| In order to study the effect of the interaction hemagglutinin and neuraminidase on the biological characteristics of the H5N6 subtype avian influenza virus. In this study, mature reverse genetics technology of influenza virus was used. With the present research group established A / Goose / Guangdong / SH7 / 2003(H5N1) for the skeleton,A / Duck / Guangdong / Y 673/2014(H5N6) and influenza virus A / Goose / Guangdong / 674/2014(H5N6) provide HA gene and NA gene, recombined two H5N6 flu virus named as r673HA674 NA and r674HA674 NA, respectively, after a series of in vitro experiments to assess the effects of HA-NA matching on their biological characteristics.The results showed that 673 HA presences of 14 amino acid positions differ from 674 HA, but potential glycosylation sites are consistent. Then the sialic acid receptor binding assay experiments were used to test two strains of the sialic acid receptor binding ability. The results showed that two recombinant flu virus main bind to ?-2,3 sialic acid receptor, Wherein 674 HA of ?-2,3 sialic acid receptor binding ability is stronger than 673 HA, but ?-2,6 sialic acid receptor binding basically the same.Plaque experiments In vitro, hemagglutination assay with red blood cell and replication of virus in cells showed that r673HA674 NA recombinant influenza virus formed plaques in MDCK cells was significantly bigger than r674HA674 NA, in guinea pig erythrocytes hemagglutination assay, r673HA674 NA recombinant influenza virus hole number reduce 11 hole, while the r674HA674 NA only changes 1 holes. showed that the r673HA674 NA recombinant influenza virus more easily dissociated in guinea pig red blood cells, and the replication test of CEF cells and MDCK cells, r673HA674 NA recombinant influenza virus replication titer are higher than r674HA674 NA in 12-60 h after infected, showed that r673HA674 NA influenza virus replication was significantly better than that of r674HA674 NA in CEF cell and MDCK cell, these experiments are described r673HA674 NA strain has better HA-NA balance than r674HA674 NA.In vivo tests, two recombinant influenza virus in SPF chickens and BALB / c mice pathogenicity was measured. The results showed that r673HA674 NA and r674HA674 NA two strains of recombinant influenza virus to SPF chickens were highly pathogenic and to 106EID50/200 u L poison attack, SPF chickens can died within 3 days, but HA-NA good balance of r673HA674 NA recombinant strains has stronger contact transmission ability than r674HA674 NA in chicken flocks, swab detoxification and volume increase, caused greater harm in chickens, The mortality rate of the r673HA674 NA recombinant influenza virus cohabitation group was 72%, while the mortality rate of the r674HA674 NA recombinant influenza virus group was 57%,but has larger MLD50 in BALB / c mice, r673HA674 NA recombinant influenza virus MLD50 was 4.5, while the r674HA674 NA recombinant influenza virus MLD50 was 3.2, weaker pathogenic to mice, replication lower titers in the lungs, the results are indicating that the BALB / c mice is not a good animal models to evaluate NA-NA balance. This is consistent with previous reports. The possible reason is respiratory tract epithelial cells in the mouse is dominated by ?-2,3 sialic acids, r674HA674 NA have a stronger binding ability of ?-2,3 sialic acid receptors and making the virus can better replicate in the respiratory tract in BALB / c mice, produced stronger pathogenic. At the same time, the composition of amino acid in the receptor binding region of the two strains of recombinant influenza virus HA may also be the cause of the stronger pathogenic of r674HA674 NA to BALB/c mice. |
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