Genetic diversity of highly pathogenic avian influenza H5N6 and H5N8 viruses in poultry markets in Guangdong, China, 2020-2022

Abstract

H5 highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/96 (Gs/Gd) lineage continue to evolve and cause outbreaks in domestic poultry and wild birds, with sporadic spillover infections in mammals. The global spread of clade 2.3.4.4b viruses via migratory birds since 2020 has facilitated the introduction of novel reassortants to China, where avian influenza of various subtypes have been epizootic or enzootic among domestic birds. To determine the impact of clade 2.3.4.4b re-introduction on local HPAI dynamics, we analyzed the genetic diversity of H5N6 and H5N8 detected from monthly poultry market surveillance in Guangdong, China, between 2020 and 2022. Our findings reveal that H5N6 viruses clustered in clades 2.3.4.4b and 2.3.4.4h, while H5N8 viruses were exclusively clustered in clade 2.3.4.4b. After 2020, the re-introduced clade 2.3.4.4b viruses replaced the clade 2.3.4.4h viruses detected in 2020. The N6 genes were divided into two clusters, distinguished by an 11 amino acid deletion in the stalk region, while the N8 genes clustered with clade 2.3.4.4 H5N8 viruses circulating among wild birds. Genomic analysis identified 10 transient genotypes. H5N6, which was more prevalently detected, was also clustered into more genotypes than H5N8. Specifically, H5N6 isolates contained genes derived from HPAI H5Nx viruses and low pathogenic avian influenza in China, while the H5N8 isolates contained genes derived from HPAI A(H5N8) 2.3.4.4b and A(H5N1) 2.3.2.1c. No positive selection on amino acid residues associated with mammalian adaptation was found. Our results suggest expanded genetic diversity of H5Nx viruses in China since 2021 with increasing challenges for pandemic preparedness.IMPORTANCESince 2016/2017, clade 2.3.4.4b H5Nx viruses have spread via migratory birds to all continents except Oceania. Here, we evaluated the impact of the re-introduction of clade of 2.3.4.4b on highly pathogenic avian influenza (HPAI) virus genetic diversity in China. Twenty-two H5N6 and H5N8 HPAI isolated from monthly surveillance in two poultry markets in Guangdong between 2020 and 2022 were characterized. Our findings showed that clade 2.3.4.4h, detected in 2020, was replaced by clade 2.3.4.4b in 2021-2022. H5N6 (n = 18) were clustered into more genotypes than H5N8 (n = 4), suggesting that H5N6 may possess better replication fitness in poultry. Conversely, the H5N8 genotypes are largely derived from the clade 2.3.4.4b wild bird isolates. As clade 2.3.4.4b continues to spread via migratory birds, it is anticipated that the genetic diversity of H5N6 viruses circulating in China may continue to expand in the coming years. Continuous efforts in surveillance, genetic analysis, and risk assessment are therefore crucial for pandemic preparedness.

Keywords: clade 2.3.4.4; genetic diversity; highly pathogenic avian influenza virus; reassortment; surveillance.

Fig 1

Monthly detection frequencies of H5, H7, and H9 AIV from retail and wholesale poultry markets in Guangdong between January 2019 and September 2022. Poultry swabs were collected every other week or monthly from a retail and a wholesale poultry market in Guangzhou, covering the period from January 2019 to September 2022, except for January to May 2020 (due to coronavirus disease-2019 lockdown) and February 2021 (retail market closure). AIV were detected by quantitative real-time RT-PCR targeting the influenza A virus M gene. AIV positive samples were further tested using primers and probes for H5, H7, and H9 subtypes by quantitative real-time RT-PCR. The number of swabs collected every month was shown in gray bars. The monthly detection frequencies of H5 (orange), H7 (blue), and H9 (green) from all sample types (oropharyngeal swabs, cloacal swabs, dressed poultry cavity, environmental swabs) were shown. Positivity rates by sample type are shown in Fig. S1.

Fig 2

Genetic analysis of the HA gene of H5N6 and H5N8 viruses isolated from poultry markets in Guangdong, 2020–2022. Time-scaled maximum clade credibility tree of clade 2.3.4.4 HA genes (n = 550) detected globally from 01 January 2014 to 30 June 2023. Subtype, host, and geographic region (location) of the tips are shown as bars on the right. Figure key of the bars depicts all the categorical variables detected with respect to the figure. Red dots indicate the 22 H5N6 and H5N8 viruses isolated in this study.

Fig 3

Genetic analysis of the N6 genes of H5N6 viruses isolated from poultry markets in Guangdong, 2020–2022. Time-scaled maximum-likelihood tree of N6 genes of A(H5N6) viruses (n = 276) detected globally from 01 January 2014 to 30 June 2023. HA clade information, host, and geographic region (location) of the tips are shown as bars on the right. Figure key of the bars depicts all the categorical variables detected with respect to the figure. Red dots indicate the 18 H5N6 viruses isolated in this study.

Fig 4

Genetic analysis of the N8 genes of H5N8 viruses isolated from poultry markets in Guangdong, 2020–2022. Time-scaled maximum-likelihood tree of N8 genes of A(H5N8) viruses (n = 158) detected globally from 01 January 2014 to 30 June 2023. HA clade information, host, and geographic region (location) of the tips are shown as bars on the right. Figure key of the bars depicts all the categorical variables detected with respect to the figure. Red dots indicate the four H5N8 viruses isolated in this study.

Fig 5

Genotype analysis of the 22 H5N6 and H5N8 viruses isolated from poultry markets in Guangdong, 2020–2022. (A) Ten genotypes were determined through identifying cluster combination using genotype progenitor strains in the phylogenetic trees (see Fig. S5 to S10). The representative strains of the gene pools are as follows: A(H5N8) 2.3.4.4b wild birds, A/Astrakhan/3212/2020(H5N8); A(H5N6) 2.3.4.4h, A/Goose/Guangdong/PO1707260055/GZH/2017(H5N6); A(H5N1) 2.3.2.1c, A/Anhui/1/2005(H5N1); A(H5Nx) 2.3.4.4, A/duck/Zhejiang/6DK19/2013(H5N2); A(H3N2), A/chicken/Ganzhou/GZ43/2016(H3N2); A(H9N2), A/Chicken/Zhejiang/HJ/2007(H9N2); and wild bird low pathogenic gene pool A/Eastern Spot-billed_Duck/Shanghai/JDS19701/2019(H4N2). The representative strains for the A(H6N6)/ A(H6N2) gene pool were A/duck/Ganzhou/GZ151/2016(H6N6), A/chicken/China/GZ1063/2014(H6N6), and A/duck/Guangdong/3111/2018(H6N2). (B) H5Nx viruses detected from the retail or the wholesale markets are shown as circles and triangles, respectively, according to genotype and date of sample collection.