Generation And Application Of Recombinant H9N2 Subtype AIV Carrying MCherry And NanoLuc Reporter Genes

The avian influenza virus(AIV) is prevalent worldwide,especially in Asia.It not only brings great harm to the poultry industry,but also can cross the host barrier to infect people,which is of great significance in human public health security.In this study,mCherry fluorescence gene and NanoLuc luciferase gene were selected as reporter genes in combination with the current method of modifying influenza virus genome to express exogenous genes.The NS genes of H9N2 subtype avian influenza virus(A/chicken/Guangdong/V/2008)were modified to construct recombinant H9N2 subtype AIV carrying mCherry and NanoLuc reporter genes respectively.The former was used to establish a method for rapid screening of effective anti-influenza drugs,while the latter used bioluminescence imaging to monitor the dynamic process of influenza virus infection in mice.NS gene can encode NS1 protein and NEP protein.Firstly,the gene sequences encoding NS1 protein and NEP protein were amplified,and the reporter gene sequences of mCherry and Nano Lcu were inserted after the NS1 gene sequence.The reporter gene sequence and NEP gene sequence were interlinked with the PTV-1 2A peptide sequence which can be recognized and cleaved by protease,and the recombinant plasmid of NS gene was constructed.V_K627-mCherry and V_K627-NanoLuc recombinant viruses were obtained by reverse genetic technique.In SPF embryo passages,reporter genes could be detected in all 4 generations of collected virus venom.Western-blot results showed that the recombinant virus NS1 protein and the reporter protein were expressed in the form of fusion.The expression of recombinant virus mCherry red fluorescent protein was detected by fluorescence and indirect immunofluorescence(IFA).The proliferation curve of recombinant virus showed that the recombinant virus replication ability was closed to that of the parent strain.Preliminary results showed that the recombinant H9N2 subtype AIV carrying mCherry and NanoLuc reporter genes was successfully rescued.The recombinant virus showed good stability and replication ability,and could express the active reporter protein.After the recombinant virus V_K627-mCherry infected MDCK cells,ribavirin,a drug known to have anti-influenza effects,was added.The fluorescence intensity of the recombinant virus decreased with the increase of drug concentration.When the concentration of ribavirin reached 50?M,the recombinant virus fluorescence was observed to disappear completely.The results showed that the effective anti-influenza drugs could be rapidly screened according to the fluorescence of the recombinant virus.After the V_K627-NanoLuc recombinant virus infected mice via nasal drops,NanoLuc luciferase substrates were injected into the eyes of mice and observed by in vivo imager.Bioluminescence signals were detected in the chest of living mice,and the fluorescence intensity was related to the infection dose of the virus.With the increase of the infection dose,the fluorescence intensity detected in mice was stronger.The dynamic process of influenza virus infection in mice was monitored by bioluminescence imaging.When the recombinant V_K627-NanoLuc virus PB2 protein 627amino acid site was mutated from K to E,the intensity of bioluminescence detected in the chest of mice was significantly reduced,and its pathogenicity to mice was also significantly reduced.The results are consistent with the results of the known influenza virus PB2 protein 627 amino acid locus on the pathogenicity of mice,indicating that the recombinant virus carrying NanoLuc reporter gene can be used to rapidly evaluate the key sites that affect the transmission or virulence of influenza virus across host.To sum up,the recombinant virus carrying mCherry fluorescence gene has provided a rapid test method for screening effective anti-flu drugs,which has certain guiding significance for the development,screening and production of new anti-flu drugs.The application of NanoLuc luciferase gene recombinant virus in vivo imaging in mice can real-time monitor the distribution of influenza virus in the body and the dynamic process of infection,which is of great significance for the study of influenza virus infection,pathogenicity and pathogenic mechanism.V_K627-mCherry and V_K627-NanoLuc recombinant viruses can provide a visual tool for influenza virus research.