SINS model in the management of biosafety level 2 laboratories: exploration and practice.

A significant number of biosafety level 2 (BSL-2) laboratories have been established in many countries for studies of various types of pathogenic agents and other infectious biological materials. The harmonized management of biological risks in such diverse laboratories thus appears as a real challenge. Zhejiang Province in China has taken the initiative to establish a comprehensively integrated laboratory biosafety management system called SINS (Standardization, Informatization, Normalization and Systematization). The SINS model system has been introduced and adopted in 1,721 BSL-2 laboratories in Zhejiang Province, and thus lead to an increase in the number of biosafety committees from 20% to more than 95% from 2007 to 2018, and the number of biosafety laboratory managers who knows biosafety-related laws and regulations increase from 52.7% to 83.7% from 2009 to 2017. Such achievements indicate that the successful implementation of SINS model has increased the effective control of biological risks in BSL-2 laboratories of the Zhejiang Province. SINS model and its main effects on leading the improvement of laboratory biosafety management was presented in this review. The SINS model helps to strengthen laboratory biosafety and thus effectively reduces occurrence of biosafety-related incidences. This model can potentially be used by other regions or countries where harmonized biosafety management system is still under-developing.

[Study on the status of infection and distribution of rabies virus in China].

OBJECTIVE: To investigate the status of infection and distribution of rabies virus (RV) in different epidemic areas in China. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Molecular evolution analysis of hantaviruses in Zhejiang Province].

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.

[Development and evaluation of TaqMan-based one-step reverse transcription-polymerase chain reaction assay for the detection of Japanese encephalitis virus].

OBJECTIVE: To establish a TaqMan based real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of Japanese encephalitis virus. METHODS: The gene sequences of Japanese encephalitis virus downloaded from the GenBank was aligned, using the biologic software. Specific primers and probes were designed in the conserved region of the C gene for Japanese encephalitis virus. The real-time RT-PCR reactive condition was optimized and the sensitivity, specificity and the stability of the assay were evaluated. Mosquitoes collected from Zhejiang province were detected by this assay. RESULTS: Mg2+, primer and probe were optimized at 5 mmol/L, 0.2 micromol/L and 0.1 micromol/L respectively. The specificity of the assay was high and there were no cross reactions with dengue virus, rabies virus, seoul virus or hantan virus. The detection limits of the assay was 0.1 TCID50. Results from preliminary application showed that TaqMan RT-PCR for Japanese encephalitis virus was sensitive, easier and faster to perform the process of traditional virus isolation and identification. It took only three hours to extract viral RNA and perform the real-time RT-PCR. CONCLUSION: This TaqMan-based one-step RT-PCR assay was a quick, sensitive and specific tool for molecular diagnosis of Japanese encephalitis virus.

[Isolation and identification of Japanese encephalitis virus from mosquitoes in Zhejiang province].

OBJECTIVE: To study the situation of arboviruses carried by mosquitoes in Zhejiang province. METHODS: Mosquitoes were collected from Zhejiang province in 2007. Virus strains were isolated by the inoculation of homogenates of the mosquitoes onto BHK-21 cell line. The isolated strains were identified by serological (IFA) and molecular methods (RT-PCR). RESULTS: Two strains were isolated from mosquitoes causing cytopathogenic effect (CPE) in BHK-21 cells. Results from serological tests showed that both of the two strains were positive for the antibody to Japanese encephalitis virus (JEV). PrM and E gene were then cloned and sequenced. Results from the phylogenetic analysis showed that the isolates belonged to genotype I JEV while through sequence analysis it showed that the homology of nucleotide sequences and amino acid sequences between the two strains were 99.0% and 98.8% respectively. Compared with the JEV vaccine strain SA14-14-2 and two strains, the homology of nucleotide sequences was up to 87.7% and homology of amino acid sequences was up to 96.4%. When comparing with the vaccine strain SA14-14-2, there were 14 common amino acid variations in all the two strains. CONCLUSION: Two strains of JEV were isolated from mosquitoes collected in Zhejiang province which was the first isolation of genotype I JEV in the province in recent years.

[Molecular characterization of hantavirus Zhejiang isolate ZT10 strain from M. fartis].

OBJECTIVE: To learn about the complete genomic sequence of the Seoul virus strain ZT10 isolated from M. fartis. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Study on the recombinant expression of Hantaan virus protein N and the establishment and application of rNP-IgM direct capture ELISA].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Pathogenic and molecular characterization of the H5N1 avian influenza virus isolated from the first human case in Zhejiang province, China.

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.

[Study on a 10-year protective effects of vaccination against hemorrhagic fever with renal syndrome].

OBJECTIVE: To evaluate the epidemiological and serological efficacy after 10 years of vaccination against hemorrhagic fever with renal syndrome (HFRS) vaccines in Zhejiang province. METHODS: 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. RESULTS: 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%. CONCLUSION: HFRS vaccine was effective on epidemiological, social and economical efficacy.

[Surveillance on natural infection of rodents with hantavirus in Shenzhen city and identification of a hantavirus strain SZ2083].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[An immunofluorescence assay for the detection of SARS associated coronavirus antibody based on recombinant nucleocapsid antigen and its application].

OBJECTIVE: To establish a new technique for SARS-CoV antibody test to detect infection of severer acute respiratory syndrome (SARS). METHODS: 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. RESULTS: 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. CONCLUSION: Immunofluorescence assay is highly specific and sensitive in detection of SARS. This reagent is safe and easy to prepare.

[Serological surveillance on vaccine against hemorrhagic fever with renal syndrome].

OBJECTIVE: To observe the serological and epidemiological efficacy of hemorrhagic fever renal syndrome (HFRS) vaccine in Zhejiang province. METHODS: Immunofluorescent antibody assay and Mcro-CPE method were used to test specific IgG antibody and the titer of neutralizing antibody. RESULTS: 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. CONCLUSION: HFRS vaccine was immunologically effective and the duration of serous antibody last long.

Severe acute respiratory syndrome-associated coronavirus genotype and its characterization.

OBJECTIVE: To study the severe acute respiratory syndrome (SARS)-associated coronavirus genotype and its characteristics. METHODS: A SARS-associated coronavirus isolate named ZJ01 was obtained from throat swab samples taken from a patient in Hangzhou, Zhejing province. The complete genome sequence of ZJ01 consisted of 29,715 bp (GenBank accession: AY297028, version: gi: 30910859). Seventeen SARS-associated coronavirus genome sequences in GenBank were compared to analyze the common sequence variations and the probability of co-occurrence of multiple polymorphisms or mutations. Phylogenetic analysis of those sequences was done. RESULTS: By bioinformatics processing and analysis, the 5 loci nucleotides at ZJ01 genome were found being T, T, G, T and T, respectively. Compared with other SARS-associated coronavirus genomes in the GenBank database, an A/G mutation was detected besides the other 4 mutation loci (C:G:C:C/T:T:T:T) involved in this genetic signature. Therefore a new definition was put forward according to the 5 mutation loci. SARS-associated coronavirus strains would be grouped into two genotypes (C:G:A:C:C/T:T:G:T:T), and abbreviated as SARS coronavirus C genotype and T genotype. On the basis of this new definition, the ZJ01 isolate belongs to SARS-associated coronavirus T genotype, first discovered and reported in mainland China. Phylogenetic analysis of the spike protein gene fragments of these SARS-associated coronavirus strains showed that the GZ01 isolate was phylogenetically distinct from other isolates, and compared with groups F1 and F2 of the T genotype, the isolates of BJ01 and CUHK-W1 were more closely related to the GZ01 isolate. It was interesting to find that two (A/G and C/T) of the five mutation loci occurred in the spike protein gene, which caused changes of Asp to Gly and Thr to Ile in the protein, respectively. CONCLUSION: Attention should be paid to whether these genotype and mutation patterns are related to the virus's biological activities,epidemic characteristics and host clinical symptoms.