A Systematic Review and Meta-Analysis of the Seroprevalence of Influenza A(H9N2) Infection Among Humans.

INTRODUCTION: Given that influenza A(H9N2) is recognized as a pandemic threat, we evaluated the overall burden of influenza A(H9N2) infections among avian-exposed human populations. METHODS: We performed a systematic search of PubMed, AGRICOLA, and CAB Abstracts databases for literature published during 1997-2013. Studies reporting serological evidence of human influenza A(H9N2) infection among avian-exposed populations were included. We used a World Health Organization (WHO)-recommended case definition for serological evidence of infection based on results of hemagglutination inhibition (HI) and microneutralization (MN) assays. We calculated overall seroprevalence through a random effects meta-analysis model. RESULTS: Seroprevalence data reported by the studies ranged from 1% to 43% (median, 9%) by HI, which was not significantly different from the seroprevalence estimated through the WHO-recommended case definition (median, 1.3%; range, 0.5%-42.6%). Reported seroprevalence by MN ranged from 0.6% to 9% (median, 2.7%), which was greater than the seroprevalence estimated through the WHO-recommended case definition (median, 0.3%; range, 0.1%-1.4%). CONCLUSIONS: A small proportion of avian-exposed humans had evidence of influenza A(H9N2) infection. As the virus has a near global distribution in poultry, it seems likely that present surveillance efforts are missing mild or asymptomatic infections among avian-exposed persons. It seems prudent to closely monitor avian-exposed populations for influenza A(H9N2) infection to provide prepandemic warnings.

Equine influenza A(H3N8) virus isolated from Bactrian camel, Mongolia.

Because little is known about the ecology of influenza viruses in camels, 460 nasal swab specimens were collected from healthy (no overt illness) Bactrian camels in Mongolia during 2012. One specimen was positive for influenza A virus (A/camel/Mongolia/335/2012[H3N8]), which is phylogenetically related to equine influenza A(H3N8) viruses and probably represents natural horse-to-camel transmission.

Evidence for subclinical H5N1 avian influenza infections among Nigerian poultry workers.

In recent years Nigeria has experienced sporadic incursions of highly pathogenic H5N1 avian influenza among poultry. In 2008, 316 poultry-exposed agricultural workers, and 54 age-group matched non-poultry exposed adults living in the Enugu or Ebonyi States of Nigeria were enrolled and then contacted monthly for 24 months to identify acute influenza-like-illnesses. Annual follow-up sera and questionnaire data were collected at 12 and 24 months. Participants reporting influenza-like illness completed additional questionnaires, and provided nasal and pharyngeal swabs and acute and convalescent sera. Swab and sera specimens were studied for evidence of influenza A virus infection. Sera were examined for elevated antibodies against 12 avian influenza viruses by microneutralization and 3 human viruses by hemagglutination inhibition. Four (3.2%) of the 124 acute influenza-like-illness investigations yielded molecular evidence of influenza, but virus could not be cultured. Serial serum samples from five poultry-exposed subjects had a ≥4-fold change in microneutralization titers against A/CK/Nigeria/07/1132123(H5N1), with three of those having titers ≥1:80 (maximum 1:1,280). Three of the five subjects (60%) reported a preceding influenza-like illness. Hemagglutination inhibition titers were ≥4-fold increases against one of the human viruses in 260 participants. While cross-reactivity from antibodies against other influenza viruses cannot be ruled out as a partial confounder, over the course of the 2-year follow-up, at least 3 of 316 (0.9%) poultry-exposed subjects had evidence for subclinical HPAI H5N1 infections. If these data represent true infections, it seems imperative to increase monitoring for avian influenza among Nigeria's poultry and poultry workers.

Serologic evidence of avian influenza virus infections among Nigerian agricultural workers.

Nigeria has had multiple incursions of highly pathogenic avian influenza A (HPAI) H5N1 virus into its poultry population since 2006. This study aimed to determine if Nigerians exposed to poultry had evidence of avian influenza virus transmission to man. Between 2008 and 2010, 316 adult farmers and open market workers and 54 age-group matched, non-animal exposed controls were enrolled in a prospective, population-based study of zoonotic influenza transmission in four towns in southeastern Nigeria. Questionnaire data and sera obtained at the time of enrollment were examined for evidence of previous infection with 10 avian influenza virus strains. Serologic studies on sera collected at the time of enrollment showed modest evidence of previous infection with three avian-origin influenza viruses (H5N1, H5N2, and H11N1) and one avian-like H9N2 influenza virus, with eight (2.4%) of animal-exposed subjects and two (3.7%) unexposed subjects having elevated microneutralization assay antibody titer levels (ranging from 1:10 to 1:80). Statistical analyses did not identify specific risk factors associated with the elevated antibody titers observed for these zoonotic influenza viruses. These data suggested only occasional virus transmission to humans in areas thought to have been enzootic for avian influenza virus. Prospective data from this cohort will help the authors to better understand the occurrence of zoonotic infections due to avian influenza viruses in Nigeria.

Influenza A(H1N1)pdm09 virus among healthy show pigs, United States.

Within 5 months after the earliest detection of human influenza A(H1N1)pdm09 virus, we found molecular and culture evidence of the virus in healthy US show pigs. The mixing of humans and pigs at swine shows possibly could further the geographic and cross-species spread of influenza A viruses.

Evidence for subclinical avian influenza virus infections among rural Thai villagers.

BACKGROUND: Regions of Thailand reported sporadic outbreaks of A/H5N1 highly pathogenic avian influenza (HPAI) among poultry between 2004 and 2008. Kamphaeng Phet Province, in north-central Thailand had over 50 HPAI poultry outbreaks in 2004 alone, and 1 confirmed and 2 likely other human HPAI infections between 2004 and 2006. METHODS: In 2008, we enrolled a cohort of 800 rural Thai adults living in 8 sites within Kamphaeng Phet Province in a prospective study of zoonotic influenza transmission. We studied participants' sera with serologic assays against 16 avian, 2 swine, and 8 human influenza viruses. RESULTS: Among participants (mean age 49.6 years and 58% female) 65% reported lifetime poultry exposure of at least 30 consecutive minutes. Enrollees had elevated antibodies by microneutralization assay against 3 avian viruses: A/Hong Kong/1073/1999(H9N2), A/Thailand/676/2005(H5N1), and A/Thailand/384/2006(H5N1). Bivariate risk factor modeling demonstrated that male gender, lack of an indoor water source, and tobacco use were associated with elevated titers against avian H9N2 virus. Multivariate modeling suggested that increasing age, lack of an indoor water source, and chronic breathing problems were associated with infection with 1 or both HPAI H5N1 strains. Poultry exposure was not associated with positive serologic findings. CONCLUSIONS: These data suggest that people in rural central Thailand may have experienced subclinical avian influenza infections as a result of yet unidentified environmental exposures. Lack of an indoor water source may play a role in transmission.

Sparse evidence of MERS-CoV infection among animal workers living in Southern Saudi Arabia during 2012.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging viral pathogen that primarily causes respiratory illness. We conducted a seroprevalence study of banked human serum samples collected in 2012 from Southern Saudi Arabia. Sera from 300 animal workers (17% with daily camel exposure) and 50 non-animal-exposed controls were examined for serological evidence of MERS-CoV infection by a pseudoparticle MERS-CoV spike protein neutralization assay. None of the sera reproducibly neutralized the MERS-CoV-pseudotyped lentiviral vector. These data suggest that serological evidence of zoonotic transmission of MERS-CoV was not common among animal workers in Southern Saudi Arabia during July 2012.

Absence of neutralizing antibodies against influenza A/H5N1 virus among children in Kamphaeng Phet, Thailand.

BACKGROUND: Influenza A/H5N1 actively circulated in Kamphaeng Phet (KPP), Thailand from 2004 to 2006. A prospective longitudinal cohort study of influenza virus infection in 800 adults conducted during 2008-2010 in KPP suggested that subclinical or mild H5N1 infections had occurred among this adult cohort. However, this study was conducted after the peak of H5N1 activity in KPP. Coincidentally, banked serum samples were available from a prospective longitudinal cohort study of primary school children who had undergone active surveillance for febrile illnesses from 2004 to 2007 and lived in the same district of KPP as the adult cohort. OBJECTIVES: We sought to investigate whether subclinical or mild H5N1 infections had occurred among KPP residents during the peak of H5N1 activity from 2004 to 2006. STUDY DESIGN: H5N1 microneutralization (MN) assay was performed on banked serum samples from a prospective longitudinal cohort study of primary school children who had undergone active surveillance for febrile illnesses in KPP. Annual blood samples collected from 2004 to 2006 from 251 children were selected based on the criteria that they lived in villages with documented H5N1 infection. RESULT: No H5N1 neutralizing antibodies were detected in 753 annual blood samples from 251 children. CONCLUSION: During 2004-2006, very few subclinical or mild H5N1 infections occurred in KPP. Elevated H5N1 MN titers found in the adult cohort in 2008 were likely due to cross-reactivity from other influenza virus subtypes highlighting the complexities in interpreting influenza serological data.

Serological evidence of equine influenza infections among persons with horse exposure, Iowa.

BACKGROUND: Equine influenza virus (EIV) is considered enzootic in North America and experimental studies have documented human EIV infections. STUDY DESIGN: This cross-sectional study examined 94 horse-exposed and 34 non-exposed controls for serological evidence of EIV infection. Sera were evaluated for antibodies against three EIV and two human H3N2 viruses using microneutralization (MN), neuraminidase inhibition (NI), enzyme-linked lectin (ELLA), and hemagglutination inhibition (HI) serological assays. Risk factor analyses were conducted using logistic regression and proportional odds modeling. RESULTS: There was evidence of previous infection by MN assay against A/equine/Ohio/2003(H3N8) but not the other 2 EIVs. Eleven (11.7%, maximum titer 1:320) horse-exposed and 2 (5.9%, maximum titer 1:160) control subjects had MN titers ≥1:80. Among the horse-exposed, 18 (19.1%) were positive by NI assay and 8 (8.5%) had elevated ELLA titers ≥1:10. Logistic regression modeling among horse-exposed revealed that having an elevated MN or ELLA titer (≤1:10) was associated with having a positive NI titer (OR=4.9; 95% CI=1.3-18.7, and OR=53.2; 95% CI=5.9-478.5, respectively). Upon proportional odds modeling, having worked as an equine veterinarian (OR=14.0; 95% CI=2.6-75.9), having a history of smoking (OR=3.1; 95% CI=1.2-7.7), and receipt of seasonal influenza vaccine between 2000 and 2005 (OR=2.3; 95% CI=1.1-5.0) were important independent risk factors for elevations in MN assay. CONCLUSIONS: While we cannot rule out confounding exposures, these data support the premise that occupational exposure to EIV may lead to human infection.

Elevated antibodies against Rift Valley fever virus among humans with exposure to ruminants in Saudi Arabia.

In 2000, an outbreak of Rift Valley fever virus (RVFV) occurred in the Kingdom of Saudi Arabia (KSA). Since then there have been sparse efforts to monitor for RVFV reemergence. During 2012, we enrolled 300 individuals with ruminant exposure and 50 age-group matched non-exposed controls in southwestern KSA, in a cross-sectional epidemiological study of RVFV. Sera from the participants were screened with an enzyme-linked immunosorbent assay (ELISA) for anti-RVFV IgG antibodies of which 39 (11.1%) were positive. Sixteen (41.0%) of those 39 were also positive by a plaque reduction neutralization assay (PRNT). The PRNT-positive subjects were further studied with an IgM ELISA and one was positive. No RVFV was detected in the 350 sera using real-time reverse transcription polymerase chain reaction. Contact with cattle (odds ratio [OR] = 3.16, 95% confidence interval [CI] 1.01, 9.90) and a history of chronic medical illness (OR = 6.41, 95% CI 1.75, 23.44) were associated with greater odds of RVFV seropositivity by PRNT. The IgM-positive participant was 36 years of age, and reported multiple risk factors for ruminant contact. Although these findings simply may be vestiges of the 2000 epidemic, KSA's frequent visits from pilgrims and importations of live animals from RVFV-endemic areas suggest that more comprehensive surveillance for imported RVFV virus in ruminants, mosquitoes, and travelers is imperative.

Seroepidemiological Study of Interepidemic Rift Valley Fever Virus Infection Among Persons with Intense Ruminant Exposure in Madagascar and Kenya.

In this cross-sectional seroepidemiological study we sought to examine the evidence for circulation of Rift Valley fever virus (RVFV) among herders in Madagascar and Kenya. From July 2010 to June 2012, we enrolled 459 herders and 98 controls (without ruminant exposures) and studied their sera (immunoglobulin G [IgG] and IgM through enzyme-linked immunosorbent assay [ELISA] and plaque reduction neutralization test [PRNT] assays) for evidence of previous RVFV infection. Overall, 59 (12.9%) of 459 herders and 7 (7.1%) of the 98 controls were positive by the IgG ELISA assay. Of the 59 ELISA-positive herders, 23 (38.9%) were confirmed by the PRNT assay (21 from eastern Kenya). Two of the 21 PRNT-positive study subjects also had elevated IgM antibodies against RVFV suggesting recent infection. Multivariate modeling in this study revealed that being seminomadic (odds ratio [OR] = 6.4, 95% confidence interval [CI] = 2.1-15.4) was most strongly associated with antibodies against RVFV. Although we cannot know when these infections occurred, it seems likely that some interepidemic RVFV infections are occurring among herders. As there are disincentives regarding reporting RVFV outbreaks in livestock or wildlife, it may be prudent to conduct periodic, limited, active seroepidemiological surveillance for RVFV infections in herders, especially in eastern Kenya.

No evidence for zoonotic transmission of H3N8 canine influenza virus among US adults occupationally exposed to dogs.

OBJECTIVES: The zoonotic potential of H3N8 canine influenza virus (CIV) has not been previously examined; yet considering the popularity of dogs as a companion animal and the zoonotic capabilities of other influenza viruses, the public health implications are great. This study aimed to determine the seroprevalence of antibodies against CIV among a US cohort. DESIGN: A cross-sectional seroepidemiological study was conducted between 2007 and 2010. SETTING: Recruitments primarily occurred in Iowa and Florida. Participants were enrolled at dog shows, or at their home or place of employment. SAMPLE: Three hundred and four adults occupationally exposed to dogs and 101 non-canine-exposed participants completed a questionnaire and provided a blood sample. MAIN OUTCOME MEASURES: Microneutralization and neuraminidase inhibition assays were performed to detect human sera antibodies against A/Canine/Iowa/13628/2005(H3N8). An enzyme-linked lectin assay (ELLA) was adapted to detect antibodies against a recombinant N8 neuraminidase protein from A/Equine/Pennsylvania/1/2007(H3N8). RESULTS: For all assays, no significant difference in detectable antibodies was observed when comparing the canine-exposed subjects to the non-canine-exposed subjects. CONCLUSION: While these results do not provide evidence for cross-species CIV transmission, influenza is predictably unpredictable. People frequently exposed to ill dogs should continually be monitored for novel zoonotic CIV infections.

Little evidence of avian or equine influenza virus infection among a cohort of Mongolian adults with animal exposures, 2010-2011.

Avian (AIV) and equine influenza virus (EIV) have been repeatedly shown to circulate among Mongolia's migrating birds or domestic horses. In 2009, 439 Mongolian adults, many with occupational exposure to animals, were enrolled in a prospective cohort study of zoonotic influenza transmission. Sera were drawn upon enrollment and again at 12 and 24 months. Participants were contacted monthly for 24 months and queried regarding episodes of acute influenza-like illnesses (ILI). Cohort members confirmed to have acute influenza A infections, permitted respiratory swab collections which were studied with rRT-PCR for influenza A. Serologic assays were performed against equine, avian, and human influenza viruses. Over the 2 yrs of follow-up, 100 ILI investigations in the cohort were conducted. Thirty-six ILI cases (36%) were identified as influenza A infections by rRT-PCR; none yielded evidence for AIV or EIV. Serological examination of 12 mo and 24 mo annual sera revealed 37 participants had detectable antibody titers (≥1∶10) against studied viruses during the course of study follow-up: 21 against A/Equine/Mongolia/01/2008(H3N8); 4 against an avian A/Teal/Hong Kong/w3129(H6N1), 11 against an avian-like A/Hong Kong/1073/1999(H9N2), and 1 against an avian A/Migrating duck/Hong Kong/MPD268/2007(H10N4) virus. However, all such titers were <1∶80 and none were statistically associated with avian or horse exposures. A number of subjects had evidence of seroconversion to zoonotic viruses, but the 4-fold titer changes were again not associated with avian or horse exposures. As elevated antibodies against seasonal influenza viruses were high during the study period, it seems likely that cross-reacting antibodies against seasonal human influenza viruses were a cause of the low-level seroreactivity against AIV or EIV. Despite the presence of AIV and EIV circulating among wild birds and horses in Mongolia, there was little evidence of AIV or EIV infection in this prospective study of Mongolians with animal exposures.

Comparison of commercial influenza A virus assays in detecting avian influenza H7N9 among poultry cloacal swabs, China.

BACKGROUND: Avian H7N9 virus emerged in China in February 2013 and has since spread widely among China's poultry, causing numerous human infections. OBJECTIVES: To compare World Health Organization (WHO) and US commercial influenza assays in detecting avian H7N9 virus in poultry cloacal specimens. STUDY DESIGN: Between April 6 and July 15, 2013, 261 cloacal swabs were collected from commercial poultry in Nanjing and Wuxi City, Jiangsu Province, China. Swabs were screened with the WHO's influenza A and H7N9 real-time RT-PCR (qRT-PCR) assays. A blinded panel of 97 specimens (27 H7N9-positive and 70 influenza A-negative) was then used to compare 3 antigen based commercial assays (Remel Xpect Flu A&B, Quidel Quickvue influenza, and Quidel Sofia Influenza A+B), and 2 molecular commercial assays (Quidel Molecular Influenza A+B assay and Life Technologies VetMAX™-Gold SIV Detection Kit). None of these commercial assays were approved for use with poultry specimens. RESULTS: Considering the WHO H7N9 qRT-PCR assay as the gold standard, all assays except the Quidel Quickvue influenza assay had high specificity (ranging from 96 to 99%). Regarding sensitivity, the Life Technologies VetMAX™-Gold SIV Detection Kit (100%; 95% CI 87-100%) and the Quidel Molecular Influenza A+B assay (85%; 95% CI 66-96%) performed the best. The sensitivities of the non-molecular antigen detection assays were either unable to detect small amounts of H7N9 viral RNA or were inhibited by specimen type. CONCLUSIONS: The Life Technologies VetMAX™-Gold SIV Detection Kit and the Quidel Molecular Influenza A+B assay are comparable in performance to the WHO H7N9 qRT-PCR assay in detecting H7N9 from poultry cloacal specimens.

Little evidence of human infection with equine influenza during the 2007 epizootic, Queensland, Australia.

BACKGROUND: Equine influenza virus (EIV) is considered enzootic in Europe (except Iceland), Asia, North Africa, and North and South America. When EIV outbreaks occur they may severely impact the equine and tourist industries. Australia faced its first EIV outbreak beginning in August of 2007. The outbreak was concentrated in New South Wales and Queensland, with more than 1400 confirmed EIV infections in horses during the first month. Rapid response from the equine industry and the federal government was successful and Australia was declared free from EIV by the end of 2007. OBJECTIVES: This cross-sectional study was designed to examine associations between exposure to EIV-infected horses and evidence of EIV infection in humans. STUDY DESIGN: Employing informed consent, between October 2007 and April 2008, 100 subjects (89 with horse exposures and 11 non-exposed) were enrolled during equine events and at the University of the Sunshine Coast. All subjects provided a blood sample and were asked to complete an online questionnaire including health history, animal exposure and demographic information. Sera samples were tested for the presence of antibodies against two H3N8 EIV strains using microneutralization, hemagglutination inhibition, and enzyme-linked lectin assays. RESULTS: Evidence for H3N8 infection was sparse, with only 9 study participants having any indication of H3N8 infection and the seroreactivity seen was low and easily explained by cross-reactions against human influenza strains or vaccines. CONCLUSIONS: These data provide little evidence to support the premise that EIV infections occurred among humans exposed to EIV-infected horses during the 2007 Australian epizootic.

A prospective study of Romanian agriculture workers for zoonotic influenza infections.

BACKGROUND: In this prospective study we sought to examine seroepidemiological evidence for acute zoonotic influenza virus infection among Romanian agricultural workers. METHODS: Sera were drawn upon enrollment (2009) and again at 12 and 24 months from 312 adult agriculture workers and 51 age-group matched controls. Participants were contacted monthly for 24 months and queried regarding episodes of acute influenza-like illnesses (ILI). Cohort members meeting ILI criteria permitted respiratory swab collections as well as acute and convalescent serum collection. Serologic assays were performed against 9 avian, 3 swine, and 3 human influenza viruses. RESULTS: During the two-year follow-up, a total of 23 ILI events were reported. Two subjects' specimens were identified as influenza A by rRT-PCR. During the follow-up period, three individuals experienced elevated microneutralization antibody titers ≥1∶80 against three (one each) avian influenza viruses: A/Teal/Hong Kong/w312/97(H6N1), A/Hong Kong/1073/1999(H9N2), or A/Duck/Alberta/60/1976(H12N5). However, none of these participants met the criteria for poultry exposure. A number of subjects demonstrated four-fold increases over time in hemagglutination inhibition (HI) assay titers for at least one of the three swine influenza viruses (SIVs); however, it seems likely that two of these three responses were due to cross-reacting antibody against human influenza. Only elevated antibody titers against A/Swine/Flanders/1/1998(H3N2) lacked evidence for such confounding. In examining risk factors for elevated antibody against this SIV with multiple logistic regression, swine exposure (adjusted OR = 1.8, 95% CI 1.1-2.8) and tobacco use (adjusted OR = 1.8; 95% CI 1.1-2.9) were important predictors. CONCLUSIONS: While Romania has recently experienced multiple incursions of highly pathogenic avian influenza among domestic poultry, this cohort of Romanian agriculture workers had sparse evidence of avian influenza virus infections. In contrast, there was evidence, especially among the swine exposed participants, of infections with human and one swine H3N2 influenza virus.

High rate of A(H1N1)pdm09 infections among rural Thai villagers, 2009-2010.

BACKGROUND: Pandemic influenza A(H1N1)pdm09 emerged in Thailand in 2009. A prospective longitudinal adult cohort and household transmission study of influenza-like illness (ILI) was ongoing in rural Thailand at the time of emergence. Symptomatic and subclinical A(H1N1)pdm09 infection rates in the cohort and among household members were evaluated. METHODS: A cohort of 800 Thai adults underwent active community-based surveillance for ILI from 2008-2010. Acute respiratory samples from ILI episodes were tested for A(H1N1)pdm09 by qRT-PCR; acute and 60-day convalescent blood samples were tested by A(H1N1)pdm09 hemagglutination inhibition assay (HI). Enrollment, 12-month and 24-month follow-up blood samples were tested for A(H1N1)pdm09 seroconversion by HI. Household members of influenza A-infected cohort subjects with ILI were enrolled in household transmission investigations in which day 0 and 60 blood samples and acute respiratory samples were tested by either qRT-PCR or HI for A(H1N1)pdm09. Seroconversion between annual blood samples without A(H1N1)pdm09-positive ILI was considered as subclinical infection. RESULTS: The 2-yr cumulative incidence of A(H1N1)pdm09 infection in the cohort in 2009/2010 was 10.8% (84/781) with an annual incidence of 1.2% in 2009 and 9.7% in 2010; 83.3% of infections were subclinical (50% in 2009 and 85.9% in 2010). The 2-yr cumulative incidence was lowest (5%) in adults born ≤ 1957. The A(H1N1)pdm09 secondary attack rate among household contacts was 47.2% (17/36); 47.1% of these infections were subclinical. The highest A(H1N1)pdm09 secondary attack rate among household contacts (70.6%, 12/17) occurred among children born between 1990 and 2003. CONCLUSION: Subclinical A(H1N1)pdm09 infections in Thai adults occurred frequently and accounted for a greater proportion of all A(H1N1)pdm09 infections than previously estimated. The role of subclinical infections in A(H1N1)pdm09 transmission has important implications in formulating strategies to predict and prevent the spread of A(H1N1)pdm09 and other influenza virus strains.

Little evidence of subclinical avian influenza virus infections among rural villagers in Cambodia.

In 2008, 800 adults living within rural Kampong Cham Province, Cambodia were enrolled in a prospective cohort study of zoonotic influenza transmission. After enrollment, participants were contacted weekly for 24 months to identify acute influenza-like illnesses (ILI). Follow-up sera were collected at 12 and 24 months. A transmission substudy was also conducted among the family contacts of cohort members reporting ILI who were influenza A positive. Samples were assessed using serological or molecular techniques looking for evidence of infection with human and avian influenza viruses. Over 24 months, 438 ILI investigations among 284 cohort members were conducted. One cohort member was hospitalized with a H5N1 highly pathogenic avian influenza (HPAI) virus infection and withdrew from the study. Ninety-seven ILI cases (22.1%) were identified as influenza A virus infections by real-time RT-PCR; none yielded evidence for AIV. During the 2 years of follow-up, 21 participants (3.0%) had detectable antibody titers (≥ 1:10) against the studied AIVs: 1 against an avian-like A/Migratory duck/Hong Kong/MPS180/2003(H4N6), 3 against an avian-like A/Teal/Hong Kong/w312/97(H6N1), 9 (3 of which had detectible antibody titers at both 12- and 24-month follow-up) against an avian-like A/Hong Kong/1073/1999(H9N2), 6 (1 detected at both 12- and 24-month follow-up) against an avian-like A/Duck/Memphis/546/74(H11N9), and 2 against an avian-like A/Duck/Alberta/60/76(H12N5). With the exception of the one hospitalized cohort member with H5N1 infection, no other symptomatic avian influenza infections were detected among the cohort. Serological evidence for subclinical infections was sparse with only one subject showing a 4-fold rise in microneutralization titer over time against AvH12N5. In summary, despite conducting this closely monitored cohort study in a region enzootic for H5N1 HPAI, we were unable to detect subclinical avian influenza infections, suggesting either that these infections are rare or that our assays are insensitive at detecting them.

Recovery of live virus after storage at ambient temperature using ViveST™.

BACKGROUND: A major impediment to performing virological field studies in developing nations is the lack of ultra-low freezers as well as the expense and difficulty of shipping frozen samples. A commercially available product, ViveST™, was developed to preserve nucleic acids at ambient temperature for use in specimen storage and transportation. However, its applications as a viral storage, transport and recovery device have not been evaluated. OBJECTIVE: To examine the ability of ViveST to preserve live virus following storage at ambient temperature. STUDY DESIGN: A panel of six viruses was stored at ambient temperature (~22°C) in ViveST with fetal bovine serum (FBS), or ViveST with minimal essential media (MEM) and compared with virus stored in universal transport media (M4RT), MEM, and FBS alone. Stored viruses included: human adenovirus (14p), dengue virus 2 (16608), echovirus 3 (Morrisey), human rhinovirus 15 (1734), Coxsackie virus B5 (Faulkner), and herpes simplex virus 1 (HF). After 7 days storage at ambient temperature, virus recovery was measured via titration using viral plaque assays or focus-forming unit assays. RESULTS: Viral titer studies indicate that ViveST with either FBS or M4RT preserved/recovered 5 different viruses for 1 week at ambient temperature. MEM preserved 4 viruses while FBS and ViveST with MEM preserved 3 viruses each. Statistical analyses indicate that M4RT and ViveST with FBS preserved significantly more virus than the other treatments. CONCLUSIONS: These data suggest that ViveST with either FBS or M4RT may be useful in field specimen collection scenarios where ultra-cold storage is not available.

Sparse evidence for equine or avian influenza virus infections among Mongolian adults with animal exposures.

In recent years, Mongolia has experienced recurrent epizootics of equine influenza virus (EIV) among its 2·1 million horses and multiple incursions of highly pathogenic avian influenza (HPAI) virus via migrating birds. No human EIV or HPAI infections have been reported. In 2009, 439 adults in Mongolia were enrolled in a population-based study of zoonotic influenza transmission. Enrollment sera were examined for serological evidence of infection with nine avian, three human, and one equine influenza virus strains. Seroreactivity was sparse among participants suggesting little human risk of zoonotic influenza infection.