Objective To prepare a polyclonal antibody against the outer membrane protein OspC and the flagellin FlaB of Borrelia burgdorferi (also called Lyme disease spirochete) after prokaryotic expression, and to test its immunogenicity. Methods The genomic DNA of B.garinii strain BgNMJW1 was used as a template to amplify the gene segments of OspC and FlaB using polymerase chain reaction (PCR); then the PCR products were subcloned into the expression vector pGEX-6p-1 and transformed into the expression strain Rosetta. The fusion proteins were expressed after induction with isopropyl thiogalactoside (IPTG) and purified using glutathione transferase (GST) column or via gel cutting. The purified proteins were then used to immunize New Zealand white rabbits to obtain polyclonal antiserum. Results The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that compared with the control, the vector carrying the target genes had obvious expression of recombinant proteins after induction, and the sizes of the recombinant proteins were about 49×10 ³ and 63×10 ³, respectively. The induction result showed that the expression of the induced proteins reached its peak level if 1 mmol/L IPTG was added when the bacteria were cultured to an absorbance ( A) of 0.4, followed by inducing at 25℃ for 10 hours. By immunizing New Zealand white rabbits with the purified fusion proteins, the polyclonal antiserum was obtained and used to detect OspC and Flab of B.garinii strain BgNMJW1 and B.burgdorferi strain BbB31A3 in Western blot, then the clear detection bands were obtained. Conclusion The combined application of OspC and FlaB can play an effective role in the diagnosis of Lyme disease.

Objective To analyze the sequence of Hantavirus(HV) from Zhejiang, Fujian provinces and Shanghai municipal. Methods The sequence of M segment of several HV strains from coastal areas of Zhejiang, Fujian province and Shanghai municipal were collected and analyzed by Mega 4.0 and DNAStar software, phylogenetic trees were built by neighbor joining method (NJ) and analyzed to compare the similarity of HV strains isolated from Zhejiang, Fujian provinces and Shanghai municipal. Results The phylogenetic tree revealed that the similarity of isolates in the same province is much higher than the other, meanwhile, in same province or city, the nucleotide similarity of the same region is higher than the other regions. The distributions of the isolates from closer regions share higher sequence similarity in the phylogenetic branches. The ZH53 strain of HTN isolated from Fujian province, Gou3 strain and ZJ5 strain of SEO isolated from Jiande region in Zhejiang province formed a distinct phylogenic lineage in HTNV clade and SEOV clade respectively, and these strains were different from other variants and international standard strains with the similarity of 82.7%-86.3% and 84.0%-85.3% respectively. The SEOV strains in Zhejiang were isolated from field mouse indicating the phenomenon of “host spillover”. Conclusion The sequence similarity and the phylogeny of HV in southeast costal area of Zhejiang, Fujian provinces and Shanghai is closed related to the isolated regions, indicating geographic distribution of HV.

Objective To analyze the prevalence and genotypes of Hantavirus (HV) among rodents in the epidemic areas of hemorrhagic fever with renal syndrome (HFRS) in Zhejiang province, China from 2008 to 2011. Methods Direct immunofluorescence assay and RT-PCR were used to detect the antigen and nucleic acid of HV. Mongolian gerbils and Vero-E6 cells were used to isolate viruses from the HV-positive rodents, and the M segments of HV isolates were subjected to sequence analysis. Results Apodemus agrarius was the dominant species of the rodents captured in the epidemic areas of HFRS in Zhejiang province from 2008 to 2011. Six strains of HV were isolated from the lungs of this rodent species. The sequence analysis showed that the six isolates belonged to Hantaan virus (HTNV), wherein Z521, 524, and 534 were classified as H7 subtype, and TT-27, T-43, and R88 might be classified as a new subtype. The six HV isolates showed high gene homology between them and with the strains previously isolated in Zhejiang province. Conclusion HTNV was prevalent in rodent in the epidemic areas of HFRS in Zhejiang province from 2008 to 2011.

Objective To provide a preliminary evaluation of the self?made horseradish peroxidase (HRP) marked Hantavirus (HV) recombinant N protein (rNP) rNP?IgM direct capture ELISA diagnostic kit. Methods Assessment of the specificity and stability of the kit and comparison of clinical results with similar products were performed to evaluate the sensitivity of the kit in the detection of serum anti?HV?IgM. Results (1) The kit was only responsive to anti?HV?IgM positive serum, and irresponsive to anti?varicella?zoster virus?IgM (anti?VZV?IgM), anti?Japanese encephalitis virus?IgM (anti?JEV?IgM), anti?dengue virus?IgM (anti?DV?IgM), anti?hand, foot and mouth EV71 virus?IgM (anti?EV71?IgM) positive sera. No obvious reduction in serum anti?HV?IgM detecting capability was noticed after placement at 37 ℃ for 3 d. (2) In 144 sera samples in 120 patients with suspected hemorrhagic fever with renal syndrome, inconsistency was observed only in the anti?HV?IgM test results in 12 sera of 12 patients between the two kinds of kits, in which 8 primary sera samples were detected positive by the kit and negative by commercial ones (the secondary sera samples were positive for both kits); 3 primary samples (the secondary samples unavailable) were at the critical value for the kit and negative for commercial kits. Meanwhile, one other primary serum sample was positive for the kit and negative for the commercial ones (the secondary and tertiary ones positive for both). Conclusion The laboratory developed capture ELISA anti?HV?IgM diagnostic kits had favorable specificity, stability and sensitivity, suitable for the clinical diagnosis and epidemiological surveillance of HV infections.

【Abstract】 Objective To clarify the natural infection situation of rodents by Hantavirus (HV) and HV strains in Zhejiang province in 2007，and provide science evidence for the prevention and control of hemorrhagic fever with renal syndrome (HFRS)．Methods Lung tissue and serum from rodent were sampled, and IgG antibody from serum was tested by indirect immunofluorscence assay and direct immunofluorscence assay was adopted to test HV antigens from lung tissues. HV was isolated by Vero?E6 cells, and HV strains were identified by Anti?McAb．Results A total of 1129 rodents were captured in 2007．The dominant specie was Apodemus agrarius, and the positive rate of HV antigen in rodent lungs was 3.0%. The IgG antibody of 57 blood samples was positive, and the positive rate was 8.0%.  Six  strains  of  hantaan (HTN)  virus  were  isolated,  five  strains  from A.agrarius  and  one  from  Rattus norvegicus.  Conclusion There  are  natural  foci  of  HFRS  which  main  infection  sources  are A.agrarius in Zhejiang province, and HTN strain could be the main prevalence strains of HV.

Objective To investigate the epidemiology of the hemorrhagic fever with renal syndrome(HFRS) and the serotype of hantavirus(HV).Methods Direct immunoflorscence assay(DFA) was adopted to detect HFRS antigen and the virus was isolated from the young Meriones unguiculata inoculated with the rat lung tissues infected by HV.The isolated virus were continuously propagated in the Vero-E6 cell and identified by DFA using Anti-HV McAb.Results Three strains of HV were isolated from the eight lung tissues infected by HV which belonged to the hantaan type.The virus titer of three strains could reach 10 ^4.75-10 ^5.25 CCID ₅₀/ml in the Vero-E6 cell.Conclusion Three strains of hantaan virus were successfully isolated from the lung tissue of rats,which could be the candidate strain for HFRS vaccine.

Objective To construct hantavirus Z10 envelope glycoprotein gene G2 recombinant and study its application in IFA. Methods Using a pair of primer based on hantavirus Z10 G2 gene sequence. PCR products were obtained by PCR from the plasmid pUCm-Z10M,then digested and cloned into vector pUCm-T which were transformed into E.coli DH5a to construct plasmid pUCm-Z10-G2. The digested product were obtained from plasmid pUCm-Z10-G2 and cloned into Baculovirus transport vestor pFastBacHTa to construct recombinant plasmid pFastBacHTa-Z10-G2 which then were transformed into E.coli DH10Bac. After white E.coli DH10Bac fungus had been cultured and selected for two times,the plasmid pFastBacHTa-Z10-G2 were abstracted again and transfected into insect cells sf9 to express G2 protein,subsequently G2 protein were verified and used to diagnose hantavirus infection by IFA. Results The recombinant baculovirus expressing G2 antigen was constructed and expressed in insect cells. The specific fluorescence in insect cells was observed by IFA. Conclusion The recombinant baculovirus expressing G2 antigen was constructed successfully and expressed in insect cells. The G2 protein as an specific and safe antigen can be used to diagnose hantavirus infection by IFA.