Isolation And Identification Of Two Strains Of H9N2 Swine Influenza Virus And Theirs Pathogenicity On Mice

Nowdays,Swine Influenza have been distributed all over the world.The species barrier of pigs is relatively low, it forms a transmission chain of "humen-swine-avian",if infected two kinds of influenza virus at the same time,it's easily lead to gene reassortment and produce new influenza virus subtypes. All of these indicate the influenza virus of H9N2 subtype is invading human life and to be a serious threat to public health and safety.The study used RT-PCR and embryonated eggs to discuss biological characteristics and isolated virus,molecular genetics and pathogenicity test on mice of the two strains of H9N2 subtype SI which isolated from pig farms of Baise Leye county,GuangXi in 2011.According to the BLAST result on NCBI,the two isolated strains determined to the influenza virus of H9N2 subtype that the laboratory name them SW/GX/P2/2011 and SW/GX/P3/2011/The ORF of nucleotide of PB2,PB1,PA,HA,NP,NA,M and NS respectively are 2280bp,2274bp,2151bp,1683bp,1497bp,1410bp,982bp and 838bp. Two isolates compare homology with A/Canine/Guangxi/1/2011 and A/Equine/Guangxi/3/2011 that is above 99.0%.Sequences analysis of HA protein in 109,161,163,202 and 203 sites were very conservative,the 234th is leucine(Leu) and it had the receptor SAa,6Gal specificity. The cleavage site of HA is-RSSRGLF-and no multiple amino acid insertion,indicating two isolates is belong to low pathogenic strains.SW/GX/P2/2011 and SW/GX/P3/2011 had no amino acid deletion in 63-65th of NA. The previous research found this three-amino-acid absence help to increase the virus neuraminidase activity and release from infected cells.NA in 151,119,276,292 and 294 sites did not mutate,indicating that SW/GX/P2/2011 and SW/GX/P3/2011 are still sensitive to the neuraminidasedrug.S31N in M2 protein exhibited SW/GX/P2/2011 and SW/GX/P3/2011 were resistant to amantadine drugs.In internal genes,sites associated with pathogenicity that 627th and 701th in PB2,622th and 709th in PB 1,315th and 515th in PA,92th in NS and 319th in NP both no difference with reference strains,indicating SW/GX/P2/2011 and SW/GX/P3/2011 in these site is very conservative.Phylogenetic tree and homology analysis indicated that HA gene from Y280-like,NA gene from G9-like,M gene and PB2 gene from G1-like,PB1 gene,PA gene,NS gene and NP gene from F98-like.The LgEID50/0.1mL value of SW/GX/P2/2011 and SW/GX/P3/2011 were 5.02 and 5.23.The result according to the pathogenicity in mice show that the mice appeared mainly pathologic changes in lung.The virus can replicate in the lungs and turbinate,but there are certain differences of the level replication between them,and the turbinate better than the lungs.Two isolates could reduce the weight of mice most as lowest as 6%,but not lethal to them,and than the weight return to normal in the 8th day.