Generation Of Recombined H5 Subtype Aiv With Different NA And The Study Of Biological Characteristic Of Recombined Viruses

To understand the influence of NA gene of H5 Nx subtype AIV?Avian influenza virus? the biological characteristics, with a goose source of H5N1 subtype of avian influenza virus A/ goose/Guangdong/SH7/2013?H5N1?, referred to SH7 strain as the carrier, using reverse genetic technology to replace NA gene segment of A/chicken/Hebei/LZF / 2014?H5N2?, A/duck/Guangdong / 673/2014?H5N6?, A/ goose/Guangdong / 674/2014?H5N6? and A/ goose/Guangdong/Js1306/2014?H5N8? strains respectively, we saved for the parental strain rSH7?H5N1? and 4 strains with different NA H5 subtype recombinant AIVs, named rSH7- LZFNA?H5N2?, rSH7-673 NA?H5N6?, rSH7-674?H5N6? and rSH7- Js1306NA?H5N8? respectively. HA gene segment of SH7 strains belongs to 2.3.4.4 clade. The NA stem?58-68? site of rSH7-674 NA strain misses 11 amino acids, N6 is short stalks, whereas the NA of rSH7-673 NA strain does not exists the above phenomenon, whose NA is a long stem N6. 105 EID₅₀ doses of the virus infected BALB/c mice respectively, and the results show that the pathogenicity in mice of different replacement NA of H5 subtype avian influenza virus is variant significantly. rSH7, rSH7-Js1306 NA and rSH7-673 NA AIVs are highly pathogenic, and can make mice death at low dose levels, and the MLD₅₀ are 2.5, 2.5 and 2.5 lg EID₅₀; rSH7-674 NA AIV is medium pathogenicity, can make mice death in relatively moderate dose, and the MLD₅₀ is 3.16 lg EID₅₀; rSH7- LZFNA presents low pathogenicity, only at the highest dose to make mice death, and the MLD₅₀ is 3.3 lg EID₅₀. SPF chicken pathogenic test demonstrated that contacting transmission ability of different NA H5 subtype AIVs are variant significantly, and with the 105 EID₅₀ dose of inoculation, groups of cohabitation animal mortality are follows respectively: rSH7?100%?, rSH7-673 NA?100%?, rSH7-674 NA?85.7%?, rSH7-Js1306NA?71%? and rSH7- LZFNA?57%?, and the pathogenicity of virus from big to small in turn is N1, N6, the N8 and N2 subtype NA, which is matched with the statistics of the H5 subtype AIV strain distribution from 1996 to 2015 various areas in China. the statistics demonstrates: H5N1 subtype AIVs are widely distributed all over the country; H5N2 subtype AIVs are distributed limitedly; the newly collected H5N6 subtype AIVs in clinical samples are high separation rate, and this subtupe viruses have a threat to infect people, H5N8 subtype AIVs exist great potential risks to poultry.Combining with the results in vivo experiment, this study explores influence of NA to H5 subtype AIV biological characteristics molecular mechanism. First, we determined the 5 strains of recombinant virus proliferation in CEF and DF1 and MDCK cells, and found that in CEF cell the virus titer was not different significantly, and in DF1 cell the N2 subtypes virus titer is low, and in MDCK cell virus titer from high to low in turn is the N8, short-stemmed N6, N1, long-stemmed N6 and N2 subtype NA. The above results matches with MLD₅₀ results of mice, and this illustrates potentially that NA gene segment which througth enhancing virus replication ability in mammalian cells increases virulence to mice.Secondly, in view of the function of the NA, we determined NA enzyme activity of the virus, NA enzyme activity from high to low in turn is the N8, N2, short-stemmed N6 and long-stemmed N6 subtype NA, and viruses on guinea pig RBC deflocculation ability results show that the long-stemmed N6 and N8 NA match with 2.3.4.4 clade of HA in this research most. In order to observe NA effects on virus replication ability more intuitively, we also determined the size of the virus to form plaque in MDCK cell, and the size of the virus plaque from big to small in turn is the N8, short-stemmed N6, long-stemmed N6, N1, and N2 subtype NA. From the above results, we can get a conclusion that the function of NA may be associated with NA enzyme activity, but NA enzyme activity is not the decisive factor; Short-stemmed NA can enhance ability of proliferation in mammalian cells; NA function is also associated with the suitability of the HA.