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Since the reemergence of highly pathogenic avian influenza virus H5N1, it caused disease in 20 people with 13 deaths in mainland of China. On February 21, 2006, the first suspected human case in Zhejiang province was reported. Pathogenic analyses, including reverse transcriptase polymerase chain reaction (RT-PCR), real-time RT-PCR, and virus isolation, were carried out to confirm the pathogen from tracheal aspirate specimen. In addition, antibody in serum sample was detected using hemagglutination-inhibition (HI). Results revealed that nucleic acid extracted from the tracheal aspirate specimen was positive for H5N1 avian influenza virus and influenza virus type A. The H5N1 virus strain named A/Zhejiang/16/06 (H5N1) was isolated. The titers of HI antibody for H5N1 avian influenza virus were 1:320 and 1:640, respectively. The sequenced genes were all avian origin. Phylogenetic analyses between the A/Zhejiang/16/06 and other H5N1 influenza viruses were also included.

In order to analyze the molecular epidemiology of Hantavirus (HV) in Zhejiang Province, the complete M and S genome sequences of 12 HV strains from different hosts and locations in Zhejiang Province of China during the period of 1981-2007 were analyzed on genetic evolution by DNAstar and MEGA 4.0 software in this research. Phylogenetic analyses revealed that HTN and SEO strains were co-circulating in Zhejiang Province, and the difference in sequence similarity and the phylogeny was closely related to the isolated regions, but had no distinct relationship with the isolate year and the host, indicating a relationship between epidemiology of HFRS and the distribution region, especially in HTNV. The isolates in the same region could be assigned in same or near phylogenetic clade sharing high sequence similarity. Interestingly, the Gou3 strain and ZJ5 strain isolated from Jiande region in Zhejiang Province formed a distinct phylogenetic lineage in SEOV clade, and different from the other SEOV variants outside China. We believed that the special SEOV variants were distributed in Jiande region.

To investigate the status of infection and distribution of rabies virus (RV) in different epidemic areas in China.Brain specimens from animals and suspected patients were collected at the districts of high-, medium- and low incidence rates of human rabies and detected by both direct Immunofluorescence assay (DFA) and RT-PCR.254 of 3007 specimens of dog brains showed RV positive by DFA (positive rate of 8.4%). Among these 254 samples, 78 showed positive (positive rate of 30.7%) by RT-PCR. 93 specimens from dogs and cats that had attacked human beings, 63 of them showed positive by DFA (positive rate of 67.7%) and all of them were also positive by RT-PCR. In addition, RV could also be detected in Apodemus agrarius, ferret badger, and suspected patients specimens from the districts under survey. There was no statistical difference between the infection rates of RV in different provinces and regions with different incidence of rabies.There might be a relatively high infection rate of RV among the domestic dogs/cats in the endemic areas in China. Wild animals might have been infected with RV in the districts under survey.

To evaluate the epidemiological and serological efficacy after 10 years of vaccination against hemorrhagic fever with renal syndrome (HFRS) vaccines in Zhejiang province.One county was randomly chosen as the research unit with all the healthy people between 16 and 60 years old were equally divided into study and control groups. The study group was vaccinated. Immunofluorescent antibody assay was used to test specific IgG antibody and Mcro-CPE method was used to test the titer of neutralizing antibody.Two weeks after the full-course immunization, the seroconversion rate became 100% (67/67, with 95% CI as 96.3%-100%) by immunofluorescent antibody test (IgG) and 44.4% (8/18 with 95% CI as 22.0%-69.0%) by neutralization test with GMT titers as 72.1 and 4.6 respectively. Booster immunization was provided one year later. Time span as two weeks prior to, one year, one and half years, two years, three years and five years after booster immunization, the rates of seroconversion on immunofluorescent antibody using IFAT method, were 28.6%, 83.3%, 75.0%, 53.1%, 22.6%, 10.0% and 55.0% respectively, and rates of seroconversion of neutralizing antibody by Mcro-CPE method were 14.8%, 55.6%, 35.0%, 31.3%, 26.0%, 10.0% and 50.0% respectively. Nine years after the reinforcement, the rates of seroconversion of immunofluorescent antibody by IFAT method was only 7.1%. The vaccinated group had no patient seen but the control group appeared 34 patients including 3 deaths. According to the ten-year observation, the vaccine seemed effective with the protection rate in population reached 100%.HFRS vaccine was effective on epidemiological, social and economical efficacy.

To learn about the complete genomic sequence of the Seoul virus strain ZT10 isolated from M. fartis.The total RNA was extracted from the infected Vero E6 cells and amplified by RT-PCR. The purified PCR products were cloned into T-vector and sequenced.The results demonstrated that the complete genome of ZT10 was comprised of L(6530), M(3651) and S(1753) segments which encoded 2151-1133 and 429 amino acids respectively.Analysis of sequence revealed that the ZT10 belonged to Seoul virus. The nucleotide sequence identity of the M gene with Seoul virus was 84.0%-96.3%. The identity with Hantan vrisu (Prospect Hill virus, Tula virus) isolated from M. fartis was 57.5%-60.9%. The sequence identity of the S gene with Seoul virus was 87.9%-96.0% at nucleotide level and 96.9%-97.9% at amino acid level.

To clone the gene encoding nucleocapsid protein (NP) of hantavirus strain Z10 (HV-Z10), to construct its prokaryotic expression system as well as to establish a rNP-IgM direct capture ELISA based on HRP-labeled recombinant NP (rNP), in order to detect serum samples of patients suffering from hemorrhagic fever with renal syndrome (HFRS) and to evaluate the effects of detection.Gene encoding NP of strain HV-Z10 was amplified by PCR and then its prokaryotic expression system pET28a-Z10N-E. coli BL21DE3 was constructed, using routine genetic engineering method. SDS-PAGE was applied to measure the expression of rNP and ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product. Western blot assay was used to determine the specific immuno-reactivity of rNP while HRP-labeled rNP-IgM direct capture ELISA was established to detect the serum samples from 95 cases of confirmed HFRS patients. The detection effect was compared with that by routine HV-IgM indirect capture ELISA method.pET28a-Z10N-E. coli BL21DE3 was able to express rNP with high efficiency. The purified rNP only showed a single protein fragment in the gel after SDS-PAGE. HV-IgG could efficiently recognize rNP and hybridize with the recombinant protein. 94.73% (90/95) of HFRS patients' serum samples were positively confirmed by rNP-IgM direct capture ELISA, while a positive rate of 92.63% (88/95) in the same samples was confirmed by HV-IgM indirect capture ELISA. The distributions of A450 values of the serum samples detected by the two IgM capture ELISAs as well as the changes of the A450 mean values from several serum samples with different dilutions were similar.We successfully constructed a high efficient prokaryotic expression system of NP encoding gene of hantavirus strain HV-Z10. The rNP-IgM direct capture ELISA that established in this study could be used as a new serological test for HFRS diagnosis because of its simplicity, safety, with high sensitivity and specificity.

For clarifying the situation of the natural infection of rodents having hemorrhagic fever with renal syndrome (HFRS) virus and to type Hantavirus (HV) using molecular technique in Shenzhen city in 2005, and offering guidance for prevention and control of HFRS.Data on the host animals was collected from the city of Shenzhen. ELISA and indirect immunofluorscent antibody(IFA) test were applied to the specific antibodies against HV in the sera of captured rats. Direct immunofluorscece assay was adopted to determine HFRS antigens and the lung tissues of the HV infected rats were inoculated into Meriones unguiculata to isolate HV. The whole viral RNA was extracted from the lung tissues of the HV infected rats and amplified the partial M fragments with RT-nested-PCR, using the HV genotype specific primers. The amplified genes were then sequenced, and subjected to genotyping and homology analysis.472 rodents were captured from Shenzhen in 2005. Surveillance on rats demonstrated 9.96% rats carrying HV (with a density of 8.25%) and the main host was Rattus norvegicus. In the blood samples of rats, anti-HV IgG antibodies were detectable in 56 cases by IFA, and proved to be positive in 76 cases by ELISA. We successfully isolated a HV strain designated as SZ2083 from Rattus norvegicus for the first time in Shenzhen and was identified to SEO type by RT-nested-PCR. Compared with the coding region of the M gene of HV L99 virus strain, the homologies of nucleotide among them were 97%, but the homology was 76% of the SZ2083 with HTN 76-118 virus strain.Results showed the existence of natural epidemic areas of HFRS in Shenzhen city. Based on the results of sequencing, it is possible that the Seoul strain of HV might be the predominant serotype of virus harbored.

To establish a new technique for SARS-CoV antibody test to detect infection of severer acute respiratory syndrome (SARS).Nucleocapsid gene was obtained by reverse transcription and polymerase chain reaction from a SARS patient and inserted into the vector pFastBacHTa expressing baculovirus. Insect Sf9 cells were transfected with the recombinant baculovirus expressing SARS nucleocapsid antigen and then cultured, fixed by acetone so as to make SARS-specific antigen. Immunofluorescence assay (IFA) technique and plaque reduction neutralization test (PRNT) were used to detect 7 samples of sera of 4 newly diagnosed SARS patients collected in different days, 48 samples of convalescent sera of SARS patients, 24 serum samples of healthy person undergoing physical examination, and 40 serum samples from non-SARS patients with fever by double blind test.The recombinant SARS-specific antigen reacted only with SARS positive sera but not with normal sera. Double blind test showed that 45 of the 46 PRNT positive sera were IFA positive with an accordance rate of 97.8%. 7 samples of sera from 4 SARS patients in acute progressive stage in Guangdong province were all IFA positive. SARS antibody could be detected since the sixth day after onset, and the titer increased from 1:40 to 1:600 on the ninth day.Immunofluorescence assay is highly specific and sensitive in detection of SARS. This reagent is safe and easy to prepare.

To observe the serological and epidemiological efficacy of hemorrhagic fever renal syndrome (HFRS) vaccine in Zhejiang province.Immunofluorescent antibody assay and Mcro-CPE method were used to test specific IgG antibody and the titer of neutralizing antibody.Two weeks after the injection of the third dose, the sero-conversion rates by both immunofluorescent antibody test (IgG) and neutralization test were 100.0% (67/67) (95% CI: 96.3 - 100.0) and 44.4% (8/18)(95% CI: 22.0 - 69.0) with geometric mean titers (GMTs) 72.1 and 4.6 respectively. The rates of seroconversion of immunofluorescent antibody by immunofluorescence antibody assay (IFA) were 28.6%, 83.3%, 75.0%, 53.1%, 22.6%, 10.0% and 55.0% before reinforcement, two weeks, one year, one year and a half years, two years, three years and five years after reinforcement. The rates of neutralizing antibody seroconversion by the Mcro-CPE method were found as 14.8%, 55.6%, 35.0%, 31.3%, 26.0%, 10.0% and 50.0% respectively. We found some antibody dependent immunization enhancement phenomenon among the inoculated population, but further observation was needed.HFRS vaccine was immunologically effective and the duration of serous antibody last long.