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Analysis of avian influenza virus H5N1 genome and construction of its infectious clones

WANG Ge-fei,LI Wei-zhong,ZHANG Heng,et al.(Dept.of Microbiology and Immunology,The Key Immunopathology Laboratory of Guangdong Province,Shantou University Medical College,Shantou 515041,China)  
To identify the genetic background of an avian H5N1 influenza virus and construct a system for reverse genetics of avian influenza,the H5N1 avian influenza virus isolated in Guangdong province,was cultured in chicken embryo eggs and purified by plaque test,and its proliferative characters were detected in MDCK cells.The 8 fragments of viral full genome were cloned by RT-PCR using H5N1 virus universal primer and sequenced.And then 8 fragments of H5N1 viral genome were inserted into the reverse genetic vector to construct the infectious clones of H5N1 avian influenza virus.The results showed that this H5N1 strain could proliferate effectively without trypsin and produce typical CPE in MDCK cells.The 8 fragments(PB2,PB1,PA,HA,NP,NA,M,NS) of this H5N1 strain are cloned by RT-PCR and identified by sequencing and analysis.The viral sequence reveal some novel mutations in the genome.The HA connecting peptides are multiple successive basic amino acid,can effective duplicating without trypsin,consistent with viral proliferative characters result in cell.Neither oseltamivir nor rimantadine resistant mutation in the genome is found.PCR and sequencing results indicated that the infectious clones of this H5N1 strain,which inserting 8 full-length genome fragments,are constructed.These researches identify the viral genetic background,establish reverse genetics system of avian influenza and provid research basis for further vaccines development of avian influenza.
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