Investigation Of H5N6 Influenza A Viruses In Pig Farms And The Pathogenicity Analysis Of H1N1 Recombinant Strain In Mices

Influenza A viruses are known to infect a wide variety of animals,including humans,pigs,horses,birds,dogs,cats and sea mammals.Pigs own?-2,3 and?-2,6 sialic acid receptors in the respiratory epithelial cells,indicating sensitivity to both human and avian influenza viruses?AIVs?,which play an important role for interspecies transmission and generation of new influenza viruses.The new reassortant influenza virus may have a high virulence in human and avian,thus a long term monitor for influenza viruses circulating in pig herbs is extremly necessary.In order to investigate the circulation of pdm09H1 and H4,H5,H6 and H9 subtype avian influenza virus in pig farms in Guangdong province,a total of 2744 pig sera-samples were collected from 20 pig farms during Oct 2014 to Oct 2015 and dectected by the hemagglutination inhibition?HI?tests.The seroprevalence of EAH1N1,H3N2 SIV and pdm09H1N1 virus were 30.35%?95%CI:28.97%-31.76%?,21.19%?95%CI:19.97%-22.46%?and 9.11%?95%CI:8.06%-10.25%?,respectively.The antibody positive rate of co-infection with SIVs showed that:EAH1N1+pdm09H1 was 6.13%?95%CI:5.12%-7.28%?,EAH1N1+H3N2 was 3.58%?95%CI:2.8%-4.4%?,pdm09H1+H3N2was1.90%?95%CI:1.34%-2.59%?and EAH1N1+pdm09H1+H3N2 was 1.50%?95%CI:1.01%-2.13%?.The positive rate of antibodies against avian influenza virus subtype H9N2was 2.52%?95%CI:1.96%-3.18%?.The antibodies against to H7,H4,H5 or H6 avian influenza were not detected in the samples.The ponds and lakes,where some kinds of wild waterfowl and birds,were observed in the pig farms in Guangdong province,which caused serious threat to public health.Monitoring the trend of swine influenza viruses in Guangdong provides scientific basis for the prevention and control of influenza.A total of 4980 nasal swabs were collected in the pig farms in Guangdong province.Four novel isolates were detected in this program,including 1 H1N1 strain,1 H5N6 strain isolated in piglet lung and 2 H5N6 strains isolated in the environment from pig farms,which were named as A/swine/Guangdong/0326/2015?H1N1,Sw0326?,A/swine/Guangdong/G3/2015?H5N6,SwG3?,A/enviroment/Guangdong/F3/2015?H5N6,EnvF3?andA/enviroment/Guang dong/F4/2015?H5N6,EnvF4?,respectively.Phylogenetic analysis showed that the hemaggl-utinin?HA?gene of H5N6 viruses belonged to clade 2.3.4.4 lineage and the neuraminidase?NA?gene shared high identities with those of Guangdong poultry H6N6 strains,which were reported potentially cross the species barrier to infect human.The internal fragments of SwG3 strain belonged to Lineage I and the PB2 gene of EnvF3 strain belonged to Lineage II,and the PB2 and PB1 genes of EnvF4 belong to Lineage II,both of env-isolates of the PB2 genes were from reassortment of the genes of type H6 viruses.The PB1 gene of EnvF4strain may derived from duck H3N6 with a high homology?96.3%?.The other internal segments of EnvF3 and EnvF4 belong to Lineage I.Phylogenetic analysis showed that the HA,NA,M and NS genes of Sw0326 strain derived from Eurasian avian-like lineage and the other internal segments?PB2,PB1,PA and NP?derived from pdm09-like lineage.L226Q and S228G were fairly conserved in all HA genes,indicating that the viruses had an?-2,3-SA receptor binding preference.There were a T160A mutation in HA of SwG3,EnvF3and EnvF4,and a I155T mutation in HA of EnvF3 and EnvF4,indicating that they had obtained the ability of human-like receptor binding properties,which are protential infections for humans and pigs.In addition,our study of the molecular properties of the virus also show that a T215A mutation in M1 proteinand a P42S mutation in NS1 protein were observed in Sw0326.In order to further research the pathogenicity the,the 6-week-old female Babl/c mice were challenged with dose of 10⁶EID₅₀/50?L of the recombinant Sw0326 virus.The body of the mice decreased obviously.The virus could be detected in the brain(3d:2.25±1.3log₁₀EID₅₀/m L and 6d:2.83±1.8log₁₀EID₅₀/mL)and lungs(3d:4.3±0.66 log₁₀EID₅₀/mL and6d:6.4±0.57log₁₀EID₅₀/mL).The pathological histology observations show that there were injury and pathological changes in lungs and brain tissues of mice.And the immuno-histochemical results showed that the virus could be detected in the lungs and brains in day 6 post-inoculation.In couclusion,there was systematic infection and it showed strong pathogenic to mice.Further research about the virulence and the spread ability of the recombinant virus shoud be taken.