Major Global Migratory Flyways – Credit FAO
#16,833Influenza viruses are broadly categorized by their two surface glycoproteins - hemagglutinin (HA) and neuraminidase (NA) - hence we often talk about seasonal H1N1 or H3N2, or avian H5N1 or H9N2 viral subtypes.
But each subtype is further divided into clades; broad categories based on the genetic makeup of the HA gene. Over time, dozens of clades can emerge for a given subtype, and while some are supplanted by more `biologically fit' strains, multiple clades can co-circulate.
Clades can be further divided into sub-clades, and subclades (e.g. 22.214.171.124b) into genotypes (and variants within genotypes), producing a myriad of quasi-similar viruses - sometimes with differing characteristics (see Differences In Virulence Between Closely Related H5N1 Strains) - but still often referred to (outside of scientific journals) as a single subtype.
Simply put, you can't tell an HxNx virus by its subtype alone.
The deadly Asian H7N9 virus is a far cry from the relatively benign North American H7N9 virus that hit a few farms in 2017. Just as our current H5N1 epizootic differs greatly from the H5N1 outbreaks of a decade ago.
With this diversity - driven by influenza's ability to reassort and mutate - comes considerable uncertainty.
All of which brings us to a fascinating, and detailed, look at the growing diversity of avian H5N1 clade 126.96.36.199b viruses around the world. Among their findings, they report that H5N1 viruses are becoming better adapted to mammals (something we've discussed often, including here, here, and here), and could become more dangerous to humans over time.
Global dissemination of H5N1 influenza viruses bearing the clade 188.8.131.52b HA gene and biologic analysis of the ones detected in China
Evolution of the globally circulating H5N1 viruses bearing the clade 184.108.40.206b HA
Pengfei Cui, Jianzhong Shi, Congcong Wang, Yuancheng Zhang, Xin Xing, Huihui Kong, show all
Accepted author version posted online: 14 Jun 2022
H5N1 avian influenza viruses bearing the clade 220.127.116.11b 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.
H5N1 viruses bearing the clade 18.104.22.168b HA gene have become the predominant strains causing global avian influenza outbreaks since October 2021. In this study, we performed phylogenic analyses of 220 H5N1 viruses that were reported in 27 countries together with 13 viruses we isolated in China, and found that the globally circulating H5N1 viruses have formed 16 different genotypes. The G1 viruses are the most widely circulating strains having been detected in wild birds or domestic poultry in 22 countries across Europe, Africa, Asia, and North America. The 13 H5N1 viruses isolated in China belong to the G1, G7, G9, and G10 genotypes, and viruses of all four of these genotypes replicated efficiently in multiple organs of mice, although their pathogenicity varied among strains. Moreover, the newly detected H5N1 viruses antigenically matched the H5-Re14 vaccine seed virus that is currently used in China to prevent infection with H5 viruses bearing clade 22.214.171.124b HA.
Reassortment is the major mechanism for influenza virus evolution. During their circulation in nature, H5N8 viruses bearing clade 126.96.36.199b HA reassorted with four different low pathogenic viruses and generated the G1 H5N1 viruses, and these G1 H5N1 viruses further reassorted with at least 10 different avian influenza viruses, thereby formed 15 more H5N1 virus genotypes. Of note, our analyses indicated that 14 of the 16 genotypes emerged between September 2021 and January 2022 (Figure 3, Table S2), which further demonstrates that the avian influenza viruses are more active in winter.
Gu et al. detected different genotypes of H5N6 viruses in China and found that the H5N6 viruses were reassortants that derived their HA genes from H5N8 viruses, NA genes from different duck H5N6 viruses, and certain internal genes from different low pathogenic viruses . Most of the donor viruses of the NA and internal genes of the H5N6 viruses were previously detected in China, suggesting that the H5N6 reassortants were generated in ducks in China . However, it seems that the four genotypes of the H5N1 viruses detected in China have not undergone any further reassortment in domestic birds in China. Among the four genotypes of H5N1 viruses in China, three of them (G1, G7, and G10) previously appeared in other countries; the G9 viruses have only been detected in China. Since the donor of the NA gene of the G9 viruses was previously detected in wild ducks in South Korea (Figure 3(b)), it is reasonable to speculate that the G9 virus was originally generated in wild birds in this region and then spread to domestic ducks in China, although there have been no reports of G9 virus detection in wild birds.
The H5 avian influenza viruses are becoming progressively more virulent in the mammalian mouse model. Some of the earliest H5N1 viruses could not replicate in mice , but more and more strains have now acquired this ability and some have become lethal in mice [4,10,15]. A series of amino acid substitutions in different proteins of avian influenza virus have been reported to increase its pathogenicity to mice [37-53], and many of these substitutions have been detected in the widely circulating H5N1 viruses, which explains why the H5N1 viruses we tested in this study were able to replicate in and kill mice. The PB2 E627K substitution was found to increase the pathogenicity of H5N1, H7N9, and H9N2 viruses in mice [42,54,55], and to promote the respiratory droplet transmission of H1N1, H5N1, H7N9, and H9N2 viruses in ferrets [54-57].
The widely spread G1 viruses have caused human infection in the United Kingdom and fox infection in Ireland, Estonia, and the Netherlands, and the virus isolated from a fox in Ireland had PB2 627K (GISAID accession # EPI1998135). These facts suggest that the H5N1 viruses could become more dangerous if they have the opportunity to replicate in mammals, and therefore, they should be carefully monitored and evaluated in mammals.
H5N1 viruses have caused severe damage to the global poultry industry, with more than 70 million domestic poultry having been destroyed in efforts to contain the disease . Of note, about 30 million poultry have been destroyed in the United States alone . After detection of H5N8 viruses bearing clade 188.8.131.52b HA in China , an updated H5/H7 trivalent vaccine was produced with the H5-Re13, H5-Re14, and H7-Re4 seed viruses and has been used in China since January 2022 [30,35]. The H5-Re14 seed virus was developed to protect against H5 viruses bearing the clade 184.108.40.206b HA gene, and a challenge study by Zeng et al.  showed that the novel H5/H7 trivalent vaccine can provide solid protection against challenge with H5N1, H5N6, and H5N8 viruses bearing clade 220.127.116.11b HA. Our antigenic analysis in this study demonstrated that H5-Re14 antigenically matches well with the H5N1 viruses of four different genotypes. Given the wide circulation of H5N1 viruses in wild birds, it is highly likely that these viruses will continue to harm the poultry industry and pose a threat to human public health. Therefore, we strongly recommend that high-risk countries vaccinate their poultry to protect them against highly pathogenic H5 avian influenza.
While none of this guarantees that HPAI H5N1 clade 18.104.22.168b will ever become a serious public health threat - the greater its diversity - the more chances it has to hit upon the right combination of genetic changes it needs to become a contender.