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.

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.

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.

Background: Two human cases of avian influenza A (H3N8) virus infection were reported in China in 2022. Aim: To characterise H3N8 viruses circulating in China in September 2021−May 2022. Methods: We 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. Results: In 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. Conclusion: Avian 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 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.