Partial Biological Characteristics Of H6 Avian Influenza Viruses In China Between 2014 And 2018

H6 avian influenza virus（AIV）is one of the most prevalent influenza virus subtypes in the world,and are commonly found in wild birds and domestic poultry.Moreover,H6 subtype AIVs have the ability to infect other species,occasionally.In recent years,H6 AIVs have been isolated from live poultry markets as well as some farms in China.In order to understand the epidemics and evaluate the risk of H6 AIVs to infect mammals,and analysis the changes of antigenicity of HA genes,we sequenced the surface genes of 409 H6 AIVs isolated from 2014 to 2018 in China.According to the time,location and host of the isolated viruses,40 viruses（28 viruses were isolated farms,11 viruses were isolated from live poultry markets and one virus from wild bird）were chosen to conduct research of biological characteristics of H6 subtype viruses isolated from 2014 to 2018in China.The HA genes of H6 viruses showed great diversity and formed phylogenetically different groups,the mean substitution rate of H6 HA gene was 3.569×10^-3 substitutions/site/year,some viruses had an amino acid insertion at position 141 or 2-amino acid deletion at position 157 to 158.NA genes contained N1,N2,N6 and N8 subtype,among the four NA subtypes in H6 AIVs,the mean substitution rate of N1,N2 and N6 were approximative,the mean substitution rate of N8 was slowest.N6 is the dominant subtype of NA genes.The 40 H6 AIVs in this study were divided into 36genotypes,there were extensive reassortments of internal genes between H6 viruses and the H1,H3,H4,H5,H7,H9,H10,H11 and H12 viruses isolated from wild birds or domestic poultry.These results indicate that complicated reassortments of H6 AIVs have occurred in the farms and of live poultry markets China.To fully understand the antigenic relationships of H6 viruses that were isolated between 2014and 2018,we selected 27 viruses based on the divergence of amino acid of HA and investigated their cross-reactivity with chicken antisera induced by each of 27 viruses by using the hemagglutinin inhibition（HI）test.These antigenic data were also analyzed quantitatively by using the antigenic map.The 27 viruses formed at least five different antigenic clusters and there were huge divergence among different antigenic clusters.Of the 40 H6 viruses in this study,we selected 19 viruses to evaluate replicative ability and virulence in BALB/c mice.all 19 viruses tested were detected in the lungs of mice,most of the viruses also were detected in the nasal turbinates of mice except two viruses;mice lost their body weight for few days and then gained again.There were no obvious clinical symptoms in mice after challenge with the viruses,indicated the low virulence to mice.It is worth noting that one virus,GS/GD/S40190/2017（H6N2）,was detected in the brain in one of three mice with the titer of 1.25log₁₀ EID₅₀/m L.To investigate whether mutations occurred in the PB2 gene of GD190 during its replication in mouse,we sequenced the virus recovered from the brain on day 3 p.i.and found E677G mutation.Another virus,DK/Hu N/S41299/2017（H6N2）was detected in kidney in one of three mice with the titer of 1.75 log₁₀ EID₅₀/m L and R368Q was found in PB2 gene of the virus recovered from the kidney.We selected 17 viruses based on the molecular characteristics of HA genes and replicate efficiency in mouse respiratory organs to investigate their receptor binding properties.We found that except CK/AH/S1224/2017（H6N6）and CK/JX/SE2578/2017（H6N6）,the other 15 viruses tested could bind to theα-2,6-linked glycans,although most of their affinity forα-2,3-linked glycans was higher except WB/XJ/S1541/2015（H6N6）,which was isolated from wild bird,another virus was DK/GX/S30803/2017（H6N6）,with HA had 2-amino acid deletion at position 157 to 158.These results implying that H6 AIVs have the potential to directly infect humans.During the surveillance of 2014-2018,two 141V insertion at HA were found,.CK/He N/S40124/2017（H6N8）and CK/SC/S4230/2016（H6N8）.Since this is the first time that such viruses were found,in order to investigate whether the 141V insertion influence the biological characteristics of H6 AIVs,we deleted one of the two viruses,CK/He N/S40124/2017（H6N8）,the141V at HA through reverse genetics approach and rescued the recombinant virus r He N124-HAΔ141.The results of further study indicated the 141V insertion does not affect the virulence to mice and the receptor binding specificity,but reduced the neutralization activity of m Ab against virus.In conclusion,we extensively characterized 40 H6 avian viruses in China,and found that H6viruses circulating in avian species in nature have undergone frequent reassortment and formed complicated genotypes.There were huge divergence among different antigenic clusters.Although most of the viruses were isolated from live poultry markets,the viruses isolated from farms have undergone frequent reassortment.H6 AIVs have the potential to directly infect humans.Hence,in order to provide data support and theoretical foundation,we should continue to monitor and study about H6 AIVs.