assessed daily for cytopathology (cpe) through day 7 but none was observed. two-hundred microliters of the supernatant was removed on day 7 and used for rna extractions. an additional 100 μl of supernatant was blind passaged onto vero cells at 70 to 80% confluency. cells were incubated for one hour at 37˚c, washed twice with sterile pbs and 2% fbs-dmem added. on day seven, supernatant was removed and trizol extractions performed for rna recovery. serum was treated as such in an attempt to amplify viral load and increase assay sensitivity serum may not be the most sensitive diagnostic sample [23] [24] [25] [26] . if any serum was remaining it was directly used for trizol rna extractions. serum samples remained from aj-z2 at 2 dpi, and aj-z3 and aj-z4 at 5 dpi. no serum remained from aj-z1. roche real time ready rna virus master kit (roche, indianapolis, in) was used on rna extracted from serum-cell supernatants, serum, urine and tissue to assay for zikv rna according to manufacturers' instructions. primers used were zikv 1086 (ccgctgcccaa cacaag) and zikv 1162c (ccactaacgttcttttgcagacat). probe was zikv 1107-fam (agcctaccttgacaagcagtcagacactcaa) [55] . two-hundred nanograms of sample rna was added to each reaction. reactions were performed in duplicate. standards were a non-infectious clone of full length zikv strain prvabc59 by which concentration was determined through optical density. molecular weight of the genome sequence was used to calculate copy number [56] . a log 10 dilution series of the standard was made and linear regression used to determine copy number equivalents of positive samples. amplification was performed according to manufacturers' protocol for roche real time ready rna virus master kit (roche diagnostics corporation, indianapolis, in) with pcr conditions as follows: 8 min at 50˚c, 30 s at 95˚c, and 45 cycles of 10 s at 95˚c, 20 s at 60˚c and 10 s at 72˚c. tissues fixed in 10%-buffered formalin were cut in and submitted to colorado state university veterinary diagnostic laboratory (csu vdl, fort collins, co) for paraffin embedding, sectioning and staining with hematoxylin and eosin, as well as immunohistochemistry (ihc). tissues cut in on bats to assess for histology included: heart, lung, liver, kidney, testes, prostate, urinary bladder and brain. additionally, for aj-z3 and aj-z5 mandibular salivary gland was cut in. aj-z4 had esophagus and lymphoid tissue that included palatine salivary gland cut in. antibody for ihc was a polyclonal rabbit antibody that targets prem and e proteins of zikv and was provided by csu vdl's pathology department. the bond-iii automated instrument (leica biosystems, wetzlar, germany) was used for ihc staining. all slides were blindly read by a diplomat of the american college of veterinary pathologists. brain tissues was prepared for immunohistochemical and immunofluorescence staining as previously reported [57] . tissue was dehydrated by using a graded ethanol series of 70% ethanol for 2 h, 80% overnight, 90% for 2 h and 100% for 2 h. brain tissues were then post-fixed in dimethylbenzene for 30 min and embedded in dimethylbenzene-paraffin at 60˚c for 2 h, after which samples were embedded in a metal frame. sagittal sections were collected at 5um thick. all dewaxing, antigen retrieval and immunofluorescence staining was automated using a leica bond rxm. in short, sections were dewaxed using ethanol and then boiled in antigen retrieval solution for 10 minutes. the cooled sections were incubated in 3% h2o2 for 15 min at room temperature and then blocked with 2% donkey and goat serum (millipore sigma) for 1 hour. rabbit anti-iba1 (wako chemicals usa, irvine, ca) and 4g-2 flavivirus e specific monoclonal antibodies (cdc, fort collins) were diluted in tbs to final concentrations of 1:250 and 1:50, respectively. sections were incubated in primary antibodies concurrently at room temperature for one hour. following removal of unbound primary antibodies by washing, goat anti-rabbit secondary (alexafluor-555) and donkey anti-mouse secondary (alexafluor-647) was added and incubated for 1 hour at room temperature. finally, dapi counterstain (vector laboratories, burlingame, ca) was applied and sections were washed with tbs prior to cover slipping for imaging. stained sections were imaged on a ziess lsm 800 with airyscan laser-scanning confocal microscope (ziess, oberkochen, germany) using a 63× oil immersion objective. each field of view was imaged as a z-stack (8-10 planes, .5-μm step size) transformed into a single maximum projection image using the ziess zen (blue) imaging software. zika virus. i. isolations and serological specificity zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria zika virus: history, emergence, biology, and prospects for control zika virus outbreak on yap island, federated states of micronesia rapid spread of emerging zika virus in the pacific area zika virus outbreak, bahia, brazil. emerg infect dis first report of autochonous transmission of zika virus in brazil zika cases and congenital syndrome associated with zika virus reported by countries and territories in the americas potential for zika virus to establish a sylvatic transmission cycle in the americas yellow fever and zika virus epizootics and enzootics in uganda molecular evolution of zika virus during its emergence in the 20th century studies on viruses in east african bats (chiroptera). 1. haemagglutination inhibition and circulation of arboviruses studies on arboviruses and bats (chiroptera) in east africa. ii. isolation and haemagglutination-inhibition studies on bats collected in kenya and throughout uganda effect of zika virus and bwamba virus in the cave bat (myotis lucifugus) transmission studies of hendra virus (equine morbillivirus) in the fruit bats, horses and cats pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission antibody-mediated immune response in the bat, pteropus giganteus detection of specfic antibody responses to vaccinatin in variable flying foxes (pteropus hypomelanus) the little brown bat, m. lucifugus, displays a highly diverse vh, dh, jh repertoire but little evidence of somatic hypermutation tacaribe virus cases fatal infection of an ostensible reservoir host, the jamaican fruit bat replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) transcriptomic signatures of tacaribe virus-infected jamaican fruit bats assay optimization for molecular detection of zika virus a rhesus macaque model of asian-lineage zika virus infection zika virus testing considerations: lessons learned from the first eighty real-time rt-pcr-positive cases diagnosed in new york state detection of zika virus in urine long-term kinetics of zika virus rna and antibodies in body fluids of a vasectomized traveller returning from martinique: a case report persistence of zika virus in body fluids-preliminary report zika virus causes testis damage and leads to male infertility in mice zika virus infection damages the testes in mice a mouse model of zika virus pathogenesis zika viral infection and neutralizing human antibody response in a blt humanized mouse model notes from the field: evidence of zika virus infection in brain and placental tissues from two congenitally infected newborns and two fetal losses-brazil zika virus damages the human placental barrier and presents marked fetal neurotropism pathology of congenital zika syndrome in brazil: a case series zika virus infection of rhesus macaques leads to viral persistence in multiple tissues fetal brain lesions after subcutaneous inoculation of zika virus in a pregnant nonhuman primate nonhuman primate models of zika virus infection, immunity, and therapeutic development zika viral dynamics and shedding in rhesus and cynomolgus macaques overview of the current status of zika virus pathogenesis and animal related research axl mediates zika virus entry in human glial cells and modulates innate immune responses microglia/macrophage-specific protein iba1 binds to fimbrin and enhances its actin-bundling activity entry sites of venezuelan and western equine encephalitis viruses in the mouse central nervous system following peripheral infection detection of zika virus in saliva biology of zika virus infection in human skin cells denge virus in mexican bats neotropical bats that co-habit with humans function as dead-end hosts for dengue virus detection of dengue virus neutralizing antibodies in bats from costa rica and ecuador sylvatic transmission of arboviruses among bornean orangutans zika virus, vectors, reservoirs, amplifying hosts, and their potential to spread worldwide: what we know and what we should investigate urgently a sero-epidemiological survey for certain arboviruses (togaviridae) in pakistan investigating the potential role of north american animals as hosts for zika virus. vector borne zoonotic dis mammalian species: artibeus jamaicensis chimeric igg-binding receptors engineered from staphylococcal protein a and streptococcal protein g genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia rapid and specific detection of asian-and african-lineage zika viruses fluoxetine prevents lps-induced degeneration of nigral dopaminergic neurons by inhibiting microglia-mediated oxidative stress the authors would like to than brent davis, cdc, fort collins, co for supplying anti-zikv polyclonal and specific monoclonal antibodies. key: cord-002179-v8lpw4r7 authors: viktorovskaya, olga v.; greco, todd m.; cristea, ileana m.; thompson, sunnie r. title: identification of rna binding proteins associated with dengue virus rna in infected cells reveals temporally distinct host factor requirements date: 2016-08-24 journal: plos negl trop dis doi: 10.1371/journal.pntd.0004921 sha: doc_id: 2179 cord_uid: v8lpw4r7 background: there are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. a better understanding of the host pathogen interaction is required to develop effective therapies to treat denv. in particular, very little is known about how cellular rna binding proteins interact with viral rnas. rnas within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication. methodology/principal findings: seventy-nine novel rna binding proteins for dengue virus (denv) were identified by cross-linking proteins to dengue viral rna during a live infection in human cells. these cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in denv amplification. knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. their requirement by denv for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. the protein abundances of these host factors were not significantly altered during denv infection, suggesting their interaction with denv rna was due to specific recruitment mechanisms. however, at the global proteome level, denv altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated. conclusions/significance: the method for identification of host factors described here is robust and broadly applicable to all rna viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral rna within cells. this study significantly increases the number of cellular factors known to interact with denv and reveals how denv modulates and usurps cellular proteins for efficient amplification. dengue is a mosquito-borne viral disease that infects 50-100 million people annually, resulting in dengue fever that is either asymptomatic or flu-like. however, tens-of-thousands of people develop the more severe, and sometimes fatal, dengue hemorrhagic fever/shock syndrome (dhf/dss) [1] . denv is found in most tropical and many subtropical areas with more than 125 countries being endemic for denv [2] . there is no approved vaccine or antiviral therapeutic available for this life-threatening disease. given the seriousness of infection, the expanding geographical range of the denv, and the limitations in the existing measures of control and prevention, there is a pressing need to better understand the biology and pathogenesis of denv. denv is a single-stranded positive-sense rna virus that belongs to the flaviviridae family. it has a 5' cap, no poly(a) tail, highly structured 5'-and 3'-untranslated regions (utr), and a single open reading frame (orf) [reviewed in [3] ]. following virus entry, the viral rna is released into the cytoplasm. viral translation and replication occur in membranous assembly "factories" localized in the perinuclear region of endoplasmic reticulum (er) [4] . the positivestranded rna molecules are encapsidated; virions are further processed as they are transported through the secretory pathway to the cell surface and released extracellularly [reviewed in [3] ]. in addition to the viral proteins, cellular proteins, termed host factors, participate in most, if not all, steps of the denv life cycle, including entry, translation, replication, virion assembly, and release [5] . since viruses require host factors for efficient amplification, targeting host factors can provide an effective antiviral target for which the virus has no genetic control over. therefore, it may be more difficult for viruses to evolve escape mutants that can replicate efficiently in the absence of the host factor [5, 6] . several cellular proteins are known to impact denv infection. for example, the polypyrimidine-tract-binding protein (ptbp1) is relocalized from the nucleus to the cytoplasm following denv infection where it enhances denv amplification by binding to the denv 3'utr and to ns4a, a viral protein required for the formation of the viral replication complex [7] [8] [9] [10] . ptbp1 may also stimulate denv translation [8] , although this is still controversial [7, 9] . while most of the known denv rna binding proteins enhance viral amplification, several reduce denv titers [10] [11] [12] . one such factor, ybx1, inhibits viral translation [12] . although previous studies have laid a foundation for establishing critical interactions between viral rna and cellular proteins [ [13] and reviewed in [14] ], the host factors identified thus far likely represent only a fraction of the total network of denv host factors. previously, we have described a high-throughput mass spectrometry method termed tux-ms (thiouracil cross-linking mass spectrometry) to identify host factors that interact with viral rna during a live infection in cell culture [15] . importantly, tux-ms allows for identification of proteins that are bound directly to the viral rna in living cells. briefly, during a viral infection in cell culture, thiouridine is biosynthetically incorporated into the viral rna to serve as a zero-distance cross-linker upon exposure to ultraviolet (uv) light. thus, proteins that are bound directly to the viral rna during a live infection are cross-linked to the rna prior to disruption of cellular compartmentalization. this is particularly valuable for the identification of denv host proteins, since denv amplification is tightly associated with cellular membrane structures [4] . following cross-linking, the viral rna together with cross-linked proteins are isolated under denaturing conditions and identified by mass spectrometry-based proteomics. using tux-ms, we reported previously the successful identification and validation of host factors for poliovirus, pointing to a low false discovery rate of < 12% [15] . here, we expanded the tux-ms methodology for use with other types of rna viruses, and investigated rna-protein interactions during denv infection. we modified the method to use virus-specific dna oligos to capture the viral rna and cross-linked proteins. furthermore, we used metabolic labelling with stable isotopes to accurately quantify relative protein levels. this quantitative thiouridine cross-linking mass spectrometry (qtux-ms) analysis identified 79 novel host proteins, which were not previously shown to be involved in denv infection. we placed these findings in the context of whole proteome changes upon denv infection, and further validated and functionally analysed a subset of the novel denv host factor candidates. overall, validation of the qtux-ms identified factors using secondary assays indicates a low rate of false positives (17%), suggesting that the majority of the other identified qtux-ms factors may also play significant roles in denv viral amplification. hela uprt cells expressing uracil phosphoribosyltransferase (uprt) were generated previously by transduction of hela cells (atcc, ccl-2) with uprt-gene containing lentivirus [15] . huh7.5 uprt cells were generated by transduction of huh7.5 cells (a kind gift from charles m. rice, rockefeller university) with the same lentiviral construct as in [15] . hela uprt and huh7.5 uprt cells were cultured at 37°c and 5% co 2 in complete dulbecco's modified minimum essential medium (dmem) supplemented with 10% fbs (fetal bovine serum; atlanta biologicals) and penicillin-streptomycin and grown with 1 mg/ml g418 (sigma) to select for the uprt gene; huh7.5 uprt cells were additionally supplemented with non-essential amino acids (cellgro). for silac labelling huh7.5 uprt cells were passed 1:10 at least twice in silac dmem (thermo scientific) with 10% dialyzed heat-inactivated fbs (thermo), l-proline (200 mg/l) and penicillin-streptomycin, and either 50 mg/l 'heavy' (13c6 l-lysine and 13c6-15n4 l-arginine; cambridge isotope laboratories, inc.) or 40 mg/l 'light' l-lysine and l-arginine amino acids [16] . dengue virus serotype 2 (denv2), strain 16681 (genebank accession number u87411) was kindly provided by dr. robert striker (university of wisconsin-madison). denv2 was propagated in the mosquito c6/36 cells at 28°c and 5% co 2 in advanced dmem supplemented with 10% fbs, penicillin-streptomycin (cellgro), l-glutamate and 10% tryptose phosphate broth (20 g/l tryptose; 2 g/l glucose; 5 g/l sodium chloride and 2.5 g/l disodium hydrogen phosphate). titers for denv2 were determined using plaque assays in bhk cells. for infections, cells were incubated with virus containing media for 2 hours, washed twice with the dmem media after removal of the virus and incubated in the serum-free dmem for the indicated time. infections and titer determination of adenovirus 5 (ad5) were performed exactly as in [15] . the denv antisense biotin-labelled dna fragments were generated using pcr and primers listed in (s1 table) from the denv2 complementary dna (cdna) and correspond to positions 4350-4914 and 4740-4914 of denv genome. pcr was followed by removal of the unlabelled dna strand according to the nanolink streptavidin magnetic beads (solulink) manual. the mixture of two biotinylated single-stranded dna fragments of 174 base pairs (bp) and 564 bp long were bound to nanolink streptavidin magnetic beads magnetic beads according to the manufacturer's protocol. 1-3 x 10 7 human hepatoma huh7.5 uprt cells labelled with either 'light' or 'heavy' silac media were infected with denv2 (moi = 10) or mock-treated, respectively. then, virus was replaced with silac media with 1 mm 4-thiouracil and 10% fbs. at 28 hours post-infection (hpi) the cells were washed with pbs and irradiated at 365 nm uv light for 20 min, collected, cell pellets were frozen on dry ice and stored at -80°c. cell pellets were lysed in the lysis buffer (50 mm tris-hcl ph 8, 4 mm magnesium chloride, 150 mm sodium chloride, 0.1% tween-20, 5 mm dithiothreitol, recombinant rnasin ribonuclease inhibitor [500 units/ml; promega], 1× complete protease inhibitor cocktail [roche]), with a half volume of 465-600 μm glass beads (sigma) by shaking at frequency of 30 hz for 1 min using a retsch mm200 mixer mill. an aliquot of 'light' and 'heavy' cell lysates were removed and the remaining lysates were incubated with the streptavidin magnetic beads labelled with denv antisense dna fragments for 15 to 30 min allowing for viral rna to bind. the beads were washed twice with wash buffer i (50 mm tris-hcl ph 8, 500 mm potassium chloride, 0.1% tween-20), once with wash buffer ii (50 mm tris-hcl ph 8, 150 mm sodium chloride, 0.5% sodium deoxycholate) and once with 10 mm tris ph 7.6. the samples were eluted at 65°c for 2 min in 20 μl of 10 mm tris ph 8.0. the eluted 'light' and 'heavy' samples were mixed at a 1:1 ratio, rna was degraded with 0.5 ng/μl bovine rnase a (fisher scientific) at 25°c for 24 hrs and subjected to quantitative mass spectrometry-based proteomic analysis. quantitative mass spectrometry-based proteomic analysis of rnainteracting proteins (qtux-ms) protein eluates and their respective mixed light/heavy input lysates were subjected to in-solution enzymatic digestion using a filter-aided sample preparation approach [17] , then analyzed by nlc-ms/ms, as described in s1 methods and in [18] . light (denv2) and heavy-labelled (mock) cell pellets were lysed in 100 mm abc containing 5% sodium deoxycholate at 95°c to ensure denaturation and virus inactivation. the protein concentrations were determined by the bca assay and mixed in equal protein amounts (100 μg total). proteins were subjected to in-solution digestion with trypsin, then fractionated and analyzed by nlc-ms/ms as described in the s1 methods. proteomic data analysis qtux-ms, its respective mixed input lysate, and the whole cell silac instrument raw data were separately processed using the maxquant software (ver. 1.5.3.8), configured with default settings, except for experiment-specific parameters, which are described in the s1 methods. the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride [19] partner repository with the dataset identifier pxd003593. using the filtered list of protein identifications, unique gene symbols were used for downstream functional ontology analyses. the gene ontology annotations from uniprot were used to generate and assign the denv2 rna interacting candidates into broader functional categories. for the whole cell silac protein expression study, genes and their associated ratios were analyzed by panther gene enrichment (panther database ver 10.0, 2015-05-15) [20] using the panther pathway and protein class ontologies. significant differential protein abundance was determined as a function of ontological classification versus the overall population (bonferroni-corrected p-value < 0.05). for specific functional protein ontologies that were differentially regulated, a subset were selected for analysis by the reactome functional interaction (fi) network cytoscape plugin [21] . the reactome fi plugin was used to visualize candidate host factors identified by qtux-ms. sirna transfections 2 x 10 6 hela uprt cells were transfected with 350 pmol of silencer select negative control (ambion) or the specified sirnas (s1 table) using the xtreamgene sirna transfection reagent system (roche). 24 hrs later, 4 x 10 5 cells/well were plated for infection. 48 hours post transfection cells were infected (moi = 0.1) with denv2 or ad5 (n = 3). at either 30 (ad5) or 40 (denv2) hpi, virus titers were determined by plaque assays using 911 or bhk cells, respectively. knockdown efficiency was measured 48 hrs post transfection by rt-qpcr. experiments were performed in two biological replicates for each host factor. statistical analysis was performed using student's t-test. cell viability and proliferation assay 24 hrs post sirna transfection equal numbers of cells (either 2 x 10 3 or 8 x 10 3 ) were seeded in 96-well plates. cell viability was measured 48 hours after sirna-mediated knockdown of individual host factors using the vybrant mtt [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide] assay kit (invitrogen) according to the manufacturer's protocol and reported relative to the negative-control sirna (set to 100%; n = 3). statistical analysis was performed using student's t-test. cdna was generated from 1μg trizol (ambion) purified total rna using moloney murine leukemia virus (mmlv) reverse transcriptase (promega) as described by the manufacturer using random primers (invitrogen). qpcr was performed using iq sybr green supermix (bio-rad) with the primers listed in the s1 table. the amplification efficiency for each primer set was 100±10% as determined using a standard curve. development of a quantitative thiouridine cross-linking mass spectrometry (qtux-ms) method for identification of proteins associated with the denv rna tux-ms can be used to identify host factors by incorporating 4-thiouridine (4su), a zero-distance cross-linker, into the viral rna (vrna) to enable cross-linking of proteins bound to vrna during a live infection in cell culture [15] . cross-linking is carried out under physiological conditions prior to cell lysis to ensure specificity and reduce false-positives from non-specific rna protein interactions that occur upon loss of compartmentalization. vrna is isolated under denaturing conditions and cross-linked proteins are identified using liquid chromatography-tandem mass spectrometry (lc-ms/ms). to improve quantification of the tux-ms identified host proteins (qtux-ms), a silac (stable isotope-labelled amino acids in cell culture) approach [16] was used to label the uninfected (mock) and infected cells with either 'heavy' or 'light' amino acids, respectively ( fig 1a) . when 4-thiouracil (4tu) is present in the medium, huh7.5 uprt human hepatoma cell lines stably expressing uprt (uracil phosphoribosyltransferase) convert 4tu to ump. then, the ump is converted to thiouridine triphosphate (4sutp) by cellular kinases [22] . both cellular and viral rna polymerases use 4sutp as a substrate during rna synthesis, which serves as a zero-distance cross-linker, covalently binding proteins to rnas upon exposure to long wave uv-light. importantly, protein-protein crosslinking is very inefficient at long uv wavelengths, ensuring that only proteins in direct contact with the reactive thiol group of the 4su-containing rna will be cross-linked [23] . we have shown previously that immunoisolation of candidate vrna-binding proteins identified by tux-ms (and confirmed by western) could be specifically co-isolated with viral rna [15] . together, this study established that tux-ms can identify bona fide interactions between host proteins and viral rna. the tux-ms method was originally developed to capture polyadenylated rna using oligo(dt) beads [15] . however, as denv rna is not polyadenylated, we modified the method to use sequence specific capture of the vrna using magnetic beads. following crosslinking in huh7.5 uprt cells infected with denv at 28 hpi and affinity capture of the vrna, the ribonucleoprotein complexes were eluted from the beads, 'heavy' and 'light' eluates were mixed, and rnase a was used to degrade the rna. the proteins were digested insolution with trypsin and subjected to quantitative ms-based proteomics ( fig 1b) . the median 'light' to 'heavy' peptide and protein ratios were calculated, reflecting the specificity of vrnaprotein capture. we identified several classes of proteins, including denv proteins, known denv host factors, and putative rna-interacting host proteins, but most of the qtux-ms identified factors have not been previously identified through interactions with denv (s2 table) . consistent with previous knowledge of flaviviral rna [24] [25] [26] [27] , our qtux-ms analysis identified several viral proteins-c, e, ns3, ns4a and ns5-as associated with vrna. in addition, qtux-ms identified six known denv host factors: polypyrimidine tract-binding protein 1 (ptbp1), interleukin enhancer-binding factor 3 (ilf3), calreticulin (calr), calnexin and heterogeneous nuclear ribonucleoproteins hnrnp h1 and hnrnp k, as well as a known denv anti-viral protein-eukaryotic initiation factor 4a (eif4a)-and 12 other proteins previously shown to associate with denv rna or proteins (s2 table) . altogether, since several known host factors were identified using qtux-ms, this suggests that the adapted method is effective at identifying host factors for denv. for identification of novel host factors, cellular proteins with a denv/mock silac ratio of 1.5-fold (n 3 quantified peptides) were considered putative denv vrna interactions. this threshold was selected when considering the median variance in the silac ratio (for (blue) amino acids are infected with denv or treated with mock, respectively, in the presence of 4tu. 4su is incorporated into cellular and denv rnas and proteins are uv cross-linked to the contacting thio-containing rna (represented as either balls to indicate native conformation or curved lines to indicate denatured proteins) in living cells at 28 hpi prior to cell lysis under denaturing conditions. viral ribonucleoprotein complexes were isolated using dna molecules complementary to denv rna bound to magnetic beads, the rna was degraded with rnase a and the proteins were identified by mass spectrometry. (b) workflow for quantitative proteomic analysis of rna-bound host factors isolated in (a). isolated proteins were mixed between mock and virus-infected samples, digested into peptides, and analysed by mass spectrometry. relative 'light' and 'heavy' peptide abundances were quantified to determine the specificity of interaction. host factor candidates were identified and subjected to functional validation. doi:10.1371/journal.pntd.0004921.g001 proteins with > 3 quantified peptides), which was approximately 25%. therefore, we opted for a conservative cut-off at 50%, representing twice this median value or 1.5-fold. common environmental and non-human cell culture contaminants were excluded, since they existed in only the 'light' silac state. in addition, our qtux-ms samples also contained histones: h3, h4, h2a, h2b, h1.5 and macroh2a.1. in a previous study, histones were shown to play roles in dengue infection [28] . however, their functions were mediated through an interaction with a viral capsid protein and were shown to be independent of rna. in addition, histones are primarily nuclear and highly abundant proteins commonly detected (> 50%) in control affinity purifications compiled across diverse protein-protein interaction studies [29] . for these reasons, histones likely represent non-specific associations rather than denv rna binding factors, and thus were excluded from further analysis. in total, 93 cellular proteins passed these selection criteria, 79 of which have not been previously shown to associate with denv (s2 table, s1 dataset). several of the known denv host factors were enriched but did not meet the stringent inclusion criteria (s2 table) , suggesting that there may be additional host factors below our enrichment threshold (see s1 dataset). importantly, the subset of 79 host factors represents a significant potential expansion in the number of known denv host factors, providing a valuable resource to test for pro-viral or antiviral activities during denv infection. it is well recognized that viral infections can induce significant changes in cellular proteomes [30] [31] [32] and an increase in protein levels during denv infection may contribute to the increased protein capture measured by qtux-ms. to address this, we used silac-ms to quantify proteome (i.e., total protein abundance) changes following denv infection. comparison of qtux-ms and proteome silac ratios showed that the protein abundances for the 93 qtux-ms-identified vrna-binding factors remained largely unchanged (fig 2a, s1 fig and s1 dataset). on average, for these proteins, the denv-induced changes in whole cell abundance were ±1.1-fold, suggesting that their identification by qtux-ms was not due to an increase in their abundance in the cell following denv infection. noteworthy, a retrospective qualitative comparison of the qtux-ms identified factors for denv with those identified in the tux-ms analysis on poliovirus revealed less than 10% were identified for both viruses [15] ( fig 2b) . since the identified proteins are largely denv specific, qtux-ms is not biased towards identifying a sub-set of cellular rna binding proteins. taken together, our results indicate that the enrichment ratios measured by qtux-ms is predominantly due to their specific association with the denv rna. while proteins that bound denv rna did not show significant changes in abundance upon infection, we performed bioinformatics analysis on the complete proteome dataset of whole cell abundance to determine the global proteome effects of denv infection under these conditions. in total, 4,907 host proteins were quantified by silac ms in biological duplicates (s1 dataset). the abundance ratios between biological duplicates were reproducible, with only~2% of the ratios varying by > 50% (fig 3a) . from these duplicates, an average abundance ratio was calculated and the respective proteins were analyzed by panther gene enrichment analysis (s2 fig) [20] . this analysis found systematic up regulation of proteins in the ubiquitin proteasome pathway (upp), comprising 18 members of the 26s proteasome as well as various ubiquitinconjugating enzymes (s3 fig). many of these enzymes are linked to ubiquitin-dependent proteasome degradation, consistent with the current knowledge that the upp is important for production of infectious virions [31, 33, 34 ]. yet, other enzymes, such ube2n and ube2v2, catalyze polyubiquitination at lys-63, which does not lead to proteasome degradation but rather participates in transcriptional activation of target genes and may promote innate immune signaling [35, 36] . in contrast, proteins in the transporter protein class were on average down regulated (fig 3b and s2 fig). assembly of the annotated proteins into reactome protein networks identified several subnetworks with various transporter activities (s4 fig). while the abundances of mitochondrial transporters and nucleoporins were the most consistently decreased, not all transporters were down regulated; for example, the lipoprotein (apo) transporters were increased in expression (fig 3c) . interestingly, the rna binding protein class was significantly down regulated (fig 3b and s2 fig) , though the effect was not as pronounced as the transporter class (fig 3b) . the overall down-regulation of rna binding proteins appears to be driven by changes in cytoplasmic and mitochondrial ribosomal subunits, and proteins involved in rna degradation and processing (s5 fig). nevertheless, the relative protein abundance for the set of 93 (known and putative) denv binding factors identified by qtux-ms was largely unchanged, despite being enriched in rna processing and translation factors (fig 2a) . overall, the quantitative proteome analysis suggests that denv selectively alters the abundance of proteins, and reveals several pathways that could be directly or indirectly modulated in the host response to denv infection. to gain insight into potential molecular mechanisms and biological processes of the 93 qtux-ms identified factors, we performed a functional network-based analysis using the curated human pathway relationships from the reactome database. this analysis revealed a high degree of connectivity, with 62 proteins forming a large interconnected network (fig 4) . the densest network connectivity included proteins involved in rna processing/translation (orange nodes) and dna binding/transcription (blue nodes). several additional proteins with rna and/or translational activities were also identified, but lacked annotation in reactome (orange single nodes). overall, our bioinformatic evaluation further supports the ability of qtux-ms to capture vrna-bound host factors and points to their possible function in denv amplification. since most of the factors were associated with rna processing in the reactome analysis (fig 4) , we focused on these factors for functional analysis of their roles in dengue infection. we have randomly selected six qtux-ms identified host proteins with functions in rna processing/translation, which were enhanced in the denv sample ranging from 1.32-to 2.26-fold (denv/mock). thus, by validating factors that are only modestly enhanced in the qtux-ms analysis this will indicate if the qtux-ms identified factors that are near the cut-off of 1.5-fold are bona fide host factors or not. we assessed the effect of sirna knockdown of these factors (fig 5a) on viral production. hela uprt cells were used for sirna silencing experiments due to higher sirna transfection efficiency compared with huh7.5 uprt cells. knockdown of five out of six qtux-ms identified candidates: non-pou domain-containing octamer-binding protein (nono), embryonic stem cell-specific 5-hydroxymethylcytosine-binding protein (hmces), rbmx (rna-binding motif protein, x chromosome), hnrnp m and hnrnp f significantly decreased denv production, while knockdown of hnrnp l had no effect on denv titer ( fig 5b; s5 fig) . knockdown of ptbp1, a positive control [7, 8] , also resulted in decreased viral titers (fig 5) . for negative controls, we selected two rna binding proteins (ddx39 and hnrnp a0) that were not identified by qtux-ms. denv titers were not altered following silencing of these two proteins, suggesting that only specific rna binding proteins are used by denv. altogether, our data suggests that rbmx, nono, hmces, hnrnp m, hnrnp f are required for viral production. these results demonstrate that qtux-ms is a robust method with a low false discovery rate for high-throughput identification of viral host factors. reduced viral amplification could be due to compromised cell fitness rather than a specific requirement of the virus for a particular host factor. using an mtt assay, we confirmed that knockdown of these factors did not impact cell viability (fig 5c) . as a positive control, knockdown of g3bp2 did reduce cell fitness, as previously shown (s6 fig) [37] . to more rigorously rule out any potential effects of host factor knockdown on cell fitness that would affect viral amplification, an unrelated virus (adenovirus 5), was amplified following knockdowns of the candidate factors. adenovirus 5 replication was not significantly decreased by rbmx, nono, hnrnp m, hnrnp f or hmces sirna knockdown (fig 5b and s6c fig) demonstrating that knockdown of these factors does not affect cell fitness for viral amplification. given that these proteins bind directly to viral rna and are required for viral amplification, rbmx, nono, hnrnp m, hnrnp f and hmces are novel denv host factors. to determine if the host factor is required for a step prior or subsequent to viral replication, vrna was quantified by qrt-pcr in the denv infected cells knocked down for rbmx, nono, hnrnp m, hnrnp f or hmces (fig 6) . as a positive control, knockdown of ptbp1, which is required for denv replication [7] , reduced denv rna levels. similarly, the intracellular vrna levels were reduced in cells knocked down for either hnrnp f, rbmx or hmces. the decrease in dengue rna levels (fig 6) is consistent with the decrease in viral titers ( fig 5b) . thus, hnrnp f, rbmx or hmces are required for the early steps in the viral replication cycle, such as translation, replication or rna stability. in contrast, knockdown of hnrnp m and nono did not change intracellular viral rna levels despite the dramatic decrease in viral titers, suggesting they may play a role downstream of replication. altogether, we have identified and validated five novel host factors for denv, demonstrating that qtux-ms can identify factors that function at different stages of the virus life cycle. an estimated 40% of the world's population is at risk from dengue for which vaccines or antivirals are not yet available. since diagnostic tests can detect denv infection at early stages, administration of antivirals could significantly improve survival rates as viral load is correlated with symptom severity [38] . using antivirals that target host factors may limit the appearance of drug-resistant viruses and may be effective for all denv serotypes and possibly for multiple flaviviruses [5, 6, 39, 40] . the qtux-ms analysis identified 79 novel cellular proteins, for which the majority are distinct from those previously identified for poliovirus using a similar approach [15] . this suggests that unrelated rna viruses have evolved to utilize distinct host rna binding proteins. interestingly, ptbp1 and nono, which were identified in both the poliovirus and denv tux-ms analyses, were shown to be required for production of both viruses (this study, figs 5 and 6) [15, 41, 42] . further analysis of virus-specific and shared host factors will reveal whether unrelated viruses utilize similar or diverse mechanisms to control viral rna replication, processing and packaging within cells. the host factors that enhance amplification of both poliovirus and dengue, (fig 2b) could serve as attractive targets for the development of broad-spectrum antivirals. the novel denv rna interactions identified in our study reveal a large network of cellular proteins which belong to different functional classes primarily associated with the nucleic acid metabolism, including numerous components of splicing, rna processing and translation machineries. these factors likely play direct roles in denv translation, replication or packaging. in addition, we have detected multiple components of cell signaling and stress response, such as several members of 14-3-3 adapter proteins, heat shock proteins and β-catenin. these factors are known to regulate diverse pathways, including host innate immune and cellular homeostasis [43] [44] [45] suggesting their possible role in host antiviral response or viral strategies to subvert the innate immune response. we demonstrated that the majority of the qtux-ms factors that we selected for validation were required for efficient denv amplification (fig 5) . specifically, we found that hnrnp f, hmces and rbmx are required for the early steps in the viral life cycle. in contrast, hnrnp m and nono appear to act downstream of viral rna replication (fig 6) , which may be significant given that both have been shown to be in a complex together [46] . nono and its binding partners are predominantly nuclear, bind rna, and are involved in pre-mrna processing, splicing, and rna transport, as well as in transcriptional activation and repression [47] [48] [49] . interestingly, other nono binding partners: psf/sfpq (polypyrimidine tract-binding protein (ptb)-associated splicing factor) and matrin3 were identified by qtux-ms as well. many of the qtux-ms identified cellular proteins are hnrnps, which encompass a large class of rna binding proteins that either localize to the nucleus or shuttle between the nucleus and the cytoplasm in order to perform multiple functions in rna metabolism, from transcription to rna turnover [50] [51] [52] . importantly, the vast majority of these factors have established roles in viral infections or in modulating the antiviral host response to various viruses [53] [54] [55] [56] , including denv (s2 table and references within). our study establishes that rbmx (hnrnp g), hnrnp f and hnrnp m are required for efficient denv amplification (fig 5b) . since several hnrnps, such as ptbp1 (hnrnp i), hnrnp a1 and hnrnp k, were previously shown to re-localize from the nucleus to the viral replication sites during denv infection [8, 57, 58] , rbmx, hnrnp f, hnrnp m and possibly other nuclear qtux-ms identified factors are also likely to be either actively recruited to the viral replication sites or retained in the cytoplasm upon denv infection. interestingly, the qtux-ms identified hnrnps affect different steps in the denv life cycle (fig 6) , suggesting that they have distinct functions during infection. this is consistent with studies that have suggested that denv rna structures are dynamic during the viral life cycle [59] and may suggest that host factors play an important role in these structural changes. furthermore, we showed that knockdown of some hnrnps (hnrnp a0 and hnrnp l) did not affect dengue viral titers significantly demonstrating that only certain hnrnps are required for denv amplification. since some of hnrnps are known to modulate cellular gene expression in response to dengue infection [60, 61] , we can not rule out that some of the observed effects on virus titers derive from their roles in regulating host mrnas. among the numerous qtux-ms identified factors of interest, our study is the first to demonstrate the involvement of hmces (or c3orf37) in viral infection. while the cellular role of human hmces is currently unknown, the mouse homologue was suggested to be an rnabinding protein and predicted to contain a putative peptidase domain [62, 63] . interestingly, it is possible that the nucleic acid binding domain enhances the protease activity or visa versa as has been shown for other such proteins [64] [65] [66] . for example, adenovirus uses a nucleic acid binding protease to localize the protease activity to the viral substrates [64] . it has been suggested that the protease is recruited to the empty capsid as an inactive protease, then it becomes fully activated once bound to the viral dna inside the virion. using the dna as a guide wire, it moves along the nucleic acid, searching for capsid and core proteins to cleave, which is required to render the viral particle infectious [64, 67, 68] . however, since we observed that knockdown of hmces resulted in a decrease in viral rna, it seems more likely that it might participate in translation, replication or the switch from translation to replication as has been shown for other nucleic acid binding proteases [65, 69] . only one of the qtux-ms identified factors was increased at the protein level in whole cells following denv infection, suggesting that qtux-ms identifications derived from the specific associations to vrna rather than changes in protein abundances. our analysis of the host cell proteome upon infection also revealed interesting changes, including up regulation of proteins in the ubiquitin pathway and down regulation of transporter proteins. the ubiquitin-proteasome pathway is one of two major cellular pathways used to degrade 80 to 90% of proteins. previous studies on denv infected cell lines and patient samples showed that the ubiquitin pathway was upregulated [31, 39, 70] . many groups have consistently shown that the ubiquitin proteasome pathway is critical for amplification of a number of flaviruses, including denv and west nile virus [31, 34, 71, 72] . however, it remains controversial as to which step in viral amplification is affected by ubiquitination, but it appears to be early during internalization or viral genome release [33, 39, 73] . further studies will be required to understand how denv up-regulates the pathway and the mechanism that ubiquitination has in denv amplification. altogether, our study has significantly increased the number of cellular proteins known to interact with the denv rna during a live infection in cells. we have also placed these interactions in the context of proteome abundance changes in the infected cells. a recent study by phillips and colleagues [13] exploited a cross-linking label-free ms approach to identify denv rna associating proteins in cell culture by cross-linking the rna to the proteins using short wavelength uv light and isolating denv rna bound proteins by anti-sense dna affinity capture [74] . while their method identified several denv host factors [12, [75] [76] [77] , the qtux-ms method reported here resulted in improved identification of known dengue host factors and putative denv rna interacting proteins. there could be several reasons for these results, such as, the qtux-ms approach achieves greater cross-linking efficiency by using long wave uv light to form crosslinks to 4-thio-uridines compared to short-wavelength uv light, which is inherently inefficient [78] . moreover, since thio-uridine is a zero distance cross-linker for rna-bound proteins at long uv wavelengths and protein-protein cross-links are not formed at long uv wavelengths, qtux-ms may also have achieved improved specificity, as only proteins in direct contact with the viral rna would be captured [23] . additionally, qtux-ms used an ms-based silac approach to determine which host proteins were specifically enriched in the vrna isolations versus mock. though isotope-labelling is not applicable in all model systems, it does afford greater quantitative accuracy compared to label-free ms strategies [16] . overall, the qtux-ms method identified 93 cellular proteins that bind to denv rna, which include 14 previously known or putative interactions. importantly, five out of the six qtux-ms identified novel factors that were tested were shown to be bona fide host factors. we used robust assays to show that the identified host factors were specifically required for denv amplification and did not merely result in a decrease in cell fitness for viral amplification. future studies will reveal whether the identified factors may also be required for other flavivirus infections that cause life-threatening illnesses, such as yellow fever, west nile, zika, japanese and tick-borne encephalitis. therefore, our data demonstrates that qtux-ms is an effective technique for identifying novel virus host factors that can be used for a broad spectrum of rna viruses by simply designing antisense dna oligonucleotides to allow for efficient sequence-specific isolation of the vrna. supporting information s1 table containing all proteins quantified by qtux-ms, including proteins enriched in denv infection (red highlighted rows) and those that did not meet the specificity threshold. for each protein group, the following are provided (from left to right), uniprot accession number, gene name, protein name, the linear and log2 denv/ mock qtux-ms enrichment ratios (columns d and e), the qtux-ms ratio variability, the number of qtux-ms quantified peptides, the average log2 denv/mock whole cell relative abundance silac ratio, whether this ratio was up (u) or down (d) regulated by more than ± 1.75-fold, the average silac ratio variability, the average number of quantified peptides, the number of razor+unique peptides and sequence coverage for qtux-ms, the protein's molecular weight and sequence length, and the complete fasta header entry for the primary protein group member. columns h-k are cross-referenced from the respective whole cell data (b). nd = not detected. (b) table containing all proteins quantified in whole cell lysates by silac. for each protein group, the following are provided (from left to right), uniprot accession number, gene name, protein name, the log2 denv/mock relative abundance ratios for replicates 1, 2, and the average (columns d and e), whether this ratio was up (u) or down (d) regulated by more than ± 1.75-fold. for each replicate, the following are provide: the silac ratio variability, the number of quantified peptides, the number of razor+unique peptides, and the number of unique peptides, total protein intensity for light (denv) and heavy (mock) conditions, and the overall sequence coverage. the complete fasta header entry is listed for the primary protein group accession number. (xlsx) s1 (a) hela uprt cells were transfected with either control or specific sirnas. 24 hours post transfection cells were counted and seeded (4 x 10 5 cells/well) in 6-well plates in triplicates. 48 hours post transfection cells were infected with denv2 at moi 0.1 and virus released to the media collected 40 hours post infection. denv2 titers were measured using plaque assays. the bars represent average values from triplicate, a standard error is reported. the representative data from one of at least two independent experiments is shown. (b) to verify sensitivity of the mtt assay, we performed sirna knockdown of g3bp2, which is known to bind denv and was previously shown to affect cell viability [37, 75] . relative viability of non-infected cells was measured using an mtt assay (invitrogen) and represented exactly as described in fig 5c. cell viability or proliferation is decreased by knockdown of g3bp2 compared with control sirna (p<0.01). sirna transfection was performed as described in the methods section using previously published sirna sequence [37] . in cells treated with g3bp2-specific sirna but not control sirna, g3bp2 protein was knocked down to the levels undetectable by western analysis using antibodies against g3bp2 (abcam, ab86135). (c). hela uprt cells were seeded 4.0 x 10 5 cells in 60 mm plates and transfected with either control or specific sirnas. 24 hours post transfection cells were infected with ad5 at moi 0.1 and collected 30 hours post infection. sirna knockdown efficiency was determined by measuring respective mrna levels in comparison to β-actin mrna abundance as described in the experimental section and reported relative to control sirna transfection (upper panel). ad5 titers were determined using plaque assays on 911 cells. the bars represent average values from triplicate, a standard error is reported. the representative data from one of at least three independent experiments is shown. 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requires the functional activities of the proteasome appraising the roles of cbll1 and the ubiquitin/proteasome system for flavivirus entry and replication antisense-mediated affinity purification of dengue virus ribonucleoprotein complexes from infected cells quantitative mass spectrometry of denv-2 rna-interacting proteins reveals that the dead-box rna helicase ddx6 binds the db1 and db2 3' utr structures a)-binding protein binds to the non-polyadenylated 3' untranslated region of dengue virus and modulates translation efficiency. the journal of general virology role of human heterogeneous nuclear ribonucleoprotein c1/c2 in dengue virus replication detecting rna-protein interactions by photocross-linking using rna molecules containing uridine analogs we would like to thank those who generously supplied reagents: lncx uprt-myc, tat, and vsvg plasmids (edward mocarski), denv2 (robert striker), and huh 7.5 cells (charlie rice). we would like to thank quynh-mai trinh for technical assistance. conceptualization: ovv tmg imc srt. key: cord-001074-qevosik3 authors: selvarajah, suganya; sexton, nicole r.; kahle, kristen m.; fong, rachel h.; mattia, kimberly-anne; gardner, joy; lu, kai; liss, nathan m.; salvador, beatriz; tucker, david f.; barnes, trevor; mabila, manu; zhou, xiangdong; rossini, giada; rucker, joseph b.; sanders, david avram; suhrbier, andreas; sambri, vittorio; michault, alain; muench, marcus o.; doranz, benjamin j.; simmons, graham title: a neutralizing monoclonal antibody targeting the acid-sensitive region in chikungunya virus e2 protects from disease date: 2013-09-12 journal: plos negl trop dis doi: 10.1371/journal.pntd.0002423 sha: doc_id: 1074 cord_uid: qevosik3 the mosquito-borne alphavirus, chikungunya virus (chikv), has recently reemerged, producing the largest epidemic ever recorded for this virus, with up to 6.5 million cases of acute and chronic rheumatic disease. there are currently no licensed vaccines for chikv and current anti-inflammatory drug treatment is often inadequate. here we describe the isolation and characterization of two human monoclonal antibodies, c9 and e8, from chikv infected and recovered individuals. c9 was determined to be a potent virus neutralizing antibody and a biosensor antibody binding study demonstrated it recognized residues on intact chikv vlps. shotgun mutagenesis alanine scanning of 98 percent of the residues in the e1 and e2 glycoproteins of chikv envelope showed that the epitope bound by c9 included amino-acid 162 in the acid-sensitive region (asr) of the chikv e2 glycoprotein. the asr is critical for the rearrangement of chikv e2 during fusion and viral entry into host cells, and we predict that c9 prevents these events from occurring. when used prophylactically in a chikv mouse model, c9 completely protected against chikv viremia and arthritis. we also observed that when administered therapeutically at 8 or 18 hours post-chikv challenge, c9 gave 100% protection in a pathogenic mouse model. given that targeting this novel neutralizing epitope in e2 can potently protect both in vitro and in vivo, it is likely to be an important region both for future antibody and vaccine-based interventions against chikv. chikungunya virus (chikv) is a mosquito-borne alphavirus first isolated in tanzania in 1952 [1] that has caused sporadic outbreaks of predominantly rheumatic disease every 2-50 years, primarily in africa and asia. the largest epidemic of chikv disease ever recorded took place during 2004-2011, and involved an estimated 1.4 to 6.5 million cases and the first autochthonous chikv infections in europe (italy in 2007 and france in 2010) [2, 3] . imported cases were also reported in nearly 40 countries, including european countries, japan, and the usa. the epidemic was associated with the emergence of a new clade of viruses, which were efficiently transmitted by aedes albopictus, a mosquito vector that has seen a dramatic global expansion in its geographic distribution [4] [5] [6] . chikv disease is characterized by acute and chronic polyarthritis/polyarthralgia, which is usually symmetric and often incapacitating and occasionally protracted [4] [5] [6] [7] [8] . other symptoms, such as fever, rash, myalgia, and/or fatigue, are often also present during the acute phase. the recent epidemic was also associated with atypical and severe clinical forms of chikv disease and some fatalities, which appeared to be restricted to the very young and elderly patients with comorbidities [8, 9] . chikv virions contain three major structural proteins: glycosylated el and e2 envelope (env) proteins embedded in the viral membrane, and a non-glycosylated nucleocapsid protein. based on similarity to other alphaviruses, e2 mediates receptor attachment, while e1 is a class ii viral fusion protein. a third glycoprotein, e3, is associated with mature virions in some alphaviruses [10] , but not others [11] , while 6k protein, a membrane-associated peptide created by cleavage of the poly-protein to release e2 and e1, is incorporated into particles at a low level [12, 13] . the organization of alphavirus surface glycoproteins in virions has been defined using cryo-electron microscopy (cryo-ems) [14] , while the atomic structure of chikv glycoproteins was recently solved by x-ray crystallography [15] , both for mature particles and for immature p62 env precursor polyprotein prior to furin cleavage. 240 copies each of three glycoproteins (e3/e2/e1) come together to form a protein coat with icosahedral symmetry and containing 80 spikes [15] . the folding, transport to the surface and function of these glycoproteins relies on their correct interactions with each other. e1 consists of three b-sheet domains, termed i, ii and iii, while e2 contains three immunoglobulin-like domains (a, b and c, with a being at the n-terminus). in the complex, domain b lies at the membrane distal end and contacts e3, domain c is closest to the viral membrane and domain a is in the center. e1 interacts laterally with e2 all along domain ii, along with additional points of contact from other regions of e1. e1 contains an internal fusion loop at the tip of domain ii, which in the mature structure exists as a b-hairpin lodged in a groove between domains a and b of e2 [15] . e3 also plays a role in protecting the fusion loop from premature exposure. treatment of chikv rheumatic disease usually involves nonsteroidal anti-inflammatory drugs (nsaids) and/or simple analgesics, which can provide relief but is often inadequate [8] . although a number of vaccine strategies have been, or are being, explored [16] [17] [18] [19] , there are currently no licensed human vaccines [20] . nevertheless, it is clear that chikv neutralizing antibodies from infected humans or vaccinated monkeys can mediate protection prophylactically, or soon after exposure. polyclonal immunoglobulins derived from humans recovered from chikv infection, when passively transferred into neonatal and interferon a/b receptor deficient (ifnar 2/2 ) mice, protected these animals from chikv-induced viremia and mortality [21] . purified total igg from monkeys immunized three times with a chikv viruslike-particle vaccine (containing e1 and e2) similarly protected ifnar 2/2 mice from chikv viremia and mortality [17] . a recent study described two monoclonal antibodies (mabs), 5f10 and 8b10, which were isolated from chikv infected individuals. these mabs specifically neutralized chikv and o'nyong'nyong virus (onnv, a virus closely related to chikv), but none of the other alphaviruses tested [22] . the 5f10 and 8b10 mabs, when used in escape mutant studies were shown to recognize key residues in e2 (v216) and e1 (t101), respectively [23] . the combination of 5f10 and 8b10 were also shown to significantly delay chikv-driven lethality in mice deficient in ifna/b and ifnc receptors, and mature b and t cells [24] . similarly, a group of mouse-derived mabs, clustering close to the putative receptorbinding domain of e2 [25] , were also found to be protective to various degrees in mouse models of chikv [26] . additionally, an immunodominant linear epitope at the n-terminus of e2 is also a target for protective antibodies [27] . herein, we describe the isolation and characterization of two human mabs, c9 and e8, from patients that were infected with chikv and recovered. we also report the characterization of antibody binding epitopes using a library of alanine scanning mutants of chikv envelope covering 910 residues (.98%) of the chikv e1 and e2 glycoproteins. although the binding epitopes for c9 and e8 both mapped to chikv e2, c9 was able to potently neutralize chikv, while e8 was not. the neutralizing epitope bound by c9 mapped to the acid-sensitive region (asr) that is critical for the rearrangement of chikv e2 during fusion and viral entry into host cells. purified human c9 antibody, when used prophylactically in an adult wild-type mouse model of chikv disease, completely protected against viremia and arthritis. written informed consent was obtained from recovered chik donors in italy and reunion, france and collection complied with relevant human subjects research protocols approved by the institutional review boards of the university of bologna and the centre hospitalier universitaire, respectively. animal work was conducted in accordance with good animal practice (nhmrc, australia), and was approved by the qimr animal ethics committee. additional murine studies were performed at blood systems research institute with approval of the institutional animal care and use committee at isis services llc (san carlos, ca) and following the recommendations of the national research council's institute of laboratory animal resources as published in their guide for the care and use of laboratory animals. chikv mab c9 variable chains were sequenced by mc labs (south san francisco, ca). for mammalian expression, c9 variable heavy (vh) and light (vl) chain cdnas were synthesized by genscript (piscataway, nj). the closest human germline signal sequences (ss), vh5 5a and vkiii a27, were used to ensure efficient processing and secretion. ss-vh cassettes were cloned into a pcaggs mammalian expression vector as ecori-nhei fragments, upstream of the human igg1 heavy chain constant region. ss-vl cassettes were cloned as ecori-bsiwi fragments upstream of the human kappa light chain constant region. chikv fab cap101a.e8 variable heavy and light chain cdnas bearing human il-2 signal sequences were synthesized by genscript. il-2ss-vh and il-2ss-vl cassettes were cloned as mfei-nhei and mfei-bsiwi fragments upstream of their respective constant regions, as described above. chikv is characterized by acute and chronic polyarthritis/ polyarthralgia that can be debilitating and protracted. currently there are no fda-approved vaccines or specific antiviral treatments for chikv. we thus identified and characterized human monoclonal antibodies directed against chikv that could be utilized in prophylactic and immediate post-exposure settings. such patient derived monoclonal antibodies could also provide useful information on critical antigens and epitopes for development of future vaccines and other biologics. we describe here the identification of two monoclonal antibodies (c9 and e8) isolated from recovered patients. c9 potently inhibited chikv infection in cells and prevented viremia and arthritis in a mouse model of chikv disease. the epitope for this antibody includes an amino-acid residue in a key acidsensitive region of the e2 glycoprotein of chikv. rearrangement of this region following exposure to low ph is critical for uncovering portions of the companion e1 glycoprotein, required for successful entry of chikv into cells. we hypothesize that binding of antibodies to this region stabilizes the native complex and thus prevents such rearrangements. chikv wild-type envelope pseudovirion production chikv envelope (e3/e2/e1) in a pcaggs vector was used for pseudoparticle preparation as described previously [28] . lentiviral pseudotypes were produced essentially as described [29] by using 10 mg of luciferase reporter plasmid, (pnl-luc, based on pnl4-3-r-e-) [30] and 30 mg of plasmid encoding viral envelope. virions were concentrated by ultracentrifuge concentration at 100,0006g in a sw28 rotor (beckman) through a 20% sucrose cushion for 1.5 h at 4uc. the pellets were resuspended overnight in hbss at 4uc.vsv-g and alphavirus envelopes expressing the rrv, sfv and sinv were used as controls for pseudovirion neutralization assay [31] . chikv wild-type pseudovirus neutralization assay hek 293t cells were plated at 2610 4 cells/well in dmem (hyclone) containing additives and incubated at 37 c in 5% co 2 overnight. the following day, serial dilutions of antibody and virus pre-incubated for 45 min were added to the hek 293t cells. a spin infection was performed at 1,2006g for 60 min and cells incubated for an additional 3 hours at 37uc. the antibody-virus mix was removed by aspiration and replaced with 100 ml of prewarmed fresh media. the cells were incubated for 48 hrs before samples were recovered for measurement of luciferase activity in the cell lysates as per manufacturers protocol (promega). chik wild-type virus production, plaque assay and 80% prnt assay chikv was obtained from atcc (atcc # vr-64), from a strain originally isolated in 1953 from the serum of a patient in east africa and expanded in suckling mice. replication competent chikv was grown in vero cells. vero cells (0.5610 5 ) were plated in a 6-well (costar) plate overnight. serial dilution of the virus stock (250 ml) was incubated with cells for 1 hr at 37uc. one hour after incubation, an overlay of 4% agarose (life technologies) in dmem supplemented with 2% fbs was added to cells and incubated at 37uc for 72 hrs. subsequently, wells were fixed with 4% formaldehyde and stained with 0.1% crystal violet in methanol: ethanol. plaques were counted against a white background. vero cells (0.5610 5 ) were plated in a 6-well (costar) plate overnight. serially diluted monoclonal antibodies were mixed with chik live virus diluted to 400 pfu/ml and pre-incubated for an hour at 37uc. following this 250 ml of the antibody-virus mixture was added to the confluent vero cell monolayer for an additional hour. subsequently, the virus was removed and an overlay of 4% agarose in dmem supplemented with 2% fbs was added and cells were incubated at 37uc for 72 hrs. the plaques were stained and counted as described above. the prnt titer is calculated as the reciprocal of the serum dilution, where $80% reduction in the number of plaques is compared to the negative control in the presence of media and no mabs. the pbmcs for ebv transformed b cell isolation were obtained from two chikv infected and recovered individuals. b cells were isolated using the miltenyi macs switched memory b cell isolation kit (130-093-617) according to the manufacturer's protocol. the cells were plated at 30 cells per well in 96 u-bottom plates. pbmc from unrelated donors were treated with mitomycin c and used as feeder cells at 5610 4 cells per well. the cells were cultured in rpmi supplemented with 7% fbs, 1000 iu/l il-2 (roche) and 2.5 mg/ml r848 peptide (invivogen) [32] . filtered b95-8 ebv supernatants (diluted 1 in 3) were added to each well and incubated for one week before being replaced with fresh media. ebv transformed b cell supernatants expressing chikv specific antibodies were screened for chikv pseudotype neutralization potential [28] . the cells from the positive wells were clonally isolated by limiting dilution followed by expansion and cloning. an immune fab phage display library was constructed from peripheral blood donated by three chikv-infected individuals. all three individuals were infected in réunion island, france, during the 2006 outbreak. peripheral blood samples were drawn 2-3 years after infection and serum was analyzed for the presence of neutralizing antibodies using chikv reporter virus pseudotypes. total rna was prepared using tri-reagent (sigma) with standard protocols. rna was converted to cdna using super script first-strand synthesis system for rt-pcr (invitrogen) following the manufacturer's instructions. construction of the library was performed by genscript (piscataway, nj) as previously described [33] . the final library was transformed into e.coli tg1 cells (invitrogen) using electroporation, and the quality of the library was assessed by sequence analysis of 100 randomly picked clones. all biosensor studies were performed at 25uc using a fortebio octet red biosensor system (fortebio, menlo park, ca). chikv vlps were loaded onto amine-reactive biosensor tips (ar2g) using an immobilized human antibody against chikv (e26d9.02, a gift from dendritics, lyon, france). briefly, ar2g tips were activated for 5 minutes with a mixture of 20 mm edc (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, sigma, st. louis, mo) and 10 mm sulfo-nhs (n-hydroxysulfosuccinimide, sigma, st. louis, mo) in water. e26d9.02 diluted to 25 mg/ml in 10 mm sodium acetate, ph 5.5, was allowed to react for 10 minutes and then the tips were deactivated for 5 minutes with 1 m ethanolamine (sigma, st. louis, mo). after a brief rinse, chikv vlps diluted to 20 mg/ml were loaded for 45 minutes followed by a 10 minute stabilization. tips were then transferred to pbs buffer supplemented with 1 mg/ml bsa (pbs-b) for subsequent antibody binding studies. c9 was prepared as a twofold serial dilution (starting at 20 mg/ml) plus buffer blanks. antibody association was measured for 5 minutes followed by a 50 minute dissociation in buffer. non-specific binding was assessed using sensor tips without vlps. data analysis was performed using octet data analysis v6.4 (fortebio, menlo park, ca). binding kinetics were analyzed using a standard 1:1 binding model. a chikv env expression construct (s27 strain) with a cterminal v5 tag was subjected to high-throughput alanine scanning mutagenesis to generate a comprehensive mutation library. primers were designed to mutate each residue within the e2, 6k, and e1 regions of env to alanine, while alanine codons were mutated to serine. in total, 910 chikv env mutants were generated (98.5% coverage), sequence confirmed, and arrayed into 384-well plates. each env mutant was transfected into hek-293t cells and allowed to express for 22 hrs. cells were stained for 1 h with human mabs c9 (0.42 mg/ml), e8 (2 mg/ml), ckv061 (0.75 mg/ml, isolated from phage display library in identical manner to e8), e26d9.02 (0.5 mg/ml, a gift from dendritics), and rabbit polyclonal antibody (1:2000 dilution, a gift from ibt bioservices) diluted in 10% ngs (sigma). mabs were detected using 3.75 mg/ml alexafluor488-conjugated secondary antibody in 10% ngs (jackson immunoresearch laboratories) for 1 h. mean cellular fluorescence was detected using the intellicyt high throughput flow cytometer (htfc, intellicyt). antibody reactivities against each mutant env clone were calculated relative to wild-type env protein reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type env-transfected controls. mutations were identified as critical to the epitope if they did not support reactivity of the test human mab, but did support reactivity of the other chikv antibodies. this counter-screen strategy facilitates the exclusion of env mutants that are misfolded or have an expression defect [34] . critical amino acids required for antibody binding were visualized on chikv env crystal structures (monomer pdb id #3n41 and trimer pdb id #2xfc, [15] ), to obtain 3d epitope maps. female c57bl/6 mice (6 weeks old) were inoculated with chikv as described previously [35] . briefly, mice were inoculated with chikv {10 4 log 10 50% cell culture infectivity dose (ccid 50 )} in 40 ml rpmi 1640 (supplemented with 2% fetal calf serum) by shallow subcutaneous injection into the top, towards the lateral side, of each hind foot in the metatarsal region, injecting toward the ankle. mice (n = 4 mice per group) were injected with (i) pbs; (ii) purified c9 mab or (iii) purified control human mab at 0.5 mg/mouse by the intraperitoneal route one day (day 21) prior to infection on day 0 with chikv. in order to avoid stimulating non-specific immune responses that may interfere with chikv infection of adult mice [35] , c9 and control antibodies with endotoxin levels below 10 eu/mg were used. arthritis was monitored by measuring the height and width of the metatarsal area of the hind feet using digital calipers [35] . the data is presented as a group average of the percentage increase in foot height6width for each foot compared with the same foot on day 0. viremias were measured by collecting 40 ml of blood from a tail vein into 0.8-ml minicollect serum separation tubes (greiner bio-one gmbh, kremsmunster, austria). the tubes were spun at 4,0006g for 2.5 min on a bench-top microcentrifuge. serum was collected and viral titers were determined as described previously and expressed as ccid 50 per ml [35] . the ability of antibodies to protect against the lethal effects of chikv infection were evaluated in a murine model as previously described [21] . all animal experiments were performed with approval of the institutional animal care and use committee at isis services llc (san carlos, ca). briefly, c57bl/6j mice were purchased from jackson laboratories (sacramento, ca) and bred at bsri. breeder pairs were housed under specific-pathogen free conditions in micro-isolator cages (innovive inc., san diego, ca). mice were checked daily and the date when litters were first observed was considered day 0. on day 9, litters with their mothers were transferred to static disposable cages (innovive inc.) and transferred a bsl-3 facility for infection and treatment. neonatal c57bl/6j mice were infected with 5610 5 pfu of chikv (s27 strain) intradermally in the ventral thorax. some mice were also intraperitoneally injected with c9 or control human igg/mouse in 0.2 ml phosphate-buffered saline (pbs) immediately prior to chikv infection. the control igg used in this experiment was purified igg from human serum (sigma-aldrich). results were analyzed using kaplan-meier survival curves using aable version 3.06 software (gigawiz ltd., co., ok). the significance of differences was determined using log-rank chisquare analysis with the results not adjusted for multiple comparisons. results are considered significant if p#0.05. the anti-chikv human monoclonal antibody c9 was isolated by ebv transformation of b cells from a chikv infected and recovered individual identified during a 2007 outbreak of chikv in northern italy [36] . chikv pseudovirus [28] neutralization was used as the primary screening assay for the selection of b cell clones and heavy and light chains were subsequently sequenced from the clones. separately, a fab fragment (e8) was isolated from a phage display library constructed from multiple chikv infected and recovered individuals from the 2005-6 epidemic on la réunion as described in the materials and methods. a virus-like particle (vlp) binding assay, using vlps produced from chikv capsid and e3/e2/e1 envelope (env) glycoprotein expression was used as the primary screen for panning phage, followed by use of the chikv pseudovirus (hiv-backbone based, without chikv capsid) neutralization assay for downstream characterization. subsequently, the antibody heavy and light chains for c9 and e8 were sequenced and cloned into human full length igg vectors for protein production and evaluation. c9 and e8 were tested in neutralization assays performed in hek 293t cells using chikv pseudoviruses bearing an envelope from the prototypical west african, asian, and east/central/ south african (ecsa) chikv strain, s27. the c9 and e8 igg antibodies neutralized chikv pseudoviruses at approximately 0.1 mg/ml and 1.0 mg/ml (ic 50 ) respectively (figure 1 ). pseudoparticles produced using envelopes derived from the lr2006 opy-1 strain from the la reunion outbreak were similarly sensitive to neutralization, with ic 50 values of 0.4 mg/ml and 10 mg/ml for c9 and e8 respectively ( figure s1 ). similar neutralization was observed regardless of the cell type used (data not shown). neutralization was specific to chikv, with no binding of anti-chikv antibody c9 to intact chikv vlps was detected using the fotebio octetred biosensor. c9 mab (66.6 nm) binding to chikv vlps or a nonparticle surface control is used to show binding specificity of mab to intact chikv vlp. (b) c9 dose response curve for binding intact chikv vlps. raw data curves for antibody associating and dissociating from captured chikv vlps are shown in black and fitted curves are shown in red. data were fitted to a 1:1 binding model to determine association rate (k on ) and dissociation rate (k off ), and equilibrium binding affinity (k d ) was calculated. c9 binds chikv vlps with 1.21 nm apparent affinity. doi:10.1371/journal.pntd.0002423.g002 detectable cross-reactivity to pseudoviruses expressing other alphavirus envelopes from rrv, sfv and sinv, as well as vsv-g (figure 1 ). the mab also neutralized chikv envelopes with a naturally occurring mutation at a critical site near the fusion loop in e1 (a226v) that is associated with increased chikv infectivity for, and transmission by, the mosquito vector, aedes albopictus (c9, ic 50 0.1 mg/ml; e8, ic 50 1.0 mg/ml for s27[a226v]) ( figure 1 ) [37] . when tested in a replication competent chikv plaque reduction neutralization test (prnt) using the s27 strain, c9 exhibited a prnt80 value of approximately 0.3 mg/ml. a comparable level of neutralization was also observed with the lr2006 opy-1 strain. in contrast to the weak neutralization observed with the chikv pseudovirus assay (figure 1 ), e8 failed to neutralize replication competent chikv, even at concentrations up to 20 mg/ml. similarly, little to no inhibition by e8 was noted utilizing vesicular stomatitis virus-based pseudotypes (rather than hiv-based) or in a cell-cell fusion assay, while c9 maintained similar neutralizing and inhibitory activity (data not shown). based on these findings, c9 can be categorized as a strongly neutralizing antibody, with similar potency to other human mabs [22] , while e8 is a non-neutralizing, or weakly neutralizing, antibody of live virus. in order to determine how strongly each mab interacts with the native virion, intact chikv vlps were captured onto the surface of fortebio octet red biosensor tips (fortebio, menlo park, ca) and antibody binding to the immobilized particles was measured using biolayer interferometry (fortebio, menlo park, ca). whereas c9 bound to vlps with an apparent affinity of 1.2 nm (figure 2a & b) , e8 failed to recognize chikv envelope protein on intact vlps (data not shown), suggesting that the e8 epitope may be occluded in the native e1/e2 conformation on virions. this finding is consistent with the inability of e8 to neutralize live chikv. c9 and e8 antibodies recognized envelope derived from chikv vlps under semi-native conditions (protein run in sds-page gels without reducing agent), suggesting that both c9 and e8 recognize conformation specific epitopes that are dependent on disulfide bonds ( figure s2) . chikv envelope proteins were immunostained with e8 antibody. clones with reactivity ,20% relative to wild-type chikv env were identified as critical for e8 binding. mutation of six individual e2 residues to alanine (y69, f84, v113, g114, t116, and d117) significantly reduced e8 binding (red bars) but did not affect binding of c9 (green bar) or other control antibodies (gray bars). residues are numbered according to e2 in pdb entry #3n41 [15] . (b) critical binding residues for e8 (shown in green) were visualized on the chikv env crystal structure. the e1, e2, and e3 envelope protein subunits in the monomer (pdb entry #3n41) are depicted in yellow, red, and blue, respectively and the fusion loop is shown in silver (left panel). in the side-view and top-down trimeric representations (center, and right panels, pdb entry #2xfc), e3 is not in the structure. in the side view trimeric representation (center panel), the viral membrane is positioned at the bottom of the figure. doi:10.1371/journal.pntd.0002423.g003 mab epitope mapping using shotgun mutagenesis in order to identify residues in the binding epitope of c9 and e8, the mabs were screened against a comprehensive chikv mutation library in which nearly every residue within the e2, 6k, and e1 envelope subunits (encompassing 910 amino-acid residues with 98.5% coverage) were individually mutated to an alanine (alanines were mutated to serines). each clone was expressed in hek-293t cells and assessed for c9 and e8 antibody binding using immunofluorescence staining. mean fluorescence was determined by high-throughput flow cytometry and antibody reactivity to each mutant was calculated relative to reactivity to wild-type (wt) chikv env. clones were identified as critical for binding if they had low reactivity to c9 or e8 but high reactivity to other chikv e2-specific control antibodies (ckv061, e26d9.02, and rabbit polyclonal antibody, described in materials and methods). this counter-screen strategy facilitates the exclusion of env mutants that are globally or locally misfolded or that have an expression defect [34] . residues identified in this way are the energetically critical contributors of an epitope, the so-called 'hot-spots' of mab binding [38, 39] . six amino acids clustered within the e2 domain a were identified as critical for e8 binding. residues e2-y69, e2-f84, e2-v113, e2-g114, e2-t116, and e2-d117, when mutated to alanine, all reacted at less than 20% of wt reactivity when screened with e8, but had high reactivity compared to three other antibodies (ckv061, e26d9.02 and rabbit polyclonal antibody), suggesting that the mutant envelope proteins are expressed and properly folded ( figure 3a) . the e8 epitope appears to be partially occluded when visualized on the native trimer structure ( figure 3b ), which likely accounts for the poor neutralization exhibited by e8. similar epitope mapping studies using shotgun mutagenesis alanine scanning identified residue e2-a162, located in the bconnector region between domains a and b of chikv e2, as a chikv envelope proteins were immunostained with c9 antibody. clones with reactivity ,20% relative to wild-type chikv env were identified as critical for c9 binding. mutation of residue a162 in e2 to serine significantly reduced c9 binding (green bar) but did not affect binding of e8 (red bar) or other control antibodies (gray bars). residues are numbered according to e2 in pdb entry #3n41 [15] . (b) a162s and a162v pseudoviruses were tested for c9 inhibitory potency. the infectivity of the mutants compared to wt was tested (inset graph), indicating that the mutants did not hinder chikv env folding or function. average raw luminescence units are shown for each construct and an env-minus negative control. (c) the critical residue a162 (shown in green) was visualized on the chikv env crystal structure. the e1, e2, and e3 env protein subunits in the monomer (pdb entry #3n41) are depicted in yellow, red, and blue, respectively and the fusion loop is shown in silver (left panel). in the side-view and top-down trimeric representations (center and right panels, pdb entry #2xfc), e3 is not in the structure. in the side view trimeric representation (center panel), the viral membrane is positioned at the bottom of the figure. doi:10.1371/journal.pntd.0002423.g004 critical residue required for c9 recognition (figure 4) . the e2-a162 residue is solvent exposed and is predicted to be easily accessible when chikv env is in the native trimer conformation (figure 4) . the e2-a162 residue is in the acid-sensitive region (asr), sandwiched in a critical pocket between chikv e1, e2 and e3, as determined by the chikv envelope crystal structure [15, 40] . interestingly, the asr, along with the e2 domain b, was also recently described for alphaviruses as being unstructured following acid ph triggering [40] . in our study we found that residue e2-a162, when mutated to serine, reacted at 12% of wt reactivity against c9 but reacted at greater than 70% of wt reactivity against other anti-chikv antibodies, strongly suggesting that the e2-a162s mutant is properly folded and involved in the c9/envelope binding interaction ( figure 4a ). other residues are also likely to be involved in the c9 epitope, but either contribute weakly to the interaction or their individual mutation to alanine does not sufficiently disrupt mab binding to be detected as 'critical'. using pseudovirions, no virus entry defects were observed with e2-a162s, further indicating that the mutant envelope is properly folded. to confirm the importance of this residue in c9 binding, infection experiments were conducted with wild type and mutant pseudovirions. e2-a162s pseudovirions were inefficiently neutralized by c9, with a 490-fold increase in the c9 ic 50 , demonstrating that this residue is required for potent c9 inhibition (figure 4 ). in contrast, wild type e2 and e2-a162v, a naturally occurring variant [41] , remained fully sensitive to c9. to assess the potential protective activity of mab c9 in vivo, we used an adult (6 week old) wild-type mouse model of chikv disease [35] . mice received an intra-peritoneal injection of purified c9 igg (0.5 mg/mouse or approximately 20-25 mg/kg) the day before being infected with the reunion island isolate of chikv (lr2006-opy-1) [35] . a control monoclonal antibody that did not recognize chikv (produced in the same fashion as c9) and pbs were used as negative controls. infected mice were monitored for viremia and foot swelling as described previously [35] . in both control groups, chikv infection of 6-week old mice resulted in a 5-6 day viremia and increased foot swelling similar to that described previously in control animals [35] . in contrast, in the same experiment, 6 week old mice injected with c9 igg 24 hours prior to exposure to virus, showed no detectable viremia or foot swelling ( figure 5 ). these results demonstrate that the c9 antibody completely protected adult animals prophylactically against viremia and arthritic disease. in order to evaluate the therapeutic potential of c9 mab, we inoculated c57bl/6j neonate mice with 5610 5 pfu of chikv and monitored the survival rate. mice infected with chikv survived for 5 days, while mice given control human igg survived for 4 days ( figure 6 ; table 1 ). in contrast 100% of the neonate mice injected with c9 at 100 mg (,25 mg/kg) co-incident with infection survived (p#0.001 compared to virus alone or human igg). we also observed that c9 (100 mg/mouse or 25 mg/kg), when administered therapeutically at 8 hours and 18 hours after chikv challenge, completely protected 100% of mice (table 1) . therefore, c9 is a potent neutralizing antibody that can therapeutically protect wild-type neonate mice from a lethal dose of virus at 8 and 18 hours post chikv inoculation. protection in wild-type mice from virus infection in vivo by a monoclonal neutralizing antibody is thought to require close to the ic 90 levels of antibodies in the serum [42, 43] . however, there is very little known for chikungunya protection in mice with human anti-chikv mabs. we performed in vivo antibody titration experiments and dosed neonates immediately before chikv challenge via intraperitoneal injection with c9 mab at 100, 20, 10, 4, 1 and 0.1 mg/mouse (or approximately equaling 25, 5, 2.5, 1.0, 0.25 and 0.025 mg/kg respectively). mice were fully protected with as little as 1 mg/kg of c9 mab, while over 50% of mice survived after receiving 0.25 mg/kg of c9 mab concurrent with viral challenge (figure 6 ). mice that were given 0.025 mg/kg of c9 mab succumbed to chikv-driven lethality similarly to control groups. our results demonstrate that with a potent neutralizing antibody such as c9 (ic 90 of 0.3 mg/ml) we can protect 100% of mice with 1 mg/kg of c9 mab and about half of all neonatal mice with only 0.25 mg/kg of c9. this study describes the isolation and characterization of two human monoclonal antibodies, c9 and e8, from chikv infected and recovered individuals. we previously developed a chikv pseudovirus assay [28] that we found amenable in our current study for high-throughput screening and selection of b-cell clones expressing chikv neutralizing antibodies. c9 neutralizes both chikv pseudoviruses and replication-competent viruses with high potency. the e8 monoclonal antibody shows less dramatic neutralization of pseudovirus and does not neutralize live virus at the highest concentration tested (20 mg/ml). this suggests that although chikv antibody selection can be carried out using the high-throughput pseudovirus assays for greater convenience, a live virus-prnt assay is the more reliable confirmatory assay for chikv neutralization. we also report the development of a novel, comprehensive chikv envelope site-directed mutation library in which nearly all of the 910 residues of the full-length e1/e2 chikv envelope protein were individually mutated to alanine in order to identify critical amino acids that are recognized by human mabs c9 and e8. e8 recognized 6 spatially proximal residues (y69, f84, v113, g114, t116a and d117) in e2 domain a, however the nonneutralizing nature of the e8 antibody suggests that the residues are not easily accessible on the native chikv envelope on live virus, and indeed the epitope appears to be partially occluded when visualized on the native trimer crystal structure. the sitedirected mutagenesis mapping studies, confirmed by neutralization escape mutant studies, revealed that e2-a162 is a critical all mice were infected with chikv at hour 0. data on chikv infected mice without antibody treatment and mice treated with control or c9 antibody at hour 0 are also shown in figure 6 . residue required for c9 mab recognition. interestingly, based on the crystal structure of the chikv envelope, the e2-a162 residue is located in the asr of e2 that encompasses amino acids 159-171 and 231-258 [15] . the asr in e2, along with domain b, is a highly conserved functional region among alphaviruses and is involved in the conformational rearrangements triggered by acid ph that lead to the exposure of the fusion loop in e1 and finally results in membrane fusion [15, 40] . it is possible that neutralizing antibodies such as c9 that bind to the asr region could fully or partially prevent the disordering and dissociation of e2 from e1 following ph triggering, thereby reducing fusion efficiency and chikv entry. residues within the asr have previously been reported to be critical for efficient particle formation and stability [44] , highlighting the delicate conformational balance that this region brings to e2. the critical e2-a162 residue is highly conserved among different chikv strains and is represented in the 1950's west african isolates 37997 and s27, as well as in the more recently circulating lr2006 opy-1 strain. however, a previous study described a strain (ag41855) isolated from uganda during a 1982 outbreak that has a valine at the e2-162 position [41] . mutating e2-a162 to valine did not result in a loss of c9 potency, suggesting c9 should be active against most currently circulating strains of chikv and other strains that arise in the future with that particular amino-acid. the fact that e2 proteins bearing the aliphatic, hydrophobic amino-acids alanine or valine did not prevent c9 neutralization, while e2 with a polar serine residue at position 162 escaped neutralization, suggests that c9 forms a critical hydrophobic interaction with that sidechain position. neutralizing antibodies have been shown to be critical for recovery from alphavirus infections and a number of neutralizing epitopes have been characterized, albeit only a handful for chikv. of particular note, antibody r6/r13 is specific to sinv and has been previously documented to have an escape mutant at position k159n (equivalent to chikv residue e2-t160) in the asr of sinv e2 glycoprotein [45] . the isolation and characterization of additional chikv mabs should offer insight into the proportion of antibodies elicited against this particular epitope in chikv infected individuals and the timing at which they appear. for example, an elegant study recently described that a predominant proportion of the very early response to chikv envelope is composed of igg3 antibodies directed against the nterminal sequence in e2 (e2ep3) [27] . there is very little known concerning chikungunya protection with human monoclonal antibodies. in order to elucidate whether strong in vitro c9 neutralization would translate to protection in vivo, we used the c9 antibody in an adult wild-type mouse model of chikv disease. in contrast to control mice, mice pretreated with c9 antibody had no detectable chikv viremia or arthritis. in a previous study, human igg purified from the pooled plasma of chikv recovered individuals was titrated in a pathogenic neonate mouse model of chikungunya (similar to one of the models used in our current study), and was shown to fully protect only when present at a concentration of 10-25 mg per mouse [21] . another recent study described two monoclonal antibodies (5f10 and 8b10) that were protective in vivo in ifndeficient mice. these two antibodies protected 100% of the mice at ,12.5 mg/kg (250 mg per mouse) [24] . however, the antibodies failed to protect mice at ,1.25 mg/kg (25 mg per mouse) and were only able to delay chikv-driven lethality. in the in vivo antibody titration experiments described here we demonstrate that 100% of the mice were protected with 1 mg/kg of c9 mab (in vitro ic 90 of 0.3 mg/ml), while over 50% of mice were protected with only 0.25 mg/kg of c9. the data with c9 is consistent with what has been determined for in vivo protection from viral infection by neutralizing antibodies [42] , while c9 at 0.25 mg/kg could also protect over half of animals from chikvdriven lethality. in order to evaluate the therapeutic potential of c9 mab we inoculated wild-type (c57bl/6j) neonate mice with a robust dose of chikv (5610 5 pfu) and monitored the survival rate. we observed that c9 mab (100 mg/mouse), when administered therapeutically at 8 and 18 hours after chikv infection, completely protected 100% of mice. this report demonstrates prophylactic and therapeutic protection against viremia in vivo by a neutralizing antibody that targets the acid-sensitive region in chikv e2. although passive antibodies likely would not be utilized for protection from chikv on a regular basis, one can envisage a scenario where a potent antibody like c9 can be manufactured and used for protecting highly susceptible individuals such as pregnant women, infants and older individuals during a chikv epidemic, or for protecting travelers or military inadvertently exposed to the virus. while, the isolation and epitope characterization of c9 antibody and demonstration of its potent neutralization in vitro and in vivo are invaluable to future studies aimed at identification of neutralization epitopes in order to produce effective antigens for vaccines, we hypothesize that the epitope recognized by the c9 antibody is also an important region to target for antibody-based intervention in future anti-chikv strategies. recent studies have demonstrated that the use of combinations of mabs is advantageous for in vivo protection by limiting development of resistance [26] and different mechanisms of viral spread [24] . given that c9 is directed against a region of chikv e2 not targeted by other mabs, it will be particularly useful as a partner in such combinations. figure s1 human mabs c9 and e8 neutralization. neutralization of pseudovirus bearing chikv lr2006-opy-1 strain (orange lines) chikv s27 strain (green lines) and vsv-g control (magenta lines) envelope by (a) c9 or (b) e8 mabs. antibody concentration is shown in the x-axis. the results are expressed as the percentage of no antibody control and represent mean of triplicate wells, and is representative of three experiments. (tiff) figure s2 western blot of reduced and non-reduced chikv vlps using c9 and e8 mabs. antibodies (1 ug/ml) were tested for reactivity against 5 ug chikv vlps that were treated with dtt/heat (+) or not (2) . hrp signal was detected using luminescence by adding a 1:1 ratio of femto supersignal. (tiff) text s1 supporting methods. (docx) an epidemic of virus disease in southern province, tanganyika territory, in 1952-53. i. clinical features infection with chikungunya virus in italy: an outbreak in a temperate region chikungunya virus, southeastern france chikungunya, an epidemic arbovirosis how did chikungunya reach the indian ocean? chikungunya epidemic in india: a major public-health disaster persistent chronic inflammation and infection by chikungunya arthritogenic alphavirus in spite of a robust host immune response arthritogenic alphaviruses-an overview systemic involvements and fatalities during chikungunya epidemic in india formation of the semliki forest virus membrane glycoprotein complexes in the infected cell biochemical studies of the maturation of the small sindbis virus glycoprotein e3 the sindbis virus 6k protein can be detected in virions and is acylated with fatty acids fate of the 6k membrane protein of semliki forest virus during virus assembly mapping the structure and function of the e1 and 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analyses at pseudo atomic resolution of chikungunya virus and antibodies show mechanisms of neutralization development of a highly protective combination monoclonal antibody therapy against chikungunya virus early neutralizing igg response to chikungunya virus in infected patients targets a dominant linear epitope on the e2 glycoprotein characterization of chikungunya pseudotyped viruses: identification of refractory cell lines and demonstration of cellular tropism differences mediated by mutations in e1 glycoprotein characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes ross river virus glycoprotein-pseudotyped retroviruses and stable cell lines for their production clonal dissection of the human memory b-cell repertoire following infection and vaccination phage display: a laboratory manual atomic-level mapping of antibody epitopes on a gpcr chikungunya virus arthritis in adult wild-type mice the 2007 epidemic outbreak of chikungunya virus infection in the romagna region of italy: a new perspective for the possible diffusion of tropical diseases in temperate areas a single mutation in chikungunya virus affects vector specificity and epidemic potential anatomy of hot spots in protein interfaces the atomic structure of protein-protein recognition sites structural changes of envelope proteins during alphavirus fusion epistatic roles of e2 glycoprotein mutations in adaption of chikungunya virus to aedes albopictus and ae. aegypti mosquitoes the antiviral activity of antibodies in vitro and in vivo preand postexposure prophylaxis of ebola virus infection in an animal model by passive transfer of a neutralizing human antibody a specific domain of the chikungunya virus e2 protein regulates particle formation in human cells: implications for alphavirus vaccine design antigenic and genetic characterization of sindbis virus monoclonal antibody escape mutants which define a pathogenesis domain on glycoprotein e2 the authors acknowledge bridget puffer for assistance in phage display library construction, simona jusyte for assistance with phage display library panning, surabhi srinivasan and nick distasio for assistance with epitope mapping, joe couto and silveria rodriguez for dna plasmid engineering and construction, and dr. marina fomin for mouse breeding. the authors would like to thank james robinson (tulane university) for advice and reagents for b-cell cloning. we also thank dendritics for kindly providing an anti-chikv monoclonal antibody and ibt bioservices for provision of the rabbit anti-chikv polyclonal antibody. key: cord-308343-crjjhpl1 authors: graef, geneva; hurst, natalie j.; kidder, lance; sy, tracy l.; goodman, laura b.; preston, whitney d.; arnold, samuel l. m.; zambriski, jennifer a. title: impact of confinement housing on study end-points in the calf model of cryptosporidiosis date: 2018-04-25 journal: plos negl trop dis doi: 10.1371/journal.pntd.0006295 sha: doc_id: 308343 cord_uid: crjjhpl1 background: diarrhea is the second leading cause of death in children < 5 years globally and the parasite genus cryptosporidium is a leading cause of that diarrhea. the global disease burden attributable to cryptosporidiosis is substantial and the only approved chemotherapeutic, nitazoxanide, has poor efficacy in hiv positive children. chemotherapeutic development is dependent on the calf model of cryptosporidiosis, which is the best approximation of human disease. however, the model is not consistently applied across research studies. data collection commonly occurs using two different methods: complete fecal collection (cfc), which requires use of confinement housing, and interval collection (ic), which permits use of box stalls. cfc mimics human challenge model methodology but it is unknown if confinement housing impacts study end-points and if data gathered via this method is suitable for generalization to human populations. methods: using a modified crossover study design we compared cfc and ic and evaluated the impact of housing on study end-points. at birth, calves were randomly assigned to confinement (n = 14) or box stall housing (n = 9), or were challenged with 5 x 10(7) c. parvum oocysts, and followed for 10 days. study end-points included fecal oocyst shedding, severity of diarrhea, degree of dehydration, and plasma cortisol. findings: calves in confinement had no significant differences in mean log oocysts enumerated per gram of fecal dry matter between cfc and ic samples (p = 0.6), nor were there diurnal variations in oocyst shedding (p = 0.1). confinement housed calves shed significantly more oocysts (p = 0.05), had higher plasma cortisol (p = 0.001), and required more supportive care (p = 0.0009) than calves in box stalls. conclusion: housing method confounds study end-points in the calf model of cryptosporidiosis. due to increased stress data collected from calves in confinement housing may not accurately estimate the efficacy of chemotherapeutics targeting c. parvum. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 cryptosporidium is a genus of protozoal parasites that infect a wide range of hosts including wild and domestic mammals, amphibians, and reptiles [1] . the most pathogenic species are c. hominis and c. parvum, which primarily affect people and cattle, respectively. c. parvum, which is zoonotic, represents an important public health threat. in resource-poor countries this is of particular concern for children who live in close proximity to cattle, and in resourcerich countries the zoonosis impacts agricultural laborers on commercial dairy farms, where herd prevalence can be as high as 80% [2, 3] . in both human and cattle hosts, disease is characterized by villus atrophy, intestinal crypt inflammation, and malabsorptive, maldigestive diarrhea that may be secretory in nature [4, 5] . in addition, large numbers of infective oocysts are shed in the stool, which impacts environmental parasite loading and propagates fecal-oral transmission [6] . diarrhea has been identified as a leading cause of death in children < 5 yrs globally [7, 8] . in resource-poor countries, cryptosporidiosis is the second leading cause of diarrhea in children (after rotavirus) and is associated with an increased risk of death in the first 2 yrs of life [7] . individuals that are immunocompromised due to other etiologies, such as hiv or severe malnutrition, are more likely to develop life-threatening diarrhea [1, 7, 9] . pediatric cryptosporidiosis has also been associated with cognitive impairment and stunting, regardless of the development of diarrhea [9] [10] [11] . despite the adverse impact on human lives there are no treatments or vaccines that are consistently effective against cryptosporidium [12] [13] [14] [15] [16] . nitazoxanide is the only therapy currently approved for the treatment of cryptosporidiosis in people. it has been demonstrated to have modest efficacy in hiv-seronegative children, but not hivseropositive children [16] . similarly, in veterinary patients there is a dearth of treatment options. halofuginone is the only drug licensed for use in cattle to control cryptosporidiosis, but it is not an ideal treatment option. it is registered in a few countries, excluding the united states, is only effective when used prophylactically and not therapeutically, has a very narrow margin of safety, and is not cost-effective [17] [18] [19] . given the substantial impact of cryptosporidiosis on child health in resource-poor settings and the lack of options to prevent and treat disease in human and animal populations, there is an urgent need to identify and test new chemotherapeutic agents. to develop effective chemotherapeutics, repeatable and reliable in vivo and in vitro models are essential. cryptosporidium is notoriously difficult to culture and study in vitro, though advances are being made using hollow fiber technology, and in vivo models either fail to achieve comparable severity of clinical illness or are cumbersome to execute [20, 21] . murine models of cryptosporidiosis are commonly used because they permit induction of infection with c. parvum. although c. parvum is not host-adapted to mice, this model is widely applied because it is tractable. cryptosporidium species do not induce severe clinical illness in mice. this includes c. muris, which is host-adapted to mice [22] . studies in the mouse model report weight loss, listlessness, and occasional pasty stool, but do not report fulminant diarrhea [23, 24] . the inability to induce measureable clinical illness is a major limitation of mouse models, because we must be able to measure a response to treatment in order to evaluate chemotherapeutic efficacy. the calf model of cryptosporidiosis is the best approximation of human disease because calves experience natural infection and clinical illness that mirrors human symptomology. however, the model is not consistently applied across research studies, which may impact evaluation of study end-points such as fecal oocyst shedding, severity of diarrhea, and degree of dehydration. the type of housing system used in calf model studies of cryptosporidiosis is dictated by the desired method of fecal sampling. the two methods most commonly used to collect and evaluate stool from calves experimentally infected with c. parvum are the complete fecal collection (cfc) method and the interval collection (ic) method. for cfc the total amount of stool produced by a calf is collected every 24 hours, blended for homogenization, measured either by weight or by volume, and an aliquot (~0.2g) is removed for evaluation [25, 26] . in the interval collection method, a small fecal sample (~10 g) is collected directly from the rectum of the calf every 12 or every 24 hours [14, [27] [28] [29] . historically, cryptosporidium challenge studies conducted in human volunteers have used cfc [30, 31] . for human participants cfc is considered to be the gold-standard for evaluating reduction of diarrhea in response to treatment. another potential advantage to cfc is that homogenization of the stool sample prior to oocyst enumeration mitigates the risk of variation in oocyst counts associated with intermittent oocyst shedding or diurnal fluctuations. though there is a report of possible diurnal variation in oocyst excretion in confinement housing, it is not clear whether this would be an issue in box stall housing or in all situations of confinement housing [26] . a major disadvantage to cfc is that it is cumbersome and difficult to execute in animal models. with human volunteers, a variety of sample collection methods exist to permit simple, non-invasive collection of cfc. with neonatal calves collection of cfc is a formidable challenge requiring use of confinement housing that severely restricts calf movement and their ability to engage in natural behaviors, such as grooming. calves may rise or lay down in sternal or lateral recumbancy, but they cannot turn around or ambulate. the use of this degree of confinement is necessary in order to direct the fecal matter expelled by the calf directly into collection pans. enlarging the confinement system to let the calf ambulate or to allow sufficient space for the calf to turn around would not permit collection of the entire stool sample, especially at the peak of illness when calves may pass stool 1-3 times per hour. this housing system, originally developed for use in the veal industry, is not ideal for calf welfare and may not be acceptable to institutional animal care and use committees, particularly those at european institutions. in contrast to cfc, the use of the ic method allows calves to be housed in box stalls where they can move freely. it also has the added advantage of reduced labor requirements for sample collection and calf husbandry. in addition to required use of confinement housing, implementation of cfc is expensive to maintain, labor intensive, and age restricted. calf enrollment cannot exceed 10 days as calves older than 10 days are too large for confinement housing. the "guide for the care and use of agricultural animals in research and teaching" specifically states that calves can not be placed in confinement after day 10 of life [32] . thus the window of data collection in confinement housing is limited to 10 days. in addition, this limits study efficiency, as it is difficult to manage large numbers of calves, which typically restricts enrollment to 4-6 animals. there is also a risk that data collected during this period may not be representative. it has been demonstrated that calves are more likely to have higher levels of cortisol in the first 10 days of life, indicating higher susceptibility to stress [33, 34] . however, the impacts of confinement housing on cortisol levels have not been evaluated and the impacts of elevated cortisol on study end-points have also not been assessed. no studies have been conducted comparing cfc and ic, nor are there studies comparing confinement housing and box stalls in the calf model of cryptosporidiosis, which is a substantial knowledge gap considering how integral this model is to cryptosporidium drug development. it is unknown if the ic method is an acceptable alternative to cfc with respect to relevant study end-points such as diarrheal severity and magnitude of fecal oocyst shedding. it is also unknown if these end-points are impacted by calf confinement. lastly, there are no studies that compare cortisol levels and other negative health outcomes, such as risk of calf injury and need for supportive care, in association with housing type. therefore, the objective of this study was to determine if data collected via ic is comparable to data collected via cfc, and if confinement housing impacts the study end-points used to evaluate chemotherapeutic agents targeting c. parvum in the calf model. this study was reviewed and approved by the washington state university institutional animal care and use committee (asaf04679). the study adhered to the guidelines put forth by the animal welfare act and specifically the animal welfare act & regulations blue book for usda animals. a total of 25 holstein bull calves were enrolled contemporaneously on a commercial dairy farm located in pasco, wa. calves were enrolled for a period of 25 days. to ensure that calves were not exposed to cryptosporidium and to limit exposure to other microorganisms, all calvings were attended and assisted. the perineum of the dam was cleaned thoroughly with a povidone-iodine scrub, and each calf was delivered using a single-use plastic sheet or a designated wheelbarrow that was thoroughly cleaned and disinfected between each calving. a physical exam was conducted and each calf was weighed. calves with normal physical exam findings that weighed more than 29.5 kg were enrolled. upon enrollment, the umbilicus of each calf was dipped in an iodine tincture, and 3 ml of vitamin e and selenium (bose, intervet/ merck animal health, germany) was administered subcutaneously. calves received 4 l of !50 g igg/l commercial colostrum replacer within 3 hours of birth (bovine igg, colostrum replacement, land o'lakes inc., st. paul, mn) via an esophageal tube feeder. calves were transported to washington state university in a dedicated trailer bedded with sterile straw. animals were randomly assigned to box stall (n = 9), confinement housing (n = 14), box stall negative control (n = 1), or confinement housing negative control (n = 1) treatments and housed in a bsl 2 isolation facility. negative control calves served as sentinels to aid in monitoring for inadvertent transmission of parasite between calves. inadvertent transmission would result in some calves receiving a larger parasite inoculum than others and could impact severity of clinical illness. calves in box stalls were housed individually in approximately 12.2 m 2 (40 ft 2 ) of space, bedded in sterile wood shavings, and had a mirror placed at eye-level for environmental enrichment. calves in the confinement housing group were placed inside of commercially manufactured elevated calf stalls (wenke manufacturing, pender, nebraska) [25, 26] . to provide environmental enrichment, 2 stalls were placed in each bsl 2 room and calves were faced toward each other at a distance of 0.6 m (2 ft) to prevent cross-contamination. the surface area of each stall was 2.7 m 2 (8.75 ft 2 ) (152.4 cm long by 53.3 cm wide), and each stall was raised 27.9 cm off of the ground (s1 fig) . stalls were constructed from 1.9 cm (0.75 in) galvanized square framed tubing with an open-grate rubber coated floor to permit feces to fall below the stall into collection bins. in compliance with the "guide for the care and use of agricultural animals in research and teaching," on day 10 of life, calves randomized to confinement housing were removed from confinement and placed in box stalls [32] . all calves were followed for 25 days post-infection. over the course of enrollment, calves were fed every 12 h via nipple buckets with a commercial, non-medicated milk replacer containing 20% crude protein and 20% fat (maxicare, land o'lakes, shoreview, mn). water was provided ad libitum. calves were randomly assigned to inoculation (n = 23) or negative control groups (n = 2). calves in the inoculation group were challenged with a commercially available iowa laboratory isolate of cryptosporidium parvum within 48-72 h from birth at a dose of 5 × 10 7 oocysts (bunch grass farms, deary, id). oocysts were administered within 1 month of original isolation and were cleaned for one minute in 0.6% sodium hypochlorite to inactivate possible viruses and bacteria co-purified with the oocysts, then washed four times with phosphate buffered saline to remove the sodium hypochlorite. the oocysts were delivered orally in a 5 ml suspension via the rigid oral portion of an esophageal feeder, followed by approximately 120ml of water to ensure that the entire oocyst suspension was given to the calf. negative control calves were sham challenged to maintain blinding. all calves in confinement housing underwent cfc every 24 h and had an ic sample collected every 12 h. calves in box stalls had an ic sample collected every 24 h. for ic sampling, up to 10 g of stool was collected directly from the rectum of the calf via digital manipulation with a gloved hand. cfc methods previously described for stool collection were used [25] . briefly, a fecal collection pan was placed beneath each calf, and urine was diverted to a 12 h adult diaper to prevent contamination of the fecal pans. all stool from the fecal pan was collected every 24 h and blended for homogenization to ensure uniform oocyst distribution. the contents of the blender were then weighed (kg). after homogenization, a 0.2 g aliquot of feces was removed from all ic and cfc samples for enumeration via real-time quantitative polymerase chain reaction (qpcr). oocysts counts were interpolated by qpcr using serial dilutions of commercially purified c. parvum oocysts (waterborne, inc., new orleans, la). total nucleic acid was extracted from supernatants of 200 mg of fecal sample, oocyst suspension, or negative control homogenized in 400 μl of pbs using a magnetic bead based automated procedure (am1840, applied biosytems, foster city, ca). an exogenous control (ms2 phage) was added to the lysis buffer to control for extraction efficiency [35] . qpcr for cryptosporidium spp. 18s rrna was performed using a previously described assay on the applied biosystems 7500-fast placfcrm using an inhibitor-optimized master mix (part 95134, quantabio, beverly, ma), 300 nm forward primer, 900 nm reverse primer and 120 nm of probe (labeled with fam) [36] . for both cfc and ic samples, the count was standardized by the fecal dry weight percentage. dry weight analysis of fecal samples was obtained by taking a 5-10 g portion of each original fecal sample, drying it at 108˚c for a minimum of 24 h (squaroid vaccuum oven, labline, kochi, india), then weighing it directly (scout pro sp202, ohaus corporation, pine brook, nj) [37] . the calves were examined and evaluated every 2-12 h. a complete physical exam was conducted every 12 h at feeding time. at the time of physical exam any new injuries or clinical signs were documented, this included abrasions and wounds, as well as swellings, areas of localized pain, and fever. in order to minimize inter-observer bias, standardized and validated rubrics were used as previously described [14, 27, 28, 37] . in addition, all research personnel underwent training to maximize inter-observer agreement. appetite, fecal consistency, mentation (state of mental activity and responsiveness), and hydration status were each individually evaluated on an ordinal scale of 1-3, where a score of 1 represented normal clinical findings and a score of 3 was consistent with severe clinical illness in the specified category (s1 table) . qualitative measures of hydration such as skin turgor and degree of enophthalmos were used. monitoring for enophthalmos was necessary for identifying calves at risk for development of corneal ulcers. as severity of dehydration worsens in calves and the globes of the eyes recede further into the orbit, the eyelids roll inward causing the eyelashes to rest directly on the cornea, abrading the surface. hydration status was also assessed quantitatively via measurement of urine production every 12 h. to determine urine production without the use of urinary catheters, an adult diaper was weighed, secured over the calf's penis, and re-weighed to determine the difference (kg). blood samples were collected via jugular venipuncture on days 2, 4, 5, and 9 post-infection. all samples were collected at 10:00 a.m. samples were obtained by individuals trained in blood collection using a vacutainer system. for all enrolled calves, to reduce the risk of falsely elevating cortisol levels, efforts were made not to create excessive stress prior to blood collection, e.g., loud noises, changes in lighting, use of excessive restraint, more than 2 venipuncture attempts. calves in confinement housing were already restrained within the housing system. additional restraint of these animals was not required and could have resulted in additional stress. blood was collected from calves in confinement housing by reaching directly through the bars of the housing mechanism. calves in box stalls were restrained in sternal recumbancy to visualize the jugular vein. restraint was for less than 120 sec and attempts were made to avoid chasing calves to capture them in the stalls. however, calves in box stalls were generally more energetic and therefore ambulated more frequently prior to blood collection. cortisol was extracted from calf plasma by solid phase extraction and quantified using ultra performance liquid chromatography in tandem with mass spectrometry (uplc-ms/ms). cortisol and cortisol-d 4 were purchased from cerilliant (round rock, tx) and stock solutions were separately prepared in 25% methanol at concentrations of 20 μg/ml and 750 ng/ ml, respectively. a calibration curve was generated in calf plasma at nominal concentrations of 1, 5, 15, 20, 40, 50, 75, 100, 150, and 200 ng/ml. quality control (qc) samples were prepared at nominal concentrations of 3, 90, and 175 ng/ml. 150 μl of calibration and qc samples were added to a 96-well plate along with the study samples. next, 20 μl of the cortisol-d 4 solution was added to each well followed by addition of 300 μl methanol. samples were aspirated and then centrifuged at 3600 rcf for 20 minutes. following centrifugation, 300 μl of the resulting supernatant was removed and placed in a 96 well plate. 900 μl of 4% phosphoric acid was added to each 300 μl aliquot, and the samples were mixed thoroughly. samples were then placed in an oasis prime hlb μelution plate (waters, milford, ma) and washed with 2 aliquots of 200 μl 25% methanol. next, 2 aliquots of 90/10 acetonitrile:methanol were added to each sample, and the resulting eluate was diluted with 25 μl water. 7.5 μl of each sample was injected onto an acquity uplc in tandem with a waters xevo tq-s. a zorbax eclipse plus c18 column (agilent, santa clara, ca) heated to 40˚c and mobile phases a: water with 0.1% formic acid and b: acetonitrile with 0.1% formic acid were used with the following gradient: 90%-80% a over the first minute, 80%-10% a from 1 to 3.5 minutes, a further decrease to 5% a until 5 minutes, followed by an increase back to 90% a until 7 minutes. the flow rate of the solvents was 0.5 ml/min. the transitions (m/z) 363.2>121.1 and 367.2>121.1 were used to quantify cortisol and cortisol-d 4 , respectively. endogenous cortisol observed in the calf serum was used to generate the calibration curve, and the amount of naturally occurring cortisol was subtracted from each calibration curve and qc sample. all qc samples were within 15% of their expected values, and the coefficients of variation (cv%) were < 15%. descriptive and inferential methods were used. the shapiro-wilk test was used to determine if the data was non-gaussian. hypothesis tests for normally distributed continuous data were analyzed using the student's t test or analysis of variance. categorical variables were tested via chi-square or fisher's exact. non-normal continuous variables were either log-transformed or analyzed via the wilcoxon rank sum test. the wilcoxon rank sum was used to assess differences in fecal oocyst counts and fecal dry matter percentage between ic and cfc samples for confinement housing calves and between ic samples in confinement housing and box stall calves. the wilcoxon rank sum test was also used to evaluate the differences in plasma cortisol, daily weight gain, daily milk replacer consumption, volume of fluid therapy, and frequency of non-fluid therapy treatments in confinement housing and box stall calves. chi-square or fisher's exact tests were used to describe the association between frequency of injury and housing type. the frequency of injury was counted once at the time of diagnosis. the frequency of non-fluid therapy treatment was counted with every intervention following diagnosis. the frequency of injury and non-fluid therapy treatments were recorded in this manner to enable the ability to track the occurrence of new injuries as well as to assess the amount of researcher effort required to subsequently treat and manage those injuries. bivariate analysis was used to evaluate the relationship between log oocysts per gram of fecal dry matter and plasma cortisol. in order to identify the best predictors of total fecal output, simple backward stepwise regression was carried out to achieve the most parsimonious model. individual explanatory variables with a p-value 0.2 in bivariate analysis were retained. data were analyzed using jmp pro 11.0 (sas institute inc., cary, nc). enrolled calves weighed an average of 42.1 ± 4.9 kg at birth (range 36.8-59.3 kg). the mean serum total protein measurement at 48 h of life was 5.8 ± 0.4 (g/dl) (range 5.1-6.4 g/dl). there were no differences in serum total protein across groups (p = 0.7), and no calves experienced failure of passive transfer. all calves challenged with c. parvum oocysts developed fecal oocyst shedding within 3 days post-infection (pi) (45% at 1 day pi, 41% at 2 days pi, and 14% at 3 days pi). the median duration from parasite challenge to onset of fulminant diarrhea and clinical illness consistent with cryptosporidiosis was 3 days post-infection (3.2 ± 0.4). on day 10 pi, all calves were still shedding oocysts in their stool. stool was tested via qpcr for coinfection. all enrolled calves were negative for e. coli k 99, salmonella spp., rotavirus, and corona virus. the two negative control calves did not develop cryptosporidiosis. in order to prevent calves from turning around in the stall and urinating in the fecal collection bin, we applied the head-catch to 2 animals. both calves died after they became cast in the stall and were unable to rise. these animals were removed from the study. final analysis included 12 confinement housed calves. for calves housed in confinement housing (n = 12), the mean total fecal output was 4.8 ± 3.8 kg/day (range 0.1-15.7 kg/day) and a mean total urine output was 1.8 ± 1.4 kg/day (range 0.1-5.8 kg/day) during the 10 day study period ( table 1 ). the uninfected control calf in confinement housing had a mean total fecal output of 0.5 ± 0.6 kg/day (range 0.1-2.1 kg/day) and a total mean urine output of 1.7 ± 1.0 kg/day (range 0.4-3.9 kg/day) ( table 1) . over the 10 day period of confinement, there were no significant differences in mean log oocysts enumerated per gram of fecal dry matter between cfc (10.8 ± 9.8) and ic samples (11.7 ± 9.5) (p = 0.6), nor were there any diurnal variations in oocyst shedding for morning and evening ic fecal samples (p = 0.1) (fig 1) . the fecal dry matter percent was significantly lower in cfc samples (p = 0.01) ( table 1) . table 1 . descriptive summary of daily and stool and urine production for calves (n = 12) in confinement housing from days 0 to 10 post-infection. stool production/day (kg) mean ± sd 4.8 ± 3.8 -urine production/day (kg) mean ± sd 1.8 ± 1.4 --iqr 0.5-1.9 fecal dry matter percentage/day mean ± sd 9.8 ± 9.5 ã 12. when comparing across calves in confinement housing (n = 12) and calves in box stalls (n = 9), calves in confinement housing shed significantly more oocysts in their stool (p = 0.05) (fig 2) . the mean peak in oocyst shedding for confinement housing calves was log 7.5 oocysts/ gram fecal dry matter, which is nearly 3 orders of magnitude greater than the mean peak in box stall calves. fecal dry matter percentage was significantly lower in cfc samples from confinement housing calves as compared to ic samples from box stall calves (p = 0.0003), however, there was no significant difference (p = 0.13) in fecal dry matter percentage for ic samples collected from confinement housing and box stall calves (tables 1 and 2) . for days 0-10 post-infection, calves in confinement housing experienced significantly more severe clinical outcomes in comparison to box stall calves across all study end-points except for average daily weight gain, even though calves in confinement housing consumed less milk replacer (p = 0.0007) ( table 2) . calves in confinement housing required significantly more fluid therapy to maintain their hydration status both during the 10 day period of confinement (p = 0.009) and after being removed from confinement and placed in box stalls (p = 0.01) ( table 3 ) (s2 fig). on average, for days 0-10 post-infection, confinement housing calves required 3.5 l/day of parenteral or oral fluid therapy while box stall calves required 0.25 l/day (table 3) . calves in confinement housing were also more likely to become injured. the frequency of abrasions or lacerations was significantly greater (p = 0.002) in confinement housing calves as was the frequency of pressure sores (p = 0.03) ( table 4 ). confinement impact of confinement housing in the calf model of cryptosporidiosis housing calves received significantly more non-fluid therapy supportive care (p = 0.01) in the form of administered pain medication, topical eye and skin treatments, wound care, and bandage changes. plasma cortisol samples from infected confinement housing calves (n = 10) and box stall calves (n = 5) were evaluated on days 2, 4, 5, and 9 post-infection. the distributions of plasma cortisol and log oocysts per gram of fecal dry matter were plotted and 2 outliers in the confinement housing group were identified and removed from analysis. the first measured plasma cortisol value for each of these calves was greater than 110 ng/ml. testing was limited to 10 calves in confinement and 5 calves in box stalls due to sample loss. mean plasma cortisol levels were significantly higher in confinement housing calves (53.6 ± 31 ng/ml) than in box stall calves (32.2 ± 21 ng/ml) (p = 0.002) (fig 3) . as the mean plasma cortisol level increased, the mean log oocysts per gram of fecal dry matter significantly increased (p < 0.0001) (fig 4) ( table 5 ). for every 25 point increase in plasma cortisol there was a 1.0 log increase in oocyst shedding (fig 4) . both housing groups initially had similar plasma cortisol levels, but the table 2 . descriptive summary of study end-points for calves in confinement housing (n = 12) and box stalls (n = 9) from days 0 to 10 post-infection. table 3 . descriptive summary of supportive care administered to calves in confinement housing (n = 12) and box stalls (n = 9). daily to determine the best predictors of total fecal output, backward stepwise regression was carried out. the following explanatory variables were entered into the model: plasma cortisol, percent of milk replacer consumed, log oocysts per gram of fecal dry matter, and fecal dry matter percentage. also included were the interactions for log oocysts per gram of fecal dry matter and plasma cortisol, and for log oocysts per gram of fecal dry matter and fecal dry matter percentage. the interaction terms were found to be non-significant and were removed from the model. the remaining variables were retained. log oocysts per gram of fecal dry matter was found to be non-significant (p = 0.3) and was removed. the best predictors of total fecal output were plasma cortisol (p = 0.02), percent of milk replacer consumed (p = 0.03), and fecal dry matter percentage (p = 0.01) ( table 6 ). recent large multicenter studies investigating etiologies of diarrhea in children have demonstrated that cryptosporidium spp. is one of the main pathogens contributing to the global burden of pediatric diarrhea, yet there are no vaccines to prevent infection and the only licensed drug for treatment has poor efficacy [7, 8] . efforts to accelerate identification of a lead drug candidate have generated increased demand on the calf model of cryptosporidiosis. this surge in demand has underscored the need to critically evaluate and refine the calf model of cryptosporidiosis in order to garner the best data possible for application in veterinary medicine and translation to human patients. alarmingly, as the model has been more frequently applied, inconsistencies in model execution have come to light. one of the most pronounced inconsistencies pertains to use of confinement housing for research calves. the desire to mimic human challenge studies and collect the total fecal output from research calves has necessitated the use of confinement housing [30, 31, 38, 39] . use of confinement housing conflicts with the five freedoms of animals, which are commonly used to define parameters for animal welfare and to determine if animal housing, environment, and handling are adequate [40] [41] [42] . in the case of confinement housing, the freedom to express natural behaviors is not observed. in research settings, the five freedoms may be compromised if there is scientific justification or if no other acceptable alternative exists. to date, no one has evaluated confinement housing side-by-side with box stall housing to determine if box stall housing is an acceptable alternative in the calf model of cryptosporidiosis. our study demonstrates that use of confinement housing is unnecessary to measure study end-points such as total oocyst excretion and diarrhea quantity and quality. moreover, the use of confinement housing appears to stress calves and lead to increased oocyst excretion that may confound data and underestimate the efficacy of the chemotherapeutics being studied. impact of confinement housing in the calf model of cryptosporidiosis our findings challenge the assumption that cfc is required to get accurate measurements of oocyst excretion or the quantity and quality of diarrhea. first, we did not find a difference in log fecal oocysts enumerated per gram of fecal dry matter in cfc or ic samples among calves in confinement. previously, it was thought that homogenization of the cfc sample would result in more accurate oocyst enumeration [25] , but this was not supported by our data. to the contrary, there was greater variation in oocyst enumeration for cfc samples, as is evidenced by the larger standard error bars (fig 1) . since these counts are standardized to the fecal dry matter percentage, this would indicate that these differences are attributable to increased water content of the stool sample. it has also been suggested that cfc must be collected and homogenized due to possible diurnal variations in fecal oocyst shedding [25, 26] , but we observed no significant difference in the number of oocysts enumerated in samples collected in the morning or evening. it is important to note that while cfc permits capture of the total fecal output, the entire cfc sample is not analyzed. for both cfc and ic a 200 mg aliquot of stool is homogenized with equal amounts of pbs. after qpcr is performed, oocyst counts are standardized by fecal dry matter percentage. therefore, our findings indicate that a representative stool sample can be attained via ic and collection of the total fecal output is not necessary in order to attain a representative stool sample for oocyst enumeration via qpcr. the fecal dry matter percentage was significantly lower in cfc samples as compared to ic collected from calves in confinement. this was controlled for in oocyst enumeration by standardizing to the fecal dry matter percentage. the increased water content in cfc samples could be from more severe diarrhea or improved ability to measure water content due to sample homogenization. alternately, it could be due to urine contamination of the fecal bin which has been previously described or to increased urine production secondary to increased frequency of fluid therapy [25] . although adult diapers were secured to the calves using duct tape (3m, st. paul, mn) and elastikon (johnson & johnson, new brunswick, nj) diapers did slide out of place with calf movement and exposed the penis. in addition, during the first 4-5 days of confinement, calves were small enough to turn around in the stall, thus there were instances when they defecated in the urine bin, which would falsely lower the fecal dry matter percentage of stool collected from the fecal bin and falsely elevate the volume of urine collected (s1 fig). if the lower fecal dry matter percentage detected in confinement housing calves is a true finding, indicating that cfc better detects increased water content in the stool, this would indicate that confinement exacerbates severity of diarrhea. this is a possibility, given that calves in confinement housing required significantly more supportive care than calves in box stalls. calves in confinement housing received an average of 3.5 l of fluid therapy each day, as compared to box stall calves, which only received 0.25 l/day. thus, confinement housing calves may experience greater volume of fluid elimination due to greater volume of fluid support. however, when comparing ic samples from confinement housing calves to ic samples from box stall calves, there is no significant difference in fecal dry matter percentage. thus, it is probable that the difference in fecal dry matter percentage between cfc and ic samples for confinement housing calves is at least partially attributable to urine contamination of the fecal bin. calves in confinement housing shed significantly more oocysts in their stool. at peak shedding, this difference was 2 orders of magnitude greater in confinement housing calves. since there was no difference in shedding detected in ic or cfc samples from confinement housing calves, it stands to reason that the difference in shedding detected between box stall and confinement housing calves is a true effect. moreover, since all qpcr data was standardized to the fecal dry matter percentage, this data is not susceptible to dilutional effects of increased water content in the stool sample. it is possible that this difference in fecal oocyst shedding can be explained by the significant difference in plasma cortisol concentration for confinement housing and box stall calves. in healthy calves, plasma cortisol concentration is reported to be high at birth (40-50 ng/ml) and gradually decreases over the first 20 days of life (15-20 ng/ml) [34, 43] , which is consistent with our findings for box stall calves. calves in confinement housing impact of confinement housing in the calf model of cryptosporidiosis did not follow this pattern. plasma cortisol was elevated at 4-5 days post-infection (62 ng/ml) in confinement housing calves, coinciding with the observed peak in fecal oocyst shedding. thus, it is feasible that increased stress due to confinement exacerbated the degree of fecal oocyst shedding in confinement housing calves. this conclusion is supported in the bivariate analysis in which plasma cortisol level and log oocysts per gram fecal dry matter were significantly associated. it could be argued that confinement housing effectively creates an immunosuppressed calf model of cryptosporidiosis. this may be deemed desirable by cryptosporidium researchers, as many children infected with cryptosporidium are immunocompromised due to other comorbidities. however, unlike immune suppressed mouse models, where diminished immune function is intentionally induced, the presumed reduction in immune function due to hypercortisolemia for calves in confinement housing is a pathological state. while chemotherapeutic efficacy in the face of immunocompromise is desirable, when testing chemotherapeutics in animal models, the degree of immune suppression induced must be predictable and repeatable. our findings also indicate that calves in confinement housing were substantially more physically and metabolically decompensated than calves in box stalls. confinement housing calves required more fluid therapy and ate significantly less milk replacer. reduced milk replacer consumption is an indication of reduced calf wellbeing. in the calf model of cryptosporidiosis this has additional relevance, as failure to meet daily fluid requirements through consumption of milk replacer puts calves at greater risk for severe dehydration and development of metabolic acidosis, which can result in calf death or removal from the study. confinement housing calves also received significantly more non-fluid treatments. not only does this indicate reduced calf welfare, it also indicates increased time demands on study personnel for provision of care to sick calves. box stall calves experienced no abrasions, lacerations, or pressure sores. these types of injuries only occurred in confinement housing calves, as they were more likely to injure themselves on the metal framework of the calf stall, and the lack of shock absorption when lying down predisposed calves to repeated injury over their joints and bony prominences. the severity and duration of these injuries may have been impacted by elevated plasma cortisol levels. mice placed in confinement to induce repeated restraint stress experienced impaired wound healing, partially through stress-induced glucocorticoid release [44] . given these findings, we wanted to determine if variables that could be collected without the use of confinement housing would accurately predict the total fecal output produced by a calf experimentally infected with c. parvum. in our regression model, holding other variables constant, plasma cortisol, the percent of milk replacer consumed, and the fecal dry matter percentage from ic samples were the best predictors of the total fecal output measured via cfc. these variables accounted for the greatest amount of inter-calf variation in total fecal output. this indicates that when used in combination, these study end-points are better measures of a treatment effect than total fecal output alone. the regression model also suggests that the total fecal output is sensitive to hypercortisolemia. interestingly, the log oocysts per gram of fecal dry matter was not a good predictor of total fecal output. a previous study has shown that the magnitude of oocyst shedding tracks with the ordinal measures of severity of diarrhea [36] . when considered alongside our data, this indicates that ordinal measures of severity of diarrhea and total fecal output are not synonymous, and that fecal dry matter percentage is a better proxy measure of total fecal output. the findings of this study suggest that confinement housing of calves may affect study endpoints that are important for the evaluation of novel chemotherapeutics targeting cryptosporidium. fecal oocyst shedding, appetite, and frequency of fluid therapy are study end-points that were all significantly impacted by the use of confinement housing. our results indicate that representative data can be captured via ic from calves housed in box stalls, and that this data is less likely to be impacted by the immunomodulatory effects of stress hypercortisolemia. while collection of total fecal output mirrors methodologies used in human challenge studies, use of confinement to capture total fecal output confounds data used to assess other study end-points and reduces overall comparability to the human challenge model. given that confinement housing calves experience increased severity of illness, higher plasma cortisol levels, greater risk of injury, and higher requirements for fluid therapy, use of confinement housing to evaluate novel chemotherapeutics targeting cryptosporidium spp. may result in reduced ability to detect a treatment effect. that said, our study is limited by the fact that we did not compare outcomes in the two housing systems during a trial of an actual cryptosporidium therapeutic. however, box stall housing has recently been used successfully in a trial evaluating a novel drug targeting cryptosporidium [45] . our study was also subject to potential bias due to the inability to blind observers to housing system. for this reason, we did not report and evaluate subjective observer assessments in this study, such as mentation. these assessments were restricted to use in determining need for supportive care. to reduce risk of bias in evaluation of injuries, severity was not assessed. instead assessments of injuries, such as abrasion or pressure sore, were dichotomous: present or not present. blinding was maintained for laboratory analysis of stool and blood samples. while we were able to report an association between plasma cortisol and oocyst shedding, this study was not designed to assess stress as a primary end-point. cortisol is just one marker of stress. a more complete assessment of stress, including behavioral assessment, and other markers such as substance p, would aid in the interpretation of the data. another concern is the loss of calves in the confinement group due to death, which could result in imprecise estimates. however, we feel that imprecise estimates are the minor concern relative to the major concern of compromised research animal welfare. when considered together, the risk of imprecise measurements due to loss as well as reduced welfare resulting in death and injury, further substantiates the need to revise the methods used for studying cryptosporidiosis in the calf model. lastly, while the study authors have extensive experience using box stalls, this was our first study using confinement housing. it could be argued that in more experienced hands the study findings may have been different. to address this, we consulted with researchers currently executing these studies in confinement and followed protocols identical to those previously published [25] . in conclusion, it is our opinion that box stall housing should be preferentially used in the calf model of cryptosporidiosis, as confinement housing compromises calf welfare, alters important outcomes, appears to stress calves, and leads to sicker and more heavily infected calves. supporting information s1 table. rubric for the evaluation of appetite, mentation, fecal consistency, and hydration. cryptosporidium: a water-borne zoonotic parasite association between management practices and within-herd prevalence of cryptosporidium parvum shedding on dairy farms in southern ontario prevalence of cryptosporidium parvum infection in southwestern ontario and its association with diarrhea in neonatal dairy calves cryptosporidium spp. and cryptosporidiosis. microbiol rev impact of confinement housing in the calf model of cryptosporidiosis plos neglected tropical diseases quantitative risk assessment of cryptosporidium species infection in dairy calves burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, gems): a prospective, case-control study pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (mal-ed). lancet glob health asymptomatic and symptomatic cryptosporidiosis: their acute effect on weight gain in peruvian children parasite prevalence and the worldwide distribution of cognitive ability effects of cryptosporidium parvum infection in peruvian children: growth faltering and subsequent catch-up growth prophylactic use of decoquinate for infections with cryptosporidium parvum in experimentally challenged neonatal calves paromomycin is effective as prophylaxis for cryptosporidiosis in dairy calves effect of nitazoxanide on cryptosporidiosis in experimentally infected neonatal dairy calves prophylactic and therapeutic efficacy of nitazoxanide against cryptosporidium parvum in experimentally challenged neonatal calves effect of nitazoxanide on morbidity and mortality in zambian children with cryptosporidiosis: a randomised controlled trial efficacy of halofuginone lactate in the prevention of cryptosporidiosis in suckling calves effect of halofuginone lactate on the occurrence of cryptosporidium parvum and growth of neonatal dairy calves european public assessment report: halocur (halofuginone) continuous culture of cryptosporidium parvum using hollow fiber technology novel bioengineered three-dimensional human intestinal model for long-term infection of cryptosporidium parvum. infect immun infectivity of cryptosporidium muris (strain rn 66) in various laboratory animals immune responses to cryptosporidium muris and cryptosporidium parvum in adult immunocompetent or immunocompromised (nude and scid) mice protein malnutrition impairs intestinal epithelial cell turnover, a potential mechanism of increased cryptosporidiosis in a murine model antibody fusions reduce onset of experimental cryptosporidium parvum infection in calves novel bumped kinase inhibitors are safe and effective therapeutics in the calf clinical model for cryptosporidiosis cryptosporidium parvum: determination of id50 and the dose-response relationship in experimentally challenged dairy calves description of fecal shedding of cryptosporidium parvum oocysts in experimentally challenged dairy calves effect of nutritional plane on health and performance in dairy calves after experimental infection with cryptosporidium parvum the infectivity of cryptosporidium parvum in healthy volunteers infectivity of cryptosporidium parvum in healthy adults with pre-existing anti-c. parvum serum immunoglobulin g federation of animal science societies. guide for the care and use of agricultural animals in research and teaching comparison of four methods of calf confinement. i. physiology neonatal changes in the plasma levels of cortisol, cortisone and aldosterone in the calf use of bacteriophage ms2 as an internal control in viral reverse transcription-pcr assays correlation between diarrhea severity and oocyst count via quantitative pcr or fluorescence microscopy in experimental cryptosporidiosis in calves a comparison of fecal percent dry matter and number of cryptosporidium parvum oocysts shed to observational fecal consistency scoring in dairy calves cryptosporidium parvum: intensity of infection and oocyst excretion patterns in healthy volunteers cryptosporidium hominis: experimental challenge of healthy adults report of the technical committee to enquire into the welfare of animals kept under intensive livestock husbandry systems moving beyond the "five freedoms" by updating the "five provisions" and introducing aligned updating animal welfare thinking: moving beyond the "five freedoms" towards "a life worth living hemato-biochemical and cortisol profile of holstein growing-calves supplemented with vitamin c during summer season restraint stress impairs early wound healing in mice via alpha-adrenergic but not beta-adrenergic receptors a cryptosporidium pi(4)k inhibitor is a drug candidate for cryptosporidiosis the authors would like to thank the undergraduate and veterinary students who tirelessly cared for the calves and collected data points, amber adams-progar for essential inputs on housing and calf behavior, renee anderson for qpcr technical support, mike kluzik for operational and logistical assistance, the office of the campus veterinarian, in particular, gay lynn clyde, nina woodford, and gwen anderson for providing care to the calves, the animal resource unit, and specifically jan luft and josh steele, who insured excellent husbandry and facility management, and wesley van voorhis and douglas call for their thoughtful review and editing of the manuscript. we also wish to express our gratitude to jaime andrade and 5d dairy farm for their unwavering support of this research, and to lazaro and the maternity pen team for their patience, flexibility, and good humor. lastly, we want to thank the calves and to acknowledge their invaluable contribution to the advancement of science and medicine. conceptualization: geneva graef, tracy l. sy, jennifer a. zambriski. key: cord-322943-lvdl7puw authors: lardon, zélie; watier, laurence; brunet, audrey; bernède, claire; goudal, maryvonne; dacheux, laurent; rotivel, yolande; guillemot, didier; bourhy, hervé title: imported episodic rabies increases patient demand for and physician delivery of antirabies prophylaxis date: 2010-06-22 journal: plos negl trop dis doi: 10.1371/journal.pntd.0000723 sha: doc_id: 322943 cord_uid: lvdl7puw background: imported cases threaten rabies reemergence in rabies-free areas. during 2000–2005, five dog and one human rabies cases were imported into france, a rabies-free country since 2001. the summer 2004 event led to unprecedented media warnings by the french public health director. we investigated medical practice evolution following the official elimination of rabies in 2001; impact of subsequent episodic rabies importations and national newspaper coverage on demand for and delivery of antirabies prophylaxis; regular transmission of epidemiological developments within the french antirabies medical center (armc) network; and armc discussions on indications of rabies post-exposure prophylaxis (rpep). methodology/principal findings: annual data collected by the national reference center for rabies nrcr (1989–2006) and the exhaustive database (2000–2005) of 56 armc were analyzed. weekly numbers of patients consulting at armc and their rpepand antirabies-immunoglobulin (arig) prescription rates were determined. autoregressive integrated moving-average modeling and regression with autocorrelated errors were applied to examine how 2000–2005 episodic rabies events and their related national newspaper coverage affected demand for and delivery of rpep. a slight, continuous decline of rabies-dedicated public health facility attendance was observed from 2000 to 2004. then, during the summer 2004 event, patient consultations and rpep and arig prescriptions increased by 84%, 19.7% and 43.4%, respectively. moreover, elevated medical resource use persisted in 2005, despite communication efforts, without any secondary human or animal case. conclusions: our findings demonstrated appropriate responsiveness to reemerging rabies cases and effective newspaper reporting, as no secondary case occurred. however, the ensuing demand on medical resources had immediate and long-lasting effects on rabies-related public health resources and expenses. henceforth, when facing such an event, decision-makers must anticipate the broad impact of their media communications to counter the emerging risk on maintaining an optimal public health organization and implement a post-crisis communication strategy. media-communicated health alerts are being used more-and-more frequently by public health decision-makers to prevent consequences of a sudden event, such as, emerging and episodic zoonotic diseases. the medical community must now consider these communications to be preventive intervention tools for public health officials [1] [2] [3] . obviously, as during any effective health intervention, undesired effects may also occur, such as rapidly rising numbers of potential cases to treat, leading, in turn, to health-resource saturation, especially if the pathogen involved is rare [4, 5] . rabies is a viral encephalitis [6] that is considered to be a reemerging zoonosis throughout much of the world [7] . in western europe, rabies in non-flying terrestrial mammals was a well-known illness that has now become a rare disease, because many countries have succeeded in eradicating it. the major risk of rabies is now due to translocation of infected animals, mainly dogs, from rabies-enzootic areas and humans with rabies infection acquired abroad [8] . although untreated rabies is invariably fatal, death can be avoided by proper administration of rabies postexposure prophylaxis (rpep), e.g., antirabies vaccine, with or without antirabies immunoglobulins (arig), before disease onset [6] . thus, rapid identification of individuals potentially exposed to rabies is critical and media alerts can be extremely useful to identify people who were in contact with the rabid animal. in france (60,000,000 inhabitants, 675,417 km 2 ), primary health-care management of patients seeking rpep is delivered through an official national network of antirabies medical centers (armc), which are distributed throughout the country. rpep is administered, predominantly according to the zagreb schedule, to people bitten by an animal suspected of being infected with rabies or exposed to its saliva. clinicians conduct a risk assessment for each exposed patient, and decide to administer rpep according to the general recommendations, epidemiological data and grade of the bite [9] . the french network for rabies prophylaxis provides exhaustive national data collected by armc [10] , and laboratory diagnoses of humans suspected of having rabies [11] and animals suspected contaminating humans. from 1968 to 1998, a period during which rabies was endemic in french foxes, more than 45,600 animals were diagnosed as rabid. in 2001, france was declared free of rabies in non-flying terrestrial mammals based on world organisation for animal health (oie) criteria and, as a consequence, the number of rpep began to decline progressively. however, in summer 2004, one imported rabid dog generated unprecedented media communications by the public health director, whose official press release, dated 31 august 2004, warned, ''at least, nine people are at risk of death and are actively and intensively being sought by the health authorities…'' during this episode, antirabies vaccine stocks in armc were almost exhausted, leading to a temporary marketing license for the multidose verorab vaccine (sanofi pasteur), which had not previously been authorized in france. that arig supplies were dangerously low is illustrated by the postponement of arig injections in some armc until day 7 after starting rpep [12, 13] for several patients. controlling rabies reintroduction and communicating the risk of rabies spread remain a challenge to public health officials in rabies-free areas. in this study, we analyzed why and how the french rabies-control organization became so oversaturated. in particular, we examined the impact of newspaper reports on the numbers of patients consulting at armc, and their rpep and arig prescriptions. this research has complied with the french national guidelines and institut pasteur policy. the analysis of data collected by the national reference center for rabies (nrcr) from the amrc was done anonymously and approved by the commission french veterinary and human authorities work in close collaboration to detect cases and organize the medical responses to rabies (figure 1) , with a territorial network of 96 veterinary services and 74 armc disseminated throughout continental france, in 2004 ( figure 2 ). on the one hand, each animal responsible for human exposure is confined under veterinary surveillance. if dead and for whatever the reason, diagnostic laboratory tests are conducted at the nrcr, institut pasteur, paris, france. on the other hand, armc are the only primary care centers allowed to prescribe rpep. for each patient, a standard case-report form (table s1 ) is systematically filled out describing important epidemiological features, such as geographic location, consultation date, type of exposure, animal species, contact date with the animal, medical decision concerning rpep. based on the data collected by armc, annual reports are written, which describe the patients visiting armc and those receiving rpep (http://www.pasteur.fr/sante/clre/cadrecnr/rage/rageactualites.html). our analysis of the behavior patterns of patients consulting armc, and the rpep and arig prescribed to them between 1989 and 2006 was based on those annual data. among the 74 french armc, 56 systematically entered their data into the nrcr database between 2000 and 2005. the following statistical analysis is based on the exhaustive weekly information provided by these 56 armc. the armc network also constitutes an effective communication infrastructure coordinated by the nrcr, including conference calls and regular exchanges of information via the internet. when rabies is suspected in a human, biological specimens are sent to the nrcr. articles on rabies-related news published in three major national daily newspapers, le monde, le figaro and libération, were retrieved from the french association for auditing media circulation: an on-line service: http://www.factiva.fr. weekly numbers of patients consulting at armc, as a function of the date each was in contact with a potentially rabid animal, were used to construct times series. autoregressive moving average (arma) [14] modeling was used to determine the significance of event-associated modification of armc weekly patient numbers and its duration. because several known events could have affected the series, a step-by-step procedure was undertaken [15, 16] . before the onset of event #2, trend and/or seasonality were estimated and removed, so that the time series was obtained in a stationary mode and, autoregressive integrated moving-average (arima) modeling was done using box-jenkins procedure from sas/ets [17] ). the model was then used to predict armc consultations and their 95% confidence intervals (95% ci). an event was considered to have an impact when the number of consultations during 2 consecutive weeks exceeded the upper 95% ci. observed values were then replaced by forecasts, to obtain analyses of the subsequent weeks. similarly, 2 consecutive weeks within the 95% ci defined the end of the event's impact period. relative differences between observed and predicted values were calculated. for impacting events, the number of cases attributed to the event (ncae) was estimated by subtracting the prediction from the observed data during the impact period. an increase rate rabies has been eliminated from a large part of the european union and, thus, any newly imported cases threaten its reemergence. the 2000-2005 data derived from the exhaustive surveillance system implemented in france was analyzed to evaluate the impact on demand for and delivery of antirabies prophylaxis following introduction of five rabies-infected dogs and one infected human into this rabies-free area. using these events, we were able to illustrate the difficulties encountered in reducing the demand for and prescription of postexposure rabies prophylaxis in this context of episodic importation. moreover, we highlighted the need for public health decision-makers to anticipate the broad spectrum of consequences of their media communications and to prepare appropriate responses (in terms of health resources) to maintain an optimally effective public health organization after importation of an exotic infectious agent or its emergence. these responses are particularly relevant in the context of limited availability of rabies post-exposure prophylaxis, especially antirabies immunoglobulin. (ir) was then calculated as the ratio of the ncae/number predicted for the impact period. with the aim of evaluating potential repercussions of an identified event impacting on rpep prescriptions, two other time series were investigated: the weekly rpep rate, defined as the number of rpep prescribed/the number of consulting armc patients, e.g. rabies vaccine with or without arig; and the weekly arig rate, corresponding to the ratio of the number of arig/the number of consulting armc patients. during the period associated with modified armc weekly numbers, weekly rpep and arig rates and mean numbers of consultations were analyzed using regression with autocorrelated errors to account for the regression residuals (arima procedure). to explore whether care provided by the armc might be influenced by experience in previous french endemic enzootic areas, we divided the country into three areas based on the french administrative regions: area 1, the former enzootic rabiesinfected-fox region from 1968 to 1998; area 2, a region that has always remained rabies-free, and area 3, the region where event #6 occurred ( figure 2 ). all analyses were performed using r (www.r-project.org) and sas software. after the reintroduction of rabies into france in 1968, the number of rabid animal cases increased to reach a maximum of 4,212 cases in 1989 [18] , followed rapidly by a maximum of 9,763 rpep prescribed for 15,948 patients consulting at armc recorded in 1990 ( figure 3 ). in 2001, france was declared free rabies reemergence and antirabies prophylaxis www.plosntds.org of rabies in non-flying terrestrial mammals based on oie criteria [19] and, as a consequence, the number of patients consulting armc and receiving rpep began to decline progressively to respective minima of 7,788 and 3,378 in 2003 ( figure 3 ). however, the numbers of patients consulting at armc and given rpep suddenly rose in 2004. therefore, 2000-2005 data were further investigated using arima modeling to describe in greater detail the trends observed. between 1 january 2000 (week 1) and 31 december (week 312) 2005, five rabid dogs illegally imported from morocco and one rabies-infected human from gabon were detected in france. during the period examined, the first event #1 dog (5 months old) was confirmed as being rabid in may 2001 (week 74) and the second, event #2 dog (3 months old) in september 2002 (week 139); they entered france from morocco, 2 months and 2 weeks before their deaths, respectively. the human case (event #3) was a 5-year-old boy, who traveled from gabon and died 2 months later, in october 2003 (week 199) [20] . event #4, #5 and #6 dogs were diagnosed as being rabid, respectively, in february 2004 (week 213), may 2004 (week 229), and august 2004 (week 243) [21] . event #6 was a 4-month-old puppy, illegally imported by car from morocco to bordeaux, france, via spain, who died of rabies in august 2004 (week 243); he was not officially vaccinated. between 1 january 2000 and 31 december 2005, 56,924 rabiesexposed individuals in france (all patients exposed abroad were excluded from the analysis) consulted in an armc, among whom 56,446 had valid exposure dates and bite/contact locations. among them, 50,930 had valid consultation dates and 56,406 had valid treatment information (figure 4 ). because the data presented 52-week seasonality, the time preceding event #1 was too short to be analyzed. in such a case, box and jenkins recommend using at least two seasonality periods to calibrate the model [14] . data analyses concerning events #1, #2, #4 and #5, corresponding to rabid dog importations, were simple and rapidly done, as these dogs had had no known contact with animals and humans other than their owners during their communicable risk periods. as a consequence, events #2, #4 and #5 were not reported in the major national newspapers and were not associated with any significant increase of armc activity. in contrast, events #3 and #6 were reported in 6 and 54 published articles retained for this study, respectively, and significantly affected the numbers of patients consulting at an armc ( figure 5 ). until event #3 (october 2003), the weekly number of patients consulting an armc declined significantly (slope = 20.34; p,0.0001), with 52-week seasonality that peaked during the summer ( figure 5 ). in october 2003, the weekly number of armc patients was significantly higher than the predicted number during the 6 weeks surrounding event #3 (weeks 198-203), with an estimated ncae of 355 (ir = 54.7%, 95% ci = 30.0-83.0). furthermore, event #3 was followed by a significant flattening of the decreasing slope of armc activity (20.23 versus 20.34; p = 0.0003). no rpep-or arig-rate modification associated with event #3 was observed. in the summer of 2004 (event #6), the weekly number of armc patients differed significantly from the predicted number during the 26 weeks surrounding it (weeks 238-263). the total 26week number of additional armc patient load was estimated at 2,928 (ir = 84.0%, 95% ci = 57.0-123.3) over the model predicted 3,486 ( figure 5 ). during that period, the observed mean rpep and arig rates were significantly higher than those recorded during the period preceding event #6, ir = 19.7% and 43.4%, respectively ( table 1) . the slopes of the armc-consultation decline after week 263 and before week 238 were estimated at 20.12 and 20.23, respectively; p,0.001. surprisingly, between weeks 264 and 312, the mean rpep rate remained persistently and significantly higher rabies reemergence and antirabies prophylaxis www.plosntds.org than before the reference period, as did the arig rate, which was more than two-fold higher than before week 237 ( table 1 ). the increased number of patients consulting at an armc in response to the newspaper articles concerning event #6 peaked at the same time as the media coverage in the three different french areas defined according to their rabies experience ( figure 6a ). in area 3, the exposure dates reported by armc patients corresponded to the risk period coinciding with the dog's movements and infectivity, whereas in areas 1 and 2, patients reported exposure dates more compatible with newspaper coverage than with the risk period ( figure 6b ). france progressively eliminated rabies in foxes and became rabies-free for indigenous non-flying terrestrial mammals in 2001 [19] . consequently, use of public health facilities dedicated to the disease decreased steadily from 1990 until 2003, suggesting a continuous impact of rabies elimination on related public health resources and expenses. however, the very mild decline of the 2000-2003 slope probably reflects the difficulties in convincing the public and adapting medical practice to the changing risk. although elimination of rabies in foxes reduced the number of rabid pets and other domestic animals, and thus exposure to rabies, pet bites continue. importation of rabid animals and infected travelers returning from abroad also regularly challenge the french public health organization of rabies control. therefore, the number of rpep prescriptions and the associated costs will not decline significantly until there is adequate assurance that the probability of a pet being rabid is sufficiently low that such therapy is not warranted, even when the pet's status cannot be verified [22, 23, 24] . regardless of potential french specificities, public health decision-makers are obliged to consider such potential events and their ensuing demand on medical community resources when attempting to predict and maintain the efficacy of rabiescontrol policies even in rabies-free countries [24] [25] [26] [27] [28] . among the six rabies events occurring during 2000-2005 in france, only two significantly affected armc activities and rpep rates. the human case imported from gabon in 2003 (event #3) was associated with enhanced armc activity during a brief period and also changed armc's declining activity, which had been observed since 2000. the boy's demise was reported 6 times in the newspapers, further confirming that ''death makes news'' for rare and acute diseases [29] . in contrast, the illegally imported . rabies-exposure notifications to armc and numbers of rpep prescribed to exposed patients in france, 1989 france, -2006 . these data are from the annual nrcr report (http://www.pasteur.fr/sante/clre/cadrecnr/rage/rage-actualites.html). doi:10.1371/journal.pntd.0000723.g003 rabies reemergence and antirabies prophylaxis www.plosntds.org rabid dog from morocco in august 2004 (event #6) had a significant and rapid impact on rabies public health resources. indeed, the critical shortage of prophylactic drugs resulted from the 84% ir of patients consulting at an armc with a 62.5% rpep rate for those patients over 26 weeks. this influx explains the bottleneck observed in armc. similarly, laboratory rabiesdiagnosis workload for animals increased by .40% during the same period (data not shown). to comply with the threatened shortage of rpep and arig due to the cumulative effect of enhanced patient influx and their more frequent prescriptions, a specific communication strategy was established for the armc network to provide information concerning the evolution of the epidemiological situation and to recall the indications of rpep. this information was disseminated via the websites of the nrcr, the ministry of health (moh), the national institute for health surveillance and the ministry of agriculture, which were regularly updated as of 28 august, fax on 2 september, and phone conferences on 3 and 9 september. to complete this plan, temporary licensing of a multidose vaccine (verorab, sanofi pasteur) was accorded and arig injections were postponed, as necessary, in accordance with who guidelines [12] . unfortunately, it was not feasible to quantitatively analyze the extent of that adaptation. however, rpep and arig never became completely unavailable. notably, the risk of a potential arig shortage in the event of an unplanned increase of demand or a limitation of supply is shared by many countries in europe and on other continents [30, 31] . compared to similar events occurring during 2000-2005 in france, event #6 has several particularities. while only restricted contacts with humans (owners, neighbors…) were suspected for cases #2, #4 and #5, the event #6 dog traveled through southwestern france during the communicable risk period, and had been roaming unleashed at three large summer music festivals, each with at least 10,000-20,000 participants [21] . according to immediate inquiries made by veterinary and medical services, this trajectory potentially led to extensive contacts between the rabid dog and humans and animals. therefore, the public health authorities' concern triggered extensive media alerts. first, the moh wanted to identify and contact each individual with confirmed contact with the event #6 dog. national and local authorities coordinated several news conferences and newspaper reports to inform the french rabies reemergence and antirabies prophylaxis www.plosntds.org population about the risk and recommendations concerning errant dogs in general, and how to react to potential exposure to a rabid dog. a european-wide alert was launched through the european warning and response system. second, beginning in early september 2004, this intensive communication frenzy of 54 newspaper articles heightened public awareness of the rabies risk. third, additional public concern might also have been heightened by controversies surrounding the crisis management. notably, event #6 occurred just before the annual opening of hunting season, in a strongly traditional hunting region. an initial decision was made to forbid hunting with dogs in the counties where the rabid dog had traveled during his infectious period. that restriction led to a passionate public debate, angering hunters and ending with hunting organizations successfully blocking the ban. fourth, public health authorities decided to eradicate freeroaming dogs. finally, press releases issued by the minister of rural affairs and the moh were contradictory concerning the implementation of mandatory antirabies vaccination of dogs and cats. the constant media attention drawn by these different players during event #6 may have contributed to enhancing the sense of rabies risk, thereby prompting people to associate dog bites with we only examined national newspaper stories available in factiva but not local newspaper reporting or television, radio and internet stories, and, thus, probably underestimated the global coverage of these episodes. in response to national newspaper coverage, people who are far from the event location can become concerned and start taking precautions as if they were in the affected area [3, 4, 32] . this phenomenon is particularly well illustrated by event #6, for which exposure dates reported by patients consulting at amrc in areas 1 and 2 corresponded to the period of newspaper coverage rather than to the risk-oftransmission period during the dog's movements. lastly, long-term modifications of armc activity and rpepand arig-prescription rates were observed. in particular, 2005 rpep and arig rates (arima study herein) and even those for 2006 had not yet returned to 2003 levels. this finding strongly suggests a persistent and unjustified heightened perception of the risk by individuals and physicians, even those specialized in rabies treatment, and this despite regular information provided by the nrcr to the armc network and a rapidly controlled situation with no recorded secondary animal and human cases during the following 2 years. in conclusion, event #6 and its associated national newspaper coverage profoundly perturbed health services, with excessive consulting at armc and durably increased antirabies drug rates for several months, along with more animal diagnostic testing. this crisis highlighted a lack of experienced manpower and insufficient vaccine stocks. outbreaks of emerging and/or deadly infections, like severe acute respiratory syndrome [34] [35] [36] [37] [38] , anthrax [39, 40] and rabies (herein), have shown that media messages dramatically influence both the public's and health-care workers' perceptions of the risk with potential implications for health-care resources. our observations underscore to what extent, under such circumstances, public health decision-makers have to anticipate the depth and scope of potential consequences of emerging or reemerging infectious diseases and their related press communications, and the need to prepare appropriate responses to keep the public health organization effective. it also illustrated that, despite communication efforts implemented by the french public health authorities and messages released through the armc network, long-term modifications of armc activities and prescriptions were observed, further emphasizing that a post-crisis communication strategy is essential. table s1 case-report form for human exposure to rabies used in france. since 2006, collection and dissemination of information are made by filling out questionnaires available at a centralized online site named voozanoo (http://www2.voozanoo.net/tikiindex.php?page = what%27s+voozanoo). found at: doi:10.1371/journal.pntd.0000723.s001 (0.07 mb doc) best practices in public health risk and crisis communication communicating the threat of emerging infections to the public six propositions on public participation and their relevance for risk communication the public's response to severe acute respiratory syndrome in toronto and the united states communicating the risks of a new, emerging pathogen: the case of cryptococcus gattii rabies and other lyssavirus diseases estimating the public health impact of rabies rabies in europe in 2005 expert consultation on rabies epidemiology and prophylaxis of rabies in humans in france: evaluation and perspectives of a twenty-five year surveillance programme a reliable diagnosis of human rabies based on analysis of skin biopsy specimens rabies vaccines what is an acceptable delay in rabies immune globulin administration when vaccine alone had been given previously time series analysis : forecasting and control revue méthodologique de quelques techniques spécifiques à l'analyse des séries temporelles en épidémiologie et santé publique a time series construction of an alert threshold with application to s. bobimorbificans in france the theory and pratice of econometrics que penser de la rage en 1990? bulletin epidémiologique de la fox rabies in france la rage : une maladie encore présente en france! an imported case of canine rabies in aquitaine: investigation and management of the contacts at risk rabies postexposure prophylaxis in returned injured travelers from france, australia, and new zealand: a retrospective study rabies postexposure prophylaxis potential cost savings with terrestrial rabies control economics of human and canine rabies elimination: guidelines for programme orientation cost effectiveness of rabies post exposure prophylaxis in the united states rabies control in the republic of the philippines: benefits and costs of elimination rabies exposures, post-exposure prophylaxis and deaths in a region of endemic canine rabies death makes news: the social impact of disease on newspaper coverage is there a need for anti-rabies vaccine and immunoglobulins rationing in europe appropriateness of rabies postexposure prophylaxis treatment for animal exposures the power of the pen: medical journalism and public awareness what are the roles and responsibilities of the media in disseminating health information media effects on students during sars outbreak the impact of the sars epidemic on the utilization of medical services: sars and the fear of sars sars epidemic in the press responding to global infectious disease outbreaks: lessons from sars on the role of risk perception, communication and management representations of sars in the british newspapers anthrax-related panic is more dangerous than the disease anthrax 2001: observations on the medical and public health response the authors thank all the armc personnel, who collected and send their data to the nrcr, for their contribution. we are grateful to janet jacobson for expert editing of the manuscript. conceived and designed the experiments: dg hb. performed the experiments: zl dg hb. analyzed the data: zl lw ab cb dg hb. contributed reagents/materials/analysis tools: lw mg ld yr dg hb. wrote the paper: zl lw ld dg hb. key: cord-002248-92pzqj35 authors: terasaki, kaori; ramirez, sydney i.; makino, shinji title: mechanistic insight into the host transcription inhibition function of rift valley fever virus nss and its importance in virulence date: 2016-10-06 journal: plos negl trop dis doi: 10.1371/journal.pntd.0005047 sha: doc_id: 2248 cord_uid: 92pzqj35 rift valley fever virus (rvfv), a member of the genus phlebovirus within the family bunyaviridae, causes periodic outbreaks in livestocks and humans in countries of the african continent and middle east. rvfv nss protein, a nonstructural protein, is a major virulence factor that exhibits several important biological properties. these include suppression of general transcription, inhibition of ifn-β promoter induction and degradation of double-stranded rna-dependent protein kinase r. although each of these biological functions of nss are considered important for countering the antiviral response in the host, the individual contributions of these functions towards rvfv virulence remains unclear. to examine this, we generated two rvfv mp-12 strain-derived mutant viruses. each carried mutations in nss that specifically targeted its general transcription inhibition function without affecting its ability to degrade pkr and inhibit ifn-β promoter induction, through its interaction with sin3-associated protein 30, a part of the repressor complex at the ifn-β promoter. using these mutant viruses, we have dissected the transcription inhibition function of nss and examined its importance in rvfv virulence. both nss mutant viruses exhibited a differentially impaired ability to inhibit host transcription when compared with mp-12. it has been reported that nss suppresses general transcription by interfering with the formation of the transcription factor iih complex, through the degradation of the p62 subunit and sequestration of the p44 subunit. our study results lead us to suggest that the ability of nss to induce p62 degradation is the major contributor to its general transcription inhibition property, whereas its interaction with p44 may not play a significant role in this function. importantly, rvfv mp-12-nss mutant viruses with an impaired general transcription inhibition function showed a reduced cytotoxicity in cell culture and attenuated virulence in young mice, compared with its parental virus mp-12, highlighting the contribution of nss-mediated general transcription inhibition towards rvfv virulence. rift valley fever virus (rvfv), a member of the genus phlebovirus within the family bunyaviridae, causes periodic outbreaks in livestocks and humans in countries of the african continent and middle east. rvfv nss protein, a nonstructural protein, is a major virulence factor that exhibits several important biological properties. these include suppression of general transcription, inhibition of ifn-β promoter induction and degradation of doublestranded rna-dependent protein kinase r. although each of these biological functions of nss are considered important for countering the antiviral response in the host, the individual contributions of these functions towards rvfv virulence remains unclear. to examine this, we generated two rvfv mp-12 strain-derived mutant viruses. each carried mutations in nss that specifically targeted its general transcription inhibition function without affecting its ability to degrade pkr and inhibit ifn-β promoter induction, through its interaction with sin3-associated protein 30, a part of the repressor complex at the ifn-β promoter. using these mutant viruses, we have dissected the transcription inhibition function of nss and examined its importance in rvfv virulence. both nss mutant viruses exhibited a differentially impaired ability to inhibit host transcription when compared with mp-12. it has been reported that nss suppresses general transcription by interfering with the formation of the transcription factor iih complex, through the degradation of the p62 subunit and sequestration of the p44 subunit. our study results lead us to suggest that the ability of nss to induce p62 degradation is the major contributor to its general transcription inhibition property, whereas its interaction with p44 may not play a significant role in this function. importantly, rvfv mp-12-nss mutant viruses with an impaired general transcription inhibition function showed a reduced cytotoxicity in cell culture and attenuated virulence in young mice, rift valley fever virus (rvfv) is the pathogen causing rift valley fever, which affects both humans and domestic ruminants, primarily in countries of the african continent and middle east. the virus is an arbovirus and circulates between mosquito vectors and ruminants in endemic areas. rvfv causes high mortality rates in young ruminants and a high rate of abortions in pregnant ruminants [1] . humans are infected with the virus either by mosquito bite or by direct contact with materials of infected animals. the majority of patients show influenzalike symptoms but few develop hemorrhagic fever, neurological symptoms, and ocular disease [2] . due to its major impact on public health, rvfv is classified as a category a priority pathogen by the national institute of allergy and infectious diseases. currently there is no approved vaccine available for humans and animals in non-endemic areas. rvfv belongs to the family bunyaviridae, genus phlebovirus. rvfv is an enveloped virus and carries 3 segmented rna genomes, the l, m and s segments, which are of negative or ambisense polarity. the l segment encodes l protein, a viral rna-dependent rna polymerase. m rna encodes 78kda protein, nsm protein, gn protein and gc protein, the latter two of which are major envelope glycoproteins and generated by co-translational cleavage of precursor gn/gc polyprotein. 78kda protein is dispensable for virus replication [3] , whereas it plays important roles in virus dissemination in mosquitoes [4, 5] . nsm is a viral anti-apoptotic protein [6, 7] and also is important for efficient virus replication in macrophage cell lines [5] . s rna expresses a nucleocapsid (n) protein and a nonstructural protein nss by using an ambisense coding strategy. the n protein encapsidates the viral rna and forms a ribonucleocapsid complex with l protein [8] . rvfv nss protein is a phosphoprotein with an apparent molecular weight of 31 kda and is localized in both the cytoplasm and nucleus [9] . in the nucleus, nss forms filament-like structures by self-dimerization through its c-terminal domain [10] . rvfv nss protein is a major virus virulence factor and has various important biological functions, which are important for countering the host antiviral response. one of the nss functions is suppression of ifn-β mrna transcription. nss binds to sin3-associated protein 30 (sap30), a subunit of a corepressor complex, and maintains ifn-β promoter in a transcriptionally silent state, leading to suppression of ifn-β mrna transcription [11] . in addition to the specific inhibition of ifn-β transcription, nss suppresses general transcription; it has been proposed that nss exerts suppression of general transcription by interacting with subunits of transcription factor ii h (tfiih) complex, p44 and p62 [12, 13] . a recent study showed that the nss-mediated general transcription inhibition contributes to the inhibition of ifn-β mrna transcription [14] . although nss-mediated transcription suppression has been considered to be important in suppressing the host antiviral response, the exact effects of the nss-mediated general transcription inhibition on virus virulence have not been defined. nss promotes the degradation of doublestranded rna-dependent protein kinase r (pkr), an antiviral ifn-stimulated gene product, through a proteasome pathway to prevent phosphorylation of eif2-α triggered by rvfv infection [15] [16] [17] [18] . furthermore, nss contributes to cellular stress responses such as an increase in reactive oxygen species, activation of dna damage signaling, cell cycle arrest, and activation of the p53 signaling pathway [19] [20] [21] [22] . although how nss induces the cellular stresses remains largely unknown, these stress responses may contribute to rvfv-induced cell death. to elucidate the mechanisms of the different functions of nss, it would be of great value to characterize a series of nss mutants, each of which specifically lacks one of these nss functions but retains other functions. however, introducing any short in-frame deletion in the rvfv nss resulted in loss of all functions [23] , suggesting to us that the nss protein structure is important for its biological activity. hence, it has been challenging to generate nss mutants that lack a specific biological function but retain its other functions. the lack of these nss mutants has prevented a detailed mechanistic analysis of each biological function of the nss. in this study, we generated two rvfv mutants, each carrying mutations in nss, that specifically targeted its general transcription inhibition function, to delineate the mechanism of its inhibition by nss. furthermore, we examined the importance of the nss-mediated inhibition of general transcription on virus-induced cytotoxicity and tested its role in rvfv virulence by using a young mouse model. all mouse studies were performed in facilities accredited by the association for assessment and accreditation of laboratory animal care in accordance with the recommendations in the guide for the care and use of laboratory animals (institute of laboratory animal resources, national research council, national academy of sciences, 1996). the animal protocol (protocol number, 1105023a) was approved by the institutional animal care and use committee of the university of texas medical branch. a standard recombinant pcr method, in which pprot7-s encoding antiviral-sense s rna [24] served as a template, was used to generate pprot7-s carrying mutations in nss coding region. the nss coding region of pprot7-s was amplified by using primers carrying mfe i or a not i site. after digestion with mfe i and not i, the pcr fragment was cloned into eco ri and the not i site of a pcaggs plasmid, resulting in the plasmids expressing nss protein. for expression of human fbxo3 isoform 1 (fbxo3/1) [14] , intracellular rnas of mrc-5 cells were subjected to cdna synthesis, and the fbxo3/1 gene was amplified with primers carrying mfe i or the not i site. after addition of an n-terminal v5 tag, the pcr product was cloned into ecor i and the not i site of pcaggs. for expression of human sap30, pcr product encoding human sap30 with n-terminal v5 tag was cloned into ecor i and the xho i site of pcaggs. all of the constructs were confirmed by sequencing. bsr-t7/5 cells which stably express t7 rna polymerase [25] were maintained in glasgow's minimal essential medium (mem) (lonza) containing 10% fetal bovine serum (fbs), 10% tryptose phosphate broth, mem amino acids solution and geneticin (1 mg/ml). vero e6 cells were maintained in dulbecco's modified mem (gibco) containing 5% fbs. mrc-5 cells were maintained in eagle's mem (emem) (gibco) containing 10% fbs, mem non-essential amino acids solution (gibco), and 1% sodium pyruvate (sigma). hela cells were maintained in emem containing 10% fbs. mp-12 is a highly attenuated rvfv strain obtained after 12 serial passages of an rvfv zh548 strain in the presence of 5-fluorouracil [26] . a recombinant mp-12 strain and other mp-12-derived mutants were rescued from cdnas as described previously [24] , except that bsr-t7/5 cells were used in place of bhk/t7-9 cells. titers of the rescued viruses were determined by using a plaque assay [24] . for the virus carrying r16h/ m250k mutations in nss, passage 0 (p0) virus obtained from plasmid-transfected bsr-t7/5 cells were serially diluted and inoculated into veroe6 cells. the highest titer of p1 virus was selected and used for the study. anti-pkr rabbit polyclonal antibody, anti-flag tag mouse monoclonal antibody, anti-flag tag rabbit monoclonal antibody, and anti-v5 tag rabbit monoclonal antibody were purchased from cell signaling technology. anti-gtf2h1 (p62) mouse monoclonal antibody, anti-gtf2h2 (p44) mouse polyclonal antibody, and anti-xpd mouse monoclonal antibody were purchased from abcam. anti-tfiih p44 (n-17) goat polyclonal antibody, anti-β-actin goat polyclonal antibody and horseradish peroxidase (hrp)-labeled anti-mouse, anti-goat, and anti-rabbit secondary antibodies were purchased from santa cruz biotechnology. anti-gst-n rabbit polyclonal antibody was generated by inoculating a rabbit with gst-n fusion protein (the entire n protein was fused with the c terminus of gst protein) followed by affinity purification of the serum [3] . anti-mp-12 mouse serum was provided by dr. robert b. tesh at the university of texas medical branch. the monoclonal antibody h2k k d k (h2k), which is against major histocompatibility complex class i antigen, was obtained from dr. paul gottlieb at the university of texas at austin. cells were washed with phosphate-buffered saline (pbs) and suspended in sds polyacrylamide gel electrophoresis (sds-page) sample buffer. samples were boiled for 3-5 min and subjected to sds-page. proteins were electroblotted onto polyvinylidene difluoride membranes (immune blot: bio rad). after blocking with skim milk, the membranes were incubated with the primary antibody for 1 h at room temperature and with the secondary antibody for 1 h at room temperature. the proteins on the membrane were detected by using an ecl western blotting detection reagent (ge healthcare life sciences) or ecl plus western blotting substrate (pierce). vero e6 cells were infected with either mp-12 or its mutants at a multiplicity of infection (m.o. i.) of 3. at 16 h post infection (p.i.), the culture media was replaced with methionine/cysteinefree medium. after starvation for 30 min, the infected cells were labeled with 100 μci/ml of 35 s-methionine/cysteine (1,000 ci/mmol; mp biomedicals) for 1 h. the radiolabeled cells were suspended in 2x sds-page sample buffer, resolved by sds-page and visualized by coomassie blue staining or autoradiography. the click-it rna alexa fluor 488 imaging kit was purchased from thermo fisher scientific. vero e6 cells were infected with recombinant viruses at an m.o.i. of 3 and incubated with 1 mm of 5-ethynyl uridine (5eu) for 1 h at 16 h p.i. cellular rna was stained with alexa fluor, 488-coupled azide for 30 min by following the manufacturer's protocol. for a negative control, mock-infected cells were treated with 5 μg/ml of actinomycin d (actd) for 30 min prior to 5eu treatment and for 1 h during 5eu treatment. after the click reaction, rvfv n protein was stained with anti-gst-n rabbit polyclonal antibody followed by alexa fluor 594 conjugated anti-rabbit antibody. images were captured on a zeiss axiophot 2 fluorescence microscope with a 40x magnification lens and processed with the imagej software [27] . for flow cytometry analysis, the infected cells were detached from the dish by accumax (innovative cell technologies) after the 5eu treatment and suspended in culture media. after washing with pbs containing 1% bovine serum albumin (bsa), cells were fixed with 2% formaldehyde/pbs for 30 min at room temperature and blocked with blocking buffer (pbs containing 0.2% saponin and 1% bsa) for 15 min on ice. cellular rna was stained by alexa fluor 488-coupled azide for 30 min in the presence of 0.5% saponin and 1% bsa. after washing with the blocking buffer, rvfv n protein was stained by anti-gst-n rabbit polyclonal antibody for 30 min on ice followed by alexa fluor 594 conjugated anti-rabbit antibody. the cells were washed with pbs containing 1% bsa, passed through a cell strainer (bd falcon), and analyzed on an lsrii fortessa (bd biosciences). single cells were gated based on their forward scatter and side scatter profile. more than 30,000 count of the gated single cells were analyzed for each experiment. confluent vero e6 cells grown in 96-well plates were either mock infected or with mutant virus at an m.o.i. of 3. cell viability was determined by using viral toxglo (promega), which measures cellular atp. at various times p.i., atp detection reagent was added to each well. after incubation for 10 min, luminescence was measured by spectramax m5e (molecular devices). total rnas were extracted by using trizol reagent (invitrogen) and subjected to northern blot analysis as described previously [28] . strand-specific digoxigenin-labeled rna probes and a digoxigenin (dig) system (roche) were used to detect rna. the 564-nucleotide-long, 293 cell-derived, and dig-labeled ifn-β riboprobe [29] was used for ifn-β mrna detection. cells cultured on chamber slides were fixed in 4% paraformaldehyde for 15 min. the fixed cells were permeabilized with 0.2% tritonx-100 for 15 min and blocked with 1% bsa in pbs for 30 min. after the blocking, the cells were stained with primary antibody diluted with the blocking solution, followed by incubation with alexa fluor 488 or 594-conjugated secondary antibodies (molecular probes). images were captured by a zeiss axiophot 2 fluorescence microscopy and processed with imagej software [27] . to detect the interaction between nss and p44, we infected hela cells with mp-12-nss-flag, which expresses nss with the c-terminal flag tag or its mutant viruses at an m.o.i. of 3. at 8 h p.i., the cells were lysed in lysis buffer [50 mm tris-hcl, ph 7.6, 0.1% np-40, 150 mm nacl, 1 mm edta, protease inhibitors (sigma), and 100 u/ml benzonase (sigma)]. after 3 cycles of freeze-thaw, the cell lysate was cleared by centrifugation at 4°c and 100,000 x g for 1 h and incubated with dynabeads protein g (life technologies) conjugated with anti-gtf2h2 (p44) mouse monoclonal antibody or h2k antibody according to the manufacturer's protocol. precipitates were washed with the lysis buffer with 0.1% tween 20 and analyzed by western blotting using the anti-p44 goat polyclonal, and anti-p44 mouse monoclonal and anti-flag mouse monoclonal antibodies. to detect the interaction between nss and sap30, we transfected hela cells with pcaggs-v5-sap30 which encodes human sap30 carrying an n-terminus v5 tag by using fugene hd transfection reagent (promega). at 16 h post transfection, the cells were infected with mp-12-nss-flag or its mutant viruses. the cells were lysed with lysis buffer (50 mm tris-hcl, ph 7.5, 100 mm nacl, 1% np-40, 1 mm edta, and protease inhibitor cocktail) [11] at 8 h p.i. the cell lysate was cleared by centrifuge at 21,000 x g and 4°c for 15 min and incubated with anti-v5-tag mab-magnetic beads (mbl international) according to the manufacturer's protocol. precipitates were washed with the lysis buffer containing 450 mm nacl and analyzed by western blotting by using anti-v5 tag rabbit monoclonal and anti-flag mouse monoclonal antibodies. for detection of interaction between nss and p62, hela cells were infected with mp-12-nss-flag or its mutant viruses at an m.o.i. of 3. at 5 h p.i., the cells were lysed in lysis buffer [20 mm tris-hcl, ph 7.6, 0.1% tritonx-100, 150 mm nacl, 1 mm edta, protease inhibitors (sigma), and 100 u/ml benzonase (sigma)] [13] . after 3 cycles of freeze-thaw, the cell lysate was cleared by centrifugation at 4°c and 100,000 x g for 1 h and incubated with dynabeads protein g conjugated with anti-gtf2h1 (p62) mouse monoclonal antibody according to the manufacturer's protocol. precipitates were analyzed by western blotting using the anti-p62 mouse monoclonal and anti-flag mouse monoclonal antibodies. to detect the interaction between nss and fbxo, we transfected hela cells with pcaggs-v5-fbxo3/1, which encodes fbxo3/1 carrying an n-terminus v5 tag, by using fugene hd transfection reagent. at 16 h post transfection, the cells were infected with mp-12-nss-flag or its mutant viruses and lysed with lysis buffer (50 mm tris-hcl, ph 7.5, 0.2% np-40, 5% glycerol, 100 mm nacl, 1.5 mm mgcl 2 , and protease inhibitor cocktail) [14] at 5 h p.i. the cell lysate was cleared by centrifugation at 21,000 x g and 4°c for 15 min and incubated with anti-v5-tag mab-magnetic beads (mbl international) according to the manufacturer's protocol. precipitates were washed with the lysis buffer and analyzed by western blotting by using the anti-v5 tag rabbit monoclonal and anti-flag mouse monoclonal antibodies. hela cells prepared in 12-well plates were transfected with 0.25 μg of the pcaggs-based plasmid encoding either nss or mutant nss or an empty vector along with 0.1 μg of prl-sv40 (promega) which expresses renilla luciferase under control of an sv40 promotor. renilla luciferase activities in the transfected cells were measured by using a renilla luciferase assay system (promega) at 24h post transfection. eighteen-day-old cd-1 mice were intraperitoneally inoculated with 10 4 pfu of mp-12, mp-12-m250k or mp-12-r16h/m250k or with hank's balanced salt solution (hbss). the animals were observed for survival for 22 days post inoculation. we previously generated and characterized an rvfv mp-12 strain-derived mutant virus, which is deficient for efficient virus genome co-packaging due to a large deletion in the 5' untranslated region of m rna segment [30] . to obtain virus variants, carrying mutations that can compensate for this deficiency, we serially passaged this mutant virus in type i ifn-incompetent vero e6 cells; the virus inoculum, used for each passage, was diluted 10 times and the released viruses were harvested at 4 days p.i. the titer of the mutant virus progressively increased with each passage; at passage 18, the virus titer was 2.9 x 10 6 pfu/ml, which was 2 logs higher than the initial titer of 2.0 x 10 4 pfu/ml prior to the serial passage. we isolated 34 plaque-cloned viruses from passage level 18, and 28 had a large deletion in the nss gene in the s segment, whereas 6 plaque-cloned viruses retained the full-length nss gene. we determined the full genome sequence of 5 clones that retained the full-length nss gene and found that all of the isolated viruses had several mutations in the l, m, and s segments. table 1 shows mutations found in the nss genes of 5 plaque-cloned viruses and the uncloned passage 18 virus. all five plaque-cloned viruses and the passage 18 virus had an amino-acid substitution at position 250, including m250k or m250t mutations. two plaque-cloned viruses and the passage 18 virus had a d100g substitution. other mutations found in plaque-cloned viruses were r16h and a75e. although k202n mutation was found in the uncloned passage 18 virus sample, this mutation was absent in the plaque-cloned viruses. to test whether the mutations affect nss functions, we generated mp-12-based mutant viruses, each carrying r16h, r16h+m250k (nss-r16h/m250k), a75e, d100g, d100g+m250t, m250k (nss-m250k), or m250t mutations in the nss, by using a reverse genetics system [24] . accumulations of nss protein in veroe6 cells infected with mp-12 carrying the d100g mutation or the d100g+m250t mutations were significantly lower than those in mp-12 infected cells (fig 1) , indicating that the d100g mutation affected efficient nss accumulation. due to the poor accumulation of nss, we excluded the mp-12 mutant carrying the d100g mutation and that carrying the d100g+m250t mutation from subsequent analysis. in cells infected with the other mutants, levels of nss protein accumulation were similar to that in mp-12 infected cells. in this study, we refer to mp-12 nss as wild type nss (wt nss). to evaluate host transcriptional shut-off activities of these nss mutants, levels of rna synthesis in virus-infected veroe6 cells were measured by labeling with 5eu from 16 h to 17 h p.i. mock-infected cells alone, as well as those treated with actd, and mp-12-infected cells and cells infected with mp-12δnss, which lacks the nss gene [24] , served as controls. 5eu-labeled rna, which was detected as a fluorescence signal, was mainly observed in the nucleus but not in the cytoplasm, indicating that viral rna synthesis, which occurs exclusively in the cytoplasm, was not very active at 16h post infection. as shown in fig 2a, strong signals of fluorescent-labeled rna were observed in the nucleus of mock-infected cells and mp-12δnssinfected cells, demonstrating active host rna synthesis. in contrast, actd-treated cells showed very weak fluorescent signals, demonstrating actd-mediated host transcriptional shut-off. mp-12-infected cells showed weaker fluorescent signals in the nucleus, compared with those in mp-12δnss-infected cells, demonstrating an inhibition of host transcription by nss. fluorescent intensity observed in cells infected with virus carrying mutation m250t, r16h or a75e in nss was similar to that in mp-12-infected cells, suggesting that host transcription was still inhibited by these nss mutants. in contrast, cells infected with mp-12 carrying nss-r16h/ we next used flow cytometry analysis to quantitatively measure the levels of 5eu-labeled rnas in mp-12-m250k-infected cells and in mp-12-r16h/m250k-infected cells. we used the same controls as described above. fig 2b shows the results using density plots, wherein 5eu incorporation and n protein levels are shown on the x and y axes, respectively. in mockinfected and actd-treated samples, most of the cells were found in quadrant 3 (q3) and q4, respectively, demonstrating active host transcription in the former, but not in the latter ( fig 2b-a and 2b-b) . in all infected samples, 86.8-93.4% of cells were rvfv n protein positive (q1+q2), demonstrating that most of the cells were infected. the level of 5eu-labeled rna in mp-12δnss-infected cells was similar to that in mock-infected cells, demonstrating similar rna synthesis activity in these two samples (fig 2b-a and 2b-f ). mp-12-infected cells showed lower levels of 5eu-labeled rna compared with those in mp-12δnss infected cells, demonstrating its lower rna transcription activity (fig 2b-c and 2b-f ). mp-12-r16h/m250kinfected and mp-12δnss-infected cells showed similar density plot patterns, suggesting to us that host transcription inhibition did not occur in mp-12-r16h/m250k-infected cells (fig 2be and 2b-f). mp-12-m250k-infected cells showed lower levels of 5eu-labeled rna compared with those in mp-12δnss-infected cells (fig 2b-d and 2b-f) . however, the percentage of mp-12-m250k-infected cells with low transcription activity was 43.0% (q1/q1+q2), while in the mp-12 samples, it was 56.6%. these results suggested that mp-12-m250k replication suppressed host general transcription, and yet the inhibitory activity of mp-12-m250k was weaker than that of mp-12. we next examined the effect of differences in the transcription inhibitory activities of the mutant viruses on global protein synthesis by incorporation of 35 s-methionine/cysteine into newly synthesizing proteins in mock-infected cells and in cells infected with mp-12, mp-12δnss, mp-12-m250k, or mp-12-r16h/m250k (fig 2c) . consistent with our previous study [24] , mp-12 replication, but not mp-12δnss replication, suppressed host protein synthesis. mp-12-r16h/m250k replication did not inhibit host protein synthesis, while mp-12-m250k replication moderately inhibited host protein synthesis. taken together, these analyses showed that nss-r16h/m250k lost the general transcription suppression activity, leading to efficient host protein synthesis in infected cells, while nss-m250k exerted inefficient general transcription suppression activity, causing moderate levels of host protein synthesis inhibition in infected cells. analysis of replication kinetics of mp-12, mp-12δnss, mp-12-r16h/m250k and mp-12-m250k showed that all viruses replicated efficiently with similar replication kinetics in vero e6 cells (fig 3a) . mp-12 formed clear plaques in vero e6 cells, whereas mp-12-r16h/m250k and mp-12-m250k formed turbid-type plaques like mp-12δnss (fig 3b) , indicating that the r16h/ m250k mutation and the m250k mutation in nss affected virus-induced cytotoxicity. cell viability assays showed that mp-12 replication caused a 90% reduction in cell viability as compared with mock-infected cells at 72 h p.i. (fig 3c) , whereas mp-12δnss-infected cells and mock-infected cells showed similar cell viabilities throughout the course of the experiments. mp-12-r16h/m250k-infected cells and mp-12-m250k-infected cells showed an 11% and 39% decrease in cell viability, respectively, as compared with that in mock-infected cells at 72 h p.i., demonstrating that both of the mutant viruses were less cytotoxic than mp-12. mp-12, mp-12-m250k and mp-12-r16h/m250k had similar replication kinetics, regardless of their differential ability to induce cytotoxicity (fig 3) , implying that virus-induced cytotoxicity did not have a significant impact on rvfv replication kinetics in vero e6 cells. because others reported that mp-12 replication induces nss-dependent p53 stabilization, which contributes to virus-induced cell death [21] , we next examined stabilization of p53 in cells infected with mp-12 and other mutant viruses (fig 3d) . amounts of p53 protein significantly increased in mp-12-infected cells, but not in mp-12δnss-infected cells, confirming the nss-dependent p53 stabilization in the infected cells. accumulation of p53 also occurred in mp-12-m250k-infected cells, whereas the accumulation levels were lower than those in nss interacts with pkr and triggers degradation of the pkr by a proteasome pathway [15] [16] [17] [18] . to investigate whether the nss mutants retain the ability for pkr degradation, we compared the total amount of pkr in virus-infected cells. similar levels of reduction in the amounts of pkr occurred in cells infected with mp-12, mp-12-m250k or mp-12-r16h/250k, suggesting that nss-r16h/m250k and nss-m250k promoted pkr degradation as efficiently as wt nss (fig 4) . le may et al. proposed that interaction of nss with sap30 interferes with the recruitment of co-activator protein cbp at activation sites on the ifn-β promotor, leading to the inhibition of ifn-β mrna transcription [11] . to investigate the ability of mp-12-r16h/m250k or mp-12-m250k to suppress ifn-β mrna transcription, we examined the levels of ifn-β mrna in mrc-5 cells infected with mp-12, mp-12-δnss, mp-12-m250k or mp-12-r16h/250k at various times p.i. (fig 5a) . mp-12δnss replication induced robust ifn-β mrna transcription, whereas ifn-β mrna was not detectable in mp-12-or mp-12-m250k-infected cells. in contrast, low levels of ifn-β mrna accumulation occurred in mp-12-r16h/m250k-infected cells, demonstrating that the nss-r16h/m250k was unable to efficiently inhibit ifn-β mrna transcription. mp-12-m250k replicated as efficiently as did mp-12 in mrc-5 cells, whereas the virus titer of mp-12δnss was significantly lower than that of mp-12 at 72 h p.i. (fig 5b) , demonstrating the efficient inhibition of ifn-β production in the former, but not in the latter. although mp-12-r16h/m250k replicated better than mp-12δnss, it replicated less efficiently mp-12-r16h/m250k-flag). cells, transiently expressing a v5-tagged sap30, were infected with these viruses, and the co-localization of the mutated nss protein with sap30 was examined by fluorescence microscopy analysis. like wt nss, both nss mutants formed filament-like structures in the nucleus (fig 5c) . the expressed sap30 was mainly observed in the nucleus and was co-localized with wt nss and both mutated nss in the filaments. we also performed co-immunoprecipitation analysis to examine nss-sap30 interaction. as a negative control, v5-tagged venus was expressed in place of v5-tagged sap30. anti-v5 antibody co-precipitated wt nss and both nss mutants along with v5-tagged sap30, while the amounts of nss-m250k that were co-immunoprecipitated with sap30 were lower than those of wt nss ( fig 5d) . anti-v5 antibody did not co-precipitate wt nss along with v5-tagged venus (fig 5d) . these data demonstrate that both nss mutants bound to sap30 in infected cells. taken together, these data indicated that nss-r16h/m250k was unable to completely block ifn-β transcription despite its ability to bind to sap30. both nss mutants retained some nss functions, including degradation of pkr and sap30 binding, and yet these nss mutants and wt nss showed different levels of general transcriptional suppression activities in virus-infected cells. transcriptional suppression activities of the mutated nss were also examined in cells transiently expressing nss along with renilla luciferase from co-transfected plasmids. luciferase activity was strongly inhibited in the cells coexpressing wt nss and renilla luciferase (fig 6a) . consistent with the data obtained from infected cells (fig 2) , nss-r16h/m250k expression did not inhibit luciferase activity, whereas nss-m250k expression moderately inhibited the luciferase activity. western blot analysis of nuclear and cytoplasmic fractions from virus-infected cells showed that like wt nss, nss-r16h/m250 and nss-m250k were also distributed in both the nucleus and the cytoplasm (s1 fig) , demonstrating that these mutations did not affect the subcellular localization of nss. these data prompted us to further examine the interplay between these nss mutants and a form of host transcription machinery, tfiih, as other studies have shown an association of tfiih with nss in the nss-induced host transcription shut-off. rvfv nss binds to p44, a subunit of tfiih, and interferes with the formation of the tfiih complex by inhibiting the subsequent interaction of p44 and xpd [12] . the reduction in the abundance of xpd and p44 also occurs upon rvfv infection, although the mechanisms that govern the reduction of these proteins are unclear [12] . in addition, nss binds to both p62, a tfiih subunit, and fbxo3, a component of e3 ubiquitin ligase, which leads to p62 degradation [13, 14] . we first examined the abundance of tfiih components, including p44, p62 and xpd, in infected cells. substantial reduction of p62 abundance and moderate reductions in the abundances of p44 and xpd occurred in cells infected with mp-12, or mp-12-m250k at 16 h p.i. (fig 6b, left panels) . in contrast, there was no substantial reduction in the abundance of these tfiih components in cells infected with mp-12δns or mp-12-r16h/m250k. the same results were obtained when viruses carrying flag-tagged nss were used. to exclude the possibility that different transcription suppression activities of these viruses affected the results, we repeated the experiments in the presence of actd and obtained similar results (fig 6b, right panels) . next, we tested the interaction of mutated nss with p44 and p62. cells infected with mp-12 or its mutants, all of which carried flag-tagged nss, were subjected to co-immunoprecipitation analysis using anti-p44 antibody (fig 6c) . anti-p44 antibody co-immunoprecipitated wt nss and both mutant nss along with p44, whereas control anti-h2k antibody precipitated neither p44 nor nss. these data demonstrated that both nss mutants bound to p44. to examine the interaction of p62 and nss, cells were infected with the viruses as described above, and cell extracts were prepared at 5 h p.i., when p62 was still detectable in mp-12-nss-flag-infected cells (fig 6d) . anti-p62 antibody, but not anti-h2k antibody, co-immunoprecipitated wt nss and both mutant nss along with p62, demonstrating binding of the mutant nss proteins to p62. we noted that the amounts of nss-m250k that were co-immunoprecipitated with p44 and with p62 were lower than those of wt nss, implying that the binding efficiencies of nss-m250k for p44 and for p62 were lower than those for wt nss. fbxo3 is an interactor of rvfv nss that is engaged in the degradation of p62; nss interacts with the full-length fbxo3 protein (fbxo3/1) as well as with a shorter splice variant of the fbxo3 that lacks the c-terminal acidic domain and poly(r) region [14] . because nss-r16h/m250k did not induce p62 degradation regardless of its ability to bind to p62 (fig 6d) , we suspected that nss-r16h/m250k would not interact with fbox3. to test this possibility, cells transiently expressing the v5-tagged fbxo3/1 were mock infected or infected with mp-12-nss-flag, mp-12-m250k-flag, or mp-12-r16/m250k-flag. at 5 h p.i., cell extracts were prepared and subjected to co-immunoprecipitation analysis by using anti-v5 antibody ( fig 6e) . all of the nss proteins, including nss-r16/m250k, were co-immunoprecipitated with fbxo3/1. table 2 summarizes interactions of the nss mutants with p44, p62 and fbxo3. nss is a major virulence factor of rvfv [31] . the nss-mediated inhibition of type i ifn production is thought to contribute to the virulence of the virus, yet it remains unclear whether the general transcription suppression function of nss contributes to this virulence. we examined the importance of the nss-mediated transcription inhibition in rvfv virulence by using a young mouse model. although mp-12 is known as an attenuated strain, the intraperitoneal inoculation of 10 4 pfu of mp-12 into 18-day-old cd1 mice resulted in the death of 55% of the mice within 13 days p.i. (fig 7) . under the same experimental conditions, none of the mp-12-r16h/m250k-inoculated mice and 20% of the mp-12-m250k-inoculated mice died. no obvious clinical signs, including neurological symptoms, were observed. these data demonstrated that the mp-12-r16h/m250k lacked virulence, and mp-12-m250k was less virulent than mp-12 in this young mouse model. in this study, we demonstrated that the m250k and r16h/m250k mutations in nss differentially reduced its inhibitory activity on host transcription without affecting its ability to inhibit ifn-β transcription, through its interaction with sap30, and induced pkr degradation. nss-r16h/m250k, which completely lacked the activity to inhibit general transcription, correspondingly lost the ability to promote the degradation of p62. unexpectedly, nss-r16h/ m250k was able to interact with fbox3 and p62 (fig 6) . these data possibly suggest that the fbxo3-nss-p62 interaction may not be sufficient to trigger p62 degradation. nss-m250k exhibited a partially reduced activity to inhibit general transcription. the results of co-immunoprecipitation assays showed that a reduced amount of nss-m250k co-precipitated with sap30, p62 and p44, compared with that in wt nss (figs 5 and 6 ). however, the slightly impaired ability of nss-m250k to inhibit general transcription could not be solely attributed to the lower binding efficiency of nss-m250k to p44 because nss-m250k efficiently suppressed ifn-β transcription and induced p62 degradation, despite its lower efficiency of binding to sap30 and p62. moreover, nss-r16h/m250k, which lacked transcription suppression activity, efficiently interacted with p44 ( fig 6) . these data bring into question the importance of nss-p44 interaction for its host transcriptional shut-off function. one possibility is that the nss-p44 interaction may only make a modest contribution towards its transcription inhibition activity and possibly could have additional, as yet unidentified, biological function(s). a second possibility is that only wt nss, but not the mutated nss, is able to interfere with the formation of an active tfiih complex. nss competes with xpd for binding to p44, resulting in inhibition of the tfiih complex formation [12] . although both nss-m250k and nss-r16h/m250k retained the ability to bind to p44, it is possible that the binding of these mutated nss to p44 did not exclude the binding of xpd. the r16h single mutation did not affect the ability of nss to inhibit transcription (fig 2) . although the m250k mutation is in close proximity to the ωxav motif located at the c-terminal region of nss, which is essential for p62 degradation [32] , mp-12-m250k still retained the ability to degrade p62 (fig 6) . however, the r16h/m250k double mutant lost the ability to induce p62 degradation. these results imply that the combined mutations, r16h and m250k, induced an unfavorable structural alteration in nss, which abolished its function to degrade p62. mp-12-r16h/m250k replication induced low levels of ifn-β mrna (fig 5) , indicating that nss-r16h/m250k was not able to block ifn-β mrna synthesis as efficiently as wt nss despite its ability to interact with sap30 (fig 5) . it has been reported that nss-induced p62 degradation contributes to the inhibition of ifn-β production [14] . accordingly, our data suggested that mp-12-r16h/m250k was unable to completely block ifn-β mrna synthesis due to a lack of ability to promote the degradation of p62 (fig 6) . although mp-12-r16h/m250k replication induced ifn-β mrna synthesis, the level of the ifn-β mrna was significantly lower than that in mp-12δnss-infected cells (fig 5) , suggesting the importance of interaction of nss with sap30 for ifn-β inhibition. taken together, the data shown here and those of others [11, 14] strongly imply that nss-sap30 interaction and nss-induced general host transcriptional suppression function are both necessary for efficient inhibition of ifn-β mrna transcription. mp-12δnss or mp-12 encoding a reporter gene in place of the nss gene causes less prominent cytopathic effects than does mp-12, demonstrating the contribution of the nss towards the induction of cytotoxicity [24] . mp-12 replication induces nss-dependent p53 stabilization, which contributes to virus-induced cell death [21] , and yet it was unclear which function of the nss contributed to the induction of the p53-mediated cytotoxicity. as shown in fig 3, mp-12-infected cells showed the lowest cell viability, followed in order by mp-12-m250k, which moderately suppressed host general transcription, and mp-12-r16h/m250k, which did not suppress host transcription. hence, there was a correlation between the strength of nss-mediated, host transcriptional shut-off activity and cell viability in the infected cells (table 3) . likewise, accumulation of p53 was the highest in mp-12-infected cells, followed in order by that in mp-12-m250k-infected cells and mp-12-r16h/m250k-infected cells, suggesting to us that p53 stabilization also correlates with the transcription inhibition activities of the different nss mutants. these results indicate that the nss-mediated host transcriptional shut-off triggered p53 stabilization, leading to p53-mediated cell death. although mp-12 is an attenuated rvfv strain, 55% of 18-day-old cd1 mice died after intraperitoneal inoculation with 10 4 pfu within 13 days p.i. (fig 7) . as the immune system is not yet fully developed in young mice, it likely failed to prevent systemic infection by mp-12. consistent with this notion, intraperitoneal inoculation of mp-12 into severe combined immune deficiency mice also caused 100% mortality [33] . mp-12-r16h/m250k carrying nss that lacks the host transcription inhibition function was completely attenuated in 18-day-old cd1 mice (fig 7) . mp-12-m250k carrying nss with an impaired ability to inhibit transcription also exhibited reduced virulence when compared to that in its parental virus mp-12. these data highlight the role of the host transcription inhibition function of nss in rvfv virulence. type i ifn has been shown to play a critical role in protecting the host from rvfv-induced disease in animal models [34] . notably, both the nss mutants, carrying either the m250k single mutation or the r16h/m250k double mutation, retained the ability to bind to sap30, the factor that is targeted by nss to inhibit ifn-β mrna transcription. however, the induction of ifn-β mrna synthesis was not completely blocked in mp-12-r16h/m250k-infected mrc-5 cells, most probably due to the lack of inhibition of host transcription in these cells expressing the mutated nss. these data suggest the possibility that the inability of mp-12-r16h/m250k to completely block the production of ifn-β in infected mice could have contributed towards its attenuation. in addition, the production of other antiviral and/or proinflammatory cytokines could have also contributed towards the attenuated phenotype of both of these mutant viruses, carrying nss with an impaired ability to block host transcription. it is also possible that the lower levels of virus-induced cytocidal effects might have contributed to the lower virulence of these nss mutant viruses, as both mutant viruses caused less severe cytopathic effects and cytotoxicity than did mp-12 in cultured cells (fig 3) . we believe that experiments using rvfv mutants, carrying the m250k single mutation and the r16h/m250k double mutation, in animal models would yield valuable information about the role of nss-mediated host transcription inhibition in regulating host cytokine responses and its impact on the pathogenesis of rvfv. mp-12 is an attenuated live vaccine candidate, but it still harbors residual virulence in a young mouse model. although further studies are required to test the immunogenicity and protective efficacy of mp-12-m250k and mp-12-r16h/m250k, there is a potential for developing these mp-12-derived nss mutant viruses as safer live attenuated rvfv vaccine candidates. we found that the serial passage of an mp-12-derived mutant virus having a large deletion in the 5' untranslated region of m rna segment [30] in vero e6 cells resulted in accumulation of variant viruses that were able to replicate better than the original mutant virus. most of viruses in passage 18 had a large internal deletion in the nss gene, which may mean their nss proteins are biologically inactive. others have reported that rvfv carrying large deletions in the nss gene start accumulating from the 15 th serial passage in bhk cells of the rvfv p strain, which is defective in ifn-α/β signaling [35] . in our experiment, the large deletions in the nss gene were detected after the 5 th serial passage of the virus. although the cell lines used in the studies were different, the deletion of the nss gene as early as after 5 passages implied that there was a selective pressure to remove the nss gene from the virus genome during the passaging of the mutant virus. the full-length nss gene of the uncloned passage 18 viruses had m250k, m250t, k202n and d100g mutations (table 1 ). in the cells infected with mp-12 carrying nss with a d100g mutation, the accumulation of nss was poor (fig 1) , indicating that the d100g mutation affects the efficient accumulation of nss. as two out of the five plaque-cloned viruses (clones 3 and 4, table 1 ) also had the d100g mutation in nss, this possibly affected its accumulation in infected cells. the remaining three clones carried nss with a m250k or r16h/m250k mutation. our results showed that these mutations in nss partially or completely abolished its host transcription inhibition function without affecting its ability to interact with sap30 to inhibit ifn-β mrna synthesis. these data implied that the host transcription function of nss was unfavorable for the mutant virus, carrying a deletion in the 5' utr, to replicate well in veroe6 cells. mp-12-m250k and mp-12-r16h/m250k replication induced low cytotoxicity due to its lower inhibitory activity on host transcription (figs 2 and 3) . we suspect that the lack of nss-induced host transcription suppression created a favorable cellular environment for the mutant virus, thereby allowing the emergence and accumulation of mutant viruses that lacked this function. further studies are required to delineate the importance of nss-mediated host transcription inhibition in the virus life cycle. with mp-12-nss-flag or its mutants, at an m.o.i. of 2 and at 6 h p.i., the cells were lysed using the lysis buffer (10 mm tris-hcl, ph 7.5, 10 mm kcl, 1.5 mm mgcl 2 , 0.5% triton x-100, protease inhibitor cocktail) followed by incubation on ice for 10 min. after centrifugation at 2,000 x g for 2 min, the resulting supernatant was collected as the cytoplasmic fraction (c). the pellet from this centrifugation was washed once with the lysis buffer (without triton x-100), suspended in 1x sds sample buffer and denoted as the nuclear fraction (n). the subcellular fractions were analyzed by western blotting using anti-flag, anti-hsp90 and anti-lamin a antibodies. (tif) breaking the chain: rift valley fever virus control via livestock vaccination the pathogenesis of rift valley fever nsm and 78-kilodalton proteins of rift valley fever virus are nonessential for viral replication in cell culture deletion of the nsm virulence gene of rift valley fever virus inhibits virus replication in and dissemination from the midgut of aedes aegypti mosquitoes the rift valley fever accessory proteins nsm and p78/nsm-gn are distinct determinants of virus propagation in vertebrate and invertebrate hosts nsm protein of rift valley fever virus suppresses virusinduced apoptosis the c-terminal region of rift valley fever virus nsm protein targets the protein to the mitochondrial outer membrane and exerts antiapoptotic function recent advances in the molecular and cellular biology of bunyaviruses the rift valley fever virus nonstructural protein nss is phosphorylated at serine residues located in casein kinase ii consensus motifs in the carboxy-terminus the carboxy-terminal acidic domain of rift valley fever virus nss protein is essential for the formation of filamentous structures but not for the nuclear localization of the protein a sap30 complex inhibits ifnbeta expression in rift valley fever virus infected cells tfiih transcription factor, a target for the rift valley hemorrhagic fever virus nss protein of rift valley fever virus promotes posttranslational downregulation of the tfiih subunit p62 virulence factor nss of rift valley fever virus recruits the f-box protein fbxo3 to degrade subunit p62 of general transcription factor tfiih rift valley fever virus nss protein promotes post-transcriptional downregulation of protein kinase pkr and inhibits eif2alpha phosphorylation nss protein of rift valley fever virus induces the specific degradation of the double-stranded rna-dependent protein kinase protein kinase r degradation is essential for rift valley fever virus infection and is regulated by skp1-cul1-f-box (scf) fbxw11-nss e3 ligase nss virulence factor of rift valley fever virus engages the f-box proteins fbxw11 and beta-trcp1 to degrade the antiviral protein kinase pkr reactive oxygen species activate nfkappab (p65) and p53 and induce apoptosis in rvfv infected liver cells induction of dna damage signaling upon rift valley fever virus infection results in cell cycle arrest and increased viral replication p53 activation following rift valley fever virus infection contributes to cell death and viral production nonstructural nss protein of rift valley fever virus interacts with pericentromeric dna sequences of the host cell, inducing chromosome cohesion and segregation defects functional analysis of rift valley fever virus nss encoding a partial truncation rescue of infectious rift valley fever virus entirely from cdna, analysis of virus lacking the nss gene, and expression of a foreign gene generation of bovine respiratory syncytial virus (brsv) from cdna: brsv ns2 is not essential for virus replication in tissue culture, and the human rsv leader region acts as a functional brsv genome promoter mutagen-directed attenuation of rift valley fever virus as a method for vaccine development nih image to imagej: 25 years of image analysis rift valley fever virus nonstructural protein nss promotes viral rna replication and transcription in a minigenome system severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells mechanism of tripartite rna genome packaging in rift valley fever virus characterization of clone 13, a naturally attenuated avirulent isolate of rift valley fever virus, which is altered in the small segment a ωxav motif in the rift valley fever virus nss protein is essential for degrading p62, forming nuclear filaments and virulence recombinant rift valley fever vaccines induce protective levels of antibody in baboons and resistance to lethal challenge in mice interplay between the virus and host in rift valley fever pathogenesis host alternation is necessary to maintain the genome stability of rift valley fever virus we thank robert tesh for anti-mp12 antibody; paul gottlieb for the monoclonal antibody h2k k d k ; mark griffin (flow cytometry and cell sorting core, the university of texas medical branch) for support with the flow cytometry analyses; and krishna narayanan for critical reading of the manuscript. conceptualization: kt sm. key: cord-001812-ov1qssnu authors: fan, yi-chin; chiu, hsien-chung; chen, li-kuang; chang, gwong-jen j.; chiou, shyan-song title: formalin inactivation of japanese encephalitis virus vaccine alters the antigenicity and immunogenicity of a neutralization epitope in envelope protein domain iii date: 2015-10-23 journal: plos negl trop dis doi: 10.1371/journal.pntd.0004167 sha: doc_id: 1812 cord_uid: ov1qssnu formalin-inactivated japanese encephalitis virus (jev) vaccines are widely available, but the effects of formalin inactivation on the antigenic structure of jev and the profile of antibodies elicited after vaccination are not well understood. we used a panel of monoclonal antibodies (mabs) to map the antigenic structure of live jev virus, untreated control virus (ucv), formalin-inactivated commercial vaccine (ficv), and formalin-inactivated virus (fiv). the binding activity of t16 mab against nakayama-derived ficv and several strains of fiv was significantly lower compared to live virus and ucv. t16 mab, a weakly neutralizing jev serocomplex antibody, was found to inhibit jev infection at the post-attachment step. the t16 epitope was mapped to amino acids 329, 331, and 389 within domain iii (ediii) of the envelope (e) glycoprotein. when we explored the effect of formalin inactivation on the immunogenicity of jev, we found that nakayama-derived ficv, fiv, and ucv all exhibited similar immunogenicity in a mouse model, inducing anti-jev and anti-edii 101/106/107 epitope-specific antibodies. however, the ediii 329/331/389 epitope-specific igg antibody and neutralizing antibody titers were significantly lower for ficv-immunized and fiv-immunized mouse serum than for ucv-immunized. formalin inactivation seems to alter the antigenic structure of the e protein, which may reduce the potency of commercially available jev vaccines. virus inactivation by h(2)o(2), but not by uv or by short-duration and higher temperature formalin treatment, is able to maintain the antigenic structure of the jev e protein. thus, an alternative inactivation method, such as h(2)o(2), which is able to maintain the integrity of the e protein may be essential to improving the potency of inactivated jev vaccines. introduction inactivated sa-14-14-2 vaccines are formulated with aluminum-hydroxide-adjuvant (ic51 or ixiaro). ic51 vaccine has been licensed for use in adult and children older than 2 months [21] . in addition, a live-attenuated jev sa14-14-2 vaccine, developed in china, is used in some asian countries such as china, india, and nepal [22] [23] [24] . the vaccine effectiveness has been estimated to be 85% to 90% after two doses of inactivated nakayama vaccine, and 91% after one dose of the live-attenuated sa14-14-2 vaccine [25] [26] [27] . unlike the live-attenuated vaccine, the formalin-inactivated jev vaccines require boost immunization to retain the protective neutralizing antibodies [22, 28] . significant numbers of jev endemic countries still depend on the locally produced, mouse brain-derived formalin-inactivated giii jev vaccine to control je epidemics [19] . formalin is the chemical most commonly used for inactivation to manufacture viral vaccines such as hepatitis a virus, polio, influenza virus, rabies virus, and simian immunodeficiency virus [29] [30] [31] [32] [33] [34] . formalin reacts with amino acids of target proteins to form reversible schiff-base adducts and non-reversible methylene bridges. it has also been used as isotopic agent to label protein by introducing isotope to specific amino acid and as a cell and tissue fixation agent. formalin functions chemically when it is used to inactivate virus, and the chemical reaction may modify the antigenic structure of the virion [35, 36] . it has been shown formalin inactivation alters antigenic properties and reduces the immunogenicity of vaccines, such as hepatitis a and b virus, polio virus, bovine herpes virus 1 and influenza virus in mouse models [37] [38] [39] [40] [41] . formalin-inactivated jev vaccine remains the most widely distributed vaccine used to control je epidemics. however, the potential effects of formalin on the antigenic structure of jev and the antibody profile elicited by this vaccine remain unclear. the use of a low concentration of formalin and short inactivation time can yield antigens capable of inducing high neutralizing titers in mice, but the association between these inactivation procedures and the alteration of antigenic structure of e and the antibody profile elicited by this vaccine remain undetermined [42] . in this study, we used a panel of e-specific, murine monoclonal antibodies (mabs) to analyze the effect of epitope modification of jev e protein in a formalin-inactivated commercial vaccine (ficv) and laboratory grown, formalin-inactivated giii and gi viruses (fiv). we showed that formalin-inactivation, indeed altered the binding pattern of a jev-derived, serocomplex cross-reactive neutralizing antibody, t16. interestingly, antibodies recognizing formalin-modified epitope were significantly lower in titer and had weaker neutralizing activity in serum from mice vaccinated with ficv and fiv-nakayama than with untreated control nakayama virus (ucv-nakayama). h 2 o 2 inactivated jev and was a superior approach that retained the antigenic reactivity of the virus with all tested mabs including t16 as compared to conventional inactivation methods such as formalin and uv. animal experiments were approved by the institutional animal care and use committee (iacuc) of national chung hsing university, taiwan (approval no: 101-88), and performed according to a protocol, which adhered to principles in the guide for the care and use of laboratory animals (nrc 2011) and meet the requirement in an association for assessment and accreditation of laboratory animal care (aaalac). the serum samples used in this study were collected from anonymous children who had received jev vaccination and were without jev infection during 2010; they were part of an already-existing collection housed at tungs' taichung metroharbor hospital in taichung. the clinical protocol was reviewed and approved by the institutional review board of the hospital (99006) for serum sample collection. serum was recovered from blood after clotting and then centrifuged, and stored at -70°c until use. vero, cos-1, and c6/36 cells (kindly provided from dr. chang gj of us cdc, fort collins, co) were grown in dulbeco's modified eagle's minimal essential medium (dmem, gibco) containing 5%, 10%, and 10% heat-inactivated fetal bovine serum (fbs, gibco), respectively. bhk cells (kindly provided from dr. chen wj of chang gung university, taiwan) were grown in minimum essential medium (mem, gibco) with 10% heat-inactivated fbs. the jev vaccine strains used were the giii strains nakayama and sa14-14-2, naturally attenuated giii t1p1 isolate [43] and gi circulating strain yl2009-4 [44] . the ficv used in this study was the mouse brain-derived, formalin-inactivated nakayama virus vaccine manufactured by adimmune corp. in taiwan. monoclonal antibodies (mabs) used for antigenic characterization were flavivirus group cross-reactive mabs (4g2, 6b3b-3, 6b6c-1 and 23-2), jev serocomplex cross-reactive mabs (t16, 2b5b-3, 6b4a-10, 1b5d-1 and 7a6c-5) and jev-specific mabs (2h4 and 2f2) [45] [46] [47] . vero cells, infected with strains of jev, namely nakayama, sa14-14-2, t1p1, and yl2009-4, at a multiplicity of infection of 1 (moi = 1), were grown in serum-free medium (sfm4megavir; hyclone, logan, ut) for 4 days. supernatant was clarified by centrifugation at 10,000 rpm for 30 min; virion particles in the supernatant were pelleted by a second centrifugation at 19,000 rpm for 16 hr. viral pellets from the second centrifugation were resuspended in 1x phosphate buffered saline (pbs). these concentrated viruses were used to derive fiv. an amount of 37% formaldehyde (sigma-aldrich, st. louis, mo) was diluted with 1x pbs to 0.5% and adjusted to ph 7.2 with 10 n naoh (sigma-aldrich, st. louis, mo). the mixture was added to concentrated jev viruses give a final formalin concentration of 0.05%. the formalin-treated virus (fiv) or untreated virus (untreated control virus; ucv) was incubated at 4°c for 49 days (the manufacture procedure for ficv provided by adimmune corporation in taiwan), or at 22°c for 10 days [42] . fiv and ucv samples incubated at 4°c were collected every week and stored at -70°c for analysis. nakayama virus specimens were inactivated by short-wavelength uv light at a distance of 3 cm on ice for 30 min or with a final concentration of 3% h 2 o 2 (fisher scientific), ph 7.2, at 22°c from 2 to 8 hr, then stored at -70°c. the residual infectious viral titers of fiv, ucv or uv-or h 2 o 2 -treated viruses were assessed by microplaque assay. antigen-capture (ag) enzyme-linked immunosorbent assay (elisa) antigen-capture elisa (ag-elisa), described previously [48] , was used to estimate e proteins concentrations in samples with anti-jev mouse hyper-immune acitic fluid (mhiaf) (immunized with purified and live jev) and determine the binding activity of mabs. briefly, a 96-well plate (sigma-aldrich, st. louis, mo) was coated with rabbit anti-jev polyclone (generated from rabbit immunized with pvax-jei vlp-expressing plasmid [49, 50] , and obtained from dr. chang gj of us cdc, fort collins, co) at 37°c for 1 hr, blocked with startblock blocking buffer (pierce, rockford, ill.), then antigen was added at 40 ng per well for incubation at 4°c overnight. antigen was incubated with mabs and mhiaf, diluted with 5% skim milk, at 37°c for 1 hr, then peroxidase-conjugated goat anti-mouse igg (h+l) (jackson immunoresearch, west grove, pa) at 37°c for 1 hr. finally, 3,3',5,5'-tetramethylbenzidine substrate (tmb; neogen corp., lexington, ky) was added at 100 μl per well for the color reaction and reactions were stopped with 2n h 2 so 4 added at 50 μl per well; the od 450 values were recorded. the antigen concentration of ucv and fiv was estimated by the od 450 of the mhiaf. the mab binding activities for ucv or fiv were determined by percentage reactivity estimated by the od 450 of ucv or fiv at the time relative to that at 0-day, respectively. all binding activities were adjusted by fold difference of antigen concentration, estimated by the od450 of mhiaf against ucv or fiv at the time relative to that at 0-day, respectively and shown as mean±sd of two duplicates of two independent assays. jevs and jev virus-like particles (vlps) were mixed with 5x sds non-reducing sample buffer (315 mm tris, ph 6.8, 50% glycerol, 5% sds, 0.025% bromophenol blue), then loaded onto a 10% sds gel. after separation, proteins were transferred to a nitrocellulose membrane, which was blocked with 5% skim milk. the proteins on the membrane were detected by use of mouse anti-jev polyclonal antibody, mabs or mhiaf and visualized by incubation with peroxidaseconjugated goat anti-mouse igg (h+l) (jackson immunoresearch, west grove, pa); bands were developed by use of the lumigold ecl western blotting detection kit (signagen laboratories, gaithersburg, md). the intensities of bands were calculated by use of imagej version 1.44 (nih, bethesda, md). the binding activity of anti-edii 101/106/107 and anti-ediii 329/ 331/389 antibodies against vlp was eliminated by introducing 101/106/107 and 329/331/389 mutations on vlp, respectively. jev vlps were produced with the pvax-jei plasmid derived from the pcbje plasmid [50] , which encodes the prm and e protein regions of the sa14 strain genome. this plasmid was also used as the template for introducing mutations into the e protein by use of a site-directed mutagenesis kit (stratagene, la jolla, ca) as described [50, 51] . the pvax-jei 101/106/107, 306, 329, 331, 332 and 389 amino acid mutants were introduced by mutagenesis primers (s1 table) , according to the manufacturer's protocols, and mutation was confirmed by sequencing. the jev vlp-expressing plasmids were electroporated into cos-1 cells by use of a 0.4-cmelectrode-gap cuvette and a bio-rad gene pulser ii (bio-rad laboratories, hercules, ca) at 250 v and 975 μf; electroporated cells were recovered overnight at 37°c and incubated at 28°c to enhance vlp secretion. the secreted vlps were analyzed by ag-elisa and used to evaluate the presence of epitope-specific antibodies. to measure the neutralizing activity of the mabs or in serum samples, briefly, 2.48×10 4 vero cells were added into 96-well plates for 24 hr at 37°c with 5% co 2 . mabs in pre-attachment assay or serum samples were inactivated at 56°c for 30 min, diluted in a two-fold series, mixed with 100 pfu jev nakayama strain for 1 hr, then shaken every 20 min. monolayers of vero cells were infected with the virus-antibody mixture for 1 hr at 37°c with 5% co 2 ; in contrast, in post-attachment assay, virus was bound on monolayers of vero cells at 4°c for 1 hour, then incubated with a two-fold series diluted and inactivated mabs at 4°c for 1 hour, and then was shift into 37°c incubator with 5% co2 for 1 hour. after incubation, 1% methyl cellulose in dmem containing 2% fbs was added to the 96-well plates for incubation for 36 hr at 37°c with 5% co 2 , then plates were washed with pbs, fixed with 75% acetone, and air-dried in a hood. the fixed cells were stained with anti-jev mhiaf for 40 min at 37°c. after a washing, peroxidase-conjugated goat anti-mouse igg (jackson immunoresearch, west grove, pa) was added for 40 min, and virus-infected foci were identified by use of a vector-vip peroxidase substrate kit sk-4600 (vector laboratories, burlingame, ca). the foci were counted manually under microscopy and used to calculate a sigmoidal dose-response for the focus reduction micro-neutralization test (frμnt 50 ) titers with use of graphpad prism v5.01. mouse immunization and epitope-specific antibody response groups of 6-week old balb/c mice (n = 5 mice per group) were vaccinated with three doses of freund's incomplete adjuvanted ucv-nakayama, fiv-nakayama or ficv. the first booster vaccination was given 2-week after the primary immunization and followed by a final booster at 4 weeks after the first booster vaccination. serum samples were collected 2 weeks after the final booster vaccination. an igg antibody-capture elisa (gac-elisa) described previously [52] was used to determine the titer of the epitope-specific antibodies in immunized mouse serum. briefly, goat antimouse igg (h+l) (kpl, gaithersburg, md) was coated on 96-well plates at 37°c for 1 hr, then plates were blocked with startblock blocking buffer (pierce, rockford, ill.). serum samples were serially diluted with wash buffer and added to plates at 37°c for 90 min. after a washing, 40 ng of the jev antigens, wild-type (wt) or mutant jev vlps was added and mixtures were incubated at 4°c overnight. the igg-capture antigens were detected by use of rabbit anti-jev polyclonal antibody and peroxidase-conjugated goat anti-rabbit igg (h+l) (jackson immu-noresearch, west grove, pa) to detect antigen-bound rabbit anti-jev polyclonal antibodies. the above steps were described for the ag-elisa. total anti-immunogen igg antibody was determined as endpoint titer. the epitope-specific antibody response was determined by the decreased reactivity titer against mutant vlp compared to wt vlp and was calculated as endpoint titer of (wt-mutant vlp). the epitope-specific neutralizing antibody activity was determined as follows. the pvax--jei wt and ediii 329/331/389 mutant plasmids were electroporated into cos-1 cells, cultured for 24 hr, and cells were resuspended in pbs. diluted mouse serum samples were mixed with 10 7 transformed cos-1 cells at 37°c on a shaker for 2 hr. then the transformed cos-1 cells were removed by centrifugation at 3,000 rpm for 10 min and the supernatant containing unbound antibodies was serially diluted and mixed with 100 plaque-forming units (pfu) of jev nakayama strain at 37°c for 1 hr. the neutralizing activity was determined by frμnt assay as described previously and neutralizing activity (%) calculated by [ the pvax-jei wt and ediii 329/331/389 mutant plasmid-transformed cos-1 cells were seeded into wells of a chamber slide (millipore, billerica, ma), and cultured at 37°c overnight, then wells were fixed with 4% paraformaldehyde (sigma, st. louis, mo, usa) in pbs at room temperature for 20 min and washed with pbs. the fixed cells were made permeable by treatment with 0.1% triton x-100 at 4°c for 5 min and washed with pbs, then wells were blocked with 3% bovine serum albumin (sigma, st. louis, mo, usa) in pbs at 37°c for 1 hr. wells were stained with anti-jev mhiaf and reacted with fitc-conjugated goat anti-mouse igg (kpl, gaithersburg, md) in 1% evans blue. images were viewed under an olympus ckx41 microscope. data are presented as mean±sd from two repeated experiments. two-tailed student's t test was used for all analyses, and statistical significance was set at p <0.05. most of the antibodies elicited by jev infection or immunization are conformation-dependent and, for the most part, recognize the viral e protein and are able to help prevent virus infection [15, 53] . formalin inactivation of several human vaccines has been shown to result in antigenic alteration to the viral particles, which can be measured by the binding activity of specific mabs [38, 40] , but the effect of formalin inactivation on commercially available jev vaccines has not been evaluated. previously, an established ag-elisa protocol was successfully used to determine the antigenic structure of jev using a panel of anti-e protein mabs [49, 53] . first, we evaluated the antigenic differences between the live nakayama virus and ficv by ag-elisa using a panel of eleven anti-flavivirus e-protein mabs [45] [46] [47] . the same antigen concentration (estimated by ag-elisa using jev-specific mhiaf) of live virus and ficv was used throughout the experiments. live nakayama virus and ficv showed similar binding pattern for ten of the eleven tested mabs, with the exception being t16 mab (fig 1) . the binding activity with t16 mab was significantly lower for ficv than for the live nakayama virus (p<0.05) with end-point titers of 10 5.39 and 10 3.48 for the live nakayama virus and ficv respectively. the mab binding pattern suggests that the antigenic structure of ficv differs from that of the live nakayama virus. the decrease in binding activity of t16 mab against ficv might be due to procedure variation during vaccine manufacture, which include differences in the formalin inactivation, differences in the virus purification process, changes in the sub-strains used and differences in the passage history of the nakayama virus used between the vaccine production virus strain and the live nakayama virus used in this experiment. to rule out the potential influence of sub-strain differences on the e structure and focus on the effect of formalin treatment on antigenic modification of nakayama virus, we subjected laboratory-grown concentrated nakayama virus to either formalin inactivation (fiv-nakayama) or without formalin at 4°c for 49 days (untreated control virus-nakayama virus, ucv-nakayama). the antigenic reactivity of fiv-nakayama and ucv-nakayama, as determined by ag-capture elisa with anti-jev mhiaf, remained constant (fig 2a) . the infectivity of fiv-nakayama decreased drastically to below the detection limitation after 7 days treatment under these conditions; however, the infectivity of ucv-nakayama decreased gradually over time and only became undetectable after 49 days (fig 2b) . ten of the eleven mabs, with the exception of t16, showed similar binding activity with fiv-nakayama and ucv-nakayama preparations collected at most time points by ag-elisa ( fig 2c) . the binding activity of the jev serocomplex cross-reactive t16 mab against fiv-nakayama was significantly decreased at the 14-day collection point (14-dc) with this sample having only 83% of the binding activity of the 0-dc sample. this decrease in binding of t16 against fiv-nakayama was time-dependent; with only 55% binding activity remaining at 49-dc (fig 2c, panel e) . unlike t16, 2b5b-3 and 2f2 binding against fiv-nakayama declined at an early time point compared to ucv-nakayama, but this was not observed at later time points. this result is consistent with the observation that only t16 exhibiting a decreased binding activity against ficv by ag-elisa (fig 1) . therefore, we believed that the decreased binding activity of t16 mab against ficv is the result of formalin inactivation and is not related to potential antigenic differences related to the sub-strain of virus or associated with the passage history of virus. to rule out formalin-induced antigenic modification occurring at strain-specific amino acids [36, 54] , we prepared three different strains of jev, the sa14-14-2 giii vaccine-strain virus, the t1p1 naturally attenuated giii virus and the yl2009-4 gi virus [44] , and then applied the same formalin inactivation procedures to all three viruses; this was followed by measurement of their mab binding by ag-elisa using a subset of eight mabs. the pattern of mab binding activity obtained with these viruses was similar to that obtained with the nakayama virus with or without formalin treatment (fig 3 and s1 fig) . again, at 49-dc, the binding activity of t16 mab was significantly decreased to 75%, 75%, and 72% for fiv-sa14-14-2, fiv-t1p1, and fiv-yl2009-4, respectively (fig 3a) . to further confirm that the decrease in binding activity of t16 mab against the e protein was due to formalin inactivation, these viral preparations with or without formalin treatment were analyzed by non-reducing sds page followed by western blotting using the t16. 4g2 and 7a6c-5 mabs, which have similar ag-elisa binding activity against the fiv-jev and the ucv-jev antigens, were included for comparison (figs 2c and 3 and s1 fig). the intensity of the e protein band, when detected by 4g2 and 7a6c-5 of the various fiv-jev and ucv-jevs, including nakayama, sa14-14-2, t1p1, and yl2009-4, were similar; however, the intensity detected by t16 was lower against the fiv-jevs than against the ucv-jevs (fig 3b) . by way of comparison, at 49-dc, the formalin-treated nakayama, sa14-14-2, t1p1, and yl2009-4 viruses were found to have reduced t16 binding intensities of only 36%, 38%, 57%, and 40% of the ucv-jevs, respectively ( fig 3c) . therefore, formalin inactivation, when it affects the antigenic structure of jev e protein, would seem not to be viral strain-specific and is likely to occur at the virion level, perhaps affecting the e monomer containing disulfide bonds. to localize the formalin-modified epitope on e protein, we mapped the epitope recognized by t16 mab. the antigenic structure of non-infectious jev vlp is similar to that of the virion particle [50, 51] . t16 mab is a jev serocomplex cross-reactive antibody and we previously found that amino acid residues 101, 104, and 106, which are present in edii, and amino acid residues 315, 331 and 389, which are present in ediii, are important for the binding of jev serocomplex cross-reactive mabs [49, 53] . thus we used a vlp-expressed plasmid to locate the formalin-modified epitope recognized by t16 mab. jev vlps with ediii amino acid substitutions s329a, s331k, and d389g, but not jev vlps with amino acid substitutions w101g/ g106k/l107d, e138k, e306g, a315g and d332r, showed decreased binding to t16 mab ( fig 4a) . amino acids 329 and 331 are located within the bc loop of the ediii of jev and amino acid 389 is located within the fg loop of the ediii of jev; these three amino acids are likely candidates to undergo modification during formalin inactivation (fig 4b) . the bc loop of ediii contains critical residues recognized by neutralizing mabs against jev and west nile virus, while the fg loop of ediii is involved in host tropism [55] [56] [57] . therefore, we analyzed the neutralizing ability of t16 by frμnt assay (fig 5a) . it was found that the frμnt 50 potency of t16 mab was 21.8 μg/ml. 4g2 mab neutralizes and inhibits flaviviral infection at the post-attachment step [58] . thus, we used both 4g2 and t16 mabs to determine the mechanism of viral neutralization. mab was used to bind to jev before infecting vero cells in order to carry out a pre-attachment assay. alternatively, mab was added to jevbound cells in order to carry out a post-attachment assay. the neutralizing patterns of 4g2 and t16 mabs were similar regardless of whether either mab was added before or after viral attachment (fig 5b) , which indicates that t16 mab seems to inhibit jev at a post-attachment step. to evaluate the influence of the t16 epitope (ediii 329/331/389) on the immunogenicity of formalin-treated jev antigens, we further investigated igg antibody responses and the properties of antibodies against ediii 329/331/389 in vaccinated mice. female balb/c mice were vaccinated with ucv-nakayama, fiv-nakayama, or ficv and then post-vaccination serum samples were analyzed by the igg-capture elisa using wild-type, ediii 329/331/389-mutated and edii 101/106/107-mutated vlps. the edii 101/106/107-mutated vlps eliminate the immunodominant b-cell epitope, conserved in all flaviviruses as well as inducing cross-reactive, non-neutralizing and/or low-neutralizing antibodies [49, 59] . the total jev-specific igg elicited by all three immunogens were similar (p>0.05) with the average titer end-points being 8.5×10 3 , 1.5×10 4 , and 1.1×10 4 for ucv-nakayama, fiv-nakayama, and ficv-immunized mice, respectively (fig 6a, panel a) . we determined the antibody responses that recognized the edii 101/106/107 epitope and the ediii 329/331/389 epitope by calculation the decreased reactivity titer against edii 101/106/107-mutated and ediii 329/331/389-mutaed vlp characteristics of untreated control nakayama virus (ucv-nakayama) and formalin-inactivated nakayama virus (fiv-nakayama) at 4°c for 49 days. (a) antigenic reactivities of ucv-and fiv-nakayama were monitored by ag-elisa using mhiaf. data are od 450 of mean±sd from two duplicates. (b) infectious viruses were determined by plaque assay after formalin treatment. (c) mabs binding activity of ucv-nakayama and fiv-nakayama were evaluated by ag-elisa. the binding activities were adjusted to antigen concentration according to the od 450 of mhiaf, compared to day 0 (as 100%). data are mean±sd from two duplicates, and the significant difference was indicated as an asterisk (p<0.05). doi:10.1371/journal.pntd.0004167.g002 compared to the wild-type vlp. the titer of antibodies recognizing the edii 101/106/107 epitope was similar (p>0.05) for all serum from mice vaccinated with ucv-nakayama (10 3.9 , range 10 3.2 -10 4.3 ), fiv-nakayama (10 4.1 , range 10 3.7 -10 4.5 ), and ficv (10 3.9 , range 10 3.1 -10 4.5 ) (fig 6a, panel b) . in contrast, the titer of ediii 329/331/389 epitope-specific antibodies was significantly lower (p<0.05) for serum from mice vaccinated with fiv-nakayama (10 2.7 , fiv and ucv jevs detected with t16, 4g2 and 7a6c-5 mabs and (c) quantification of t16 mab binding activity with e of the ucv jevs by the value of ucv jevs shown as 100% of the fiv jevs value. data are mean±sd from two duplicates, and the significant difference was indicated as an asterisk (p<0.05). doi:10.1371/journal.pntd.0004167.g003 (fig 6a, panel c) . based on the above, we suspected that ficv-immunized children might produce a similarly lower proportion of ediii 329/331/389 epitope-specific antibody. twelve ficv-immunized children serum samples were found to show a lower level for the ediii 329/331/389 epitopespecific igg antibodies, namely 23% (10-46%) (s2 table) ; this result closely resembles the antibody reactions elicited in the fiv-nakayama-immunized and ficv-immunized mice. to confirm the results obtained by epitope-specific igg elisa, the fiv-nakayama and ficv immunized mouse serum samples were examined by western blot analysis using the same concentration of wt, edii 101/106/107-mutated vlp and ediii 329/331/389-mutated vlp ( fig 6b) and the results quantified against standardized protein concentrations (fig 6c) . the prm protein of all of the jev vlps, including the wt antigen, the edii 101/106/ 107-mutated antigen and the ediii 329/331/389-mutated antigen, were equally recognized by the anti-jev mhiaf. furthermore, the edii 101/106/107-mutated vlp and ediii 329/331/ 389-mutated vlp could not be recognized or showed significantly decreased recognition with the 4g2 and t16 mabs, namely <1% and 36% reactivity, respectively. the serum collected from mice vaccinated with ucv-nakayama was less able to bind to the ediii 329/331/ 389-mutated vlp (35%) than fiv-nakayama and ficv (65% and 64%, respectively), but this was not true for the edii 101/106/107-mutated vlp (12%, 11%, and 17%, respectively) (fig 6b and 6c ). the results of the epitope-specific igg elisa and western blot analysis are consistent and indicate a stronger immunogenicity of the ediii 329/331/389 epitope on ucv-nakayama than that on fiv-nakayama and ficv. therefore, formalin-inactivated nakayama virus or vaccine in immunized mice was only able to affect the induction of antibodies recognizing the ediii 329/331/389, but was not able to affect the induction of antibodies recognizing the edii 101/106/107 epitope. the protective efficacy of vaccines against jev infection is positively associated with the presence of neutralizing antibodies in mice [60] . based on this we evaluated the correlation between the pre-adsorption neutralizing antibody titers of mouse serum immunized with ucv-nakayama, fiv-nakayama or ficv were similar, with frμnt 50 titers of 52 , 46 , and 52 , respectively (fig 7a) . we then measured the post-adsorption frμnt 50 titers (fig 7b) to determine the contribution of the ediii 329/331/389-specific antibodies to the viral neutralizing activity. the neutralizing antibody titers were lower for serum postadsorbed with the wt jev vlp-expressing cos-1 cells than for serum post-adsorbed with normal cos-1 cells using serum samples elicited by all three vaccines (fig 7b) . however, the post-adsorption serum specimens using jev ediii 329/331/389-mutant vlp-expressing cos-1 cells showed a significant reduction in their neutralizing antibody titers activity when serum from either fiv-nakayama-immunized mice or ficv-immunized mice was used, but not when the serum from ucv-nakayama-immunized mice was used. the differences in neutralizing activity of the serum samples after adsorption with cos-1 cells expressing the wt vlp or ediii 329/331/389-mutant vlp may have been due to the contribution made by ediii 329/331/389-specific antibodies. when the results were fitted using non-linear regression analysis (fig 7c) , this showed that the contribution of ediii 329/ 331/389-specific antibodies to neutralizing antibody activity was proportionally higher (69%, range 62-78%) using the serum from ucv-nakayama-immunized mice than when the serum from fiv-nakayama-immunized mice (38%, range 31-48%) or ficv-immunized mouse serum (44%, range 35-58%) was used. thus, formalin modification of the ediii 329/331/389 epitope would seem to affect the production of neutralizing antibodies. a search for alternative methods to be used for the production of inactivated jev vaccine a previous report has suggested that jev inactivation by formalin at 22°c for 10 days might be more immunogenic than inactivation at 4°c for 49 days [42] . therefore, we asked if inactivation temperature (4°c vs. 22°c) and inactivation duration (49 days vs. 10 days, respectively) is able to influence t16 modification. we measured the t16 mab binding activity of the je nakayama, sa14-14-2, t1p1, and yl2009-4 viruses treated with formalin at 4°c or 22°c for 10 days (fig 8a) . at 10-dc, the t16 mab binding activity against fiv-nakayama, fiv-sa14-14-2, fiv-t1p1, and fiv-yl2009-4 were all lower at 75%, 77%, 63%, and 43% at 22°c than at 4°c, where the results were 94%, 98%, 120%, and 94%, respectively. thus t16 epitope modification is present on fiv-jevs treated either at 22°c for 10 days (remaining 43-77% of t16 mab binding activity) or at 4°c for 49 days (remaining 55-75% of t16 mab binding activity) (figs 2c and 3a) . uv has also been used to inactivate viruses in the past and such uv-inactivated viruses are able to induce protective humoral immunity [61, 62] . surprisingly, uv-inactivated nakayama virus only weakly bound anti-jev mhiaf and t16 mab when assessed by western blot analysis, which indicates that the antigenic structure of nakayama virus might be severely altered by uv irradiation (fig 8b) . hydrogen peroxide (h 2 o 2 ) can be used as a biocide and is known to interact with amino or sulfhydryl groups on antigens. amanna et al. recently reported that h 2 o 2 -inactivated viruses are still able to induce protective cellular and humoral immunity [63, 64] . therefore, we followed their protocol and inactivated the nakayama virus with 3% h 2 o 2 at 22°c from 2 to 8 hr. two-hours of h 2 o 2 treatment reduced viral infectivity by at least 42000-fold to under the detection limitation (fig 8c) . ag-capture elisa revealed that the binding activities of the t16 and other cross-reactive mabs against the ucv-nakayama and the h 2 o 2 -treated nakayama virus were the same after 2-hr of treatment at 22°c. this suggests the antigenic structure of the nakayama virus remained intact after h 2 o 2 inactivation (fig 8d) . several countries, including japan, south korea, and taiwan, have successfully reduced the number of je clinical cases by using inactivated jev vaccines, but more effective and safe alternative vaccines are still needed [65] [66] [67] . the factors that affect the effectiveness and safety of jev vaccines are the virus strain, method for viral cultivation, vaccine purity and vaccine formulation [68] ; however, the effect of formalin inactivation on the quality of vaccine has never been studied. this is important because formalin-induced hypersensitivity has been found associated with risk of enhanced disease during subsequent infection with respiratory syncytial virus (rsv), and formalin inactivation altered the antigenicity of poliovirus [38, 41, 69] . antigenic characterization of formalin-inactivated poliovirus vaccine by using a panel of mabs revealed that modification of antigenicity is time-dependent [38] . using a previously collected panel of anti-flavivirus mabs and the established ag-elisa [49, 53] , we found that only the t16 mab binding domain was time-and temperature-dependently altered by formalin inactivation. this observation suggested it might be valuable to evaluate the effect of residual formalin in formulated bulk on vaccine shelf life in the future. importantly, regardless of the jev strain used, formalin treatment altered the t16 epitope of all tested je viruses. in contrast, epitopes recognized by 2b5b-3 and 2f2 mabs on fiv-nakayama were temporarily modified for specimen collected at early time point. modification of these two epitopes was nakayama strain-specific and was reversible since this phenomenon was only observed in the early time point specimen of formalin-treated nakayama alone. among anti-flavivirus antibodies, most of the virus-specific, non-cross-reactive, and ediiirecognizing antibodies have strongly neutralizing activity, and most of the cross-reactive and edii-or edi-recognizing antibodies have weak or no neutralizing activity [16, 70] . t16 mab is a jev-derived, jev-serocomplex cross-reactive antibody. it shows weakly neutralizing activity at the post-attachment step in vitro. however, antibodies that comprise a large portion of the antibody response after wnv infection have only weak neutralizing activity in vitro but still provide therapeutic protection in vivo via the immune complement system [71] . the amino acid residues in both edii and ediii of the e protein are important to the binding of jev serocomplex cross-reactive mabs [49, 53] . we determined the binding of t16 mab to jev vlps by the amino acid positions edii-104, -329, -331, and -389 but used only ediii 329/331/ 389-mutated vlps to analyze epitope-specific antibody responses because edii-104-mutated vlps showed reduced secretion. interestingly, the t16 epitope overlaps with the jev-specific highly neutralizing e3.3 mab epitope [56] . this result provides additional support that most e-protein epitopes within flaviviruses are overlapping [53] . formalin is known to mainly react with the amino and thiol groups of amino acids to form methylol groups, which is followed by the formation of schiff-base adducts; this reaction is reversible. these schiff-bases adducts can cross-link to functional groups of various amino acids, such as arginine, tyrosine, tryptophan, histidine, glutamine, lysine, and cysteine, forming non-reversible methylene bridges [35] . thus, the epitope of t16 mab, namely glycine 104, serine 329, serine 331 and aspartic acid 389, are likely not directly modified by formalin but are possibly influenced by nearby amino acids, including those at 105, 335, 336, 387, 390, and 391, and such cross-linking might directly or indirectly affect the conformational structure of the t16 epitope. formalin treatment did not alter t16-overlapped epitopes recognized by 4g2 and 6b6c-1. t16 mab might be more sensitive to this formalin-generating modification on the non-overlapped residue(s) essential for t16 recognition. currently we still do not know residues specifically reacting with formalin. structural differences and amino acid variation in flavivirus immunogens, such as whether the virions are mature or immature, vlps, or ediii alone, may also affect the immunogenicity, antibody profile, and neutralizing potency elicited [72] [73] [74] [75] . for example, ediii-reacting antibodies show high neutralizing potency, but the recombinant ediii immunogen induces low avidity and low titers of neutralizing antibodies against the virus [72] . in this study, we found that formalin inactivation altered the structure of the jev e protein and thus affected the profile of induced antibodies. in this study, t16 epitope was the only epitope affected by the formalin inactivation; however, whether the t16 epitope is the only e structure alteration affecting the profile of antibodies elicited by formalin-inactivated vaccines/viruses is unknown because the t16 epitope, ediii 329/331/389, was not directly reactive with formalin. the formalin-modified ediii 329/331/389 region was found less immunogenic and had less of a contribution to the neutralizing activity, despite non-significant differences in neutralizing antibody titers among ucv-nakayama-immunized mice and fiv-nakayama-immunized mice. weak-neutralizing and non-neutralizing epitopes were located in the fusion peptide, and the introduction of mutations into the fusion peptides of the vlp disrupted the binding activity of anti-fusion loop mabs. the fusion peptide mutant reduced the immunogenicity of the fusion peptide but retained its ability to evoke neutralizing antibodies [76, 77] . thus, the formalin-modified region affects the profile of vaccine-induced antibodies and alters the distribution of neutralizing antibodies. we did not determine the effect of formalin inactivation on the tcell response, which needs to be addressed because a negative effect of formalin-inactivation on the influenza-virus t-cell response has been documented and t-cell immunity plays a role in how vaccines protect against jev infection [37, 78, 79] . the use of epitope scaffolds or deglycosylation has successfully exposed immunorepressive and cryptic epitopes and enhanced immunogenicity in hiv or redirected the antibody response in simian immunodeficiency virus [80, 81] . we found the titers of ediii 329/331/ 389-reactive antibodies higher among ucv-nakayama-than fiv-nakayama-or ficv-immunized mice and use of edii 101/106/107-reactive antibodies gave similar results. previously, we found that edii 101/106/107 and ediii 329/331/389 form an overlapped epitope for flavivirus group cross-reactive mabs, such as 4g2 and 6b6c-1 [53] . thus, the edii 101/106/107 region may be less likely to cooperate with the ediii 329/331/389 region in inducing an antibody response when the immunogen been modified by formalin. the formalin-inactivated, lactate dehydrogenase-elevating, virus-elicited antibodies differ from antibodies after natural infection. formalin-inactivated influenza virus could not induce a t-cell response and was less protective in mice against homologous and heterologous influenza virus challenge as compared with γ-ray-inactivated virus [37, 82] . however, another study indicated that the use of low formalin concentrations, short inactivation period, and high incubation temperature improved the immunogenicity of formalin-inactivated jev vaccine and elicited high titers of neutralizing antibodies in mice [42] . here, we showed that the binding activity of t16 mab was reduced more by virus inactivation at 22°c than 4°c for the same treatment duration. surprisingly, uv-inactivated nakayama virus failed to be recognized by mhiaf and t16. adjusting the condition for uv irradiation may maintain the antigenic structure of jev. uv light inactivates virus by cross-linked viral nucleic acid and viral proteins. cross-linked by oxidation between the amino acid residues may increase the susceptibility of protease cleavage [83, 84] , and degradation of aromatic side chain of amino acid and disulfide bond forming cysteine in protein has been indicated after uv treatment [85, 86] . the loss of viral antigenicity was also observed in uv-inactivated virus including poliovirus (showing both antigenic and morphologic change), and influenza a virus (exhibiting low hemagglutination activity) [37, 87] . murray valley encephalitis virus, belonging to jev serocomplex, inactivated with uv showed lower immunogenicity compared to non-infectious vlp but the uvinduced antigenic change wasn't described [88] . in conclusion, formalin and uv inactivation alter the antigenic structure of e protein in jev and reduce the immunogenicity of associated vaccines. h 2 o 2 inactivation seems to be a better alternative for jev vaccine production. it maintained the antigenic structure of e protein, measured by a panel of mabs. further study should focus on identifying an optimal inactivation procedure and testing the immunogenicity of h 2 o 2 -inactivated jev vaccine. finally, to prevent unexpected modification of the various epitopes on the jev vaccine during inactivation, a non-infectious jev vlp or dna vaccine should be developed. formalin inactivation introduces an antigenic modification that affects the ediii of jev and thus distorts the profile of vaccine-induced neutralizing antibodies. antigenic-stable inactivation methods are needed to develop better-inactivated jev vaccines. table. epitope-specific antibody response in serum samples collected from ficv-immunized children. 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heterologous neutralization antibody responses after immunization with japanese encephalitis vaccine among taiwan children manipulation of immunodominant dengue virus e protein epitopes reduces potential antibody-dependent enhancement sculpting humoral immunity through dengue vaccination to enhance protective immunity protective mechanisms induced by a japanese encephalitis virus dna vaccine: requirement for antibody but not cd8(+) cytotoxic t-cell responses immunization with plasmid dna encoding the envelope glycoprotein of japanese encephalitis virus confers significant protection against intracerebral viral challenge without inducing detectable antiviral antibodies envelope deglycosylation enhances antigenicity of hiv-1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies elicitation of structure-specific antibodies by epitope scaffolds formalin inactivation of the lactate dehydrogenase-elevating virus reveals a major neutralizing epitope not recognized during natural infection oxidation of virus proteins during uv(254) and singlet oxygen mediated inactivation uv-photolysis of amino acids and peptides. cleavage of peptide bond during laser irradiation identification of a single tryptophan residue as critical for binding activity in a humanized monoclonal antibody against respiratory syncytial virus ultraviolet illumination-induced reduction of alpha-lactalbumin disulfide bridges biophysical properties of poliovirus particles irradiated with ultraviolet light cross-protective and infection-enhancing immunity in mice vaccinated against flaviviruses belonging to the japanese encephalitis virus serocomplex we thank dr. chen, li-kuang (college of medicine, tzu-ch university, hualien, taiwan) for kindly providing the t16 mab used in this study, and dr. ralph kirby (national yang-ming university, taiwan) for the assistance on english writing while preparing this manuscript. conceived and designed the experiments: ssc gjjc ycf. performed the experiments: ycf ssc. analyzed the data: ycf ssc. contributed reagents/materials/analysis tools: hcc lkc. wrote the paper: ycf ssc gjjc. key: cord-001365-6u80p5sj authors: weger-lucarelli, james; chu, haiyan; aliota, matthew t.; partidos, charalambos d.; osorio, jorge e. title: a novel mva vectored chikungunya virus vaccine elicits protective immunity in mice date: 2014-07-24 journal: plos negl trop dis doi: 10.1371/journal.pntd.0002970 sha: doc_id: 1365 cord_uid: 6u80p5sj background: chikungunya virus (chikv) is a re-emerging arbovirus associated with febrile illness often accompanied by rash and arthralgia that may persist for several years. outbreaks are associated with high morbidity and create a public health challenge for countries affected. recent outbreaks have occurred in both europe and the americas, suggesting chikv may continue to spread. despite the sustained threat of the virus, there is no approved vaccine or antiviral therapy against chikv. therefore, it is critical to develop a vaccine that is both well tolerated and highly protective. methodology/principal findings: in this study, we describe the construction and characterization of a modified vaccinia virus ankara (mva) virus expressing chikv e3 and e2 proteins (mva-chik) that protected several mouse models from challenge with chikv. in particular, balb/c mice were completely protected against viremia upon challenge with chikv after two doses of mva-chik. additionally, a129 mice (deficient in ifnα/β) were protected from viremia, footpad swelling, and mortality. while high anti-virus antibodies were elicited, low or undetectable levels of neutralizing antibodies were produced in both mouse models. however, passive transfer of mva-chik immune serum to naïve mice did not protect against mortality, suggesting that antibodies may not be the main effectors of protection afforded by mva-chik. furthermore, depletion of cd4(+), but not cd8(+) t-cells from vaccinated mice resulted in 100% mortality, implicating the indispensable role of cd4(+) t-cells in the protection afforded by mva-chik. conclusions/significance: the results presented herein demonstrate the potential of mva to effectively express chikv e3-e2 proteins and generate protective immune responses. our findings challenge the assumption that only neutralizing antibodies are effective in providing protection against chikv, and provides a framework for the development of novel, more effective vaccine strategies to combat chikv. chikungunya virus (chikv; togaviridae, alphavirus), the etiologic agent of chikungunya fever is an emerging pathogen that has recently caused several severe outbreaks throughout africa and southeast asia [1] [2] [3] . large outbreaks have occurred on la réunion island (an overseas department of france), mauritius, sri lanka, and india, among others [4] . in addition, autochthonous transmission has been seen in europe, with outbreaks occurring in both italy and mainland france [5] , suggesting temperate climates can support virus transmission. furthermore, as of january 2014, the european centre for disease control has confirmed 70 cases of chikv on the caribbean islands of st. martin, martinique, guadeloupe, and saint barthelemy, with many more suspected, indicating spread to continental america is possible. chikv is transmitted to humans by aedes aegypti and aedes albopictus mosquitoes, the latter of which has been an important vector in many of the recent outbreaks due to mutations in the envelope genes of the virus that allow for more efficient transmission [6] [7] [8] [9] . chikv causes a dengue-like illness associated with fever, rash and joint pain and was first described in modern day tanzania in 1952 [10] . the term chikungunya is derived from the makonde word meaning ''that which bends up'' and describes the posture of an infected individual [11] . recently, the u.s. army developed a live-attenuated vaccine candidate, called chik 181/clone 25 or 181/25, but it caused transient arthralgia in a small number of volunteers during phase ii clinical trials [12, 13] . experimental subunit [14] , recombinant viruses [15] [16] [17] , and vlp [18] based vaccines have also been described which are currently at various stages of preclinical or clinical development, however there currently are no licensed vaccines or antiviral treatments available for chikv. an alternative approach for developing chikv vaccines is the use of viral vectors. a complex adenovirus expressing the complete chikv structural poly-protein has been described and was shown to be immunogenic and protective in mice [19] . however, safety concerns regarding adenovirus based-vectors may limit this approach [20] . in contrast, modified vaccinia virus ankara (mva) has been tested in over 120,000 humans and was proven to be highly safe and effective in protecting against smallpox [21, 22] . mva was attenuated by over 500 passages of vaccinia virus (vacv) in chicken embryo fibroblasts (cefs), which resulted in large deletions of its genome that restricted its host-range [23, 24] . during passaging, mva lost the ability to productively infect mammalian cells, leading to abortive replication [25] . vacv, which is far more reactogenic than mva [26, 27] , has been used previously against other alphaviruses, namely sindbis and venezuelan equine encephalitis (veev) viruses, providing robust immunity against the latter, suggesting poxviruses as suitable vectors against alphaviruses [28] [29] [30] [31] [32] [33] . despite the presence of high levels of neutralizing antibodies elicited by most of the vacv vectored veev vaccine candidates, they were ineffective in providing protection against airborne infection, suggesting they were unable to elicit a sufficient t cell mediated immune response, which has been shown to be critical for protection against lethal veev encephalitis [34, 35] . mva, like its parent virus vacv, has been extensively tested as a vaccine vector, expressing viral, bacterial or parasite antigens and has been shown to induce both humoral and cell-mediated protective immune responses [25, [36] [37] [38] [39] [40] [41] [42] . furthermore, mva can be delivered effectively by different routes and has much greater stability than most other live virus based vaccine approaches [43] . additionally, because mva undergoes only abortive replication in mammalian cells, vector stability is not a problem as only one infection cycle occurs [25] .therefore, an mva vectored chikv vaccine would be an attractive option for resource-limited countries, where the majority of chikv infections occur. additionally, volz and sutter (2013) have targeted mva as an ideal vector for safe next generation vaccines, with chikv being mentioned specifically as an attractive candidate pathogen [44] . furthermore, garcia-arriaza et al. (2014) successfully used mva for expression of the entire chikv structural protein as a vaccine candidate, indicating mva can provide effective immunity against this virus [45] . in this report, we describe the construction and immunological evaluation of an mva-based chikv candidate vaccine based on the e3-e2 proteins. the vaccine was tested in two mouse modelsone immunocompetent and the other lacking a/b interferon signaling (a129)-and was uniformly protective. mice deficient in a/b interferon signaling have been used for many viruses and have been shown repeatedly to be a good model for chikv, providing many similarities to human infection [46] [47] [48] . complete protection against mortality was provided when mva-chik was administered in a prime-boost regimen. in addition, it provided 80% protection against mortality in this highly immunocompromised mouse model after only 11 days post-vaccination. surprisingly, while playing a role, neutralizing antibodies did not appear to be necessary for protection against chikv, contrary to other recent reports [49, 50] . most importantly, depletion of cd4 + t cells in vaccinated mice resulted in loss of protection, with 100% succumbing to infection upon challenge with wild-type chikv, indicating an indispensable role of mva-chik immune cd4+ t cells in protection. this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the iacuc protocol (protocol #v01380) was approved by the institutional animal care and use committees of the university of wisconsin. african green monkey kidney cells (vero, atcc #ccl-81) and baby hamster kidney cells (bhk-21, atcc # ccl-10) cells were maintained in dulbecco's modified eagle medium (dmem; gibco, carlsbad, ca) supplemented with 5% fetal bovine serum (fbs), 100 u/ml of penicillin, 100 mg/ml of streptomycin, and 2.5 mg/ml amphotericin b, and incubated at 37uc in 5% co 2 . chicken embryo fibroblasts (cefs) were obtained from charles river (charles river laboratories international, inc., wilmington, ma) and maintained in optimem (invitrogen, carlsbad, ca) supplemented with 10% fbs under the same conditions. chikungunya virus strain la reunion (chikv-lr; genbank: dq443544.2) was used for both the construction of recombinant poxviruses and challenge experiments. the virus was kindly provided by dr. scott weaver (university of texas medical branch, galveston, texas). the chik-ires vaccine candidate has been previously described [51] and was used in this study as a positive control. briefly, the vaccine was constructed by replacing the subgenomic promoter in a cdna chikv clone with an internal ribosome entry site from encephalomyocarditis virus (emcv-ires) and has shown to be effective and safe in mice [51] and non-human primates [52] . the mva virus used in these studies was obtained through bei resources (niaid, nih,ref # nr-727). a recombinant mva virus expressing gfp (herein called mva-gfp) was based on the wild-type mva and its construction has been previously described [37, 53] . for the production of recombinant mva vaccines, the chikv e2 and e3 genes (hereafter called p62) were pcr amplified using phusion high-fidelity dna polymerase (new england biolabs, ipswich, ma) and cloned into a specially modified poxvirus transfer vector, pi2-red [37] . e2 was chosen because it has been previously demonstrated that most neutralizing antibodies mapped to epitopes in this region [49, 54, 55] . e3 has not been strongly associated with protection; however, it was included to allow proper folding of the e2 protein [56] . the expression of p62 was controlled by a synthetic early/late vaccinia virus promoter and used the vaccinia virus transcription terminator, which have been previously described [57] . the vector used in this study contained flanking sequences that when transfected into mva-gfp infected cells can recombine into the deletion iii region of the viral chikungunya virus (chikv) has recently re-emerged from africa to cause disease outbreaks in asia, europe, and more recently the caribbean. the virus is transmitted by aedes mosquitoes and causes a disease that is characterized by high fever and incapacitating joint pain that can cause great personal and economic loss. at present, no approved vaccine or antivirals are approved against chikv. in this study, we developed a novel chikv vaccine that is vectored by modified vaccinia virus ankara (mva), an attenuated vaccine vector which has been shown to be safe in humans and induce a strong immune response. the vaccine expresses the e3 and e2 proteins of chikv, the latter of which is thought to be the main mediator of protection. the vaccine was effective in two mouse models and protected against all markers of disease tested despite the absence of high levels of neutralizing antibodies, the gold standard of protection. depletion of cd4+ t cells from vaccinated mice resulted in loss of protection, implicating these cells in the protection induced by the vaccine. genome [58] . this vector contains dsred under the control of a late p11 promoter to allow for visual based selection and permits an easy distinction between recombinant (red) and wild-type (green) viruses (fig. 1a) . recombinant mva-chik viruses were generated as described previously [59, 60] . briefly, cefs were seeded into six-well plates the day before transfection and then infected at a multiplicity of infection (moi) of 0.05 pfu/cell for 1.5-2 hours with mva-gfp. cells were then washed with 16 pbs and transfected with appropriate transfer vectors using fugene hd (roche diagnostics, indianapolis, in) following manufacturer's protocol. cells were monitored for the presence of red fluorescence 24 hours after transfection. at 48-72 h post-transfection, monolayers were harvested, centrifuged at 5006g for 5 min at 4uc and cells disrupted by freeze-thaw (3 times) followed by sonication (2 times for 15 s using a cup sonicator). the disrupted cell extracts containing recombinant viruses expressing dsred were plated onto fresh cef cells and overlaid with 0.8% agarose. after 48-72 h, recombinant virus-generated plaques were detected by observing fluorescence and picked into 300 ml media with a sterile filter pipette tip. the cell/virus samples were subjected to freeze thaw and sonication (as described above) and plated to continue passaging. plaques were passaged until no wild-type (gfp expressing) virus was observed at which point pcr was performed to confirm the presence of only recombinant virus [61] . high titer virus stocks were prepared from pcr positive cultures and recombinant mva-chik viruses were further characterized. pcr analysis was performed on final virus stocks to ensure genetic homogeneity and stability. dna was extracted using the quick-gdna miniprep kit from zymo research per manufacturer's instructions (zymo research, irvine, ca). pcr was then performed using phusion polymerase with deletion iii specific primers (forward 59-atgcggcacctctcttaa-39, reverse 59-tgggctccttataccaagca-39). western blot analyses were used to determine the in vitro expression patterns of mva-chik constructs. for this purpose, bhk-21 cells were seeded at 3.0610 5 cells/well into six-well plates and 24 hours (hr) later infected at an moi of 5 pfu/cell in optimem in the absence of fbs. at 24 hr after infection, cells were harvested and lysed using radio-immunoprecipitation assay buffer (ripa; 150 mm nacl, 1.0% np-40, 0.5% sodium deoxycholate, 0.1% sds, and 50 mm tris, ph 8.0) at 4uc for 30 minutes (min) under gentle agitation. lysates were then centrifuged for 20 min at 12,000 rpm and supernatant was collected for further analysis. supernatants were diluted into 26 laemmli buffer (bio-rad, richmond, ca), heated at 95uc for 5 min and 50 ml were loaded into a bio-rad 4-20% precast gel. gels were transferred to a nitrocellulose membrane using trans-blot turbo blotting system following manufacturer's instructions. polyclonal serum obtained from specific-pathogenfree (spf) rabbits inoculated with a vaccine strain of chikv was used to probe blots at a dilution of 1:5000 and developed using bcip/nbt alkaline phosphatase system (bio-rad). bhk-21 cells were used for immunocytochemistry (icc) experiments. one day prior to infection, 2610 5 cells were plated onto glass coverslips, which had been placed inside of 24 well tissue culture plates. the following day, the cells were infected at an moi of 10 pfu/cell. infection was allowed to progress for 24 hrs and then fixed using 2% paraformaldehyde (pfa) solution in phosphate buffer. for permeabilization, cells were treated with 0.5% triton x-100 for 10 min at 4uc, followed by 50 mm nh 4 cl in pbs for 10 min at room temperature (rt), and then blocked with pbs containing 10% bovine serum albumin (bsa) for 3 to 4 hr at rt. cells were then incubated overnight at 4uc with a 1:1000 dilution of anti-chikv polyclonal rabbit serum. cells were subsequently washed three times with pbs containing 0.05% tween and then incubated with a 1:2000 dilution of anti-rabbit igg alexafluor-488 for 45 min at rt. cells were mounted using vectashield mounting medium (vector laboratories). images were acquired using an evos microscope with attached camera. elisas were performed as described by brewoo et al., 2010 [37] . briefly, 96-well elisa plates were coated with purified chikv (0.5 mg in 100 ml carbonate buffer, ph 9.6 per well) at 4uc overnight. coated plates were washed twice with 0.05% tween-20 in pbs (washing buffer) and incubated with blocking buffer (1% bsa in pbs) at rt for 1 hr. serum samples then were serially diluted from 1:100-1:12,800 in elisa diluent (0.1% bsa in washing buffer) and added in triplicate to the prepared elisa plates and plates were incubated at rt for 1 hr. known negative (uninoculated) and positive serum samples from mice inoculated with chik-ires from previous studies were used as controls. after washing, 100 ml per well of a 1:10,000 dilution of horseradish peroxidase (hrp)-conjugated rabbit anti-mouse igm+g (abcam inc, cambridge, ma) was added to each well and incubated at rt for 1 hr. plates were washed, and 100 ml per well of tetra-methyl-benzidine (tmb) chromogen (sigma, st louis, mo) was added to each well and incubated in the dark for 5 min. the reaction was then stopped by adding 100 ml per well of 2 mm h 2 s0 4 . colorimetry was measured using an elx800 absorbance microplate reader (biotek, winooski, vt) at test wavelength of 450 nm and a reference wavelength of 630 nm. the highest dilution that was positive (exceeded the mean of known negative serum samples plus three standard deviations) was considered the endpoint, and its reciprocal value was recorded as the titer. groups of four-to six-week-old female balb/c mice (harlan sprague dawley, indianapolis, in) or six-to ten-week-old mixed the different areas where deletion occurred in the vaccinia virus genome to create mva are shown. mva-chik was created by transfecting the plasmid containing chikv p62 under control of a se/l promoter which also expressed dsred2 under control of a p11 promoter. this plasmid contains flanking regions to deletion iii (deliii) which is where the recombinant gene was inserted into the genome with a fluorescent marker (a). pcr analysis of the deliii region. viral dna was extracted from purified, final virus stocks of mva-chik, mva-gfp and mva-wt. pcr was performed using primers specific for the deliii flanking regions. a dna ladder is included for comparison of size (b). monolayers of cef cells were infected with recombinant mva-chik viruses at moi of 5 or 10 pfu/cell for western blot or immunostaining, respectively. after 24 h post infection, cells were harvested and subjected to sds-page followed by western blot analysis (c) or fixed with 2% pfa as described in the methods. both the cellular pellet and supernatant were analyzed for expression of chikv e3/e2. the order is the same for both and is as follows, wild-type chikv (1), mva-chik (2), mva-gfp (3), and mock infected cells (4) . following fixation, cells for immunostaining were either allowed to remain intact or permeabilized with triton x-100 (d). anti-chikv polyclonal rabbit was used for primary staining for both assays. green represents chikv e2 positive cells, red is dsred protein produced by the virus and blue is nuclear staining with hoechst. doi:10.1371/journal.pntd.0002970.g001 gender a/b interferon signaling deficient mice (a129) received either primary only or primary and booster immunizations (28 days apart) with each vaccine candidate via intradermal (i.d.) injection into the hind, left footpad. a dose of 1610 7 tcid 50 units in 50 ml was used for all mva vaccinations. the dose chosen is either lower than or consistent with many previous reports of mva vectored vaccines [62] [63] [64] . negative control groups were immunized with mva-gfp at the same dose. as a positive control, mice were vaccinated with experimental control vaccine virus chik-ires [17] at a dose of 10 4 tcid 50 units. at either 11 days post-prime or two weeks post-boost (where applicable), all animals were challenged with wild-type chikv-lr by i.d. inoculation of 10 4 (balb/c) or 10 2 (a129) tcid 50 units in 50 ml into the hind, left footpad. mice were bled prior to boost and prior to challenge following vaccination to monitor levels of neutralizing and anti-virus antibodies. passive transfer studies were performed using a129 mice as previously described [37] . briefly, serum was collected from vaccinated balb/c or a129 mice and equal volumes from each sample were used to create a pool of sera for each mouse strain separately. 100 ml (balb/c) or 200 ml (a129) of this inoculum were then injected into naive mice intraperitoneally (i.p.). serum from chik-ires vaccinated a129 mice (100 ml) was used as a positive control. 24 hours after passive transfer, mice were challenged in the same manner as described above. following challenge, mice were bled three days consecutively to monitor viremia via the maxillary vein. with the exception of the balb/c experiment, all animal experiments were repeated at least once, with similar results. for depletion studies, groups of six-to ten-week-old a129 mice the depletion efficiency in pbmcs on the day of infection was more than 99% for both t cell subsets as assessed by flow cytometry by staining with anti-mouse cd4 fitc (rm4-5), antimouse cd8a percp (53-6.7) mabs (from bd bioscience) (data not shown). mice were monitored for morbidity and mortality for two weeks. histopathology was performed on tissues collected from a129 mice 7 days post infection (d.p.i.). footpad and leg tissues were severed from euthanized mice, cut in half to expose the tissues, and fixed in 4% buffered pfa for two days. hind limbs were then decalcified in 4% pfa containing 14% edta for 3-4 weeks, changing decalcification solution weekly. tissues were paraffin embedded, sectioned and stained with hematoxylin and eosin (h&e). pictures were taken using a sony nex-5n camera attached to a nikon microscope. blood samples collected from mice following challenge were used to assess viremia. viremia was assessed via end point dilutions in 96 well plates seeded with vero cells (seeded the night before at 2610 4 cells/well) and was expressed as tissue culture infectious dose 50 (tcid 50 ) per ml. for prime only studies a serum sample was taken ten days post-vaccination to measure neutralizing antibodies. for studies with prime and boost serum samples were collected on days 28 and 42 post-primary vaccination. neutralization titers were determined using a tcid 50 based assay that was modified from grosfeld et al. [65] . briefly, sera were incubated at 56uc for 60 min to inactivate complement and then serially diluted two-fold starting at a dilution of 1 in 5. dilutions were performed in dmem as described above except containing only 2% fbs. subsequently, 100 tcid 50 units of chikv were added to each well in the same dilution media. the virus/serum mixture was then incubated at 37uc for 1 hr. following incubation, 100 ml of the mixture was added to 96 well plates seeded with vero cells and monitored for 3-4 days for presence of cytopathic effects (cpe). t cell responses were analyzed as previously described [50, 53] . briefly, a129 mice were euthanized 2 weeks post-boost. after red blood cell (rbc) lysis, single-splenocyte suspensions were resuspended in rpmi-1640 medium supplemented with 10% fbs 16 penicillin/streptomycin and 0.14 mm of b-mercaptoethanol. splenocytes were stimulated with three chikv peptide pools that cover the entire protein sequences of e3, e2 and nsp2, respectively. the peptide pools were used at a concentration of 1 mg/well separately in 200 ml total volume for 16 h in the presence of brefeldin a. peptide pools were a generous gift from dr. daniel streblow [66] and were synthesized by thermo fisher scientific. cells were stained intracellularly for ifn-c apc (xmg1.2), il-2 pe (jes6-5h4), tnf-a pe (mp6-xt22) and cd40l (mr1) after surface staining of cd4 fitc (rm4-5) or anti-mouse cd8a percp (53-6.7). the samples were acquired on a bd facscalibur and analyzed with flowjo v7.6.5 (tree star). graphpad prism 6 software (la jolla, ca) was used for all statistical analyses. statistical analysis of viremia levels were performed using an unpaired t-test with welch's correction for unequal variance. survival analysis was performed to assess vaccine effectiveness against; reported p-values are from the mantel-cox test. genetic homogeneity of final virus constructs was analyzed by pcr. purified viral dna was used as a template for amplification by primers specific for the flanking regions of deletion iii. the presence of e3/e2 was confirmed in mva-chik by a band at the expected size of roughly 3 kb (fig. 1b) which indicated the maintenance of the insertion throughout plaque selection. mva-gfp (,1500 bp) and wild-type mva (657 bp) were included as controls. in addition, dna sequencing confirmed that no genetic alterations were present in the final construct (data not shown). the expression of p62 antigen by the mva-chik recombinant viruses and wild-type chikv was monitored by immunoblot analysis. cell pellet lysates and supernatants from infected cefs were tested for protein expression (fig. 1c) . the p62 (62 kd) protein was detected at 24 hr p.i. in both wild-type chikv and mva-chik infected cells, but not in mva-gfp or mock infected cells. interestingly, a cleaved e2 protein (,50 kd) was detected in wild-type infected cells, but not in the mva-chik lysates, suggesting the lack of furin cleavage and release of the e3 peptide from the p62 precursor protein. expression of e2 was observed in the supernatant of chikv infected cells, but there prior to boost (where applicable) and challenge, mice were bled and serum was monitored for both total ig(g+m) and neutralizing activity by tcid 50 was no detection of e2 expression in mva-chik, suggesting that the protein was not being secreted. to determine the cellular localization of chikv proteins, mva-chik infected cells were fixed and then probed for production of chikv e3/e2 proteins using polyclonal serum. in order to determine if the e2 protein was reaching the surface of the cells, a single cell set was permeabilized while the other set remained intact. permeabilized cells stained much brighter than those that were not (fig. 1d) . this indicated that chikv e3/e2 proteins were being maintained inside the cell and not reaching the cell surface as is observed with wild-type chikv infection. to evaluate the protective efficacy of the mva-chik vaccine, an active immunization study was conducted in a129 mice. this mouse strain is more sensitive to chikv infection and experience high viremia, footpad swelling and succumb to infection [51] compared to immunocompetent strains of mice. mice were vaccinated as described previously and then challenged with wt-chikv. mva-chik or mva-gfp vaccinated mice had background levels of neutralizing antibodies, whereas all chik-ires vaccinated mice seroconverted (fig. 2a) . serum collected from these mice post-prime and post-boost was tested for total anti-virus ig(g+m) antibodies by elisa. a clear booster effect was seen in mva-chik vaccinated mice, as the differences between post-prime and post-boost samples were highly significant (p,0.001) (fig. 2b) . this difference was not as profound with chik-ires vaccinated mice as they had high levels of anti-virus antibodies even prior to boost. mva-chik and chik-ires groups were protected 100% from lethal challenge after prime and boost (fig. 2c ). in addition, mva-chik mice were completely protected against both viremia and footpad swelling (figs. 2d-e) . next, we compared by histopathology the footpads of mva-chik and mva-gfp vaccinated a129 mice. vaccinated mice were challenged in the same way described above and subsequently were euthanized seven d.p.i. to assess local tissue damage. following infection, footpads from mva-chik vaccinated mice displayed mild inflammation with limited muscle damage (fig. 3a) . in contrast, footpads from mva-gfp mice exhibited severe necrotic muscle degeneration with edema, consistent with previous reports from unvaccinated mice (fig. 3b) [47, 67] . the immunocompetent balb/c mouse model was selected to evaluate the immunogenicity of mva-chik as it has been used previously with mva and chikv [37, 53, 68] . following vaccination, low levels of neutralizing antibodies were detected in mice immunized with mva-chik (fig. 4a) . immunized mice were then challenged with wild-type chikv to determine protection against viremia. viremia was not detected in any of the mva-chik vaccinated mice (n = 6) (fig. 4b) . in contrast, all mva-gfp immunized mice had significant levels of viremia (p = 0.027). to determine whether antibodies present in balb/c (avg. neut. titer of 9.2) or a129 (undetectable neut. titer) vaccinated mice were sufficient for protection against chikv, pooled immune serum was passively transferred into naïve a129 mice. as a positive control, an additional group of mice was treated in the same manner with a pool of serum (avg. neut. titer of 64) from a129 mice vaccinated with chik-ires candidate vaccine. all of the mice treated with either anti-mva-chik or mva-gfp immune serum had high levels of viremia and succumbed to infection (fig. 4c-d) . viremia in mice passively transferred serum from mva-chik vaccinated a129 was slightly reduced on day 2 post-infection, however this was not significant (p = 0.11). in contrast, chik-ires immune serum provided full protection against viremia and mortality (figs. 4c-d) . to determine the role of t cells in protection we first investigated whether mva-chik could elicit a t cell response in a129 mice by measuring cytokine production. upon in vitro restimulation with a chikv peptide pool that covers the protein sequence of e2, mva-chik immune cd4 + t cells produced the cytokine ifnc and upregulated co-stimulatory molecule cd40l at significantly higher levels as compared to mva-gfp vector control (p = 0.02 and 0.01, respectively) ( fig. 5a-b) . no response was observed when mva-chik splenocytes were stimulated with peptide pools against either e3 or nsp2 (viral protein negative control) (fig. 5c-d) , suggesting the immune response is specific to e2. in addition, there were no significant differences in other cytokines monitored, including tnfa, il-2, il-4, il-6, il-10, and il-17a (data not shown). furthermore, none of the cytokines tested showed a difference between the two groups in immune cd8+ t cells (data not shown). similar results were obtained when mva-chik immune splenocytes were stimulated with live or inactivated whole virus preparations (data not shown). to test whether mva-chik-specific immune cd4 + or cd8 + t cells were indeed protective, we depleted cd4 + or cd8 + t cells from vaccinated mice prior to challenge with wt-chikv. mice depleted of cd8 + t cells or control non-depleted mice had no footpad swelling and survived the challenge (fig. 6a-b) . in contrast, cd4 + depleted mice along with mva-gfp controls succumbed to infection (fig. 6b) . mice depleted of cd4 + t cells also had high levels of viremia and footpad swelling, similar to the mva-gfp controls (fig. 6a-c) . interestingly, cd8 + t cell depleted vaccinated mice remained healthy without footpad swelling throughout the duration of the study despite developing a low level of viremia three d.p.i. the recent re-emergence of chikv has resulted in several explosive outbreaks and underscores the need for an effective vaccine. several groups have recently employed varying strategies to construct effective vaccines against chikv. live-attenuated, inactivated, adenovirus vectored, dna, and vlp based strategies all have been tested and have been shown to be effective in providing protection against chikv [12, 13, 68, 69] . despite promising results, these vaccine candidates all have some drawbacks. safety, stability, and production of a broad long-lived immune response are necessary for any effective vaccine. safety concerns remain for both live-attenuated and adenovirus based approaches. inactivated and vlp based approaches require adjuvants and normally elicit weak t cell responses. in contrast, mva has been shown to be safe in thousands of human patients micro-neutralization assay (a) and elisa (b), respectively. prime only mva-gfp data is not shown because it does not differ with the prime & boost groups. mice were monitored for 14 days following challenge for survival (c) and footpad swelling (d). the first three days following challenge viremia levels were measured via tcid 50 (e). the dotted line indicates the limit of detection of the assay. doi:10.1371/journal.pntd.0002970.g002 and also in every animal tested [21, 22, 58] . furthermore, mice deficient in a/b/c interferon signaling experienced no clinical symptoms following vaccination with mva-chik (data not shown) and we have previously shown other mva constructs to be safe in highly immunodeficient scid mice [37] . in addition, the stability of poxviruses and mva's ability to be lyophilized are optimal for use in developing countries where the vaccine is needed most and cold-chains cannot be reliably maintained. mva also has been shown to stimulate a broad immune response, including the induction of cellular immunity [62, 70] . here, we describe the construction, expression, and preclinical efficacy and safety of a novel mva-chik vaccine candidate. balb/c mice, which are immunocompetent, were 100% protected from viremia upon challenge. a129 mice, which are deficient in a/b interferon signaling, and therefore highly susceptible to chikv infection, were also completely protected against challenge with wtchikv after prime and boost vaccinations. in addition, 80% of mice were protected from lethal challenge following prime only vaccination with mva-chik. despite the robust protection observed, mice did not produce a strong neutralizing antibody response following vaccination. although vaccinated a129 mice developed undetectable levels of neutralizing antibodies, they did produce a significant amount of anti-virus antibodies, which for other alphaviruses have been shown to be protective [71] [72] [73] . a129 mice passively transferred mva-chik immune serum from either balb/c or a129 mice were not protected against mortality, or footpad swelling following challenge, despite a slight reduction in viremia 2 days postinfection in mice transferred a129 mva-chik immune serum (p = 0.11). this suggests antibodies induced by vaccination were not sufficient to provide protection in this manner. this could be due to the fact that passively administered immune serum is significantly diluted in the circulation of naïve mice. further evidence that antibodies are not sufficient for protection alone was provided by depletion studies conducted in mva-chik vaccinated mice. when immune cd4 + t-cells were depleted, despite the presence of full levels of circulating anti-virus antibodies, mice were 100% susceptible to lethal infection. if anti-virus antibodies were essential for protection a reduction in mortality or viremia would be expected in the cd4+ depleted mice, which was not observed. these data suggest that mva-chik is providing protection by a novel mechanism that is not based solely on vaccine induced serum circulating antibodies. while antibodies do not protect via passive transfer or during depletion, it remains unclear what role they may be playing during infection of an mva-chik immunized mouse. immunocytochemistry and western blot analysis suggested chikv p62 protein was being maintained inside of the cell and that furin cleavage was not occurring to separate e3 from e2, respectively. p62 was chosen due the presence of immunodominant epitopes and the fact that previous reports have shown that it could be an effective immunogen against other alphaviruses [71, 74] . in addition, it has been previously observed that when expressed by a plasmid, the presence of e1 is not a requirement to allow e2 to be placed on the outside of the plasma membrane [75, 76] . barth et al. (1997) also demonstrated that in the context of an e1 deleted semliki forest virus, p62 is not transported efficiently to the plasma membrane while e2 alone is [77] . therefore, absence of e2 on the surface of infected cells is not completely unexpected and may have proven to be serendipitous in allowing the production of a more robust cd4 + immune response. this could provide an explanation for the lack of a strong humoral immune response, as antibodies would have limited access to this intracellular protein. in addition, an improperly folded protein would be expected to produce many virus binding antibodies but few that are neutralizing, which our data suggest. a recent report by metz et al. (2013) , suggested that e2 produced in combination with e1 in a baculovirus system is more immunogenic (i.e., produces higher neutralizing antibodies) than either protein alone [78] . this might suggest that e1 and e2 are more efficient together to obtain a robust neutralizing antibody response and might explain why mva-chik produces few neutralizing antibodies. analysis of ex vivo cellular responses indicated that mva-chik induced antigen-specific cd4 + t cells but not cd8 + t cells, suggesting that immune cd4 + t cells are the main effector cells. this is supported by the cellular depletion studies that showed the protective role of cd4+ t-cells. t-cell mediated protection has previously been demonstrated with another alphavirus, veev [79] . yun et al. (2009) were able to adoptively transfer cd4 + t-cells and obtain protection after multiple immunizations, where passive transfer of hyper-immune serum was not protective. interestingly, they did not observe a difference in viral titers in the brain from surviving mice, while mva-chik vaccination completely protected against viremia in our studies. recent studies based on another vaccine candidate, chik-ires, have shown that a correlate of protection based on antibodies can be established for chikv [50] . herein, we present data that suggest that this is specific to the vaccine being used and that there may be more than one way to provide protection against chikv. mva-chik selectively expresses e2-e3 proteins. it could be argued that the processing of this virus is substantially different than the live attenuated chik-ires vaccine or wildtype chikv. as a result, during infection a different set of epitopes are generated that may well be protective via alternative mechanisms. in another report, using mice deficient in either bcells or cd4 + t cells, data were presented that suggested that antibodies are essential in controlling chikv infection and that cd4 + t cells may exacerbate disease [49, 80] . hawman et al. (2013) found similarly, that mice deficient in both b and t-cells had less severe tissue pathology early after chikv infection when compared to wild-type controls [81] . however, in the same report it was demonstrated that virus was inefficiently cleared in the absence of lymphocytes, suggesting a possible dual role for these cells during early chikv infection where some tissue damage is allowed in order to control viral dissemination. while we observe very little evidence of tissue damage in vaccinated mice, it is possible that mva-chik induced specific cd4 + t-cells which are able to effectively control viral replication while minimizing collateral tissue injury. it remains possible that cd4 t-cells are required for recall responses to produce antibodies following challenge, although this is unlikely as mice would be expected to have early viremia, which we did not observe in vaccinated mice (fig. 2e) . currently, we are conducting studies to determine the role of cd4 + t cells in protection. our data are also contrary to the previous report by teo et al. (2013) , where no footpad swelling was observed upon antibody depletion of cd4 + t cells during chikv infection [80] . however, their studies were performed in naïve mice with a functional interferon system, in contrast, our studies used vaccinated mice without proper ifn a/b signaling, and thus might account for the differences observed. additionally, recent studies in humans show that there is a strong cd4+ ifnc positive t-cell response directed against the e2 protein, suggesting it might be playing a role in the protective immune response against the virus [82] . therefore, the data presented here may be relevant to human infection, and suggest that antibodies may not be the only mediator of protection against alphaviruses. a recently published report by garcia-arriaza et al. (2014) also has used mva as a vector as a chikv vaccine candidate [83] . their vaccine, which expressed the entire structural protein is effective at inducing a high level neutralizing antibody response accompanied by a strong cd8+ t-cell specific cell mediated response, in direct contrast to the results reported here. they report that the e2 protein can be found in both the plasma membrane and the cytoplasm, in contrast to our results which clearly showed e2 is not on the surface of the cell (figure 1 ). this could explain why the immune responses are so drastically different between the two vaccine candidates. while depletion studies indicated cd4+ t cells are indispensable for protection for mva-chik in this report, the mechanism of protection is unclear in their report, although high levels of neutralizing antibodies prior to challenge, balb/c mice were bled and serum was monitored for neutralizing activity by tcid 50 microneutralization assay (a). mice were bled two days following challenge and viremia levels were measured via tcid 50 (b). for passive immunization, prime and boost vaccinated balb/c and a129 mice were bled and serum taken from each mouse was pooled with equal volumes into separate pools from each strain. pools of serum (100 ml for balb/c and 200 ml for a129) were then injected i.p. into a129 mice which were challenged 24 hrs later with 10 2 tcid 50 units of wild-type chikv. viremia was measured 2 d.p.i (c) and survival was monitored for two weeks post-challenge (d). the dotted line indicates the limit of detection of the assay. doi:10.1371/journal.pntd.0002970.g004 figure 5 . mva-chik elicited a chik-e2-specific cd4 + t cell response in a129 mice. two weeks following boost, splenocytes from mva-gfp or mva-chik immunized mice were stimulated with chikv nsp2, e2 or e3 peptide pools (1 mg/well). t-cells were then stained for intracellular ifnc (a and c) or cd40l (b and d). representative dot plots (for only one mouse per group) are shown for both cytokines with mva-chik and mva-gfp stimulated with e2 peptide pools or background media control (a and b). for stimulation with e2, e3 and nsp2 peptide pools data are presented as the mean+sd of the percentages cytokine-positive cells among gated cd4 + t cells (3 mice/group) with background subtracted (c and d). stimulation was significantly higher for ifnc and cd40l in the mva-chik immune group, as compared to mva-gfp control (p = 0.02 and p = 0.01, respectively). no cytokine production in cd8 + t cells was observed (data not shown). doi:10.1371/journal.pntd.0002970.g005 induced by their vaccine are likely protective [50] . additionally, the vaccine constructed by garcia-arriaza et al. (2014) uses the entire structural protein consisting of capsid-e3-e2-6k-e1. in contrast, our vaccine contains only the e3-e2 portion. recent reports have shown that the capsid protein contains immunodominant epitopes for both b and t cells [55, 82] , and was therefore excluded from our vaccine construct in favor of e2 with its accessory protein e3. furthermore, we used a footpad injection which more closely mimics intradermal vaccination a human might be expected to receive. garcia-arriaza et al. (2014) used intraperitoneal vaccinations, which are not given to humans and therefore could potentially induce a vastly different immune response delivered by another route. in conclusion, these studies demonstrate the potential of mva to effectively express chikv e3-e2 proteins and generate protective immune responses despite the presence of low or absent neutralizing antibody responses. the mva construct expressing chikv p62 protein was effective in several mouse models and provided protection against two critical markers of disease, viremia and joint swelling. the vaccine was protective despite low levels of neutralizing antibodies which are normally considered to be the golden standard for protection against alphaviruses. therefore the results reported here challenge the assumption that only these antibodies are effective in providing protection against chikv or other alphaviruses. future studies combining a t-cell targeted vaccine, like the one presented here, along with a more 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infection is controlled by the adaptive immune response identical strength of the t cell responses against e2, nsp1 and capsid chikv proteins in recovered and chronic patients after the epidemics of 2005-2006 in la reunion island a novel poxvirus-based vaccine (mva-chikv) is highly immunogenic and protects mice against chikungunya infection we are grateful to andrea trujillo and attapon kamlangdee for their technical assistance and help with data analysis. key: cord-003389-0yh5k6jk authors: patton, john b.; bennuru, sasisekhar; eberhard, mark l.; hess, jessica a.; torigian, april; lustigman, sara; nutman, thomas b.; abraham, david title: development of onchocerca volvulus in humanized nsg mice and detection of parasite biomarkers in urine and serum date: 2018-12-12 journal: plos negl trop dis doi: 10.1371/journal.pntd.0006977 sha: doc_id: 3389 cord_uid: 0yh5k6jk background: the study of onchocerca volvulus has been limited by its host range, with only humans and non-human primates shown to be susceptible to the full life cycle infection. small animal models that support the development of adult parasites have not been identified. methodology/principal findings: we hypothesized that highly immunodeficient nsg mice would support the survival and maturation of o. volvulus and alteration of the host microenvironment through the addition of various human cells and tissues would further enhance the level of parasite maturation. nsg mice were humanized with: (1) umbilical cord derived cd34(+) stem cells, (2) fetal derived liver, thymus and cd34(+) stem cells or (3) primary human skeletal muscle cells. nsg and humanized nsg mice were infected with 100 o. volvulus infective larvae (l3) for 4 to 12 weeks. when necropsies of infected animals were performed, it was observed that parasites survived and developed throughout the infection time course. in each of the different humanized mouse models, worms matured from l3 to advanced fourth stage larvae, with both male and female organ development. in addition, worms increased in length by up to 4-fold. serum and urine, collected from humanized mice for identification of potential biomarkers of infection, allowed for the identification of 10 o. volvulus-derived proteins found specifically in either the urine or the serum of the humanized o. volvulus-infected nsg mice. conclusions/significance: the newly identified mouse models for onchocerciasis will enable the development of o. volvulus specific biomarkers, screening for new therapeutic approaches and potentially studying the human immune response to infection with o. volvulus. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 onchocerciasis, caused by the parasitic filarial nematode onchocerca volvulus, remains a significant source of morbidity throughout sub-saharan africa [1] . o. volvulus infection is commonly diagnosed through the presence of microfilariae in skin snips, but skin samples can be further analyzed by qpcr for enhanced sensitivity [2] . antibody tests (e.g. ov16 [3, 4] ) are available but do not have the ability to differentiate between past and present infections which is problematic in areas where the infection is endemic [5] . recently, a limited number of biomarkers have been identified in the urine that can distinguish between o. volvulus-infected and non-infected individuals [6] [7] [8] . a significant obstacle for studying the biology of o. volvulus and for the development of new therapeutics and diagnostics has been the absence of small animal models. the only susceptible animal hosts for o. volvulus are chimpanzees [9, 10] and mangabey monkeys [11] . chimpanzees infected with o. volvulus had patent infections that lasted between 6 to 9 years with adult-worm bundles located in deep tissues with microfilariae in skin snips being detected 12-18 months post infection [12, 13] . while immunologically intact mice are resistant to infection with the infective larvae (l3) of o. volvulus [14] , adult worms within nodules have been successfully transplanted into scid mice (nod.cb17-prkdc scid /j) with the worms surviving greater than 20 weeks [15] . this observation was confirmed by the successful transplantation of adult onchocerca ochengi into scid mice [16] . as an alternative approach, o. volvulus l3 were implanted in primates and rodents within diffusion chambers that consist of a lucite ring enclosed with permeable membranes, allowing for migration of cells and other humoral factors into the diffusion chamber with simultaneous containment of the parasites. o. volvulus in diffusion chambers implanted in multiple species of rodents and primates showed limited growth of larvae in all host species [17] . several strategies have been employed to overcome murine resistance to infection with various human pathogens. the collaborative cross (cc) is a large group of inbred mouse strains that were developed to address many of the different shortcomings found within the existing experimental mouse populations, including small numbers of homozygous strains, limited genetic diversity, and non-ideal population structures. based on the hypothesis that there is a genetic basis for mouse susceptibility and resistance to infection, novel strains of cc mice have been identified that are susceptible to specific bacteria, viruses, and parasites of humans [18] [19] [20] [21] [22] [23] . as an alternative approach to overcome murine resistance to infection, mice have been developed with greatly diminished immune responses. scid mice were shown to be susceptible to infection with brugia malayi while immunocompetent mice were resistant to the infection [24] . nod.cg-prkdc scid il2rg tm1wjl /szj (nsg) mice are a highly immune-compromised strain of mice that have profound defects in the adaptive and innate immune responses [25] . the most notable defects include those in: 1) macrophages, dendritic cells and in the complement cascade [26] [27] [28] ; 2) maturation of t and b cells [29] ; 3) nk cells; 4) signaling of 6 different cytokines [30] and 5) the presence of eosinophils in the peripheral circulation and in tissue [31] . nod-rag1 tm1mom il2rg tm1wjl mice (nrg) are phenotypically similar to nsg mice with disruption in the b and t-cell production [32] . interestingly, nsg mice can support the complete lifecycle of the human nematode strongyloides stercoralis, whereas immunologically intact mice cannot [31] . a third strategy to enhance pathogen survival in mice has been to add human-derived cells required for survival and growth [33] . nsg mice have the unique ability to support several different xenografts including human hematopoietic stem cells (cd34 + stem cells) that allow for the development of an immature partially-functional human immune system [25, 27, 30, 34] . nod.cg-prkdc scid il2rg tm1wjl tg (cmv-il3, csf2, kitlg)1eav/mloyszj (sgm) mice have an additional three human genes il3, csf2, and kitlg under the cmv promoter to enhance the overall microenvironment for the development of human xenografts. this results in increased numbers of cd33 + myeloid cells, b-cells, t-cells, and hematopoietic stem cells [35] . humanized blt mice are nsg mice that received a xenograft of human cd34 + stem cells and a transplant of human fetal thymus and liver implanted under the kidney capsule, which results in the formation of a "human immune organ" [36] . control and potential elimination of onchocerciasis has been significantly impeded by the limited number of available drugs and the development of resistance to those therapies [37, 38] . in addition, biomarkers to assess the infection status of treated individuals or macrofilaricidal activity are sorely lacking [6] [7] [8] . one of the critical barriers blocking drug and biomarker development has been the absence of suitable small animal hosts for experimentation. hence, this study was focused on the development of a small animal model that would support the growth and maturation of o. volvulus and could serve to identify parasite specific biomarkers. to this end we tested multiple genetically defined mouse strains and xenografted (with human cells) immunodeficient mice to identify those microenvironments suitable for o. volvulus development. in so doing, we were able to develop convenient and tractable murine models that support the development of o. volvulus l3 into advanced larval stages. moreover, we were able to use these o. volvulus-infected animals to identify parasite-derived biomarkers measurable in both urine and serum. the parasite material was collected during the years 1994-1999 in the research facility at the tropical medicine research station, kumba, cameroon. the procedures used for the production of o. volvulus forest strain third-stage-larvae (l3) were approved by an nih accredited institutional review board of the medical research council kumba, cameroon (protocol 001). the protocol was reviewed and approved annually. l3 were collected from black flies (simulium damnosum) that were fed on consenting infected donors. after seven days the flies were dissected and the developed l3 were collected, cleaned and cryopreserved. the cryopreserved l3 were shipped to the new york blood center in liquid nitrogen and upon arrival in new york were stored in liquid nitrogen. all protocols using the l3 cryopreserved samples in this study were approved by the new york blood center's irb (protocol 321 and protocol 603-09). all l3 samples were anonymized. all experimental procedures in mice were performed in compliance with the ethical and regulatory standards set by the nih for animal experimentation. the animal use protocol (01469) was approved by the thomas jefferson university institutional animal care and use committee. the animal care and use protocol adhered to the "guide for the care and use of laboratory animals" published by the national research council, usa. cryopreserved l3 were prepared as previously described [39] [40] [41] . briefly, black flies (simulium damnosum) were fed on consenting donors infected with o. volvulus, and after 7 days the developed l3 were collected from dissected flies, cleaned, and cryopreserved in dimethyl sulfoxide and sucrose using biocool ii computerized freezing equipment (fts systems inc., stone ridge, ny) [42] . cryopreserved l3 were removed from liquid nitrogen storage and placed on dry ice for 15 minutes followed immediately by a 37˚water bath. the l3 were then washed 5 times in a 1:1 mixture of nctc-135 and iscove's modified dulbecco's medium (sigma, st. louis mo) supplemented with 100 u penicillin, 100 μg streptomycin (corning, tewksbury ma) 100 μg gentamicin and 30 μg chloramphenicol per ml (sigma). l3 isolated from different collection days were tested first for viability in diffusion chambers implanted in balb/byj for 21 days, as previously described [17] . batches of l3 with viabilities greater than 50% at 21 days post implantation were used in these studies. one hundred worms (except where noted) were then counted and loaded into 1 ml tuberculin syringes with 21 g needle for subcutaneous injection of the larvae into the nape of the neck. all mice were housed in micro-isolator boxes in a pathogen-free room at the laboratory animal science facility at thomas jefferson university (philadelphia, pa). collaborative cross (cc) mouse strains, cast/eij, il16680 (cc055/tauunc), au8052 (cc052/geniunc), au8049 (cc038/geniunc) and or13067 (cc003/unc), were purchased and imported from the systems genetics core facility of university of north carolina (unc-chapel hill). information about the cc strains can be found on the unc systems genetics website at http:// csbio.unc.edu/ccstatus/index.py. the mice were kept under temperature, humidity and light cycle-controlled conditions and fed autoclavable rodent chow and given water ad libitum. nod-scid il2rg null (nsg), nod-rag1 null il2rg null (nrg), and nod-scid il2rg null -3/ gm/sf (sgm) mice were obtained from the jackson laboratories (bar harbor, me). an nsg, nrg, and sgm mouse breeding colony was maintained in the laboratory animal science facility, at thomas jefferson university with breeding trios given acidified water and low fat 5k52 animal chow (labdiet, st. louis, mo). the following human cell types were individually transferred into nsg mice: (1) human keratinocytes (hacat) (atcc, manassas, va), (2) bovine embryo skeletal muscle cells (besm) in the initial experiments, mice were injected with 5×10 6 besm, hacat, lec, or huskmc cells subcutaneously weekly throughout the experiment. the frequency of injection was subsequently determined by in vivo imaging experiments. genes encoding green fluorescent protein (gfp) and luciferase were inserted into huskmc cells using lentiviral vectors following the manufacturer's recommendations (cell biolabs, inc, san diego, ca). cells expressing gfp were isolated using the gfp marker by fluorescence-activated cell sorting using a bd facs aria (bd biosciences, franklin lakes, nj). the isolated cells were grown in huskmc media as described above and 5×10 6 cells were injected subcutaneously into nsg mice. mice were injected with vivoglo (promega, madison, wi) and imaged following manufacturer's recommendations on an ivis lumina xr (promega). two approaches were used to create humanized mice containing multiple human-cell types [25, 36] . human umbilical cord blood was obtained through collaboration with thomas jefferson university hospital department of obstetrics and gynecology from full term natural deliveries. cd34 + stem cells were isolated from cord blood using magnetic assisted cell sorting (macs) and cryopreserved until use. sgm, nrg and nsg mice were humanized with cd34 + umbilical cord derived stem cells by intrahepatic injection of 5x10 5 cd34 + stem cells into 48-hour old pups that were irradiated with 1.5 gray. six weeks following injection of the stem cells, peripheral blood from the mice was screened for the presence of human cells and mice with counts greater than 600 human cd45 + hematopoietic cells per μl of whole blood were used for experimentation, following previously published protocols [31] . blt mice were purchased from the jackson laboratories or were prepared following previously established protocols [27] . briefly, nsg mice (4-to 6-week old) were implanted with 1 mm 3 sections of fetal thymus and liver (advanced biomedical resources, alameda, ca) under the kidney capsule. two weeks post-implantation the mice were treated with busulfan (sigma) (20 mg/kg ip) and were injected retro-orbitally with 5x10 5 cd34 + stem cells, isolated from the donor fetal liver using macs, (miltenyi biotec inc. auburn, ca). eight weeks following the stem cell xenograft the peripheral blood from the blt mice was screened for the presence of human cells. blt mice were screened and selected using the same protocol described above for the cd34 + cord blood mice. pcr screening for o. volvulus dna was performed on all the infected mouse tissues to identify the presence of current or past o. volvulus larvae in that location. mice were anesthetized and exsanguinated, and the internal organs were removed, and the skin was removed from the muscle. the muscle and skin were then divided into 100 different sections and individually frozen in 1.7 ml eppendorf tubes. dna was extracted from the tissue sections using the promega genomic dna kit a1125 following the manufacturer's directions. realtime pcr was performed using custom taqman probes (integrated dna technologies, coralville, ia) against the ov-150 [44] [45] [46] repeats and an abi onestep-plus (thermofisher). mice were necropsied following previously established protocols for the isolation of filarial worms from tissues [47, 48] . briefly, mice were anesthetized using isoflurane gas and exsanguinated. the head was removed from the body of the mouse and discarded. the remaining internal organs and skin were removed from the muscles, and the muscle was divided into upper and lower sections at the bottom of the rib cage. all portions of the mouse (muscle, skin, and all internal organs with the exception of the head) were soaked overnight in rpmi containing 10% fbs and with 100 u penicillin, 100 μg streptomycin (corning), and emerging parasites were then collected and enumerated. infected mice were evaluated using two criteria: 1) percent established, measured the proportion of mice in a group of infected animals from which live parasites were recovered; and 2) the geometric mean number of live worms recovered per mouse within the group. recovered worms were placed in boiling fixative consisting of 95% ethanol (deacon labs, king of prussia, pa) and 5% glycerol (fisher, fair lawn, nj). after allowing the alcohol to evaporate, glycerol was added, and the worms were transferred to glycerin jelly (gelatin 10 g, ddh 2 o 60.0 ml, glycerine 70.0 ml, phenol 1.0 ml). fixed worms were measured using an olympus szx16 dissecting scope connected to a dp26 camera (olympus, center valley, pa). cellsens dimensions software (olympus) was used to measure the length of the recovered worms. serum and urine were collected and frozen as terminal procedures during necropsy. serum was thawed on ice and 25 μl removed for processing from each mouse. sera from 4 mice from each group/strain were pooled for maximizing the protein identifications. abundant proteins were depleted using an affinity chromatography (mars-ms-3, agilent) according to the manufacturer's directions. urine was thawed on ice, centrifuged and then filtered through a 0.22 μm filter (corning). serum and urine samples were prepared for mass spectrometry by digestion using the filter-assisted sample preparation (fasp) method [49] . briefly, the samples brought to 1% sodium deoxycholate (sdc), 50 mm tris-hcl, ph 7.6, 3 mm dithiothreitol, sonicated briefly, and incubated in a thermo-mixer at 90 o c, 1,000 rpm for 20 min. samples were centrifuged to clarify and the supernatant was transferred to a passivated 30 kd mwco device (millipore, merck kgaa, darmstadt, germany) and centrifuged at 13,000g for 30 min. the remaining sample was buffer exchanged with 1% sdc, 100 mm tris-hcl, ph 7.6, then alkylated with 15 mm iodoacetamide. the sdc concentration was reduced to 0.1%. samples were digested using trypsin at an enzyme to substrate ratio of 1:100, overnight, at 37 o c in a thermo-mixer at 1,000 rpm. digested peptides were collected by centrifugation and the filter washed with 0.5 nacl to elute electrostatically bound peptides. digested peptides were desalted using reversed phase stop-and-go extraction tips [50] . peptides were eluted with 80% acetonitrile, 0.5% formic acid and lyophilized in a speedvac (thermo savant, holbrook, ny) to near dryness, approximately 1 h. each digestion mixture was analyzed by ultra-high performance liquid chromatography tandem mass spectrometry (uhplc-ms/ms). lc was performed using an easy-nlc 1000 uhplc system (thermo fisher scientific, waltham, ma). mobile phase a was 97.5% milliq water, 2% acetonitrile, 0.5% formic acid. mobile phase b was 99.5% acetonitrile, 0.5% formic acid. the 240 min lc gradient ran from 0% b to 35% b over 210 min, then to 80% b for the remaining 30 min. samples were loaded directly to the column. the column was 50 cm x 75 um i.d. and packed with 2 μm c18 media (thermo easy spray pepmap). the lc was interfaced to a quadrupole-orbitrap mass spectrometer (q-exactive, thermo fisher scientific, waltham, ma) via nano-electrospray ionization using a source with an integrated column heater (thermo easy spray, thermo fisher scientific, waltham, ma). the column was heated to 50˚c. an electrospray voltage of 2.2 kv was applied. the mass spectrometer was programmed to acquire, by data-dependent acquisition, tandem mass spectra from the top 10 ions in the full scan from 400-1200 m/z. dynamic exclusion was set to 15 s, singly-charged ions were excluded, isolation width was set to 1.6 da, full ms resolution to 70,000 and ms/ms resolution to 17,500. normalized collision energy was set to 25, automatic gain control to 2e5, max fill ms to 20 ms, max fill ms/ms to 60 ms and the underfill ratio to 0.1%. mass spectrometer raw data files were converted to mzml format using msconvert [51] . mgf files were generated from mzml using the peak picker hires tool, part of the openms framework [52] . all searches were performed on amazon web services-based cluster compute instances using the proteome cluster interface. detailed search parameters are printed in the search output xml files. briefly, all searches required 10 ppm precursor mass tolerance, 0.02 da fragment mass tolerance, strict tryptic cleavage, up to 2 missed cleavages, fixed modification of cysteine alkylation, variable modification of methionine oxidation and protein-level expectation value scores of 0.0001 or lower. proteome cluster builds species-and genus-specific protein sequence libraries from the most current uniprotkb distribution [53] . mgf files were searched using the most recent protein sequence libraries available from uniprotkb using x!tandem [54] and omssa [55] . xml output files were parsed and non-redundant protein sets determined using proteome cluster based on previously published rules [56] . ms1-based isotopoic features were detected and peptide peak areas were calculated using the featurefindercentroid tool, part of the openms framework [52] . proteins were required to have 1 or more unique peptides across the analyzed samples with e-value scores of 0.0001 or less. geometric means (gm) were used as measures of central tendency. data were analyzed for larval growth by multifactorial analysis of variance anova with post-hoc fisher's least significant difference (lsd) testing in systat v.11 (systat inc., evanstown, il, usa). probability values less than 0.05 were considered statistically significant. all experiments were performed a minimum of 2 times. to determine if there was an underlying genetic basis for the resistance of mice to infection with o. volvulus [14] , 5 mouse strains having a wide range of genetic diversity from the cc (cast/eij, il16680, au8052, au8049 and or13067) [57] were screened for their susceptibility to o. volvulus. five mice from each of the different strains were infected with 100 o. volvulus l3 and necropsied at 4-weeks post infection. none of the 5 strains of cc mice tested was susceptible to infection with o. volvulus l3. to assess the role of the mouse immune system in mediating resistance to o. volvulus, immunodeficient mouse strains were assessed for their susceptibility to infection with o. volvulus. in a preliminary set of studies, 250 o. volvulus l3 were injected into 2 nsg mice. after 4-weeks the skin and muscle from the mice was divided into 100 anatomically distinct sections and qpcr for o-150 was performed on extracted dna from each section. twenty-three of the sections were positive from, spatially widespread regions of the body. it was concluded that parasites survived in nsg mice and had the ability to migrate extensively. this indicated that all regions of the mice had to be inspected for the presence of parasites following infection. nsg mice were infected with 100 o. volvulus l3 and necropsied at 4-and 8-weeks following infection. infected mice had an established infection rate of 63% with a gm worm recovery of 2.0 (range 1 to 4) worms recovered at 4-weeks following infection. at 8-weeks, 75% of mice had an established infection, with a gm recovery of 1.4 (range 1 to 3) worms per mouse ( fig 1a) . the recovered worms were measured and found to be significantly increased in size (p<0.0001; min: 677 μm, max 843 μm, gm: 717 μm) at 4-weeks compared to l3. they also significantly increased in size between 4 and 8 weeks (p<0.0001) reaching a maximum of 1,085 μm (min: 700 μm, gm: 1034 μm) (fig 2) . table) . mice engrafted with lec had 20% established infection and gm of 4 worms recovered per mouse with a range of 1 to 4 worms per mouse (s1 table) . hacat cell engrafted mice had a 60% established infection and gm recovery 1.7 worms recovered per mouse with between 1 and 2 worms per mouse (s1 table) . huskmc cell engrafted mice had an 81% estab(fig 1b) . at each time point tested the parasites in huskmc-engrafted mice demonstrated continued parasite growth. at 4-weeks recovered parasites had gm lengths of 713 μm (min: 592 μm, max: 788 μm), at 8-weeks lengths of 751 μm (min: 642 μm, max: 1,081 μm), and at 12-weeks the gm length was 1,121 μm with a maximum length of 2,086 μm observed (min: 748 μm), representing a 4-fold increase in length over l3 (fig 2) . these growth rates represented significant changes between l3 and 4-week worms (p = 0.0010) and between 8-and 12-week worms (p<0.0001). sgm, nrg and nsg (hunsg) mice humanized with cd34 + umbilical cord derived stem cells were infected with 100 o. volvulus l3. each of these humanized mice had human hematopoietic lineage cells at a concentration greater than 600 cells per μl of blood. while a complete picture of the cell populations in these specific mice was not determined, based upon flow analysis during the screening process all humanized mice had both human b and t-cells present in their blood. at 4-weeks post-infection humanized sgm mice contained a gm of 2.4 (range 1-7) worms/mouse, humanized nrg mice had a gm of 1.7 (range 1 to 4) worms/ table) . hunsg at 4-weeks post-infection had a 69% established infection rate with a gm of 3.4 (range 1-14) worms/mouse, and at 8-weeks post infection hunsg mice had a 46% established infection rate and a gm of 2.6 (range 1-18) worms/mouse (fig 1c) . larval growth in hunsg mice was comparable to that seen in nsg and in nsg mice engrafted with huskmc (fig 2) . a significant increase in length was seen between the l3 and 4-week recovery 12 ). after 4-, 8-or 12-weeks the percent established (the proportion of mice in a group of infected animals from which live parasites were recovered) (fig 1a-1d ) and the geometric mean number of live worms recovered per mouse within the group was determined (fig 1e-1h) . https://doi.org/10.1371/journal.pntd.0006977.g001 humanized nsg (hunsg) mice: nsg mice that had received a human cd34 + stem cell transfer, (4) blt: nsg mice that had been engrafted with human fetal liver derived cd34 + stem cells and engrafted with fetal thymus and liver tissues. after 4, 8 or 12-weeks, animals were necropsied and worms were recovered and measured. solid colored bar is the geometric mean of the lengths of larvae recovered. solid black line is the geometric mean of the length of l3 recovered from black flies and dotted line is the 95 th confidence interval. � asterisk represents statistical difference, p value � 0.05, in length of larvae recovered from mice. complete statistical analyses for all groups are included on s2 blt mice humanized with fetal thymus, liver and cd34 + stem cells, which display an enhanced repertoire of the developing human cells [58] , were infected with 100 o. volvulus l3 and then necropsied at 4-and 8-weeks post infection. at 4-weeks post infection blt mice had an established infection rate of 77% with a gm of 4.8 (range 2-10) worms/mouse. at 8-weeks post infection blt mice had an established infection rate of 60% with a gm of 2.3 (range 2-3) worms/mouse (fig 1d) . growth of the parasites was equivalent to that seen in the nsg mice and the other humanized models with the maximum length reaching 1,080 μm at 4-weeks (min: 391 μm, gm: 623 μm) and 1,448 μm at 8-weeks (min: 730 μm, gm: 949 μm) (fig 2) . the overall growth was significant between the l3 and worms 4-weeks post infection (p<0.0001) and between worms recovered at the 4-and 8-week time points (p<0.0001). during necropsy, mice were sectioned into 4 groupings: upper muscles, lower muscles, skin, and the complete set of internal organs. no nodules were found in any of these tissues upon necropsy. worms were recovered from all four of the different tissue groupings with no apparent preference for any region of the animal in all of the mice tested. detailed morphological analyses focused on worms recovered from blt mice infected for 8-weeks and huskmc humanized mice infected for 12-weeks. although there were clear differences in lengths of these worms, their morphological characteristics were similar. no differences in the ratio of males and female worms recovered from the mice was observed, however it was noted that most of the longer worms were females. both the anterior and posterior ends in both sexes were bluntly rounded and only slightly tapered (fig 3a, 3b and 3f ). other than growth in length, the major change from l3 was development of the reproductive systems. in the l3, both the female and male systems are rudimentary genital primordia consisting of only several cells. in the 8-12-week old worms, the female ovejector had formed and had attached to the body wall. the ovejector was ovoid in shape, relatively large and filled the body cavity, and had a distinct lumen (fig 3c and 3d) . rudimentary cellular growth of the reproductive tubes was also evident (fig 3d) . in males, the testis, located at approximately mid-body, had become elongate in shape and had looped posteriorly to form a classic shepherd's crook (fig 3e) . in addition, the spicule pads were well developed and demarcated (fig 3f) but were still oval in shape and had not yet started to take on the shape of the spicules nor was there any evidence of cuticularization. these observations are consistent with parasite development into advanced fourth stage larvae (l4). global proteomic analyses were performed with serum and urine collected from blt mice infected for 8-weeks and huskmc humanized mice infected for 12-weeks. a total of 7,430 proteins were identified based on the spectral matching to a combined protein database of human, mouse, o. volvulus and its wolbachia (wov) endosymbiont. because of the ambiguity in distinguishing certain spectral matches for proteins commonly found in both humans and mice, these proteins were grouped as non-o. volvulus proteins (4,743 in serum, 2,836 in urine, s1 development of onchocerca volvulus in nsg mice serum and urine of the infected mice, the blt and huskmc mice had 5 proteins (ovoc115 56, ovoc835, ovoc10244, ovoc4009 and ovoc9087) in common in the serum and 5 (ovoc7220, ovoc4139, ovoc224, ovoc8249 and ovoc9267) in the urine (fig 4b) . almost all the proteins identified have been shown through rnaseq to be transcribed by various stages of the o. volvulus parasite (fig 4b) . the objective of this project was to identify small animal models that would support the development of black-fly derived o. volvulus l3 into advanced mammalian-adapted stages of the parasite. these small animal models are critically needed for identifying biomarkers released from the early stages of the infection and for screening potential new anthelmintics. published findings on the susceptibility of mice to infection with the l3 of o. volvulus suggest that mice are resistant to infection when the larvae were injected subcutaneously [14] . however, o. volvulus l3 have been shown to survive and develop in mice when implanted within diffusion chambers at rates comparable to those seen in susceptible primates [17] . genetic traits can play a significant role in the susceptibility of animals to infection as has been clearly demonstrated by the diversity of susceptibility of different mouse strains to infection with litomosoides sigmodontis [59] . the cc mouse project was developed to produce an extremely diverse set of inbred animals that could be used for mapping different genetic traits [57] . five different cc mouse strains, selected based on their diverse genetic backgrounds, were tested for susceptibility to o. volvulus and were completely resistant to the infection. this observation suggests that either mice are missing some integral factors required for parasite growth or the immune responses in mice are effective at eliminating the infection. the question remains as to why parasites are recovered live from diffusion chambers implanted in mice but not when injected into the tissues. there are several possible explanations including: (1) the diffusion chamber acts as a barrier from the immune response creating an immune privileged site, (2) the larvae within the diffusion chamber are blocked from migrating through the tissue releasing excretory and secretory products and thereby eliciting an immune reaction, or (3) the diffusion chamber attracts host components to the parasite microenvironment that are beneficial for parasite development. to test the hypothesis that mouse-intrinsic immune responses control o. volvulus infections, nsg mice that lack both functional innate and adaptive immune systems were infected with o. volvulus l3. advanced stages of the parasites were consistently recovered from the infected nsg mice, and parasites survived and developed over the 8-week time course into advanced l4. these findings demonstrate that the mouse immune response was capable of controlling infection with o. volvulus, with elements of the mouse immune response eliminating the infection in immunologically intact mice. many mechanisms have been described for innate immune control of nematode infections in mice [39, [60] [61] [62] [63] [64] [65] [66] [67] all or some of which may be effective against o. volvulus. interestingly, tissues and cells in nsg mice provide required factors for parasite development and based on pcr analyses, the larvae actively migrated far from the infection site. the present study did not identify the point at which o. volvulus ceased surviving and developing in nsg mice. studies with the human parasite s. stercoralis have demonstrated that the entire parasite life cycle will develop in nsg mice within 4-weeks [31] . given the significant difference in time that it takes for o. volvulus adults to develop (12-15 months in chimpanzees [9, 10] ) and their size as mature adults (females are 50 cm [68] ) it is unlikely that the entire o. volvulus life cycle, including mating and development of microfilariae, will occur in nsg mice. in vitro studies on the development of filarial worms including o. volvulus [69, 70] and brugia malayi [71] have demonstrated that host cells are needed in the culture wells to optimize parasite growth and development. furthermore, optimal development and survival of larval t. spiralis in mice requires the presence of mouse eosinophils [33] . it was thus hypothesized that adding human cells to the nsg mice, from the tissues that the parasites are normally found juxtaposed in humans, might provide additional required nutritional or developmental elements found in humans required for parasite development and survival. four different single cell xenografts were screened: huskmc, lec, hacat and besm. of these four cell lines, huskmc was found to support the highest average percent survival of the implanted worms and consistent infection rates over a 12-week time period (figs 1 and 2) as an alternative to adding single cell populations to the nsg mice, multipotential umbilical cord stem cells were transferred to the immunodeficient mice. nsg, nsg-sgm, and nrg mice, all of which lack functional immune responses, were humanized with cd34 + umbilical cord stem cells. the humanization of the various immunocompromised mouse strains resulted in the development of an immature human immune system. the developing immune system in these mice displays a t-independent response, limited antigen-specific igm responses, and the presence of multiple innate immune cells has been noted [25] . when humanized nsg, nsg-sgm, and nrg mice were infected with o. volvulus l3, higher gm parasite recoveries were observed when compared to nsg mice without any human cells but these enhancements were not significant. although nsg-sgm did have consistent infection rates with o. volvulus we had significant difficulties establishing reliable engraftment of human cells in this strain of mice. extending this concept further, blt mice which contain cd34 + stem cells in addition to fetal tissue were used. blt mice are known to develop a more mature version of a human immune system. limited t-dependent recognition is seen within these mice and better overall functionality of both the b and t-cells has been documented [36] . blt mice supported the highest average parasite recoveries of any mouse model tested at 4-weeks, although the average fell to be in line with the hunsg and huskmc models by 8-weeks post infection. the mean lengths of the parasites recovered from 4-and 8-week time points from the nsg mice were comparable to those recovered from blt mice. this suggests a link between the enhanced survival, may be related to the presence of the human immune cells, but the growth of the parasites that survive may not be related to the human cell byproducts. although cellular engraftments levels in blt and hunsg mice were not rechecked at the end of the experiment previously published data have shown that these animals reliably hold their engraftments for extended periods of time [72, 73] . the mouse models developed in this study offer a number of advantages over the models that currently exist. l3 implanted within diffusion chambers in both non-human primates and mice successfully molt into l4 but the lengths of the recovered parasites were significantly shorter than those recovered at 8 and 12 weeks post infection from the mice tested in this study [17] . while non-human primates can support the development of the parasites from l3 to microfilariae producing adults, cost and ethical considerations limit the utility of primates for drug discovery and antigen identification [10, 14] . nodules containing adult worms recovered from humans have been implanted into scid mice and the female worms survived and released microfilariae [15] . this method allows for identification of adult antigens and biomarkers, but lacks the intermediate l3 and l4 stages of development. finally, an alternative model using adult male o. ochengi implanted into scid mice has been developed for the identifying filaricides [16] , which may also be effective against o. volvulus. the nsg models developed in this study have the critical advantage of working with o. volvulus, thereby allowing species-specific screening of filaricides and identification of biomarkers. it was of interest to note that the immune response found in normal mice was highly efficient at eliminating the l3 of o. volvulus, but the human immune cells in nsg mice were supportive rather than destructive of the parasites. it is possible that the mouse immune cells were evolutionally adapted to eliminate the parasites, whereas the human cells were evolutionally adapted to support the parasites as observed in nature. a comparison between mouse and human immune reactions to the worms might yield important new insights into the etiology of resistance and susceptibility to infection with o. volvulus. the source of the l3 used in these studies was from parasites cryopreserved in liquid nitrogen. after defrosting, 100 individual parasites were selected, attempting to identify only the viable/undamaged l3. it is reasonable to predict that a percentage of larvae exiting from the mouth parts of a black fly have the potential to resume development in the human host and that the cryopreservation process damaged some of those worms. approximately 50 percent of the larvae recovered after cryopreservation survived in diffusion chambers implanted in mice, which may explain the overall number of worms recovered from the different mouse models in this study. while minor differences in the overall larval recovery levels were observed between individual batches of cryopreserved larvae, multiple batches were combined before implantation to help ensure a consistent viability level going into the animal hosts. even with the combination of multiple batches of larvae it cannot be ruled out that the overall viability of the injected larvae played a role in the observed recovery rates. the recovery rates of o. volvulus larvae after developing in nsg mice with or without human cells was in the same order of magnitude as that reported for the recovery of adult filarial worms, where infections were initiated by larvae recovered directly from the insect vector. these infections included brugia pahangi in cats [74] , brugia malayi in leaf-monkey [75] , and onchocerca ochengi in cattle [76] . in the final analysis, optimal parasite recovery was observed in mice humanized with huskmc (maximum of 10 worms), hunsg (maximum of 18 worms) and blt mice (maximum of 10 worms). it was clear that worms increased in size during the infection period with individual worms achieving up to 4 times the size of the original l3. growth of worms within a single mouse was not consistent and suggests that there is significant variability within the infecting larval population. it does verify, however, that humanized mice have the potential to support extended development of o. volvulus. both male and female worms grew in length and resumed their sexual development. although no cast cuticles were observed, it was evident from organ development that the parasites had molted into fourth-stage larvae. urine and serum was collected from humanized mice infected for 8-12-weeks with o. volvulus for the identification of biomarkers. infected huskmc mice or blt mice were selected for this analysis so the biomarkers identified would develop in the presence of human cells thereby potentially enhancing their specificity. several o. volvulus-specific peptides were identified in the serum and urine of blt and huskmc mice (s3 table) , however, no o. volvulusspecific proteins were found in both urine and serum from either mouse source. the most likely useful biomarkers were the proteins listed in fig 4b. though most of the proteins were identified by one or more unique peptide(s), among the proteins with unknown function (ovoc9087, ovoc835, ovoc224), ovoc9087 does not have orthologues in other filarial species and hence would likely be able to distinguish o. volvulus from other filarial infections. because of the expected low number and abundance of o. volvulus-specific protein identification in the serum and urine from any given mouse in the current system, mass spectrometry was carried out with pooled serum and urine samples from each group of mice. in conclusion, novel small-animal hosts, nsg mice, have been identified that support the survival and development of o. volvulus l3 into advanced l4 mammalian stages. humanized mice have also been shown to be effective at identifying biomarkers for early o. volvulus infections. it is anticipated that these small-animal hosts for o. volvulus will also be useful as part of the 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infections in cattle as a model for human onchocerciasis: recent developments we thank the consenting infected donors from the villages around kumba, cameroon (marumba i, marumba ii, boa bakundu, bombanda, and bombele) who participated in the study. we also thank dr. peter enyong and the staff at the tropical medicine research station for their technical support in the production and cryopreservation of the o. volvulus l3s.we thank dr. timothy manser, thomas jefferson university, for his support and encouragement for using human-stem cell engrafted humanized mice; dr. judy sakanari, university of california san francisco for her gift of bovine embryo skeletal muscle cells and dr. fernando pardo manuel de villena, ms. darla r. miller, and dr. linda d. siracusa for guidance with collaborative cross strain import and husbandry. key: cord-003243-u744apzw authors: michael, edwin; sharma, swarnali; smith, morgan e.; touloupou, panayiota; giardina, federica; prada, joaquin m.; stolk, wilma a.; hollingsworth, deirdre; de vlas, sake j. title: quantifying the value of surveillance data for improving model predictions of lymphatic filariasis elimination date: 2018-10-08 journal: plos negl trop dis doi: 10.1371/journal.pntd.0006674 sha: doc_id: 3243 cord_uid: u744apzw background: mathematical models are increasingly being used to evaluate strategies aiming to achieve the control or elimination of parasitic diseases. recently, owing to growing realization that process-oriented models are useful for ecological forecasts only if the biological processes are well defined, attention has focused on data assimilation as a means to improve the predictive performance of these models. methodology and principal findings: we report on the development of an analytical framework to quantify the relative values of various longitudinal infection surveillance data collected in field sites undergoing mass drug administrations (mdas) for calibrating three lymphatic filariasis (lf) models (epifil, lymfasim, and transfil), and for improving their predictions of the required durations of drug interventions to achieve parasite elimination in endemic populations. the relative information contribution of site-specific data collected at the time points proposed by the who monitoring framework was evaluated using model-data updating procedures, and via calculations of the shannon information index and weighted variances from the probability distributions of the estimated timelines to parasite extinction made by each model. results show that data-informed models provided more precise forecasts of elimination timelines in each site compared to model-only simulations. data streams that included year 5 post-mda microfilariae (mf) survey data, however, reduced each model’s uncertainty most compared to data streams containing only baseline and/or post-mda 3 or longer-term mf survey data irrespective of mda coverage, suggesting that data up to this monitoring point may be optimal for informing the present lf models. we show that the improvements observed in the predictive performance of the best data-informed models may be a function of temporal changes in inter-parameter interactions. such best data-informed models may also produce more accurate predictions of the durations of drug interventions required to achieve parasite elimination. significance: knowledge of relative information contributions of model only versus data-informed models is valuable for improving the usefulness of lf model predictions in management decision making, learning system dynamics, and for supporting the design of parasite monitoring programmes. the present results further pinpoint the crucial need for longitudinal infection surveillance data for enhancing the precision and accuracy of model predictions of the intervention durations required to achieve parasite elimination in an endemic location. we report on the development of an analytical framework to quantify the relative values of various longitudinal infection surveillance data collected in field sites undergoing mass drug administrations (mdas) for calibrating three lymphatic filariasis (lf) models (epifil, lym-fasim, and transfil), and for improving their predictions of the required durations of drug interventions to achieve parasite elimination in endemic populations. the relative information contribution of site-specific data collected at the time points proposed by the who monitoring framework was evaluated using model-data updating procedures, and via calculations of the shannon information index and weighted variances from the probability distributions of the estimated timelines to parasite extinction made by each model. results show that data-informed models provided more precise forecasts of elimination timelines in each site compared to model-only simulations. data streams that included year 5 post-mda microfilariae (mf) survey data, however, reduced each model's uncertainty most compared to data streams containing only baseline and/or post-mda 3 or longer-term mf survey data irrespective of mda coverage, suggesting that data up to this monitoring point may be optimal for informing the present lf models. we show that the improvements observed in the predictive performance of the best data-informed models may be a function of temporal changes in inter-parameter interactions. such best data-informed models may also produce plos mathematical models of parasite transmission, via their capacity for producing dynamical forecasts or predictions of the likely future states of an infection system, offer an important tool for guiding the development and evaluation of strategies aiming to control or eliminate infectious diseases [1] [2] [3] [4] [5] [6] [7] . the power of these numerical simulation tools is based uniquely on their ability to appropriately incorporate the underlying nonlinear and multivariate processes of pathogen transmission in order to facilitate plausible predictions outside the range of conditions at which these processes are either directly observed or quantified [8] [9] [10] [11] . the value of these tools for guiding policy and management decisions by providing comparative predictions of the outcomes of various strategies for achieving the control or elimination of the major neglected tropical diseases (ntds) has been highlighted in a series of recent publications [8, 11, 12] , demonstrating the crucial role these quantitative tools are beginning to play in advancing policy options for these diseases. while these developments underscore the utility of transmission models for supporting policy development in parasite control, a growing realization is that these models can be useful for this purpose only if the biological processes are well defined and demographic and environmental stochasticity are either well-characterized or unimportant for meeting the goal of the policy modelling exercise [9] [10] [11] [13] [14] [15] [16] . this is because the realized predictability of any model for a system depends on the initial conditions, parameterizations and process equations that are utilized in its simulation such that model outcomes are strongly sensitive to the choice of values used for these variables [17] . any misspecification of these system attributes will lead to failure in accurately forecasting the future behaviour of a system, with predictions of actual future states becoming highly uncertain even when the exact representation of the underlying deterministic process is well established but precise specification of initial conditions or forcing and/or parameter values is difficult to achieve [17, 18] . this problem becomes even more intractable when theoretical models depend on parameter estimates taken from other studies [5, 17, 19] . both these challenges, viz. sensitivity to forcing conditions and use of parameter estimates from settings that are different from the dynamical environment in which a model will be used for simulation, imply that strong limits will be imposed on the realized predictability of any given model for an application [9, 10, 20] . as we have shown recently, if such uncertainties are ignored, the ability of parasite transmission models to form the scientific basis for management decisions can be severely undermined, especially when predictions are required over long time frames and across heterogeneous geographic locations [4, 5, 7] . these inherent difficulties with using an idealized model for producing predictions to guide management have led to consideration of data-driven modelling procedures that allow the use of information contained within observations to improve specification and hence the predictive performance of process-based models [9, 10, 14, [21] [22] [23] . such approaches, termed model-data fusion or data assimilation methods, act by combining models with various data streams (including observations made at different spatial or temporal scales) in a statistically rigorous way to inform initial conditions, constrain model parameters and system states, and quantify model errors. the result is the discovery of models that can more adequately capture the prevailing system dynamics in a site, an outcome which in turn has been shown to result in the making of significantly improved predictions for management decision making [9, 10, 14, 24] . initially used in geophysics and weather forecasting, these methods are also beginning to be applied in ecological modelling, including more recently in the case of infectious disease modelling [9, 10] . in the latter case, the approach has shown that it can reliably constrain a disease transmission model during simulation to yield results that approximate epidemiological reality as closely as possible, and as a consequence improve the accuracy of forecasts of the response of a pathogen system exposed to various control efforts [4-7, 21, 25-27] . more recently, attention has also focused on the notion that a model essentially represents a conditional proposition, i.e. that running a model in a predictive mode presupposes that the driving forces of the system will remain within the bounds of the model conceptualization or specification [28] . if these driving forces were to change, then it follows that even a model well-calibrated to a given historical dataset will fail. new developments in longitudinal data assimilation can mitigate this problem of potential time variation of parameters via the recursive adjustment of the model by assimilation of data obtained through time [22, 29, 30] . apart from allowing assessment of whether stasis bias may occur in model predictions, such sequential model calibration with time-varying data can also be useful for quantifying the utility of the next measurement in maximizing the information gained from all measurements together [31] . carrying out such longitudinal model-data analysis has thus the potential for providing information to improve the efficiency and cost-effectiveness of data monitoring campaigns [24, [31] [32] [33] , along with facilitating more reliable model forecasts. a key question, however, is evaluating which longitudinal data streams provide the most information to improve model performance [33] . indeed, it is possible that from a modelling perspective using more data may not always lead to a better-constrained model [34] . this suggests that addressing this question is not only relevant to model developers, who need observational data to improve, constrain, and test models, but also for disease managers working on the design of disease surveillance plans. at a more philosophical level, we contend that these questions have implications for how current longitudinal monitoring data from parasite control programmes can best be exploited both scientifically and in management [31] . specifically, we suggest that these surveillance data need to be analysed using models in a manner that allows the extraction of maximal information about the monitored dynamical systems so that this can be used to better guide both the collection of such data as well as the provision of more precise estimates of the system state for use in making state-dependent decisions [2, [35] [36] [37] . currently, parasite control programmes use infection monitoring data largely from sentinel sites primarily to determine if an often arbitrarily set target is met [3] . little consideration is given to whether these data could also be used to learn about the underlying transmission dynamics of the parasitic system, or how such learning can be effectively used by management to make better decisions regarding the interventions required in a setting to meet stated goals [2, 4] . here, we develop an analytical framework to investigate the value of using longitudinal lf infection data for improving predictions of the durations of drug interventions required for achieving lf elimination by coupling data collected during mass drug interventions (mdas) carried out in three example field sites to three existing state-of-the-art lymphatic filariasis (lf) models [4, 6, 21, [38] [39] [40] [41] [42] [43] . to be managerially relevant to current who-specified lf intervention surveillance efforts, we evaluated the usefulness of infection data collected in these sites at the time points proposed by the who monitoring framework in carrying out the present assessment [44] . this was specifically performed by ranking these different infection surveillance data streams according to the incremental information gain that each stream provided for reducing the prediction uncertainty of each model. longitudinal pre-and post-infection and mda data from representative sites located in each of the three major regions endemic for lf (africa, india, and papua new guinea (png)) were assembled from the published literature for use in constraining the lf models employed in this study. the three sites (kirare, tanzania, alagramam, india, and peneng, png) were selected on the basis that each represents the average endemic transmission conditions (average level of infection, transmitting mosquito genus) of each of these three major extant lf regions, while providing details on the required model inputs and data for conducting this study. these data inputs encompassed information on the annual biting rate (abr) and dominant mosquito genus, as well as mda intervention details, including the relevant drug regimen, time and population coverage of mda, and times and results of the conducted microfilaria (mf) prevalence surveys (table 1) . note each site also provided these infection and mda data at the time points pertinent to the existing who guidelines for conducting lf monitoring surveys during a mda programme [44] , which additionally, as pointed out above, allowed the assessment of the value of such infection data both for supporting effective model calibration and for producing more reliable intervention forecasts. the three existing lf models employed for this study included epifil, a deterministic monte carlo population-based model, and lymfasim and transfil, which are both stochastic, individual-based models. all three models simulate lf transmission in a population by accounting for key biological and intervention processes such as impacts of vector density, the life cycle of the parasite, age-dependent exposure, density-dependent transmission processes, infection aggregation, and the effects of drug treatments as well as vector control [4, 21, 38-40, 42, 43, 49] . although the three models structurally follow a basic coupled immigration-death model formulation, they differ in implementation (e.g. from individual to population-based), the total number of parameters included, and the way biological and intervention processes are mathematically incorporated and parameterized. the three models have been compared in recent work [8, 12] , with full details of the implementation and simulation procedures for each individual model also described [6, 8, 12, 21, 39, 42, 43, 49, 50] . individual model parameters and fitting procedures specific to this work are given in detail in s1 supplementary information. we used longitudinal data assimilation methods to sequentially calibrate the three lf models with the investigated surveillance data such that parameter estimates and model predictions reflect not only the information contained in the baseline but also follow-up data points. the available mf prevalence data from each site were arranged into four different temporal data streams to imitate the current who guidelines regarding the time points for conducting monitoring surveys during an mda programme. this protocol proposes that infection data be collected in sentinel sites before the first round of mda to establish baseline conditions, no sooner than 6 months following the third round of mda, and no sooner than 6 months following the fifth mda to assess whether transmission has been interrupted (defined as reduction of mf prevalence to below 1% in a population) [44, 51] . thus, the four data streams considered for investigating the value of information gained from each survey were respectively: scenario 1-baseline mf prevalence data only, scenario 2-baseline and post-mda 3 mf prevalence data, scenario 3-baseline, post-mda 3, and post-mda 5 mf prevalence data, and scenario 4-baseline and post-mda 5 mf prevalence data. in addition to these four data streams, a fifth model-only scenario (scenario 0) was also considered where no site-specific data was introduced. in this case, simulations of interventions were performed using only model-specific parameter and abr priors estimated for each region. the first step for all models during the data assimilation exercises reported here was to initially simulate the baseline infection conditions in each site using a large number of samples (100,000 for epifil and transfil, and 10,000-30,000 for lymfasim) randomly selected from the parameter priors deployed by each model. the number of parameters which were left free to be fitted to these data by each model range from 3 (lymfasim and transfil) to 21 (epifil). the abr, a key transmission parameter in all three models, was also left as a free parameter whose distribution was influenced by the observed abr (table 1) and/or by fits to previous region-specific datasets (see s1 supplementary information for model-specific implementations). the subsequent steps used to incorporate longitudinal infection data into the model calibration procedure varied among the models, but in all cases the goodness-of-fit of the model outputs for the site-specific mf prevalence data was assessed using the chi-square metric (α = 0.05) [52] . epifil used a sequential model updating procedure to iteratively modify the parameters with the introduction of each subsequent follow up data point through time [6] . this process uses parameter estimates from model fits to previous data as priors for the simulation of the next data which are successively updated with the introduction of each new observation, thus providing a flexible framework by which to constrain a model using newly available data. fig 1 summarizes the iterative algorithm used for conducting this sequential model-data assimilation exercise [6] . lymfasim and transfil, by contrast, included all the data in each investigated stream together for selecting the best-fitting models for each time series-i.e. model selection for each data series was based on using all relevant observations simultaneously in the fitting process [30, 53, 54] . although a limitation of this batch estimation approach is that the posterior probability of each model is fixed for the whole simulation period, unlike the case in sequential data assimilation where a restricted set of parameters is exposed to each observation (as a result of parameter constraining by data used in the previous time step)which thereby yields models that give better predictions for different portions of the underlying temporal process-here we use both methods to include and assess the impact that this implementation difference may have on the results presented below. for all models, the final updated parameter estimates from each data stream were used to simulate the impact of observed mda rounds and for predicting the impact of continued mda to estimate how many years were required to achieve 1% mf prevalence. interventions were modelled by using the updated parameter vectors or models selected from each scenario for simulating the impact of the reported as well as hypothetical future mda rounds on the number of years required to reduce the observed baseline lf prevalence in each site to below the who transmission threshold of 1% mf prevalence [44] . when simulating these interventions, the observed mda times, regimens, and coverages followed in each site were used (table 1) , while mda was assumed to target all residents aged 5 years and above. for making mf prevalence forecasts beyond the observations made in each site, mda simulations were extended for a total of 50 annual rounds in each site at an assumed coverage of 65%. while the drug-induced mf kill rate and the duration of adult worm sterilization were fixed among the models (table 1) , the worm kill rate was left as a free parameter to be estimated from post-intervention data to account for the uncertainty in this drug efficacy parameter [4, 7, 21] . the number of years of mda required to achieve the threshold of 1% mf prevalence was calculated from model forecasts of changes in mf prevalence due to mda for each modeldata fusion scenario. the predictions from each model regarding timelines to achieve 1% mf for each fitting scenario were used to determine the information gained from each data stream compared to the in all scenarios, the initial epifil models were initialized with parameter priors and a chi-square fitting criterion was applied to select those models which represent the baseline mf prevalence data sufficiently well (α = 0.05). the accepted models were then used to simulate the impact of interventions on mf prevalence. the chi-square fitting criterion was sequentially applied to refine the selection of models according to the post-mda mf prevalence data included in the fitting scenario. the fitted parameters from selection of acceptable models at each data point were used to predict timelines to achieve 1% mf prevalence. the scenarios noted in the blue boxes indicate the final relevant updating step before using the fitted parameters to predict timelines to achieve 1% mf in that data fitting scenario. information attributable to the model itself [14, 33, 55] . the relative information gained from a particular data stream was calculated as i d = h m -h md where h measures the entropy or uncertainty associated with a random variable, h m denotes predictions from the model-only scenario (scenario 0) which essentially represents the impact of prior knowledge of the system, and h md signifies predictions from each of the four model-data scenarios (i.e. scenarios [1] [2] [3] [4] . the values of i d for each data scenario or stream were compared in a site to infer which survey data are most useful for reducing model uncertainty. the shannon information index was used to measure entropy, h, as follows: is the discrete probability density function (pdf) of the number of years of mda predicted by each fitted model to reach 1% mf, and is estimated from a histogram of the respective model predictions for m bins (of equal width in the range between the minimum and maximum values of the pdfs) [14, 56] . to statistically compare two entropy values, a permutation test using the differential shannon entropy (dse) was performed [57] . dse is defined as |h 1 -h 2 | where h 1 was calculated from the distribution of timelines to achieve 1% mf for a given scenario, y 1 , and h 2 was calculated from the distribution of timelines to achieve 1% mf for a different scenario, y 2 . the list of elements in y 1 and y 2 were combined into a single list of size y 1 + y 2 and the list was permuted 20,000 times. dse was then recalculated each time by calculating a new h 1 from the first y 1 elements and a new h 2 from the last y 2 elements from each permutation, from which p-values may be quantified as the proportion of all recalculated dses that were greater than the original dse. model predictions of the mean and variance in timelines to lf elimination were weighted according to the frequencies by which predictions occurred in a group of simulations. in general, if d 1 , d 2 ,. . .,d n are data points (model predictions in the present case) that occur in an ensemble of simulations with different weights or frequencies w 1 ,w 2 ,. . .,w n , then the weighted mean, here, n is the number of data points and n 0 is the number of non-zero weights. in this study, the weighted variance of the distributions of predicted timelines to achieve 1% mf prevalence was calculated to provide a measure of the precision of model predictions in addition to the entropy measure, h. a similar weighting scheme was also used to pool the timeline predictions of all three models. here, predictions made by each of the three models for each data scenario were weighted as above, and a composite weighted 95% percentile interval for the pooled predictions was calculated for each data stream. this was done by first computing the weighted percentiles for the combined model simulations from which the pooled 2.5 th and 97.5 th percentile values were quantified. the matlab function, wprctile, was used to carry out this calculation. the extent by which parameter constraints are achieved through the coupling of models with data was evaluated to determine if improvements in such constraints by the use of additional data may lead to reduced model prediction uncertainty [33] . parameter constraint was calculated as the ratio of the mean standard deviation of all fitted parameter distributions to the mean standard deviation of all prior parameter distributions. a ratio of less than one indicates the fitted parameter space is more constrained than the prior parameter space [33] . this assessment was carried out using the epifil model only. in addition, pairwise parameter correlations were also evaluated to assess whether the sign, magnitude, and significance of these correlations changed by scenario to determine if using additional data might alter these interactions to better constrain a model. for this assessment, spearman's correlation coefficients and p-values testing the hypothesis of no correlation against the alternative of correlation were calculated, and the exercise was run using the estimated parameters from the epifil model. epifil was used to conduct a sensitivity analysis investigating whether the trend in relative information gained by coupling the model with longitudinal data was dependent on the interventions simulated. the same series of simulations (for three lf endemic sites and five fitting scenarios) were completed with the extended mda coverage beyond the observations given in table 1 set here at 80% instead of 65% to represent an optimal control strategy. as before, the timelines to reach 1% mf prevalence in each fitting scenario were calculated and used to determine which data stream provided the model with the greatest gain of information. the results were compared to the original series of simulations to assess whether the trends are robust to changes in the intervention coverages simulated. epifil was also used to perform another sensitivity analysis expanding the number of data streams to investigate if the who monitoring scheme is adequate for informing the making of reliable model-based predictions of timelines for achieving lf elimination. to perform this sensitivity analysis, pre-and post-mda data from villupuram district, india that provide extended data points (viz. scenario 1-4 as previously defined, plus scenario 5-baseline, post-mda 3, post-mda 5, and post-mda 7 mf prevalence data, and scenario 6-baseline, post-mda 3, post-mda 5, post-mda 7, and post-mda 9 mf prevalence) were assembled from the published literature [47, 58] . the timelines to reach 1% mf prevalence and the entropy for each of these additional scenarios were calculated to determine whether additional data streams over those recommended by who are required for achieving more reliable model constraints, which among these data might be considered as compulsory, and which might be optional for supporting predictions of elimination. differences in predicted medians, weighted variances and entropy values between data scenarios, models and sites were statistically evaluated using kruskall-wallis tests for equal medians, f-tests for equality of variance, and dse permutation tests, respectively. p-values for assessing significance for all pairwise tests were obtained using the benjamini-hochberg procedure for controlling the false discovery rate, i.e. for protecting against the likelihood of obtaining false positive results when carrying out multiple testing [59] . here, our goal was twofold. first, to determine if data are required to improve the predictability of intervention forecasts by the present lf models in comparison with the use of theoretical models only, and second, to evaluate the benefit of using different longitudinal streams of mf survey data for calibrating the three models in order to determine which data stream was most informative for reducing the uncertainty in model predictions in a site. table 2 summarises the key results from our investigation of these questions: these are the number of accepted best-fitting models for each data stream or scenario in the three study sites (table 1) , the predicted median and range (2.5 th -97.5 th percentiles) in years to achieve the mf threshold of 1% mf prevalence, the weighted variance and entropy values based on these predictions, and the relative information gained (in terms of reduced prediction uncertainty) by the use of longitudinal data for constraining the projections of each of the three lf models investigated. even though the number of selected best-fit models based on the chi-square criterion (see methods) differed for each site and model, these results indicate unequivocally that models constrained by data provided significantly more precise intervention predictions compared to model-only predictions ( table 2 ). note that this was also irrespective of the two types of longitudinal data assimilation procedures (sequential vs. simultaneous) used by the different models in this study. thus, for all models and sites, model-only predictions made in the absence of data (scenario 0) showed the highest prediction uncertainty, highlighting the need for data to improve the predictive performance of the present models. the relative information gained by using each data stream in comparison to model-only predictions further support this finding, with the best gains in reducing model prediction uncertainty provided by those data constraining scenarios that gave the lowest weighted variance and entropy values; as much as 92% to 96% reductions in prediction variance were achieved by these scenarios in comparison to modelonly predictions between the three models ( table 2 ). the results also show, however, that data streams including post-mda 5 mf survey data (scenarios 3 and 4) reduced model uncertainty (based on both the variance and entropy measures) most compared to data streams containing only baseline and/or post-mda 3 mf survey data (scenarios 1 and 2) ( table 2) . although there were differences between the three models (due to implementation differences either in how the models are run (monte carlo deterministic vs. individual-based) or in relation to how the present data were assimilated (see above)), overall, scenario 3, which includes baseline, post-mda 3, and post-mda 5 data, was shown to most often reduce model uncertainty the greatest. additionally, there was no statistical difference between the performances of scenarios 3 and 4 in those cases where scenario 4 resulted in the greatest gain of information (table 2) . it is also noticeable that the best constraining data stream for each combination of site and model also produced as expected the lowest range in predictions of the numbers of years of annual mda required to achieve the 1% mf prevalence in each site, with the widest ranges estimated for model-only predictions (scenario 0) and the shorter data streams (scenario 1). in general, this constriction in predictions also led to lower estimates of the median times to achieve lf elimination, although this varied between models and sites ( table 2) . the change in the distributions of predicted timelines to lf elimination without and with model constraining by the different longitudinal data streams is illustrated in fig 2 for the kirare site (see s2 supplementary information for results obtained for the other two study villages investigated here). the results illustrate that both the location and length of the tail of the prediction distributions can change as models are constrained with increasing lengths of longitudinal data, with inclusion of post-mda 5 mf survey data consistently producing a narrower or sharper range of predictions compared to when this survey point is excluded. fig 3 compares the uncertainty in predictions of timelines to achieve elimination made by each of the three models without (scenario 0) and via their constraining by the data streams providing the lowest prediction entropy for each of the models per site. note that variations in scenario 0 predictions among the three models directly reflect the different model structures, parameterizations, and the presence (or absence) of stochastic elements. the boxplots in the figure, however, show that for all three sites and models, calibration of each model by data the lowest entropy scenario for each site is bolded and shaded grey. additional scenarios shaded grey are not significantly different from the lowest entropy scenario. data assimilation in filarial model predictions greatly reduces the uncertainty in predictions of the years of annual mda required to eliminate lf compared to model-only predictions, with the data streams producing the lowest entropy for simulations in each site significantly improving the precision of these predictions ( table 2 ). this gain in precision, and thus the information gained using these data streams, is, as expected, greater for the stochastic lymfasim and transfil models compared to the deterministic epifil model. note also that even though the ranges in predictions of the annual mda years required to eliminate lf by the data streams providing the lowest prediction entropy differed statistically between the three models, the values overlapped markedly (e.g. for kirare the ranges are 10-18, 9-14, 9-15 for epifil, lymfasim and transfil data assimilation in filarial model predictions respectively), suggesting the occurrence of a similar constraining of predictive behaviour among the three models. to investigate this potential for a differential model effect, we further pooled the predictions from all three models for all the data scenarios and evaluated the value of each investigated data stream for improving their combined predictive ability. the weighted 95% percentile intervals from the pooled predictions were used for carrying out this assessment. the results are depicted in fig 4 and indicate that, as for the individual model predictions, uncertainty in the collective predictions by the three lf models for the required number of years to eliminate lf using annual mda in each site may be reduced by model calibration to data, with the longitudinal mf prevalence data collected during the later monitoring periods (scenarios 3 and 4) contributing most to improving the multi-model predictions for each site. the boxplots show that by calibrating the models to data streams, more precise predictions are able to be made regarding timelines to achieve 1% mf prevalence across all models and sites. the results of pairwise f-tests for variance, performed to compare the weighted variance in timelines to achieve 1% mf prevalence between model-only simulations (scenario 0) and the lowest entropy simulations (best scenario) (see table 2 ), show that the predictions for the best scenarios are significantly different from the predictions for the model-only simulations. significance was determined using the benjamini-hochberg procedure for controlling the false discovery rate (q = 0.05). for epifil, lymfasim and transfil, the best scenarios are scenarios 4, 3, and 4 for kirare, scenarios 4, 4, and 3 for alagramam, and scenarios 3, 3, and 3 for peneng, respectively. we attempted to investigate if model uncertainty in predictions by the use of longitudinal data was a direct function of parameter constraining by the addition of data. given the similarity in outcomes of each model, we remark on the results from the fits of the technically easier to run epifil model to evaluate this possibility here. the assessment of the parameter space constraint achieved through the inclusion of data was made by determining if the fitted parameter distributions for the model became reduced in comparison with priors as data streams were added to the system [33] . the exercise showed that the size of the estimated parameter distributions reduced with addition of data, with even scenario 1 data producing reductions for kirare and peneng (fig 5) . in the case of alagramam, however, there was very little, if any, constraint in the fitted parameter space compared to the prior parameter space. this result, together with the fact that even using all the data in kirare and peneng produced up to only between 2.5 to 5% reductions in fitted parameter distributions when compared to the priors, indicate that the observed model prediction uncertainty in this study may be due to other complex factors connected with model parameterization. table 3 provides the results of an analysis of pairwise parameter correlations of the selected best-fitting models for data scenario 1 compared to those selected by the data stream that gave the best reduction in epifil prediction uncertainty for alagramam (scenario 3). these results show that while the parameter space was not being constrained with the addition of more data, the pattern of parameter correlations changed in a complex manner between the two constraining data sets. for example, although the number of significantly correlated parameters did not differ, the magnitude and direction of parameter correlations were shown to change between the two data scenarios ( table 3) . the corresponding results for kirare and peneng are shown in s3 supplementary information , and indicate that a broadly similar pattern of changes in parameter associations also occurred as a result of model calibration to the sequential data measured from those sites. this suggests that this outcome may constitute a general phenomenon at least with regards to the sequential constraining of epifil using longitudinal mf prevalence data. an intriguing finding (from all three data settings) is that the most sensitive parameters in this regard, i.e. with respect to altered strengths in pairwise parameter correlations, may be those representing the relationship of various components of host immunity with different transmission processes, including with adult worm mortality, rates of production and survival of mf, larval development rates in the mosquito vector and infection aggregation (table 3) . this suggests that, as more constraining data are added, changes in the multidimensional parameter relationships related to host immunity could contribute to the sequential reductions in the lf model predictive uncertainty observed in this study. the lf elimination timeline predictions used above were based on modelling the impacts of annual mda given the reported coverages in each site followed by an assumed standard coverage for making longer term predictions (see methods). this raises the question as to whether the differences detected in the case of the best constraining data stream between the present study sites and between models ( table 2 ) could be a function of the simulated mda coverages in each site. to investigate this possibility, we used epifil to model the outcome of changing the assumed mda coverage in each site on the corresponding entropy and information gain trends in elimination predictions made from the models calibrated to each of the site-specific data scenarios/streams investigated here. the results of increasing the assumed coverage of mda to 80% for each site are shown in fig 6 and indicate that the choice of mda coverage in this study are unlikely to have significantly influenced the conclusion made above that the best performing data streams for reducing model uncertainty for predicting lf elimination pertains to data scenarios 3 and 4. however, while the model-predicted timelines to achieve the 1% mf prevalence threshold using the observed mda coverage followed by 80% mda coverage showed that the data stream which most reduced uncertainty did not change from the impact of using the observed mda coverage followed by 65% mda coverage modelled for kirare and peneng (table 2, fig 6) , this was not the case for alagramam, where data from scenario 3 with a 80% coverage resulted in the greatest reduction in entropy compared to the original results using 65% coverage which indicated that scenario 4 data performed best (table 2, fig 6) . notably, though, the entropy values of predictions using the data scenario 3 and 4 constraints were not statistically different for this site (p-value < 0.05) (fig 6) . epifil was also used to expand the number of calibration scenarios using a dataset with longer term post-mda data from villupuram district, india. this dataset contained two addition data streams: scenario 5 which included baseline, post-mda 3, post-mda 5, and post-mda 7 mf data, and scenario 6, which included baseline, post-mda 3, post-mda 5, post-mda 7, and post-mda 9 mf data. scenario 6 thus contained the most post-mda data and was demonstrated to be the most effective for reducing model uncertainty, but this effect was not statistically significantly different from the reductions produced by assimilating data contained in table 3 . spearman parameter correlations for scenarios 1 (lower left triangle) and 3 (upper right triangle) for alagramam, india. data assimilation in filarial model predictions scenarios 3 and 5 ( table 4 ). the inclusion of more data than are considered in scenario 3 therefore did not result in any significant additional reduction in model uncertainty. epifil was used to evaluate the accuracy of the data-driven predictions of the timelines required to meet the goal of lf elimination based on breaching the who-set target of 1% mf for all sites, either scenario 3 or 4 had the lowest entropies, and scenario 4 was not significantly different from scenario 3 for kirare and alagramam. these results were not statistically different from the results given 65% coverage (see table 2 ), suggesting that the data stream associated with the lowest entropy is robust to changes in the interventions simulated. scenarios where the weighted variance or entropy were not significantly different from the lowest entropy scenario are noted with the abbreviation ns. significance was determined using the benjamini-hochberg procedure for controlling the false discovery rate (q = 0.05). https://doi.org/10.1371/journal.pntd.0006674.g006 table 4 . predictions of timelines to achieve 1% mf in villupuram district, india, considering extended post-mda data. reporting those scenarios which are statistically significantly different from each other by numbers (0-4) as superscripts. for example, the weighted variance for scenario 0 has the superscript numbers (1) (2) (3) (4) (5) (6) to indicate that the weighted variance for scenario 0 is significantly different from the weighted variance for scenarios 1-6. significance was determined using the benjamini-hochberg procedure for controlling the false discovery rate (q = 0.05) in all pairwise statistical tests. + information gained by each data stream (scenario1-6) are presented in comparison to the information contained in the model-only simulation (scenario 0) https://doi.org/10.1371/journal.pntd.0006674.t004 data assimilation in filarial model predictions prevalence. this analysis was performed by using the longitudinal pre and post-infection and mda data reported for the nigerian site, dokan tofa, where elimination was achieved according to who recommended criteria after seven rounds of mda (table 5 ). the data from this site comprised information on the abr and dominant mosquito genus, as well as details of the mda intervention carried out, including the relevant drug regimen applied, time and population coverage of mda, and outcomes from the mf prevalence surveys conducted at baseline and at multiple time points during mda [60] . the results of model predictions of the timelines to reach below 1% mf prevalence as a result of sequential fitting to the mf prevalence data from this site pertaining to scenarios 0-4 (as defined above) are shown in table 6 . note that in the post mda 3, 5 and 7 surveys, as no lf positive individuals were detected among the sample populations, we used a one-sided 95% clopper-pearson interval to determine the expected upper one-sided 95% confidence limits for these sequentially observed zero infection values data assimilation in filarial model predictions using the "rule of three" approximation after k empty samples formula [61] . the results show that model constraining by scenario 2, which includes baseline and post-mda 3 data, and scenario 3, which includes baseline, post-mda 3, and post-mda 5 data, resulted in both the least entropy values and the shortest predicted times, i.e., from as low as 2 to as high as 7 years, required for achieving lf elimination in this site ( table 6 ). the data in table 5 show that the first instance the calculated one-sided upper 95% confidence limit in this setting fell below 1% mf prevalence also occurred post mda 7 (i.e after 7 years of mda). this is a significant result, and indicates that apart from being able to reduce prediction uncertainty, the best data-constrained models are also able to more accurately predict the maximal time (7 years) by which lf elimination occurred in this site. our major goal in this study was to compare the reliability of forecasts of timelines required for achieving parasite elimination made by generic lf models versus models constrained by sequential mf prevalence surveillance data obtained from field sites undergoing mda. a secondary aim was to evaluate the relative value of data obtained at each of the sampling time points proposed by the who for monitoring the effects of lf interventions in informing these model predictions. this assessment allowed us to investigate the role of these data for learning system dynamics and measure their value for guiding the design of surveillance programmes in order to support better predictions of the outcomes of applied interventions. fundamentally, however, this work addresses the question of how best to use predictive parasite transmission models for guiding management decision making, i.e. whether this should be based on the use of ideal models which incorporate generalized parameter values or on models with parameters informed by local data [10] . if we find that data-informed models can reduce prediction uncertainty significantly compared to the use of theoretical models unconstrained by data, then it is clear that to be useful for management decision making we require the application of model-data assimilation frameworks that can effectively incorporate information from appropriate data into models for producing reliable intervention projections. antithetically, such a finding implies that using unconstrained ideal models in these circumstances will provide only approximate predictions characterized by a degree of uncertainty that might be too large to be useful for reliable decision making [14, 33, 62] . here, we have used three state-of-the-art lf models calibrated to longitudinal human mf prevalence data obtained from three representative lf study sites to carry out a systematic analysis of these questions in parasite intervention modelling (see also walker et al [63] for a recent study highlighting the importance of using longitudinal sentinel site data for improving the prediction performances of the closely-related onchocerciasis models). further, by iteratively testing the reduction in the uncertainty of the projections of timelines required to achieve lf elimination in a site made by the models matching each observed data point, we have also quantified the relative values of temporal data streams, including assessing optimal record lengths, for informing the current lf models. our results provide important insights as to how best to use process models for understanding and generating predictions of parasite dynamics. they also highlight how site-specific longitudinal surveillance data coupled with models can be useful for providing information about system dynamics and hence for improving predictions of relevance to management decision-making. the first result of major importance from our work is that models informed by data can significantly reduce predictive uncertainty and hence improve performance of the present lf models for guiding policy and management decision-making. our results show that these improvements in predictive precision were consistent between the three models and across all three of our study sites, and can be very substantive with up to as much as 92% to 96% reductions in prediction variance obtained by the best data-constrained models in a site compared to the use of model-only predictions ( table 2 ). the practical policy implications of this finding can also be gleaned from appraising the actual numerical ranges in the predictions made by each individual model for each of the modelling scenarios investigated here. in the case of epi-fil, the best data-informed model (scenario 3 in peneng) gave an elimination prediction range of 7-12 years, while the corresponding model-only predictions for this site indicated a need for between 6-29 years of annual mda (table 2 ). these gains in information from using data to inform model parameters and hence predictions were even larger for the two stochastic models investigated here, viz. lymfasim and transfil, where ranges as wide as 7-28 years predicted by model-only scenarios were reduced to 9-14 years for the best data-informed models in the case of lymfasim for kirare village, and from as broad as 8-48 years to 7-22 years respectively in the case of transfil for peneng (table 2 ). these results unequivocally indicate that if parasite transmission models are used unconstrained by data, i.e. based on general parameter values uninformed by local data, it would lead to the making of predictions that would be marked by uncertainties that are likely to be far too large to be meaningful for practical policy making. if managers are risk averse, this outcome will also mean their need to plan interventions for substantially much longer than necessary, with major implications for the ultimate cost of the programme. note also that although statistically significant changes in the median years of mda required to achieve lf elimination were observed for the best datainformed models for all the three lf model types in each site, these were relatively small compared to the large reductions seen in each model's predictive uncertainly (table 2, fig 3) . this result highlights that the major gains from constraining the present models by data lies in improving their predictive certainty rather than in advancing their average behaviour. however, our preliminary analysis of model predictive accuracy suggests that the best data-constrained models may also be able to generate more accurate predictions of the impact of control ( table 6 ), indicating that, apart from simply reducing predictive uncertainty, such models could additionally have improved capability for producing more reliable predictions of the outcomes of interventions carried out in a setting. the iterative testing of the reduction in forecast uncertainty using mf surveillance data measured at time points proposed by the who (to support assessment of whether the threshold of 1% mf prevalence has been reached before implementation units can move to post-treatment surveillance [44]) has provided further insights into the relative value of these data for improving the predictive performance of each of the present lf models. our critical finding here is that parameter uncertainty in all three lf models was similarly reduced by the assimilation of a few additional longitudinal data records (table 2 ). in particular, we show that data streams comprising baseline + post-mda 3 + post-mda 5 (scenario 3) and those comprising baseline + post-mda 5 data (scenario 4) best reduced parameter-based uncertainty in model projections of the impact of mdas carried out in each study site irrespective of the models used. although preliminary, a potential key finding is that the use of longer-term data additional to the data measured at the who proposed monitoring time points did not lead to a significant further reduction in parameter uncertainty (table 4) . also, the finding that the who data scenarios 3 and 4 were adequate for constraining the present lf models appears not to be an artefact of variations in the mda coverages observed between the three study sites (fig 6) . these results suggest that up to 5 years of post-mda mf prevalence data are sufficient to constrain model predictions of the impact of lf interventions at a time scale that can go up to as high as 7 to 22 years depending on the site and model, and that precision may not improve any further if more new data are added ( table 2, table 4 ). given that the who post-mda lf infection monitoring protocol was developed for the purpose solely focussed on supporting the meeting of set targets (e.g. the 1% mf prevalence threshold) and not on a priori hypotheses regarding how surveillance data could be used also to understand the evolution and hence prediction of the dynamical parasitic system in response to management action, our results are entirely fortuitous with respect to the value of the current lf monitoring data for learning about the lf system and its extinction dynamics in different settings [31] . they do, nonetheless, hint at the value that coupling models to data may offer to inform general theory for guiding the collection and use of monitoring data in parasite surveillance programmes in a manner that could help extract maximal information about the underlying parasite system of interest. our assessment of whether the incremental increase in model predictive performance observed as a result of assimilating longitudinal data may be due to parameter constraining by the addition of data has shed intriguing new light on the impact that qualitative changes in dynamical system behaviour may have on parameter estimates and structure, and hence on the nature of the future projections of system change we can make from models. our major finding in this regard is that even though the parameter space itself may not be overly constrained by the best data stream (scenario 3 in this case for alagramam village), the magnitude and direction of parameter correlations, particularly those representing the relationship of different components of host immunity with various transmission processes, changed markedly between the shorter (scenario 1) and seemingly optimal data streams (scenario 3). this qualitative change in system behaviour induced by alteration in parameter interactions in response to perturbations has been shown to represent a characteristic feature of complex adaptive ecological systems, particularly when these systems approach a critical boundary [64] [65] [66] . this underscores yet another important reason to incorporate parameter information from data for generating sound system forecasts [67] . the finding that additional data beyond 5 years post-mda did not appear to significantly improve model predictive performance in this regard suggests that pronounced change in lf parameter interactions in response to mda interventions may occur generally around this time point for this parasitic disease, and that once in this parameter regime further change appears to be unlikely. this is an interesting finding, which not only indicates that coupling models to at least 5 years post-mda will allow detection of the boundaries delimiting the primary lf parameter regions with different qualitative behaviour, but also that the current who monitoring protocol might be sufficient to allow this discovery of system change. although our principal focus in this study was in investigating the value of longitudinal data for informing the predictive performance of the current lf models, the results presented here have also underscored the existence of significant spatial heterogeneity in the dynamics of parasite extinction between the present sites ( table 2, fig 3) . in line with our previous findings, this observed conditional dependency of systems dynamics on local transmission conditions means that timelines or durations of interventions required to break lf transmission (as depicted in table 2 ) will also vary from site to site even under similar control conditions [3] [4] [5] 21] . as we indicated before, this outcome implies that we vitally require the application of models to detailed spatio-temporal infection surveillance data, such as that exemplified by the data collected by countries in sentinel sites as part of their who-directed monitoring and evaluation activities, if we are to use the present models to make more reliable intervention predictions to drive policy and management decisions (particularly with respect to the durations of interventions required, need for switching to more intensified or new mda regimens, and need for enhanced supplementary vector control) in a given endemic setting [64] . as we have previously pointed out, the development of such spatially adaptive intervention plans will require the development and use of spatially-explicit data assimilation modelling platforms that can couple geostatistical interpolation of model inputs (eg. abr and/or sentinel site mf/ antigen prevalence data) with discovery of localized models from such data in order to produce the required regional or national intervention forecasts [5] . the estimated parameter and prediction uncertainties presented here are clearly dependent on the model-data fusion methodology and its implementation, and the cost function used to discover the appropriate models for a data stream [20] . while we have attempted to evaluate differences in individual model structures, their computer implementation, and the data assimilation procedures followed (e.g. sequential vs. simultaneous data assimilation), via comparing the collective predictions of the three models versus the predictions provided by each model singly, and show that these factors are unlikely to play a major role in influencing the current results, we indicate that future work must address these issues adequately to improve the initial methods we have employed in this work. currently, we are examining the development of sequential bayesian-based multi-model ensemble approaches that will allow better integration of each model's behaviour as well as better calculation of each model's transient parameter space at each time a new observation becomes available [30] . this work also involves the development of a method to fuse information from several indicators of infection (e.g. mf, antigenemia, antibody responses [21] ) together to achieve a more robust constraining of the present models. as different types of data can act as mutual constraints on a model, we also expect that such multiindicator model-data fusion methods will additionally address the problem of equifinality, which is known to complicate the parameterization of complex dynamical models [24, 68] . of course, the ultimate test of the results reported here, viz. that lf models constrained by coupling to year 5 post-mda data can provide the best predictions of timelines for meeting the 1% mf prevalence threshold in a site, is by carrying out the direct validation of our results against independent observations (as demonstrated by the preliminary validation study carried out here using the dokan tofa data (tables 5 and 6)). we expect that data useful for performing these studies at scale may be available at the sentinel site level in the countries carrying out the current who-led monitoring programme. the present results indicate that access to such data, and to post-treatment surveillance data which are beginning to be assembled by many countries, is now a major need if the present lf models are to provide maximal information about parasite system responses to management and thus generate better predictions of system states for use in policy making and in judging management effectiveness in different spatiotemporal settings [24, 31] . given that previous modelling work has indicated that if the globally fixed who-proposed 1% mf prevalence threshold is insufficient to break lf transmission in every setting (and thus 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deteriorating environments practical limits for reverse engineering of dynamical systems: a statistical analysis of sensitivity and parameter inferability in systems biology models multi-sensor model-data fusion for estimation of hydrologic and energy flux parameters. remote sensing of environment key: cord-340569-f1odmjcs authors: hossain, mohammad sorowar; siddiqee, mahbubul h.; siddiqi, umme ruman; raheem, enayetur; akter, rokeya; hu, wenbiao title: dengue in a crowded megacity: lessons learnt from 2019 outbreak in dhaka, bangladesh date: 2020-08-20 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008349 sha: doc_id: 340569 cord_uid: f1odmjcs nan until it reached its peak in august (having more than 50% of the total case reported from january to december) [3] . then gradually, unlike all the previous outbreaks in bangladesh, the 2019 outbreak started to spread across the country. we hypothesize that mass movement of people who are already infected with the virus can accelerate the spreading of the disease to areas where the burden of dengue fever was otherwise low. daily dengue case data were collected from the dghs, while daily rainfall and temperature data during 2012-2019 were obtained from the bangladesh meteorological department. population consensus (2011) was obtained from the bangladesh bureau of statistics. time series analysis was performed to see the patterns of temperature and rainfall during and around the outbreak compared to the historical averages. a simple autoregressive integrated moving average (arima) model (1,0,0) was developed after controlling time series auto-correlation to explore if there was sufficient structure in the time series of dengue incidences to be explained by human movement. a district-level difference map between the incidence of one week after eid and one week before eid was created and visualized using arcmap (version 10.6). in july 2019 (prior to eid), over 81% of all dengue cases (16,253) were reported within the dhaka city [4] . before eid exodus (first week of august 2019), an interesting pattern of frequency of dengue case reporting was observed: during the week prior to eid, 57% (n = 7,952) of all the cases were reported inside dhaka compared to 43% (n = 5,927) throughout the rest of bangladesh, indicating the outbreak was mostly localized up until that point ( table 1 , fig 1) . however, this pattern started to reverse during the eid week, when the number of cases outside dhaka started to rise to nearly 55% (n = 7,663) of the total cases. the same trend sustained after eid weeks. while the incidence of dengue cases decreased sharply in dhaka city after the eid week (as much as about 24 times compared to pre-eid week), a net increase (around 4--7-fold) was observed in some districts of bangladesh, including narail, pirojpur, manikgonj, and faridpur (fig 2) . time series cross-correlation and autoregressive model showed that the dengue within dhaka city appeared to play a significant role in the transmission of dengue outside of dhaka (s1 fig, s1 table) . in bangladesh, all the dengue outbreaks since 2000 were primarily confined to dhaka city [7] . however, it is expected to spread in other areas because of the presence of aedes mosquitoes throughout bangladesh [6] . based on a survey conducted in 2014-2015, both a. aegypti and a. albopictus were found in dhaka city and in other urban areas. while the prevalence of a. aegypti was higher in urban areas, a. albopictus was more abundant in rural areas across bangladesh [6] . therefore, any scenario that increases migration of dengue patients and the carriers from dhaka to other regions could have the virus picked up by a new host (i.e., a. albopictus). we observed that the timing of changing the spatial pattern coincided with the eid. just before this time, nearly half of the population in dhaka (over 10 million) leave the city and travel across the country, usually within a week (to reunite with their family members) [8] . hence, based on the data analyzed from the 2019 outbreak, it can be hypothesized that the mass mobility during eid might have contributed to the spread of the outbreak. in this regard, the potential effects of climate factors on mosquito breeding were also considered. changes in climatic conditions could be associated with dynamic changes in the temporal distribution of dengue. however, bangladesh is a relatively small flat country, and with dhaka being geographically positioned in the middle, climatic factors like temperature and rainfall usually do not vary significantly [9] . therefore, while high temperatures during august 2019 might have influenced the largest outbreak in dhaka (potentially by influencing the extrinsic incubation period [eip] in the mosquitoes), a similar trend would have been seen for both dhaka and outside dhaka as the similar high temperature was reported all over bangladesh. however, a delayed rise of incidence in dengue fever outside dhaka at the time when incidence within dhaka was decreasing suggests that the temperature was most likely not the key factor in this case. instead, the geographic expansion of dengue in new populations was more likely facilitated by the travel of infected individuals to non-endemic areas. previous studies showed that transport vehicles could disperse aedes mosquitoes within a country [10] . lack of nationwide epidemiological studies made it difficult to verify whether the rise in dengue cases outside dhaka was due to a large number of infected individuals traveling as asymptomatic carriers. however, a number of investigative reports published in the national daily newspapers could provide an essential clue in this regard; stories of clustered infections among people not having a history of traveling to dhaka during one month before onset was frequent. this eventually suggests that transmission of dengue virus (or both virus and mosquito) during the mass migration before the eid was likely the most significant factor behind the geographical distribution of the diseases. from a public health standpoint, such mass mobility events (e.g., eid exodus, hajj pilgrimage, migration during the chinese new year) can sometimes be reasons for major concern if their location and timing coincide with major outbreak [11] . such unexpected coincidence could facilitate outbreaks (especially with highly contagious viruses) morphing into an epidemic or even pandemic (depending on other factors like ease of transmission and favorability of climate factors). the ongoing outbreak of coronavirus in wuhan, china, can be a recent example of this. transmission and spread of infectious diseases, including dengue and other aedes mosquito-borne diseases (chikungunya and zika), especially during an outbreak can therefore present an immense challenge for public health safety, as there is no vaccine against dengue. this is especially true for the countries that have high population density and have a positive and negative values in the legend represent increase and decrease of weekly dengue incidence, respectively. to calculate weekly incidence, total number of dengue cases for the week for each district was divided by the total population of the respective district and multiplied by 100,000. https://doi.org/10.1371/journal.pntd.0008349.g002 limited capacity to manage an increasing number of patients beyond the urban areas where the healthcare facilities are relatively well equipped. therefore, efforts taken by the regulatory authorities to restrain the disease need to take this mass migration-associated risk into account. public health preparedness can be an effective tool to reduce mortality and morbidity. in this regard, alongside conventional measures (e.g., the establishment of a comprehensive disease surveillance system, integrated vector control and management through community awareness, implementation of early diagnosis, prompt treatment, and effective patient referral protocol through trained caregivers at all levels of healthcare facilities), unconventional measures, particularly limiting or prohibiting mass migration (depending on the level of risk), could be considered. attempts at conducting risk assessment based on early warning signs therefore need to incorporate social factors and mass movement events along with the climate factors. supporting information s1 table. climate change and vector-borne viral diseases world urban areas & population projections disease control and research) dghs (director general of health services) co-circulation of dengue virus type 3-genotype i and type 2-cosmopolitan genotype largest dengue outbreak of the decade with high fatality may be due to reemergence of den-3 serotype in dhaka, bangladesh, necessitating immediate public health attention nationally-representative serostudy of dengue in bangladesh allows generalizable disease burden estimates. elife eid exodus: asia's muslims on the move at the end of ramadan climate of bangladesh direct evidence of adult aedes albopictus dispersal by car dengue fever in saudi arabia: a review of environmental and population factors impacting emergence and spread key: cord-345315-y3bdjnhg authors: dai, yaoyao; wang, jianming title: identifying the outbreak signal of covid-19 before the response of the traditional disease monitoring system date: 2020-10-01 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008758 sha: doc_id: 345315 cord_uid: y3bdjnhg new coronavirus cases and related deaths are continuing to occur worldwide. early identification of the emergence of novel outbreaks of infectious diseases is critical to the generation of timely responses. we performed a comparative study to determine the feasibility of the early detection of the covid-19 outbreak in china based on influenza surveillance data and the internet-based baidu search index to evaluate the timelines of the alert signals compared with the traditional case reporting and response systems. an abnormal increase in the number of influenza-like illnesses (ili) occurred at least one month earlier than the clinical reports of pneumonia with unknown causes and the conventional monitoring system. the peak of the search volume was 20 days earlier than the issuance of the massive official warning about the epidemic. the findings from this study suggest that monitoring abnormal surges of ili and identifying peaks of online searches of key terms can provide early signals of novel disease outbreaks. we emphasize the importance of broadening the potential of syndromic surveillance, internet searches, and social media data together with the traditional disease surveillance system to enhance early detection and understanding of emerging infectious diseases. synopsis: early identification of the emergence of an outbreak of a novel infectious disease is critical to generating a timely response. the traditional monitoring system is adequate for detecting the outbreak of common diseases; however, it is insufficient for the discovery of novel infectious diseases. in this study, we used covid-19 as an example to compare the delay time of different tools for identifying disease outbreaks. the results showed that both the abnormal spike in influenza-like illnesses and the peak of online searches of key terms could provide early signals. we emphasize the importance of testing these findings and discussing the broader potential to use syndromic surveillance, internet searches, and social media data together with traditional disease surveillance systems for early detection and understanding of novel emerging infectious diseases. introduction new coronavirus cases and related deaths are continuing occur worldwide. [1] the who, on march 11, 2020 , declared the coronavirus disease 2019 (covid-19) outbreak a global pandemic. this pandemic dates back to december 2019, when a cluster of unexplained pneumonia cases was identified, which were linked to a seafood market in wuhan, china. [2] subsequent investigations determined that a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was the causative agent now at the heart of the pandemic of an emerging infectious disease (eid). the virus jumped from the transportation hub to other areas during the peak seasonal travel periods of the winter holiday and the traditional spring festival. [3] to control the spread and mitigate the risk of the virus, a series of strong, unprecedented measures were taken by the chinese government. these measures included the mandatory wearing of face masks in public, canceling of mass events, closing of scenic attractions, suspending of long-distance buses, and asking hundreds of millions of chinese citizens to stay indoors to break the transmission chain. [4, 5] despite the rapid increase in the number of covid-19 cases in january, china has now passed the peak of the epidemic and has effectively controlled the disease. [4] no new infections of the novel coronavirus were reported on march 18 in wuhan, the epicenter of the epidemic in china, marking a notable first success in the months-long battle with the virus and showing hope of suppressing the pandemic. because this is an infectious disease caused by a new virus, it took approximately one month from the initial detection of unexplained pneumonia cases to the definite conclusion of "human-to-human transmission" and the inclusion of the disease in the management of statutory infectious diseases by the national health commission, china. the traditional disease monitoring system is useful for detecting the outbreak of common infectious diseases, but it is insufficient for the discovery of new diseases. [6] how to build a comprehensive early warning system of public health emergencies from multiple sources has become the focus of attention of all countries. to compensate for the shortcomings of the traditional disease monitoring system, some scholars have tried to use digital data streams, [7] network density, [8] and google trends (gt) [9] as early warning indicators; these attempts have achieved remarkable results; nevertheless, the roles of these indicators in covid-19 remain unclear. in this study, we performed a comparative study to discuss the early warning capability, timelines, and validity of alert signals for the first wave of the covid-19 outbreak in china based on the surveillance data of influenza-like illness (ili) and the baidu search index (bsi) compared with the traditional case reporting system. covid-19 data from china were obtained from the center for disease control and prevention of china and national health commission of china as well as the report of the who-china joint mission on coronavirus disease 2019 (https://www.who.int) and vital surveillances report on the epidemiological characteristics of an outbreak of covid-19-china, 2020. [10] the data source of influenza-like illnesses we extracted data regarding ili reported from january 2015 to may 2019 from the national health commission of china. after the 2003 sars epidemic, the chinese government built the world's most extensive internet-based disease reporting system, called the china information system for disease control and prevention (cisdcp). [11] cases of infectious diseases, categorized as class a, b, and c, are required to be reported through the cisdcp within a limited time. we compared the monthly morbidity of ili during the last five years and plotted a line chart to describe the long-term trend. we also compared the peak of ili with the onset of the covid-19 in the late 2019 in china. we used the baidu search engine (http://index.baidu.com/v2/#/) to analyze the bsi for searches of the keywords of "pneumonia" and "sars" from november 1, 2019, to february 1, 2020. baidu is the world's largest chinese search engine and china's largest internet integrated service company. the bsi reflects active searches by internet users. we compared the timeline of peak searches for these key terms with the time of official response to the epidemic. data were entered into excel and analyzed using spss 25 (ibm, ny, usa). the ili cases across several years were compared using the analysis of variance. the dunnet method was used for pairwise comparison. the test level for significance was set at 0.05. data of this study were extracted from a public database. no individual information was published in this paper. therefore, this study is exempt from ethical approval. on december 29, 2019, the department of health of hubei province and wuhan city received a report from a local hospital regarding patients with unexplained pneumonia, all of whom were employees of the south china seafood wholesale market. on december 31, the national health commission and cdc sent a team of experts to wuhan. the investigators excluded several suspected causes, including influenza, avian influenza, adenovirus, severe acute respiratory syndrome coronavirus (sars-cov), and the middle east respiratory syndrome coronavirus (mers-cov). on january 1, 2020, the local government closed this seafood market and disinfected the area. on january 3, 2020, the chinese government informed the who of the outbreak of unexplained pneumonia. on january 7, 2020, the pathogen was identified as a new type of coronavirus, and then, the full genome sequences of this new virus were shared. on january 10, an expert group and a who team were invited to visit wuhan for a field investigation. by january 19, 198 novel coronavirus cases have been reported in wuhan. as of january 19, the risk of human-to-human transmission of this new virus had not been determined, and officials have not realized the potential global epidemic risk. on january 20, the novel coronavirus pneumonia was incorporated as a notifiable disease under the infectious disease law and health and quarantine law in china. on january 23, the whole city of wuhan was locked down, and all the residents were required to stay at home. two days later, the chinese government made the highest-level commitment to mobilize all forces to stop the epidemic. [12] as of january 28, 2020, there were more than 5900 confirmed cases and more than 9000 suspected cases of covid-19 across 33 chinese provinces or municipalities. [13] human-to-human transmission of the pathogen was also confirmed. [14] huang et al. analyzed laboratory-confirmed covid-19 cases in wuhan and showed that the symptom onset date of the first patient was december 1, 2019. [14] it is estimated that the origin of covid-19 was most likely earlier than december 2019. as shown in fig 1a, it took more than one and a half months for the traditional surveillance system to trigger the alert of the outbreak of this eid. as shown in fig 1b, there was a search peak for the terms of "pneumonia" (39641 times) and "sars" (297864 times) on december 31, 2019, mainly in wuhan (pneumonia: 11304 times; sars: 53887 times), where the outbreak of covid-19 occurred. with the official announcement of the exclusion of sars and the absence of apparent human to human transmission, the number of searches decreased rapidly the following day. until around january 20, the bsi of these two terms began to rise again, resulting in a second search peak, which was consistent with the increase in confirmed covid-19 cases countrywide (fig 1b) . overall, there were differences in the number of ili in 2014-2019 (f = 8.03, p<0.001). as shown in fig 2a, the ili case numbers in 2019 were significantly higher than those reported in the previous years of 2014-2018 (p<0.05). we observed an early spike in ili in winter of 2019, with a fast-growing period from november to december (fig 2b) . this observation suggests that covid-19 cases may have occurred before december 2019. the signal of the abnormally rapid increase in ili cases was earlier than the report of clinical cases of pneumonia with unknown causes through the official routine disease monitoring system. early identification of the emergence of an outbreak of a novel infectious disease is critical to generating a timely response. the traditional monitoring system is adequate for detecting the outbreak of common diseases; however, it is insufficient for the discovery of novel eids. in this study, we used covid-19 as an example to compare the delay time of different tools for identifying disease outbreaks. the results showed that both the abnormal spike in ili and the peak of online searches of key terms could provide early signals of novel eids. for centuries, infectious diseases have been among the leading causes of death and have presented growing challenges to human health. the threat is further increased by the continued emergence of new and unrecognized infectious disease epidemics. [15] due to the lack of sensitive and specific diagnostic tools, infections are often undiagnosed and therefore untreated, or are diagnosed at late stages. early detection of infectious diseases plays a crucial role in all treatment and prevention strategies. a crucial goal of infectious disease surveillance is the early detection of epidemics, which is essential for disease control. in china, the current surveillance system is based on confirmed case reports. [16] it is not practical for health units to perform laboratory tests to confirm a novel infectious disease. most infectious disease outbreaks start with clinicians noticing unusual patterns. patients may present with patterns of symptoms that are similar to those of more common diseases but which, after repeated observation and diagnostic testing, may deviate in scale, seasonality, or severity. [17] the discovery of covid-19 is an example. in december 2019, clinicians from wuhan city reported several patients with unexplained pneumonia, all of whom were employees of south china seafood wholesale market. bronchoalveolar lavage samples were collected and sequenced for the whole genome. bioinformatic analyses indicated that the pathogen was a novel coronavirus, showing the closest relationship with the bat sarslike coronavirus strain batcov ratg13. on january 8, 2020, the novel coronavirus was confirmed as the cause of unexplained pneumonia. however, at that time, people did not realize the potential risk of an epidemic (even less a pandemic) caused by this new pathogen. it was not until mid-to late-january that the risk of widespread transmission was taken seriously. in other words, clinical symptom monitoring and case reporting can help identify new diseases; however, these practices do not provide timely signals of an epidemic. internet-derived information has recently been recognized as a valuable tool for epidemiological investigation. [18] timeliness and precision in the detection of infectious disease outbreaks from the information published on the web are crucial for prevention of their spread. arsevska et al. retrieved data from a corpus of relevant documents and compared them with african swine fever (asf) outbreaks from the google search engine and the pubmed database. [19] the results showed that relevant documents could serve as a source of terms to detect infectious animal disease emergence on the web. walker et al. used google trends (gt) to investigate whether there was a surge in searches for information related to the covid-19 epidemic. these authors observed a strong correlation between the frequency of searches for smellrelated information and the onset of covid-19 infection in italy, spain, uk, usa, germany, france, iran and netherlands. [20] li et al. demonstrated that the data obtained from gt, bsi and the sina weibo index on searches for the keywords 'coronavirus and 'pneumonia' correlated with the published daily incidence of covid-19, with the maximum r > 0.89. [21] however, few studies explored the role of web-based search index in detecting the first occurrence of the covid-19. in this study, we used the bsi to explore the correlation between the internet search index and the outbreak of covid-19. the bsi is a public sampling database of search queries users entered into the predominant search engine (baidu) in china. unlike gt, the bsi reflects the absolute baidu search volume and is not displayed as normalized values. [22] one important issue that emerges from web-based searches is that they tend to underestimate the real epidemiological burden when the general population has poor knowledge of the disease. [18] additionally, the bsi can be influenced by media clamor. therefore, the real scientific usefulness of the so-called "digital epidemiology" remains questionable, at least when using gt or bsi. although the source of information cannot be taken for granted or even replace the "real life" epidemiological data, mining the web is an intriguing perspective for eids. when and where sars-cov-2 originated remains unclear. the similarity between covid-19 and influenza symptoms makes it possible that the excess ili cases were due to covid-19 cases. the presence of sars-cov-2-positive swabs in the patients supports this possibility. [23] the predominant symptoms associated with covid-19 are fever, cough, and sore throat; that is, patients often present with an ili. at the early stage of the epidemic, covid-19 cases may have been misdiagnosed as influenza or other respiratory diseases. thus, we hypothesized that ili surveillance data could be used as a tool for early detection of covid-19. kong et al. analyzed 640 throat swabs collected from patients with ili in wuhan from october 6, 2019, to january 21, 2020, and found that nine samples were positive for sars-cov-2, suggesting community transmission of sars-cov-2 in wuhan in early january 2020. [24] the dramatic increase in ili in wuhan in early december further supported this hypothesis. [24] spellberg et al. observed a seasonal spike in ili in los angeles, usa. [25] among patients with mild ili, 5% were tested positive for sars-cov-2. such transmission is consistent with the countywide unusual third ili spike that occurred late in the season and with declining rates of influenza positivity. [25] however, seasonal influenza activity was lower in 2020 than in previous years in japan. [26] it may have been affected by temperature or virulence and by measures taken to constrain the sars-cov-2 outbreak. [26] the coinfection of covid-19 and influenza a reported in iran also highlighted the importance of considering sars-cov-2 pcr assay regardless of positive findings for other pathogens during the epidemic. [27] silverman et al. explored how ili outpatient surveillance data could be used to estimate the prevalence of covid-19, and they found a surge in noninfluenza ili above the seasonal average in march 2020 and showed that this surge correlated with covid-19 across states. [28] in our study, several potential limitations should not be neglected. first, the web-based search for key terms or ili surge counts in relation to the emergence of covid-19 may be attributed to potential confounders. second, the observed ili surge may represent more than just sars-cov-2-infected patients. whether ili surveillance data could be used for the signal of the eids without dominant features of covid-19, such as cough and fever, is unclear. third, the web-based search can be affected by media coverage, the population's knowledge or the degree of information disclosure. in conclusion, monitoring abnormal surges in ili and identifying online search peaks of key terms can provide early signals of novel disease outbreaks. we emphasize the importance of testing these findings and discussing the broader potential to use syndromic surveillance, internet searches, and social media data together with traditional disease surveillance systems for early detection and understanding of eids. the covid-19 epidemic. tropical medicine & international health what we know so far: covid-19 current clinical knowledge and research a novel coronavirus outbreak of global health concern successful containment of covid-19: the who-report on the covid-19 outbreak in china the positive impact of lockdown in wuhan on containing the covid-19 outbreak in china new technologies in predicting, preventing and controlling emerging infectious diseases an early warning approach to monitor covid-19 activity with multiple digital traces in near real-time detecting early signals of covid-19 global pandemic from network density applications of google search trends for risk communication in infectious disease management: a case study of the covid-19 outbreak in taiwan the novel coronavirus pneumonia emergency response epidemiology team zhonghua liu xing bing xue za zhi = zhonghua liuxingbingxue zazhi emergence and control of infectious diseases in china gaps remain in china's ability to detect emerging infectious diseases despite advances since the onset of sars and avian flu genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding clinical features of patients infected with 2019 novel coronavirus in wuhan emerging and neglected infectious diseases: insights, advances, and challenges establishing a web-based integrated surveillance system for early detection of infectious disease epidemic in rural china: a field experimental study tracking virus outbreaks in the twenty-first century is google trends a reliable tool for digital epidemiology? insights from different clinical settings identification of terms for detecting early signals of emerging infectious disease outbreaks on the web. computers and electronics in agriculture use of google trends to investigate loss-of-smell-related searches during the covid-19 outbreak retrospective analysis of the possibility of predicting the covid-19 outbreak from internet searches and social media data, china, 2020. euro surveillance: bulletin europeen sur les maladies transmissibles = european communicable disease bulletin correlations of online search engine trends with coronavirus disease (covid-19) incidence: infodemiology study pubmed central euro surveillance: bulletin europeen sur les maladies transmissibles = european communicable disease bulletin sars-cov-2 detection in patients with influenza-like illness community prevalence of sars-cov-2 among patients with influenzalike illnesses presenting to a los angeles medical center pubmed central seasonal influenza activity during the sars-cov-2 outbreak in japan co-infection of coronavirus disease 2019 and influenza a: a report from iran using influenza surveillance networks to estimate state-specific prevalence of sars-cov-2 in the united states key: cord-340939-ikomc19t authors: van doremalen, neeltje; lambe, teresa; sebastian, sarah; bushmaker, trenton; fischer, robert; feldmann, friederike; haddock, elaine; letko, michael; avanzato, victoria a.; rissanen, ilona; lacasse, rachel; scott, dana; bowden, thomas a.; gilbert, sarah; munster, vincent title: a single-dose chadox1-vectored vaccine provides complete protection against nipah bangladesh and malaysia in syrian golden hamsters date: 2019-06-06 journal: plos negl trop dis doi: 10.1371/journal.pntd.0007462 sha: doc_id: 340939 cord_uid: ikomc19t nipah virus (niv) is a highly pathogenic re-emerging virus that causes outbreaks in south east asia. currently, no approved and licensed vaccine or antivirals exist. here, we investigated the efficacy of chadox1 niv(b), a simian adenovirus-based vaccine encoding niv glycoprotein (g) bangladesh, in syrian hamsters. prime-only as well as prime-boost vaccination resulted in uniform protection against a lethal challenge with niv bangladesh: all animals survived challenge and we were unable to find infectious virus either in oral swabs, lung or brain tissue. furthermore, no pathological lung damage was observed. a single-dose of chadox1 niv(b) also prevented disease and lethality from heterologous challenge with niv malaysia. while we were unable to detect infectious virus in swabs or tissue of animals challenged with the heterologous strain, a very limited amount of viral rna could be found in lung tissue by in situ hybridization. a single dose of chadox1 niv(b) also provided partial protection against hendra virus and passive transfer of antibodies elicited by chadox1 niv(b) vaccination partially protected syrian hamsters against niv bangladesh. from these data, we conclude that chadox1 niv(b) is a suitable candidate for further niv vaccine pre-clinical development. introduction chadox1-vectored vaccines fulfil all these requirements, making this a promising platform. the chadox1 vector is a replication-deficient simian adenovirus vector which has been used to produce several vaccines which are now in clinical development. a common feature of these vaccines is their low reactogenicity, strong immunogenicity, and the absence of vector replication after immunization, an important safety feature. in pre-clinical studies a single dose of chadox1 vectored vaccines has been shown to be protective against infection with rift valley fever virus, middle east respiratory syndrome coronavirus, mycobacterium tuberculosis and zika virus [18] [19] [20] [21] . large scale manufacturing has been performed for replicationdeficient adenoviral vectored vaccines for ebola, with one vaccine now licensed and another in advanced clinical development [22, 23] . further, a simple thermostabilization process allows for vaccine storage at ambient temperatures [24] , removing the need for a cold chain for storage and shipping. we now report on pre-clinical immunogenicity and efficacy testing of chad-ox1 niv b . animal experiment approval was received from the institutional animal care and use committee (iacuc) at rocky mountain laboratories. experiments were performed in an association for assessment and accreditation of laboratory animal care-approved facility by certified staff, following the guidelines and basic principles in the nih guide for the care and use of laboratory animals, the animal welfare act, united states department of agriculture and the united states public health service policy on humane care and use of laboratory animals (protocol # 2017-033e and 2018-035e). the institutional biosafety committee (ibc) approved work with infectious niv and hendra virus (hev) strains under bsl4 conditions and sample inactivation was performed according to ibc-approved standard operating procedures for removal of specimens from high containment. henipavirus isolates were obtained from the special pathogens branch of the centers for disease control and prevention, atlanta, ga or public health agency, winnipeg, canada. niv bangladesh (genbank no. ay988601), niv malaysia (genbank no. af212302), and hev (genbank no. af017149) have been passaged three, four, and three times in veroe6 cells respectively. all virus propagation in this manuscript was performed in veroe6 cells in dulbecco's modified eagle's medium (dmem, sigma) supplemented with 2% fetal bovine serum (gibco), 1 mm l-glutamine (gibco), 50 u/ml penicillin (gibco), and 50 μg/ml streptomycin (gibco) (2% dmem). veroe6 cells were maintained in dmem supplemented with 10% fetal bovine serum, 1 mm l glutamine, 50 u/ml penicillin and 50 μg/ml streptomycin. the glycoprotein (g) gene from nipah virus (bangladesh outbreak 2008-2010, genbank accession number: jn808864.1) was codon optimized for humans and synthesized by geneart (thermo fisher scientific). the synthesized g gene was cloned into a transgene expression plasmid comprising a modified human cytomegalovirus immediate early promoter (cmv promoter) with tetracycline operator (teto) sites and the polyadenylation signal from bovine growth hormone (bgh). the resulting expression cassette was inserted into the e1 locus of a genomic clone of chadox1 using site-specific recombination [25] . the virus was rescued and propagated in t-rex-293 cells (invitrogen). purification was by cscl gradient ultracentrifugation, and the virus was titered as previously described [26] . doses for vaccination were based on infectious units (iu). female golden syrian hamsters (4-6 weeks old) were purchased from envigo. animals were vaccinated i.m. with 50 μl of 10 8 iu of vaccine or injected i.m. with 50 μl of saline, in each thigh (100 μl total volume). for the homologous challenge vaccine experiment, animals were vaccinated at d-70 and/or d-42. for the heterologous challenge experiment, animals were vaccinated at d-28. three days prior to vaccination and virus challenge animals were bled via orbital sinus puncture. all animals were challenged with 1000ld 50 of virus in 500 μl dmem via i.p. inoculation: niv bangladesh = 5.3 x 10 5 tcid 50 ; niv malaysia = 6.8 x 10 4 tcid 50 ; hev = 6.0 x 10 3 tcid 50 . we chose the i.p. route as a uniformly lethal challenge route and to be able to compare with previously conducted vaccine experiments [27] . for each study group, 10 hamsters were utilized. of these, four animals were euthanized 4 (hev) or 5 (niv) days post inoculation and the remaining six animals were followed for 28 days post challenge. weight was recorded daily up to 10 days post infection, and oropharyngeal swabs were taken daily up to 7 days post inoculation in 1 ml of dmem. animals were euthanized when >20% of weight loss was recorded, or severe disease signs (e.g. difficulty breathing or paralysis) were observed. upon euthanasia, blood and tissues were collected and subsequently analyzed for virology and histology as approved by iacuc. female golden syrian hamsters (4-6 weeks old) were purchased from envigo. fifteen animals were vaccinated with either chadox1 niv b or chadox1 gfp as described above at 56 and 28 days before serum collection. serum was collected via cardiac puncture, pooled per vaccine group and iggs were purified using the mabtrap kit (sigma) according to manufacturer's instructions from 10 ml of serum. purified iggs were filtered through an 0.45μm filter and diluted to 4.5 ml in sterile pbs. ten hamsters were immunized via i.p. injection using 400 μl per hamster. all animals were challenged as described above one day post treatment. for each study group, 10 hamsters were utilized. of these, four animals were euthanized 5 days post challenge and the remaining six animals were followed for 56 days post challenge. weight was recorded daily up to 10 days post challenge, and oropharyngeal swabs were taken daily up to 7 days post inoculation in 1 ml of dmem. animals were euthanized when >20% of weight loss was recorded, or severe disease signs (e.g. difficulty breathing or paralysis) were observed. upon euthanasia, blood and tissues were collected and subsequently analyzed for virology and histology as approved by iacuc. virus titrations were performed by end-point titration in veroe6 cells, which were inoculated with tenfold serial dilutions of virus swab media or tissue homogenates. after 1hr incubation at 37˚c and 5% co 2 , tissue homogenate dilutions were removed, washed twice with pbs and replaced with 100 μl 2% dmem. cytopathic effect was scored at 5 dpi and the tcid 50 was calculated from 4 replicates by the spearman-karber method [28] . added to veroe6 cells and incubated at 37˚c and 5% co 2 . at 5 dpi, cytopathic effect was scored. the virus neutralization titer was expressed as the reciprocal value of the highest dilution of the serum which still inhibited virus replication. niv-g malaysia (residues e144-t602, gene accession number nc_002728) was cloned into the phlsec mammalian expression vector [29] and niv-f malaysia (residues g26-d482, gene accession number ay816748.1) was cloned into the phlsec vector containing a c-terminal gcnt trimerization motif [30] . the constructs were transiently expressed in human embryonic kidney (hek) 293t cells in roller bottles, as described previously [29] . supernatant was harvested 96 hours after transfection and diafiltrated using the akta flux system (ge healthcare) against either pbs, ph 7.4 (niv-g) or buffer containing 10 mm tris and 150 mm nacl, ph 8.0 (niv-f). the proteins were further purified by ni-nta immobilized metal-affinity chromatography using his-trap hp columns (ge healthcare) followed by size exclusion chromatography. niv-g was purified using a superdex 200 10/300 increase gl column (ge healthcare) equilibrated in pbs ph 7.4 and niv-f was purified using a superose 6 increase 10/ 300 gl column (ge healthcare) equilibrated in 10 mm tris and 150 mm nacl ph 8.0. maxisorp plates (nunc) were coated overnight at 4˚c with 5 μg of g or f protein per plate in carb/bicarb binding buffer (4.41 g khco 3 and 0.75 g na 2 co 3 in 1 l distilled water). after blocking with 5% milk in pbs with 0.01% tween (pbst), serum (2x serial diluted starting at 100x dilution) in 5% milk in pbst was incubated at rt for 1 hr. antibodies were detected using affinity-purified antibody peroxidase-labeled goat-anti-hamster igg (fisher, 14-22-06) in 5% milk in pbst and tmb 2-component peroxidase substrate (seracare) and read at 450 nm. all wells were washed 3x with pbst in between steps. prior to using f and g proteins based on niv malaysia, we established that cross-reactivity with niv bangladesh antibodies was sufficient for usage in elisa by testing sera known to be positive for niv bangladesh antibodies. necropsies and tissue sampling were performed according to ibc-approved protocols. harvested tissues were fixed for a minimum of 7 days in 10% neutral-buffered formalin and subsequently embedded in paraffin. hematoxylin and eosin (h&e) staining and in situ hybridization (ish) were performed on tissue sections and cell blocks. detection of niv and hev viral rna was performed using the rnascope ffpe assay (advanced cell diagnostics inc., newark, usa) as previously described [31] and in accordance with the manufacturer's instructions. briefly, tissue sections were deparaffinized and pretreated with heat and protease before hybridization with target-specific probes for niv or hev. ubiquitin c and the bacterial gene, dapb, were used as positive and negative controls, respectively. whole-tissue sections for selected cases were stained for niv and hev viral rna, ubc and dapb by the rnascope vs ffpe assay (rnascopevs, newark, usa) using the ventana discovery xt slide autostaining system (ventana medical systems inc., tucson, usa). a board-certified veterinary anatomic pathologist evaluated all tissue slides. statistical analysis was performed by the log-rank (mantel-cox) test to compare survival curves, and by welch-corrected one-tailed unpaired student's t-test to compare infectious virus titers in tissue. sem was calculated for all samples. p-values < 0.05 were significant. to determine efficacy of the chadox1 niv b vaccine, we vaccinated groups of 10 hamsters with either a single dose at d-42 or a prime-boost regime at d-70 and d-42. as control groups, we either injected hamsters with chadox1 gfp at d-70 and d-42 or saline at d-42 ( fig 1a) . virus neutralizing antibodies could be detected after a single dose of chadox1 niv b and increased upon a secondary dose (average vn titer ± sem = 30.5 ± 5.7 after single dose, 91 ± 21 after boost). in contrast, no neutralizing antibodies could be detected in serum obtained from the control groups ( fig 1b) . all hamsters were challenged with a lethal dose of niv bangladesh (1000 ld 50 ) via intraperitoneal inoculation on d0 ( fig 1a) . all vaccinated animals survived challenge and did not show signs of disease, such as weight loss, at any stage throughout the experiment. this was in contrast to the control groups in which all animals succumbed to disease between d6 and d10 and exhibited weight loss (fig 1c and 1d) , as well as respiratory and/or neurological signs, including labored breathing and paralyzed hind legs. statistical analysis demonstrated that survival in the vaccinated groups was significant compared to both control groups (p < 0.0001). oropharyngeal swabs were taken daily and assessed for infectious virus by limiting dilution titrations. none of the vaccinated animals shed virus at any timepoint. in contrast, control animals from both groups were found to shed virus at d5 and d6 (fig 1e) . four animals of each group were euthanized at d5 and lung and brain tissue were harvested. infectious virus could only be detected in lung tissue of animals from both control groups (average titer ± sem = 3.3 x 10 4 ± 2.5 x 10 4 tcid 50 /g of tissue) and was not detected in any tissue of the vaccinated animals ( fig 1f) . we did not observe any differences between the two control groups. lung and brain tissue harvested at d5 were then evaluated for pathological changes. none of the vaccinated animals displayed pulmonary pathology and no viral rna was detected in lung tissue by ish. control animals developed pulmonary lesions that were indistinguishable between the two groups. these hamsters developed bronchointerstitial pneumonia that was characterized by multifocal inflammatory nodules that were centered on terminal bronchioles and extend into adjacent alveoli. the nodules were composed of large numbers of foamy macrophages and fewer neutrophils and lymphocytes admixed with small amounts of necrotic debris. in most cases hemorrhage, fibrin and edema admixed with inflammatory cells was observed. edema and fibrin often were extended into surrounding alveoli. alveoli that were adjacent to areas of inflammation were thickened by fibrin, edema and small numbers of macrophages and neutrophils as previously observed in niv infected hamsters [32] . there was abundant viral rna demonstrated by ish in areas of inflammation (brown staining). the viral rna was predominantly found in type i pneumocytes but was also multifocally present in vascular and bronchiolar smooth muscle and endothelial cells (fig 2) . to determine efficacy of chadox1 niv b against niv malaysia and hev, groups of 10 hamsters were vaccinated with a single dose of chadox1 niv b or a single dose of chadox1 gfp at d-28 ( fig 3a) . as before, virus neutralizing antibodies could be detected after vaccination with chadox1 niv b but not upon injection with chadox1 gfp (average vn titer ± sem = 68.6 ± 13.6) ( fig 3b) . subsequently, hamsters were challenged with either niv malaysia or hev (1000 ld 50 ) via intraperitoneal inoculation on d0 (fig 3a) . all vaccinated animals challenged with niv malaysia survived with no signs of disease such as weight loss at any stage throughout the experiment. in contrast, animals challenged with niv malaysia that received chadox1 fgp all succumbed to infection between d5 and d6. these animals experienced weight loss and respiratory and neurological signs (fig 3c and 3d) . statistical analysis demonstrated that survival in the vaccinated group was significantly different from the control group (p = 0.0012). oropharyngeal swabs were taken daily and assessed for infectious virus. none of the vaccinated animals challenged with niv malaysia shed virus at any timepoint. in contrast, control animals challenged with niv malaysia were found to shed virus at d5 and d6 (fig 3e) . four animals from both groups were euthanized at d5 and lung and brain tissue were harvested. infectious virus could only be detected in lung and brain tissue of animals from the control group (average virus titer lung ± sem = 1.5 x 10 5 ± 5.2 x 10 4 tcid 50 /g, brain ± sem = 6.8 x 10 1 ± 4.4 x 10 1 tcid 50 /g) and was not detected in any tissue of the vaccinated animals ( fig 3f) . four out of six vaccinated animals challenged with hev succumbed to disease between d5 and d7. the two survivors showed minimal weight loss (<2%) and no signs of disease. animals that received chadox1 fgp all succumbed to hev infection between d4 and d6. these animals showed weight loss as well as respiratory and neurological signs (fig 3c and 3d) . logrank (mantel-cox) test demonstrated that survival in the vaccinated group was significant (p = 0.0476) compared to the control group. oropharyngeal swabs were taken daily and assessed for infectious virus. none of the vaccinated animals challenged with hev shed virus at any timepoint. in contrast, control animals challenged with hev were found to shed virus at d4, d5 and d6 (fig 3e) . four animals from both groups were euthanized at d4 and lung and brain tissue were harvested. infectious virus was detected in three out of four lungs of the vaccinated animals and all lungs of the control animals (average virus titer ± sem = 5.2 x 10 5 ± 3.6 x 10 5 and 4.4 x 10 6 ± 2.2 x 10 6 tcid 50 /g tissue for vaccinated and control animals, respectively). no statistical difference in infectious virus titer was found between the two groups using an unpaired onetailed student's t-test (p = 0.0674). infectious virus was only detected in brain tissue of animals from the control group (average titer ± sem = 4.6 x 10 2 ± 2.0 x 10 2 tcid 50 /g) and not in vaccinated animals (fig 3f) . harvested lung tissue was then evaluated for pathological changes. all four groups of hamsters developed pulmonary lesions. all animals challenged with hev and control animals challenged with niv malaysia developed bronchointerstitial pneumonia which was indistinguishable from the lesions described for the control animals in the homologous challenge study. vaccinated hamsters challenged with niv malaysia developed mild to moderate bronchointerstitial pneumonia and did not display any evidence of pulmonary edema, fibrin or hemorrhage. ish demonstrated viral rna predominantly in type i pneumocytes and rarely in vascular and bronchiolar smooth muscle and endothelial cells in animals challenged with hev and control animals challenged with niv malaysia. in vaccinated animals challenged with niv malaysia, however; there was very little rna present and only in type i pneumocytes in areas of inflammation (fig 4) . finally, we wanted to assess the protective effect of antibodies elicited after chadox1 niv b vaccination. two groups of 15 hamsters were either vaccinated with chadox1 niv b or injected with chadox1 fgp at d-56 and d-28. all animals were bled at d0 and we collected 13 and 15 ml respectively. igg was purified from 10 ml pooled serum. ten animals per group were then injected peritoneally with purified igg. animals were challenged with a lethal dose of niv bangladesh (1000 ld 50 ) one day post passive transfer ( fig 5a) . we were unable to detect neutralizing antibodies in serum obtained at d5 from four hamsters from each group. however, serum from animals treated with niv antibodies was positive by elisa against niv g protein, albeit with a lower reciprocal titer than antibodies in serum obtained from single-dose vaccinated animals (fig 5b) . one out of six animals treated with niv antibodies succumbed to disease on d11. no weight loss was observed, however the animal showed severe neurological signs. none of the other niv antibody-treated animals experienced weight loss or signs of disease. four out of six animals treated with gfp antibodies succumbed to disease between d6 and d8. these animals showed weight loss and respiratory or neurological signs. the two surviving animals did not show any signs of disease throughout the experiment. one of these animals did not seroconvert as measured by elisa against niv f and g protein, and it was suspected this animal was not infected. therefore, this animal was excluded from the survival curve. the log-rank (mantel-cox) test demonstrated that survival in the treated group was significant (p = 0.0168) compared to the control group (fig 5c and 5d) . oropharyngeal swabs were taken daily and assessed for infectious virus. shedding was minimal and found in one animal treated with niv antibodies on d5, and five animals treated with gfp antibodies between d4 and d6 (fig 5e) . four animals from both groups were euthanized at d5 and lung and brain tissue were harvested. infectious virus could only be detected in lung tissue of animals treated with gfp antibodies and was not detected in any tissue of the animals treated with niv antibodies (fig 5f) . lung tissue harvested at d5 was then evaluated for pathological changes. both groups of hamsters developed pulmonary lesions similar to those described in the homologous challenge study, however; the niv antibody-treated hamsters developed mild to moderate pulmonary lesions whereas the control animals developed severe lesions. additionally, none of the niv antibody-treated hamsters displayed any pulmonary fibrin, edema or hemorrhage. ish demonstrated viral rna in type i pneumocytes in areas of inflammation. abundance of viral rna was notably less in animals treated with niv antibodies (fig 6) . niv is a re-emerging infectious disease which causes outbreaks with a high case-fatality rate. no licensed vaccine against niv currently exists, and it is therefore key that a safe and effective vaccine be developed. several vaccine candidates have been explored in different animal models. these can be categorized as subunit vaccines or live-vectored vaccines that target the niv outer membrane proteins g and/or f. protection against disease and lethality has been shown in hamsters [27, 33] , pigs [34, 35] , african green monkeys [36] [37] [38] , cats [39] , and ferrets [40, 41] . efficacy is thought to be mediated by neutralizing antibodies, as passive transfer of chadox1 nivb efficacy in the syrian golden hamster antibodies in naive animals also results in protection against disease [27, 42] . these approaches are promising, but no vaccine candidates have so far been moved into clinical trials. in the studies presented here, we tested the efficacy of a vaccine based on niv bangladesh g protein in a replication-deficient simian adenovirus vector in syrian hamsters. a primeonly as well as a prime-boost regime protected syrian hamsters against challenge with a lethal dose of niv bangladesh and niv malaysia, and partially protected against hev challenge. furthermore, antibodies elicited by vaccination alone provided partial protection against a niv bangladesh challenge. two genetic lineages of niv have been described; niv malaysia and niv bangladesh [10] [11] [12] . although niv malaysia has not caused an outbreak in humans since 1999, the virus was isolated from pteropus vampyrus, pteropus hypomelanus and pteropus lylei in malaysia and cambodia [43] [44] [45] and another spillover event could occur. having one vaccine that protects against both lineages of niv virus would be the easiest and cheapest countermeasure. a singledose vaccination with chadox1 niv b , which is based on niv bangladesh, fully protected syrian hamsters against lethal disease caused by niv malaysia. the g proteins of the niv strains used in this study are 95.5% pairwise identical on the amino acid level, with 27 amino acid differences scattered throughout the protein. although we did not see sterile protection against niv malaysia, none of the vaccinated animals showed signs of disease and all were protected against lethal disease. these results suggest that chadox1 niv b could protect against both lineages of niv. like niv, hev is a species in the henipavirus genus and thus we investigated cross-protection of chadox1 niv b against a lethal challenge with hev in syrian hamsters. the g protein of the hev strain used in this study was 78.2% identical to the chadox1 niv b g protein; 133 amino acids differ between the two proteins. chadox1 niv b only protected partially against hev challenge; four out of six animals did not survive challenge. we observed a non-significant decrease in infectious hev titer in lung and brain tissue of vaccinated animals compared to control animals. it is possible that disease progression in vaccinated animals is delayed compared to control animals. this is supported by the delay in time to death; whereas the average time to death is 5 days in control animals, it is 6 days in vaccinated animals. cross-protection of niv or hev vaccines has been studied by other groups as well. an adeno-associated virus vaccine expressing niv g protein offered 50% protection against a lethal challenge with hev in hamsters [46] . in contrast, vaccines based on hev provide full protection against niv in the ferret and nhp model [36, 41, 47] . likewise, high levels of crossprotective antibodies were found in sera from hev-infected individuals, whereas cross-protective antibodies were limited in niv-infected individuals [48] . this might be caused by induction of a more robust and cross-reactive immune response by native hev protein compared to niv protein, as suggested by bossart et al. [48] . human cases of hev are associated with direct contact with infected horses, the intermediate animal host of hev, and direct contact with bats or their products has not yet been associated with hev infection in humans [49] . it is therefore likely that prevention of hev in horses will completely prevent human cases. currently, a hev vaccine (equivac) is available for horses and fully protects against hev [50] . furthermore, the total number of human cases that contracted hev is relatively low at 7 [13] . thus, the requirement of a human vaccine for hev is therefore less urgent than that of a niv vaccine. previous work has shown that the humoral immune response to niv vaccination is sufficient to protect syrian hamsters against a lethal challenge with niv [27, 42] . likewise, administration of a human neutralizing monoclonal antibody (m102.4) provided full protection against both hev and niv in multiple animal models [51, 52] . administration of purified igg obtained from chadox1 niv b vaccinated hamsters provided partial protection against niv challenge. furthermore, infectious virus could only be detected in the lungs of control animals and not in the lungs of vaccinated animals, and thus as in previous studies, chadox1 niv belicited antibodies are able to provide protection against a lethal challenge with niv. although we were able to detect niv g protein-specific antibodies in serum obtained from niv antibody-treated animals, the reciprocal titer was much lower than that detected in serum from syrian hamsters after a single dose of chadox1 niv b . it is possible that administering a higher dose of igg would have led to uniform protection. two animals treated with igg purified from animals which received injections with chad-ox1 fgp survived a lethal challenge with niv bangladesh. occasional survival has been observed in the syrian hamster model [33] . the increased survival rate might however also reflect a non-specific effect of treatment with igg, which has been reported previously [53] . as the survival rate was significantly different between the niv igg-treated group and the control igg-treated group, the passive transfer experiment shows that antibodies elicited by chad-ox1-niv b are sufficient for protection against a lethal challenge with niv. animals in the passive transfer experiment were observed for 56 days, to ensure that the two animals that survived would not succumb to disease after 28 days. the syrian hamster is a suitable initial small animal model to investigate the efficacy of niv vaccines, followed by the african green monkey [54] . the immune system of african green monkeys is more like humans than that of hamsters and is therefore seen as a more relevant animal model to test niv vaccines. based on the results presented in the current manuscript, future studies are planned to test chadox1 niv b in african green monkeys, supported by the coalition for epidemic preparedness innovations (cepi). we show that chadox1 niv b provides complete protection against lethal disease in syrian hamsters challenged with niv bangladesh. furthermore, chadox1 niv b vaccination results in complete survival but with limited evidence of viral replication after niv malaysia challenge, and partial protection against hev. passive transfer of antibodies elicited by chadox1 niv b vaccination provide partial protection against lethal challenge with niv bangladesh. nipah virus: a recently emergent deadly paramyxovirus pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission pathogenic differences between nipah virus bangladesh and malaysia strains in primates: implications for antibody therapy nipah virus outbreaks in the who south-east asia region person-to-person transmission of nipah virus in a bangladeshi community nipah virus transmission from bats to humans associated with drinking traditional liquor made from date palm sap, bangladesh date palm sap 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green monkeys from nipah virus challenge single-dose live-attenuated vesicular stomatitis virus-based vaccine protects african green monkeys from nipah virus disease recombinant measles virus vaccine expressing the nipah virus glycoprotein protects against lethal nipah virus challenge a recombinant subunit vaccine formulation protects against lethal nipah virus challenge in cats single injection recombinant vesicular stomatitis virus vaccines protect ferrets against lethal nipah virus disease vaccination of ferrets with a recombinant g glycoprotein subunit vaccine provides protection against nipah virus disease for over 12 months nipah virus: vaccination and passive protection studies in a hamster model characterization of nipah virus from naturally infected pteropus vampyrus bats isolation of nipah virus from malaysian island flying-foxes nipah virus in lyle's flying foxes, cambodia protection against henipavirus infection by use of recombinant adeno-associated virus-vector vaccines feline model of acute nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine neutralization assays for differential henipavirus serology using bio-plex protein array systems changing resource landscapes and spillover of henipaviruses hendra virus vaccine, a one health approach to protecting horse, human, and environmental health a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection a neutralizing human monoclonal antibody protects african green monkeys from hendra virus challenge a protective monoclonal antibody targets a site of vulnerability on the surface of rift valley fever virus development of an acute and highly pathogenic nonhuman primate model of nipah virus infection we would like to thank the animal care takers for their excellent care of the animals, and anita mora for assistance with figures. benjamin carrasco provided outstanding assistance with the preparation for animal experiments. greg saturday, kimberly meade-white and kathleen cordova were instrumental in assistance during the animal studies. we thank benhur lee for kindly providing the c-dna for niv-f. key: cord-327799-ngzvdd8c authors: chaumont, claire; kamara, kimberly; baring, elisa; palacio, karen; power, ana; lancaster, warren title: the sars-cov-2 crisis and its impact on neglected tropical diseases: threat or opportunity? date: 2020-09-21 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008680 sha: doc_id: 327799 cord_uid: ngzvdd8c nan the current global priority is to protect people from attaining the covid-19 infection and to attend to those infected, resulting in the disruption of other activities of the health sector, such as neglected tropical disease (ntd) control and elimination programs, which, across the globe, have postponed mass drug administration (mda) campaigns. the impact of this disruption, combined with that of the pandemic, will ripple for years to come. this commentary reflects on how the current crisis modifies the future of the ntd sector focused on the five diseases treated through preventative chemotherapy (often called pc-ntds): soil-transmitted helminths, schistosomiasis, lymphatic filariasis, onchocerciasis, and trachoma. the most obvious impact of the crisis on ntd programs relates to its effect on infection prevalence due to delayed mass drug administration campaigns. the world health organization (who) developed a new roadmap that was meant to officially launch in june 2020 that includes specific disease targets to control and eliminate ntds by 2030 [1] . with sars-cov-2, not only is the launch of the roadmap postponed, but all ntd activities were postponed to prevent risk of additional transmission of covid-19 [2] . with the moratorium slowly being lifted, tools and guidelines are emerging to help countries undertake the necessary steps for ntd interventions to not fall too far behind its disease control and elimination goals. all five pc-ntds require annual mass treatment with high treatment coverage to reduce infection rates. delays or cancelation of such campaigns can lead to infection resurgence. a recent modelling exercise has shown that these delays will particularly impact diseases with short elimination timelines (three to five years), such as trachoma or lymphatic filariasis, and areas with high levels of schistosomiasis infections where interruption of set cycles could erase years of effort [3] . delays and interruptions to treatment cycles are not unknown to the ntd space, due to setbacks in the supply chain, unseasonal rains, conflicts, political campaigning, or even reduced funding. ntd programs are remarkably adept at making the necessary accommodations to adjust for such delays [4] . regardless, many countries may need to "catch up" with intermediate rounds of mdas to mitigate the effect of the enforced pause on infection rates in communities. in addition, delays to monitoring and evaluation activities may further impact the ability of the community to allocate stretched resources as efficiently as possible. this is especially relevant for schistosomiasis, considering recent global efforts to develop more precise maps to identify hot spots. prior to the sars-cov-2 outbreak, ntd prevention through mda was described as the "best buy in public health" [5] . despite the high disease-adjusted life years (dalys) burden, the five pct diseases risk being deprioritized if resources are stretched due to their low mortality rate when compared to diseases such as malaria [6] . the prioritization of sars-cov-2 stalled ntd programming and stretched staff, including community health workers (chws), who are often redeployed to support other pressing health matters. some ntd funding, both internal and external, was repurposed in the short term in order to contribute to the sars-cov-2 response for acute services or products, and it is unclear whether such funds will be replenished [7] . long-term funding for ntds may also be impacted. this is especially true if a resurgence of infection pushes elimination goals further in the future, if the costs of ntd interventions significantly increase due to the inclusion of needed safety measures, or if additional mda rounds are needed to limit disease resurgence. the sars-cov-2 crisis will also profoundly change the design and delivery of ntd programs. shifts in human capital availability and transmission dynamics of sars-cov-2 will require national ntd programs to adopt different implementation approaches to ensure availability and use of appropriate personnel, as well as preventive measures, including social distancing, safe hygiene practices, and face masks and/or coverings. health personnel at national and subnational levels are joining the pandemic response. human resources for ntd activities are thus shifting due to economic crisis and health system priorities. this will result in reduced capacity and turnover to focus on restarting the planning of interventions, both for preventive and curative services. in addition, preventative chemotherapy has historically relied on community drug distributors, voluntarily or paid. in the future, there may be less willingness for individuals in these roles to support mda due to an overwhelmed system and economic strains that stymie volunteerism. a reevaluation of human capacity needs will be required to determine the appropriate approach to restart activities. the increased need for social distancing will also modify ntd program delivery. house-tohouse treatment may need to be favored over fixed-point distribution, to minimize people coming together in crowds. this will impact how community drug distributors or teachers are mobilized for distribution (e.g., teachers usually involved in school-based distribution may have to reach children in their houses). when distribution must be organized at fixed points, rules limiting the number of people gathering will have to be put in place. in addition, new protocols may need to be put into place for morbidity management or surgical activities to ensure patients can safely receive these services. reengaging communities on access to free treatment and case management services will be another hurdle for ntd programs to overcome. mda campaigns are dependent on a high level of community acceptance to ensure high coverage levels of the campaign. however, amidst injunctions of social distancing, communities may be skeptical of gathering. an overall distrust of health services and personnel may exist, a situation that arose post the ebola outbreak in west africa. in 2015, the ministry of health and social welfare (mohsw) of liberia conducted a post-ebola ntd readiness assessment, which outlined the need to improve community awareness and understanding of the treatment [8] . these lessons guided the scaleup of ntds post-ebola and the revisions to social mobilization to ensure the success of the program. as ntd programs assess restarting activities, revising communication strategies will be key for community and health worker uptake. the sars-cov-2 pandemic seriously threatens the gains made by ntd programs in recent years. at the same time, the sector is endeavoring to use this moment to think more synergistically to adapt this extraordinary vertical campaign platform to new circumstances to further serve populations affected by ntds and fight the pandemic along the way. first, the way the ntd sector responds to delays is an opportunity to modify or accelerate treatment strategies (for example, through additional mda rounds, expanded treatments, or more targeted services for high-transmission areas). this goes along with the growing realization that a greater investment should be made in securing consistently high coverage and intake of preventative chemotherapy. leveraging and learning from other delivery platforms' experiences during other campaigns, such as polio, malaria, or human african trypanosomiasis (which have all leveraged geospatial data to identify untreated areas, map out all settlements and plot efficient routes, and used gps to confirm delivery), could also strengthen the ntd platform. the need to "catch up" because of covid-19-related delays may spur further treatment innovations or adaptations, including in research & development for drugs and diagnostics. in parallel, there is an opportunity to try different delivery models to ensure the success of ntd activities restarting while mitigating potential risks. control and elimination activities for the five pct ntds traditionally utilize a campaign model through community-and school-based platforms. an alternative could be returning to a former approach used for the control of onchocerciasis, community directed treatment with ivermectin (cdti), in which the treatment was conducted by community members over a longer period of time [9] . this approach could better mitigate the risk of sars-cov-2 transmission, but community acceptance and costs would need to be carefully considered, as well as its potential impact on coverage. another approach would be to explore further the integration of ntds into routine primary health care services, such as the delivery of drugs in antenatal care clinics or nutrition programs. coordinating treatment alongside food distribution efforts, particularly for schistosomiasis control, could also be a positive outcome in a time when economic crisis may lead to further food insecurity for vulnerable populations at risk of ntds. taking the medication with food would ease any side effects of the drug praziquantel and could increase overall compliance. there are also opportunities for expanding the use of mhealth technology for payment transfer, monitoring, and supervision of ntd activities to maximize "touchless" approaches. finally, restarting ntd programming is an opportunity to reconsider social mobilization strategies. information campaigns will require additional efforts put towards discouraging mass gathering for treatment and safety measures for health or community worker-led treatments. it will be critical to educate drug distributors and the communities on covid-19 symptoms to ensure they are not mistaken for serious adverse events. sensitization of treatment will also require additional channels of communication as the predominant health communication is currently focused on pandemic-related news. programs should consider conducting surveys to better understand if there is a need to increase efforts to mobilize and generate demand among communities. the covid-19 pandemic will have a long-lasting economic, social, and health impact across the globe. in the field of ntds, it may lead to reinfections due to delayed care. in this brave new world, the ntd community has a responsibility to advocate for continued prioritization of ntds on the global health agenda, in alignment with the likely transformed political and funding landscape. this could be a pivotal moment to further strengthen health systems with embedded horizontal platforms for ntd prevention. ending the neglect to attain the sustainable development goals. a road map for neglected tropical diseases 2021-2030. geneva world health organization. who | covid-19: who issues interim guidance for implementation of ntd programmes world health organization the potential impact of programmes interruptions due to covid-19 on 7 neglected tropical diseases: a modelling-based analysis. meeting report. gates open research neglected tropical diseases as a 'litmus test' for universal health coverage? understanding who is left behind and why in mass drug administration: lessons from four country contexts country leadership and collaboration on neglected tropical diseases. in: uniting to combat ntds asymmetries of poverty: why global burden of disease valuations underestimate the burden of neglected tropical diseases exclusive: dfid pauses "some new decisions" as aid budget expected to fall. in: devex [internet commentary: restarting ntd programme activities after the ebola outbreak in liberia who | setting up community-directed treatment with ivermectin (cdti): how it works and who does what world health organization key: cord-280030-neqycg6v authors: sewlall, nivesh h.; richards, guy; duse, adriano; swanepoel, robert; paweska, janusz; blumberg, lucille; dinh, thu ha; bausch, daniel title: clinical features and patient management of lujo hemorrhagic fever date: 2014-11-13 journal: plos negl trop dis doi: 10.1371/journal.pntd.0003233 sha: doc_id: 280030 cord_uid: neqycg6v background: in 2008 a nosocomial outbreak of five cases of viral hemorrhagic fever due to a novel arenavirus, lujo virus, occurred in johannesburg, south africa. lujo virus is only the second pathogenic arenavirus, after lassa virus, to be recognized in africa and the first in over 40 years. because of the remote, resource-poor, and often politically unstable regions where lassa fever and other viral hemorrhagic fevers typically occur, there have been few opportunities to undertake in-depth study of their clinical manifestations, transmission dynamics, pathogenesis, or response to treatment options typically available in industrialized countries. methods and findings: we describe the clinical features of five cases of lujo hemorrhagic fever and summarize their clinical management, as well as providing additional epidemiologic detail regarding the 2008 outbreak. illness typically began with the abrupt onset of fever, malaise, headache, and myalgias followed successively by sore throat, chest pain, gastrointestinal symptoms, rash, minor hemorrhage, subconjunctival injection, and neck and facial swelling over the first week of illness. no major hemorrhage was noted. neurological signs were sometimes seen in the late stages. shock and multi-organ system failure, often with evidence of disseminated intravascular coagulopathy, ensued in the second week, with death in four of the five cases. distinctive treatment components of the one surviving patient included rapid commencement of the antiviral drug ribavirin and administration of hmg-coa reductase inhibitors (statins), n-acetylcysteine, and recombinant factor viia. conclusions: lujo virus causes a clinical syndrome remarkably similar to lassa fever. considering the high case-fatality and significant logistical impediments to controlled treatment efficacy trials for viral hemorrhagic fever, it is both logical and ethical to explore the use of the various compounds used in the treatment of the surviving case reported here in future outbreaks. clinical observations should be systematically recorded to facilitate objective evaluation of treatment efficacy. due to the risk of secondary transmission, viral hemorrhagic fever precautions should be implemented for all cases of lujo virus infection, with specialized precautions to protect against aerosols when performing enhanced-risk procedures such as endotracheal intubation. viral hemorrhagic fever (vhf) is an acute systemic illness classically involving fever, a constellation of initially nonspecific signs and symptoms, and a propensity for bleeding and shock. vhf may be caused by more than 25 different viruses from four taxonomic families: arenaviridae, filoviridae, bunyaviridae, and flaviviridae. transmission of hemorrhagic fever viruses is through direct contact with blood and bodily fluids during the acute illness. although patient isolation and specific vhf precautions (consisting of surgical mask, double gloves, gown, protective apron, face shield, and shoe covers) are advised for added security, experience has shown that routine universal and contact precautions are protective in most cases [1] . aerosol precautions, such as the use of n95 particulate filters, are only recommended when performing specific potentially aerosol-generating procedures, such as endotracheal intubation. south africa has often played a role of ''sentinel'' for vhf in countries further to the north through the travel and admission of undiagnosed patients to south african hospitals, often with subsequent nosocomial transmission to healthcare workers. for example, cases of marburg and ebola hemorrhagic fevers have been reported in johannesburg in persons initiating travel in zimbabwe [2] and gabon [3] , respectively. in 2008 a nosocomial outbreak of five cases of vhf occurred in johannesburg [4, 5] (figure 1). the primary patient was a tour operator who was evacuated from lusaka, zambia. the etiologic agent was determined to be a novel arenavirus and the name ''lujo virus'' was proposed. the source of the patient's infection is unknown, but assumed to be a rodent, as with all other pathogenic arenaviruses. recent field studies of small mammals in zambia did not result in isolation of lujo virus, although another novel arenavirus was discovered [6] . arenaviruses are divided into two groups: the new world (or tacaribe) complex, and the old world (or lymphocytic choriomeningitis/lassa) complex, with various members of both groups causing vhf in south america and africa, respectively [7] lassa virus, the distribution of which is confined to west africa, is the only other old world arenavirus associated with vhf [8] . lujo virus is only the second pathogenic arenavirus to be recognized in africa and the first in over 40 years. some arenavirus infections, especially lassa fever, have shown benefit with the use of the nucleoside analogue ribavirin [9] . because of the remote and resource poor locations where lassa fever typically occurs, as well as the history of civil unrest in west africa in recent decades, there have been few opportunities to undertake in-depth study of the clinical manifestations or pathogenesis of lassa fever or other vhfs, or the response of these infections to treatment options typically available in industrialized countries. we describe the clinical features of the five recognized cases of lujo hemorrhagic fever (lhf) in the 2008 outbreak in south africa and summarize their clinical management, as well as providing additional epidemiologic detail, with a focus on the risks for secondary transmission. the initial description of the outbreak [4] was published primarily under the auspices of the south african national institute for communicable diseases, which had a blanket ethics author summary viral hemorrhagic fever is a syndrome often associated with high fatality and risk of secondary transmission. in 2008, an outbreak of a novel hemorrhagic fever virus called lujo occurred in johannesburg, south africa, with secondary transmission from the index patient to four healthcare workers. four of the five patients died. lujo belongs to the arenavirus family and is only the second pathogenic arenavirus, after lassa virus, to be recognized in africa and the first in over 40 years. because most viral hemorrhagic fevers occur in remote, resource-poor settings, few in-depth controlled studies of their clinical manifestations, transmission dynamics, pathogenesis, or response to treatment options are possible. we describe the clinical features of the five cases in this outbreak and summarize the clinical management, as well as providing additional epidemiologic detail. lujo virus causes a clinical syndrome remarkably similar to lassa fever. the treatment options used in these five cases are discussed as well as the recommended precautions to prevent secondary transmission. approval for use of all the patients' data. the same data set has been used for this publication, with ethics committee approval, with the exception of further data collated on the one survivor, who provided written consent for use of data and images related to her illness. case descriptions case 1. the initial case and primary patient (patient 1) was a 36 year old white female who lived on the outskirts of suburban lusaka, zambia. she kept horses, dogs and cats at her house and evidence of rodents was found in her stables (personal communication, r. swanepoel). the patient fell sick on september 2 (illness day [id]-1) with the abrupt onset of fever, myalgia, sore throat, and headache, for which she took over-the-counter antipyretics and analgesics. the next day she described nonbloody diarrhea and vomiting. a mild erythematous rash appeared on id-5 on her chest and upper arms. fever up to 39uc continued intermittently, escalating on id-7, accompanied by retrosternal chest pain and worsening sore throat, after which she presented to a clinic in lusaka (id-8), where she was given broad spectrum antibiotics. by id-9 the rash covered her entire body. myalgias became more prominent and her face was noticeably swollen. rapid deterioration occurred on id-10 with progressive confusion and generalized tonic-clonic seizures. she was intubated with some difficulty using only succinylcholine and started on further antibiotics, including ceftriaxone, ciprofloxacin and ampicillin. the patient was evacuated by air ambulance to a private tertiary care hospital in johannesburg on september 12 (id-11). the glasgow coma score was 3/10, with contracted non-reactive pupils and absent corneal reflexes but no papilledema-findings consistent with transtentorial brain herniation syndrome and damage to the pontine tegmentum from diffuse cerebral edema. generalized edema, including of the face and neck, was present. there was no visible hemorrhage. a fine macular rash was observed over her torso and legs. an eschar resembling a tick bite was visible on her right foot. diffuse interstitial infiltrates with bibasal atelectasis was noted on chest radiography. the patient received a tentative diagnosis of tick bite fever (rickettsia africae) and was started on intravenous (iv) cefepime, clarithromycin, and linezolid, along with lactated ringers solution and dobutamine. mechanical ventilation was continued (fio 2 0.8; bipap 20/ 10 mmhg; rate 14). the p a o 2 /f i o 2 ratio was 160. on id-12 progressive organ failure occurred. oliguria was followed by a high anion gap metabolic acidosis and worsening generalized edema. continuous veno-venous hemodialysis was commenced. a ct scan of the brain showed extensive cerebral edema with compression of the brainstem ( figure 2 ). an eeg showed diffuse slowing. blood tests on id-11 demonstrated leukocytosis (27610 9 /l), thrombocytopenia (42610 9 /l), elevated hepatic transaminases (ast 1,029 iu/l, alt 386 iu/l) and lactate dehydrogenase (ldh 2,432 iu/l), and mildly elevated c reactive protein (crp) (27 mg/l). the wbc rose to 58610 9 /l the next day . blood cultures remained negative, as well as tests for malaria, typhoid fever, brucellosis, syphilis, and autoimmune disease. rapidly progressive hemodynamic collapse and death occurred on id-13 despite inotropic and vasopressor therapy. case 2. patient 2, a 33 year old white male, was the paramedic who accompanied patient 1 on the medical evacuation flight from zambia to johannesburg, subsequently returning to lusaka. he participated in the intubation of patient 1 at the referring hospital wearing disposable gloves but no gown, mask or face visor. no specific exposure to blood or other bodily fluids was noted. on september 21 (id-1), nine days after last contact with the index case, patient 2 noted the abrupt onset of fever, headache and myalgias. three days later (id-4) he was admitted to a hospital in lusaka for a possible upper respiratory infection and treated with oral amoxicillin and antipyretics. on id-4 he developed a diffuse, erythematous skin rash, sore throat, and worsening myalgia and his fever rose to 40uc. intravenous fluids and antibiotics were begun. on id-7 he was transferred to the same hospital in johannesburg as patient 1. initial evaluation showed him to be fully awake and alert with a diffuse maculopapular eruption on his chest, arms, legs and back, sub-conjunctival hemorrhage, face and neck swelling, and pharyngitis, with ecchymoses on the hard and soft palates. he began to have non-bloody diarrhea. clinical laboratory examination revealed thrombocytopenia (52,000/ml); leucopenia (2610 8 /l); elevated transaminases (ast 969 iu/l, alt 293 iu/l), ldh (2040 iu/l), and procalcitonin (2,0 ng/ ml); marginally elevated crp (27) ; a positive d dimer (.10 mg/ ml); and microscopic hematuria. the inr was 1.42 and the partial thromboplastin time. (ptt) was elevated to 90 seconds. tests for malaria, rickettsia, and salmonella were negative. a presumptive diagnosis of thrombotic thrombocytopenic purpura was made and plasmapheresis initiated on id-8. prominent bleeding from the central vein insertion site was noted. on id-9 the patient was seen by the intensive care unit (icu) physician who cared for patient 1 and an epidemiologic link was noticed. vhf precautions were immediately implemented. given the history, possible filovirus infection was considered and contact tracing of the first patient was commenced by members of the hospital infection control team. modest improvement in patient 2's condition was noted after plasmapheresis, with the platelet count increasing to 86,000/ml. however, rapid clinical deterioration began on id-10, including altered mental status, oliguria, metabolic acidosis, and progressive generalized edema. sustained low efficiency dialysis was begun and the patient was intubated due to worsening ards (p a o 2 / f i o 2 ratio 100). fulminant hepatitis (ast 3,763 iu/l; alt 1,107 iu/l; ldh 7,207 iu/l), encephalopathy, and shock ensued and the patient died on id-12 despite inotropic and vasopressor support. case 3. on october 2, the day of patient 2's death, contact tracing revealed that an icu nurse (patient 3) who cared for patient 1 was admitted to a private hospital west of johannesburg, close to her family home. patient 3 was a 34 year old black female who became ill on september 25 (id-1), nine days after caring for patient 1 (a previous publication on this outbreak erroneously cites this patient's first day of illness as september 23) [4] . she was primarily involved in turning and cleaning patient 1, including washing the corpse and removing the dialysis catheter after her death. infection control precautions in the care of patient 1 included providing care in an isolation room and wearing of surgical gowns, latex gloves, surgical masks, and plastic visors. no needle stick injuries or splashes of blood or bodily fluids were reported. patient 3's illness began with headache and myalgia followed by sore throat, high fever, and rigors on id-5. oral amoxicillin and antipyretics were started by her general practitioner. worsening headache and fever prompted hospitalization and isolation on id-6, where nausea, abdominal cramps, non-bloody vomiting, and dysphagia were reported and a fine, macular rash noted on her trunk. (nb: although paweska et al. [4] reported that no rash was seen in the black patients with lhf, subsequent review of the treating physician's notes confirmed that a rash was indeed seen in this patient.) clinical laboratory testing on admission was limited but demonstrated thrombocytopenia (78,000/ml) and normal transaminases (ast 18 iu/l, alt 24 iu/l). renal function was normal. initial therapy consisted of iv fluids and ceftriaxone, fluconazole, and omeprazole. the patient's condition worsened on id-7 with non-bloody diarrhea, worsening rash, and peri-orbital and facial swelling. subconjunctival hemorrhage was noted. clinical laboratory analysis showed leukocytosis (13,000/ml), worsening thrombocytopenia (38.000/ml), and drastic elevations of liver enzymes (ast 2,182 iu/l; alt 748 iu/l; ldh 3,421 iu/l). the quantitative d-dimer was markedly elevated(.10.0 ug/l). oral ribavirin (1,800 mg loading dose followed by 800 mg q8 hours) and iv gancyclovir (5 mg/kg q12 hr) were begun, the latter to cover the possibility of disseminated herpes virus infection. nevertheless, the patient's condition worsened on id-9, with continued diarrhea and facial edema, progressive mental obtundation, thrombocytopenia (47,000/ml) and persistently elevated transaminases (ast 2,486 iu/l; alt 804 iu/l). a decision not to institute intensive care was taken collectively by the provincial outbreak investigators given the circumstances at the time and the facilities available at the hospital. the patient became comatose and died on id-10. case 4. patient 4 was a 38 year old black female with a history of aids and a cd4 count of 250. she worked as a cleaner and was involved in the disinfection of the hospital room where patient 1 died, which was performed wearing a scrub gown, surgical mask, plastic visor and surgical latex gloves. no specific exposures to blood or bodily fluids were reported. patient 4 fell ill on september 27 (id-1), 13 days after cleaning patient 1's room. initial complaints included headache, dry cough, rhinitis, sore throat, myalgias and left sided chest pain. she visited her general primary care practitioner where a fever of 38.5uc was recorded and amoxicillin and diclofenac were prescribed. five days later (id-6), she presented to the infectious disease clinic at her local hospital. on the basis of fevers, sweating and an abnormal chest radiograph, outpatient therapy for tuberculosis was started. however, her condition continued to deteriorate and she was admitted to her local hospital on id-8. at this point, the contact tracing team had located her and she was transferred to a tertiary academic hospital where she was noted to be confused with photophobia, nausea and vomiting. physical exam showed candidiasis and generalized lymphadenopathy. lumbar puncture and cerebrospinal fluid analysis showed five neutrophils and no lymphocytes, markedly elevated protein (.5 g/dl) and elevated glucose (7 mmol/l), which were considered consistent with a diagnosis of tuberculous meningitis. clinical laboratory analysis revealed thrombocytopenia (23,000/ml), elevated transaminases (ast 549 iu/l, alt 237 iu/l), mild renal dysfunction, a high anion gap metabolic acidosis, and a positive hepatitis b surface antigen. the patient's confusion worsened and fatal cardiac arrest occurred on id-10. case 5. patient 5 was a 47 year old white female who worked as an icu nurse caring for patient 2 from september 27-29. she had significant exposure to blood and bodily fluids, including cleaning up vomitus and changing bloody dressings over the insertion site of the central catheter on september 27. although there was not yet a particular concern of vhf when the nurse was caring for patient 2, she reported wearing plastic aprons, disposable gloves, and surgical masks, although she admits to potential lapses in the consistent wearing of this apparel. along with other contacts, patient 5 was placed on twice daily temperature monitoring. on october 10 (id-1), ten days after her last exposure to patient 2, she noted a temperature of 38.4uc along with retro-orbital headache, nausea, and significant anxiety and was admitted to the hospital. blood tests revealed thrombocytopenia (91,000/ml), leucopoenia (1,300/ml), normal levels of hepatic transaminases and an elevated d-dimer (2.84 mg/ml). a diagnosis of probable vhf was made (this was 2 days before an etiologic agent was identified). since iv ribavirin was not available, oral ribavirin (2 g loading dose followed by 1 g q6 hrs) was begun on id-2 along with atorvastatin (80 mg qd) and nacetylcysteine (800 mg q8), both for their immunomodulatory and anti-inflammatory effects [10, 11] , and anxiolytics. on id-2 myalgias became prominent and thrombocytopenia worsened (61,000/ml). on id-3 the temperature was 38.6uc and non-bloody diarrhea and vaginal bleeding began, despite the patient being midcycle. laboratory tests on id-4 show a leukocyte count of 7,100/ml, elevated transaminases (ast 192 iu/l, alt 81 iu/l), and a prolonged ptt of 60 seconds (control 31 seconds). drowsiness and exudative pharyngitis, including a peritonsillar pseudo-membrane, were present. on id-5 the patient complained of odynophagia and facial edema and a resting tremor were noted ( figure 3a ). despite being clinically hypovolemic, relative bradycardia (hr 68/minute) was present. thrombocytopenia (48,000/ml) and transaminitis (ast 209 iu/l, alt 82 iu/l) worsened. intravenous recombinant factor viia (1.2 mg q6 hrs) was begun and the n-acetylcysteine was switched to iv administration (1 g q8 hrs). on id-6, the facial edema was slightly improved but palatal ecchymoses were noted along with conjunctival injection. to cover possible bacterial or fungal superinfection, iv cefepime (2 g q12 hrs) and fluconazole (400 mg q12 hrs) were started. non-bloody diarrhea and hypotension (bp 80/40 mmhg) with relative bradycardia (hr 64/min) persisted on id-7. the patient became tachypnoeic, with basilar crackles noted on auscultation. a decision was made to intubate the patient but the procedure, although ultimately successful, proved difficult due to the swollen airway with a pseudo-membrane extending to the glottic folds. multiple, coalescent hemorrhagic areas were present in the hypopharynx. minor contact bleeding followed suctioning. a central line was inserted, with significant bleeding around the insertion site. ribavirin was continued via naso-gastric tube until a supply of iv ribavirin was finally obtained and administered at 20 mg/kg q6 hrs in place of the oral drug. the patient's condition improved on id-8, with better hemodynamic parameters and reduction in mechanical ventilation (fio 2 0.4). no focal neurologic deficits were noted on interruption of sedation. subconjunctival hemorrhage was noted and a fine, blanching, erythematous, maculopapular rash appeared on her trunk, arms and legs, sparing the palms and soles ( figure 3b and 3c). the patient continued to steadily improve, with the nadir of thrombocytopenia on id-12 (23,000/ml) coinciding also with the peak transaminase level (ast 235 iu/l, alt 119 iu/l). the rash resolved by id-14. distal neuropathic weakness appeared on id-13 and hepatomegaly and splenomegaly on id-15. she was weaned from mechanical ventilation on id-15. however, on id-17 she developed sinus tachycardia (130/min) associated over the following four days with basal crackles and an s3 gallop rhythm treated with diuretics and carvedilol. this finding was attributed to myocarditis, a conclusion supported by the finding of an elevated nt-pro bnp level (1000 pg/ml). on id-18, the ten day course of iv ribavirin was completed and ribavirin, recombinant factor viia, and n-acetylcysteine were stopped. no further bleeding was noted. thrombocytopenia improved (86,000/ml) and the ast was down to 177 iu/l, although the ptt was still elevated (50 sec) on id-39. the russell's viper test for lupus anticoagulant was weakly positive. the patient recovered slowly and was discharged from hospital on november 2 (id-42). neurologic features were prominent during the patient's recovery. anxiety, mood fluctuation, and confusion were considered consistent with post-traumatic stress disorder for which she was treated with antidepressants and anxiolytics, which were slowly weaned after one year. distal critical illness peripheral neuropathy and myopathy, tremors, and weakness persisted for at least 6 months after hospital discharge. no hearing loss was noted, although formal audiometry was not performed. her sinus tachycardia resolved by id-52. complete nonscarring alopecia developed from id-83 and resolved slowly over a four month period. repeat tests for lupus anticoagulant were negative. the five patients' ages ranged from 33 to 47 years. there were two white females, two black females, and one white male. the incubation periods of the 3 secondary and 1 tertiary cases ranged from 9-13 days. four of the five patients died (cfr 80%). signs and symptoms. the signs and symptoms of the five patients are presented in table 1 . in all cases, the clinical illness began with the abrupt onset of common and nonspecific symptoms, including fever, malaise, headache, and myalgias, that would not particularly raise suspicion of vhf. sore throat (in one case accompanied by pharyngeal exudates), non-bloody diarrhea, and nausea and vomiting readily ensued, sometimes accompanied by retrosternal or epigastric pain. a blanching erythematous maculopapular rash on the torso extending to the limbs, but sparing the palms and soles, appeared toward the end of the first week of illness in 4/5 patients and seemed to coalesce before fading and disappearing in the sole survivor by id-14. subconjunctival injection or hemorrhage and swelling of the face and neck appeared slightly after the rash in most cases, around the end of the first week of illness. neurological signs were less frequent, but included tremors and seizures, the latter in the end stages of disease and accompanied by cerebral edema noted on ct scan. hepatomegaly and splenomegaly developed in the survivor by id-15 persisting until id-40. no episodes of major hemorrhage were noted, although minor hemorrhage was common in the later stages of disease, including the aforementioned sub-conjunctival hemorrhage, palatal ecchymoses, and bleeding at injection sites. rapid clinical deterioration consistent with shock and multi-organ system failure was noted between ids 7-10, with death a mean of nine days (range 6-12 days) in the four fatalities. the simplified acute physiology score ii (a predicted mortality score derived from measurement of various physiologic parameters 24 hours after icu admission) for the four fatal cases ranged from 4.7% to 73%, compared to 28.5% for the surviving patient. convalescence was protracted for the survivor. clinical laboratory findings. clinical laboratory findings for the five patients are presented in table 2 . typical findings included early leucopenia and lymphocytopenia followed later by leukocytosis, thrombocytopenia, and elevated ldh and transaminases, with ast generally 2-3 times greater than alt. elevated d-dimer levels and prolonged ptt consistent with disseminated intravascular coagulopathy (dic) were noted in three patients. no red cell fragmentation was seen but microscopic hematuria was documented in 3/5 patients. other notable laboratory results included mildly elevated bun (3/5 patients) and creatinine (2/5 patients) and normal or slightly elevated levels of crp and procalcitonin. clinical management. although epidemiological links were made between many of the patients as the outbreak progressed, the diagnosis of arenavirus infection was not made until october 13 (id-3 of patient 5's illness). furthermore, the five patients were hospitalized at three different centers in south africa and treated by different healthcare workers. thus, there was little opportunity for uniformity of clinical approach. management of the nonsurvivors included iv fluids (4/4); broad spectrum antibiotics (4/ 4); transfusion of packed red blood cells, platelets, and fresh frozen plasma (2/4); hemodialysis (2/4); mechanical ventilation (2/4); plasmapheresis (1/4); and oral ribavirin (1/4, but the patient received only three doses before death). the surviving patient received many of these same treatments. distinguishing characteristics of her care which could have played a role in her survival include rapid commencement of ribavirin (oral ribavirin was begun on id-1 with conversion to iv on id-8), and the administration of recombinant factor viia, n-acetylcysteine, and atorvastatin on id 2. based on the five cases of lhf recognized to date, the clinical disease associated with lhf is remarkably similar to lassa fever [7] . surprisingly, the two viruses are genetically quite distinct (up to 38.1% on the nucleotide level), with lujo virus grouping much closer genetically to old world arenaviruses not associated with vhf [5] lassa fever classically begins with non-specific signs and symptoms including fever, general malaise, headache, myalgia, chest or retrosternal pain, and sore throat with progressive diarrhea and other gastrointestinal involvement [7, 9] . severe cases may progress to a capillary leak syndrome with septic shock, rash, facial and neck swelling, and multi-organ system failure. the facial and neck swelling seen in both lhf and lassa fever appear to be specific to old world arenavirus infection and may help differentiate it from other african vhfs. like in lassa fever (and despite the slight misnomer ''vhf''), major bleeding was not a prominent feature in the patients with lhf, although minor bleeding was common. the ast and alt are typically elevated in lassa fever, with ast much greater than alt and high levels of ast associated with a poor prognosis [7] . this same pattern was seen in all five patients with lhf, with the only survivor manifesting the lowest peak ast and ast: alt ratio. some distinctive features of lhf relative to typical lassa fever were the abrupt disease onset (typically indolent in lassa fever) and the presence of dic, which is generally not considered to be part of the pathogenesis of lassa fever, although the matter has not been extensively studied [9] . although rash is consistently seen in light-skinned persons with lassa fever, for unknown reasons it is almost never seen in blacks. all of the white patients and one of the two black patients with lhf manifested a very prominent rash. interestingly, the black patient without rash was hiv infected, suggesting that the rash of lhf may be immune mediated. patient 5 also had relative bradycardia, an interesting finding given reports of depressed cardiac function in an animal model of arenavirus infection [12] . the cfr associated with this outbreak of lhf was 80%. the cfr of hospitalized patients with lassa fever is typically in the 20-30% range, ranging up to 50% in some nosocomial outbreaks [13] . however, mild and asymptomatic lassa virus infection is thought to be common, with mortality rates less than 5% when infection in the community is considered [7, 14] . no antibody survey of case contacts or community members in the region of origin of the index case in zambia has been conducted to determine if mild or asymptomatic infection with lujo virus occurs. the four nosocomial infections of lujo virus illustrate the risk to healthcare workers. although no specific exposures were reported and some degree of personal protective equipment was worn by all four secondary or tertiary cases, it appears that strict barrier nursing practices were not always maintained and full vhf precautions were often implemented late in the course of treatment, if at all. furthermore, the four infected healthcare workers generally had very close and sometimes prolonged contact with the patient, including in closed settings, such as the medical evacuation flight of patient 1, augmenting the possibility of exposure to blood and bodily fluids. they also performed procedures that are often considered to be high risk, such as endotracheal intubation, insertion of indwelling intravascular catheters, and dialysis. the transmissibility of other emerging viruses such as sars and mers coronaviruses has similarly been enhanced when such procedures have been performed [15] . in addition to the 4 secondary/tertiary cases, another 94 persons were identified as contacts and monitored, including support staff (kitchen, laundry, cleaning), laboratory and radiography technicians, and nursing staff. we did not categorize contacts in terms of risk at the time, but now estimate that at least 30 of these would be reasonably categorized as high risk. nevertheless, no suspected cases of lhf were noted in this group. we suspect that the degree of transmissibility of lujo virus is likely analogous to that of lassa virus, for which, although reliable reproduction numbers and secondary attack rates are difficult to ascertain, they are generally thought to be low. nevertheless, occasional outbreaks with secondary and tertiary cases are sometimes seen, especially when barrier nursing practices are not maintained [16, 17] . until the matter can be studied more thoroughly, vhf precautions should certainly be implemented for all suspected and confirmed cases of lhf, with specialized precautions to protect against aerosols when performing endotracheal intubation [1] . despite the high prevalence of hiv infection in many areas of sub-saharan africa, including some areas where vhf is common, data are scarce on hiv and hemorrhagic fever virus co-infection, such as was the case with our patient 4. she was also infected with hepatitis b virus. a 68 year old sierra leonean man with a history of hiv infection and chronic progressive neurological deterioration was infected with lassa virus in 2006 [18] the patient survived despite severe disease requiring intubation and mechanical ventilation. in the 2000-2001 outbreak of ebola virus in uganda, the cfr was not statistically different between those who were hiv positive and negative [19] . the samples were anonymously tested and no clinical data were reported. although the clinical data on patient 4 are also sparse, there were no obvious differences in the clinical manifestations of lhf in this patient compared to the others, with the exception of the aforementioned absence of rash. it is also interesting to note that her peak fever (38.5uc) and leukocyte count (14610 9 /l) were not particularly high, consistent with her compromised immune system. there have been very few controlled studies on the management of vhf. most recommendations represent the informal consensus of experienced clinicians and investigators. supportive therapy is the mainstay [20] . the pathogenesis of severe cases of vhf is thought to be similar to severe sepsis, with a severe inflammatory response syndrome mediated in part by various soluble cytokines and chemokines and nitric oxide [21] . therefore, the basic management principles of shock are also recommended for vhf [20, 22] however, since most vhfs occur in resourcepoor areas with little access to advanced icu medicine, opportunities to use and make observations on the efficacy of these or other advanced treatment options are rare. although obviously not a controlled trial, we were nevertheless able to make some detailed observations on the management of five patients with lhf, who were often treated in more advanced healthcare settings. the most detailed data are from patient 5, who was the only patient for whom a specific diagnosis of vhf was considered and confirmed early in the course of disease. despite receiving ribavirin at disease onset, patient 5's clinical status deteriorated and her illness was severe and prolonged. although these results could be interpreted as lack of efficacy of ribavirin against lujo virus, this is unlikely considering the drug's proven efficacy in other arenavirus infections [8, [23] [24] [25] of greater importance was probably the fact that ribavirin was administered orally for the first 6 days of treatment. efficacy of oral ribavirin for arenavirus infection has not been definitively shown and, in light of the significant first-pass hepatic metabolism resulting in an oral bioavailability of only ,50%, it is unlikely that oral administration reliably reaches the minimum inhibitory concentration for arenaviruses in serum [26] serum levels are undoubtedly further diminished by decreased gut absorption, vomiting, and diarrhea in these severely ill patients. various adjunctive therapies with demonstrated or theoretical efficacy in severe sepsis were administered to patient 5 and a few of the other patients, including hmg-coa reductase inhibitors (statins), n-acetylcysteine [27, 28] , recombinant factor viia, [29, 30, 31] mechanical ventilation, plasmapheresis, and hemodialysis. animal models of sepsis have suggested that statin drugs may improve outcomes in septic shock [32, 10] . furthermore, a large, population-based cohort analysis in canada showed reduced risk of sepsis in patients with cardiovascular disease who were treated with statins [11] . patient enrolment is currently ongoing for prospective trials of statin therapy after the development of sepsis. n-acetylcysteine is an antioxidant and free radical scavenger that resulted in decreased nuclear factor-kb and interleukin-8 in patients with sepsis, suggesting a blunting of the inflammatory response [28] . recombinant factor viia is a prohaemostatic agent thought to act at the local site of tissue injury and vascular wall disruption by binding to exposed tissue factor to promote generation of thrombin and platelet activation. [29] . the drug has been used in hemophilia and other coagulation disorders, as well as in liver disease, reversal of anticoagulant therapy, and for episodes of excessive or life threatening bleeding related to surgery or trauma [30, 31] . other therapies being explored for sepsis and, in some cases specifically for vhf, such as the recombinant inhibitor of the tissue factor/factor viia coagulation pathway, rnapc2, and activated protein c, were not used in this outbreak due to lack of availability and/or risk of bleeding. the seemingly counterintuitive use of anticoagulants like rnapc2 stemmed from work with an ebola virus animal model to ameliorate the effects of tissue factor resulting in dic [21] . it is difficult to assess the contribution of the various therapies to the patient outcomes. although hemofiltration has been suggested in patients with refractory hemodynamic septic shock, with a significant decrease in icu mortality in responders [33] , and plasmapheresis appeared to have a brief positive effect in patient 2, we are reluctant to advocate treatments or procedures that potentially increase healthcare worker exposure to blood. in fact, one explanation for the high secondary attack rate associated with this outbreak could be that such high-risk procedures were frequently undertaken. many of the drugs employed in the management of patient 5 are already clinically approved. investigation of many of these compounds in animal models of vhf is warranted, including in lhf model using strain 13/n guinea pigs [34] . ideally, controlled clinical trials in humans would also be undertaken, although the feasibility of this is dubious for most vhfs, with the possible exception of lassa fever, for which many infections occur across west africa, or perhaps through a ''multicenter'' approach through advanced planning with ministries of health and other partners in endemic areas for vhfs [35, 36, 21] . until controlled efficacy data are available, and considering the high cfr often associated with vhf, we feel that it is both logical and ethical to explore the use of these approved compounds in treatment of patients with vhf when possible. treating clinicians should make a concerted effort to collect and publish detailed, repeated, and systematic clinical observations to facilitate objective evaluation of their efficacy. the pace of discovery of arenaviruses has increased considerably in recent years, with over ten new viruses being isolated since 2000. pathogenic arenaviruses will almost certainly continue to be discovered. furthermore, rapid population growth, especially in africa, and incursion for both economic and leisure activities into natural habitats harboring rodents will likely put humans at risk. the clinical findings and management experience reported here will be of use to clinicians faced with patients with arenavirus infections and as well as other vhfs. the day of illness that the value was noted is in parentheses. *patients 1, 2, and 5 received transfusions of packed red blood cells, platelets, and fresh frozen plasma during the course of their illnesses. abbreviations: alt-alanine aminotransferase, ast-aspartate aminotransferase, crp-c reactive protein, esr-erythrocyte sedimentation rate, hb-hemoglobin, hcthematocrit, inr-international normalized ratio, ldh-lactate dehydrogenase, nd-not done, pct-procalcitonin, ptt-partial thromboplastin time, wbc-white blood cell count. doi:10.1371/journal.pntd.0003233.t002 infection control for viral haemorrhagic fevers in the african health care setting outbreak of marburg virus disease in johannesburg unexpected ebola virus in a tertiary setting: clinical and epidemiologic aspects nosocomial outbreak of novel arenavirus infection, southern africa genetic detection and characterization of lujo virus, a new hemorrhagic feverassociated arenavirus from southern africa novel arenavirus, zambia arenavirus infections lassa fever. effective therapy with ribavirin a case-control study of the clinical diagnosis and course of lassa fever hmg-coa reductase inhibitor simvastatin profoundly improves survival in a murine model of sepsis statins and sepsis in patients with cardiovascular disease: a population-based cohort analysis physiological and immunologic disturbances associated with shock in a primate model of lassa fever lassa fever, a new virus disease of man from west africa. i. clinical description and pathological findings a prospective study of the epidemiology and ecology of lassa fever aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review an outbreak of lassa fever on the jos plateau review of cases of nosocomial lassa fever in nigeria: the high price of poor medical practice lassa fever in france ebola hemorrhagic fever: novel biomarker correlates of clinical outcome 389: viral hemorrhagic fevers treatment of marburg and ebola hemorrhagic fevers: a strategy for testing new drugs and vaccines under outbreak conditions surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock treatment of bolivian hemorrhagic fever with intravenous ribavirin brief report: treatment of a laboratory-acquired sabia virus infection treatment of argentine hemorrhagic fever review of the literature and proposed guidelines for the use of oral ribavirin as postexposure prophylaxis for lassa fever mechanism of action and value of n-acetylcysteine in the treatment of early and late acetaminophen poisoning: a critical review the effect of n-acetylcysteine on nuclear factor-kappa b activation, interleukin-6, interleukin-8, and intercellular adhesion molecule-1 expression in patients with sepsis the activation of factor x and prothrombin by recombinant factor viia in vivo is mediated by tissue factor efficacy and safety of recombinant factor viia for treatment of severe bleeding: a systematic review low-dose recombinant factor viia for trauma patients with coagulopathy simvastatin decreases nitric oxide overproduction and reverts the impaired vascular responsiveness induced by endotoxic shock in rats highvolume hemofiltration as salvage therapy in severe hyperdynamic septic shock severe hemorrhagic fever in strain 13/n guinea pigs infected with lujo virus new opportunities for field research on the pathogenesis and treatment of lassa fever lassa fever in guinea: i. epidemiology of human disease and clinical observations the authors would like to thank cecilia gonzales, landon vom steeg and rene kleyn for assistance preparing the manuscript. checklist s1 strobe checklist. (pdf) key: cord-303647-c4umbcvn authors: reed, patricia e.; mulangu, sabue; cameron, kenneth n.; ondzie, alain u.; joly, damien; bermejo, magdalena; rouquet, pierre; fabozzi, giulia; bailey, michael; shen, zhimin; keele, brandon f.; hahn, beatrice; karesh, william b.; sullivan, nancy j. title: a new approach for monitoring ebolavirus in wild great apes date: 2014-09-18 journal: plos negl trop dis doi: 10.1371/journal.pntd.0003143 sha: doc_id: 303647 cord_uid: c4umbcvn background: central africa is a “hotspot” for emerging infectious diseases (eids) of global and local importance, and a current outbreak of ebolavirus is affecting multiple countries simultaneously. ebolavirus is suspected to have caused recent declines in resident great apes. while ebolavirus vaccines have been proposed as an intervention to protect apes, their effectiveness would be improved if we could diagnostically confirm ebola virus disease (evd) as the cause of die-offs, establish ebolavirus geographical distribution, identify immunologically naïve populations, and determine whether apes survive virus exposure. methodology/principal findings: here we report the first successful noninvasive detection of antibodies against ebola virus (ebov) from wild ape feces. using this method, we have been able to identify gorillas with antibodies to ebov with an overall prevalence rate reaching 10% on average, demonstrating that ebov exposure or infection is not uniformly lethal in this species. furthermore, evidence of antibodies was identified in gorillas thought previously to be unexposed to ebov (protected from exposure by rivers as topological barriers of transmission). conclusions/significance: our new approach will contribute to a strategy to protect apes from future ebov infections by early detection of increased incidence of exposure, by identifying immunologically naïve at-risk populations as potential targets for vaccination, and by providing a means to track vaccine efficacy if such intervention is deemed appropriate. finally, since human evd is linked to contact with infected wildlife carcasses, efforts aimed at identifying great ape outbreaks could have a profound impact on public health in local communities, where ebov causes case-fatality rates of up to 88%. emerging infectious disease (eid) epidemics and pandemics arise without warning, even with global efforts aimed at tracking pathogens early and at the source, a fact most recently evidenced by the swift global spread of influenza h1n1 [1, 2] and a current outbreak of ebolavirus affecting multiple west african countries simultaneously [3] . most major human eids are of zoonotic origin and include viral infections of both global (hiv-1, hiv-2, h1n1) and localized significance (ebolavirus, monkeypox, marburgvirus, nipah virus, severe acute respiratory syndrome [sars]-associated coronavirus) [2] . systematic monitoring of people and wildlife at hotspots of eid is one strategy for preventing human pathogens of animal origin from reaching a pandemic state [4] . by detecting animal pathogens before or just as they emerge in humans, it may be possible to mitigate against their worldwide spread [2] . furthermore, in the case of some diseases such as ebola virus disease (evd), the monitoring of wildlife disease serves as a critical component of early warning systems aimed at preventing the transmission of zoonotic diseases to humans [5, 6] . evd has repeatedly passed from infected apes to hunters, leading to multiple epidemics and 360 human deaths (463 cases) in gabon and the republic of congo (roc) alone since 1994 [5, [7] [8] [9] . more significantly, human epidemics are often preceded by observed animal outbreaks, underlining the human health implications of surveillance and control of epizootics [5, 6] . the international union for conservation of nature (iucn) currently lists the western lowland gorilla (g. gorilla gorilla) as critically endangered and cites infectious disease as one of the top two threats to this species [10] . ebolavirus is lethal in humans and nonhuman primates and has been described as a significant threat to the survival of western lowland gorillas and chimpanzees (pan troglodytes) in central africa [7, 10, 11] . data from ecological surveys in central african ape habitats illustrate declines in ape signs (nests, feces, prints) temporally and spatially linked with confirmed human evd outbreaks [12] [13] [14] . mathematical modeling suggests that, between 1983 and 2000, gorilla numbers in gabon dropped by more than 56%, and it is hypothesized that infectious pathogens, including ebolavirus and bacillus anthracis, may contribute to gorilla mortality in africa [10, 12, 15] . despite the significance to both human and wildlife health, direct evidence of great ape exposure to ebolavirus or other pathogens (either by pathogen or immune response detection) is scant, complicating our ability to monitor epizootics. therefore, to fill this gap, there is a need for prospective epidemiologic studies combining ecological data with laboratory screening. most currently available data regarding primate pathology and immune response comes from experimentally infected laboratory macaques [16, 17] . in direct response to the challenges associated with collecting blood or tissue from wildlife, non-invasively collected biological samples such as feces have been used for wildlife disease screening [18, 19] . primate feces have been screened for the presence of viral nucleic acids due to shedding of simian immunodeficiency virus (siv), circoviruses, enteroviruses and hepatitis viruses [20] [21] [22] [23] . for siv, feces have also shown the presence of virus-specific antibodies [23] . we developed a non-invasive immunological assay to detect ebolavirus antibodies in great ape feces, allowing us more insight into wild ape ebolavirus infections and their surveillance, and leading the way to identifying the best approaches for their protection. in addition, this new assay may prove valuable in the development and employment of prospective epidemiological ebolavirus studies in wild great ape populations. eighty gorilla fecal samples were collected in 5 different habitats in the roc. in zone a, 20 gorilla fecal samples were opportunistically collected while following habituated gorillas roughly 2 and 3 years after ebolavirus infection was confirmed in ape carcasses at that site using a combination of rt-pcr, immunohistochemistry and antigen capture [5, 9] . in june 2007, 15 samples were collected in zone b1 during a reconnaissance walk survey (recces) composed of eight ,10 km linear recces radiating every 30u from a central point with terminal ends of every other pair connected by 8 km recces. this zone is southwest of the mambili river in the southeastern-most area of odzala-kokoua national park (oknp), and samples were collected two years after two gorilla carcasses found in this area tested positive for ebov using rt-pcr and antigen capture assays [9, 24] . for these surveys, two teams operated simultaneously, each averaging 5.6 km per day over 5 days, and following pre-determined global positioning system (gps) points. a continuous gps track log was maintained and uploaded to a garmin 12xl gps (www.garmin.com) with a position recorded every 1 km. three missions occurred in zone b2. the first occurred from 30th august to 8th september 2005 when a 45 km closed loop survey was conducted on the northeast side of the mambili river. this search was for evidence that would indicate that the abovedescribed may 2005 epizootic southwest of the mambili river had also affected wildlife on the opposite side of the waterway. ten gorilla fecal samples were collected and a continuous gps track log was maintained and uploaded to a garmin 12 xl unit, with points taken every 5 km. also, in 2005, a large-scale ecological and large mammal survey was conducted throughout oknp under the auspices of the wildlife conservation society and the projet espèces phares of the european union [25] . from september 5 th to 11 th , 2005, five gorilla fecal samples were collected during these missions by means of reconnaissance walk surveys and of a systematic unbiased line transect design aimed to estimate animal abundance derived from the density of animal sign, multipliers decay rate and production, and the area of the survey zone; both designed with and analyzed by the distance software program [26] [27] [28] . lastly, in june 2007, the original 45 km loop described above was repeated during which 5 gorilla fecal samples were collected. in november-december 2007 (zone d) and march-april 2008 (zone c), reconnaissance walk surveys, similar to the approach applied in zone b1, were conducted in great ape habitats that, by the end of the study period, had no reported disease outbreaks. the purpose of these missions was to estimate ape abundance by recording all ape nests. gps points were taken every 5 km and 25 samples were collected. ebolavirus causes deadly outbreaks in wild great apes, and has been reported as a significant threat to the survival of wild lowland gorillas in central africa. improved knowledge of basic information regarding geographic distribution of ebolavirus in great ape populations, including the identification of immunologically naïve populations and the determination of whether apes survive virus exposure, will be needed in order for protective interventions such as immunization to be effective. however, monitoring ebolavirus infection in wild gorillas by current methods is challenging because of the difficulty in obtaining diagnostic samples from these elusive primates. additionally, there are limitations associated with the available laboratory assays used to document ebolavirus infection. here we report the first successful noninvasive detection of ebov immunity in wild great apes, demonstrating survival in this species. this tool will be useful in a comprehensive strategy aimed at the protection of this endangered species and improved prevention of evd outbreaks in human populations. sample collectors wore disposable latex gloves and surgical masks while collecting feces. approximately 20 g of fresh feces was placed in 20 ml of rnalater (qiagen gmbh, hilden, germany) in a 50 ml plastic screw-top vial (corning incorporated, corning, new york, usa), sealed with parafilm (pechiney, menasha, wi, usa), and placed in zip-closure plastic bags and stored at ambient temperature (,28uc or 82uf). samples collected in zone b1 were placed in liquid nitrogen vapor in a dry shipper (arctic express dual 10, thermolyne) at the end of each day and maintained in this state until arrival at the analyzing laboratory. feces were determined to be that of gorillas when recovered under one of the following conditions: post-observation collection (after seeing gorillas) or post-audition collection (after hearing gorillas), in association with gorilla nests or in association with gorilla trails [29, 30] . genotype studies have demonstrated that feces collected using these methods are accurately classified as gorilla feces 98% of the time [30] . in addition, the presence of long tri-lobed sections, ample fiber, and abundant green leafy material further classified these samples as gorilla dung [31, 32] . only feces estimated to be less than 24 hours old using published criteria [32] were collected. the plasmid encoding ebov np is a p1012 derivative [16] . to purify the recombinant viral protein, plasmid p1012np was tagged at the c-terminus by site-directed mutagenesis with the quick-change xl site-directed mutagenesis kit (stratagene, la jolla, ca, usa). p1012np was provided with the hexa-histidine tag. the tagged plasmids were transfected into human embryonic kidney cells (freestyle 293-f cells, catalog no. r790-07) obtained from invitrogen (carlsbad, ca) and grown in a shaking flask at 37uc under 8% co 2 with freestyle 293 expression medium (invitrogen). the ebov his-tagged np was purified by nickel-affinity gel, ni sepharose 6 fast flow (ge healthcare, piscataway, nj), and eluted with 400 mm imidazol. the concentration of purified np protein was measured with quick start bradford protein assay reagent (biorad, hercules, ca, usa) and used in the western blot assay. to screen nonhuman primate fecal samples for ebolavirus antibodies, we adapted an existing enhanced chemiluminescent western blot assay [23] . feces were vigorously mixed in rnalater (ambion life technologies, grand island, ny, usa), 1.5 ml of the mixture diluted in 7.5 ml of pbs-tween-20 (0.05%), heated at 60uc for 60 minutes, centrifuged at 35006g for 20 minutes and dialyzed in pbs 1x with stir bar at 4uc for 18 to 24 hours to resuspend fecal immunoglobulins that normally precipitate in rnalater. purified or cell lysate np protein was denatured in sample reducing agent (invitrogen nupage), heated at 70uc for 10 minutes, separated by 4-12% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) (0.25 mg per well) (invitrogen np0321, carlsbad, california, usa), and followed by transfer to nitrocellulose membrane (invitrogen lc2001, carlsbad, california, usa) which was blocked with 5% nonfat milk in pbs-tween (0.3%) and bovine albumin (2.4%). membranes were then cut into strips and incubated overnight in fecal extract on a rocking plate. specific np-bound antibody was detected with goat-anti-human igg peroxidase conjugate and the blot was visualized using an enhanced chemiluminescence detection system. the films were exposed to the immunoblot strips then scanned using an epson perfection 4870 photo scanner. to define a cut-off of positivity we used the image j program (imagej, nih, bethesda, maryland, usa) that allowed us to subtract the background in each strip and to compute the integrated density of the band that is the sum of the values of the pixels in the selection in the blot. specimens which showed no visible specific band in the blots were scored as negative whereas those which showed specific band (reactivity with a protein of approximate molecular mass of 115 kda corresponding to ebov nucleoprotein np) were regarded as positive if their integrated density was in excess of the mean of integrated density plus 3 standard deviations of the negative blots. blots with a weak specific visible band and an integrated density below this cutoff were classified as uncertain. a subset of samples collected in 2005 (the year of the last evd outbreak in the region) was also screened for the presence of filovirus rna using a nested rt-pcr. fifty nanograms of total rna isolated from rnalater preserved fecal samples were extracted using the rnaqueous 4pcr kit (ambion life technologies, grand island, ny, usa) and used in a onestep rt-pcr, followed by a nested pcr step. we used degenerate primer pairs in order to amplify a 245 bp fragment of the l polymerase gene from any filovirus. the one step rt-pcr primers are: 59-atmgraayttttcyttytcatt-39 and 59rytataawartcactracatgcat-39; the nested pcr primers are 59-ttyccwagyaayatgatggt-39 and 59-ggdattrdrwartgcatcca-39. to assess the quality of the total rna from the fecal sample, we amplified a housekeeping gene for each sample, the ß-glucuronidase gene (gus, accession number af084552) using a nested pcr assay. the gus primers used for the one step rt-pcr were 59-gcttaccacccagtttgag39 and 59-tgggga-tacctggtttcattg-39, whereas the nested primers were 59-tcagagcgagtatggagc39 and 59-gcactttttggttgtctc-39. we generated a 253 bp fragment. positive and negative controls were included to ensure that cdna product could be amplified and that no contamination from cdna or previous pcr products occurred. we compared antibody prevalence between sampling locations using a log-likelihood ratio test (g-test) [33] . a 95% confidence interval (ci) was constructed for the prevalence. in order to examine ebolavirus exposure in wild great apes we sought to develop a strategy of detection in samples collected by non-invasive methods that would be sensitive and specific enough to detect multiple ebolavirus species with minimal false positive results. it has been shown previously that an enhanced chemiluminescent western immunoblot assay is able to successfully detect specific antibodies in rnalaterpreserved feces from simian immunodeficiency virus-infected chimpanzees (sivcpz) [23] . the sensitivity and the specificity of sivcpz antibody detection in fecal samples were estimated to be 92% and 100%, respectively. viral sivcpz nucleic acid could be amplified in an immunoblot-positive fecal sample, confirming sivcpz infection [23] . furthermore, a similar approach was used to diagnose simian foamy virus infection in wild chimpanzees (sfvcpz). the sensitivities of sfvcpz antibody and viral nucleic acid detection in fecal samples from captive chimpanzees were 73% and 75% respectively, and assay specificities were 100% [34] . these studies show the potential of assessing rnalater-preserved fecal samples to document wild apes' exposure to viruses. given the success of this approach, we developed a fecal western blot assay to detect ebolavirus antibodies. we chose purified ebov np as the antigen for antibody detection since it is one of the most abundant structural proteins produced during infection and a major target of the host immune response. this is supported by previous studies showing that humans who have survived natural ebov infection developed strong antibody responses mostly against np [35] [36] [37] . in addition, the np sequence is well conserved among ebolavirus species (figure s1 ), making it useful for detection of antibodies against multiple ebolavirus species [38, 39] and potentially increasing the breadth of this detection method. we first assessed the ability to detect np antibodies in rnalater-preserved fecal samples from captive cynomolgus macaques. fecal specimens were experimentally spiked with different dilutions of positive serum containing polyclonal immunoglobulin from a monkey that was vaccinated with a genetic vaccine encoding np [16] . serum from this vaccinated monkey displayed antibody reactivity with np by both elisa and western blot analysis (not shown). extracts from these positive serum-spiked feces were then used to incubate immunoblot strips containing immobilized np. anti-np antibodies were detected by enhanced chemiluminescent western blot immunoassay in fecal samples at seropositive nonhuman primate (nhp) plasma dilutions of up to 10 5 -fold (figure 1) , indicating a high sensitivity of the assay for fecal antibody detection. a similar level of sensitivity was observed for detection of anti-siv and anti-hiv antibodies by western immunoblots using plasma samples from sivsm-infected nhp diluted up to 10 24 and plasma samples from hiv-1 infected individual diluted up to 10 26 [40] . in contrast, fecal extracts from captive and uninfected nonhuman primates (cynomolgus macaque and western lowland gorilla species) treated in the same way showed no reactivity in the np immunoblot, demonstrating low background for the assay and lack of cross-reactivity with serum antibodies directed against irrelevant proteins. these results demonstrated that np antibodies present in primate fecal samples can be extracted and detected by immunoblotting. to evaluate whether wild apes show evidence of previous ebolavirus exposure, we screened 80 fecal samples from gorillas living in the roc for ebolavirus antibodies. fecal samples were opportunistically collected from great ape habitats using one of two survey methodologies. the first method employed a systematic unbiased line transect design aimed to estimate animal abundance or the density or size of wildlife [26] [27] [28] . the second consisted of reconnaissance walks to provide a general overview of large animal distributions and investigate animal trails where animal dung is likely to be encountered [41] . fecal samples were collected from two regions within or adjacent to oknp in western roc near the border with gabon ( figure 2 ). the first is an evd diagnostically confirmed outbreak (dco) region where human cases were laboratory confirmed between 2001 and 2005 [13] , and ape carcasses collected between 2002 and 2005 tested positive for ebov [5, 9, 24] . the presence of long-term and functioning wildlife disease surveillance programs and gorilla habituation and research studies in the roc allowed for immediate access to the dco region during and after evd epidemics which facilitated the collection of 35 samples from gorillas with a high likelihood of previous exposure to ebov, and samples were collected at this site within 25-43 months of confirmed great ape evd cases being found. the second region is an area with no reported outbreaks at that time (nro). here, there were no reported human cases, observable signs of epidemics, ebov-positive animal samples, or significant losses in ape numbers despite repeated visits up until the end of this study in april 2008. routine and systematic reconnaissance missions for table 1) . all human evd outbreaks that had previously occurred in our sampling zone study are thought to be the result of handling infected wild animal carcasses, including gorillas [13] . samples from carcasses were used to document ebov outbreaks in gorillas by rt-pcr, antigen detection elisa and immunohistochemistry [5] . to explore whether ebolavirus antibodies could be detected in fecal samples obtained from wild apes we focused initially on the dco region (zones a and b1; figure 3 ) in order to maximize the likelihood of obtaining fecal samples from apes that had been exposed to ebov. among 35 fecal samples collected from the dco region, 5 tested positive for np antibodies by immunoblot ( figure 4 , table 1 ). two ebov antibody positive fecal samples out of 20 (10%, ci: 0-26.3%) came from zone a where, in late 2002 and early 2003, ebov was laboratory confirmed in great ape carcasses at the lossi sanctuary [5, 24] (figure 2 ). of 15 samples collected in zone b1 in 2007, two samples were uncertain (defined in methods) and three were positive for np antibodies (23.3%, ci: 0-47.9%). samples were collected from zone b1 during a mission two years after ebolavirus was detected in ape carcasses at the site [5, 24] (figure 2 ). we also tested ape fecal samples obtained from the outbreakfree (nro) region to explore whether np antibody detection can be used as a potential surveillance tool. the nro region contains zones b2, c and d. zone b2 is adjacent to b1, yet separated from it by the relatively large mambili river. in the fall of 2005, fifteen ape fecal samples were collected in zone b2 to determine whether a may 2005 epizootic had also affected wildlife on the opposite side of the waterway. two years later, in june 2007, the original 45 km closed loop track was repeated to explore any temporal changes in ape density or np seropositivity. three positive fecal samples out of twenty (15%, ci: 0-30.6%) were found in zone b2 (table 1 ) and one sample was uncertain. twenty-five fecal samples were collected in zone c (march and april 2008) and zone d (november and december 2007). the zone c mission followed the discovery of one chimpanzee carcass that later tested negative for ebov (e. leroy, personal communication, april 30, 2008) . no antibodies were found in the fecal samples from zones c and d, where no outbreaks had been reported. altogether, eighty fecal samples from wild great apes were analyzed by western blot and eight (10%) were found to be np antibody positive (table 1) . three samples (one from zone b2 and 2 from zone b1) had blots with a weak specific visible band and an integrated density below the cutoff, and were thus classified as uncertain (not shown). the remaining 69 fecal samples showed no detectable np-specific antibodies and were classified as antibody negative. roughly half of the samples were collected in the dco region and 14.2% of these samples were found to be antibody positive, whereas a smaller proportion (6.6%) of samples collected in the nro region were positive. the difference between the nro and dco regions is not statistically significant (log likelihood ratio statistic (g) = 1.3925, x-squared df = 1, p-value = 0.238) ( table 1) , but overall the data show that anti-np antibodies are present in fecal samples from wild ape populations even in areas with no prior reports of human or wild great ape outbreaks. these data demonstrate that the screening of wild gorilla feces by western blot for the purpose of monitoring ebolavirus exposure was successful in detecting np antibodies. this study represents the first time that ebolavirus antibodies have been detected in wild great ape fecal samples, and carries important implications for the future management and survival of these primates. this is especially relevant because intervention strategies to protect apes against future evd infections are being actively explored, including vaccination since ebolavirus vaccines have been shown to protect laboratory monkeys from disease [42, 43] . there have been no studies or observations involving great apes that have described immune response, clinical signs, precise mortality rates or whether survivorship provides long-term immunity, and little is known regarding the overall ebolavirus serological status of apes in central africa. to date, serum samples from gorillas (n = 30) and chimpanzees (n = 256) in central africa have been screened for antibodies against ebov [7] . most of these animals were sampled while living in captive settings (pets, rescue centers, primate centers); four subjects were free-ranging and sampled directly from the wild in oknp but were seronegative. obtaining samples from free-ranging wildlife is needed to improve our understanding of infectious agents circulating in the environment. all other health data related to ebolavirus from free-ranging apes comes from necropsies performed during wildlife die-offs and, in those cases, the vast majority of samples collected are too degraded to have diagnostic value [5] . as expected, there is also nothing known regarding potential co-infections involved in great ape evd which may modify the host immune response, alter pathogenesis, increase mortality or influence the effectiveness of any future prophylactic plans, such as the administration of a vaccine once available. this is due to the difficulty in acquiring diagnostic samples from wild populations. capture and subsequent blood collection for serological screening is costly, time consuming, and carries some risk to the animals while providing information on only a few individuals. in fact, despite the disappearance of a staggering number of great apes in gabon and the roc and 6 years of sustained and active surveillance in these countries during the course of this study, only 37 carcasses were recovered, with confirmed ebov infection in 16 of those individuals [9] [10] [11] 24] . moreover, finding animal carcasses in vast tracts of rain forest is difficult; it requires intensive searching and often results in the acquisition of highly degraded samples, which are not suitable for detection of viral antigens or nucleic acid and are more prone to negative results [5] . this newly developed approach for non-invasive sampling of great apes has allowed the successful detection of anti-ebov antibodies in fecal samples, yielding a seroprevalence rate of 10% in gorillas. since genetic identification of individual fecal samples was not performed, we cannot rule out the possibility of resampling, so the prevalence rate is an upper limit for this data set. however, recent genetic analysis of gorilla and chimpanzee samples collected during iron-cross recces (the type of surveillance executed in sites b1, c, and d) from 2006-2010 suggest a low resampling rate. of 162 samples, three were identified with genetic identity the same as three previously sampled individuals, yielding a 2% resampling rate for sites in which the same site was revisited with the shortest interval of seven-months apart; the resampling rate in the currently study could be higher because two sites were sampled one-month apart and two locations, a and b2, were not iron-cross recces (personal communication, k.j. lee) in addition to estimating ebolavirus exposure in nhp, this technique of screening feces by western blot is in fact a multipurpose tool. it provides the potential to employ serial fecal collections to detect a temporal change in incidence exposure in a given zone. for instance, we saw a trend toward a decrease in ebolavirus fecal antibodies in zone b1/b2 from 20% in 2005 to 12% in 2007, which can be tested in the future using formal prospective studies. fecal antibody screening can also be used before and after vaccination to demonstrate vaccine-induced immune responses developed in great ape populations, noting that antibody levels in vaccinated non-human primates are an immune correlate of protection [42] . finally, this approach will facilitate the identification of immunologically naïve populations for largescale vaccination trials, thereby improving cost-effectiveness by identifying communities that could benefit the most from vaccination efforts. along these lines, it provides us with the first real possibility to investigate patterns of evd emergence in wild apes independent of animal mortality and the role natural barriers, such as rivers, may have in mitigating its spread. this ability to map exposure patterns across central africa may also provide insight into how this virus spreads within and between ape populations, a question that has generated two disparate theories: multiple virus introductions and a single spreading outbreak [10, 11, 24, 44] . key to pandemic prevention is disease surveillance at the human/wildlife interface, especially considering the fact that the majority of emerging infectious diseases events (over 60%) are of animal origin and that those caused by wildlife pathogens are increasing [6, 45] . the strategy described herein will be valuable in providing zoonotic information of public health concern from regions where resources are poor and help counter the emergence of diseases which have potential to become the next pandemic. monitoring diseases in animals using methods such as those we describe here allows for the identification and surveillance of many pathogens, including those with potential to adapt and spread in humans, like hiv and plasmodium parasites [23, 46, 47] . these findings also illustrate the role in situ conservation organizations can play in disease surveillance programs. adapting these tools for use in other wildlife species may provide information regarding the transmission of ebolavirus and other emerging infectious diseases to human populations. recent concerns surround the role pigs play in the emergence of diseases such reston ebolavirus and h1n1 [1, 48] . central africa's forests are home to tens of thousands of wild pigs, including the giant forest hog (hylochoerus meinertzhageni) and the red river hog (potamochoerus porcus), and are characterized as emerging disease hotspots [45] . although no evidence has emerged supporting speculation of ebolavirus-associated wild pig die-offs in africa, employing this assay in these species may address whether pigs are amplifiers, victims or carriers of the virus [48] . it is noteworthy that in the case of influenza pigs are considered ''mixing vessels'' for viruses and capable of generating new strains transmissible to humans [49] . the extensive bush meat trade in africa provides ample opportunity for pathogen transmission from pigs to humans, and underlines the importance of disease surveillance in this species. wildlife managers frequently perform wide scale ecological surveys, simultaneously collecting biological samples and data on the density and distribution of wildlife. with the benefit of the new diagnostic capacity and sampling strategies described herein, different fecal sampling approaches can be integrated into these surveys to provide information that has thus far eluded us concerning the distribution, ecology and epidemiology of ebolavirus. for the first time, both the logistical and diagnostic capacities are available to immunologically screen large popula-tions of wild great apes for previous exposure to ebolavirus and even estimate and monitor prevalence rates. figure s1 ebolavirus nucleoprotein sequences. sequence alignment of the nucleoprotein np from zaire ebolavirus (ebov, accession no. np_066243), tai forest ebolavirus (tafv, accession no. aci28629), reston ebolavirus (restv, accession no. bab69003), sudan ebolavirus (sudv, accession no. aad51107) and bundibugyo ebolavirus (bdbv, accession no. aci28620). the numbering of the amino acids is according to their position in the sequence. ''*'', identical residues; '':'' conserved residues; ''.'', semi-conserved residues. 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fernandes, geisa ferreira; araujo, leticia mendes; della terra, paula portella; dos santos, priscila oliveira; pereira, sandro antonio; schubach, tânia maria pacheco; burger, eva; lopes-bezerra, leila maria; de camargo, zoilo pires title: proteomics-based characterization of the humoral immune response in sporotrichosis: toward discovery of potential diagnostic and vaccine antigens date: 2015-08-25 journal: plos negl trop dis doi: 10.1371/journal.pntd.0004016 sha: doc_id: 276850 cord_uid: tnlyk0wz background: sporothrix schenckii and associated species are agents of human and animal sporotrichosis that cause large sapronoses and zoonoses worldwide. epidemiological surveillance has highlighted an overwhelming occurrence of the highly pathogenic fungus sporothrix brasiliensis during feline outbreaks, leading to massive transmissions to humans. early diagnosis of feline sporotrichosis by demonstrating the presence of a surrogate marker of infection can have a key role for selecting appropriate disease control measures and minimizing zoonotic transmission to humans. methodology: we explored the presence and diversity of serum antibodies (igg) specific against sporothrix antigens in cats with sporotrichosis and evaluated the utility of these antibodies for serodiagnosis. antigen profiling included protein extracts from the closest known relatives s. brasiliensis and s. schenckii. enzyme-linked immunosorbent assays and immunoblotting enabled us to characterize the major antigens of feline sporotrichosis from sera from cats with sporotrichosis (n = 49), healthy cats (n = 19), and cats with other diseases (n = 20). principal findings: enzyme-linked immunosorbent assay-based quantitation of anti-sporothrix igg exhibited high sensitivity and specificity in cats with sporotrichosis (area under the curve, 1.0; 95% confidence interval, 0.94–1; p<0.0001) versus controls. the two sets of sporothrix antigens were remarkably cross-reactive, supporting the hypothesis that antigenic epitopes may be conserved among closely related agents. one-dimensional immunoblotting indicated that 3-carboxymuconate cyclase (a 60-kda protein in s. brasiliensis and a 70-kda protein in s. schenckii) is the immunodominant antigen in feline sporotrichosis. two-dimensional immunoblotting revealed six igg-reactive isoforms of gp60 in the s. brasiliensis proteome, similar to the humoral response found in human sporotrichosis. conclusions: a convergent igg-response in various hosts (mice, cats, and humans) has important implications for our understanding of the coevolution of sporothrix and its warm-blooded hosts. we propose that 3-carboxymuconate cyclase has potential for the serological diagnosis of sporotrichosis and as target for the development of an effective multi-species vaccine against sporotrichosis in animals and humans. sporothrix schenckii was originally described in 1898 as the causal agent of a subcutaneous disease in humans in the mid-atlantic usa [1] . subsequently, the disease was reported in rats naturally infected in southeastern brazil [2] and later in a wide range of animals including dogs, cats, horses, cows, camels, dolphins, goats, mules, birds, pigs, and armadillos. several sporothrix spp., previously reported to be closely related to s. schenckii, are known to establish infections in various hosts, with dissimilar virulence traits [3, 4] and responsiveness to antifungal treatment [5] . the s. schenckii complex consists of at least four closely-related species [6, 7] , ranging from geographically restricted agents such as s. brasiliensis [8, 9] to cosmopolitan pathogens such as s. schenckii s. str. and s. globosa [7, 10, 11] . sporothrix spp. are endowed with an extraordinary ecological diversity [12] [13] [14] [15] ; they are frequently recovered from soil, plants debris, and insects (coleoptera: scolytidae). phylogenetic data support a recent habitat shift within sporothrix from plants to cats [9] that culminated in the largest epizootic transmission in southeastern brazil [16] [17] [18] [19] . feline sporotrichosis emerged in the 1990s, with s. brasiliensis recovered from many outbreaks [8, 20] . more recently, s. brasiliensis has been recognized as a threat to humans [21] [22] [23] due to the massive zoonotic transmission in southeastern brazil that affects thousands of patients regardless of whether they are immunocompetent or immunocompromised [9, [24] [25] [26] . cats have been a source of sporothrix spp. infection transmitted to humans and other animals [18, 19, 27] . most human cases occurred in housewives and professionals who had contact with infected animals and a history of scratches or bites [21, 28] . the largest epidemic of sporotrichosis due to zoonotic transmission was reported in the state of rio de janeiro, brazil [18, 19, 21, 23, 28] ; since then, the incidence of sporotrichosis among animals, particularly cats, has increased [8, 28, 29] . more than 4,000 humans and 4,124 cats were diagnosed at instituto nacional de infectologia (ini) evandro chagas /fundação oswaldo cruz by 2012 [30] . pereira et al. [29] observed that the majority of cats with sporotrichosis in rio de janeiro between 2005 and 2011 were male, mongrel, and unneutered, had a median age of 24 months, and presented with three or more cutaneous lesions in non-adjacent locations. this mycosis in cats is hard to treat and generally requires a long period of daily care; these animals do not always respond well to antifungal treatment [30] . sporothrix is widely distributed in nature, and traumatic inoculation of plant organic matter is classically associated with infection [31] . feline habits render cats more susceptible to the fungal agent because they are constantly in contact with soil, plant debris, and other cats that may be infected. in cats, a broad spectrum of clinical presentation ranges from single lesions to fatal systemic forms. after monitoring the feline epidemic for 4 years, schubach et al. [18] observed that the lymphocutaneous form occurred less frequently than did involvement of the mucous membranes of the respiratory tract and upper digestive tract and multiple cutaneous lesions. some animals present involvement of skin and various internal organs [32] . cats are susceptible to a variety of fungal infections, including blastomycosis [33] , histoplasmosis [34] , cryptococcosis [35] , candidiasis [36] , dermatophytosis [37] , aspergillosis [38] , coccidioidomycosis [39] , and sporotrichosis. misdiagnosis results in ineffective treatment, which further worsens outcome. major contributors toward misdiagnosis include the small number of affordable and effective treatment techniques as well as other social issues. definitive diagnosis of feline sporotrichosis is based on mycological culture, micromorphological characterization, and mold-to-yeast conversion. histopathological methods and cytopathological examination are useful tools for the presumptive diagnosis of sporothrix infection in cats [40] . detection of specific anti-sporothrix antibodies offers a rapid alternative for accurate diagnosis [41] [42] [43] . we recently proposed a serological approach that employs an enzymelinked immunosorbent assay (elisa) to diagnose feline sporotrichosis [44] using purified antigen (the s. schenckii cona binding fraction) and crude antigen, with high sensitivity and specificity. there is a lack of information about feline sporotrichosis and the antigenic components involved in infection; therefore, the present study aimed to explore the diversity of molecules expressed by closely related species (s. brasiliensis and s. schenckii) and that are recognized by immunoglobulin g (igg) in sera from cats naturally infected with s. brasiliensis. we found remarkable cross-reactivity among s. brasiliensis and s. schenckii antigens, and we identified an immunodominant molecule that is an important biomarker in feline sporotrichosis, irrespective of clinical manifestation. here, we show that, although s. brasiliensis and s. schenckii may infect different warm-blooded hosts, infection result in highly similar igg-mediated response in cats compared to humans, what is important for serodiagnosis and to the development of prophylactic or therapeutic vaccine against the enormous health burden of sporotrichosis in endemic areas. this knowledge may enable selection of potential biomarkers that can be used in seroepidemiological studies, diagnosis, and vaccine development, and may contribute to understanding of the pathogenesis of this infection in cats and humans. this study was performed in strict accordance with recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the protocol was approved by the ethics in research committee of the fundação oswaldo cruz, rio de janeiro, brazil, under license number l-041/06. this study was also approved by the institutional ethics in research committee of the federal university of são paulo under protocol number 0244/ 11. s. brasiliensis and s. schenckii s. str. from cats and humans were used for protein extraction ( table 1 ). the dimorphic nature of sporothrix spp. was demonstrated by converting the fungus to its yeast form at 36°c in brain-heart infusion medium (difco) and observing typical oval multibudding yeast cells. molecular identification was performed and confirmed via dna sequencing of the gene encoding calmodulin [25] . isolates were selected because they had been previously characterized at the molecular level [3, 9, 45, 46] ; crude exoantigen (cbs 132974 = ss118; s. schenckii s. str.) was successfully used to diagnose feline sporotrichosis via elisa [44] . sporothrix spp. was grown on sabouraud medium (difco) agar slants at room temperature (20-25°c) for 7 days. approximately 2x10 6 conidia (85% viable cells) were used to inoculate 500-ml flasks containing 150 ml of brainheart infusion broth. cultures were incubated at 36°c in a rotary shaker (multitron ii, infors ht) with constant orbital agitation at 110 rpm for 7 days. whole extracts of sporothrix yeast cells were obtained as described elsewhere [47] . briefly, yeast cells (4 ml of each culture) were collected via centrifugation at 5000 x g for 10 min at 4°c and washed three times in ultrapure water. pellets were frozen in liquid nitrogen and disrupted by gridding. cells were macerated with a pestle until a fine powder was obtained. this cellular powder was vortexed for 30 min at 4°c in tris-ca 2+ buffer (20 mm tris-hcl ph 8.8, 2 mm cacl 2 ) containing a commercial cocktail of protease inhibitors (1:1000; ge healthcare), rnase and dnase (1:1000; ge healthcare), and 600-μm glass beads (1:1; sigma). cell debris and glass beads were removed via centrifugation at 5000 x g for 10 min, and dithiothreitol (final concentration 20 mm) was added to the supernatant [48] . protein concentrations were determined using the bradford method [49] . cell extracts were kept at -80°c until use. sera from cats with definitive diagnoses of sporotrichosis (via s. brasiliensis isolation in culture; n = 49) were obtained from ini/fundação oswaldo cruz, rio de janeiro, brazil. the distribution and number of skin lesions of the cats were classified as l1 (cutaneous lesion in only one place), l2 (cutaneous lesion in two non-adjacent places), or l3 (cutaneous lesions in three or more non-adjacent places). during examination, blood was collected via vein puncture and stored in an incubator for 1 h; serum was obtained via centrifugation and stored at -20°c until use. sera from 19 cats with no evidence of sporotrichosis or other diseases (the control group) were obtained from são paulo as described elsewhere [44] . sera from 20 cats with other diseases were also studied to verify cross-reactions with feline infectious peritonitis/coronavirus (5 sera), feline leukemia virus (3 sera), feline immunodeficiency virus (2 sera), leptospirosis (3 sera), rickettsiosis (2 sera), erlichiosis (3 sera), and leishmaniasis (2 sera) as previously described [44] (s1 diagram). all sera were stored at -20°c until use. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and 1d immunoblotting s. brasiliensis and s. schenckii protein extracts (2 μg) were analyzed via sds-page with 10% gels [50] and silver-stained [51] . the relative molecular weights of the fractions were estimated using standard broad-range molecular weight markers (protein benchmark, invitrogen). for immunoblotting, proteins (10 μg) from strains cbs 132990, cbs 132021, cbs 132974, and cbs 132984 were resolved with sds-page and transferred onto 0.45-μm polyvinylidenedifluoride membranes (bio-rad) at 20 v for 30 min with transfer buffer (25 mm tris base, 192 mm glycine, 20% methanol, ph 8.3) [52] using a trans-blot sd semi-dry device (bio-rad). electrotransference was confirmed by staining with 0.15% ponceau s and 1% acetic acid [vol/vol]. membranes were destained and free binding sites were blocked overnight in phosphate-buffered saline blocking buffer (1% bovine serum albumin supplemented with 0.05% [vol/vol] tween 20, 5% [wt/vol] skim milk, ph 7.6) at 4°c. to determine the best dilution of serum, one sample was tested at four dilutions (1:100, 1:200, 1:500, and 1:1000) against yeast extracts. afterward, for all sera, membranes were probed individually with primary antibody diluted 1:500 at 25°c for 2 h. membranes were washed three times with tris-buffered saline (ph 7.5) containing 0.05% [vol/vol] tween-20 for 10 min and incubated with horseradish peroxidase-conjugated goat anti-feline igg (1:1000) for 2 h at room temperature. membranes were then washed with tris-buffered saline (ph 7.5) containing 0.05% [vol/vol] tween-20 and signal was detected with an enhanced chemiluminescence detection kit (ge healthcare). blots were imaged in a transilluminator (uvitec cambridge). allience 4.7 software was used to take several images at different time exposures, from 2 s each to a total of 10 images over 2 s. proteins were separated via 2d gel electrophoresis as previously described [45, 47] . briefly, proteins (300 μg) were precipitated using the 2d clean-up kit (ge healthcare) and resuspended in rehydration buffer (7 m urea, 2 m thiourea, 2% chaps, 1.2% destreak, 2% vol/vol isoelectric focusing buffer ph 4-7, and trace amounts of bromophenol blue) to a final volume of 250 μl. immobilized ph gradient strips (ph 4-7, 13 cm; ge healthcare) were rehydrated at 30 v for 12 h. isoelectric focusing was performed at 20°c using a multiphor iii system (ge healthcare) as follows: 200 v for 2 h, 500 v for 2 h, 1000 v for 5 h, and a gradient applied from 1000 to 5000 v for 2 h. finally, the voltage was set to 5000 v for 60,000 vhr. after 1d isoelectric focusing, the ipg strips were reduced for 15 min with 1.5% dithiothreitol and alkylated for 15 min with 2.5% iodocetamide in equilibration buffer (6 m urea, 50 mm tris-hcl ph 6.8, 30% glycerol, and 2% sodium dodecyl sulfate). second-dimension separation was carried out by placing equilibrated ipg strips onto 10 % polyacrylamide gels, sealing them with 0.5 % [wt/vol] low-melting-point agarose, and separating the proteins at 10°c using a hoefer se 600 unit (15 ma/gel for 30 min and then 23 ma/gel until the dye front reached the bottom of the gel). proteins were developed with silver staining [51] or were directly transferred for immunoblotting. for 2d immunoblotting, proteins were transferred onto 0.45-μm polyvinylidenedifluoride membranes at 25 v for 1 h with transfer buffer [52] using the trans-blot sd semi-dry system. the success of electrotransference was evaluated by staining with 0.15% ponceau s and 1% acetic acid 1% [vol/vol]. membranes were destained and free binding sites were blocked overnight in phosphate-buffered saline blocking buffer (1% bovine serum albumin supplemented with 0.05% [vol/vol] tween 20, 5% [wt/vol] skim milk, ph 7.6) at 4°c. membranes obtained from 2d gels were probed with 1:500 primary antibody (gold standard pooled feline sera; n = 10) under the conditions used for 1d immunoblotting. immunoreactive antigens were detected using an enhanced chemiluminescence detection kit (ge healthcare). 2d immunoblots were imaged using the method used for 1d immunoblots. diagnostic values included sensitivity, specificity, positive predictive value, and negative predictive value. receiver operating characteristic (roc) curves were prepared and analyzed to determine the sensitivity and specificity of each antigen preparation for elisa. the area under the curve (auc) for roc analysis was calculated to evaluate the diagnostic value of elisa. we assumed that a test lacked diagnostic power when the roc curve was linear with an auc of 0.5 (the roc curve coincided with the diagonal). a powerful test was assumed to yield an auc of~1.0, indicating the absence of both false-positives and false-negatives (the roc curve reached the upper left corner of the plot). to measure the degree of concordance of the results from preparations from strains cbs 132990, cbs 132021, cbs 132974, and cbs 132984, we calculated the kappa statistic and its 95% confidence interval (ci). kappa values were interpreted as follows: 0.00-0.20, poor agreement; 0.21-0.40, fair agreement; 0.41-0.60, moderate agreement; 0.61-0.80, good agreement; 0.81-1.00, very good agreement [53] . p-values 0.05 were considered statistically significant. all calculations were performed with medcalc statistical software version 14.8.1 (medcalc software bvba; http://www.medcalc.org). findings are reported in line with the stard checklist for studies of diagnostic accuracy (s1 checklist). we previously reported a high prevalence of s. brasiliensis in feline sporotrichosis outbreaks [8, 9, 20] . based on this information, the main goal of the present investigation was to evaluate the presence and diversity of serum-derived antibodies against s. brasiliensis antigens in naturally infected cats. further, we previously proposed the existence of a convergent humoral response in human sporotrichosis against antigens from s. brasiliensis, s. schenckii, and s. globosa [45] . to establish whether s. brasiliensis and s. schenckii express different antigens, we assessed whole cellular protein extracts from two strains of s. brasiliensis plus two strains of s. schenckii s. str. that were previously characterized by molecular [8, 9, 25, 54] and serological [3, [44] [45] [46] [47] methods. remarkably, and in support of our hypothesis that immunological distance increases with phylogenetic distance, sera from these cats reacted similarly, with no significant differences in titer between elisa plates coated with proteins from s. brasiliensis or s. schenckii (fig 1) . elisa . further information about elisa statistics appears in s1 table. doi:10.1371/journal.pntd.0004016.g001 s. schenckii cbs 132984, median 1.028 od, 95% ci 1.027-1.294 od (s1 table) . when using the assay to diagnosis cats with sporotrichosis, the area under the roc curve was 1.0 (95% ci 0.94-1.000; p<0.0001; fig 2) , indicating excellent performance. the control group of 19 non-sporothrix infected animals was associated with lower medians and smaller ranges: s. brasiliensis cbs 132990, median 0.2640 od, 95% ci 0.2592-0.3098 od; s. brasiliensis cbs 132021, median 0.2590 od, 95% ci 0.2517-0.2942 od; s. schenckii cbs 132974, median 0.2730 od, 95% ci 0.2512-0.3136 od; and s. schenckii cbs 132984, median 0.2670 od, 95% ci 0.2567-0.2907 od (s1 table) . differences between the absorbance values for the infected and noninfected groups were statistically significant (p<0.0001). sera from cats with other infections were non-reactive. similar cutoff values yielded 100% specificity and sensitivity: s. brasiliensis cbs 132990, 0.377 od; s. brasiliensis cbs 132021, 0.363 od; s. schenckii cbs 132974, 0.407 od; and s. schenckii cbs 132984, 0.346 od (s1 fig). elisa results showed very good agreement for the antigens assayed (kappa = 1.0). to diagnosis feline sporotrichosis via elisa, we recommend the use of antigen preparations of s. brasiliensis, since this is the most prevalent species in feline sporotrichosis outbreaks. antigen diversity was assayed with 1d immunoblots using the four antigen preparations of s. brasiliensis and s. schenckii tested via elisa. proteins extracts were evaluated according to the amount of protein extracted, the diversity of bands, the integrity of the samples, and the reproducibility of extraction. approximately 2 μg of sporothrix yeast whole-cell extracts were resolved by sds-page; silver staining revealed numerous proteins ranging from 10 kda to 160 kda in size, with different intensities. the tris-ca 2+ extraction protocol [45, 47] was suitable for the study of sporothrix antigenic molecules during feline sporotrichosis, yielding samples with high amounts of protein and no degradation. as expected, antibodies from cats with sporotrichosis reacted with a wide variety of s. brasiliensis and s. schenckii proteins 20kda to >160kda in size (fig 3) . cat-to-cat variation resulted in characteristic banding patterns for each animal (fig 3) ; supporting the hypothesis that in a genetically diverse population, the antibody repertoire is expected to vary among individual cats. on the other hand, we detected minor or no differences in igg-reacting banding patterns between antigen preparations (fig 3) , consistent with the close genetic distance between s. brasiliensis and s. schenckii [8, 9] . despite this variation, all cats produced antibodies against a 60-kda molecule in the s. brasiliensis proteome and a 70-kda molecule in the s. schenckii proteome. the major antigenic s. brasiliensis molecules (cbs 132990 and cbs 132021) recognized by feline igg consisted of the following sizes: 60 kda (100% and 100%, respectively), 90 kda (92% and 92%, respectively), 100 kda (86% and 86%, respectively), 38 kda (60% and 56%, respectively), 40 kda (56% and 58%, respectively), 45 kda (44% and 42%, respectively), 30 kda (36% and 26%, respectively), 52 kda (30% and 32%, respectively), and 110 kda (28% and 30%, respectively) (fig 4a and 4c ). minor molecules recognized by feline igg had sizes of 80 kda, 25 kda, 28 kda, 120 kda, 160 kda, 35 kda, 20 kda, 55 kda, 85 kda, and 23 kda (fig 4a and 4c) . the major antigenic s. schenckii molecules (cbs 132974 and cbs 132984) recognized by feline igg had sizes of: 70 kda (100% and 100%, respectively), 90 kda (86% and 88%, table. doi:10.1371/journal.pntd.0004016.g004 respectively), 100 kda (76% and 82%, respectively), 38 kda (74% and 62%, respectively), 40 kda (64% and 56%, respectively), 52 kda (58% and 56%, respectively), 30 kda (50% and 34%, respectively), 55 kda (48% and 48%, respectively), and 45 kda (40% and 30%, respectively) (fig 4b and 4d) . the minor s. schenckii molecules recognized by feline igg had sizes of 25 kda, 80 kda, 28 kda, 110 kda, 120 kda, 23 kda, 35 kda, 160 kda, 85 kda, and 20 kda (fig 4b and 4d ). sera from uninfected cats did not react with s. brasiliensis or s. schenckii antigens. sera from cats with other infections were also non-reactive in the immunoblot assay. the frequencies at which sporothrix molecules were recognized in the antigen preparations are presented in s2 table. there was no association between the number of bands recognized by each serum and the distribution and number of skin lesions on cats with sporotrichosis. we previously reported that the 60-kda and 70-kda proteins in s. brasiliensis and s. schenckii, respectively, are related to virulence profiles and are the main antigenic molecules during murine [3] and human [45] sporotrichosis. we also determined that this protein undergoes post-translational modification and is present as isoforms and glycoforms in the s. brasiliensis and s. schenckii proteomes [45] . we therefore investigated whether antibodies present in cat sera recognize all six isoforms in the s. brasiliensis proteome, as previously shown with human antibodies [45] . s. brasiliensis proteins were therefore resolved via 2d electrophoresis and immunoblotted with pooled sera from cats with sporotrichosis (n = 10) and optimal antibody titers according to elisa. serum-derived antibodies in naturally infected cats mainly recognized all six isoforms of gp60 (fig 5) . the present results confirm that s. brasiliensis 3carboxymuconate cyclase is a highly polymorphic protein [45] with sizes of 55-62 kda and with isoelectric points of 4.45-4.80. 2d immunoblotting revealed less diversity and more weakly reacting spots than 1d immunoblotting, perhaps due to protein loss during sample preparation for 2d electrophoresis (compared to the crude extracts used in 1d immunoblotting and elisa) and serum dilution during pooling. in the past, sporotrichosis was reported in horses more frequently than in other animals [55, 56] . although the disease has been reported in several animal species, cats are currently the most frequently affected domestic animal. outdoor cats are exposed to the fungus through contact with natural environmental sources or other sick cats. cats have gained importance in the zoonotic transmission of sporothrix to humans [8, 28-30, 57, 58] . presently, the all-time high number of feline sporotrichosis cases reported in brazil has reached epidemic proportions [8, 20, 29, 59] . unlike humans, cats are highly susceptible to this fungus due to the low frequency of granulomas and the richness of fungal elements observed during skin histopathology [18] . moreover, unlike human-disseminated sporotrichosis, which classically affects immunocompromised individuals [24, 26] , systemic disease in cats occurs frequently and is not associated with immunodeficiency caused by feline immunodeficiency virus and/or feline leukemia virus [18] . in this scenario, absence of an efficient host immune response is a key factor in disease progression. the low frequency of granulomas and uncontrolled fungal growth suggest that a lack of adequate cellular immunity underlies disease severity and pathology. elisa has been achieved with various antigen preparations, thus enabling serodiagnosis of human [41-43, 60, 61] and feline [44] sporotrichosis. the present investigation suggests that elisa-based quantitation of anti-s. brasiliensis igg is remarkably sensitive for the detection of feline sporotrichosis (cutoffs of 0.346-0.407 od); as expected, this strategy does not differentiate between s. brasiliensis or s. schenckii as the agent of infection. despite its high prevalence in south and southeast brazil, s. brasiliensis infection in cats is not an exclusive host-pathogen association, since the sibling agent s. schenckii s. str. also occurs in cats in brazil [8, 9, 20] and malaysia [62] , albeit with significantly lower frequency. our serology-based observations of a convergent antigenic response are similar to previous 2d immunoblotting results for human sporotrichosis [45] . interestingly, in other thermally dimorphic fungi such as paracoccidioides brasiliensis and p. lutzii (onygenales), antigen composition seems to vary considerably between species, an observation that supported development of a differential diagnosis system based on titration of serum-derived antibodies from humans infected with distinct strains of paracoccidioides [63] . it is likely that similarities in antigen profiling among clinical sporothrix spp. (s. brasiliensis, s. schenckii, and s. globosa) could be related to a recent speciation event and therefore be less susceptible to variability; however, this hypothesis requires further investigation. in addition, factors related to environment or to host association may impose strong selection pressures on sporothrix antigen profiles. to date, no information about the humoral response in feline sporotrichosis has been reported in the literature; it is a completely unknown area in veterinary medicine that merits exploration. here, sera from cats with sporotrichosis displayed immunoblotting patterns of sporothrix-specific immunogenic molecules that were markedly different from patterns from sera from uninfected cats. these immunogenic components were 20-160 kda in molecular weight. the main molecules recognized by cat sera were a 60-kda protein in the s. brasiliensis proteome and a 70-kda protein in s. schenckii s. str., followed by molecules weighing 100 kda, 90 kda, 40 kda, and 38 kda. the variety of antigenic components recognized by each serum may be due to specific antigens secreted by individual fungal strains [3, 64] as well as to different mechanisms of activation of each host's immune system [45, 65] . however, variation in antibody repertoire seems reasonable in a genetically diverse host population [66] . we interpreted published data lacking taxonomic information or protein identification via matrix-assisted laser desorption/ionization time of flight (maldi-tof)/mass spectrometry (ms) and identified an immunodominant fraction that oscillated between 60 kda and 70 kda in various studies (table 2 ). in this scenario, the humoral immune response may be influenced by the infection strain and the antigen preparation used to detect the humoral response. regarding human sporotrichosis, mendoza et al. [67] observed that 147-kda, 90-kda, 74-kda, 55-kda, and 40-kda molecules are commonly recognized by sera from patients with sporotrichosis, and scott & muchmore [68] showed that 70-kda, 40-kda, 36-kda, and 22-kda molecules are immunodominant in human sporotrichosis. a 70-kda molecule was previously highlighted as immunodominant during murine sporotrichosis [69] [70] [71] , with igg1 and igg3 predominant [71] . this 70-kda molecule was originally described as an adhesin molecule for fibronectin and laminin in s. schenckii s. str. [69, 70] that localized to the fungal cell wall [46] . more recently, we identified this molecule via 2d immunoblotting followed by maldi-tof/ ms as 3-carboxymuconate cyclase (genbank: kp233225), the major antigen of human sporotrichosis [45] . based on 1d electrophoresis [3, 46] and 2d electrophoresis [45] , the molecular weight (55-73kda) and isoelectric point (4.33-4.85) of 3-carboxymuconate cyclase vary intraand interspecifically [3, 45, 46, 64] . variation in gp60/gp70 may be related to differential glycosylation patterns and amino-acid substitution along the protein core, since all glycoforms/isoforms display identical maldi-tof/ms spectra [45] (table 2) . here, the intensity of the recognition of distinct molecules differed among sera from different cats, but serum from an individual cat displayed little variation when probing different antigen preparations. these data suggest that the antibody response differs between cats and that there are few qualitative variations in the expression of cellular antigens by s. brasiliensis and s. schenckii s. str. in this study, the clinical presentation of sporotrichosis in cats corresponded mainly to multiple skin lesions; we observed no association between the distribution and number of skins lesions and the number or type of molecules recognized by antibodies in the sera. although it remains to be clarified whether the antibodies produced during active infection in feline sporotrichosis are protective, antibodies against 3-carboxymuconate cyclase appear to inhibit fungal adhesion to the host in a dose-dependent manner [70, 73] . in another dimorphic fungus, p. brasiliensis, passive administration of monoclonal antibodies against the immunodominant antigen gp43 or against the recombinant protein before and after intratracheal or intravenous infections reduced fungal burden and decreased pulmonary inflammation in mice [75, 76] . gaining insight into host-parasite interplay in the immunological context is essential for understanding the emergence of feline sporotrichosis and is critical to serodiagnosis and the development of vaccines. here, we demonstrated that antigens derived from yeast cell extracts of s. brasiliensis and s. schenckii s. str. yielded excellent results in elisa and immunoblotting. the variety of molecules recognized by sera may be related to certain characteristics of the isolate, such as virulence, or even related to immune-system activation in each individual host. during infection, sporothrix antigens elicit an igg-mediated response; 3-carboxymuconate cyclase (gp60 in s. brasiliensis and gp70 in s. schenckii) is the immunodominant molecule in feline sporotrichosis, similar to murine and human disease. therefore, this molecule may also be useful as a marker in the diagnosis of feline sporotrichosis and is a promising candidate for the development of therapeutic vaccines to tackle sporotrichosis in highly endemic areas. supporting information s1 checklist. stard checklist. (doc) s1 table. summary statistics for antigen detection via elisa with four antigenic extracts. (doc) s2 table. frequencies of antigen recognition on immunoblotting with serum-derived antibodies against s. brasiliensis and s. schenckii proteins in naturally infected cats (n = 49). on refractory subcutaneous abscesses caused by a fungus possibly related to the sporotricha on a mycosis observed in men and mice: contribution to the knowledge of the socalled sporotrichosis characterization of virulence profile, protein secretion and immunogenicity of different sporothrix schenckii sensu stricto isolates compared with s. globosa and s. brasiliensis species different virulence levels of the species of sporothrix in a murine model genetic diversity and antifungal susceptibility profiles in causative agents of sporotrichosis molecular phylogeny of sporothrix schenckii global its diversity in the sporothrix schenckii complex phylogenetic analysis reveals a high prevalence of sporothrix brasiliensis in feline sporotrichosis outbreaks emerging sporotrichosis is driven by clonal and recombinant sporothrix species phylogeography and evolutionary patterns in sporothrix spanning more than 14,000 human and animal case reports sporothrix globosa, a pathogenic fungus with widespread geographical distribution phylogeny of the ophiostoma stenoceras-sporothrix schenckii complex multi-gene phylogenies define ceratocystiopsis and grosmannia distinct from ophiostoma emerging lineages in the ophiostomatales sporothrix chilensis sp. nov. 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proteins during the assembly of the head of bacteriophage t4 improved silver staining of plant proteins, rna and dna in polyacrylamide gels electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications practical statistics for medical research. london: chapman and hall genotyping species of the sporothrix schenckii complex by pcr-rflp of calmodulin systemic mycoses in dogs and cats sporotrichosis in a horse feline sporotrichosis: transmission to man sporotrichosis in man and animal feline sporotrichosis in the southern region of rio grande do sul, brazil: clinical, zoonotic and therapeutic aspects a comparative serological study of the sscbf antigenic fraction isolated from three sporothrix schenckii strains development of an enzyme-linked immunosorbent assay for the serodiagnosis of several clinical forms of sporotrichosis molecular typing of sporothrix schenckii isolates from cats in malaysia serology of paracoccidioidomycosis due to paracoccidioides lutzii heterogeneity of proteins expressed by brazilian sporothrix schenckii isolates differential induction of th1-prone immunity by human dendritic cells activated with sporothrix schenckii of cutaneous and visceral origins to determine their different virulence identification of the feline humoral immune response to bartonella henselae infection by protein microarray production of culture filtrates of sporothrix schenckii in diverse culture media immunoblot analysis of antibody responses to sporothrix schenckii cell surface expression of adhesins for fibronectin correlates with virulence in sporothrix schenckii passive immunization with monoclonal antibody against a 70-kda putative adhesin of sporothrix schenckii induces protection in murine sporotrichosis humoral immune response against soluble and fractionate antigens in experimental sporotrichosis host organism defense by a peptide-polysaccharide extracted from the fungus sporothrix schenckii isolation and some properties of a glycoprotein of 70 kda (gp70) from the cell wall of sporothrix schenckii involved in fungal adherence to dermal extracellular matrix detection of 2 immunoreactive antigens in the cell wall of sporothrix brasiliensis and sporothrix globosa the monoclonal antibody against the major diagnostic antigen of paracoccidioides brasiliensis mediates immune protection in infected balb/c mice challenged intratracheally with the fungus saccharomyces cerevisiae expressing gp43 protects mice against paracoccidioides brasiliensis infection key: cord-294798-ji3p0l4j authors: white, sarah k.; mavian, carla; elbadry, maha a.; beau de rochars, valery madsen; paisie, taylor; telisma, taina; salemi, marco; lednicky, john a.; morris, j. glenn title: detection and phylogenetic characterization of arbovirus dual-infections among persons during a chikungunya fever outbreak, haiti 2014 date: 2018-05-31 journal: plos negl trop dis doi: 10.1371/journal.pntd.0006505 sha: doc_id: 294798 cord_uid: ji3p0l4j in the context of recent arbovirus epidemics, questions about the frequency of simultaneous infection of patients with different arbovirus species have been raised. in 2014, a major chikungunya virus (chikv) epidemic impacted the caribbean and south america. as part of ongoing screening of schoolchildren presenting with acute undifferentiated febrile illness in rural haiti, we used rt-pcr to identify chikv infections in 82 of 100 children with this diagnosis during may—august 2014. among these, eight were infected with a second arbovirus: six with zika virus (zikv), one with dengue virus serotype 2, and one with mayaro virus (mayv). these dual infections were only detected following culture of the specimen, suggesting low viral loads of the co-infecting species. phylogenetic analyses indicated that the zikv and mayv strains differ from those detected later in 2014 and 2015, respectively. moreover, chikv and zikv strains from co-infected patients clustered monophyletically in their respective phylogeny, and clock calibration traced back the common ancestor of each clade to an overlapping timeframe of introduction of these arboviruses onto the island. introduction chikungunya virus (chikv) (family togaviridae, genus alphavirus), is the causative agent of chikungunya fever. after first isolation of chikv in 1952 in present-day tanzania, outbreaks and epidemics were limited to regions of asia, africa, and the pacific islands. in 2013, chikv emerged for the first time in the americas, with sustained autochthonous transmission and rapid spread through the region [1] [2] [3] . the acute symptoms of chikv infection are similar to those of infection with other arbovirus species, including dengue virus (denv), zika virus (zikv), and mayaro virus (mayv), each presenting with a constellation of symptoms including fever, headache, and myalgias/arthralgias. long-term, chikv infections have been linked with persistent arthralgias in a subset of cases [3] ; it has also been reported that upwards of 90% of chikv-infected individuals are symptomatic, in contrast to findings with zikv, where it is estimated that only 20% of infected persons are symptomatic [4, 5] . the similarity of the clinical presentation of acute-phase arbovirus infections is further complicated by the potential for simultaneous infection with multiple arboviruses. in a recent literature review, co-infections with chikv and denv ranged from 1% to 34% of patients [6] . however, virtually no data are available on frequency of co-infection with chikv and arboviruses other than denv. even where good laboratory diagnostic facilities are available, identification of co-infections often does not occur, as there is a tendency to cease investigation once an initial pathogen has been identified, and/or identification of a second pathogen may require facilities for virus isolation, which may not be available. as part of ongoing studies of acute undifferentiated febrile illness in a cohort of school children in rural haiti, we identified 82 children with rt-pcr-confirmed chikv infections during may-august 2014, corresponding with the time period when the caribbean chikv epidemic was moving through haiti. specimens were also simultaneously screened by rt-pcr for denv1-4, then additionally for zikv. aliquots of the plasma specimens were then inoculated onto cell cultures for the isolation of additional pathogens of potential interest [6] . we report here results of these studies, focusing on rates of arbovirus co-infection in our patient cohort and potential sources of origin of the co-infecting strains. blood specimens were collected from schoolchildren with acute febrile illness seen at the christianville school clinic in gressier, haiti, beginning in may 2014. this clinic serves as the primary source of healthcare for approximately 1,250 children at four school campuses (the main lasalle campus [campus a] and three small satellite elementary school campuses [campuses b, c, and d]) operated by the christianville foundation in the gressier/leogane region; schools are located within a radius of approximately 10 miles. the clinic has facilities for short-term stays of a few hours for sick children, with those requiring hospitalization referred to the local community hospital. as previously described, acute undifferentiated febrile illness was defined as occurrence of fever in a child with no obvious source of infection (i.e., no respiratory symptoms, symptoms of uti, or evidence of malaria or typhoid) [7] . we have previously reported isolation of zikv [8] , denv [9] , human coronavirus nl63 [10] , and enterovirus d68 [11] from children in this school cohort; however, the cases/outbreaks in these prior publications did not include chikv, or cases within the may-august, 2014, time frame of the current study. clinical features of these cases are reported elsewhere [12] . whole blood (ca. 0.5-2 ml) was collected in k 2 edta tubes (bd vacutainer, becton, dickinson and company, franklin lakes, nj). as part of the initial diagnostic evaluation, plasma was screened for chikv viral rna (vrna) by molecular methods [13, 14] . all virus isolations and rna extractions on chikv-positive specimens were performed in a bsl-3 facility at the university of florida (uf) emerging pathogens institute in gainesville, florida. the uf irb and the haitian national irb approved all protocols, and written informed consent was obtained from parents or guardians of all study participants. to assess the sensitivity of rt-pcr tests and for confirmation purposes, chikv isolations from plasma specimens were attempted in epithelioid cells derived from african green monkey kidneys (vero e6, atcc crl-1586). the vero e6 cells used for virus isolation were maintained in cell culture medium comprised of admem (advanced dulbecco's modified essential medium) supplemented with 10% low antibody, heat-inactivated, gamma-irradiated fetal bovine serum (fbs, hyclone, ge healthcare life sciences, pittsburgh, pa), l-alanine and lglutamine supplement (glutamax, invitrogen, carlsbad, ca), and 50 μg/ml penicillin, 50μg/ ml streptomycin, 100μg/ml neomycin (psn antibiotics, invitrogen) with incubation at 37˚c in 5% co 2 . confluent cell cultures were split into 25cm 2 flasks 24 hours prior to inoculation to attain 60% confluent cell monolayers the following day. prior to inoculation, the culture medium was removed and inoculum containing 100μl of plasma that had been filtered through a sterile 0.45 filter and mixed with 400μl admem with 3% fbs, glutamax, and psn antibiotics was added to the monolayer. the inoculated monolayer was incubated at 37˚c in 5% co 2, and rocked every 15 minutes for 1 hour. a negative control (mock-infected) cell culture was inoculated with 500ul of dmem without virus or plasma and handled in parallel with the other cultures. after allowing for virus adsorption for 1 hr, the inocula were removed and replaced with 3ml of admem with 3% fbs, glutamax, and psn antibiotics, and thereafter incubated at 37˚c in 5% co 2 . cell cultures were refed every 3 days by the replacement of 1.5ml of spent media with admem with 3% fbs. the cultures were observed for up to 21 days' post-inoculation, or until chikv-induced cytopathic effects (cpe) were observed in the cell monolayers using an inverted microscope. when cpe were observed throughout 50% of the monolayer, a final collection of 2ml spent media, and 1ml of scraped cells in spent media, were cryopreserved at -80˚c for future tests. total rna was extracted from both plasma specimens and spent media, and tested by realtime rt-pcr (rtrt-pcr) following published protocols for chikv vrna [13] for confirmation of previous tests performed in haiti. rna extracted from plasma specimens were then screened for denv serotypes 1-4 [15] and zikv [16] vrnas by rtrt-pcr. cycle threshold (ct)-values under 38 were considered positive. viral genomic rna that was extracted from chikv, denv1-4, and zikv strains that were obtained from the biodefense and emerging infections research resource repository (bei resources, manassas, va) were used as positive control materials for rtrt-pcr. cell cultures were also tested for the presence of denv and zikv vrnas by rtrt-pcr, even if the corresponding plasma specimen tested negative. additionally, spent media from cultures displaying non-chikv cpe, that were denv and zikv negative by rtrt-pcr, were screened with a duplex rt-pcr for the vrnas of other alphaviruses (venezuelan equine encephalitis -, eastern equine encephalitis -, western equine encephalitis -, aura-and mayaro viruses) and flaviviruses (yellow fever -, saint louis encephalitis -, bussaquara -, ilheus -, and rocio viruses) [17] . the denv-1 strain from bei and the 2015 mayv sample from our laboratory [18] were used as the flavivirus and alphavirus positive controls, respectively, in the duplex rt-pcr protocol. whole genome sequence data from 10 of the chikv samples (7 from children with co-infections, 3 from selected randomly mono-infections) were obtained by sanger sequencing and a primer-walking approach, as previously described [14, 19] . similarly, we designed sequencing primers for mayv and zikv that also amplify approximately 800bp overlapping segments, and used a primer walking method for whole genome sequencing of those viruses [8, 18] . for the sequencing of denv, primers described by christenbury et al were utilized [20] . amplification of each segment was performed using an accuscript high-fidelity first-strand cdna synthesis kit (agilent technologies, santa clara, ca) followed by pcr with phusion polymerase (new england biolabs, ipswich, ma). the 5' and 3' ends of the viral genomes were obtained using rna-ligase mediated (rlm) systems for 5' and 3' rapid amplification of cdna ends (race) per the manufacturer's protocols (life technologies, carlsbad, ca). amplicons were purified, sequenced bi-directionally, then the sequences assembled with the use of sequencher dna sequence analysis software v2.1 (gene codes, ann arbor, mi), and subsequently analyzed in comparison to denv, mayv, and zikv sequences available in genbank for nucleotide sequence comparisons. the vrna sequences we obtained differed from those of the corresponding viruses in our repository, confirming the newly analyzed sequences did not arise from laboratory contamination. alignments for each virus-specific dataset (chikv, denv-2, mayv, and zikv), including full genome sequences selected to represent the major clades described so far for each virus were obtained using the muscle algorithm implemented in mega7 (http://www.mega software.net/) [21] [22] [23] . genbank accession numbers (ac), geographical location, and year associated with isolation of each strain are reported in s1 table. based on previous evidence of recombination reported for mayv, potential presence of recombination was also investigated for the new 2014 mayv isolate (ky985361) [24] with algorithms implemented in the rdp4 [25] software (http://web.cbio.uct.ac.za/~darren/rdp.html), as previously described [24] . the phylogenetic signal in each virus-specific data set was evaluated by likelihood mapping using treepuzzle (http://www.tree-puzzle.de/) [26] , and substitution saturation plots using dambe6 ((http://dambe.bio.uottawa.ca/dambe/dambe.aspx) [27] . maximum likelihood (ml) trees were inferred by iq-tree using the best-fitting nucleotide substitution model chosen according to bayesian information criterion (bic) (s2 fig) [28, 29] . statistical robustness for internal branching order in each phylogeny was assessed by ufboot-ultrafast bootstrap (bb) approximation (2,000 replicates) implemented in iq-tree [30] , and strong statistical support along the branches was defined as bb>90%. ufboot was eployed as it accelerates computing and reduces overestimating branch support [30] . alignments files are available from the authors upon request. the temporal signal for each dataset was assessed using ml trees with tempest v1.5 (http:// tree.bio.ed.ac.uk/software/tempest/) [31] . bayesian inference of time-scaled phylogenies were carried out with beast v1.8.4 (http://beast.bio.ed.ac.uk/) [32, 33] by enforcing either a strict or relaxed molecular clock [34] and the sdr06 substitution model with empirical base frequencies and gamma distribution of site-specific rate heterogeneity. two demographic priors were also tested for each analysis: constant population size or non-parametric bayesian skyline plot (bsp). bayesian markov chain monte carlo (mcmc) were run for 50-200 million generations (sampled at fixed intervals to obtain posterior distributions with 10,000 data points), depending on the data set, in order to assure proper mixing of the mcmc, which was assessed on the basis of the effective sampling size (ess) of each parameter estimate, accepting only ess values >200. the best clock/demographic model for each virus-specific data set was chosen by comparing marginal likelihood estimates (mle) [35] , obtained using path sampling (ps) and stepping-stone sampling (ss) methods [33, 36] with bayes factor (bf) (s2 table) . in practice, the bf natural logarithm was used for comparison with lnbf<2 indicating no evidence against the null hypothesis (i.e. less parameter-rich model), 2-6-weak evidence, 6-10-strong evidence, and >10 very strong evidence [37] . for each data set, the maximum clade credibility (mcc) tree (tree with the largest product of posterior clade probabilities) was selected from the posterior tree distribution of the best fitting clock/demographic model, after appropriate burn-in, using treeannotator v1.8.4 implemented in the beast software package. the final trees were manipulated in figtree v1.4.3 for display purposes (http://tree.bio.ed.ac.uk/software/figtree/). xml files for the beast runs are available from the authors upon request. with one exception, all rtrt-pcr-confirmed chikv infections in our student cohort occurred in the 9-week period between may 23 and july 25, 2014; the one "outlier" case occurred in mid-august, 2014. among the 100 plasma specimens taken from febrile schoolchildren in the may-august time period, 82 tested positive via rtrt-pcr for chikv, but were negative for denv1-4 and zikv. among the 18 specimens from febrile schoolchildren during this time period that were not positive for chikv, two gave equivocal results in the rtrt-pcr assay and were excluded from the analysis, two were denv type 1 positive, and 14 were negative in all assays. cultures were not performed on these 18 specimens due to insufficient specimen volume, reflecting the large number of assays that had been conducted in the initial screening of what were relatively small blood samples from each child. the mean age of children from whom specimens were collected was 9.6 years. ninety-two of the 100 specimens were from students at campus a of the school; 7 were from campus b, and one was from campus c. gender, average age, and school level for the 82 children whose samples were positive for chikv by rtrt-pcr are included in table 1 . of the 82 rtrt-pcr chikv-positive specimens, attempts were made to isolate virus from 62 specimens, with the remainder not inoculated onto cell cultures due to insufficient specimen volume. typical chikv-induced cpe, consisting of cell membrane blebbing, cell lysis, and apoptosis, were observed in cultures from 43 of the 62 samples on average 5 days post inoculation (dpi), with some specimens displaying advanced cpe as early as 2 dpi and others not until 20 dpi (fig 1) ; target vrna's were positive by rtrt-pcr for chikv but negative for denv and zikv [15] [16] [17] . all negative control cell cultures lacked cpe and tested negative by rtrt-pcr for the target vrnas of this work. eleven (15%) of the 62 samples cultured did not display any cpe during the period of culture and tested negative for target vrnas in these studies, despite initial positive chikv results by rtrt-pcr. eight cultures displayed cpe inconsistent with those expected for chikv (fig 2) . the mixed cpe in these latter cultures was associated with co-infection with chikv together with another virus, as determined by molecular tests of the cultured specimens: co-infecting arboviruses included zikv (n = 6), denv type 2 (n = 1), and mayv virus (n = 1). sex and age of patients were comparable for those with chikv mono-inections and the subset of chikv cases with co-infections. seven of the 8 co-infections were from campus a of the school; one co-infection (chikv and zikv) was in a student from campus b. (fig 3) using the best fitting molecular clock/demographic model (s2 table) , were inferred for each data set. bayesian and ml reconstructions displayed very similar topologies and suggested three independent phylogenetic lineages of chikv in haiti, possibly the result of three separate introductions (figs 3a and s3a) . the chikv strains from the current study were in a well-supported monophyletic clade which was most closely related to a strain from el salvador (fig 3a) . within this clade, six chikv strains were from patients co-infected with zikv and one with denv-2. molecular clock median estimate of the time of the most recent common ancestor (tmrca) for the clade was august 2013, with 95% highest posterior density (95%hpd) intervals between april 2013 and january 2014 ( fig 3a) . as shown in fig 3, two other 2014 chikv isolates, not collected as part of this study but previously reported by lanciotti (kr559476 and kr559478) from mono-infected haitian patients, clustered within a second distinct clade related to different central and south american strains, but separate from our gressier strains. one of the 10 sequenced chikv strains from our cohort, from a randomly selected mono-infection in a child from campus b, was in yet a third clade, which clustered most closely with a strain from the dominican republic. the six zikv sequences obtained in june 2014 from the chikv co-infected patients were highly similar (99.9%) to each other and also cluster within a well-supported monophyletic clade, which, interestingly, includes a divergent strain isolated in 2016 from guadaloupe (figs 3b and s3b) . the primary clade of co-infecting zikv strains is closely related to another zikv clade including isolates from the 2016 usa florida outbreak [38] . in contrast, previously reported [8] haitian strains from this same school cohort in december of 2014 (ku509998), and haitian strains from early 2016 (kx051563 [39] and kx269878 [40] ) belonged to distinct phylogenetic lineages, consistent with multiple independent introductions of zikv to haiti. the median origin of the haitian zikv co-infection strains was estimated by molecular clock in april 2014 with a 95%hpd between january and may 2014 (fig 3b) , a timeframe overlapping with the estimates of the corresponding chikv co-infections clade. the specimen from the chikv and denv-2 co-infection was obtained on june 11, 2014. the denv-2 isolate was closely related to a previously reported strain isolated in early 2016 (kx702404) from a us traveler returning from haiti [39] and they both cluster within a wellsupported clade including brazilian and peruvian subclades (figs 3c and s3c) . according to the time-scaled phylogeny (fig 3c) , the tmrca of the denv-2 isolate from the chikv coinfected patient traced back to february 2008 with 95%hpd between may 2007 and october 2008. finally, the mayv strain from a chikv co-infected patient seen on june 4, 2014 is the earliest documented case of mayv in haiti to date. its genome is highly similar (99.4%) to a strain from brazil isolated in 1960, and phylogenetic analysis cluster both strains in a well-supported monophyletic clade (figs 3d and s3d) , within genotype l [24, 40] , with a tmrca dating back to 1958 (95%hpd interval of 1949-1960). it should be noted, however, that since we only have one mayv strain clustering with the brazilian 1960 strain, the tmrca is unlikely to represent the date of introduction of mayv in haiti. another, previously reported haitian strain (kx496990) [18, 24] , clusters in a different well-supported clade within genotype l, a scenario once again consistent with multiple independent introductions of the virus in the caribbean. despite increasing recognition of the frequency with which simultaneous co-infection with multiple arbovirus species occurs [19, 39, [41] [42] [43] , co-infection dynamics are not well understood, and the clinical, pathologic, and immunologic significance of such co-infections remains to be determined. as a starting point in unraveling these dynamics, we were interested in better quantifying the frequency of such infections, and exploring possible origins of co-infecting strains. to optimize our ability to identify co-infections (and following standard practices in our laboratory [8, 17, 19, 39] ), we continued diagnostic studies even after initial identification of chikv or another pathogen in a patient sample. interestingly, while we identified multiple co-infecting viruses in cell culture, the original patient rtrt-pcr in this study was, in each instance, negative for the second pathogen, suggesting that viral loads for the co-infecting species were low. we also had instances where viral cultures were negative in the setting of a positive rtrt-pcr, possibly reflecting the presence of non-viable virus in the sample, and/or strains that have mutated or which do not grow well in vero cells. if we are to better understand the role of co-infections in disease occurrence, the diagnostic significance of both rtrt-pcr negative/culture positive and rtrt-pcr positive/culture negative samples will require further study. co-infections with chikv and denv are well described, with reported rates of co-infection ranging from 1% to 34% [6] ; the highest reported co-infection rates were from madagascar (26%) and nigeria (34%). another study from the colombian-venezuelan border reports a chikv/denv co-infection prevalence of 7.6% [43] . in our study, in contrast, the co-infection rate was only 1%, with only two additional dengue cases (both denv-1) identified among chikv-negative patients; this may reflect a low level of circulating denv in the community during the chikv epidemic, and/or a high existing level of population immunity to the virus. we also identified one chikv/mayv co-infection. we have previously reported a mixed mayv and denv-1 infection that occurred outside of the time frame of the chikv epidemic (january, 2015) in the same school cohort [18] . the high genomic similarity of this haitian mayv isolate with a brazilian strain isolated in 1960 corroborates our hypothesis that mayv has been criptically circulating in the human population at a sub-epidemic level, most likely in brazil, for years and that it was introduced just recently onto hispaniola [24] . what was unexpected in our data set was the relatively high frequency of zikv/chikv coinfections. this occurred in a setting in which we were only screening plasma samples. as we have previously reported [39] , urine can be positive for zikv for a longer period of time than plasma; if we had also screened urine, there is the possibility that we would have identified additional zikv-positive patients. we, and others, have previously reported cases of zikv/ chikv co-infection [19, 42, 44, 45] , so finding the two together is not surprising. the zikv case cluster in the current study would appear to have been relatively widespread within the community (i.e., not a point source at the school), as one of the six co-infections was from a child at a different school campus, ca. 5 miles from the main campus. interestingly, our molecular clock analysis indicates that the strains of chikv and zikv found in our patients were both introduced in haiti within the same short time frame, each one giving rise to a well-supported monophyletic clade, one including all chikvs from chikv/zikv co-infected subjects, the other one including all zikvs from the same co-infected subjects. the simultaneous emergence of these two clades in the haitian population is compatible with a simultaneous coinfection scenario or indicate, at the very least, the sequential infection of a patient with two arboviruses within a relatively short period of time. in light of recent reports that mosquito coinfection with zikv and chikv allows simultaneous transmission without affecting vector competence [46] , it is plausible that the two viruses were co-transmitted by the same mosquito. given that these are among the earliest, if not the earliest, documented zikv infections in the hemisphere, the observation that they appeared together with epidemic chikv raises interesting questions about the possible role of chikv in the initiation of the zikv epidemic in the americas. because our work draws on student populations from four schools located within a radius of about 10 miles, we have some feel for disease prevalence within the immediate study area; however, generalizability of these data to larger regions of haiti may be limited. nonetheless, our findings are consistent with the idea that there were multiple introductions of chikv into haiti between 2013 and 2014, with identification of three distinct phylogenetic lineages, each clustering with strains from different parts of the caribbean and/or south america. this was also the case for zikv: the zikv co-infecting strains did not cluster with the zikv strains previously obtained in haiti in 2014 and 2016, consistent with multiple introductions or re-introductions of this arbovirus in haiti between 2014 and 2016. although for denv-2 and mayv the data are more limited, the tmrcas for denv-2 suggested that the introduction in haiti happened between 2008 and 2014, a time interval again overlapping with the estimated introduction of both chikv and zikv co-infecting strains circulating in haiti. clustering of both the zikv and denv-2 strains together with zikv and denv-2 strains obtained in florida in 2016 [38, 47] , as well as the multiple independent introductions of chikv, zikv, and mayv to haiti inferred from the phylogenies, demonstrate the potential role of the caribbean as a node for arbovirus infections connecting south, central, and north america. whole-genome sequencing analysis from the chikungunya virus caribbean outbreak reveals novel evolutionary genomic elements emergence of human arboviral diseases in the americas chikungunya virus infection: an overview movement of chikungunya virus into the western hemisphere zika virus: history, emergence, biology, and prospects for control co-distribution and co-infection of chikungunya and dengue viruses spectrum of outpatient illness in a school-based cohort in haiti, with a focus on diarrheal pathogens zika virus outbreak in haiti in 2014: molecular and clinical data complete genomic sequence of dengue virus 1 isolation of coronavirus nl63 from blood from children in rural haiti: phylogenetic similarities with recent isolates from malaysia isolation of an enterovirus d68 from blood from a child with pneumonia in rural haiti: close phylogenetic linkage with new york strain clinical and epidemiologic patterns of chikungunya virus infection and coincident arboviral disease in a school cohort in haiti chikungunya virus in us travelers returning from india complete genome sequences of chikungunya viruses isolated from plasma specimens collected from haitians in 2014 analytical and clinical performance of the cdc real time rt-pcr assay for detection and typing of dengue virus genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia duplex reverse transcription-pcr followed by nested pcr assays for detection and identification of brazilian alphaviruses and flaviviruses mayaro virus in child with acute febrile illness zika and chikungunya virus coinfection in a traveller returning from colombia, 2016: virus isolation and genetic analysis a method for full genome sequencing of all four serotypes of the dengue virus mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets muscle: multiple sequence alignment with high accuracy and high throughput muscle: a multiple sequence alignment method with reduced time and space complexity emergence of recombinant mayaro virus strains from the amazon basin rdp4: detection and analysis of recombination patterns in virus genomes tree-puzzle: maximum likelihood phylogenetic analysis using quartets and parallel computing dambe: software package for data analysis in molecular biology and evolution iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies w-iq-tree: a fast online phylogenetic tool for maximum likelihood analysis ultrafast approximation for phylogenetic bootstrap exploring the temporal structure of heterochronous sequences using tempest (formerly path-o-gen) bayesian phylogenetics with beauti and the beast 1.7 beast: bayesian evolutionary analysis by sampling trees exploring the demographic history of dna sequences using the generalized skyline plot improving marginal likelihood estimation for bayesian phylogenetic model selection improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty zika virus evolution and spread in the americas coinfection with zika and dengue-2 viruses in a traveler returning from haiti, 2016: clinical presentation and genetic analysis evolutionary and ecological characterization of mayaro virus strains isolated during an outbreak dengue and chikungunya virus coinfection in a german traveller zika, dengue, and chikungunya co-infection in a pregnant woman from colombia co-circulation and simultaneous co-infection of dengue, chikungunya, and zika viruses in patients with febrile syndrome at the colombian-venezuelan border co-infections with chikungunya and dengue viruses coinfections of zika and chikungunya viruses in bahia, brazil, identified by metagenomic next-generation sequencing mosquito co-infection with zika and chikungunya virus allows simultaneous transmission without affecting vector competence of aedes aegypti complete genome sequence of dengue virus type 2 from a resident of north-central florida with locally transmitted dengue fever estimation of the number of nucleotide substitutions in the control region of mitochondrial dna in humans and chimpanzees we thank sonese chavannes and gina anilis of the christianville school clinic, the laboratory staff, and the christianville foundation, for their dedication and support. key: cord-285772-4xt4anq5 authors: huang, rui; zhu, li; xue, leyang; liu, longgen; yan, xuebing; wang, jian; zhang, biao; xu, tianmin; ji, fang; zhao, yun; cheng, juan; wang, yinling; shao, huaping; hong, shuqin; cao, qi; li, chunyang; zhao, xiang-an; zou, lei; sang, dawen; zhao, haiyan; guan, xinying; chen, xiaobing; shan, chun; xia, juan; chen, yuxin; yan, xiaomin; wei, jie; zhu, chuanwu; wu, chao title: clinical findings of patients with coronavirus disease 2019 in jiangsu province, china: a retrospective, multi-center study date: 2020-05-08 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008280 sha: doc_id: 285772 cord_uid: 4xt4anq5 limited data are available for clinical characteristics of patients with coronavirus disease 2019 (covid-19) outside wuhan. this study aimed to describe the clinical characteristics of covid-19 and identify the risk factors for severe illness of covid-19 in jiangsu province, china. clinical data of hospitalized covid-19 patients were retrospectively collected in 8 hospitals from 8 cities of jiangsu province, china. clinical findings of covid-19 patients were described and risk factors for severe illness of covid-19 were analyzed. by feb 10, 2020, 202 hospitalized patients with covid-19 were enrolled. the median age of patients was 44.0 years (interquartile range, 33.0–54.0). 55 (27.2%) patients had comorbidities. at the onset of illness, the common symptoms were fever (156 [77.2%]) and cough (120 [59.4%]). 66 (32.7%) patients had lymphopenia. 193 (95.5%) patients had abnormal radiological findings. 11 (5.4%) patients were admitted to the intensive care unit and none of the patients died. 23 (11.4%) patients had severe illness. severe illness of covid-19 was independently associated with body mass index (bmi) ≥ 28 kg/m(2) (odds ratio [or], 9.219; 95% confidence interval [ci], 2.731 to 31.126; p<0.001) and a known history of type 2 diabetes (or, 4.326; 95% ci, 1.059 to 17.668; p = 0.041). in this case series in jiangsu province, covid-19 patients had less severe symptoms and had better outcomes than the initial covid-19 patients in wuhan. the bmi ≥ 28 kg/m(2) and a known history of type 2 diabetes were independent risk factors of severe illness in patients with covid-19. during december 2019, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) emerged in wuhan, china and spread among humans in china and other countries [1, 2] . global attention was soon focused on this virus due to the rapidly increasing number of confirmed cases [3] . sars-cov-2 infection may result in severe and even fatal respiratory diseases [4] . as of march 13, 2020, 132,536 confirmed cases have been reported in 123 countries with 4,947 deaths [5] . despite the rapid spread of the disease worldwide, the clinical characteristics of coronavirus disease 2019 (covid-19) remain largely unclear. furthermore, there are no directly antiviral drugs for covid19 . several studies have reported the clinical characteristics of covid-19 patients who were hospitalized in wuhan (the outbreak center of the infection) [4, 6, 7] . huang et al. first reported 41 cases of covid-19 and most patients had a history of exposure to huanan seafood market in wuhan [6] . fever and cough were the most common symptoms [6] . 13 (32%) patients were admitted to an intensive care unit (icu) due to the severity of disease and six (15%) patients died [6] . chen et al. conducted a retrospective, single-center study which included 99 confirmed cases of covid-19 in wuhan and found that the virus was more likely to infect older men with comorbidities, and the mortality rate was as high as 11% [4] . another single-center study which analyzed 138 hospitalized patients with confirmed covid-19 in wuhan, found that 26% of patients received icu care and the mortality rate was only 4.3% [7] . although several studies have reported the clinical manifestations and short-term prognosis of covid-19, the source of cases in majority of these studies was from a single center in wuhan. the characteristics of the disease and risk factors of severe illness among inpatients in other parts of china outside wuhan were still lacking. chang et al. reported early clinical features of 13 patients with confirmed covid-19 who were admitted to hospitals in beijing, china [8] . in their study, all the patients recovered indicating milder clinical presentation caused by infections [8] . however, the sample size was very small with only inclusion of 13 patients and the study was limited by the lack of detailed data since patients were transferred to the designated hospitals after diagnosis [8] . xu et al. summarized the clinical characteristics of 62 patients in zhejiang province, which revealed that the symptoms of patients were relatively mild [9] . however, only 62 patients were included in the study and risk factors for severe illness could not be analyzed due to the limitation of sample size [9] . nevertheless, these studies provide important evidence that the clinical characteristics of patients outside wuhan may differ from those initially reported in wuhan. in this multi-center study, we aimed to describe the clinical characteristics of covid-19 and to identify the risk factors of severe illness among inpatients with confirmed covid-19 in jiangsu province, which is located in the east of china. 1) . data of the confirmed covid-19 patients were collected from january 22, 2020 to february 10, 2020. the 8 designated hospitals are responsible for the treatment of covid-19 clinical findings of covid-19 patients in jiangsu patients in jiangsu province, china, assigned by the chinese government. all confirmed patients were diagnosed based on the criterion of world health organization (who) interim guidance [10] . the study was approved by the institutional ethics board of these hospitals, with a waiver of informed consent. all the medical records of confirmed cases were reviewed by more than two health care workers in each medical center. the data of epidemiology, clinical laboratory, radiology, treatment, and prognosis were collected from medical records. the demographic characteristics, comorbidities, exposure history, symptoms, signs, laboratory data, radiological data, treatment data, and outcomes were collected. all data were entered into a computerized database for further analysis. different researchers preformed the cross check of the data to avoid errors. unclear information was further clarified by directly contacting the specific clinicians who were responsible for the patients. the diagnostic criteria of acute respiratory distress syndrome (ards), acute cardiac injury, acute renal injury and acute liver injury were based on the corresponding guidelines [11] [12] [13] [14] . additionally, treatment medication and outcomes of the enrolled cases were also collected based on the medical records. the criteria for discharge was based on the guidelines for the diagnosis and treatment of novel coronavirus infection by the chinese national health commission (trial version 5) [15] . patients were grouped into severe and non-severe covid-19 according to the same guidelines [15] . all patients were confirmed by throat swab sample obtained from the upper respiratory tract and detected by a real-time reverse transcriptase polymerase chain reaction (rt-pcr) in accordance with the protocol by the world health organization [16] .the positive throat swab samples were confirmed by both hospitals and local centers for disease control and prevention. these routine laboratory tests were detected according to the state of disease, including blood routine tests, liver function, renal function, coagulation function, inflammatory biomarkers and myocardial enzymes. radiological assessments including chest x-ray or chest computed tomography (ct) were performed for each patient. continuous variables were described as medians (interquartile range (iqr)), and categorical variables were presented as the counts and percentages. continuous variables were compared by the independent group t tests (normal distribution) and mann-whitney u (non-normal distribution). categorical variables were compared by chi-square or fisher exact test. the risk factors for severe illness were analyzed by binary logistic regression. variables having p values <0.05 in the univariate analysis were further used for a multivariate logistic regression analysis. p<0.05 was regarded to be statistically significant. the data analysis was performed by spss version 22.0 software (spss inc., chicago, il, united states). distribution map was plotted by qgis version 3.10.2 (qgis development team, open source geospatial foundation project). a total of 202 admitted patients who were identified as covid-19 were included in the study. the median age of the patients was 44.0 (iqr 33.0-54.0) years ( although only 3 (1.5%) patients had direct exposure to huanan seafood market, 84 (41.6%) patients visited hubei province and 92 (45.5%) patients had contact with people who visited hubei province after the onset of the covid-19 epidemic. 98 (48.5%) patients had known contact with suspected or confirmed cases. however, 32 (15.8%) patients did not report any known contact with hubei. the median time from symptom onset to admission was 5.0 days (iqr 2.0-7.0 days). age ( 23 (11.4%) of the patients developed severe illness in our study. the age of severe patients was comparable with non-severe patients (median age 49.0 yr vs. 44.0 yr, p = 0.066) and the bmi index of severe patients was higher than non-severe patients (median bmi, 26.4 kg/m 2 vs. 24.2 kg/m 2 , p = 0.004). nearly half (44.4%) of the severe patients were obese (bmi � 28 kg/ m 2 ) compared to only 10.4% of the non-severe patients. furthermore, more severe patients had history of type 2 diabetes (34.8% vs. 6.1%, p<0.001). more non-severe patients contacted with suspected or confirmed patients than severe patients (51.4% vs. 26.1%, p = 0.022). the most common symptoms at onset of illness were fever (156 [ compared to non-severe patients, severe patients presented higher percentage of shortness of breath (7.3% vs. 26.1%, p = 0.004), lower lymphocytes (median 0.8 ×10 9 /l vs. 1.2×10 9 /l, p<0.001) and albumin (alb) levels (median 38.1 g/l vs. 41.1 g/l, p = 0.002). however, prothrombin time was not significantly different between severe patients (median 12.3 s) and non-severe patients (median 12.8 s, p = 0.089). the proportion of patients with crp < 10mg/ l, 10-50mg/l, and � 50mg/l were 40.0%, 30.0%, and 30.0% in severe patients, while patients with crp < 10mg/l, 10-50mg/l, and � 50mg/l account for 65.3%, 30.6%, and 4.0% in nonsevere patients, respectively. more patients showed elevated crp in severe patients than nonsevere patients (p<0.001). furthermore, severe patients had higher fasting blood glucose (median 6.5 mmol/l) as compared with non-severe patients (median 5.7 mmol/l, p = 0.009). on admission, abnormalities of chest ct examinations were detected in 193 (95.5%) patients. out of 193 patients, 158 (78.2%) had bilateral involvement, and 149 (73.8%) had ground glass opacity (table 2) . bilateral pneumonia was more commonly observed in severe patients (95.7%) than non-severe patients (76.0%, p = 0.031). representative chest ct findings for a 30-year-old man on admission to the hospital were shown in fig 2. bilateral ground-glass opacities were observed in both lungs on admission (fig 2) . oxygen therapy was required in 109 (54.0%) patients and non-invasive mechanical ventilation was required in 9 (4.5%) patients. none of the patients required intubation and invasive mechanical ventilation. 196 (97.0%) patients received antiviral therapy (lopinavir/ritonavir, 89.1%; interferon α-2b, 59.9%; arbidol, 29.2%; oseltamivir, 15.8%). 149 (73.8%) patients were administered with empirical antibiotic treatment. additionally, sixty-four (31.7%) patients were given corticosteroids and 31 (15.3%) patients were given gamma globulin. (table 4 ). our study provides a comprehensive description of the clinical characteristics of laboratoryconfirmed cases of covid-19, and the risk factors for severe covid-19 in 202 cases from 8 designated hospitals in 8 cities of jiangsu province, china. consistent with the study by wang et al., about half of the patients in our study were male [7] . these data differ from the recent report by huang et al. and chen et al. which showed sars-cov-2 is more likely to infect male [4, 6] . one possible explanation is that more covid-19 patients in the previous report had an exposure history of the huanan seafood market in wuhan, and most of patients tended to be male workers [7] . however, only 1.5% of patients in our study had known contact with huanan seafood market. the median age of the patients was 44 years and only 12.9% of the patients aged over 60 years. 27.2% of the patients had chronic comorbidities. several studies suggested that sars-cov-2 is more likely to infect elder adult males with chronic comorbidities [4, 7, 17] . however, our study indicated that patients at a wide age range can be infected by sars-cov-2. recently, wei et al. even reported nine infants infected by sars-cov-2 [18] . although 15.8% of the patients did not report any known contact with wuhan-related people, the majority of patients in our study were wuhanrelated which indicated the epidemiology are important for the diagnosis of covid-19 for patients outside wuhan. consistent with two recent reports, fever and cough were the common symptoms whereas other symptoms such as diarrhea were much rare [4, 6] . however, our study found that 22.8% of the patients were afebrile on admission. the percentage of patients with fever was lower than the previous study which was ranged from 83% to 98.6% [7] . thus, the afebrile patients with an epidemiological link of the disease should be also suspected for covid-19. compared with symptoms of non-severe patients, shortness of breath was more common in severe patients. the most common laboratory abnormalities observed in this study were decreased white blood cells and lymphocyte counts as well as increased crp and ldh levels. compared to non-severe patients, severe patients had laboratory abnormalities such as lower lymphocytes and alb levels as well as higher ldh and cpr levels. the onset of symptoms and laboratory abnormalities on admission may help the physicians identify the covid-19 patients who likely develop severe illness and provide better supportive care. currently, except for meticulous supportive care, no specific treatment has been recommended for covid-19. although antibacterial agents are ineffective for covid-19, over 70% of the patients in this study still receive antibacterial agents. about 90% of the patients received antiviral therapy, such as atomized inhalation of interferon α-2b, lopinavir/ritonavir, arbidol, and oseltamivir. the benefit of the antiviral agents for covid19 is not yet clear and deserves further investigation. 31.7% of the patients used corticosteroids during the treatment, especially for severe patients (78.3%). although some experts recommend the prudently use of short courses of corticosteroids at low-to-moderate doses for critically ill patients with covid-19, the use of corticosteroids remains controversial in covid-19 [19] . moreover, the optimal time, dose and duration of corticosteroids for patients with covid19 are not yet clear and need to be evaluated by randomized controlled trials to provide a more solid evidence for treatment recommendations in the future. gamma globulin was also used for the treatment of covid-19 and severe patients were more likely to receive gamma globulin treatment. however, the efficacy of gamma globulin for covid-19 remains controversial. 23 (11.4%) patients were identified as severe illness cases in our study which is lower than the previous study (15.7%) [20] . the icu admission (5.4%) was also lower as compared with the previous studies which ranged from 26.1% to 32% [6, 7] . as of february 10, 2020, no patients died in our study. the mortality rate in our study is significantly lower than patients in wuhan as previously reported which ranged from 4.3% to 15% [6, 7] . wu et al. also reported only 1 of 62 patients was transferred to an icu and no patient died in zhejiang province [9] . the possible interpretation of the mild disease and better outcomes in our study may be that the patients were younger than previous reports. the elderly people may have an increased risk of coexisting disorders and are more susceptible to developing severe forms of disease than younger people [21, 22] . indeed, less patients had comorbidities (27.2%) in our study as compared with the previous reports (32.0%-46.4%) [4, 6, 7] . the median time from symptom onset to admission was 5.0 days which is also shorter than the previous studies (7.0 days) [6, 7] . thus, early diagnosis and prompt care of the covid-19 patients might also together contribute to the significant reduction in mortality rate in our study [20] . early identification of patients with severe illness is important for the management of patients with covid-19. we conducted univariate and multivariate analysis for risk factors of covid-19 patients with severe illness on admission. for the first time, we identified the obesity (bmi over 28 kg/m 2 ) and a history of type 2 diabetes as two independent risk factors of severe illness, suggesting that the metabolic conditions was associated the severity of covid-19. in the previous study, metabolic syndrome-related conditions such as diabetes and obesity were also demonstrated to be etiologically linked to middle east respiratory syndrome coronavirus (mers-cov) pathogenesis [23] . these disorders were reported to down regulate some key mediators of host immune response to pathogenesis [23, 24] . previous study found that a known history of type 2 diabetes was also an independent predictor of death and morbidity in patients with severe acute respiratory syndrome (sars) [25] . whether the obesity and known history of type 2 diabetes could predict the fatal outcome of patients with covid-19 deserves further investigation. this study has several limitations. first, only 202 patients with confirmed covid-19 were included from eight cities of jiangsu province. it would be better to include as many as possible patients in jiangsu province to get more comprehensive understanding of covid-19 outside wuhan. moreover, we only included patients with laboratory confirmed cases. the suspected but undiagnosed cases were not included in the present study. as of february 10, 2020, 515 confirmed cased have been reported in jiangsu province [5] . in fact, about a half of the confirmed cases were included in our present study. thus, our study is very representative. second, clinical outcomes of the remaining 165 hospitalized patients were not available at the time of analysis. thus, we could not assess the risk factors for poor outcomes of patients. however, this multicenter study provides an early assessment of the epidemiological and clinical characteristics of covid-19 outside wuhan, china. in conclusion, covid-19 patients in jiangsu province were less severe and have improved prognosis than the initial covid-19 patients as previously reported in wuhan indicating that mild cases might already have occurred. a bmi > 28 kg/m 2 and a history of type 2 diabetes are independent risk factors for severe illness of covid-19. sustained intensive control efforts remain urgently needed to minimize the risk of covid-19. a novel coronavirus from patients with pneumonia in china severe acute respiratory syndrome-related coronavirus: the species and its viruses-a statement of the coronavirus study group a novel coronavirus outbreak of global health concern epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study novel coronavirus 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commission (trial version laboratory diagnostics for novel coronavirus early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia novel coronavirus infection in hospitalized infants under 1 year of age in china on the use of corticosteroids for 2019-ncov pneumonia clinical characteristics of 2019 novel coronavirus infection in china severe human influenza infections in thailand: oseltamivir treatment and risk factors for fatal outcome motivational strategies to prevent frailty in older adults with diabetes: a focused review prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis connecting type 1 and type 2 diabetes through innate immunity. cold spring harb perspect med plasma glucose levels and diabetes are independent predictors for mortality and morbidity in patients with sars conceptualization: rui huang, chuanwu zhu, chao wu. key: cord-355469-ojop6i4k authors: egawa, kazutaka; shimojima, masayuki; taniguchi, satoshi; nagata, noriyo; tani, hideki; yoshikawa, tomoki; kurosu, takeshi; watanabe, shumpei; fukushi, shuetsu; saijo, masayuki title: virulence, pathology, and pathogenesis of pteropine orthoreovirus (prv) in balb/c mice: development of an animal infection model for prv date: 2017-12-14 journal: plos negl trop dis doi: 10.1371/journal.pntd.0006076 sha: doc_id: 355469 cord_uid: ojop6i4k background: cases of acute respiratory tract infection caused by pteropine orthoreovirus (prv) of the genus orthoreovirus (family: reoviridae) have been reported in southeast asia, where it was isolated from humans and bats. it is possible that prv-associated respiratory infections might be prevalent in southeast asia. the clinical course of prv is not fully elucidated. methods: the virulence, pathology, and pathogenesis of two prv strains, a human-borne prv strain (isolated from a patient, who returned to japan from bali, indonesia in 2007) and a bat-borne prv (isolated from a bat [eonycteris spelaea] in the philippines in 2013) were investigated in balb/c mice using virological, pathological, and immunological study methods. results: the intranasal inoculation of balb/c mice with human-borne prv caused respiratory infection. in addition, all mice with immunity induced by pre-inoculation with a non-lethal dose of prv were completely protected against lethal prv infection. mice treated with antiserum with neutralizing antibody activity after inoculation with a lethal dose of prv showed a reduced fatality rate. in this mouse model, bat-borne prv caused respiratory infection similar to human-borne prv. prv caused lethal respiratory disease in an animal model of prv infection, in which balb/c mice were used. conclusions: the balb/c mouse model might help to accelerate research on the virulence of prv and be useful for evaluating the efficacy of therapeutic agents and vaccines for the treatment and prevention of prv infection. prv was shown for the first time to be a causative virus of respiratory disease on the basis of koch’s postulations by the additional demonstration that prv caused respiratory disease in mice through their intranasal inoculation with prv. pteropine orthoreovirus (prv), a member of genus orthoreovirus in the family reoviridae, was originally isolated from the heart blood of a grey-headed flying fox (pteropus poliocephalus) in australia in 1968 [1] . prv was isolated from a patient with respiratory tract infection (rti) as a causative agent in malaysia in 2006 [2] . seven patients with symptoms of influenza-like illness, such as fever, cough, and sore throat, caused by prv were reported between 2006 and 2017 [2] [3] [4] [5] [6] [7] [8] . three patients with prv infection were reported in malaysia in 2006 and 2010 [2, 3, 5, 8] . four cases of prv infection imported from indonesia to japan and hong kong were identified in 2007, 2009, and 2010 [4, 6-8] . the presence of these rti cases in southeast asia suggests that prv might be the causative viral pathogen of rti. some patients with prv also showed the symptoms of abdominal pain, watery diarrhea, and vomiting [3] [4] [5] 8] . antibodies to prv were detected in 13% of the residents of tioman island, malaysia [2] , and 4.4% of patients with nonspecific symptoms in central vietnam [9] . furthermore, prv genomes were detected in 17% of patients with rtis in negeri sembilan state, malaysia [10] . these reports raise the concern that the prevalence of human prv infection in southeast asia might be higher than previously thought. however, the disease spectrum and the pathogenesis of prv infection in humans also remain unclear. fourteen strains of prv have been isolated from fruit bats (pteropus poliocephalus, p. hypomelanus, p. vampyrus, rousettus leschenaultia, eonycteris spelaea, and r. amplexicaudatus) in australia, malaysia, indonesia, pr china, and the philippines from 1968 to date [1, 8, [11] [12] [13] [14] [15] . the indonesia/2010 strain was isolated from the salivary swab of p. vampyrus imported from indonesia to italy in 2010 [14] . prv-neutralizing antibodies were also detected in 83% of fruit bat species (r. amplexicaudatus, e. spelaea, and macroglossus minimus) in the philippines, suggesting that prv is generally prevalent in some species of wild bats in southeast asia [15] . it is still not known whether prv causes illnesses in fruit bats [8] , whereas bat-borne prv is a potentially pathogenic to humans. therefore, it is important to characterize both humanborne and bat-borne prv. a prv strain isolated from a patient with rti was found to be lethal in c3h mice, but the virulence and pathology of this strain in mice were not investigated in detail [16] . in the present study, the virulence, pathology, and pathogenesis of prv in balb/c mice were elucidated to validate respiratory disease caused by prv and to develop an animal model of prv infection. two prv strains that were isolated in previous studies [7, 15] were used in this study. the prv strain miyazaki-bali/2007 (prv-mb) was isolated from a patient with prv infection, who returned to japan from bali, indonesia in 2007 [7, 17] . the prv strain samal-24 (prv-samal-24) was isolated from e. spelaea in the philippines in 2013 [15] . the nucleotide sequences of the 10 segments of each of these two prv strains are deposited in genbank (table 1) . prvs were propagated in human embryonic kidney-derived 293ft cells (thermo fisher scientific, inc.) for the preparation of the working virus solution. cells infected with each strain of prv were cultured at 37˚c in dulbecco's modified eagle's medium (dmem; sigma-aldrich co., llc) supplemented with 5% heat-inactivated fetal bovine serum (fbs) and 1% antibiotics (penicillin and streptomycin; pen-strep, thermo fisher scientific, inc.) (dmem-5fbs). after 2 days of culture, the medium was centrifuged at 800 × g for 5 min to remove cellular debris. the supernatant was overlaid onto 20% sucrose in a 50 ml tube (becton dickinson, ltd.) and centrifuged at 100,000 × g for 2 h to concentrate the virus. the concentrated viruses were dissolved with dmem with 2% fbs and 1% pen-strep (dmem-2fbs), and the aliquots were stored at -80˚c until use. the infectious dose of each virus was determined in a plaque assay in vero cell (atcc, ccl-81) monolayers as described previously [7] . the cells were inoculated with a serially diluted virus solution of prv-mb or prv-samal-24 and incubated for 1 h at 37˚c for adsorption. the cell monolayers were washed with phosphate buffered saline solution (pbs), and the cells were cultured with dmem-5fbs supplemented with 0.8% agarose for 2 days at 37˚c. plaque was visualized by staining the cells with neutral red solution. plaques were counted, and the virus titers were calculated in plaque-forming units per milliliter (pfu/ml). nine-week-old female balb/c mice (japan slc, inc.) were used. the mice used were healthy and weighed approximately 20 g. the mice, which were anesthetized with a combination of ketamine (100 mg/kg) and xylazine (4 mg/kg) in 0.9% sodium chloride solution, were inoculated with each strain of prv. five table 1 . genbank accession numbers for the nucleotide sequences of the 10 rna genome segments of the prv-mb and prv-samal-24 strains used in this study. virus genome segment mice per group were intranasally inoculated with 1.0 × 10 3 to 1.0 × 10 6 pfu of each prv strain in 20 μl dmem-2fbs. the clinical signs and body weight of the mice were monitored for 14 days, and the 50% lethal dose (ld 50 ) of prv (for mice) was calculated according to the method of reed and muench [18] . mice that were intranasally inoculated with 20 μl dmem-2fbs (vehicle) were used as the control. the changes in body weight and the survival rates were plotted using the graphpad prism software program (graphpad software, inc.) and were analyzed statistically by a one-way anova. quantitative detection of the prv genome in organs and blood five mice were intranasally inoculated with 1.0 × 10 5 pfu of the prv-mb or prv-samal-24 strain as described above. the mice were sacrificed on the 5th or 6th day post-infection (dpi), and then blood and the organs (the head including the brain and nasal cavity, trachea, lung, liver, kidney, spleen, and intestine) were collected. the viral rna load in each organ and blood was determined by a quantitative real-time rt-pcr (qrt-pcr) as described below. determination of viral rna load with a quantitative real-time rt-pcr blood samples were collected from the mice (5 per group) infected with each strain of prv by cardiac puncture after euthanasia. each of the blood samples was mixed with isogen ls (wako pure chemical industries, ltd.), and total rna was extracted from each blood sample according to the manufacturer's instructions. the organs and tissues; the brain, nasal cavity, trachea, lung, heart, liver, spleen, kidney, and intestine were collected. these samples were immediately submerged in rnalater (ambion, life technologies, inc.) and stored at -80˚c until use. total rna was extracted using isogen (wako pure chemical industries, ltd.) according to the manufacturer's instructions. the viral copy numbers were determined with a qrt-pcr as follows. the forward and reverse primers and probe were specifically designed according to the nucleotide sequence of the outer-capsid protein (ocp) region in the s4 segment of prv-mb or to that of prv-samal-24. the sequences of the forward primer, the reverse primer, and the probe for the amplification of the prv-mb genome were 5'-cattgtcactccgatcatgg-3', 5'-tgggagtgtgcagagcatag-3' (eurofins genomics, inc.), and fam/5'-gtaggtatg ccactcgtggaatcc-3'/tamra (sigma-aldrich co. llc.), respectively. the sequences of the forward primer, the reverse primer, and the probe for the amplification of the prv-samal-24 genome were 5'-caatttccactcgttcgttg-3', 5'-gatggtgtggaaacgg atac -3' (eurofins genomics, inc.), and fam/5'-gaccagaccagatacgtggaatcc -3'/tamra (sigma-aldrich co. llc.), respectively. the qrt-pcrs were performed using a light cycler 96 system (roche diagnostics, ltd.) with a quantitect probe rt-pcr kit (qiagen, ltd.). the light cycler experimental protocol was as follows: reverse transcription (50˚c for 30 min), denaturation (95˚c for 15 min), and 45 cycles of amplification and quantification (94˚c for 15 s and 60˚c for 60 s), followed by a final cooling step at 40˚c for 30 s. in this study, the standard controls for prv-mb and prv-samal-24 were 10-fold serial dilutions of the plasmid dna containing the s4 segments of prv-mb and prv-samal-24, respectively. the viral copy numbers in the samples were calculated as the ratio of the copy numbers of each standard control. the viral copy numbers were plotted using the graphpad prism software program, and the results were statistically analyzed by a one-way anova. the viral rna detection limits in the blood, trachea, and other tissues were determined to be 2.5 × 10 3 copies/ml, 1.6 × 10 3 -5.0 × 10 3 copies/0.1 g, and 2.5 × 10 3 copies/0.1 g, respectively. one pfu was equivalent to 2.9 copies of viral rna. two mice were intranasally inoculated with 1.0 × 10 6 pfu of the prv-mb strain or of prv-samal-24 strain as described above. the mice were sacrificed on the 4th dpi, and organs (the head including the brain and the nasal cavity, trachea, lung, liver, kidney, and intestine) of the mice were collected. the infectious virus titer in each organ was determined with a plaque assay as described below. each organ collected was immediately submerged in dmem-2fbs, homogenized and centrifuged at 800 × g for 5 min to remove debris. the supernatant fraction was collected and stored at -80˚c until use. the virus titer in the supernatant fraction was determined in a plaque assay in vero cell monolayers as described previously [7] . the virus titers were plotted using the graphpad prism software program. the virus titer detection limit was determined to be 2.4 × 10 1 pfu/0.1 g. the mice intranasally infected with 1.0 × 10 3 pfu of prv-mb (prv-mb-1.0×10 3 pfu mice) and those intranasally infected with 1.0 × 10 5 pfu of prv-mb (prv-mb-1.0×10 5 pfu mice) were sacrificed by exposure to excess isoflurane, and the lungs were collected on the 1st, 3rd, and 5th dpi (5 mice per group each day). the viral rna loads in the lungs were determined with qrt-pcr. pathological analyses of the lungs were performed by immunohistochemical (ihc) analysis as described below. the collected tissues were stained with hematoxylin and eosin (h&e) for histopathology. an ihc analysis was performed for the detection of prv antigen in the tissues. the ihc analysis methods were the same as those described previously except for the antigen detection antibody [19] . the sections were deparaffinized by placing them in a retrieval solution (ph 6) (nichirei biosciences, inc.), followed by heat-treatment with an autoclave at 121˚c for 10 min. the polyclonal antibody to the ocp (s4 segment) of prv-mb raised in a rabbit by immunization with the antigen (ocp antibody) was used for the ihc detection of prv antigen [20] . the ocp antibody used in the ihc analysis reacted specifically with ocp antigens of prv [20] . to validate whether the ocp antibody reacts with the mouse lungs non-specifically, the lung tissues of 6-month-old balb/c mice infected with severe acute respiratory syndrome coronavirus (sars-mouse-lung), in which severe inflammation was shown, and those of the mice inoculated with mock solution (mock-mouse-lung) were tested by ihc analysis [21] . the samples showed a negative reaction in the ihc analysis (s1 fig), indicating that the ocp antibody does not react non-specifically with the mouse lungs with inflammation and that the positive signals detected in the ihc analysis indicate the presence of the ocp of prv. as the negative control, normal rabbit serum (nrs; dako, ltd.) was used in ihc analysis. after treatment, the sections were reacted with the ocp antibody or nrs and then washed with pbs. the sections were incubated with nichirei-histofine simple stain mouse max po (r) (nichirei biosciences, inc.) according to the manufacturer's instructions. the peroxidase activity was detected with 3, 3'-diaminobenzidine (sigma-aldrich co. llc.), and the sections were counterstained with hematoxylin. five mice were intranasally inoculated with either dmem-2fbs containing 1.0 × 10 3 pfu (non-lethal dose) of prv-mb or dmem-2fbs (control). serum was separated from the blood collected through the caudal vein on the 27th dpi by centrifugation. the serum was tested for the prv-mb neutralizing antibody titers as described previously [9, 15] . in addition, mice that were pre-inoculated with prv-mb or control were re-inoculated with 1.0 × 10 5 pfu (lethal dose) of prv-mb on the 35th day after the first inoculation with prv-mb or control. the clinical signs and body weight were monitored for 14 days. the mice that showed >25% initial body weight loss were euthanized. their body weight changes and survival rates were plotted using the graphpad prism software program. twenty-five mice were intranasally inoculated with 1.0 × 10 3 pfu (non-lethal dose) of prv-mb followed by a second intranasal inoculation with 1.0 × 10 5 pfu of prv-mb 3 weeks after the first infection. the mice were then intranasally inoculated once more with 1.0 × 10 5 pfu of prv-mb 3 weeks after the second infection. on the 5th day after the third inoculation, the mice were sacrificed and blood was collected by cardiac puncture. serum was separated by centrifugation. mouse serum, which was collected from the 25 control mice without inoculation with prv-mb, was used as the control serum. the serum was diluted 4-fold with pbs. five mice per group were intranasally infected with 1.0 × 10 5 pfu of prv-mb, and then the diluent of the serum (100 μl) was administered once daily until the mice showed >25% initial body weight loss for a maximum of 5 days. serum was administered at just after 1 h after inoculation, or on the 1st, 2nd, 3rd, and 4th dpi. the diluent of the control serum (100 μl) was used for mock treatment. the mice that showed >25% initial body weight loss were euthanized. the body weight changes and survival rates were plotted using the graphpad prism software program. the animal studies were carried out in strict accordance with the guidelines for proper conduct of animal experiments of the science council of japan and in strict compliance with animal husbandry and welfare regulations. all animal experiments were approved by the committee on experimental animals at the national institute of infectious diseases (niid) in japan (approval nos. 215016, 116086, and 116082). all of the animals infected with prv were handled in biosafety level 3 animal facilities, in accordance with the guidelines of the niid. the mice were inoculated with virus solution under proper anesthesia, and all efforts were made to minimize any potential pain and distress. after inoculation, the animals were monitored once a day during the study period. a humane endpoint was introduced for all mice with >25% initial body weight loss. the prv-mb-1.0×10 5 pfu mice or the prv-mb-1.0×10 6 pfu mice developed symptoms (piloerection, slowness in movement, anorexia, and weight loss) from the 2nd dpi. all of the mice died by the 6th dpi (fig 1) . the severity of the symptoms in the prv-mb-1.0×10 4 pfu mice was less than that in the prv-mb-1.0×10 5 pfu mice or the prv-mb-1.0×10 6 pfu mice, and 3 of the 5 mice died by the 8th dpi. the extent of body weight loss in the prv-mb-1.0×10 3 pfu mice was greater than that in the control mice. the prv-mb-1.0×10 3 pfu mice did not show any symptoms other than body weight loss. the ld 50 of prv-mb in the balb/c mice was determined to be 6.8 × 10 3 pfu/head. the level of viral rna in the lungs (average level, 6.9 × 10 8 copies/0.1 g) was higher than those in the other organs (fig 2a, left panel) . viral rna was detected in the blood (maximum level of 7.5 × 10 6 copies/ml) (fig 2a, right panel) . in contrast, viral rna was not detected in the brain, heart, liver, spleen, kidney, and intestine. the infectious virus was detected mainly in respiratory organs (fig 2b) . the titer in the lungs (average virus titer, 6.4 × 10 4 pfu/0.1 g) was the highest among the organs tested. a pathological examination revealed tissue damage and inflammation (i.e., necrosis and the accumulation of inflammatory cells including lymphocytes) in the lower respiratory tract, including the bronchiole and alveoli, in which viral antigens were detected in ihc analysis by using the ocp antibody, on the 4th dpi (fig 3a, left and middle panels) . neutrophils and type ii pneumocytes infiltrated to the alveoli and alveolar walls, and tissue damage in the lungs was detected (fig 3b) . the prv antigen-positive lesions revealed in the ihc analysis by using the ocp antibody showed negative reaction in the ihc analysis using nrs (fig 3a, right panels) . no pathological changes or viral antigens were detected in the other tissues examined. the viral rna in the lungs of the prv-mb-1.0×10 3 pfu mice or the prv-mb-1.0×10 5 pfu mice was determined throughout the course of infection (fig 4) . on the 1st dpi, the viral rna load in the lungs of the prv-mb-1.0×10 5 pfu mice was similar to that of the prv-mb-1.0×10 3 pfu mice. in contrast, on the 3rd and 5th dpi, the viral rna load in the lungs of the prv-mb-1.0×10 5 pfu mice was significantly higher in comparison to the prv-mb-1.0×10 3 pfu mice. the presence of viral antigens in the lungs of the prv-mb-1.0×10 3 pfu mice and the prv-mb-1.0×10 5 pfu mice was investigated immunohistochemically on the 1st, 3rd, and 5th dpi. viral antigens were detected in the bronchial epithelium of the prv-mb-1.0×10 5 pfu mice on the 1st dpi (fig 5b, upper panel) , in the alveolar duct, alveoli, and bronchial epithelium on the 3rd dpi, and in the alveolar area on the 5th dpi (fig 5b, middle and lower panels) . cellular damage characterized by positive nuclear aggregation, cellular atrophy, and cellular debris was detected in the terminal bronchioles, which was also positive for prv-mb antigen (fig 6) . prv-mb caused extensive and massive pulmonary infection in the prv-mb-1.0×10 5 pfu mice. in contrast, few viral antigens were detected in the bronchial epithelium of the prv-mb-1.0×10 3 pfu mice on the 1st and 3rd dpi (fig 5a, upper and middle panels, respectively), and no viral antigens were detected in the bronchial epithelium or alveoli on the 5th dpi ( fig 5a, lower panel) . the serum neutralizing antibody titers induced in the mice inoculated with 1.0 × 10 3 pfu (non-lethal dose) of prv-mb on the 27th dpi were between 640 and 2560. the mice were then challenged with 1.0 × 10 5 pfu of prv-mb on the 35th day after the first inoculation with a non-lethal dose of prv-mb. all of these mice survived, whereas all of the control mice died by the 6th dpi (fig 7) . a protective effect: the survival rate of the anti-serum-treated mice was 60%, whereas all of the control mice died (fig 8) . when the antiserum treatment was initiated on the 1st or 2nd dpi, taking the day on which the mice were infected with prv-mb as day 0, 40% of the mice survived, whereas the control mice and the mice in which the treatment was initiated on the 3rd dpi or later died by the 6th dpi. the body weight reduction in these groups was similar to that of the control group. nine-week-old balb/c mice were infected with a graded dosage (1.0 × 10 3 -1.0 × 10 6 pfu) of prv-samal-24. the intranasal inoculation of the mice with prv-samal-24 led to fatal outcomes. the ld 50 of prv-samal-24 for balb/c mice was determined to be 4.2 × 10 3 pfu/ head. among the respiratory organs, viral rna was detected in the lungs of the mice infected with prv-samal-24; the viral rna copy numbers in the lungs were up to 3.7 × 10 8 copies/0.1 g on average (fig 9a, left panel) . viral rna was also detected in the blood (maximum level, 1.8 × 10 6 copies/ml) (fig 9a, right panel) . in addition, the infectious virus was isolated from the respiratory tract organs (fig 9b) . the infectious dose of prv-samal-24 was the highest in the lungs among the tissues tested with the dose being up to 9.5 × 10 3 pfu/0.1 g on average. pathological examination of the prv-samal-24-1.0×10 6 pfu mice revealed inflammatory lesions in the lungs (by h&e staining), and viral antigens were also detected, especially from the bronchioles to the alveoli (by ihc staining) as was observed in the mice infected with prv-mb (s2 fig). the present study showed through virological and pathological examinations that the lung was the principle target organ of prv replication after intranasal inoculation in balb/c mice. prv mainly replicated in the bronchiolar epithelium by the 3rd dpi. the bronchiolar epithelium is composed of ciliated and nonciliated cells, such as clara cells and goblet cells, which are classified as secretory cells [22] . morphologically, the prv antigen-positive cells were likely to be clara cells and goblet cells. prv infection caused severe inflammation in the lungs of the mice on the 4th dpi (acute phase). morphologically, prv mainly replicated in the pneumocyte-like cells, and the prv antigen-positive cells were likely to be type i pneumocytes, which are involved in the process of gas exchange between the alveoli and blood [23] . mammalian orthoreovirus, which is classified to the genus orthoreovirus in the family reoviridae, was reported to replicate in type i pneumocytes and was shown to induce severe pneumonia in some rodent species, including mice and rats [24, 25] . type i pneumocytes might be a critical replication site for prv. it was assumed that fatal outcomes were induced in mice infected with a lethal dose of prv due to a decrease in respiratory function that occurred as a result of the destruction of the bronchiolar epithelial cells and pneumocytes. in this study, the cell types, in which prv replicated, were identified only by morphological observation. further studies are needed to elucidate the primary target cells, which are infected with prv and in which prv replicates. the high-titer prv genome and infectious prv were detected in the lungs of the mice infected with a lethal dose of prv on the 4th to 6th dpi (acute phase) (figs 2 and 9 ). balb/c mice were susceptible to prv and developed rtis, similarly to humans. demonstration of infectious prv in lungs indicates that prv definitely replicated there (figs 2b and 9b). koch's postulates (i.e., isolation of prv from patients with rtis, induction of rti in mice by infection with prv, and detection of infectious prv in respiratory organs of mice infected with prv) support a causal role of prv infection in the development of respiratory tract diseases in humans [26] . although all of the cases of prv infection in humans showed symptoms associated with rti, it is evident that the clinical characteristics of prv infections in humans have not been fully elucidated. it is possible that prv causes more severe infections than have previously been reported. we evaluated the utility of the newly developed mouse model of prv infection. immunity to prv was induced by non-lethal infection, and it protected the mice from lethal infection with prv (fig 7) . the early initiation of antiserum treatment was effective in the treatment of lethal prv infection (fig 8) . these results suggest that balb/c mice may serve as a useful animal model for evaluating the efficacy of vaccines and therapeutic agents for prv. the pathogenicity of prv-samal-24 was evaluated in this mouse model. similarly to prv-mb, prv-samal-24 caused viremia and respiratory disease in the balb/c mice. the amino acid identities (encoded by each gene segment) between prv-samal-24 and prv-mb were as follows: cell attachment protein region of the s1 segment, 82%; p10 region of the s1 the organs and the blood samples were obtained from the prv-samal-24-1.0×10 5 pfu mice on the 5th dpi (5 mice segment, 100%; p17 region of the s1 segment, 94%; inner-capsid protein region of the s2 segment, 97%; sigma ns region of the s3 segment, 97%; ocp region of the s4 segment, 97%; minor inner-capsid protein region of the m1 segment, 94%; major outer-capsid protein region of the m2 segment, 95%, mu ns region of the m3 segment, 91%, guanylyltransferase region of the l1 segment, 94%, rna polymerase region of the l2 segment, 98%, and major inner-capsid protein region of the l3 segment, 98%. prv-samal-24 and prv-mb were also reported to show cross-reactivity in an immunofluorescence assay [15] . as both the in vivo and in vitro characteristics of prv-samal-24 are similar to prv-mb, it is possible for bat-borne prv to cause illness in humans. in conclusion, a balb/c mouse model of prv infection, in which prv caused acute rti, was developed. immunocompetent balb/c mice were sensitive to prv, when the mice were infected with prv intranasally. this model might be useful for analyzing the pathogenicity of prv in mice and for evaluating the efficacy of vaccines and therapeutic agents that will be developed to prevent and treat prv infection. this model is also useful for further studies on prv infections in vivo. the sars-mouse-lung (left panels) and mock-mouse-lung (right panels) [21] were examined by ihc with using the ocp antibody. the ihc signals in the lung at 100× magnification (upper panels), the bronchiole at 200× magnification (middle panels), and the alveolus at 1000× magnification (lower panels) are shown. no signal, which indicates non-specific reaction of the ocp antibody, was detected in the sars-mouse-lung, in which severe inflammation was found on h&e staining [21] , and mock-mouse-tissue. the scale bars in upper and middle panels indicate 200 μm, whereas those in lower panels indicate 20 μm. the lungs were obtained from prv-samal-24-1.0×10 6 pfu mice on the 4th dpi. h&e staining (left panels) and ihc with an ocp antibody (right panels) were performed. the h&e staining and ihc with an ocp antibody of the lung at 100× magnification (upper panels) and of a bronchiole and an alveolus at 400× magnification (lower panels) are shown. the black boxes in the upper panels were shown at higher magnification in the lower panels. the black arrows in the lower panels indicate the bronchiolar epithelial cell necrosis, which was positive for prv-samal-24 antigen. the red arrows in the lower-right panel indicate the prv-samal-24 antigen-positive pneumocytes. the scale bars in the upper panels indicate 500 μm, whereas those in the lower panels indicate 100 μm. (tif) nelson bay virus. a novel reovirus a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans identification and characterization of a new orthoreovirus from patients with acute respiratory infections a novel reovirus isolated from a patient with acute respiratory disease investigation of a potential zoonotic transmission of orthoreovirus associated with acute influenza-like illness in an adult patient virulence potential of fusogenic orthoreoviruses imported case of acute respiratory tract infection associated with a member of species nelson bay orthoreovirus pteropine orthoreovirus: an important emerging virus causing infectious disease in the tropics serological evidence of human infection with pteropine orthoreovirus in central vietnam pteropine orthoreovirus infection among out-patients with acute upper respiratory tract infection in malaysia pulau virus; a new member of the nelson bay orthoreovirus species isolated from fruit bats in malaysia xi river virus, a new bat reovirus isolated in southern china characterization of a novel orthoreovirus isolated from fruit bat a new member of the pteropine orthoreovirus species isolated from fruit bats imported to italy. infection first isolation and characterization of pteropine orthoreoviruses in fruit bats in the philippines. archives of virology reverse genetics for fusogenic bat-borne orthoreovirus associated with acute respiratory tract infections in humans: role of outer capsid protein sigmac in viral replication and pathogenesis rapid whole genome sequencing of miyazaki-bali/2007 pteropine orthoreovirus by modified rolling circular amplification with adaptor ligation-next generation sequencing determination of 50% endpoint titer using a simple formula neuropathogenicity of two saffold virus type 3 isolates in mouse models serologic assays for the detection and strain identification of pteropine orthoreovirus mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice comparison of nonciliated tracheal epithelial cells in six mammalian species: ultrastructure and population densities knowns and unknowns of the alveolus. proceedings of the respiratory infection of mice with mammalian reoviruses causes systemic infection with age and strain dependent pneumonia and encephalitis reovirus infection in rat lungs as a model to study the pathogenesis of viral pneumonia 100th anniversary of robert koch's nobel prize for the discovery of the tubercle bacillus we thank dr. singh harpal and dr. aiko fukuma (department of virology 1, niid), and ayako harashima and yuko sato (department of pathology, niid) for their technical per group). four prv-samal-24-1.0×10 5 pfu mice were sacrificed on the 5th dpi due to their reaching the humane endpoint. another mouse was found dead on the 5th dpi; therefore, a blood sample could not be obtained. therefore, the viral rna loads in each organ and those in the blood were determined for 5 and 4 mice, respectively. the left and right panels indicate the viral rna in each organ and the blood of the prv-samal-24-1.0×10 5 pfu mice, respectively. (b) the organs were obtained from the prv-samal-24-1.0×10 6 pfu mice on the 4th dpi (2 mice per group). udl, under detection limit.https://doi.org/10.1371/journal.pntd.0006076.g009development of an animal infection model for prv key: cord-281456-3dlsbr7c authors: al-alimi, abdullah ahmed; ali, syed a.; al-hassan, faisal muti; idris, fauziah mohd; teow, sin-yeang; mohd yusoff, narazah title: dengue virus type 2 (denv2)-induced oxidative responses in monocytes from glucose-6-phosphate dehydrogenase (g6pd)-deficient and g6pd normal subjects date: 2014-03-13 journal: plos negl trop dis doi: 10.1371/journal.pntd.0002711 sha: doc_id: 281456 cord_uid: 3dlsbr7c background: dengue virus is endemic in peninsular malaysia. the clinical manifestations vary depending on the incubation period of the virus as well as the immunity of the patients. glucose-6-phosphate dehydrogenase (g6pd) deficiency is prevalent in malaysia where the incidence is 3.2%. it has been noted that some g6pd-deficient individuals suffer from more severe clinical presentation of dengue infection. in this study, we aim to investigate the oxidative responses of denv2-infected monocytes from g6pd-deficient individuals. methodology: monocytes from g6pd-deficient individuals were infected with denv2 and infection rate, levels of oxidative species, nitric oxide (no), superoxide anions (o(2) (−)), and oxidative stress were determined and compared with normal controls. principal findings: monocytes from g6pd-deficient individuals exhibited significantly higher infection rates compared to normal controls. in an effort to explain the reason for this enhanced susceptibility, we investigated the production of no and o(2) (−) in the monocytes of individuals with g6pd deficiency compared with normal controls. we found that levels of no and o(2) (−) were significantly lower in the denv-infected monocytes from g6pd-deficient individuals compared with normal controls. furthermore, the overall oxidative stress in denv-infected monocytes from g6pd-deficient individuals was significantly higher when compared to normal controls. correlation studies between denv-infected cells and oxidative state of monocytes further confirmed these findings. conclusions/significance: altered redox state of denv-infected monocytes from g6pd-deficient individuals appears to augment viral replication in these cells. denv-infected g6pd-deficient individuals may contain higher viral titers, which may be significant in enhanced virus transmission. furthermore, granulocyte dysfunction and higher viral loads in g6pd-deificient individuals may result in severe form of dengue infection. dengue infection is among the leading causes of morbidity and mortality in the tropics and subtropics where as many as 100 million people are infected with 22,000 deaths yearly [1] . dengue infection is caused by dengue virus (denv), an rna virus of the family flaviviridae. there are four serotypes of the virus which are referred to as denv1, denv2, denv3 and denv4. all four serotypes can cause the full spectrum of disease [2] . denv is primarily transmitted to humans by the bite of infected aedes mosquitoes, particularly aedes aegypti. other aedes species that transmit the disease include a. albopictus, a. polynesiensis, and a. scutellaris [3] . although uncommon, denv can also be transmitted via infected blood products and through organ transplantation [4, 5] . a large percentage (,80%) of people infected with denv show only mild symptoms such as fever. on the other hand, some patients experience more severe illness such as dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss), which can be life threatening [6] . several factors have been associated with the development of severe dengue including prior infection with a heterotypic serotype, the strain of infecting virus, age and gender, and the genetic background of the patient. denv-related illness is more common in children, and in contrast to other infections, well-nourished children are more predisposed to severe illness [7] . polymorphism in genes coding for tumor necrosis factor alpha (tnfa), mannan-binding lectin 2 (mbl2), cytotoxic t-lymphocyte antigen 4 (ctla4), transforming growth factor b (tgfb), dc-sign, and human leukocyte antigen (hla) class i and ii alleles have been linked with an increased risk of severe dengue complications [8] . yet another genetic abnormality reported to have a link with dhf/dss is the deficiency of glucose-6-phosphate dehydrogenase (g6pd), a ubiquitous xlinked enzyme which is part of the innate defense mechanisms [9] . g6pd deficiency is the most common enzymopathy worldwide, with highest prevalence among sub-saharan african countries [10] , [11] . high frequencies (6.0-10.8%) of g6pd deficiency have also been reported for southeast asian countries [12] . g6pd deficiency affects the production of reactive nitrogen (rns) and oxygen species (ros) such as nitric oxide (no), superoxide (o 2 2 ), and hydrogen peroxide (h 2 o 2 ) resulting in alterations of normal redox state of the cells [13] . cells of immune system employ rns/ ros to kill invading pathogens. a reduction of the redox state of immune cells may render immune cells less effective against invading organisms, resulting in an increased severity of the infection [14] . it is found that monocytes from g6pd-deficient patients show an increased susceptibility to denv2 infection with higher replication ability than those from normal controls [9] . although there appears to be a connection between g6pd deficiency and enhanced denv replication [15] , no studies have been carried out to elucidate the molecular mechanism behind this observation. in the present study, we aim to find out whether it is the altered redox state of monocytes from g6pd-deficient individuals, which is responsible for the increased susceptibility of monocytes to enhanced denv replication. this study was approved by the research and ethics committee of clinical research center (crc), ministry of health and research, and ethics committee, universiti sains malaysia. the study was carried out between january 2010 and december 2011. written informed consent was obtained from all the blood donors who agreed to participate in the study. standard inclusion criterion for blood donors was followed and only male donors were included. additionally, donors with prior history of denv infection were excluded. after blood was donated, a portion (2.5 ml) of blood from the tubing of the blood bag was taken. the blood was screened for g6pd deficiency using ultraviolet test (cat #: sqmmr 500, r&d system, athena-greece) and g6pd activity was assayed using g6pd kit (cat #: pd2616, randox laboratory antrim, uk) following manufacturers' protocols. donors without g6pd deficiency on screening were selected as controls. the sera of g6pd-deficient and g6pd-normal donors were screened for denv-reactive antibodies using igg/igm capture elisa kits (igm cat #: e-den01m and igg cat #: e-den02g, panbio, brisbane, australia) according to the manufacturer's protocol. the c6/36 mosquito a. albopictus cell line (crl-1660 atcc, usa) was used to propagate denv2 (d2my00-22563), kindly provided by prof shamala of university of malaya, in leibovitz culture medium (l-15) (sigma, usa) supplemented with 1% lglutamine (sigma, usa), 19% tryptose phosphate broth (hi media, india), 1% of penicillin/streptomycin (gibco, grand island, usa) and 5% fetal bovine serum fbs (gibco, grand island, usa). denv2 in conditioned medium was titrated using a plaque assay on vero cells (ccl-81 atcc, usa) essentially as described previously [16] and stored at 280uc in aliquots. peripheral blood mononuclear cells (pbmcs) were isolated from both g6pd-deficient and normal controls' blood by density gradient centrifugation using lymphoprep medium (cat #: n07-1114547 axis shield, norway) following manufacturer's protocol. pbmcs were then used to isolate primary monocytes using a macs kit system ii (cat #: 130-091-153, miltenyibiotec gmbh, gladbach, germany). the purified monocytes were enumerated and cell viability was determined by trypan blue exclusion assay. the cells were then seeded in 24-well plates (costar, corning, usa) at 2610 5 cells/well and maintained in humidified incubator at 37uc in the presence of 5% co 2 . monocytes from g6pd-deficient and normal controls were infected with denv2 at multiplicity of infection (moi) 0.1 for 3 hours at 37uc/5% co 2 as described previously [9] . for optimal virus contact to the monocytes, the plates were gently agitated every 15 min. after 3 hours of incubation, the cells were washed twice with serum-free medium, re-suspended in complete growth medium and cultured at 37uc/5% of co 2 for five days. mockinfected monocyte cultures were set simultaneously as negative controls. conditioned media were harvested at various time points (24, 48, 72 , 96, 120 hours) post-infection and the number of infected cells and virus titers were determined using flow an estimated 50 to 100 million cases of dengue fever occur each year worldwide. among these, there are 200,000 to 500,000 cases of life-threatening dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss). factors contributing to the development of dhf/dss are not yet fully identified. glucose-6-phosphate dehydrogenase (g6pd) deficiency is prevalent in southeast asian countries where dengue is also endemic. besides affecting normal function of erythrocytes, g6pd deficiency also affects other cells by causing abnormal cellular redox. altered redox state of cells may render them less effective in clearing up microbial and viral infections. here we confirm previous findings that monocytes from g6pd-deficinet individuals support better dengue virus replication. in addition, we show that reduced production of reactive oxygen, and nitrogen species and elevated levels of oxidative stress are responsible for the enhanced viral replication. we suggest that redox imbalance observed in infected monocytes from g6pd-deficient individuals may facilitate dengue transmission and affect clinical outcome. however, a handful of studies carried out in areas where both g6pd deficiency and dengue are endemic, reveal no statistically significant correlation between severity of dengue and g6pd deficiency. well-designed studies are needed to demonstrate that g6pd-deficient individuals are at risk of severe dengue. cytometry and plaque assay respectively. virus-containing conditioned media were stored in aliquots at 280uc. infected and mock-infected monocytes were harvested and washed twice with cold phosphate buffered saline (pbs) containing 0.1% nan 3 (sigma-usa). surface labeling was performed by incubating the cells with 100 ml (1:10 diluted) of cd14-pe mab (cat#: 347497 becton dickinson, san jose, ca, united states) for 30 min on ice in dark. cells were fixed and permeabilized in 200 ml cytofix/cytoperm solution (bd biosciences, san diego, ca) for 15 min at room temperature followed by 26washing in cytoperm/ cytowash solution. cells were stained with 100 ml (1:20 diluted) of denv2 e protein-specific monoclonal antibody (abcam # ab41349) and incubated for 1 hour on ice in dark and washed twice with cytoperm/cytowash solution. secondary fitc-labeled goat anti-mouse-igg (cat #: 349031, becton dickinson, biosciences usa) antibody was added to a final concentration of 3.5 mg/ ml and incubated for 30 minutes on ice in dark. finally, the cells were washed twice in cytoperm/cytowash solution and resuspended in 0.5 ml of 1% paraformaldehyde containing 0.1% nan 3 and subjected to flow cytometry. mock-infected monocytes were used as negative controls and were treated and run simultaneously with the infected groups (g6pd-deficient and g6pd-normal monocytes) to improve gating, setup of compensation and the precision of measurement. a minimum of 10,000 events were acquired using a facs calibur ii flow cytometer (becton dickinson, biosciences, usa). data were analyzed using flowing software ver 1.0 (turku center for biotechnology, university of turku, finland http://www.flowingsoftware.com/). isotypematched antibody was used as a negative control. the percentage of denv2 positive cells was determined from fitc fluorescence histograms using a region that was defined based on the analysis of the mock-infected control cells. dengue virus particles were titrated by plaque assay as previously described by roehrig jt et al. [16] . briefly, vero cells were seeded in 12-well plates at a density of 2610 5 cells/well in complete dmem medium (without phenol red) and incubated overnight at 37uc/5% co 2 . serial 10-fold dilutions of viruscontaining supernatant in dmem/2% fbs were added to the wells and incubated at 37uc/5% co 2 for 90 minutes with gentle agitation every 15 minutes. the medium was carefully removed and the cells were overlaid with 1.0 ml of dmem/10% fbs containing 0.5% carboxymethylcellulose (cmc) (sigma -usa) and incubated at 37uc/5% co 2 for 7 days. cmc was removed by washing the wells twice with 2.0 ml pbs/well. cells were fixed with freshly prepared cold 4% paraformaldehyde and stained with 1% crystal violet (amresco, usa) in 20% ethanol for 30 minutes. the viral titers were expressed as plaque forming units (pfu)/ml = [(number of plaques per well)6(dilution)]/ (inoculums volume). nitric oxide (no) released into the conditioned media of denv2 and mock-infected monocytes was determined by measuring the stable nitrite using a greiss reagent assay kit (cat #: r&d system) according to the manufacturer's protocol. the absorbance was measured at 540 nm using bio mate 3 (thermo scientific, usa) spectrophotometer. all measurements were performed in duplicates. the intracellular superoxide anion and ros production were quantified in the denv2 infected and control cells using a total ros/superoxide detection kit (cat #: enz 51010, enzo life sciences inc., usa) following manufacturer's protocol. briefly, denv2 and control cells were harvested at 24, 48, 72, 96, and 120 hours and washed using ros buffer supplied with the kit. the positive, negative, denv2-infected and control cells were then incubated with 500 ml ros/superoxide detection reagents for 30 minutes at 37uc/5% of co 2 in rpmi-1640/10% fbs and subjected to flow cytometry analysis. data analysis and anticipated results were obtained by generating a log fl1 (x-axis) versus a log fl2 (y-axis) dot plot with quadrants added to it. monocytes with increased production of superoxide demonstrated a bright orange fluorescence and detected using the fl2 channel appeared in the two upper quadrants of a log fl1 (x-axis) versus a log fl2 (y-axis) dot plot. monocytes with high production of oxidative stress demonstrated a bright green fluorescence and registered in fl1 channel appeared in the upper and lower right quadrants of a log fl1 (x-axis) versus a log fl2 (y-axis) dot plots. results of experiments are presented as a percentage of the cells with increased superoxide and ros production or as an increase in the mean fluorescence of induced samples versus controls. results were reported as mean 6 sd. data were analyzed using spss software version 11.5. the p-value was calculated using student's t test. a p-value less than 0.05 were considered significant. error bars were expressed as means 6 sd. four hundred blood donors were screened for g6pd enzyme deficiency and 16 donors were found to be g6pd deficient by the ultraviolet test. out of 384 g6pd-normal donors, 16 were matched for age (33.1864.28 years for g6pd-normal and 34.9564.34 years for g6pd-deficient) and selected as normal controls (g6pd-normal donors). the g6pd deficient (n = 16) and age-matched g6pd-normal donors (n = 16) were then subjected to g6pd enzyme activity assay. g6pd-normal donors will be referred to as normal controls from hereafter. all 16 g6pd-deficient individuals had less than 10% of normal g6pd activity as measured by the fluorometric assay. the mean g6pd activity for g6pd-deficient individuals was 0.28560.26 iu/g hb, which was significantly (p,0.0001) lower than the mean activity of g6pd-age matched normal controls (13.5662.02 iu/g hb) as shown in fig. 1 . in g6pd-deficient monocyte cultures, increased denv2 infected cell rates were detected by flow cytometry until 48 hours post-infection, when peak values were reached. after this peak, the percentage of denv2 infected cells decreased (fig. 2a) . monocytes from 16 g6pd-deficient subjects showed mean virus infection percentage of 40.86867.330% at 48 hours post infection. in normal control monocytes culture, the frequency of denv2 infected cells increased until 72 hours post-infection, when peak values were reached followed by a decrease (fig. 2a) . monocytes from 16 normal controls showed mean virus infection percentage of 24.3%64.6% at 72 hours post infection. these results indicate that the mean percentage of denv2-infected monocytes from g6pd-deficient subjects exceeded the percentage of denv2-infected monocytes from normal controls. this difference of infected cells from two groups was statistically significant (p,0.0001). moreover, the peak infection was delayed by 24 hours in monocytes from normal controls compared to monocytes from g6pd-deficient subjects. this data was further verified by measuring the cell-free denv2 in the supernatant of infected monocyte cultures (fig. 2b) . in g6pd-deficient monocyte cultures, increased denv2 titers were detected by plaque assay until 48 hours post infection, when peak values were reached. after this time point, denv2 titers decreased (fig. 2b) . denv2 released from 16 g6pd-deficient monocyte cultures showed mean peak titers of 37610 2 pfu/ml on the 48 hours post infection. in normal control monocyte cultures, increased denv2 titers were detected until 72 hours post infection, when peak values were reached. after this time point, denv2 titers decreased (fig. 1b) . denv2 released from 16 normal control monocyte cultures showed mean peak titers of 22610 2 pfu/ml on the 72 hours post infection. these results indicate that significantly (p,0.0001) more denv2 was released by the infected monocytes from g6pddeficient subjects compared to infected monocytes from normal controls. the peak denv2 titers were detected at an earlier time point (48 hours post infection) in g6pd-deficient monocyte cultures compared to normal control monocyte cultures (72 hours post infection). as illustrated in fig. 3 , denv2 infection induced the production of no in both normal control and g6pd-deficient monocytes in a time dependent manner. in g6pd-deficient monocyte cultures, increased no produced until 48 hours post infection, when peak values were reached followed by a decrease (fig. 3) . no released from 16 g6pd-deficient monocyte cultures showed mean peak levels of 603.75 mm l 21 at 48 hours postinfection. on the other hand, in the normal control monocytes, increased no produced until 72 hours post-infection, when peak values were reached (fig. 3) . no released from the normal control monocytes showed mean peak levels of 1050 mm l 21 at 72 hours post-infection. the difference in no levels produced between g6pd-deficient and normal control monocytes was significant (p,0.0001) and the peak no levels were detected at an earlier time point (48 hours post infection) in g6pd-deficient monocyte cultures compared to normal control monocyte cultures (72 hours post infection). the lower levels of endogenous no production induced after the infection of g6pd-deficient monocytes correlate with the accelerated replication of denv2 in these cells. as shown in fig. 4 , denv2 infection of monocytes induced generation of superoxide anions (o 2.2 ) in both normal control and g6pd-deficient monocytes (p,0.001) in a time dependent manner. in g6pd-deficient monocyte cultures, increased o 2.2 levels were observed until 24 hours post infection, when peak values of 23.3% were reached (fig. 4) . on the other hand, in normal control monocyte culture, o 2 .2 levels increased until 48 hours post-infection, when peak value reached to 70.3% (fig. 4) . these results indicate that significantly (p,0.0001) less o 2 .2 was generated by the infected monocytes from g6pddeficient subjects compared to infected normal control monocytes. oxidative stress in monocytes of g6pd-deficient and normal controls as displayed in fig. 5 , denv2 infection of monocytes induced oxidative stress accumulation in both normal control and g6pddeficient monocytes (p,0.001) in a time dependent manner. in g6pd-deficient monocyte cultures, oxidative stress accumulation increased until 72 hours post infection, when peak levels of 84.4% were reached (fig. 5) . conversely, in normal control monocyte culture, oxidative stress accumulation increased until the 96 hours post-infection, when peak values of 63.2% were reached (fig. 5) . these results indicate that significantly (p,0.0001) more oxidative stress was accumulated by the infected monocytes from g6pddeficient subjects compared to infected monocytes from normal control. the peak oxidative stress accumulation was noticed at an earlier time point (72 hours post infection) in g6pd-deficient monocyte cultures compared to normal control monocyte cultures (96 hours post infection). a higher and earlier accumulation of oxidative stress in g6pd-deficient monocytes appears to be a result of greater viral replication in these cells compared to normal control monocytes. there was a significant strong, positive correlation (r = 0.702; p = 0.001) between the denv2-infected cells and no levels in monocyte from g6pd-deficient subjects (fig. 6a) , compared to monocytes from normal controls (fig. 6b) . there was a significant moderate, positive correlation (r = 0.476; p = 0.040) between the denv2-infected cells and o 2 .2 levels in monocyte from g6pddeficient subjects (fig. 6a) , compared to monocytes from normal controls (fig. 6b) . there was also a significant moderate, positive correlation (r = 0.368; p = 0.121) between the denv2-infected cells and o 2 .2 levels in monocyte from g6pd-deficient subjects (fig. 6a) , compared to monocytes from normal controls (fig. 6b) . these correlation studies further support the hypothesis that oxidative state of the cell may contribute to enhanced denv infection. denv causes infection which may range from mild to severe in affected patients. the severe form is associated with dhf and dss, which can be life threatening. the factors that define progression towards severe forms (dhf/dss) of dengue infection remain to be elucidated. one possible correlate though may be the innate g6pd deficiency, which is the most common enzymopathy worldwide. g6pd, through the pentose phosphate pathway (ppp), provides the reduced form of nadph for various cellular reactions including glutathione (gsh) recycling, superoxide anion production via nadph oxidase, nitric oxide (no) synthesis, and cholesterol synthesis [12] . inhibition of g6pd results in the generation of less reactive oxygen and nitrogen species (super-oxide anion, hydrogen peroxide, hydroxyl radical, and no) in the granulocytes and endothelial cells [17] , [18] . recurrence of microbial infections in g6pd deficient individuals has been reported [19] . the effect of cellular redox state on the course of viral infections is also well documented. for instance, replication of corona virus, coxsackie virus, rhinovirus, influenza, hiv, hepatitis, and enterovirus 71 virus is modulated by the redox milieu [20] , [21] , [22] , [23] , [24] , [25] . coxsackie viruses found replicate to a higher titer in c3h/jhe mice fed with diets deficient in selenium (se), vitamin e or both than in mice given a normal diet [26] . similarly, glutathione administration exhibited antiviral effect on influenza virus [22] . these findings suggest that redox imbalance may be conducive to enhanced replication and virulence of certain viruses. chao et al., [9] reported that monocytes from g6pd-deficient patients, using an ex vivo culture system, were more readily infected with the two denv2 strains-(1) the new guinea c strain from the df patient or (2) the 16681 strain from the dhf figure 5 . oxidative stress accumulation in denv-infected monocytes from g6pd-deficient and normal controls. the accumulation of oxidative stress in monocytes of both normal controls and g6pd-deficient donors increased significantly (p,0.001) after denv2 infection. in a timedependent manner, monocytes from g6pd-deficient subjects accumulated significantly (p,0.001) higher oxidative stress compared to monocytes from normal controls. doi:10.1371/journal.pntd.0002711.g005 figure 6 . correlation between denv2 replication and no, o 2.2 , and oxidative stress in monocytes from g6pd-deficient and normal controls. a significant moderate to strong correlation was found between the % infected monocytes and oxidative state (no, o 2.2 , and oxidative stress) for monocytes from g6pd-deificient individuals (a) compared to normal controls (b). doi:10.1371/journal.pntd.0002711.g006 patient than with those from normal controls. however, the underlying mechanism of this enhanced susceptibility was not investigated. here we confirm chao's findings and show in addition that reduced production of no and o 2 2 as well as earlier accumulation of oxidative stress contribute significantly to enhanced denv2 infectivity in monocytes from g6pd-deficient subjects. enhanced infection of g6pd-deficient monocytes by denv may be attributable to increased viral receptors on these cells or greater production of viral particles or a combination of both. we have not studied the up-regulation of viral receptors but did clearly see an enhanced production of viral particles in these cells. based on our findings and those reported by others, we propose a model for the association between the redox status of the host cells and denv. following entry into the cell, denv fuses with endosomal membrane to release its nucleocapsid into the cytoplasm where viral rna is replicated and translated into proteins. these processes may be affected by the cellular redox state, being more efficient in an oxidizing environment, thus resulting in enhanced virus production. findings of chao et al., [9] and the ones presented here suggest that the high competency for denv infection in monocytes of g6pd-deficient individuals may result in increased replication and higher virus yield. higher viral loads in g6pd-deficient individuals may increase the probability of dengue transmission to others via infected mosquitos. several reports have shown a correlation between severe dengue and high viral loads [27] , [28] , [29] , [30] , [31] . g6pd deficiency is reported to cause granulocyte dysfunction [18] , [32] . a combination of granulocyte dysfunction and enhanced replication of denv in monocytes of g6pd-deficient individuals may prevent the clearing of the primary infection thus predisposing infected individuals to severe form of disease. it remains to be seen whether g6pd deficiency-mediated enhanced viral replication has any outcome on severity of dengue infection in areas where both dengue infection and g6pd deficiency are endemic. in thailand, the prevalence of g6pd deficiency in the general population is approximately 14%. a cohort of 89 males diagnosed with dhf was studied to determine if g6pd deficiency was related to occurrence and/or course of dengue infection. out of 89, a total of 17 (19.1%) dhf patients had g6pd deficiency, thus no significant association established between g6pd deficiency and dhf [15] . however, this study is based on only a modest number of hospitalized patients and therefore provides little information on the incidence of severe dengue in g6pd deficient individuals. high prevalence of g6pd is also reported in african population [33] but a low incidence of severe dengue is noted in populations of african origin in a couple of studies conducted in cuba [34] and haiti [35] . since the outcome of these studies is inconclusive, well-designed studies are needed to demonstrate whether g6pd-deficient individuals are at risk of severe dengue with statistical significance. such studies would be potentially beneficial in providing added knowledge of host defense mechanism, and may be clinically important for g6pd-deficient individuals travelling to or living in denv endemic areas. world health organization impact of 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leucine 61 in glucose-6-phosphate dehydrogenase leads to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections the global prevalence of glucose-6-phosphate dehydrogenase deficiency: a systematic review and meta-analysis ethnicity and difference in dengue virus-specific memory t cell responses in cuban individuals race: a risk factor for dengue hemorrhagic fever we wish to thank the staffs of blood bank, penang hospital (hpp) and cluster of immunology, amdi for the assistance rendered. we also wish to thank professor francois villinger of yerkes national primate research centre, emory university, and professor vincent c bond of morehouse school of medicine, atlanta ga, for their critical review of this manuscript and constructive suggestions. conceived and designed the experiments: saa fmah nmy. performed the experiments: aaaa saa syt. analyzed the data: saa syt. contributed reagents/materials/analysis tools: fmi nmy. wrote the paper: aaaa saa. key: cord-305890-mdwjrfzp authors: bönsch, claudia; kempf, christoph; mueller, ivo; manning, laurens; laman, moses; davis, timothy m. e.; ros, carlos title: chloroquine and its derivatives exacerbate b19v-associated anemia by promoting viral replication date: 2010-04-27 journal: plos negl trop dis doi: 10.1371/journal.pntd.0000669 sha: doc_id: 305890 cord_uid: mdwjrfzp background: an unexpectedly high seroprevalence and pathogenic potential of human parvovirus b19 (b19v) have been observed in certain malaria-endemic countries in parallel with local use of chloroquine (cq) as first-line treatment for malaria. the aims of this study were to assess the effect of cq and other common antimalarial drugs on b19v infection in vitro and the possible epidemiological consequences for children from papua new guinea (png). methodology/principal findings: viral rna, dna and proteins were analyzed in different cell types following infection with b19v in the presence of a range of antimalarial drugs. relationships between b19v infection status, prior 4-aminoquinoline use and anemia were assessed in 200 png children <10 years of age participating in a case-control study of severe infections. in cq-treated cells, the synthesis of viral rna, dna and proteins was significantly higher and occurred earlier than in control cells. cq facilitates b19v infection by minimizing intracellular degradation of incoming particles. only amodiaquine amongst other antimalarial drugs had a similar effect. b19v igm seropositivity was more frequent in 111 children with severe anemia (hemoglobin <50 g/l) than in 89 healthy controls (15.3% vs 3.4%; p = 0.008). in children who were either b19v igm or pcr positive, 4-aminoquinoline use was associated with a significantly lower admission hemoglobin concentration. conclusions/significance: our data strongly suggest that 4-aminoquinoline drugs and their metabolites exacerbate b19v-associated anemia by promoting b19v replication. consideration should be given for choosing a non-4-aminoquinoline drug to partner artemisinin compounds in combination antimalarial therapy. human parvovirus b19 (b19v) is a nonenveloped icosahedral virus with a single-stranded dna genome which has been classified within the erythrovirus genus of the parvoviridae family. the virus is readily transmitted via the respiratory route and has a worldwide distribution. seroprevalence increases with age and 50%-80% of adults have detectable b19-specific antibody. since its discovery in 1975 [1] , b19v has been associated with an expanding range of clinical disorders that reflect the patient's immunologic and hematologic status. in healthy individuals, b19v typically causes a mild childhood febrile illness known as erythema infectiosum or fifth disease. more severe manifestations of b19v infection are arthropathies, aplastic anemia, hydrops fetalis and fetal death [2] . viremia occurs during the first week of infection. the virus has a predilection for bone marrow erythroid progenitor cells. at the height of viremia, there is an abrupt fall in the reticulocyte count and anemia can supervene. although this is rarely apparent in healthy patients, it can have serious clinical consequences where there is pre-existing anemia [2] . a case in point is malaria. b19v co-infection has been considered a significant risk for severe anemia in children living in malaria-endemic regions [3] . studies examining the inter-relationship between malaria, b19v infection and anemia have, however, produced inconsistent results. in a retrospective study from papua new guinea (png) [4] , 60% of children ,2 years of age and 90% of 6 year-olds were b19v seropositive. b19v infection was significantly associated with severe anemia even in the absence of risk factors including malaria. similar results were obtained in children from the republic of niger [5] . however, studies from malawi and kenya did not show evidence that b19v infection contributes to anemia in children and the seroprevalence of b19v was relatively low [6, 7] . at the time the studies were performed in png [4] and niger [5] , chloroquine (cq) was used as first-line treatment for malaria in these countries. in addition, a serosurvey performed in eritrea at a time when cq was the first-line agent also revealed an unusually high b19v seroprevalence [8] . by contrast, cq had been discontinued in malawi because of resistance of local strains of plasmodium falciparum and its use was declining in kenya when the b19v seroprevalence studies were conducted [6, 7] . although cq has, in addition to antimalarial efficacy, broad antiviral activity [9, 10] , it also enhances semliki forest virus (sfv) and encephalomyocarditis virus (emcv) infection in mice [11] , and epstein-barr virus (ebv) expression [12] . the latter effect is thought to play a role in the higher incidence of ebv-induced burkitt's lymphoma in malaria-endemic areas where cq is in common use [13] . we hypothesize, therefore, that geo-epidemiological differences in b19v seroprevalence and pathogenic potential result from cqassociated enhanced replication. to test this hypothesis, we examined the effect of cq and other commonly-used antimalarial drugs on b19v replication in three different cultured cell lines. in addition, we examined the relationship between b19v infection and use of 4-aminoquinoline drugs in a sample of children from png who were hospitalized with severe anemia. the results provide evidence that cq and aq aggravate b19v-associated anemia by promoting b19v replication. all patients were participants in a prospective observational and genetic study of severe pediatric infections (http://www.malariagen. net/home/). written informed consent was obtained from each parent/guardian. ethical approval for the study was obtained from both the png institute of medical research institutional review board and the medical research advisory committee of the png department of health. the study was conducted in accordance with the helsinki declaration. a b19v-infected plasma sample was obtained from our donation center (genotype 1; csl behring ag, charlotte, nc) and was concentrated by ultracentrifugation through 20% (w/v) sucrose. ut7/epo cells were cultured in rpmi, 10% fcs and 2 u/ml of recombinant human erythropoietin (epo; janssen-cilag, midrand, south africa). hepg2 cells were cultured in mem supplemented with 10% fcs. bone marrow mononuclear cells (bmmcs) were obtained as frozen stocks from stemcell technologies (vancouver, bc, canada) and were cultured in imdm, 10% fcs and 2 u/ml of epo. all cells were incubated at 37uc and in an atmosphere of 7.5% co 2 . all drugs were purchased from sigma (st. louis, miss). chloroquine diphosphate (cq), primaquine diphosphate (pq) and amodiaquine dihydrochloride dihydrate (aq) were dissolved in water, piperaquine (ppq) in 5% lactic acid, mefloquine hydrochloride (mq) in dmso, lumefantrine (lft) in dimethylformamide and artesunate (at) and pyrimethamine (pm) in ethanol. the final drug concentration ranges used in the b19v infectivity assay were 5-100 mm for pq and lft, aq and pm, 0.05-20 mm for mq, 2.5-100 mm for at, 1-100 mm for ppq and 0.05-100 mm for cq. the highest concentration of the drugs did not exceed 0.2% of total culture volume. b19v infectivity was assessed in two different cell lines, namely megakaryoblastic leukemia ut7/epo cells and the human hepatocellular liver carcinoma cell line hepg2, and in primary bone marrow mononuclear cells (bmmcs). ut7/epo cells are the most susceptible cell line to b19v infection [14] . although viral rna, dna and proteins can be detected in b19v-infected ut7/ epo cells, viral replication is restricted to a level that does not normally allow production of virus progeny. the hepg2 cell line was chosen because it allows virus binding and probably internalization, but it is non-permissive for b19v infection [15] . bmmcs have been shown to support b19v infection, although only a minor subset of these cells is permissive for b19v [16] . ut7/epo, hepg2 and bone marrow mononuclear cells (bmmcs) (3610 5 ) were infected with 20,000 dna-containing b19v particles per cell (5,000 for bmmcs), corresponding to an moi of approximately 20 (5 for bmmcs) in the presence of the pre-determined concentrations of the selected drugs. for viral rna and dna analysis, cells were collected at different postinfection times as indicated in the figure legends. total poly (a) + mrna was isolated and viral ns1 mrna quantified as previously described [17] . total dna was extracted and viral dna was quantified using established methods [17] . ut7/epo cells were infected as specified above in the presence of 0 or 25 mm cq. at increasing post-infection times (see figure 1c ), cells were lysed in protein loading buffer and total proteins were resolved by sodium dodecyl sulfate (sds)-10% polyacrylamide gel electrophoresis (page). after transfer to a pvdf membrane, the blot was probed with a mouse antibody against b19v structural proteins (1:2,000 dilution; us biologicals, swampscott, ma), followed by a horseradish peroxidase-conjugated secondary antibody (1:20,000 dilution). the viral structural proteins were visualized with a chemiluminescence system (pierce, rockford, il). additionally, viral protein expression was examined by immunofluorescence, as previously described [17] . human parvovirus b19 (b19v) is typically associated with a childhood febrile illness known as erythema infectiosum. the infection usually resolves without consequence in healthy individuals. however, in patients with immunologic and/or hematologic disorders, b19v can cause a significant pathology. the virus infects and kills red cell precursors but anemia rarely supervenes unless there is pre-existing anemia such as in children living in malariaendemic regions. the link between b19v infection and severe anemia has, however, only been confirmed in certain malaria-endemic countries in parallel with chloroquine (cq) usage. this raises the possibility that cq may increase the risk of severe anemia by promoting b19v infection. to test this hypothesis, we examined the direct effect of cq and other commonly used antimalarial drugs on b19v infection in cultured cell lines. additionally, we examined the correlation between b19v infection, hemoglobin levels and use of cq in children from papua new guinea hospitalized with severe anemia. the results suggest strongly that cq and its derivatives aggravate b19v-associated anemia by promoting b19v replication. hence, careful consideration should be given in choosing the drug partnering artemisinin compounds in combination antimalarial therapy in order to minimize contribution of b19v to severe anemia. chloroquine promotes b19v infection www.plosntds.org with pbs to remove unbound virus and incubated at 37uc in the presence or absence of antimalarials. at increasing post-internalization times from 1 to 7 h, the cells were washed 2 times with pbs and the amount of intact viral dna was quantified as specified above. we studied 111 children ,10 years of age with severe anemia (hemoglobin ,50 g/l) and 89 community-based age and sexmatched healthy control children with a hemoglobin .100 g/l. those with severe anemia represented a subset (15.9%) of all 697 children admitted to modilon hospital, madang province on the north coast of png with any severe illness during the period of study. modilon hospital is a referral hospital and the only provincial facility able to manage severely ill children. all such children were given treatment as recommended under png national treatment guidelines including intramuscular artemether for malaria infection. the healthy controls were recruited from the same villages as the patients and were slide-negative for malaria. as well as a hemoglobin concentration (haemocueh, angelholm, sweden) at presentation, plasma was assayed for b19v igm by eia kit (biotrin international) and, in those with severe anemia, for viral dna using two specific oligonucleotide primers [4] . we did both tests because viremia starts to decline once specific igm is produced around day 9 after inoculation, while virus-induced marrow suppression can last for another 2-3 weeks [4] . thus, although the simultaneous detection of b19v igm and dna is strongly indicative of acute infection, we did not want to exclude children with evidence of recent but resolving infection as a contributor to severe anemia. in those who were b19v igm or pcr positive, plasma was assayed for chloroquine and amodiaquine and their respective active desethyl metabolites using a validated high performance liquid chromatography assay [18] . the assay had a limit of quantitation of 1 mg/l for each analyte. effect of cq on b19v replication in ut7/epo cells cq increased the production of b19v ns1 gene transcription after 24 h incubation. at cq concentrations ranging from 10 to 75 mm, ns1 rna increased up to 1,170% ( figure 1a) . similarly, kinetic studies in the presence of 25 mm of cq showed that viral dna synthesis was more rapid and extensive than in untreated cells ( figure 1b) . the expression of structural viral proteins in extracts of infected ut7/epo cells was also increased in the presence of 25 mm cq ( figure 1c ). viral protein expression was immunofluorescence experiments showed, that in the presence of cq a larger number of cells were infected by b19v (fig. 1d ). in the absence of cq, only a minor amount of viral dna synthesis was observed starting at 120 h post-infection. no viral rna could be detected, confirming the poor permissiveness of this cell line for b19v infection. however, in the presence of increasing concentrations of cq, viral dna synthesis increased progressively reaching 2,290% at cq concentrations of 60 mm (figure 2a) . kinetic studies showed that, in the presence of cq (25 mm), viral dna was detected earlier than in untreated cells ( figure 2b ). viral ns1 rna was only detectable in cq-treated cells ( figure 2c ). the presence of cq (25 mm) accelerated b19v rna synthesis. however, in cq-treated cells, viral rna transcription ceased abruptly and was followed by progressive degradation resembling apoptosis ( figure 2d ). detection of phosphatidyl serine-anexin v complexes by fluorescence microscopy confirmed that the infected bmmcs entered the apoptotic pathway (data not shown). therefore, the effect of cq in cultured bmmcs could not be evaluated at stages later that 10 h post-infection. the apoptotic effects were not observed in the cell lines ut7/epo and hepg2 at concentrations up to 60 mm (data not shown). with the exception of the cq-analogue aq which enhanced b19v infection at concentrations above 5 mm, no other antimalarial drug had a significant effect on b19v infection ( figure 3 ). mild inhibition was observed in the presence of at, while mq inhibited the infection at concentrations .10 mm. these effects were also observed when the drugs were added 4 to 7 h post-infection (data not shown), raising the possibility that b19v infectivity was reduced by a drug-specific cytotoxicity. the enhancement of b19v infection by cq decreased progressively with increases in the time at which cq was added, with no detectable effect at 8-9 h post-infection ( figure 4a ). these data indicate that cq acts early in b19v infection. at progressive times after internalization of b19v, the cells were washed and the viral dna was quantified. in untreated cells, a progressive degradation of the incoming viral dna was evident. however, in the presence of cq (25 mm) or aq (20 mm), degradation of incoming particles was prevented or minimized ( figure 4b ). [18, 19] , this suggests that most of these children had been treated with either cq or aq within the previous 6 weeks. hemoglobin concentrations by b19v igm/pcr and 4aminoquinoline status are shown in figure 5 . the lowest concentrations were in the 5 children who were both igm and pcr positive (i.e. had acute b19 infections). these children had a similar mean age (53 vs 57 months), body weight (15 kg in both groups) and spleen size (5 vs 7 cm) to those children who were either igm or pcr positive (p.0.37 by mann-whitney u test) and the percentages with malaria were similar (40.0 vs 44.4%; p = 0.63 by fisher's exact test). in patients who were igm or pcr positive (indicating a recent but not necessarily acute infection or one which was acute but early in its course), 4-aminoquinoline use was associated with a significantly lower admission hemoglobin concentration (p = 0.037). although the number of patients treated with aq was small and restricted to children who were igm positive but pcr negative, they had some of the highest hemoglobin concentrations in this subgroup. this suggests that, consistent with the in vitro data, cq had a greater suppressive effect on bone marrow than aq in our patients. severe anemia is a common and life-threatening complication of malaria in children living in endemic areas [20] . b19v coinfection has been identified as a major factor in its pathogenesis [3] , but there are significant regional differences in its seroprevalence and resulting clinical impact [4] [5] [6] [7] [8] despite the fact that b19v infection is a common childhood illness. because b19vassociated severe anemia appears to parallel local use of cq as first-line treatment for malaria, we hypothesized that cq promotes b19v replication and that, as a consequence, it contributes indirectly to severe anemia. although a more profound clinical study would be necessary, the present results provide already a strong evidence for this hypothesis. apart from its antimalarial effects, cq has a wide antiviral activity. one of the most important mechanisms of action against viruses is the alkalinization of the endosomal vesicles. in this way, cq is active against viruses that require a low ph step for cell entry, such as flavivirus, retrovirus and coronavirus [9] . all parvoviruses studied to date also depend on endosomal acidification for cell entry because it facilitates capsid structural transitions [21, 22] , and in particular the externalization of the n-terminal region of vp1 which is required for endosomal escape and nuclear targeting [23] . accordingly, cq inhibits parvovirus infections. however, we have previously shown that b19v is unique among parvoviruses in that n-vp1 is already externalized on receptor binding [17] and thus not dependent on a low endosomal ph for this critical conformational change. b19v is also unique among parvoviruses for its higher sensitivity to acid degradation [24] . consequently, cq-associated alkalinization of endosomal vesicles would be expected to minimize the acidic degradation of incoming b19v particles. our data confirm that the intracellular degradation of b19v is prevented or minimized in the presence of cq or aq. however, other lysosomotropic drugs such as ammonium chloride or bafilomycin a1, which also raise the endosomal ph, had an inhibitory effect on b19v infection in our in vitro system (data not shown). therefore, the mechanism underlying the stabilization of b19v by cq or aq is likely to extend beyond ph-neutralizing activity to destabilization of endosome/lysosome membranes typically observed in cq-treated cells. in this way, cq would facilitate the endosomal escape of b19v before it reaches the degradative lysosomal compartment and increase the number of particles that can target the nuclei for replication. this is of particular importance since nuclear targeting chloroquine promotes b19v infection www.plosntds.org has been identified as a major limiting factor in parvovirus infections [22, 25] . the plausibility of our in vitro observations as an explanation of epidemiological data depends on the pharmacological properties of cq, especially tissue concentrations. the in vitro enhancement of b19v infection by cq was achieved at concentrations (10-75 mm) that were well above those achieved in plasma after therapeutic doses in children (typically ,5 mm) [18] . however, chloroquine promotes b19v infection www.plosntds.org b19v does not replicate in plasma but in tissues, primarily the bone marrow. cq concentrations in bone marrow are substantially higher than in plasma [26] and have been measured at approximately 100 mm in animal studies [27] . this could reflect, in part, concentration of the drug within precursor cells such as has been observed in circulating erythrocytes [28] . the long terminal elimination half-life of cq (around 10 days in children) [18] means that conditions favorable to b19v viral replication in bone marrow may persist for several weeks after dosing. in many malaria-endemic regions, antimalarial therapy is given empirically to febrile children without blood smear confirmation. ironically this might include fever due to b19v itself. the administration of frequent courses of cq may mean that a child spends long periods of each year at risk of cq-associated enhanced b19v viremia and its consequences such as anemia. aq is a long half-life 4-aminoquinoline compound like cq and also promoted b19v replication in our in vitro experiments. other drugs tested, including primaquine (an 8-aminoquinoline) and mefloquine (a methanol quinoline), did not influence b19v infection in vitro, suggesting that the effect is specific to 4aminoquinoline compounds. some other viral infections (sfv, figure 3 . effect of different antimalarial drugs on b19v infection. ut7/epo cells were infected with b19v at 4uc for 2 h. the cells were washed with pbs to remove unbound virus and incubated at 37uc in the presence of different drugs. all drugs were used in concentrations ranging from 0 to 100 mm. after 24 h, the amount of b19v ns1 rna was quantified. the data are expressed as the percentage of the value obtained in untreated cells (dotted line) averaged for two independent experiments. sd bars are shown. doi:10.1371/journal.pntd.0000669.g003 parvovirus-like particles in human sera clinical aspects of parvovirus b19 infection parvovirus infection, malaria, and anemia in the tropics -a new hidden enemy? parvovirus b19 infection contributes to severe anemia in young children in papua new guinea human parvovirus infection in children and severe anemia seen in an area endemic for malaria parvovirus b19 infection does not contribute significantly to severe anemia in children with malaria in malawi severe anemia in children living in a malaria endemic area of kenya seroprevalence of viral childhood infections in eritrea effects of chloroquine on viral infections: an old drug against today's diseases? recycling of chloroquine and its hydroxyl analogue to face bacterial, fungal and viral infections in the 21st century chloroquine enhances replication of semliki forest virus and encephalomyocarditis virus in mice chloroquine enhances epstein-barr virus expression antimalarial drugs and burkitt's lymphoma development of an improved method of detection of infectious parvovirus b19 hepg2 hepatocellular carcinoma cells are a non-permissive system for b19 virus infection human b19 erythrovirus in vitro replication: what's new? interaction of parvovirus b19 with human erythrocytes alters virus structure and cell membrane integrity pharmacokinetics and efficacy of piperaquine and chloroquine in melanesian children with uncomplicated malaria pharmacokinetic and pharmacodynamic study of amodiaquine and its two metabolites after a single oral dose in human volunteers pathophysiology of severe malaria in children intracellular route of canine parvovirus entry low phdependent endosomal processing of the incoming parvovirus minute virus of mice virion leads to externalization of the vp1 n-terminal sequence (n-vp1), n-vp2 cleavage, and uncoating of the full-length genome parvoviral host range and cell entry mechanisms molecular mechanism underlying b19 virus inactivation and comparison to other parvoviruses exploitation of microtubule cytoskeleton and dynein during parvoviral traffic toward the nucleus tissue distribution of chloroquine, hydroxychloroquine, and desethylchloroquine in the rat tissue distribution of subcutaneously administered chloroquine in the rat pharmacokinetics of chloroquine in thais: plasma and red-cell concentrations following an intravenous infusion to healthy subjects and patients with plasmodium vivax malaria guidelines for the treatment of malaria. geneva: world health organization we are grateful to the png children and their families for their participation, staff at modilon hospital for assistance with collection of clinical data and samples, dr. anna rosannas for pcr assays, and emeritus prof. ken ilett and dr. madhu page-sharp for 4-aminoquinoline and metabolite assays. emcv and ebv) [11, 12] are enhanced by cq but not by other 4-aminoquinoline antimalarial drugs.we were able to obtain preliminary human data that are consistent with our laboratory findings. in our 200 unselected png children who were participants in a case-control study of severe pediatric infections, we confirmed previous reports that b19v seropositivity is associated with severe anemia and that the lowest hemoglobin concentrations are in those children who had acute infections (i.e. both igm and pcr positive) [4] . although there were limited numbers, there was some evidence that prior 4aminoquinoline, especially cq, use exacerbates b19v-associated severe anemia apart from in those igm-and pcr-positive cases who were presumably at the stage of maximal viral replication and consequent bone marrow suppression. properly designed epidemiological studies in larger, non-convenience samples are, however, needed to confirm these findings.although cq is a safe and inexpensive antimalarial drug, the increasing emergence of resistant p. falciparum and p. vivax has seen its use decline throughout the tropics. our data suggest that the prevalence of severe malarial anemia should also fall as a result. however, when an effective b19v vaccine becomes available, this should be considered a priority intervention where pediatric b19v seroprevalence rates are high and other causes of anemia such as nutritional deficiency and intestinal parasitic infection are present. artemisinin-based combination therapy (act) is the current who-recommended first-line treatment for uncomplicated malaria [29] . our data suggest that, pending more definitive in vivo data including appropriately designed clinical trials, a non-4aminoquinoline drug should be preferred to partner the artemisinin derivative so that the contribution of b19v to severe anemia is minimized. conceived and designed the experiments: cb im tmed cr. performed the experiments: cb lm ml cr. analyzed the data: cb ck im tmed cr. wrote the paper: tmed cr. key: cord-288202-r3r2bc7v authors: morel, noelia; lassabe, gabriel; elola, susana; bondad, mauricio; herrera, silvia; marí, carlos; last, jerold a.; jensen, oscar; gonzalez-sapienza, gualberto title: a monoclonal antibody-based copro-elisa kit for canine echinococcosis to support the paho effort for hydatid disease control in south america date: 2013-01-10 journal: plos negl trop dis doi: 10.1371/journal.pntd.0001967 sha: doc_id: 288202 cord_uid: r3r2bc7v cystic echinococcosis is still a major concern in south america. while some regions show advances in the control of the disease, others have among the highest incidence in the world. to reverse this situation the pan american health organization (paho) has launched a regional project on cystic echinococcosis control and surveillance. an early concern of the program was the lack of a standardized diagnostic tool to monitor infection in dogs, a key target of control programs. under this premise, we have developed a new copro-elisa test after extensive screening of a large panel of monoclonal antibodies (mabs) and polyclonal sera, which performs with high standards of sensitivity (92.6%) and specificity (86.4%) as established by necropsy diagnosis of dogs. the key component of the test, mabeg9 has a convenient igg isotype and reacts with a periodate-resistant epitope found in high molecular weight components of the worm. time-course analysis of experimentally infected dogs showed that even animals with a very low number of parasites could be detected as early as day 20 post infection. the test was formulated in a ready-to-use kit format with proven stability of each component for a minimum of 3 months at room temperature. this characteristic facilitates its standardized use and shipping to other laboratories, which was demonstrated by the identical results obtained by two different laboratories in peru and our own laboratory when a large number of field samples were analyzed independently in a blind fashion. cystic echinococcosis, caused by infections with echinococcus granulosus, is one of the main zoonoses related to dogs and is affecting most parts of the globe [1] . different control programs aimed to decrease the impact of the disease have been instrumented in many countries. although the rate of success has been highly variable, it has become evident that a tight control of dog infections is the key element to arrest the life cycle of the parasite. this has created a demand for reliable diagnostic tests for canine echinococcosis as a tool to obtain epidemiological baseline information and help in the surveillance of hydatid control [2] [3] [4] [5] . however, accurate diagnosis of dog infection is complex and challenging, and other than careful necropsy of dogs, there is no perfect gold standard [6] . parasitological examination of eggs is unsafe and not very useful because e. granulosus are morphologically difficult to distinguish from other taeniae eggs, and appear late (after the first month) of infection. traditionally, screening of dogs for e. granulosus has been done by arecoline purgation, followed by examination of the purge for parasites by trained personnel. the method is highly specific, but it is tedious, biohazardous, unpopular among dog owners, and its sensitivity is modest, particularly when the parasite burden is low and bowel evacuation is incomplete [7, 8] . as an alternative, different laboratory tests for ante-mortem diagnosis of canine echinococcosis have been developed, including detection of antibodies in serum, polymerase chain reaction (pcr) amplification of parasite dna and immunological detection of antigens (coproantigens) in fecal samples. different studies have been carried out to explore the potential use of dog serology to diagnose the disease. however, the systemic immune response to the parasite is poor and consequently the sensitivity attained is also low [4] . parasite dna excreted with eggs, proglottids or cells has been detected in fecal specimens by pcr amplification using specific primers derived from mitochondrial dna [9, 10] . the method has provided the high specificity of pcr, but due to the presence of inhibitors the dna extraction procedure is cumbersome and the technique requires expensive reagents and specialized laboratories [11] . in addition, the pre-patent period of the infection, when there is no egg production, is a critical time-window that challenges the sensitivity of the test. introduced at the beginning of the 1990's, the detection of parasite antigens in fecal samples by immunoassays became a widely accepted diagnostic test [12, 13] . the major attractive features of the method include its simplicity, the possibility of detecting parasite components even in the prepatent period, and the fact that the target antigens (coproantigens) are highly stable. samples can be collected in 1% of 40% formaldehyde and kept at room temperature for extended periods of time, facilitating the logistics of large-scale studies in remote areas [7, [14] [15] [16] . the study of a large group of infected dogs necropsied at the end of the pre-patent period demonstrated a superior sensitivity for the copro-elisa (83%), when compared to copro-pcr (26%) or arecoline purgation (43 and 77% after one or two doses, respectively). most failures to detect positive infected dogs occurred when the number of worms was less than one hundred [5] . despite its proven utility and the numerous reports of copro-elisa developments, the availability of commercial or homemade tests is often a major obstacle to the implementation and evaluation of echinococcosis control programs. the need to overcome this difficulty has been recognized as an early goal of the southern cone sub-regional project on cystic echinococcosis control and surveillance: argentina, brazil, chile and uruguay, and paho (pan american health organization), a joint and collaborative effort to battle the disease in the region. in this framework, we have developed an immunoassay for e. granulosus copro-antigen detection under the premise that in addition to performing with high standards of proven sensitivity and specificity, it had to be robust, standardized and developed in a kit format to be available for its use in regional programs for the control of the disease. all activities involving animals were performed with special care to establish high standards of biosafety and assure animal welfare. all protocols were performed according to the international guiding principles for biomedical research involving animals, (cioms) and were approved by the comisión nacional de zoonosis and the research department of the ministry of health of the province of chubut. nineteen adult dogs, of mixed breeds and sex, were vaccinated against distemper encephalomyelitis, parvovirosis, rabies, leptospirosis, hepatitis and coronavirus (merial, france), treated orally with praziquantel 5 mg/kg, pyrantel embonate 15 mg/kg and febantel 15 mg/kg (disper, uruguay) to eliminate worm parasites, and topically with a solution of fipronil and methoprene (merial) to arrest the development of ectoparasites. dogs were purchase from local suppliers and maintained on commercial dog food and water ad libitum. nineteen fecal samples (n1-n19), corresponding to non-infected dogs, were collected two days after deworming. then, ten dogs (identified as p1-p10) were infected orally with 5,000-150,000 viable (.80%) protoscoleces from ovine fertile hydatid cysts. two additional dogs (identified as th1 and th2) were infected orally with 1 and 3 larvae of taenia hydatigena, respectively. parasite material was obtained from sheep slaughter in local abattoirs in paysandú, uruguay. stool samples from each dog were collected daily during the experimental period, at the end which (27-30 days post-infection (dpi)), the animals infected with e. granulosus were euthanized for necropsy diagnosis by intramuscular injection of acepromazine maleate (0.11 mg/kg) and ketamine (20 mg/kg) (laboratorio holliday, argentina), following by an intravenous overdose of sodium thiopental (laboratorio crusur vet, uruguay). the dogs infected with t. hydatigena were purged at 70 dpi with arecoline bromhydrate (crescent chemical co. inc., new york, usa), following by treatment with anti-parasitic drugs. three additional samples from dogs experimentally infected with e. granulosus (26 dpi) and two with t. hydatigena (about 55 dpi) from previous studies [14, 16] were obtained as a kind gift from dr. carlos carmona. in addition, 85 unwanted dogs from the province of chubut, argentina, and 11 unwanted dogs from junin, peru, were euthanized following the procedure described above, and the small intestines were removed post mortem, opened longitudinally and examined directly for the presence of parasites following the recommendations of the who/oie [17] . all activities were performed following the local legislation and the international guiding principles for biomedical research involving animals (http://www.cioms.ch/). stool samples from each dog were collected daily during the experimental infection period, or from the rectum upon necropsy, in 1% of 40% formaldehyde, phosphate buffered saline (pbs), in a 1:4 v/v ratio. samples were vigorously shaken to obtain homogeneous slurries, and were then boiled for 20 min in a water bath, to eliminate their biological risks. after centrifugation for 10 min at 2,2006 g the supernatants were aliquoted and kept frozen at 220uc until used. the parasite antigens were prepared essentially as described by casaravilla et al. [16] . cystic echinococcosis, caused by infection with the larval stage of the echinococcus granulosus tapeworm, is a lifethreatening zoonosis of worldwide distribution. the adult worm parasitizes the small intestine of dogs, which become infected after eating offal of an animal contaminated with the parasite, and releases eggs into the environment that can be accidentally ingested by domestic animals or humans, maintaining the life cycle of the parasite. deworming of dogs is a major component of control programs, and simple and reliable methods are needed to monitor the base-line infection in the canine population. the lack of these tests was recognized as a major obstacle to the paho effort to control the disease in south america. this paper describes the development of a diagnostic assay that detects parasite antigens in dog feces. the key component is a monoclonal antibody carefully selected to attain high levels of sensitivity and specificity, which were established with a large panel of field fecal samples obtained from animals diagnosed by necropsy. several aspects of the long-term stability of the test were optimized to facilitate its shelf-life and transference to other laboratories. unit with an ym-10 membrane (millipore, usa), followed by dialysis against pbs. e. granulosus somatic antigen (sm) was obtained by sonication of adult parasites in pbs supplemented with ultra protease inhibitor tablets (roche, indianapolis) on ice, centrifugation and filtration (0.22 mm). antigens from t. hydatigena were similarly prepared. protein content was determined using a bca kit (pierce, rockford, illinois). to prepare polyclonal antibodies, 100 mg of sm or e/s antigens were dissolved in 250 ml of pbs and vigorously mixed with 250 ml of freund's complete adjuvant (pierce, illinois) to form a thick emulsion. this emulsion was then injected subcutaneously into several points on the back of new zealand white rabbits. after 4 and 8 weeks, the animals were immunized intramuscularly with additional doses of 100 mg of antigen emulsified in freund's incomplete adjuvant. ten days after the final booster the animals were bled. the igg fraction was prepared by precipitation of the sera with ammonium sulfate, affinity purified on protein g agarose (amersham, uppsala, sweden), and kept at 220uc until used. for monoclonal antibody (mab) preparation, balb/c mice were primed intraperitonally with 40 mg of e. granulosus sm or e/s antigen in freund's complete adjuvant, and boosted after 3 and 6 weeks with the corresponding antigen using freund's incomplete adjuvant. three days after the last booster mice were sacrificed and the splenocytes fused with sp2/0 cells using standard procedures. cultures producing mabs reactive with the corresponding antigen were selected by elisa. we initially performed a fusion experiment using mice immunized with alternate doses of e/s and sm antigens, and additional selection in a sandwich format using fecal samples. mab cpr39 was selected due to its convenient isotype and performance. several fusion experiments were performed to produce a large panel of monoclonal antibodies. the screening of the monoclonal antibodies was initially performed as described before, and then each of the double-positive supernatants (13 clones) was tested in a sandwich format in combination with the different polyclonal antibodies. however, this time instead of using fecal samples from heavily infected dogs, each supernatant was tested against the following: i) a negative sample from a non-infected dog, ii) a sample collected from a low-burden dog at 28 dpi (dog p9, 74worms), iii) a sample from a heavily infected dog taken at an early stage (10 dpi) of the pre-patent period (dog p8, 3459 worms), and iv) a sample from dog th2 taken at 56 dpi that was positive in the cpr39 copro-elisa. out of this complex screening clone mabeg9, in combination with polyclonal antibody pabc4 (prepared from a rabbit immunized with e/s), was chosen because this antibody pair produced high signals with samples (ii) and (iii), and low readings with samples (i) and (iv). parasite preparations were resolved by sds-page 12% under reducing conditions and then transferred onto nitrocellulose sheets (bio-rad, hercules, california, usa). the nitrocellulose was blocked with 3% non-fat milk powder in pbs 1 h at 37uc and was incubated for 1 h at 37uc with a 1:500 dilution of the rabbit antisera in pbs containing 0.05% of tween 20 (pbs-t), 3% non-fat milk, or directly with the culture supernatants of the mabs antibodies. the membranes were then incubated for 1 h at 37uc with alkaline phosphatase-conjugated to anti-rabbit igg or antimouse igg (pierce) (diluted 1/2500). after extensive washing, a substrate solution containing 5-bromo-4-chloro-39-indolylphosphate p-toluidine salt (bcip) and nitro-blue tetrazolium chloride (nbt) was added according to the manufacturer's instructions (sigma). five mg/ml of polyclonal igg (100 ml/well) (pab cg10 or pabc4 for the mab cpr39 or mabeg9 copro-elisa, respectively) was dispensed into microtitration plates (greiner, germany) and incubated overnight at 4uc. the plates were then blocked with 5% non-fat milk (pbs) and washed with pbs-t. alternatively, the plates were further treated with pbs, 0.1% bovine serum albumin (bsa), 5% sucrose, 0.02% sodium azide, flapped repeatedly against adsorbent paper, dried in a 40% relative humidity chamber for 4 h, and kept in sealed aluminum foil bags containing adsorbent packets (sigma) until used. samples were analyzed in triplicates, after a 1:2 dilution in pbs; 100 ml/well were incubated for 1 h at room temperature, the wells washed and loaded (100 ml/well) with a 1:50 dilution of the mab supernatants and incubated for 1 h at room temperature. finally, the wells were incubated (1 h upon necropsy at 27-30 dpi, 9 of the experimentally infected dogs p1-p10 dogs harbored e. granulosus worms. the number of worms recovered from each dog varied widely, from 74 to 8,500, and showed no correlation with the initial number of protoescoleces used for infection. no other parasites were observed in addition to the e. granulosus worms. adult worms (typically less than 1 m long) were recovered from dogs th1 and th2 infected with t. hydatigena. after careful washing, the recovered parasites were kept in culture to prepare excretion/secretion antigens, or were sonicated as described to obtain somatic antigens. the sds-page analysis of these antigens is shown in figure 1a ; a protoescoleces extract was also included as a reference antigen. all preparations possessed a complex composition and the sds-page profile did not reveal any obvious similarities among the individual components of the different preparation. initially, the reactivity of polyclonal antibody pabcgb10 raised against the sm antigen was characterized, figure 1b . there was a strong response to a large number of components of the immunizing antigen, as well as to high molecular weight components of ege/s and t hydatigena. destruction of sugar epitopes by periodate treatment significantly decreased the reactivity of this antibody with the somatic antigen, as has been observed before [2, 19] , but had little effect in the case of the e/s preparation. the marked cross-reactivity of the polyclonal antibodies with e/s components of t. hydatigena evidences how difficult it is to obtain a specific assay using these antibodies as reagents. for that reason we concentrated our efforts in the preparation and selection of monoclonal antibodies after initial screening against e/s and sm and further selection using pab cgb10 for capture and the hybridoma supernatants for detection, four clones providing the best signal/noise ratios with fecal samples were selected. among these, clone mabcpr39, secreting an igg2b, was chosen. this elisa was used to examine fecal samples obtained at the end of the infection, days 27-30 for e. granulosus or at selected dpi (highest cross-reactivity) for t. hydatigena ( figure 2) . the assay performed with negligible reactivity with samples from noninfected dogs and moderate cross-reactivity with t. hydatigena infected animals. all e. granulosus infected dogs were positive, however, the readouts of the samples corresponding to dogs with small parasite burdens were significantly higher, but too close to the mean value of the negative dogs to give unequivocal results. the assay was then used to study the time course of coproantigen excretion during the experimental infection period ( figure 3) . using a cut-off derived from the analysis of field samples (see below), we found that dogs with high parasite burdens became positive after two weeks, and in spite of some fluctuations remained so thereafter. on the other hand, dogs with less than 200 worms at necropsy, presented low readouts throughout the experiment, becoming positives only at the end of the experimental infection. along the time course of the examined period, the dogs harboring t. hydatigena worms presented very low values (below the cut-off), showing positive values only in very few days after 40 dpi (not shown). due to the limited sensitivity of the cpr39 copro-elisa, new antibodies were prepared. the best combination resulted when pab c4 was used as capture antibody in combination with mab eg9. the reactivity of pabc4 with different parasite antigens in western blot was similar to that of pabcgb10 (figure 1b) and it is not shown. mabeg9 is an igg; its reactivity with different parasite extracts is displayed in figure 1c . the antibody reacted with a large number of the egsm bands, mostly in the middle to high molecular weight range. two components of 100 and 160 kda are the most prominent immunoreactive bands of the e/s antigens, which appear to be also present in the sm preparation. the crossreactivity with th e/s antigens was negligible (as was also the case with protoscoleces antigens, not shown). treatment with periodate had no effect upon the reactivity of mabeg9 with any of the antigens, which was unexpected because previous studies have shown that the sugar epitopes have a major role in the immune response against e. granulosus coproantigens [2, 19] . to rule out technical artifacts, the efficacy of the periodate treatment was tested in parallel using the monoclonal antibody e492/g1 that defines a sugar epitope highly expressed in e. granulosus protoscoleces preparations [20] , figure 1d . when the experimentally-infected dog samples were analyzed with the new assay, it was evident that the eg9 copro-elisa produced stronger signals with samples from dogs with small numbers of worms, allowing a better discrimination between noninfected and infected dogs ( figure 4) . this is an important improvement because most of the reported tests failed to detect these kind of samples. cross-reactivity with t. hydatigena coproanti-gens did not seem to be a major problem when the time course of coproantigen excretion was study with the eg9 copro-elisa ( figure 5 ). using the cut-off estimated by the receiver operating characteristic (roc) curves from the analysis of field samples (see below), only on very few occasions and after 40 dpi were some of the samples from t. hydatigena infected dogs positive. the eg9 copro-elisa allowed an earlier detection of infected dogs. animals with 200 or more parasites became positive between 12-15 dpi and by dpi 20 all infected dogs were positives and remained so thereafter. curiously, coproantigens in dogs p7 and p8 samples could be detected very early. this is more remarkable in the case of dog p7 that harbored a rather small number of worms (128) at necropsy, and may be the result of the spontaneous expulsion of worms a few days after infection. the cut-off and cross-reactivity of the assays were evaluated with field samples collected from 96 unwanted dogs in the provinces of chubut, argentina and junin, peru. upon necropsy, 9 of 85 dogs from chubut, and 6 of 11 dogs from junin were positive for e. granulosus, 48 were infected with other parasites and 33 were found negative, table 1. fecal samples from these dogs were tested using the cpr39 and eg9 copro-elisas and the cutoff selected from receiver operating characteristic (roc) curves [18] to have a convenient balance between sensitivity and specificity as follows: cpr39 elisa = 0.224 au and eg9 elisa = 0.340 au. using these values, the sensitivity of the tests were 80.0 and 86.6% respectively, which is in agreement with the results obtained with the experimental infection of dogs, figure 4 . the specificity of the eg9 test was also superior 86.4% versus 82.7% for the cpr39 elisa. in addition, the cpr39 test produced readings close to the cut-off for an important number of samples, figure 6 , which may be problematic for the use of a cutoff internal calibrator, because small inter-assay variations in the reading of the calibrator may have an important effect upon the number of samples that are classified in either category. actually, if samples that fall between 610% of the cut-off are considered as undetermined (a common practice), none of the samples analyzed with the eg9 test would fall in this category, while four of the samples analyzed with the cpr39 would. while it was not possible to obtain an exact counting of parasites in the dogs from the province of chubut, this was feasible in the case of the animals necropsied in junin. although most dogs had a large number of worms, two of them did not, and these two dogs were exaustively examined. we found only 9 e. granulosus and 2 ascaris lumbricoides, and 47 e. granulosus and very few dipylidium caninum worms in the first and second dogs, respectively. interestingly, the average absorbance readings of the fecal samples obtained from these animal were 1.16 and 2.99 au, which represent strong positive results, indicating the sensitivity of the test and confirming the capacity of the assay to detect small numbers of parasites that had been observed in the experimentally infected dogs. in this regard, when the e. granulosus positive dogs from the experimental infection are included, the overall sensitivity of the test rises to 92.6%. despite the fact that mabeg9 was selected for its reduced cross-reactivity with the thsm antigen, most of its cross-reactivity was against dogs infected with t. spp (mostly hydatigena) alone or together with other parasites. only one dog infected with t. canis (1.44 au) and one of the non-infected dogs (0.694 au) from the province of chubut were classified as positive with the eg9 elisa, figure 6 . the relatively high readings of these two samples are unexpected. based on the results of the experimental infection of dogs and the fact that all other samples (19) from dogs harboring t. canis alone or together with dipylidium caninum showed very low readings, a misdiagnosis at necropsy can not be discarded. it is worth noting that cross-reactivity was essentially against taenia. despite the fact that we could not identify to species all field samples, the great majority of samples from chubut and all from junin corresponded to t. hydatigena; hence most false positive results will be related to dogs eating offal. development of the elisa test in a kit format and interlaboratory analysis of blind samples the eg9 test was then formulated in a kit format to facilitate its use and transference to other laboratories. initially, we studied different conditions for coating and drying the plates and found that using sugars as additives during drying have the best effect on the stability of the capture antibody. trehalose and sucrose showed the best results and the latter was chosen because of its much lower cost. figure 7a shows that dried plates showed similar capacity as fresh ones to discriminate between weak positive and negative samples over a one year period of storage at 4uc. similar results were obtained after 2 years of storage (not shown). to set up the value of the cut-off an internal standard was prepared by adjusting the dilution of the e. granulosus sm antigen in order to produce a readout equal to the value of the cut-off. the stability of this cut-off calibrator solution and other components of the assay (in their ready-to-use dilution) were tested using different diluent buffers at 4uc, room temperature, and 37uc, and some representative results are shown in figure 7b-d. different formulations of the calibrator solution were stable over a 6 month period, even at 37uc; only the results for the sm antigen diluted in pbs, 5% glycerol, 0.1% kathon (dow chemicals, midland, michigan) are shown. the reactivity of mab eg9 started to diminish after a few days at 37uc, but was highly stable over the 6 month period at 4uc (not shown) or at room temperature, figure 7c . the most critical component was the secondary antibody. for several different formulations (only two shown in figure 7d ) all of them rapidly lost the enzymatic activity at 37uc, but using pbs, 0.1% bsa, 0.1% kathon, 0.1 mm tmb, the conjugate remained stable for up to 150 days at room temperature (or 4uc, not shown). overall, we can conclude that even at room temperature the kit has a safe shelf life of 3 months, which can surely be extended upon refrigeration, which is a convenient advantage for the shipment and use of the kit in remote places. due to the availability of commercial ready-to-use tmb substrate solutions, the preparation of this kit component was not pursued in this study. in case of high demand of the kit, it will be necessary to prepare new pab. in our experience, this is a critical component of the test, but we have selected similarly performing pabs from a panel of 3 to 4 immunized rabbits, after initial selection with fecal samples from dogs harboring low number of e. granulosus parasites and counter selection with samples from t. hydatigena infected dogs. the reproducibility of the results obtained with the copro-elisa kit and the feasibility of its transference were demonstrated by the analysis of blind samples. to this end, 52 fecal samples collected in the province of junin, peru were processed as described, and aliquots were distributed to two laboratories (ins and digesa) in lima, peru, where the copro-elisa kit had been transferred, and to our laboratory in uruguay (facultad de química). the samples were independently analyzed by the three laboratories and classified as positive, negative or uncertain if their readout felt within the calibrator absorbance reading 610% range. twenty four samples were positive, indicating a prevalence of 46.1% in the studied areas (canchayllo and jauja). the kit performed with excellent reproducibility and all individual samples were equally classified by the three laboratories with the exception of two of the samples with readings close to the cut-off, one of them categorized as negative by two of the labs and uncertain by the third one, and another sample classified as (weak) positive by two of the labs and uncertain by the remaining one. two tests were developed and the difference in their performance highlights the importance that careful selection of antibody pairs has to attain a high standard of sensitivity and specificity. the eg9 test performed with high sensitivity (92.6%) and good specificity (86.4%). these parameters are closely similar to previously reported values for other tests. however, it is very important to keep in mind that the only trustworthy comparison of tests is that obtained with a common panel of samples assayed under identical conditions, which highlights the need of interlaboratory studies to compare the performance of existing test. the test cross-reactivity was low, and in addition, since it was basically restricted to t. hydatigena, most false positive results will still indicate access of dogs to offal. a major contribution of our study is the use of a well-characterized mab that assures availability and batch-to-batch reproducibility, as well as the formulation of the assay in a kit format with extended shelf life. the eg9 test is being shared with other control programs in the region and we hope that it will help to monitor the progress of control programs and standardize the epidemiological baseline information in the region. checklist s1 stard checklist for the eg9 copro antigen test. flowchart s1 stard flow chart detailing the method used to assess the diagnostic performance of the eg9 copro antigen test. (docx) partial characterisation of carbohydrate-rich echinococcus granulosus coproantigens control of hydatidosis echinococcosis: disease, detection and transmission screening for echinococcus granulosus in dogs: comparison between arecoline purgation, coproelisa and copropcr with necropsy in pre-patent infections echinococcosis: diagnosis and diagnostic interpretation in population studies coproantigens in taeniasis and echinococcosis detection of echinococcus granulosus coproantigens in australian canids with natural or experimental infection copro-diagnosis of echinococcus granulosus infection in dogs by amplification of a newly identified repeated dna sequence polymerase chain reaction for detection of patent infections of echinococcus granulosus (''sheep strain'') in naturally infected dogs copro-dna tests for diagnosis of animal taeniid cestodes coproantigen detection for immunodiagnosis of echinococcosis and taeniasis in dogs and humans detection of echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes coproantigen detection in dogs experimentally and naturally infected with echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay detection of echinococcus granulosus coproantigens in faeces from naturally infected rural domestic dogs in south eastern australia production and characterization of monoclonal antibodies against excretory/secretory products of adult echinococcus granulosus, and their application to coproantigen detection manual on echinococcosis in humans and animals:a public health problem of global concern the meaning and use of the area under a receiver operating characteristic (roc) curve echinococcus granulosus coproantigens: chromatographic fractionation and characterization modulation of the cellular immune response by a carbohydrate rich fraction from echinococcus granulosus protoscoleces in infected or immunized balb/c mice we thank dr. carmona and dr. cecilia casaravilla for their advice and provision of samples. we are also grateful to the medical veterinarians and staff of the comisión nacional de zoonosis, uruguay, for help with the experimental infection of dogs. the personnel and medical veterinarians from the dirección regional de salud de huancayo, peru, are also acknowledged for their help with the pilot study of dog infection. key: cord-332473-ec8lu2a3 authors: amorim, raquel; temzi, abdelkrim; griffin, bryan d.; mouland, andrew j. title: zika virus inhibits eif2α-dependent stress granule assembly date: 2017-07-17 journal: plos negl trop dis doi: 10.1371/journal.pntd.0005775 sha: doc_id: 332473 cord_uid: ec8lu2a3 zika virus (zikv), a member of the flaviviridae family, is the most recent emerging arbovirus with pandemic potential. during infection, viruses trigger the host cell stress response, leading to changes in rna translation and the assembly of large aggregates of stalled translation preinitiation complexes, termed stress granules (sgs). several reports demonstrate that flaviviruses modulate the assembly of stress granules (sg). as an emerging pathogen, little is known however about how zikv modulates the host cell stress response. in this work, we investigate how zikv modulates sg assembly. we demonstrate that zikv negatively impacts sg assembly under oxidative stress conditions induced by sodium arsenite (ars), a treatment that leads to the phosphorylation of eif2α. by contrast, no measurable difference in sg assembly was observed between mock and zikv-infected cells treated with sodium selenite (se) or pateamine a (pata), compounds that trigger eif2α-independent sg assembly. interestingly, zikv infection markedly impaired the phosphorylation of eif2α triggered in ars-treated infected cells, and the abrogation of sg assembly in zikv-infected cells is, at least in part, dependent on eif2α dephosphorylation. these data demonstrate that zikv elicits mechanisms to counteract host anti-viral stress responses to promote a cellular environment propitious for viral replication. zika virus (zikv) is transmitted to humans primarily through mosquito bites, but there have also been cases of sexual, perinatal, and suspected blood transfusion transmission. it has been associated with fetal malformations and neurological disorders in adults. the rising concern about this pathogen led the world health organization to declare it as a public health emergency of international concern regarding neurological disorders. there is an urgent global scientific effort underway to better understand zikv biology and define interactions that occur between the virus and the host cell. we evaluated how zikv infection counteracts the assembly of dynamic aggregates of rna and proteins called stress plos neglected tropical diseases | https://doi.org/10.1371/journal.pntd.0005775 july 17, 2017 1 / 20 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 zika virus (zikv) is a positive-sense, single-stranded rna virus that belongs to the genus flavivirus of the family flaviviridae, which also includes yellow fever (yfv), west nile (wnv), dengue (denv) and japanese encephalitis viruses (jev) [1] . the genome of zikv encodes a large polyprotein precursor that is co-and post-translationally processed by viral and cellular proteases into three structural proteins [capsid (c), precursor of membrane (prm), and envelope (e)] and seven nonstructural proteins [(ns1, ns2a, ns2b, ns3, ns4a, ns4b and ns5)] that are involved in virus replication, which takes place in the cytoplasm of the host cell [2] . like other flavivirus members, zikv relies mainly on arthropods such as mosquitoes or ticks for transmission and thus is classified as an arthropod-borne virus (arbovirus). the main arthropod vectors of zikv are aedes sp. mosquitoes (a. aegypti or a. albopictus) [3] . along with the vector-borne transmission, other routes of zikv transmission have been demonstrated, including sexual transmission, transplacental and perinatal transmission and blood transfusion [4] , raising the concern about the global spread of the disease. zikv was first isolated from a rhesus monkey in the zika forest (uganda) in 1947 [5] . for more than 50 years, zikv was rarely reported to cause disease in humans and was commonly associated with mild illness. in 2007, there was an outbreak in the federated states of micronesia [6] , followed by outbreaks in french polynesia in 2013-14, in which severe neurological complications were reported [7] . since then, zikv is considered to be the most recent emerging arbovirus with pandemic potential [8] . in 2015, autochthonous transmission of zikv was confirmed in the northeastern region of brazil [9] . a dramatic increase in reported cases of microcephaly in the affected brazilian regions suggested an association between zikv infection and fetal malformations [10] and neurological disorders in adults, including guillain-barré syndrome and meningoencephalitis [11] . in february 2016, the world health organization declared a public health emergency of international concern regarding neurological disorders associated with the rapid emergence of zikv in oceania and the americas [12] . in response to conditions of environmental stress, eukaryotic cells activate kinases (hri, gcn2, pkr and perk) that phosphorylate eif2α (eukaryotic initiation factor 2 alpha) to ease cellular injury or, alternatively, to induce apoptosis. phosphorylation of eif2α reduces global translation by impairing the formation of the ternary complex eif2-gtp-trna met , allowing cells to conserve resources and to initiate a reconfiguration of gene expression to effectively manage stress conditions [13] . protein synthesis arrest triggers the assembly of stress granules (sg), that are large ribonucleoprotein (mrnp) aggregates formed by stalled translation preinitiation complexes [14, 15] . the major components of sg are untranslated mrnas, eukaryotic translation initiation factors (eif4e, eif4g, eif4a, eif2), the 40s ribosomal subunit and rnabinding proteins such as the poly(a) binding protein (pabp), t-cell intracellular antigen 1 (tia-1), tia-1-related protein (tiar), and ras gtpase activating protein-binding protein 1 (g3bp1) [16] . distinct cell host processes are interrupted or co-opted during viral infection, leading to the activation of cell stress responses on many levels. sg assembly lowers the cytosolic availability of components of the cellular translation machinery and functions as a platform that connects stress and antiviral innate responses, implying an overall antagonistic relationship between viruses and sgs [17] . in this sense, viruses have evolved a plethora of strategies to guarantee their replication by preventing or blocking sg assembly in infected cells, for example by co-opting rna granule factors and/or blockage of activation of eif2α kinases, such as pkr [18] . cellular stress responses are essential in eliciting immune detection and in the cell's ability to shut down viral gene expression in response to viral infection. so far, little is known about how zikv modulates stress responses in infected cells. recently, it was shown that zikv infection triggers a potent repression of host cell translation initiation, while viral protein synthesis remains unaffected [19] . the interplay between viral replication and the cellular stress response may contribute to the exacerbated pathogenesis seen in the current epidemic. elucidation of the interaction of viral components with host factors involved in sg assembly will provide new insight into the pathology of zikv infection. in this work, we investigated how zikv infection modulates sg assembly. green african monkey kidney (vero) (atcc) cells and human osteosarcoma-derived u2os containing g3bp1-gfp (a kind gift from dr. paul anderson and nancy kedersha, harvard medical school [20] ) cells were maintained at 37˚c and 5% co 2 atmosphere in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs) (hyclone) and 1% penicillin/streptomycin (life technologies). cell viability was evaluated by trypan blue exclusion cytotoxicity assay [21] to produce viral stocks, vero cells were infected with zikv strain prvabc59/2015 at a multiplicity of infection (moi) of 0.01 and incubated for 3 days at 37˚c. viral supernatants were then harvested, centrifuged at 300 x g for 10 minutes at 4˚c and filtered on a 45 μm syringe filter. viral titers were determined by plaque forming assay using culture media supplemented with carboxymethylcellulose (sigma) as described previously [22] . a stock with a viral titer of 2 x 10 7 was used in the experiments. for immunofluorescence assays, 7.5 x 10 4 vero or u2os cells were seeded on 18 mm diameter coverslips the day prior infection. for western blotting analysis, 7.5 x 10 4 vero or u2os cells were seeded in each well of a 12-well plate. then, cells were incubated for 1 hour with zikv diluted in dmem at an moi of 0.5 [23] . after this period, the viral inoculum was removed by aspiration and cells were incubated in complete culture media for the periods specified in each experiment. vero cells were seeded on 18 mm coverslips and infected as described above. viral rna was labeled as described in [24] . briefly, cells were treated for 30 minutes with 1 μg/ml actinomycin d (sigma) to block host cellular transcription. then, cells were transfected with 10 mm 5-bromourudine 50-triphosphate (brutp) (sigma) using lipofectamine 2000 reagent (invitrogen). after 1 hour, cells were fixed and processed for indirect immunofluorescence analysis. stress was induced using 500 μm sodium ars (naaso 2 ; sigma-aldrich) for goat anti-tiar (santa cruz biotechnology) was used for indirect immunofluorescence microscopy at a dilution of 1:500; rabbit anti-eif4g (santa cruz biotechnologies) was used for indirect immunofluorescence at 1:500; mouse anti-zika ns1 (biofront technologies) was used at 1:500 for indirect immunofluorescence and 1:1,000 for western blotting; mouse anti-brutp (enzo life sciences) was used for indirect immunofluorescence at 1:100; rabbit antiphospho eif2α (ser51) (cell signaling technology) was used for indirect immunofluorescence and 1:500 and for western blotting at 1:1,000; mouse anti-eif2α (cell signaling technology) was used for western blotting at 1:1,000; and mouse anti-actin (abcam) was used for western blotting at 1:10,000; rabbit anti-gadd34 (thermo fisher scientific) was used for western blotting at 1:1000; rabbit anti-perk antibody (cell signaling technology) was used for western blotting at 1:1000. horseradish peroxidase-conjugated secondary antibodies were purchased from rockland immunochemicals and used at 1:5,000, and alexafluor secondary antibodies were purchased from life technologies and used at 1:500. cells were lysed in np40 lysis buffer (50 mm tris ph 7.4, 150 mm nacl, 0.5 mm edta, 0.5% np40). equal amounts of protein were separated by sds-page and transferred to a nitrocellulose membrane (bio-rad). blocking was performed using 5% nonfat milk in tris-buffered saline with 0.1% tween 20 (tbst) for 1 hour at room temperature. membranes were probed with the indicated primary and appropriate horseradish peroxidase-conjugated secondary antibodies. for detection of total and phosphorylated forms of proteins, samples were run in duplicate gels and transferred to independent membranes for western blotting. membranes were probed for actin and protein levels were normalized in both membranes for the downstream densitometry analysis [26] . proteins were detected using western lightning plus-ecl (perkinelmer). for quantitation, the pixel intensity of each band was determined using the imagej program (nih) and then normalized to the indicated control. cells were prepared for indirect immunofluorescence as described previously [27] . briefly, cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% triton x-100. to prevent nonspecific binding, the cells were blocked using roche blocking solution for 30 minutes at room temperature. primary antibodies were applied followed by incubation with the appropriate secondary antibody in blocking solution. stained cells were mounted in prolong gold antifade reagent with dapi (life technologies). laser scanning confocal microscopy was performed using a leica dm16000b microscope equipped with a wavefx spinning disk confocal head (quorum technologies) using a 40x objective lens. images were acquired with a hamamatsu imageem em-charges coupled device (ccd) camera and collected as z-stacks that were rendered for image reconstruction using the imaris software (v. 8.1.3, bitplane, inc.). twenty-four hours after infection, vero cells were treated with 500 μm ars for 1 h, 2mm dtt for 1 h or 50 nm pata for 1 h or u2os cells were treated with 1 mm se for 2 h and then processed for immunofluorescence as described above. infected cells were identified by detection of viral protein ns1 or brutp labeled rna, and sg-positive cells were defined as having at least 3 sg as determined by colocalized g3bp1 and tiar or eif4g and tiar puncta. at least 150 cells were analyzed per condition in 10 to 15 fields in 3 independent experiments and the data are presented as the percentage of cells containing sg. all experiments were performed in triplicate, and the data are presented as the mean ± standard deviation (sd). a p-value <0.05 in a two-way anova test was considered statistically significant. graphpad prism 6 (graphpad software inc.) was used to conduct statistical analyses and create graphs. zikv is a positive-strand rna virus that replicates in the cytoplasm but little is known about redistribution of host proteins in zikv infected cells. sequestering sg components to sites of viral replication is a strategy used by viruses to impair sg assembly in infected cells [18] . to determine whether zikv replication altered the distribution of sg markers, vero cells were infected with zikv with an moi of 0.5 and 6, 12 and 24 hours after, nascent viral rna was labeled with brutp and detected by indirect immunofluorescence. in zikv-infected cells, viral protein or rna was not detectable to 12 hpi. tiar was evenly distributed throughout mock-infected cells (fig 1a and 1b) , and eif4g was distributed homogeneously in the cytoplasm (fig 1a and 1b) . however, in infected cells, tiar was still found in both the cytoplasm and nucleus but also concentrated in foci in the perinuclear region ( fig 1a and 1b) , colocalizing with the zikv rna ( fig 1a) and viral nonstructural protein, ns1 ( fig 1b) . no change in eif4g distribution was observed between mock and infected cells (fig 1a and 1b) . these findings suggest that zikv infection induces the redistribution of tiar to sites of viral rna replication. cleavage of proteins that nucleate sg assembly has been reported to be a strategy employed by viruses to overcome cellular stress response [28] . we next evaluated whether zikv replication alters the levels of sg markers. cells were mock infected or infected with zikv and at 24 hpi cells lysates were collected and analyzed by sds-page followed by western blotting. no alteration was observed in the levels of g3bp-1, tiar and pabp between mock and infected cells ( fig 1c) . these results indicate that zikv infection does not induce changes in the levels of sg-nucleating proteins. several viruses, including many members of flaviviridae family, have the ability to modulate sg assembly to keep the cell environment favorable to their own replication [29, 30]. we investigated whether zikv can interfere with the assembly of sg in infected cells. vero cells were infected with zikv or mock-infected and treated with sodium arsenite (ars) at 24 hours post-infection to induce cellular stress. ars is an oxidative agent that rapidly induces sg assembly through phosphorylation of eif2α [31] . sg assembly was determined by indirect immunofluorescence of tiar and eif4g and infected cells were identified by the presence of the viral protein ns1. in the absence of stress, mock-infected (blue arrows) and zikv-infected cells (red arrows) exhibited sg assembly at a rate of 0.61% and 0%, respectively (fig 2a, top panels and fig 2b) , indicating that zikv infection does not induce the assembly of sg. in mock-infected cells, ars treatment induced abundant sg assembly as expected, with 81.4% of the cells presenting tiar and eif4g co-localized in cytoplasmic puncta (fig 2a and 2b ). in contrast, zikv infected cells presented sg at a rate of only 21.6% (fig 2a, bottom panel and fig 2b) . similar results were observed when u2os cells were used in place of vero cells (s1 fig). these results indicate that zikv infection blocks the assembly of type i sgs. zikv does not block eif2α-independent assembly of stress granules pateamine a (pata) is a natural product isolated from a marine sponge that disrupts the translation initiation by hyperactivating the eif4a helicase and disrupting the eif4f complex, leading to the assembly of sg in an eif2α-independent manner [32] . to test whether zikv infection was also capable of blocking pata-induced sgs, vero cells were mock-infected or infected with zikv and at 24 hpi were treated with pata. sg assembly was determined by colocalized puncta of tiar and eif4g and infected cells were identified by the presence of the viral protein ns1. pata treatment, as expected, induced a robust sg assembly in 97.2% of the mock-infected cells (fig 3a, top panels, and 3b). interestingly, zikv infection did not impair pata-induced sg assembly, as 97.5% of the infected cells presented tiar and eif4g puncta ( fig 3a, top panels, and 3b). sodium selenite (se) promotes the assembly of type ii sg that differ from canonical sgs in their morphology, composition and mechanism of assembly, mainly by disrupting the eif4f complex formation through 4ebp1 [33] . to test whether zikv infection alters se-induced sg assembly, u2os cells (s1 fig) stably expressing gfp-g3bp1 were mock-infected or infected with zikv and at 24 hpi were treated with se. u2os cells were used in place of vero cells due to the high toxicity of se to the latter ones. sg assembly was determined by colocalized puncta of tiar and g3bp-1 and infected cells were identified by the presence of the viral protein ns1. similarly to pata-induced sg, no significant difference was observed in the assembly of sg between mock and infected cells treated with se ( fig 3a, bottom panels, and 3b). these findings indicate that zikv infection blockage of sg assembly is eif2α-dependent. many viruses modulate p-eif2α levels during replication to assure viral protein synthesis and avoid cellular stress responses. for example, coronaviruses can induce gadd34 expression to enhance pp1 activity and consequently the dephosphorylation of eif2α [34], and herpesviruses encode a viral protein that mimics the function of gadd34 [35] . we examined the phosphorylation status of eif2α in zikv infected untreated or ars treated cells. protein lysates were analyzed by western blotting using an antibody specific for eif2α phosphorylation at s51. as shown in fig 4a and 4b , little phosphorylation of eif2α was detected in mock-infected and untreated vero cells, with a slight increase in p-eif2α in zikv-infected cells. as expected, high levels of eif2α phosphorylation (40-fold increase) were observed in extracts of mock-infected cells treated with ars. however, in zikv-infected and ars treated cells, levels of eif2α phosphorylation were zikv recruits tiar to sites of viral rna replication. vero cells were infected with zikv with an moi of 0.5 and a. at 24 hpi, cells were treated with actinomycin d and the nascent viral rna was labeled with brutp and detected by immunofluorescence/laser scanning confocal microscopy (if/lscm) using a 40x objective lens; b. at 24 hpi, cells were fixed and the viral protein ns1 was detected by immunofluorescence followed by confocal microscopy. representative of 2 experiments; c. at 24 hpi, cells were lysed and lysates were analyzed for g3bp-1, tiar, pabp, ns1 and β-actin by sds-page followed by western blotting. a. vero cells were infected with zikv with an moi of 0.5 or mock-infected and treated at 24 hpi with 500 μm ars for 1 h to induce cellular stress. the sg markers tiar and eif4g were observed by if/lscm and infected cells were identified by the consistently lower (10.5-fold increase). zikv replication was confirmed by the detection of the viral protein ns1 in cell extracts. the amount of total eif2α was similar under all conditions tested (fig 4a) , indicating that zikv replication does not alter its expression. to further confirm that zikv-infected cells exhibit lower levels of p-eif2α under arsenite treatment, phosphorylation of eif2α was also analyzed by if/lscm. as shown in fig 4c, phosphorylation of eif2α is strongly induced in the cytoplasm of non-infected cells (blue arrows). in contrast, in zikvinfected cells, the phospho-eif2α signal is visibly weaker (red arrow). upon arsenite treatment, the fluorescence intensity of p-eif2α in infected cells was in average 30% lower in zikv-infected cells in comparison to mock-infected cells (fig 4d) . these results indicate that zikv infection impairs eif2α phosphorylation triggered by oxidative stress. our findings show that zikv infection blocks sg assembly and phosphorylation of eif2α triggered by ars, an hri activator. to investigate whether this blockage is dependent on the eif2α kinase activated upon stress, vero cells were mock-infected or infected with zikv and at 24 hpi were treated with dtt, an endoplasmic reticulum (er) stressor that activates perk. sg assembly was determined by tiar puncta and infected cells were identified by the presence of ns1. dtt treatment induced sg assembly in 81.8% of the mock-infected cells (fig 5a and 5b ). in contrast, only 28.6% of zikv-infected cells presented sg (fig 5a and 5b) . the blockage of sg assembly correlates with lower levels of p-eif2α upon dtt treated in zikvinfected cells (fig 5c, lane 4) when compared to mock-infected cells (fig 5c, lane 2) . interestingly, the activation of perk in dtt-treated cells, demonstrated by an increased perk mobility, was similar in mock and zikv-infected cells (fig 5c, compare lanes 2 our results suggest that zikv infection might abrogate sg assembly by blocking eif2α phosphorylation. to test this further, vero cells were infected with zikv and at 24 hpi, the levels of gadd34, a pp1a cofactor, were evaluated. our results show that are gadd34 levels are significantly higher in zikv-infected cells as compared to uninfected cells (fig 6a and 6b) . to evaluate the role of gadd34/pp1a activity on zikv-infected cells, we treated cells with salubrinal and its derivative sal003, small molecules that selectively inhibit the pp1/gadd34-mediated dephosphorylation of phospho-eif2α [36, 37] . vero cells were treated with 75 μm of salubrinal or 10 μm of sal003 for 3 h prior to the addition of ars to the cells. the phosphorylation status of eif2α was evaluated by western blotting analysis (fig 6c) . in cells treated with salubrinal prior to ars-induced stress, zikv-infected cells present higher levels of phospho-eif2α as compared to mock-infected control (fig 6c, compare lanes 6 and 8) . similar results were obtained with sal003 [37] (s2 fig). the assembly of sg in the distinct conditions was monitored by indirect immunofluorescence. sgs were induced in 23.1±6.5% of zikv-infected cells treated with ars. this value increased to 47.8±7.0% in cells that were treated with salubrinal prior to ars-induced stress and was not significantly different from mock-infected cells (fig 6d and 6e ). no significant difference was observed between control or salubrinal pre-presence of the viral protein ns1. blue arrows: uninfected cells; red arrows: infected cells. b. at least 150 cells in each condition were analyzed. cells with at least 3 sg were considered positive. data are presented as mean ± sd from 3 independent experiments. https://doi.org/10.1371/journal.pntd.0005775.g002 zika virus inhibits stress granule assembly treated mock-infected cells (fig 6e) . hence, inhibiting eif2α dephosphorylation reduces the ability of zikv infection to block ars-induced sg assembly. these results indicate that eif2α dephosphorylation is differentially modulated during zikv replication and that this feature can contribute to zikv-mediated blockage of sg assembly. to further confirm the importance of modulating eif2α for zikv replication, vero cells were infected with zikv and after 1 hpi, salubrinal was added to culture media in increasing concentrations. after 24 h, supernatants of each condition were collected and viral titer was determined by plaque forming assay and cells were lysed and lysates were processed by sds-page followed by western blotting. treatment of cells with salubrinal led to a dose-dependent decrease in the production of infectious particles released to the culture media (fig 7a, bar graph and 7b). cells treated with 75 μm of salubrinal produce only 4.9% of the infectious viral particles produced by control cells (fig 7a, bar graph and 7b ). salubrinal had no toxic effects on treated cells (fig 7a, line graph) . finally, a dose-dependent decrease in ns1 expression was observed in salubrinal treated cells (fig 7c) . the relationship between viruses and the cellular stress response is a multifaceted and complex phenomenon that depends on the structural and genetic characteristics of the virus and the host cell [38] . infection by several types of rna and dna viruses results in changes in the cellular environment as viral replication co-opts several cellular pathways, including nutrient, energy and macromolecular synthesis, to produce infectious particles. in this process, viruses trigger the host cell stress response, which can lead to the assembly of sgs [17] . since viral replication relies on the host translational machinery, most viruses suppress the stress response pathway and sg assembly at some point of their replicative cycle [18] . interactions between stress proteins and viral components have been described in a large variety of experimental models at different stages of the viral lifecycle, depending on the type of virus and host cell [29, 39] . zikv has emerged as a global public health threat over the last decade. many aspects of the molecular mechanisms involved in the pathogenesis of this emerging virus remain unclear and require further investigation. in this work, we described that zikv replication does not induce sg assembly in vero cells (fig 1) . this contrasts with the results recently published by roth and colleagues [19] that describe the assembly of sg-like structures on huh-7 cells infected with zikv. it is possible that those distinct findings are due to the usage of distinct cell lines. in our work, we also show that zikv and blocks sg assembly triggered by treatment of cells with ars (fig 2) and dtt (fig 5) . these finds are similar to the ones described recently by roth and colleagues, in which they describe that flaviviruses block sg assembly independently of the eif2α kinase activated by stress [19] . interestingly, during the review process of this manuscript, basu and colleagues [40] reported that zikv-mediated blockage of sg assembly was specific for oxidative stress induced by arsenite. the reasons why these differences a. vero cells were infected with zikv with an moi of 0.5 or mockinfected and treated at 24 hpi with 50 nm pata for 2 h to induce cellular stress. b. u2os gfp-g3bp1-expressing cells were infected with zikv or mock-infected and treated at 24 hpi with 1 mm se for 2 hours. sg assembly was determined by if/lscm staining for the sg markers tiar and eif4g (vero cells) or g3bp-1 and tiar (u2os cells) and infected cells were identified by the presence of the viral protein ns1. blue arrows: uninfected cells; red arrows: infected cells. c. at least 150 cells in each condition were analyzed. cells with at least 3 sg were considered positive. data are presented as mean ± sd from 3 independent experiments. https://doi.org/10.1371/journal.pntd.0005775.g003 a. vero cells were infected with zikv with an moi of 0.5 or mock-infected and treated at 24 hpi with 500 μm ars for 1 h to induce cellular stress. lysates were analyzed for s51-phospho(p)-eif2α, eif2α (total) and ns1 by sds-page followed by western blotting. b. densitometry quantification of p-eif2α was determined by imagej analysis. values presented in the graph are normalized against the total amount of eif2α in the cell lysate and represent fold change with the untreated mock-infected cells being arbitrarily set to 1. asterisks represent the statistically significant difference between mock and zikv-infected cells (two-way anova; p < 0.05). c. cellular stress was determined by if/lscm staining for sg markers, tiar and phosphor-eif2α and infected cells were identified by the presence of the ns1 viral protein. were observed remain to be determined. several reports have shown that members of the flaviviridae family modulate sg assembly in infected cells. the 3' stem loop from the viral minus strand of wnv and denv captures tia-1 and tiar to promote viral genome rna synthesis and inhibit sg assembly [29, 41] . jev capsid protein interaction with caprin-1 leads to the sequestration of several sg components, such as g3bp1 and usp10, in the perinuclear region of infected cells, resulting in impairment of sg assembly [30] and bovine viral diarrhea virus (bvdv) blocks ars-mediated sg assembly [42] . tia1 and tiar are recruited to tick-borne encephalitis virus (tbev) sites of replication [43] . finally, hepatitis c virus (hcv) replication leads to oscillating sg assembly/disassembly in infected cells through controlling the phosphorylation of eif2α and co-opting tia-1, tiar and g3bp1 [44, 45] . more recently, roth and colleagues [19] demonstrated that denv and zikv uncouple translation suppression from the stress response by a mechanism that is yet to be identified. we demonstrate that zikv infection did not lead to a blockage in pata or se-induced sg (fig 3) . the assembly of sg triggered by both molecules is independent of the phosphorylation of eif2α, suggesting that zikv blocks stress granules assembly mainly via eif2α signaling. interestingly, this does not seem to be a general feature of flaviviral infections, as it has been demonstrated by roth and colleagues that denv inhibits sg assembly induced by hippuristanol, an inhibitor of eif4a rna binding [19] . the phosphorylation of eif2α is a key regulator of mrna translation initiation, and the level of phospho-eif2α is modulated by the activities of kinases and phosphatases [46] . oxidative stress induced by ars culminates on eif2α phosphorylation by hri [31], which prevents the recycling of the eif2-gtp-trna met ternary complex, leading to polysome disassembly and consequent translational arrest and sg assembly [14] . regulation of protein synthesis by eif2α phosphorylation plays an important role in the cellular defense against viral infection, thus viruses evolved diverse strategies to prevent it. our results show that zikv attenuates eif2α phosphorylation triggered by ars (fig 4) and dtt ( fig 5) and this ability is, at least in part, a consequence of modulating its dephosphorylation, as supported by the observation that treatment of cells with salubrinal reverses the zikvmediated blockage of sg assembly induced by ars (fig 6) . similar to the finding of wang and colleagues using coronavirus [34], we demonstrated that zikv infection induces a moderate increase in gadd34 expression (fig 6a and 6b) . recently, buchman and colleagues [47] described a mechanism by which trehalose modulates p-eif2α levels and stress granule assembly/disassembly by enhancing the expression of gadd34 and crep. the increase in the cellular levels of the pp1 phosphatase subunits could lead to faster dephosphorylation of p-eif2α and disassembly of sgs, thereby rendering the cells able to recover more quickly from stress. it is possible that the enhanced levels of gadd34 found in zikv-infected cells play a similar role in response to stress. treatment of cells with salubrinal causes an accumulation of phospho-eif2α through an inhibition of pp1/gadd34-mediated dephosphorylation of eif2α without increasing eif2α kinase activity [36] . modulation of pp1 activity by viral infection was demonstrated for human cytomegalovirus [48] , african swine fever virus [49] , newcastle disease virus a. vero cells were infected with zikv with an moi of 0.5 or mock-infected and treated at 24 hpi with 2mm dtt for 1 h to induce cellular stress. the sg marker tiar was observed by if/lscm and infected cells were identified by the presence of ns1. blue arrows: uninfected cells; red arrows: infected cells. b. at least 150 cells in each condition were analyzed. cells with at least 3 sg were considered positive. data are presented as mean ± sd from 3 independent experiments. c. after dtt treatment, cells were lysed and lysates were analyzed for perk, s51-phospho(p)-eif2α, eif2α (total) and actin by sds-page followed by western blotting. densitometry quantification of p-eif2α was determined by imagej analysis. values presented are normalized against the total amount of eif2α in the cell lysate and represent fold change with the untreated mock-infected cells being arbitrarily set to 1. https://doi.org/10.1371/journal.pntd.0005775.g005 [50] , papillomavirus [51] and herpes simplex virus [52] . icp34.5 is a protein homologous to gadd34 encoded by hsv that is essential for hsv replication in some cell types. it binds cellular pp1 and promotes eif2α dephosphorylation, ensuring viral replication despite activation of pkr [35] . treatment of hsv-infected cells with salubrinal inhibits viral replication in a dose-dependent manner and leads to higher phospho-eif2α levels [36, 52] . similarly, our results demonstrate that zikv replication is severely impaired in salubrinal-treated cells (fig 7) , indicating that zikv relies to some extent on eif2α dephosphorylation for its replication. these findings are distinct from the model proposed by roth and colleagues [19] , in which modulation of sg assembly in zikv-infected cells was independent of eif2α dephosphorylation promoted by elevated gadd34 levels. cell typefig 6. zikv modulates eif2α dephosphorylation. a. vero cells were infected with zikv at an moi of 0.5 or mock-infected. at 24 hpi, cells were lysed and cells were lysed and lysates were analyzed for gadd34 and actin by sds-page followed by western blotting. b. densitometry quantification of gadd34 and actin were determined by imagej analysis. values presented are normalized against the total amount of gadd34 in the cell lysate and represent fold change with the mock-infected cells being arbitrarily set to 1. c. vero cells were infected with zikv or mock-infected and treated at 24 hpi with 75 μm salubrinal for 3 h to block the dephosphorylation of eif2α and then treated with 500 μm ars for 1 h to induce cellular stress. lysates were analyzed for s51-phospho(p)-eif2α and eif2α (total) by sds-page followed by western blotting. values of p-eif2α fold change were normalized by the corresponding eif2α levels of the same condition. d. vero cells were infected with zikv or mock-infected and at 24 hpi were treated with 75 μm salubrinal for 3 h and then oxidative stress was induced by treatment with 500 μm ars for 1 h. sg assembly was determined by if/lscm staining for the sg markers tiar and eif4g and infected cells were identified by the presence of the viral protein ns1. blue arrows: uninfected cells; red arrows: infected cells.e. at least 150 cells in each condition were analyzed. cells with at least 3 sg were considered positive. data are presented as mean ± sd from 3 independent experiments and asterisks represent the statistically significant difference between mock and zikv-infected cells (two-way anova; p < 0.05). https://doi.org/10.1371/journal.pntd.0005775.g006 specificities can be responsible for those contrasting results. it remains to be determined whether treatment with salubrinal has secondary effects on the infected cells that could act in synergy with the pp1/gadd34 inhibition and the mechanism by which zikv modulates this activity. in conclusion, our work provides new insights into the zikv biology by demonstrating that zikv inhibits sg assembly in a phospho-eif2α dependent way. this ability may reflect one of the many strategies that zikv has evolved to control the host stress response and demonstrate that zikv elicits mechanisms to counteract host anti-viral stress responses to promote a cellular environment propitious for viral replication. elucidation of the interaction of viral components with host factors involved in sg assembly may provide new insights into the pathology of zikv infection and lead to the identification of novel targets for therapeutic intervention. a. u2os cells were infected with zikv with an moi of 0.5 or mock-infected and treated at 24 hpi with 500 μm ars for 1 h to induce cellular stress. the sg markers g3bp-1 and eif4g were observed by if/ lscm and infected cells were identified by the presence of the viral protein ns1. blue arrows: uninfected cells; red arrows: infected cells. b. at least 150 cells in each condition were analyzed. cells with at least 3 sg were considered positive. data are presented as mean ± sd from 3 independent experiments. c. u2os cells were infected with zikv with an moi of 0.5 or mock-infected and treated at 24 hpi with 500 μm ars for 1 h to induce cellular stress. lysates were analyzed for s51-phospho(p)-eif2α and eif2α (total) by sds-page followed by western blotting. b. densitometry quantification of p-eif2α was determined by imagej analysis. values presented in the graph 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salubrinal in herpes simplex virus-1 infected cells we would like to thank gary pignac-kobinger, nancy kedersha, paul anderson, jerry pelletier and colin crist for generous provision of cell lines, viruses and reagents, meijuan niu for technical help, and shringar rao and alessandro cinti for helpful comments on the manuscript. key: cord-309587-xc4jaw31 authors: lembo, tiziana; hampson, katie; kaare, magai t.; ernest, eblate; knobel, darryn; kazwala, rudovick r.; haydon, daniel t.; cleaveland, sarah title: the feasibility of canine rabies elimination in africa: dispelling doubts with data date: 2010-02-23 journal: plos negl trop dis doi: 10.1371/journal.pntd.0000626 sha: doc_id: 309587 cord_uid: xc4jaw31 background: canine rabies causes many thousands of human deaths every year in africa, and continues to increase throughout much of the continent. methodology/principal findings: this paper identifies four common reasons given for the lack of effective canine rabies control in africa: (a) a low priority given for disease control as a result of lack of awareness of the rabies burden; (b) epidemiological constraints such as uncertainties about the required levels of vaccination coverage and the possibility of sustained cycles of infection in wildlife; (c) operational constraints including accessibility of dogs for vaccination and insufficient knowledge of dog population sizes for planning of vaccination campaigns; and (d) limited resources for implementation of rabies surveillance and control. we address each of these issues in turn, presenting data from field studies and modelling approaches used in tanzania, including burden of disease evaluations, detailed epidemiological studies, operational data from vaccination campaigns in different demographic and ecological settings, and economic analyses of the cost-effectiveness of dog vaccination for human rabies prevention. conclusions/significance: we conclude that there are no insurmountable problems to canine rabies control in most of africa; that elimination of canine rabies is epidemiologically and practically feasible through mass vaccination of domestic dogs; and that domestic dog vaccination provides a cost-effective approach to the prevention and elimination of human rabies deaths. rabies is a viral zoonosis caused by negative-stranded rna viruses from the lyssavirus genus. genetic variants of the genotype 1 lyssavirus (the cause of classical rabies) are maintained in different parts of the world by different reservoir hosts within 'host-adaptive landscapes' [1] . although rabies can infect and be transmitted by a wide range of mammals, reservoirs comprise only mammalian species within the orders carnivora (e.g. dogs, raccoons, skunks, foxes, jackals) and chiroptera (bats). from the perspective of human rabies, the vast majority of human cases (.90%) result from the bites of rabid domestic dogs [2] and occur in regions where domestic dogs are the principal maintenance host [3] . over the past three decades, there have been marked differences in efforts to control canine rabies. recent successes have been demonstrated in many parts of central and south america, where canine rabies has been brought under control through large-scale, synchronized mass dog vaccination campaigns [4] . as a result, not only has dog rabies declined, but human rabies deaths have also been eliminated, or cases remain highly localized [5] . the contrast with the situation in africa and asia is striking; here, the incidence of dog rabies and human rabies deaths continue to escalate, and new outbreaks have been occurring in areas previously free of the disease (e.g. the islands of flores and bali in indonesia [6] ; http://wwwn.cdc.gov/travel/ contentrabiesbaliindonesia2008.aspx). in this paper, we identify four major reasons commonly given for the lack of effective domestic dog rabies control including (1) low prioritisation, (2) epidemiological constraints, (3) operational constraints and (4) lack of resources (table 1) , focussing on the situation in africa. we address each of these issues in turn, using outputs from modelling approaches and data from field studies to demonstrate that there are no insurmountable logistic, practical, epidemiological, ecological or economic obstacles. as a result, we conclude that the elimination of canine rabies is a feasible objective for much of africa and there should be no reasons for further delay in preventing the unnecessary tragedy of human rabies deaths. this paper compiles previously published data (see references below) and additional analyses of those data, but we present a brief summary of the data collection methods below. hospital records of animal-bite injuries compiled from northwest tanzania were used as primary data sources. these data informed a probability decision tree model for a national disease burden evaluation [7] , which has since been adapted for global estimates of human rabies deaths and disability-adjusted life years (dalys) lost due to rabies [3] , a standardized measure for assessing disease burden [8, 9] . hospital records were also used to initiate contact tracing studies [10] [11] [12] , whereby bite-victims were interviewed to obtain more detail on the source and severity of exposure and actions taken, allowing subsequent interviews with other affected individuals (not documented in hospital records) including owners of implicated animals. statistical techniques applied to these data for estimating epidemiological parameters and inferring transmission links are described elsewhere [10, 12] . rabies monitoring operations including passive and active surveillance involving veterinarians, village livestock field officers, paravets, rangers and scientists were used to collect samples from carcasses (domestic dogs and wildlife whenever found), which were subsequently tested and viral isolates were sequenced [10, [13] [14] [15] [16] , with results being used to inform estimates of rabies-recognition probabilities [7] and for phylogenetic analyses [10, 16] . operational research on domestic dog vaccination strategies was carried out in a variety of settings [14, 17] . household interviews were also used for socio-economic surveys and to evaluate human:domestic elimination of canine rabies has been achieved in some parts of the world, but the disease still kills many thousands of people each year in africa. here we counter common arguments given for the lack of effective canine rabies control in africa presenting detailed data from a range of settings. we conclude that (1) rabies substantially affects public and animal health sectors, hence regional and national priorities for control ought to be higher, (2) for practical purposes domestic dogs are the sole maintenance hosts and main source of infection for humans throughout most of africa and asia and sufficient levels of vaccination coverage in domestic dog populations should lead to elimination of canine rabies in most areas, (3) the vast majority of domestic dog populations across sub-saharan africa are accessible for vaccination with community sensitization being of paramount importance for the success of these programs, (4) improved local capacity in rabies surveillance and diagnostics will help evaluate the impact of control and elimination efforts, and (5) sustainable resources for effective dog vaccination campaigns are likely to be available through the development of intersectoral financing schemes involving both medical and veterinary sectors. dog ratios, levels of vaccination coverage achieved and reasons for not bringing animals to vaccination stations [17, 18] . the study was approved by the tanzania commission for science and technology with ethical review from the national institute for medical research (nimr). this retrospective study involved collection of interview data only, without clinical intervention or sampling, therefore we considered that informed verbal consent was appropriate and this was approved by nimr. permission to conduct interviews was obtained from district officials, village and sub-village leaders in all study locations. at each household visited, the head of the household was informed about the purpose of the study and interviews were conducted with verbal consent from both the head of the household and the bite victim (documented in a spreadsheet). approval for animal work was obtained from the institutional animal care and use committee (iacuc permit #0107a04903). (a) there is not enough evidence to define rabies control as a priority a principal factor contributing to a low prioritization of rabies control has been the lack of information about the burden and impact of the disease [19, 20] . data on human rabies deaths, submitted from ministries of health to the world health organization (who), are published in the annual world surveys of rabies and through the who rabnet site (www.who.int/ rabies/rabnet/en). for the who african region (afro) comprising 37 countries, these surveys report an average of 162 human deaths per year between 1988 and 2006. it is therefore unsurprising that for national and international policy-makers, rabies pails into insignificance in comparison with other major disease problems. this perceived lack of significance of human rabies is reflected in the absence of any mention of rabies in either of the two published global burden of disease surveys [21, 22] , which assessed more than 100 major diseases. these surveys adopted the metric of the daly which is widely used as the principal tool for providing consistent, comparative information on disease burden for policy-making. until recently no estimates of the daly burden were available for rabies. official data on human rabies deaths submitted to who from africa are widely recognized to greatly under-estimate the true incidence of disease. the reasons for this are manifold: (1) rabies victims are often too ill to travel to hospital or die before arrival, (2) families recognize the futility of medical treatment for rabies, (3) patients are considered to be the victims of bewitchment rather than disease, (4) clinically recognized cases at hospitals may go unreported to central authorities, and (5) misdiagnosis is not uncommon. the problems of misdiagnosis were highlighted by a study of childhood encephalitis in malawi, in which 3/26 (11.5%) cases initially diagnosed as cerebral malaria were confirmed as rabies through post-mortem tests [23] . several recent studies have contributed information that consistently demonstrates that the burden of canine rabies is not insubstantial. human rabies deaths. estimates of human rabies cases from modeling approaches, using the incidence of dog-bite injuries and availability of rabies post-exposure prophylaxis (pep), indicate that incidence in africa is about 100 times higher than officially reported, with ,24,000 deaths in africa each year [3, 7] . consistent figures have subsequently been generated from detailed contact-tracing data: in rural tanzanian communities with sporadic availability of pep (a typical scenario in developing countries), human rabies deaths occur at an incidence of ,1-5 cases/100,000/year (equivalent to 380-1,900 deaths per year for tanzania) [11] . similarly, a multi-centric study from india reported 18,500 human rabies deaths per year [24] , consistent with model outputs of 19,700 deaths for india [3] . a crude comparison of annual human deaths for a range of zoonotic diseases is shown in figure 1 (top). while diseases such as severe acute respiratory syndrome (sars), rift valley fever and highly pathogenic avian influenza cause major concerns as a result of pandemic potential and economic losses, these figures provide a salutary reminder of the recurrent annual mortality of rabies and other neglected zoonoses, such as leishmaniasis and human african trypanosomiasis (hat). decision-tree models applied to data from east africa and globally indicate that the daly burden for rabies exceeds that of most other neglected zoonotic diseases (figure 1 -bottom) [3, 25, 26] . human animal-bite injuries and morbidity. most of the rabies daly burden is attributed to deaths, rather than morbidity because of the short duration of clinical disease. the daly burden for rabies is particularly high, because most deaths occur in children and therefore a greater number of years of life are lost [25, 27] . daly estimates incorporate non-rabies mortality and morbidity in terms of adverse reactions to nerve-tissue vaccines (ntvs) [3] , which are still widely used in some developing countries such as ethiopia, however rabies also causes substantial 'morbidity' as a direct result of injuries inflicted by rabid animals, and this is not included in daly estimates. contact-tracing studies suggest an incidence as high as 140/ 100,000 bites by suspected rabid animals in rural communities of tanzania [11] . thus, for every human rabies death there are typically more than ten other rabid animal-bite victims who do not develop signs of rabies, because they obtain pep (figure 1 bottom) or are simply fortunate to remain healthy. the severity of wounds has not yet been quantified, but case-history interviews suggest that injuries often involve multiple, penetrating wounds that require medical treatment. economic burden. the major component of the economic burden of rabies relates to high costs of pep, which impacts both government and household budgets. with the phasing out of ntvs, many countries spend millions of dollars importing supplies of tissue-culture vaccine (,$196 million usd pa [3] ). at the household level, costs of pep arise directly from anti-rabies vaccines and from high indirect (patient-borne) costs associated with travel (particularly given the requirement of multiple hospital visits), medical fees and income loss [3, 28] . indirect losses, represent .50% of total costs ( figure 2 ). total costs have been estimated conservatively at $40 us per treatment in africa and $49 us in asia accounting respectively for 5.8% and 3.9% of annual per capita gross national income [3] . poor households face difficulties raising funds which results in considerable financial hardship and substantial delays in pep delivery [11, 28] . shortages of pep, which are frequent in much of africa, further increase costs as bite victims are forced to travel to multiple centres to obtain treatment, also resulting in risky delays [11] . additional economic losses relate to livestock losses derived from an incidence of 5 deaths/100,000 cattle estimated to cost $12.3 million annually in africa and asia [3] . however, substantially higher incidence has been recorded in tanzania, with 12-25 cases/100,000 cattle reported annually in rural communities (hampson, unpublished) . canine rabies introduced from sympatric domestic dog populations is also recognized as a major threat to endangered african wild dogs (lycaon pictus) and ethiopian wolves (canis simensis) [29] [30] [31] [32] . potential losses of tourism revenue may be substantial; african wild dogs are a major attraction in south africa national parks with the value of a single pack estimated at $9,000 per year [33] and ethiopian wolves are a flagship species for the bale mountains national park. psychological impact. an important, but often underappreciated component of disease burden is the psychological impact on bite-victims and their families. in rural tanzania, .87% of households with dog bite victims feared a bite from a suspected rabid animal more than malaria [28] because malaria can be treated whereas clinical rabies is invariably fatal and malaria treatment is generally affordable and available locally in comparison to pep. when human rabies cases occur, the horrifying symptoms and invariably fatal outcome result in substantial trauma for families, communities and health care workers [34] . increasing incidence of rabies in africa has prompted concerns that the epidemiology of the disease may be more complex, involving abundant wildlife carnivores that may sustain infection cycles [13, [35] [36] [37] [38] . there is also uncertainty about the level of vaccination coverage needed to control rabies particularly in rapidly growing domestic dog populations [39, 40] . to eliminate infection, disease control efforts need to be targeted at the maintenance population [41] . this is clearly demonstrated for fox rabies in western europe, whereby control of rabies in foxes (through mass oral vaccination) has led to the disappearance of rabies from all other 'spill-over' hosts [42] . despite the predominance of domestic dog rabies in africa, the role of wildlife as independent maintenance hosts has been debated, and many perceive the abundance of wildlife as a barrier to elimination of canine rabies on the continent. it has also been argued that the predominance of dog rabies is an artefact of poor surveillance and under-reporting in wildlife populations [43] . in the wildlife-rich serengeti ecosystem in tanzania, evidence suggests that domestic dogs are the only population essential for maintenance [10, 13, 16] : (1) phylogenetic data showed only a single southern africa canid-associated variant (africa 1b) circulating among different hosts [16] ; (2) transmission networks suggested that, for wildlife hosts, within-species transmission cannot be sustained [16] ; and (3) statistical inference indicated that cross-species transmission events from domestic dogs resulted in only relatively short-lived chains of transmission in wildlife with no evidence for persistence [10] . the conclusion that domestic dogs are the only maintenance population in such a species-rich community suggests that elimination of canine rabies through domestic dog vaccination is a realistic possibility, and provides grounds for optimism for wider-scale elimination efforts in africa. in other parts of central and west africa, transmission of rabies appears to be driven by domestic dogs [44] . an outstanding question relates to southern africa. earlier and recent evidence indicate that jackal species (canis mesomelas and c. adustus) and bat-eared foxes (otocyon megalotis) may maintain the canid variant in specific geographic loci in south africa and zimbabwe [2, [36] [37] [38] [45] [46] [47] [48] [49] [50] , but it is still not clear whether these cycles can be sustained over large spatial and temporal scales in the absence of dog rabies [13, 51, 52] . independent wildlife cycles may preclude continent-wide elimination of this variant through dog vaccination alone and wildlife rabies control strategies, in conjunction with dog vaccination, may need to be considered in specific locations [38] . a critical proportion of the population must be protected (p crit ) to eliminate infection and this threshold can be calculated from the basic reproductive number (r 0 , defined as the average number of secondary infections caused by an infected individual in a susceptible population) [53] . vaccinating a large enough proportion of the population to exceed p crit will not only protect the vaccinated individuals but will reduce transmission such that, on average, less than one secondary infection will result from each primary case (effective reproductive number, r e ,1), which can ultimately lead to elimination. vaccination has eliminated canine rabies in many countries demonstrating the success of this concept [54] . however, theory suggests that r 0 increases with population density [39] and thus higher coverage will be needed in higher density populations. however, evaluation of historical outbreak data from around the world and recent data from tanzania indicate that r 0 in domestic dog populations is consistently low (between 1.0 and 2.0) [12] , confirming the feasibility of rabies elimination through vaccination in african domestic dog populations. an important conclusion of this study was that in populations with rapid turnover (such as those in many african countries) at least 60% of the population must be vaccinated during annual campaigns to prevent coverage falling below p crit between campaigns. data from africa clearly show that very few control efforts have reached these levels of coverage [ table 2 ], which is why rabies remains a persistent problem [12] . although emergence of new variants maintained in wildlife also remains a possibility, as shown in the usa, where wildlife rabies now dominates since elimination of canine rabies [55] . for africa, these questions are likely only to be resolved with large-scale intervention involving mass vaccination of dogs. several arguments are given for why mass vaccination campaigns have failed to achieve the high levels of coverage that are necessary to interrupt rabies transmission. we counter these arguments below: a perception of many inaccessible stray/ownerless dogs. a common claim is that the majority of dogs in africa are unowned 'stray' animals, and therefore inaccessible for parenteral vaccination. it is not hard to see why this perception has arisen -unrestrained dogs, without any apparent evidence of ownership, are commonly observed. further investigation, however, usually reveals that the vast majority are owned, and at least one household claims some responsibility, including presentation for vaccination. published studies in africa, which quantify the proportion of unowned dogs, are admittedly sparse, but all support this observation [56] [57] [58] . capture-mark-recapture methodologies and household questionnaires used in african settings have all found consistently low estimates (tunisia ,7% [57] , 1%, 8% and 11% in three sites in n'djamena, chad [56] , and 1% in a peri-urban site in tanzania [58] ). notably, the tanzanian site was selected specifically on the basis of reports of many unowned dogs. while mark-recapture methods yield reliable estimates of unowned dog numbers, their implementation and analysis is not trivial and efforts are underway to develop simpler, yet robust methodologies [59] . certainly in traditional africa, i.e. most of sub-saharan africa, the issue of roaming dogs seems not to be one of a lack of ownership, but rather an inability or unwillingness by owners to confine their dogs. unwillingness/inability to bring dogs for vaccination. published studies tend to refute the idea that owners are often unable or unwilling to restrain their dogs for parenteral vaccination. a multi-country who-commissioned study (tunisia, sri lanka and ecuador) concluded that ''dogs which are not catchable by at least one person are rare and represent generally less than 15% of the dog population'' [57] . similarly a study from nepal found that 86-97% of dogs were accessible to parenteral vaccination [60] . although an early study in turkey concluded that 48% of all free-roaming owned dogs could not be captured by their owners [61] , more recent surveys found that most unvaccinated dogs could be handled (only 16% could not) and that a much larger proportion (56%) resulted from a lack of information about the campaign -a much easier problem to remedy (unpublished data). in africa, very similar figures were obtained in a multi-site study in urban and rural tanzania, where only 15% of vaccination failures were due to a reported inability by the owner to handle the dog, while 53% of cases were due to poor information dissemination [17] . however, there may be settings in transitional africa (e.g. parts of southern africa including kwazulu natal [2] ) where handling of dogs is more difficult due to a break-down in traditional animal husbandry and other social factors, and more intensive efforts may be required for these special cases. given that most dogs are accessible for parenteral vaccination, high coverage can be achieved with well-planned vaccination campaigns. during pilot programmes in urban and rural africa which have not charged owners for vaccination, coverages obtained have exceeded 60% [14, 17, 56] . pastoral communities pose particular challenges due to remote locations and seminomadic lifestyles, but .80% coverages can still be achieved through house-to-house delivery strategies or community-based animal health workers [17] . young pups usually make up a large proportion (.30%) of african dog populations [62] and there is a widespread perception among veterinary authorities and dog owners that they should not be vaccinated, which leads to insufficient coverage [17] . however, rabies vaccines can safely be administered to pups ,3 months of age [63] , and in village campaigns in tanzania, vaccines consistently induced high levels (.0.5 iu/ml) of rabies virus neutralizing antibody [64] . the issue of inclusion of pups can effectively be addressed through appropriate advertising before campaigns. cost-recovery, through charging dog owners for rabies vaccination, is widely promoted for sustainable programmes and to encourage responsible dog ownership. however, charging for a vaccination that represents a public rather than a private good, can be counterproductive, resulting in low turnouts and coverage (,30%) with little or no impact [65] . charging for vaccination may indeed be the principal reason why owners are unwilling to bring dogs for vaccination. ineffective campaigns that achieve ,30% coverage are a waste of resources and can be highly demoralising for veterinary staff and communities. when resources are spread thinly, such that only low coverage is achieved or only small pockets are well vaccinated, then large-scale failure is inevitable. a more epidemiologically sensible strategy is to focus resources into a single (preferably well-bounded) area where high coverage can be consistently achieved. uncertainty about dog population sizes and ecology for effective design and planning of vaccination campaigns. official figures used for planning frequently underestimate true population sizes. for example, gsell [58] found that the owned dog population in a municipality in tanzania was six times larger than official records. although standard survey methodologies for estimating dogs/household or dog:human ratios [57, [66] [67] [68] are not without problems (for example, double ownership of dogs), a rough estimate of owned dog populations can be derived from national (human) population censuses, and can be corrected for different demographic and ecological settings [18, 69] . more detailed studies can be conducted to identify key household determinants of dog ownership (for example, religion, age and sex of household heads, household size, socio-economic level, and livestock presence/absence [18, 28, 70, 71] . such determinants have been used to generate a 'dog density' map of tanzania, for assistance in planning national rabies vaccination campaigns ( figure 3 ). the above factors are all generally described as obstacles that ultimately lead to a lack of investment into rabies control and surveillance. we suggest that investment would actually reap multiple benefits including economic ones, if appropriate strategies are implemented overcoming the constraints described. a lack of surveillance and diagnostic capacity for rabies detection. poor surveillance and diagnosis capacity means that (1) data is insufficient to demonstrate disease burden and motivate policy-makers, and (2) impacts of control efforts cannot be evaluated. considerable progress has been made in the development of simple and inexpensive techniques for sample preservation and rapid post-mortem diagnosis suitable for laboratories with limited storage and/or diagnostic resources with potential to increase incountry capabilities for surveillance. a new direct rapid immunohistochemical test (drit) requires only light microscopes [72] , which are widely available. the test is simple and can be performed by a range of operators if appropriate training is provided. field evaluation studies in africa demonstrated that this assay has characteristics equivalent to those of the direct fluorescent antibody (dfa) test, the global standard for rabies diagnosis, including excellent performance on glycerolated field brain material [15, 73] , the preservative of choice under field conditions [74, 75] . other simple field-diagnostics that allow rapid screening, including enzyme immunoassays [76] , dot blot enzyme immunoassays [77] and lateral-flow immunodiagnostic test kits [78, 79] are being evaluated. these tools offer hope of extending diagnostic capacity in resource-limited settings. animal-bite injury data from hospitals are an easily accessible source of epidemiological information and have been verified as reliable indicators of animal rabies incidence and human exposures [11, 14] . furthermore, increasing availability of communication infrastructure through mobile phone network access in remote areas could enhance surveillance by allowing real-time reporting. costs of effective dog vaccination campaigns are beyond the budget of veterinary services. veterinary services in africa usually report very limited budgets and often have to divert resources during outbreaks of other diseases [80, 81] . this is clearly the most significant constraint to effective rabies control. however, with increasing human and dog populations, dog rabies incidence, human exposures to rabies and the costs required to prevent human rabies deaths through pep will invariably continue to rise unless rabies can be controlled at the source, i.e. in domestic dog populations [82] . many countries in asia, such as thailand, vietnam and sri lanka have greatly reduced human rabies deaths through increased pep use, but at a very high cost [83] . in vietnam, for example, deaths fell from 285 in 1996 to 82 in 2006 with administration of .600,000 pep courses per year at an estimated cost of ,$27 million/year [84] . although domestic dog populations need to be targeted for the effective control of rabies, this is usually deemed to be the responsibility of veterinary services even though many of the benefits accrue to the medical sector. in rural tanzania, dog vaccination campaigns led to a rapid and dramatic decline in demand for costly human pep [14] . in pastoral communities, vaccination not only reduced rabies incidence, but has now resulted in a complete absence of exposures reported in local hospitals for over two years (figure 4) . large-scale campaigns can therefore translate into human lives and economic savings through reduced demand for pep. costs per dog vaccinated are generally estimated to be low (rural tanzania ,$1.73 [17] , philippines ,$1.19-4.27 [85] , tunisia ,$1.3 [86] , thailand ,$1.3 [86] and urban chad ,$1.8 [87] ) and preliminary studies suggest that including dog vaccination in human rabies prevention strategies would be a highly cost-effective intervention at ,us $25/daly averted (s. cleaveland, unpublished data; see also 82) . developing joint financing schemes for rabies prevention and control across medical and veterinary sectors would provide a mechanism to use savings in human pep to sustain rabies control programs in domestic dogs. although conceptually simple, the integration of budgets across different ministries is likely to pose political and administrative challenges. however, given sufficient political will and commitment, developing sustained programmes of dog vaccination that result in canine rabies elimination should be possible. in conclusion, here we show that a substantial body of epidemiological data have now been gathered through multiple studies demonstrating that: (1) rabies is an important disease that exerts a substantial burden on human and animal health, local and national economies and wildlife conservation, (2) domestic dogs are the sole population responsible for rabies maintenance and main source of infection for humans throughout most of africa and asia and therefore control of dog rabies should eliminate the disease, (3) elimination of rabies through domestic dog vaccination is epidemiologically feasible, (4) the vast majority of domestic dog populations across sub-saharan africa are accessible for vaccination and the few remaining factors compromising coverage can be addressed by engaging communities through education and awareness programs, (5) new diagnostic and surveillance approaches will help evaluate the impact of interventions and focus efforts towards elimination, and (6) dog rabies control is 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kathmandu valley report of the 3 rd consultation on oral immunization of dogs against rabies. geneva: world health organization (who/rab dog rabies and its control is it possible to vaccinate young canids against rabies and to protect them? proceedings of the sixth southern and eastern african rabies group/world health organization meeting welfare and health assessment of tanzanian dogs and its relationship to immunological response to rabies vaccination owner valuation of rabies vaccination of dogs a dog ecology study in urban and semi-rural area of zambia the epidemiology and control of domestic dog rabies ecological and epidemiological data requirements for the planning of dog rabies control colombo humane dog population and rabies management project aspects of dog ownership and canine rabies control in africa and asia dog ecology and demography information to support the planning of rabies control in machakos district standard operating procedure for the direct rapid immunohistochemistry test for the detection of rabies virus antigen. national laboratory training network course. atlanta: us department of health and human services rabies diagnosis for developing countries simplified technique for the collection, storage and shipment of brain specimens for rabies diagnosis. who/rab.res/88 simple technique for the collection and shipment of brain specimens for rabies diagnosis development and evaluation of an enzyme immunoassay for rapid diagnosis of rabies in humans and animals rapid diagnosis of rabies in humans and animals by a dot blot enzyme immunoassay evaluation of a rapid immunodiagnostic test kit for rabies virus a simple and rapid immunochromatographic test kit for rabies diagnosis proceedings of the sixth southern and eastern african rabies group/world health organization meeting proceedings of the seventh southern and eastern african rabies group/world health organization meeting transmission dynamics and economics of rabies control in dogs and humans in an african city preventing the incurable: asian rabies experts advocate rabies control report to the world health organization. dog rabies elimination demonstration project rabies control in the republic of the philippines: benefits and costs of elimination economics of human and canine rabies eliminationguidelines for programme orientation costdescription of a pilot parenteral vaccination campaign against rabies in dogs in n'djamã©na, chad we are indebted to the ministries of livestock development and fisheries and of public health and social welfare, tanzania national parks, tanzania wildlife research institute, ngorongoro conservation area authority, tanzania commission for science and technology, and national institute for medical research for permission and collaboration; the frankfurt zoological society and the mwanza and arusha veterinary key: cord-286255-ded5t1ai authors: tomashek, kay m.; lorenzi, olga d.; andújar-pérez, doris a.; torres-velásquez, brenda c.; hunsperger, elizabeth a.; munoz-jordan, jorge luis; perez-padilla, janice; rivera, aidsa; gonzalez-zeno, gladys e.; sharp, tyler m.; galloway, renee l.; glass elrod, mindy; mathis, demetrius l.; oberste, m. steven; nix, w. allan; henderson, elizabeth; mcquiston, jennifer; singleton, joseph; kato, cecilia; garcía gubern, carlos; santiago-rivera, william; cruz-correa, jesús; muns-sosa, robert; ortiz-rivera, juan d.; jiménez, gerson; galarza, ivonne e.; horiuchi, kalanthe; margolis, harold s.; alvarado, luisa i. title: clinical and epidemiologic characteristics of dengue and other etiologic agents among patients with acute febrile illness, puerto rico, 2012–2015 date: 2017-09-13 journal: plos negl trop dis doi: 10.1371/journal.pntd.0005859 sha: doc_id: 286255 cord_uid: ded5t1ai identifying etiologies of acute febrile illnesses (afi) is challenging due to non-specific presentation and limited availability of diagnostics. prospective afi studies provide a methodology to describe the syndrome by age and etiology, findings that can be used to develop case definitions and multiplexed diagnostics to optimize management. we conducted a 3-year prospective afi study in puerto rico. patients with fever ≤7 days were offered enrollment, and clinical data and specimens were collected at enrollment and upon discharge or follow-up. blood and oro-nasopharyngeal specimens were tested by rt-pcr and immunodiagnostic methods for infection with dengue viruses (denv) 1–4, chikungunya virus (chikv), influenza a and b viruses (flu a/b), 12 other respiratory viruses (orv), enterovirus, leptospira spp., and burkholderia pseudomallei. clinical presentation and laboratory findings of participants infected with denv were compared to those infected with chikv, flu a/b, and orv. clinical predictors of laboratory-positive dengue compared to all other afi etiologies were determined by age and day post-illness onset (dpo) at presentation. of 8,996 participants enrolled from may 7, 2012 through may 6, 2015, more than half (54.8%, 4,930) had a pathogen detected. pathogens most frequently detected were chikv (1,635, 18.2%), flu a/b (1,074, 11.9%), denv 1–4 (970, 10.8%), and orv (904, 10.3%). participants with denv infection presented later and a higher proportion were hospitalized than those with other diagnoses (46.7% versus 27.3% with orv, 18.8% with flu a/b, and 11.2% with chikv). predictors of dengue in participants presenting <3 dpo included leukopenia, thrombocytopenia, headache, eye pain, nausea, and dizziness, while negative predictors were irritability and rhinorrhea. predictors of dengue in participants presenting 3–5 dpo were leukopenia, thrombocytopenia, facial/neck erythema, nausea, eye pain, signs of poor circulation, and diarrhea; presence of rhinorrhea, cough, and red conjunctiva predicted non-dengue afi. by enrolling febrile patients at clinical presentation, we identified unbiased predictors of laboratory-positive dengue as compared to other common causes of afi. these findings can be used to assist in early identification of dengue patients, as well as direct anticipatory guidance and timely initiation of correct clinical management. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 as malaria incidence continues to decrease throughout the tropics, a new area of research has focused on identifying etiologies of non-malaria, acute febrile illness (afi) [1, 2] . knowledge is limited in this area, in large part because afis often have similar non-specific clinical presentations early in the clinical course when most patients are likely to present for care. in addition, rapid point-of-care diagnostics are often not readily available. surveillance for afis, if done, is largely passive and relies on clinical identification of cases and voluntary case reporting. therefore, burden of disease for the etiologic agents of afi are likely underestimated. an improved understanding of the major causes of afi is important to guide clinical management, develop diagnostics, inform public health policy, and direct prevention efforts [3] . in mexico, south and central america, and the caribbean, afi are common among patients of all age groups. in the last four decades, dengue, a mosquito-borne afi caused by four genetically distinct dengue viruses (denv 1-4), has become an increasingly common cause of afi [4, 5] . the burden of dengue is thought to be less in latin america than in southeast asia [6] ; however, several studies have found that the incidence of dengue is likely underestimated in latin america due to reliance on passive case surveillance [6] [7] [8] . understanding region-specific etiologies of afi and estimating the true incidence of dengue is necessary to plan large scale interventional trials for assessing the impact of mosquito control measures and vaccines. in addition, collecting clinical signs and symptoms from afi patients of all ages with identified etiologic agents has utility in developing unbiased case definitions and identifying early clinical predictors to guide clinical management. prospective studies enrolling patients with afi provide a methodology to systematically identify causes of afi in a population and describe variation in the clinical course by patient age and etiologic agents. since 2000, nine such studies have evaluated afis including dengue among both pediatric and adult patients [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . while these studies are comparable in many ways, the studies differ in that several excluded either infants and young children [9] [10] [11] [12] [13] 16] , older adults [12, 13] , severe or hospitalized cases [13, 16] , or cases with a known source of fever [9] [10] [11] [12] 14] . in addition, most studies were conducted in low resource settings in southeast asia where malaria is still endemic [9, 10, [12] [13] [14] [15] [16] [17] [18] . in fact, two studies enrolled based on a potential participant meeting national eligibility criteria for malaria testing [12, 13] , and two other studies excluded cases based on malaria blood smear positivity [14, 15] . in this manuscript, we describe a 3-year prospective study of afi among all age groups that used a pre-defined diagnostic testing algorithm for denv 1-4 and 21 other pathogens. we conducted this study in puerto rico, where malaria was eradicated in 1962 [19] and dengue has been endemic since the late 1960s [20] . we describe the frequency of dengue and other afis, and the distribution of these diseases in terms of person, place and seasonality. last, we describe clinical predictors of dengue by timing of presentation compared to other afis. the study was conducted in southern puerto rico at saint luke's episcopal hospital (sleh)-ponce, a 425-inpatient bed, tertiary care teaching hospital during may 7, 2012-may 6, 2015; and sleh-guayama, a 161-inpatient bed hospital during february 1, 2013-may 6, 2015. sleh-ponce is one of four hospitals serving 481,708 residents of ponce and 11 neighboring municipalities [21] . sleh-ponce has about 50,000 emergency department (ed) visits and 11,000 inpatient admissions annually. sleh-guayama is one of two hospitals that serve 96,439 residents of guayama and three adjacent municipalities. sleh-guayama has 35,000 ed visits and 6,000 inpatient admissions annually. patients presenting to the ed or as a direct hospital admission were eligible for enrollment if fever was present (defined by a body temperature of !38.0˚c [oral] or !38.5˚c [axillary]) or they reported a history of fever of 7-day duration. after informed consent was administered, vital signs, signs and symptoms of current afi, history of exposures and chronic disease, and clinical laboratory results were recorded on an enrollment case report form (crf). a physician examined the participants and recorded the clinical diagnosis on the crf. participants discharged to home after enrollment were asked to return 7-10 days post-illness onset (dpo). at the follow-up visit, a second completed crf included a report of any healthcare services received and signs and symptoms experienced since enrollment. participants admitted to the hospital upon enrollment had their hospital course summarized on a standardized data collection form that included treatment received, results of clinical laboratory and radiologic investigations, and disease manifestations. prior to enrollment, informed consent was administered in accordance with puerto rico law (article 13, section 13, regulation 7617 of the office of patient ombudsman, act #194). specifically, written informed consent was obtained from eligible adults >20 years old and emancipated minors 14-20 years old. written informed assent was obtained from non-emancipated minors 14-20 years old and written informed consent was obtained from the parents or guardians. verbal informed assent was obtained from children 7-13 years old and written informed consent was obtained from the parents or guardian, and the parents or guardian of children <7 years old. the institutional review boards at the centers for disease control and prevention (cdc) and ponce health sciences university approved the study protocol. all study participants had blood (5 ml in edta, 7 ml whole blood), urine (15 ml), nasopharyngeal (np), and oropharyngeal (op) specimens collected at enrollment. convalescent blood (5 ml in edta, 5 ml whole blood) and urine (10 ml) were collected at the follow-up visit or hospital discharge. np and op specimens were placed in a vial containing viral transport medium. serum, blood, and urine specimens and inoculated vials were kept at 4˚c until transported to cdc dengue branch (cdc-db) in san juan, puerto rico. molecular diagnostic testing for denv 1-4, influenza a and b viruses (flu a/b), and 12 other respiratory viruses (orv) including adenovirus (adv), human respiratory syncytial virus (hrsv), human metapneumovirus (hmpv), parainfluenza virus 1-4 (piv-1-4), human rhinovirus (hrv), and four human coronaviruses (hcov) (229e, oc43, nl63 and hku1), was performed at cdc-db. however, testing for hrv, piv-2, piv-4, and the four hcov was discontinued after the first year because of low yield (i.e., only 1 piv-2, 37 hcov and 4 hcov co-infections identified). in brief, rna was extracted from np and op specimens and tested for orv and flu a/b viral genome by real time, reverse transcriptase-polymerase chain reaction assay (rrt-pcr) [22] . serum specimens collected 6 dpo were tested by denv-serotype specific rrt-pcr [23, 24] , and those collected !4 dpo were tested by an antibodycapture enzyme-linked immunosorbent assay (mac-elisa) (inbios international, inc., seattle, wa) [25] [26] [27] . beginning in may 2014, specimens collected 6 dpo were tested by chikv-specific real-time rt-pcr [28], and those collected !6 dpo were tested by anti-chikv mac-elisa [25] . remaining serum, whole blood, and urine were stored at -70˚c until shipped to cdc in atlanta, georgia. at cdc, serum specimens collected 3 dpo were tested in the picornavirus laboratory by a pan-enterovirus real-time rt-pcr assay that targets the vp1 region [29]; positive specimens were sequenced. paired serum specimens from enrollment and the follow-up visit or hospital discharge were tested for leptospira spp., and burkholderia pseudomallei at the bacterial special pathogens branch laboratory. specimens were tested by microscopic agglutination test (mat) for 20 leptospira reference antigens representing 17 serogroups [30]. all convalescent serum specimens were tested for presence of burkholderia pseudomallei and leptospira antibodies by an indirect hemagglutination assay (iha) [31] and mat respectively, and acute specimens were tested in cases for which the corresponding convalescent specimen was positive. the first 250 patients with leptospira spp. and burkholderia pseudomallei negative specimens and for which paired specimens were available were tested by ifa for rickettsia spp., ehrlichia spp., and coxiella spp. at the rickettsial zoonoses branch laboratory. whole blood and/or acute serum from cases with a reactive ifa were assessed for c. burnetii, r. rickettsii, r. typhi, and/or e. chaffeensis dna by pcr. a laboratory-positive dengue case had denv nucleic acid or anti-denv igm detected in a single specimen. a laboratory-negative dengue case had no anti-denv igm detected in serum collected !6 dpo. a laboratory-positive influenza case was defined by presence of flu a/b nucleic acid in a np or op specimen. laboratory-positive hmpv, hrsv, adeno, piv-1, piv-2, piv-3, piv-4, hrv, and hcov cases had the respective viral nucleic acid present in a np or op specimen. a laboratory-positive leptospirosis case was defined by !4-fold increase in mat titers in paired specimens, or mat titer !800 in a single specimen. a laboratory-positive melioidosis case was defined by presence of burkholderia pseudomallei nucleic acid in a clinical specimen and/or a !4-fold rise in iha titer in paired specimens. a laboratory-positive enteroviral case was defined by presence of enterovirus nucleic acid in serum collected 3 dpo. a laboratory-positive ehrlichiosis case was defined by presence of ehrlichia chaffeensis igg reciprocal titer >1:128 by ifa, a !4-fold rise in igg titer in paired serum specimens, or a positive pcr on an acute whole blood or serum specimen. a laboratory-positive rickettsia case was defined by presence of r. rickettsii or r. typhi igg titer >1:128 by ifa, a !4-fold rise in igg titer in paired serum specimens, or a positive pcr in a whole blood or serum specimen. a laboratory-positive coxiella case was defined by presence of c. burnetii igg titer >1:128 by ifa, a !4-fold rise in igg titer in paired serum specimens, or positive pcr on an acute whole blood or serum specimen. leukopenia was defined as a white blood cell count 5,000 cells/μl. thrombocytopenia was defined as a platelet count 100,000/μl. severe hemoconcentration was defined by a hematocrit !20% above the u.s. population mean hematocrit for age and sex, and moderate hemoconcentration was defined by a hematocrit >97.5 th percentile for age and sex to less than the cut-off for severe hemoconcentration [32] . a skin bleed was defined by presence of skin bruising and/or petechiae. mucosal bleeds included epistaxis, gingival bleed, hematemesis, melena, hematochezia, menorrhagia, or hematuria (>5 red blood cells per high powered field) in a male or non-menstruating female. frequencies were calculated for demographic characteristics and medical history by study year. clinical and laboratory features were compared by sex, age group, and laboratory diagnostic groups including infection with denv, flu a/b, orv, and chikv. differences in proportions were tested by applying the chi-square test, and medians were compared using the mann-whitney-wilcox test. bonferroni correction was used to account for simultaneous multiple comparisons. the woolf test was performed to evaluate the homogeneity of odds ratio across dpo group for death among adult participants by sex, and the mantel-haenzel test was used to determine significance. multiple imputation was used to predict an independent plausible value for missing values using generalized linear regression on non-missing variables to create 40 imputed complete data sets [33] . to identify predictors of laboratory-positive dengue as compared to all other afi cases, stepwise akaike information criterion (aic) variable selection was used for each imputed data set. variables retained at least once in the 40 models were included in a pooled logistic regression model [34] . odds ratios (or) and 95% confidence intervals (ci) were calculated for significant early (<3 dpo) and late (3) (4) (5) predictors. data were analyzed using the "mi" and "mass" packages from r software (v3.3.0, r foundation for statistical computing, vienna, austria). during the study, sites recorded 234,221 ed visits of which 43,567 (18.6%) patients had fever or reported fever (fig 1) . enrollment was offered to 11,505 afi case-patients, and 10,039 (87.3%) gave their consent/assent to participate. however, 1,043 (10.4%) of those enrolled withdrew from the study or were withdrawn due to noncompliance with study enrollment procedures. of the remaining 8,996 participants, 2,999 (33.3%) had follow-up forms completed and 24.9% had follow-up specimens collected. half (50.3%) of the 8,996 participants were female, and the median age was 12.8 years (range 0-103) ( table 1 ). one-third (33.7%) of participants reported having a chronic medical condition; a higher proportion of participants enrolled in the first year reported a co-morbidity than those enrolled in subsequent years. the most common co-morbid conditions were asthma (18.6%), high blood pressure (10.7%), diabetes (7.5%), high cholesterol (4.7%), coronary heart disease (4.4%), and thyroid disease (4.2%). participants resided in 49 of the 78 municipalities in puerto rico; however, most (76.3%) were residents of five municipalities: ponce (43.0%), guayama (15.6%), juana díaz (8.2%), salinas (4.9%), and villalba (4.6%). most (71.8%) participants were enrolled <3 dpo (median dpo at enrollment = 1, range: 0-8 days) ( table 2 ). the timing of presentation did not differ by sex but did differ by age, with a higher proportion of child participants (i.e., <20 years old) presenting <3 dpo than adult participants (74.9% child vs. 68.2% adult females, p <0.001; and 73.4% child vs. 67.4% adult males, p <0.001). one quarter (24.9%) of participants were admitted to the hospital at enrollment. adult participants were less likely to be admitted than child participants; a higher proportion of female adult participants than male adult participants were sent home after enrollment (78.3% vs. 74.6% respectively, p <0.05). however, a higher proportion of male versus female adult participants died after enrollment (0.8% vs. 0.2% respectively), in fact, adult males were five times more likely to die than adult females when adjusting by dpo (or = 5.4, prospective study of acute febrile illness in puerto rico ci: 1.5-19.0). there were no statistical significant differences between female and male participants <20 years old in terms of the timing of presentation and disposition. the most common signs and symptoms (aside from fever) at enrollment were tiredness/lethargy (73.5%), anorexia (65.0%), chills (64.5%), headache (64.3%), muscle, bone or back pain (60.0%), cough (53.4%), red conjunctiva (49.2%), rhinorrhea (49.1%), nausea (48.9%), and joint pain (48.9%). slightly more than half (54.8%, 4,930) of the 8,996 participants had a pathogen detected ( fig 2) . chikv was detected in 1,635 (18.2%) participants and was the most common pathogen the distribution of pathogens causing afi varied by age (fig 2) . the proportion of chikungunya cases increased with age, accounting for 9.3% of all afi cases in participants <5 years old versus 33.4% in participants !50 years old. in contrast, the contribution of orv to afi cases decreased with age, making up 21.6% of afi cases in participants <5 years old, 6.4% in participants 5-19 years old, 3.7% in participants 20-49 years old, and 4.1% in participants !50 years old. dengue was the most common cause of afi in participants 5-19 years old, accounting for 20.3% of all cases; 2.8% of afi cases in participants <5 years old were dengue, 9.8% in participants 20-49 years old, and 7.4% in participants !50 years old. the contribution of influenza was similar among age groups making up 8.4% of afi cases in participants <5 years old, 13.9% in 5-19 years old, 15.4% in 20-49 years old, and 9.7% in !50 year-old participants. analysis of the temporal disease trends demonstrated that a dengue epidemic occurred in 2012 and continued through 2013, during which a total of 921 dengue cases were detected (fig 3) . in comparison, few (n = 49) dengue cases were detected in 2014 to the end of the study period in 2015. the first chikungunya case was detected in may of 2014, and was followed by a six-month outbreak during which 1,558 cases were detected. few (n = 61) chikungunya cases were detected in 2015. a large bimodal influenza epidemic took place in 2013 with increased case detection in the dry months of january-april (n = 225), and during the rainy season, july-october (n = 302). fewer influenza cases (n = 356) were detected in 2014 and 2015, and those detected occurred primarily in dry months with no obvious bimodal distribution. an increase in afi cases due to orv was detected at the same time influenza cases were detected, with the exception of 2013 when the peak time of orv case detection appeared to follow that of influenza. prospective study of acute febrile illness in puerto rico subject demographics at enrollment differed by subsequent laboratory diagnosis (table 4) . a lower proportion of participants with dengue and orv illness were females when compared with participants with chikungunya. participants with orv illness were significantly younger (median age = 3.2 years, p <0.001) than participants with dengue (15.4 years), chikungunya (24.3 years), or influenza (14.1 years). in contrast, the median age of participants with chikungunya was significantly greater than participants in all other diagnostic groups, and they were more likely to report having a chronic medical condition. a higher proportion of participants with dengue reported having a household member with dengue at enrollment than participants with other diagnoses (11.8% of dengue cases versus 5% in other diagnostic groups, p <0.001). over half of all participants reported having mosquito bites in the 30 days before enrollment; however, a higher proportion of participants with chikungunya reported mosquito bites than participants with other laboratory diagnoses (73.9% versus <55% in other diagnostic groups, p<0.001). clinical presentation and disposition varied by laboratory diagnostic group (table 4) . participants with laboratory-positive dengue presented later (median = 3 days), and a higher proportion were admitted at enrollment than participants with other laboratory diagnoses; nearly half (46.6%) of dengue cases were admitted compared with 27.3% of participants with orv illness, 18.8% with influenza, and 11.2% with chikungunya. a significantly higher proportion of participants with dengue had chills, signs of poor circulation, eye pain, headache, dizziness, anorexia, nausea, abdominal pain, and diarrhea at enrollment than participants with influenza, orv illness or chikungunya. a higher proportion of participants with dengue versus these other diagnoses had thrombocytopenia and leukopenia. compared to influenza and orv illness cases, a significantly higher proportion of dengue cases had a skin rash, pruritic skin, any bleeding, a skin bleed, and muscle, bone, back, and joint pain, whereas a higher proportion of chikungunya versus dengue cases had these findings. a higher proportion of dengue versus influenza and orv illness cases had facial and/or neck erythema and mucosal bleeding. in contrast, a significantly higher proportion of participants with influenza and orv illness than dengue had cough, rhinorrhea, and sore throat. among 6,349 participants who presented early (<3 dpo) in the clinical course, leukopenia, thrombocytopenia, headache, eye pain, nausea, and dizziness were significant positive predictors of laboratory-positive dengue as compared to all other afi cases across all age groups ( table 5 ). presence of rhinorrhea and irritability predicted non-dengue afi. age group had a statistically significant effect on multiple predictors (table 6) . rash was a positive early predictor of dengue among participants <5 years old, and being male was a positive predictor among adults 20-49 years old. chills and cough were positive predictors for those >50 years old while cough was a negative predictor among those <20 years old. muscle, bone or back pain was a negative predictor in those >50 years old. pruritic skin as a predictor varied by age group, but most significantly between the <5 and 50+ year-old groups. among the 2,146 participants who presented 3-5 dpo, thrombocytopenia, leukopenia, facial and/or neck erythema, nausea, eye pain, signs of poor circulation, and diarrhea were significant positive predictors of dengue across all age groups ( table 7) . presence of rhinorrhea, red conjunctiva and cough predicted non-dengue afi. again, age group significantly affected multiple predictors (table 8) . abdominal pain was a positive predictor for participants 20-49 years old. red and/or swollen joints was a positive predictor among participants <5 years old but a predictor of non-dengue afi among participants !50 years old. leukopenia was a prospective study of acute febrile illness in puerto rico significant positive predictor across all age groups, but to varying degrees. chills; muscle, bone, back and joint pain; and any bleeding as predictors varied depending on the age group. as a clinical syndrome, afis are a diagnostic challenge for clinicians especially early in the clinical course when anticipatory guidance and supportive care may pre-empt medical complications. our study identified the afi etiology in over half (55%) of participants and most were infected with one of nine viral pathogens. this detection frequency was higher than that of other recent prospective afi studies that tested for multiple pathogens (55% versus 36-41%) [9] [10] [11] [12] 15] . this difference may in part be explained by the greater contribution of chikungunya in our study than in the other studies that tested for this pathogen [9, 10, 17] . however, unlike other studies, we were unable to detect any evidence of disease caused by rickettsia or coxiella spp., which made up 4-13% of all afis in other studies [10] [11] [12] [13] ; and unlike other areas, malaria [9, [11] [12] [13] 18] and typhoid fever [9, 10, 12, 13, 15] were not part of our diagnostic algorithm as they are only occasionally detected among travelers returning to puerto rico. while denv was not as commonly identified as chikv or flu a/b, participants with dengue were more likely to be admitted to the hospital at enrollment. the proportion of afi cases with dengue in our study was comparable to other recent studies which found 4-9% of afi cases had dengue [9] [10] [11] [12] [13] [15] [16] [17] [18] . one exception to this was a study that found 34% of afi patients had dengue; however, the study's eligibility criteria likely enhanced enrollment of dengue cases [14] . in our study, dengue incidence varied by age group with a nearly a 10-fold difference between participants <5 years old and 10-19 years old (3% versus 27%), which may be due to differences in likelihood of seeking medical care in primary versus secondary denv infections [35] . of note, 6% of participants !65 years old had dengue as a cause of afi, a finding comparable to a puerto rico study in which 5% of 17,666 laboratory-positive dengue cases detected by surveillance were !65 years old [36] . in contrast, other recent prospective studies chikungunya, the most commonly identified afi overall, was least likely to result in hospital admission, although two male participants with chikv infection died. these cases were older individuals (>75 years old) who had underlying co-morbidities which may have complicated their clinical course. nonetheless, since autopsy was not performed for either case, ascertaining whether chikv infection played a role in either fatality is difficult. however, in our study chikungunya was disproportionally identified among older participants, with positivity increasing from <10% of pre-school aged children to about one-third of participants !50 years old. this pattern of disease has been seen in other areas with recent chikv emergence [18] , and may be due to older individuals having an increased likelihood of complications due to preexisting co-morbidities [44, 45] . co-infections confirmed by molecular assays were detected among 1% of our participants, most commonly involving enteroviral or orv infections; less than one-third of all co-infections included a denv or chikv infection. another recent prospective study found that 1% of afi participants had co-infections involving molecularly diagnosed dengue or influenza, malaria, and positive blood culture [9] . interestingly, we did not detect any co-infections involving chikv and denv. an analysis of island-wide surveillance data from puerto rico during the same time period found only one chikv/denv co-infection among approximately 1,000 specimens tested by rt-pcr for both denv and chikv [46] . these findings are consistent with another prospective afi study conducted in sri lanka [17] . although a recent study has shown that aedes aegypti can be infected with as many as three arboviruses simultaneously and can likely transmit these viruses to humans [47] , the frequency of co-or tri-infection of mosquitoes in the wild depends upon the geographic spread and degree of circulation of each virus. during our study, denv transmission decreased significantly before chikv transmission peaked, making co-infections less likely. in addition, in puerto rico, where aedes aegypti is the sole vector for chikv and denv, viral interaction and viral prospective study of acute febrile illness in puerto rico interference within the mosquito may reduce the likelihood of co-infection [48] [49] [50] . however, rt-pcr positive denv/chikv co-infections have been documented at higher rates in five countries [51] . we identified differences in clinical predictors of laboratory-positive dengue by timing of presentation and age group highlighting the importance of considering these factors when developing prediction algorithms for clinical management [52] [53] [54] [55] [56] [57] [58] [59] [60] . we found, as have others [61] , that even early (<3 dpo) in the clinical course leukopenia and thrombocytopenia are predictive of dengue across all age groups, and thrombocytopenia strengthened as a predictor over time. in our study, headache and eye pain were the only "aches and pains" that were predictive of dengue for all age groups [62] . eye pain was a predictor early and later in the clinical course, a finding consistent with pediatric [63] and adult [56] prospective cohort studies, as well as a surveillance study conducted in puerto rico [52] . we also found that rash among children <5 years old presenting early and erythema on the face and/or neck in all age groups presenting 3-5 dpo, were positive predictors of dengue. while the presence of skin rash has been found to be a predictor of dengue in several prospective studies [61] , few studies have evaluated erythema as a predictor [18, 64, 65] . last, like other prospective studies [56, 66] , we found that nausea is an early predictor for dengue. we were also able to show that adults aged 20-49 years presenting 3-5 dpo were more likely to have abdominal pain than those with other afis, and dengue cases of all ages presenting 3-5 dpo were also more likely to have diarrhea and poor circulation in addition to nausea, findings that lend support to the idea that warning signs for severe dengue develop after the early phase of the illness. our study, which enrolled all patients presenting with fever regardless of age, sex, or clinical characteristics, may be limited in generalizability. the study was conducted in southern puerto rico which may differ from neighboring islands and other parts of the island with regard to population demographics, preexisting immunity to denv and other flaviviruses, and exposure to infections. second, while we enrolled nearly 600 older adults (!65 years old), we were unable to adequately evaluate predictors of dengue among this population because we had only 36 dengue cases and most presented early in the clinical course. last, we did not systematically collect stool and test for potential enteric pathogens, and bacterial infections were likely under recognized because blood cultures were only done on patients in whom sepsis was suspected. while our study identified an etiology in over half of all afi cases, the etiology of 45% of afi remained unknown even after extensive testing and the majority of diagnosed cases were caused by one of nine viral pathogens that typically do not require empiric therapy. in fact, we were unable to find any cases of rickettsia spp., ehrlichia spp., and coxiella spp., and only sporadic cases of melioidosis and leptospirosis were identified. our findings demonstrate that dengue is not only one of the leading causes of afi in puerto rico, but results in greater morbidity than other afis as measured by need for hospitalization. moreover, dengue affects people of all ages including older adults who may be at higher risk of developing medical complications. clinicians should include dengue on the differential diagnosis of afi among older adults so that timely anticipatory guidance can be offered. we found that the presence of leukopenia and thrombocytopenia were the best predictors of dengue in both time periods overall and for all age groups. our findings suggest that eye pain should be reevaluated as a predictor of dengue. future studies should focus on improving clinical diagnosis of afi including dengue by timing of presentation and age of the patient. supporting information s1 checklist. strobe statement. (docx) we thank saint luke's episcopal hospital patients for their participation in this study. we would like to thank physicians, nurses, clinical laboratory staff and administrative personnel at the saint luke's episcopal hospitals in ponce and guayama for their assistance to recruiting potential participants and implementing study procedures. in addition, we would like to acknowledge the medical management information offices from saint luke's episcopal hospitals for facilitating the review of medical records for admitted participants. we would also like to thank dr. brad biggerstaff from the cdc's division of vector-borne diseases for his critical review of the data analysis and manuscript. in addition, we would like to thank cdc staff members at the dengue branch, polio and picornavirus laboratory branch, rickettsial zoonoses branch, and bacterial special pathogens branch (zoonoses and select agent laboratory) for processing and testing of all clinical specimens. last, we would like to acknowledge the technical support of ponce health sciences university. without their interest in and support of this project, the sedss sites would have never been established and this study would never have been possible. mapping the aetiology of non-malarial febrile illness in southeast asia through a systematic review-terra incognita impairing treatment policies transition in the cause of fever from malaria to dengue, northwestern ecuador, 1990-2011. emerging infectious diseases rapid diagnostic tests for non-malarial febrile illness in the tropics. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases the global distribution and burden of dengue infectious etiologies of acute febrile illness among patients seeking health care in south-central cambodia. the american journal of tropical medicine and hygiene etiologies of acute undifferentiated febrile illness in thailand etiology of acute undifferentiated febrile illness in the amazon basin of ecuador. the american journal of tropical medicine and hygiene causes of non-malarial fever in laos: a prospective study. the lancet global health acute undifferentiated febrile illness in rural cambodia: a 3-year prospective observational study dengue as a cause of acute undifferentiated fever in vietnam etiology of acute, non-malaria, febrile illnesses in jayapura, northeastern papua, indonesia. the american journal of tropical medicine and hygiene unsuspected dengue and acute febrile illness in rural and semi-urban southern sri lanka. emerging infectious diseases chikungunya as a cause of acute febrile illness in southern sri lanka contribution of dengue fever to the burden of acute febrile illnesses in papua new guinea: an age-specific prospective study. the american journal of tropical medicine and hygiene revista panamericana de salud publica = pan american journal of public health the american journal of tropical medicine and hygiene population division rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-pcr using degenerate primers and probes containing deoxyinosine residues analytical and clinical performance of the cdc real time rt-pcr assay for detection and typing of dengue virus standardization of immunoglobulin m capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections the 2005-2006 outbreak on reunion journal of clinical virology: the official publication of the pan american society for clinical virology surveillance for chikungunya and dengue during the first year of chikungunya virus circulation in puerto rico. the journal of infectious diseases impact of simultaneous exposure to arboviruses on infection and transmission by aedes aegypti mosquitoes viral interference and persistence in mosquito-borne flaviviruses evaluation of simultaneous transmission of chikungunya virus and dengue virus type 2 in infected aedes aegypti and aedes albopictus (diptera: culicidae) dissecting vectorial capacity for mosquito-borne viruses. current opinion in virology co-distribution and co-infection of chikungunya and dengue viruses clinical and laboratory features that differentiate dengue from other febrile illnesses in an endemic area-puerto rico differences in dengue severity in infants, children, and adults in a 3-year hospital-based study in nicaragua. the american journal of tropical medicine and hygiene clinical features and differences between child and adult dengue infections in rayong province, southeast thailand. the southeast asian journal of tropical medicine and public health the differences of clinical manifestations and laboratory findings in children and adults with dengue virus infection the early clinical features of dengue in adults: challenges for early clinical diagnosis early clinical features of dengue infection in puerto rico four dengue virus serotypes found circulating during an outbreak of dengue fever and dengue haemorrhagic fever in jakarta, indonesia, during differences in clinical and laboratory characteristics and disease severity between children and adults with dengue virus infection in taiwan risk factors and clinical features associated with severe dengue infection in adults and children during the 2001 epidemic in chonburi clinical and laboratory features that distinguish dengue from other febrile illnesses in endemic populations dengue: guidelines for diagnosis, treatment, prevention and control. geneva: who early clinical features of dengue virus infection in nicaraguan children: a longitudinal analysis sensitivity and specificity of a novel classifier for the early diagnosis of dengue distinguishing dengue fever from other infections on the basis of simple clinical and laboratory features: application of logistic regression analysis early clinical and laboratory indicators of acute dengue illness key: cord-350533-fp1ctpax authors: tchesnokov, egor p.; bailey-elkin, ben a.; mark, brian l.; götte, matthias title: independent inhibition of the polymerase and deubiquitinase activities of the crimean-congo hemorrhagic fever virus full-length l-protein date: 2020-06-04 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008283 sha: doc_id: 350533 cord_uid: fp1ctpax background: the crimean-congo hemorrhagic fever virus (cchfv) is a segmented negative-sense rna virus that can cause severe human disease. the world health organization (who) has listed cchfvas a priority pathogen with an urgent need for enhanced research activities to develop effective countermeasures. here we adopted a biochemical approach that targets the viral rna-dependent rna polymerase (rdrp). the cchfv rdrp activity is part of a multifunctional l protein that is unusually large with a molecular weight of ~450 kda. the cchfv l-protein also contains an ovarian tumor (otu) domain that exhibits deubiquitinating (dub) activity, which was shown to interfere with innate immune responses and viral replication. we report on the expression, characterization and inhibition of the cchfv full-length l-protein and studied both rna synthesis and dub activity. methodology/principle findings: recombinant full-length cchfv l protein was expressed in insect cells and purified to near homogeneity using affinity chromatography. rdrp activity was monitored with model primer/templates during elongation in the presence of divalent metal ions. we observed a 14-mer full length rna product as well as the expected shorter products when omitting certain nucleotides from the reaction mixture. the d2517n mutation of the putative active site rendered the enzyme inactive. inhibition of rna synthesis was studies with the broad-spectrum antivirals ribavirin and favipiravir that mimic nucleotide substrates. the triphosphate form of these compounds act like atp or gtp; however, incorporation of atp or gtp is markedly favored over the inhibitors. we also studied the effects of bona fide nucleotide analogues 2’-deoxy-2’-fluoro-ctp (fdc) and 2’-deoxy-2’-amino-ctp and demonstrate increased inhibitory effects due to higher rates of incorporation. we further show that the cchfv l full-length protein and the isolated otu domain cleave lys48and lys63-linked polyubiqutin chains. moreover, the ubiquitin analogue cc.4 inhibits the cchfv-associated dub activity of the full-length l protein and the isolated dub domain to a similar extent. inhibition of dub activity does not affect elongation of rna synthesis, and inhibition of rna synthesis does not affect dub activity. both domains are functionally independent under these conditions. conclusions/significance: the requirements for high biosafety measures hamper drug discovery and development efforts with infectious cchfv. the availability of full-length cchfv l-protein provides an important tool in this regard. high-throughput screening (hts) campaigns are now feasible. the same enzyme preparations can be employed to identify novel polymerase and dub inhibitors. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the crimean-congo hemorrhagic fever virus (cchfv) is a segmented negative-sense rna virus that belongs to the bunyavirales order, family nairoviridae [1] . cchfv is either transmitted directly from ticks to humans, indirectly via a wide range of infected animals or from person-to-person by contact with infectious body fluids. infection can cause severe human disease and is endemic in asia, the middle east, africa, and in parts of southern europe [2] . cchfv is viewed as an emerging threat at the asian/european interface where turkey reports 700-1.300 cases per year [3] . effective vaccines or treatments are not available. for these reasons, the world health organization (who) has listed cchfv as a priority pathogen with an urgent need for enhanced research & development (r&d) activities [4] . viral polymerases are validated drug targets. however, inhibition of the rna-dependent rna polymerase (rdrp) activity of negative-sense rna viruses has been challenging. previous drug discovery and development efforts focused on the influenza virus, which also contains a segmented genome [5] [6] [7] . the rdrp inhibitor favipiravir (t-705) is approved for the treatment of influenza in japan [8] . this compound mimics the structure of a purine that is intracellularly activated to a nucleoside triphosphate (ntp) with a pseudo base. biochemical studies with purified influenza rdrp demonstrated ambiguous base-pairing with both cytidine and uridine, which can lead to lethal mutagenesis or premature termination of rna synthesis [6] . similar mechanisms have earlier been proposed for the structurally related ribavirin [9] . both drugs show a broad spectrum of activities against rna viruses, including cchfv [10] [11] [12] [13] . although some studies have indicated a clinical benefit, a small randomized trial did not reveal a significant difference between the groups of patients who received the drugs plus supportive treatment or supportive treatment only [14] . evaluation of the drugs in mice models suggests that favipiravir is more potent when compared with ribavirin [11, 12] . 2'-deoxy-2'fluorocytidine (fdc) was also shown to inhibit cchfv replication, and this compound seems to exceed the potency of both ribavirin and favipiravir [15] . fdc was originally identified as an inhibitor of hepatitis c virus (hcv) replication, but its development as an antiviral drug has been limited due to off-target activities [16] . biochemical tools that facilitate screening for cchfv rdrp inhibitors have yet to be developed. the cchfv rdrp activity is part of a multifunctional l protein that is currently not available for biochemical or structural studies [17, 18] . the study of enzymes from other segmented negative-sense rna viruses provided important insight into structure and function (fig 1) . the polymerase of influenza (orthomyxoviridae) is a heterotrimeric complex composed of an rdrp subunit, a cap-snatching endonuclease, and a cap-binding domain (fig 1, top) [19, 20] . the structure of the la crosse virus (lacv) rdrp shows a similar overall arrangement in a single chain (fig 1, middle) [21] . lacv belongs to the peribunyaviridae family and is therefore more closely related to the cchfv. the full-length cchfv l-protein is unusually large with~4000 amino acids and a molecular weight of~450 kda, which is commonly not conducive to heterologous expression. rdrp and cap-snatching endonuclease motifs have been identified [22] , although recent attempts to express the l protein or the isolated nuclease domain have failed to yield active enzyme [23] . recently, a functional cap-binding domain has been identified in the c-terminus of the rift valley fever virus (rvfv) l protein [24] ; however it is still unknown whether it is also present in the cchfv l protein. the cchfv l-protein also contains an ovarian tumor (otu) domain that exhibits deubiquitinating (dub) activity (fig 1, bottom) . structural information is limited to the isolated cchfv otu domain [25] . the cchfv otu domain is a cysteine protease capable of removing ubiquitin (ub) from proteins, which interferes with innate immune responses [10, 26] . the ubiquitin variant cc.4 binds to the otu domain with high affinity and inhibits cchfv dub activity [27] . cc.4 was also shown to affect infectivity and replication [28] . hence, the availability of recombinant cchfv full-length l protein would provide an important tool for drug discovery and development efforts. here, we utilized the baculovirus expression system and generated active full-length cchfv l protein. rdrp activity is seen with model rna substrates in the presence of catalytic, divalent metal ions. we confirm that the triphosphate (tp) forms of ribavirin, favipiravir, and 2'-deoxy-2'-fluoro-ctp are recognized as substrates and inhibit rna synthesis. moreover, we identified 2'-deoxy-2'-amino-ctp as a novel inhibitor of cchfv rdrp. we further show that cc.4 inhibits the cchfv-associated dub activity of the full-length l protein and the isolated dub domain to a similar extent. inhibition of rdrp activity does not affect dub activity and inhibition of dub activity does not affect rdrp activity, which provides strong evidence to show that both domains are functionally independent. all rna primers and templates used in this study were purchased from dharmacon (lafayette, co, usa). ribavirin-tp was purchased from jena bioscience (jena, germany). favipiravir-tp was purchased from santa cruz biotechnology (santa cruz, ca, usa). 2'deoxy-2'-fluoro-ctp (fdc-tp) and 2'-deoxy-2'amino-ctp (2'd-2'amino-ctp) were purchased from trilink (san diego, ca, usa). ntps were purchased from ge healthcare (cranbury, nj, usa). [α-32 p]-gtp and-ctp were purchased from perkinelmer (boston, ma, usa). c-terminus derived ubiquitin conjugated with 7-amido-4-methylcoumarin (ubiquitin-amc) was purchased from boston biochem (cambridge, ma, usa). the pfastbac-1 (invitrogen, burlington, on, canada) plasmid with the codon-optimized synthetic dna sequences (genscript, piscataway, nj, usa) coding for cchfv l protein (aie16126) with n-terminal strep-and 8xhistidine (his)-tags was used as a starting material for protein expression in insect cells (sf9, invitrogen, burlington, on, canada). we employed the multibac (geneva biotech, indianapolis, in, usa) system for protein expression in insect cells (sf9, invitrogen, burlington, on, canada) according to published protocols [29, 30] . cchfv l protein was initially purified using the strep-or his-tag-affinity chromatography according to the manufacturer's specifications (iba, (goettingen, germany), and thermo scientific, rockford, il, usa, respectively). his-tag purification yielded generally higher amounts of protein. the identity of the purified cchfv l protein was confirmed by mass spectrometry (ms) analysis (dr. jack moore, alberta proteomics and mass spectrometry, edmonton, ab, canada). the mass spectrometry peptide fragments included residues starting from n6 to r3935 out of 3945 residues of the full length cchfv l protein, which corresponds to 99.6% coverage. residues 1-217 of the cchfv l protein were inserted into vector pet-49b(+) and expressed as a gst-tagged fusion. the cchfv otu domain was purified form escherichia coli, and the gst tag removed using methods describe previously [25] . expression and purification of cc.4 was performed as previously described [27] . data acquisition and quantification were done as previously reported by us [31, 32] . the amount of cchfv l used in the rna synthesis assay was optimized such that incorporation of [α32 qualitative analysis of polyub chain hydrolysis using sds-page. 600 ng of either k48or k63-linked polyubiquitin chains (ub 3 -ub 7 ; boston biochem) were incubated with either purified cchfv l protein, or cchfv otu at a final concentration of 20 nm in a final volume of 20 μl. the cchfv otu-specific inhibitor cc.4 was incubated with cchfv l protein or otu at a final concentration of 100 nm. reaction were performed in in 50 mm tris ph 8.5, 100 nacl, 5 mm dtt at 37˚c. at pre-determined time points, 6 μl of each reaction were removed stopped with the addition of 2x sds-page loading buffer. reactions were resolved by electrophoresis on a 10% tris-tricine gel and visualized using a pierce silver stain kit. quantitative analysis of dub activity using fluorescene-based assay. a c-terminusderived ubiquitin peptide conjugated with 4-amino-4-methylcoumarin (amc) was used as a fluorogenic substrate for 5 nm cchfv l protein or isolated otu domain in a reaction buffer containing tris-hcl (ph 8, 25 mm) and tcep (2 mm). reactions were started by the addition of ubiquitin-amc (500 nm) in a final reaction volume of 25 μl. reactions were conducted in a black 384-well plate (greiner-784900, greiner bio-one, kremsmünster, austria). once amc is cleaved from the conjugated substrate it can emit fluorescence at 445 nm upon excitation at 355 nm acquired on a spectramax m5 (molecular devices, san jose, ca, usa). slopes of linear portions of fluorescent signal formation were used to determine the velocity of substrate cleavage in the presence and absence of ubiquitin variant cc.4 (cc. 4) which has been shown previously to inhibit dub activity of cchfv isolated otu-domain [27] . dub activity velocities were plotted versus cc.4 concentrations and fitted to a log(inhibitor)-versus-normalized-response-(variable slope) equation using graphpad prism 7.0 (graph-pad software, inc., san diego, ca, usa) to determine the ic 50 values for the inhibition of dub activity by cc.4. the baculovirus expression system has been successfully used to generate recombinant l-protein from segmented negative-sense rna viruses, including influenza b and la crosse [19, 21] . here we employed this approach to express recombinant cchfv l protein with an n-terminal his-tag (fig 2) . we have also generated an active site mutant that contains a d to n change at position 2517. the aspartic residue is located in motif c and is responsible for coordinating the catalytic metal ions [31, 33, 34] . motif c is conserved among rdrp enzymes of segmented rna viruses (fig 2a) . sds-page analysis of the protein preparation revealed a band above the 250 kda molecular weight marker (fig 2b) , which corresponds to the full-length cchfv l protein. both proteins were purified to near homogeneity and unambiguously identified through mass spectrometry (methods, s1 and s2 figs). initiation of rna synthesis involves conserved sequences at the 5'-and 3'-untranslated regions of the rna genome as demonstrated for influenza and other segmented negative sense rna viruses [19, 20, 35] . transitioning to elongation follows after a few nucleotide additions. the elongation mode is largely independent of the sequence and facilitates the study of rna synthesis inhibition with nucleotide analogues. hence, we devised short model primer/ templates that adequately mimic the elongation state. originally used for the study of respiratory syncytial virus (rsv) rdrp [36] , these type of sequences were recently utilized to quantify nucleotide incorporation events with various rdrp enzymes from ebola virus (ebov), hepatitis c virus (hcv), zika virus (zikv), influenza b virus (flub), as well as middle east respiratory syndrome coronavirus (mers-cov) [31, 34] . in this study we employed the same biochemical approach for comparative purpose. the 14-mer template ( fig 2c) is designed such that [α-32 p]ctp is utilized as the first nucleotide substrate and incubation with specific ntp cocktails would be expected to yield defined reaction products. the data show that mg 2+ as well as mn 2+ can serve as catalytic metal ions and promote extensions of a 4-mer primer. limited rna synthesis in the presence of [α-32 p]ctp alone revealed the expected 5-mer product. the presence of [α-32 p]ctp and atp yields a 6-mer product, a mixture of [α-32 p]ctp, atp and utp allows formation of a 7-mer and the addition of all four ntps yields a 14-mer full-length rna product. template-independent nucleotide additions are not observed. mg 2+ was used throughout this study as this is the preferred ion with respect to the yield of the fulllength product. the d2517n active site mutant was inactive when tested for rna synthesis (fig 2d) . previous biochemical studies have shown that both favipiravir-tp and ribavirin-tp are substrates for several rdrp enzymes and compete with atp or gtp for incorporation into the growing rna chain [5, 6, 9, 37] . here we studied possible effects of these inhibitors on rna synthesis by cchfv rdrp (fig 3) . we employed a 4-mer primer and three different rna templates to assess efficiency of incorporation opposite template u, c, and a at position 6 ( fig fig 2. purification and characterization of the rdrp activity of recombinant cchfv l wild type and d2517n mutant proteins. (a) a partial sequence alignment of rdrp of segmented viruses shows the conserved sdd sequence within motif c. residue d2517 within cchfv l protein is highlighted in purple. (b) sds page migration pattern of the purified enzyme preparation stained with coomassie brilliant blue g-250 dye. the band migrating above the 250 kda molecular weight marker (lane 1) contains cchfv l wild type or d2517n mutant full-length protein (as indicated) confirmed by mass spectrometry (see s1 and s2 figs for details). lane 2 contains 20x less protein than lane 1. the protein band is still visible, suggesting that there is~20 times more of the cchfv l protein than impurities. this corresponds to~95% pure protein preparation. (c) rna primer/template used in the rna synthesis assays is shown above the gel. template and primer were both mono-phosphorylated (p) at their 5'-ends. c indicates incorporation of the radiolabeled nucleotide opposite template position 5. rna synthesis was monitored with purified wild type cchfv l protein in the presence of [α-32 p]ctp, rna primer/templates and ntp combinations designed to generate either intermediate or full template length products. note that 10x less of wild type cchfv l protein was used in reactions started with mncl 2 . lane m illustrates the migration pattern of the radiolabeled 4 nucleotide-long primer. (d) rna primer/template used in the rna synthesis assays is shown above the gel. template and primer were both phosphorylated (p) at their 5'-ends. g indicates incorporation of the radiolabeled nucleotide opposite template position 5. rna synthesis was monitored with purified cchfv l wt and d2517n mutant proteins in the presence of [α-32 p]gtp, rna primer/template and ntp combinations designed to generate full template length products. lane m illustrates the migration pattern of the radiolabeled 4 nucleotide-long primer. lane "[α-32 p]gtp-only' illustrates the background signal associated with the [α-32 p]gtp preparation in the absence of enzyme. https://doi.org/10.1371/journal.pntd.0008283.g002 3, top). limited rna synthesis in the presence of [α-32 p]ctp yields the labeled 5-mer product for all three templates. the presence of [α-32 p]ctp and atp yields the expected 6-mer product rna and the presence of all four ntps allows full-length product formation (fig 3, left) . the omission of atp resulted in minor abortive products that likely reflect limited mis-incorporations and extensions. the same reactions were performed with ribavirin-tp and favipiravir-tp, respectively. the presence of [α-32 p]ctp and ribavirin-tp, instead of atp, also yields a 6-mer product. the additional presence of utp and gtp shows limited ongoing primer extensions. full-length product formation is not observed under these conditions, which indicates inhibition of rna synthesis. we obtained similar results with favipiravir-tp. both inhibition of the crimean-congo hemorrhagic fever virus full-length l-protein ribavirin-tp or favipiravir-tp are also used as substrates opposite template c (fig 3, middle) . in contrast, the inhibitors are not incorporated opposite template a (fig 3, right) . together the data show that cchfv rdrp incorporates ribavirin and favipiravir opposite template u and c as described for other rdrp enzymes. following incorporation of these compounds, rna synthesis is severely compromised. partial chain-termination is a likely mechanism of inhibition. incorporation and in turn inhibition by ribavirin-tp or favipiravir-tp requires that the nucleotide analogues are able to compete with atp or gtp. quantitative data can be obtained by measuring enzyme kinetic parameters and compare the efficiency of incorporation of the inhibitor with their natural counterparts. for this purpose, the natural nucleotide or the inhibitor was added at increasing concentrations at a fixed time point following the addition of [α-32 p]ctp (fig 4a and 4c) . a graphic representation of the data is shown in fig 4b and 4d . it is evident that product formation is much more efficient with the natural substrates atp and gtp. steadystate kinetic parameters k m and v max are provided in table 1 . favipiravir-tp and ribavirin-tp show reduced v max and increased k m values when compared with atp or gtp. both factors lead to reductions in the efficiency of incorporation v max / k m . the selectivity of nucleotide incorporation is defined as v max / k m (ntp)/ v max / k m (inhibitor) and helps to translate these values into quantitative terms. the selectivity values for ribavirin are relatively high when measured against atp (304) and gtp (696), respectively. the values for favipiravir are slightly more favorable against both atp (86) and gtp (329). together, the data suggest that the incorporation of atp or gtp is markedly favored over the analogues, which could limit their antiviral effects. we next studied the effects of bona fide nucleotide analogues with an intact base moiety. as a starting point, we selected two compounds with bioisosteric replacements for the 2'-hydroxyl group that is commonly recognized by rdrp enzymes: 2'-deoxy-2'-fluoro-ctp (fdc) and 2'deoxy-2'-amino-ctp. the 2'-fluoro and the 2'-amino groups may not prevent incorporation, but could potentially inhibit rna synthesis. we devised an rna template that allowed us to monitor incorporation of these compounds at position 8 (fig 5a, top) . limited elongation of the 4-mer primer in the presence of [α-32 p]gtp, atp and utp yields a 7-mer product and the addition of ctp allows formation of the expected 14-mer full-length rna product ( fig 5a) . the same reactions were also performed with fdc-tp and 2'-amino-ctp, respectively. both compounds are incorporated to yield 8-mer products. further primer extensions and full-length product formation is substantially reduced. kinetic parameters and selectivity values are provided in table 2 . reductions in v max are not as pronounced as seen with ribavirin-tp or favipiravir-tp. the main contribution to overall reductions in the efficiency of incorporation is here driven primarily by moderate increases in k m values. as a consequence, selectivity values for 2'-deoxy-2'-fluoro-ctp (23) and 2'deoxy-2'-amino-ctp (15) are relatively small when compared with ribavirin-tp or favipiravir-tp. thus, the bona fide nucleotide analogues are better substrates for cchfv rdrp. we also tested incorporation and inhibition with 2'-deoxy-2'-fluoro-ctp in the presence of its natural competitor ctp (fig 6a) . the extent of 2'-deoxy-2'-fluoro-cmp incorporation and subsequent chain-termination of rna synthesis depends on the competing ctp concentration as illustrated by increasing ic 50 values for 2'-deoxy-2'-fluoro-cmp (fig 6b) . the cchfv-associated dub activity was previously studied exclusively with the isolated otu domain. crystal structures of the viral otu domain bound to ubiquitin provided important information about the active site and substrate binding [25] . requirements for dub activity and its inhibition in this context may now be compared with the full-length l protein. for an initial qualitative comparison of the two enzymes, we assessed deubiquitination with lys48-and lys63-linked polyubiqutin chains (fig 7) . the starting materials contains mixtures of polymers with 3-7 ubiquitin units. time course experiments show that the full-length protein is able to process the polymeric chains to monomeric units. similar results were obtained with lys48-linked (fig 7a, left) and lys63-linked substrates (fig 7b, left) . inhibition of these reactions was assessed with the cchfv otu-specific inhibitor cc.4 [27] . these conditions yield longer products predominantly with 1-4 ubiquitin units (fig 7a and 7b, right) . most importantly, we obtained almost identical results with the isolated otu domain (fig 7c and 7d) . we then employed a fluorescence-based assay to translate these findings into quantitative terms (fig 8) . the assay involves the fluorogenic substrate ubiquitin-amc that is released in the presence of dub activity as schematically shown in fig 8a [38] . here we show that the inhibition of the crimean-congo hemorrhagic fever virus full-length l-protein cchfv l full-length protein and the isolated otu domain cleave ubiquitin-amc in a time dependent manner with similar velocities illustrated by the slopes of the initial linear portion of product formation (fig 8b) . addition of otu-specific inhibitor cc.4 blocked dub activity of both enzymes. ic 50 values for the inhibition of corresponding dub activities are 0.010 inhibition of the crimean-congo hemorrhagic fever virus full-length l-protein ±0.0031 nm and 0.0048±0.0015 nm, respectively (fig 8c) . the minor 2-fold difference suggests that the isolated otu domain is functionally equivalent to the otu domain of the fulllength l-protein. to study directly a putative interdependency of rdrp and dub activities, we compared formation of the 14-mer full-length rna product in absence and presence of cc.4 (fig 9) . the data show that that~900-fold excess of cc.4 over the enzyme concentration generated only a 25% decrease in the levels of rna synthesis, which is comparable to the effect generated by the equivalent volume of the buffer only (fig 9a and 9b ). to study the putative effect of rna synthesis or rna synthesis blockage on the otu domain, we monitored dub activity in the presence of all four ntps in the absence and in the presence of nucleotide analogues 2'deoxy-2'-fluoro-ctp and 2'-deoxy-2'-amino-ctp. inhibition of dub activity is not seen under these conditions (fig 9c) . moreover, the rdrp active site mutant d2517n retained dub activity (fig 9d) . together the data suggest that rdrp and dub activities are functionally independent. effective treatments for cchfv infection are currently not available and the requirement for high biosafety measures hamper drug discovery and development efforts with infectious virus. the identification of small molecule compounds that inhibit viral rna synthesis relies on the availability of recombinant l protein, which is a multifunctional protein with a high molecular weight of~450 kda. here we report the expression of full-length cchfv l protein that possess active rdrp and dub domains. the initiation of rna synthesis associated with segmented negative sense rna viruses is a sequence-dependent process that commonly involves conserved regions at the 5' and 3' termini in the untranslated regions of the genome [17, 18, 39, 40] . as rna synthesis proceeds the rdrp enzyme is expected to form an elongation complex capable of transcribing the rna template largely independent of the sequence [41] . we devised model primer/template substrates that mimic the elongation state. gel-based biochemical assays demonstrate that the cchfv full-length l protein is able to extend a short 4-mer primer in the presence of either mg 2+ or mn 2+ . the divalent metal ions likely catalyze the nucleotidyl transfer following a twometal ion mechanism as described for other polymerases [42, 43] . the presence of all four ntps yielded a full-length rna product corresponding to the length of the 14-mer template. limited rna synthesis in the presence of only one, two, and three ntps yielded the expected shorter products. hence, the use of a heteropolymeric primer/template substrate allowed us to study the efficiency and fidelity of single nucleotide incorporation events. in contrast, de novo initiation of rna synthesis in the absence of a primer or the use of shorter rna primers can lead to multiple reaction products [44, 45] , which makes it difficult to translate the data into quantitative terms. as demonstrated for other polymerases [5, 6, 13] , the cchfv l-protein can accommodate favipiravir-tp and ribavirin-tp. both inhibitors act as atp-or gtp-analogues. once incorporated, these compounds compromise further incorporation events and cause partial chaintermination. however, the efficiency of incorporation of the natural counterparts is evidently favored. incorporation of atp (gtp) over ribavirin-tp is approximately 300-(700-)fold more efficient, which likely provides a significant competitive advantage under biologically relevant conditions. the high selectivity for natural nucleotide pools prevents frequent inclusions of the inhibitor into the growing rna chain. this bottleneck provides a plausible explanation for the lack of any clinical benefit with ribavrin. selectivity values for atp(gtp) over favipiravir-tp are slightly more favorable (~90 (330)). favipiravir was also shown to exceed the antiviral effects of ribavirin in a mouse model and in cell-based assays using a cchfv reporter virus [11, 12] . 2'-deoxy-2'-fluorocytidine is another broad spectrum antiviral that was previously identified and assessed in the same assay [15] . this nucleotide analogue showed the lowest ec 50 value of all three compounds. 2'-deoxy-2'-fluorocytidine-tp also shows higher rates of incorporation in our biochemical assay. incorporation of ctp over the inhibitor is much more favorable as compared to ribavirin and favipiravir. we measured a selectivity value of 23. of note, we identified 2'-deoxy-2'-aminocytidine-tp as a new inhibitor of the cchfv lprotein and this compound showed the most favorable selectivity value (~15). it is currently unknown whether the non-phosphorylated mother compound or any derivative may also affect cchfv replication. structural data with the rdrp of human norovirus, which is a fig 6. competition between ctp and 2'deoxy-2'fluoro-ctp. (a) rna primer/template used in the rna synthesis assays to test competition of ctp and 2'deoxy-2'fluoro-ctp is shown above the gel. g indicates incorporation of the radiolabeled nucleotide opposite template position 5. template g allows incorporation of ctp or 2'deoxy-2'fluoro-ctp and their competition for incorporation when both nucleotides are present in the reaction mixture. rna synthesis was monitored with purified cchfv l protein in the presence of [α-32 p]gtp, rna primer/template, 5 mm mgcl 2 and 3.7, 11, 33 and 100 μm atp, ctp, utp and increasing concentrations of 2'deoxy-2'fluoro-ctp. the presence of three natural ntps in the absence of 2'deoxy-2'fluoro-ctp allows full-length product formation up to position 14. lane m illustrates the migration pattern of the radiolabeled 4 nucleotide-long primer. (b) full-lengthtemplate product was quantified as a fraction of total signal in the lane, normalized to the full-template-length product fraction in the absence of 2'deoxy-2'fluoro-ctp and plotted versus log concentrations of the 2'deoxy-2'fluoro-ctp. data were fitted to a dose response function in graphpad (prism 6.0) to determine the concentration of 2'deoxy-2'fluoro-ctp at which the amount of full-length-template product decreased by 50% (ic 50 ). https://doi.org/10.1371/journal.pntd.0008283.g006 positive-sense rna virus (caliciviridae family), showed that the compound binds to the active site and causes reorientations of several amino acids [46] . the otu domain of the cchfv l-protein is another attractive target for the development of intervention strategies [27] . here we demonstrate that the full-length enzyme and the isolated otu domain show very similar levels of dub activities. both enzymes cleave lys48-and these results enabled us to study the relationship or possible interdependency of rdrp and otu domains. first of all, the otu protease activity is not required to obtain active rdrp enzyme. sds-page and ms analysis revealed a single band that was identified as the fulllength cchfv l protein with > 95% peptide coverage. autocatalytic processing is not observed and the otu domain remains attached to the rdrp domain. rna synthesis mediated by the l-protein was previously also demonstrated in cell-based minigenome systems [10] . western analysis likewise revealed that the l-protein is not further processed by the otu domain. a mutation in the otu catalytic site (c40a) did not affect minigenome replication suggesting that the otu protease activity of the l-protein is not required for rna synthesis. conversely, scholte and colleagues reported more recently that the same mutation abolished viral propagation [26] . the presence of the otu inhibitor cc.4 also caused inhibition of cchfv replication in addition to enhancing host antiviral responses [28] . the authors considered different scenarios that help to reconcile this data. the interaction between the otu domain and cc.4 may affect formation of higher order complexes containing rna, l-protein and the nucleoprotein (np). alternatively, the bound inhibitor may render the polymerase inactive. our biochemical data show that the presence of cc.4 does not affect elongation of rna synthesis. moreover, inhibition of rdrp activity in the presence of nucleotide analogues does not affect dub activity. these results demonstrate that rdrp and otu activities are functionally independent. however, it is conceivable that the ubiquitin variant affects the intracellular recruitment of other host or viral factors to the replication complex. we can also not exclude that dub activity and the specific initiation of rna synthesis are somehow linked. structural data with flub rdrp revealed domain rearrangements that may also play a role for the cchfv rdrp during initiation [19, 47] . the expression of recombinant full-length cchfv l-protein provides an important tool for novel drug discovery and development efforts. screening of large libraries of small molecules is now feasible, given that biochemical rdrp assays do not require high biosafety measures. non-nucleoside inhibitors of cchfv rdrp are currently unknown and high-throughput screening (hts) campaigns have the potential to identify such compounds [48, 49] . for this purpose, the gel-based assays utilized in this study need be transferred ideally into plate-based assays with a non-radioactive readout [50] . the few known nucleotide-like inhibitors against cchfv show a broad spectrum of activities against several rna viruses. the availability of the full-length l-protein enables the discovery of more selective compounds with a greater competitive advantage over natural nucleotide pools. although dub activities in the context of the full-length l-protein and the isolated domain are very similar, it is conceivable that the structural environment of the l-protein may also facilitate the discovery of small molecule inhibitors with high selectivity over human dub orthologs. recent advances in understanding crimean-congo hemorrhagic fever virus crimean-congo hemorrhagic fever and expansion from endemic regions crimean-congo haemorrhagic fever in travellers: a systematic review the who r&d blueprint: 2018 review of emerging infectious diseases requiring urgent research and development efforts biochemical evaluation of the inhibition properties of favipiravir and 2'-c-methyl-cytidine triphosphates against human and mouse norovirus rna polymerases the ambiguous base-pairing and high substrate efficiency of t-705 (favipiravir) ribofuranosyl 5'-triphosphate towards influenza a virus polymerase mechanism of action of t-705 against influenza virus japanese surveillance systems and treatment for influenza the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen crimean-congo hemorrhagic fever 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polymerase can perform rna synthesis with modified primers and nucleotide analogs we thank dr. jack moore at the alberta proteomics and mass spectrometry facility for mass spectrometry analysis, dr. sigurd willbanks (otago university, new zealand) for suggesting a simple method to illustrate protein purity as shown in fig 2b, and dr. sachdev sidhu and wei zhang from university of toronto for supplying the cc.4 ubv clone. key: cord-352178-irjhmxsg authors: saxton-shaw, kali d.; ledermann, jeremy p.; borland, erin m.; stovall, janae l.; mossel, eric c.; singh, amber j.; wilusz, jeffrey; powers, ann m. title: o'nyong nyong virus molecular determinants of unique vector specificity reside in non-structural protein 3 date: 2013-01-24 journal: plos negl trop dis doi: 10.1371/journal.pntd.0001931 sha: doc_id: 352178 cord_uid: irjhmxsg o'nyong nyong virus (onnv) and chikungunya virus (chikv) are two closely related alphaviruses with very different infection patterns in the mosquito, anopheles gambiae. onnv is the only alphavirus transmitted by anopheline mosquitoes, but specific molecular determinants of infection of this unique vector specificity remain unidentified. fifteen distinct chimeric viruses were constructed to evaluate both structural and non-structural regions of the genome and infection patterns were determined through artificial infectious feeds in an. gambiae with each of these chimeras. only one region, non-structural protein 3 (nsp3), was sufficient to up-regulate infection to rates similar to those seen with parental onnv. when onnv non-structural protein 3 (nsp3) replaced nsp3 from chikv virus in one of the chimeric viruses, infection rates in an. gambiae went from 0% to 63.5%. no other single gene or viral region addition was able to restore infection rates. thus, we have shown that a non-structural genome element involved in viral replication is a major element involved in onnv's unique vector specificity. o'nyong nyong virus (onnv) is an arthropod-borne virus (arbovirus) associated with a small number of large-scale epidemics. one such epidemic began in 1959 in uganda, lasted three years and affected over 2 million people [1] . serological evidence of onnv transmission indicated circulation in kenya until the late 1960s [2] , additional serological surveys in [1974] [1975] showed circulation in west africa [3] , but onnv did not cause another epidemic until 1996, when 400 people were sickened in the rakai district in southern uganda [4] . the known distribution of onnv mirrors that of the mosquito vectors that transmit the virus, anopheles gambiae and anopheles funestus [5] . humans are the only currently known reservoir of onnv [6] . onnv infection in humans is usually selflimiting, but does cause a low grade-fever, joint pain, lymphadenopathy, and a generalized papular or maculopapular rash [7] . chikungunya (chikv) virus is a closely related alphavirus which has caused millions of cases of disease throughout countries in and surrounding the indian ocean since its re-emergence in 2004 [8] [9] [10] . additional cases occurred in travelers returning from affected areas to asia, north america, and to europe, where a few small epidemics have since occurred due to autochthonous transmission [11] [12] [13] [14] . humans are infected with chikv when bitten by infected aedes aegypti or, during epidemics, aedes albopictus mosquitoes. patients infected with chikv suffer from clinical symptoms similar to those infected with onnv except that the fever is a higher, there is typically no lymphadenopathy, and the arthralgia is both incapacitating and chronic. chikv and onnv diverged from a common ancestor thousands of years ago [15] and despite their genetic similarity, onnv and chikv have distinct ecologies. in particular, they are not transmitted by the same mosquito vectors [6] . in fact, onnv is the only alphavirus to be vectored by anopheline mosquitoes [5] while chikv is transmitted by culicine mosquitoes. the differential mosquito infectivities of chikv and onnv have been characterized in the laboratory where an. gambiae mosquitoes have been shown to be highly susceptible to onnv infection and refractory to chikv infection [6] . thus, this mosquito serves as an ideal system to examine the molecular determinants of infection using hybrids of the two viruses. members of the genus alphavirus contain a single-stranded, positive-sense rna genome (,11.7 kb) that contains two regions, a non-structural domain making up the 59 two-thirds of the rna and a structural domain at the 39 end making up the remaining one-third of the genome [16] . the non-structural domain encodes four viral non-structural proteins: nsp1, nsp2, nsp3, and nsp4 which are essential for replication and polyprotein processing. in addition to copying the rna genome, the non-structural proteins synthesize 26s subgenomic mrna which is capped and polyadenylated and which ultimately produces five individual structural proteins (capsid, e3, e2, 6k, e1) [17] . previous studies with chimeric alphaviruses have shown that viral components in both the structural and non-structural portions of the venezuelan equine encephalitis genome contribute to infection phenotypes in guinea pigs [18] . similar results were seen with chimeric eastern equine encephalitis viruses, with both structural and nonstructural proteins contributing to neurovirulence and viral tissue tropism in mice [19] . chimeric viruses are also useful for studying virusvector interactions as seen in a study that mapped mosquito infection determinants specifically to the e2 envelope glycoprotein region of venezuelan equine encephalitis [20] . earlier studies using chimeric onnv suggested that all of the viral structural proteins are necessary for onnv to infect an. gambiae mosquitoes [21] ; however these studies used limited sample sizes and looked only at the structural regions of the genome. the current study expanded upon this earlier work to examine the contribution of each specific viral region or gene to virus-vector specificity. all plasmid clones used in this study were designed and constructed in house. the full length clone ponn.ap3 was constructed from onnv strain sg650 [5] (genbank accession number af079456) while pchik.b was constructed from chikv strain 37997 (genbank accession number ay726732). these two full -length parental clones were used to construct 15 chimeric viruses as shown in figures 1 and 2 . all plasmid clones designed to evaluate structural regions of the genome were constructed in a similar fashion, with the substituted region produced from a pcr product and the backbone region produced from the parental plasmid clones described previously. for example, to construct pchik/onn e2, the e2 region of onnv was amplified from parental ponn.ap3 by pcr with pfu turbo polymerase (stratagene, la jolla, ca). the onnv amplicon and pchik.b were digested with the same restriction enzymes (table 1) . when necessary, appropriate restriction enzyme sites were added to pchik.b using a quikchange xl site-directed mutagenesis kit (stratagene) according to the manufacturer's instructions. modifications were performed so that no amino acid changes were introduced and all viruses generated from constructs with introduced mutations were tested to insure they replicated in a manner comparable to the parental viruses. both restriction digests were run on an agarose gel at low voltage for at least 8 hours. the doubly-digested insert and plasmid backbone were cut from the gel and purified from the agarose using the minelute gel extraction kit (qiagen, valencia, ca). the backbone vector was treated with antarctic phosphatase (neb, beverly, ma) to remove the 59 phosphate groups, thus preventing self-ligation of the plasmid. prepared plasmid backbone and insert were ligated overnight with t4 dna ligase (neb) and then electroporated into xl-1 blue electrocompetent cells (stratagene). transformed cells were grown on yt plates with 50 mg/ml ampicillin and incubated overnight at 37uc. colonies were picked and screened for confirmation of onnv insert by pcr. plasmid clones were confirmed by sequencing the entire construct. plasmid clones designed to evaluate non-structural regions of the genome were engineered as exact gene substitutions by using a series of subclones, as described in the protocol s1 and in figures s1, s2, s3, s4, s5, s6. briefly, chikv regions flanking the desired non-structural protein, and containing convenient restriction sites were amplified from pchik.b by pcr with pfu turbo polymerase (stratagene). primers used for amplification added a type ii restriction enzyme site to the outside of each desired insert product. these two pcr products and a modified cloning vector, puc19m2 were each double digested using type i enzymes exterior to the type ii engineered sites. a 3-way ligation then produced the first subclone (puc19m2 with the chikv sequence flanking where the desired non-structural protein sequence would subsequently be inserted). the desired onnv non-structural protein was amplified using primers which added the same type ii enzyme site as was added to the pcr products. the first subclone and the onnv pcr product were both digested with the same type ii enzyme, which cuts itself out upon digestion. ligation of the two digested products produced a second subclone (puc19m2 with the entire and exact onnv nonstructural protein, flanked by chikv sequence). this second subclone and pchik.b were then digested using the convenient restriction sites already present in the flanking chikv sequence. ligation of the doubly-digested pchik.b backbone and the insert obtained from the second subclone produced the final construct. colonies were screened and verified by complete genome sequencing and plasmid dna was prepared as described above. templates for in vitro transcription were generated by linearizing each full-length clone with a unique not i restriction site present downstream of the poly (a) tail. linearized plasmids were treated with proteinase k (invitrogen, carlsbad, ca) to digest any endogenous rnases or dnases. dna was purified using a phenolchloroform extraction followed by an ethanol precipitation. a 20 ml aliquot of linearized and treated dna was then transcribed in vitro by incubating the dna with 0.4 ml (100 mm) each rntp (promega, madison, wi), 0.4 ml bsa (10 mg/ml) (neb, beverly, ma), 2 ml dtt (100 mm) (promega, madison, wi), 8 ml (5x) transcription buffer (promega, madison, wi), 1.3 ml (15 u/ml) t7 rna polymerase (promega, madison, wi), and 4 ml (10 mm) acap-structure analog (neb, beverly, ma) for one hour at 39uc. transcribed rna (10 ml) was mixed with 400 ml bhk-21 cells (1610 7 cells/ml) in a 2 mm gap cuvette (btx:harvard apparatus, inc., holliston, ma) and electroporated twice using a btx electrocell manipulator with the following settings: 460volts, 725ohms, 75 mf [22] . after electroporation, the cells were transferred to a t-25 tissue-culture flask. dulbecco's modified eagle medium (dmem) with 10% by volume fetal bovine serum and 1% by volume penicillin/streptomycin was added to the flask o'nyong nyong virus (onnv) is unique in that it is the only alphavirus, and one of few viruses in general, to be transmitted to humans by the bite of an anopheline mosquito. the genetics responsible for this unique vector specificity would be useful information in helping to develop antivirals, vaccines, and other methods for interrupting virus transmission. previous research using other arboviruses has shown that specific viral genomic regions, amino acid sequences, or even single nucleotide mutations can have a profound effect on virus growth, infection, and virulence characteristics. using chimeric viruses that substitute a gene from one virus with a gene from a closely related virus is a proven method of evaluating the relative contribution of each gene to a given phenotype. our study analyzed both structural and non-structural regions of the onnv genome using chimeric viruses and artificially infected anopheles gambiae mosquitoes. when onnv non-structural protein 3 (nsp3) replaced nsp3 from chikungunya virus in one of the chimeric viruses, infection rates in an. gambiae went from 0% to 63.5%. no other single gene or viral region addition was able to restore infection rates. that onnv nsp3 is largely responsible for onnv's unique ability to infect an. gambiae is especially interesting since the exact mechanisms and functions of this highly-variable protein remain poorly understood. before incubation at 37uc. tissue culture supernatant was harvested approximately 72 hours post-transfection or when cytopathic effects (cpe) were observed. supernatant was aliquoted and stored at 280uc until later use. each time a virus was generated, the entire virus was sequenced to verify fidelity to the original sequence. viral rna was extracted using qiaamp viral rna mini kit (qiagen). extracted rna was then added to a reverse-transcriptase pcr reaction using the titan one tube rt-pcr system (roche, indianapolis, in). complementary dna for sequencing the 59 end of each viral genome was generated using a firstchoice rlm-race kit (ambion, austin, tx). this complementary dna was then sequenced using virus-specific primers with the big dyev3.1 kit on an abi 3130xl genetic analyzer (applied biosystems, foster city, ca). sequence files were aligned and analyzed for sequence quality and genome coverage using lasergene suite software (dnastar, madison, wi). virus rescued from clones was titered by plaque assay. ten-fold virus dilutions from 10 21 to 10 27 were added to individual well of 6-well plates covered in monolayers of vero cells. plates were incubated at 37uc, with 5% co 2 . cells were fixed 48-72 hours later using a solution of 40% methanol and 0.25% crystal violet in water. plaques were then counted and titers were calculated as plaque forming units per milliliter (pfu/ml). the ability of each chimeric virus to infect mosquitoes was evaluated using the g3 strain of an. gambiae, originally obtained from the national institute of health. this strain has been maintained as a colony in our lab with rearing conditions that include a 12:12 hour light:dark cycle in chambers maintained at 28uc with approximately 95% humidity [23] . infectious blood meals were prepared from equal volumes of packed, calf erythrocytes, 10% sucrose in fetal bovine serum, and 4.4-6 log 10 pfu/ml of virus. mosquitoes were allowed to feed on the warmed infectious blood meal for one hour through an artificial membrane feeder (hemotek, accrington, uk). fully engorged females were separated and maintained for an incubation period of up to 12 days. mosquitoes were sacrificed at days 4, 8, and 11 or 12 post-infectious-blood meal. heads and bodies were separated into individual tubes and stored at 280uc until subsequent processing. infection rates were determined using results from the 2-3 replicate feeds were combined and the titer (log 10 pfu/ml) shown is an average (for titers that were within 0.5 log 10 pfu/ml of one another). infection rate is determined using mosquito bodies while dissemination rate is derived from heads. doi:10.1371/journal.pntd.0001931.g001 individual bodies and dissemination rates were calculated as the number of positive heads among the positive bodies. at least two replicate infectious feeds were done for each chimeric virus, with replicate feeds performed entirely independent of one another. no less than 140 mosquitoes were tested for any one chimeric virus. individual frozen mosquito bodies and heads were triturated in 300 ml of dmem supplemented with (by volume): 10% heatinactivated fetal bovine serum, 1% penicillin/streptomycin, 0.1% gentamicin, and 0.1% fungizone. the mosquito homogenates were passed through a 0.2 mm gelman acrodisc filter (krackeler scientific inc., albany, ny) to remove potential bacterial or fungal contaminates. filtrate from each body or head was added to a single well of a 96-well flat-bottom tissue-culture plate, along with 50 ml of prepared bhk-21 cell suspension (approximately 4.6 log 10 cells/well). inoculated tissue-culture plates were incubated at 37uc for 5 days. cells were observed daily for cpe due to virus replication. virus replication in mosquito body samples indicated that virus had infected the mosquito's midgut, while replication in the mosquito heads showed a disseminated infection. to confirm that all constructed viruses were comparably replication competent, growth curves were performed in cell culture on all rescued viruses. briefly, 24-well plates (corning, corning, ny) were seeded with vero (african green monkey) cells. monolayers at 90% confluency were infected with virus at a multiplicity of infection (moi) of 0.1. at specified times post infection, supernatant was removed from two wells for each virus and placed in a screw-cap cryovial at 270uc until titration by plaque assay. titration results for each virus were compared at all time points by the student t-test. to confirm virus replication (and not just persistence of the input virus) within the mosquito, five females that had fed on the onnv infectious blood meal were sacrificed every other day post-infectious feed. each body and head was processed separately, as described earlier. rna was extracted from homogenized mosquitoes using qiaamp viral rna mini kit (qiagen). the amount of rna in each head and body was determined using the quanti tect probe rt-pcr kit (qiagen) and a taqman real-time pcr assay as previously described [24] , except that onnv sg650 specific primers were used (10692 fwd-59 gca ggg agg cca gga cag t, 10840 rev-59 gcc cct ttt tcy ttg agc cag ta) . the real-time probe was labeled with a 59 end hex reporter dye and a 39 end bhq1 quencher dye (10759 fwd-59 aaa gac cag cgg cag gag caa tac ac) and pcr results are reported here as pfu-equivalents/mosquito by comparison with known concentration standards. fisher's exact probability test was employed to evaluate whether infection rates with chimeric viruses were statistically different from those with parental viruses. the infection rate was defined as significantly different from parental chikv if the two-tailed pvalue was ,0.007. the two-tailed p-value had to be ,0.01 to be statistically different from parental onnv infection rates. both adjusted alphas were obtained using the bonferroni correction for multiple comparisons to ensure an overall type i error of 0.05. computations were made using freely-available software [25] . this study built upon previously established disparate infectivity patterns for two closely related alphaviruses, chikv and onnv, in an. gambiae [5, 6] and infection rates with the parental viruses generated from our full-length infectious clones were concordant with those previously reported. by day 12, up to 91% of an. gambiae mosquitoes were infected with onnv, whereas a maximum of only 6% were infected with chikv. these values are similar to previously published work [6] . with this highly significant difference (p,0.0001) between the two viruses, characterization of individual viral gene substitutions was likely to reveal which elements were involved in mosquito infection. prior to initiating these experiments with chimeric viruses in an. gambiae, viral replication (and not just persistence of input virus) within both cell culture and in the mosquito was confirmed. cell culture growth curves of all of the chimeras were performed in vero cells to confirm that all viruses were indeed replication competent and replicated in a manner similar to their parental viruses ( figure 3 ). the structural change viruses all replicated efficiently and replication was virtually identical among all chimeras sharing non-structural genes. in general, those viruses with the onnv non-structural genes grew to peak titers of 10 6.5 -10 7 pfu/ ml while those with chikv non-structural genes had peak titers of 10 7.5 -10 8 pfu/ml. all non-structural chimeric viruses grew similarly well, rapidly increasing in titer from 1000 pfu/ml to 10 7 -10 8.5 pfu/ml. no consistent statistical differences were observed among the non-structural substitution viruses. the quantity of onnv rna present in individual mosquito bodies and heads through 11 days post-infectious feed adhered to the expected pattern of decrease during the extrinsic incubation period followed by a rise in virus replication at later time points as determined by qrt-pcr. moreover, after 5 days post infection, the five mosquito bodies tested at each of the subsequent time points had more rna copies than could have been initially imbibed in the blood meal indicating replication of the virus was indeed occurring (figure 4) . nine unique chimeric hybrids of chikv and onnv were constructed using convenient restriction enzyme sites to produce substitutions in the structural region of the viral genome and to examine the contribution of each of these specific regions to virusvector specificity (figure 1 ). six additional non-structural chimeric viruses were also constructed using a novel type ii restriction enzyme cloning strategy to examine the broader genome with respect to onnv's unique vector specificity for an. gambiae mosquitoes (figure 2) . having confirmed viral replication and infections rates of parental onnv in an. gambiae, infection and dissemination rates with each of the 15 chimeric viruses were determined. each time a virus was generated through in vitro transcription for this study, it was sequenced completely prior to use in an infectious feed. mosquitoes containing replicating virus in the body, as shown by cpe analysis, were defined as being positive for viral infection. mosquito heads were analyzed separately from bodies to determine dissemination rates. each chimeric virus constructed from the parental chikv genome maintained a chikv-like infection profile (,10% infection rate), with one exception. when allowed to feed on a blood meal containing approximately 5.5 log 10 pfu/ml of chik/onn nsp3 virus, 63.5% (n = 85) of mosquitoes had replicating virus when harvested on day 8 post infection ( figure 2) . none of the onnv substitutions made to the structural regions of the chikv parental genome produced infection results deviating from those seen with the complete chikv parental genome ( figure 1 ). three of the 5 chimeric viruses constructed from the parental onnv genome retained onnv-like infection rates at day 8 in an. gambiae, while the remaining two viruses showed significantly lower infection rates. only 11.1% (n = 135) of mosquitoes feeding on onn/chik 39str and 53.2% (n = 77) of onn/chik 59str were shown to be infected at day 8. infection rates for mosquitoes sacrificed at days 4, 11, or 12 corroborate day 8 results (data not shown). dissemination rates for each of the viruses in an. gambiae was very low and all were comparable for both day 8 and day 11 samples. only 5 viruses showed any dissemination (figures 1 and 2): parental onn.ap3,onn/chik e2, onn/chik estr, onn/ chik 39nsp4-59c, chik/onn 39str. the rest of the viruses showed no dissemination. a panel of 15 chimeric viruses were developed here to study specific elements of the onnv genome and to determine which of these regions are necessary for onnv to infect an. gambiae mosquitoes. as chikv virus primarily infects aedes species and onnv primarily infects anopheles species, these two closely related viruses provide an ideal opportunity to study these viral genetic determinants of infection. this study is the first to look at the importance of onnv non-structural proteins in an. gambiae infection. of the ten chikv-backbone chimeras constructed and tested, only the one containing onnv nsp3 produced infection rates closer to parental onnv than to the parental chikv. the ability of onnv nsp3 to up-regulate infection rates so substantially shows that onnv nsp3 is the main determinant of onnv vector specificity for an. gambiae. interestingly, the reciprocal chimeric virus (full length-onnv with the chikv nsp3) was not able to be rescued from cdna in either mammalian or insect cells. this would further suggest that nsp3 plays a critical role in viral replication that is distinct in these two closely related viruses that exhibit 81% and 72% amino acid and nucleotide identity respectively in nsp3. that nsp3 should be found to be essential to infection is especially interesting given the fact that the precise functions of this protein are not fully defined. it is required for the correct formation and localization of replication complexes and does provide essential functions in both minus strand and subgenomic rna synthesis, but specific mechanisms are not yet resolved [21] [22] [23] . to further add to the intrigue of this protein, it has been shown that some members of the alphavirus family actually contain inserts of foreign genetic material within nsp3. an eight amino acid sequence from the carboxyl-terminus of chikv nsp3 maps to a putative zinc finger protein in ae. aegypti, the main vertebrate vector for that virus [26] . in semliki forest virus, a 7 amino acid sequence corresponds to elements found in a wide-range of cellular proteins [26] . numerous other examples of what may be inserts of foreign genetic material been shown by sequencing nsp3 from the following alphaviruses: chikv, eastern equine encephalitis virus, semliki forest virus, and venezuelan equine encephalitis virus [26] . alphavirus nsp3 can be clearly divided into two distinct domains. the macro domain, or amino-terminal region, is highly conserved, not just among alphaviruses but also among coronaviruses, hepatitis e virus, rubella virus and even cellular proteins [27, 28] . the carboxyl-terminus domain of the alphavirus nsp3 is highly variable in size and sequence and is devoid of any predicted secondary structure [29, 30] . chimeric viruses were constructed using the natural division between the conserved and nonconserved regions of nsp3 to engineer two additional chimeric viruses to determine if the region conferring specificity to an. gambiae could be attributed solely to either of the distinct domains within nsp3. interestingly, the addition of just the carboxylterminus of onnv nsp3 did produce a small, although not a statistically significant, increase in infection rates as compared with parental chikv in an. gambiae. the carboxyl-terminus of nsp3, which has been subject to rapid alteration during alphavirus evolution, may also be involved in the optimization of replication in diverse host cell types [29] . studies with sindbis showed that deletions in the carboxyl-terminus rendered mutants defective at initiating a productive infection, generating plaques in mosquito cells at only 1-2% the efficiency of the parental virus [31] . a recent study noted a carboxyl-terminus, proline-rich sequence motif, the pipppr motif, shared by many alphavirus nsp3 proteins and demonstrated that even a single mutation in this region of semliki forest virus or sindbis virus greatly impaired rna synthesis by disrupting binding with host cell amphiphysins [32] . it is possible that this motif also modulates onnv vector specificity since onnv and chikv do differ from one another by one amino acid in this pipppr region. attenuated virulence and reduced rates of rna synthesis and virus replication were also seen in vertebrate cells with semliki forest virus mutants lacking some portion of the carboxyl-terminus of nsp3 [33] . yet, studies in mammalian cell lines showed that a 34 amino acid deletion in this region of nsp3 in venezuelan equine encephalitis had no detectable effect on replication [34] . collectively, these studies support the current finding that nsp3 can be vital for productive infection, but in a manner that is host and virus specific. another interesting characteristic of the carboxyl-terminus of nsp3 is that it is phosphorylated at multiple serine and threonine residues [35, 36] . the role of this phosphorylation is not exactly clear, except that it does seem to modulate the efficiency of minusstrand rna synthesis [37, 38] . determination of the exact mechanisms of this modulation and the mechanisms for the host-specific effects seen with nsp3 mutants in this and other studies would be extremely valuable information and allow for design of further studies. furthermore, our studies show that an intact onnv nsp3 is required for onnv-like infection rates, and that dividing the region either disrupts a vital interaction between the two or removes an element necessary for an. gambiae infection. the former seems more probable since substituting chikv for either half of onnv nsp3 results in infection rates not significantly different from rates with parental chikv. while molecular determinants residing in nsp3 did turn out to be the most dramatic finding of our study, we did also examine the structural regions of the genome. previously published studies by another group had suggested that all of the viral structural proteins are necessary for onnv to infect an. gambiae mosquitoes [21] . our study was able to provide critical fine tuning to this conclusion. in our experiments with chikv-backbone chimeras containing various regions of the onnv structural proteins, each maintained parental chikv-like infection profiles despite containing portions of the onnv genome. in fact, even an intact onnv structural region was not sufficient for infection of an. gambiae, as shown with the chimera chik/onn estr (figure 1 ). the reciprocal chimeras, substituting sections of chikv structural regions for the like section of onnv structural genes, in most cases, did not greatly reduce mosquito infection rates. the notable exceptions were in the chimeras that divided the onnv structural region in half. both onn/chik 59str and onn/ chik 39str were significantly less infectious to an. gambiae than was parental onnv. however, since the reciprocal chimeras, chik/onn 59str and chik/onn 39str, did not show upregulated infection rates, the drop in infection with the chimeric viruses is likely due to disruption of one or more virus-virus or virus-host interactions. in alphaviruses, the extreme 39 terminus of the genome, just preceding the poly(a) tail, has a sequence which is highly conserved among all alphaviruses and which is absolutely required for efficient virus replication [39, 40] . this 19-nucleotide sequence is identical in chikv and onnv so this could not have played a role in the decreased infection rates seen with onn/chik 39str. however, studies using sindbis mutants with large deletions in the 39non-translated region (ntr) have shown that the rest of the 39 ntr is also important for virus replication in a host-specific manner [40] . onnv is 156 additional nucleotides shorter in the 39ntr when compared to chikv; this size difference alone could result in conformational changes resulting in the inability of the virus to interact with itself or with host proteins. of note is the design of our estr and 39str structural clones, which contain the indicated structural region as well as the 39 ntr from the non-parental virus ( figure 1) ; this design is different from those previously described [21] and may suggest the possibility of multiple interactions within the proteins or gene sequences of the virus itself that may have a minor role in the overall ability of a chimeric virus to replicate within the mosquito. it is further possible that the differences in chikv and onnv conserved sequence elements [41] are sufficient to undercut rna stability, resulting in greatly reduced mosquito infection patterns. studies with chimeric viruses must be viewed in the overall context of the virus' life cycle. when substitutions are made to construct chimeric viruses, numerous aspects of the virus-host interactions and virus-virus regulatory functions can be disrupted resulting in reduced infection rates. reduced infection rates may be a direct consequence of missing the essential genomic region or may be an indirect result of disrupting an essential regulatory interaction. conversely, when the addition of a specific region increases mosquito infection rates, we must conclude the region itself to be essential for infection. interestingly, because there was such a low dissemination rate of all viruses within this study, elements involved in dissemination throughout the mosquito may be distinct from those important in initial infection. however, this study has shown that onnv nsp3 is directly responsible for onnv infection of an. gambiae. there are also numerous interactions within nsp3 itself, within the two halves of the structural region, and possibly the 39 ntr which, when disrupted, can eliminate mosquito infection. figure s1 illustration of exact nsp3 substitution made to create chik/onn nsp3. (tiff) figure s2 construction of chikv nsp3 receiving plasmid. pcr primers were designed to generate two amplicons flanking the dna insertion sites and extend outward to include unique restriction enzyme sites and inward to include a unique type ii restriction site. amplification with these primers, subsequent digestion with pcii/saci or ecori/saci, followed by a 3-part ligation produce a puc-based vector containing chikv sequence flanking the site where onnv nsp3 will later be inserted. (tiff) figure s3 amplifying onnv nsp3. pcr primers were designed to amplify the desired dna insert, with the addition of type ii restriction enzyme sites to the termini. type ii sites were oriented such that they will be removed upon later digestion. (tiff) figure s4 expanded sequence of assembled chikv nsp3 receiving plasmid (top). termini of onnv nsp3 amplicon (bottom). the lines indicate the cut sites for the type ii restriction enzymes. (tiff) figure s5 products produced after digestion with appropriate type ii restriction enzymes. these products were ligated to build the chikv/onnv nsp3 cassette plasmid. (tiff) figure s6 construction of final clone, chik/onn nsp3. chikv/onnv nsp3 cassette plasmid and pchik.b were digested with spei and avrii. the resulting products were ligated to generate the final clone with the complete onnv nsp3 gene replacing the like gene in chikv. protocol s1 methods for construction of gene specific clones. (docx) o'nyong-nyong fever: an epidemic virus disease in east africa. 8. virus isolations from anopheles mosquitoes the epidemiology of o'nyong-nyong in the kano plain viral infections in travellers from tropical africa emergence of epidemic o'nyong-nyong fever in southwestern uganda infection patterns of o'nyong nyong virus in the malaria-transmitting mosquito, anopheles gambiae differential infectivities of o'nyong-nyong and chikungunya virus isolates in anopheles gambiae and aedes aegypti mosquitoes o'nyong nyong fever: an epidemic virus disease in east africa. iii. some clinical and epidemiological observations in the northern province of uganda seroprevalence of chikungunya virus (chikv) infection on lamu island seroprevalence of chikungunya virus infection on grande comore island, union of the comoros chikungunya fever: an epidemiological review of a re-emerging infectious disease infection with chikungunya virus in italy: an outbreak in a temperate region autochthonous chikungunya virus transmission may have occurred in bologna, italy, during the summer 2007 outbreak first cases of autochthonous dengue fever and chikungunya fever in france: from bad dream to reality re-emergence of chikungunya and o'nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships the alphaviruses: gene expression, replication, and evolution a new role for ns polyprotein cleavage in sindbis virus replication the use of chimeric venezuelan equine encephalitis viruses as an approach for the molecular identification of natural virulence determinants structural and nonstructural protein genome regions of eastern equine encephalitis virus are determinants of interferon sensitivity and murine virulence vector infection determinants of venezuelan equine encephalitis virus reside within the e2 envelope glycoprotein determinants of vector specificity of o'nyong nyong and chikungunya viruses in anopheles and aedes mosquitoes intracellular immunization of mosquito cells to lacrosse virus using a recombinant sindbis virus vector manual for mosquito rearing and experimental techniques virulence variation among isolates of western equine encephalitis virus in an outbred mouse model lineage replacement accompanying duplication and rapid fixation of an rna element in the nsp3 gene in a species of alphavirus evolution and taxonomy of positive-strand rna viruses: implications of comparative analysis of amino acid sequences evolutionary conservation of histone macroh2a subtypes and domains functions of alphavirus nonstructural proteins in rna replication amino acid mutations in the replicase protein nsp3 of semliki forest virus cumulatively affect neurovirulence deletion and duplication mutations in the c-terminal nonconserved region of sindbis virus nsp3: effects on phosphorylation and on virus replication in vertebrate and invertebrate cells sh3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsp3 promotes viral rna replication deletions in the hypervariable domain of the nsp3 gene attenuate semliki forest virus virulence in vitro synthesis of infectious venezuelan equine encephalitis virus rna from a cdna clone: analysis of a viable deletion mutant semliki forest virusspecific non-structural protein nsp3 is a phosphoprotein phosphorylation of sindbis virus nsp3 in vivo and in vitro elimination of phosphorylation sites of semliki forest virus replicase protein nsp3 phosphorylation site analysis of semliki forest virus nonstructural protein 3 deletion mapping of sindbis virus di rnas derived from cdnas defines the sequences essential for replication and packaging mutagenesis of the 39 nontranslated region of sindbis virus rna the 39 untranslated region of sindbis virus represses deadenylation of viral transcripts in mosquito and mammalian cells we would like to thank andrea peterson for maintaining the an. gambiae, g3 colony used in this study. we would also like to thank dr. kimberly keene for modifying infectious clones. thanks to dr. mark delorey for his guidance in choosing appropriate statistical analysis tools. a special thanks to dr. carol wilusz and dr. susan knudson for their valuable insight. the findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention. key: cord-296226-ugeupo3u authors: sim, shuzhen; ng, lee ching; lindsay, steve w.; wilson, anne l. title: a greener vision for vector control: the example of the singapore dengue control programme date: 2020-08-27 journal: plos negl trop dis doi: 10.1371/journal.pntd.0008428 sha: doc_id: 296226 cord_uid: ugeupo3u vector-borne diseases are a major cause of morbidity and mortality worldwide. aedes-borne diseases, in particular, including dengue, chikungunya, yellow fever, and zika, are increasing at an alarming rate due to urbanisation, population movement, weak vector control programmes, and climate change. the world health organization calls for strengthening of vector control programmes in line with the global vector control response (gvcr) strategy, and many vector control programmes are transitioning to this new approach. the singapore dengue control programme, situated within the country’s larger vision of a clean, green, and sustainable environment for the health and well-being of its citizens, provides an excellent example of the gvcr approach in action. since establishing vector control operations in the 1960s, the singapore dengue control programme succeeded in reducing the dengue force of infection 10-fold by the 1990s and has maintained it at low levels ever since. key to this success is consideration of dengue as an environmental disease, with a strong focus on source reduction and other environmental management methods as the dominant vector control strategy. the programme collaborates closely with other government ministries, as well as town councils, communities, the private sector, and academic and research institutions. community engagement programmes encourage source reduction, and house-to-house inspections accompanied by a strong legislative framework with monetary penalties help to support compliance. strong vector and epidemiological surveillance means that routine control activities can be heightened to specifically target dengue clusters. despite its success, the programme continues to innovate to tackle challenges such as climate change, low herd immunity, and manpower constraints. initiatives include development of novel vector controls such as wolbachia-infected males and spatiotemporal models for dengue risk assessment. lessons learnt from the singapore programme can be applied to other settings, even those less well-resourced than singapore, for more effective vector control. vector-borne diseases (vbds) are a major cause of morbidity and mortality in the tropics and subtropics, accounting for more than 17% of the global burden of infectious diseases [1] , with over 80% of the world's population at risk from at least one vbd [2] . aedes-borne diseases, including dengue, chikungunya, yellow fever, and zika, are increasing at an alarming rate driven by urbanisation, local and global population movement, and climate change [3] [4] [5] . aedes aegypti mosquitoes lay eggs in a wide variety of artificial containers and structures, including those that occur in discarded plastic containers, water storage containers, flowerpots, tyres, poorly constructed concrete structures in the ground, and gutters, which abound in urban environments. the main tool we have for controlling vbds is vector control [6] . although vector control has been hugely successful against some vbds such as malaria [7] , weak implementation of sustainable programmatic vector control has struggled to control aedes-borne diseases in many parts of the world in the past 20 years. margaret chan, former director-general of the world health organization (who), famously said in her 2016 world health assembly address that the zika epidemic was "the price being paid for a massive policy failure that dropped the ball on mosquito control in the 1970s" [8] . in order to strengthen vector control, who member states called on who to develop the global vector control response 2017-2030 (gvcr) [9] . this strategic document includes a framework of key priorities for vector control strengthening. the gvcr calls for collaboration within and outside the health sector, increased engagement of communities, scaling up and integration of vector control tools and approaches, and improved surveillance and monitoring and evaluation (fig 1) . this is supported by a foundation of capacity and capability strengthening and by increased basic and applied research and innovation. also important are factors including country leadership, advocacy, resource mobilisation, and regulatory, policy, and normative support. who member states are reorienting their programmes in line with the gvcr [10] , but good examples of the gvcr principles in action are currently limited. one exception is the dengue control programme in singapore, which in fact predates the gvcr but adopts many gvcr principles. this manuscript aims to document the key features of the programme, highlight how they sit within the gvcr framework, and draw out important lessons that can be applied by other vector control programmes, including those less well-resourced than singapore's. the republic of singapore is an island city-state in southeast asia with a land area of 724.2 km 2 and a population of 5.64 million [11, 12] . singapore is located at the southern tip of the malay peninsula, connected to peninsular malaysia via two causeways and by ferry services with indonesia's riau islands to the south. singapore is a travel and business hub with 18.5 million visitors in 2018 [13] . the city-state is highly urbanised and has a high population density with almost 8,000 people per km 2 , and typically tens of thousands of people per km 2 in the main residential areas [14] . since 1963, singapore has been working towards the vision of creating a 'city in a garden', where greenery and biodiversity are seen as solutions to improving health and well-being of its citizens [15] [16] [17] . seventy-nine percent of singapore's residents live in high-rise apartments built by the government's housing and development board (hdb), with the remainder living in privately owned condominiums (16%) or landed property (5%) [18] . the climate in singapore is tropical and it is hot and humid all year round. singapore is endemic for dengue, with all four dengue virus (denv) serotypes circulating, frequent emergence of various genotypes, and a cyclical pattern of outbreaks every 5-7 years [19] [20] [21] [22] . in 2005 in , 2007 in , 2013 , and 2016, singapore experienced explosive dengue fever outbreaks that resulted in 14,032, 8,287, 22,170, and 12,848 indigenous cases, respectively, with incidence rates of 322.5, 180.6, 404.9, and 229.1 per 100,000 population [23] [24] [25] [26] . in 2019, singapore also experienced a large dengue outbreak with more than 16,000 cases [27]. there is low herd immunity, particularly among younger generations, due to decades of low dengue transmission [28] [29] [30] . the primary dengue vector is a. aegypti, and although aedes albopictus is also present, it is considered a weak secondary vector [31] . exposure to chikungunya is low in singapore, with seroprevalence of 1.9% among adults [32] . the country has experienced small local outbreaks of chikungunya, with the largest (1,011 cases and an incidence rate of 18.7 per 100,000) occurring in 2013, a year when aedes densities were high [25] . singapore's first zika outbreak occurred in 2016, and although it occurred at the same time as the epidemic in brazil and other latin american countries, the zika strain was found to have originated in southeast asia [33] . malaria was eliminated in singapore in 1981 [34] , but there are still sporadic imported cases and competent vectors including anopheles epiroticus (formally anopheles sundaicus), anopheles maculatus, and the emerging vector anopheles sinensis [35, 36] . since independence, singapore has recognised that a clean and green environment is necessary not only to ensure good health and a good quality of life for its people but also for economic competitiveness. the government embarked on projects to clean up the land and waterways and invested heavily in critical sanitation and environmental infrastructure, including drainage development projects, sewerage and used water treatment infrastructure, and solid waste management. the environmental management put in place to implement this high standard of public cleanliness has greatly benefited singapore's efforts to tackle vbds. underscoring the view that aedes-borne diseases are environmental diseases, dengue control in singapore is led by the national environment agency (nea), a statutory board of the ministry of the environment and water resources (mewr). in view of the importance of infrastructure maintenance and design, environmental sanitation, people's behaviours, and use of technologies on dengue prevention, the nea collaborates closely with other government ministries (e.g., health, national development, education, finance), town councils (responsible for management and maintenance of the common property of public housing estates, including vector control), community associations, research and academic institutions, and the private sector (fig 2) . intersectoral activities are coordinated by an inter-agency dengue taskforce which meets regularly, and monthly during outbreaks. in the event of a severe dengue outbreak, the taskforce membership is escalated to minister level. in addition to regular meetings, taskforce members are also in close contact via email and telephone to exchange feedback and information-for example, on unusual aquatic habitatsin a timely manner. resources for government agencies to carry out dengue control activities are allocated by the ministry of finance, to which each agency justifies its own funding needs. if necessary, nea supports these justifications, especially in an outbreak year when nea has called on agencies to step up dengue control measures. nea may also offer subsidies to town councils to support enhanced dengue control measures in public housing estates. for example, an important partner of the nea is the hdb, the statutory board responsible for public housing. in high-rise apartments in singapore, laundry is typically hung out to dry on bamboo poles fixed into tube-shaped bamboo pole holders on the side of the building. discovery of substantial numbers of aedes larvae in these holders led to provision of caps to cover them when not in use. later, the clothes-drying system was redesigned such that the bamboo poles now rest on brackets instead, eliminating the need for holders (fig 3) . as mounting the bamboo poles in the holders required residents to lean out of the window, replacing the holders with brackets not only removed a mosquito breeding habitat but was also safer for residents. antimosquito valves that allow water to drain away but prevent escape of mosquitoes are installed in gully drains in hdb apartments, and hdb blocks are constructed without roof gutters, since these are difficult to access and maintain at height and therefore can become aedes habitats. nea works closely with town councils who are responsible for vector control around the hdb blocks. construction sites play an important role as a driver of sustained dengue transmission [37] , since rainwater-filled land excavation holes, construction materials, and equipment (e.g., water tanks, skips, canvas sheeting) can become aedes habitats. the singapore contractors association limited (scal) is therefore an important partner for nea. construction sites are mandated to engage an environmental control officer (large sites may even have their own dedicated officer) who ensures appropriate action is taken to reduce vector proliferation, including larviciding with bacillus thuringiensis israelensis (bti) (fig 4) . the land transport authority is responsible for good housekeeping and vector control on their infrastructure construction sites and the urban redevelopment authority conducts vector control along roads and their car parks. within the nea, the department of public cleanliness maintains a system of daily litter collection and its mandate is cleaning to 'public health standards'. approximately 60% of singapore's waste is recycled and the remainder incinerated, with the waste ash deposited on an offshore landfill site, semakau island, helping expand the island. this serves to reduce artificial containers in the environment which could otherwise become habitats for aedes mosquitoes. within the mewr, the nea works alongside the public utilities board (pub), which is responsible for ensuring a sustainable and efficient water supply and responsible for drainage system in singapore. vector control activities of the pub include designing drains for maintainability (accessible and with sufficient gradient to prevent water pooling) and regular flushing of drains monthly or bimonthly. the department of public cleanliness conducts regular cleaning of drains to remove any debris or refuse. the national parks board (nparks) also works closely with nea to maintain a pleasant environment with trees, plants, and water features such as ponds and streams, while keeping vector densities to a minimum. vector control measures implemented include weekly bti treatment of ponds and regular removal of floating vegetation. if vectors do become a problem, surveillance and vector control measures are quickly put in place. for example, in the first quarter of 2019, ornamental papyrus beds in bishan-ang mo kio park encouraged the proliferation of biting midges and a. sinensis. nea's identification of the vectors responsible led to control measures including bti application, intermittent drying of the papyrus beds, and the nea corporate communications department is responsible for mass and social media communications, whereas the 3p (people, private, public) network division leads nea's education and publicity initiatives and programmes for the 3p sectors, across all environmental initiatives, not just dengue prevention. community engagement makes use of existing structures including the people's association (the statutory board responsible for promoting social cohesion) and grassroots organisations under the people's association, such as citizens' consultative committees, residents' committees in hdb estates, and neighbourhood committees in private housing estates. these groups encourage bottom-up participation by seeking residents' cooperation in checking for mosquito breeding in homes. nea also trains members of the public and the people's association's community emergency response teams as dengue prevention volunteers, who educate fellow residents on dengue prevention. each year the 3p network division organises the national dengue prevention campaign, with the timing of the launch dependent on the dengue forecast provided by the environmental health institute (ehi), the research arm of nea. 3p work through the mayors of each district, grassroots members, and dengue prevention volunteers who mobilise their communities to conduct source reduction according to the '5-step mozzie wipeout' by doing house-to-house visits, distributing educational materials, and organising block parties and other events. the '5-step mozzie wipeout' targets the top five most common habitats in residential premises in singapore. information materials are available in all four written national languages (fig 6) . the '5-step mozzie wipeout' recommends to (1) turn the pail, (2) tip the vase, (3) flip the flowerpot plate, (4) loosen the hardened soil, and (5) clear the roof gutter and place bti larvicide inside. for those interested in aedes control outside singapore, it is important to note that these aquatic habitats may not be the top five habitats in other countries, so it is important to survey local sites to identify the major sources of a. aegypti. in singapore, messaging is directed towards people taking personal responsibility for mosquito prevention and explaining that by conducting source reduction, they can protect their families from dengue. 3p uses different methods to evaluate knowledge, attitudes, and practices of communities over time and looks for fresh angles to prevent fatigue from regular public health messaging. besides nationwide general messaging, community engagement strategies also target specific population groups to encourage them to play a greater role in mosquito prevention, including domestic helpers, construction workers, the elderly, and school children. for example, domestic helpers and construction workers are targeted with behaviour change messaging through outreach and roadshows at dormitories, shopping malls, and other places of congregation. since these groups are often migrants and therefore transient populations, behaviour change materials are produced in the relevant languages (e.g., bahasa, hindi, indonesian, tagalog) and outreach is conducted regularly. dengue prevention roadshows are also conducted at elderly care corners and videos are available in local chinese dialects used by many elderly residents who originate from mainland china. the 3p network division partners with the ministry of education to include dengue prevention in school curricula at primary, secondary, and tertiary levels. timely information on the number and location of dengue cases is made publicly available on the nea website and myenv mobile app. nea also implements a community dengue alert system, in the form of colour-coded banners placed in high-visibility locations in dengue clusters to inform residents about the dengue situation in their neighbourhood and the corresponding actions they can take (fig 7) . the banners use the widely recognisable 'traffic light' colour code of red (high alert), yellow (medium alert), and green (low alert) to allow for easy interpretation of the situation. communities are engaged and regularly give feedback about, for example, increased mosquito numbers, littering, and other environmental issues either online or via a 24-hour nea hotline. globally, aedes indices such as the house index (hi, percentage of houses positive for larvae and or pupae) are traditionally used for aedes vector monitoring, with a value of 1% often being used as an arbitrary dengue transmission threshold [38, 39] . the singapore dengue control programme has brought the hi to very low levels (from 50% in the 1960s to 0.3% in the 2000s). despite this low hi, singapore still experiences regular outbreaks, suggesting that the hi is no longer sensitive for dengue risk assessment [40, 41] . with low vector populations, the singapore programme now uses in-house-developed gravitraps, which are designed to lure and trap gravid female aedes on the sticky lining [31] . since 2017, a system of 50,000 gravitraps have been deployed in public housing estates nationwide, and the scheme will be expanded to landed housing by end of 2019 [31] . monitoring of the traps every 2 weeks is currently outsourced, although an automated trap is currently under development by nea. gravitrap indices are plotted on a geographical information system (gis) and used to target intensified source-reduction campaigns. areas with a high gravitrap index are also published on the nea website to motivate the local community to take action to reduce dengue transmission. in keeping with the country's green aspirations, insecticides are used judiciously, with bti and temephos used as larvicides and pirimiphos-methyl as adulticide. choice of insecticide is guided by resistance monitoring, which is performed every few years. as well as aedes, surveillance is also conducted for culicines (potential japanese encephalitis vectors) and anophelines using the in-house-developed night catcher, a modified cdc light trap that keeps caught adult mosquitoes fresh and alive for analysis and segregates hourly-caught mosquitoes in separate containers. the ministry of health is responsible for case surveillance and clinical management of dengue patients. dengue is a notifiable disease, meaning that medical practitioners and clinical laboratories must report clinically suspected and laboratory-confirmed cases. if a patient presents at a health facility with suspected dengue, then blood samples are taken for diagnostic testing at a hospital or private laboratories using either rapid diagnostic tests against nonstructural protein 1 (ns1) and immunoglobulin m (igm) antibodies or reverse transcriptase polymerase chain reaction (rt-pcr) for acute cases. alternatively, samples can be sent to the ehi for diagnosis, and through this system, ehi is able to monitor circulating dengue serotypes [42] . an epidemiological investigation of each case, conducted by telephone or in person, is triggered by the environmental public health operations department at nea, and case locations are plotted on a gis interface. a dengue cluster is formed when two or more cases have onset within 14 days and are located within 150 m of each other (based on residential and workplace addresses, as well as movement history collecting during the epidemiological investigation). dengue clusters are graded and the situation communicated to the public via the colour-coded community dengue alert system: high-risk area 10 or more cases (red); high-risk area with fewer than 10 cases (yellow); and no new cases, under surveillance for the next 21 days (green) (fig 8) . once a cluster is formed, this triggers the nea environmental public health operations team to ramp up vector control activities in the cluster. entomological indices and dengue case numbers are not reliable measures for assessing the long-term impact of vector control programmes because of changes in surveillance and diagnostic capabilities over time [30] . instead, nea uses blood bank igg seroprevalence to estimate the force of infection (foi) over time [30] . the singapore dengue programme relies predominantly on source reduction and larviciding using bti, which are implemented throughout the year through community and programme efforts. in the first 6 months of 2019, approximately 60% of vector habitats were found in residential premises, rising to 70% in cluster areas. house inspections are conducted routinely and at increased frequency in cluster areas by the environmental public health operations team (fig 8) . field staff follow a strict protocol including identifying themselves clearly and ensuring that the resident witnesses the taking of any samples for species identification at the ehi. use of insecticides is seen as a short-term solution, so fogging with organophosphates is restricted to clusters during outbreaks only. source-reduction activities are facilitated by legislation and law enforcement, which singapore uses in addition to community engagement to enhance public compliance. according to the control of vectors and pesticides act (cvpa, the main legislation dealing with mosquito breeding), the operations team can enter homes to conduct inspections and vector control, and residents are fined at least s$200 (us$145) if aquatic stages of vectors are found on their premises [43] . inspections of construction sites and other premises are also conducted by a dedicated nea team. if aquatic stages of the vector are found, then an order can be served for vector control to be implemented within a specific time period by a registered vector control personnel. in the event that environmental management is found wanting, the cvpa allows nea to issue an order to stop any work being undertaken on the premises indefinitely or until vector control to remove favourable habitats has been carried out. a list of construction sites that currently have stop work orders are available on the nea website (www.nea.gov.sg). failure to comply with the order can lead to a fine of up to s$20,000 (us$14,500) and, in the case of a second or subsequent conviction, to a fine of up to s$50,000 (us$36,000) and/or imprisonment for up to 6 months. the cvpa also covers pesticides registration, as well as licensing and certification of private-sector vector control operators [43] . to address challenges such as climate change, increasing urbanisation, and the manpowerintensive nature of source-reduction activities, the singapore programme invests in research and development of new vector control technologies. for example, ehi is evaluating the use of wolbachia, a maternally inherited endosymbiotic intracellular bacterium, which when artificially introduced into a. aegypti can suppress dengue infections. singapore opted for a population-suppression strategy whereby only male wolbachia mosquitoes are released. when male wolbachia mosquitoes mate with wild-type females, the eggs produced will not hatch, because of a phenomenon called cytoplasmic incompatibility. use of suppression was favoured over a population-replacement strategy, in which both males and females are released, since it is in line with the long-term programme strategy of source reduction. wolbachia testing has followed a phased approach in two main release sites, including gaining an understanding of the basic biology of the released male wolbachia mosquitoes in the field, such as how far and how high they fly. the programme has also collaborated with the international atomic energy agency (iaea) to irradiate wolbachia mosquitoes at the pupal stage to render infertile any remaining females, so as to avoid replacement of the wild-type population with wolbachia a. aegypti mosquitoes. pilot testing has been accompanied by a careful community engagement campaign developed by ehi to emphasise that wolbachia is safe and that male mosquitoes do not bite, and to encourage continued source reduction. the programme also works with private-sector collaborators to develop and evaluate technologies for wolbachia testing and implementation. ehi works with orinno technology, a local start-up company, to develop new automated equipment to facilitate their work, including a larvae counting system, pupae sorting and counting system, and mosquito launcher to simplify and speed up the releases of the wolbachia mosquitoes by field staff. the programme also works with verily life sciences, an alphabet, inc., affiliate, to field-test the company's automated sorting and release technologies. the dengue control programme has developed a novel indicator for entomological risk assessment known as the a. aegypti breeding percentage [44] . routine larval surveillance is not uniform spatially and temporally and so would be biased if used for risk assessment. instead, the breeding percentage expresses the number of a. aegypti-positive habitats over the total number of aedes-positive habitats (a. aegypti and a. albopictus) to cancel out the sampling error from nonsystematic inspection and cryptic breeding sites. predicting dengue outbreaks is difficult and the ehi has, in partnership with academic and research institutions including the national university of singapore, developed several different risk models to enable better resource planning and preparedness for outbreaks. for example, a risk map is prepared each year to guide resource allocation to different areas [45] , a temporal model is used to predict dengue cases up to 3 months in advance [46] , and a spatiotemporal model integrating climate, vector density, population demographics (connectivity using public transport and mobile phone data), cases, infrastructure (age of building and number of units), and satellite data (vegetation) is used for high-resolution prediction and realtime allocation of resources [45] . staff personal and professional development is a focus of the nea. for example, staff undertake continuing professional development courses organised by the internal training arm of nea, the singapore environment institute. rotations between departments are encouraged and staff can receive financial support and leave of absence to attend further education. there are possibilities for both vertical and horizontal movement within the organisation, recognising the need for both specialists and generalists. the ehi partners with academic and research institutions, and staff members have obtained phd and other degrees through their research work conducted at the ehi. the ehi of nea has been a who collaborating centre for reference and research of arbovirus and their associated vectors since 2011. this involves consulting and advising (e.g., the director of ehi sits on the who strategic technical advisory group on neglected tropical diseases and strategic advisory group of experts working group for dengvaxia, the sanofi dengue vaccine), enhancing global outbreak preparedness (e.g., cross-border virus surveillance through unitedengue consortium, evaluation of diagnostics, etc.) and capacity building (e.g., training and sharing of best practice). the singapore dengue control programme is one of the best in the world, but what makes it so successful, and how can the lessons learnt be applied to other vector control programmes? although in many countries dengue control sits under the ministry of health, unusually in singapore it sits within the mewr. this is in line with the programme view that dengue is an environmental disease. dengue vector control uses mainly environmental management approaches, such as proactive source reduction, and environmental management including drainage and house improvements contributed to the elimination of malaria from singapore in 1981 [34] . key individuals including the director-general of public health, could be trained as engineers or other professionals, not medical doctors. a strong sense of environmentalism stems from singapore's founding father, lee kuan yew, who oversaw the transformation of singapore after independence and in 1967 championed the idea of the 'garden city' [47] . lee kuan yew promoted a green environment that was free of litter in order to create good living conditions for singapore's residents, but also to simultaneously encourage tourism, investment, and trade. strong political will and long-term political stability (the people's action party have been the only party to form a government since independence in 1965) means that this vision and trajectory has been maintained over time, for example, in the current sustainable singapore blueprint [15] . despite sitting within mewr, the programme collaborates closely with the ministry of health with efficient systems for sharing information on confirmed cases to allow rapid intervention by the nea environmental public health operations team. adopting a similar environmental approach to vector control could enable more effective control of vbds worldwide (and incidentally was largely responsible for the success of vector control in the early and mid-1900s before the advent of ddt [6] ). for example, progress in controlling malaria is stalling in many high-burden countries due to weak vector control programmes, and potentially also insecticide resistance [48, 49] . since malaria is primarily a disease caused by standing water, proactively tackling immature vectors by using environmental management could be a synergistic addition to predominantly insecticide-based adult anopheline control. increasingly complicated and multidimensional public issues including climate change, globalisation, public health, and infectious disease outbreaks call for a transformation in public administration. since the 1990s, singapore has adopted a whole-of-government approach, which 'denotes public service agencies working across portfolio boundaries to achieve a shared goal and an integrated response to particular issues' [50] [51] [52] . the whole-of-government culture, propagated for decades in singapore, facilitates the view of dengue control as a shared responsibility across agencies. for example, cross-sectoral collaboration for dengue control is facilitated by the inter-agency dengue taskforce. further, rotation of leadership between different government departments and agencies means that so-called 't-shaped' managers have not only specialist expertise but a broader perspective on issues and can help to break down departmental or agency silos [53] . this coordinated whole-of-government approach is exemplified by the singapore response to a dengue outbreak that coincided with the start of the covid-19 pandemic in early 2020 (box 1). singapore also employs collaborative governance, whereby governing is based on collaboration between government and nongovernment stakeholders. this is exemplified by the important role of the people's association in bridging between government and communities. community outreach initiatives enjoy broad political support, as they provide an opportunity for the government to directly connect with the community. local politicians are also invested in preventing outbreaks in their constituencies and often communicate the importance of dengue prevention to residents during walkabouts. another success factor may be the use of a 'carrot and stick' approach to source reduction with community engagement and behaviour change campaigns led by the 3p network division, backed up by strong legislation and enforcement. a recent study conducted by ehi shows that houses that have more frequent inspections have a lower number of reported mosquito larval habitats [58] , lending support to the system of house inspections. a lack of corruption (singapore is rated as one of the least corrupt nations in the world by transparency international [59] ) also supports the penalty system for vector habitats. the clear accountability of mosquito breeding offences under the law makes it easier for stakeholders to understand their respective roles, thus facilitating collaborative action against mosquito breeding. strict enforcement of the law is another push factor that encourages joint efforts to take preventive measures. imposition of fines for vector habitats may not be possible in all vector control programmes globally but should be considered. singapore's dengue control response in 2020, which is taking place amid the covid-19 pandemic, offers a case study of intra-and intersectoral collaboration to combat twin environmental public health threats. detection of dengue threat and raising the alert more than 4,000 dengue cases were reported in the first quarter of 2020, double that for the same time period in 2019 [54] . in early 2020, ehi's risk models, incorporating case data from the ministry of health, forecasted a dengue surge in the coming months. ehi's analysis of serotype trends further detected an increase in the proportion of denv-3 cases, with denv-3 overtaking denv-2 as the predominant serotype in early 2020. as denv-3 has not been predominant in singapore for nearly three decades, population immunity to this serotype is likely low, further increasing the risk of an outbreak. given this outlook for 2020, nea alerted relevant government agencies and set in motion an enhanced and coordinated dengue control response. with support from political and grassroots leaders, nea brought forward the national dengue prevention campaign (typically held just before the traditional midyear peak dengue season) to late march 2020 [54] , with the intention of raising awareness and rallying the public to conduct preemptive measures early on. this kicked off island-wide community-led outreach efforts, helmed by grassroots leaders and supported by dengue prevention volunteers, to encourage residents to carry out dengue prevention practices. at the organisational level, the nea-led inter-agency dengue taskforce met in january and march 2020 to coordinate the response across sectors and continues to meet regularly. emphasis has been placed on enhancing vector control in assets managed by various agencies (such as buildings, reservoirs, drains, and parks), especially if these are located in areas with high mosquito populations or within dengue clusters. singapore reported its first case of covid-19 on 23 january 2020 [55] . to curb local covid-19 spread, nea in february 2020 launched "sg clean" [56] , a whole-of-government campaign to rally individuals and businesses to keep public areas clean (such areas include toilets, hawker centres, community spaces, and other premises). dengue control messaging was woven into 'sg clean'. a key campaign message was that enhanced public cleanliness, such as maintaining clean premises and not littering, eliminates mosquito breeding habitats and hence helps to reduce the spread of dengue in addition to covid-19. in april 2020, singapore implemented a 'circuit breaker' to curb covid-19 transmission, which involved stringent social distancing measures and cessation of nonessential work activities [57] . given the high-risk dengue outlook for 2020, nea worked at the whole-of-government level to include vector control activities as an essential service to what continues to drive transmission in singapore, where there is a well-resourced and effective dengue control programme? the dengue incidence rate in singapore has increased dramatically in the last 25 years, but this is likely because of improved diagnostics, increased referral for testing by medical practitioners, and increased awareness among the public [30] . a better indicator of the true infection rate is dengue foi: between the 1960s (when singapore first implemented environmental management and vector control programmes) and the 1990s, the dengue foi in singapore dropped 10-fold to approximately 0.01 (10 per 1,000 individuals per year) and has since held steady at this low level [30] . the decline in foi can probably be attributed to the effectiveness of the dengue control programme (along with an increasingly ageing population). this success in reducing disease transmission has resulted in a lowered herd immunity against dengue, leaving singapore's population vulnerable to outbreaks despite a low vector population. another challenge is the high level of population movement in and out of the country, which is known to facilitate the co-circulation of denv serotypes [60] . singapore has 1,107 km of expressways and 199.3 km of mass rapid transit (mrt) lines across the island; over 300,000 people commute into the island state from malaysia each day [61] ; and in 2018, there were 65.63 million passenger movements in and out of changi airport from 380 cities in 100 countries and territories worldwide [62, 63] . in order to reduce the foi further, the programme is evaluating innovative strategies such as wolbachia, novel community engagement mechanisms, and risk mapping for more effective intervention targeting. as well as better implementation of current tools and approaches, new vector control tools are urgently needed to combat vbds worldwide. in conclusion, the dengue control programme in singapore provides an excellent example of the gvcr in action. important elements include strong collaboration across government departments and between government and nongovernment actors, integration of tools and approaches, effective surveillance, and community engagement. as a high-income country, singapore is in an enviable position of having reliable government funding for dengue control. nevertheless, aspects such as working across sectors or implementation of environmental management do not need to be expensive, and the former can even save costs for the vector control programme. adoption of these elements could lead to more effective vector control programmes worldwide to reduce the intolerable burden of vbds. be continued during the 'circuit breaker' period. businesses and owners of premises are expected to ensure that adequate vector control activities continue at their premises (including offices, commercial buildings, schools, and construction sites), even if regular operations are on hold. nea continues to carry out island-wide routine inspections and enforcement, with precautions taken to minimise covid-19 transmission. these precautions include ensuring that officers carrying out inspections are healthy, wear masks, and practise good personal and hand hygiene. despite the efforts of the nea, dengue control has been particularly challenging in 2020 because of the switch to denv-3, warm weather, and high numbers of people staying at home during the 'circuit breaker' period, which can increase aedes aquatic habitats and provides easy access to blood meals for female aedes mosquitoes. we thank the following individuals for their insights: dulcie chan, rama chandramogan, khoo seow poh, christina liew, sueanne mocktar, ong chin soon, tai ji choong, tony teo, and grace yap of singapore's national environment agency and nanthini elamgovan of singapore's national parks board. thanks also to manuela bernardi for her assistance in producing the figures. • vbds are environmental diseases-situation of programmes within the ministry of environment and/or strengthening environmental management is encouraged. • whole-of-government approaches (working across government ministries) and collaborative governance (collaboration between government and nongovernment) support vbd control. • strong vector and epidemiological surveillance enables targeting of vector control interventions. • consider the role of legislation and enforcement in reducing vector habitats. • stable financing supports effective vector control. • innovation and science-based approaches should be harnessed to support surveillance and control. top five papers world health organization. vector-borne diseases fact sheet integrating vector control across diseases the global burden of dengue: an analysis from the global burden of disease study urbanization and globalization: the unholy trinity of the 21st century the current and future global distribution and population at risk of dengue the importance of vector control for the control and elimination of vector-borne diseases the effect of malaria control on plasmodium falciparum in africa between address to the sixty-ninth world health assembly by director-general of the world health organization world health organization. global vector control response world health organization. framework for a national vector control needs assessment geneva: who online database-total land area of singapore world bank. population density (people per sq. km of land area) ministry of national development-centre for liveable cities singapore. sustainable singapore blueprint forging a greener tomorrow: singapore's environmental journey from slum to eco-city singapore's journey towards environmental and water sustainability singapore: department of statistics singapore forecast of dengue incidence using temperature and rainfall epidemiological aspects of an outbreak of dengue fever/dengue haemorrhagic fever in singapore the 1973 dengue haemorrhagic fever outbreak in singapore and its control dengue in singapore from 2004 to 2016: cyclical epidemic patterns dominated by serotypes 1 and 2 communicable diseases surveillance in singapore communicable diseases surveillance in singapore communicable diseases surveillance in singapore communicable diseases surveillance in singapore environment agency. quarterly dengue surveillance data-q1 2019, q2 2019, q3 2019 and q4 2019 dengue seroprevalence of healthy adults in singapore: serosurvey among blood donors seroepidemiology of dengue in the adult population of singapore force-of-infection and true infection rate of dengue in singapore-its implication on dengue control and management gravitraps for management of dengue clusters in singapore seroprevalence of antibodies against chikungunya virus in singapore resident adult population hard lessons in surveillance and response from the singapore zika experience eradication of malaria from singapore risk of anopheles sinensis as an emerging malaria vector in singapore a study on anopheles maculatus and anopheles sundaicus in singapore construction sites as an important driver of dengue transmission: implications for disease control critical examination of aedes aegypti indices: correlations with abundance assessing the relationship between vector indices and dengue transmission: a systematic review of the evidence dengue prevention and 35 years of vector control in singapore who regional office for the western pacific. guidelines for dengue surveillance and mosquito control. manila: who regional office for the western pacific dengue in singapore from 2004 to 2016: cyclical epidemic patterns dominated by serotypes 1 and 2 control of vectors and pesticides act a novel entomological index, aedes aegypti breeding percentage, reveals the geographical spread of the dengue vector in singapore and serves as a spatial risk indicator for dengue. parasit vectors mapping dengue risk in singapore using random forest three-month realtime dengue forecast models: an early warning system for outbreak alerts and policy decision support in singapore memoirs of lee kuan yew. singapore: times media private limited connecting government: whole of government responses to australia's priority challenges. canberra: management advisory committee (australian government) a primer on implementing whole of government approaches. dublin: centre for effective services reviewing whole-of-government collaboration in the singapore public service introducing t-shaped managers. knowledge management's next generation nea brings forward national dengue prevention campaign and rolls out additional new tools to combat dengue, with increasing evidence of a sustained switch in dengue virus serotype confirmed imported case of novel coronavirus infection in singapore: multi-ministry taskforce ramps up precautionary measures campaign launched to rally public and businesses to work together to keep singapore clean circuit breaker to minimise further spread of covid-19 the effectiveness of inspections on reported mosquito larval habitats in households: a case-control study transparency international secretariat increasing airline travel may facilitate cocirculation of multiple dengue virus serotypes in asia dengue outbreaks in singapore and malaysia caused by different viral strains key: cord-345494-8lcdx719 authors: chao, chien-chung; belinskaya, tatyana; zhang, zhiwen; ching, wei-mei title: development of recombinase polymerase amplification assays for detection of orientia tsutsugamushi or rickettsia typhi date: 2015-07-10 journal: plos negl trop dis doi: 10.1371/journal.pntd.0003884 sha: doc_id: 345494 cord_uid: 8lcdx719 sensitive, specific and rapid diagnostic tests for the detection of orientia tsutsugamushi (o. tsutsugamushi) and rickettsia typhi (r. typhi), the causative agents of scrub typhus and murine typhus, respectively, are necessary to accurately and promptly diagnose patients and ensure that they receive proper treatment. recombinase polymerase amplification (rpa) assays using a lateral flow test (rpa-nfo) and real-time fluorescent detection (rpa-exo) were developed targeting the 47-kda gene of o. tsutsugamushi or 17 kda gene of r. typhi. the rpa assay was capable of detecting o. tsutsugamushi or r. typhi at levels comparable to that of the quantitative pcr method. both the rpa-nfo and rpa-exo methods performed similarly with regards to sensitivity when detecting the 17 kda gene of r. typhi. on the contrary, rpa-exo performed better than rpa-nfo in detecting the 47 kda gene of o. tsutsugamushi. the clinical performance of the o. tsutsugamushi rpa assay was evaluated using either human patient samples or infected mouse samples. eight out of ten pcr confirmed positives were determined positive by rpa, and all pcr confirmed negative samples were negative by rpa. similar results were obtained for r. typhi spiked patient sera. the assays were able to differentiate o. tsutsugamushi and r. typhi from other phylogenetically related bacteria as well as mouse and human dna. furthermore, the rpa-nfo reaction was completed in 20 minutes at 37(o)c followed by a 10 minute incubation at room temperature for development of an immunochromatographic strip. the rpa-exo reaction was completed in 20 minutes at 39(o)c. the implementation of a cross contamination proof cassette to detect the rpa-nfo fluorescent amplicons provided an alternative to regular lateral flow detection strips, which are more prone to cross contamination. the rpa assays provide a highly time-efficient, sensitive and specific alternative to other methods for diagnosing scrub typhus or murine typhus. rickettsial pathogens are among the leading causes of morbidity and mortality during military operations. in recent years, emerging rickettsial diseases have been reported throughout the world and are a significant medical concern for local and deployed personnel and travelers [1] [2] [3] . these pathogens include spotted fever group rickettsia (sfgr, far eastern spotted fever, japanese spotted fever, siberian and queensland tick typhus, and thai tick typhus just to name a few), typhus group rickettsia (tgr, epidemic and murine typhus) and orientia tsutsugamushi (scrub typhus, st). due to the high mortality rate of untreated rickettsial infections, early treatment with appropriate antibiotics is critical [4] . doxycycline is the drug of choice, except in cases of pregnancy and tetracycline hypersensitivity. symptoms of rickettsial infections are nonspecific and can be confused with a variety of other pathogens (e.g., dengue, malaria, leptospirosis) that require different treatment regimens. to ensure that appropriate treatment is initiated promptly, early diagnosis of rickettsial infections is critical. currently, the diagnosis of rickettsial diseases relies mainly on serological methods based on antibody detection [5, 6] . however, antibody based assays may not be adequate for the diagnosis of disease in the acute phase, as antibody levels may not be detectable at the onset of illness. therefore, antigen/pathogen detection before the rise of antibody levels is important. almost all antigen/pathogen detection assays for rickettsial diseases are based on polymerase chain reaction (pcr [7, 8] ), quantitative real-time pcr (qpcr) or nested pcr targeting different genes, including 56 kda [9] , 47kda [10] , groel [11] of o. tsutsugamushi and ompb [7] , 17 kda [9] , and glta [12, 13] of rickettsia. however, all these assays require end user training to operate the thermocycler, and a functional and well-calibrated thermocycler is difficult to obtain and maintain in resource poor settings. recently, loop-mediated isothermal amplification has been developed [14] [15] [16] [17] [18] [19] [20] as a potential alternative for pcr based tests. in addition to groel as the target [11] , huber et al. [21] demonstrated the use of the conserved 47 kda gene as the target for the lamp assay, which achieved similar sensitivity to that of qpcr. recombinase polymerase amplification (rpa) uses a mixture of prokaryotic recombinases to guide synthetic oligonucleotide primers to targets in the sample [22, www. twistdx.co.uk for detail description]. a strand displacing dna polymerase (large fragment from bacillus subtilis pol i, bsu) is used for primer extension [22] . the method is highly sequence specific in complex nucleic acid mixtures and in urine without the need for additional preparation of samples [23] . it offers amplification of the target sequence by reiterative oligonucleotide-primed dna synthesis without the need to denature dna at a high temperature. the assay can be performed between 24°c to 45°c [22] with very high efficiency so that detection of product from a single molecule is possible in 20 minutes [22] . the successful application of rpa is evident as shown in many recent publications. rt-rpa has been developed to detect hiv [24] , rift valley fever virus [25, 26] , ebola virus, sudan virus and marburg virus [26] , mers-cov [27] , foot-andmouth disease virus [28] , and bovine coronavirus [29] . additionally, assays have been developed for detection of chlamydia trachomatis in urine samples [23] , diagnosis of cryptosporidiosis in animal and patient specimens [30] , and detection of neisseria gonorrhoeae, salmonella enterica, methicillin-resistant staphylococcus aureus (mrsa) [31] , francisella tularensis [32] , and group b streptococci [33] . furthermore, the assay has been used in combination with elisa for food analysis [34] . while rpa is similar to lamp, in that no thermocycler is needed, the optimal reaction temperatures for rpa and lamp are 37°c and 60°c, respectively. in addition, rpa is potentially easier to adapt to a multiplex format [22] than lamp as rpa requires only one pair of primers rather than three like lamp, in which the mixing of multiple pairs of primers may result in non-specific primer interactions and may limit the total amount of primer able to be added to the reaction. finally, the incorporation of a probe can increase the specificity of the assay. this is particularly important, as the lamp assay is known to result in non-specific amplification [35] . these advantages suggest that rpa may be a viable pointof-care nucleic acid detection method that can be utilized in resources-limited areas where these diseases are endemic. in this report, we demonstrated that a lateral flow end point detection rpa (twistamp-nfo kit, hereafter rpa-nfo) could be performed at 37°c in 20 minutes to detect dna from either o. tsutsugamushi or r. typhi. similarly, a real-time fluorescent signal detection (twistamp-exo kit, hereafter rpa-exo) could be performed at 39°c to detect dna from either o. tsutsugamushi or r. typhi. a detection limit similar to that of the gold standard qpcr was achieved by both rpa-exo and rpa-nfo. the specificity was evaluated using genomic dna from closely related microorganisms at 1000 folds excess in copy number. the detection limit was established using a range of target genomic dna from as high as 500 copies per reaction to less than 10 copies per reaction. further evaluation was done using dna extracted from liver, lung and spleen collected from o. tsutsugamushi infected mice, dna extracted from clinical samples obtained from confirmed st patient sera, or r. typhi-spiked normal human plasma. a cross contamination proof (xcp) lateral flow cassette was used to detect rpa-nfo amplicons. this method eliminates the need to open the reaction tube at the end of the reaction, thus minimizing the chance of contamination. the animal study protocol (#d11-06) was reviewed and approved by the naval medical research center institutional animal care and use committee (iacuc) in compliance with all applicable federal regulations governing the protection of animals in research. the experiments reported herein were conducted in compliance with the animal welfare act and in accordance with the principles set forth in the "guide for the care and use of laboratory animals," institute of laboratory animals resources, national research council, national academy press, 1996. oligonucleotide primers used for the rpa assays were manually designed based on the 47 kda gene sequence from the karp strain of o. tsutsugamushi (47-rpa) and the 17 kda gene sequence from the wilmington strain of r. typhi (17-rpa) per recommendation by twistdx (cambridge, uk). a minimum of 5 sets of primers and probes for each target were designed for different end-point detection methods. all primers and probes were synthesized by eurofins mwg operon (huntsville, al) and biosearch technologies (petaluma, ca), respectively. the probes were hplc purified. rpa-nfo and rpa-exo kits were both purchased from twistdx (cambridge, uk, www. twistdx.co.uk). the amplicons generated by the rpa-nfo were detected using a lateral flow strip or cassette (fig 1a) . rpa-exo was used to monitor fluorescent signals in real time using a fluorimeter ( fig 1a) . fig 1b shows pcr product of the 47 kda gene sequence of o.tsutsugamushi karp strain and the 17 kda gene from r. typhi wilmington strain was cloned into a vr1012 vector and puc vector, respectively, following standard molecular biology technology. the plasmid was extracted from a 3 ml culture using the qiagen plasmid mini kit (qiagen, ca) following the manufacturer's instruction. the concentration was determined using a nanodrop (thermo fisher scientific, ca) and the expected copy number of the target gene was calculated based on the size of the plasmid (http://cels.uri.edu/gsc/cndna.html). the purified plasmid was used as a template to optimize the rpa assays. genomic dna from multiple strains of o. tsutsugamushi, including karp, afsc4, afsc7, garton, ikeda, and boryong, tgr and sfgr was extracted from renografin gradient purified organisms using qiaamp mini dna kit (qiagen, ca) as previously described [36] . similarly, dna from cultured leptospira, coxiella burnetii, and bartonella bacillifomis was extracted by the same method. extraction of dna from st confirmed patient blood, from mice infected by o. tsutsugamushi, and from normal human plasma (nhp) spiked with cultured r. typhi dna extracted from blood of patients with confirmed st was provided to us by dr. yupin suputtamongkol of siriraj hospital, mahidol university, bangkok, thailand. the animal protocol to perform the mouse experiments (idd-11-06) was approved by nmrc iacuc. according to the approved protocol, mice were challenged intraperitoneally (ip) by the karp strain of o. tsutsugamushi and observed for up to 21 days. at the indicated time post infection, mice were sacrificed, and various organs were collected which included the lungs, liver and spleen. genomic dna was extracted from these organs following the manufacturer's instructions of the qiaamp blood and tissue mini dna kit (qiagen, ca). to prepare cultured r. typhi-spiked nhp, the number of r. typhi present in each of the three independently prepared r. typhi cultures was determined. to do this, dna was extracted from 100 μl each of the three r. typhi cultures using the qiaamp blood and tissue dna kit following the manufacturer's instructions. the bacterial load was determined by qpcr to be within the range of 10 10 -10 11 copies/ml. an initial dilution was made by adding cultured r. typhi into nhp at a 1:100 dilution followed by a serial dilution of 1:10 using nhp as a diluent to ensure that the final concentration of the 200 μl of spiked nhp was 2,000 copies/ml of cultured r. typhi. the dna from each 200 μl r. typhi-spiked nhp was extracted using the qiaamp blood and tissue dna kit. the dna was used as template for the 17-rpa assay to evaluate its performance and qpcr was used to quantitate the copy number of the 17 kda gene. each forward primer was mixed with different reverse primers separately to examine which pair performed the best using plasmid dna (10-1000 copies) as template. different reaction times, concentrations of primers and probes, and temperatures ranging from 37°c to 42°c were evaluated to select the most sensitive combination for the rpa reactions as per the manufacturer's recommendations (twistdx, cambridge, uk). each combination of parameters was performed at least twice. the temperature range was varied every 0.5 degrees within the range recommended by the manufacturer. the evaluation was done using the rpa-nfo for the rpa reaction and the results were evaluated using milenia genline hybridetect-1 (mgh) strips by millenia biotec gmbh (gieben, germany). based on the evaluation performed in previous section, a final condition to carry out the recombinase polymerase amplification is described below. the reaction mixture was 50 μl using the rpa-exo or rpa-nfo. the reaction mixture contained rehydration buffer, recombinases, and bsu strand displacing polymerase with the addition of 420 nm each of the forward and reverse primers, 120 nm of the fam-tagged probe, at a total volume of 42.5 μl. after mixing these components, 2.5 μl of 280 mm magnesium acetate was pipetted into the tube lids, and 5 μl of dna was added to the reaction mixture unless otherwise indicated. the lids were closed and the magnesium acetate was spun down into the reaction mixture to initiate the reaction. for rpa-exo, the tubes were placed into an esequant tube scanner device (qiagen, ca) and the reaction was conducted at 39°c for 20 minutes. the tubes were briefly agitated at 4 minutes after the initiation of the reaction, spun down and placed back into the tube scanner. the reaction in each tube was monitored in real time following the increase of fluorescent signals. for rpa-nfo, tubes were placed into a heating block at 37°c for 20 minutes. the tubes were briefly agitated at 4 minutes after the reaction was initiated, spun down and placed back into the heating block. the mgh strips were used to evaluate results according to the manufacturer's instruction after the reaction was completed. for each reaction, 1 μl of sample amplicon was used for strip development. as an alternative detection method, after the reaction was completed, the entire reaction tube was inserted into the xcp cassette (bioustar, hongzhou, china) and the cassette was closed per the manufacturer's instruction. the presence of a signal at the t line was an indication of positive detection of dna using a 5' fam labeled probe. the copy number of genomic dna extracted as previously described was determined by qpcr (below). the dna was diluted to the desired copy number in a total volume of 5 μl to perform the rpa as described previously. the reaction was performed a minimum of 6 times for each amount of genomic dna. due to the limited availability of extracted dna from scrub typhus confirmed patients, only 47-rpa-nfo was performed with 1 μl of dna. for the dna from o. tsutsugamushi-infected mice, the dna was extracted from liver, spleen and lungs of these mice as described previously. the evaluation of 47-rpa-exo and 47-rpa-nfo was performed as described above using 5 μl of extracted dna. quantitative pcr was performed to compare and confirm the detection limit of the rpa assay. whenever the copy number of a sample is provided, it was obtained via qpcr using a standard curve built with a plasmid of known copy number. the 7500 fast real-time pcr system (applied biosystems, foster city, ca) was used to perform qpcr reactions and analyze the results. the primers used here are the same as those previously used for amplifying the 47kda gene sequence of the karp strain of orientia and 17 kda gene sequence of r. typhi [10, 37] . a total reaction mixture of 20 μl contained 1 μm each of forward primer and reverse primer targeting the 47kda gene of orientia or 17 kda gene of r. typhi, 1x rt 2 sybr green qpcr mastermix (sabiosciences, frederick, md), and dna template. an initial 5-minute activation step at 95°c was followed by 40 cycles of 10 seconds at 95°c, 30 seconds at 60°c, and a melting curve determination cycle. to determine the amount of mouse genomic dna in the total dna extracted from organs of o. tsutsugamushi infected mice, qpcr was performed as described by sunyakumthorn et al [38] . after running the rpa-nfo reaction with different primer and probe combinations, incubation temperatures and reaction time as mentioned in materials and methods, we applied several performance factors including total signal strength, absence of background signal on the mgh strip and detection limit to determine which combination of primer and probe, including concentration, reaction temperature and reaction time provided the lowest limit of detection. we used qpcr as a gold standard to quantitate the dna to establish the detection limit. a primer and probe set that provided the best detection limit for the individual detection of o. tsutsugamushi or r. typhi dna was identified (table 1) . using a lateral flow test to detect rpa amplicons of o. tsutsugamushi (47-rpa-nfo) or r. typhi (17-rpa-nfo) dna provided a similar level of detection to that of qpcr the 47-rpa-nfo appeared to be detecting 53 copies/reaction which is slightly higher than that by qpcr (10 copies/reaction). different strains and isolates of o. tsutsugamushi including karp, kato, gilliam, afsc4, afsc7, ikeda, garton, and boryong were tested. while the region (156 bp) of the 47 kda sequence that rpa primers targeted shared greater than 90% homology among these 8 strains (s1 fig), the detection limit ranged from 10 to 400 copies per reaction. similarly, the detection of r. typhi dna using different copy numbers of the 17 kda gene is representatively shown in fig 2b. the 17-rpa-nfo was able to detect around 20 copies per reaction which is slightly higher than that by qpcr (6 copies per reaction). specificity of the rpa methods to detect only o. tsutsugamushi or r. typhi dna to further evaluate whether the 47-rpa or 17-rpa only detected o. tsutsugamushi or r. typhi, respectively, purified genomic dna from phylogenetically related organisms was tested. when dna from r. typhi, r. bellii, r. rickettsii, r. conorii, leptospira, c. burnetii, b. bacilliformis was used in the 47-rpa-exo, none were positive (fig 4a) . consistent with this observation, the 17-rpa-exo did not show any reactivity with dna from o. tsutsugamushi, leptospira, c. burnetii or b. bacilliformis (fig 4b) . the same results were obtained using 47-rpa-nfo. it is noted that when 17-rpa-nfo was used to detect r. conorii and r. rickettsii, positive results were only observed when at least 10 4 copies/reaction of dna were present (s2 fig), suggesting that the assay was much less sensitive to detect these two sfgr. other sfgr, such as r. honei and r japonica were also evaluated to show that they were not detectable by 17-rpa-nfo unless 10 4 or more copies of dna were present, same results were obtained using 17-rpa-exo. additionally, the performance of 17-rpa-exo was also evaluated using dna extracted from r. prowazekii. the assay consistently detected as low as 80 copies per reaction. taken together, these results are consistent with the notion that this 17-rpa is better suited to detect tgr. detection limit of the rpa methods using extracted genomic dna different amounts of extracted genomic dna were used to evaluate the detection limit of the two rpa methods running at least 6 replicates for each assay. the 47-rpa-nfo and 47-rpaexo had different detection limits whereas the 17-rpa-nfo and 17-rpa-exo had very similar detection limits. as shown in table 2 , when testing 47-rpa-exo, the assay detected 100%, 77% and 42% of samples with 100-120 copies, 40-60 copies and 10-12 copies per reaction, respectively. when 47-rpa-nfo was used, the assay was positive for 100%, 67%, 54% and 0% of samples with 500-550 copies, 200-250 copies, 100-120 copies and 40-60 copies per reaction, respectively. based on these results, 47-rpa-exo had a significantly lower detection limit from that of 47-rpa-nfo. the 17-rpa-exo detected 88%, 91% and 43% of samples ranging from 100-120 copies, 40-60 copies and 5-15 copies per reaction, respectively. the 17-rpa-nfo, on the other hand, detected 100%, 73%, 71% and 57% of samples ranging from 100-120 copies, 40-60 copies, 20-25 copies and 5-15 copies per reaction, respectively. there was no statistical significant difference ( evaluation of 47 kda based rpa to detect o. tsutsugamushi dna isolated from nested pcr confirmed patient blood samples and qpcr confirmed laboratory, o. tsutsugamushi-infected mouse samples the 47-rpa was evaluated using a total of 10 positive and 10 negative human samples, the results showed that 8 out of 10 positives were 47-rpa-nfo positive while all 10 negatives were negative (fig 5) , similar to what was observed using the lamp method [21] . even though we only ran 47-rpa-nfo using 1 μl instead of 5 μl dna due to the limited availability of extracted dna, nevertheless, the limited number of clinically confirmed st patients shows that 47-rpa exhibited 80% sensitivity and 100% specificity. the 47-rpa was also evaluated using extracted dna from liver, spleen and lung from laboratory, o. tsutsugamushi-infected mice. these samples were confirmed qpcr negative before day 4 post infection and then a gradual increase in quantity was seen as shown in table 3 . the same extracted dna was used as a template for both 47-rpa-exo and 47-rpa-nfo. among these samples, 3 qpcr negative samples were determined negative by 47-rpa-exo and 47-rpa-nfo. among the 9 qpcr positive samples, 7 of them were detected positive by 47-rpa-exo and 47-rpa-nfo. these results demonstrated that 78% sensitivity and 100% specificity was achieved using dna extracted from organs obtained from o. tsutsugamushi-infected mice. taken together, it is concluded that 47-rpa provides around 80% sensitivity and 100% specificity. evaluation of 17-rpa to detect r. typhi dna isolated from normal human plasma spiked with cultured r. typhi due to the lack of qpcr confirmed r.typhi positive patient samples, the clinical performance of the 17-rpa was evaluated by spiking normal human plasma with cultured r. typhi. the results of the qpcr quantitation, 17-rpa-exo, and 17-rpa-nfo are shown in table 4 . all qpcr positive samples were positive by 17-rpa-exo and 17-rpa-nfo. based on the amount of r. typhi dna spiked into nhp and the final copies of r. typhi in the extracted dna, the dna extraction resulted in average dna recovery of 81% (48%-104%). using the xcp cassette to replace the mgh strips at the end of the rpa-nfo reaction, the fam labeled probes showed signal at the t line (fig 6) , just like the strips. these results demonstrated the potential usage of this xcp cassette as a replacement for the mgh strips for the detection of rpa-nfo amplicons. as the xcp cassette does not require opening the reaction tube and uses all of the reaction volume for detection, it is conceivable that the usage of the xcp cassette to detect the end product could be more sensitive, and present less opportunity for contamination than the usage of mgh strips to detect the rpa-nfo product. the data presented here shows that rpa is a method that could be used to detect plasmid dna or dna extracted from patient samples, infected mice, and pure organisms with a detection limit of tens of copies, within 20 minutes. this is generally comparable to that of qpcr (figs 1 and 2 and table 3 ). this detection limit observed is not likely due to the limitation of the rpa assay as it has been shown to detect product amplified from a single molecule [22] . rather, it is conceivable that the detection limit can be improved if different genes are selected for assay development. in addition to having a similar detection limit, the rpa method has several advantages over qpcr, making it an attractive alternative. firstly, a heating block that is capable of maintaining a temperature of 37°-39°c for 20 minutes is sufficient to perform the reaction. secondly, reaction mixtures are pre-made and provided by twistdx. with the addition of water, template, primers and probe, the reaction is then initiated upon mixing, thus minimizing the potential for contamination often observed with other nucleic acid amplification methods. thirdly, the method offers multiple end-point detection options with similar detection limits that could fit into many different laboratory settings, thus making one assay applicable in a well-equipped laboratory, a mobile laboratory or a rural area where instruments and infrastructure may be limited. finally, successful detection of o. tsutsugamushi from clinical and mouse samples known to be infected, demonstrated the potential clinical application of the assay. the rpa method was developed almost 10 years ago. however, it has not been accepted as well as some other isothermal amplification methods, i.e., lamp. although the method is relatively easy to perform with all the advantages described previously, one difficulty lies in the design of the primers and probes. there is no software available to assist in properly designing primer and probe sets. furthermore, the requirement of extra length primers and probes has made the manual design more difficult than other methods, such as qpcr, pcr and lamp which have well established software for primer and probe design. the relatively rare modifications on the probes also add cost to the synthesis. consequently, it may prevent the evaluation of large numbers of potential primers and probes to lead to the most sensitive and specific assay possible. nevertheless, the advantages seem to outweigh these minor drawbacks as we and many others [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] were able to demonstrate the utility of the assay for detection of closely related pathogens without sacrificing sensitivity or specificity. in addition to the development of rpa for detection of o. tsutsugamushi and r. typhi, the rpa-nfo method was developed c. burnetii dna [39] . the detection limit of these assays was comparable to that of qpcr. in the detection of o. tsutsugamushi using rpa methods, there appeared to be a slight difference in detection limit between rpa-nfo (54% of 100-120 copies per reaction) and rpaexo (77% of 40-60 copies per reaction). on the contrary, the difference in detection limit was less pronounced for r. typhi detection as shown in table 2 . due to the observations that rpanfo requires higher copies of target gene for positive detection in general, the rpa-nfo assays for both targets were evaluated using higher copies/reaction or additional ranges of copies/ reaction to best estimate the detection limit of rpa-nfo assay. the test results using varying ranges of genomic dna for 47-rpa and 17-rpa-demonstrated differences in percent positive detection, for most tested ranges, the assay provided around 50% positivity regardless of whether it was rpa-nfo or rpa-exo. additionally, these results are consistent with the notion that lateral flow strips are generally regarded as not-as-sensitive as other detection methods, such as fluorescent signal detection. the use of strips to evaluate the results of rpa-nfo requires the reaction tubes to be opened to remove samples after the reaction is completed, thus adding extra post reaction time and increasing the possibility of cross contamination due to the presence of abundant amplicons. the cassette form of the lateral flow strips essentially eliminates cross contamination yet still requires additional time to obtain the final results. it is worth noting that both mgh strips and xcp cassettes are commercially available and they were used without any attempt to optimize the performance. thus, it is conceivable that further optimization of these devices could improve the sensitivity and eliminate the difference in sensitivity between 47-rpa-nfo and 47-rpa-exo. the target genes, 47 kda and 17 kda, were selected for o. tsutsugamushi and r. typhi dna detection, respectively, based on their relatively conserved nature in sequence. the 47 kda gene codes one of the major protein antigens recognized by sera from patients infected with many strains of o. tsutsugamushi [40] . the sequences of 47 kda proteins have been compared to show greater than 96% identity [41] between strains with the exception of one recently identified strain, o. chuto [42] , that shared only about 83% identity [41] . the gene has been used as the target for the development of pcr, qpcr, and lamp assays and has shown consistent results in sensitivity, specificity, and broad reactivity toward many different strains of o. tsutsugamushi [10, 21] . while the region (156 bp) of 47 kda that rpa targeted shared greater than 90% homology among the 8 strains tested (s1 fig), it is noted that a different detection limit was observed between strains. in spite of the difference in detection limit observed for different strains, these detection limits still fall within the broad expected level of o.tsutsugamushi circulating in patients' blood [43] . since the level of o. tsutsugamushi present in patient's blood is also associated with the disease severity, it is likely that the current 47-rpa-exo and 47-rpanfo need further improvement to achieve lower detection limits in order to be more clinically applicable. similarly, the 17 kda gene has been used to confirm the presence of rickettsia dna in potentially infected samples [9, 37] . additional targets were used to delineate whether the infection was due to tgr or sfgr [7, 8] . it is noted that the designed primers and probe for 17-rpa, which are located at the latter half of the 17 kda sequence, showed a lower detection limit for r. typhi dna while it required a copy number greater than 10 4 of r. conorii and r. rickettsii dna to be detectable by 17-rpa-nfo (s2 fig). same observation was made for r. honei and r. japonica. the 17 kda genes from sfgr that were tested share 89% of the identity with the full length 17 kda gene of r. typhi. the 17 kda gene of r. typhi, shares 95% of the identity with the 17 kda gene of r. prowazekii, another member of the tgr. the 17 kda gene of r. conorii, r. rickettsia, r honei, and r. japonica show no more than 87% of the identity with that of r. typhi within the region where the primers and probe are located (134 bp), the identity between r. typhi and r. prowazekii is 97%. since the sensitivity is clearly different between r. typhi and the two species in the sfg rickettsia using just one set of primers and probe, it suggests that 87% identity is not enough to use one set of primers and probe to detect both tg and sfg rickettsia with equal sensitivity. therefore, another set of primers and probe is needed for sfgr in order to have a similar detection limit to that for r. typhi. this difference in reactivity toward tgr and sfgr suggests that the assay is highly specific. while the detection limit using nhp spiked with cultured r. typhi was about 2000 copies/ml (table 4) , it is higher than the median of 210 dna copies/ml in blood of confirmed murine typhus patients [44] , suggesting that the assay may not be sensitive enough to be clinical useful. since no evaluation of 17-rpa was done using clinical samples, it is warranted that additional evaluations should be done to further characterize the clinical sensitivity and specificity of the 17-rpa. both rpa-exo and rpa-nfo kits are specific only to the targeted organisms. this is demonstrated in fig 3a and 3b , and table 3 . in fig 3, it is shown there was no amplicon unless the target dna (i.e., o. tsutsugamushi or r. typhi) was present even though the non-target dna was present in 1000 folds excess compared to the target gene. the presence of excessive human dna and mouse dna apparently did not interfere with the detection of the target gene, suggesting that the assay provided sufficient specificity for clinically relevant samples. this is supported by the results showing that 8 out of 10 pcr confirmed o. tsutsugamushi positives were positive and 10 out of 10 pcr confirmed o. tsutsugamushi negatives were negative. while we achieved 80% sensitivity and 100% specificity using a limited number of clinical samples, we also evaluated the specificity and sensitivity of the 47-rpa-exo and-nfo more closely using samples from mice infected by live o. tsutsugamushi. as shown in table 3 , no samples collected prior to day four post infection had qpcr detectable o. tsutsugamushi. as expected due to the similarity in sensitivity between qpcr and rpa, none of these samples were positive by either 47-rpa-nfo or 47-rpa-exo. on the contrary, as the infection progressed, o. tsutsugamushi dna became detectable by qpcr and 47-rpa as early as day four post infection in all organs evaluated, even though the number of o. tsutsugamushi detected was low. for those samples with low copy of o. tsutsugamushi dna by qpcr, not all of them were 47-rpa detectable. overall, both 47-rpa-exo and-nfo showed consistent results of 78% sensitivity and 100% specificity, similar to the observation made using dna extracted from scrub typhus patients. it is noted that the number of o. tsutsugamushi was undetectable before day 4 post infection or the number was very low at day 4 post infection. this is probably related to the number of live organisms injected into the mice, the naturally-slow growth of o. tsutsugamushi and the route and rate of dissemination of o. tsutsugamushi once it enters into the mice. the amount of mouse dna was not the same for all samples, so we normalized the copy number of o. tsutsugamushi to a mouse complement factor d gene [cfd, 38] . the ratio of mouse dna to o. tsutsugamushi dna ranged from as low as 1.9 (lung, day 11) to as high as 76800 (lung, day 4). these results suggested that the ratio of non-target dna to target dna did not affect the detection of target dna with a 10 4 dynamic range and that either 47-rpa-nfo or 47-rpaexo has the potential applicability in clinical samples for diagnosis of infection. nevertheless, the number of samples evaluated in this report is limited and the evaluation of additional clinical samples is needed to better describe the clinical sensitivity and specificity. in this work, we took advantage of the similarity between lamp and rpa-nfo in detecting fam labeled amplicons, provided a reverse primer is labeled with biotin. the bioustar xcp cassette was designed specifically for the detection of lamp amplicons when a fam labeled loop primer along with a biotin labeled reverse primer were used together in the reaction. in the original design, the c-line signal indicates an internal control containing a dig labeled loop primer while the t-line signal indicates a positive sample containing a fam labeled loop primer (www.bioustar.com). the results in fig 6 clearly demonstrated that the assay was only positive at the t line when correspondingly labeled probe was present together with all primers and dna templates. while it is true that the xcp cassette was not designed to detect rpa-nfo products, our approach confirmed the possibility of using the xcp cassette for rpa-nfo to provide an end point readout with no cross contamination. furthermore, this provides a single xcp cassette that can be used for the detection of practically all fam (or dig) labeled probe amplicons, including qpcr, lamp and rpa-nfo in a pick-and-choose manner to select those assays for target organisms that are most relevant to the local area where the assay will be performed. in conclusion, the work presented here demonstrates the development of rpa-nfo and rpa-exo for the detection of o. tsutsugamushi or r. typhi dna. the assay had a detection limit similar to that of qpcr. the specificity of the assays was evaluated using excessive amounts of mouse dna and this did not affect the reaction. the assays were also evaluated using extracted dna from human patient samples, demonstrating around 80% sensitivity and 100% specificity using limited clinical samples. finally, the ease with which the cross-contamination-proof lateral flow cassette can be used to detect multiple amplicons from various nucleic acid amplification methods makes it promising for wide-ranging use in the field. typhi dna detection in rpa-nfo could not detect r. conorii and r. rickettsii dna with similar detection limit. the rpa-nfo was performed as described in the materials and methods using dna extracted from r. typhi, r. conorii and r. rickettsii with different copy number as determined by qpcr. lane n: negative, lanes 1-4 contained 425, 170, 85 and 40 copies/reaction of r. typhi dna, respectively, lane 5 contained 10 4 r. rickettsii dna, and lanes 6-7 contained 10 4 and 10 3 copies/reaction of r. conorii dna. 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authors wish to thank mrs. erin atkins and dr. temitayo awoyomi for reviewing and editing the manuscript and dr. yupin suputtamongkol and her associates for providing extracted dna. these dna samples were confirmed as positive or negative using pcr. the opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the department of the navy, the naval service at large, the department of defense, or the u. s. government. authors chien-chung chao and wei-mei ching are employees of the u. s. government. this work was prepared as part of official duties. title 17 u.s.c. §105 provides that 'copyright protection under this title is not available for any work of the united states government.' title 17 u.s.c. §101 defines a u.s. conceived and designed the experiments: ccc wmc. performed the experiments: tb zz. analyzed the data: ccc tb zz. contributed reagents/materials/analysis tools: ccc tb zz. wrote the paper: ccc tb wmc. key: cord-287582-ya81rc2n authors: viennet, elvina; ritchie, scott a.; williams, craig r.; faddy, helen m.; harley, david title: public health responses to and challenges for the control of dengue transmission in high-income countries: four case studies date: 2016-09-19 journal: plos negl trop dis doi: 10.1371/journal.pntd.0004943 sha: doc_id: 287582 cord_uid: ya81rc2n dengue has a negative impact in lowand lower middle-income countries, but also affects upper middleand high-income countries. despite the efforts at controlling this disease, it is unclear why dengue remains an issue in affluent countries. a better understanding of dengue epidemiology and its burden, and those of chikungunya virus and zika virus which share vectors with dengue, is required to prevent the emergence of these diseases in high-income countries in the future. the purpose of this review was to assess the relative burden of dengue in four high-income countries and to appraise the similarities and differences in dengue transmission. we searched pubmed, isi web of science, and google scholar using specific keywords for articles published up to 05 may 2016. we found that outbreaks rarely occur where only aedes albopictus is present. the main similarities between countries uncovered by our review are the proximity to dengue-endemic countries, the presence of a competent mosquito vector, a largely nonimmune population, and a lack of citizens’ engagement in control of mosquito breeding. we identified important epidemiological and environmental issues including the increase of local transmission despite control efforts, population growth, difficulty locating larval sites, and increased human mobility from neighboring endemic countries. budget cuts in health and lack of practical vaccines contribute to an increased risk. to be successful, dengue-control programs for high-income countries must consider the epidemiology of dengue in other countries and use this information to minimize virus importation, improve the control of the cryptic larval habitat, and engage the community in reducing vector breeding. finally, the presence of a communicable disease center is critical for managing and reducing future disease risks. pathogens and vectors can now be transported rapidly around the world. consequently, new infections are emerging and some old infections, including dengue, have re-emerged. dengue notwithstanding differences in reporting methods, the economic impacts of dengue on a variety of measures are considerable for hics. these impacts can be direct, such as the costs of treatment, hospitalization, and prevention, or indirect, such as the loss of productivity related to absence, disability, or death and the effects on tourism [21] . table 1 presents epidemiological data for dengue infection in queensland (australia), florida (us), singapore, and taiwan. 41, 48] 2015, broward county denv-1, -2 85 1 ---[49] (continued) cycles were occurring every 5-6 years with peaks in july and august. infection and mortality in singapore are reported principally among adults [92] . a retrospective case-control study was performed for the period 01 january to 31 september 2004 and included all cases of dengueassociated mortality treated in tan tock seng hospital, singapore [93] . of the seven patients who died, five (71.4%) were male [93] . in 2005, 14,006 cases were reported, and about 80% were among adults [9] . after small numbers of cases in 2006, singapore experienced another large denv outbreak in 2007, with 8,826 cases [94] (fig 2c) . that same year, the serotype denv-2 re-emerged with a clade replacement and overtook denv-1, which had been circulating since 2004, and remained the predominant serotype until 2013 [57] . a change in the predominant denv serotype may have triggered a spike in dengue cases because of a lack of serotype-specific immunity [9] . in 2013, there was a record 22,248 reported dengue cases, with 842 dengue cases in a single week [95] . the risk of acquired dengue for nonimmune visitors ranged from 42/100,000 during a low season of a nonepidemic year to 1,700/100,000 during the high season of an epidemic year in singapore [96] . taiwan. in taiwan, several denv outbreaks occurred before 1945, and then there were no outbreaks until 1981 [97] . before 2014, the largest and most severe outbreak happened in 2002 (5,388 and 241 dhf cases) [72] . in 2002, there were fewer imported cases in the southern (n = 13) compared with the northern (n = 28) parts of taiwan probably because of fewer visits from travelers and fewer workers from endemic countries in the south. however, 98% of locally acquired cases occurred in the south where ae. aegypti is most abundant [98] . since 2011, the centers of disease control, roc (taiwan), has reported more than 800 imported cases and 18,500 locally acquired dengue cases, with a significant increase in 2014 [99, 100] (fig 2d) . for the first time since formal records were first kept in 1981, the largest denv outbreak occurred in 2015 with 42,916 reported cases (42,572 locally-acquired, 209 deaths) [75, 77] . most of these cases (53.4%) occurred in tainan city and 44.5% in kaohsiung city located in southern taiwan [75, 77] . outbreaks of dengue usually peak in the summer and autumn [101] . despite repeated dengue outbreaks caused by importation to the southern part of the island, taiwan is not currently considered dengue-endemic [72] . based on a study of 9,939 cases of dengue fever and dhf from 2000 to 2007, the duration of illness for dengue fever and dhf (excluding fatalities) was 8.4 and 10.8 days, respectively [102] . active surveillance from 2004-2007 reported a denv seroprevalence of 1.1% (of 42,150 total cases) and 77% of cases as primary dengue [72] . estimates of the economic burden of dengue are calculated by adding the direct and indirect costs. the direct costs consist of health care costs such as those related to the provision of health care, non-health care costs such as those related to consumption of resources like transport, and household expenditures. the indirect costs are those associated with the loss in productivity from illness and death [103] . a robust assessment of the economic burden helps to i) identify information gaps, research needs, and refinements to the national statistical reporting systems; ii) argue that policies on dengue control and prevention should be given a high priority on the public health policy agenda; and iii) provide a baseline measure to determine the cost effectiveness of dengue policies and programs [104] . however, few studies have assessed the economic burden of dengue [105] . the estimation of the true extent of dengue, its incidence, and its associated costs have substantial uncertainty, partly because of unreported and unrecognised apparent denv infections [8] . in 2013, there were an estimated total of 58.40 million symptomatic denv infections globally, including 13,586 fatal cases [8] . the annual global cost of dengue illness averaged us $8,900 million. globally, 18% of cases are admitted to the hospital, 48% remain ambulatory, and 34% do not seek medical care. costs per case are generally higher in hics, especially in australia and the us [8] . queensland, australia. the average work time lost was 10.5 days among people who recalled a dengue-like illness during the 1993 dengue epidemic in charters towers, australia [106] . from 1990 to 2008, the cost of lost work and the control cost averaged an estimated us$36.18 million (2.01 million per annum) [107] . however, this calculation does not include physical and psychological suffering. a delayed response to dengue outbreaks of 4-6 weeks would result in 86 times (or us$13 million in 2003) and 346 times (or us$382 million in 2009) higher dengue illness costs versus a scenario with active surveillance and response within 2 weeks, respectively [108] . therefore, effective dengue surveillance and response lowers costs associated with dengue. florida, us. cost has not been estimated in florida specifically, but estimates have been made for the us and many central and south american countries [109] . shepard et al. (2011) estimated the aggregate annual total cost of dengue in the americas to be about us$2.1 billion for the period 2000-2007 [109] . the us had the highest estimated average total cost per dengue case (ambulatory cost added to hospitalized cost = us$19,413) among the selected countries. singapore. among the 12 countries analysed by shepard et al. [4] (bhutan, brunei, cambodia, east timor, indonesia, laos, malaysia, myanmar, the philippines, singapore, thailand, and vietnam), which did not include the us, australia, or taiwan, the predicted values of direct and indirect unit costs per dengue case were the highest in singapore (direct costs: $2,455, indirect costs: $1,821 in 2010 us dollars). in singapore, carrasco et al. [6] investigated the cost-effectiveness of future vaccination programs and estimated that, from 2000 to 2009, the average economic impact of dengue illness in constant 2010 us dollars ranged from $0.85 billion to $1.15 billion, of which control costs constituted 42%-59% [6] . taiwan. in taiwan, it cost about us$227 (2011) to treat a patient with dengue fever [110] . the probability of being infected by denv due to climate change ranges from 12% to 43% to 87%, which represents low, mid, and high probabilities when the temperature increased by 1.8°c [111] . people would pay us$22.8, us$102.3, and us$162.28 (in 2008), respectively, per year in order to avoid the increased probabilities of being infected with denv. visitor numbers for september 2015 were 50% lower than the same period in 2014, due at least in part to dengue [112] . nonclimatic factors include the epidemiology, ecology, and distribution of the vector, socioeconomic, and environmental factors; climatic factors include weather variability, climate change, and extreme events. these factors are discussed in detail in the following sections. ae. aegypti is most prevalent in highly urbanized areas, whereas ae. albopictus is present mainly in rural, suburban, and vegetated urban areas [113] [114] [115] . abiotic factors including urban landscape, temperature, humidity, and rainfall are strongly associated with the abundance of both species [116] . displacement of ae. aegypti by ae. albopictus because of resource competition among larvae and crossmating between ae. albopictus males and ae. aegypti females might reduce the overall risk of dengue transmission [115, 117, 118] . continued vector surveillance is crucial in susceptible areas currently free of ae. aegypti and ae. albopictus. ae. albopictus and ae. aegypti are present in queensland, florida, singapore, and taiwan. the former is not yet established in monroe county, florida [119] or in mainland queensland, where ae. aegypti is prevalent. in singapore, ae. aegypti predominates [120] . in taiwan, ae. aegypti only occurs south of the tropic of cancer (23°26 0 n 120°8 0 e, chiayi and hualien counties), and ae. albopictus is distributed throughout the country and below an elevation of 1,500 m above sea level [98] . the movement of ae. albopictus from the torres strait islands, queensland, to the mainland, with subsequent potential expansion southwards, could increase the area that is receptive to denv epidemics [121, 122] and also expand the area receptive to transmission of other viruses including chikungunya and zika. this is also the case in central, northern, and eastern parts of taiwan, where only ae. albopictus exists and outbreaks occur rarely. ae. aegypti is thus the main vector, but ae. albopictus can transmit dengue in the absence of ae. aegypti [71] . the number of locally acquired cases is higher in the southern than in other parts of taiwan, despite a lower number of imported cases. therefore, the presence of ae. aegypti may be essential to outbreak initiation. in southern florida, [116, 119] ae. aegypti is more abundant in the early dry season than in the late wet season and can spread during the early wet season in the absence of ae. albopictus [116] . high denv virus replication rates are associated with a high vertical transmission rate in mosquitoes [123] . fewer ae. aegypti than ae. albopictus progeny are infected vertically, although the latter species may sustain denv during interepidemic periods [123, 124] . vertical transmission of denv-1 from infected ae. aegypti female mosquitoes to their eggs may have served as an interepidemic reservoir between outbreaks in successive years in key west, florida [125] , as could low-level horizontal transmission, especially if not detected. in taiwan, although continuation of outbreaks through winter is rare, three overwinter outbreaks occurred from1987-2010: 100,000 cases from 1987-1988; more than 5,000 cases from 2001-2002; and more than 1,400 cases from 2009-2010 [101] . many dengue importations into australia are presumably undetected or unreported [120] . in charters towers, queensland, subterranean breeding (e.g., in septic tanks, sump pits, telecommunication pits, mine shafts, and wells) contributed significantly to ae. aegypti populations and therefore denv-2 transmission during the 1993 outbreak [126] . many larval sites are cryptic and difficult to locate, particularly subterranean sites and elevated sites such as rainwater tanks and roof gutters [127] . subterranean sump pits and wells can support large populations of ae. aegypti, especially during the winter season when surface containers are dry [127] [128] [129] . a reduction in the number of large water storage containers likely reduced ae. aegypti distribution in the past [130] . in singapore, refuse is commonly an aedes oviposition site [131] . the main breeding habitats in homes are domestic containers, flower pot plates, ornamental containers, and plants, and in public areas discarded receptacles, closed perimeter drains, gully traps, housing and development board (hdb) corridor drain/gullies, and plants [132] . with increased transport of materials and people, the potential for vector transportation and virus transmission and spread has increased substantially [133] . in queensland, florida, and taiwan, the arrival of viremic travelers is currently necessary for local transmission. in australia, dengue is now more often diagnosed than malaria in ill travelers who have returned from tropical regions (except africa) [134] . international arrivals to townsville and cairns airports commenced in 1980 and 1984, respectively, and consequently the number of travelers from dengue-endemic countries has increased significantly [135] . because queensland, florida, singapore, and taiwan are popular tourist destinations, this increases the likelihood of virus introduction and subsequent local transmission. during the 1997-1999 epidemic in north queensland, the movement of viremic individuals facilitated multiple initiation foci [23] . today in the us, miami, florida is the most used airport in terms of the number of international passengers boarding american carriers [136] . key west is a tourist destination with more than 2 million visitors annually [137] . in 2013, changi airport, the main airport in singapore, handled more than 53.7 million passengers with an annual growth rate of 4.7% [138, 139] . about 203 million travelers and 68 million conveyances were reported to cross the border between the southern malaysian state of johor and singapore in 2013 [140] . during the second largest denv outbreak in taiwan in 2014, a total of 44.8 million passengers (an increase of 12.6% over 2013) were traveling on international, cross-strait, and transit flights [141] . as a travel hub, singapore [56] and taiwan experience continuous importation of denv. proximity to endemic regions and the increased number of dengue cases in neighboring countries presents a significant risk for countries with endemic vectors [142] . in australia, among cases with a known source, 23% originated overseas from 1991 to 1999 and 64% from 2000 to 2012. between 1995 and 2011, the main sources of dengue importation in australia were from neighboring dengue-endemic countries such as indonesia (24.6%), papua new guinea (23.2%), thailand (13.4%), east timor (8.9%), and the philippines (6.7%) [33]. florida is also very close to dengue-endemic countries in the caribbean (e.g., puerto rico), central america (honduras), and south america (brazil) and is the top us destination for puerto rican migrants [143] . in singapore from 2000 to 2014, the neighboring dengue-endemic countries indonesia and malaysia were the two main sources of dengue importations, contributing 38% and 26%, respectively, of the total imported cases in singapore [50] [51] [52] [53] 55, 58, [60] [61] [62] [63] [64] [65] 68] . virus importation by tourists and migrant workers (e.g., from china in 2002 [144] ) from dengue-endemic countries and decreasing herd immunity have contributed significantly to the failure of dengue control in singapore [145] . over the past two decades in taiwan, imported cases have played an important role in the major outbreaks of dengue [146] . between 1981 and 2010, imported dengue cases primarily involved travellers from the philippines, vietnam, thailand, and indonesia [101] . in many tropical regions, urbanization, lack of dependable water systems, overcrowding, and poor housing contribute to large dengue epidemics [80] . this is less of a problem in hics. however, in north queensland and key west, florida, dengue transmission has been associated with old unscreened housing, allowing access to ae. aegypti [147, 148] . typical "queenslander" houses, designed with architectural characteristics suitable to a tropical or subtropical climate such as large sprawling timber structure on stumps, and an extensive deep shaded verandah, are being replaced by new apartment blocks designed with screening and air conditioning in cairns and other far north queensland centers. these should limit dengue transmission [127] because avoidance of mosquito bites effectively prevents transmission [149] . in queensland, sites such as backpacker hostels are identified as "ignition" or starting points, whereas centers where communities congregate, such as schools and churches, are identified as "dissemination" points [23] from which the virus may spread to multiple geographic foci [23] . between 2006 and 2011, australia, particularly queensland, experienced rapid population growth [150, 151] , with a total of 121,000 new queensland residents over the five years [152] . the queensland average annual population growth was 2%, [150, 151] primarily driven by overseas immigration [153] . the 2006 census shows that the main countries of origins of new cairns residents were the united kingdom (uk), japan, philippines, korea, and papua new guinea, whereas other regional areas, such as townsville and mackay, feature migrants prominently from the uk, india, the philippines, and south africa [152] . singapore is highly urbanized with a population of 5.47 million in 2014, an increase of 1.3% from 2013, and a population density of 7,615 persons per km 2 [154] ; high population density increases dengue risk. in singapore, about 80% of the houses are managed by the hdb [155]. before the mid-2000s, most houses had bamboo pole holders outside the kitchen windows that were used to dry laundry, but also provided good breeding grounds for mosquitoes [156] . now, the bamboo poles can be covered [157] , supported from both ends, or replaced by a laundry rack. although these changes should help prevent the breeding of aedes mosquitoes, no studies have confirmed this. over the period 2002-2014, residents in hdb compound houses, free standing properties (including shops and houses), and condominiums constituted 70%, 19%, and 11%, respectively, of the dengue cases. incidence rate was higher in those living in free standing properties [51, 53, 55, 58, [60] [61] [62] [63] [64] [65] 68] (fig 3) . in taiwan, from 1998 to 2002, a higher level of urbanization was also associated with increased risk of dengue fever at the township level [158] . poor environmental hygiene usually goes with rapid urbanization, which tends to increase vector breeding. it is unclear why the 2015 outbreak clustered intensively in tainan city, but a persistent problem of illegal dumping of garbage in the local parks possibly contributed through pooling of water and consequent mosquito oviposition [159] . climate change may contribute to the emergence and re-emergence of dengue through its interactions with biotic and abiotic factors. however, due to the complex interaction of climate and other factors, the magnitude and direction of changes in incidence are difficult to predict. australian annual average daily mean temperatures have increased progressively by 0.9°c since 1910, and the frequency of extreme weather has changed, with more heat and fewer cool extremes [160] . substantial warming has occurred in the three oceans (pacific, indian, southern) surrounding australia [161] . by 2070, australian temperatures are projected to rise by 1-5°c, depending on global co 2 emissions. consequently, climate sensitive diseases are expected to increase in incidence [162] . in southern florida, climatic factors may be the cause of competition between ae. albopictus and ae. aegypti [163] . the presence of ae. albopictus was significantly lower after the dry season than during the wet season, whereas the ae. aegypti presence was consistent [163] . however, local coexistence of these species is possible when a warm dry climate favors ae. aegypti and reduces the competition from ae. albopictus by increasing mortality of ae. albopictus eggs [163] . in the laboratory, dry periods cause disproportionately greater mortality of ae. albopictus eggs compared to ae. aegypti eggs [163] . in singapore, from 1972 to 2014, the annual mean temperature has increased from 26.6°c to 27.7°c, and rainfall has become more intense [164] . temperature, relative humidity, and el niño southern oscillation were significantly and independently associated with dengue cases in singapore [165] . moreover, in singapore and taiwan, rainfall, temperature, and dengue fever incidence are positively related [166, 167] . health officials in singapore reported a surge in dengue cases in december 2015, which coincided with warmer weather associated with the el niño phenomenon [69] , although no study has confirmed this. in taiwan, dengue transmission typically begins with importation in the summer and ends in the winter. the combination of its subtropical climate, neighboring hyperendemic countries, the hot rainy summer (average temperature, 28°c) and dry cold winter (average 20°c), and the coexistence of two vectors facilitates dengue transmission in the summer and autumn [72] . in 2007, after an initial wave of dengue infections in taiwan, the temperature was reduced by two typhoons, and fewer cases were reported. however, in early autumn, the subsequent increase in moisture and temperature caused a second wave of dengue fever [168] . the exact causes of the large outbreak in kaohsiung, taiwan, in 2014 are not known, but it is believed that higher mean temperatures than in previous years (+0.5°c-1.3°c from june-september 2014) contributed [75] . the causes of the intensive and clustered 2015 outbreak in tainan are also poorly understood. large areas of standing water accumulated in some parks after flooding and only drained for two to three weeks may have been the primary breeding areas for mosquitoes that sustained the epidemic [159] . drought together with population and economic growth have caused anthropogenic environmental changes. these include limited water supply relative to demand in queensland cities, despite flooding, which has led to an increase in rainwater tank usage. regulations and guidelines mandate or recommend measures to prevent mosquito breeding in tanks, including flap valves and screens with mesh size of no more than 1 mm [169] . nonetheless, an increase in domestic water storage could increase future mosquito populations and consequently dengue risk [170] . in north queensland, dengue transmission is also associated with unscreened rainwater tanks. drought and consequent changes in water storage practices are projected to increase the ae. aegypti range [170] . under climate projections, shade-dependent evaporation interacts with container size and biological characteristics, such as resistance to cold and egg dessication, to determine geographic range for ae. aegypti [171] . simulation modelling indicates that climate warming may lead to increases or decreases in dengue vector abundance, depending on the level of carbon emissions [172] . queensland, australia. since the 1992-1993 dengue epidemic, public health authorities in north queensland have intensified surveillance, control, and education programs (e.g., the "tip it, store it, throw it" message), and the development and use of predictive tools [27] . strategies are applied at three different activity levels: ongoing prevention; response to sporadic cases; and outbreak response [127] . when a case is notified, officials interview the patient and complete a case report form [27] . control activities include larval and adult control within a 200 m radius of the case residence and high-risk contact areas [27] . the use of indoor residual spraying targeting resting sites of ae. aegypti has been shown to significantly decrease dengue transmission in cairns [173] and has become the primary adulticiding method used. regulation of water storage to minimize mosquito breeding is particularly important for detecting and eliminating the most productive sites [174] . florida, us. following the introduction of west nile virus in the northeastern us in the summer and autumn of 1999, the us centers for disease control and prevention (cdc) and the department of agriculture cosponsored a meeting of experts to develop guidelines for the surveillance, prevention, and control of west nile virus and other arboviruses [175] . dengue became a nationally notifiable disease in the us in 2009 because of concerns about the increasing number of importations and the consequent risk for local transmission and transfusion transmission [176] . during the key west outbreaks, the florida keys mosquito control district (fkmcd) used all means available to reduce the populations of ae. aegypti. response strategies in key west included public meetings and communication, education at schools, and tourism council press releases [42] . tourism, public health, and vector control officials collaborated in response. health department personnel conducted outreach visits to clinicians. vector control activities include spraying adulticides within a 200 m radius around homes of casepatients, outdoor residual and spatial insecticide treatments, door-to-door campaigns to educate the public, and finding and eliminating mosquito breeding sites [42] . florida law mandates inspections and the elimination of breeding sites; however, fines are not usually imposed and violations are frequent [42] . since the implementation of response strategies, autochthonous dengue infections still occur in key west, and a 2012 survey of the decision makers involved in the control of the outbreak revealed the need to focus prevention strategies on educational campaigns [177] . singapore. the vector-control programs instigated in the late 1960s have progressively reduced dengue transmission and thus herd immunity. as a consequence, the incidence of dengue has surged since the 1990s, mainly in young adults [178] . low herd immunity and a shift from domestic to nondomestic transmission (e.g., schools, hospitals, and workplaces) are the main issues in singapore [179] . the number of potential breeding sites in nondomestic settings is large, and these are poorly identified and localized. the national environment agency (nea) adopts a multipronged approach to control dengue comprising preventive surveillance and control, public education, and community involvement, enforcement, and research. the strategies include active surveillance in areas prone to dengue or where mosquito populations are high, and source reduction when cases are reported [180] . in singapore, pyrethroids were commonly used for fogging in the past and are still commonly used as insecticide by the pest control industry. consequently, ae. aegypti has become pyrethroid-resistant in singapore [181] . crossresistance between dichlorodiphenyltrichloroethane and pyrethroids occurs [182] . therefore, viable alternatives including biological control are urgently needed. however, biocontrol approaches might impose evolutionary selective pressures on the virus and vectors. the viral diversity resulting from the increase in dengue transmission over recent years and reduced mosquito population because of vector control may also have contributed to selection pressure and to the evolution of a virus lineage with better fitness; i.e., denv that would spread better through ae. aegypti population [91] . although new environmentally friendly biocontrol strategies show promise, an improved understanding of the biology and ecology of the vectors is crucial [130] . the singapore government introduced a fine in 2005, which doubled in 2008, for all homes found to be breeding mosquitoes [183] . for example, in 2012, 900 mosquito-breeding offenses were detected on construction sites, with 626 being first-time offenders [184] . to implement an effective vector-control program, more officers were recruited to conduct routine checks of homes within dengue-active zones in 2013. when residents did not respond to notices, the nea used its legislative powers to gain entry to homes [185] . the nea fined a town council us$160 (in 2013 us dollars) for letting mosquitoes breed in water tanks. town councils which break the law twice could be fined up to us$1,500. as of august 2014, the nea has inspected 1.9 million houses or premises and has deployed more than 1,000 gravitraps in dengue clusters for mosquito-control purposes, in addition to regular fogging and space spraying in certain hotspots. the nea has prosecuted 14 contractors and issued 62 stop-work orders [186] . since march 14, 2016, the owners of homes found to be breeding mosquitoes are fined us$148 (in 2016 us dollars), regardless of whether the home is within a dengue cluster [187] . since 2015, the nea has also increased its epidemiological investigation to identify potential sources of infection other than registered residential addresses, such as workplaces and areas of congregation. nea improved its dengue control by i) increasing inspections by creating dedicated construction site teams, ii) publishing the list of sites issued with stop-work orders on the dengue microsite to serve as a deterrent to contractors, iii) requesting contractors to put in place a temperature-screening regime to identify cases earlier, iv) encouraging application of insect repellent and, v) for dengue-infected workers, to sleep under bed nets or in air-conditioned sick bays to prevent further transmission [188] . taiwan. before 2009, the taiwan centers for disease control (tcdc) used only a clinical case definition for reporting dengue, which may have led to over-reporting. the tcdc's current dengue case definition, established in 2009 and in use since, requires laboratory confirmation for reported cases [189] . dengue must be reported to tcdc within 24 hours [72] . a central command center was established to provide resources and personnel from various ministries (education, interior, national defense) and agencies (department of health, environmental protection administration, government information office). the aim was to foster close cooperation with local government units and public education to reduce vector breeding. these efforts have been associated with reduced case numbers from 2010 to 2012 [70] . however, it is difficult to confirm this trend because of the short period. when aedes larvae are found or when an individual refuses control measures, a significant fine is imposed [190] . taiwan had also established airport fever screening [191] . questionnaire screening was used from 1998 to 2002, but following the 2003 severe acute respiratory syndrome outbreak, fever screening at the airport was added. fever screening at the airport has been successful in identifying 45% of 542 known imported dengue cases with fever [191] . however, the intensity of local transmission is not proportional to the number of imported cases [191] . pyrethroids are also often used to control ae. aegypti and thereby suppress epidemics in taiwan. however, ae. aegypti has also become pyrethroid-resistant in taiwan [192] . singapore has in place community-based programs for the removal of potential breeding habitats [193] , which involve the community in mosquito control through environmental management, health education, and community ownership ("bottom-up" approach) (e.g., "do the mozzie wipeout" campaign, "do the 5-step mozzie wipeout") [194] . this is essential for avoiding community complacency. however, the community-based approach can be slow or demotivating and, as a consequence, unsustainable [195] . bottom-up and top-down approaches should be combined [195] . in addition to the proactive measures against mosquito-breeding sites, efforts have been made to increase opportunities for people to interact through social media focused on dengue transmission. examples include "stop dengue now" and "amy khor" in singapore and "follow the moz" or "eliminate dengue" facebook pages in queensland. unfortunately, the latter two are no longer updated. although the use of these forms of online social media cannot provide a solution for reducing dengue transmission, it empowers citizens by giving them easy access to knowledge and by proposing a form of participatory surveillance involving mutuality and sharing. singapore citizens actively voice opinions on social media about dengue. for example: "(. . .) each time an nea officer knocks on my door, i cooperate with them but at the same time, i wonder whether the door-to-door checks aren't a waste of time when my hdb neighbourhood is strewn with litter such as empty cigarette packs, plastic bags, and dirty food containers. each piece of litter is a breeding ground (. . .)" [196] , "residents must take greater responsibility to rid their homes of mosquitoes breeding. i have often witnessed households not responding to nea officers knock (. . .)" [197] . local residents in singapore also have the opportunity to volunteer in efforts to control dengue. dressed in yellow vests and red shirts, they go from door to door encouraging their neighbors to assist in controlling dengue [198] . a smartphone application designed to encourage citizens to identify and report potential breeding sites of aedes mosquitoes is also available (e.g., trashwatch, http://www.vectoranalytica. com/inc/products/trashwatch/). however, some countries, including australia, do not have access to this application. from 2012-2013, the queensland government budget cuts impacted hospital and health services in queensland, mainly in metro north (-22.5 million australian dollars) and metro south (-18.8 million) areas in brisbane, gold coast (-9.2 million), townsville (-7.8 million), sunshine coast (-7 million), and cairns (-6.5 million) [199] . these cuts have impaired dengue prevention and control programs [200] . moreover, unlike the us, singapore, and taiwan, australia lacks a single center, such as the cdc, for dissemination of information to the public, relevant organizations, and government agencies. in the us, public health budget cuts have also impaired vector surveillance and control programs [201] . in 2011, the florida department of health infectious disease control branch budget was reduced by us$3.8 million with consequent loss of 172 full-time positions in health departments [202] . in early 2016, the president of the us asked congress to allocate us$1,900 million to surveillance and control of the zika virus, ae. aegypti, and ae. albopictus, but, to date, lawmakers had not acted [203] . in florida, the mosquito control budget varies greatly between counties. lee county, home to fort myers and 700,000 people [204] , funded by local property taxes, spends $16 million a year and has a staff of 88, ten helicopters, and four propeller planes for spraying [205] . in contrast, vasquez, an entomologist and director of the mosquito-control district of florida's miami dade county, florida's biggest county with 2.6 million people, spends a $1.6 million budget a year, enough for 15 employees. moreover, his inspectors are not always welcomed by the community and have to deal with denial, angry homeowners, and, sometimes, local politics [203] . early recognition and notification of dengue cases [33] and timely initiation of appropriate supportive care is critical to reduce medical complications and mortality among patients with severe forms of dengue [206] . in florida, at least until 2010, medical and public health practitioners tended to have limited experience with dengue, and the failure of diagnosis and reporting led to delayed detection of denv introduction [7] . in the key west 2009-2010 outbreak, initial cases were attributed by local physicians to nonspecific viral illness without consideration of dengue [207] . although singapore generally has an efficient clinical service, hospitals are sometimes crowded [208] . because mosquitoes sufficient to infect an entire neighborhood can breed in a small number of negligently maintained properties, citizen cooperation is indispensable [209] . however, cooperation between citizens and public officials is often lacking, and there may be outright obstruction of mosquito-control efforts. unfortunately, control is often seen as the exclusive responsibility of local and state mosquito-control agencies [89] . transfusion-transmitted dengue is rare [210] , but no licensed donor-screening test for denv is available worldwide. consequently, many blood donations are unable to be collected during epidemics [211] . rigorous selection of donors offers protection but is labour intensive, costly, may affect sufficiency of supply, and may not be possible. what do these areas have in common? the countries and states presented in this review have in common i) a competent mosquito vector, ii) introduction of denv by travelers, iii) increased local transmission of dengue coinciding with population growth and increased mobility, iv) recent budget cuts impacting public health services, and v) a largely nonimmune population. the four countries and states have areas where ae. aegypti is established. outbreaks rarely occur where only ae. albopictus is present (e.g., central, northern, and eastern parts of taiwan and the torres straits islands in australia). the four countries and states are surrounded by dengue-endemic countries and have air connections with dengue-endemic countries. in queensland, florida, and taiwan, the arrival of viremic travelers is a prerequisite for local transmission. both taiwan and singapore are highly populated and urbanized, increasing dengue transmission risk. both florida and queensland have experienced past budget cuts that affected public health services. florida and queensland are particularly at risk for autochthonous dengue outbreaks due to its largely nonimmune populations. the countries and states presented in this review also display unique features, such as i) their dengue epidemic/hyperendemic status, and consequent mortality rate, ii) the frequency, amplitude, and duration of outbreaks, iii) their vector control program and community engagement. dengue is hyperendemic in singapore and taiwan, but epidemic in queensland and florida, and the rarity or absence of serious outcomes in australia and florida may be due to the infrequency of consecutive infections with different serotypes. dhf and deaths are rare in australia, and absent in florida, but occur often in singapore and taiwan. mortality is usually linked to delayed provision of supportive treatment or presence of comorbidities [212] . the recent epidemiology of dengue in singapore was characterized by a 5-6-year cycle [9, 56] . however, since 2013 in singapore, the incidence rates have markedly increased, exceeding 10,000 cases each year. dengue transmission in florida is still reasonably limited, and the majority of dengue cases reported are travel-associated cases [88] . in queensland, the "tip it, store it, throw it" message is now being heard by the local community according to queensland health's cairns acting director of tropical public health services [35] . in far north queensland, the number of dengue fever cases in 2015 was 33% lower than the total dengue cases in 2014 [36] . compare to the other three countries and states, singapore citizens have more opportunities to engage on social media about dengue, to be aware of the problem, and thus to be proactive. however, despite the hard work of contractors engaged by the nea to clean out certain private estates and public areas throughout singapore, littering remains an environmental and social problem linked to economic and health issues [131] . this review summarises key determinants for dengue transmission, which could be better managed to help reduce transmission ( table 2) . some are relevant for many countries and states (e.g., proximity to endemic countries, population growth and movement, lack of awareness, and engagement of residents/tourists) and some are country/region specific (e.g., high level of urbanization, budget cuts, housing structure). the world's first dengue vaccine, dengvaxia (cyd-tdv) by sanofi pasteur, first registered in mexico early december 2015, and later approved by the philippines and brazilian authorities, has provided some hope [213] . cyd-tdv is a live recombinant tetravalent dengue vaccine that has been evaluated as a three-dose series on a 0/6/12 month schedule in phase iii clinical studies, and is for use in individuals aged 9-45 years living in endemic areas [213] . this vaccine is currently being reviewed by around 20 countries in asia and latin america [214] . the phase iii trials showed a variation in vaccine efficacy according to the age at vaccination and serostatus before vaccination [213, 215] . five additional vaccine candidates are under evaluation in clinical trials [213] . recommendations concerning the cyd-tdv vaccine will be published by the world health organization in july 2016 [213] . in 2012, the fkmcd announced that it had partnered with the british firm oxitec to release large numbers of genetically modified ae. aegypti males in key west, florida [89] . this project was considered but rejected by some residents, and the use of genetically modified male mosquitoes in key west is still in negotiations and is awaiting the us food and drug administration approval [89, 216] . the re-emergence of dengue in florida as well as the threat posed to the us from other emerging mosquito-borne arboviruses (e.g., chikungunya, zika viruses) emphasizes the need for strong vector-borne disease surveillance and mosquito-control infrastructure to rapidly identify and control outbreaks of dengue or other mosquito-borne diseases. transinfection of ae. aegypti with the endosymbiotic bacterium wolbachia pipientis is undergoing field trials in australia, indonesia, vietnam, brazil, and columbia [217] . some strains of wolbachia can reduce ae. aegypti lifespan and reproduction and thus interfere with denv replication and transmission [218, 219] . the virulent wolbachia strain wmelpop has been shown to cause widespread degeneration of tissues (brain, retina, muscle) and early death • travel-re lated risks need to be better managed and incorporated in national strategies for nonendemic countries that experience, or are at risk for, epidemics. tourism bodies need to be involved in disease prevention in order to diminish possible opposing viewpoints. • education of the public and the medical profession is central to prevention. • to avoid institutional memory lost when key employees leave, ("brain-drain" effect), transition to their replacements should be prepared to preserve this information. • a robust assessment of the economic burden (direct and indirect costs) of dengue infections is highly needed for those countries to justify investing in dengue control programs. in ae. aegypti adults [220] and eggs [221] but failed to establish following field releases. releases of the more benign wmel strain led to establishment in seven cairns suburbs (infection frequency > 90%), and this strain has persisted for at least five years [222, 223] . a field trial was later launched in townsville, north queensland, in october 2014 [224] . in singapore, the nea's environmental health institute has tested the use of wolbachia bacteria in the laboratory, but there have not been field trials. reliance on insecticides for dengue control has been excessive [225] , and these compounds are costly and sometimes ineffective in urban areas. such deficiencies and limitations of current vector-control strategies have necessitated the development of predictive mathematical, statistical, or spatial models for dengue early warning systems [225] [226] [227] [228] . the development of a climate-driven spatiotemporal prediction model and the use of geographic information system in dengue surveillance are essential to inform disease prevention and control interventions. yu et al. (2011) [166] proposed a spatiotemporal climate-based model of early dengue fever warning in southern taiwan, based on stochastic bayesian maximum entropy analysis. the analysis provides the required "one-week-ahead" outbreak warnings based on spatiotemporal predictions of dengue fever distributions, that can be used by the taiwan disease control agency to timely identify, control, and prevent dengue fever transmission [166] . hii et al. (2012) [229] developed a weather-based dengue forecasting model that allows warning 16 weeks in advance of dengue epidemics in singapore with high sensitivity and specificity. the authors demonstrate that models using temperature and rainfall could be simple, precise, and low cost tools for dengue forecasting and could be used to enhance decision making on the timing and scale of vector control programs [229] . however, these tools also have limitations because the factors used for prediction may interact nonlinearly [230] . models may also under-or overestimate the projected incidence, and they are often regionally specific and consequently not suitable for global projections. similarly, global models [231] are not necessarily appropriate for small-scale projections. geographic information system technology is a useful tool for creating a user-friendly map to show the physical location of dengue cases from authoritative sources such as the nea (e.g., outbreak, http:// outbreak.sgcharts.com/). finally, traveller behaviour, vector evolution, biocontrol impacts, and other important risks are unpredictable. changes in climatic conditions, patterns of human settlement, movement, and population density, distribution of the two main mosquito vectors, and water-management technologies have all influenced the occurrence of dengue outbreaks since the mid-20th century. the competitive relationship between the two main vectors, ae. aegypti and ae. albopictus, may lead to future changes in the epidemiology of the disease. this review has considered dengue transmission in queensland in australia, singapore, taiwan, and florida in the us. we have identified important epidemiological issues in these regions, such as population growth in the four settings, increases in local transmission despite control efforts in singapore and taiwan, increased human mobility from neighboring endemic countries (especially in taiwan and singapore), lack of citizen engagement in the four settings, and highly populated urban areas (in taiwan and singapore). budget cuts in health and the current absence of a practical vaccine contribute to an increased risk. in the hics discussed, dengue has become an increasing challenge, despite active vector control programs. however, it would be naïve to assume a single cause, or even a small set of causes, is adequate to explain the change in dengue epidemiology in any or all of the countries discussed. host, agent, and environmental factors all interact. these include demography, travel, strain variation, and changes in housing and the global environment. because the factors affecting dengue incidence and transmission are diverse, a single model for disease control is unlikely to be applicable in all settings. assuming ongoing global economic growth, other countries where ae. aegypti and/or ae. albopictus are established (e.g., france and japan) may, in future experience, regulate dengue transmission [232, 233] . the epidemiology of dengue will continue to change. although ae. aegypti is the dominant urban vector, ae. albopictus can also cause outbreaks, especially in less tropical areas. flexibility, ingenuity, and imagination will be required to control dengue in the face of these challenges. we are indebted to the private and public health laboratories and health departments in the states and territories of australia that collect the data provided by the national notifiable diseases surveillance system, australian government, department of health, and queensland • increased human mobility from neighboring endemic countries. • lack of awareness and engagement of residents/tourists/migrant workers (excess of litter is a recurrent issue). • resistance to pyrethroids insecticides. • budget cuts in health. the global distribution and burden of dengue world health organization. global health estimates 2014 summary tables: deaths by cause, age and sex, by world bank income group category updated income classifications economic and disease burden of dengue in southeast asia epidemiology of dengue: past, present and future prospects economic impact of dengue illness and the cost-effectiveness of future vaccination programs in singapore potential social, economic, and health impacts of dengue on florida the global economic burden of dengue: a systematic analysis the 2005 dengue epidemic in singapore: epidemiology, prevention and control dengue-a re-emerging infectious disease in singapore measuring urban form: a comparative analysis of south east queensland and south florida. the 3rd world planning schools congress administration noaa world map of köppen-geiger climate classification updated four degrees of latitude: mosquito control on the "right" coast of australia and florida, usa? 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mosquitoes in dengue fight community urged to stay vigilant despite drop in dengue cases aedes aegypti and aedes aegypti-borne disease control in the 1990s: top down or bottom up each time an nea officer knocks on my door, i cooperate with them but at the same time, i wonder whether the door-to-door checks aren't a waste of time when my hdb neighbourhood is strewn with litter such as empty cigarette packs, plastic bags, and dirty food containers. each piece of litter is a breeding ground residents must take greater responsibility to rid their homes of mosquitoes breeding. i have often witnessed households not responding to nea officers knock forty-six jobs axed as federal government cuts health budget cumulative list of funding and staffing cuts to services, staff, funding and programs by campbell newman lnp government doh layoffs hit some harder only a dozen inspectors stand between zika and miami florida's mosquito control forces mobilize against zika threat the impact of a program 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degeneration and early death a virulent wolbachia infection decreases the viability of the dengue vector aedes aegypti during periods of embryonic quiescence limited dengue virus replication in field-collected aedes aegypti mosquitoes infected with wolbachia eliminate dengue our challenge eliminate dengue our challenge a simulation model of the epidemiology of urban dengue fever: literature analysis, model development, preliminary validation, and samples of simulation results skeeter buster: a stochastic, spatially explicit modeling tool for studying aedes aegypti population replacement and population suppression strategies dynamic life table model for aedes aegypti (diptera: culicidae): analysis of the literature and model development dynamic life table model for aedes aegypti (diptera: culicidae): simulation results and validation forecast of dengue incidence using temperature and rainfall weather-driven variation in dengue activity in australia examined using a process-based modeling approach dengue fever epidemic potential as projected by general circulation models of global climate change comparison of clinical characteristics and laboratory findings of malaria, dengue, and enteric fever in returning travelers: 8-year experience at a referral center in tokyo bilan de la surveillance épidémiologique renforcée du chikungunya et de la dengue key: cord-312223-qgwzgazd authors: shafagati, nazly; narayanan, aarthi; baer, alan; fite, katherine; pinkham, chelsea; bailey, charles; kashanchi, fatah; lepene, benjamin; kehn-hall, kylene title: the use of nanotrap particles as a sample enrichment method to enhance the detection of rift valley fever virus date: 2013-07-04 journal: plos negl trop dis doi: 10.1371/journal.pntd.0002296 sha: doc_id: 312223 cord_uid: qgwzgazd background: rift valley fever virus (rvfv) is a zoonotic virus that is not only an emerging pathogen but is also considered a biodefense pathogen due to the threat it may cause to public health and national security. the current state of diagnosis has led to misdiagnosis early on in infection. here we describe the use of a novel sample preparation technology, nanotrap particles, to enhance the detection of rvfv. previous studies demonstrated that nanotrap particles lead to both 100 percent capture of protein analytes as well as an improvement of more than 100-fold in sensitivity compared to existing methods. here we extend these findings by demonstrating the capture and enrichment of viruses. results: screening of nanotrap particles indicated that one particle, nt53, was the most efficient at rvfv capture as demonstrated by both qrt-pcr and plaque assays. importantly, nt53 capture of rvfv resulted in greater than 100-fold enrichment from low viral titers when other diagnostics assays may produce false negatives. nt53 was also capable of capturing and enhancing rvfv detection from serum samples. rvfv that was inactivated through either detergent or heat treatment was still found bound to nt53, indicating the ability to use nanotrap particles for viral capture prior to transport to a bsl-2 environment. furthermore, both np-40-lysed virus and purified rvfv rna were bound by nt53. importantly, nt53 protected viral rna from rnase a degradation, which was not observed with other commercially available beads. incubation of rvfv samples with nt53 also resulted in increased viral stability as demonstrated through preservation of infectivity at elevated temperatures. finally, nanotrap particles were capable of capturing veev and hiv, demonstrating the broad applicability of nanotrap particles for viral diagnostics. conclusion: this study demonstrates nanotrap particles are capable of capturing, enriching, and protecting rvfv virions. furthermore, the use of nanotrap particles can be extended to a variety of viruses, including veev and hiv. rift valley fever virus (rvfv) belongs to the genus phlebovirus and family bunyaviridae. rvfv is composed of a tripartite singlestranded rna genome with large (l), medium (m), and small (s) segments [1, 2, 3, 4] . rvfv particles have icosahedral symmetry and are 90-110 nm in diameter [4] . the envelope is made up of a lipid bilayer that is embedded with the gn and gc glycoproteins. these glycoproteins, which are the most exposed components of the virus during infection, play a crucial role in the entry of the virus into the host cell. rvfv is a highly pathogenic arthropod-borne virus that is primarily transmitted by mosquitoes, particularly after heavy rainfall. although it can infect a wide range of vertebrate hosts, rvfv primarily affects livestock and humans [2] . animals are infected through mosquito bites and other arthropod vectors. humans are typically affected when they come in close contact with infected bodily fluids or tissues, but transmission via mosquito bites, as well as aerosolization may also occur. however, humans are dead-end hosts [1, 5] . since being first identified in 1930 in the rift valley of kenya, outbreaks have led to high mortality rates as well as significant economic loss [3] . rvfv has remained endemic in sub-saharan africa, causing major outbreaks throughout the continent over the last century [6] . in 1976, 200,000 individuals were infected and 600 fatal cases were reported in egypt [5] . most likely due to international livestock trade, it has since crossed the arabian peninsula into saudi arabia and yemen. over 30 mosquito species, mostly aedes and culex are vectors for rvfv [5] . of particular concern is that the aedes species is widely distributed in the eu countries and many of those countries (turkey, greece, italy, spain, portugal, and france) have high-risk vector habitat areas that may serve as emergent sites. moreover, in the unites states, this species has been found in 23 states [7] . since rvfv is capable of utilizing a wide range of mosquito vectors, the virus has the potential to spread further into non-endemic areas [5, 8] . mortality rates are dependent on species and age. in livestock, mortality rates are as high as 30%. mortality rates can reach as high as 95% in newborns and the young, while abortion rates are as high as 100% [5] . symptoms in humans are usually mild and include febrile illness resembling the flu, with a small percentage developing serious clinical manifestations such as retinal lesions, meningoencephalitis, hepatitis, severe hemorrhagic fever, coma and death. in recent years an increase in mortality amongst humans from 2% to 45% has been reported, suggesting evolving mechanisms of virulence and mutations [3] . due to its transmission via aerosolization, high pathogenicity, and classification as a group iii (bioterrorism potential) category a emerging infectious disease by the niaid, work with rvfv requires bsl-3 containment. it is highly suggested that laboratory staff working with rvfv be vaccinated. therefore, diagnosis of rvfv is restricted to a small number of laboratories. this limitation has led to some delay in diagnostics associated with virus isolation and identification techniques that may pose a problem for healthcare authorities in the event of an rvfv epidemic. there is a crucial need for rapid detection and identification of the virus [3, 5] . nanotrap particles are a novel technology that can address all the critical analytical challenges for pathogen identification and measurement. they are homogenous hydrogel particles of about 800 nanometers in size that have a shell made of polymers of nisopropylacrylamide (nipam) and co-monomers such as acrylic acid (aac) and allylamine (aa) with cross links of n,n9methylenebisacrylamide (bis). this shell can be modified to alter permeability or porosity by increasing or decreasing the percentage of bis [9, 10] . charge-based affinity baits are incorporated into the nanotrap particles by copolymerization and covalent binding to the shell [10] . the nanotrap particles are temperature-and ph-sensitive, decreasing in size with increased temperature and low ph. the molecular sieving properties of the particles depend on several aspects. the degree of cross-linking within the particles provides inclusion and exclusion of high abundance large molecules (e.g. albumin). affinity baits further facilitate the capture and concentration of the target protein, and prevent it from exiting the particle. they may be of negative or positive charge, therefore attracting analytes of opposite charge. this was seen in an early experiment performed by luchini et al. where the incubation of particles containing anionic affinity baits captured myoglobin, a protein with a positive charge [9] . some nanotrap particles are composed of nipam shells, and a few of these shelled nanotrap particles are also coated with vinyl sulfonic acid (vsa) [11] . nanotrap particles are able to perform three functions in one step: molecular size sieving, target analyte affinity sequestration, and complete protection of captured analytes from degradation. furthermore, nanotrap particles help to bridge the gap between detection and the limits of sensitivity. mass spectrometry (specifically liquid chromatography coupled with tandem mass spectrometry) is a favored technique for the discovery of candidate biomarkers in biological fluids. however, this technique only accepts a small input volume and complex solutions often lead to decreased sensitivity. the nanotrap particles concentrate protein analytes in small volumes to effectively amplify the sensitivity of mass spectrometry. in addition, their promiscuity allows for multiple analytes to be harvested from a single sample [9] . experiments conducted by luchini et al. demonstrated the capture and enrichment of small molecules spiked in complex solutions such as whole blood and serum [9, 10] . a 2011 study by douglas et al. on the detection of lyme disease demonstrated that nanotrap particles can improve sensitivity more than 100-fold (over existing methods) as well as lead to 100 percent capture and 100 percent elution yield of low abundance antigens in biofluids. lyme disease antigens at low abundance were detected in both urine samples as well as from a single infected tick [12] . the current library of commercially available nanotrap particles has been designed to specifically harvest proteins, peptides, metabolites and small molecules. we hypothesized that nanotrap particles would be able to capture whole virus through the interaction of the nanotrap particle with the positively charged residues on the surface of rvfv. our study demonstrates that nanotrap particles are capable of capturing whole virus, and can be assayed with both qrt-pcr and plaque assays. importantly, serial dilution studies and studies in serum indicate that nanotrap particles increase detection sensitivity at lower viral titers. furthermore, the virus can be inactivated with either heat or detergent, while the virus captured can still be detected with qrt-pcr. importantly, the nanotrap particles protect purified viral rna as well as stabilize the infectivity of rvfv. the studies described here expand upon the nanotrap particles repertoire to characterize the capture of viruses. the nipam/aa nanotrap particles were provided by ceres nanoscience, manassas, va. the vero cell line (kidney epithelial cells) was grown in dulbecco's modified eagle medium (dmem) supplemented with 10% fbs, 1% penicillin/streptomycin, and 1% glutamax there is a dire need for fast and efficient diagnosis of many viral diseases. our research specifically looked at rvfv, a virus that can only be worked with in biosafety level 3 (bsl-3) laboratories, and its capture with nanotrap particles. nanotrap particles are hydrogel particles that contain internal affinity baits. they have previously been used in the capture of several analytes, but never in the capture of whole virus particles. we were not only able to capture and detect rvfv at very low titers from both media and serum, but we were also able to inactivate the virus, which allows for its safe transport to bsl-2 laboratories. while there are other commercially available beads that can also capture virus, nanotrap particles are the only beads that can protect the viral rna from enzymatic degradation. furthermore, we demonstrated that whole virus detection with nanotrap particles is not limited to only rvfv, but that nanotrap particles can be used to detect other viruses such as human immunodeficiency virus (hiv) and venezuelan equine encephalitis virus (veev). (dmem+++). the j1.1 cell line, which are jurkat e6.1 suspension cells chronically infected with the la1 strain of hiv-1, were grown in medium containing advanced rpmi-1640, 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% l-glutamine. all cell lines were cultured in a humidified environment containing 5% co 2 at 37uc. the experiments used a live attenuated vaccine derived from the rvfv zh548 strain, known as mp-12, which had been isolated in 1977 from a patient with uncomplicated rvfv. the virus was generated by 12 serial passages in mrc5 cells, inducing 25 nucleotide changes across the viral genome [13] . both rvfv zh548 and mp12 strains were anonymized. mp12 was propagated by infecting vero cells at 80-90% confluency at an moi of 0.1 in dmem+++. cell culture medium was collected from the cells when ,75% cytopathic effect was observed (typically 72 hours post-infection (hpi)). cell culture medium was centrifuged at 10,000 rpm for 10 minutes to pellet the cellular debris. cell free-viral supernatants were then filtered using a 0.22 mm filter and viral titer determined by plaque assays. screening experiments for venezuelan equine encephalitis virus (veev) used the live attenuated vaccine tc-83, which had been derived from the trinidad donkey (trd) strain by 83 serial passages in fetal guinea pig hearts. this induced changes at 12 nucleotide positions across the viral genome [14] . the viral supernatant of chronically infected j1.1 cells was used in the hiv-1 screening experiments. the lav strain of hiv-1 had previously been used to infected jurkat e6 cells at a multiplicity of infection of 0.1 to 0.01 for 2 hours at 27uc and cultured for two weeks. the cells that survived the cytopathic effects of virus infection were cloned and the supernatant from growth-positive wells were screened for reverse transcriptase (rt) activity [15] . the j1.1 cells express viral rna and proteins at low levels. six commercially available beads -deae-sephadex (sigma-aldrich), dynabeads m-280 streptavidin (invitrogen), sephacryl s-200 beads (ge healthcare), biorex 70 resin (bio-rad laboratories), sp sephadex c-25 (ge healthcare), and bio-gel htp hydroxyapatite (bio-rad laboratories) were used to compare their capture to nt53. each bead was washed four times with water and a 33% percent slurry with water was prepared. according to a protocol standardized by ceres nanoscience, 100 microliters (ml) of sample was incubated with 75 ml of nanotrap particles for 30 minutes at room temperature. the sample was centrifuged at 10,000 rpm for 5 minutes and the supernatant was discarded. the pellet was washed with 100 ml of rnase-and dnase-free water four times. the pellet was then resuspended in the appropriate buffer. for lysis of mp12 with np-40, 1% np-40 was added to 100 ml of mp12 and incubated at room temperature for 30 minutes. a standard nanotrap particle incubation was performed followed by a qrt-pcr assay. for the rnase treatment, purified mp12 rna was treated with rnase a and incubated for one hour at 37uc. vero cells were plated in 6 well plates at 1.0e+06 cells/ml in order to achieve 100% confluency. after nanotrap particle incubation and subsequent washes, the pellet was resuspended in 100 ml of supplemented dmem and serial dilutions performed. four hundred ml of the serial dilution was added to each well in duplicate and incubated for 1 hour. three hundred milliliters (ml) of a primary overlay known as the cv mixture containing equal parts 0.6% agarose in distilled water and media containing 2x emem, 5% fbs, 1% minimum essential amino acids, 1% sodium pyruvate, and 1% glutamax was added directly to each well. the cells were fixed with 10% formaldehyde in water after 72 hpi. the cells were stained with 1% crystal violet in 20% ethanol and water. after two hours, the crystal violet stain was washed off and the plaques formed were counted to determine the plaque forming units per milliliter (pfu/ml). after nanotrap particle incubation and subsequent washes, the pellet was resuspended in 180 ml of lysis/binding solution (life technologies) containing guanidinium thiocyanate and incubated on ice for thirty minutes. the samples were spun at 13,000 rpm for 5 minutes at room temperature. the supernatant was transferred to a 96-well plate and rna extraction was performed with ambion's magmax 96-well viral rna extraction kit according to manufacturer's instructions. in order to determine the number of viral genomic copies produced, qrt-pcr with viral specific primers was performed using rna ultrasense onestep quantitative rt-pcr system (life technologies). the experiment was performed according to a standardized protocol using fifteen ml of master mix containing enzyme mix, 5x reaction mix, 50 mm magnesium sulfate (excluded for veev qrt pcr), rox reference dye, 10 mm taqman fluorogenic probe, 10 mm forward primer (aaaggaacaatg-gactctggtca), and 10 um reverse primer (cacttct-tactaccatgtcctccaat) added to five ml of extracted rna. the samples were heated at 50uc for 15 minutes, 95uc for 2 minutes, and at 95uc and 60uc for 40 cycles. rvfv was spiked into 100% bovine, sheep, and donkey serum (purchased from innovative research) at 1.0e+05 pfu/ml. one ml of spiked serum was used in a standard nanotrap particle incubation with nt53. after a standard nanotrap incubation was performed with nt53 and rvfv, the samples were incubated at room temperature with 0.1, 0.5, or 1% np-40 for 1 hour or heated at 57uc for 0.5, 1, or 2 hours. the samples were then analyzed by plaque assays and qrt pcr. a standard nanotrap particle incubation with one ml of hiv-1 supernatant from infected j1.1 cells and nanotrap particles was performed. an rna extraction was performed (as described above). a master mix was then prepared with the following components for a 25 ml reaction: superscript iii rt/platinum taq mix -0.5 ml, 2x reaction mix with rox -12.5 ml, and 0.5 ml of forward and reverse primer mix (ltr forward primer cgagcttgctacaagggact and ltr reverse primer gagattttccacactgactaaaag) at 10 mm. five ml of sample, 6.5 ml of water, and 13.5 ml of master mix were aliquoted out into pcr tubes. the pcr was conducted with the following cycles: 15 min at 50uc (cdna synthesis), 2 min at 95uc (prime reaction), and 35 cycles at 30 seconds at 95uc (denature), 30 seconds at 51uc (annealing), 30 seconds at 72uc (extend), 72uc for 10 min, and a hold at 4uc. a dna gel was prepared using 1% agarose powder in 1x tae buffer with the addition of ethidium bromide for a final concentration of 0.5 mg/ml. the gel was visualized on an ultraviolet transilluminator and the volume of each band was quantified. nanotrap particles can capture whole virus nanotrap particles have previously been shown to capture proteins. we hypothesized that the rvfv glycoproteins would be figure 1 . rvfv capture by nanotrap particles. a) seven different types of nanotrap particles were incubated with viral supernatants containing rvfv (1e+7 pfu/ml) for 30 minutes at room temperature and washed 4 times with water. viral rna was extracted from the particles with ambion's magmax viral rna extraction kit and quantitated by qrt-pcr assays. b) percent detected virus was calculated compared to a sample processed without nanotrap particle incubation. c) viral supernatants were incubated with nt46, nt53, and nt69 for 30 minutes at room temperature and washed 4 times with water. serial dilutions followed by plaque assays were performed to determine if full virus was bound by the particles. doi:10.1371/journal.pntd.0002296.g001 capable of interacting with the nanotrap particles, facilitating capture in a fashion similar to the way in which protein biomarkers interacted with the nanotrap particles. to test this hypothesis, seven different nanotrap particles were tested with rvfv. four nanotrap particles possessed shells (nt53, nt55, nt69, and nt71) whereas three did not (nt45, nt46, and nt75) ( table 1) . we incubated culture supernatants from rvfvinfected veros with each nanotrap particle. all seven nanotrap particles successfully captured virus, averaging 6.8e+07 genomic copies per reaction. specifically, nt46, nt53, and nt69 captured higher genomic copies than the other nanotrap particles, with each capturing approximately 1.0e+08 genomic copies per reaction ( figure 1a ). this corresponds to 78-83% capture ( figure 1b) of a sample containing a high titer of virus. in order to determine if the amplification observed in the qrt-pcr assay was due to the nanotrap particle capturing intact viral particles or association of viral rna (presumably due to lysed virus) with the particles, plaque assays were performed. if the particles captured viral rna or lysed virus, no plaques should be observed. plaque assays were performed on the three best candidates from the qrt-pcr screening. captured viruses were not eluted off of the nanotrap particles, but rather the samples were diluted and added directly to the vero cells during the plaque assay procedure. we hypothesized that the viral glycoproteins would have a greater affinity for the cellular receptor than the nanotrap particles and thus would enter the cells. nt53, which contains a cibacron blue bait with a shell, captured infectious rvfv virion six-and four-fold more than nt46 or nt69, respectively ( figure 1c ). these plaques were not due to cell death induced by the nanotrap particles themselves, as nanotrap particles alone did not produce plaques. therefore nt53 was chosen for all future experiments with rvfv. experiments were performed to determine potential elution methods that would release the virus without affecting the viral particle integrity. it had previously been found that sodium chloride (nacl) concentrations between 0.5m and 2m could effectively elute various analytes from cibacron blue dyes by disrupting electrostatic interactions between cibacron blue dyes (the bait molecule found within nt53; [16] ). we hypothesized that incubating the rvfv-bound nanotrap particles on ice would allow the particles to swell and, with the aid of vortexing, the virus would disassociate from the nanotrap particles. therefore, we tested a nacl based elution method coupled with an ice-swelling method. plaque assays were performed to determine the amount of virus eluted from the nanotrap particles and the amount that remained bound to the particles after a high salt elution. after nt53 incubation with rvfv, the pellets were resuspended in 2.0 m nacl in dmem and placed on ice for 30 minutes with vortexing every ten minutes. both the eluates and pellets were analyzed by plaque assay ( figure s1 ). the results showed that 5.5% of rvfv was detected after elution with 2.0m nacl. the addition of nacl coupled with incubation on ice only slightly released rvfv virions, demonstrating the virus' strong affinity for the nanotrap particles. however, as seen in figure 1b , rvfv-bound nanotrap pellets directly added to vero cells during the plaque assays procedure were capable of producing plaques. based on these results, we opted not to elute rvfv from the nanotrap particles, but rather to add the rvfv bound to the nanotrap particles directly during the plaque assay procedure. we next wanted to determine the limit of detection of rvfv in plaque assays with nt53. nt53 was incubated with rvfv at decreasing titers, from 2.5e+6 to 2.5e+1 pfu/ml, and plaque assays performed (figure 2a) . captured virus was detected down to 2.5e+1 pfu/ml for rvfv. these results show that nanotrap particles are capable of capturing whole virus even at low viral titers. we next determined the percentage of rvfv captured by nt53 in comparison to the total input amount. rvfv at 1.0e+6 and 1.0e+3 pfu/ml were added to nt53. at 1.0e+6 pfu/ml, 99.35% of the virus was bound to the nanotrap particles whereas at 1.0e+03 pfu/ml, ,100% of the virus appeared bound to the nanotrap particles ( figure 2b ). the results confirm that the nanotrap particles are efficient at capturing rvfv, especially at a lower titer. interestingly, the results also suggest that a small volume of rvfv can be captured with nanotrap particles and then recultured to grow more virus. rvfv detection is more sensitive with nanotrap particle incubation in clinical instances of infection, the viral titers in circulation during very early stages after exposure are expected to be low and therefore, hard to detect [17] . we wanted to determine if viral enrichment by the nanotrap particles (nt53) would enhance detection of rvfv when compared to detection in the absence of enrichment afforded by the nanotrap. we specifically wanted to see the enrichment potential at lower viral titers when detection would be most difficult. for these assays, we chose to utilize qrt-pcr based detection due to its increased sensitivity over plaque assays. to this end, we spiked rvfv into cell culture media that contained 10% fbs at various concentrations from 1.0e+5 to figure 2 . characterization of rvfv nanotrap particle capture. a) viral supernatants were serially diluted (2.5e+6 to 2.5e+1 pfu/ml) and incubated with nt53 for 30 minutes at room temperature. the pellets were washed 4 times with water and then particles were tested in plaque assays to determine if full virus was bound by the particles. b) viral supernatants at 1.0e+6 and 1.0e+3 pfu/ml were incubated with nt53 for 30 minutes at room temperature. the sample was spun at 10,000 rpm for 5 minutes and the unbound viral supernatant was saved separately. nt53 was washed 4 times with water and then particles were tested in plaque assays to determine how much virus were bound verses unbound by the particles. the percentage of bound virus at 1.0e+6 and 1.0e+3 pfu/ml was graphed. doi:10.1371/journal.pntd.0002296.g002 1.0e+1 pfu/ml. nt53 was then added to 1 ml of the spiked media. viral capture with and without nanotrap particles gave similar yields at higher viral titers ( figure 3a) . however, at lower viral titers, there was a significant increase in viral capture with the use of the nanotrap particles compared to samples without nanotrap particle capture. there was greater than a 100-fold increase of viral detection with the use of nt53 at 1.0e+1 pfu/ml. we next wanted to determine if we could capture and enrich virus from a clinically relevant matrix. rvfv was spiked into 100% bovine, donkey, and sheep sera at 1.0e+05 pfu/ml. incubation of rvfv spiked sera with nt53 resulted in enrichment by 13-, 3-, and 52-fold for bovine, sheep, and donkey sera, respectively ( figure 3b ). these results demonstrate that nt53 not only captures but also enriches virus found in complicated matrices such as animal sera. the complex analytes (e.g. albumin) found in the sera are likely excluded by the nanotrap particles and do not interfere with whole virus capture. however, we speculate that since the serum from each of the three animals contains different analytes, there may be interfering proteins that would lead to the observed enrichment differences. nanotrap particles are capable of capturing inactivated rvfv viral inactivation is crucial for its transport from the field or a bsl-3 facility to a bsl-2 environment for downstream analysis. however, after inactivation the virus may be susceptible to degradation. therefore, we wanted to determine if rvfv would remain bound to the nanotrap particles in an inactivation scenario. after nanotrap particle incubation with rvfv and subsequent washes, np-40 detergent was used to inactivate the virus (figure 4 ). plaque assays were performed to confirm viral inactivation. plaque assays demonstrated that 0.1% np-40 did not fully inactivate the virus incubated with or without nt53 ( figure 4a ). higher concentrations of np-40 (0.5% and 1%) fully inactivated rvfv in the presence or absence of nt53. while the plaque assays confirmed inactivity of rvfv, qrt-pcr data demonstrated that rvfv was still captured following np-40 addition ( figures 4b). in the presence of np-40 the levels of capture with nt53 were decreased as compared to the controls. this is likely due to the interference of the nanotrap particle binding to rvfv due to the presence of detergent. we next tested the ability of nt53 to function in another commonly employed viral inactivation procedure. the samples were heat inactivated at 57uc for three different time pointsthirty minutes, one hour, and two hours -and plaque assays were performed to confirm viral inactivation. at thirty minutes approximately 1.0e+4 and 1.0e+2 pfu/ml of rvfv with and without nt53, respectively, were still detectable. interestingly, rvfv was more resistant to heat inactivation in the presence of nt53, suggesting the nanotrap particles may have a slight protective effect on the virus. however, complete inactivation was achieved at one hour ( figure 4c ). while the plaque assays confirmed inactivation of rvfv, qrt-pcr data demonstrated the ability of nt53 to detect rvfv nucleic acids after heat inactivation ( figure 4d ). these data demonstrate that it is possible to fully inactivate the virus before applying downstream assays such as qrt-pcr. furthermore, these two experiments demonstrate the ability to inactivate a sample and transport it as a noninfectious sample, while still retaining capture. nanotrap particles are capable of capturing and protecting viral rna as we observed viral capture in the presence of np-40, we hypothesized that the virus was being lysed and the released viral rna recaptured with the nanotrap particles. if the nanotrap particles were not providing protection of the viral rna, there will be no possibility for any downstream assays using inactivated material, which is a critical step in diagnostics. to test this hypothesis, we first lysed the virus with 1% np-40 and followed by adding nt53 to the lysed material. results in figure 5a indicate that nt53 was able to capture the lysed virus. however, there was a 100-fold decrease in lysed virus with the addition of nt53 compared to the control (no nt53) with and without np-40. these results mirrored what was observed in figure 4b , where nt53 was capable of capturing virus to a lesser extent in the presence of np-40. these results demonstrated that rvfv could be initially inactivated by traditional inactivation methods and then captured with nanotrap particles. by incubating with the nanotrap particles, the viral rna will be protected and hence, can be used for downstream rna detection. in order to directly show that nt53 was able to capture and protect viral rna, we performed a nanotrap experiment with purified viral rna. we first incubated nanotrap particles with purified rna, and performed qrt-pcr assays. results in figure 3 . rvfv enrichment with nt53 incubation. a) rvfv was spiked into cell culture media (dmem+++) at various concentrations from 1.0e+5 to 1.0e+1 pfu/ml. nt53 was added to 1 ml of media and captured according to standardize protocols. the pellet was washed 4 times with water, followed by processing with ambion's magmax 96well viral rna extraction kit. rvfv-spiked media without nt53 were processed in parallel. viral rna was quantitated by qrt-pcr with viral specific primers. b) rvfv was spiked into 100% bovine, sheep, and donkey serum at 1.0e+5 pfu/ml. nt53 were added to 1 ml of serum and captured according to a standardize protocol. the pellet was washed 4 times, followed by processing with ambion's magmax 96-well viral rna extraction kit. serum without nt53 was processed in parallel. viral rna was quantitated by qrt-pcr. doi:10.1371/journal.pntd.0002296.g003 figure 5b demonstrate that nt53 is capable of capturing purified viral rna, albeit with less affinity than whole virus capture. nt53 was able to capture 0.01% of the input viral rna. while we screened the nanotrap particles for whole virus capture, we did not screen the nanotrap particles for rvfv viral rna capture. there is likely a nanotrap particle that captures viral rna with greater efficiency than nt53. as previous studies have shown that proteins captured by nanotrap particles were protected from trypsin degradation, we next aimed to determine if nanotrap particle capture could protect viral rna from rnase degradation [9, 10] . samples with and without nt53 were treated with rnase a at 140 or 1400 units/ml and incubated for one hour at 37uc. interestingly, the rna incubated with nt53 was protected from rnase a degradation, whereas the rna controls were subject to complete rnase a degradation ( figure 5b ). at 140 units/ml of rnase a, the captured rna was detected at the same level as the rnaseuntreated sample. even at a substantially higher rnase concentration (1400 units/ml), 1% of the viral rna input was still detected. our results demonstrate that the nanotrap particles are capable of capturing and protecting viral rna from enzymatic degradation. in some situations, it may be important to retain the infectivity of the captured virus to enable the virus to be propagated for further characterization. therefore, we evaluated the ability of the nanotrap particles to capture and preserve the infectivity of rvfv following capture. rvfv was spiked into bovine serum and incubated with or without nt53 at 25uc for 48 or 72 h. in the absence of nt53, the infectivity of rvfv was decreased by ,3 logs ( figure 5c ). in contrast, samples incubated with nt53 displayed only ,1.5 log decrease by 48 h. although ,3.5 log decrease was observed with nt53 at 72 h, this still resulted in increased virus detected as compared to the control samples due to the enrichment afforded by nt53. the infectivity of rvfv was also assessed for samples that were incubated at 37uc, which would likely result in a more rapid decline in viral infectivity and thus 24, 48, and 72 h time points were examined. as suspected, a ,4 log decrease was observed in samples incubated at 37uc for 24 h without nt53. in contrast, samples captured by nt53 only displayed ,2 log decrease as compared to the control nt53 sample. at extended time points a further decrease in infectivity was observed with and without nt53, but in all cases a higher amount of infectious virus could be rescued from samples . viral inactivation following nt53 capture. panels a and b: nt53 was incubated with viral supernatants containing rvfv at 2.7e+8 pfu/ml for 30 minutes at room temperature. samples were then incubated at room temperature with 0.1, 0.5 or 1% np-40 for 1 hour (np-40). control samples were not treated with np-40 nor captured with nt53. samples treated with np-40 without nt53 were processed in parallel (black bars). viral inactivation was assayed by plaque assays (a) and viral rna was extracted from the particles with ambion's magmax 96-well viral rna extraction kit and quantitated by qrt-pcr (b). panels c and d: nt53 was incubated with viral supernatants containing rvfv at 2.7e+8 pfu/ml for 30 minutes at room temperature. samples were then incubated at 57uc for 30 minutes, one hour, or two hours. control samples were not heat treated nor captured with nt53. samples heat-treated without nt53 were processed in parallel (black bars). viral inactivation was assayed by plaque assays (c) and viral rna was extracted from the particles with ambion's magmax 96-well viral rna extraction kit and quantitated by qrt-pcr (d comparison of capture efficacy of nanotrap particles and commercially available beads for rvfv capture nanotrap particles have unique properties not demonstrated in other beads that are used for protein purification and albumin exclusion such as dye baits that make them an ideal candidate in virus capture. therefore, we wanted to directly compare the ability of other beads to capture rvfv with nt53's rvfv capture capability. the capture of rvfv was tested with nt53 and six commercially available beads used in various assays. deae-sephadex beads are used in ion exchange chromatography for purifying and isolating proteins; dynabeads m-280 streptavidin are used for isolating nucleic acids and antibodies; sephacryl s-200 beads are used to purify protein and macromolecules; biorex 70 resin beads are used for purification and fractionation of peptides, proteins, and other cationic molecules; sp sephadex c-25 beads are used in chromatography to separate and purify protein, polypeptides, and other charged molecules; and bio-gel htp hydroxyapatite beads are used in chromatography to separate and purify proteins, nucleic acids, viruses, and other macromolecules. these calcium phosphate beads work by the cationic interaction of the ca 2+ functional groups with the carboxylate residues located on the protein surface and the anionic interaction of the po 4 22 functional groups with the basic protein residues. rvfv was incubated with each of these beads and plaque assays were performed to determine whole virus capture. bio-gel htp hydroxyapatite (htp) captured rvfv the most efficiently, averaging 1.0e+7 pfu/ml, while nt53 performed the second best averaging 2.5e+6 pfu/ml ( figure 6a ). the other four beads captured rvfv around or below 1.0e+5 pfu/ml. as we have demonstrated that nt53 not only captures intact rvfv, but can also capture and protect viral rna from rnase a degradation, we tested the ability of htp to act in a similar capacity. nt53 was able to fully protect the viral rna against rnase a degradation and genomic copies for nt53 with and without nt53 treatment were similar. however, htp beads were figure 5 . nt53 protects viral rna from degradation and preserves viral infectivity. a) rvfv was lysed with 1% np-40 for 30 minutes at room temperature and then incubated with nt53 for 30 minutes at room temperature. the viral rna was extracted from the particles with ambion's magmax 96-well viral rna extraction kit and quantitated by qrt-pcr. samples without nt53 and samples without np-40 were processed in parallel. b) nt53 was incubated with purified rna at 2.0e+8 genomic copies for 30 minutes at room temperature. following water washes, the samples were resuspended in water and treated with 140 or 1400 units/ml of rnase a. samples with no rnase a treatment were processed in parallel. the viral rna was extracted from the particles with ambion's magmax 96-well viral rna extraction kit and quantitated by qrt-pcr. samples without nt53 were processed in parallel (black bars). c) rvfv (1.4+e7 pfu/ml) was spiked into 1 ml of bovine serum, nt53 added, and samples incubated at 25uc (for 48 or 72 hr) or 37uc (for 24, 48, or 72 h). samples without nt53 were processed in parallel. samples were assayed for viral infectivity by plaque assay. control samples are samples that were processed immediately for plaque assays with no incubation period. doi:10.1371/journal.pntd.0002296.g005 unable to provide protection against rnase degradation, and no viral rna was detected ( figure 6b) . a control experiment with rna alone demonstrated that our rnase treatment was effective. we next compared the ability of nt53 and htp to capture rvfv during an inactivation scenario. for these experiments nt53 or htp were added to the samples followed by viral inactivation through treatment with 1% np-40 or heating at 57uc. the amount of virus captured was quantitated by qrt-pcr ( figure 6c ). as was observed in previous experiments, viral inactivation with either np-40 or heat treatment resulted in some loss of rvfv binding to the nanotrap (1.2 and 0.8 log, respectively). however, htp rvfv capture was more dramatically affected, resulting in a 2.5 log decrease with the np-40 treated samples and a 2.7 log decrease in the heat inactivated samples. collectively, our experiments demonstrate that while htp is capable of capturing whole virus, it cannot protect viral rna against rnase degradation and it displays a reduced ability to capture rvfv during an inactivation scenario. in contrast, nt53 is capable of capturing and protecting rvfv as well as capturing rvfv in samples that have been inactivated by heat or detergent treatment. we next asked the question if nanotrap particles were capable of capturing other viruses. for this, we selected veev and hiv-1. veev, which at approximately 70 nm in diameter is a smaller virus than rvfv, which is approximately 100 nm in diameter. veev viral supernatants were incubated with various nanotrap particles shown in table 1 and capture was measured by qrt-pcr. our data indicated that all six nanotrap particles successfully captured veev, averaging 9.9e+06 genomic copies per reaction ( figure 7a) , with a slight preference observed with nt45, nt46, and nt55 capturing 1.3e+07, 1.1e+07, and 1.1e+07 genomic copies per reaction, respectively. nanotrap particles capture was also tested using hiv-1. hiv-1 supernatants from infected j1.1 cells were incubated with nanotrap particles. rna extraction was performed, cdna was synthesized, and rt-pcr was performed. a semi-quantitative analysis shown in figure 7b , demonstrated that all seven nanotrap particles were able to capture hiv-1 with nt46 and nt53 demonstrating the best capture ( figure 7b ). these results indicate that nanotrap particles are capable of capturing multiple viruses. (7) for 30 minutes at room temperature. the sample was washed 4 times with water and then particles were tested in plaque assays to determine how much virus was bound by the particles. b) nt53 or htp was incubated with purified rna at 1.0e+7 genomic copies for 30 minutes at room temperature. following water washes, the samples were resuspended in water and treated with 380 units/ml of rnase a. samples with no rnase a treatment were processed in parallel. the viral rna was extracted from isolated particles and quantitated by qrt-pcr (black bars). samples without nt53 were processed in parallel (gray bars). c) nt53 and htp beads were incubated with viral supernatants containing rvfv at 1.7e+8 pfu/ml for 30 minutes at room temperature. samples were then inactivated by incubation at 57uc for one hour or incubating in the presence of 1% np-40 at room temperature for 1 hour. a ''no bead'' control processed in parallel was included for each condition. viral rna was extracted from the particles and quantitated by qrt-pcr. doi:10.1371/journal.pntd.0002296.g006 viral infections in nature do not occur in isolation and are often accompanied by other co-infections (bacterial and/or viral); therefore we sought to determine if the nanotrap particles could capture rvfv in a ''mixed'' infection setting. to this end, bovine serum was spiked with rvfv only or with both rvfv and hiv, followed by nt53 viral capture and quantification as measured by qrt-pcr. results indicated that nt53 was capable of capturing and enriching rvfv from samples that contained only rvfv or both rvfv and hiv ( figure 7c ). seven-fold enrichment was observed in samples containing rvfv only and 5-fold enrichment from samples containing both rvfv and hiv. these data provide evidence that the nanotrap particles could be used with clinical samples. therefore, in conclusion, the results demonstrate that nano-trap particles can capture and enrich rvfv from both cell culture media and clinically relevant matrices. the captured virus can then be inactivated and viral rna protected from enzymatic degradation. the bound rvfv can be eluted off the nanotrap particles, and used in downstream assays such as plaque assays and qrt-pcr. furthermore, nanotrap capture can be extended to other viruses as well, including veev and hiv. rift valley fever virus is a zoonotic virus that primarily affects livestock but has the potential to cause severe disease in humans. rvfv has led to outbreaks in egypt and the arabian peninsula with the potential to spread to the united states and europe. changes in climate, travel, and trade have made rvfv an emerging disease that can have deadly economic and social consequences. furthermore, rvfv is of biodefense interest due to its potential spread via aerosolization. there are currently no fda-approved vaccines, so there is a reliance on sensitive and specific diagnostics early on in infection. the current state of rvfv diagnostics includes virus isolation, nucleic acid techniques, and antibody detection. current rt-pcr-based assays require a critical amount of the virus circulating in the system. this can lead to misdiagnosis, especially falsenegative results, of the disease early on in infection. in contrast, our results demonstrate the ability of nanotrap particles to enrich for rvfv from both cell culture supernatants as well as more complex matrices such as animal serum. the capability of nanotrap particles to enrich virus is crucial early on in infection during which the virus can go undetected using other diagnostic methods. in our serum sample studies we noted different levels of enrichment depending on the source of the serum. for example, nanotrap particles incubated in donkey serum resulted in a 52fold increase in rvfv detection sensitivity, whereas incubation in sheep serum only displayed a 3-fold increase. the presence of other analytes found in serum may be competing for capture with nanotrap particles, which likely will differ between species as well figure 7 . capture of other viruses with nanotrap particles. a) six different types of nanotrap particles were incubated with viral supernatants containing veev for 30 minutes at room temperature and washed 4 times with water. viral rna was extracted and quantitated by qrt-pcr assays. b) seven different types of nanotrap particles were incubated with 1 ml j1.1 supernatant for 30 minutes at room temperature and washed 4 times with water. the pellets were diluted in 100 ul water and rna extracted. a cdna synthesis using 150 ng of each sample was performed and followed by pcr using 10 ul cdna. a dna gel was run to determine viral capture. a sample with no reverse transcriptase added and a sample with just water were used as negative controls. the volume of each band was quantified and graphed. c) bovine serum was spiked with rvfv (1.0e+6 pfu/ml) only or both rvfv (1.0e+6 pfu/ml) and hiv (100 ml of j.1. supernatants). samples were incubated with nt53 for 30 minutes, viral rna extracted and quantitated by qrt-pcr. samples without nt53 were processed in parallel (black bars). doi:10.1371/journal.pntd.0002296.g007 as between individual animals. importantly we have also demonstrated the ability to capture rvfv in samples that also contained hiv. this is an important area of investigation, as clinical samples will likely contain multiple pathogens, providing further competition for nanotrap binding. we hope to extend our spiked serum sample studies to experiments with serum samples taken from animals exposed to rvfv and human clinical samples. these studies will allow further optimization of the nanotrap particle collection. in our current study we used very stringent wash conditions to ensure that the virus captured was tightly bound to the nanotrap particles. in clinical samples, it may be necessary to decrease the number of wash steps to ensure that the maximum amount of virus is being captured from more complex samples. alternatively, different wash buffers (altering salt and detergent concentrations) could be utilized to allow more selective binding of analytes. due to the complex nature of clinical samples, it may also be necessary to increase the amount of nanotrap particles added to prevent saturation. nonetheless, our studies provide an important first step in the application of nanotrap particles as a sample preparation and enrichment process to improve diagnostics from serum samples. one important advantage of utilizing nanotrap particles is their ability to protect analytes from degradation. previous studies have indicated that protein captured by nanotrap particles are protected from trypsin degradation [9, 10] . in these experiments pdgf was incubated with an excess of trypsin. following incubation the majority of the trypsin was found outside of the nanotrap particles. however, even though some of the trypsin entered the particles, pdgf was completely protected from degradation. in the current study we extend these findings to demonstrate that viral rna was protected from degradation in the presence of rnase a. sample preservation is critical for stabilization of sample integrity both during field collection and during transported to diagnostic facilities. bio-gel htp hydroxyapitite, while able to capture rvfv was unable to protect viral rna from degradation, further demonstrating the advantage of using nanotrap particles over other commercially available chromatography beads. another critical aspect of the nanotrap particles is the ability to collect viral samples and inactivate them to render them noninfectious, while still retaining the ability to detect the analyte of interest. this was demonstrated by captured of rvfv by nt53 followed by inactivation of rvfv with np-40 (determined by plaque assays). following inactivation, viral genetic material was still detected with qrt pcr and to a higher level than that observed with htp beads. this is of particular importance in the transport of rvfv from a bsl-3 environment or field sample collection setting to a bsl-2 laboratory for diagnostic testing. given that bsl-3 laboratories are both difficult to access and work in a bsl-3 environment is time-consuming and expensive, the inactivation method will allow for fewer lapses in time between obtaining the samples and the determining the results. in addition, as the nanotrap particles allow the capture of the whole virus, the samples can then be analyzed with a variety of downstream analysis methods such as elisa for the nucleoprotein of rvfv, western blotting, plaque assays, and qrt-pcr. the exact mechanism of nanotrap binding to rvfv is unclear at this point. we hypothesize the nanotrap particle capture is occurring through interactions with rvfv's glycoproteins, gn and gc. gn and gc are the only viral proteins available for capture by virtue of being exposed on the outside of the virion. the fact that the binding observed with htp beads slightly exceeded nt53 in binding rvfv may provide insight into the mechanism of binding. htp is known to bind primarily through electrostatic interactions and similarly, cibacron blue, the affinity bait component of nt53 binds through electrostatic, hydrophobic or a combination of surface and electrostatic interactions. based on these results, we expect that electrostatic interactions may provide the dominant mode of nt53 binding to rvfv. due to the size of the virus (90-100 nm) as compared to the size of the nanotrap particles (800 nm), it is unclear if the viruses are entering inside the core of the nanotrap particle or binding to the outside of the nanotrap particles. we have observed preferentially binding of rvfv with nanotrap particles containing cibracon blue baits, suggesting that the bait plays at least a partial role in the binding. even if rvfv binding is partially or primarily found on the surface of the nanotrap particles, the particles provide a unique advantage over other commercially available beads, which is sequestration of analytes within the nanotrap particles. this is important as many enzymes (proteases, rnase,etc.) found in serum can rapidly digest protein and rna. however, the nanotrap particles can bind to small molecular weight proteins (such as trypsin) rendering them inactive [9] , thereby providing protection for other proteins and/or viruses captured by the nanotrap particles. nanotrap particles can be engineered with increased pore sizes to facilitate capture of rvfv inside the nanotrap particles. this approach has the added advantage of ensuring capture within the particles themselves, which would be predicted to further increase the stability of the virus as well as increase the viral binding capacity of the nanotrap particles. one potential disadvantage of larger pores sizes would be the loss of some of the sieve sieving capabilities of the particles. the size sieving is important for more complex samples (whole blood, sera, etc.), which would benefit from the ability of the nanotrap particles to enrich for certain analytes (i.e. viruses) while excluding high abundant proteins such as bsa, which may interfere with downstream assays or mask lower abundant molecules such as low levels of viruses. however, it may be possible to obtain a balance of larger pores with efficient size sieving if the appropriate level of cross-linking could be achieved. this is an active area of research and warrants further investigation. our results demonstrated that nanotrap particle capture was not limited to rvfv, but could be extended to other viruses including veev and hiv. all three of these viruses are enveloped rna viruses. future studies will focus on the capture and enrichment of different viral classes, including dna viruses and non-enveloped viruses. that capture of a wide range of viruses is especially important when multiple viruses cause the same type of disease and/or the symptoms of infection are very general. for example, respiratory infections can be attributed to multiple pathogens, including influenza a and b viruses, coronaviruses, and adenoviruses. for this type of application, the promiscuity of the nanotrap particles is particularly important, as it will allow the capture and enrichment of multiple viruses from the same sample. therefore, we believe that increasing sensitivity for respiratory viral infections is an important diagnostic issue that the nanotrap particles could address. in conclusion, our results have demonstrated that nanotrap particles are able to capture and enrich whole virus. while other commercially available beads can also capture virus, only nano-trap particles are capable of protecting the integrity of the virus after inactivation with detergent or exposure to rnase a. however, further research is needed to determine the exact mechanism by which the nanotrap particles capture and protect the virus. figure s1 elution with nacl as demonstrated by plaque assays. rvfv supernatants were incubated with nt53 for 30 minutes at room temperature. the sample was spun at 10,000 rpm for 5 minutes. nt53 was washed 4 times with water and then particles were eluted with dmem+2m nacl and incubated on ice for 30 minutes with vortexing every 10 minutes. the particles were tested in plaque assays to determine how much virus was eluted off of the particles. (tif) rift valley fever in rural northern senegal: human risk factors and potential vectors nonstructural nss protein of rift valley fever virus interacts with pericentromeric dna sequences of the host cell, inducing chromosome cohesion and segregation defects rift valley fever virus(bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention molecular biology of rift valley fever virus an inhibition enzyme-linked immunosorbent assay for the detection of antibody to rift valley fever virus in humans, domestic and wild ruminants rift valley fever virus infection in african buffalo (syncerus caffer) herds in rural south africa: evidence of interepidemic transmission identification and geographical distribution of the mosquitos of north america, north of mexico the pathogenesis of rift valley fever smart hydrogel particles: biomarker harvesting: one-step affinity purification, size exclusion, and protection against degradation nanoparticle technology: addressing the fundamental roadblocks to protein biomarker discovery multifunctional core-shell nanoparticles: discovery of previously invisible biomarkers the use of hydrogel microparticles to sequester and concentrate bacterial antigens in a urine test for lyme disease mapping of the mutations present in the genome of the rift valley fever virus attenuated mp12 strain and their putative role in attenuation attenuation of venezuelan equine encephalitis virus strain tc-83 is encoded by the 59-noncoding region and the e2 envelope glycoprotein an hiv-1-infected t cell clone defective in il-2 production and ca2+ mobilization after cd3 stimulation human serum albumin chromatography by cibacron blue f3ga-derived microporous polyamide hollow-fiber affinity membranes core-shell hydrogel particles harvest, concentrate and preserve labile low abundance biomarkers the authors thank dr. sina bavari (usamriid) for providing rvfv mp-12. the authors thank dr. cynthia de la fuente for critical reading of the manuscript. the following reagent was obtained through the nih biodefense and emerging infections research resources repository, niaid, nih: venezuelan equine encephalitis virus tc-83 (subtype ia/b), nr-63. conceived and designed the experiments: an fk cb bl kkh. performed the experiments: ns ab cp kf. analyzed the data: ns an kkh. contributed reagents/materials/analysis tools: bl. wrote the paper: ns an kkh. key: cord-352771-s0hfsxzb authors: barriga, gonzalo p.; villalón-letelier, fernando; márquez, chantal l.; bignon, eduardo a.; acuña, rodrigo; ross, breyan h.; monasterio, octavio; mardones, gonzalo a.; vidal, simon e.; tischler, nicole d. title: inhibition of the hantavirus fusion process by predicted domain iii and stem peptides from glycoprotein gc date: 2016-07-14 journal: plos negl trop dis doi: 10.1371/journal.pntd.0004799 sha: doc_id: 352771 cord_uid: s0hfsxzb hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. to enter cells, hantaviruses fuse their envelope membrane with host cell membranes. previously, we have shown that the gc envelope glycoprotein is the viral fusion protein sharing characteristics with class ii fusion proteins. the ectodomain of class ii fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. these fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain iii (diii) and the stem region. such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. based on our previous homology model structure for gc from andes hantavirus (andv), here we predicted and generated recombinant diii and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. recombinant andv diii was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. using diii and the c-terminal part of the stem region, the infection of cells by andv was blocked up to 60% when fusion of andv occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. furthermore, the fragments impaired andv glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from puumala virus (puuv). the gc fragments interfered in andv cell entry by preventing membrane hemifusion and pore formation, retaining gc in a non-resistant homotrimer stage, as described for diii and stem peptide inhibitors of class ii fusion proteins. collectively, our results demonstrate that hantavirus gc shares not only structural, but also mechanistic similarity with class ii viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses. stage, as described for diii and stem peptide inhibitors of class ii fusion proteins. collectively, our results demonstrate that hantavirus gc shares not only structural, but also mechanistic similarity with class ii viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses. the genus hantavirus of the family bunyaviridae comprises diverse viruses that are highly pathogenic to humans. in asia and europe the hantaan, seoul and puuv viruses cause hemorrhagic fever with renal syndrome, while in america andv and sin nombre virus can lead to hantavirus pulmonary syndrome with mortality rates above 30% [1] [2] [3] [4] [5] . hantaviruses are currently the most lethal human pathogenic viruses known to occur in america and, due to the lack of food and drug administration (fda)-approved preventive or therapeutic measures [6, 7] , they have been classified as category a pathogens. like other members of the bunyaviridae family, hantaviruses have a tri-segmented single strand rna genome of negative polarity packaged by the nucleoprotein into viral ribonucleocapsids, which are also associated to the viral rna-dependent rna polymerase [8] . a lipid membrane, which further envelopes the viral ribonucleocapsids, anchors the viral gn and gc glycoproteins. this viral envelope is derived from a host cell membrane during the budding process of the virus [9, 10] . to infect new cells, hantaviruses bind to cell surface receptors [11] [12] [13] [14] , and are subsequently taken up by endocytosis [15, 16] . a crucial step in viral cell entry is the fusion of the virus with an endosomal membrane of the host, escaping from its degradation in lysosomes. yet, little is known about the fusion process of hantaviruses; however, our recent data show that the low ph of endosomes triggers a non-reversible fusion process, in which the gc protein inserts into target membranes and forms a highly stable post-fusion homotrimer [17] . in general, virus-cell membrane fusion is thought to be accomplished by multiple steps [reviewed in 18, 19] . after the activation, viral fusion proteins insert a fusion peptide or fusion loop into a target membrane. at this intermediate stage, the fusion peptide is located at one end of the fusion protein while the opposite end is anchored to the viral envelope membrane by a transmembrane region, thereby bridging the viral and cellular membranes. by undergoing additional conformational changes, the fusion protein is thought to pull both anchors together, until it reaches a hairpin-like conformation in which both membrane-inserted domains are located at the same end of the protein. once the opposed membranes have been brought into a close distance by the introduction of local membrane curvature, the fusion of the outer leaflets of the membranes produces a hemifusion intermediate, followed by the full fusion of the membranes. the fusion culminates in the formation of a fusion pore through which the virus can deliver its ribonucleocapsids into the cell cytosol to initiate replication. viral fusion proteins are currently grouped into at least three different classes based on their molecular structures: class i fusion proteins have a high alpha helical content, class ii proteins consist principally of beta sheets, while class iii fusion proteins include characteristics from the first two classes [reviewed in [19] [20] [21] . early in silico and in vitro analyses suggested that the gc glycoprotein of hantaviruses shares structural similarity with class ii fusion proteins [22, 23] . this notion has also been proposed for other members of the bunyaviridae [24] [25] [26] [27] [28] , and the crystal structure of gc from rift valley fever virus (rvfv) ultimately confirmed this notion for phleboviruses [29] . class ii fusion proteins are composed of three domains (i-iii) and a stem region that connects the ectodomain to the transmembrane anchor [30] [31] [32] [33] [34] . to adopt a hairpin-like structure, diii moves towards the fusion loop [35, 36] , while the stem region, which is connected to the transmembrane anchor, is thought to follow the movement of diii by folding against the trimeric core formed by the fusion protein [37] [38] [39] . the extensive conformational changes that occur during the fusion process offer opportunities to disrupt the fusion cascade, thereby blocking viral infection. ligands that bind selectively to an intermediate form of the fusion protein preceding its post-fusion conformation can delay or inhibit viral entry. in the case of human immunodeficiency virus 1, which has a fusion protein with a class i fold, there is a licensed drug based on a 20-residue peptide [reviewed in 40, 41] . this peptide comprises a partial sequence of the outer layer of the trimeric post-fusion hairpin conformation of gp41 and binds to the trimeric core of the fusion protein in its extended intermediate conformation, preventing the foldback reaction [reviewed in 42, 43] . among class ii proteins, diii and the stem region form the outer layer of the trimeric post-fusion conformation [35] [36] [37] . liao & kielian (2005) showed that the addition of soluble diii with or without the stem region of semliki forest virus e1 protein and soluble diii of dengue virus type 2 (dv2) e protein inhibit the entry of the respective virus into cells and confirmed a common inhibitory mechanism of class i and class ii fusion proteins [44] . other studies have shown that peptides derived from the stem region of the fusion protein of flavi-and phleboviruses also inhibit viral cell entry [45, 46] . the binding of stem peptides to fusion proteins is thought to prevent the post-fusion conformation as in the case of diii; however, their amphipathic characteristics seem to allow binding to the virus before attachment to the cell, and are hence thought to be carried on the virus into endosomes [47, 48] . this characteristic provides an advantage for the delivery of the inhibitor to the site of virus-cell membrane fusion when this process occurs in a closed endosomal compartment. here, we hypothesized that if hantavirus gc shares mechanistic similarity with class ii fusion proteins, then it should be inhibited with strategies used for other class ii fusion proteins. to test this hypothesis, we predicted and produced diii and the stem region of andv gc and assessed them for andv inhibition. our results show that indeed both, recombinant diii and synthetic stem peptides, interfered with the andv infection, acting at late stages of the andv fusion process. for the pcr amplification of the predicted diii and diiis sequences we used the cloned cdna of the m segment from andv isolate chi-7913 [49] and from puuv strain k27 cloned into pwrg/puuv-m(s2) expression plasmid [50] (kindly provided by jay hooper, usamriid, usa), genbank accession numbers aao86638 and l08754, respectively. the pcr products were cloned into pet28a, which gave rise to fusion proteins with an n-terminal tag of 34 residues including polyhistidine (his-tag). for the preparation of diii without the his-tag, the pcr product of andv diii was cloned into pgst-parallel-1 [51] . the expression product of this plasmid contained an n-terminal tag of 314 residues including a glutathione s transferase (gst) affinity tag followed by a cleavage site for the tobacco etch virus (tev) protease. after cleavage with tev protease, 7 residues from the gst-fusion protein remained fused to the n-terminal of diii, corresponding to the sequence gamdpef. the expression of recombinant diii proteins was performed as established before [52] . in brief, his-tagged fusion proteins were expressed in escherichia coli (e.coli) bl21, which was transformed with the different pet28a/diii plasmids. the isopropyl-beta-d-thiogalactopyranosideinduced bacteria were lysed by sonication in buffer containing 20 mm tris, 50 mm nacl, and 0.2 mm phenylmethanesulfonylfluoride, ph 8. lysis was performed by sonication on ice and soluble proteins were purified through the ion exchange resin bio-rex 70 (bio-rad laboratories) followed by tandem ultrafiltration on devices with 30 kda and 3 kda of molecular weight cut-off. recombinant, untagged diii was produced following a similar purification procedure described elsewhere [53] , with some modifications. briefly, e. coli origami 2(de3) (novagen), were transformed with pgst-parallel-1/diii, and expression was induced, and bacteria were pelleted and lysed as described above. the clarified supernatant was purified on glutathione-sepharose 4b (ge healthcare), and the bound protein was eluted with 20 mm reduced glutathione (sigma-aldrich). his6-tagged tev protease was used to cleave off the gst moiety, and the gst moiety and the tev protease were removed by sequential passage through glutathione-sepharose 4b and ni-nta (qiagen) resins. eventually, diii was concentrated using an ultrafiltration device with 3 kda cutoff, and further purified on a hiload 16/60 superdex 200 prep grade column (ge healthcare). proteins were analyzed by standard tris-glycine sds-polyacrylamide gel electrophoresis using 4-12% or 15% gels. for irreversible reduction, 8 mm dithiothreitol was added, the proteins were heated to 95°c for 3 min, and subsequently alkylated by incubation with 24 mm iodoacetamide for 15 min at 37°c. circular dichroism measurements were performed using a spectropolarimeter (jasco-j600), and 5 scans were recorded at room temperature between 190 and 260 nm, using a 1 mm optical pass cuvette. measured values of ellipticity were converted into ellipticity per amino acid residue. for deconvolution of the spectra, different databases of contin and cdsstr servers [54] [55] [56] were used. peptides spanning the predicted stem region of andv gc were synthesized (new england peptides). the n-terminal r1 peptide comprised the sequence hlervtgfnqidsdkvydd gapp, and the c-terminal r2 peptide comprised the sequence tfkcwftksgewllgiln gn. two additional peptides were used, r2.1 and r2.2, comprising either the first ten residues or the last ten residues of the r2 peptide, respectively. to avoid the introduction of additional charges, the c-terminal of r2.1 was amide-modified, and the n-terminal of r.2.2 was acidmodified, comprising the sequences tfkcwftksg and ewllgilngn, respectively. the nn peptide comprising the sequence qlvtarqklkdaekavevdpddvnkstlqrraav stletklg, derived from the andv nucleoprotein, was used as control. such nucleoprotein-based peptide was chosen to limit the possibility of inter-or intramolecular interactions with the fusion protein that may occur during its conformational changes in the fusion process. this nn peptide of 42 residues was used as control of both short r peptides and longer diii fragments. all peptides were soluble in hne buffer (10 mm hepes, 130 mm nacl, 0,1 mm edta, ph 7.4), except peptides r2 and r2.2, which were dissolved in 10 mm borate buffer (ph 9). andv isolate chi-7913 (kindly provided by héctor galeno, instituto de salud pública, chile) was propagated in vero e6 cells (attc) as described before [57] . all work involving the infectious virus was performed under biosafety level 3 conditions (centro de investigaciones médicas, pontificia universidad católica de chile, chile). 293ft cells (invitrogen) were propagated in dmem supplemented with 10% fetal calf serum (fcs). cho-k1 cells (attc) were grown in f12-k medium containing 10% fcs. the infection of vero e6 cells by andv (multiplicity of infection (moi) = 0.1) was quantified by flow cytometry as formerly established [57] . briefly, cells were incubated with andv for 1 h at 37°c in the presence and absence of protein or peptide inhibitor candidates. subsequently, cells were washed and infection was allowed to proceed for 16 h. cells were next fixed with 2% (w/v) paraformaldehyde for virus inactivation, and permeabilized with 0.1% triton x-100. for immunofluorescence staining, cells were incubated for 45 min with anti-andv n monoclonal antibody (mab) 7b3/f6 [58] and then the primary antibody was detected with goat antimouse immunoglobulin conjugated to alexa 555 (life technologies). !10.000 cells of each condition were analyzed using a flow cytometer (facs can ii, becton dickinson). the standard deviation of at least n = 3 experiments is indicated as the error bar of each value. production of simian immunodeficiency virus (siv) vectors pseudotyped with vesicular stomatitis virus (vsv) g protein or andv glycoproteins and transduction siv vectors bearing the vsv glycoprotein were prepared as indicated elsewhere [59] . briefly, 293ft cells were transfected with 8 μg of plasmid coding for siv gag-pol (psiv3+), 8 μg of plasmid encoding gfp as an rna minigenome (pgae1.0) [60] , and 4 μg of plasmid coding for the envelope protein g of vsv (pvsv-g, clontech). alternatively, the plasmid pi.18/gpc coding for andv glycoproteins was used to generate siv vectors pseudotyped with andv glycoproteins. at 48 h post-transfection, supernatants containing pseudotyped particles were concentrated by centrifugation at 100,000 g for 75 min. different dilutions of vsv-g pseudotyped siv vectors were incubated for 1 h with vero e6 cells in the presence and absence of protein or peptide inhibitor candidates. three days later, the expression of gfp in transduced cells was analyzed by flow cytometry (facscan, becton dickinson). !10.000 cells were counted for each experimental condition. a cell proliferation assay was used to assess cytotoxicity of the gc domains and peptides as described by the manufacturer (celltiter96, promega). briefly, gc domains and peptides were incubated with vero e6 cells for 18 h at 37°c, and the conversion of tetrazolium to formazan was assessed by measuring the absorbance at 570 nm in a microplate reader (synergy 4, biotek). a three-color based cell-cell fusion assay was performed as established before [22] . briefly, 48 h after the transfection of vero e6 cells with pi.18/gpc [59] or pwrg/puuv-m(s2) [50] coding for the glycoproteins of andv and puuv, respectively, the cells were incubated with low ph media (e-mem, 20 mm sodium succinate, ph 5.5) for 5 min at 37°c. three hours later, the cell cytoplasm was stained with 5-chloromethylfluorescein diacetate (celltracker green cmfda, invitrogen), cell nuclei with dapi (life technologies), and gc was labeled by anti-gc mab 2h4/f6 [61] , and anti-mouse immunoglobulin conjugated to alexa555 (invitrogen). the fusion index of gc-expressing cells was calculated by the formula: for each sample, approximately 200 nuclei per field were counted (20x magnification), and the mean fusion index of five fields was calculated (n = 3). vero e6 cells were pre-chilled on ice for 10 min with 20 mm nh 4 cl. the adsorption of andv (moi = 0.2) was performed at 4°c for 1h. next, cells were washed, and the fusion of the virus with the plasma membrane was triggered by incubation in low ph media (e-mem, 20 mm sodium succinate, ph 5.5) for 5 min at 37°c in the presence and absence of inhibitor candidates. next, cells were washed, and infection was followed by incubation for 16 h at 37°c in the presence of 20 mm nh 4 cl. subsequently, viral infection was assessed as described above. the multimerization state of gc was assessed by sucrose gradient centrifugation as previously established [17] . briefly, andv was treated at ph 5.5 to allow for the rearrangement of glycoproteins on the viral envelope. where indicated, diii or r2 were added to the virus during its low ph incubation during 30 min at 37°c. subsequently, viral glycoproteins were extracted by 1% triton x-100 and separated in a gradient of 7-15% (w/v) sucrose by centrifugation at 150,000 g for 16 h. fractions were collected, and the presence of gc in each fraction was assessed by western blot analysis using anti-gc mab 2h4/f6 [61] . the molecular mass of each fraction was assessed by the coomassie staining of a molecular marker (gel filtration standard, bio-rad) that was applied to the same sedimentation gradient. the experimental molecular mass of the marker was next plotted against the log of its theoretical molecular mass in the panel above the western blots of the gradient. the stability of the gc homotrimer was further tested by trypsin digestion as indicated before [17] . briefly, well-characterized vlps projecting andv glycoproteins were prepared as described elsewhere [62] and were incubated for 30 min at ph 5.5. after the acidification, vlps were incubated with tcpk trypsin (sigma) for the indicated times. finally, digestion was stopped by adding sample buffer and heating to 95°c for 10 min. the digestion of gc was tested by western blot analysis, using anti-gc mab as described above. in this assay, the transfer of monosialotetrahexosylganglioside (gm1) from an effector cell to a target cell was measured as described before for snare proteins [63] . to adapt this assay to measure the andv glycoprotein-mediated gm1 transfer, 293ft cells (gm1 + ) were transfected with pi.18/gpc [59] using lipofectamine 2000 (invitrogen), as indicated by the manufacturer. forty-five h post-transfection, the cells were detached from the plates, and resuspended in supplemented dmem. at the same time, target cho-k1 cells (gm1 -) were trypsinized and labeled with 40 μm 7-amino-4-chloromethylcoumarin (celltracker blue cmac, invitrogen) in supplemented 12-k medium for 40 min at 37°c. the excess dye was then removed by replacing the dye-containing media with supplemented f12-k medium and subsequent incubation for 30 min. after washing with pbs, target cells were resuspended in supplemented dmem. next, effector cells (transfected 293ft cells) and cmac-labeled target cells (cho-k1 cells) were combined in a ratio of 1:4 (effector:target) for 3 h. then, the fusion protein was activated by incubating the cells in low ph media (dmem, sodium succinate 20 mm, 10% fcs, ph 5.5) for 5 min at 37°c. this medium was replaced with supplemented dmem, ph 7.4 and after 30 min of incubation the cells were fixed with 4% paraformaldehyde. for fluorescence staining, cells were incubated for 30 min with 5 μg/ml of cholera toxin β-subunit (ctx) conjugated to alexa fluor 488 (invitrogen) at 37°c, washed with pbs, and mounted with dabco. cells were examined by confocal microscopy (fluorview fv1000, olympus and single plane images from 10 different microscopic fields were taken in each condition. the percentage of gm1 transfer was calculated using the formula: where n a corresponds to the number of cells positive for cmac and gm1, while n b corresponds to the number of cells positive for gm1 that are furthermore in contact with at least one target cell. cells were considered positive for gm1 transfer when the fluorescent label was detected at the full circumference of the cells. the standard deviation of at least n = 3 experiments was calculated and was indicated as an error bar for each value. a student´s t-test was performed for statistical evaluation of n!3 independent epxeriments: ããã , p < 0.00025; ãã , p < 0.0025; ã , p < 0.025. for the hantavirus gc protein, we predicted diii and the stem region by sequence alignments with known class ii fusion proteins and subsequent model derivation. these proteins included among others the more recently crystallized rvfv gc [29] . none of the new sequence alignments achieved a higher sequence identity, greater cysteine match or model validation scores compared to the alignment used for the original gc model structure [23] , which is based on the pre-fusion structure of the tick-borne encephalitis virus (tbev) e protein (pdbid: 1svb) [31] . for this reason, we used the alignment from the original andv gc model [23] to identify a putative diii in andv gc (fig 1a and 1b) . the sequence that was derived from this model (residues asp315-leu414) was termed andv diii, and served as template to predict a putative diii in gc of other hantaviruses such as puuv. we subsequently defined the putative stem region in andv gc as the sequence encompassing residues leu414-asn456, which corresponded to the region between the c-terminal end of the predicted diii and the predicted gc transmembrane region obtained by the tmpred server [64] (fig 1c) . the production of diii from different class ii fusion proteins, including those of flaviviruses and alphaviruses, has been previously established in e. coli [52, [67] [68] [69] [70] [71] . the feasibility of preparing soluble diii from flavi-and alphaviruses in a prokaryotic expression system retaining the native structure may be related to its globular igg-like fold and lack of glycosylation [44] . the purification of diii is generally achieved from inclusion bodies followed by refolding [44, 71] , or from the supernatant of the cell lysate [52, [67] [68] [69] [70] . here, we prepared three recombinant diii proteins; diii derived from andv gc with or without n-terminal his-tag (andv hdiii, andv diii), and diii from puuv gc with n-terminal his-tag (puuv hdiii). the proteins were obtained from the supernatant of e. coli bl21 or origami 2(de3) cells, and eluted after their purification as a single peak detected by absorbance at 280 nm. fig 2a shows an elution profile example of purified andv diii from the last size exclusion chromatography column together with the homogeneity of the preparation assessed by sds-page. because the predicted andv and puuv diii sequences contain eight highly conserved cysteine residues, the diii proteins were next characterized for the presence of disulfide bridges by reduction and subsequent alkylation. an increase in the electrophoretic mobility could be detected for reduced andv diii, andv hdiii and puuv hdiii compared to its unreduced control (fig 2b) , indicating that the cysteines seemed to be arranged in disulfide bridges. we next explored the presence of secondary structure elements in diii from hantaviruses by circular dichroism. the spectra showed a unique negative maximum at 209 nm (fig 2c) , confirming the presence of secondary structure. deconvolution of the circular dichroism spectra into four components by different servers [54] [55] [56] indicated that diii contained 40-41% β-sheets,~60% random coils and turns with an α-helical content close to zero. this composition coincides with the high content of β-sheets and turns observed in diii of class ii fusion proteins [31] . taking these data together, the monomeric form of recombinant andv diii in solution, the presence of disulfide bridges, the secondary structure content, and the solubility of the recombinant protein (>20 mg/ml) indicate that diii was folded. in addition, we also produced a protein spanning the predicted diii and stem region of andv gc, including an n-terminal his-tag (andv hdiiis). however, we did not obtain enough andv hdiiis for its purification, presumably due to the poor solubility of this protein. to overcome this difficulty, we generated the predicted gc stem region separately in two synthetic peptides, one spanning the n-terminal part (r1 peptide) and the other spanning the cterminal part (r2 peptide) (fig 1c) . we also used a negative control peptide comprising a region of the andv nucleoprotein (nn peptide). once the predicted diii and stem fragments were synthesized, purified, and characterized, we measured their inhibitory activity against andv during virus cell entry via the native, endosomal infection route. for this purpose, andv was incubated with vero e6 cells in the presence of the gc fragments. after 1 h incubation, the cells were washed and the infection monitored after 16 h based on an earlier established protocol [57] . andv diii and andv hdiii reduced andv infection up to 60%, at 3-4 μm (fig 3a) . the n-terminal his-tag did not further improve inhibition of viral infection, as observed for the alpha-and flavivirus diii proteins [44] . interestingly, puuv hdiii did not show any cross-inhibition of andv. this result was unexpected since cross-reactivity by diii has been reported within the alphavirus and flavivirus genera [44, 72] . it is likely that the absence of cross-inhibition was due to the presence of the n-terminal his-tag in puuv diii (see results below; fig 4c) . next, we tested whether the andv stem peptides also impair andv infection. the r2 peptide, comprising the c-terminal part of the predicted gc stem region, blocked the infection of cells by andv up to 55% at 20 μm (fig 3b) . in contrast, the r1 peptide spanning the n-terminal part of the predicted gc stem, did not show any reduction of andv infection, similar to the negative control nn peptide (fig 3b) . to test whether a specific region of r2 was required for inhibition, we further tested two different parts of r2 (see fig 1c) . the n-terminal r2.1 and c-terminal r2.2 peptides both showed similar reducing effects on viral infection (fig 3b) . to further explore the specificity of the gc fragments on viral inhibition, we used as a model the unrelated vesicular stomatitis virus (vsv). while vsv enters cells by endocytosis and low ph-triggered fusion, this virus achieves fusion by a different class of fusion protein (class iii). to analyze vsv-mediated entry, siv vectors [24] were pseudotyped with the envelope glycoprotein g of vsv and the transduction of cells by this vector was evaluated by the expression of the gfp reporter gene. neither andv diii nor the andv stem peptides altered cell transduction at any tested concentrations up to 6 μm and 60 μm, respectively (fig 3c) . in contrast, when the ph of the endocytic route was neutralized with the weak base ammonium chloride, vsv-g mediated transduction was blocked up to 80% (fig 3c) . on the other hand, when siv vectors were pseudotyped with andv glycoproteins, the diii and r2 fragments produced~40% and~30% of inhibition of these vectors at 6 μm and 20 μm, respectively ( fig 3d) , corroborating that the inhibitory fragments were active in this system. none of the gc-derived peptides and recombinant domains abrogated the viability of cells when they were incubated with vero e6 cells (fig 3e) , indicating that the reduction of andv infection was not due to a cytopathic effect of the gc-derived fragments. it was previously reported that flavivirus stem peptides bind to the virus before it enters the cell, helping its delivery into endosomal compartments [47] . based on this observation, we compared the inhibition of andv by (a) pre-incubation of the virus with the r2 peptide, or (b) co-incubating the r2 peptide with the virus during its adsorption to cells. comparing the results of both experimental designs, a similar dose-dependent inhibition could be observed (fig 3f) , coinciding with the results obtained for flaviviruses (47) . based on this result, it seems plausible that binding of the r2 peptide to andv may occur very fast, making longer incubation times unnecessary. taken together, our data demonstrate that the exogenous fragments derived from andv gc, comprising the predicted diii or the c-terminal part of the predicted stem region, abrogate the entry of andv into cells. the inhibitory potential of the gc fragments was next tested in a cell-cell fusion assay driven by the andv glycoproteins [22] . therefore, we transfected vero e6 cells with a plasmid in the presence of exogenous diii or stem peptides. transduction was assessed by gfp reporter gene expression using flow cytometry, and 100% transduction defined as transduction in the absence of gc-derived fragments (mock). (e) cytotoxicity analysis of gc diii proteins and stem peptides on vero e6 cells. (f) andv inhibition by the r2 stem peptide through pre-incubation (pre-r2) or co-incubation (co-r2). for pre-incubation, andv was coding for the andv glycoproteins, and incubated the cells 48 h post-transfection with low ph media for 5 min at 37°c, as described in materials and methods. the acidification of the media allows the activation of the viral gc fusion protein on the plasma membrane of cells, thereby triggering the formation of syncytia. when the andv gc fragments were incorporated in the low ph incubation step, andv diii and andv hdiii diminished the glycoprotein-mediated fusion activity by~70% at 25 μm and 50 μm, respectively (fig 4a) . in concordance with the results obtained with infectious andv (fig 3a) , the puuv hdiii did not achieve cross-inhibition of andv glycoproteins (fig 4a) . on the other hand, the andv r2 stem peptides impaired the andv glycoprotein fusion activity up to 75% at 20 μm (fig 4b) . the andv-derived gc fragments were likewise tested for their potential to cross-inhibit other hantaviruses such as puuv. therefore, we performed a cell-cell fusion assay driven by the puuv glycoproteins. in this assay, puuv hdiii blocked the puuv glycoprotein-mediated fusion process, reducing fusion up to 65% at 25 μm (fig 4c) . this result coincides with the concentration range in which the andv diii proteins block the andv glycoprotein fusion activity and confirms the activity of puuv hdiii. when we tested the andv diii proteins for cross-inhibition, we found that andv diii, but not andv hdiii, blocked puuv glycoprotein-mediated fusion up to 50% (fig 4c) . these results, together with the data on absent crossreaction of puuv hdiii with andv, suggest that the hantavirus diii proteins without the nterminal his-tag have cross-inhibiting activity; however the n-terminal his-tag of these domains seems to interfere in the inhibition. thus so far, this notion remains to be corroborated with puuv diii lacking the n-terminal his-tag. when we tested the andv stem peptides to block puuv-mediated fusion, we found that they also had a cross-inhibition function (fig 4d) . among them, the r2.1 peptide reached the highest cross-reduction result of~70% at 20 μm, in line with the observation that this peptide also achieved the highest inhibition value of andv-mediated cell-cell fusion. collectively, these results on hantavirus cross-inhibition suggest that residues in diii and stem fragments are involved in intramolecular interactions. some of these residues seem to be conserved between andv and puuv (fig 1d) , allowing for the cross-interaction with exogenous diii and stem fragments from a different hantavirus. andv cell entry can be blocked at different steps such as receptor binding and membrane fusion. for hantaviruses, the envelope glycoprotein or the specific domain involved in binding to receptors has not yet been identified. to discard a possible effect of the andv inhibitors in steps preceding virus-cell membrane fusion, we incubated the cells with andv diii or the r2 peptide for 1 h before the addition of the virus. unbound fragments were subsequently washed out, and cells were then infected with andv. the addition of neither diii nor r2 at 10 and 20 μm, respectively, before the cells were incubated with andv led to a significant decrease in virus infection (fig 5a) . these data emphasize that the pre-incubation of diii and stem fragments did not abrogate early steps in virus infection such as receptor binding or cellular signaling pathways. furthermore, the results coincide with data obtained with stem peptides derived from dv2, where the pre-incubation of cells with these peptides also did not affect infection by dv2 [47] . next, we asked whether the diii and stem fragments interfered directly in the virus-cell fusion process. to test this, we assessed inhibition in a fusion infection assay, fusing andv with the plasma membrane. therefore, andv was pre-bound to cells at 4°c and then andv diii or r2 were added during the 5 min low ph pulse that triggers fusion. the blockade of this viral entry pathway was highly efficient in the case of the r2 peptide, reaching over 95% of inhibition at 20 μm (fig 5b) . recombinant diii led to a lower inhibition efficiency of 70% at 20 μm, result that in comparison to that obtained with r2 could be explained by the short incubation time in this experimental design. compared to inhibition of the normal entry route of the virus, higher concentrations of andv diii might have been necessary for inhibition since more input virus was used to reach similar levels of infection (moi = 0.2). taking these data together, our results confirm that the exogenous diii and stem fragments function specifically during the viral fusion process. since the fusion process involves multiple steps, we next assessed at which specific stage the gc fragments interfere in this process. to this end, we started analyzing the trimerization of gc using an earlier protocol [17] . andv was therefore incubated at neutral or low ph with or without diii. subsequently, the viral glycoproteins were extracted from the virus and applied to a sucrose gradient (7-15%) to evaluate their molecular mass. after ultracentrifugation, each fraction was examined by western blot analysis for the presence of gc. gradient sedimentation at ph 7.4 led to the detection of gc in fractions corresponding to the molecular mass of gc monomers of~50 kda (fractions 5-7), in the presence or absence of andv diii (fig 6a) . two gc migration bands could be observed in the reducing electrophoretic system, which may correspond to different oxidation forms of gc as described earlier [73] . only the lower molecular mass band was previously found to shift from gc monomers at neutral ph to gc trimers at low ph [17] . when we incubated andv at ph 5.5, in the presence or absence of the diii inhibitor, the lower molecular mass band of gc shifted from the fractions corresponding to monomers and was found in fractions corresponding to gc homotrimers (fractions 11-12; fig 6a) . this result indicates that andv diii abrogated neither gc fusion activation, nor gc trimerization. the post-fusion conformation of andv gc corresponds to a highly stable homotrimer [17] . in this context, we next explored the stability of the gc homotrimer formed in the presence of exogenous diii and stem fragments by assessing its resistance to protease digestion. for this experimental approach we used andv-like particles (vlps) [62] , since they can be purified to higher concentrations than the virus. these vlps resemble the viral envelope by exposing the gn and gc glycoproteins. to test their trypsin resistance, the vlps were first incubated at neutral or low ph in the presence or absence of the inhibitors, and subsequently subjected to digestion with trypsin for different incubation times. as expected, the neutral ph form of gc was fully degraded within 30 min, while the low ph form of gc was largely resistant to digestion (fig 6b) . in contrast, gc lost its trypsin resistance and digestion could be observed when andv diii or the r2 peptide was co-incubated with vlps during the low ph incubation step, indicating that they interfered in the formation of a stable gc homotrimer (fig 6b) . some residual gc could be detected, most likely because the diii or stem fragments did not block all gc molecules, which coincides with the inhibitory results (figs 3a and 3b and 5b) . the formation of a stable homotrimer was however not prevented when the control peptide nn was added before acidification, nor when either the nn peptide, diii or the r2 peptide was added after the low ph incubation, confirming the specificity of the assay. together, these data suggest that the exogenous diii and stem fragments prevented the formation of a stable post-fusion hairpin structure, presumably by direct binding to gc in its extended intermediate conformation. the presence of exogenous gc fragments interfered neither with the activation of andv gc nor with its homotrimerization; however it did not allow the formation of a stable post-fusion structure. the hemifusion intermediate is a stage that occurs between these fusion steps, in which the outer membranes of the virus and cell have fused, while the inner leaflets still remain apart. in order to test if the inhibition of andv by exogenous diii occurs before or after the hemifusion intermediate state, we developed a hemifusion assay for the andv glycoproteins. for this purpose, we took advantage of a previously developed cell-cell fusion assay between 293ft cells expressing the andv glycoproteins (effector cells) and cho-k1 cells (target cells) [17] . in this assay, the low ph-triggered transfer of gm1 from effector cells to target cells was analyzed by confocal microscopy, which allows detection of gm1 at the cell surface in a single focal plane, with minimal sample intervention (fig 7a) . to allow unambiguous identification of gm1 transfer from effector cells to target cells, cho-k1 cells were only defined as gm1 + when the label was detected on their full circumference (fig 7a, red arrows) . conversely, target cells in contact with effector cells, but showing only partial or no gm1 stain, were defined as gm1 (fig 7a, yellow arrows) . although this assay allows for the detection of lipid mixing between cells, it does not discriminate between full fusion and hemifusion of membranes. to obtain a quantitative measure of gm1 transfer from effector to target cells, we quantified the percentage of transfer in each condition. at ph 7, a background level of transfer around 20% was observed in all conditions (fig 7b) . when glycoproteins were activated at ph 5.5, gm1-transfer from effector cells to target cells was detected in~70% of mock control treatment ( fig 7b) . however, when andv diii was incorporated in the low ph incubation, the transfer of gm1 appeared to be less frequent, decreasing to below 40% (fig 7b) . this value is still higher than the background level observed for gm1 transfer at neutral ph, indicating that this domain reduced lipid mixing in an incomplete manner. most probably, exogenous andv diii did not make contact with all fusion proteins during the short low ph incubation, which coincides with the results for blocking cell-cell fusion (see fig 4a) . the impairment of gm1-transfer was highly specific, since the incorporation of puuv hdiii, which has no crossinhibition activity against andv (see figs 3 and 4a), did not abrogate the acid-induced gm1 transfer of~70% (fig 7a and 7b ). in summary, our results show that andv diii arrests the fusion process after gc trimerization, but before reaching a hemifusion intermediate. in this context, it seems likely that gc fragments prevent the movement of the endogenous diii or the stem region towards the core of the gc trimer as described for other class ii fusion proteins [39, 44] . the fusion of the viral membrane with a host cell membrane is a crucial step in the entry of enveloped viruses into cells. in the present study we demonstrated that predicted diii and stem fragments blocked acid-induced fusion of andv within the endosomal entry pathway and with the cell surface. fusion was allowed to proceed until gc trimerization, but prevented membrane hemifusion and fusion pore formation. these results not only provide novel information about inhibitory strategies against andv and other hantaviruses, but also provide a proof of concept that gc shares structural similarity with the overall fold of class ii fusion proteins. comparing the inhibition of hantaviruses by exogenous diii with that of other class ii fusion proteins, andv and puuv hantaviruses were blocked by andv diii without additional n-terminal his-tag or c-terminal residues, although containing seven n-terminal residues derived from the gst-tag. while the addition of an n-terminal his-tag achieved a 100-fold improvement in blocking fusion of semliki forest virus [44] , andv diii -with or without n-terminal his-tag -achieved similar inhibitory results. for sfv diii it has been proposed that this n-terminal tag may mimic the domain i-diii linker region, thereby stabilizing the interaction with the fusion protein core [44] . on the other hand, it has been reported that the presence of c-terminal residues derived from the stem region is necessary for inhibition by exogenous diii of dv2 and chikungunya virus e proteins [44, 72] . more specifically, for chikungunya virus it has been shown that nine residues from the e stem region are required for diii to bind to the fusion protein domain i-domain ii core. hence, the potency of inhibiting the andv fusion process through andv and puuv diii may be further improved in future studies by adding n-or c-terminal residues. the andv diii, including the stem region, was largely insoluble and therefore this larger gc fragment could not be tested in the andv entry assays. the r1 peptide spanning the first 17 n-terminal residues of the 44-residue stem region did not affect andv entry, while the r2 peptide spanning the last 20 c-terminal residues blocked andv infection to a similar extent as diii. when we tested the two peptides r2.1 and r2.2, comprising either the n-or c-terminal half of the andv r2 stem peptide, the inhibitory activity against andv was largely retained by both peptides. hence, the r2 peptide seems to contain residues in these two regions that participate similarly during inhibition. similar to the c-terminal region of the andv stem region, the c-terminal region, but not the n-terminal, of dv2 e protein binds and blocks the fusion protein [47] . interestingly, inhibitory peptides derived from membrane proximal regions (or stem regions) of diverse fusion proteins such as those of rvfv, dv2, sars-coronavirus, influenza virus, and hepatitis c virus have been found likely to interact with membrane interfaces by a hydropathy segment [74] , which is predicted by the wimley-white interfacial hydrophobicity scale (wwihs) for the transfer of a peptide from an aqueous environment to a palmitoyl-oleoyl-phosphatidyl choline interface [75, 76] . a need for such a membrane-binding property for inhibitory activity coincides with studies on peptides derived from the stem region of alphavirus fusion proteins; these peptides generate wwihs below values of 1 (s1 table) , indicative of weak membrane binding [74] , and fail to block alphavirus fusion activity [44, 72] . in the case of the dv2 e protein, such a membrane-binding sequence is found at the c-terminal end of the peptide [48] with a high wwihs value (s1 table) . in fact, the stem peptides of the dv2 e protein have been shown to interfere with the fusion of the virus in the endosomal compartment by a two-step mechanism: first by binding to the viral membrane outside the cell, and next by binding against the e core trimer once fusion has been triggered in the endocytic compartment (47) . using the wimley-white scale [77] , an interfacial hydropathy segment with a wwihs vale >5 was predicted for the andv gc stem region that coincides with the sequence of the r2 peptide (s1 table) , indicative for moderate membrane partitioning [74] . if the andv r2 peptide would interact with membranes by a hydropathy segment, then such an interaction did not favor andv inhibition sufficiently in the endosomal route, since fusion impairment was most efficient when directly present in the fusion compartment; when applied at the same concentration, the r2 stem peptide blocked 45% of andv infection when entry occurred via the endocytic pathway, while r2 reached over 95% inhibition of andv infection when fusion occurred at the plasma membrane. in this sense, modifications to the r2 peptide that favor membrane interaction, such as those introduced to peptides derived from the west nile virus e stem region [48] or the membrane proximal region of influenza virus hemagglutinin and hiv gp41 [78, 79] , may help in the future to improve its inhibitory activity and to direct it towards the closed environment of endosomes. the andv diii and stem peptides blocked not only fusion mediated by andv glycoproteins, but also the fusion activity of the glycoproteins of another hantavirus (puuv). this result is in accordance with the high sequence identity of 72% between diii of these viruses and also with reports on the cross-inhibition activity of diii within the genus alphavirus [72] , where diii conservation is as high as 50%. however, the n-terminal his-tag of andv hdiii seemed to prevent the cross-inhibition of puuv fusion activity, indicating that this tag may interfere in specific binding to gc from heterologous species. it is likely that the histidines of the tag become positively charged in the low ph environment, which in turn may induce repulsion with positively charged residues in the gc of hantaviruses. such repulsion may be overcome by a higher binding affinity of diii to gc from the same hantavirus, but not to heterologous viruses. in addition to the diii of andv, peptides derived from the stem region of andv also cross-inhibited the puuv fusion activity, which further corroborates the presence of conserved residues among hantavirus gc proteins that are involved in the likely binding of this peptide. cross-inhibition of fusion proteins by stem peptides has been previously reported for viruses from the genus flavivirus; dengue virus stem peptides blocked different dengue virus serotypes but not other flaviviruses. [48] . the absence of cross-inhibition in that case was related not to a poor interaction with the respective e protein, but rather to a poor interaction with the viral membrane [48] . finally, stem peptides derived from the rvfv gc protein have been reported to block the three different classes of viral fusion proteins [46] , acting as a broad-spectrum fusion inhibitor [80] . for the exogenous stem peptides from andv we did not observe cross-inhibition of other fusion proteins such as that of vsv at concentrations up to 60 μm. therefore, it is more likely that the andv stem fragments may be applied to inhibit similar viruses within the same genus, but not other viral fusion machineries. taken as a whole, our results demonstrate that strategies employed against class ii fusion proteins allow for the inhibition of hantaviruses such as andv and puuv. although targeting the endosomal site of virus fusion has not yet been optimized, it was possible to block fusion and infection under 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fusion inhibitor dramatically increases its antiviral potency broad-spectrum antivirals against viral fusion we thank héctor galeno of instituto de salud pública de chile for providing the andes virus, isolate chi-7913. our special acknowledgements also to the centro de investigaciones médicas, pontifícia universidad católica de chile for supporting this work by providing access to its bsl-3 facility.