Development Of High-growth Canine Influenza Reassortant Virus And The Research Of The Fragment End Sequences Of The Influenza Virus Genes Deciding Transcription Level

The influenza virus is a kind of important viruses harm canine, In this paper, we used influenza virus reverse genetics rescue a series of H3N2subtype canine influenza virus strains A/canine/Zhejiang/01/2010A (H3N2)(referred ZJCIV) and PR8(A/Puerto Rico/8/34(H1N1)) of the recombinant viruses, obtained by screening an highly efficient expression of the recombinant virus of H3N2subtype canine influenza virus surface antigen;We used PR8virus micro-replication subsystem to study the fragment end sequences of the eight genes of the influenza virus impact on the reporter gene (the Firefly luciferase) transcription in the case of the highest packaging efficiency, At the same time, a comparative study of different packaging efficiency of the M gene end sequence impact on the reporter gene transcription.H3N2subtype of canine influenza outbreak in recent years pose a great threat to different kinds of dogs, there is no effective vaccine for the virus control now. Wild H3N2subtype of canine influenza isolates in the chick embryo and cell proliferation are very poor, In order to obtain highly efficient expression of recombinant viruses of the H3N2subtype of canine influenza virus surface antigen, the experimental use of reverse genetics, the HA and NA genes of ZJCIV and PR8virus internal genes and mutation gene of NP (G132A) and NS2(E67/74S) artificial restructuring, rescued and received four canine influenza reassortant virus CIV-PR8, CIV-NP, CIV-NS and CIV-NP-NS.9-11day-old SPF chicken embryos inoculated with the virus containing100EID50. CIV-NS, CIV-NP-NS inoculated chicken embryos for36h, allantoic fluid of HA titer of the highest, CIV-NP and CIV-PR8reached the highest after48h, allantoic fluid HA titer of NS average of up to213.33.2×103TCID50virus infection in MDCK cells, CIV-NS cell supernatant HA titer, up to210, CIV-NP-NS, followed by29, wild virus ZJCIV only24; Both by amplification in chick embryo or MDCK cell, the TCID50of the four recombinant canine influenza virus are higher than ZJCIV, CIV-NS TCID50significantly higher than other strains. In this study, we have successfully rescued four recombinant canine influenza virus which proliferation was markedly higher than the wild strain ZJCIV, CIV-NS both in the chick embryo and MDCK cells, proliferation was markedly better than other strains, this recombinant virus is expected to become canine influenza vaccine development candidate virus.The influenza virus gene fragments of the3’end and5’noncoding region and part of the coding region sequence is not only play the role of packaging signals, and these sequences determine the amount of influenza virus gene expression. In this study, PR8influenza virus strains of the highest packaging efficiency eight gene segments5’and3’ end sequences were cloned between the RNA polymerase I promoter and RNA polymerase I terminator, and designed restriction sites in the5’and3’end and then inserted the reporter gene (the Firefly luciferase) in the restriction sites, and5’end coding region of the initiation codon ATG was mutated to the GTG, the gene only transcripted and expressed the open reading frame of report genes. The constructed plasmids were co-transfected into293T cells with the expression plasmids of PB1, PB2, PA, NP gene of influenza virus and the expression plasmid of the internal reference Renilla luciferase, We used the dual luciferase reporter gene system to compare the packaging efficiency of the highest case, the influenza virus of8gene fragments terminal sequence impact on the transcription of the reporter gene (the Firefly luciferase). The study found that the PA gene terminal sequence transcription at the the highest level, the minimum at the terminal sequence of the M gene. We selected the the end of the five kinds of packaging efficiency of the M gene sequence, the recombinant plasmids in the same way, the constructed plasmid and expression plasmids of the influenza virus PB1, PB2, PA, NP and the internal reference Renilla luciferase expression plasmid were co-transfected into293T cells, used the dual luciferase reporter gene system to compare the different packaging efficiency, the terminal sequence of the influenza virus M gene impact on transcription of the reporter gene (the Firefly luciferase). The results showed the highest transcription level of vRNA of the5’end220nucleotides and the3’end of0nucleotides. The above research laid the foundation for constructing highly efficient expression of the influenza virus vectors of the influenza virus genes and exogenous genes.