Discussion
Highly pathogenic AIVs have been affecting the world’s poultry industry and also brought enormous challenges to the public health security since they accidentally moved from animals to human, causing a first outbreak in Guangdong, China, in 1996(Shortridge et al., 1998; Webster & Govorkova, 2014; Xu et al., 1999). Fortunately, there is no evidence of human-to-human transmission of the avian influenza virus. The H5 avian influenza virus has evolved rapidly from Clade 0 to the present Clade 2.3.4.4 and Clade 2.3.2.1(Group, 2012), and frequent reassortments of AIVs have been the leading cause of several epidemics(Bi et al., 2016; Karo-karo et al., 2019; Qi et al., 2018; Shi et al., 2018). The highly pathogenic avian influenza virus prevalence in China is an extraordinarily complicated problem to solve. We focused on the prevalence, gene differentiation, and reassortment of H5N6 in China, based on the available data for 2016−2019. We gathered evidence on a new reassortment virus complex diversity produced by frequent reassortment of different subtypes AIVs. Cases of both poultry and human infections are largely related to the complexity of the reassortment.
The rate of evolution of avian influenza also deserves close attention. The rate of evolution of H5N6 HA and NA genes is 7.3262E-03 and 6.9073E-03, respectively. And the rate of evolution of H9N2 HA and NA is 2.19E-03-2.83E-03, and is 3.3E-03-3.7E-03, respectively(Jin et al., 2020). H5N6 compared to H9N2 has a faster rate of evolution in HA and NA genes.This has a lot to do with the clade of HA and NA genes rapid differentiation is closely linked. And internal gene is relatively complicated, due to the restructuring of the LAIVs, especially the part of basic internal genes from H6N6, H6N2, H9N2, most notably the two H6N6 viruses from which the immune pressure is relatively small. According to the study of Bi et al(Bi et al., 2016), the PB1 gene of H5N6 is basically derived from H5N1, and PB2 is partly derived from H6N6, which may be the reason why our study showed that PB1 has significant gene differentiation compared with other genes.
In this study, a total of 16 gene sequences from humans [Figure 6 A], including eight strains isolated during 2016 (six isolates from January to April and two from November and December), two isolated since November, seven of eight strains were new reassortment H5N6 viruses that contain the internal genes fragments from H9N2 [Figure 6 A], two strains isolated during 2017 which are of the G1 genotype, and six strains isolated during 2018. These last six strains comprise three strains with the G1 genotype and three with the G1.4 genotype, and differed from G1 due to a PA gene from other AIVs. We propose that the virus can move from poultry to humans after new reassortment. The LPAIV provides the gene segments for the reassortment event, which is a problem that cannot be ignored. The current epidemic strain is of the G1 genotype, which is of great importance because G1 can also infect humans. From our mice experimental data, all of the strains are H5N1, except one, three H5N6 belong to the G1, G1.2, and G1.4 genotypes, and show differences virulence in mice. Apparently, one strain is unable to represent all of the genotype fully, but what caught our attention is that H5N6 shows high virulence, which potentially increases the risk of infection to humans.
Waterfowl have long been considered the natural reservoir of avian influenza, and our research results show that the duck, goose, and wild bird waterfowl contain numerous genotypes, respectively 8, 3, and 3 [Figure 6B]. Two major epidemic genotypes, G1 and G2.1, which are distributed in the three kinds of waterfowl. There were no G2.1 genotypes in human and chicken sources, and we only found H9N2 gene reassortment strains isolated from chicken, people, and the environment. We speculate that the virus could have spread from ducks to chickens with H9N2 reassorting and the production of the G2.2, G3.1, and G3.3 new genotypes. The virus could then have spread to people through chickens or the environment. G1.4 and G3.2 are distributed only in ducks, humans, and the environment, and from ducks or the environment can spread to people [Figure 6B]. The pandemic genotypes G1 and G2 are distributed in wild birds, which probably means that the virus continues to circulate in wild birds and waterfowl. The contact between waterfowl and landfowl mainly occurs in LPMs, which are the main place for virus reassortment and cross-host transmission of avian influenza. Both previous research and the work of our group confirm this scenario.
H5N1 and H5N6 can be highly pathogenic and induce high mortality in chicken. Vaccination is one of our main measures against the highly pathogenic avian influenza(Alarcon et al., 2018; Arai et al., 2019; Shi et al., 2018). The Chinese vaccine is upgraded continuously, following the evolution of the influenza virus, and the vaccine can protect poultry effectively, reducing the virus isolation rate greatly. However, there are still outbreaks of HPAIV every year, according to statement from the Ministry of Agriculture. Our laboratory has received from the farm of clinically suspected cases of AIV. Through analysis, we confirmed that the part of the clinically suspected case really is HPAIV, and most isolated strains from the clinically suspected cases are reassortment viruses, which may explained why there are still bits of outbreaks of HPAIV. Some may unuseed vaccines,and these reassortment strains may escape the vaccine’s immune response. A large number of reassortment viruses can be detected each year. And the vaccine obviously cannot resist all of the new reassortment viruses. Even so, occasional outbreaks can be addressed by rapid culling, disinfection, and landfills, and the effect of preventing the spread of the virus is also significant. Thus, we need to raise enough awareness about the occasional outbreak, and only quick and effective prevention measures can prevent a possible outbreak.