Molecular Characteristics Of PB1-F2 Of Influenza A And Its Association With Human IFITM3 Gene

Objiective:At present,the type of influenza infected human were mainly influenza A and B.Influenza A mainly include seasonal H3N2,H1N1,while scattered in the occurrence of human infection H7N9 subtype avian influenza.Compared with influenza B,influenza A has the characteristics of high morbidity,high severity of severe death,and can cause widespread and epidemic in the world by escaping from antigen.PB1-F2 protein of influenza A is an important virulence factor of the virus and play important role in the pathogenesis.In addition to viral factors,the host genetic heterogeneity is also an importan factor affecting the clinical outcome of influenza patients.Dysfunction of IFITM3 protein often indicates patients with adverse outcomes.In this study,we collected hospitalization cases of influenza A in Jiangning Hospital and the genetic variation characteristic and evolution trend of PB1-F2 were analyzed.The frequency of distribution of rs 12252 on IFITEM3 gene in different population were analyzed to explore the association between susceptibility IFITM3 gene and infection of Influenza A.The possible pathogenic effects of influenza A will be explored from both viral and host genetic susuceptibility.In addition,we also analyzed the changes of serum cytokines and blood biochemical indexes of influenza A in order to provide scientific basis for follow up study of influenza A.Methods:The swallow swabs and peripheral blood samples were collected from the department of respiratory disease in Jiangning Hospital during September 2016 to march 2017.Epidemiological case study were also conducted that including general characteristics of the cases,clinical manifestation and the blood biochemical indicators.1.Throat swabs were used to extract nucleic acid and conduct real-time quantitative PCR to identify virus type.Positive influenza A samples were cultured in vitro and PB1 gene were amplified.Downloading PB1 sequences of nucleotide during 2016 to 2017 from NCBI influenza Genbank.MEGA 5.2 was used to make the comparation of mutations on nucleotides and amino acids.Fasttree was used to cnstrusct the phylogenetic trees.Datamonkey software(http.7www.datamonkey.org)was used to make PB1 amino acid selection pressure analysis online.2.The following databases:Pubmed,Web of Science,Elsevier,Wanfang and CNKI were used to search case-control or cohort study that conducted the association of rs 12252 polymorphism with the susceptibility of influenza.Keywords were used to search.Two reviewers selected the literature accoding to into and exclude criteria for independent.Meta analysis was performed using Stata 12.0 software.The odds ratio and 95%CI of allele C exposed to the allele T were calculated from the case group and the control group.Cochrane Q test and I2 value were used to test the consistency between the result.3.Influenza A positive patients were put in case group,while negative patients were put into influenza symptom group.Health control group were selected from 1000 Genomes.DNA were extracted from blood and rs 12252 site sequence were amplified to identify genotype.SPSS20.0 anSTATA.12 software were used to analyze HWE in control group and different genetic models were established to analyze the association between susceptibility gene IFITM3 and infection of influenza A.4.Serum samples were selected from influenza A,influenza symptom and health control group and enzyme-linked immune sorbent assay was used to detect expression level of cytokine(IL-6、IL-10、IL-18、IL-1β、IFN-γ、TNF-α).Epidata3.2 software was used to construct the database and SPSS20.0 was used to analyze the difference in blood biochemical level.Result:1.5621 cases of ILI were cumulatively reported during April 2016 to March 2017 in Nanjing.733 positive strains were isolated and the positive rate was 13.04%.Seasonal H3N2 and influenza B were the main epidemic strains.Influenza was mainly prevalent in winter and spring.The age aged 5-25 and 25-60 were the main infected population.102 cases of influenza swabs were collected in Jiangning Hospital.19 cases of positive influenza and the positive rate was 18.63%.17 strains of seasonal H3N2 subtype and 2 strains of influenza B isolated in winter and spring.The prevalence and distribution of influenza in Jiangning area are consistent with the distribution in Nanjing.2.The sequence of PB1 gene of seasonal H3N2 subtype isolated from 2016-2017 was similar to that of vaccine strain.The nucleotide and amino acid homology of Jiangning isolation were 98.56%-98.88%and 99.47%-99.74%.The homology of nucleotide and amino acid of PB1 of H1N1 influenza were 97.95%-99.06%and 98.93%-99.60%respectively with the vaccine strain A/Califomia/7/2009(H1N1).The mutation of amino acid T152S and Y166F were found in PB1 fragment of the isolates in global and Jiangning area.Mutation of amino acid of I181V,T257A,N328T,S375N and I667T occurred.Amino acid mutation of G154D,I397M and I435T occurred on all the H1N1 influenza in the world and P437L,Y467C occurred frequently.The positive pressure site 428 was observed of global H3N2 isolates by using IFEL model.The positive pressure site 539 was observed of Jiangning isolates by using FEL and REL models.The phylogenetic tree of H3N2 was divided into six branches.13 Jiangning isolates were located in branchⅢ and another 4 strains located in branch IV.Influenza H1N1 influenza strains were mainly evolved into two branches.3.A total of 9 articles were selected into the meta analysis which including a total of 1074 cases of influenza and 3510 cases of health control.The rs12252 allele of IFITM3 gene was significant associated with the infection of influenza.The allele model,dominant gene model and recessive gene model showed statistically significant results(C vs T:OR=1.626,95%CI:1.395-1.895;CC+CT vs TT:OR=1.680,95%CI:1.362-2.072;CC vs CT+ TT:ORF=2.155,95%CI:1.624-2.860).Subgroup analysis showed that C allele were associated with increasing the risk of influenza among Caucacian population and allele C was statistically correlated with mild and severe influenza(severe influenza C vs T:ORF=1.637,95%CI:1.039-2.579;mild influenza C vs T:OR=1.373,95%CI:1.036-1.820).Allele C was significantly associated with severe influenza in Asian population,independent of mild influenza(severe influenza C vs T:OR=2.272,95%CI:1.671-4.397;mild influenza C vs T:ORF=1.200,95%CI:0.928-1.553).4.The frequency of IFITM3 gene rs 12252 C allele in East Asia,Africa,America and Europe were 52.78%,26.02%,17.72%and 4.08%.The frequency of C allele in influenza A,influenza,flu-like symoptom and severe influenza group were 52.94%,57.89%,54.22%and 77.78%.The allele of the control group were in accordance with the Hardy-Weinberg genetic balance.The result of C allele model,dominant gene model and recessive gene model showed that C allele of rs 12252 were not statistically associated with susceptibility of influenza A.However,The C allele was significantly associated with severe influenza(OR=1.548,95%CI:1.187-2.019).5.The level of serum TNF-a,IL-6,IFN-γ and IL-18 in influenza A patients were(38.59±23.93)ng/L,(7.75±4.37)ng/L,(22.27+14.78)ng/Land(74.70±43.80)ng/L,and all that were significantly higher than those in control group.The level of serum TNF-α,IL-6 and IFN-γin influenza group were(32.64 ±22.03)ng/L,(7.07 ±2.96)ng/L and(24.61 ±20.30)ng/L,and all that were significantly higher than those in control group.6.The level of HBG,HCT,PLT and Baso in influenza A patients were(127.84 ±20.10)g/L,(39.54±4.75),(185.94±47.22)×109/L,(1.60±0.70)×109/L and(0.01±0.01)×109/L and were significantly lower than those of control group.CRP level was(28.86 ± 24.27)mg/L,which was higher than that of the control group.The level of HBG and LYM in influenza symptom were(132.20±17.74)g/L and(1.74±0.72)×109/L,which were significantly lower than those in control group.The CRP level of influenza symptom group was higher than that in control group.The level of TP and ALB in influenza A were(64.27士5.23)g/L and(37.56±4.99)g/L,which were lower than those in control group.The level of GGT were higher than that of control group.Conclution:1.PB1 gene fragment of influenza A virus is conserved.The evolution trend of PB1 fragment of H3N2 influenza which isolated from Jiangning during 2016-2017 were consistent with those isolated in other place over the world.PB1-F2 rupture protein strains occupy a certain proportion.The global influenza A(H1N1)virus strain express the PB1-F2 protein with a length of 11aa.No highly pathogenic mutation was found on PB1-F2 protein of influenza isolated in Jiangning.The epidemiology and clinical significance of its amino acid mutation were to be further studied.2.The results of the meta analysis showed that the rs 12252 locus C allele of the SNP of IFITM3 gene was associated with susceptibility to influenza.In Caucasian population,allele C was associated with mild and severe influenza,whereas in the Asian population,allele C was associated with severe influenza and was not associated with mild influenza.3.According to the analysis of allele model,dominant model and recessive model,influenza A and pulmonary infection with influenza symptom was not correlated with the polymorphism of rs 12252 of IFITM3 gene.The association of severe influenza associated with rs12252-C allele of IFITM3 gene.3.The level of serum cytokines TNF-α,IL-6,IFN-γ and IL-18 of influenza A patients increased.The level of HBG,HCT and PLT slightly decreased and CRP significantly increased with mild anemia.The level of TP and ALB decreased and the level of GGT increased.The infection of influenza virus could cause liver injury.