Generation And Characterization Of Transgenic Animal Model With A Targeted Disruption Of Influenza A Virus Hemagglutinin

Influenza A virus,which is pathogenic and detrimental,continuously poses a threat to poultry,animals and public health as well.Antigenic drift and antigenic shift contribute to a global rise in drug-resistant influenza virus strains,and bring more and more difficulties in the prevention and treatment of the virus.Thus,development of novel anti-influenza strategies has become an urgent task.In this study,we constructed two expression vectors(sh-HA-1 and sh-HA-2)capable of stably expressing short hairpin RNA(shRNA)that specifically targets conserved sequences of influenza A virus hemagglutinin(HA).Then the interference efficiency of sh-HA-1 and sh-HA-2 was tested by western blotting in A549 cells.The vector sh-HA-1 was found to have a better interference effect on HA expression and was then integrated into mouse genome,which generated the genetically modified mice that stably express the specific shRNA.We found that HA expression was dramatically disrupted in the lungs of transgenic(TG)mice after both WSN and PR8 virus infection.Morphological characteristics,body weight changes,the survival rate and organ lesions were observed and analyzed between wild type(WT)and TG mice.In addition,plaque assay was performed to measure virus titer in lungs,and leukocytes in peripheral blood were evaluated.Moreover,we also determined the ability of genetically engineered mice to pass influenza virus to other mice.The results are shown as follows:(1)A549 cell lines stably expressing shRNAs targeting HA or luciferase control were infected with WSN or PR8 virus and harvested at 8h and 16h post infection.Both sh-HA-1 and sh-HA-2 exhibited good interference effect on HA expression as compared with that of control.Remarkably,sh-HA-1 had better interference efficiency than sh-HA-2.(2)TG mice with a targeted disruption of influenza A virus HA were generated.The progeny of TG mice were genotyped by PCR,and the ratio of positive generations was about 50%.(3)A dramatic decrease of influenza virus HA was found in the TG mice infected with WSN or PR8 virus compared with that in WT mice.The TG mice were more resistant to influenza virus infection,exhibiting lower weight loss,longer survival time,and less severe organ damage.Furthermore,infection with either WSN virus or PR8 virus resulted in a significant decrease in white blood cells of WT mice,while those in TG mice were close to the normal level after same challenge with the viruses.(4)The TG mice showed a reduced ability in transmission of influenza virus.These results reveal that we successfully generated TG mice stably expressing shRNA that causes the animals reduced susceptibility to influenza virus infection through disrupting expression of viral HA.In particular,our approach will provide a theoretical basis and technical support for the generation of other animals that are resistant to virus infection.