Isolation,Identification And Molecular Evolution Of 3 H9N2 Swine Influenza Viruses Isolated From FuJian Province

Swine influenza (SI)is a pig respiratory disease caused by swine influenza viruse(SIV).Clinical signs include high fevers,coughing,dyspnea ,muscle aching and causing abortions and deaths among the breeding sows.At the present,the disease have became widespread among swine herds in the world. Pigs serve as an intermediate host for genetic reassortment of human and avian viruses because they can be infected by either avian or human viruses and are more frequently involved in interspecies transmission of influenza A viruses than other animals. In addition,the situation of avian influenza has become more complicated in the mainland of China recently.Therefore, the studing of the biological ,genetic properties of the main recent epidemical strains circulating among China pigs will not only provide useful information for the disease prediction,promote healthy development of the livestock,but also provide useful data for human influenza pandemic plan. 3 swine Influenza viruses were isolated from 28 nasal swabs obtained from Fujian Province. They were identified as A influenza viruses of H9N2 subype using hemagglutination inhibition(HAI) tests and neuraminidase inhibition(NI) tests, and molecular evolution of the 3 strains has been analyzed in this study.Viruses were isolated in embryonated chicken eggs as described in ref.133. Each virus was biologically cloned by three rounds of limiting dilution in embryonated specific-pathogen-free(SPF) chichen eggs. After that, allantoic fluids were harvested. Store the isolates at -75℃.The nucleic acids of the 3 influenza viruses conducted in the research were extracted from the viruses propagated in specific-pathogen-free chichen embryos. All of the eight segments were amplified by RT-PCR,and the purified PCR products were done cycle sequencing with specific primers,furthermore,the sequencing products were purified and run gel on CEQ 8000 DNA sequencing machine.The specific primers of the eight genes for PCR and cycle sequencing were designed using the Oligo (6.0 version) software.Finally, the DNAstar software were used for data analysis,sequence translation,phylogenetic tree drawing and molecular evolution analysis. The results of this investigation displayed that all of 3 SIVs isolated from Fujian province are AIVs. And they have high homology and a close phylogenic relationship with AIVs of Y280-like sublineage,.Nearly all genes of 3 SIVs belonged to Y280-like sublineage,except the NA genes of sw/Fujian/02/03(FJ02) as well as sw/Fujian/10/03(FJ10),and PA,M genes of sw/Fujian/09/03(FJ09). The NA genes of FJ02,FJ10 were situated in G9-like sublineage, and PA,M genes of FJ09 posses high homology with an avian influenza virus of H3N8 subtype.