Zika virus has been documented since 1952, but been associated with mild, self-limiting disease. Zika virus is classified as an arbovirus from a family of Flaviviridae and primarily spread by Aedes Aegypti mosquitos. However, in a large outbreak in Brazil in 2015, Zika virus has been associated with microcephaly.In this review we applied the Bradford-Hill viewpoints  to investigate the association between Zika virus and microcephaly. We examined historical studies, available data and also compared historical rates of microcephaly prior to the Zika virus outbreak. The available evidence was reviewed against the Bradford Hill viewpoints.All  the nine criteria were met to varying degrees: strength of association, consistency of the association, specificity, temporality, plausibility, coherence, experimental evidence, biological gradient and analogy.Conclusion: Using the Bradford Hill Viewpoints as an evaluation framework for causation is highly suggestive that the association between Zika virus and microcephaly is causal. Further studies using animal models on the viewpoints which were not as strongly fulfilled would be helpful. Until recently, Zika virus has been thought to cause a mild, self-limiting disease. However, in a large outbreak in the Americas in 2015, Zika virus has been implicated as a cause of complications such as microcephaly, Guillaine Barre syndrome (GBS) and myelitis.,Microcephaly is defined as babies born with abnormally small skulls and underdeveloped brains relative to gestational age and sex.After experiencing an increase in Zika virus infections, Brazil reported approximately 6158 suspected cases of microcephaly from late 2015, of which 745 cases were confirmed as zika.,Since this time the association between Zika virus and microcephaly has been debated by leading health agencies, but only a few studies have applied viewpoints for causation in a systematic way.The Bradford-Hill Viewpoints are used in public health to determine causation of diseases.Using the Bradford Hills nine viewpoints we analysed data from a 2013 outbreak in French Polynesia and the 2015 Brazilian outbreak to establish a causal link between Zika virus and microcephaly. We searched major databases including PubMed, Medline and Embase from 2000 onwards using search words, such as Zika virus, Microcephaly and Flavivirus. We also searched news reports and grey literature for information on the outbreaks. We examined historical studies and previous data and also we compared historical rates of microcephaly prior to the Zika virus outbreak.Available data and studies were used to determine whether each of the nine Bradford Hill viewpoints were fulfilled. For strength of association, we sought studies which calculated measures of association for Zika virus and microcephaly. Where the calculation was not done, but suitable data available, we calculated a measure of association. We utilised data from a prospective cohort study conducted in Brazil “Zika virus infection in pregnant women in Rio de Janeiro”,The assessment of each viewpoint was qualitative/descriptive, as there was an element of subjectivity in applying quantitative scoring. Evidence collected for each viewpoint is presented in the results with a final judgement as to whether the viewpoint was fulfilled or not.The evidence for each of the Bradford-Hill viewpoints:Viewpoint #1: Strength of association,Ecological evidence supports the association between Zika virus and microcephaly with several reports from WHO and CDC, illustrating an approximate 20-fold increase in numbers of microcephaly cases in countries which are currently affected with Zika virus outbreak such as Brazil, Colombia, Panama and Martinique.In 2013-2014, Zika virus caused a large outbreak in French Polynesia, where it was estimated to have infected 66% (95% CI 62–70) of the general population.Standardized inclusion criteria were used, where all babies born with head circumference of at least 2 standard deviations or below the mean for age, sex and ethnicity of the normal head circumference were considered to have microcephaly. Pregnant women with a clinical picture of Zika infection or with positive PCR for Zika virus were included.It was also found that the estimated rate of microcephaly in this population during the Zika virus outbreak was substantially higher than the baseline of approximately 2 per 10 000 neonates (0.02%).This study found a very strong association between Zika virus infection and microcephaly. With a large sample size from serological and surveillance data obtained from the outbreak, the authors illustrate using mathematical modelling the probability of microcephaly occurrence. The overall risk ratio of 20.8 further strengthens the association between Zika virus and the occurrence of microcephaly in infected pregnant mothers.In addition, a prospective cohort study was conducted in Brazil with 88 pregnant women with a history of clinical symptoms of Zika virus, out of which 72 women tested positive for the virus.The viewpoint is met.Viewpoint #2: Consistency of the association,,To date there are two epidemiological studies investigating the association between Zika virus and microcephaly. Of those available studies, there is consistency between studies finding an association. As mentioned above a study conducted using data from the outbreak in French Polynesia in 2013-2014 and a study conducted in Brazil among pregnant women in Rio de Janeiro in 2015 both found a positive association. A case control study in Brazil compared 32 babies born with microcephaly and 62 babies born without microcephaly, for every case born two control cases were matched from the same hospital with area of residence and date of delivery.The viewpoint is met.Viewpoint #3: SpecificityThe criterion for specificity generally refers to one exposure causing one disease. However, when considered more broadly, if there is an association in a specific population group with the same environmental exposure this strengthens the argument for causation.,,,,,This viewpoint should be assessed in combination with the strength of association. Microcephaly is a rare outcome and is estimated to occur in approximately 2 to 12 babies per 10,000 births in the United States of America (USA). The aetiology of microcephaly is usually exposure of the fetus in utero to an external agent or a gene mutation that is capable of causing cell death.The viewpoint is met.Viewpoint #4: TemporalityIn Brazil Zika virus infection was recognised in late 2014 as a febrile illness and the first cases were confirmed by RT-PCR in April 2015, with microcephaly following in November 2015, so that exposure to Zika virus preceded the occurrence of microcephaly.A study in the State of Paraiba, Brazil, further illustrated the temporality where zika virus was detected in amniotic fluid in two pregnant women with fetal microcephaly.A study published in the New England Journal of Medicine (NEJM), reported a possible link between Zika virus and microcephaly which illustrated the temporality. The study, illustrated a case of vertical transmission of Zika virus from a woman in Brazil where she presented with clinical features of Zika virus infection while pregnant. It was also reported that a 33-year-old Finish woman became infected while on holiday with her husband in Central America.,,The epidemiological studies mentioned both show an increase in the number of microcephaly cases following an outbreak of Zika virus.Viewpoint #5: Biological gradients or dose-response relationshipBiological gradients can be illustrated by the level of exposure to a given factor and corresponding level of outcomes.This viewpoint is met.Viewpoint #6: Plausibility,,Flaviviruses in general are known to have some neurotropic effects. The majority of arbovirus cause fever and non-specific symptoms but at times have been shown to cause more severe outcomes such as neurological syndrome and haemorrhagic symptoms, as well as abortion and congenital anomalies in animals and mice.A case report recently published described a pregnant woman who tested positive for Zika virus and had ultrasounds which indicated malformation in the fetus. Testing of the foetal brain tissue showed the presence of Zika virus as well as the presence of Zika RNA in the placenta and other fetal organs and tissues.,,Studies conducted to understand the neuropathology of Zika virus illustrate its ability to affect neurons cells and to cause cell death resulting in abnormal head size. This was illustrated by the study of the effect of Zika virus on progenitor cells and immature cortical neurons.The viewpoint is met.Viewpoint #7: Coherence,,,The current studies reporting an association between Zika virus and microcephaly do not conflict with previously reported evidence. A recent study conducted in Brazil to demonstrate the effect of Zika virus on the growing neurosphere and brain organoid using human neural stem cells, illustrate the ability of Zika virus to induce death of human neural cells and impair formation of neurosphere hence causing microcephaly.The viewpoint is met.Viewpoint #8: Experimental evidenceScientists working to understand Zika virus have used a cyro-EM structure of mature Zika virus and compared this with other Flavivirus.Tang et al, conducted a study using human induced pluripotent stem cells and also on human embryonic stem cells and immature cortical neuron cells to investigate the effect of Zika virus on cells.In an experimental study the Brazilian Zika strain was seen to cause microcephaly with other neurodevelopment anomalies in mice models.These experiments further support the association between Zika virus and microcephaly. The above studies illustrated cell structure, infectivity rate and its effects on the human pluripotent stem cells and embryonic cells for better understanding of its ability to induce malformation in neural development. However, more needs to be done in terms of animal models especially in pregnant animals to further elaborate the effects of Zika virus in neural development anomalies.This viewpoint is met.Viewpoint #9: Analogy,,,The criteria for analogy was described originally by Hill as analogous examples of exposures that may have led to the same outcome. The examples of thalidomide and rubella were used by Hill as exposures that could each lead to congenital malformations.,,In a study done on 151 patients from 2014-2015 in a hospital in Brazil who presented with infection from arbovirus, 6 patients developed autoimmune disorders of which 4 patients had GBS and two patients had Acute demyelinating encephalomyelitis (ADEM).,,A study was done in Guadeloupe on a 15 year old girl who presented with signs of myelitis and tested positive for Zika infection.The viewpoint is met.,,,We found that all the Bradford Hill viewpoints were met for the association of microcephaly with zika virus infection, thus adding to the evidence for a causal relationship. The framework of the Bradford Hill Viewpoints is useful for evaluating causation, and can be drawn upon as a systematic method for evaluation of new associations between exposure and disease. Until recently, Zika virus has been generally associated only with a mild febrile illness.,,,,,,,,,,To date, a small number of studies have been conducted to investigate the possible causal relationship between the outcomes of microcephaly with exposure to Zika virus infection in pregnancy.,Previously two studies have applied the Bradford Hill viewpoints to systematically analyse the evidence for a causal association between Zika infection in pregnancy and microcephaly.An alternative approach to the Bradford Hill viewpoints is Shepherd’s criteria of teratogenicity, which have also been used to analyse the link of Zika infection in pregnancy and microcephaly.Over time since the initial identification of the Brazilian epidemic, more studies and evidence has become available. We systematically applied the accumulated evidence from the available studies to analyse a possible causal relationship between Zika virus infection and microcephaly using Bradford Hill’s viewpoints. The evidence is overwhelmingly in favour of causation. The establishment of causation and increase in certainty aids public health policy and disease control efforts, including travel advisories for pregnant women. In complex outbreaks where causation is unknown, the use of the Bradford Hill viewpoints can assist with systematic decision making and public health policy.The authors have declared that no competing interests exist.All data in this review are publicly available in previously published papers, as noted in the Methods section.Professor C. Raina MacIntyre. Email: r.macintyre@unsw.edu.au


Flaviviridae, and infection of humans with ZIKV through mosquito bites may result in a disease known as Zika fever. Most Zika fever cases are asymptomatic and thus go unreported, but if symptoms do appear, they are usually mild in nature , and self-resolve within one week (Chen and Hamer Zika virus (ZIKV) is a mosquito-borne pathogen from the family ZIKV was first isolated from a rhesus macaque in Uganda during 1947. Subsequent serological surveys showed that humans in Africa have long been exposed to the virus and many are seropositive for ZIKV-specific antibodies, suggesting that the virus had circulated amongst the human population for a considerable length of time   was used, in conjunction with IFN receptor-specific antibodies to weaken the host immune response (Govero, Esakky et al. Cell paper, while a clinical isolate of ZIKV was used, Ifnar1/−− mice were used to observe the results (Ma, Li et al. What do these results mean from a public health perspective? While the results of these studies are no doubt concerning, they are not a cause for panic. It should be kept in mind that these findings, while novel and unexpected, are results derived from experiments with immunosuppressed mice. In the What these results do mean, however, is the need to broaden and deepen our understanding of ZIKV pathogenesis, but in a way that is more relatable to humans. The first step to achieve this would be to develop an animal model that more accurately recapitulates important aspects of clinical ZIKV disease as observed in humans. In a previous study, a group inoculated rhesus and cynomolgus macaques subcutaneously with ZIKV. While the animals showed no signs of disease, the virus was detected in the blood, urine, cerebrospinal fluid, semen and saliva, and transiently in vaginal fluid (Osuna, Lim et al.


Autophagy is a homoeostatic process by which cytoplasmic material is targeted for degradation by the cell. Viruses have learned to manipulate the autophagic pathway to ensure their own replication and survival. Although much progress has been achieved in dissecting the interplay between viruses and cellular autophagic machinery, it is not well understood how the cellular autophagic pathway is utilized by viruses and manipulated to their own advantage. In this review, we briefly introduce autophagy, viral xenophagy and the interaction among autophagy, virus and immune response, then focus on the interplay between NS-RNA viruses and autophagy during virus infection. We have selected some exemplary NS-RNA viruses and will describe how these NS-RNA viruses regulate autophagy and the role of autophagy in NS-RNA viral replication and in immune responses to virus infection. We also review recent advances in understanding how NS-RNA viral proteins perturb autophagy and how autophagy-related proteins contribute to NS-RNA virus replication, pathogenesis and antiviral immunity. Macroautophagy  is an evolutionarily conserved process that includes the immersion and transport of cytosolic contents to the lysosome for degradation . AccordiOriginally autophagy was identified as a response to starvation, and it was thought of as a non-selective digestion process; However, it is now evident that autophagy specifically degrade aggregated proteins and damaged cellular organelles via autophagy receptors that link cargo to growing autophagosomal membranes . An autoTable 1), several distinct features are displayed as below:Negative-strand (NS)-RNA viruses encompass some of the most significant human and animal pathogens extant, such as Ebola virus (EBOV), influenza virus (IAV), Nipah virus, Newcastle disease virus (NDV) and rabies virus (RABV). Similar to other viral pathogens, NS-RNA viruses overcome the host cell’s defense to ensure their own survival and propagation. Not surprisingly, autophagy plays a critical role in NS-RNA virus replication and/or infection. By dissecting the known interactions between NS-RNA viruses and the cellular autophagy machinery ((i) Some NS-RNA viruses induce autophagy in the absence of viral replication. It is reported that Vesicular stomatitis virus (VSV), either wild-type or UV-inactivated, activated autophagy in Drosophila S2 cells . Further(ii)Rhabdoviridae, lessened SRV9 prompted more autophagosomes to gather rather than pathogenic CVS-11 in an in vitro model, suggesting a role of autophagy in RABV pathogenesis. The autophagic induction pattern may differ between attenuated and virulent NS-RNA virus strains. In the case of measles virus (MeV), attenuated MeV strain induced a first transient wave of autophagy immediately upon infection via a CD46-Cyt-1/GOPC pathway . InteresNotably, while the majority of the NS-RNA viruses induce autophagy for their own benefit during virus infection, some of them block autophagy especially at two checkpoints of the process, i.e., early during autophagosome formation and at the stage of autophagosome fusion with late endosomes or lysosomes. One example is the segmented RNA virus IAV which inhibits autophagosome maturation, leading to increased apoptotic cell death of infected cells . The inhRhabdoviridae, SVCV glycoprotein, instead of viral replication, stimulates the autophagy pathway  by which NS-RNA viruses perturb autophagy remains elusive. A large body of evidence indicates that viral proteins play a critical role in NS-RNA virus-induced autophagy. For paramyxoviruses, viral glycoprotein-mediated membrane fusion triggers autophagy . In addi pathway . Of impo pathway . A numbeFigure 1 the pro-viral and anti-viral functions of autophagy during negative-stranded RNA viral infection), although this conclusion is largely based on in vitro investigations. Therefore in vivo studies on the role of autophagy in NS-RNA virus infection and pathogenesis, especially how autophagy regulates innate and adaptive immune responses to these pathogens, are of great interest and importance, particularly for those most significant human and animal pathogens, such as IAV and NDV. In the following sections, we discuss recent progresses in understanding the role of autophagy in NS-RNA viral replication cycle, ranging from virus entry up to egress.Given the known interplay between NS-RNA viruses and autophagy, how does autophagy impact on NS-RNA virus infection? Also, how do NS-RNA viruses benefit from autophagy? Accumulating evidence indicates that for most NS-RNA viruses, autophagy functions as a proviral mechanism in infected cells  induced autophagy in cell culture model, which plays a role in NDV replication  pathways , a regulator chaperone-mediated autophagy, sequestered EBOV and Marburg (MARV) viruses VP40 away from the site of budding at the plasma membrane, thereby counteracting the function of VP40 in promoting efficient egress and spread of virus particles . In addiin vivo studies. Below we discussed how three important viral proteins from IAV, MeV and VSV play critical roles in virus replication and/or pathogenesis via the interaction with the cellular autophagic machinery.Growing evidence indicates that both viral proteins and host cellular autophagy-related proteins play critical roles in the interplay between NS-RNA virus and autophagy, thereby contributing to virus replication and/or pathogenesis. In one hand, viral proteins such as IAV proteins including M2 have been shown to interact with autophagy-related proteins to affect the life of virus in infected cells. IAV proteins M2, HA, and NS1 take part in starting the formation of autophagosomes in infected cells . Using yin vitro  demonstrated typical primary antibody reactions following immunization against influenza, but they did not produce protective secondary antibody reactions when challenged with IAV, causing high viral loads, broad lung damage, and raised the fatality rate  with IAV, these mice failed to generate protective secondary antibody responses although normal primary antibody responses after immunization against IAV was induced, suggesting that autophagy is necessary for the survival of virus-specific memory B cells in mice and the maintenance of protective antibody responses required to combat IAV infection in vivo. (ii) How different NS-RNA viruses perturb autophagy to augment replication and pathogenesis? Reversely, how autophagy regulates the various aspects of NS-RNA virus replication and propagation? Obviously, our current understanding of the role of the autophagy machinery in the propagation and control of virus infections, the ability of viruses to co-opt the cellular autophagic pathway to establish virulence in vivo is in its infancy. For example, for NS-RNA viruses, the role of autophagy in the assembly of viral components and budding of viral particles remains largely unknown. (iii) It is not known whether the in vitro or animal trials of autophagy modulators can be translated into useful therapeutics against viral infections in humans. Particularly, such approaches should not only enhance augment direct antiviral activity against NS-RNA virus infection, but could also augment natural acquired immune responses and vaccination strategies. Taking these points into consideration, targeting autophagy may lead to the development of a new class of specific antiviral therapies for the treatment of NS-RNA viruses-related diseases. Most of the data regarding the interplay between NS-RNA viruses and host autophagy machinery are obtained in cell cultural model; the question remained whether such interactions are present or relevant during infections SM designed the study. YW, KJ, and SM wrote the manuscript and reviewed the manuscript. KJ and QZ edited the manuscript. CD participated in developing the hypothesis. All authors listed approved the version to be published and have made a substantial and intellectual contribution to the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alouatta caraya) captured in northeastern Argentina. We used 90% plaque-reduction neutralization tests (PRNT90) to analyze 108 serum samples for antibodies to WNV, SLEV, YFV, DENV (serotypes 1and 3), ILHV, and BSQV. Virus genome detection was performed using generic reverse transcription (RT)-nested PCR to identify flaviviruses in 51 antibody-negative animals. Seventy animals had antibodies for one or more flaviviruses for a total antibody prevalence of 64.8% (70/108). Monotypic  and heterotypic  patterns were differentiated. Specific neutralizing antibodies against WNV, SLEV, DENV-1, DENV-3, ILHV, and BSQV were found. Unexpectedly, the highest flavivirus antibody prevalence detected was to WNV with 9 (8.33%) monotypic responses. All samples tested by (RT)-nested PCR were negative for viral genome. This is the first detection of WNV-specific antibodies in black howlers from Argentina and the first report in free-ranging non-human primates from Latin-American countries. Given that no animals had specific neutralizing antibodies to YFV, our results suggest that the study population remains susceptible to YFV. Monitoring of these agents should be strengthened to detect the establishment of sylvatic cycles of flaviviruses in America and evaluate risks to wildlife and human health.Several medically important mosquito-borne flaviviruses have been detected in Argentina in recent years: Dengue (DENV), St. Louis encephalitis (SLEV), West Nile (WNV) and Yellow Fever (YFV) viruses. Evidence of Bussuquara virus (BSQV) and Ilheus virus (ILHV) activity were found, but they have not been associated with human disease. Non-human primates can act as important hosts in the natural cycle of flaviviruses and serological studies can lead to improved understanding of virus circulation dynamics and host susceptibility. From July–August 2010, we conducted serological and molecular surveys in free–ranging black howlers ( Alouatta caraya), a monkey that is highly susceptible to the virus. During 2010 we tested 108 black howlers from Northeastern Argentina for flaviviruses. Most of these animals were negative for Yellow Fever virus but had antibodies to several other flaviviruses. Unexpectedly, the highest specific neutralizing antibody prevalence was to West Nile Virus; these results represent the first evidence of West Nile Virus circulation in a new host in Argentina. Detection of Dengue virus antibodies in black howlers highlights the potential for establishment of a dengue sylvatic cycle, not yet demonstrated in the Americas. We call for strengthening the monitoring of flaviviruses to evaluate risk for wildlife and human health in the region.Flaviviruses are responsible for a growing disease burden in Argentina and other countries in the Americas. Non-human primates, such as monkeys, can be important hosts in the natural cycle of several flaviviruses. Yellow Fever virus outbreaks occurred in Argentina during 2007–2009 in areas of Misiones and Corrientes provinces inhabited by black howlers ( Flavivirus [Emerging and re-emerging diseases are one of the main threats to global public health; 60% of these diseases are zoonoses  and the majority originated from wildlife . The incavivirus . They usSeveral of the most prominent and medically important mosquito-borne flaviviruses were detected in Argentina in recent years: Dengue virus (DENV), St. Louis encephalitis virus (SLEV), WNV and Yellow Fever virus (YFV). During 2016, Zika virus was also detected in Argentina with autochthonous circulation restricted to Tucuman Province .Dengue viruses have emerged as the most important human arboviral pathogens from non-human primate enzootic reservoirs to humans resulting in an urban endemic transmission cycle. In Africa and Southeast Asia the viruses have been maintained in a sylvatic cycle, most likely involving non-human primates as reservoirs. These cycles have not been recognized in South America, but serological studies have suggested a possible secondary amplification cycle involving mammals other than non-human primates. The question of whether mammals maintain DENV in enzootic cycles and can play a role in its reemergence in human populations remains to be answered , 9. ArgeSince 2002 Argentina has experience the re-emergence of SLEV, with febrile illness and encephalitis outbreaks in humans, mainly in temperate areas of the country –14. GenoCulex mosquitoes, but it is not well known in Argentina. Recent studies of vector competence showed that Argentine Culex are competent vectors, but they were characterized as moderately efficient vectors of WNV and less susceptible to infection than US mosquito strains [The isolation of WNV from equines in Argentina in 2006 was the first direct evidence of its circulation in the Southern Cone. Nucleotide sequences showed that the virus belonged to clade 1a of lineage 1 and clustered in a subclade with American strains isolated during 1999–2002 , 22. Det strains .Cebus, Alouatta, and Callithrix. The susceptibility of monkeys to lethal infections of YFV in America has been considered a major indicator for enzootic disease outbreaks in forest areas [Alouatta caraya) [Yellow fever is an infectious disease that remains endemic or enzootic in rainforests of South America and sub-Saharan Africa. The sylvatic yellow fever cycle is maintained by viral circulation between monkeys and diurnally active mosquitoes that breed in tree holes in the forest canopy. Many species of non-human primates are hosts of this cycle. The species most commonly involved in virus transmission are New World monkeys of the genera st areas –27. Sylvst areas , 29. Blast areas –32. Epiz caraya) , 34. Thi caraya) , 36.Viruses and viral disease outbreaks play an ecological role increasingly recognized in populations of wild animals. At least 27 viruses have been reported to infect both humans and wild primates and most of them are classified as emerging threats to human populations , 38. TheDue to the impact of recent yellow-fever epidemics, there was special concern about the status of the black howler, which is the monkey species most affected by epizootics in Argentina. We conducted serological and molecular tests to detect flavivirus circulation in free-living black howlers in Northeast Argentina in 2010.The study was carried out in July–August 2010 in San Cayetano (SC), Corrientes province ; Isla del Cerrito (IC) , and Isla Brasilera (IB), Chaco province  in northeastern Argentina . San CayCaptured black howlers were immobilized with methomidine hydrochloride combined with ketamine hydrochloride, administered via a dart driven by compressed air. To maintain body temperature at optimum conditions, animals were covered with blankets and warm water bottles were placed with them throughout the procedure. From 109 captured black howlers, we collected 108 blood samples. Distribution by provinces was 51 (51.5%) captures in Chaco and 58(48.5%) in Corrientes. Sex, weight, and measurements were recorded . Of the This research complied with the Code of Best Practices for Field Primatology , the guidelines for the ethical treatment of primates (IACUC protocol 09267) and the laws of Argentina . Titers ≥10 were considered positive. Monotypic or heterotypic patterns were differentiated according to whether the animal was positive to one or several flaviviruses, respectively. In heterotypic patterns, interpretation of PRNT data was as follows: animals with a neutralizing antibody titer (PRNT90) ≥ four-fold higher than the other flavivirus titers were considered positive for antibody to that virus.Serum samples were heat inactivated at 56°C for 30 min. Two-fold serial dilutions from 1:10 to 1:2560 of each sample were incubated with 100 plaque-forming units (PFU) of WNV (strain ChimeriVax TM WNV), SLEV (strain ChimeriVax TM SLEV), DENV-1 (strain Hawaii), DEN-3 (strain H87), YFV (vaccine strain 17D-YEL), ILHV  and BSQV . Vital dAnimals with neutralizing antibody titers against multiple viruses without four-fold difference in titer were considered flavivirus antibody positive with no specific virus identified and labeled as “undetermined” flavivirus.90 antibody negative for all the flaviviruses in our panel and 24 animals that were YFV antibody negative. Viral RNA was extracted from 140 uL of serum using QIAamp viral RNA extraction kit  and then generic reverse transcription (RT)-nested PCR was used to identify flaviviruses. This procedure was used to amplify a specific 143-bp fragment of the NS5 gene [The molecular approach was performed on sera from 27 animals that were PRNTNS5 gene . The ampOf the 108 black howlers studied, 64.8% (70/108) had evidence of past flavivirus infection. Monotypic  and heterotypic  patterns were differentiated. The remaining 42% of antibody-positive animals was classified as undetermined for virus identification.90 titers are shown in We identified specific neutralizing antibodies against WNV, SLEV, DENV-1, DENV-3, ILHV and BSQV. Antibody prevalences were 22.2% (24/108 with 9 monotypic responses) for WNV, 10.2% (11/108) for SLEV, 1.85%  for DENV (1/108 DENV-3 and 1/108 DENV-1), 0.93%  for ILHV, and 0.93%  for BSQV. Distribution of PRNTThere were no statistically significant differences in the prevalences of infection for each flavivirus between sexes or among study sites or habitat classes. When antibody distribution was analyzed by age, a statistical difference was observed only for WNV antibody prevalence in adult black howlers (p = 0.0075).In 30 of 70 animals (43%) results were inconclusive because of neutralizing antibody titers against multiple viruses without fourfold difference; those were considered positive for an undetermined flavivirus . We obseFor molecular studies, we selected serum samples from 51 animals. These were 27 animals without flavivirus antibodies and 24 that were YFV antibody negative. All animals analyzed by (RT)–nested PCR were negative for flavivirus genome.90 used to identify specific antibodies in black howler serum samples indicated a variable prevalence for one or more of six of the seven flaviviruses tested including WNV, SLEV, DENV-1, DENV-3, ILHV, and BSQV. No specific immune response to YFV was detected.Our goal was to understand the potential role of free-living black howlers as hosts in the natural cycles of flaviviruses in Argentina. The PRNTThirty-four groups of black howlers have been identified in the study area, in about 3,000 ha, several of these groups have been under behavioral study since 2000 , 44 in uAlouatta guariba clamitans) and the black howler. In these epizootics, the deaths of 65 monkeys were detected in Misiones and Corrientes provinces [Yellow fever outbreaks occurred near this region in November 2007–October 2008, seriously affecting the populations of two howler monkey species: the brown howler , SLEV (1), DENV-3 (1) and ILHV (1); we couldn´t use 4 fold criteria because the lowest dilution studied was 1:10. When individuals with no previous exposure to a flavivirus are infected with one, a monotypic response to the infecting virus is demonstrated in serological tests, such as PRNT, and the etiologic agent can be accurately identified. Interpretation of heterotypic patterns is complex and 4-fold difference in titers could be a limited criterion to provide distinction of the most recent infection. The antibody response in sequential experimentally infected animals illustrates the difficulty for a serologic diagnosis of WNV infection in animals (or humans) with preexisting flavivirus immunity . SequentPrevalence of antibody to WNV antibody was the highest among the flaviviruses evaluated. The monotypic WNV prevalence was 8.33% (9/108) and represents the clearest serologic evidence of WNV activity in a new host in Argentina. We found WNV-positive black howlers at all sampling sites, demonstrating a widespread distribution in the study region.Higher WNV antibody titers were detected in the group with a heterotypic pattern. This type of response could reflect sequential infections or cross reactions originated by the higher antibody titers. We also detected WNV reactivity in 22 black howlers in the group classified as undetermined flavivirus, thus increasing the WNV antibody prevalence. Because of conservation considerations, we avoided capturing immature individuals or pregnant females. Thus, the higher prevalence in adults may reflect this capture bias. However, we should not rule out the possibility that this result could be due to undetected circulation of WNV in Argentina for some time before its isolation in 2006 .infections with SLEV with prevalences of 35% (by hemagglutination test) and 32% (by PRNT). Studies in 2004–2005 demonstrated SLEV antibody prevalence  in free ranging black howlers in the upper Parana River basin in southern Brazil [Prevalence of detectable antibody to SLEV in our study was 11% 12/108) without monotypic reaction. This value would be increased if we considered the animals that were SLEV positive among the group classified as “undetermined flavivirus”. Investigations in black howlers from this zone in 2001 confirmed n Brazil ,18. St. /108 withn Brazil , 14. OurOnly four animals had low neutralizing antibody titers for YFV despite recent epizootics, two of those had specific heterotypic reactions for WNV and SLEV and the others were in the positive group for undetermined flavivirus. These results could represent cross-serologic reaction or past infection. On the other hand, 51 animals were negative for viral genome. We did not detect YFV circulation in black howlers. This small and fragmented population suffers habitat destruction, hunting pressure, and cyclical yellow fever and thus, is at risk of disappearing in the long term . The low90 against DENV-3 (0.93%) and DENV-1 (0.93%). Titers obtained were low but serum samples were previously heat inactivated to eliminate nonspecific reactions. These dengue serotypes were selected because their previous circulation was confirmed in human cases detected in Misiones, Corrientes, and Chaco provinces in 2000–2008. Besides, 47% of the animals were positive to DENV-1 and 53% for DENV-3 in the group of animals labeled us undetermined. As we mentioned previously, these results could be originated by cross reaction or they could represent limited spillback through contact with human environment, but we have to consider that other studies suggest the presence of sylvatic DENV in the Americas [We detected monotypic reactions in PRNTAmericas , 49, 50.Ilheus virus is believed to be maintained in zoonotic cycles between birds and mosquitoes and has been isolated in Central and South America primarily from mosquitoes but also from sentinel monkeys and birds . There aOur work evidences circulation of WNV, SLEV, DENV-1, DENV-3, ILHV, and BSQV in wild, non-human primate populations of Corrientes and Chaco provinces, Argentina. Future studies might include ZIKV detection, which has been recognized in South America since 2015. To our knowledge this is the first detection of WNV-specific antibodies in black howlers from Argentina and the first report in free-ranging non-human primates from Latin America. Additionally, our results show that our study population remains susceptible to YFV as no specific neutralizing antibodies were detected. The black howler population remains tentative in the upper Paraná Atlantic Forest. Recurrence of YFV circulation or other pathogens could be detrimental to the population’s existence. Improved monitoring of these agents is needed to evaluate risk to wildlife and human health in the region.


With limited and low-genetic barrier drugs used for the prevention of mother-to-child transmission (PMTCT) of HIV in sub-Saharan Africa, vertically transmitted HIV-1 drug-resistance (HIVDR) is concerning and might prompt optimal pediatric strategies.The aim of this study was to ascertain HIVDR and viral-tropism in majority and minority populations among Cameroonian vertically infected children.A comparative analysis among 18 HIV-infected children (7 from PMTCT-exposed mothers and 11 from mothers without PMTCT-exposure) was performed. HIVDR and HIV-1 co-receptor usage was evaluated by analyzing sequences obtained by both Sanger sequencing and ultra-deep 454-pyrosequencing (UDPS), set at 1% threshold.10 copies/mL, and 526 (282–645) cells/mm3, respectively. All children had wild-type viruses through both Sanger sequencing and UDPS, except for 1 PMTCT-exposed infant harboring minority K103N (8.31%), born to a mother exposed to AZT+3TC+NVP. X4-tropic viruses were found in 5 of 15 (33.3%) children (including 2 cases detected only by UDPS). Rate of X4-tropic viruses was 0% (0/6) below 5 years , and became relatively high above 5 years  versus CD4 >15% ; similarly for CD4 ≤200 (3/4 [75%]) versus CD4 >200  age, viremia, and CD4 count were 6 (4–10) years, 5.5 (4.9–6.0) logNGS has the ability of excluding NRTI- and NNRTI-mutations as minority species in all but 1 children, thus supporting the safe use of these drug-classes in those without such mutations, henceforth sparing ritonavir-boosted protease inhibitors or integrase inhibitors for the few remaining cases. In children under five years, X4-tropic variants would be rare, suggesting vertical-transmission with CCR5-tropic viruses and possible maraviroc usage at younger ages. From these observations, we postulated that minority DRMs in HAART-naïve children might grow-up through selective drug-pressure and populate plasma in a short-frame, herein justifying the rapidly emerging DRMs we observed at failure. Although not yet clinically endorsed, pediatric minority DRMs might be more concerning in the context of PMTCT, henceforth underscoring an unmet clinical need.,11 Coupled to previous knowledge on the detection of DRMs by next-generation sequencing (NGS),–14 we thus hypothesized that using NGS to assess DRMs in vertically infected HAART-naïve children would contribute in designing long-term HAART strategies for SSA-children.As the footprint of long-term HAART depends largely on the effectiveness of first-line drugs in sustaining viral suppression, establishing adequacy between pediatric HAART and DR-mutations (DRMs) would be clinically relevant. As HAART would be reaching 1.5 million children by 2020, as high as 20% virological failure (VF) is expected, favored by high-viremia and poor adherence in children.,16 Without optimal strategies, VF would quickly overcome HAART success, maintaining children vulnerable.Current pediatric HAART-regimens consist of lamivudine (3TC), abacavir (ABC), or zidovudine (AZT), associated to ritonavir-boosted lopinavir (LPV/r) or NVP. LPV/r is recommended to overcome PMTCT-resulting non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance, whereas NVP matches with postnatal prophylaxis.,8 Although not yet approved for under 16 years, the CCR5 antagonist—maraviroc—might represent a suitable antiretroviral alternative for children, pending proof-of-concept towards relevant pediatric clinical trials. Particularly, there are limited evidence on the potential effectiveness of maraviroc for SSA-children in PMTCT, initial-HAART and/or following treatment-failure.–21 With rising concerns of minority variants on response to several classes of antiretrovirals, a genuine delineation of HIV-1 tropism, considering both minority and majority quasi-species,,23 could rationalize maraviroc suitability for pediatric HAART-policies in SSA.Moreover, pediatric HAART options are limited in SSA, urging the quest for a wider therapeutic portfolio.Based on these assumptions, we aimed to ascertain DRMs and HIV-1 co-receptor usage, in majority and minority viral populations, from children according to maternal PMTCT-exposure in a resource-limited setting (RLS).22.1A comparative study was conducted in 2015 among 18 HIV-1 vertically infected Cameroonian children, all HAART-naïve, stratified according to maternal antiretroviral exposure during pregnancy: control-group  versus case-group (7 children from mothers exposed to reverse transcriptase inhibitors [RTIs]). For each child, a plasma sample was collected to perform both Sanger- and 454 ultra-deep pyrosequencing (UDPS).2.2 Briefly, viral RNA was extracted from plasma using QIAamp Viral RNA minikit , following manufacturer's instructions. PR/RT-containing region was then reverse-transcribed and amplified using SuperScript One-Step for long templates reverse transcriptase polymerase chain reaction (RT-PCR) of Invitrogen kit , with an eventual second-round seminested PCR. Direct sequencing was then performed using 7 overlapping primers.Protease (PR)/RT Sanger sequencing was performed as previously described. Briefly, viral RNA containing the V3-loop region was reverse-transcribed and amplified using an RT/Taq mix, with an eventual second-round seminested PCR. Direct sequencing was then performed using 4 overlapping primers.V3 loop Sanger sequencing was performed as previously described.2.32O DNase RNase free, 1 μL forward primer (10 μmol/L), 1 μL reverse primer (10 μmol/L), 1 μL RNase Out (40 U/μL Invitrogen) and 1.2 μL RT/TAQ, for a final volume of 50 μL. RT-PCR conditions were the following: 1 cycle 50°C, 30 minutes; 1 cycle 94°C, 2 minutes; 40 cycles ; a final extension 68°C, 10 minutes. Forward and reverse primers were respectively 5’GACAGGCTAATTTTTTAGGG3’  and 5’GATAAATTTGATATGTCCATTG3’ . Nested-mid PCR was then performed with the Fast Start HiFi PCR system  using 5 pairs of barcoded-modified forward and reverse primers for each amplicon , 8 μL MgSO4 (5 mmol/L), 2.8 μL H2.4Ten microliters viral RNA were reverse transcribed with 1-step RT-PCR system using forward  and reverse  primers, under the following conditions: 1 cycle 50°C, 30 minutes; 1 cycle 94°C, 2 minutes; 35cycles ; a final extension 68°C, 10 minutes. A nested mid-PCR was then performed with the Fast Start HiFi PCR system  as previously described.2.5PR/RT PCR products  and V3 loop (one fragment of 367 bps) were purified using Agencourt AMPure PCR purification beads  and quantified with Quant-iT PicoGreen double-stranded DNA assay kit  on a GloMax multidetection system . Phylogenetic analyses excluded any possible sample contamination (data not shown).Pooled purified PCR products were clonally amplified by emulsion PCR and pyro-sequenced on the 454 GS junior platform  as previously described.2.6The entire PR (amino acid position: 1–99), RT (1–251) and the entire V3 loop (1–35) sequences obtained after 454-pyrosequencing were de-multiplexed and then quantified using the SFF tool Roche. Using a home-made Perl script and SHORAH package 0.5.1, sequences were filtered and corrected for homopolymeric region-associated errors and aligned against HIV-1 consensus B. Final alignments were manually checked for insertion or deletion in homopolymeric regions that could result in a frame shift. Nucleotidic/aminoacidic variants were evaluated and quantified by a home-made pearl script, and sequences were considered reliable when showed an intra-patient frequency ≥1% in both forward and reverse strands.2.7http://hivdb.stanford.edu/pages/download/resistanceMutations_handout.pdf) and geno2pheno.v2.5 (http://coreceptor.geno2pheno.org/), respectively. Using a quantitative interpretation, viruses were considered CXCR4-tropic (X4-variants) by UDPS when ≥2% viral species had a false-positive rate (FPR) ≤3.5%, or by Sanger sequencing when FPR was ≤10%, describing the probability of classifying an R5-virus falsely as an X4-variant.PR/RT DRMs and HIV-1 co-receptor usage were interpreted using Stanford HIVdb list  available at 2.9HIV-1 DRMs and coreceptor usage were compared between the two PMTCT-groups. Coreceptor results by Sanger sequencing and UDPS were considered concordant if viral-tropism was identical from both sequencing technologies. Viral-tropism was explored according to age and CD4 count.P values <.05 were considered statistically significant.All statistical analyses were performed using the statistical open source environment R.v.3.1.1. 2.10Ref. N°034/NEC/SE), proxy-informed consent was provided, unique identifiers were used for privacy and confidentiality, and a material transfer agreement was established.Ethical clearance was obtained from the Cameroon National Ethics Committee  cells/mm3, respectively, without any significant difference between the 2 groups (data not shown). In the control, neither children nor their mothers had any antiretroviral exposure. Antiretroviral history of children belonging to the case-group, considered at higher risk of HIVDR, is described in Table Overall, median (interquartile range [IQR]) age, viremia, and CD4 count were 6 (4–10) years, 5.5 (4.9–6.0) log3.2HIV-1 subtyping revealed 50% CRF02_AG (9/18), 33.3% F (6/18), 11.1% CRF01_AE (2/18), and 5.6% CRF11.cpx (1/18).3.3PR/RT sequences were successfully obtained both through Sanger sequencing and UDPS for 17/18 children. The median UDPS coverage was of 1642 (IQR: 1269–5193) reads. In the entire covered PR/RT regions, the 2 sequencing technologies showed total concordance in variants detection, and all UDPS variants with frequencies <20% were not detected by Sanger sequencing , an accessory polymorphism weakly selected under etravirine (ETR) and rilpivirine (RPV), was found in a child aged 8 years from the control group.By using UDPS, 1 (aged 1 year) of 7 children 14.3%) from the case-group harbored viruses with K103N , a nonpolymorphic mutation causing high-level resistance to NVP and efavirenz (EFV). This infant was born from an RTI-treated mother (AZT + 3TC + NVP). Thus, Sanger sequencing and UDPS were performed also for the mother (ID-18613). UDPS revealed a virus harboring 2 major DRMs: L74 V at minority-level (2.5%), causing high- and intermediate-level resistance respectively to didanosine and to ABC; Y181C at population-level (96.7%), causing high- and intermediate-level resistance respectively to NVP and to EFV, ETR, and RPV , a polymorphic accessory mutation selected under EFV, in a child aged 6 years Table .Other variants, found even at RTI-associated drug resistance positions, were with minimal or no effect on drug susceptibility or virological response. Of note, in either group, no major DRMs to ritonavir-boosted protease inhibitors (PI/r) were found by both Sanger sequencing and UDPS.3.4V3 loop sequencing was successful by both Sanger sequencing and UDPS for 15 of 18 children and the mother ID-18613, with an overall viral-tropism concordance of 87.5% 14/16) between Sanger sequencing and UDPS (Table /16 betweX4-tropic viruses were found in 5 of 15 (33.3%) children (including 2 cases detected only by UDPS), all aged above 5 years. Specifically, in 1 child (ID-11621) UDPS provided an added value in tropism-determination compared to Sanger sequencing. Indeed, a clinically relevant quantity of minority X4-tropic variants (frequency: 3.9%) was detected by UDPS in this child . In another child (ID-10196), despite an R5-tropism (FPR = 79.7%) determined by Sanger sequencing, a discordant tropism was observed through UDPS with a high percentage of X4-tropic variants , because of insertions detected only at minority levels.P = .040). As expected, X4-tropic viruses were higher with CD4 ≤15% (4/9 [44.4%]) versus CD4 >15% ; similarly for CD4 ≤200 (3/4 [75%]) versus CD4 >200 . No statistical difference was found in X4-variants between the 2 PMTCT-groups: 2 of 7 (28.6%) case group versus 3 of 8 (37.5%) control group, P = 1.000.Of relevance, the rate of X4-tropic viruses was 0% (0/6) among children under 5 years , and became significantly higher as from 5 years and above , known to be associated with resistance to NNRTIs used both for PMTCT and first-line HAART in SSA, was found in a PMTCT-exposed infant, thus suggesting NNRTI-sparing regimens for such children.,30,34 Discrepancy in DRMs between mother and infant would be due to sample collection later after delivery (at the moment of infant HIV diagnosis), with possible selection following prophylaxis/breastfeeding; as previously reported in similar RLS . This infant (aged 1 year), compared to the median age of the study population (6 years), suggests that circulating DRMs might have fade-up with increasing age.,33 NNRTI mutations (E138A and V179D), found in children without PMTCT-exposure, are known as polymorphisms with little or no effect on drug susceptibility or virological response. The ability of NGS in excluding minority RTI-mutations  supports the safe use of NNRTIs/NRTIs in those without such mutations, thus sparing from inappropriate switch to PI/r- or integrase inhibitor-containing regimens.,17,33–35In this high CRF02_AG-infected population,,37 as well as a baseline FPR <60 as previously demonstrated.,39 Further investigations might help in establishing novel public health strategies for an eventual usage of maraviroc in children.,40 As current PMTCT-practice might not be an independent factor for viral-tropism , CCR5-antagonist (maraviroc) could be a useful therapeutic weapon for pediatric HAART.,18,40Coreceptor usage in these children provides a clue for clinical application. Indeed, X4-variants appeared to be associated with older ages and lower CD4 cells, suggesting limited vertical transmission by CXCR4-tropic viruses, and later appearance of X4-variants with chronicity, immunological impairment,,39 Interestingly, by detecting minority insertions associated with a complete discrepant result on Sanger sequencing, UDPS appears very useful in validating tropism determination for non-B subtypes.Of the two children showing discordant results between the two sequencing techniques, the added value of UDPS in detecting X4-tropic minority variants is in accordance with previous reports.,22,41Therefore, UDPS might provide additional information in detecting DRMs and viral-tropism, confirming the added value of this technology for both clinical diagnostics and management of non-B HIV-infected children.,43In spite of this added value of UDPS, implementing NGS is more challenging in RLS , suggesting the need for simpler and affordable approaches integrating minority variants (point-of-care or pragmatic sequencing).,13,44–46 This study therefore provides relevant data to be used as base for further/enlarged studies.A potential study limitation could be the relatively small sample size, which makes the study probability relatively large. Also, in the PMTCT-exposed group, only 3 of 7 were exposed to triple ART, calling for subsequent investigations with scale-up of option B+. Moreover, HIV-1 variants were investigated only in plasma compartment, suggesting the need for exploring HIV variability in several compartments  and the impact on treatment and monitoring strategies in SSA.In a nutshell, NGS could help in identifying PMTCT-exposed children harboring minority NNRTI-DRMs, therefore serving for a timely switch of treatment and limiting failure rate. NGS also reveals a possible absence of X4-variants among children below 5 years, thus suggesting possible public health approaches using maraviroc. These preliminary evidences, generated on a small sample of mainly CRF02_AG-infected individuals, merit further investigations for improved pediatric-HAART strategies in RLS.Conceptualization: A. Nanfack, C-F. Perno, C. Fokunang, C. Tangimpundu, D. Armenia, D. Takou, E. Temgoua, F. Ceccherini-Silberstein, G. Cappelli, J. Fokam, M-M. Santoro, P. Koki, V. Colizzi.Data curation: D. Armenia, F. Ceccherini-Silberstein, J. Fokam, L. Carioti, M. Bellocchi, M-M. Santoro.Formal analysis: D. Armenia, F. Ceccherini-Silberstein, J. Fokam, L. Carioti, M. Bellocchi, M-M. Santoro.Funding acquisition: C-F. Perno, J. Fokam, V. Colizzi.Investigation: A. Ndjolo, A. Nanfack, C-F. Perno, C. Fokunang, C. Tangimpundu, D. Takou, E. Temgoua, F. Ceccherini-Silberstein, G. Cappelli, J. Fokam, J. Torimiro, M-M. Santoro, P. Koki, V. Colizzi.Methodology: D. Armenia, D. Takou, F. Continenza, F. Ceccherini-Silberstein, J. Fokam, L. Carioti, M. Bellocchi, M-M. Santoro.Project administration: A. Ndjolo, C-F. Perno, C. Fokunang, F. Ceccherini-Silberstein, G. Cappelli, J. Fokam, J. Torimiro, P. Koki, V. Colizzi.Resources: A. Ndjolo, C-F. Perno, J. Fokam, V. Colizzi.Software: D. Armenia, D. Takou, F. Continenza.Supervision: A. Ndjolo, C-F. Perno, C. Fokunang, F. Ceccherini-Silberstein, G. Cappelli, M-M. Santoro, P. Koki, V. Colizzi.Validation: A. Ndjolo, C-F. Perno, C. Fokunang, C. Tangimpundu, D. Armenia, E. Temgoua, F. Continenza, F. Ceccherini-Silberstein, G. Cappelli, J. Fokam, J. Torimiro, L. Carioti, M. Bellocchi, M-M. Santoro, P. Koki, V. Colizzi.Visualization: A. Nanfack, C-F. Perno, C. Tangimpundu, D. Takou, E. Temgoua, F. Continenza, J. Fokam, M-M. Santoro.Writing – original draft: C-F. Perno, F. Ceccherini-Silberstein, J. Fokam, M-M. Santoro.Writing – review & editing: A. Ndjolo, A. Nanfack, C. Fokunang, C. Tangimpundu, D. Armenia, D. Takou, E. Temgoua, F. Continenza, G. Cappelli, J. Torimiro, L. Carioti, M. Bellocchi, P. Koki, V. Colizzi.We are appreciative to our institutional staff that participated locally in the enrolment and in sample processing. We thank Domenico Di Carlo for statistical analyses.


We identified influenza A(H9N2) virus G1 lineage in poultry in Burkina Faso.Urgent actions are needed to raise awareness about the risk associated withspread of this zoonotic virus subtype in the area and to construct a strategyfor effective prevention and control of influenza caused by this virus. Since their detection in China in 1992, influenza A(H9N2) viruses have caused largeeconomic losses to the poultry industry and have occasionally been transmitted tomammalian species, including humans. Three main genetic lineages were described amongthe Eurasian H9N2 subtype viruses: G1, Y280, and Y439 (Korean) lineage . All samples were collected from a layer farm that wasexperiencing decreased egg production and respiratory signs among its flock; the animalswere suspected to have infectious bronchitis virus (IBV).Molecular analyses of the animal samples showed negative results for IBV and indicatedthat animals from the farm were infected with avian influenza A(H9N2) virus. The 8 genesegments were obtained for 1 representative virus by using a MiSeq Platform . Sequences were submitted to GenBank under accession numbersMF510849–56.The maximum-likelihood phylogenetic tree of the hemagglutinin (HA) gene showed that theH9N2 subtype virus from Burkina Faso belonged to the G1 lineage, which has remarkablezoonotic potential. This virus clustered with H9N2 subtype viruses isolated in Moroccoin 2016 (99.2% similarity) and with an H9N2 subtype virus identified in the United ArabEmirates in 2015 (A/chicken/Dubai/D2506.A/2015) (98.7% similarity)  Figure 1Phylogeographic analysis  identified multiple introductions ofinfluenza A(H9N2) virus into North Africa from the Middle East and Pakistan. The H9N2subtype virus identified in Burkina Faso seems to have originated from Morocco, althoughwe cannot rule out the possibility that H9N2 subtype viruses were circulating inunsampled locations .Analyses of the deep sequencing data showed that ≈50% of the virus populationin the tracheal swab specimen had leucine at position 226 (H3 numbering) of the HAreceptor binding site ,which enables preferential binding to human-like α2–6-linked sialicacid receptors  have been widely circulatingsince the beginning of 2015, is a concern because of animal health implications,negative effects on local economies, and possible emergence of reassortant viruseswith unknown biological properties. Reassortment events between H9N2 and highlypathogenic H5N1 subtype viruses were reported in China in 2005 and 2016  virus, Burkina Faso.


Encephalitis causes significant global morbidity and mortality. A large number of viruses cause encephalitis, and their geographic and temporal distributions vary. In many encephalitis cases, the virus cannot be detected, even after extensive testing. This is one challenge in management of the encephalitis patient. Since cytokines are pivotal in any form of inflammation and vary according to the nature of the inflammation, we hypothesized cytokine levels would allow us to discriminate between encephalitis caused by viruses and other aetiologies. This pilot study was conducted in a tertiary care hospital in Dhaka, Bangladesh. Viral detection was performed by polymerase chain reaction using patient cerebrospinal fluid. Acute phase reactants and cytokines were detected in patient serum. Of the 29 biomarkers assessed using the Wilcoxon rank-sum test, only vascular endothelial growth factor (VEGF) was significantly higher (P = 0.0015) in viral-positive compared with virus–negative encephalitis patients. The area under the curve (AUC) for VEGF was 0.82 . Serum VEGF may discriminate between virus-positive and virus-negative encephalitis. Further study will be needed to confirm these findings. In developing countries, the situation is more severe; in India, the incidence is 20.2 cases/100,000/year5, and in Vietnam, 30% of hospitalized children with encephalitis die and 25% develop sequelae6. In 59–89.9% of encephalitis cases in developing countries, the aetiology is unknown10. Indeed, this is also true in industrialized countries for 53.3–69.8% cases14. Although >100 viruses are known to cause encephalitis15, their distribution varies across regions. In North America, the UK, Australia, and other areas in the West, herpes simplex virus (HSV) followed by varicella-zoster virus (VZV) are primarily responsible for encephalitis15. However, this profile is changing in some countries. For example, in France, the primary encephalitis-causing virus is enterovirus followed by HSV16, and in the US, cases of autoimmune N-methyl-D-aspartate receptor (NMDAR) encephalitis are now outnumbered by those caused by infectious agents17. In developing countries, the primary contributing virus also differs. For example, the primary cause is dengue virus followed by HSV type-1 (HSV-1) in Brazil18, adenovirus followed by mumps virus in Malawi19, Japanese encephalitis virus followed by enterovirus in Vietnam6, enterovirus followed by measles virus in India7, and adenovirus followed by human bocavirus (HBoV) in Sri Lanka10. Therefore, determining the aetiology for viral encephalitis is a challenge that requires comprehensive improvement. New methods such as virus macroarray20 and next-generation sequencing22 are promising approaches; however, these methods do have limitations such as the need for rapid diagnostics in resource-poor settings. Therefore, a simple, practical biomarker is needed to aid in the diagnosis of viral encephalitis.Acute encephalitis is an inflammation of the brain that requires hospitalization and can lead to severe sequelae, as well as high mortality rates. In industrialized countries, the incidence of encephalitis ranges from 5.23–7.3 cases/100,000 population/year23. Cytokines levels usually exhibit remarkable association with certain human diseases, which highlights their potential utility as encephalitis biomarkers. Many central nervous system (CNS) infections induce common pro-inflammatory cytokines, including interferon-γ (IFN-γ), tumour necrosis factor (TNF)-α, IFN-γ induced protein (IP)-10, monokine induced by gamma interferon (MIG)23. The cytokine spectrum in CNS-infections varied in different studies possibly due to the differences in etiology, age, stage of infection, degree of inflammation, source of sample tested , or method used for measuring cytokines . The pattern of cytokine elevation may differ depending on the type of viral encephalitis. For example, IFN-γ and TNF-α are elevated in HSV encephalitis, whereas interleukin (IL)-1b is increased in enterovirus encephalitis23. However, IFN-γ is not elevated in influenza encephalopathy and human herpesvirus (HHV)-623.Cytokines constitute promising and relevant biomarker candidates for detecting viral encephalitis because they are central to any inflammatory process, and encephalitis is a brain parenchyma inflammation caused by infection, post-infection complication, or an autoimmune processTo date, no cytokines that can differentiate between virus-positive and –negative cases have been identified. Such a test would likely have a considerable impact on patient management. Therefore, we hypothesized that differences between certain cytokines may differentiate between encephalitis induced by viruses compared with encephalitis induced by other causes. The purpose of the present pilot study was to identify cytokines and determine the serum levels that would allow us to discriminate between virus-positive and-negative encephalitis cases.A total of 120 children with acute encephalitis were enrolled in the study. CSF and serum samples were able to be collected from 102 children at the acute stage of infection. However, complete demographic and clinical data only were available for 97 samples; therefore, these were used in the present study. The male to female ratio was 1.7:1. The children ranged in age from 2–114 months, with a mean age of 46.9 months.A total of 18 (18.5%) infection causes were identified , 51 for procalcitonin, and 37 each for the other cytokines. The median and the 25th and 75th percentile values of the concentration of each biomarker in virus-positive and virus-negative samples are presented in Table Receiver operating characteristic (ROC) curve analysis was conducted to obtain the area under the curve (AUC) and validate the ability of VEGF to discriminate between virus-positive and virus-negative samples. The overall AUC for VEGF was 0.82  and procalcitonin in meningitis diagnosis have been previously reported23.Using only clinical features without laboratory support, the specific etiologic agents of encephalitis cannot be determined. Furthermore, the microbiology of encephalitis is not static, and findings from one study period may not be reproducible among the same population in future years23. In the present study, these cytokines were also elevated compared with normal physiological levels (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6029.pdf), indicating inflammation and host immune reaction to disease. The novel finding of this study is that VEGF serum level is significantly higher in patients with virus-positive compared with virus-negative encephalitis.Many CNS infections induce common pro-inflammatory cytokines such as Th1-related  and other cytokines, including interleukin (IL)-1ra, IL-1b, IL-6, and IL-8, are often elevated, suggesting activation of the lymphocytes in the CNS that participate in viral clearancein vitro, VEGF increases the permeability of brain microvascular endothelial cell monolayers by downregulating tight junction proteins, disrupting cell-cell adhesion, and inducing endothelial fenestrations25. In addition, it remains unclear whether increased VEGF during viral encephalitis is a host induced protective mechanism for blood-brain barrier disruption because VEGF also plays important roles in wound healing and tissue cytoprotection by stimulating vascular angiogenesis, permeability, and remodelling26.It remains unclear whether the VEGF increase in viral encephalitis is induced by viruses as a part of the pathogenesis. However, 27, which is consistent with our observation. Other evidence indicates that VEGF in CSF significantly increases in bacterial meningitis, including active tubercular meningitis29.To our knowledge, the utility of serum VEGF in differentiating between virus-positive and virus-negative encephalitis has not been previously reported. However, compared with serum from uninfected controls, VEGF has been reported to be significantly increased in tick-borne encephalitis28. Another limitation was that we did not use a next-generation sequencer to detect novel viruses. However, we expect that the number of novel viruses among the total cases would have been too small to affect our overall findings30.One limitation of our study was that, due to the insufficient number of samples we were unable to determine the level of cytokines in CSF. Therefore, the dynamics of VEGF in CSF during encephalitis remain unclear. Contrary to our finding of viral encephalitis, serum VEGF is decreased in bacterial meningitis28 in the clinical course and outcome of virus-positive encephalitis. Larger studies are needed to validate the usefulness of serum VEGF concentrations in accurately predicting the presence of viruses in the CSF of encephalitis patients.We identified serum VEGF as a possible biomarker for differentiating between virus-positive and virus-negative encephalitis. This distinction may facilitate patient management by guiding decisions about administering antiviral drugs. Our observation also supports the need to determine the effects of anti-VEGF treatmentWe conducted an observational, non-interventional study of paediatric patients from April 2010 through August 2012 at the Institute of Child and Mother Health (ICMH) Hospital in Matuail, Dhaka, Bangladesh. This tertiary care hospital serves approximately 4.8 million people in its catchment area and has 200 beds, among which, 85 are dedicated to paediatrics. The study was approved by the ICMH ethics committee. All experiments were performed in accordance with relevant guidelines and regulations. Children with encephalitis admitted to the paediatric ward during the study period were enrolled. Encephalitis was defined as presence of fever, convulsion, and unconsciousness with or without signs of meningeal irritation. Samples were collected from children whose guardians provided verbal consent for their participation. Only samples remaining after the completion of routine tests were used for this study.CSF samples were subjected to macroscopic examination, total and differential white blood cell (WBC) counts, bacterial culture, Gram staining and measurement of protein and glucose levels. Blood was cultured for bacteria and examined for total and differential WBC counts, ESR, and haemoglobin and CRP levels.Genomic DNA and RNA were extracted using a QIAmp viral RNA mini kit  and Trizol reagent  according to the manufacturers’ instructions.10. Dengue, Japanese encephalitis, West Nile, yellow fever and tick-borne encephalitis viruses were tested using universal flavivirus primers10. Nipah, measles, mumps, parainfluenza, respiratory syncytial and metapneumoviruses were tested using Paramyxovirinae and Pneumovirinae primers10. Chikungunya, Sindbis, Semliki forest, Eastern and Western equine encephalitis viruses were tested using generic alphavirus primers10. Poliovirus, coxsackievirus, and echovirus were tested using human enterovirus-specific primers. Rabies virus, Chandipura virus, rotavirus, astrovirus, HBoV, norovirus, and enteric adenovirus were detected using specific primers10. Bufavirus30, Merkel cell polyomavirus31, WU virus32, KI polyomavirus32, trichodysplasia spinulosa-associated polyomavirus33, cardiovirus34, human parechovirus36, and Liao ning virus37 were also tested using respective primers. Bacteria were detected via PCR using primers for the 16S rRNA gene10.Altogether, we tested for 41 encephalitis-causing viruses using previously published methods. HSV-1, HSV-2, cytomegalovirus, VZV, (Human herpes virus) HHV-6, HHV-7, and HHV-8 were tested using universal HHV primersNucleotide sequences of the PCR products were determined to confirm the results and distinguish viral types. The amplicons were sequenced with an ABI Prism 3130 Genetic Analyzer  using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), according to the manufacturer’s instructions.Serum levels of human IL-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, eotaxin, fibroblast growth factor basic, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-CSF, IFN-γ, chemokine (C-X-C motif) ligand (CXCL)-10 (IP-10), chemokine (C-C motif) ligand (CCL)-2 [Monocyte chemoattractant protein (MCP)-1], CCL-3 [Macrophage inflammatory protein (MIP)-1a, CCL-4 (MIP-1b), platelet derived growth factor, CCL-5 (RANTES), TNF-α, and VEGF in 14 virus-positive and 24 virus-negative patients were measured in duplicate using a multiplex sandwich immunoassay-based protein array system  according to the manufacturer’s instructions.http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) using hierarchical clustering, and then visualized using Java TreeView (http://jtreeview.sourceforge.net/).To reveal cytokine profiles in patients with viral encephalitis, we performed cluster analysis on the virus status of samples (positive or negative) and serum cytokine levels. This analysis employed clustering in two ways based on: (i) serum cytokine level and (ii) presence or absence of any virus in the samples. The results are represented as a heatmap. Data were formatted using Microsoft Excel . Clustering was done by Cluster 3.0 . For Wilcoxon rank-sum tests, we considered a difference significant if the obtained P-value was smaller than the Bonferroni-adjusted α of 0.0017.The ROC curve was used to obtain the AUC in order to evaluate whether each cytokine can be used to make a distinction between the two etiological groups. In addition, for significant candidates, the Youden index was calculated to identify the point of maximum sensitivity and specificity and to describe its potential effectiveness. All statistical analyses were performed using Stata 14.0 .


Recognizing that infectious agents readily cross international borders, the International Health Regulations Emergency Committee issues Temporary Recommendations (TRs) that include vaccination of travelers from countries affected by public health emergencies, including serotype 1 wild polioviruses (WPV1s). This analysis estimates the costs and benefits of TRs implemented by countries with reported WPV1 during 2014–2016 while accounting for numerous uncertainties. We estimate the TR costs based on programmatic data and prior economic analyses and TR benefits by simulating potential WPV1 outbreaks in the absence of the TRs using the rate and extent of WPV1 importation outbreaks per reported WPV1 case during 2004–2013 and the number of reported WPV1 cases that occurred in countries with active TRs. The benefits of TRs outweigh the costs in 77% of model iterations, resulting in expected incremental net economic benefits of $210 million. Inclusion of indirect costs increases the costs by 13%, the expected savings from prevented outbreaks by 4%, and the expected incremental net benefits by 3%. Despite the considerable costs of implementing TRs, this study provides health and economic justification for these investments in the context of managing a disease in advanced stages of its global eradication. Between May 2014 and the end of 2016, the IHREC for polio issued TRs to five countries experiencing WPV1 transmission  Few studies estimate the costs and benefits of traveler recommendations for infectious diseases 2We focus on the costs and benefits of the TRs during the 3 years 2014–2016 because the IHREC for polio issued the first TRs for polio in May 2014. We consider the possibility of prevented outbreaks (defined as one or more reported polio cases linked to a WPV1 importation into a previously polio-free country and excluding circulating vaccine-derived poliovirus (cVDPV) outbreaks) for up to 10 years  and the expected lifetime societal benefits of prevented polio cases. We report all monetary outcomes in year 2015 US dollars ($) and discount using a rate of 3% We dynamically and probabilistically account for the relationships depicted in We assume that the number of WPV1 importation outbreaks in any given month follows a Poisson distribution with a rate equal to the number of reported WPV1 cases in countries that implemented TRs , multiplWe performed 1000 stochastic iterations of the model with a monthly time step for the outbreak simulation. Each iteration involves both random realizations from all uncertain model inputs and random realizations of outbreaks, which depend on the realized outbreak rate per reported WPV1 case and the delay between exportations and onset of paralysis of the first case.3With all model inputs at their best estimates , the dirWe conducted several univariate sensitivity analyses on the expected INBs (including indirect costs). We observed the greatest impact from changing the basis for outbreak simulations from the last 10 years to 5 years before 2014, during which more outbreaks occurred per reported WPV1 case, but these outbreaks generally remained smaller and shorter in duration see . This ch4The full characterization of the costs of implementing TRs reveals significant expected direct costs of over $20 million over 3 years . For perspective, in 2016, Pakistan reported total annual immunization costs of $235 million Besides the quantified expected net benefits of the TRs demonstrated in this analysis, the TRs also provide a means to account for the negative externalities that countries that sustain WPV1 transmission impose on other countries by increasing the global risk of WPV1 importation outbreaks. Although countries may not perceive benefits of implementing the TRs within their borders, doing so produces real benefits for other countries. In addition, countries that aggressively implement TRs may also reap some benefits within their borders by effectively reaching seasonal migrant populations that play an important role in sustaining WPV1 transmission .Although during 2014–2016 no outbreaks occurred in polio-free countries due to virus exported from countries that implemented TRs, we cannot know whether outbreaks would have occurred without implementation of the TRs, thus introducing inherent uncertainty. Similarly, we cannot know whether TRs will continue to prevent them. If WPV1 circulation continues, then it appears likely that eventually exportations may occur in spite of the TRs . Further, if WPV1 circulation continues in endemic countries and if polio-free countries do not sustain high enough vaccination coverage to protect themselves, then any delay in importations associated with the TRs in endemic countries will imply potentially more explosive outbreaks when the importation occurs . Thus, despite the expected benefits of TRs, the most important strategy to prevent WPV1 importations remains sustaining high enough population immunity to transmission in all countries.The TRs differentiate “states currently exporting wild poliovirus or cVDPV,” which must “ensure that all residents and long-term visitors (i.e. >four weeks) of all ages, receive a dose of oral poliovirus vaccine (OPV) or inactivated poliovirus vaccine (IPV) between four weeks and 12 months prior to international travel” from “states infected with wild poliovirus or cVDPVs but not currently exporting,” which should merely “encourage residents and long-term visitors to receive a dose of OPV or IPV four weeks to 12 months prior to international travel We highlight several limitations with a potential large impact (see The global removal of all serotype 2-containing OPV launched an era of unprecedented low population immunity to serotype 2 transmission


Zika virus is normally transmitted by mosquitos, but cases of sexual transmission have been reported. We describe a patient with symptomatic Zika virus infection in whom the virus was detected in semen for 92 days. Our findings support recommendations for 6 months of barrier contraceptive use after symptomatic Zika virus infection. Aedes aegypti mosquitos and causes symptoms in ≈20% of persons infected   (Seventeen days after the initial testing, Zika virus IgM and IgG were detected in a new serum sample from the patient. The patient and his partner were planning to conceive and were reluctant to wait the 6 months recommended by Public Health England (In this case, Zika virus RNA was detected in the semen of a previously healthy, immunocompetent adult who contracted his infection during a short visit to Rio de Janeiro, Brazil, in February 2016. RNA was present in semen samples until 92 days after the onset of symptoms and was subsequently undetectable on 2 occasions.This report follows several others from the current outbreak in which Zika virus RNA has been detected in semen >90 days after symptom onset in an immunocompetent and previously healthy adult. Previous reports include a man who had visited Haiti, in whom Zika virus RNA was persistently identified in semen until day 188 after symptom onset (This case is important because it reinforces the possibility raised by Nicastri et al. (An important caveat, however, is that virus could not be cultured from semen samples from this patient or from the patients reported by Nicastri et al. (An inability to detect Zika virus RNA in semen has been assumed to equate to a lack of infectivity by the sexual route (Published studies report an enormous variability in how long after infection Zika virus RNA can be detected in semen, which makes advising patients on the risks for sexual transmission difficult. In this case, testing serial semen samples for evidence of Zika virus RNA helped guide patient management, and doing this routinely in returning travelers in non-Zika virus–endemic countries might be warranted. In such a context, a positive result should be interpreted as indicating possible ongoing risk for sexual transmission.Further studies on the kinetics of virus isolation and Zika virus RNA detection from semen are needed to help inform guidelines on Zika virus sexual transmission and how to manage the risks. These studies would be especially useful in managing asymptomatic patients in whom evidence of Zika virus RNA detection, virus isolation, and sexual transmission is almost totally lacking.


Cardiovirus, family Picornaviridae, with two known serotypes. It is spread worldwide and infects a huge range of vertebrate hosts with zoonotic potential for humans. The pig is the mammal most likely to be impacted on with the disease, but EMCV occurrence has also been reported in non-human primates and in a variety of domestic, captive and wild animals. Until now, human cases have been very rare and the risk appears to be almost negligible in spite of human susceptibility to the infection.The Encephalomyocarditis virus (EMCV) is a small, non enveloped, positive sense single-stranded RNA virus in the genus Between September and November 2012 a fatal Encephalomyocarditis virus outbreak involving four Barbary macaques and 24 crested porcupines occurred at a rescue centre for wild and exotic animals in Central Italy. In this open-field zoo park located near Grosseto, Tuscany about 1000 animals belonging to different species, including various non-human primates were hosted at that time. Sudden deaths were generally observed without any evident symptoms or only with mild nonspecific clinical signs. The major gross change was characterised by grey-white necrotic foci in the myocardium and the same EMCV strain was isolated both in macaques and crested porcupines. Phylogenetic analysis has confirmed that only one EMCV strain is circulating in Italy, capable of infecting different animal species.This report confirms the susceptibility of non-human primates to the EMCV infection and describes the disease in porcupine, a common wild Italian and African species. No human cases were observed, but given the zoonotic potential of EMCV these findings are of importance in the context of animal-human interface. Cardiovirus, family Picornaviridae, with two known serotypes [The Encephalomyocarditis virus (EMCV) is a small, non enveloped, positive sense single-stranded RNA virus in the genus erotypes . A probaerotypes . It is serotypes , 3, 4.The pig is the mammal most likely to be impacted on with the disease , 6, but EMCV can cross the species barrier  and the This study describes the occurrence of EMCV infection in macaques and crested porcupines housed in a WWF Rescue Centre for Wild and Exotic Animals, in Central Italy. The Centre is 20 ha in size and at the time of the epidemic hosted about 1000 animals belonging to different species including various primates, felids, reptiles, parrots and birds of prey, mainly seized by Police Authorities because of illegal detention.Macaca sylvanus) and 24 crested porcupines (Hystrix cristata) died.The disease outbreak occurred between September and November 2012, when four Barbary macaques (n = 22) were housed in three different external hutches with seven or eight animals in each; the crested porcupines (n = 25) were housed in a hutch within a greenhouse and about 800 m from the macaques.The macaques  cells and BHK21 (Baby hamster kidney 21) cells . Cell cuMacaca and Hystrix. After RNA extraction and reverse transcription, a PCR End Point protocol [https://www.ncbi.nlm.nih.gov/genbank/) using the Nucleotide Blast (BLASTn) program. For virus typing the complete P1 (capsid-encoding) gene region was amplified and sequenced. Viral RNA extraction was performed on samples both from macaque and porcupine using the commercial kit QIAamp viral RNA (Qiagen). The VP1 coding sequences were amplified by One Step RT-PCR kit (Qiagen) and using two specific primers . A portion of 1070 bp was sequenced using Big Dye terminator Kit (Applied Biosystems) with the same primers used for amplification, and with two internal primers in addition . The obtained sequences were assembled and edited . Phylogenetic tree was generated with Neighbour Joining algorithm with Kimura-2 parameters and Bootstrap 1000 repeats phylogenetic test. Alignment and phylogenetic analysis were made by MEGA6 software.Finally, biomolecular analysis was performed on samples from both protocol  was usedDuring necropsy, similar lesions were observed in both macaques and porcupines Table . Some neBacteriological investigations evidenced bacteria of secondary infection and toxicological analysis were negative.Virus was isolated in all sampled organs from macaques and crested porcupines. VERO and BHK21 cell cultures showed a cytopathic effect after 48–72 h post-inoculation of homogenates of different organs. The virus was identified as EMCV by using the IEM technique   and two country mice (Mus musculus) were found dead in the area close to the macaques enclosure but nor lesions nor EMCV were detected. Dormice [No information is available on the possibility of EMCV having been introduced by another species in the Rescue Centre. During the outbreak three rats (two  Dormice  and squi Dormice  which shUnfortunately, to avoid causing unnecessary and potentially damaging stress, no blood samples were collected from the surviving animals to verify their seropositivity or their status of persistently infected, but EMCV is known to be a potential issue for other zoological park animals as it may involve several species , 3, 7, 8The results of this report indicate that EMCV is responsible of myocarditis and sudden asymptomatic death both in Barbary macaque and crested porcupine. Phylogenetic analysis confirms that only one EMCV strain is circulating in Italy, capable of infecting different animal species.No human cases were observed, but given the zoonotic potential of EMCV, these findings are of importance in the context of animal-human interface.


Zika virus is a mosquito-borne flavivirus that causes clinical symptoms similar to those observed in dengue and chikungunya virus infections. The Korea Centers for Disease Control and Prevention initiated laboratory testing using a real-time reverse transcription-polymerase chain reaction in January 2016. More than 1,000 suspected cases of infection were tested and nine were confirmed as imported cases of Zika virus infection from January to July 2016. The travel destinations of the infected individuals were Brazil, Philippines, Viet Nam, Guatemala, Puerto Rico, and the Dominican Republic. Phylogenetic analysis based on the partial envelope gene indicated that the viruses belonged to the Asian genotype circulating in South America. We further investigated the duration for which the viral RNA and virus-specific antibodies were detectable after the symptom onset. After the day of symptom onset, Zika virus was detectable until 6 days in serum, 14 days in urine and saliva, and 58 days in semen. Immunoglobulin M against Zika virus was detected as early as 2 days after the symptom onset and was maintained at these levels until 41 days, whereas Immunoglobulin G was detectable from 8 days after the symptom onset and was maintained until 52 days. These findings would help diagnostic laboratories improve their testing programs for Zika virus infection. Flavivirus in the family Flaviviridae. The virus was first isolated from a sentinel monkey in the Zika forest in Africa in 1947 [Zika virus is a mosquito-borne virus belonging to the genus  in 1947 . The tra in 1947 . Since tT) number ≤ 40 was considered to be positive for Zika virus infection. We additionally performed qRT-PCR according to the protocol described by Lanciotti et al. (1086/1163/1108FAM set), as a reference method [The Korea Centers for Disease Control and Prevention (KCDC) designated Zika virus infection as a notifiable disease and initiated laboratory testing for this disease in January 2016. All persons who had travelled to countries with active Zika virus transmission in the 2 weeks prior to the onset of symptoms were suspected to be infected with the Zika virus, and laboratory testing was recommended. Sexual partners of persons with suspected or confirmed Zika virus infection were also recommended to undergo laboratory testing. The KCDC used a custom-made real-time reverse transcription-polymerase chain reaction (qRT-PCR) kit, namely the genesig Zika Virus polyprotein standard kit  for detection of viral RNA in various types of samples. To detect the virus, viral RNA was extracted from 140 μL of sample using the QIAamp Viral RNA Mini kit  according to the manufacturer’s instructions. No treatment, such as concentration or filtration of the sample, was done prior to RNA extraction. The contents of genesig Zika Virus polyprotein standard kit are as follows: virus specific primer/probe mixture, positive control template (in vitro-transcribed RNA transcript), nuclease-free water, and 2× qRT-PCR Mastermix. Sequences of the primers and probe were not disclosed according to the company’s policy. The qRT-PCR mixture consisted of 10 μL 2× qRT-PCR Mastermix, 1 μL primer/probe mixture, 4 μL nuclease-free water, and 5 μL RNA sample. Thermal cycling conditions for reverse transcription were as follows: 55 °C for 10 min, enzyme activation at 95 °C for 2 min followed by 45 cycles of PCR . The amplification was performed using the ABI StepOne Plus or 7500 instrument . A sample with a threshold cycle  gene from Zika-positive samples . The comWe further investigated the duration for which the viral RNA was detectable in various specimens , as number of days after the symptom onset. Specimens were collected until 85 days after the symptom onset at 1-week intervals. We also conducted serological testing for serum samples to reveal the antibody kinetics in patients with Zika virus infection, using three commercial kits: Quantitative Human Zika Virus IgM ELISA kit , Zika virus IgM/IgG Ab Rapid Test , and Anti-Zika Virus IgM/IgG ELISA . Among the 3 kits, the performance of only Euroimmun ELISA kit  was previously reported [From January to July 2016, more than 1,000 suspected cases of Zika virus infection were tested and nine were confirmed as cases of imported Zika virus infection. The destinations of travel were Brazil (1 case), Philippines (3 cases), Viet Nam (2 cases), Guatemala (1 case), Puerto Rico (1 case), and the Dominican Republic (1 case). The mean time ± standard deviation between the symptom onset and first sample collection was 3.6 ± 2.1days . The onset of symptoms was defined based on the memory of each patient and it was recorded by physicians Viral RNA was detected in urine samples of all 9 patients (100%), but was detected in serum samples of only 3 out of the 9 patients (33.3%) . The sequence similarities between the seven samples were 97.4–99.7% and 99.1–100% at the nucleotide and amino acid sequence levels, respectively . Further, a phylogenetic tree based on the partial E gene sequences obtained from different countries was constructed . Anti-Zika Virus IgM and IgG ELISA kits from Euroimmun Inc. (Germany) were successfully used for detection of anti-Zika virus antibodies in 8 of the 9 (88.9%) patients Fig. . HoweverThe cross-reactivity of anti-Zika virus IgM antibody with other flaviviruses was investigated using flavivirus-specific ELISA kits. IgM cross-reactivity to other flaviviruses was shown in 2 of the 9 (22.2%) confirmed patients. Serum of the patient 2 showed a positive result in West Nile ELISA, and serum of the patient 6 exhibited a positive result in all the ELISAs . The results are consistent with those of the previous studies describing the high cross-reactivity in the flavivirus serology .In conclusion, we reported 9 travel-associated Zika cases in South Korea between January – July 2016. We found that a custom-made qRT-PCR kit and Euroimmun Anti-Zika Virus IgM/IgG ELISA kit were acceptable for routine laboratory testing for Zika virus infection. Additionally, we described virus and antibody kinetics in cases of Zika virus infection using various sample types. These findings should enable diagnostic laboratories to improve their testing programs for Zika virus infection.