Studies On Molecular Epidemiology Of Hantavirus In Tianjin And GP Multi-epitope Antigen Gene Construction And Expression

Hantavirus(HV) belongs to the family Bunyaviridae,and the genome isasingle-stranded and negative-sense RNA including a tripartite genome,large (L) gene,medium (M) gene and small (S) gene coding for the viral ploymerase,two envelopeglycoprotein (G 1 and G2) and nucleocapsid protein (NP),respectively.Hemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonarysyndrome (HPS) are two infectious diseases caused by hantaviruses.Hantaviruscauses HFRS mostly in China.90% of the total cases worldwide happened in China,and estimated 20,000-50,000 cases annually.It has been a significant public healthproblem in China.The incidence rates were increasing gradually since 1999 andreached to 4.05/10~5 in 2002,the highest in Tianjin.To understand the epidemical type,nucleotide sequence and phylogeneticcharacteristics of hantaviruses isolated in Tianjin,the molecular epidemiologic studywas carried out on HV from patients and rodents.Because of the importance of G2protein of HV,G2 protein of L99 strain was analyzed by bioinformatic softwares,andthe multi-epitope antigen was constructed,expressed and purified.The MEA-ELISAmethod was used to test the HFRS patients.PartⅠMolecular epidemiology of Hantavirus in TianjinObjective:For the epidemic situation of HFRS in recent years,molecular epidemiologicalsurvey was carried out on hantaviruses from HFRS patients and the host animals.Atthe same time,it was studied on the early laboratory testing methods for HFRSpatients,and the testing methods for rat lung.Methods:(1) The sera were collected from 16 patients suffering from HFRS in Tianjin area.The specific IgM and IgG of the patients were tested with ELISA and IFArespectively.The hantaviruses from the patients were tested by RT-Nested PCR.PCRproducts of HFRS patients were sequenced and analyzed.(2) The 243 lung specimens of rodents from Tianjin were tested by using the new IFAmethods.The positive specimens were genotyped by RT-Nested PCR.The PCR products were sequenced and analyzed.At the same time,the sequence homology ofHVs from patients and rodents was analyzed.(3) The HTN type HV was isolated from lung specimen,and the S and M completefragments were acquired by RT-PCR and sequencing,and the homology andphylogenetics were analyzed.Results:(1) The hantaviruses from HFRS patients were all SEO type in Tianjin.And thenucleotide homologies among them were 93.7%～100%.The positive rates of HFRSpatients were 52.65%,52.65% and 68.75% by IFA,ELISA and RT-Nested PCRDetection,respectively.The positive rate was 91.67% at early stage((?)＜5 days) byRT-Nested PCR detection.(2) The 10 positive specimens were detected from 243 lung specimens of rodentsand genotyped.The results showed that nine specimens were infected with SEO virus,and another one was infected with HTN virus.The sequence homologies among thenine SEO type HV were 95.4%～100%.(3) The HV sequence homology from patients and rodents was analyzed.It was100% that the fragment (nt1343～1629) homology among the HV strains from theBC0207 specimen dissected from mice and specimen lyf,ssg and hcy from HFRSpatients.It confirmed the existence of HV epidemic strain in Tianjin.(4) HTN virus was isolated from a lung specimen,and named as TJJ16.Thehomology and phylogenetic analysis of the S and M fragments showed that,thenucleotide homology between TJJ16 and Q32 strain were 96.1% and 99.4%,theamino acid homology were 97.9% and 99.6%.Conclusion:The hantaviruses from HFRS patients all belong to SEO type,and showedobvious characteristics of the regional aggregation in Tianjin.The SEO and HTNviruses were all detected from rodents in Tianjin,and showed obvious characteristicsof the regional aggregation too.The SEO viruses all belong to the same subtype.Thehomology analysis showed that TJJ 16 and Q32 strain belong to the same subtype.PartⅡThe construction and expression of GP multi-epitope antigen gene of HV Objective:To construct the multi-epitope antigen gene of G2 glycoprotein of HV L99 strains,and express the multi-epitope antigen in Escherichia coli,and The MEA-ELISAmethod was used to test the HFRS patients.Methods:The M gene and its encoded glycoprotein G2 were analyzed comprehensively bybioinformatic softwares.Five dominant B cell epitopes were selected after theanalysis,including hydrophilicity,surface accessibility,antigenicity and proteinsecondary structure.Multi-epitope antigen (mea) was constructed by connecting fiveepitopes with GPG (Gly-Pro-Gly).And the MEA gene was synthesized byoverlapping PCR,and expressed by pET prokaryotic expression system.Therecombinant protein MEA was purified by Nickel affinity chromatography.The eukaryotic expression plasmid,pcDNA3.1-G2 was constructed,and inoculatedinto BALB/c mice.And then,anti-G2 serum was acquired.The anti-G2 serum wasused to detect MEA and rNP.The IgG from HFRS patients and healthy people weretested by MEA-ELISA.Results:(1) The five possible B cell epitopes were selected;the multi-epitope fusion genewas constructed.(2) The multi-epitope fusion gene was expressed with pET prokaryotic expressionsystem successfully.The yield of multi-epitope antigen was 0.14mg/mL afterpurification.(3) The eukaryotic expression plasmid,pcDNA3.1-G2,was constructed,and used toDNA immune BALB/c mice.The anti-serum against the protein HVG2 was acquired.(4) The antigencity analysis on MEA showed that,the anti-G2 serum can act withMEA,but not rNP.(5) The 76.67% HFRS patients were detected by MEA-ELISA,and all health peoplewere not detected.Conclution:The GP multi-epitope antigen gene of HV was constructed successfully,andexpressed by pET system.MEA was the highly specific recombinant protein antigens, and had the glycoprotein antigenicity of HV.It can be applied to detect the specificantibody of HFRS patients,and to research on the new epitope vaccine of HV.