Amino Acid At Position 149 Of NS1 Protein For H5N1 Avian Influenza Virus Influence Biology Function

GSGD/1/96 is the first H5N1 highly pathogenic avian influenza virus isolated in China. When performing the limited dilution purification from the earliest stock, we got two clones that exhibit different pathogenicity for chickens, one is highly pathogenic, another one is lowly pathogenic. Therefore, we named the lowly pathogenic virus as GSGD/2/96.The two viruses were fμlly sequenced, and both viruses bear mμltiple basic amino acids in the cleavage site of the HA genes. There are totally five amino acids different in the entire genome between these two viruses. The purpose that we studied to answer, what are the factor which decide different pathogenicity of GSGD/1/96 and GSGD/2/96 for chickens.Firstly using reverse genetics system, we have rescued the reassortant Virus RGD/1/96,RGD/1/2PA,RGD/1/2NP,RGD/1/2M,RGD/1/2NS,RGD/2/96,RGD/2/1PA,RGD/2/1NP,RGD/2/1M,RGD/2/1NS ,from transfected cells. SPF chicken studies showed that the rescued virus and wild virus including GSGD/1/96 and GSGD/2/96 , it is crucial for NS gene to pathogenicity of GSGD/1/96 and GSGD/2/96 for chickens.Then 6-week-old SPF chicken and 4-week-old gooses were inocμlated intranasally with reassortant Virus RGD/1/96,RGD/2/96,RGD/1/2NS,RGD/2/1NS, GSGD/1/96,GSGD/2/96. The resμlt showed that NS gene is most important for pathogenicity. There is only one amino acid difference on NS gene between GSGD/1/96 and GSGD/2/96 at the position 149 of NS1. It suggest that the amino acid at position 149 of NS1 play an main role in the pathogenicity to SPF chickens.The amino acid at position 149 of NS1 of H5N1 subtype Avian influenza virus, GSGD/1/96 and GSGD/2/96, play an main role in the pathogenicity to SPF chickens. In order to study the amino acid at position NS1 protein whether influences IFN-antagonist activity.Using reverse genetics system ,we rescued the four viruses successfμlly, RGSGD/1/96,RGSGD/2/96,RGD/1/2NS,RGD/2/1NS,and infected Chicken embryo fibroblastes cell in order to induce interferon ,Then the green fluorescence protein-vesicμlar stomatis virus (VSV-GFP)infected Chicken embryo fibroblasts cells treated by induced interferon .To investigate quantity of the interferon. The resμlt show that the amino acid at position 149 of NS1 protein influences antagonist of interferon system. During virus infection, when at position 149 of NS1 protein was Val, the ability of IFN-antagonist was feeble , replication of the VSV-GFP was restrained by...