新型冠状病毒2019-nCoV与动物冠状病毒进化关系分析
新型冠状病毒(2019-nCoV)感染肺炎疫情自2019年12月在湖北省武汉市发生以来,已在我国和世界多个国家传播,引发全球关注。为阐明2019-nCoV与已知动物冠状病毒的关系,从冠状病毒的宿主分布、遗传进化分析、宿主受体分析等角度,对2019-nCoV的可能来源进行了综合分析。结果显示:2019-nCoV与从云南省中菊头蝠中检测到的冠状病毒分离株RaTG13基因组同源性最高,为96.2%,两者遗传进化关系也较为接近;从我国广东省和广西壮族自治区查获的走私穿山甲中检测到的冠状病毒则与2019-nCoV遗传关系相对稍远,与2019-nCoV基因组同源性分别为90.4%和85.5%;2019-nCoV与已知家畜、家禽,以及犬猫等宠物中检测到的冠状病毒基因组同源性≤54.2%,遗传进化关系较远。结合监测结果,基本排除2019-nCoV来源于已知家畜、家禽以及犬猫等宠物冠状病毒的可能性。Phylogenetic Analysis between the Novel Coronavirus(2019-nCoV)and Animal CoronavirusesSince the outbreak of pneumonia caused by the novel coronavirus(2019-nCoV)in Wuhan City of Hubei Province in December 2019,it has spread in China and many other countries around the world,which is the focus of global attention. In order to clarify the relationship between 2019-nCoV and the known animal coronaviruses,the comprehensive analysis was performed to find the possible source of 2019-nCoV based on the host distribution,genetic phylogenetic analysis,host receptor analysis of coronaviruses in the study. The results showed that the genome homology between 2019-nCoV and the coronavirus RaTG13 detected from Rhinolophus affinis in Yunan Province was the highest(96.2%),and 2019-nCoV was closely related to RaTG13 in genetic phylogenetic analysis. The genetic relationship between 2019-nCoV and the two coronaviruses detected from seized smuggling pangolin in Guangdong and Guangxi were relatively longer compared with RaTG13,and the genome homology of the 2019-nCoV with coronaviruses detected from seized smuggling pangolin in Guangdong and Guangxi was 90.4% and 85.5%,respectively. Genetic distance between 2019-nCoV and coronaviruses obtained from known domestic livestock,poultry,pets including dogs and cats was genetically long,and the genome homology of 2019-nCoV with them was≤54.2%. Combined with the published surveillance results,the possibility of 2019-ncov originating from the known coronaviruses of domestic livestock,poultry,pets including dogs and cats is basically excluded. 全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202003002&v=MjI3MTVUcldNMUZyQ1VSN3FmWk9kdEZ5amtWTC9QUHlyUGViRzRITkhNckk5RlpvUjhlWDFMdXhZUzdEaDFUM3E=
2020-03-12
猪源冠状病毒监测简报
目前,已知感染猪群的冠状病毒有6种,分别是猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪呼吸道冠状病毒(PRCV)、猪急性腹泻综合征冠状病毒(SADS-CoV)、猪血凝性脑脊髓炎病毒(PHEV)和猪δ冠状病毒(PDCoV)。在冠状病毒科α、β、γ、δ等4个属中,PEDV、TGEV、PRCV、SADS-CoV均为α冠状病毒,PHEV为β冠状病毒,PDCoV为δ冠状病毒。PRCV在临床上引发仔猪呼吸道症状,PHEV感染会造成仔猪呕吐和神经症状,其他4种病毒均可引起猪群腹泻。爱彩彩票对2011年以来收集自全国16个省份的2 915份临床腹泻猪群样品(肠道、粪便等)进行了PEDV、TGEV检测,检出PEDV阳性1 223份、TGEV阳性109份,PEDV、TGEV检出率分别为41.96%、3.74%;对2014年以来收集自14个省份的12 430份临床健康猪组织样品(淋巴结、扁桃体等)进行了PEDV检测,检出阳性325份,检出率为2.61%。针对引发新型冠状病毒肺炎的病原2019新型冠状病毒(2019-nCoV),对2018—2019年收集自15个省份的2 658份生猪组织样品开展了追溯性检测,结果均为阴性。对2019-nCoV与猪源冠状病毒的亲缘关系进行了分析。基于全基因组的遗传进化分析表明:2019-nCoV与同为β冠状病毒属的PHEV核苷酸同源性仅为54%,与α、δ属猪源冠状病毒亲缘关系更远。基于以上数据与分析,可初步排除2019-nCoV来源于已知猪源冠状病毒的可能性Brief Report on the Surveillance of Swine-origin CoronavirusesCurrently,there are six kinds of coronaviruses known to infect pigs,namely porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),porcine respiratory coronavirus(PRCV),swine acute diarrhoea syndrome coronavirus(SADS-CoV),porcine hemagglutinating encephalomyelitis virus(PHEV),porcine deltacoronavirus(PDCoV). Among the four genera of the family Coronaviridae,PEDV,TGEV,PRCV and SADS-CoV belong to Alphacoronavirus,PHEV belongs to Betacoronavirus,PDCoV belongs to Deltacoronavirus. Clinically,PRCV causes respiratory symptoms in piglets,PHEV leads to vomit and neurological symptoms in piglets,while the other four kinds of viruses cause diarrhea in swine.Since 2011,a total of 2 915 samples(intestines or feces)of pigs with diarrhea symptom from 15 provinces in China have been tested for PEDV and TGEV by China Animal Health and Epidemiology Center,and 1 223 samples were PEDV-positive and 109 were TGEV-positive. The proportion of PEDV-positive samples and that of TGEV-positive samples of all the sample tested were 41.96%,3.74% respectively. Since 2014,a total of 12 430 tissue(lymph nodes or tonsils)samples of pigs in slaughterhouses from 14 provinces have been tested for PEDV,and 325 samples were PEDV-positive,the proportion of which was 2.61%. Retrospective detection of 2019-nCoV,which causes novel coronavirus pneumonia,was undertaken. A total of 2 658 tissue samples of pigs collected from 15 provinces during 2018 to 2019 were tested to be all 2019-nCoV negative.The homology between 2019-nCoV and swine-origin coronaviruses was analyzed. The phylogenetic evolutionary analysis based on genome indicated that the nucleotide homology between 2019-nCoV and PHEV both of which belong to Betacoronavirus was only 54%,the other swine-origin coronaviruses which belong to Alphacoronavirus or Deltacoronavirus exhibited a longer genetic distance from 2019-nCoV. According to the data and analysis above,the possibility of 2019-nCoV originating from the known swine-origin coronaviruses could be preliminarily ruled out. 全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202003001&v=MTkzMjJyUGViRzRITkhNckk5RlpZUjhlWDFMdXhZUzdEaDFUM3FUcldNMUZyQ1VSN3FmWk9kdEZ5amtVYnJKUHk=
2020-03-12
基于RPA的鲤春病毒血症病毒核酸检测技术的建立与评价
重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA )是近几年新发展并普及应用的一种核酸扩增技术,能够实现检测设备小型化,从而满足现场诊断(point-of-care testing,POCT)的需求。本研究以鲤春病毒血症病毒(spring viraemia of carp virus,SVCV) 为检测靶标,建立了SVCV的RPA检测技术,并分析了该方法的灵敏性、特异性和稳定性,评判了该技术应用于POCT的可能性。结果表明:基于RPA的检测方法灵敏度较高,检测下限可达4×103 PFU/mL,但比以Taq酶为基础的RT-PCR技术略低;特异性好,未发现对其他水生动物疫病病原,如传染性造血器官坏死病病毒(IHNV)、病毒性出血性败血症病毒(VHSV)、传染性胰腺坏死病病毒(IPNV)、传染性鲑鱼贫血病病毒(ISAV)等发生非特异性反应;稳定性良好,3次重复性试验的结果近乎一致。试验证明,该方法能够替代RT-PCR技术,应用于POCT的试剂和仪器设备研发。 Establishment and Evaluation of a Nucleic Acid Detection Technique for Spring Viraemia of Carp Virus Based on RPARecombinase polymerase amplification(RPA)is a kind of nucleic acid amplification method developed and widely used in recent years,which could make relevant detection equipments miniaturized to satisfy the requirements of point-of-care testing(POCT). In this study,based on spring viraemia of carp virus(SVCV),the RPA method for detection of SVCV was established,and its sensitivity,specificity and stability were analyzed,then the possibility of applying this method to POCT was evaluated. The results showed that the method was with high sensitivity,and its lower limit detection was 4×103 PFU/mL which was slightly lower than that of Taq enzyme based RT-PCR;its specificity was good,no any non-specific reaction with the pathogens of other aquatic animal was found,such as IHNV,VHSV,IPNV and ISAV;and the results of three repeated tests were almost the same due to its good stability. In conclusion,the RPA method could be used in research of relevant instruments and reagents for POCT.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202002022&v=MDM0MjZHNEhOSE1yWTlIWm9SOGVYMUx1eFlTN0RoMVQzcVRyV00xRnJDVVI3cWZaT2R0RnluaFc3M1BQeXJQZWI=
2020-03-12
鸡传染性支气管炎病毒实时荧光RT-PCR检测方法的建立与初步应用
为建立一种快速准确检测鸡传染性支气管炎病毒的方法,根据鸡传染性支气管炎病毒的核衣壳蛋白基因进行引物和探针设计,建立了一种能够检测鸡传染性支气管炎病毒的实时荧光RT-PCR方法,并对该方法进行特异性和灵敏度检测,同时与国家标准检测方法进行对比。结果显示,本检测方法特异性好,不与其他相关病毒发生交叉反应,灵敏度可达到10-4 ng/μL,与国标检测方法的符合率达100%。结果表明,本研究建立的检测方法可以准确鉴定鸡传染性支气管炎病毒,具有良好的应用前景。 Establishment of a Real-time RT-PCR Assay for Detecting Avian Infectious Bronchitis Virus and Its Preliminary ApplicationIn order to establish a rapid and accurate method for detecting avian infectious bronchitis virus(AIBV),the primers and probes were designed according to nucleocapsid protein genes of AIBV,and a real-time RT-PCR assay was established,then its specificity and sensitivity were tested and compared with national standard. The results showed that no any cross-reaction with other relevant viruses was observed by this assay,and it was with good specificity,high sensitivity(up to 10-4 ng/μL),and the compliance rate between the assay and the national standard was 100%. A conclusion was given that the established assay could accurately identify AIBV,and it would have a good prospect in practice.全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202002019&v=MTE2MDdTN0RoMVQzcVRyV00xRnJDVVI3cWZaT2R0RnluaFViL09QeXJQZWJHNEhOSE1yWTlFYllSOGVYMUx1eFk=
2020-03-12
猪瘟病毒抗体纳米荧光检测试纸条的临床应用
为探究猪瘟病毒抗体纳米荧光检测试纸条的临床应用效果,对181份未免疫猪瘟疫苗、71份已免疫猪瘟疫苗的临床猪血清,采用本研究试纸条和北京世纪元亨公司的ELISA试剂盒分别进行抗体检测,对4头存在母源抗体的不同免疫时间仔猪血清、不同阶段的猪血清,采用本研究试纸条进行抗体检测,并对检测结果进行分析统计。结果显示,本研究试纸条与北京世纪元亨公司的ELISA试剂盒的阴性符合率为100%,阳性符合率为95.77%,经Kappa检验两者一致性良好;初生仔猪首免后,猪瘟抗体滴度有所下降,30 d后加强免疫,抗体滴度逐渐升高,试纸条检测结果与猪瘟抗体衰减变化过程呈正相关;不同阶段猪群的猪瘟抗体阳性率均为100%,抗体平均值较高,离散度较整齐,与规模化养猪场合理免疫程序相吻合。综合表明,本试纸条的临床应用效果较好,可用于实时监控猪群的猪瘟抗体变化,适用于基层规模化猪场的猪瘟抗体快速检测。Clinical Application of Nanofluorescence Strip for Detecting Antibodies against Classical Swine Fever VirusIn order to evaluate clinical effects of nanofluorescence strip for detecting antibodies against classical swine fever virus(CSFV),the clinical serum samples were collected from 181 pigs without vaccination against CSF and 71 pigs with vaccination to detect corresponding antibody by the researched nanofluorescence strips and ELISA kits made by Beijing Anheal Laboratories Co.,Ltd,respectively,serum samples from four piglets with maternal antibody at different vaccination time and at different stages were detected by the strip,then the results were analyzed as follows:the negative coincidence rate of the two methods was 100%,their positive coincidence rate was 95.77%,and their consistency was good by Kappa identity test;the antibody titer of CSF in newborn piglets decreased after their first vaccination,and gradually increased after 30 days when booster vaccination was implemented,and the results by strip detection were positively correlated with the attenuation process of antibodies against CSFV;the positive rate of antibody in pig herds at different stages was 100%,the average value of antibody titer was high,and the dispersion was neat,which was consistent with the reasonable immunization procedure in large-scale swine farms. Therefore,the changes of antibodies against CSFV in pig herds could be monitored in real time by this nanofluorescence strip due to its good clinical effects,and it was suitable for rapid detection of CSFV antibody in large-scale farms at the grass-roots. 全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202002018&v=MzIwNjZZUzdEaDFUM3FUcldNMUZyQ1VSN3FmWk9kdEZ5bmhVcnpJUHlyUGViRzRITkhNclk5RWJJUjhlWDFMdXg=
2020-03-12
湖南省4株猪繁殖与呼吸综合征病毒全基因组序列测定与分析
为了解湖南省猪繁殖与呼吸综合征病毒(PRRSV)的遗传变异情况,对湖南省2016—2019年分离的4株PRRSV(Hunan/2016/075、Hunan/2017/085、Hunan/2018/123、Hunan/2019/055)进行全基因组测序,对其ORF5、NSP2基因以及全基因组核苷酸序列进行分析。结果显示,分离的4株病毒株均为美洲型,全基因组同源性为83.1%~98.7%,其中3株属于高致病型,1株为属于类NADC30型。Hunan/2016/075、Hunan/2017/085毒株全长序列与谱系5中亚系5.1的代表性毒株BJ-4亲缘关系相近,聚为一簇;Hunan/2018/123毒株与谱系8.7中的tjnh1501毒株聚为一簇;Hunan/2019/055毒株独立成一簇,与谱系1亲缘关系相近。本研究掌握了湖南省PRRSV的遗传变异情况,从而为制定有效的防控策略提供了依据。 Determination and Analysis on the Complete Genomic Sequence of Four Strains of PRRS Virus in Hunan ProvinceIn order to recognize the genetic variation of porcine reproductive and respiratory syndrome virus(PRRSV)in Hunan Province,4 strains of PRRSV(Hunan/2016/075,Hunan/2017/085,Hunan/2018/123 and Hunan/2019/055)isolated from 2016 to 2019 in the province were sequenced,and their ORF5 and Nsp2 genes and nucleotide sequences of their complete gene were analyzed. The results showed that all the four strains belonged into North American(NA)type,with the homology of complete genome of 83.1% to 98.7%,in which,3 strains were highly pathogenic and 1 strain was classified as NADC30-like strain. In view of the genetic relationship,the full-length sequences of Hunan/2016/075 and Hunan/2017/085 were similar to BJ-4 strain,the representative one of 5.1 substrain of 5 family,which were clustered into one cluster;Hunan/2018/123 was clustered together with tjnh1501 strain of 8.7 family;and Hunan/2019/055 formed one cluster by itself,which was similar to 1 family concerning its genetic relationship. In short,the status of genetic variation of PRRSV in Hunan Province was identified,which would provide some references for developing effective control measures. 全文下载链接:https://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFDAUTO&filename=ZGDW202002016&v=MTE1NzZWNzNMUHlyUGViRzRITkhNclk5RVlvUjhlWDFMdXhZUzdEaDFUM3FUcldNMUZyQ1VSN3FmWk9kdEZ5bmc=
2020-03-12
禽源冠状病毒监测简报
冠状病毒(Coronaviruses)在分类地位上属于套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)正冠状病毒亚科(Orthocoronavirinae),包括α、β、γ和δ等4个属,可感染包括人类在内的多种动物。其中,α冠状病毒属主要感染人和猪、犬、猫、蝙蝠等,β冠状病毒属主要感染人和牛、马、猪、鼠、蝙蝠等哺乳动物,γ冠状病毒属主要感染鸡、火鸡、鸭、鹅等禽类,δ冠状病毒属主要感染野禽和猪。引起此次新型冠状病毒肺炎疫情的病毒(2019-nCoV)属于冠状病毒科β冠状病毒属。可感染禽类的冠状病毒主要来自γ冠状病毒属和δ冠状病毒属。鸡传染性支气管炎病毒(IBV)等家禽中分离到的冠状病毒均为γ冠状病毒属。家禽冠状病毒感染危害较大的是IBV。此外,鸭冠状病毒、鹅冠状病毒和鸽冠状病毒等在家禽中也有检出。一些野禽可感染δ属冠状病毒(如鹅口疮冠状病毒HKU12等)。2013年以来,爱彩彩票对全国17省份共计5 249份家禽样品进行了禽源冠状病毒检测,共检测到IBV 446株,新型鸭冠状病毒和新型鸽冠状病毒等新型禽冠状病毒共277株,IBV、新型鸭冠状病毒、新型鸽冠状病毒的检出率分别为8.50%、2.04%、3.24%。对2019-nCoV与禽源冠状病毒的亲缘关系进行分析发现,2019-nCoV与已知的禽源冠状病毒核苷酸同源性较低;基于冠状病毒1ab基因保守区域的进化分析表明,2019-nCoV与禽源冠状病毒亲缘关系较远。基于上述分析,初步排除2019-nCoV来源于已知家禽冠状病毒的可能性。Overview on the Surveillance of Avian-orgin CoronavirusesCoronaviruses belong to Nidovirales,family Coronaviridae,Orthocoronavirinae,which are divided into four genera:Alphacoronavirus,Betacoronavirus,Gammacoronavirus and Deltacoronavirus,and can infect many animals including humans. Among them,Alphacoronavirus mainly infects humans and pigs,dogs,cats,bats,etc.,Betacoronavirus mainly infects humans,cattle,horses,pigs,mice,bats and other mammals,and Gammacoronavirus mainly infects chickens,turkeys,ducks,geese and other birds,Deltacoronavirus mainly infects wild birds and pigs. The emerging 2019-nCoV causing novel coronavirus pneumonia(NCP)outbreak in China belongs to the Betacoronavirus of Coronaviridae. Coronaviruses that can infect birds are mainly from the Gammacoronavirus and Deltacoronavirus of Coronaviridae. Coronaviruses isolated from poultry such as avian infectious bronchitis virus(IBV)are all Gammacoronavirus. In the avian coronaviruses,IBV is more harmful to the poultry. In addition,duck coronavirus,goose coronavirus and pigeon coronavirus were also detected. Some wild birds can be infected with Deltacoronavirus(such as thrush coronavirus HKU12,etc.). Since 2013,a total of 5 249 poultry samples from 17 provinces in China have been detected by China Animal Health and Epidemiology Center,a total of 446 avian infectious bronchitis viruses,277 of novel duck coronaviruses and novel pigeon coronaviruses have been detected. The positive rates of avian infectious bronchitis virus,novel duck coronavirus,and novel pigeon coronavirus were 8.50%,2.04% and 3.24%,respectively.Analysis of the relationship between 2019-nCoV and avian coronavirus found that the nucleotide homology between 2019-nCoV and known avian-orgin coronaviruses was quite lower compared with mammal-origin coronaviruses. Evolutionary analysis based on the conserved region of the coronavirus 1ab gene shows that 2019-nCoV is far from avian coronavirus. According to the above analysis results,the possibility of 2019-nCoV originating from the known avian-origin coronaviruses can be preliminarily ruled out.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20200206.1649.002.html
2020-03-12
鱼类病原性弧菌血清学诊断芯片技术构建
针对鱼类致病性肠道弧菌(Vibrio ichthyoenteri)、鳗弧菌(Vibrio anguillarum)、副溶血弧菌(Vibrio parahaemolyticus)、溶藻弧菌(Vibrio alginolyticus)、河流弧菌(Vibrio fluvialis)和哈维氏弧菌(Vibrio harveyi),制备了牙鲆(Paralichthys olivaceus)抗血清,提取了各弧菌的外膜蛋白、胞外产物、鞭毛蛋白和全菌蛋白,分析了牙鲆抗血清与各抗原蛋白的免疫反应,发现各弧菌抗血清与各自的抗原反应最强,与其他弧菌抗原有不同程度的交叉反应。将6种弧菌的抗原组分固定于芯片载体形成微阵列,构建了其血清学诊断抗原芯片,使用辣根过氧化物酶标记的抗牙鲆IgM单抗2D8作为检测抗体,确定了检测结果判读方法;将抗原芯片应用于牙鲆和大菱鲆(Scophthalmus maximus)的弧菌病诊断,并利用ELISA技术,验证了该抗原芯片用于弧菌病诊断的准确性。本研究为鱼类弧菌病的血清学诊断提供了有力工具。Construction of Serological Diagnostic Chip for Pathogenic Vibrio in FishThe antiserum samples of Paralichthys olivaceus were prepared respectively against 6 kinds of pathogenic vibrios,including Vibrio ichthyoenteri,V. anguillarum,V. parahaemolyticus,V. alginolyticus,V. fluvialis and V. harveyi. The outer membrane protein,flagellum protein,extracellular products and whole-cell bacterial protein of the strains were extracted and purified,then the immune reactions between the antigen components and the antiserum samples were analyzed,it was found that,for all Vibrios,the immune reaction between its antigen and its own antiserum was the strongest,besides,there were also cross-reactions in different extent between the antiserum with the antigen of other Vibrios. Further,the antigen components of the 6 strains were fixed on the chip carrier to form an antigen microarray so as to construct the serological diagnostic antigen chip,the method for interpreting detection results was determined by using monoclonal antibody 2D8 of IgM marked with horseradish peroxidase as the detection antibody;the antigen chip was applied to diagnose vibriosis in Paralichthys olivaceus and Scophthalmus maximus,and its accuracy was verified by ELISA. As a conclusion,a useful tool was provided for serological diagnosis of fish vibriosis.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191206.0907.036.html
2019-12-25
羊种布鲁氏菌Rev.1株VjbR基因的克隆、原核表达及生物信息学分析
本研究旨在构建表达载体pET-30a-VjbR,进而表达布鲁氏菌VjbR蛋白;利用生物软件分析该蛋白生物信息学信息,为进一步研究该蛋白奠定基础。以布鲁氏菌Rev.1株基因组为模板,扩增VjbR基因序列,将其克隆至原核表达载体pET-30a(+)中,获得重组质粒pET-30a-VjbR,并进行原核表达、Western-blot检测及该基因的生物信息学分析。结果显示:本试验成功克隆并表达了VjbR基因;经对表达产物进行SDS-PAGE分析纯化,发现本试验得到较纯蛋白;经Western-blot检测,发现该基因表达蛋白可与布鲁氏菌羊阳性血清发生特异性反应,具有良好的反应原性;通过生物信息学系列软件统计分析,发现VjbR蛋白无跨膜区,无信号肽,且该蛋白共有15个抗原决定簇,在二级结构中,α-螺旋的氨基酸有131个,占比为49.81%。布鲁氏菌VjbR基因的成功表达与纯化,为进一步制备该蛋白的单克隆抗体及iELISA诊断试剂盒的研制奠定了基础。Cloning,Prokaryotic Expression and Bioinformatics Analysis of VjbR Gene of Brucella melitensis Rev.1 StrainIn order to construct the expression vector(pET-30a-VjbR)to express the VjbR protein of Brucella,and to analyze the bioinformatics information of the protein by biological software so as to lay a foundation for further relevant study. Taking the genome of Brucella melitensis Rev.1 strain as a template,VjbR gene sequence was amplified and then cloned into the prokaryotic expression vector,pET-30a(+),to obtain the recombinant plasmid,pET-30a-VjbR,then the prokaryotic expression,western-blot and bioinformatics analysis were respectively carried out. The results showed that VjbR gene was successfully cloned and expressed;the purer protein was received after SDS-PAGE analysis and purification for the expressed products;it was detected that the expressed protein could specially react with the positive sheep serum through western-blot,showing good reactivity;according to the analysis by series of bioinformatics software,it was found that VjbR protein was with no any transmembrane domain or signal peptide,it had 15 antigenic determinants. In the secondary structure,the number of α-spiral amino acids was 13,accounting for 49.81%. In conclusion,the successful expression and purification of VjbR gene of Brucella would lay a foundation for further preparation of monoclonal antibodies and development of iELISA kit.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191206.0907.030.html
2019-12-16
禽肾炎病毒与鸡细小病毒双重PCR检测方法的建立
为了同时检测禽肾炎病毒(avian nephritis virus,ANV)和鸡细小病毒(chicken parvovirus,ChPV),根据GenBank中ANV的ORF基因和ChPV的 NS1基因的保守序列,设计筛选了扩增片段大小分别为511 bp和242 bp的特异性引物,通过反应条件优化、特异性和敏感性试验,建立了一种双重PCR检测方法。该方法最佳引物比例为ANV上下引物各0.4 μL(20 mol/L),ChPV上下引物各0.6 μL(20 mol/L);最佳退火温度为53.8℃;灵敏度可达到55 fg(ANV)和58 fg(ChPV);对常见鸡病病原体进行检测,结果全为阴性。本研究建立的PCR方法具有特异、敏感、快速、稳定的优点,可用于同时鉴别诊断ANV和ChPV的混合感染。Establishment of a Duplex PCR Assay for Detection of Avian Nephritis Virus and Chicken ParvovirusA duplex PCR assay for simultaneous detection of both avian nephritis virus(ANV)and chicken parvovirus(ChPV)was established. Specifically,one pair of specific primers were designed according to the conserved sequences of ORF gene of ANV and NS1 gene of ChPV published in GenBank,after optimization of reaction conditions,specificity and sensitivity tests,the duplex PCR assay was established and evaluated. The results showed that the best ratio of primers was 0.4 μL(20 mol/L)respectively for upper and lower primers of ANV,and 0.6 μL(20 mol/L)respectively for those of ChPV;the best annealing temperature was 53.8 ℃;the detection limits could be up to 55 fg(ANV)and 58 fg(ChPV);then the assay was used to detect common pathogens of chicken diseases,and all detection results were negative. A conclusion was given that,the established assay was specific,sensitive,rapid and stable,and the assay could be used to identify and diagnose mixed infection of ANV and ChPV.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191206.0907.026.html
2019-12-16
羊传染性脓疱病毒NZ2毒株CBP蛋白结构分析与表位预测
本研究应用生物信息学方法,以ORFV NZ2毒株的CBP蛋白氨基酸序列为研究对象,对CBP蛋白的理化特性、亲水性、跨膜区、信号肽、二级结构、三级结构、B细胞表位和T细胞表位进行了预测分析。结果显示,CBP蛋白的相对分子质量为31.179 89 kDa,理论等电点为4.68,半衰期为30 h,稳定系数为43.06,脂肪族指数为81.78,总平均亲水性为-0.383。CBP蛋白为亲水性蛋白,无跨膜区,不属于跨膜蛋白,存在信号肽;二级结构中α螺旋、β折叠、β转角、无规卷曲分别占38.11%、33.91%、5.94%和10.48%,三级结构形似倒立的哑铃,上下对称。筛选出了6个优势B细胞表位、3个CTL细胞表位,无Th细胞表位。CBP蛋白为亲水性膜外蛋白,细胞表位较少,在细胞膜外为ORFV逃避宿主免疫反应发挥作用。本研究为CBP蛋白的功能研究与ORFV的致病机制研究提供了理论依据。Structural Analysis and Epitope Prediction of CBP of ORFV NZ2 StrainTaking the amino acid sequence of CBP of orf virus(ORFV)NZ2 strain as the research object,the physicochemical property,hydrophilicity,transmembrane domain,signal peptide,secondary structure,tertiary structure and epitopes of B cell and T cell,were predicted and analyzed by bioinformatics method. The results showed that the relative molecular weight of CBP was 31.179 89 kDa,the theoretical isoelectric point was 4.68,half-life period was 30 hours,stability coefficient was 43.06,aliphatic index was 81.78,and total average hydrophobicity was -0.383. CBP was a hydrophilic protein instead of a transmembrane one,no any transmembrane domain,but signal peptide was contained;α-helix,β-fold,β-turn and random coil accounted for 38.11%,33.91%,5.94% and 10.48% respectively in the secondary structure;the tertiary structure was similar with an inverted dumbbell in form,and it was symmetrical from top to bottom. A total of 6 dominant B cell epitopes and 3 CTL cell epitopes were screened out,and no Th cell epitope was found. CBP was a hydrophilic extracellular protein with few cell epitopes,which helped ORFV avoid host immune response outside the cell membrane. In conclusion,a theoretical basis was provided for the study on CBP' s functions and the pathogenesis of ORFV.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191206.0907.032.html
2019-12-16
非洲猪瘟病毒结构蛋白p54的原核表达与反应原性鉴定
p54蛋白为非洲猪瘟病毒(ASFV)的主要结构蛋白之一,参与病毒对靶细胞的吸附与进入。为深入研究p54结构蛋白的抗原性,根据GenBank序列号(MK128995)对应的E183L基因序列,设计1对特异性引物扩增其整个CDS区,并克隆至pET-30a(+)载体,构建了表达p54蛋白的重组质粒pET-30a(+)-p54。用BL21(DE3)转化该质粒,经IPTG诱导表达后进行SDS-PAGE电泳,可见重组质粒表达出1条分子量约为20 kDa的特异性条带,且重组表达蛋白以融合表达蛋白形式存在于上清。进一步通过His亲和层析法纯化目的蛋白,用ASFV阳性血清进行蛋白质免疫印迹反应,发现表达的重组p54蛋白能与ASFV阳性血清产生特异性反应,表明p54蛋白表达成功。本研究为ASFV抗体ELISA检测方法的建立奠定了基础。Prokaryotic Expression and Reactivity Identificationof Structural Protein p54 of African Swine Fever VirusAs one of the major structural proteins of African swine fever virus(ASFV),p54 protein helps the virus adsorb and enter into the target cells. In order to further study its antigenicity,one pair of specific primers were designed to amplify the whole CDS domain of E183L gene according to the serial number(MK128995)in GenBank. Then the amplification products were cloned into the vector pET-30a(+),and the recombinant plasmid pET-30a(+)-p54 was constructed,after transformation into BL21(DE3),induced expression by IPTG and SDS-PAGE electrophoresis. one specific band with the molecular weight of 20 kDa was obtained and the recombinant protein appeared in the supernatant in the form of fusion protein. Further,the recombinant protein was purified by His-tag affinity chromatography,and the positive serum of ASFV was used for Western blotting. it was found that the expressed recombinant p54 protein could specifically react with ASFV positive serum,indicating that p54 protein was successfully expressed. The study would lay a foundation for the development of ELISA kit for detection of ASFV.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191206.0907.028.html
2019-12-16
2018年辽宁省大连市奶牛副结核病血清流行率调查
本研究对大连市全部11个奶牛场开展抽样调查,通过设计两阶段随机抽样方案并结合问卷调查,初步了解了大连市奶牛场副结核病流行状况以及奶牛场从业人员对副结核病防控知识的掌握情况和行为特点。研究结果显示,大连市奶牛场副结核病平均个体血清流行率为17.23%(95%CI:15.99%~18.74%),场群真实流行率为100%;年龄、胎次是奶牛副结核病重要的风险因素;奶牛副结核病是导致大连市奶牛腹泻的主要原因之一;大连市奶牛场从业人员对副结核病的认知程度有限,部分规模场对本病认识不足,导致部分防控措施落实不到位。研究结果提示,大连市需重视对奶牛副结核病的防控,采取综合防控措施,阻止本病的继续蔓延并在有条件的场户开展净化;各奶牛场应严格做好生物安全工作,在引进奶牛时严格检疫,防止引进阳性牛、隐性感染牛;兽医部门应该加强科普宣传,增强奶牛场工作人员对副结核病的判断力,提高其防范副结核病的能力。Investigation on Sero-prevalence of Cow Para-tuberculosisin Dalian City of Liaoning Province in 2018In this study,all the 11 dairy farms in Dalian City were sampled and investigated,and the prevalence of para-tuberculosis,practitioners' knowledge and behaviors for control of the disease were preliminarily identified through two-stage random sampling plan and questionnaire survey. The results showed that the average individual serological prevalence of para-tuberculosis was 17.23%(95%CI:15.99%–18.74%),and the true prevalence at the farm/herd level was 100%;the risk factors of such infection included age and calving number;cow para-tuberculosis was one of the main causes of diarrhea;and some control measures could not be put in place due to lack of relevant knowledge and awareness in some scaled farms. Therefore,it was suggested that the disease should be controlled as a top priority by series of comprehensive measures to prevent any further spreading,some purification measures could be carried out in some conditional farms/households;bio-safety measures should be carried out by all farms,all introduced cows should be strictly quarantined to avoid any positive cases or suppressive infection;also,popularization and publicity should be strengthened by veterinary departments to enhance workers' judgment on the disease in the farms and to improve their ability to prevent the disease.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191206.0906.008.htmll
2019-12-16
H9亚型禽流感病毒实时荧光RT-PCR检测方法的建立与应用
为建立一种快速准确检测H9亚型禽流感病毒基因的检测方法,根据GenBank中H9亚型禽流感病毒血凝素编码基因进行荧光检测引物和探针设计,建立了一种针对H9亚型禽流感病毒的荧光RT-PCR检测方法。利用该方法进行灵敏度和特异性检测,并用本方法和国家标准中的荧光RT-PCR检测方法,同时对临床样本进行检测。结果显示,仅H9亚型禽流感病毒出现了正常荧光检测曲线,而其他病毒及阴性对照均未出现;检测灵敏度为RNA终浓度10-4 ng/μL(1.30×104 copies/μL);临床样本检测结果与国标方法一致,符合率为100%。结果表明,该方法特异性强、灵敏度高,可用于H9亚型禽流感病毒检测。Establishment and Application of a Real-time RT-PCR Assay for Detectionof H9 Subtype Avian Influenza VirusIn order to establish a method for detecting H9 subtype avian influenza virus(AIV-H9)rapidly and accurately,the fluorescence detection primers and probes were designed and synthesized according to hemagglutinin encoding gene of AIV-H9 registered in GenBank,and a fluorescence PCR assay was thereby established,and its specificity and sensitivity were tested. Then by the established assay and the RT-PCR stipulated in national standard,some clinical samples were simultaneously tested. The results showed that the normal fluorescence detection curve only appeared in AIV-H9 rather than other non-specific viruses and negative sample;its detection limit was 10-4 ng/μL(1.30×104 copies/μL)of RNA;the detection result of clinical samples by established assay was consistent with the national standard,and the coincidence rate was 100%. In conclusion,the assay could be used to detect AIV-H9 due to its strong specificity and high sensitivity.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191206.0907.024.html
2019-12-16
2018年海南省猪A型塞尼卡病毒血清流行病学调查
为了解海南省A型塞尼卡病毒(Senecavirus A,SVA)感染状况,应用间接ELISA方法,对从海南省15个市(县)112个场点采集的2 547份猪血清样品进行SVA抗体检测。结果显示:SVA血清样品抗体阳性率为10.9%,场点阳性率为50.0%;15个市(县)的场点阳性率在14.3%~100%之间,其中有6个超过50%;15个市(县)的样品阳性率在1.0%~31.7%之间,大部分市(县)小于10%;规模化养殖场场点阳性率(61.2%)高于散养户(33.3%);仔猪、母猪、后备猪、公猪、育肥猪样品阳性率分别是20.5%、15.5%、13.5%、10.7%、5.9%。结果表明:SVA已扩散至整个海南省,防控形势较严峻,需制定相应防控措施;交通枢纽地区和规模化养殖场的SVA流行面较广,仔猪和种猪群中的SVA流行率较高,应重点加强监测和相关流行病学研究。本研究摸清了海南省猪群中的SVA的感染与分布情况,找出了重点防控区域和防控对象,对于指导该地区SVA防控具有参考意义。A Seroepidemiological Investigation on Senecavirus A in Swine in Hainan Province in 2018In order to identify the prevalence of Senecavirus A(SVA)in Hainan Province,2 547 pig serum samples were collected from 112 farms in 15 cities(counties),and SVA antibodies were detected by indirect ELISA. The results showed that the average positive rates of SVA serum antibody at the individual and farm level were 10.9% and 50.0% respectively;the positive rates at the farm level in 15 cities(counties)ranged from 14.3% to 100%,and the rates in 6 out of 15 cities(counties)exceeded 50%;the positive rates at the individual level in 15 cities(counties)ranged from 1.0% to 31.7%,and the rates in most samples were less than 10%;the positive rate in scaled farms(61.2%)was higher than that in free-range households(33.3%);the positive rates of samples from piglets,sows,replacement gilts,breeding pigs and fattening pigs were 20.5%,15.5%,13.5%,10.7% and 5.9%,respectively. The results indicated that relevant control measures should be developed under such severe situation that SVA had spread across the province;monitoring and corresponding epidemiological research should be strengthened as the top priorities due to wide spread of SVA in transportation hub areas and large-scale farms and its high prevalence in piglets and breeding populations. In this study,the infection and distribution of SVA in pig populations in Hainan Province were identified,and the key areas and objects to be controlled were defined,which could provide some references for prevention and control of SVA in the province.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191206.0906.004.html
2019-12-16
安徽省规模禽场高致病性禽流感血清学横断面研究与风险因素分析
为掌握安徽省禽群的禽流感免疫抗体水平以及引起免疫不合格的主要风险因素,在2018年春季集中免疫前,对安徽省7个市开展了H5和H7亚型禽流感血清学检测和问卷调查。按照分层随机抽样方法,在7个市选择种禽场、蛋禽场和肉禽场,共140个养禽场,在每个养殖场内随机抽取35份血清样品,进行HI抗体检测,并根据检测结果,计算群体水平的真实免疫合格率/阳性率;将H5或H7亚型禽流感抗体群体合格率低于70%的养禽场定义为免疫不合格场,进行单因素风险分析。结果显示:安徽省7个市H5(Re-8株)和H7N9(Re-1株)的场群真实抗体合格率/阳性率分别为67.7%(95%CI:60.0%~75.4%)和39.8%(95%CI:31.7%~47.9%),62个养禽场的H5或H7亚型抗体未达标(<70%);存栏量越小(P=0.046)、免疫次数越少(P=0.016),H5或H7亚型免疫抗体不合格的可能性越高(P<0.05);存栏量小于4 000羽、免疫后时间≤21 d或≥91 d、家禽日龄≤26或≥2 200、免疫次数≤1、规模化饲养鹅,以及不使用“H5+H7”二价灭活疫苗(OR=2.66,95%CI:1.34~5.35)、周边3 km内有野禽栖息地(OR=2.78,95%CI:1.10~7.65)的养禽场的H5或H7亚型免疫抗体不合格风险相对较高。结果表明:2018年春季安徽省7个市H5、H7亚型禽流感免疫抗体保护率下降幅度明显,需要及时开展春季集中免疫;养禽场规模、类型以及家禽种类、是否免疫疫苗、免疫次数、养禽场周边是否有野禽栖息地等,均与群体的禽流感免疫抗体合格率显著相关,因此在制定强制免疫计划时,应视情况进行分类指导,实时补免或增加免疫次数。此外,养禽场选址时应尽量避开野禽栖息地。本研究为决策部门制定禽流感免疫方案提供了技术支撑。A Serological Cross-sectional Study on HPAI in Large-scale Poultry Farmsin Anhui Province and Analysis on Relevant Risk FactorsIn order to identify the level of antibody against highly pathogenic avian influenza(HPAI)in large-scale poultry farms in Anhui Province and to analyze major risks that might lead to unqualified immunity,serological surveillance and questionnaire survey on H5 and H7 subtype HPAI were carried out in 7 cities prior to intensive vaccination in the spring of 2018. A total of 140 poultry farms including breeding,layer and broiler farms were selected to randomly collect 35 serum samples per farm using stratified random sampling method for hemagglutination inhibition test(HI),and then the true immune qualification rate/positive rate at the farm level was calculated according to the test results;the farms of which the qualification rate of antibody against H5 or H7 subtype virus was lower than 70% were defined as the ones with unqualified immunity,and then risk analysis on single factor was carried out. The results showed that the true immune qualification rates/positive rates of H5(Re-8 strain)and H7N9(Re-1 strain)at the farm level were 67.7%(95%CI:60.0%-75.4%)and 39.8%(95%CI:31.7%-47.9%),respectively;in 62 farms,the antibody level failed to reach the standard(less than 70%);the smaller the stock(P=0.046)and the less the vaccination frequency(P=0.016),the higher the probability of disqualification of immune antibodies(P<0.05). The high risk factors were as follows:the poultry stock was less than 4 000,the period was within 21 days or above 91 days since last vaccination,the ages of live poultry were less than 26 days or more than 2 200 days,the immunization times were less than or equal to 1,raising geese in a large scale,the poultry were not vaccinated with“H5+H7”bivalent inactivated vaccines(OR=2.66,95% CI:1.34-5.35),as well as there were wild bird habitats within 3 km radius around(OR=2.78,95%CI:1.10-7.65). Results showed that,the level of antibody against H5 and H7 subtype HPAI decreased obviously in the above 7 cities in the spring of 2018,hence it was necessary to carry out intensive vaccination in spring in time. The antibody level was obviously related to the factors including the scale and type of poultry farms,the type of poultry,vaccination or not,frequency of vaccination,wild bird habitats around farms,etc.,so related vaccination should be strengthened or supplemented in real time with classification guidance as required when the compulsory immunization plan was developed. In addition,any wild bird habitat should be avoided to the greatest extent when a poultry farm was designed and constructed. Above all,some technical supports for developing AIV immunization plan were provided to decision-making departments.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191206.0906.002.html
2019-12-16
马梨形虫病病原双重荧光PCR快速检测方法的建立与应用
在序列比对分析的基础上,设计2对引物和2条MGB探针,经体系优化建立了可快速鉴别检测两种马梨形虫病病原(马泰勒虫和驽巴贝斯虫)的双重荧光PCR检测方法。该方法可分别检出22和31基因拷贝的虫体DNA,且不与其他马病病原发生交叉反应。应用建立的荧光PCR检测方法,对采自进出口马匹的10份马全血和20份马血清进行检测,结果从马全血中检出2份马泰勒虫阳性样品。使用一对马泰勒虫EMA1全基因扩增引物,从2份阳性样品中扩增了EMA1基因。克隆测序后,对该基因全序列进行分析,证实其为马泰勒虫特异基因序列;2份样品的基因序列相似性为98.9%,存在6个氨基酸变异;2个样品中的EMA1基因不完全一致,表明2个感染样品中的虫体可能为不同的马泰勒虫虫株。Establishmentand Application of a Duplex Real-time PCR Method for Detectionof Piroplasmosis Disease Two pairs ofprimers and two MGB probes were designed based on sequence alignment,after system optimization,a duplex real-time PCR method foridentification and detection of two pathogens(Theileria equiand Babesia caballi)of piroplasmosisdisease was established. The detection limits were 22 and 31 gene copiesrespectively,and no cross reaction with otherpathogens of horse diseases was observed. By the established real-time PCR,10 whole blood and 20 serum samplescollected from imported and exported horses were tested,and 2 positive samples(against Theileria equi)were detected;then EMA1 gene from the two positivesamples were amplified through a pair of amplification primers of EMA1 gene ofTheileria equi. After being cloned and sequenced,the wholesequence of the genes were analyzed,and theamplified genes were confirmed to be the specific sequences of Theileria equi;The similarity of EMA1 sequence of thetwo samples was 98.9%,with six aminoacid mutations,indicating that the worms in the twosamples might belong to different strains of Theileria equi.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191009.1108.038.html
2019-10-24
病死猪圆环病毒2型检测及全基因序列分析
为准确检测猪圆环病毒2型(PCV2)并分析其基因序列情况,在序列比对分析的基础上,设计一对简并引物和一条探针,经体系优化建立了PCV2的荧光PCR方法。该方法可检出10拷贝的PCV2质粒DNA,不与猪瘟病毒等多种病毒发生交叉反应。对送检的2批次45份病死猪脏器进行检测,检出25份阳性样品,与套式PCR方法的复合率为95.6%;对3份强阳性样品使用一对全基因组扩增引物,扩增了PCV2型全基因片段;经克隆测序后分析,发现3个病毒基因分别属于2种基因亚型(PCV2b和PCV2d)。本研究对于PCV2的准确检测及其变异特征的了解具有参考意义。Detection ofPorcine Circovirus Type 2 in Dead Pigs and Analysis on ItsComplete Genome Sequence In order toaccurately detect porcine circovirus type 2(PCV2)and to analyze its genome sequence,a pair of degenerate primers and aprobe were designed based on sequence alignment analysis,after systematic optimization,a real-time PCR method was established,which could detect 10 copies ofplasmid DNA of PCV2,with no anycross reaction with classical swine fever virus and other viruses. Then twobatches of 45 organ samples collected from dead pigs were tested by establishedmethod,and 25 samples were detected to bepositive,the coincidence rate with nested-PCRwas 95.6%;for the three strong-positive samples,their complete gene fragments of PCV2were amplified by a pair of primers,then weresequenced and analyzed,and it wasfound that the genes of the three viruses were classified into two differentsubtypes(PCV2b and PCV2d). Therefore,it was of certain significance toconfirm relevant test results and recognize any variation of PCV2 by the abovemethod. 全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191009.1107.036.html
2019-10-24
PCV2感染后JAK-STAT信号通路的基因差异表达分析
JAK-STAT信号通路是阐明细胞因子生物学功能及病毒如何发挥作用的分子基础,但对于猪圆环病毒2型(PCV2)感染及病毒复制的作用机制,目前尚无报道。本研究采用PCR列阵方法,建立PK-15细胞JAK-STAT信号通路的功能分类芯片,分析JAK-STAT信号通路中所有已知的Jak和Stat家族成员、激活JAK-STAT信号通路的受体,以及与Stat蛋白相关的核辅助因子及共同活化因子、Stat诱导蛋白及该通路的负反馈调节蛋白等基因在PCV2感染后的差异表达情况,为进一步明确PK-15细胞JAK-STAT信号通路对病毒复制力和复制机制奠定基础。Analysis onDifferential Gene Expression of JAK-STATSignaling Pathway after PCV2 InfectionThe JAK-STATsignaling pathway could help us to expound biological function of cytokines andhow viruses work,but the infection with porcinecircovirus type 2(PCV2)and virus replication mechanism havenot been reported so far. In this study,PCR array wasused to establish a functional taxonomic chip of the JAK-STAT pathway in PK-15cells,then the differential gene expressionof the pathway after PCV2 infection were analyzed,including allknown Jak and Stat family members,receptorsactivating the pathway,nuclearcofactors and coactivators related to Stat protein,Stat-inducible protein and negativeregulatory proteins,which wouldlay a foundation for further identifying how the pathway influence virusreplication and replication mechanism.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191009.1107.034.html
2019-10-24
表达羊口疮病毒B2L蛋白重组小反刍兽疫病毒的拯救
羊口疮(传染性脓疱)和小反刍兽疫的病原体分别为羊口疮病毒(orf virus,ORFV)和小反刍兽疫病毒(peste despetits ruminants virus,PPRV)。如果通过反向遗传技术进行基因修饰,PPRV可成为表达外源蛋白的有效载体。ORFV含有1个具有高免疫原性的B2L蛋白。本研究首先构建了含B2L基因开放阅读框的重组PPRV cDNA clone,然后通过反向遗传技术,拯救了一株表达B2L蛋白的重组PPRV。试验表明,该重组病毒的生长动力学类似其母源病毒;Western blot和质谱分析表明,该重组PPRV可在细胞内表达B2L蛋白。该研究为羊口疮和小反刍兽疫二联疫苗的研制奠定了基础。Rescue of Recombinant Peste des PetitsRuminants Virus for Expressing Orf Virus B2L ProteinOrf(contagiousecthyma)and peste des petits ruminants(PPR)are caused byorf virus(ORFV)and peste despetits ruminants virus(PPRV),respectively. If genetically modifiedby reverse genetics tool,the PPRV wouldbe a useful vector to express foreign proteins. The ORFV contains a highlyimmunogenic envelope protein,B2L protein.In this study,a recombinant PPRV cDNA clone,which contained the B2L gene openreading frame,was rescued using reverse genetics toexpress the B2L protein. This recombinant PPRV was demonstrated to have similargrowth kinetics to that of its parental virus. In vitro expression of the B2Lprotein by it was demonstrated by Western blot and confirmed by massspectrometry,therefore paving the way fordevelopment of a bivalent vaccine candidate against both diseases.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191009.1107.032.html
2019-10-24
以GIS为基础的禽流感预测预警研究进展
GIS是一种对地理环境等方面数据进行储存、分析、加工和呈现的综合工具。利用GIS可以把流感病例的相关数据与气候、环境和社会等数据进行整合和分析,通过统计学或数学模型,找到影响流感发生和传播的风险因素,对流感的发生趋势进行预测和可视化预警。国内外学者利用GIS在禽流感风险因素分析和预测等方面做了大量研究,包括对人感染H7N9流感、高致病性H5亚型禽流感、野禽流感风险因素和预测的研究,不同基因型病毒分布特征的研究,兽医决策辅助应用研究等。这些研究对指导兽医流行病学研究人员将GIS更好地应用于禽流感防控具有帮助意义。ResearchProgress of Forecasting and Early Warning of AvianInfluenza Based on GISGeographicinformation system(GIS)is a comprehensive tool for storing,analyzing,processing and presenting data relatedto geographic environment,which couldintegrate and analyze the data regarding influenza cases,climate,society andothers,and identify all risk factors foroccurrence and spreading of influenza by use of statistics or mathematicalmodels to forecast and visually warn any trends of influenza. In recent years,a large number of studies on risk factorsanalyzing and forecasting of avian influenza(AI)had been carried out by scholars inboth home and abroad by means of GIS,including,studies on the risk factors andforecasting of human infection with H7N9 influenza,H5 highly pathogenic avian influenza(HPAI)and wild AI,studies on the distributioncharacteristics of different genotypes of viruses,as well as theapplication researches on the supporting of veterinary decision-making. Thesestudies would provide some references for veterinary epidemiologists toeffectively control AI by GIS.全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191009.1107.028.html
2019-10-24
新疆某猪场母猪繁殖障碍病病原学检测和毒株鉴定
2018年4月,新疆某规模化繁育猪场出现大量母猪流产,产死胎、木乃伊胎等繁育障碍症状。为确诊病因,采集胎儿组织和母猪流产分泌物,通过普通PCR、RT-PCR、real-time PCR技术,对猪繁殖与呼吸综合征病毒(PRRSV)、伪狂犬病病毒(PRV)、猪细小病毒(PPV)、弓形虫4种病原进行检测。结果显示,PRRSV检测呈阳性,PRV、PPV和弓形虫检测呈阴性。通过PRRSV特异性引物NSP2进行PRRSV毒株鉴定,确定该猪场存在PRRSV类NADC30毒株。结果表明,类NADC30毒株是导致该猪场出现母猪繁殖障碍的重要病原,说明该新型毒株已经蔓延到新疆地区,并开始对该地生猪养殖业造成危害。本试验为新疆地区猪场母猪繁殖障碍的病因调查提供了依据。Pathogen Detection of Sow Reproductive Disorder Disease and Identification of Relevant Strain in a Swine Farm in Xinjiang In April 2018,some symptoms of reproductive disorders,such as abortion,stillbirth and mummified fetus,appeared in a large number of sows in a scaled breeding farm in Xinjiang. In order to identify all related causes,some fetal tissues and abortive secretions from sows were sampled to detect porcine reproductive and respiratory syndrome virus(PRRSV),pseudorabies virus(PRV),porcine parvovirus(PPV)and toxoplasma gondii through normal PCR,RT-PCR and real time PCR technology respectively. The results showed that the samples were positive for PRRSV,and negative for PRV,PPV and Toxoplasma gondii. The NADC30-like strain of PRRSV was identified in the farm by NSP2,the specific primer of PRRSV. Besides,the reproductive disorders in the farm may be mainly caused by the NADC30-like strain,indicating that the new strain had been spreading to Xinjiang and begun to bring great harm to local pig-production industry. In view of that,some references were hereby provided for investigating the causes of sow reproductive disorders in Xinjiang. 全文下载链接:http://kns.cnki.net/KCMS/detail/37.1246.S.20190903.1708.038.html
2019-09-17
2013—2017年我国小反刍兽疫病毒H基因分子演化特征
为研究我国小反刍兽疫病毒(PPRV)的分子流行特点,对2013—2017年我国37株PPRV流行毒株进行血凝蛋白(H)基因序列测定和生物信息学分析。结果显示:37个毒株H基因核苷酸序列之间的遗传距离为0~0.007 7,变异分布在41个位点,H蛋白氨基酸序列之间的遗传距离为0~0.013 2,变异分布在24个位点。与15株代表毒株进行序列比对发现,2013—2017年我国37株PPRV流行毒株H基因的13个位点发生了核苷酸序列突变,其中9个导致了氨基酸序列的改变。以最大似然法构建分子进化树,发现2013—2017年我国流行的37个毒株构成基因4系中一个独立的进化小分支。本研究阐明了2013—2017年我国PPRV H基因的分子演化特征,从而为该病控制和消灭策略的制定提供了数据支持。Molecular Evolution Characteristics of H Gene of Peste Des Petits Ruminants Virus in China During 2013 to 2017In order to study molecular characteristics of peste des petits ruminants virus(PPRV)in China,37 prevalent strains of PPRV collected from 2013 to 2017 were used for hemagglutinin(H)gene sequencing and bioinformatics analysis. The results showed that the genetic distance among nucleotide sequences of H gene of these strains was 0 to 0.007 7,involving 41 variable sites,and the genetic distance among amino acid sequences of H protein ranged from 0 to 0.013 2,with 24 variable sites. Based on the sequence alignment against 15 reference strains,it was found that the mutation of nucleotide sequences took place in 13 variable sites of H gene of all these strains,of which,9 nucleotide mutations resulted in the change of amino acid sequence. In addition,a molecular evolutionary tree was constructed by maximum likelihood,it was found that the 37 studied strains could be grouped into a distinct clade in lineage IV. In short,the molecular evolution characteristics of H gene of PPRV in China from 2013 to 2017 were stated in this paper,which would provide data supports to formulate relevant strategies for control and eradication of the disease. 全文下载链接:http://kns.cnki.net/KCMS/detail/37.1246.S.20190903.1708.010.html
2019-09-17
荧光原位杂交在布鲁氏菌和牛结核分枝杆菌检测中的应用
荧光原位杂交技术可快速鉴定病原菌,较传统病原分离鉴定方法具有明显优势。针对我国动物布鲁氏菌病和牛结核病这两个重要的人兽共患病,目前还没有标准的荧光原位杂交检测方法可供参考。为建立布鲁氏菌和牛结核分枝杆菌荧光原位杂交检测方法,快速诊断动物布鲁氏菌病和牛结核病,利用布鲁氏菌探针Bru-996和结核分枝杆菌探针MTB770,通过优化杂交温度、杂交时间和样品处理等关键条件,确定最佳检测程序;根据已知背景的菌株和临床样品,对Bru-996和MTB770探针的特异性和敏感性进行评价,最终建立了荧光复位杂交诊断方法。结果显示:反应条件优化后,该方法可在4 h内完成布鲁氏菌检测;荧光标记Bru-996探针与布鲁氏菌待检菌株的杂交结果均为阳性,而与结核分枝杆菌、禽结核分枝杆菌和大肠杆菌杂交结果均为阴性,并从5个已知背景的组织病料中成功检出布鲁氏菌。牛结核分枝杆菌检测则需要6~8 h,杂交前必须对样品用二甲苯和溶菌酶进行处理;MTB770探针可特异性识别并能从牛肺部结节中检出牛结核分枝杆菌。结果表明,荧光原位杂交方法快速、简便,而且Bru-996和MTB770探针分别在布鲁氏菌和牛结核分枝杆菌检测上具有较高的特异性,可替代传统的病原分离鉴定,作为动物布鲁氏菌病和牛结核病的实验室确诊方法。 Application of Fluorescence in Situ Hybridization in Detection of Brucella and Mycobacterium bovisCompared with traditional methods of pathogen isolation and identification,fluorescence in situ hybridization(FISH)technology could be used to rapidly identify pathogenic bacteria due to its obvious advantages. However,there has been no any formal FISH technology for two kinds of major zoonoses(animal brucellosis and bovine tuberculosis)in China. In order to develop the FISH technology for rapidly diagnosing the two diseases,the probes of Brucella(Bru-996)and Mycobacterium tuberculosis(MTB770)were used to determine the optimal detection procedure by optimizing the temperature and time of hybridization,dealing with samples and optimizing other important conditions. Then the specificity and sensitivity of the two probes were evaluated based on strains with known background and clinical samples. The results showed that Brucella could be detected within 4 hours after reaction conditions were optimized. The hybridizations between Bru-996 probe and Brucella strains were positive,and that between Bru-996 probe and Mycobacterium bovis,Mycobacterium avium and Escherichia coli were all negative;and Brucella was successfully detected from five tissue materials with known background. It would take 6 to 8 hours when Mycobacterium bovis was detected,and samples must be treated with Xylene and Lysozyme prior to hybridization. Mycobacterium bovis could be identified and detected from bovine pulmonary nodules by MTB770 probe. In view of the advantages of FISH technology of rapidity and convenience,and the high specificity of Bru-996 and MTB770 probes in detection of Brucella and Mycobacterium bovis,it was believed that the developed FISH technology could replace relevant traditional methods and be used to diagnose animal brucellosis and bovine tuberculosis in laboratories. 全文下载链接:http://kns.cnki.net/KCMS/detail/37.1246.S.20190903.1708.032.html
2019-09-17
4株猪流行性腹泻病毒的HA和HI试验
为观察猪流行性腹泻病毒(PDEV)的血凝特性,进而为建立PEDV HA和HI试验,评估疫苗免疫抗体水平奠定基础,将CV777、DR13、YN13和YN144共4株PEDV毒株,在37 ℃下,分别用0、5、10、50、100 μg/mL胰蛋白酶处理30 min,然后用鸡、兔和猪红细胞进行血凝(HA)试验;用已经鉴定为PEDV抗体阳性的血清和乳清分别进行血凝抑制(HI)试验。结果显示:4株PEDV毒株只与兔红细胞发生凝集反应;CV777、DR13毒株的HA现象需要用5 μg/mL的胰蛋白酶进行预处理,但是YN13和YN144毒株的HA现象不需要胰蛋白酶作用;该凝集现象能够被PEDV抗体特异性抑制。结果表明,PEDV能够凝集兔的红细胞,不能凝集鸡和猪的红细胞,因而可以用兔红细胞建立PEDV的HA和HI试验方法。 HA-HI Test for Four Strains of Porcine Epidemic Diarrhea VirusIn order to observe the hemagglutination(HA)of porcine epidemic diarrhea virus(PDEV)and to lay a foundation for development of HA-HI test for PEDV and evaluation of the antibody level,four strains of PEDV,including CV777,DR13,YN13 and YN144,were treated with trypsin of 0,5,10,50 and 100 μg/mL respectively for 30 minutes at 37 ℃,followed by HA test by use of red blood cells(RBC)of chicken,rabbit and pig respectively;then hemagglutination inhibition(HI)test was carried out with serum and whey that had been identified to be PEDV-positive. The results showed that four strains of PEDV only agglutinated with rabbit RBC. After CV777 and DR13 strain were pretreated with 5 μg/mL trypsin,the HA phenomenon occurred,while no trypsin was needed for YN13 andYN144 strain. In addition,HA could be specifically inhibited by antibody against PEDV. In conclusion,rabbit RBC rather than chicken and swine RBC could be agglutinated by PEDV,therefore,the HA-HI test for PEDV could be developed by use of rabbit RBC. 全文下载链接:http://kns.cnki.net/KCMS/detail/37.1246.S.20190903.1708.036.html
2019-09-17
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