[Generation of attenuated H5N1 and H5N2 subtypes of influenza virus recombinants by reverse genetics system].Wei Sheng Wu Xue Bao. 2005 Feb; 45(1):53-7.WS
The HA connecting peptide at cleavage site, PQRERRKKR / GL, of an H5N1 subtype avian influenza virus (AIV) was replaced with PQRESR / GL, and then the modified HA gene was cloned into the transcription/expression vector, pHW2000, constructing a plasmid named pHW524-HA. The NA (N1) gene from the H5N1 virus and the NA (N2) gene from an H9N2 AIV were also cloned into pHW2000 separately, resulting in plasmids pHW506-NA and pHW206-NA. With the organization of pHW524-HA, pHW506-NA or pHW206-NA, and six plasmids containing internal genes from A/WSN/33 backbone virus, two transfectants, H5N1/WSN and H5N2/WSN, were subsequently generated by eight-plasmid system. After 15 consecutive passages in embryonated eggs, the two recombinants grew up to high titers of 1:2(9) in hemagglutination test with no changes in nucleotide sequences of the surface genes detected. Both the recombinant viruses belonged to mildly pathogen when evaluated by the pathogenicity test in six-week-old SPF chickens. H5N2/WSN recombinant virus was obviously less pathogenic than H5N1/WSN virus for embryonated chicken eggs. This presentation showed that the reverse genetics system is a very useful tool for studying the construction and function of individual genes and for the generation of virus as vaccine candidate.