| Analysis of NA protein of the domestic H9N2 isolates revealed that an increasing number of NA protein of H9N2 isolates contained a potential glycosylation site at 264 amino acid residue (NA264N) between 2000 and 2013. However, the number of H9N2 isolates with NA264N is decreasing after 2013.Notably,H9N2 with NA264N is only existed in China (Except for two H9N2 isolates from Vietnam). This finding suggests that the presence and endemic of such H9N2 might result from the massive vaccination of H9N2 in domestic poultry industry. However, little is known about the roles of such mutation at NA of H9N2. In this study, two H9N2 strains, one with NA264N (A/Chicken/Jiangsu/WS1/2012(H9N2),WS1), and another without NA264N (A/chicken/Beijing/BJ/2004(H9N2), BJ) were first selected. And four recombinant viruses named rgWS1-NA264N, rgWS1-NA264H, rgBJ-NA264H and rgBJ-NA264N respectively, were rescued to evaluate the effects of NA264N on the characteristics of H9N2 both in vitro and in vivo.1.Rescue H9N2 with NA264N and its mutantsOn the basis of the finding that H9N2 isolates with NA264N is endemic in domestic poultry, in order to investigate the effect of NA264N on H9N2.Two domestic H9N2 strains,WS1 with NA264N and BJ without NA264N, were selected. And the eight reverse genetics plasmids of WS1 strain, HA and NA reverse genetics plasmids of BJ strain, were constructed through ExnaseTM Ⅱ-based in vitro recombination approach. The constructed plasmids were named as WS1-PB2, WS1-PA, WS1-PB1, WS1-HA, WS1-NP, WS1-NA264N, WS1-MP, WS1-NS, BJ-HA and BJ-NA respectively. Meantime, two plasmids with mutation at NA264 were generated through overlap PCR, and designated WS1-NA264H and BJ-NA264N respectively. And four recombinant viruses named rgWS1-NA264N, rgWS1-NA264H, rgBJ-NA264H and rgBJ-NA264N respectively, were rescued through transfection with eight plasmids for influenza reverse genetics system. The viral titer of rgWS1-NA264N, rgWS1-NA264H, rgBJ-NA264H and rgBJ-NA264N was 1.58×10∧A7,5×10∧7,1.08×10∧7 and 1.58×10∧7 of TCIDso/ml respectively. The four rescued viruses provide important materials for studying the function of NA264N of H9N2.2.Effects of NA264N on the characteristic of H9N2In order to study the roles of NA264N, the viral replication, shedding and pathogenesis of the four rescued viruses rgWSl-NA264N, rgWSl-NA264H, rgBJ-NA264H and rgBJ-NA264N were compared in vitro and in vivo. The growth kinetic analysis showed that rgWSl-NA264H and rgBJ-NA264H could grow faster than rgWSl-NA264N and rgBJ-NA264N in MDCK cell respectively. The pathogenesis study in mice demonstrated that viral titers of rgWSl-NA264H and rgWSl-NA264N in the lung from the infected mice didn’t show significant difference whereas viral titer of rgBJ-NA264H in the lung showed significantly higher than rgBJ-NA264N at day 3 post infection. And the maxi average body weight loss of mice infected with rgBJ-NA264H was up to 20% whereas that of mice infected with rgBJ-NA264N was only 10%. Chicken infection study showed that laryngeal viral shedding of chickens infected with rgWSl-NA264H and rgBJ-NA264H was more than that of chicken infected with rgWS1-NA264N and rgBJ-NA264N respectively. Notably, the laryngeal viral shedding of chickens contacted with rgWSl-NA264N and rgWSl-NA264H didn’t show obvious differences whereas the laryngeal of chickens contacted with rgWSl-NA264H shedded significantly more viruses than that of chickens contacted with rgBJ-NA264N at day 6 post contact. All these data indicate that the potential glycosylation site at NA264N plays some roles in viral replication, shedding and pathogenesis. |
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