OBJECTIVEFor 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.

METHODSData 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.

RESULTS472 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.

CONCLUSIONResults 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.

Animals ; China ; epidemiology ; Cities ; Data Collection ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect ; Genotype ; Hantavirus ; classification ; genetics ; isolation & purification ; Hantavirus Infections ; epidemiology ; veterinary ; Rats ; virology ; Rodentia ; virology

OBJECTIVETo learn about the complete genomic sequence of the Seoul virus strain ZT10 isolated from M. fartis.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONAnalysis 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.

Animals ; Antigens, Viral ; analysis ; Cell Line ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Viral ; genetics ; Fluorescent Antibody Technique, Direct ; Hantavirus ; classification ; genetics ; isolation & purification ; Phylogeny ; Polymerase Chain Reaction ; Sequence Alignment ; Sequence Analysis, DNA

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.
Animals ; China ; Disease Reservoirs ; virology ; Evolution, Molecular ; Hantavirus ; classification ; genetics ; isolation & purification ; Hantavirus Infections ; virology ; Humans ; Molecular Sequence Data ; Phylogeny ; Rodentia ; virology ; Viral Proteins ; genetics

BACKGROUNDKnee osteoarthritis (KOA) is a chronic joint disease that manifests as knee pain as well as different degrees of lower limb swelling, stiffness, and movement disorders. The therapeutic goal is to alleviate or eliminate pain, correct deformities, improve or restore joint functions, and improve the quality of life. This study aimed to evaluate the efficacy and safety of Zhuanggu joint capsules combined with celecoxib and the benefit of treatment with Zhuanggu alone for KOA.

METHODSThis multi-center, randomized, double-blind, double-dummy, parallel controlled trial, started from December 2011 to May 2014, was carried out in 6 cities, including Beijing, Shanghai, Chongqing, Changchun, Chengdu, and Nanjing. A total of 432 patients with KOA were divided into three groups (144 cases in each group). The groups were treated, respectively, with Zhuanggu joint capsules combined with celecoxib capsule simulants, Zhuanggu joint capsules combined with celecoxib capsules, and celecoxib capsules combined with Zhuanggu joint capsule simulants for 4 weeks consecutively. The improvement of Western Ontario and McMaster Universities Osteoarthritis (WOMAC) index and the decreased rates in each dimension of WOMAC were evaluated before and after the treatment. Intergroup and intragroup comparisons of quantitative indices were performed. Statistically significant differences were evaluated with pairwise comparisons using Chi-square test (or Fisher's exact test) and an inspection level of α = 0.0167.

RESULTSFour weeks after treatment, the total efficacies of Zhuanggu group, combination group, and celecoxib group were 65%, 80%, and 64%, respectively, with statistically significant differences among the three groups (P = 0.005). Intergroup pairwise comparisons showed that the total efficacy of the combination group was significantly higher than that of the Zhuanggu (P = 0.005) and celecoxib (P = 0.003) groups. The difference between the latter two groups was not statistically significant (P > 0.0167). Four weeks after discontinuation, the efficacies of the three groups were 78%, 95%, and 65%, respectively, with statistically significant differences (P < 0.0001). Intergroup pairwise comparisons revealed that the efficacy of the combination group was significantly better than that of the Zhuanggu and the celecoxib groups (P < 0.0001). The difference between the latter two groups was not statistically significant (P > 0.0167). The incidences of adverse events in Zhuanggu group, combination group, and celecoxib group were 8.5%, 8.5%, and 11.1%, respectively, with insignificant differences (P > 0.05).

CONCLUSIONSZhuanggu joint capsules alone or combined with celecoxib showed clinical efficacy in the treatment of KOA. The safety of Zhuanggu joint capsules alone or combined with celecoxib was acceptable.

TRIAL REGISTRATIONChinese Clinical Trial Registry, ChiCTR-IPR-15007267; http://www.medresman.org/uc/project/projectedit.aspx?proj=1364.

Adult ; Aged ; Celecoxib ; administration & dosage ; adverse effects ; Double-Blind Method ; Drug Therapy, Combination ; Female ; Humans ; Male ; Middle Aged ; Osteoarthritis, Knee ; drug therapy

OBJECTIVETo 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.

METHODSGene 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.

RESULTSpET28a-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.

CONCLUSIONWe 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.

Blotting, Western ; Capsid Proteins ; genetics ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Immunoglobulin M ; immunology ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Viral Core Proteins ; genetics ; immunology ; metabolism

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.

Objective To establish a TaqMan based real-time reverse transeription-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 μmol/L and 0.1 μmol/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.

Objective To investigate the sub-cellular distribution correlation between activated LIMK1 (pLIMK1Thr508) and Aurora-A in mouse oocyte meiosis,and changes in Aurora-A location and spindle structure in condition of LIMK1 inhibition.Methods Immunofluorescence staining was employed to detect the sub-cellular localization of pLIMK1Thr508and its spatial-temporal correlation with spindle organizing regulator Aurora-A in mouse oocyte meiosis; BMS-3, the specific inhibitor to LIMK1 activity, was applied to analyze the effects of LIMK1 inhibition on Aurora-A distribution and spindle formation. Results At meiotic prophase,pLIMK1Thr508was weakly detected and concentrated in the germinal vesicle(GV) in oocytes,with no signal of Aurora-A across the cytoplasm and nuclear area;as meiotic assumption approaching,pLIMK1Thr508left nuclear,aggregating as a single dense dote in the vicinity of nuclear, and being co-localized with the emerging Aurora-A; After germinal vesicle broke down (GVBD), pLIMK1Thr508and Aurora-A remained overlapped and concentrated as multi foci around the condensed chromosomes;at metaphase Ⅰ(MⅠ) and metaphase Ⅱ(MⅡ), pLIMK1Thr508was co-localized with Aurora-A on spindle poles;During anaphase Ⅰ(AⅠ) to telophase Ⅰ(Tel Ⅰ) progression, pLIMK1Thr508was detached from spindle poles and mainly concentrated on the cleavage furrow,while Aurora-A loosely congressed on spindle. In ad-dition, LIMK1 inhibition with BMS- 3 destroyed Aurora-A polar location and spindle formation. Conclusions pLIMK1Thr508is a microtubule organizing center (MTOC)-associated protein, may participate in spindle assembly and maintenance through regulating Aurora-A in mouse oocytes during meiotic progression.

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.

ObjectiveTo investigate the effects of exogenous Fusarium oxysporum and Trichoderma viride on the diversity of soil fungal community and carbon metabolic function of cultivated Panax ginseng. MethodIllumina HiSeq 2500 high-throughput sequencing combined with Biolog-ECO was used to analyze the species diversity and functional diversity of soil fungal communities in P. ginseng soil under different exogenous treatments. ResultThe results of high-throughput sequencing showed that the number and species of microorganisms in the soil were significantly changed after exogenous microorganisms were added. The soil fungi with relative abundance greater than 1% included Mortierella sp.，Fusarium sp.，Humicola sp.，and Simplicillium sp. Mortierella sp. in each treatment group significantly increased. Humicola sp. and Simplicillium sp. could be induced to increase by exogenous addition of F. oxysporum，while T. viride at a high concentration could significantly inhibit the growth of F. oxysporum. As revealed by Biolog and principal component analysis （PCA），the average well color development （AWCD） in the high-dose T. viride group （MG） was significantly higher than that in the control group （QS）and the low-dose F. oxysporum group（LD）. The utilization abilities for amino acids，carboxylic acids，polymers， and amines were enhanced in the MG group，but the microbial metabolic activity was reduced in the high-dose F. oxysporum group （LG）. There was no significant increase in the utilization of phenolic acids by soil microorganisms in both groups. ConclusionExogenous addition of F. oxysporum can lead to the growth and reproduction of other pathogenic fungi. Exogenous addition of T. viride can enhance the soil fungal community structure and metabolic diversity，inhibit the proliferation of F. oxysporum，and improve the soil microbial environment of cultivated P. ginseng.