Preparation Of VSV Pseudovirus Containing H6N2 Avian Influenza Virus HA Protein

ObjectiveH6N2 Avian influenza virus(AIVs)is a low-pathogenic avian influenza that has not attracted human attention.However,with the increasing isolation rate of H6N2 subtype AIVs,this subtype AIVs will pose a threat to human health.This study constructed a vesicular stomatitis virus(VSV)containing the HA protein of H6N2 avian influenza virus to provide technical support for further research on avian influenza receptors,neutralizing epitopes and the like.MethodsViral RNA was extracted from avian influenza virus A/green peafowl/Qinghuandao/55/12(H6N2)(GP/QHD/55),and the full-length cDNA template was reverse-transcribed.The HA gene sequence was selected,primers were designed,and the target gene was amplified.The restriction enzyme cleavage site was introduced and ligated with the vector pcDNA3.1+to transform DH5a competent cells,and the recombinant plasmid pcDNA3.1+HA was prepared and identified by restriction enzyme digestion.The positive recombinant plasmid was transfected into 293T cells and verified by Western blot.In vitro expression;293T cells transfected with HA plasmid were infected with VSV?G*-G virus to prepare VSV?G*-HA pseudovirus,and the pseudovirus was successfully packaged by fluorescence microscopy;the hemagglutination activity of pseudovirus was detected by hemagglutination experiment.Results1.A recombinant pcDNA3.1+HA recombinant plasmid was constructed using avian influenza virus A/green peafowl/Qinghuandao/55/12(H6N2)(GP/QHD/55)as a template.2.The expression of HA in 293T cells was confirmed by Western blot.The VSV?G*-HA pseudovirus was successfully packaged by fluorescence microscopy.3.Viral hemagglutination activity assay The VSV?G*-HA pseudovirus has similar hemagglutination activity as the avian influenza virus.ConclusionsUsing the VSV?G*-G virus system as a tool,a pseudovirus harboring the A/green peafowl/Qinghuandao/55/12(H6N2)(GP/QHD/55)avian influenza virus HA gene was successfully constructed.