Isolation,Identification And Pathogenicity Of H5 Subtype Avian Influenza Virus In Some Areas Of Guangdong Province From 2017 To 2018

There are many recombinants of H5 avian influenza viruses,but H5N6 viruses are the prevalent in poultry in southern China.H5N6 subtype highly pathogenic avian influenza virus were first emerged in Laos in 2013,and then spread widely in wild waterfowl and domestic in China.By February 2019,H5N6 subtype avian influenza highly pathogenic viruses had caused thirty outbreaks in China killing about 660000 poultry.In May 2014,the first fatal human case of H5N6 subtypes avian influenza virus infection was identified in Sichuang.Up to February 2019,23 laboratory-confirmed cases of human infection with H5N6 subtypes avian influenza viruses had been reported in China.The virus not only caused huge economic losses to the poultry industry,but also posed a serious threat to public health.In our study,the molecular characteristics,genetic evolution and pathogenicity of 20 H5N6 subtype avian influenza viruses isolated in Guangdong from2017 to 2018 were analyzed.In this study,the cleavage site in HA protein of the 20 H5N6 AIVs were KERRRKR?G or RERRRKR?G,which were consistent with the characteristic of H5 subtype highly pathogenic avian influenza virus.The NA stalk of all H5N6 viruses contained 11 amino acid deletions at residues 58 to 68.In our study,no substitutions in the NA protein were related to drug resistance.All viruses were E at 627 sites and D at 701 site of PB2 protein,which were molecular characteristic of the typical avian influenza viruses from birds.Some virulence-related amino acids of PB1,PA,NP and NS proteins were substituted.Only the Q32 strain showed a mutation associated with amantadine resistance(S31N)at the 31stIn this study,20 H5N6 subtype avian influenza viruses were classified into CN.JP.VN-like,Eurasian lineage,and VN 2014-like.These viruses belonged to different genotypes,which indicated genetic evolution of them were diverse.In this study,HA protein of 20 H5N6 subtype avian influenza viruses were classified to Clade 2.3.4.4.Compared with the Re-8 vaccine strain,7 strains showed 2-3 amino acids mutations and 13 strains showed 4-7 amino acid mutations in the HA protein antigen region,respectively.Therefore,the amino acids of 20 H5N6 viruses showed some mutations in HA protein antigenic region compared with the Re-8 vaccine strain.In order to understand the pathogenicity and transmissibility of the H5N6 subtype avian influenza viruses,Chickens of inoculated group were inoculated 8 viruses with the dose of 0.1 m L 10⁴EID₅₀/0.1 m L,but chickens of contact group were inoculated PBS with the dose of 0.1 m L.The results showed that except the Q80 strain,the mortality of other 7viruses in the inoculated chickens were 100%.The replication titers of 6 viruses in all tissues were 5.25-9.75 Log₁₀EID₅₀/0.1 m L at 2DPI,but the replication titers of Q80 and Q181 in all tissues were 4.50-6.92 Log₁₀EID₅₀/0.1 m L at 2DPI.The mortality in the contact group of Q95 was 60%,while the mortality in the contact group of other 7 viruses were100%.Viral shedding in contact group of 8 viruses could detected from oropharynx or cloaca swabs at 3 day post-infection(DPI).Viral titers of oropharynx and cloaca swabs in the Q135 contact group were 4.75 Log₁₀EID₅₀/0.1 m L and 4.50 Log₁₀EID₅₀/0.1 m L,respectively.Viral titers of oropharynx or cloaca swabs in other 7 viruses contact groups were lower than 2.60 Log₁₀EID₅₀/0.1 m L and 2.90 Log₁₀EID₅₀/0.1 m L,respectively.Therefore,the 8 H5N6 subtype avian influenza viruses isolated in Guangdong in2017-2018 showed high pathogenicity to chickens and strong horizontal transmissibility in chickens.