Molecular Characteristics And Pathogenicity Of H6 Subtype Avian Influenza Virus In Guangdong Province From 2014 To 2017

The H6 subtype avian influenza virus infects a wide range of hosts,and is prevalent in north American and Asian bird populations.It is also one of the common subtypes avian influenza virus of poultry and waterfowl in southern China.Since 2000,there have been reported of H6 subtype avian influenza virus isolated from poultry in China.Its epidemic and transmission are on rise,which leads to the possibility of communication is greatly increased between H6 subtype avian influenza virus and other subtype avian influenza viruses.At the same time,the use of a large number of vaccines or drugs to prevent the occurrence and spread of avian influenza virus in birds,and the changes in the living environment of the virus may lead to genetic mutations of the virus and changing its biological characteristics.Which poses a great threat to the poultry breeding economy and public health.Therefore,it is of great significance to analyze the genetic evolution and biological characteristics of H6subtype AIV in China.This study is identification and analysis about H6 subtype of avian influenza virus separated from many different live poultry market in Guangdong and epidemiology investigation as well as result analysis that includes many domestic H6 subtype avian influenza virus.The viruses isolated in 2014?2017 are selected for genetic evolution analysis research and pathogenicity studies.The isolation includes 48 strains H6N2 subtype of avian influenza virus strains and 44 strains H6N6 subtype of avian influenza virus strains.The virus isolation and identification results showed that 5 of the H6N2 subtype avian influenza virus strains isolated from chickens,and the rest isolated from waterfowl.Among the H6N6subtype avian influenza virus strains,2 of the strains isolated from pigeons,3 of the strains isolated from the environment,5 of the strains isolated from chickens,and the rest isolated from waterfowl.The amino acid sequence of PQIETR?GLF was observed near the HA amino acid lysis site of the 92 strains,indicating that all strains were consistent with typical molecular markers of low-pathogenic avian influenza virus.The amino acid sites encoding HA protein of the H6N2 subtype avian influenza virus isolates corresponded to the H3subtype avian influenza virus sequences at positions 155 and 190.Respectively,21 and 25copies of the isolates showed site variation.3 strains of the H6N6 subtype avian influenza virus isolates showed 138 site mutation,while the H6N2 subtype avian influenza virus isolates showed no 138 site mutation.A single potential glycosylation site deletion occurred in 1 of the H6N2 subtype avian influenza virus isolates.Four isolates of the H6N6 subtype of avian influenza virus were found to have a single potential glycosylation site deletion.In addition,single potential glycosylation site deletion occurred in NA amino acid fragments of7 strains of the H6N2 subtype avian influenza virus isolates,and single potential glycosylation site insertion occurred in 3 strains of the H6N2 subtype avian influenza virus isolates.In the H6N2 subtype avian influenza virus isolates,the NA amino acid fragments of4 strains showed the loss of a single potential glycosylation site.According to the basic information of the isolated virus,20 of the H6N2 subtype avian influenza viruses and 20 of the H6N6 subtype avian influenza viruses were screened out for whole-gene information acquisition and analysis.The remaining six viral gene fragments coding protein of the isolated strain did not show any mutation at the key site or change in the number of nucleotides.According to the eight different virus gene fragments in the genetic evolutionary tree distribution location,conclusions can be drawn that there are 7 different genotype combinations in separated H6N2 subtype of avian influenza virus,10 different genotype combinations in separated H6N6 subtype of avian influenza virus.It accords with the fact that the H6 subtype avian influenza virus gene exchanges frequently in south China.Two H6N2 subtypes and two H6N6 subtypes of avian influenza viruses were screened by genetic information analysis.The virus were named d752?e488?d9 and e168.10⁶EID₅₀/200?L virus dose to artificially infect specific pathogen free chickens and BALB/c mice.The result showed both the SPF chickens and the BALB/c mice infected by the virus strain presented serum antibody positive.And Different levels of virus were detected in the lapping fluid of tissues and organs such as lungs and trachea of experimental animals.A/goose/Guangdong/e488/2017(H6N2)could only replicate in the trachea of chickens in the challenge group,and the titer was 3.75±0.5 log₁₀EID₅₀/200 L.The virus strain A/pigeon/Panyu/d9/2016(H6N6)could only detect significant viral titers in the lungs of chickens in the challenge group,with a titer of 3.25 log₁₀EID₅₀/200 L.In the mouse experiment,the influence of the H6N2 subtype avian influenza virus'infection group on mice was significantly greater than that of the H6N6 subtype avian influenza virus'infection group,but both of them can caused infection and significant body weight changes in mice such as mammals.