Emergence of a triple reassortment avian influenza virus (A/H5N6) from wild birds

Dear Editor, A recent publication in this Journal reported the emergence of an H5N6 strain originating from farm dogs in China1Yao X.Y. Lian C.Y. Lv Z.H. Zhang X.L. Shao J.W. Emergence of a novel reassortant H5N6 subtype highly pathogenic avian influenza virus in farmed dogs in China.J Infect. 2023 Oct; 87: e70-e72Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar. H5N6 subtype avian influenza strains in Clade 2.3.4.4 have circulated in poultry in China since 20132Yang L. Zhu W. Li X. Bo H. Zhang Y. Zou S. et al.Genesis and Dissemination of Highly Pathogenic H5N6 Avian Influenza Viruses.J Virol. 2017 Mar 1; 91Crossref Scopus (67) Google Scholar. In 2014, highly pathogenic avian influenza H5 GsGd (Clade 2.3.4.4) caused multiple outbreaks on several continents.3Cui Y. Li Y. Li M. Zhao L. Wang D. Tian J. et al.Evolution and extensive reassortment of H5 influenza viruses isolated from wild birds in China over the past decade.Emerging Microbes & Infections. 2020; 9 (2020/01/01): 1793-1803Crossref PubMed Scopus (0) Google Scholar. Studies have shown that H5N6 AIV can infect not only poultry and wild birds, but also mammals such as pigs, cats, and even humans. China has adopted a vaccine immunization policy to prevent the spread and prevalence of influenza viruses among poultry. However, this strategy does not prevent the spread and evolution of influenza viruses in waterfowl, wild birds and mammals4Chen J. Xu L. Liu T. Xie S. Li K. Li X. et al.Novel Reassortant Avian Influenza A(H5N6) Virus, China, 2021.Emerg Infect Dis. 2022 Aug; 28: 1703-1707Crossref Scopus (7) Google Scholar, 5Shi J. Zeng X. Cui P. Yan C. Chen H. Alarming situation of emerging H5 and H7 avian influenza and effective control strategies.Emerg Microbes Infect. 2023 Dec; 122155072Crossref Scopus (30) Google Scholar. Avian influenza viruses carried by wild birds can not only cause avian influenza outbreaks but also infect companion animals that go outdoors, as in the case of dogs in Guangxi1Yao X.Y. Lian C.Y. Lv Z.H. Zhang X.L. Shao J.W. Emergence of a novel reassortant H5N6 subtype highly pathogenic avian influenza virus in farmed dogs in China.J Infect. 2023 Oct; 87: e70-e72Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 6Xu Y. Ramey A.M. Bowman A.S. DeLiberto T.J. Killian M.L. Krauss S. et al.Low-Pathogenic Influenza A Viruses in North American Diving Ducks Contribute to the Emergence of a Novel Highly Pathogenic Influenza A(H7N8) Virus.J Virol. 2017 May 1; 91Crossref Scopus (28) Google Scholar. Therefore, monitoring avian influenza viruses carried by wild birds has become important. In the course of avian influenza surveillance at the wild bird interface in the Pearl River Delta region, our laboratory isolated a wild-bird-derived H5N6 subtype AIV strain from wild bird fecal samples collected in Shenzhen in March 2023. This strain was named A/wild bird/Guangdong/SZ1/230322/ H5N6. We sequenced the whole genome of the virus and analyzed its genetic evolution. The HA phylogenetic tree showed that the isolate was located in Clade 2.3.4.4b1, which is in Clade 2.3.4.4b with the strains currently prevalent in poultry hosts on the live bird market and the vaccine strains currently found in China (Re-14 and rHN5801). SZ1 belongs to the same branch as the H5N6 strain originating from farm dogs reported by Yao et al. in GISAID in 2023. Genetic analyses showed that the cleavage site of the HA gene in A/Wildbird/Guangdong/SZ1/230322/H5N6 contained the contiguous basic amino acid REKRRKR↓GLF, which is consistent with the molecular profile of highly pathogenic strains. The SZ1 strain has mutations in the HA gene at the S137A and T192I loci, a double-site mutation that results in enhanced binding of the virus to the α-2,6-linked SAs (human-type receptors), which is characterized by enhanced binding to the mammalian receptor. 226Q and 228 G indicated that these strains have an affinity for the avian-type receptors7Shi Y. Wu Y. Zhang W. Qi J. Gao G.F. Enabling the 'host jump': structural determinants of receptor-binding specificity in influenza A viruses.Nat Rev Microbiol. 2014 Dec; 12: 822-831Crossref PubMed Scopus (0) Google Scholar, 8Yang Z.Y. Wei C.J. Kong W.P. Wu L. Xu L. Smith D.F. et al.Immunization by avian H5 influenza hemagglutinin mutants with altered receptor binding specificity.Science. 2007 Aug 10; 317: 825-828Crossref PubMed Scopus (196) Google Scholar. T33K, L89V, R477G, I495V, E627K, and 701D in the PB2 gene, L13P, D538, K578, and 614E in the PB1 gene, A150R and A184K in the NP gene, E133 and G631S in the PA gene, D97E in the NS gene, and N30D and T215A mutations in the M1 gene not only enhance receptor binding but also increase virulence in mammals9Song M.S. Pascua P.N. Lee J.H. Baek Y.H. Lee O.J. Kim C.J. et al.The polymerase acidic protein gene of influenza a virus contributes to pathogenicity in a mouse model.J Virol. 2009 Dec; 83: 12325-12335Crossref Scopus (144) Google Scholar, 10Steel J. Lowen A.C. Mubareka S. Palese P. Transmission of Influenza Virus in a Mammalian Host Is Increased by PB2 Amino Acids 627K or 627E/701N.Plos Pathog. 2009 Jan; 5Crossref Scopus (465) Google Scholar. BLAST of the GenBank database showed that the sequences of the HA and NA fragments observed in this study showed the highest nucleotide sequence identity with A/Canine/China/GX30/2023 (H5N6) and A/duck/Sichuan/SC5698/2021 (H5N6), 98.59% and 98.62%, respectively. The sequences of the NS, PA, PB2, and PB1 fragments of the internal genes were similar to the canine-derived virus from Guangxi, China. The nucleotide similarity between the M segment sequence and the human source sequence from Guangdong, China, was 99.49% (Table S1). Fig. 2 shows a schematic diagram of the triple reassortment of human, canine, and avian gene fragments it carries.Fig. 2Schematic composition of the whole gene mimicry of the SZ1 strain. The eight gene segments of the virus are, from top to bottom, Hemagglutinin (HA), Neuraminidase (NA), Polymerase B2 (PB2), Polymerase B1 (PB1), Polymerase A (PA), Nucleoprotein (NP), Nonstructural protein (NS), and Matrix protein (M). Each color represents a separate viral background.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To assess the antigenicity of the strain, a cross hemagglutination inhibition test was performed using standard World Organisation for Animal Health (WOAH) procedures. Two prevalent H5 subtype strains (B787, B732-12) and two commonly used vaccine strains (Re-14, rHN5801) were used as controls (Table S2). We found that both Re-14 and rHN5801 antisera autoreactive antibody titers were 1024. The titer of the Re-14 antiserum against SZ1 HI was 128, which is eight times lower than the titer of the self-reactive antibody. The HI titer of rHN5801 antiserum against SZ1 was 64, 16 times lower than that of the self-reactive antibody. The results showed that the antigenicity of the SZ1 strain was somewhat different from that of the Re-14 and rHN5801 vaccine strains, and that the vaccine’s immunoprotective effect was weaker. B787 and B732-12 antiserum self-reactive antibody titers were 256 and 1024, respectively. The antisera had 2–3 times lower HI antibody titers against SZ1, indicating minimal antigenic differences between them. In summary, the wild-bird-origin H5N6 strain SZ1 isolated in Shenzhen in this study belonged to Clade 2.3.4.4b1as well as the virus currently prevalent in China.The strains have similar antigenic properties. Analysis of the key HA gene loci showed that the SZ1 strain retained both high affinity for binding to the avian-type receptors (226Q and 228 G) and enhanced binding to the human-type receptors (S137A and T192I). Whole gene sequence comparison analyses revealed that the HA, NP, NS, PA, PB1, and PB2 gene fragments of the SZ1 strain were all highly similar to H5N6 isolated from Guangxi farm dogs and the M gene was highly similar to the human strain, suggesting that highly pathogenic H5N6 strains carried by wild birds have the potential to be transmitted to outdoor mammals. Shenzhen is an important stopover, sometimes referred to as a “transit refueling station,” on the Siberian–Australian wild bird migration route, and thus attracts many migratory birds every winter. Wild bird migration routes are an important means of influenza virus transmission and play an important role in the spread and evolution of the virus. Therefore, it is crucial to improve the continuous monitoring of wild bird migration routes. None. This research was supported by the China Agriculture Research System of MOF and MARA (CARS-41), China National Animal Disease Surveillance and Epidemiological Survey Program (2021-2025) (no. 202111), and the Science and Technology Program of Guangdong Province (2021B1212030015). Download .docx (.01 MB) Help with docx files Supplementary material