Isolation, Identification And Pathogenicity Of Swine Influenza Virus In Guangdong Province

Influenza A virus as a class of large branches of influenza,according to the host species diversion,can be divided into human influenza virus,avian influenza virus(AIV),swine influenza virus(SIV),Equine influenza virus(EIV)and canine influenza virus(CIV)et al..Swine influenza virus can cause respiratory diseases with mainly clinical symptoms such as sudden onset,high fever,difficult breathing,appetite loss,depression,paroxysmal coughing on pigs during the infection.The main features are the high efficiency transmission and low mortality.Although with a low mortality rate,SIV frequently found to show co-infection or secondary infection with other pathogens in complex farming environment.Under the condition just mentioned above,the mortality rate greatly increased and resulted in huge economic loss.As the main host of SIV,with the equally distribution of ?-2,3 and ?-2.6 sialic acid receptor in the pig's respiratory epithelial cells,Pig also can be infected by AIV and human influenza virus.If such three kinds of influenza A viruses co-infected one pig under natural condition,a gene recombination may happened among these viruses,and the new flu virus with resorted genome could be expected.Such new influenza virus may have a high pathogenicity on huma n and avian.Under this condition,a long term monitor for those flu viruses circulating in pig herbs is extreme necessity.The sample sites of this study covered the main pig farming areas in Guangdong Province,including Guangzhou,Shenzhen,Zhuhai,Q ingyuan,Yangjiang,Maoming,Jiangmen,Shaoguan,Zhaoqing,Yunfu and other places.A total of 2980 swine swabs were collected.Twelve strains were isolated via chick embryo and cell sorting method.Among which 9 strains were H1N1,2 strains were H1N2 and 1 strain was H3N2.In this study,three representative strains were selected and named as A/swine/Guangdong/FS4/2016(H3N2)(FS4),A/swine/Guangdong/BRT15/2016(H1N1)(BRT15)and A/swine/Guangdong/DG2/2015(H1N2)(DG2)respectively.An additional strain named A/swine/Guangdong/YJ4/2014(H1N1)(YJ4)isolated in 2014 by our lab,also enrolled in this study.Phylogenetic analysis showed that the HA and NA genes of FS4 strain derived from seasonal influenza and the other six internal fragments derived from pdm09-like strains.HA gene of DG2 derived from Eurasian avian-like influenza while NA gene derived from human seasonal influenza.The internal fragments of DG2 strain derived from pdm09.The HA,NA and M gene fragments of BRT15 and YJ4 strains were derived from Eurasian fowl,and the other internal gene fragments derived from pdm09.In summary,all of the four strains are recombinant viruses.The pathogenicity analysis of these four strains showed that all mice in the DG2 challenge group died at the 4th day post infection.The weight of the mice decreased by 50.57% and the virus could be detected in the brain and lungs.The virulence of DG2 was the strongest one among these four strains.The mice of YJ4 challenge group all died on the 5th day post infection,the individua l weight loss could reach to 44.44% and only can detect the virus in the brain.The survival rate was 28.6% and the individual weight loss up to 41.19% in the BRT15 challenge group which also can detect the virus in the brain and lungs.The survival rate o f FS4 challenge group was 100% and the weight of mice was slightly decreased after challenging and recovered quickly.The virus could be detected in lungs and brain at the early stage of infection but was cleared rapidly.The FS4 had the lowest virulence compared with other four strains.Based on the analysis of the potential glycosylation sites of the HA genes of four strains,compare with other four strains,there were seven additional potential glycosylation sites on the HA gene of the FS4 strain,which was more than other three strains.This may be one reason of resulting a lower virulence.The analysis of the amino acid sequence of the PB2 protein of these four viruses showed that there were no mutants happened in 627 and 701 site which were considered to be virulence-associated positions.