Research On The Influence Of Changers Amino Acid At Position 42 Of NS1 Protein For H5N1 Avian Influenza Virus Virulence

In the study, we targeted a H5N1 Highly Pathogenic Avian Influenza virus A/Duck/Guangxi/27/2003(H5N1()DK27),and shifted Serine to Glycine at position 42 of NS1 protein by mutating NS gene. In order to investigate whether the amino acid at position 42 of the NS1 protein have some influences on the virulence of influenza A virus, the recombinanted virus RDK27NS1S42G were successfully rescued from transinfected 293T cells by reverse genetics system , and intranasally inoculated SPF BALB/C mice .Firstly, DK27's eight genes were sequenced and cloned in PBD vector separately. The recombinanted virus RDK27 were successfully rescued from transinfected 293T cells by reverse genetics system. Comparing the results of Intranasally inoculated SPF BALB/C mice experiment with DK27and RDK27, we found that the mice have significant weight loss, and all died after 8 days, two viruses had evident highly pathogenicity on mice. There was also no obvious difference between the virulence of DK27 and RDK27.In order to study whether the amino acid at position 42 of NS1 protein influences virulence of H5N1 Avian Influenza virus, we shifted Serine to Glycine at position 42 of NS1 protein by mutating NS gene and rescued the recombinanted mutation virus RDK27NS1S42G by reverse genetics system. In intranasally inoculated BALB/c mice experiment with RDK27NS1S42G, the mice have significant weight loss, and all died after 8 days, it showed that the viruses had evident highly pathogenicity for mice. After 4,6 days of intranasal inoculation, the virus titer in the lungs of mice was the highest, more than 7.03EID50. The virus titer in the brains of mice took the second place, but it increased along with the time. The high level replicated virus were also isolated from the spleen and kidneys of mice, although the virus titer was not high. Comparing the results of Intranasally inoculated SPF BALB/C mice experiment with RDK27,the virulence of RDK27NS1S42G was not evidently different from RDK27. In conclusion, the mutation amino acid at position 42 of NS1 protein(S42G) have no evident influence on the virulence of H5N1 Avian Influenza virus.