A Combination of Serological Assays to Detect Human Antibodies to the Avian Influenza A H7N9 Virus

Human infection with avian influenza A H7N9 virus was first identified in March 2013 and represents an ongoing threat to public health. There is a need to optimize serological methods for this new influenza virus. Here, we compared the sensitivity and specificity of the hemagglutinin inhibition (HI), microneutralization (MN), and Western blot (WB) assays for the detection of human antibodies against avian influenza A (H7N9) virus. HI with horse erythrocytes (hRBCs) and a modified MN assay possessed greater sensitivity than turkey erythrocytes and the standard MN assay, respectively. Using these assays, 80% of tested sera from confirmed H7N9 cases developed detectable antibody to H7N9 after 21 days. To balance sensitivity and specificity, we found serum titers of ≥20 (MN) or 160 (HI) samples were most effective in determining seropositive to H7N9 virus. Single serum with HI titers of 20–80 or MN titer of 10 could be validated by each other or WB assay. Unlike serum collected from adult or elderly populations, the antibody response in children with mild disease was low or undetectable. These combinations of assays will be useful in case diagnosis and serologic investigation of human cases.     

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.     

Phylogeography of Highly Pathogenic H5 Avian Influenza Viruses in China

Virologica Sinica - The spread of H5 highly pathogenic avian influenza viruses poses serious threats to the poultry industry, wild bird ecology and human health. Circulation of H5 viruses between...     

Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza A H7N9 Viruses

H7N9 was a cause of significant global health concern due to its severe infection and approximately 35% mortality in humans. By screening a Fab antibody phage library derived from patients who recovered from H7N9 infections, we characterized two human monoclonal antibodies (HuMAbs), HNIgGD5 and HNIgGH8. The epitope of these two antibodies was dependent on two residues in the receptor binding site at positions V186 and L226 of the hemagglutinin glycoprotein. Both antibodies possessed high neutralizing activity.     

Newcastle Disease Virus-Vectored H7 and H5 Live Vaccines Protect Chickens from Challenge with H7N9 or H5N1 Avian Influenza Viruses

Sporadic human infections by a novel H7N9 virus occurred over a large geographic region in China. In this study, we show that Newcastle disease virus (NDV)-vectored H7 (NDV-H7) and NDV-H5 vaccines are able to induce antibodies with high hemagglutination inhibition (HI) titers and completely protect chickens from challenge with the novel H7N9 or highly pathogenic H5N1 viruses, respectively. Notably, a baculovirus-expressed H7 protein failed to protect chickens from H7N9 virus infection.     

Chinese and Global Distribution of H9 Subtype Avian Influenza Viruses

H9 subtype avian influenza viruses (AIVs) are of significance in poultry and public health, but epidemiological studies about the viruses are scarce. In this study, phylogenetic relationships of the viruses were analyzed based on 1233 previously reported sequences and 745 novel sequences of the viral hemagglutinin gene. The novel sequences were obtained through large-scale surveys conducted in 2008-2011 in China. The results revealed distinct distributions of H9 subtype AIVs in different hosts, sites and regions in China and in the world: (1) the dominant lineage of H9 subtype AIVs in China in recent years is lineage h9.4.2.5 represented by A/chicken/Guangxi/55/2005; (2) the newly emerging lineage h9.4.2.6, represented by A/chicken/Guangdong/FZH/2011, has also become prevalent in China; (3) lineages h9.3.3, h9.4.1 and h9.4.2, represented by A/duck/Hokkaido/26/99, A/quail/Hong Kong/G1/97 and A/chicken/Hong Kong/G9/97, respectively, have become globally dominant in recent years; (4) lineages h9.4.1 and h9.4.2 are likely of more risk to public health than others; (5) different lineages have different transmission features and host tropisms. This study also provided novel experimental data which indicated that the Leu-234 (H9 numbering) motif in the viral hemagglutinin gene is an important but not unique determinant in receptor-binding preference. This report provides a detailed and updated panoramic view of the epidemiological distributions of H9 subtype AIVs globally and in China, and sheds new insights for the prevention of infection in poultry and preparedness for a potential pandemic caused by the viruses.     

H5N1型禽流感病毒广谱中和单克隆抗体的筛选及其中和机制初步研究

利用杆状病毒-昆虫细胞表达H5N1型禽流感病毒的血凝素蛋白(HA),纯化后的重组蛋白HA免疫小鼠并制备杂交瘤单克隆抗体,用H5N1型禽流感病毒的全病毒进行筛选,成功地获得了抗H5N1型禽流感病毒血凝素蛋白HA的单抗.MDCK细胞微量中和试验表明,单抗8G10D7可对clade2和clade9的H5N1型禽流感病毒起中和作用.Western-blot及血凝抑制实验进一步证明了该单抗的结合位点位于HA蛋白的HA1亚基上.鸡胚感染病毒预防试验结果表明,8G10D7对禽来源的和人来源的H5N1型禽流感病毒均可达到100%的保护率;在治疗试验组中,8G10D7对禽来源的病毒感染具有较高的保护率,可达100%,对人来源的H5N1型禽流感病毒最高也可达87.5%的保护率.该抗体的获得不仅为H5N1型高致病性禽流感病毒的预防和治疗带来了希望,同时其中和位点的发现也为以后亚单位疫苗的研制提供新的思路.     

Novel Reassortant Highly Pathogenic H5N2 Avian Influenza Viruses in Poultry in China

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.     

Genomic Signatures for Avian H7N9 Viruses Adapting to Humans

An avian influenza A H7N9 virus emerged in March 2013 and caused a remarkable number of human fatalities. Genome variability in these viruses may provide insights into host adaptability. We scanned over 140 genomes of the H7N9 viruses isolated from humans and identified 104 positions that exhibited seven or more amino acid substitutions. Approximately half of these substitutions were identified in the influenza ribonucleoprotein (RNP) complex. Although PB2 627K of the avian virus promotes replication in humans, 45 of the 147 investigated PB2 sequences retained the E signature at this position, which is an avian characteristic. We discovered 10 PB2 substitutions that covaried with K627E. An RNP activity assay showed that Q591K, D701N, and M535L restored the polymerase activity in human cells when 627K transformed to an avian-like E. Genomic analysis of the human-isolated avian influenza virus is crucial in assessing genome variability, because relationships between position-specific variations can be observed and explored. In this study, we observed alternative positions that can potentially compensate for PB2 627K, a well-known marker for cross-species infection. An RNP assay suggested Q591K, D701N, and M535L as potential markers for an H7N9 virus capable of infecting humans.     

Antibodies to Antigenic Site A of Influenza H7 Hemagglutinin Provide Protection against H7N9 Challenge

Identifying major antigenic and protective epitopes of the H7 hemagglutinin (HA) will be important for understanding the antibody response to vaccines developed against the novel influenza H7N9 viruses that emerged in China in 2013. To facilitate antigenic characterization of the H7N9 HA and to develop reagents for evaluation of H7N9 candidate vaccines, we generated a panel of murine monoclonal antibodies (mAbs) to the HA of A/Shanghai/2/2013 using mammalian cell-derived virus-like particles (VLP) containing the H7 HA. Neutralizing antibodies identified an HA epitope corresponding to antigenic site A on the structurally similar influenza H3 hemagglutinin. Importantly, the neutralizing antibodies protect against A/Shanghai/2/2013 challenge. This antigenic site is conserved among many H7 viruses, including strains of both Eurasian and North American lineage, and the isolated neutralizing antibodies are cross-reactive with older H7 vaccine strains. The results indicate that the identified antigenic site is a potentially important protective epitope and suggest the potential benefit of cross-reactive antibody responses to vaccination with H7 candidate vaccines.     

Environmental Sampling for Avian Influenza A(H7N9) in Live-Poultry Markets in Guangdong,China

BackgroundTo provide an increased understanding of avian influenza A(H7N9) activity in live-poultry market in space and time and hence improve H7N9 epidemic control, an ongoing environmental sampling program in multiple live-poultry markets across Guangdong, China was conducted during March 2013–June 2014.MethodsA total of 625 live-poultry markets throughout 21 prefecture areas took part in the study. A total of 10 environmental sites in markets for sampling were identified to represent 4 different poultry-related activity areas. At least 10 environmental samples were collected from each market every month. The real time RT-PCR was performed to detect the avian influenza A(H7N9) virus. Field survey was conducted to investigate the sanitation status of live-poultry markets.ResultsThere were 109 human infections with H7N9 avian influenza in Guangdong, of which 37 (34%) died. A total of 18741 environmental swabs were collected and subjected to real-time RT-PCR test, of which 905(4.83%) were found positive for H7N9 virus. There were 201 (32.16%) markets affected by H7N9 in 16 prefecture areas. The detection of H7N9 virus in markets spiked in winter months. 63.33% markets (38/60) had no physical segregation for poultry holding, slaughter or sale zones. Closing live-poultry market significantly decreased the H7N9 detection rate from 14.83% (112/755) to 1.67% (5/300).ConclusionsThis study indicates the importance of live-poultry market surveillance based on environmental sampling for H7N9 Avian Influenza control. Improving live-poultry market management and sanitation and changing consumer practices are critical to reduce the risk of H7N9 infection.     

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.