Studies On The Molecular Mechanisms For The Pathogenicity And Transmissibility Of Avian Influenza A(H7N9) Viruses

In the spring of 2013, a novel H7N9 avian influenza virus emerged in the Yangtze River Delta region of China. As of May 17th,2015, a total of 661 H7N9 cases have been reported with fatal cases of 267. This H7N9 virus was a reassortant virus, with its HA, NA and six internal genes originated from duck H7, wild bird N9, and poultry H9N2 influenza viruses, respectively. Mammalian-adapted substitutions have been observed in some of the H7N9 strains, including HA-Q226L which was reported to be associated with the enhanced affinity of the virus to human-like receptors, and PB2-E627K/D701N which would increase pathogenicity of the avian influenza virus in mammals. After the discovery of H7N9 virus, studies on its origin, evolution and biological characterizations have been conducted. Pathogenicity and transmissibility were also evaluated on different animal models.During the evolution process, H7N9 viruses continuously reassorted with the H9N2 viruses and generated different genotypes with various internal genes. Differences on the pathogenicity and transmissibility of these genotypes were still unknown. Although several research groups have studied the impact of substitutions E627K or D701N in PB2 protein on the virulence of H7N9 viruses in BALB/c mice model, the synergistic effect of 627K and 701N dual mutations remains to be discovered. Molecular markers associated with the pathogenicity or transmissibility of the H7N9 viruses have not yet been reported on the other internal genes than PB2.In this study, nine H7N9 influenza virus strains with different genotypes were selected for the evaluation of their pathogenicity in C57BL/6 mice. Distinct virulence was observed between genotypes or strains. A more virulent strain, A/Anhui/01/2013 (AH01, H7N9), and a much less pathogenic virus, A/Shanghai/05/2013 (SH05, H7N9), were used for the study of virus transmissions in ferrets. Our results showed that AH01 viruses could not only transmit efficiently via physical contact but also via airborne exposure. However, only limited physical contact transmission has been observed in ferrets inoculated with the SH05 virus, and no airborne transmissibility occurred.Sequence comparisons found that SH05 possessed glutamic acid (E) at PB2-627, while AH01 contained lysine (K) at this position. However, substitutions of PB2-E627K or D701N could be detected within one passage of either virus in ferrets via physical contact, as determined by next generation sequencing of samples collected from the nasal washes of ferrets inoculated or exposed to SH05 or AH01. This result indicated that substitutions E627K or D701N at PB2 protein could readily occurred during the adaptation of H7N9 viruses in mammals.Impacts of four different residue combinations (PB2-627E-701D, PB2-627E-701N, PB2-627K-701D, and PB2-627K-701N) on virulence of the H7N9 viruses were then studied, using reverse genetic techniques. Our results found that PB2-627K-701N mutations conferred the highest polymerase activity and improved viral replication in mammalian cells, and also enhanced virulence in mice. PB2-627E-701D mutations were also associated with improved viral replication in mammalian cells and enhanced virulence in mice, but polymerase containing these two residues showed reduced activities when compared with the wild type virus with PB2-627K-701D, or the mutant with PB2-627E-701N.Analyses on recombinant polymerase complexes with genes deriving from A/Quail/Hongkong/G1/1997 (G1, H9N2) virus and AHO1 virus showed that NP gene of the G1 virus enhanced the polymerase activity of AHO1 virus. Residues 41V and 201D in the NP protein not only increased the AH01 polymerase activity but also would be associated with greater replication ability of the virus in lower temperature.In summary, this study found that different genotypes of H7N9 viruses possessed different pathogenicity and transmissibility. PB2-627K or 701N easily occurred during the adaptation of H7N9 viruses in mammals. These two residues were associated with increased virulence in mice, as showed in mutants carrying the PB2-627K-701N or PB2-627E-701D residues. Two novel molecular markers,41V and 210D in NP protein were identified to be associated with the enhanced replications of H7N9 viruses in vitro. Therefore, special attention should be paid to H7N9 viruses with such substitutions.