口蹄疫病毒病毒样颗粒研究进展

口蹄疫病毒病毒样颗粒(virus-like particles,VLPs)是不含口蹄疫病毒RNA的空衣壳结构,由口蹄疫病毒的4种结构蛋白体外自组装形成的,形态上与天然病毒粒子相似,具有很强的免疫原性和生物学活性。文章介绍了口蹄疫病毒衣壳的组装、口蹄疫病毒VLPs在国内外的研究进展和影响VLPs形成的因素,并对VLPs在疫苗研制等方面的应用前景作了展望。     

犬细小病毒病毒样颗粒研究进展

犬细小病毒病毒样颗粒(VLPs)是利用表达系统体外表达犬细小病毒衣壳蛋白VP2后,自我组装而成,与天然病毒具有相似的形态和空间结构,其不含有病毒的遗传物质,且具有较好的安全性和免疫原性,能有效刺激机体产生良好的免疫反应。犬细小病毒病毒样颗粒在生物医药领域的应用主要集中于研发病毒样颗粒疫苗,而应用病毒样颗粒作为生物载体亦是目前的关注热点。此外,近年来利用不同表达系统表达犬细小病毒VP2蛋白后组装获得病毒样颗粒的报道层出不穷。基于此,论文综述了犬细小病毒病毒样颗粒的获得及其应用研究进展,以期为相关科研工作者提供参考。     

诺如病毒

正诺如病毒(Norovirus)病是一种非细菌性急性胃肠炎,多数呈局部规模性暴发。近几年的规模性感染尤为严重,自从人轮状病毒疫苗的广泛应用以来,诺如病毒就成为了全球公认的病毒性胃肠炎的最主要病原体,全球约50%以上的病毒性肠炎是由诺如病毒引起的。据美国疾病预防控制中心报道,每年约有2300万由诺如病毒感染造成的胃肠炎病例,据世界卫生组织统计,全球由于诺如病毒感染而引起的死亡人     

Nipah病毒

马来西亚东部沙捞越州当局在致命的Ｎｉｐａｈ病毒感染34人后,淘汰了5千头猪。这种病毒能导致人患致命的脑炎。去年在马来西亚半岛爆发此病,结果105人死亡。沙捞越州当局在首次检测中就证明这类病毒以猪为寄主,然后将州内4个地方14个猪场进行了隔离。去年Ｎｉｐａｈ病毒爆发导致马来西亚1百万头猪被淘汰,使该国的猪场数量从1770个减少到822个。该病毒又重新爆发的报道使消费猪肉最庞大的中国人社区引起极大恐惧,而大多数马来西亚人为穆斯林,不食猪肉。上月政府因避免猪被此病毒传染,命令马来西亚半岛的15个猪场作为安全保护被隔离起来。该病…     

埃博拉病毒

埃博拉病毒是因 1 976年在扎伊尔北部埃博拉河畔的一个村落里造成第一次大规模流行而得名。当时此病毒使该村数百人神秘地死去。 1 995年 4月在基克韦特市该病毒肆虐又造成 2 5 0人丧生。 1 996年在加蓬 ,又有 45人死于此病毒感染。埃博拉病毒的危险系数甚至要高于艾滋病病毒 ,不知这种病毒原生何处 ,只知道它可通过人体血液、唾液、汗水及其它分泌物接触传染。埃博拉病毒发作迅速 ,潜伏期约为3周 ,发病后 3 d内突发高热、严重头痛及肌肉痛 ,并伴有腹泻 ;并出现皮肤紫肿和水疱 ;到第 6 d时 ,开始体内出血。发病约 9d死亡。生存率只有 1 0 %…     

羊痘病毒

羊痘为我国法定的一类传染病,目前针对羊痘病毒尚无特效药。研究人员对羊痘病毒基因组进行了深入的研究,并建立了一系列针对羊痘病毒的检测方法。文章就近年来对羊痘病毒及其基因结构和检测方法的研究进行总结,为防治羊痘病毒提供理论依据。     

蜜蜂病毒

1.慢性蜜蜂麻痹病毒(Chronic Bee-paralysis Virus 慢性蜜蜂麻痹病毒是成年蜜蜂麻痹症的病原,该病毒在全世界蜜蜂中都可出现,并引起相似的症状,未发现有别的寄主,与已知的其它蜜蜂病毒无血清学关系。大多数病毒的质粒呈椭圆形,一端常有一个小突起,另一端平滑,病毒质粒无被膜、不等轴、直径为22μm,长度变化很大(图1),在离心时     

病毒净

2005年10月28日．农业部发布560号公告，将金刚烷胺、利巴韦林、病毒灵、阿昔洛韦等抗病毒药西药及其复方制剂列入兽药地方标准废止目录，不能用于动物疫病的防制，同时人类抗病毒药也禁用于兽医临床。于是成都恩威药业有限公司本着“愿众生幸福，社会吉祥”的理念．将传统道家“清净无为，天人一体、众生平等”的理念应用到动物健康产品的开发中．研制成功了高效安全、绿色环保的新型专用禽类抗病毒药，     

羊痘病毒

羊痘病毒能引起山羊痘、绵羊痘和牛的结节性疹块病.作者着重综述山羊痘和绵羊痘.羊痘病毒的基因组较大,山羊痘病毒和绵羊痘病毒的基因组非常相似,但自然条件下一般不会发生交叉感染.临床症状主要以发热和全身性丘疹或结节为特征,结合其临床症状实验室用透射电子显微镜检测病毒粒子的典型形态即可做出快速诊断.病毒培养和分离虽有较高的特异性,但耗时长;因羊痘病毒和副痘病毒有相似的血清型,一些血清学的诊断方法如琼脂扩散试验(AGID)、间接免疫荧光试验、检测抗体的ELISA等,因会出现抗体交叉反应而无法区分这2种病毒的感染;又因羊痘感染后主要引起细胞介导免疫,动物接触病原后仅产生低水平的中和抗体,故而病毒中和试验敏感性也不高.近年来,用于ELISA检测抗原的方法已经建立,具有较高的特异性;Western印迹试验利用羊痘病毒的P32抗原与待检血清反应,具有较好的敏感性和特异性,但价格昂贵、操作难.PCR可用来检查活体或组织培养品中羊痘病毒基因组;在此基础上发展了多重PCR技术,不但敏感性和特异性更强,且大量节省了时间和精力.治疗羊痘病毒无特效药,做好疫苗接种等防制措施很关键;目前采用活疫苗和灭活疫苗来控制本病,所有被鉴定的羊痘病毒毒株都有一个相同的主要中和位点,可以交叉保护.灭活苗的免疫保护期短.以羊痘病毒基因组作为其他反刍动物病原基因的载体,生产新一代羊痘病毒基因工程疫苗,具很大潜力.     

A SYBR Green Ⅰ Based on Real-time Quantitative RT-PCR Assay for Specific Detection of Peste des Petits Ruminants Virus

The study was aimed to establish a rapid and sensitive diagnostic method for the prevention and control of peste des petits ruminants.In this study,a fragment of PPRV N gene was amplified and cloned into pMD19-T cloning vector.Real-time quantitative PCR assay was performed using SYBR premix Ex Taq.The standard curve was plotted and the specificity,sensitivity and reproducibility of the assay were assessed.The generated standard showed linearity over the entire range from 2.82×10⁰ to 2.82×10⁷ copies/μL with a linear correlation（R²）of 0.992.The specificity of the assay showed that other viruses failed to show an amplification signal.The coefficient of variation（CV）values for intra- and inter-assay variability were low,ranging from 0.27%~2.77% and 0.41%~3.39%,respectively.The lower detection limit,based on plasmid copy number,achieved was 2.82 copies/μL and was 1 000 times more sensitive than conventional PCR assay.cDNA of 12 samples were tested using this method,9 were positive,and 3 were negative.The samples were also tested using conventional PCR,7 were positive and 5 were negative,proving that the two-step SYBR Green Ⅰ based Real-time quantitative RT-PCR assay reported here were more sensitive than conventional PCR.The establishment of this detection method is of great significance to rapid and sensitive diagnosis of peste des petits ruminants and preventing the spread of peste des petits ruminants.     

实时荧光定量RT-PCR检测小反刍兽疫病毒方法的建立

本试验旨在建立一种快速、灵敏的诊断小反刍兽疫的方法。本研究通过RT-PCR方法扩增小反刍兽疫病毒N基因,连接到pMD19-T克隆载体上,构建质粒标准品。根据GenBank中中国流行毒株及Nigeria 75/1疫苗株N基因保守序列设计引物,利用SYBR Green Ⅰ法进行实时荧光定量PCR,建立标准曲线,并进行特异性试验、敏感性试验和重复性试验。结果表明,在2.82×10⁰~2.82×10⁷拷贝/μL范围内,Ct值与质粒拷贝数对数值呈良好的线性关系,标准曲线线性关系R²值为0.992;其他病毒无特异性扩增曲线,特异性良好;批内变异系数为0.27%~2.77%,批间变异系数为0.41%~3.39%,重复性较好;检测灵敏度可达2.82拷贝/μL,是普通PCR的1 000倍。用该方法对12份cDNA样品进行检测,9份为阳性,3份为阴性,而普通PCR检测,7份为阳性,5份为阴性,说明本方法比普通PCR灵敏度高。本检测方法的建立对快速、灵敏诊断小反刍兽疫,防止疫情的扩散具有重要意义。     

山羊痘病毒LAMP检测方法的建立

为了建立适用于基层养殖场快速、可视化的山羊痘病毒(GTPV)环介导等温扩增技术(LAMP)检测方法,根据GenBank公布的GTPV ORF103保守区核苷酸序列为靶序列,设计并合成一组特异性LAMP引物,优化反应条件(时间、温度)和反应体系(dNTP、内外引物浓度比),并进行特异性和灵敏度试验。结果表明,所建立的LAMP方法在65℃时反应50 min即可获得清晰的梯状条带;扩增物经10×SYBR GreenⅠ荧光染料染色,紫外线照射或日光下即可直接判定结果;灵敏度试验结果表明,该方法能够检出的GTPV核酸最低浓度为1.287 ng/mL,与常规PCR方法相比,灵敏度增高100倍;该方法特异性良好,与小反刍兽疫病毒(PPRV)、羊支原体(Mccp)、羊口疮病毒(ORFV)均无交叉反应;对10份临床样品检测结果显示,该方法与传统PCR检测结果一致。建立的LAMP检测方法可用于临床山羊痘病毒的检测。     

小反刍兽疫病毒RT-LAMP检测方法的建立

利用逆转录环介导等温核酸扩增技术(RT-LAMP)建立了小反刍兽疫病毒快速检测方法,同时评价了该方法的灵敏性和特异性。结果表明,根据小反刍兽疫病毒N基因保守区域设计的LAMP引物能够在63℃恒温下,1小时内实现目的核酸的大量扩增,由于在检测前加入荧光指示试剂,检测结果可以直接用肉眼判断,避免了由于开盖检验带来的扩增产物污染导致的假阳性。该检测体系具有较高的特异性,只能特异性地检测目的病毒,与其他同属的病毒或类似病毒等无交叉反应;具有较高的检测灵敏度,比普通RT-PCR灵敏性高10倍,与荧光RT-PCR的灵敏度相当。     

小反刍兽疫病毒反向遗传学研究进展

小反刍兽疫(PPR)是由小反刍兽疫病毒(PPRV)引起的山羊、绵羊等小反刍动物的急性、高度接触性传染病。反向遗传学是在获得生物基因信息的基础上,对基因进行突变、缺失等操作,进而研究基因变化对表型的影响。本文综述了包括PPRV在内的单股负链RNA病毒反向遗传学的最新研究进展,以期为PPRV及同科属病毒的反向遗传操作系统的建立提供新的思路。     

鸭坦布苏病毒和鸭瘟病毒二重荧光定量RT-PCR方法的建立

The study was conducted to establish the duplex Real-time PCR assay for detecting both duck tembusu virus (DTMUV) and duck plague virus (DPV). According to the sequences of DTMUV E gene and DPV UL6 gene in GenBank, two sets of specific oligonucleotide primers for DTMUV and DPV along with two TaqMan probes were designed. The duplex Real-time PCR assay was developed through optimization of reaction conditions and validation of specificity, sensitivity and repetitiveness of the method. The sensitivity of the assay were both 100 template copies for DTMUV and DPV. There was no specific bands of the same sizes were amplified from other duck pathogens, such as duck Newcastle disease virus, duck hepatitis virus, muscovy duck parvovirus, duck circovirus, H9 subtype avian influenza virus, egg drop syndrome virus. This duplex Real-time RT-PCR assay is a sensitive, quick, specific and quantitative test for detection of DTMUV and DPV, and will be useful for the control of these viruses in ducks.     

一种鉴别尼帕病毒和高致病性猪繁殖与呼吸综合征病毒二重荧光RT-PCR检测方法的建立

为建立一种快速、敏感和特异地鉴别尼帕病毒(NiV)和高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)的检测方法,本试验以NiV M基因和HP-PRRSV nsp2基因为靶序列,通过优化反应条件建立了一种二重荧光RT-PCR检测方法,并对该方法的特异性、定量线性范围、敏感性和重复性进行了评价及初步应用.结果显示,用该方法检测NiV M基因和HP-PRRSV nsp2基因的RNA标准对照(NiV-M-RNA和HP-PRRSV-nsp2-RNA),线性范围分别为4.6×10¹~4.6×10⁷和4.1×10¹~4.1×10⁸拷贝/μL;最低检出限分别为46和4.1拷贝;该方法组内试验和组间试验的变异系数均小于2.0%,显示出良好的可重复性;该方法仅对NiV和HP-PRRSV呈现特异性扩增曲线,不与猪瘟病毒(CSFV)、猪流行性腹泻病毒(PEDV)、猪流感病毒(SIV)、猪细小病毒(PPV)、猪伪狂犬病病毒(PRV)和猪圆环病毒2型(PCV2)发生交叉反应.用该方法对236份猪实际样品进行NiV和HP-PRRSV核酸检测,所有样本的NiV检测结果均为阴性,8份样本的HP-PRRSV检测结果为阳性.本研究建立的方法为猪实际样本中NiV和HP-PRRSV的鉴别检测提供了一种快速、敏感和特异的技术手段.     

Establishment of a Duplex Real-time RT-PCR Assay for the Differential Detection of Nipha Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

To establish a rapid,sensitive and specific assay for the differential detection of Nipah virus (NiV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV),a duplex Real-time RT-PCR was developed with specific primers and probes targeting to the special sequences of NiV M gene and HP-PRRSV nsp2 gene by optimization of reaction conditions.The performance of the assay was linear ranging from 4.6×10¹ to 4.6×10⁷ copies/μL for RNA standard control of NiV M (NiV-M-RNA) and from 4.1×10¹ to 4.1×10⁸ copies/μL for RNA standard control of HP-PRRSV nsp2 (HP-PRRSV-nsp2-RNA),and detection limits of the assay was 46 copies for the NiV-M-RNA and 4.1 copies for the HP-PRRSV-nsp2-RNA,respectively.The coefficients of variation (CVs) of both inter-assay and intra-assay repeatability were less than 2.0%,showing good repeatability.The assay was able to specifically detect NiV and HP-PRRSV simultaneously without cross-reaction with classical swine fever virus (CSFV),porcine epidemic diarrhea virus (PEDV),swine influenza virus (SIV),porcine parvovirus (PPV),pseudorabies virus (PRV) and porcine circovirus type 2 (PCV2).Of the 236 samples from pigs for both NiV and HP-PRRSV detection by the established assay,all the samples were negative for NiV,8 samples were HP-PRRSV positive.In conclusion,this assay offers a useful approach for the differential detection of NiV and HP-PRRSV in clinical specimens from the pigs.     

Establishment of a Real-time Fluorescent Recombinase Polymerase Amplification (RPA) for the Detection of African Swine Fever Virus

African swine fever (ASF), which caused by African swine fever virus (ASFV), is an acute, highly contagious disease characterized by high fever. It leads to serious economic losses in pig industry. 3 assemblies of primers and probes targeting were designed to amplify ASFV B646L (p72) gene using recombinase polymerase amplification (RPA) technology. The Real-time fluorescent RPA method was established after screening of primers and probes, optimization of reaction conditions, tests of sensitivity, specificity and repeatability. The results showed that the method could detect 10 copies of DNA within 20 min at 39℃. No cross-reaction was found when testing swine fever virus, porcine circovirus type 2, porcine parvovirus, pseudorabies virus. According to the fluorescence intensity from 5.5×10⁶ to 5.5×10⁰ copies/μL at each time point, the coefficient of variation was 0.38% to 28.30%. In conclusion, this method could be used for the qualitative detection of ASFV pathogen, which might provide technical support for the early diagnosis of ASFV infection in China, and was also of great significance for the development of corresponding control measures.