The Construction Of Reverse Genetics System For Avian Influenza Virus

Every year, Avian Influenza Virus(AIV) will induce very high morbidity and mortalityin Avian, and cause serious economic losses. In the same time, interspecies transmission ofthe virus, especially subtype H5N1, can make man ill or die directly, threat of human healthand safety. So, it is very significant and necessary to understand the pathogenic mechanismof AIV for therapy and defend of it.Influenza A Virus contain a genome of negative sense single-stranded RNA dividedinto eight linear segments, study on anyone of it alone has no direct meaning for the wholevirus particles. But Reverse Genetic(RG) can solve this problem,we can use RG system torescue live virus through plasmid, and it will help us to understand the pathogenicmechanism of AIV better and to develop new vaccine.The subtype of H5N1and H3N2of IAV were cloned by RT-PCR. Through theanalysis of the sequence alignment we found that the sequence of NA and HA were highvariation,but in contrary, PB1、PB2、PA and NP were conservative.We used Immunoprecipitation (IP) and bimolecular fluorescence complementation(BiFC) to test and verify the NP-NP interaction in vivo, and C terminal deletionof NP experiment indicated that it was required by the interaction and oligomer form-ation of NP.For the further discussion of the role of NP in virus replication, we inserted report geneEGFP and luc into pHH21, and then we observed green fluorescence under microscope byco-express PB1、PB2、PA and NP, the result showed that we assembled activated RNP andgave us a new method to study PB1、PB2、PA and NP near virus’s physiological state.We inserted the genome of H5N1and H3N2to the plasmid pHW2000and pHH21toconstruct reverse genetic system to rescue virus, the system we named polⅠ-polⅡand polⅠrespectively.Therefore, we generated a bidirectional transcription construct that containscDNA encoding IAV gene flanked by an RNA polymerase I (pol I) promoter for vRNAsynthesis and an RNA polymerase II (pol II) promoter for mRNA synthesis. The utility ofthis approach was proved by the generation of virus after transfecting the polⅠ-polⅡ system.The cDNA of IAV got from virus were the basis of IAV research; and the successfulconstruction of IAV reverse genetic system will help us to study the function of each genein virus level and to make new vaccine.