The Molecular Mechanism Of Mutation On Variable Region In HA/NA Gene UTR Of The New Avian Influenza Viruses Affecting The Biological Characteristics Of Virus

Objective:The polymorphism of base in the variable region genome of the untranslated region(UTR)of influenza virus affect the transcription,replication,translation of gene and affect the biological characteristics of the virus.Based on the former study using the RNA polymerase I reporting system with enhanced green fluorescence protein(EGFP)gene as the reporter gene,libraries containing random mutations at sites within the HA/NA-UTR were constructed using random mutagenesis.In this study,the mutant clones were selected from the randomized mutagenesis libraries for the HA/NA-UTR.The expression of EGFP gene in HA/NA-UTR of H5N6,H7N9 and H9N2 influenza virus was analyzed qualitatively and quantitatively.To explore the characteristics of single nucleotide polymorphism in variable region of the UTR,we select clones with upregulated expression of the reporter gene and analyze their sequence characteristics.At the same time,the bidirectional reverse genetics system was constructed to rescue H9N2 influenza virus and UTR mutated viruses.Then the infectivity of the viruses were measured and compared to study that the gene mutation within the variable region of the UTR of HA/NA gene in the influenza virus influences viral genomic expression and infection.Methods:1.Selecting clones with upregulated expression of EGFP: using the RNA polymerase I reporting system(HA/NA-3'UTR-EGFP-5'UTR),the randomized mutagenesis libraries containing random mutations at sites within the HA/NA-UTR were constructed.The mutant clones of H5/H7/H9 and N2/N6/N9 subtypes were selected from the libraries for the HA/NA-UTR and transfected into MDCK cells infected with the H1N1 influenza virus.The expression of the reporter EGFP was observed using fluorescence microscopy,and the relative fluorescence intensity was measured using a multifunctional microplate reader to analyze the expression of the reporter gene(EGFP).Moreover,the real-time PCR method was used to quantitatively detect the relative expression level of m RNA of the reporter gene.Therefore,the HA/NA-UTR mutant clones with upregulated expression of EGFP were screened out.2.Alignment and analysis of the sequence characteristics of up-regulated clones: the gene sequences of mutant clones were identified by sequencing.Then using the Mega7 software to compare the differences between the sequences of the up-regulated clones and the wild-type,and analyzing the mutations at the specific sites.Thereby,exploring the multi-site mutations in the UTR of influenza virus resulted in up-regulation of the expression of HA and NA gene.3.Construction of the bidirectional reverse genetics system and packaging of the viruses: eight-plasmid system of H9N2 influenza virus was constructed by the bidirectional reverse genetics system.The eight plasmids were transfected into 293 T and MDCK cells to rescue H9N2 influenza virus and NA-UTR mutated viruses with obvious mutation characteristics.The virus titer was measured by the hemagglutination test.And calculating the dose of virus to cause half of the tissue culture cell cytopathic effect by the TCID50 experiment,thereby comparing the their differences of infectivity.Result:1.According to the observation by fluorescence microscope,the fluorescence value measured by multi-microplate reader and the Ct value quantitatively detected by real-time PCR method,it was found that among the 18 randomly selected UTR mutants,the fluorescence intensity and relative expression of the H5-8,H7-7,H7-8,H9-8,N2-2 and N9-7 mutant clones were significantly up-regulated compared with wild type,and the statistical difference was significant.2.The sequence alignment of the up-regulated strain compared with the wild type:H5-8(3'UTR: U4?A4,G17?A17,U18?A18,U19?A19,G21?A21,A22?G22,G25?U25;5'UTR: C24?A24,C27?G27,C31?U31,G35?A35,C36?U36,C40?U40,A42?G42,U44?G44,U45?G45,U46?C46);H7-7(3'UTR:U17?A17,A22?G22,U33?C33;5'UTR: A4?G4,G5?A5,C8?A8,G11?U11,G12?C12,U14?A14,G15?A15,U16?C16,U17?C17,C28?G28,U29?G29);H7-8(3'UTR: U17?A17,A18?G18,A22?G22,A26?C26,U33?G33;5'UTR:A4?U4,G5?A5,A9?U9,G11?A11,G12?A12,U14?G14,G15?U15,U16?G16,U17?C17,A25?C25,C28?G28);H9-8(3'UTR: U4?C4,U16?C16,A17?C17,G20?C20,U21?A21,G25?U25,G26?C26;5'UTR: A8?G8,C24?A24,U27?C27);N2-2(3'UTR: U13?C13;5'UTR: C22 ? U22);N9-7(3'UTR: U4?C4,G16?A16;5'UTR: U25?A25,C26?U26,A27?C27).The up-regulated clone of N2-2 has the obvious characteristics of mutation.3.The bidirectional reverse genetics system of H9N2 was successfully constructed,and the H9N2 influenza virus and the NA-UTR mutated viruses were packaged.4.According to the results of the hemagglutination test and the TCID50 experiment,the infectivity of the N2-2-3'UTR and N2-2-3'/5UTR mutated viruses enhanced compared to the wild type(H9N2 influenza virus).Conclusion:The H5-8/H7-7/H7-8/H9-8/N2-2/N9-7 mutant clones with upregulated expression of the reporter gene were successfully selected from the randomized mutagenesis libraries and the results were statistically significant.The bidirectional reverse genetics system containing eight-plasmids of H9N2 influenza virus was successfully constructed,and the H9N2 influenza virus and the NA-UTR mutated viruses were rescued,which lay the foundation for further exploring the effects of the base polymorphisms in the variable region region within the UTR of influenza A virus on viral functions and the molecular mechanism of new avian influenza virus.