Background
Influenza A viruses are RNA viruses.To date, 18 major antigenic variants of HA and 11 of NA have been detected(Fouchier et al., 2005; Tong et al., 2012). In addition, more than 20 other mammalian host species as well as various poultry species can be infected(Wille & Holmes, 2019). The first outbreak of the highly pathogenic H5N1 avian influenza was reported in Guangdong Province in 1996. The following year human infection of the H5N1 virus was reported in Hong Kong(Xu,Subbarao,Cox, & Guo, 1999). After that, the viral genes divided rapidly and produced multiple lineages (Clades 0-9)(Duan et al., 2008). Reassortments with other low pathogenic avian influenza produced numerous genotypes. Clade 2.3.2 H5N1, which is associated with the outbreak of avian flu of Lake Qinghai, China, 2005, astonished the world, in that migratory birds were observed with the infection, and over 6,000 birds died. Then, Clade 2.3.2 viruses evolved into Clade 2.3.2.1, which diversified into 2.3.2.1a, b, and c in 2009(Bi,Chen, & Zhang et al., 2016; Bi,Chen, & Zhang et al., 2016). The first isolation of Clade 2.3.3.4 was reported in Eastern China in 2010, and then spread to all parts of the world(Lee,Bertran,Kwon, & Swayne, 2017). Bi et al. reported that H5 has reassorted with H6N6 and H9N2/H7N9 from 2013 to 2016, resulting in a large number of genotypes. In China, 34 genotypes have been identified(Bi,Chen, & Wang et al., 2016). In recent years, clade2.3.4.4 and clade2.3.2.1 still can be detected in China(Bi et al., 2016; Sun et al., 2016). According to the ministry of agriculture of China, six outbreaks of highly pathogenic avian influenza have occurred in Sichuan, Xinjiang, and Hunan, China in January and February of 2020, five of which were caused by H5N6 (including four outbreaks in wild birds) and one by H5N1. A total of 6,340 poultry died, and 20,089 were culled. Two poultry outbreaks resulted in the deaths of 6, 340 poultry, and a total of 20,089 poultry were disposed of in a bio-safety way. Four outbreaks killed 48 wild birds(http://www.moa.gov.cn/gk/yjgl_1/yqfb/).
Bi et al. studied the epidemiology of H5N6 in China from 2013 to 2016, and analyzed in detail the geographic distribution and reassortment evolution of the virus in live poultry markets(Bi et al., 2016). They and many other studies have shown that live poultry trade markets play an essential role in the proliferation of viruses, and people who work, live, or have long-term contacts with the live poultry trade market (LPM) are more likely to be infected by the virus than the general population(Gao, 2014; Kim et al., 2018; Shi et al., 2018). The LPMs have played an essential role in the evolution and reassortment of avian influenza(Qi et al., 2018; Shi et al., 2018). The constant surveillance of the LPM is an important procedure in the epidemiological study of avian influenza and public health safety.With this in mind, we collected oropharyngeal and cloacal swabs from an LPM every two weeks in the Guangdong province and samples from poultry farmers in other provinces of China during 2016-2019. We used these data to analyze in detail the genetic evolution reassortment ,prevalence and pathogenicity in mice of H5N6 avian influenza virus.