Construction Of Canine Poll Promoter-based Bidirectional Expression Vector And Cloning Of Influenza Virus Genome

Influenza viruses, members of the Orthomyxoviridae family, are divided into threegenera, influenza A, B and C viruses, based on antigenic differences between theirnucleoproteins (NPs) and matrix (M) proteins, and all of the animal influenza viruses belongto type A. Type A viruses are further subtyped on the basis of antigenic differences on theexternal glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). Currently,influenza A viruses are classified into16HA and9NA subtypes, all of which are maintainedin aquatic birds. Type A influenza viruses have been isolated from various animals, includinghumans, pigs, horses, sea mammals, cats, dogs and birds. In the past decade, the pandemicsof highly pathogenic avian influenza H5N1and the novel H1N1influenza have bothillustrated the potential of influenza viruses to rapidly emerge and spread widely in animalsand people. Influenza A viruses of the H3N8subtype are a major cause of respiratory diseasein horses. The discovery in2004that an equine origin H3N8influenza virus was the cause ofan extensive epidemic of respiratory disease in dogs in the USA came as a surprise.In recent years, reverse-genetics systems for the rescue of recombinant Influenza Avirus have proven to be of great value for influenza virus research and vaccine development.Transfection of eight plasmids under the control of an human RNA polymerase I promoterfor the production of viral RNA (vRNA) and four expression plasmids encoding the threepolymerase proteins and NP under the control of an RNA polymerase II promoter resulted inthe rescue of infectious virus. Next, Hoffman et al. reported a modification of the reversegenetics system, allowing virus to be generated from eight bidirectional RNApolI/IIplasmids instead of12plasmids. A limitation is that they can only be used on cells ofprimate origin due to species specificity of the human RNA pol I promoter.To develop a highly efficient method for direct rescue of influenza virus in MDCK cells,we amplified the canine PolI promoter sequence, by use of a standard PCR using an MDCKcell DNA template and specific primer pairs designed according to the database information.The PCR products were then cloned into pTA2and sequenced. The cloned sequencepossessed93%homology with the corresponding region of canis lupus familiaris gene forRNA polymerase I sequence.Next, we constructed the bidirectional vector pVW3000, which allows transcription of viral genomic RNA and mRNA from one template. In the first step to construct thebidirectional expression plasmid pVW3000, canine PolI promoter, two BsmBI restrictionsites and33nucleotides of the murine RNA polymerase I terminator sequence wereassembled using an overlap PCR strategy. Then,300nucleotides of canine PolI promoterexpression cassette were inserted into the BamHI/EcoRI window between the BGH poly(A)signal and the CMV promoter of pVAX1vector. Finally the bidirectional vector pVW3000was identified by restriction endonuclease reaction and sequencing.In order to determine whether the canine RNA pol I promoter sequence described aboveallowed efficient synthesis of influenza virus-like transcripts in MDCK cells, we constructedreporter plasmids pVW-M-GFP. The enhanced green fluorescence protein(EGFP) codingsequence, flanked by the5’ and3’ ends of the M segment of influenza virus, was cloned intopVW3000via BsmBI restriction sites. Then, H3N8influenza virus RNA wasreverse-transcribed with Prime Script Reverse Transcriptase, and the eight full-length viralsegments were amplified with universal primer for all influenza A viruses usingKOD-plus-neo Polymerase. The eight segments were inserted into pVW3000via BsmBIrestriction sites yielding the seven bidirectional plasmids pVW3000-PB1, pVW3000-PA,pVW3000-HA, pVW3000-NA, pVW3000-NP, pVW3000-M and pVW3000-NS. Finallyeight recombinant plasmids were identified by restriction endonuclease reaction andsequencing. In conclusion, these studies laid a solid foundation for the application of thecanine pol I-driven reverae genetics system in MDCK cells.