目的 分析2008-2016年湖南省登革热病例及病原学监测结果,阐明近年湖南省登革热流行病学和病原学特征.方法 对湖南省2008-2016年报告的输入性登革热病例信息进行流行病学三间分析,对蚊媒监测中采集的蚊虫开展登革热等虫媒病毒分离和检测,对2802例健康人群开展IgG抗体水平检测和分析,对实验室确诊病例开展登革热病毒血清型分型,对部分毒株E基因进行序列测定,构建系统发生树.结果 2008-2016年湖南省累计报告输入性登革热病例156例,20～50岁占病例总数的71.37％,14个市州均有病例分布;327600只白蚊伊蚊登革热等虫媒病毒检测均为阴性,未分离到登革热病毒;2802例健康人群登革热病毒IgG抗体阳性率为6.71％,不同年龄组之间差异有统计学意义(χ2=13.81,P=0.001);36例输入性病例存在4个血清型,DENV-1占63.89％,系统发生树提示DENV-1病毒主要来源于广东.结论 湖南省无本地登革热感染病例,输入性病例以DENV-1血清型为主,健康人群抗体阳性率较高,存在输入性病例引起本地流行的风险.

This study reports the etiological identification, clinical diagnosis, and the results of the local epidemiological surveillance of the first case of severe fever with thrombocytopenia syndrome virus (SFTSV) infection in 2014 in Hunan Province, China. The infected patient was isolated and closely monitored. The virus is a member of the Bunyaviridae sandfly family and is characterized by real-time PCR, electron microscopy, immunofluorescence, and whole-genome sequencing. We also detected IgG and IgM antibodies against SFTSV among the local human population and domestic animals in a serological surveillance. Prevalence of SFTSV-specific antibodies was monitored in the local population for two years after the identification of the first SFTS case. Approximately 5% (4/77) of the people who had direct contact with the patient were seropositive, which is significantly higher than the seropositivity of the general local population [1.57% (44/2800), P < 0.05]. Furthermore, the percentage of the general population who were seropositive was higher in 2015 than in 2014 (χ2 = 7.481, P = 0.006). The epidemiological investigation found that the SFTSV is epidemic in goats, cattle, and chickens in Hunan Province. The risk of infection of domestic animals can be minimized by feeding in pens rather than allowing foraging.

Objective To make etiological diagnosis and evaluate the protective effects of post-exposure prophylaxis (PEP) in an event of one dog injured seven persons.Methods Direct immunofluorescence assay (DFA) and nested polymerase chain reaction (PCR) were employed to detect nucleoprotein and nucleoprotein(N) gene of rabies virus in the brain tissues of the dog,the positive samples were sequenced for the full length of N gene of rabies virus,then the homology of the N gene of rabies virus was analyzed after the phylogenetic tree was constructed.Rapid fluorescent focus inhibition test (RFFIT) was applied to detect the rabies virus neutralizing antibodies (RVNA) on day 0,14 and 40 after PEP.Results The cerebral,cerebellar and hippocampal tissues were positive by DFA and nested PCR.The phylogenetic tree indicated the rabies virus belonged to the rabies virus genotype I.The homology of the nucleotide and amino acid of the rabies virus N gene were over 86％ with the vaccine strains.The titer of the RVNA increased significantly from the day 0 to day 14 after PEP,the lowest was 5.78 IU/ml and the highest was 26.15 IU/ml.On the day 40,the highest RVNA titer was 51.96 IU/ml.No rabies cases occurred in a one year follow-up visit.Conclusions Normative PEP can effectively prevent the occurrence of rabies cases.

目的 构建汉坦病毒Hunan03株核蛋白基因原核重组表达载体,在大肠杆菌中进行核蛋白表达,研究核蛋白的免疫性及免疫反应性.方法 设计特异性扩增汉坦病毒Hunan03株S基因完整开放阅读框(ORF)的引物,RT-PCR扩增,产物克隆到pGM T载体,转化感受态细胞TOP10,应用蓝白斑筛选、酶切、PCR鉴定,定向克隆到pGEX-6p-2原核表达载体,转化Ecoli.BL21 StarTM(DE3),IPTG诱导表达,SDS-PAGE、Western blot对重组蛋白进行鉴定.应用Glutathione Sepharose 4B纯化柱纯化重组蛋白,免疫新西兰兔,建立间接ELISA法对核蛋白的免疫原性与免疫反应性进行评价.结果 PCR扩增S基因ORF区域产物大小约1 306 bp,重组载体pGEX-6p-2-S经双酶切、PCR、测序鉴定提示构建成功;在37℃,IPTG浓度为0.8 mmol/L诱导5h的条件下,表达出最高量的相对分子量约74 kDa的GST-NP融合蛋白.建立的间接ELISA法检测GST-NP融合蛋白免疫后的新西兰兔血清,IgM抗体滴度达1∶8 000,IgG抗体滴度达1∶16 000.结论 成功构建了高效表达的汉坦病毒S基因重组表达载体,获得了纯度较高具有较好的免疫原性与免疫反应性的核蛋白,为后续汉坦病毒单克隆抗体的制备奠定了基础.

Objective To detect and analyze the virus infection status among laboratory animals in Hunan Province.Methods A total of 56 rats and 135 Balb/c mice were randomly collected from the laboratory animal production and use units in Hunan Province.The antibodies of rat hantannvirus,rat sendai virus,mouse sendai virus,mouse poxvirus and mouse hepatitis virus were detected by ELISA.The nucleic acids of enterovirus 71,coxsackievirus A16 and rotavirus were detected by real-time PCR.The hantannvirus M gene was determined by PCR after specific primers were designed and the positive product was sequenced and analyzed by bioinformatics methods to determine the genetype of the virus.Results The antibodies of hantannvirus and sendai virus were not detected in 56 serum samples of rats,and the antibodies of mouse sendai virus,poxvirus and hepatitis virus were not found in 135 serum samples of mice.189 blood samples from rats and mice showed negative results for nucleic acids of enterovirus 71,coxsackievirus A16 and rotavirus.One sample of SD rat was positive for hantannvirus Mgene and the product size was about 860 bp.BLAST analysis confirmed that the gene fragment belonged to hantannvirus.Further phylogenetic analysis showed it belonged to the HTN subtype of hantannvirus,and when compared to the vaccine strain Z10,the nucleotide homology and amino acid homology were 86% and 89% respectively.Conclusions The laboratory animals in Hunan Province have hantannvirus infection and should be timely immunized.Laboratory animal practitioners should receive health examination regularly.

Objective To know the laboratory animal's microbial quality in Hunan Province,and to provide the important basis for scientific management of experimental animals,to ensure the quality of laboratory animals,the experimental results and the occupational health and safety of animal science workers.Methods laboratory animals' microbiological test in accordance with GB 14922.2-2001、GB/T 14926-2001 and GB/T 14926.21-2008.Sample testing with the principle of simple random on the breeding colony in the relevant unit.Results SPF rat,SPF mice,Normal New Zealand rabbits,guinea pigs' batch qualified rate was 73.68%,77.78%,60.61% and100% respectively.Conclusions It is showed that microbiological quality of laboratory animals from 2006 to 2010 in Hunan province had a downward trend on the earlier stage,and had been improved greatly.Measures should be taken to strengthen the management of laboratory animals and to eliminate microbial infections in experimental animals.

Objective To analyze the conding region of hantanvirus S gene and predict the structure of nucleoprotein for diagnostic antigen study.Methods RT-PCR was used to amplify the S gene of hantanvirus Hunan03 strain after designing specific primers.The amplification product was cloned into pGM-T vector and then the recombinant vector was transformed into E.coli TOP10,gene sequencing was carried out after blue-white selection and PCR screening for positive clones.The database of NCBI and Swiss-Prot/TrEMBL were used to predict and analyze the structure,biological characteristics and protein structures of S gene.Results The amplification product was about 1290 bp,the pGM-T/S vector was constructed and successfully sequenced,the whole length of the open reading frame (ORF) was composed of 1290 nucleotide residues,among them the GC content was 44.11％ and the AT content was 55.89％,it was composed of 429 amino acids (20 kinds),the accession number of the sequence submitted to GenBank was JN712306,its homology of nucleotides to the 76-118 strain was 83％ and the homology of amino acids was 98％,ten nonspecific variation sites were found.The grand average of hydropathicity was-0.405.There were three transmembrane domains and four non transmembrane domains in the secondary structure of nucleoprotein including 55％ of helix structure,6.1％ of sheet structure and 38.9％ of loop structure.Conclusion The bioinformatics analysis of Hunan03 strain S gene might be important for provide the substructure data to reveal the significance of S gene characteristics on hemorrhagic fever renal syndrome (HFRS) prevention and control.

Objective To analyze the epidemic and genetic characteristics of rabies prevalent in Hunan Province,1979-2010.Methods Retrospective and descriptive epidemiology were applied to analyze the dynamic distribution in space,time and crowd on the basis of the rabies data collected in the recent 32 years.A local rabies N gene database of Hunan Province was established according to the reporting of GenBank and EMBL by PubMed-Entrez Search software,the characteristics and variation of N gene of all selected sequences were analyzed by bioinformatics methods.The antigenic and genetic of the virus N gene was analyzed after the phylogenetic tree was constructed by Clustal W 1.83 and MEGA 5.0.4 software.Results There were 12 576 rabies cases reported in the recent 32 years in Hunan Province and the total mortality rate was about 0.69/100 000,which scattered in the whole province including 124 counties.Most of the cases were centralized in the south and central areas of Hunan Province and nearly 43.23% of the cases occurred from July to October,64.35% of the cases were farmers.The average content of GC and AT was respectively 44.49% and 55.51% of the 58 strains and the average single strand molecular weight was about 409.7 KDa.The highest content of amino acid component was Leu,while the lowest content was Trp.All of Hunan strains had a relatively genetic relationship to the Guizhou and Guangxi strains and 58 strains isolated in Hunan were belonged to rabies gene typeⅠafter the phylogenetic tree was successfully built.Conclusions The rabies epidemic situation has a large fluctuation in recent 32 years in Hunan Province,the 58 isolated strains are quite stabilize and have no critical variation.

To investigate the genotype and characteristic of hantavirus M and S hemorrhagic fever with renal syndrome(HFRS),lung tissues of rats were screened by immunofluorescent antibody assay(IFA),and the total RNA was extracted from the serum of HFRS patients and IFA positive lung tissues.RT-PCR was applied to amplify the M and S gene of hantavirus after specific primers were designed.PCR positive product was recovered and purified before cloning to the pMD-18 T vector and the sequencing was carried by ABI3730 gene sequencer.The obtained sequencings were compared to the HTNV and SEOV sequences of hantavirus for nucleotide homology analysis and applied MEGA5.0.4 software to construct the phylogenetic tree.The yellow-green needle-like particles were scattered in the positive lung tissues under fluorescence microscope.The PCR products of M and S gene was about 490bp and 593bp with general primers and the further amplification with HTNV typing primers to the M and S gene was respectively 242bp and 276bp,while the amplified results of M and S gene were negative with the primers of SEOV.The sequences of Hunan01,Hunan02 and Hunan03 had a high nucleotide homology to the 84FLi and SN7 strains,which had a lower homology to the Gou3 strain.Hunan01,Hunan02 and Hunan03 were belonged to H5 subtype of HTNV.In conclusion,the technology of genotyping is appropriate for hantavirus diagnosis,and the HTNV patients and animal hosts are exist in Hunan Province.

OBJECTIVE:To analyze the etiology of rabies in Hunan province and the genetic characteristics of rabies N gene isolated from 2008 to 2009.METHODS:Direct immunofluorescence assay (DFA) and nested PCR were employed to detect the monitoring samples including brain tissues of dogs and saliva, serum or urine which were collected in 2008 to 2009, from the rabies patients. Positive samples were sequenced by ABI3730 gene analyzer for the full length of the N gene target. The homology and hpylogeography of the rabies virus were analyzed after the phylogenetic tree was constructed by Blast, Clustal W and Mega 4.0 software.RESULTS:Of the 1451 tissue samples from the dogs' brain, 31 were positive under DFA and the positive rate was 2.14%. The DFA positive samples were redetected by RT-PCR and the positive rate was 1.17%. 56 samples of saliva, serum and urine samples were detected by RT-PCR from the rabies patients, with 3 positives and the positive rate was 5.36%. The length of nest PCR products were 255 bp. The rates of homology to the nucleotide and the amino acid of rabies N gene were 87.2% - 87.9% after compared to the pasture strain. The phylogenetic tree was successfully built and 20 strains isolated lately belonged to the rabies gene type I.CONCLUSION:The epidemic situation of human and dogs rabies in Human were relatively stable, with all the isolated rabies virus belonging to genotype I, without any variation.