Sequence Analysis Of H5N6 Subtype AIV And Construction Of Vaccine Candidate

Since the first discovery of H5N6 subtype of avian influenza virus in C hina in December 2013,Heilongjiang,Guangdong,Gansu,Hubei and other provinces outbroke H5N6 subtype of highly pathogenic avian influenza epidemic,causing a large number of poultry deaths.At the same time,Sichuan occurred the first severe deaths case due to H5N6 subtype of influenza virus in May 2014.Then Guangdong,Yunnan,Jiangxi,Hunan and other places reported cases of human infection.As of 2016,a total of 16 people were infected with H5N6 subtype of influenza virus,of which 10 were killed.It could be seen H5N6 couldnot only cause poultry death seriously to restricts the development o f aquaculture,but also became another major threat to human life and health due to its high mortality rate.In order to understand the molecular characteristics and genetic evolution of H5N6 subtype AIV in recent years,10 H5N6 subtype avian influenza viruses isolated from poultry from Guangdong,Yunnan and Fujian province were chose for analysis of molecular characterization and genetic evolution based on the special geographical location and environmental climatic conditions of these province.HA gene distribution analysis showed that the HA gene of Yunnan isolate MZ151 was distributed in 2.3.2 branch,and the other 9 strains were distributed in 2.3.4.4 branches.Amino acids near the alkaline cleavage site of the HA gene of 10 strains showed two types,namely KERRRKR?GLF and RERRRKR?GLF.Both of them were composed of several continuous basic amino acids K/R,which meet the high pathogenic avian influenza typical molecular characteristics of the virus.HA protein Q226 L and G228 S were not mutated,indicating that these viruses were still mainly combined with sialic acid ?2,3-galactosyl receptor.But the mutations of I155 T,N158D,T160 A,S239P in HA proteinindicated that these viruses might also have the ability to bind to the sialic acid ?2,6-galactoside receptors.NA gene of all the 10 strains originated from the H6N6 subtype of influenza virus and existed 11 amino acid deletion in the NA stalk through NA gene sequence analysis.The results of internal gene sequence analysis showed that PB2 gene of all the 10 strains also originated from the H6N6 subtype AIV andmost of the internal genes of 10 strains derived from H5 subtype influenza virus.No E627 K or D701 N was found in PB2 proteins.However,the mutations A588 V in PB2 protein,N30 D,T215A in M1 protein and P42 S,D92E in NS1 protein indicated that these viruses might also have a certain pathogenicity to mammals.It is necessary to develop candidate vaccines for epidemic strains to counter the persistence of antigenic changes and potential epidemics of the 2.3.4.4 branch highly pathogenic H5N6 subtype avian influenza virus prevalent in the southern C hina.A/duck/ Fujian/16469/2016(H5N6)located in branch 2.3.4.4 was selected as the vaccine candidate donor strain according to the HA gene phylogenetic tree.We used the D7 vaccine strain reverse genetic vaccine development platform established by the research group and saved a candidate vaccine strain r16469 by deleting a number of continuous basic amino acids in the vicinity of the cleavage site of the HA gene of the donor strain,making it have the molecular characteristics of low pathogenic avian influenza virus.r16469 strain inactivated vaccine could induce high level of HI antibody after single immunning SPF chickens due to its good immunogenicity.We challenged with 106EID50 of H5N6 subtype virus by eye drop andnose drop after 21 days of immunization with SPF chickens.The results showed that r16469 strain could completely protect against the challenge of H5N6 subtype avian influenza virus named YN0928 and 16469.The immune chickens didn't show clinical symptoms.Besides no virus was found in the cloacal swabs.