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 genetic evolution and epidemiological pattern of Hantavirus (HV) isolated from different hosts, vectors and patients in different time from different regions of southeastern China from 1980 to 2015. Methods The mutation site and frequency in S, M segment hypervariable region of HV isolated from southeastern China,including Jiangsu province, Zhejiang province, Shanghai and Fujian province, from 1980 to 2015 were detected. Combining the information of HV, hosts, vectors, environment and epidemiology, the HV evolution laws in different regions, time, natural reservoir and important vectors were analyzed. Results The HV strains isolated from the hemorrhagic fever with renal syndrome (HFRS) patients in the southeast coastal area were mainly the Hantaan virus (HTNV), which were highly homologous with the 76-118 strain. While the Seoul virus (SEOV) carried by patients exhibited the highest sequence similarity to the Z37 strain. The southeast coastal area is the mixed type of HV-affected area, with apparent endemicity. The HV isolated from the rodents shared the highest identity to SEOV. The HV clades were related to host types and the locations of sample collection. SEOV was carried primarily by Rattus norvegicus, while HTNV was carried primarily by Apodemus agrarius. Conclusion The formation and maintenance of HFRS epidemic areas show a certain regularity.

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 epidemic situation of hemorrhagic fever with renal syndrome (HFRS) and the population distribution and virus-carrying status of the hosts in Zhejiang province, China, in 2013, and to provide scientific evidences for establishing further prevention and control measures. Methods The descriptive epidemiological method was used to analyze the three-dimensional (time, region, and population) distribution of HFRS. Rodents were captured by night trapping in five HFRS monitoring points in Zhejiang province. Lung and serum samples were collected followed by detection of hantavirus antigen and antibody using an immunofluorescence assay, and the rodent population distribution and virus-carrying status in Zhejiang province were analyzed. Results A total of 527 HFRS cases were reported in 2013 in Zhejiang province. The incidence rate was 0.9622/100 000, resulting in a 3.89% increase compared with 2012, and no death was reported. The cases were mostly seen in five cities (Ningbo, Taizhou, Shaoxing, Quzhou and Lishui), and all the prefectural level cities had cases reported except Zhoushan. There were two peaks of incidence, one in spring (May-June) and the other in winter (around December). The high-risk age group of HFRS was between 20 and 65 years, accounting for 93.17% of the overall incidence rate (491/527). A total of 13 785 effective traps were set in the five monitoring points, and 546 rodents were captured, so the average density of rodents was 3.96%. A total of 678 rodent serum samples were collected and 80 were positive (positive rate: 11.80%). A total of 669 lung samples were collected and 44 were positive (positive rate: 6.58%). There were significant differences in the positive rate of HFRS antibody and the virus-carrying rate between the five HFRS monitoring points (χ²=30.962, P<0.05 and χ²=9.83, P<0.05, respectively). Conclusion The incidence of HFRS in Zhejiang province in 2013 is on a rising trend compared with that in 2012, and the density of host animals and virus-carrying rate are both higher. Preventive measures should be taken into consideration to control the epidemic situation.

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.

【Abstract】 Objective To explore the epidemiological characteristics of hemorrhagic fever with renal syndrome(HFRS) in Zhejiang province for establishing pertinent prevention strategy. Methods Descriptive epidemiology method was applied for the analysis of epidemic situation. The antibody in the serum of HFRS patients was detected by indirect immunoflorscence method, and the HV antigen in the rodent lung was assayed by direct immunoflorscence method. Results There were 757 cases in the whole province in 2007, mainly distributed in Ningbo, Shaoxing, Taizhou, Lishui and Quzhou, accounting for about 86.79% of the total （657/757）. There were two incidence peaks in summer, winter and spring. Most of the cases were young and middle?aged peasants. Conclusion The incidence rate of HFRS in Zhejiang province has been stable in recent years, but it was still high in some counties and cities. Therefore, the prevention and control of HFRS in the main epidemic areas should be strengthened.

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 Several vaccine candidate strains of hemorrhagic fever with renal syndrome(HFRS) were isolated from HFRS epidemic area,the sero type were identified and the antigenicity were studied.Methods The virus strains were continuously propagated in brain of sucking Mongolian gerbil.The characteristics of virus multiplication,viral titers and the amounts of virus antigen after serial passages were studied.Results 9 strains virus were isolated successfully from 32 positive mouse lung,of which 6 strains were determined as hantaan virus by Anti HFRS McAb and 3 strains as seoul virus.Virus titers and antigen titers of ZJ7(Hantaan virus,HTN) can reach 6.50 lgCCID ₅₀/ml、 1∶5 120,ZJ5(Seoul virus,SEO) and 6.00 lgCCID ₅₀/ml、 1∶5 120,respectively.Conclusion These two virus strain possessed high titers and good antigen and can be vaccine candidate strain of HFRS.

Objective To observe the adaptability of Hantavirus and the rule of these strains replication in Vero cells.Methods Virus are extracted every 3 days from Vero cells culture Hantavirus infected.Cytopathogenic effect(CPE) are observed until these cells degrade.Viral titers and the amounts of virus antigen after serial passages are observed by IFAT and RPHA.Results There was no cytopathogenic effect(CPE) in Vero cells after 32 days.Viral titers and the amounts of virus antigen reach to the top at 8-11th day after Hantavirus infect Vero cells.Viral titers and the amounts of virus antigen still keep up at the level of 5.00 Lg TCID ₅₀/ml and 1∶20 at 32th day after infaction.Conclusion The results suggest that these 2 candidate strains had adapted to Vero cells possessed high titers and good antigenicity and be feasible to prepare the HFRS vaccine in Vero cells.

Rabbits were inoculated with two doses of monovalent inactivated hantavirus vaccines in primary immunization. One month later, they were immunized secondarily with one dose of another type vaccine. The cross-immunization could induce good neutralizing antibody(NA) responses to both Hantaan and Seoul viruses. It was similar to two doses of bivalent vaccine immunization in immune effects. The same type NA titres as the vaccine in primary immunization were boosted significantly after the cross-immunization. The result demonstrate that monovalent hantavirus inactivated vaccine induce cross-NA response. The cross-immunization may be a effective, reliable and economical immunizing strategy in people inoculated with monovalent vaccine.