Phylogenetic Analysis And Pathogenicity To Mouse Of H6 Subtype Avian Influenza Virus Isolated From 2014 To 2016 In Partial Area Of China

In 2013,the first case of human infection with the H6N1 subtype avian influenza virus(AIV)was reported in Taiwan,China,indicating that H6 viruses pose a potential threat to human heanlth.During the epidemiological surveillance of AIVs in poultry,H6 viruses are isolated frequently in China.To further understand the genetic evolution and related biological characteristics of H6 viruses in China,the surface gene sequence of 105 H6 subtype AIVs that were isolated in poultry in partial area of China from 2014 to 2016 was determined,and 18 representative viruses were selected for genomic sequence analysis,antigenic different analysis and risk evalution of mammals infection,which will provide data support for prevention and control of avian influenza.1.Phylogenetic analysis of H6 subtype avian influenza virus Isolated from2014 to 2016 in partial area of ChinaTo study the genetic evolution of H6 subtype AIVs,the Hemagglutininand(HA)and Neuraminidase(NA)genes of 105 H6 subtype AIV that were isolated from 2014 to 2016 in partial area of China were sequenced,18 viruses were selected for purification and sequencing of the whole genome according to the different isolation time,place,host,HA genes homology and different combinations of HA and NA subtypes in H6 subtype AIVs.The results showed that H6N6 subtype was the primary subtype combination,multiple subtype combinations of H6N2,H6N1,H6N4,and H6N8 coexisted in China from 2014 to 2016.The eight gene fragments of H6 viruses presented multiple branches on the evolutionary tree,and showed significant genetic diversity in different branches;the internal genes of different viruses were more complex and diverse,and represented the recombination phenomenon among other multiple subtypes.eighteen viruses were classified into 15 genotypes based on the criterion of gene nucleotide homology?95%.Therefore,H6 subtype AIVs isolated in China from 2014 to 2016 presented genetic diversity and complexity of genetic evolution.2.Antigenic difference analysis of H6 subtype avian influenza viruses isolatedfrom 2014 to 2016 in partial area of ChinaEighteen H6 subtype AIVs were inactivated with(3-propiolactone and the hemagglutination inhibition(HI)titers of viruses were concentrated to more than 81og2.The concentrated virus and adjuvant were mixed at a ratio of 9:1 to prepare an inactivated vaccine.Six-week-old SPF chickens were immunized in multipoint of muscle with a dose of lml/head.Blood was collected on day 28 post-immunization for serum separation and the immunized sera were used for cross HI test with these 18 viruses.The results showed that the cross-reactivity and cross-antigenicity between viruses were related to the HA homologies of the viruses.The higher homology of the HA gene,the better cross-reactivity between viruses and the smaller difference in antigenicity.However,the cross-reactivities between viruses in different HA gene branches were poor,and the antigenicities were also significantly different,there was even no reactivity between some viruses of 18 viruses.Therefore,the antigenicities of H6 viruses isoalted in China from 2014-2016 were diversity,and categorized into four antigen-groups.3.Pathogenicity to mouse of H6 subtype avian influenza viruses isolated from2014 to 2016 in partial area of ChinaGroups of eight 6-week-old female BALB/c mice were lightly anesthetized with dry ice and inoculated with 106 EID50 of H6 influenza viruses in the volume of 50?L of medium with 18 of H6 subtype AIVs,and five mice of the control Group were inoculated with 50 ?L PBS in the same way.Three of the eight mice were euthanized on day three postinoculation for titration of virus from the brains,turbinates,spleens,kidneys and lungs by inoculation of 10-day-old chicken embryos.The remaining mice in the control group and the inoculation group were observed for 14 consecutive days,and their body weights were recorded every day.The results showed that H6 viruses isolated in China from 2014 to 2016 showed low pathogenicity to mice.Most of the mice showed a transient weight decline on the day 3 to day 5 postinoculation,and after that their body weight gradually returned to normal.The results of visceral virus titration showed that most viruses could replicate efficiently in the lungs of mice without pre-adaptation,and individual viruses could replicate efficiently in mouse turbinates and lungs,suggesting that H6 subtype AIV has a potential risk for mammals infection.