A Cross-Sectional Study of Avian Influenza in One District of Guangzhou, 2013

Since Feb, 2013, more than 100 human beings had been infected with novel H7N9 avian influenza virus. As of May 2013, several H7N9 viruses had been found in retail live bird markets (LBMs) in Guangdong province of southern China where several human cases were confirmed later. However, the real avian influenza virus infection status especially H7N9 in Guangzhou remains unclear. Therefore, a cross-sectional study of avian influenza in commercial poultry farms, the wholesale LBM and retail LBMs in one district of Guangzhou was conducted from October to November, 2013. A total of 1505 cloacal and environmental samples from 52 commercial poultry farms, 1 wholesale LBM and 18 retail LBMs were collected and detected using real-time RT-PCR for type A, H7, H7N9 and H9 subtype avian influenza virus, respectively. Of all the flocks randomly sampled, 6 farms, 12 vendors of the wholesale LBM and 18 retail LBMs were type A avian influenza virus positive with 0, 3 and 11 positive for H9, respectively. The pooled prevalence and individual prevalence of type A avian influenza virus were 33.9% and 7.9% which for H9 subtype was 7.6% and 1.6%, respectively. None was H7 and H7N9 subtype virus positive. Different prevalence and prevalence ratio were found in different poultry species with partridges having the highest prevalence for both type A and H9 subtype avian influenza virus. Our results suggest that LBM may have a higher risk for sustaining and transmission of avian influenza virus than commercial poultry farms. The present study also indicates that different species may play different roles in the evolution and transmission of avian influenza virus. Therefore, risk-based surveillance and management measures should be conducted in future in this area.     

Phylogeography of Avian influenza A H9N2 in China

Background

During the past two decades, avian influenza A H9N2 viruses have spread geographically and ecologically in China. Other than its current role in causing outbreaks in poultry and sporadic human infections by direct transmission, H9N2 virus could also serve as an progenitor for novel human avian influenza viruses including H5N1, H7N9 and H10N8. Hence, H9N2 virus is becoming a notable threat to public health. However, despite multiple lineages and genotypes that were detected by previous studies, the migration dynamics of the H9N2 virus in China is unclear. Increasing such knowledge would help us better prevent and control H9N2 as well as other future potentially threatening viruses from spreading across China. The objectives of this study were to determine the source, migration patterns, and the demography history of avian influenza A H9N2 virus that circulated in China.

Results

Using Bayesian phylogeography framework, we showed that the H9N2 virus in mainland China may have originated from the Hong Kong Special Administrative Region (SAR). Southern China, most likely the Guangdong province acts as the primary epicentre for multiple H9N2 strains spreading across the whole country, and eastern China, most likely the Jiangsu province, acts as an important secondary source to seed outbreaks. Our demography inference suggests that during the long-term migration process, H9N2 evolved into multiple diverse lineages and then experienced a selective sweep, which reduced its genetic diversity. Importantly, such a selective sweep may pose a greater threat to public health because novel strains confer higher fitness advantages than strains being replaced and could generate new viruses through reassortment.

Conclusion

Our analyses indicate that migratory birds, poultry trade and transportation have all contributed to the spreading of the H9N2 virus in China. The ongoing migration and evolution of H9N2, which poses a constant threat to the human population, highlights the need for a more comprehensive surveillance of wild birds and for the enhancement of biosafety for China’s poultry industry.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1110) contains supplementary material, which is available to authorized users.     

Seroprevalence of Antibodies to Avian Influenza A (H5) and A (H9) Viruses among Market Poultry Workers, Hanoi, Vietnam, 2001

Background

The frequency of avian influenza A virus infections among poultry workers is not well understood.

Methods

A seroprevalence study of market poultry workers and persons without occupational poultry exposure was conducted during 2001 in Hanoi, Vietnam. Sera were tested for avian influenza H5 and H9 antibodies by microneutralization and Western blot assays.

Results

Seroprevalence of H5 and H9 antibodies was 4% and 3% in poultry workers and 1% and 3.5% in non-poultry workers, respectively.

Conclusions

Seroprevalence of H5 and H9 antibodies was low among Hanoi market poultry workers in 2001, but can serve as a baseline for additional studies.     

Live Poultry Exposure and Public Response to Influenza A(H7N9) in Urban and Rural China during Two Epidemic Waves in 2013-2014

Background

The novel influenza A(H7N9) virus has caused 2013 spring and 2013–2014 winter waves of human infections since its first emergence in China in March 2013. Exposure to live poultry is a risk factor for H7N9 infection. Public psychobehavioral responses often change during progression of an epidemic.

Methods

We conducted population-based surveys in southern China to examine human exposure to live poultry, and population psychological response and behavioral changes in the two waves. In Guangzhou, an urban area of Guangdong province, we collected data using telephone surveys with random digit dialing in May-June 2013 and again in December 2013 to January 2014. In Zijin county, a rural area of the same province, we used door-to-door surveys under a stratified sampling design in July 2013 and again in December 2013 to January 2014. All responses were weighted by age and sex to the respective adult populations.

Findings

Around half of the urban respondents (53.8%) reported having visited LPMs in the previous year in the first survey, around double that reported in the second survey (27.7%). In the rural surveys, around half of the participants reported raising backyard poultry in the past year in the first survey, increasing to 83.2% participants in the second survey. One third of urban subjects supported the permanent closure of LPMs in the first and second surveys, and factors associated with support for closure included female sex, higher level of worry towards H7N9, and worry induced by a hypothetical influenza-like illness.

Conclusions

Our study indicated high human exposure to live poultry and low support for permanent closure of markets in both urban and rural residents regardless of increased worry during the epidemic.     

苏州活禽市场禽流感病毒(H7N9)季节变化及HA、NA、PB2基因系统进化分析

自2013年3月中国首次发现新型禽流感病毒H7N9以来,其于2013-2014年期间发生流行,2015年也有散发性感染。该病毒的流行不仅危及家禽养殖业,还对公共卫生安全造成严重威胁。为调查活禽市场中H7N9的进化史和季节性变化,本研究于2013年7-12月在H7N9主要流行地区之一江苏省苏州市活禽市场采集2 655份鸡、鸭咽拭子样本,对样本中流感病毒核酸进行检测。结果显示,冬季样本中H7N9阳性率显著高于夏季样本,同时发现样本中存在H5、H7和H9亚型毒株之间的混合感染。进一步对H7N9阳性样本的HA、NA和PB2基因序列进行分析,结果表明阳性样本中HA、NA和PB2基因序列与新型H7N9病毒的相应基因序列同源,其在家禽体内传代时也在继续进化。特别是一些样品中PB2基因序列与H5N1病毒PB2基因序列的同源性较高。结果提示,苏州存在一种新型H7N9病毒基因重排的可能性,建议在活禽市场对所有禽流感病毒亚型进行持续监控,从而有助于流感病毒的及时防控。     

Highly pathogenic avian influenza (H5N1): pathways of exposure at the animal-human interface, a systematic review

Background

The threat posed by highly pathogenic avian influenza A H5N1 viruses to humans remains significant, given the continued occurrence of sporadic human cases (499 human cases in 15 countries) with a high case fatality rate (approximately 60%), the endemicity in poultry populations in several countries, and the potential for reassortment with the newly emerging 2009 H1N1 pandemic strain. Therefore, we review risk factors for H5N1 infection in humans.

Methods and Findings

Several epidemiologic studies have evaluated the risk factors associated with increased risk of H5N1 infection among humans who were exposed to H5N1 viruses. Our review shows that most H5N1 cases are attributed to exposure to sick poultry. Most cases are sporadic, while occasional limited human-to-human transmission occurs. The most commonly identified factors associated with H5N1 virus infection included exposure through contact with infected blood or bodily fluids of infected poultry via food preparation practices; touching and caring for infected poultry; consuming uncooked poultry products; exposure to H5N1 via swimming or bathing in potentially virus laden ponds; and exposure to H5N1 at live bird markets.

Conclusions

Research has demonstrated that despite frequent and widespread contact with poultry, transmission of the H5N1 virus from poultry to humans is rare. Available research has identified several risk factors that may be associated with infection including close direct contact with poultry and transmission via the environment. However, several important data gaps remain that limit our understanding of the epidemiology of H5N1 in humans. Although infection in humans with H5N1 remains rare, human cases continue to be reported and H5N1 is now considered endemic among poultry in parts of Asia and in Egypt, providing opportunities for additional human infections and for the acquisition of virus mutations that may lead to more efficient spread among humans and other mammalian species. Collaboration between human and animal health sectors for surveillance, case investigation, virus sharing, and risk assessment is essential to monitor for potential changes in circulating H5N1 viruses and in the epidemiology of H5N1 in order to provide the best possible chance for effective mitigation of the impact of H5N1 in both poultry and humans.

Disclaimer

The opinions expressed in this article are those of the authors and do not necessarily reflect those of the institutions or organizations with which they are affiliated.     

Prevalence of Antibodies against Avian Influenza A (H5N1) Virus among Cullers and Poultry Workers in Ho Chi Minh City, 2005

Background

Between 2003 and 2005, highly pathogenic avian influenza A (H5N1) viruses caused large scale outbreaks in poultry in the Ho Chi Minh City area in Vietnam. We studied the prevalence of antibodies against H5N1 in poultry workers and cullers who were active in the program in Ho Chi Minh City in 2004 and 2005.

Methodology/Principal Findings

Single sera from 500 poultry workers and poultry cullers exposed to infected birds were tested for antibodies to avian influenza H5N1, using microneutralization assays and hemagglutination inhibition assay with horse blood. All sera tested negative using microneutralization tests. Three samples showed a 1∶80 titer in the hemagglutination inhibition assay.

Conclusions/Significance

This study provides additional support for the low transmissibility of clade 1 H5N1 to humans, but limited transmission to highly exposed persons cannot be excluded given the presence of low antibody titers in some individuals.     

Potential Geographic Distribution of the Novel Avian-Origin Influenza A (H7N9) Virus

Background

In late March 2013, a new avian-origin influenza virus emerged in eastern China. This H7N9 subtype virus has since infected 240 people and killed 60, and has awakened global concern as a potential pandemic threat. Ecological niche modeling has seen increasing applications as a useful tool in mapping geographic potential and risk of disease transmission.

Methodology/Principals

We developed two datasets based on seasonal variation in Normalized Difference Vegetation Index (NDVI) from the MODIS sensor to characterize environmental dimensions of H7N9 virus. One-third of well-documented cases was used to test robustness of models calibrated based on the remaining two-thirds, and model significance was tested using partial ROC approaches. A final niche model was calibrated using all records available.

Conclusions/Significance

Central-eastern China appears to represent an area of high risk for H7N9 spread, but suitable areas were distributed more spottily in the north and only along the coast in the south; highly suitable areas also were identified in western Taiwan. Areas identified as presenting high risk for H7N9 spread tend to present consistent NDVI values through the year, whereas unsuitable areas show greater seasonal variation.     

Genetic and Molecular Characterization of H9N2 Avian Influenza Viruses Isolated from Live Poultry Markets in Hubei Province,Central China, 2013–2017

H9N2 subtype avian influenza virus(AIV) is an influenza A virus that is widely spread throughout Asia, where it jeopardizes the poultry industry and provides genetic material for emerging human pathogens. To better understand the epidemicity and genetics of H9 subtype AIVs, we conducted active surveillance in live poultry markets(LPMs) in Hubei Province from 2013 to 2017. A total of 4798 samples were collected from apparent healthy poultry and environment. Realtime RT-PCR revealed that the positivity rate of influenza A was 26.6%(1275/4798), of which the H9 subtype accounted for 50.3%(641/1275) of the positive samples. Of the 132 H9N2 viral strains isolated, 48 representative strains were subjected to evolutionary analysis and genotyping. Phylogenetic analysis revealed that all H9N2 viral genes had 91.1%–100% nucleotide homology, clustered with genotype 57, and had high homology with human H9N2 viruses isolated from2013 to 2017 in China. Using a nucleotide divergence cutoff of 95%, we identified ten distinct H9N2 genotypes that continued to change over time. Molecular analysis demonstrated that six H9N2 isolates had additional potential glycosylation sites at position 218 in the hemagglutinin protein, and all isolates had I155 T and Q226 L mutations. Moreover, 44 strains had A558 V mutations in the PB2 protein and four had E627 V mutations, along with H9N2 human infection strains A/Beijing/1/2016 and A/Beijing/1/2017. These results emphasize that the H9N2 influenza virus in Hubei continues to mutate and undergo mammalian adaptation changes, indicating the necessity of strengthening the surveillance of the AIV H9N2 subtype in LPMs.     

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.     

A novel peptide ELISA for universal detection of antibodies to human H5N1 influenza viruses

Background

Active serologic surveillance of H5N1 highly pathogenic avian influenza (HPAI) virus in humans and poultry is critical to control this disease. However, the need for a robust, sensitive and specific serologic test for the rapid detection of antibodies to H5N1 viruses has not been met.

Methodology/Principal Findings

Previously, we reported a universal epitope (CNTKCQTP) in H5 hemagglutinin (HA) that is 100% conserved in H5N1 human isolates and 96.9% in avian isolates. Here, we describe a peptide ELISA to detect antibodies to H5N1 virus by using synthetic peptide that comprises the amino acid sequence of this highly conserved and antigenic epitope as the capture antigen. The sensitivity and specificity of the peptide ELISA were evaluated using experimental chicken antisera to H5N1 viruses from divergent clades and other subtype influenza viruses, as well as human serum samples from patients infected with H5N1 or seasonal influenza viruses. The peptide ELISA results were compared with hemagglutinin inhibition (HI), and immunofluorescence assay and immunodot blot that utilize recombinant HA1 as the capture antigen. The peptide ELISA detected antibodies to H5N1 in immunized animals or convalescent human sera whereas some degree of cross-reactivity was observed in HI, immunofluorescence assay and immunodot blot. Antibodies to other influenza subtypes tested negative in the peptide-ELISA.

Conclusion/Significance

The peptide-ELISA based on the highly conserved and antigenic H5 epitope (CNTKCQTP) provides sensitive and highly specific detection of antibodies to H5N1 influenza viruses. This study highlighted the use of synthetic peptide as a capture antigen in rapid detection of antibodies to H5N1 in human and animal sera that is robust, simple and cost effective and is particularly beneficial for developing countries and rural areas.     

Detection and Genetic Characteristics of H9N2 Avian Influenza Viruses from Live Poultry Markets in Hunan Province,China

H9N2 avian influenza viruses (AIVs) are highly prevalent and of low pathogenicity in domestic poultry. These viruses show a high genetic compatibility with other subtypes of AIVs and have been involved in the genesis of H5N1, H7N9 and H10N8 viruses causing severe infection in humans. The first case of human infection with H9N2 viruses in Hunan province of China have been confirmed in November 2013 and identified that H9N2 viruses from live poultry markets (LPMs) near the patient’s house could be the source of infection. However, the prevalence, distribution and genetic characteristics of H9N2 viruses in LPMs all over the province are not clear. We collected and tested 3943 environmental samples from 380 LPMs covering all 122 counties/districts of Hunan province from February to April, 2014. A total of 618 (15.7%) samples were H9 subtype positive and 200 (52.6%) markets in 98 (80.3%) counties/districts were contaminated with H9 subtype AIVs. We sequenced the entire coding sequences of the genomes of eleven H9N2 isolates from environmental samples. Phylogenetic analysis showed that the gene sequences of the H9N2 AIVs exhibited high homology (94.3%-100%). All eleven viruses were in a same branch in the phylogenetic trees and belonged to a same genotype. No gene reassortment had been found. Molecular analysis demonstrated that all the viruses had typical molecular characteristics of contemporary avian H9N2 influenza viruses. Continued surveillance of AIVs in LPMs is warranted for identification of further viral evolution and novel reassortants with pandemic potential.     

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.     

Reassortant between Human-Like H3N2 and Avian H5 Subtype Influenza A Viruses in Pigs: A Potential Public Health Risk

Background

Human-like H3N2 influenza viruses have repeatedly been transmitted to domestic pigs in different regions of the world, but it is still uncertain whether any of these variants could become established in pig populations. The fact that different subtypes of influenza viruses have been detected in pigs makes them an ideal candidate for the genesis of a possible reassortant virus with both human and avian origins. However, the determination of whether pigs can act as a “mixing vessel” for a possible future pandemic virus is still pending an answer. This prompted us to gather the epidemiological information and investigate the genetic evolution of swine influenza viruses in Jilin, China.

Methods

Nasopharyngeal swabs were collected from pigs with respiratory illness in Jilin province, China from July 2007 to October 2008. All samples were screened for influenza A viruses. Three H3N2 swine influenza virus isolates were analyzed genetically and phylogenetically.

Results

Influenza surveillance of pigs in Jilin province, China revealed that H3N2 influenza viruses were regularly detected from domestic pigs during 2007 to 2008. Phylogenetic analysis revealed that two distinguishable groups of H3N2 influenza viruses were present in pigs: the wholly contemporary human-like H3N2 viruses (represented by the Moscow/10/99-like sublineage) and double-reassortant viruses containing genes from contemporary human H3N2 viruses and avian H5 viruses, both co-circulating in pig populations.

Conclusions

The present study reports for the first time the coexistence of wholly human-like H3N2 viruses and double-reassortant viruses that have emerged in pigs in Jilin, China. It provides updated information on the role of pigs in interspecies transmission and genetic reassortment of influenza viruses.     

Seasonal Patterns in Human A (H5N1) Virus Infection: Analysis of Global Cases

Background

Human cases of highly pathogenic avian influenza (HPAI) A (H5N1) have high mortality. Despite abundant data on seasonal patterns in influenza epidemics, it is unknown whether similar patterns exist for human HPAI H5N1 cases worldwide. Such knowledge could help decrease avian-to-human transmission through increased prevention and control activities during peak periods.

Methods

We performed a systematic search of published human HPAI H5N1 cases to date, collecting month, year, country, season, hemisphere, and climate data. We used negative binomial regression to predict changes in case incidence as a function of season. To investigate hemisphere as a potential moderator, we used AIC and the likelihood-ratio test to compare the season-only model to nested models including a main effect or interaction with hemisphere. Finally, we visually assessed replication of seasonal patterns across climate groups based on the Köppen-Geiger climate classification.

Findings

We identified 617 human cases (611 with complete seasonal data) occurring in 15 countries in Southeast Asia, Africa, and the Middle East. Case occurrence was much higher in winter (n = 285, p = 0.03) than summer (n = 64), and the winter peak occurred across diverse climate groups. There was no significant interaction between hemisphere and season.

Interpretation

Across diverse climates, HPAI H5N1 virus infection in humans increases significantly in winter. This is consistent with increased poultry outbreaks and HPAI H5N1 virus transmission during cold and dry conditions. Prioritizing prevention and control activities among poultry and focusing public health messaging to reduce poultry exposures during winter months may help to reduce zoonotic transmission of HPAI H5N1 virus in resource-limited settings.     

Influenza A H5N1 clade 2.3.4 virus with a different antiviral susceptibility profile replaced clade 1 virus in humans in northern Vietnam

Background

Prior to 2007, highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry and humans in Vietnam were consistently reported to be clade 1 viruses, susceptible to oseltamivir but resistant to amantadine. Here we describe the re-emergence of human HPAI H5N1 virus infections in Vietnam in 2007 and the characteristics of the isolated viruses.

Methods and Findings

Respiratory specimens from patients suspected to be infected with avian influenza in 2007 were screened by influenza and H5 subtype specific polymerase chain reaction. Isolated H5N1 strains were further characterized by genome sequencing and drug susceptibility testing. Eleven poultry outbreak isolates from 2007 were included in the sequence analysis. Eight patients, all of them from northern Vietnam, were diagnosed with H5N1 in 2007 and five of them died. Phylogenetic analysis of H5N1 viruses isolated from humans and poultry in 2007 showed that clade 2.3.4 H5N1 viruses replaced clade 1 viruses in northern Vietnam. Four human H5N1 strains had eight-fold reduced in-vitro susceptibility to oseltamivir as compared to clade 1 viruses. In two poultry isolates the I117V mutation was found in the neuraminidase gene, which is associated with reduced susceptibility to oseltamivir. No mutations in the M2 gene conferring amantadine resistance were found.

Conclusion

In 2007, H5N1 clade 2.3.4 viruses replaced clade 1 viruses in northern Vietnam and were susceptible to amantadine but showed reduced susceptibility to oseltamivir. Combination antiviral therapy with oseltamivir and amantadine for human cases in Vietnam is recommended.     

Evaluations for In Vitro Correlates of Immunogenicity of Inactivated Influenza A H5, H7 and H9 Vaccines in Humans

Background

Serum antibody responses in humans to inactivated influenza A (H5N1), (H9N2) and A (H7) vaccines have been varied but frequently low, particularly for subunit vaccines without adjuvant despite hemagglutinin (HA) concentrations expected to induce good responses.

Design

To help understand the low responses to subunit vaccines, we evaluated influenza A (H5N1), (H9N2), (H7N7) vaccines and 2009 pandemic (H1N1) vaccines for antigen uptake, processing and presentation by dendritic cells to T cells, conformation of vaccine HA in antibody binding assays and gel analyses, HA titers with different red blood cells, and vaccine morphology in electron micrographs (EM).

Results

Antigen uptake, processing and presentation of H5, H7, H9 and H1 vaccine preparations evaluated in humans appeared normal. No differences were detected in antibody interactions with vaccine and matched virus; although H7 trimer was not detected in western blots, no abnormalities in the conformation of the HA antigens were identified. The lowest HA titers for the vaccines were <1∶4 for the H7 vaccine and 1∶661 for an H9 vaccine; these vaccines induced the fewest antibody responses. A (H1N1) vaccines were the most immunogenic in humans; intact virus and virus pieces were prominent in EM. A good immunogenic A (H9N2) vaccine contained primarily particles of viral membrane with external HA and NA. A (H5N1) vaccines intermediate in immunogenicity were mostly indistinct structural units with stellates; the least immunogenic A (H7N7) vaccine contained mostly small 5 to 20 nm structures.

Summary

Antigen uptake, processing and presentation to human T cells and conformation of the HA appeared normal for each inactivated influenza A vaccine. Low HA titer was associated with low immunogenicity and presence of particles or split virus pieces was associated with higher immunogenicity.     

Epidemiological and Virological Characteristics of Pandemic Influenza A (H1N1) School Outbreaks in China in 2009

Background

During the 2009 pandemic influenza H1N1 (2009) virus (pH1N1) outbreak, school students were at an increased risk of infection by the pH1N1 virus. However, the estimation of the attack rate showed significant variability.

Methods

Two school outbreaks were investigated in this study. A questionnaire was designed to collect information by interview. Throat samples were collected from all the subjects in this study 6 times and sero samples 3 times to confirm the infection and to determine viral shedding. Data analysis was performed using the software STATA 9.0.

Findings

The attack rate of the pH1N1 outbreak was 58.3% for the primary school, and 52.9% for the middle school. The asymptomatic infection rates of the two schools were 35.8% and 37.6% respectively. Peak virus shedding occurred on the day of ARI symptoms onset, followed by a steady decrease over subsequent days (p = 0.026). No difference was found either in viral shedding or HI titer between the symptomatic and the asymptomatic infectious groups.

Conclusions

School children were found to be at a high risk of infection by the novel virus. This may be because of a heightened risk of transmission owing to increased mixing at boarding school, or a lack of immunity owing to socio-economic status. We conclude that asymptomatically infectious cases may play an important role in transmission of the pH1N1 virus.     

Influenza Virus Surveillance in Pakistan during 2008-2011

Background

There is little information about influenza among the Pakistani population. In order to assess the trends of Influenza-like-Illness (ILI) and to monitor the predominant circulating strains of influenza viruses, a country-wide lab-based surveillance system for ILI and Severe Acute Respiratory Illness (SARI) with weekly sampling and reporting was established in 2008. This system was necessary for early detection of emerging novel influenza subtypes and timely response for influenza prevention and control.

Methods

Five sentinel sites at tertiary care hospitals across Pakistan collected epidemiological data and respiratory samples from Influenza-like illness (ILI) and severe acute respiratory illness (SARI) cases from January 2008 to December 2011. Samples were typed and sub-typed by Real-Time RT-PCR assay.

Results

A total of 6258 specimens were analyzed; influenza virus was detected in 1489 (24%) samples, including 1066 (72%) Influenza type A and 423 (28%) influenza type B viruses. Amongst influenza A viruses, 25 (2%) were seasonal A/H1N1, 169 (16%) were A/H3N2 and 872 (82 %) were A(H1N1)pdm09. Influenza B virus circulation was detected throughout the year along with few cases of seasonal A/H1N1 virus during late winter and spring. Influenza A/H3N2 virus circulation was mainly observed during summer months (August-October).

Conclusions

The findings of this study emphasize the need for continuous and comprehensive influenza surveillance. Prospective data from multiple years is needed to predict seasonal trends for vaccine development and to further fortify pandemic preparedness.