Construction Of H9N2Avian Influenza Virus Vector Carrying Infectious Bronchitis Virus S1

Reverse genetics is a conception contrary to classic genetics research, which study the affect of phenotype by gene modification with site-specific mutagenesis by gene cloning or genetic recombination technology. It is a very important technology to investigate the mechanism of viral pathogenesis, and have a great contribution for the developing new vaccine.The reporter gene fluorescin GFP and Infectious Bronchitis Virus(IBV) S1gene were inserted respectively into the NA segment of H9N2subtype avian influenza virus (AIV) A/Chicken/Shanghai/F/98(C/SH/F/98, F), and three chimeric NA segments were formed. The chimeric NA segments then were inserted into the pHW2000, and transfected into293T cells with the other seven segments of F strain. After inoculating the SPF embryonated chicken eggs with transfection product293T cells, the reassortant AIVs rF/NA1245-2A-GFP, rF/NA183-GFP and rF/NA183-S1were obtained with the hemagglutination titer.1. Construction of transcription/expression vectors carrying GFP or S1Two kinds of chimeric NA vectors were designed using the NA gene of F strain as the template, which included the reporter gene fluorescin GFP or Infectious Bronchitis Virus(IBV) S1gene. The first chimeric NA vectors contained the5’non-encoding region,1245nt of encoding region, inserting2A and GFP sequences, then following157nt of encoding region and3’non-encoding region from NA gene of F strain (pHWNA1245-2A-GFP). The components that included the5’non-encoding region,183nt of encoding region, inserting GFP/S1sequences,3’non-encoding region and closed157nt of encoding region derived from NA gene F strain became the second chimeric NA vector (pHWNA183-GFP/S1). All chimeric NA vector were sequenced and inserted into pHW2000, finally the transcription/expression vectors pHWNA1245-2A-GFP, pHWNA183-GFP and pHWNA183-S1were obtained and identified by PCR or digestion.2. Generation of reassortant AIV expressing the foreign gene by reverse geneticsOne chimeric NA plasmid (pHWNA1245-2A-GFP, or pHWNA183-GFP, or pHWNA183-S1) and seven plasmids of F strain (pHW201-pB2、pHW202-pB1、 pHW203-PA、pHW204-HA、pHW205-NP、pHW207-M、pHW208-NS)were mixed at the ratio1:1respectively, and formed three different kinds of8-plasmids rescue system of reassortant viruses encoding the foreign gene. The fluorescin were observed clearly in293Tcells at50h after transfected with GFP plasmids. The10-day SPF embryonated chicken eggs were inoculated with the293T cells transfected with three different kinds of8-plasmids rescue system, and which allantoic fluid presented1:24-1:26hemagglutination titer (HA). The successful rescued viruses were called rF/NA1245-2A-GFP, rF/NA183-GFP and rF/NA183-S1. Theses rescued viruses were confirmed by PCR and electron microscope, but could not survive in next generation when inoculated10-day SPF embryonated chicken eggs or MDCK cells.