Molecular Epidemiological Investigation Of Avian Influenza Virus In Waterfowl In Guangdong Province From 2016 To 2017

The appropriate climate and geography conditions in Guangdong province have made the region a high incidence area for influenza virus.At present,many avian influenza viruses can infect mammals through species barrier through recombination of gene structure.Waterfowl,as a natural host of influenza,plays a very important role in the spread of influenza.Therefore,molecular epidemiological investigation of influenza viruses in waterfowl in Guangdong province has important public health significance.In order to investigate the epidemic of avian influenza virus in waterfowl in Guangdong Province,we collected 2143 throat swabs,suspected diseases and environmental samples from waterfowl farms in Guangdong province.The test results showed that 6 strains of H6N6subtype influenza virus and 4 strains of H9N2 subtype influenza virus were isolated from this experiment,and the overall isolation rate was 0.47%.The 8 gene fragments of H6N6 strain isolated in this experiment belonged to poultry origin and have high similarity with H6N6and H6N2 strains isolated from waterfowl in Jiangxi,Guangxi and Hunan provinces.All strains belong to the Eurasian branch.It is important to note that the HA fragments of these strainsarehighlysimilartoA/swine/Yangzhou/080/2009?H6N6?and A/swine/Guangdong/K6/2010?H6N6?.Amino acid analysis showed that there was no deletion in the neck of the NA fragment,and there was no 627E and 701D mutation in the PB2 fragment,but all H6N6 strain isolated in this experiment have occurred Q226L mutation,which makes it more likely to tend to the SA alpha-2,6Gal receptor in human respiratory epithelial cells.In addition,all M2 fragment have V27I mutation,which may cause changes in the drug resistance of the strain.The 4 strains H9N2 influenza viruses isolated in this experiment are on the same branch both belong to H 9.4.2 lineage.Amino acid analysis showed that there was no deletion in the neck of the NA fragment,and there was no 627E and 701D mutation in the PB2 fragment,but M2 fragment of these 4 strains have occured S31N mutation,suggesting that these strains have the efficacy of resistance to amantadine.In addition,the four strains H9N2 influence virus are similar with H6N6 isolated from this experiment have occurred Q226L mutation in HA fragment.This mutation is observed in different areas.It shows that influenza viruses prevalent in Guangdong's waterfowl are breaking through species barrier and have the ability to infect mammals.In order to further study the pathogenicity of H6N6,A/goose/Guangdong/25/2017?H6N6?strain was selected for intranasal drop infected test in 6-week-old BALB/c female mice After virus infected,the mental status,eating status,body weight change,etc were observe.On the 3rd,5th,and 7th days after poison infected,4 mice were euthanized from all group,1 mice were observed histopathology of the lung organ and the other 3 mice was used to measure the replication of the virus in brain,spleen,kidney and lung organ.The weight of mice in infected group decreased rapidly,depression was depressed and feed intake decreased and on the third day,the body weight gradually increased and returned to normal on 7th day.The weight of mice in control group and contact group increased gradually.Histopathological results showed that this strain could cause diffuse lesions in the lungs of mice,with fluid exudation in blood vessels and alveolar spaces,and inflammatory cell infiltration.The results of virus titer test showed that the influenza virus was not found in other organs,control group and contact group except the lung in infected group.The viral titers in lungs of infected group on 3rd,5th and 7th days were 1.76×10³ EID₅₀/m L,1.06×10⁵ EID₅₀/mL and 0 EID₅₀/m L,respectively.The above results show that the strain can replicate in the lungs of mice,but without lethality and can't transversely transmitting between mice.In the face of the continuous prevalence and variation of avian influenza virus in waterfowl,we must conduct a systematic molecular epidemiological investigation of influenza viruses in waterfowl.Due to the important role of waterfowl in the transmission of influenza virus,we need to understand the pathogenicity of influenza viruses isolated from waterfowl to mammals.In this study,molecular epidemiological investigations and pathogenicity tests in mice were used to understand the pathogen characteristics and pathogenesis of influenza virus in waterfowls and providing a theoretical basis for influenza virus control measures.