Study Of Pathogenicity Of H5N1 Influenza Virus Sw/FJ/1/01 And DK/ZJ/52/00

To demonstrate the molecular basis of cross-species transmission of H5N1 swine influenza viruses, we selected two isolates DK/ZJ/52/00(ZJ00),Sw/FJ/1/01(FJ01)as model viruses, there were only 23 amimo acids difference between the complete genomes of the two strains: ZJ00 viruses can not replicate in the lungs of mice, however FJ01 viruses can replicate in the lungs of mice and also led mice to die. We established eight-plasmid reverse genetics system of the RZJ00 and RFJ01 to evaluate the pathogenicity differences of the two viruses by BALB/c mice . We rescued 16 single gene reassortant viruses by transfected 293T cell on the background of the FJ01 and ZJ00. The results of infection of BALB/c mice with 106EID50 18 viruses respectively was that PA and M gene of ZJ00 viruses dramatically attenuated the RFJ01, respectively, and making RFJ01/ZJ00PA and RFJ01/ZJ00M couldnot kill mice ; on the other hand, PA and M gene of FJ01 viruses causes RZJ00 viruses to replicate in the lungs of mice, 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 FJ01 and ZJ00 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 ZJ00 viruses together can completely change the pathogenicity of the RFJ01 viruses, making RFJ01 could not replicate in the lungs of BALB/c mice at all; on the other hand, M and PA gene of FJ1 viruses together could make RZJ00 viruses replicate in the lungs of mice, therefore it was concluded that M and PA gene cooperate to affect the replication ability of FJ01 and ZJ00 viruses in BALB/c mice.Comparing Amino acid sequence of PA and M genes between FJ01 and ZJ00 viruses, we finded that there were only four amino acids differences in PA and only one amino acids differences in M genes.To elucidate the molecular basis of the virulence and replication discrepancy between the FJ01 and ZJ00 viruses, we generated four mutant viruses. A PAI54V substitution at position 54 in PA resulted in marked attenuation of RFJ01 virus; However, a PAV54I substitution at position 54 in PA enabled ZJ00 to replicate in lungs of mice, although viruses titer was very low. However, changes of amino acids in position 330 of PA gene could not affect the the pathogenicity of FJ01 and ZJ00 viruses in mice. It was clear that amino acids at position 54 of PA to affect the pathogenicity of FJ01 and ZJ00 viruses in mice.Using reverse genetics, we demonstrated that amino acids at position 26 of M2 gene and at position 54 of PA gene cooperate to affect the pathogenicity of FJ01 and ZJ00 viruses in mice.