Estsblishment Of Cold-Adapted Attenuated Reverse Genetic System For Influenza A Virus

Influenza is a highly contagious respiratory disease caused by Influenza virus. Influenza virus, a member of the family Orthomyxoviridae, can infect humans and many kinds of animals. Influenza A virus that had caused fourth pandemic in 20th century is a mass threaten to humans. The fragmented genome of the influenza A virus is more vulnerable to genetic variations which leads antigenic shift and drift. This phenomenon makes it's more difficult to prevent and control this disease. Compare to other measures, vaccination remains the most effective approach to prevent this disease. Currently, influenza vaccines used in humans contains"inactivated"vaccines,split vaccine and live attenuated vaccines. Live attenuated vaccines, an intranasal vaccine, can induce both local mucosa immune response and system immune response. Due to their safety and efficacy, the live attenuated vaccines are seen as viable alternatives to the"inactivated vaccines". Reverse genetic systems entirely based on cloned cDNAs of influenza virus established by Neumann, Hoffmann, et al. since 1999 is being employed to specially design"recombinant"A and B viruses, and it has opened the way to develop live attenuated vaccines. In our research , cold-adapted,temperature sensitive and attenuated influenza A virus A/Ann Arbor/6/60 (H2N2) is used as a master donor virus(MDV-A) to generate an attenuated recombinants influenza A virus. This system is also used to rescue the strains for the manufacture of seasonal influenza vaccine for 2006/2007.1. Construction of six transcription/expression plasmids contained six inner gene of MDV-APolyA signal sequence was amplified from plasmid pcDNA3.1(+)by PCR to replace the bovine growth hormone polyadenylation signals of pHW2000, the reconstructed vector named as pAD3000.Six inner fragments contained five mutations of MDV-A were artificial synthesized. This six cDNAs were separately cloned into pAD3000 to construct six transcription/expression plasmids for the purpose of synthesizing of vRNAs and mRNAs by polⅠand polⅡfrom one template after being transfected into eukaryotic cells such as COS-1. All plasmids were sequenced to ensure it's the same with the consensus MDV-A sequence except the five mutations.The COS-1 cell was cotransfected with eight plasmids which contained one plasmid of one fragment from MDV-A and seven plasmids had other seven fragments from PR8. The transfected cells were incubated at 35℃or 33℃,48 hours after transfection, the supernatant and cos-1 cells transfected were inoculated into the allantoic cavity of 10-day old specific-pathogen-free (SPF) chicken eggs . The HA titer were determined. A series 7+1 reassortant virus was rescued. It showed that all the six plasmids contained MDV-A genes were shown to be functionally expressed. With this progress, a cold-adapted,temperature sensitive and attenuated strain would be rescued next.2. Generation of cold-adapted attenuated reassortant influenza virus rMDV-A by reverse genetic and identification some of its biological characterizationsSix plasmids contained MDV-A genes were transfected into COS-1 cells in combination with plasmids incorporating the HA and NA genes of A/PR/8/34. The transfected cells were incubated at 33℃,48 hours after transfection, the supernatant and cos-1 cells transfected were inoculated into the allantoic cavity of 10-day old specific-pathogen-free (SPF) chicken eggs .The reassortant rMDV-A virus was determined by HA assay. The rescued viruses were indistinguishable from the wild-type in morphology. Recovered rMDV-A viruses was passed in eggs for four generations, and then its six inner segments were amplified by RT-PCR, each fragments was cloned into pGEM-T vector and sequenced. MDCK cells were infected with rMDV-A virus without trypsin. Temperature sensitivity of rMDV-A was determined by plaque assay on MDCK cells. The results showed that the inner gene of the fourth generation reassortant virus was the same with the consensus MDV-A sequence. Recovered rMDV-A viruses could replicate in eggs stably. The MDCK cells infected with rMDV-A became rounding, shedding and formed plaque. Plaque assay showed that rMDV-A have significant reduction in plaque at 39℃compared with 33℃. Recovered rMDV-A viruses was sensitive to temperature, lower temperature such as 33℃was more suitable for its replication. This reverse genetics system provided a new experimental tool for development of new-type vaccine candidates, for example, live-attenuated vaccine candidates.3. Rescue of the seasonal influenza vaccine composition strains for the season 2006/2007 by cold-adapted attenuated reverse genetic systemThe HA and NA gene of A/New Caledonia/20/99(H1N1),A/Wisconsin/67/2005(H3N2) were amplified by RT-PCR, each fragments was cloned into pGEM-T vector and sequenced. The correct genes were then be cloned to pAD3000. This two plasmids contained HA and NA genes were transfected into COS-1 cells in combination with six plasmid incorporating the inner genes of MDV-A respectively. The transfected cells were incubated at 33℃,48 hours after transfection, the supernatant and cos-1 cells transfected were inoculated into the allantoic cavity of 10-day old specific-pathogen-free (SPF) chicken eggs .The recovered virus named rMDV-A-H1 and rMDV-A-H3.Generation of this two ressortant virus is the base of the development of trivalent vaccine for 2006/2007.Overall, this reverse genetics system provided a new experimental tool for development of new-type vaccine candidates, for example, live-attenuated vaccine candidates.