Evolution And Pathogenicity Of H1N2, H3N2 And H5N1 Swine Influenza Viruses

Swine influenza is a highly infectious viral disease of pigs causing considerable economic impact. Three different subtypes-H1N1, H3N2 and H1N2 are circulating in swine worldwide. Pig as an intermediate host posed significance in producing a new viruses. Therefore, it has great significance to make clear which subtypes of influenza are ciculating and its pathgencity in China.To learn about regularity of veterinary epidemiology and pathogenicity of SIV, Firstly, we collected 5886 nasal swabs and 5788 serum samples of pigs in part of China, including Shanghai,Henan,Shandong,hubei,Jiangsu,Guangdong province. The result of antibody titers of 5788 serum samples was as follows: H1 positive percentage was 40%, H3 positive percentage was 90%, H5 positive percentage was 0.086%, H9 positive percentage was 0.138%. Four H1N2 subtype, six H3N2 subtype and one H5N1 subtype influenza isolates were isolated by inoculation of Madin-Darby canine kidney(MDCK) cells with nasal swabs, and no viruse isolated in SPF embryonated eggs, this results demonstrated that MDCK cell line was more sensitive than SPF embryonated eggs on isolating SIV; The results demonstrated that H1 and H3 subtype is the primary circulating isolates among pigs in China. And then swine/Guangdong/1/2006(H1N2)(SwGD1/06)and Swine/Guangdong/1/2005(H3N2)(SwGD1/05)were selected to be sequenced and compared with sequences available in GenBank, and making phylogenetic trees of the complete genes. The results of analysis indicated that SwGD1/06 (H1N2) probably origins from H1N2 swine influenza viruses circulated in North America, while SwGD1/05(H3N2) probably come from H3N2 human influenza viruses circulated in North America. Finally , to study the pathogencity of the two viruses to pigs, we selected pigs whose antibody was negative to H1,H3,H5,H9 standard antigens as experiment animal. Challenged with 106EID50 H1N2 and H3N2 viruses respectively, we could reisolate viruses from nasal swabs on first,third,fifth,seventh day post infection, respectively, and the viruses titer was highest on the third day post infection, but we could not reisolate viruses from any other organs of infected pigs. The results indicated that H1N2 and H3N2 viruses could replicate in pig's upper respiratory tract without clinical symptoms.To demonstrate the molecular basis of cross-species transmission of H5N1 swine influenza viruses, we selected two isolates DK/ZJ/52/00(DK52),Sw/FJ/1/01(FJ1)as model viruses, whose inheritance was closest, there were only 23 amimo acids difference between the complete genomes of the two strains: DK52 viruses could not replicate in the lungs of mice, however FJ1 viruses could replicate in the lungs of mice and also led mice to die. We established eight-plasmid reverse genetics system of the two viruses(RDK52,RFJ1) to evaluate the pathogenicity differences of the two viruses by BALB/C mice model. We rescued 16 single gene reassortant viruses by transfected 293 Tcell on the background of FJ1 and DK52, respectively. The results of infection of mice with 106EID50 18 viruses respectively was that PA and M gene of DK52 viruses dramatically attenuated the RFJ1(Lg EID50, 4.71 versus 1.5 ), respectively, and making RFJ1/DK52PA and RFJ1/DK52M couldnot kill mice ; on the other hand, PA and M gene of FJ1 viruses causes RDK52 viruses to replicate in the lungs of mice (LgEID50, 0, versus1.5), respectively. And other genes could not change the pathogenicity of the reassortmant viruses. This result indicated that PA and M genes could affect the replication ability of FJ1 and DK52 viruses in mice.Furtherly, we replaced M and PA genes at the same reassorments viruses using an eight-plasmid reverse genetics system, results of infection with PA and M genes substitutation viruses had shown that M and PA gene of DK52 viruses together can completely change the pathogenicity of the RFJ1 viruses, making RFJ1 could not replicate in the lungs of mice at all; on the other hand, M and PA gene of FJ1 viruses together could make RDK52 viruses replicate in the lungs of mice(LgEID50, 2.5), therefore it was concluded that M and PA gene cooperate to affect the replication ability of FJ1 and DK52 viruses in mice.Comparing Amino acid sequence of PA and M genes between FJ1 and DK52 viruses, we finded that there were only four and one amino acids differences in PA(54,330,384,459) and M2(26) genes, respectively.To elucidate the molecular basis of the virulence and replication discrepancy between the FJ1 and DK52 viruses, we generated four mutant viruses. A I-to-V (PAI54V) substitution at position 54 in PA resulted in marked attenuation of RFJ1 virus (LgEID50,4.71 versus 1.5); However, a V-to-I (PAV54I) substitution at position 54 in PA enabled DK52 to replicate in lungs of mice, although viruses titer was very low (Lg EID50, 1.5). M gene acted as PA, a P-to-L (M2S26L) substitution at position 26 in M2 resulted in marked attenuation of RFJ1 virus (LgEID50,4.71 versus 1.5); a L-to-S (M2L26S) substitution at position 26 in M2 enabled DK52 to replicate in lungs of mice, although viruses titer is very low (Lg EID50, 1.5). However, changes of amino acids in position 330,384,459 of PA gene couldnot affect the the pathogenicity of FJ1 and DK52 viruses in mice. It was clear that amino acids at position 54 of PA and position 26 of M2 cooperate to affect the pathogenicity of FJ1 and DK52 viruses in mice.In summary, we investigate the epidemiology of swine influenza viruses among pigs in part of China, the results indicated that H1 and H3 subtype was currently the primary circulating isolates among pigs in China; the results of pigs challenged with H1N2 and H3N2 virusses demonstrated that H1N2 and H3N2 virusses could replicate in pig's upper respiratory tract without clinical symptoms, therefore this study possesed great significance in veterinary epidemiology and pathogenicity of H1N2 viruses in pigs. Using reverse genetics, we demonstrated that amino acids at position 54 of PA gene and at position 26 of M2 gene cooperate to affect the pathogenicity of FJ1 and DK52 viruses in mice, this result can provided powerful proof to explain why H5N1 influenza viruses acrossed species transmiting to human. Therefore this study posed significance in human public health.