Pathogenicity On Mammals Of European Avian-alike Swine H1N1 Influenza Virus And Its Establishment Of Revers Genetics

Swine Influenza(SI)is an acute,hyperpyretic,outbreaking,and infectious respiratory disease caused by Swine Influenza Virus.At present,the main subtypes in herds are H1N1,H1N2 and H3N2,among which the European avian H1N1 subtype and the classical H1N1 subtype are the main epidemic strains in our country.In order to identify the epidemic situation of Swine Influenza in domestic herds,we took European avian H1N1 SIV(A/swine/Tianjin/6/2011(H1N1),TJ6 for short)as an antigen,the antibody levels of a total of 1080 pig serum samples from Fujian,Hebei,Heilongjiang,Shandong,Guangdong,Jiangsu,Zhejiang and Shanghai(from 2014 to 2016)were detected by haemagglutination inhibition test.The positive rates of SIV antibodies were 31.9%,19.5%,37.5%,22.2%,79.6%,56.9%,41.4% and 51.9%,respectively.The results showed that the European avian H1N1 SIVs were widely propagated and steadily existed in domestic herds.Then,MDCK cells and BALB / c mice were used as the model to study the biological characteristics of European avian H1N1 SIV.TJ6 was inoculated to MDCK cells at a dose of0.001 MOI,and the growth curve of the virus was measured on the cell.The mice were challenged with 104 TCID50 to measure the changes in body weight and the virus titer of the lung.The results showed that TJ6 induced significant cytopathic effect(CPE)on MDCK cells,and the virus titer reached the highest level in 48 hours after inoculation.The mice in TJ6 challenge group had the lowest weight on the third day,and the virus cound be detected in the ground fluid and then the body weight was increased.The virus cound cause significant changes in body weight and pathological changes in mice.In this study,we construct a reverse genetic platform.The 8 gene fragments of TJ6 were amplified by RT-PCR and separately cloned into the transcription/ expression vector pBD.The eight recombinant plasmids were purified and co-transfect into 293 T cells.The supernatant of 293 T cells were collected after 54 hours and inoculated into the MDCK cell.The supernatant was collected after the cells shed,and conducted the hemagglutination test.The titer was set at 1: 32.The eight virus fragments were amplified and sequenced.The sequencing results were consistent with the original strain,and there was no significant difference in the cell lesion,which proved that the virus was successfully saved.