Biological Characterization Of H9N2Avian Influenza Virus And Construction Of PR8Eight-plasmid Reverse Geneitc System For Preparation Of Vaccine Stock

To study the pathogenesis of H9N2avian influenza viruses (AIV) in mammalian host, wesequenced the genome of6H9N2AIV isolated in southern China during2009-2010and determinedtheir replicate ability in mice. Genome sequencing analysis demonstrated that of all six viruses,226position of HA gene is Leu, which indicates the ability to bind to-2,6-salic acid, while other sitesremains the characteristic of low pathogenic virus, position183,190,228remains Asn, Ala, Gly, andthe cleavage site amino acid is-PSR(K)SSRGLF-. The phylogenetic tree showed that some internalgene of six H9N2viruses is close to gene of subtypes, indicating the possibility of recombinant withother viruses. The mouse study showed that these H9N2viruses were low pathogenic in BALB/cmice, causing no significant change of body weight and restricting their replication in respiratoryorgans.Eight-plasmid reverse genetic system, which is based on bi-directed vector that containspromoter of both RNA polymeraseⅠand RNA polymeraseⅡ(eg. pBD, pHW2000), can translateRNA polymerase complex and transcribe virus RNA simultaneously, and thereafter rescue infectivevirus particles. Establishment of the system can substantially reduce the cycle of plasmid constructionand virus rescue. Our study cloned six internal genes of PR8to pBD vectors, and rescuedrPR8-GD322to reexamine the system. The system we constructed provided a new platform forlaboratory research.In the face of continuing accumulation of antigenic changes within Clade7highly pathogenicavian influenza viruses (HPAIVs), and with it the potential pandemic threat to poultry or even humans,it is necessary to replenish our vaccine stock according to the antigenic analysis based on surveillance.In this study, a reassortant virus, named rPR8-LN/S4092, was generated using reverse genetics. Thevirus contained HA and NA genes of an epidemic strain A/Chicken/Liao Ning/S4092/2011in thebackground of internal genes derived form A/Puerto Rico/8/34(PR8). The reassortant virus wasattenuated by removal of the multi-basic amino acid motif in the HA gene by mutation and deletion(from PQIEGRRRKR to PQRETR).Egypt avian influenza vaccine mostly depends on import. Entrust by Egypt government，A/Chicken/Egypt/A2/D/2011(H9N2) was selected as vaccine seed, and the reassortant virusrPR8-Eg/A2/D was generated using eight-plasmid reverse genetic system we established. Sequencinganalysis demonstrated that whole genome sequence of vaccine virus was exactly as expected.