Genetic pairing of the hemagglutinin and neuraminidase glycoproteins of the highly pathogenic A(H5Nx) avian influenza viruses

Abstract

During the past 3 decades, the A/Goose/Guangdong/1/1996 (Gs/Gd) lineage highly pathogenic avian influenza (HPAI) viruses of the H5 subtype have successfully evolved to gain genetic diversity, to expand the range of infected host species, and to spread globally. The early Gs/Gd-lineage A(H5N1) viruses mainly infected domestic poultry and demonstrated strong hemagglutinin (HA) and neuraminidase (NA) genetic pairing. Since the appearance of clade 2.3.4, the Gs/Gd-lineage viruses have established transmission among migratory birds, which facilitated long-distance spread and opportunities for reassortment previously restricted by geographic segregation and migratory flyways. Since 2020 - 2021, clade 2.3.4.4b viruses have successfully achieved global spread to all continents except Oceania, further expanding the genetic diversity. With the global spread of the clade 2.3.4.4b, the genetic pairing of the HA and NA may not be maintained due to increased opportunities of genetic reassortment. This thesis aims to investigate the changes in genetic diversity brought by the recent epizootic outbreaks in which it was found that the HA and NA of H5N1 clade 2.3.4.4b viruses continued to pair after 2020. Interestingly, the HA shifted to pair from a short stalk N1 NA (NA clade NA.1) to full-length N1 NA (NA clade NA.2). The dominant pairings detected after 2020 were HA clade 2.3.4.4b.1 with NA clade NA.2.2 and HA clade 2.3.4.4b.2 with NA clade NA.2.1. Geography and clade defining mutation were found to be factors differing between the pairings. To understand the impact of the re-introduction of the clade 2.3.4.4b in China on the local avian influenza virus genetic diversity, the genetic composition of H5Nx viruses isolated from two poultry markets in Guangzhou from 2020 - 2022 was analyzed. H5N6 and H5N8 were the dominant subtypes detected in which the H5N6 HA clustered in clades 2.3.4.4b and 2.3.4.4h, while the H5N8 HA exclusively clustered in clade 2.3.4.4b. The N6 NA genes were divided into two clusters, differentiated by an eleven amino acid stalk truncation, while the N8 genes clustered with clade 2.3.4.4 H5N8 viruses circulating among wild avian species. Ten transient genotypes were identified in which H5N6 isolates contained genes originating from the Gs/Gd-lineage H5Nx viruses and low pathogenic avian influenza in China, while the H5N8 isolates contained genes derived from A(H5N8) 2.3.4.4b and A(H5N1) 2.3.2.1c. Overall, the results show that genetic pairing between HA and NA has been continued among the 2.3.4.4b viruses, with geographic segregation and potential epistatic amino acid changes as potential underlying mechanisms for further investigation. The results also demonstrate the impact of re-introduction of clade 2.3.4.4b in China and the subsequent expansion on the genetic diversity of H5Nx viruses in China. Compared to its first emergence, the challenges posed by the Gs/Gd-lineage H5Nx viruses has increased today due to changes in host range, ecological factors, and viral genetic diversity.