| Background and Objective:H3N2 influenza subtype is the most predominant subtype of seasonal influenza viruses, and considered as one of the important pathogens affecting human health. Compared to H1N1 (09pdm), influenza B, it has characteristics of higher prevalence, higher proportion of severe cases and deaths. The PB1-F2 protein, is considered to be an important virulence factors of influenza viruses, can enhance its capacity of replication, promote apoptosis in infected cells and evoke secondary bacterial pneumonia. To explore evolutionary trends and genetic characteristics of the PB1-F2 protein, we analyzed the PB1-F2 genes from GenBank Influenza virus Database and strains isolated from Jiangsu Province. In order to examine the truncated and full-length PB1-F2 protein whether affects its functions, we conducted preliminary study of infected cell apoptosis、viral replication and cytokine changes. In addition, this study also established influenza virus reverse genetics system to lay a solid technical foundation for generating a mutant strain of influenza virus to explore its biological functions continuously.Methods:1. Downloading PB1-F2 sequences of protein and nucleotide and sequence-related information of avian influenza virus, human influenza virus and Swine influenza virus from GenBank influenza virus database, Using Excel, SPSS 19.0 statistical software calculated the truncated PB1-F2 protein data. Using MEGA6.0.6, DNA Sequence Polymorphisms.1, Fasttree analyzed the nucleotide sequence and amino acid sequence of PB1 and PB1-F2 segments and constructed phylogenetic trees.2.101 H3N2 influenza strains were isolated from Jiangsu influenza surveillance network laboratories. ContingExpress Aplication was used to assembly PB1 genes. MEGA 6.0.6 was used to make the comparation of mutations on nucleotides and amino acids. Fasttree was used to cnstrusct the phylogenetic trees. Datamonkey software(http:/www.datamonkey.org) was used to make PB1 amino acid selection pressure analysis online.3. Selecting strains with full-length and trucated PB1-F2 protein (S1238 and F1345) infected A549 and MDCK cells, respectively. Using Enzyme-linked Immunosorbent Assay detected virus replication efficiency between S1238 and F1345, MTT method was used to detect capacity of promoting cell apoptosis, RT-PCR was used to detect mRNA relative expression level of cytokine(IL-6、IL-8、IFNα、IFNβ、IFNγ、MCP-1).4. Site-directed mutagenesis of truncated PB1-F2 protein (F1345) made the termination codon TAA to CAA. Using 8 plasmid rescue system rescued F1345-MU (Full-length PB1-F2) to establish a reverse genetic system in our laboratory.Results:1. Distribution of the truncated PB1-F2 protein strain:the proportion of truncated PB1-F2 protein of human, avian and swine influenza virus downloading from GenBank influenza virus database was 24.35%, in which almost all strains of human H1N1 subtype were truncated PB1-F2, since 1948. Truncated human H3N2 protein was discovered in 1996, and separation rate went up gragually, its proportion accounted for 10.07%. Truncated avian influenza virus was with the lowest proportion of 6.47%.2. Genetic characteristics and phylogenetic trees of PB1-F2 genes:nucleotide homology of H3N2 and H1N1 influenza viruse were 97.02% to 100%,79.20% to 100%. Transition/Transversion Ratio of PB1-F2 gene (H1N1) was as low as 3.86. Mean segregating sites per site and mean evolutionary distance of H3N2 and H1N2 were 0.0705 and 0.0347, of H1N1 subtype were 0.0733 and 0.0609. Compared to H3N2 and H1N2, the mean distance of H1N1 was smaller. Synonymous and non-synonymous substitutions (dN/dS) of PB1-F2 of all subtypes in this study were greater than 1. The phylogenetic tree of PB1 genes can be roughly divided into seven groups, group I was human H3N2, H1N2 subtypes, group II was SIV(H1N1, H3N2, H1N2), human H2N2 subtype was in Ⅲ, IV and V groups were strains of avian influenza, belonged to America, Oceania area and Asia, European area respectively. Group VI were SIV(H1N1, H3N2) isolated from European countries and regions. Group VII was human H1N1 influenza virus.3. Surveillance of PB1-F2 genes isolated from Jiangsu H3N2 strains:193 H3N2 influenza viruses were divided into four groups in general, subtype H3N2 strains isolated in Jiangsu Province and 2002-2015 isolations were located on group IV, isolations in1968-1994 were on Ⅱ and Ⅲ groups. Compared to strains isolated from 1968-1994, the amino acid substitutions of PB1 gene(52,113,179,216,576,586,619,621,709)were observed in 2002-2014 with adaptive changes. The mutation of the key virulence sites does not appear among Human A/H3N2 influenza.4. Functional changes in truncated and full-length PB1-F2 protein:After infection with F1345 (Truncated) and S1238 (Full-length) in MDCK cell, we found a decreasing trend of two strains with time. Cell survival rate of S1238 was less than F1345 strains. SI238 strain had a high replication capacity after infecting 12h to 36h than F1345. Replication capability of F1345 significantly increased after 36h. And there were statistically significants between strains at each time point. The relative expression level of cytokines IL-8 were highest after 8h post infection. IFNα/β started to increase after 32h post infection Only Chemokines MCP-1 had a significant diffidence between F1345 and S1238 on relative expression level of mRNA.5. Establishment of reverse genetics system of influenza virus:making a mutation on Truncated PB1-F2 protein of F1345, T76C in its open reading frame, TAA stop codon mutated to glutamine CAA. Constructing 8 recombinant plasmids PHW2000-PB1 (WT), PHW2000-PB2, PHW2000-PA, PHW2000-HA, PHW2000-NA, PHW2000-M, PHW2000-NP, PHW2000-NS and a mutant PB1 plasmid PHW2000-PB1 (MU) were co-transfected 293T cells and MDCK cells to rescue wild-type truncated strain A/Nanjing/1655/2010 (H3N2) and full-length PB1-F2 mutant strain F1345 (MU). Establish influenza virus reverse genetics system, laying a solid foundation of further studying on PB1-F2 protein and its functional mechanism.Conclution:1. Proportion of truncated PB1-F2 strain in seasonal human H3N2 influenza virus were increasing year by year, showing a slow while steady evolutionary trend. This indicated that truncated PB1-F2virus is becoming an evolutionary trend of subtype H3N2 influenza virus.2. PB1-F2 genes of influenza strains isolated from Jiangsu in 2012-2015 were highly concentrated in the same clade, consistent with the evolutionary trends of PB1-F2 (H3N2) in China. And we did not find any key virulence mutations in PB1-F2 protein amino acid of Jiangsu Province isolates.3. Length Polymorphism of PB1-F2 protein in F1345 and S1238 strains which caused changes in cell proliferation, the replication of the truncated one was higher than the full-length strain, and not found significant differences in inducing cell apoptosis.4. Proinflammatory cytokines and chemokine induced by truncated and full-length PB1-F2 protein strains can not make an exacted conclusion of which would make a high cytokines and chemokine expression level. Mainly due to the influenza strains used in the experiment had a high sequence homology, but there were still differences in antigen sites, thus affecting the accuracy of the experimental results. Only through artificial, targeted gene directed mutation and high fidelity cloning operation to establish a reverse genetics system for influenza virus, to ensure that the same sequences of control and experimental strains on other genes exactly, which in order to control the other segments influence, we could analyze the impacts of the virus PB1-F2 function accurately. |
