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The emergence of novel avian influenza reassortants in wild birds in recent years is a public health concern. However, the viruses that circulate in migratory birds are not fully understood. In this study, we summarized and categorized global H11 avian influenza viruses and reported that waterfowl and shorebirds are the major reservoirs of the identified H11 viruses. The surveillance data of the 35,749 faecal samples collected from wild bird habitats in eastern China over the past seven years revealed a low prevalence of H11 viruses in birds, with a positive rate of 0.067% (24 isolates). The phylogenetic analysis of the twenty viruses indicated that H11 viruses have undergone complex reassortment with viruses circulating in waterfowl and shorebirds. These tested viruses do not acquire mammalian adaptive mutations in their genomes and preferentially bind to avian-type receptors. Experimental infection studies demonstrated that the two tested H11N9 viruses of wild bird origin replicated and transmitted more efficiently in ducks than in chickens, whereas the pigeon H11N2 virus isolated from a live poultry market was more adapted to replicate in chickens than in ducks. In addition, some H11 isolates replicated efficiently in mice and caused body weight loss but were not lethal. Our study revealed the role of waterfowl and shorebirds in the ecology and evolution of H11 viruses and the potential risk of introducing circulating H11 viruses into ducks or chickens, further emphasizing the importance of avian influenza surveillance at the interface of migratory birds and poultry.

Due to the fact that many avian influenza viruses that kill chickens are not lethal to ducks, farmers are reluctant to use avian influenza inactivated vaccines on ducks. Large numbers of unvaccinated ducks play an important role in the transmission of avian influenza viruses from wild birds to domestic poultry, creating a substantial challenge to vaccination strategies for avian influenza control. To solve this problem, we constructed a recombinant duck enteritis virus (DEV), rDEV-dH5/H7, using a live attenuated DEV vaccine strain (vDEV) as a vector. rDEV-dH5/H7 carries the hemagglutinin gene of two H5 viruses [GZ/S4184/17 (H5N6) (clade 2.3.4.4 h) and LN/SD007/17 (H5N1) (clade 2.3.2.1d)] and an H7 virus [GX/SD098/17 (H7N9)]. These three hemagglutinin genes were stably inherited in rDEV-dH5/H7 and expressed in rDEV-dH5/H7-infected cells. Animal studies revealed that rDEV-dH5/H7 and vDEV induced similar neutralizing antibody responses and protection against lethal DEV challenge. Importantly, rDEV-dH5/H7 induced strong and long-lasting hemagglutinin inhibition antibodies against different H5 and H7 viruses and provided complete protection against challenges with homologous and heterologous highly pathogenic H5 and H7 influenza viruses in ducks. Our study shows that rDEV-dH5/H7 could serve as an ideal live attenuated vaccine to protect ducks against infection with lethal DEV and highly pathogenic avian influenza viruses.

Human infections with the H7N9 influenza virus have been eliminated in China through vaccination of poultry; however, the H7N9 virus has not yet been eradicated from poultry. Carefully analysis of H7N9 viruses in poultry that have sub-optimal immunity may provide a unique opportunity to witness the evolution of highly pathogenic avian influenza virus in the context of vaccination. Between January 2020 and June 2023, we isolated 16 H7N9 viruses from samples we collected during surveillance and samples that were sent to us for disease diagnosis. Genetic analysis indicated that these viruses belonged to a single genotype previously detected in poultry. Antigenic analysis indicated that 12 of the 16 viruses were antigenically close to the H7-Re4 vaccine virus that has been used since January 2022, and the other four viruses showed reduced reactivity with the vaccine. Animal studies indicated that all 16 viruses were nonlethal in mice, and four of six viruses showed reduced virulence in chickens upon intranasally inoculation. Importantly, the H7N9 viruses detected in this study exclusively bound to the avian-type receptors, having lost the capacity to bind to human-type receptors. Our study shows that vaccination slows the evolution of H7N9 virus by preventing its reassortment with other viruses and eliminates a harmful characteristic of H7N9 virus, namely its ability to bind to human-type receptors.

H5N1 avian influenza viruses bearing the clade 2.3.2.1 hemagglutinin (HA) gene have been widely detected in birds and poultry in several countries. During our routine surveillance, we isolated 28 H5N1 viruses between January 2017 and October 2020. To investigate the genetic relationship of the globally circulating H5N1 viruses and the biological properties of those detected in China, we performed a detailed phylogenic analysis of 274 representative H5N1 strains and analyzed the antigenic properties, receptor-binding preference, and virulence in mice of the H5N1 viruses isolated in China. The phylogenic analysis indicated that the HA genes of the 274 viruses belonged to six subclades, namely clades 2.3.2.1a to 2.3.2.1f; these viruses acquired gene mutations and underwent complicated reassortment to form 58 genotypes, with G43 being the dominant genotype detected in eight Asian and African countries. The 28 H5N1 viruses detected in this study carried the HA of clade 2.3.2.1c (two strains), 2.3.2.1d (three strains), or 2.3.2.1f (23 strains), and formed eight genotypes. These viruses were antigenically well-matched with the H5-Re12 vaccine strain used in China. Animal studies showed that the pathogenicity of the H5N1 viruses ranged from non-lethal to highly lethal in mice. Moreover, the viruses exclusively bound to avian-type receptors and have not acquired the ability to bind to human-type receptors. Our study reveals the overall picture of the evolution of clade 2.3.2.1 H5N1 viruses and provides insights into the control of these viruses.

Avian influenza viruses (AIVs) of the H5 subtype rank among the most serious pathogens, leading to significant economic losses in the global poultry industry and posing risks to human health. Therefore, rapid and accurate virus detection is crucial for the prevention and control of H5 AIVs. In this study, we established a novel detection method for H5 viruses by utilizing the precision of CRISPR/Cas12a and the efficiency of RT-RPA technologies. This assay facilitates the direct visualization of detection results through blue light and lateral flow strips, accurately identifying H5 viruses with high specificity and without cross-reactivity against other AIV subtypes, NDV, IBV, and IBDV. With detection thresholds of 1.9 copies/μL (blue light) and 1.9 × 10 3 copies/μL (lateral flow strips), our method not only competes with but also slightly surpasses RT-qPCR, demonstrating an 80.70% positive detection rate across 81 clinical samples. The RT-RPA/CRISPR-based detection method is characterized by high sensitivity, specificity, and independence from specialized equipment. The immediate field applicability of the RT-RPA/CRISPR approach underscores its importance as an effective tool for the early detection and management of outbreaks caused by the H5 subtype of AIVs.

The continued evolution and emergence of novel influenza viruses in wild and domestic animals poses an increasing public health risk. Two human cases of H3N8 avian influenza virus infection in China in 2022 have caused public concern regarding the risk of transmission between birds and humans. However, the prevalence of H3N8 avian influenza viruses in their natural reservoirs and their biological characteristics are largely unknown. To elucidate the potential threat of H3N8 viruses, we analyzed five years of surveillance data obtained from an important wetland region in eastern China and evaluated the evolutionary and biological characteristics of 21 H3N8 viruses isolated from 15,899 migratory bird samples between 2017 and 2021. Genetic and phylogenetic analyses showed that the H3N8 viruses circulating in migratory birds and ducks have evolved into different branches and have undergone complicated reassortment with viruses in waterfowl. The 21 viruses belonged to 12 genotypes, and some strains induced body weight loss and pneumonia in mice. All the tested H3N8 viruses preferentially bind to avian-type receptors, although they have acquired the ability to bind human-type receptors. Infection studies in ducks, chickens and pigeons demonstrated that the currently circulating H3N8 viruses in migratory birds have a high possibility of infecting domestic waterfowl and a low possibility of infecting chickens and pigeons. Our findings imply that circulating H3N8 viruses in migratory birds continue to evolve and pose a high infection risk in domestic ducks. These results further emphasize the importance of avian influenza surveillance at the wild bird and poultry interface.

Background: Avian influenza (AI) is a disease caused by the avian influenza virus (AIV). These viruses spread naturally among wild aquatic birds worldwide and infect domestic poultry, other birds, and other animal species. Currently, real-time reverse transcription polymerase chain reaction (rRT-PCR) is mainly used to detect the presence of pathogens and has good sensitivity and specificity. However, the diagnosis requires sophisticated instruments under laboratory conditions, which significantly limits point-of-care testing (POCT). Rapid, reliable, non-lab-equipment-reliant, sensitive, and specific diagnostic tests are urgently needed for rapid clinical detection and diagnosis. Our study aimed to develop a reverse transcription recombinase polymerase amplification (RT-RPA)/CRISPR method which improves on these limitations., Methods: The Cas12a protein was purified by affinity chromatography with Ni-agarose resin and observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Specific CRISPR RNA (crRNA) and primers targeting the M and NP genes of the AIV were designed and screened. By combining RT-RPA with the Cas12a/crRNA trans-cleavage system, a detection system that uses fluorescence readouts under blue light or lateral flow strips was established. Sensitivity assays were performed using a tenfold dilution series of plasmids and RNA of the M and NP genes as templates. The specificity of this method was determined using H1-H16 subtype AIVs and other avian pathogens, such as newcastle disease virus (NDV), infectious bursal disease virus (IBDV), and infectious bronchitis virus (IBV)., Results: The results showed that the method was able to detect AIV and that the detection limit can reach 6.7 copies/μL and 12 copies/μL for the M and NP gene, respectively. In addition, this assay showed no cross-reactivity with other avian-derived RNA viruses such as NDV, IBDV, and IBV. Moreover, the detection system presented 97.5% consistency and agreement with rRT-PCR and virus isolation for detecting samples from poultry. This portable and accurate method has great potential for AIV detection in the field., Conclusion: An RT-RPA/CRISPR method was developed for rapid, sensitive detection of AIV. The new system presents a good potential as an accurate, user-friendly, and inexpensive platform for point-of-care testing applications., (© 2023. The Author(s).)

The H5 subtype highly pathogenic avian influenza viruses bearing the clade 2.3.4.4 HA gene have been pervasive among domestic poultry and wild birds worldwide since 2014, presenting substantial risks to human and animal health. Continued circulation of clade 2.3.4.4 viruses has resulted in the emergence of eight subclades (2.3.4.4a-h) and multiple distinct antigenic groups. However, the key antigenic substitutions responsible for the antigenic change of these viruses remain unknown. In this study, we analyzed the HA gene sequences of 5713 clade 2.3.4.4 viruses obtained from a public database and found that 23 amino acid residues were highly variable among these strains. We then generated a series of single-amino-acid mutants based on the H5-Re8 (a vaccine seed virus) background and tested their reactivity with a panel of eight monoclonal antibodies (mAbs). Six mutants bearing amino acid substitutions at positions 120, 126, 141, 156, 185, or 189 (H5 numbering) led to reduced or lost reactivity to these mAbs. Further antigenic cartography analysis revealed that the amino acid residues at positions 126, 156, and 189 acted as immunodominant epitopes of H5 viruses. Collectively, our findings offer valuable guidance for the surveillance and early detection of emerging antigenic variants.

BackgroundTwo human cases of avian influenza A (H3N8) virus infection were reported in China in 2022.AimTo characterise H3N8 viruses circulating in China in September 2021-May 2022.MethodsWe sampled poultry and poultry-related environments in 25 Chinese provinces. After isolating H3N8 viruses, whole genome sequences were obtained for molecular and phylogenetic analyses. The specificity of H3N8 viruses towards human or avian receptors was assessed in vitro. Their ability to replicate in chicken and mice, and to transmit between guinea pigs was also investigated.ResultsIn total, 98 H3N8 avian influenza virus isolates were retrieved from 38,639 samples; genetic analysis of 31 representative isolates revealed 17 genotypes. Viruses belonging to 10 of these genotypes had six internal genes originating from influenza A (H9N2) viruses. These reassorted viruses could be found in live poultry markets and comprised the strains responsible for the two human infections. A subset of nine H3N8 viruses (including six reassorted) that replicated efficiently in mice bound to both avian-type and human-type receptors in vitro. Three reassorted viruses were shed by chickens for up to 9 days, replicating efficiently in their upper respiratory tract. Five reassorted viruses tested on guinea pigs were transmissible among these by respiratory droplets.ConclusionAvian H3N8 viruses with H9N2 virus internal genes, causing two human infections, occurred in live poultry markets in China. The low pathogenicity of H3N8 viruses in poultry allows their continuous circulation with potential for reassortment. Careful monitoring of spill-over infections in humans is important to strengthen early-warning systems and maintain influenza pandemic preparedness.

The H10 subtypes of avian influenza viruses pose a continual threat to the poultry industry and human health. The sporadic spillover of H10 subtypes viruses from poultry to humans is represented by the H10N8 human cases in 2013 and the recent H10N3 human infection in 2021. However, the genesis and characteristics of the recent reassortment H10N3 viruses have not been systemically investigated. In this study, we characterized 20 H10N3 viruses isolated in live poultry markets during routine nationwide surveillance in China from 2014 to 2021. The viruses in the recent reassortant genotype acquired their hemagglutinin (HA) and neuraminidase (NA) genes from the duck H10 viruses and H7N3 viruses, respectively, whereas the internal genes were derived from chicken H9N2 viruses as early as 2019. Receptor-binding analysis indicated that two of the tested H10N3 viruses had a higher affinity for human-type receptors than for avian-type receptors, highlighting the potential risk of avian-to-human transmission. Animal studies showed that only viruses belonging to the recent reassortant genotype were pathogenic in mice; two tested viruses transmitted via direct contact and one virus transmitted by respiratory droplets in guinea pigs, though with limited efficiency. These findings emphasize the need for enhanced surveillance of H10N3 viruses., (© 2023 Wiley Periodicals LLC.)

H16 avian influenza viruses mainly circulate in wild migratory gulls worldwide, and the infection risks in poultry and mammals remain largely unknown. In this study, we isolated a novel H16N3 virus from migratory gulls in eastern China in 2021. Genetic analysis indicated that the H16N3 virus originated from the H16 and H13 viruses that circulated in wild birds. This H16N3 virus has not adapted to replicate in chickens, ducks, or mice, although it can be transmitted between inoculated and contacted birds. The circulation of H16Nx viruses in the Northern Hemisphere indicates that we should strengthen active surveillance to monitor their prevalence and evolution in migratory gulls and their introduction into other migratory and domestic waterfowl. IMPORTANCE Migratory wild birds are natural reservoirs of H16 viruses and play a key role in the global prevalence of these viruses. Here, we found that H16 viruses predominantly circulate in migratory gulls and that the gull H16N3 virus cannot replicate efficiently in chickens, ducks, or mice without prior adaptation. These findings contribute to our understanding of the ecology, evolution, and biological properties of H16 viruses and will guide avian influenza surveillance in birds.

The globally circulating H5N8 avian influenza viruses bearing the clade 2.3.4.4b hemagglutinin (HA) gene are responsible for the loss of more than 33 million domestic poultry since January 2020. Moreover, the H5N8 viruses have reassorted with other avian influenza viruses and formed H5N1, H5N2, H5N3, H5N4, and H5N5 viruses in Europe, Africa, and North America. In this study, we analyzed 15 H5N6 viruses isolated from poultry and seven H5N6 viruses isolated from humans, and found these viruses formed seven different genotypes by deriving the clade 2.3.4.4b HA gene of H5N8 viruses, the neuraminidase of domestic duck H5N6 viruses, and internal genes of different viruses that previously circulated in domestic ducks and wild birds in China. Two of these genotypes (genotype 3 and genotype 6) have caused human infections in multiple provinces. The H5N6 viruses isolated from poultry have distinct pathotypes in mice; some of them replicate systemically and are highly lethal in mice. Although these viruses exclusively bind to avian-type receptors, it is worrisome that they may obtain key mutations that would increase their affinity for human-type receptors during replication in humans. Our study indicates that the novel H5N6 reassortants bearing the clade 2.3.4.4b HA gene of H5N8 viruses were generated through reassortment in domestic ducks and may have spread across a wide area of China, thereby posing a new challenge to the poultry industry and human health. Our findings emphasize the importance of careful monitoring, evaluation, and control of the H5N6 viruses circulating in nature.

H5N1 avian influenza viruses bearing the clade 2.3.4.4b hemagglutinin gene have been widely circulating in wild birds and are responsible for the loss of over 70 million domestic poultry in Europe, Africa, Asia, and North America since October 2020. During our routine surveillance, 13 H5N1 viruses were isolated from 26,767 wild bird and poultry samples that were collected between September 2021 and March 2022 in China. To investigate the origin of these Chinese isolates and understand their genetic relationship with the globally circulating H5N1 viruses, we performed a detailed phylogenic analysis of 233 representative H5N1 strains that were isolated from 28 countries. We found that, after they emerged in the Netherlands, the H5N1 viruses encountered complicated gene exchange with different viruses circulating in wild birds and formed 16 genotypes. Genotype one (G1) was predominant, being detected in 22 countries, whereas all other genotypes were only detected in one or two continents. H5N1 viruses of four genotypes (G1, G7, G9, and G10) were detected in China; three of these genotypes have been previously reported in other countries. The H5N1 viruses detected in China replicated in mice, with pathogenicity varying among strains; the G1 virus was highly lethal in mice. Moreover, we found that these viruses were antigenically similar to and well matched with the H5-Re14 vaccine strain currently used in China. Our study reveals the overall picture of H5N1 virus evolution and provides insights for the control of these viruses.

Wild birds are the natural reservoirs of avian influenza viruses, and surveillance and assessment of these viruses in wild birds provide valuable information for early warning and control of animal diseases. In this study, we isolated 19 H7N7 avian influenza viruses from wild bird between 2018 and 2020. Full genomic analysis revealed that these viruses bear a single basic amino acid in the cleavage site of their hemagglutinin gene, and formed four different genotypes by actively reassorting other avian influenza viruses circulating in wild birds and ducks. The H7N7 viruses bound to both avian-type and human-type receptors, although their affinity for human-type receptors was markedly lower than that for avian-type receptors. Moreover, we found that the H7N7 viruses could replicate efficiently in the upper respiratory tract and caecum of domestic ducks, and that the H5/H7 inactivated vaccine used in poultry in China provided complete protection against H7N7 wild bird virus challenge in ducks. Our findings demonstrate that wild bird H7N7 viruses pose a substantial threat to the poultry industry across the East Asian-Australian migratory flyway, emphasize the importance of influenza virus surveillance in both wild and domestic birds, and support the development of active control strategies against H7N7 virus., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2022 Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Published by Elsevier Masson SAS.. All rights reserved.)

H6 avian influenza virus (AIV) is one of the most prevalent AIV subtypes in the world. Our previous studies have demonstrated that H6 AIVs isolated from live poultry markets pose a potential threat to human health. In recent years, increasing number of H6 AIVs has been constantly isolated from poultry farms. In order to understand the biological characteristics of H6 AIVs in the context of farms, here, we analyzed the phylogenetic relationships, antigenicity, replication in mice and receptor binding properties of H6 AIVs isolated from farms in China between 2014 and 2018. Phylogenetic analysis showed that 19 different genotypes were formed among 20 representative H6 viruses. Notably, the internal genes of these H6 viruses exhibited complicated relationships with different subtypes of AIVs worldwide, indicating that these viruses are the products of complex and frequent reassortment events. Antigenic analysis revealed that 13 viruses tested were divided into three antigenic groups. 10 viruses examined could all replicate in the respiratory organs of infected mice without prior adaptation. Receptor binding analysis demonstrated that some of the H6 AIVs bound to both α-2, 3-linked glycans (avian-type receptor) and α-2, 6-linked glycans (human-type receptor), thereby posing a potential threat to human health. Together, these findings revealed the prevalence, complicated genetic evolution, diverse antigenicity, and dual receptor binding specificity of H6 AIVs in the settings of poultry farms, which emphasize the importance to continuously monitor the evolution and biological properties of H6 AIVs in nature., (© 2021 Wiley-VCH GmbH.)

H10Nx influenza viruses have caused increasing public concern due to their occasional infection of humans. However, the genesis and biological characteristics of H10 viruses in migratory wild birds are largely unknown. In this study, we conducted active surveillance to monitor circulation of avian influenza viruses in eastern China and isolated five H10N4 and two H10N8 viruses from migratory birds in 2020. Genetic analysis indicated that the hemagglutinin (HA) genes of the seven H10 viruses were clustered into the North American lineage and established as a novel Eurasian branch in wild birds in South Korea, Bangladesh, and China. The neuraminidase (NA) genes of the H10N4 and H10N8 viruses originated from the circulating HxN4 and H5N8 viruses in migratory birds in Eurasia. We further revealed that some of the novel H10N4 and H10N8 viruses acquired the ability to bind human-like receptors. Animal studies indicated that these H10 viruses can replicate in mice, chickens, and ducks. Importantly, we found that the H10N4 and H10N8 viruses can transmit efficiently among chickens and ducks but induce lower HA inhibition (HI) antibody titers in ducks. These findings emphasized that annual surveillance in migratory waterfowl should be strengthened to monitor the introduction of wild-bird H10N4 and H10N8 reassortants into poultry. IMPORTANCE The emerging avian influenza reassortants and mutants in birds pose an increasing threat to poultry and public health. H10 avian influenza viruses are widely prevalent in wild birds, poultry, seals, and minks and pose an increasing threat to human health. The occasional human infections with H10N8 and H10N3 viruses in China have significantly increased public concern about the potential pandemic risk posed by H10 viruses. In this study, we found that the North American H10 viruses have been successfully introduced to Asia by migratory birds and further reassorted with other subtypes to generate novel H10N4 and H10N8 viruses in eastern China. These emerging H10 reassortants have a high potential to threaten the poultry industry and human health due to their efficient replication and transmission in chickens, ducks, and mice.

The H5N8 avian influenza viruses have been widely circulating in wild birds and are responsible for the loss of over 33 million domestic poultry in Europe, Russia, Middle East, and Asia since January 2020. To monitor the invasion and spread of the H5N8 virus in China, we performed active surveillance by analyzing 317 wild bird samples and swab samples collected from 41,172 poultry all over the country. We isolated 22 H5N8 viruses from wild birds and 14 H5N8 viruses from waterfowls. Genetic analysis indicated that the 36 viruses formed two different genotypes: one genotype viruses were widely detected from different wild birds and domestic waterfowls; the other genotype was isolated from a whopper swan. We further revealed the origin and spatiotemporal spread of these two distinct H5N8 virus genotypes in 2020 and 2021. Animal studies indicated that the H5N8 isolates are highly pathogenic to chickens, mildly pathogenic in ducks, but have distinct pathotypes in mice. Moreover, we found that vaccinated poultry in China could be completely protected against H5N8 virus challenge. Given that the H5N8 viruses are likely to continue to spread in wild birds, vaccination of poultry is highly recommended in high-risk countries to prevent H5N8 avian influenza., (© 2021. The Author(s).)

H9N2 avian influenza viruses are widely prevalent in birds and pose an increasing threat to humans because of their enhanced virulence and transmissibility in mammals. Active surveillance on the prevalence and evolution of H9N2 viruses in different avian hosts will help develop eradication measures. We isolated 16 H9N2 viruses from chickens, green peafowls, and wild birds in eastern China from 2017 to 2019 and characterized their comparative genetic evolution, receptor-binding specificity, antigenic diversity, replication, and transmission in chickens and mice. The phylogenetic analysis indicated that the green peafowl viruses and swan reassortant shared the same ancestor with the poultry H9N2 viruses prevalent in eastern China, while the seven wild bird viruses belonged to wild bird lineage. The chicken, peafowl, and swan H9N2 viruses that belonged to the poultry lineage preferentially recognized α-2, 6-linked sialic acids (human-like receptor), but the wild bird lineage viruses can bind both α-2, 3 (avian-like receptor) and human-like receptor similarly. Interestingly, the H9N2 viruses of poultry lineage replicated well and transmitted efficiently, but the viruses of wild bird lineage replicated and transmitted with low efficiency. Importantly, the H9N2 viruses of poultry lineage replicated in higher titer in mammal cells and mice than the viruses of wild birds lineage. Altogether, our study indicates that co-circulation of the H9N2 viruses in poultry, wild birds, and ornamental birds increased their cross-transmission risk in different birds because of their widespread dissemination.

The H7N9 avian influenza virus (AIV) that emerged in China have caused five waves of human infection. Further human cases have been successfully prevented since September 2017 through the use of an H7N9 vaccine in poultry. However, the H7N9 AIV has not been eradicated from poultry in China, and its evolution remains largely unexplored. In this study, we isolated 19 H7N9 AIVs during surveillance and diagnosis from February 2018 to December 2019, and genetic analysis showed that these viruses have formed two different genotypes. Animal studies indicated that the H7N9 viruses are highly lethal to chicken, cause mild infection in ducks, but have distinct pathotypes in mice. The viruses bound to avian-type receptors with high affinity, but gradually lost their ability to bind to human-type receptors. Importantly, we found that H7N9 AIVs isolated in 2019 were antigenically different from the H7N9 vaccine strain that was used for H7N9 influenza control in poultry, and that replication of these viruses cannot, therefore, be completely prevented in vaccinated chickens. We further revealed that two amino acid mutations at positions 135 and 160 in the HA protein added two glycosylation sites and facilitated the escape of the H7N9 viruses from the vaccine-induced immunity. Our study provides important insights into H7N9 virus evolution and control., Competing Interests: The authors have declared that no competing interests exist.

Lethal infection of wild birds with different subtypes of H5 viruses continuously occur. To investigate the genetic evolution and pathogenicity of H5 viruses in wild birds, we performed a detailed genetic and biologic analysis of 27 viruses, including H5N1, H5N2, H5N6, and H5N8 subtypes, that were responsible for avian influenza outbreaks in wild birds in China over the past decade. We found that these 27 viruses, bearing different clades/subclades of HA, were complicated reassortants and formed 12 different genotypes. Ten of the viruses tested were highly pathogenic in chickens, but showed distinct pathotypes in ducks and mice. Five of these 10 viruses, which were all from clade2.3.4.4, could bind human-type receptors. Our findings reveal the diversity of the genetic and biologic properties of H5 viruses circulating in wild birds and highlight the need to carefully monitor and evaluate the risks these viruses pose to animal and public health.

Wild and domestic aquatic birds are the natural reservoirs of avian influenza viruses (AIVs). All subtypes of AIVs, including 16 hemagglutinin (HA) and nine neuraminidase (NA), have been isolated from the waterfowls. The H5 viruses in wild birds display distinct biological differences from their highly pathogenic H5 counterparts. Here, we isolated seven H5N3 AIVs including three from wild birds and four from domestic ducks in China from 2015 to 2018. The isolation sites of all the seven viruses were located in the region of the East Asian-Australasian Migratory Flyway. Phylogenetic analysis indicated that the surface genes of these viruses originated from the wild bird H5 HA subtype and the N3 Eurasian lineage. The internal genes of the seven H5N3 isolates are derived from the five gene donors isolated from the wild birds or ducks in Eastern-Asia region. They were also divided into five genotypes according to their surface genes and internal gene combinations. Interestingly, two of the seven H5N3 viruses contributed their partial internal gene segments (PB1, M and NS) to the newly emerged H7N4 reassortants, which have caused first human H7N4 infection in China in 2018. Moreover, we found that the H5N3 virus used in this study react with the anti-serum of the H5 subtype vaccine isolate (Re-11 and Re-12) and reacted well with the Re-12 anti-serum. Our findings suggest that worldwide intensive surveillance and the H5 vaccination (Re-11 and Re-12) in domestic ducks are needed to monitor the emergence of novel H5N3 reassortants in wild birds and domestic ducks and to prevent H5N3 viruses transmission from the apparently healthy wild birds and domestic ducks to chickens., (© 2019 Blackwell Verlag GmbH.)

Background: In 2017-2018, a new highly pathogenic H5N6 avian influenza virus (AIV) variant appeared in poultry and wild birds in Asian and European countries and caused multiple outbreaks. These variant strains are different from the H5N6 virus associated with human infection in previous years, and their genetic taxonomic status and antigenicity have changed. Therefore, revision of the primers and probes of fluorescent RT-PCR is important to detect the new H5N6 subtype AIV in poultry and reduce the risk of an epidemic in birds or humans., Methods: In this study, the primers and probes including three groups of HA and four groups of NA for H5N6 influenza virus were evaluated. Then a set of ideal primer and probes were selected to further optimize the reaction system and established a method of double rRT-PCR assay. The specificity of this method was determined by using H1~H16 subtype AIV., Results: The results showed that fluorescence signals were obtained for H5 virus in FAM channel and N6 virus in VIC channel, and no fluorescent signal was observed in other subtypes of avian influenza viruses. The detection limit of this assay was 69 copies for H5 and 83 copies for N6 gene. And, the variability tests of intra- and inter-assay showed excellent reproducibility. Moreover, this assay showed 100% agreement with virus isolation method in detecting samples from poultry., Conclusion: The duplex rRT-PCR assay presented here has high specificity, sensitivity and reproducibility, and can be used for laboratory surveillance and rapid diagnosis of newly emerged H5N6 subtype avian influenza viruses.

Ducks play a key role in the maintenance and spread of avian influenza viruses (AIVs) in nature, and control of AIVs in ducks has important implications for AIV eradication from poultry. We previously constructed a recombinant duck enteritis virus (DEV), rDEVus78HA, that expresses the HA gene of an H5N1 AIV and showed that rDEVus78HA immunization provides complete protection against both DEV and H5N1 AIV challenge in specific-pathogen-free ducks. In this study, we performed a 60-week clinical trial and found that this rDEVus78HA vaccine can function as a bivalent vaccine in farmed ducks against lethal challenge with DEV and H5N1 virus. Moreover, we found that rDEVus78HA-vaccinated ducks were efficiently protected against challenges with recently isolated heterologous H5N6 and H5N8 viruses. Our results demonstrate that rDEVus78HA could be extremely valuable for the control of DEV and H5 AIVs in ducks., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Human infectious avian influenza virus (AIV) H7N9 emerged in China in 2013. The N9 gene of H7N9, which has the ability to cause death in humans, originated from an H11N9 influenza strain circulating in wild birds. To investigate the frequency and distribution of the N9 gene of the H11N9 and H7N9 influenza virus circulating in wild birds between 2006 and 2015, 35,604 samples were collected and tested. No H7N9 but four strains of the H11N9 subtype AIV were isolated, and phylogenetic analyses showed that the four H11N9 viruses were intra-subtype and inter-subtype reassortant viruses. A sequence analysis revealed that all six internal genes of A/wild bird/Anhui/L306/2014 (H11N9) originated from an H9N2 AIV isolated in Korea. The H9N2 strain, which is an inner gene donor reassorted with other subtypes, is a potential threat to poultry and even humans. It is necessary to increase monitoring of the emergence and spread of H11N9 AIV in wild birds., (© 2018 Blackwell Verlag GmbH.)

The H3N2 influenza viruses became widespread in humans during the 1968 H3N2 pandemic and have been a major cause of influenza epidemics ever since. Different lineages of H3N2 influenza viruses are also commonly found in animals. If a different lineage of H3N2 virus jumps to humans, a human influenza pandemic could occur with devastating consequences. Here, we studied the genetics, receptor-binding properties, and replication and transmission in mammals of 15 H3N2 avian influenza viruses detected in live poultry markets in China. We found that the H3N2 avian influenza viruses are complicated reassortants with distinct replication phenotypes in mice. Five viruses replicated efficiently in mice and bound to both human-type and avian-type receptors. These viruses transmitted efficiently to direct-contact guinea pigs, and three of them also transmitted among guinea pigs and ferrets via respiratory droplets. Moreover, ferret antiserum induced by human H3N2 viruses did not react with any of the H3N2 avian influenza viruses. Our study demonstrates that the H3N2 avian influenza viruses pose a clear threat to human health and emphasizes the need for continued surveillance and evaluation of the H3N2 influenza viruses circulating in nature.

The H7N9 viruses that emerged in China in 2013 were nonpathogenic in chickens but mutated to a highly pathogenic form in early 2017 and caused severe disease outbreaks in chickens. The H7N9 influenza viruses have caused five waves of human infection, with almost half of the total number of human cases (766 of 1,567) being reported in the fifth wave, raising concerns that even more human infections could occur in the sixth wave. In September 2017, an H5/H7 bivalent inactivated vaccine for chickens was introduced, and the H7N9 virus isolation rate in poultry dropped by 93.3% after vaccination. More importantly, only three H7N9 human cases were reported between October 1, 2017 and September 30, 2018, indicating that vaccination of poultry successfully eliminated human infection with H7N9 virus. These facts emphasize that active control of animal disease is extremely important for zoonosis control and human health protection.

Background: Wild birds are gaining increasing attention as gene-mixing reservoirs for influenza viruses. To investigate the molecular properties of the viruses isolated and epidemiological analysis of H9N2 subtype AIV in wild birds, we studied samples obtained over two years (2014-2015) from wetlands in Anhui province, China., Methods: A total of 4534 samples were collected from migratory waterfowl in Anhui in 2014-2015, and 8 strains of H9 subtype AIV were isolated., Results: Phylogenetic analysis showed different degrees of gene segment reassortment in H9 viruses between the Eurasian lineage and the North American lineage. Most importantly, two viruses harbored the E627K mutation in the polymerase PB2 (PB2) protein. This is the first report of the mutation of this virus from low pathogenicity to high pathogenicity in wild birds., Conclusions: The continued surveillance of wild birds, especially migratory birds, is important to provide early warning and control of AIV outbreaks. Our results highlight the high genetic diversity of AIV along the Eurasian-Australian migration flyway and the need for more extensive AIV surveillance in eastern China., (© 2018 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

H7N9 low pathogenic influenza viruses emerged in China in 2013 and mutated to highly pathogenic strains in 2017, resulting in human infections and disease in chickens. To control spread, a bivalent H5/H7 inactivated vaccine was introduced in poultry in September 2017. To monitor virus evolution and vaccine efficacy, we collected 53,884 poultry samples across China from February 2017 to January 2018. We isolated 252 H7N9 low pathogenic viruses, 69 H7N9 highly pathogenic viruses, and one H7N2 highly pathogenic virus, of which two low pathogenic and 14 highly pathogenic strains were collected after vaccine introduction. Genetic analysis of highly pathogenic strains revealed nine genotypes, one of which is predominant and widespread and contains strains exhibiting high virulence in mice. Additionally, some H7N9 and H7N2 viruses carrying duck virus genes are lethal in ducks. Thus, although vaccination reduced H7N9 infections, the increased virulence and expanded host range to ducks pose new challenges., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Binding exclusively to human-type receptors is a prerequisite for avian influenza viruses to transmit from human to human. We previously reported that 34% of H6 avian influenza viruses recognize the human-type receptor, but their affinity for the avian-type receptor remains higher than that for the human-type receptor. Here, we found that a single amino acid change from glutamine to leucine at position 226 of hemagglutinin caused a switch in receptor-binding preference from avian-type to human-type receptors and rendered A/chicken/Guangdong/S1312/2010(H6N2) capable of respiratory droplet transmission in guinea pigs., (Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

H7N7 avian influenza viruses have been widely detected in wild birds and domestic poultry since they were first detected in chickens in Italy in 1902. They can occasionally transmit to humans. Here, we isolated six H7N7 viruses in live poultry markets during routine surveillance from 2010 to 2013. Sequences analysis revealed that these viruses are reassortants bearing genes of H3N8, H7N3, H7N7, and H10N7 influenza viruses detected in wild birds and ducks, and can be categorized into three genotypes (A, B, and C). All six viruses bound to both human-type and avian-type receptors. The viruses in genotype B and C could replicate efficiently in the lungs and nasal turbinates of mice without prior adaptation, and the genotype C virus also replicated in the brain of two of three mice tested. It is important to continue to monitor the evolution of H7N7 viruses and to evaluate their potential to cause human infections., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The H5N1 avian influenza viruses emerged in Southeast Asia in the late 20th century and have evolved into multiple phylogenetic clades based on their hemagglutinin (HA)-encoding genes. The clade 7.2 viruses were first detected in chickens in northern China in 2006, and vaccines specifically targeted to the clade were developed and have been used in poultry in China since 2006. During routine surveillance and disease diagnosis, we isolated seven H5 viruses between 2011 and 2014 that bear the clade 7.2 HA genes. Here, we performed extensive studies to understand how the clade 7.2 H5 viruses have evolved in chickens in China. Full genome sequence analysis revealed that the seven viruses formed two subtypes (four H5N1 viruses and three H5N2 viruses) and four genotypes by deriving genes from other influenza viruses. All of the viruses had antigenically drifted from the clade 7.2 viruses that were isolated in 2006. Pathogenicity studies of four viruses, one from each genotype, revealed that all of the viruses are highly pathogenic in chickens, but none of them could replicate in ducks. The four viruses exclusively bound to avian-type receptors and replicated only in the turbinates and/or lungs of mice; none of them were lethal to mice at a dosage of 10 6 50% egg infective doses (EID 50 ). Our study indicates that although the clade 7.2 viruses have not been eradicated from poultry through vaccination, they have not become more dangerous to other animals (e.g., ducks and mice) and humans., Importance: Animal influenza viruses can acquire the ability to infect and kill humans. The H5N1 viruses have been a concern in recent decades because of their clear pandemic potential. We sorted H5N1 influenza viruses into different phylogenetic clades based on their HA genes. The clade 7.2 viruses were detected in chickens in several provinces of northern China in 2006. Vaccines for these viruses were subsequently developed and have been used ever since to control infection of poultry. Here, we analyzed the genetic and biologic properties of seven clade 7.2 viruses that were isolated from chickens between 2011 and 2014. We found that after nearly 9 years of circulation in chickens, the clade 7.2 viruses still exclusively bind to avian-type receptors and are of low pathogenicity to mice, suggesting that these H5 viruses pose a low risk to human public health., (Copyright © 2016 Liu et al.)

An inactivated H5N1 avian influenza (AI) vaccine (Re-6) that bears the HA and NA genes from a clade 2.3.2.1 H5N1 virus, A/duck/Guangdong/S1322/10 (DK/GD/S1322/10), has been used in domestic poultry in China and other Southeast Asian countries to control clade 2.3.2.1 H5N1viruses since 2012. The efficacy of this vaccine against H5N1 viruses isolated in recent years has not been reported. In this study, we evaluated the protection efficacy of the Re-6 vaccine in chickens against challenge with four clade 2.3.2.1 H5N1 viruses, one clade 2.3.4.4 H5N1 virus, and one clade 7.2 H5N1 virus; these viruses were isolated in mainland China, Hong Kong, and the Democratic People's Republic of Korea between 2011 and 2015. The vaccinated chickens were completely protected (no disease signs, virus shedding, or death) from the challenge with the four clade 2.3.2.1 H5N1 viruses. In the clade 7.2 virus-challenged group, all of the vaccinated chickens remained healthy and survived for the entire 2-wk observation period; virus shedding was only detected from 1 of 10 chickens on day 3 postchallenge. In the clade 2.3.4.4 virus-challenged group, 8 of the 10 vaccinated chickens remained healthy and survived the 2-wk observation period; however, virus shedding was detected from 8 of 10 chickens on day 5 postchallenge. These results indicate that the Re-6 vaccine provides solid protection against clade 2.3.2.1, good protection against clade 7.2, and poor protection against clade 2.3.4.4.

The Goose/Guangdong-lineage H5 viruses have evolved into diverse clades and subclades based on their hemagglutinin (HA) gene during their circulation in wild birds and poultry. Since late 2013, the clade 2.3.4.4 viruses have become widespread in poultry and wild bird populations around the world. Different subtypes of the clade 2.3.4.4 H5 viruses, including H5N1, H5N2, H5N6, and H5N8, have caused vast disease outbreaks in poultry in Asia, Europe, and North America. In this study, we developed a new H5N1 inactivated vaccine by using a seed virus (designated as Re-8) that contains the HA and NA genes from a clade 2.3.4.4 virus, A/chicken/Guizhou/4/13(H5N1) (CK/GZ/4/13), and its six internal genes from the high-growth A/Puerto Rico/8/1934 (H1N1) virus. We evaluated the protective efficacy of this vaccine in chickens challenged with one H5N1 clade 2.3.2.1b virus and six different subtypes of clade 2.3.4.4 viruses, including H5N1, H5N2, H5N6, and H5N8 strains. In the clade 2.3.2.1b virus DK/GX/S1017/13-challenged groups, half of the vaccinated chickens shed virus through the oropharynx and two birds (20%) died during the observation period. All of the control chickens shed viruses and died within 6 days of infection with challenge virus. All of the vaccinated chickens remained healthy following challenge with the six clade 2.3.4.4 viruses, and virus shedding was not detected from any of these birds; however, all of the control birds shed viruses and died within 4 days of challenge with the clade 2.3.4.4 viruses. Our results indicate that the Re-8 vaccine provides protection against different subtypes of clade 2.3.4.4 H5 viruses.

Unlabelled: H4 avian influenza virus (AIV) is one of the most prevalent influenza virus subtypes in the world. However, whether H4 AIVs pose a threat to public health remains largely unclear. Here, we analyzed the phylogenetic relationships, receptor binding properties, replication, and transmissibility in mammals of H4 AIVs isolated from live poultry markets in China between 2009 and 2012. Genomic sequence analysis of 36 representative H4 viruses revealed 32 different genotypes, indicating that these viruses are undergoing complex and frequent reassortment events. All 32 viruses tested could replicate in the respiratory organs of infected mice without prior adaptation. Receptor binding analysis demonstrated that the H4 AIVs bound to α-2,6-linked glycans, although they retained the binding preference for α-2,3-linked glycans. When we tested the direct-contact transmission of 10 H4 viruses in guinea pigs, we found that three viruses did not transmit to any of the contact animals, one virus transmitted to one of three contact animals, and six viruses transmitted to all three contact animals. When we further tested the respiratory droplet transmissibility of four of the viruses that transmitted efficiently via direct contact, we found that three of them could transmit to one or two of the five exposed animals. Our study demonstrates that the current circulating H4 AIVs can infect, replicate in, and transmit to mammalian hosts, thereby posing a potential threat to human health. These findings emphasize the continual need for enhanced surveillance of H4 AIVs., Importance: Numerous surveillance studies have documented the wide distribution of H4 AIVs throughout the world, yet the biological properties of H4 viruses have not been well studied. In this study, we found that multiple genotypes of H4 viruses are cocirculating in the live poultry markets of China and that H4 viruses can replicate in mice, possess human-type receptor binding specificity, and transmit between guinea pigs via direct contact. Strikingly, some H4 strains also can transmit via respiratory droplet, albeit with limited efficiency. These results clearly show the potential threat posed by H4 viruses to public health., (Copyright © 2016 Liang et al.)

A novel H10N8 influenza A virus has been detected in three humans in China since December 2013. Although this virus was hypothesized to be a novel reassortant among influenza viruses from wild birds and domestic poultry, its evolutionary path leading to human infection is unknown. Sporadic surveillance at the live poultry market (LPM) suspected to be the source of infection for the first H10N8 patient has shown a gradual increase in influenza virus prevalence culminating with a predominance of H10N8 viruses. Influenza viruses detected in the LPM up to 8 months prior to human infection contributed genetic components to the zoonotic virus. These H10N8 viruses have continued to evolve within this LPM subsequent to the human infection, and continuous assessments of these H10N8 viruses will be necessary. Serological surveillance showed that the virus appears to have been present throughout the LPM system in Nanchang, China. Reduction of the influenza virus burden in LPMs is essential in preventing future emergence of novel influenza viruses with zoonotic and pandemic potential., (Copyright © 2014 Elsevier B.V. All rights reserved.)

H9N2 subtype influenza viruses have been detected in different species of wild birds and domestic poultry in many countries for several decades. Because these viruses are of low pathogenicity in poultry, their eradication is not a priority for animal disease control in many countries, which has allowed them to continue to evolve and spread. Here, we characterized the genetic variation, receptor-binding specificity, replication capability, and transmission in mammals of a series of H9N2 influenza viruses that were detected in live poultry markets in southern China between 2009 and 2013. Thirty-five viruses represented 17 genotypes on the basis of genomic diversity, and one specific "internal-gene-combination" predominated among the H9N2 viruses. This gene combination was also present in the H7N9 and H10N8 viruses that have infected humans in China. All of the 35 viruses preferentially bound to the human-like receptor, although two also retained the ability to bind to the avian-like receptor. Six of nine viruses tested were transmissible in ferrets by respiratory droplet; two were highly transmissible. Some H9N2 viruses readily acquired the 627K or 701N mutation in their PB2 gene upon infection of ferrets, further enhancing their virulence and transmission in mammals. Our study indicates that the widespread dissemination of H9N2 viruses poses a threat to human health not only because of the potential of these viruses to cause an influenza pandemic, but also because they can function as "vehicles" to deliver different subtypes of influenza viruses from avian species to humans.

In February 2014, while investigating the source of a human infection with influenza A(H7N9) virus in northern China, we isolated subtypes H7N2 and H9N2 viruses from chickens on the patient's farm. Sequence analysis revealed that the H7N2 virus is a novel reassortant of H7N9 and H9N2 viruses. Continued surveillance is needed.

Unlabelled: Influenza viruses of the H6 subtype have been isolated from wild and domestic aquatic and terrestrial avian species throughout the world since their first detection in a turkey in Massachusetts in 1965. Since 1997, H6 viruses with different neuraminidase (NA) subtypes have been detected frequently in the live poultry markets of southern China. Although sequence information has been gathered over the last few years, the H6 viruses have not been fully biologically characterized. To investigate the potential risk posed by H6 viruses to humans, here we assessed the receptor-binding preference, replication, and transmissibility in mammals of a series of H6 viruses isolated from live poultry markets in southern China from 2008 to 2011. Among the 257 H6 strains tested, 87 viruses recognized the human type receptor. Genome sequence analysis of 38 representative H6 viruses revealed 30 different genotypes, indicating that these viruses are actively circulating and reassorting in nature. Thirty-seven of 38 viruses tested in mice replicated efficiently in the lungs and some caused mild disease; none, however, were lethal. We also tested the direct contact transmission of 10 H6 viruses in guinea pigs and found that 5 viruses did not transmit to the contact animals, 3 viruses transmitted to one of the three contact animals, and 2 viruses transmitted to all three contact animals. Our study demonstrates that the H6 avian influenza viruses pose a clear threat to human health and emphasizes the need for continued surveillance and evaluation of the H6 influenza viruses circulating in nature., Importance: Avian influenza viruses continue to present a challenge to human health. Research and pandemic preparedness have largely focused on the H5 and H7 subtype influenza viruses in recent years. Influenza viruses of the H6 subtype have been isolated from wild and domestic aquatic and terrestrial avian species throughout the world since their first detection in the United States in 1965. Since 1997, H6 viruses have been detected frequently in the live poultry markets of southern China; however, the biological characterization of these viruses is very limited. Here, we assessed the receptor-binding preference, replication, and transmissibility in mammals of a series of H6 viruses isolated from live poultry markets in southern China and found that 34% of the viruses are able to bind human type receptors and that some of them are able to transmit efficiently to contact animals. Our study demonstrates that the H6 viruses pose a clear threat to human health.

To gain insight into the ecology of avian influenza viruses (AIV), we conducted active influenza virus surveillance in domestic ducks on farms located on the flyway of migratory birds in the Dongting Lake region of Hunan Province, China, from winter 2011 until spring 2012. Specimens comprising 3,030 duck swab samples and 1,010 environmental samples were collected from 101 duck farms. We isolated AIV of various HA subtypes, including H3, H4, H5, H6, H9, H10, H11, and H12. We sequenced the entire coding sequences of the genomes of 28 representative isolates constituting 13 specific subtypes. When the phylogenetic relationships among these isolates were examined, we observed that extensive reassortment events had occurred. Among the 28 Dongting Lake viruses, 21 genotypes involving the six internal genes were identified. Furthermore, we identified viruses or viral genes introduced from other countries, viral gene segments of unknown origin, and a novel HA/NA combination. Our findings emphasize the importance of farmed domestic ducks in the Dongting Lake region to the genesis and evolution of AIV and highlight the need for continued surveillance of domestic ducks in this region.

Although vaccination is an important strategy for controlling H5N1 avian influenza virus infections, broilers (short-lived meat chickens) remain the major victims in disease-endemic countries. Inactivated vaccine usually requires 2-3weeks to establish solid protection, and recombinant vaccines, including the recombinant fowlpox virus vaccine and the recombinant Newcastle disease virus vaccine are affected by maternal antibodies against the vectors. These disadvantages compromise the protective efficacy of these vaccines in broilers. Here, we evaluated the safety and efficacy of a new recombinant duck enteritis virus that expresses the HA gene of an H5N1 virus (rDEV-re6) in specific-pathogen-free chickens and broilers. We found this new rDEV-re6 virus to be safe in chickens and to induce rapid and solid protection after a single dose. This virus may therefore serve as an ideal single-dose vaccine for broilers., (Copyright © 2012 Elsevier B.V. All rights reserved.)

During their circulation in nature, H5N1 avian influenza viruses (AIVs) have acquired the ability to kill their natural hosts, wild birds and ducks. The genetic determinants for this increased virulence are largely unknown. In this study, we compared two genetically similar H5N1 AIVs, A/duck/Hubei/49/05 (DK/49) and A/goose/Hubei/65/05 (GS/65), that are lethal for chickens but differ in their virulence levels in ducks. To explore the genetic basis for this difference in virulence, we generated a series of reassortants and mutants of these two viruses. The virulence of the reassortant bearing the PA gene from DK/49 in the GS/65 background increased 10(5)-fold relative to that of the GS/65 virus. Substitution of two amino acids, S224P and N383D, in PA contributed to the highly virulent phenotype. The amino acid 224P in PA increased the replication of the virus in duck embryo fibroblasts, and the amino acid 383D in PA increased the polymerase activity in duck embryo fibroblasts and delayed the accumulation of the PA and PB1 polymerase subunits in the nucleus of virus-infected cells. Our results provide strong evidence that the polymerase PA subunit is a virulence factor for H5N1 AIVs in ducks.

Despite substantial efforts to control H5N1 avian influenza viruses (AIVs), the viruses have continued to evolve and cause disease outbreaks in poultry and infections in humans. In this report, we analyzed 51 representative H5N1 AIVs isolated from domestic poultry, wild birds, and humans in China during 2004 to 2009, and 21 genotypes were detected based on whole-genome sequences. Twelve genotypes of AIVs in southern China bear similar H5 hemagglutinin (HA) genes (clade 2.3). These AIVs did not display antigenic drift and could be completely protected against by the A/goose/Guangdong/1/96 (GS/GD/1/96)-based oil-adjuvanted killed vaccine and recombinant Newcastle disease virus vaccine, which have been used in China. In addition, antigenically drifted H5N1 viruses, represented by A/chicken/Shanxi/2/06 (CK/SX/2/06), were detected in chickens from several provinces in northern China. The CK/SX/2/06-like viruses are reassortants with newly emerged HA, NA, and PB1 genes that could not be protected against by the GS/GD/1/96-based vaccines. These viruses also reacted poorly with antisera generated from clade 2.2 and 2.3 viruses. The majority of the viruses isolated from southern China were lethal in mice and ducks, while the CK/SX/2/06-like viruses caused mild disease in mice and could not replicate in ducks. Our results demonstrate that the H5N1 AIVs circulating in nature have complex biological characteristics and pose a continued challenge for disease control and pandemic preparedness.

A/duck/Guangxi/53/2002 (DKGX/53) and A/duck/Fujian/01/2002 (DKFJ/01) are H5N1 avian influenza viruses that are lethal in chickens. In mice, however, DKFJ/01 is highly pathogenic, whereas DKGX/53 displays low pathogenicity. In this study, we used reverse genetics to demonstrate that two amino acid residues at positions 30 and 215 of the M1 protein of these two viruses are important determinants for pathogenicity in mice. We thus firstly prove the M1 protein contributes to the virulence of H5N1 viruses in mice, and the amino acid residues shown to attenuate the virulence could be targeted in influenza virus candidates for live vaccine development.

Using plasmid-based reverse genetics, we generated a molecularly altered virus, H5N1/PR8-5B19, containing modified HA and NA genes from A/Goose/Guangdong/1/96 (GS/GD/1/96). In the H5N1/PR8-5B19 virus, the HA cleavage site was modified to resemble that of low-pathogenic avian strains and a portion of the NA stalk region was replaced by the immunodominant 5B19 epitope of the S2 glycoprotein of murine hepatitis virus (MHV). H5N1/PR8-5B19 is not lethal to embryonated eggs or chickens. Chickens immunized with the H5N1/PR8-5B19 inactivated vaccine produced high levels of HI antibody and a measurable antibody response against the MHV 5B19 epitope, and were fully protected against subsequent challenge with different highly pathogenic H5N1 avian influenza viruses. H5N1/PR8-5B19 is therefore an attractive marker vaccine candidate, eliciting a strong, protective antibody response and enabling serological discrimination between vaccinated and wild-type virus-infected chickens.

We explored the molecular basis of antigenic variation by comparing two H9N2 subtype avian influenza viruses, A/Chicken/Shandong/6/96 (CK/SD/6) and A/Chicken/Guangxi/10/99 (CK/GX/10), that react differently to a monoclonal antibody C/B3. To assess the genetic basis for this antigenic difference, we used reverse genetics to generate a series of chimera and mutants of these two viruses. We found that a single-amino-acid substitution of asparagine for serine at position 145 (S145N) in the HA protein prevents the reaction of CK/SD/6 virus with C/B3. Substitution of serine for asparagine at the same position (N145S) enables the CK/GX/10 to react with C/B3 in hemaglutinin inhibition, immunofluorescence and neutralization assays. We further demonstrated that the amino acid N145 in the H9 HA protein is glycosylated. Our results provide experimental evidence that the glycosylation of HA oligosaccharide attachment sites implicated in antibody binding could have a role in antigenic variation.

In 2001 and 2003, we isolated two H5N1 viruses, A/swine/Fujian/1/01 (SW/FJ/01) and A/swine/Fujian/1/03 (SW/FJ/03), from pigs in Fujian Province, southern China. Genetically, these two viruses are similar, although the NS gene of the SW/FJ/03 virus has a 15-nucleotide deletion at coding positions 612 to 626. The SW/FJ/01 virus is highly lethal for chickens, whereas the SW/FJ/03 virus is nonpathogenic for chickens when administrated intravenously or intranasally. To understand the molecular basis for the difference in virulence, we used reverse genetics to create a series of single-gene recombinants of both viruses. We found that a recombinant virus containing the mutated NS gene from the SW/FJ/03 virus in the SW/FJ/01 virus background was completely attenuated in chickens. We also found that viruses expressing the mutant NS1 protein of SW/FJ/03 did not antagonize the induction of interferon (IFN) protein. Conversely, only the recombinant virus containing the wild-type SW/FJ/01 NS gene in the SW/FJ/03 background was lethal in chickens and antagonized IFN protein levels. Further, we proved that the NS1 genes of the two viruses differ in their stabilities in the host cells and in their abilities to interact with the chicken cleavage and polyadenylation specificity factor. These results indicate that the deletion of amino acids 191 to 195 of the NS1 protein is critical for the attenuation of the SW/FJ/03 virus in chickens and that this deletion affects the ability of the virus to antagonize IFN induction in host cells.

H5N1 highly pathogenic avian influenza virus (HPAIV) has continued to spread and poses a significant threat to both animal and human health. Current influenza vaccine strategies have limitations that prevent their effective use for widespread inoculation of animals in the field. Vaccine strains of Newcastle disease virus (NDV), however, have been used successfully to easily vaccinate large numbers of animals. In this study, we used reverse genetics to construct a NDV that expressed an H5 subtype avian influenza virus (AIV) hemagglutinin (HA). Both a wild-type and a mutated HA open reading frame (ORF) from the HPAIV wild bird isolate, A/Bar-headed goose/Qinghai/3/2005 (H5N1), were inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain. The recombinant viruses stably expressing the wild-type and mutant HA genes were found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of the recombinant viruses in chickens induced both NDV- and AIV H5-specific antibodies and completely protected chickens from challenge with a lethal dose of both velogenic NDV and homologous and heterologous H5N1 HPAIV. In addition, BALB/c mice immunized with the recombinant NDV-based vaccine produced H5 AIV-specific antibodies and were completely protected from homologous and heterologous lethal virus challenge. Our results indicate that recombinant NDV is suitable as a bivalent live attenuated vaccine against both NDV and AIV infection in poultry. The recombinant NDV vaccine may also have potential use in high-risk human individuals to control the pandemic spread of lethal avian influenza.

Three H5N1 influenza viruses were isolated from shell washes of duck and goose eggs confiscated from travelers coming from Vietnam. All eight gene segments of these viruses share high sequence identity with the H5N1 avian influenza viruses that caused outbreaks in poultry and humans in Hong Kong in 1997. Animal studies indicate that these isolated viruses are able to replicate in mouse lung and could be found in the organs of ducks without causing any clinical signs or death. However, the viruses are highly pathogenic for chickens. Although the source of these recently isolated Hong Kong 97-like H5N1 viruses is undetermined, their detection in the egg shell of duck and goose suggests that this particular genotype of H5N1 virus may have re-emerged in nature or may have been circulating continuously.