锦鲤疱疹病毒

摘要： 为提升实验室对锦鲤疱疹病毒病(koi herpes virus disease,KHVD)病原检测的能力,加强水生动物防疫系统实验室能力建设,本实验室参加了2020年由全国水产技术推广总站组织的锦鲤疱疹病毒病病原检测能力测试.按照中国检验检疫科学研究院下发的《能力测试作业指导书》,结合水产行业标准《鲤疱疹病毒检测方法第1部分:锦鲤疱疹病毒》(SC/T 7212.1-2011)对样品进行鉴定,检测结果为满意.另外,本实验室选用3种商品化的锦鲤疱疹病毒实时荧光定量PCR检测试剂盒(A、B、C),以本次参测盲样为样品进行了试剂比对,试验结果表明:只有A与作业指导书要求的《SC/T 7212.1-2011》检测结果完全相同,B和C均未检测出阳性盲样.

摘要： 对我国江苏地区养殖锦鲤暴发性鲤疱疹病毒病(koiherpes virus disease,简称KHVD)的病原进行鉴定.2018年5月,江苏省一锦鲤养殖场暴发不明原因传染疾病,病鱼肝、脾、肾脏中未分离到细菌.透射电镜观察发现大量球状病毒粒子.提取自然发病鱼的鳃、肾、脾、肝组织DNA作为模板进行PCR检测,结果显示为阳性.Blast比对结果显示,扩增序列与锦鲤疮疹病毒(koi herpesvirus,简称KHV)胸苷激酶(thymidine kinase,简称TK)基因和KHV DNA聚合酶(SPH)基因核苷酸序列同源性为99％.对病毒株的TK基因全长序列进行系统发育分析,证实该毒株属于KHV亚洲型毒株.

摘要： 鲤浮肿病毒(Carp Edema Virus,CEV)和锦鲤疱疹病毒(Kio Herpes Virus,KHV)皆可感染锦鲤,对锦鲤养殖造成严重危害.本研究旨在建立一种可同时检测CEV和KHV的三重PCR检测方法.参考日本学者Oyamatsu等建立的nest-PCR方法和锦鲤疱疹病毒行业标准SC/T 7212.1—2011《鲤疱疹病毒检测方法第1部分:锦鲤疱疹病毒》,分别选取扩增CEV P4a基因的548 bp片段,KHV TK基因的409 bp片段和KHV Sph基因的292 bp片段的3对特异性引物进行组合,考虑多重PCR扩增过程中的影响因素,优化退火温度和引物浓度,进行敏感性和特异性试验.结果显示,该方法能特异地扩增出3条目的条带,大小分别是548 bp、409 bp和292 bp,最佳退火温度为53°C,引物浓度均为0.3μmol/L.该方法对CEV和KHV的检测限均为300 copies/μL,同时与其他水生动物病毒均无交叉反应.因此,本研究建立的方法可用于锦鲤样品中CEV和KHV的快速检测.

摘要： 为开发针对鲤疱疹病毒3型(Cyprinid herpesvirus 3,CyHV-3)[又称为锦鲤疱疹病毒(Koi herpesvirus,KHV)]的DNA疫苗,该研究将CyHV-3 ORF65插入pEGFP-N1,构建pEGFP-ORF65重组质粒,通过转染实验与活体成像实验证实pORF65-EGFP融合蛋白可以在建鲤(Cyprinus carpio var.Jian)体外、体内表达;重组质粒按照3μg·尾?1的剂量尾柄肌肉注射免疫10 g左右的建鲤鱼苗,免疫3次,ELISA检测表明pEGFP-ORF65免疫后可以显著提高血清特异性抗体水平;攻毒实验显示,CyHV-3攻毒21 d后,PBS组、pEGFP-N1组和pEGFP-ORF65组的建鲤成活率分别为25％、30％ 和90％,攻毒建鲤的成活率显著提高(P<0.01).该研究为ORF65作为DNA疫苗靶基因应用于CyHV-3的防控提供了依据.

摘要： 鲤浮肿病毒(Carp Edema Virus,CEV)和锦鲤疱疹病毒(Kio Herpes Virus,KHV)皆可感染锦鲤,对锦鲤养殖造成严重危害。本研究旨在建立一种可同时检测CEV和KHV的三重PCR检测方法。参考日本学者Oyamatsu等建立的nest-PCR方法和锦鲤疱疹病毒行业标准SC/T 7212.1—2011《鲤疱疹病毒检测方法第1部分:锦鲤疱疹病毒》,分别选取扩增CEV P4a基因的548 bp片段,KHV TK基因的409 bp片段和KHV Sph基因的292 bp片段的3对特异性引物进行组合,考虑多重PCR扩增过程中的影响因素,优化退火温度和引物浓度,进行敏感性和特异性试验。结果显示,该方法能特异地扩增出3条目的条带,大小分别是548 bp、409 bp和292 bp,最佳退火温度为53°C,引物浓度均为0.3μmol/L。该方法对CEV和KHV的检测限均为300 copies/μL,同时与其他水生动物病毒均无交叉反应。因此,本研究建立的方法可用于锦鲤样品中CEV和KHV的快速检测。

摘要： In May 2015, an unknown disease caused severe economic losses in a common carp (Cyprinus carpio) culture pond in Sichuan Province. The mortality rate was 80%. Necropsy, bacteriologic test, histological examina-tion, pathogenicity tests, virus isolation, PC assay and phylogenetic analysis were performed to explore its causes. Principal symptoms included sunken eyes, hemorrhage at the base of the pectoral and pelvic fins, severe gill nec-rosis and swollen kidney. Bacteriologic test was negative. Histopathologically, the most important changes were observed in the gills and kidneys. The respiratory epithelial cells of gill lamellae was swelling and/ or dropping and congestion of the blood vessels in the gill filaments. The base of branchial epithelium also exhibit hyperplasia, hy-pertrophy and cellular layer increased. The obvious disorder in structure, swelling, and disintegration and necrosis were found in epithelial cells of renal tubulus. Positive results were obtained from all detected samples according to a standard PCR diagnosis of koi herpesvirus (KHV/ CyHV-3) Sph gene proposed by Office International des Epizooties (OIE) by using gills and kidney tissue DNA isolated from diseased C. carpio. After filtration treatment the kidney and liver homogenate was injected intraperitoneally into 20 healthy common carp and the injected carp showed similar clinical symptoms as the fish that was naturally infected. C. carpio died acutely in the trial group with a cumulative mortality rate 90%. Tissue suspension was inoculated to the common carp brain (CCB) mono-layer cells and a typical cytopathic effect (CPE) was observed in CCB cells after three blind passages. Icosahedral viral particles were observed in cells under the electron microscope. The virus particles were spherical in shape measuring 180-200 nm in diameter. Based on Neighbor-Joining analyses of the TK gene sequences, phylogenetic tree was constructed and the result showed that the isolate belongs to Asian 1 genotype. This is the first report of KHV infection in cultured common carp associated with high mortality in Southwestern China. The result present in this study is important for virus evolution, classification, viral diagnosis and disease control.%2015年5月,四川某鲤养殖场暴发一种传染性疾病,导致鲤大面积死亡,死亡率高达80%.为研究此次疾病病因和流行规律,将病料进行解剖、细菌学检查、病理组织观察、PCR鉴定、病毒分离和系统进化分析.结果显示,发病鱼眼球凹陷,胸鳍及腹鳍出现出血斑,病鱼鳃严重坏死,肾脏肿大.细菌检查为阴性.组织病理学观察,病变最明显的是鳃和肾脏.病鱼鳃丝血管扩张充血,鳃小片呼吸上皮细胞肿胀、脱落.鳃丝基部的上皮细胞大量增生,层次增多.肾小管上皮细胞组织结构紊乱,细胞肿胀,管腔变狭小,有崩裂和坏死现象.提取病鱼的肾脏和鳃组织DNA为模板,针对世界动物卫生组织(OIE)推荐的检测锦鲤疱疹病毒(KHV)的Sph基因进行PCR检测,出现特异性扩增产物.将病鱼的肾脏和肝脏组织研磨过滤灭菌后,腹腔注射20尾健康鲤,实验组表现为急性死亡(累积死亡率为90%),出现与自然发病鱼相同的症状.将组织匀浆接种到普通鲤脑细胞系(CCB),盲传3代后可稳定地观察到典型的细胞病变.细胞培养物染色超薄切片电镜观察结果显示,病毒为有囊膜的球状,病毒粒子直径为180~200 nm.对分离株的TK基因全长序列进行系统发育分析,证实该毒株属于KHV亚洲1型毒株.本研究首次报道我国西南地区养殖鲤中KHV感染引起大面积死亡,为KHV起源进化、分类以及疾病诊断和防控提供重要依据.

摘要： To investigate the cause of sudden death of the koi(Cyprinus carpio),the moribund koi that showed typical symptoms including sunken eyes and head, rotted gill and haemorrhages on the skin were examined. No bacteria were isolated from spleen, liver and kindey of diseased fish by bacteriological examination, but a large number of viral nucleocapsid were found in gill tissue by electron microscopy observation. After filtration treat-ment, the gills, brain, liver, spleen, and kidney tissue suspension was inoculated to carp fin cells, Cytopathic ef-fects (CPE) were presented 7 days post co-cultivation. In artificial infection test, the death rate of fish by infecting tissue homogenate and culture liquid of infected cell reached 93.3% and 86.7% respectively. Histopathological analysis showed that the target organs included the gill, liver and kidney. Positive results were obtained from dis-eased koi according to a PCR diagnosis of koi herpesvirus (KHV) proposed by Office International des Epizooties (OIE), and high concentration of KHV was detected in gill and kidney tissue by fluorescence quantitative PCR. Based on neighbor-joining analyses of the TK gene sequences, phylogenetic tree was constructed and the result in-dicated that the KHV isolated in our study was Asian genotype isolate. Furthermore, according to the sequence alignment of Marker I and Marker II regions of KHV, the KHV detected in this study belongs to I++II–genotype, and was named KHV-TJ1601. The present study demonstrated that the I++II–genotype KHVwas isolated from the naturally infected fish in North China for the first time, and provided important material for virus genesis, evolu-tion, classification, and vaccine preparation.%为分析近年来天津地区养殖鲤(包括锦鲤)暴发性死亡的病因,利用病原菌分离、细胞培养、电镜观察、组织病理切片、人工感染实验、PCR和荧光定量PCR检测、基因分型等方法对患病鱼及病原进行了研究.结果显示,在患病鱼体表未发现大量寄生虫;在肝、脾、肾等内脏组织中未能分离到细菌;在鳃组织中发现大量的圆形病毒颗粒;使用患病鱼组织滤液感染锦鲤鳍条原代细胞,可观察到典型的细胞病变效应(CPE),注射患病鱼组织滤液和产生病变的细胞上清液可分别导致健康锦鲤93.3%和86.7%的死亡率;通过病理组织切片观察,主要病变组织为鳃、肝脏和肾脏.采用世界动物卫生组织(OIE)推荐的锦鲤疱疹病毒(KHV)检测方法进行PCR检测发现KHV呈阳性,且KHV在鳃组织中含量最高,肾脏次之,脑组织中最少.结合TK基因全长序列建立系统进化树和基因型分析,证实此次分离到的KHV为亚洲型毒株,属于I++II–基因型,将其命名为KHV-TJ1601株.这是我国华北地区首次报道KHV I++II–基因型的存在,可为KHV的进化分析和疫苗制备提供基础资料.

摘要： 为了对鲤疱疹病毒3型(Cyprinid herpesvirus 3, CyHV-3)ORF136基因编码蛋白进行功能研究和血清学诊断,本实验通过对ORF136基因推导的第31~157位氨基酸序列进行PCR扩增,并与原核载体pET-32a(+)连接,转化至大肠杆菌Rosetta(DE3)感受态后进行IPTG诱导表达,将纯化后的重组蛋白免疫新西兰白兔(Oryctolagus cuniculus)以制备ORF136多克隆抗体, 运用Western blot和间接免疫荧光技术对抗体进行鉴定.结果表明, 重组融合表达蛋白大小与预期一致, 约为35 kD, 且主要分布在包涵体中.Western blot分析显示, 免疫兔后获得的纯化ORF136多克隆抗体能特异性识别纯化的CyHV-3和感染CyHV-3的KS细胞; 间接免疫荧光分析进一步表明ORF136多抗能识别感染CyHV-3的KS细胞.ORF136多克隆抗体的制备为ORF136蛋白功能研究和CyHV-3血清学诊断方法的建立提供了重要基础.%Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is the etiologic agent of koi herpesvirus disease(KHVD)causing morbidity and mortality in common carp and koi Cyprinus car-pio L.populations around the world.Recently,knowledge about diagnosis and detection based on nucleic acid has been reported, but validation of serological techniques for virus infection is limited due to lack of effective anti-bodies. Additionally, fundamental research on the function of structural proteins from CyHV-3 is necessary to understand the relationship between host and virus. ORF136 is one of the predicted envelope proteins incorporated into mature virions. To study the function of proteins encoded by the ORF136 gene of CyHV-3 and to establish serological methods for detection of CyHV-3, the predicted antigenic determinant based on amino acid sequences of proteins encoded by ORF136 was amplified using PCR. Then, the recombinant prokaryotic expression vector was constructed by connecting the cloned fragment to prokaryotic expression vector pET-32a (+), used for trans-formation of E.coli Rosetta (DE3) and protein expressions induced by IPTG. New Zealand white rabbits were immunized with the recombinant proteins of pET-32a-ORF136 four times. Furthermore, the antiserums were col-lected and affinity purification was used to obtain polyclonal antibody after 66 days. Characterization of the poly-clonal antibody against the ORF136 protein was further analyzed using western blot and indirect immunofluores-cence assays. The expected product size of 381 bp was obtained and the recombinant prokaryotic expression vec-tor pET-32a-ORF136 was confirmed to be constructed as expected using enzyme digestion. As SDS-PAGE analy-sis indicated that the molecular weight of recombinant protein pET-32a-ORF136 was consistent with the expected size (about 35 kD) and was distributed in the inclusions. Western blot analysis further showed that the purified polyclonal antibody could specifically recognize purified CyHV-3 virions and KS cells infected with CyHV-3. Moreover, immunofluorescence staining showed that specifically green fluorescence was present in the cytoplasm of KS cells infected with CyHV-3, but not in the negative control, further suggesting that CyHV-3 was recognized by the polyclonal antibody. The purified recombinant proteins and effective antibodies are necessary to develop serological methods to detect antigens, except nucleic acid detection method. The purified recombinant proteins could be used as antigens to capture antibodies against CyHV-3 from common carp or koi exposed to KHVD. Similarly, development of a double antibody sandwich ELISA for detection of CyHV-3 also requires an effective polyclonal antibody to capture antigens released from the host. Antibodies produced by recombinant proteins from rabbit or mouse provides the foundation for functional research into structural and nonstructural proteins. In this study, the polyclonal antibody against the ORF136 protein was prepared and analyzed using immunoblotting and immunofluorescence techniques, which provides an essential and valid tool for further research into CyHV-3.

摘要： 为了鉴定鲤疱疹病毒3型(Cyprinid Herpesvirus 3, CyHV-3) 主要免疫原性蛋白,研究采用CyHV-3-T分离株感染CCB细胞系,运用蔗糖密度梯度超速离心方法对 CyHV-3进行纯化,纯化的病毒颗粒经 SDS -PAGE和考马斯亮蓝染色后,用锦鲤(Cyprinus carpio haematopterus)抗CyHV-3阳性血清进行Western blotting分析和液相色谱串联质谱鉴定.结果表明,透射电镜下可观察到大量完整囊膜包裹或只有裸露核衣壳的CyHV-3颗粒,Western blotting结果显示抗CyHV-3阳性血清与多种病毒蛋白具有明显特异性免疫反应,质谱鉴定表明其中4种免疫原性蛋白分别为ORF92、ORF66、ORF72和ORF81,其中ORF66和ORF72为首次鉴定的具有免疫原性衣壳蛋白.本研究将为CyHV-3血清学诊断方法的建立、亚单位疫苗或DNA疫苗的研制提供更多候选抗原.%CCB cell line was infected with CyHV-3 isolate T in this study to identify major immunogenic proteins from CyHV-3.Virus suspensions were collected after freezing and thawing repeatedly when apparent cytopathic effect (CPE) was observed and purification was performed by 20% to 66% sucrose density gradient in combination with ultracentrifuga-tion.The obtained purified virions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS -PAGE) and stained with coomassie brilliant blue.Furthermore, western blotting analysis was performed by using anti-ser-um from koi naturally infected with CyHV-3.Some protein bands showed clearly and specifically immunoreactive, they were cut for further characterization through liquid chromatography tandem mass spectrometry.Transmission electron microscope (TEM) results showed that the purified CyHV-3 virions contained intact envelope or naked nucleocapsid with high purity and typical characteristic in line with previous report.Quite some protein bands showed immunoreactive using anti-serum against CyHV-3 from koi by western blotting, 4 of which clearly immunogenic proteins were further identified as capsid protein ORF92, ORF66, ORF72 and envelope protein ORF81 through LC-MS/MS, respectively.In addition, ORF66 and ORF72 was also the newly identified capsid protein showed immunogenicity.This study might provide more antigen can-didates for development of serological diagnosis methods for CyHV-3 and DNA vaccine or subunit vaccine preparation.

摘要： 探讨合适的锦鲤疱疹病毒(Koi Herpesvirus KHV)保存方法,促进KHV流行病学调查与防控以及疫苗开发等相关工作的开展.方法比较了5种不同保存条件对锦鲤疱疹病毒的影响,包括1):-80°C超低温保存;2):液氮超低温保存;3):50％磷酸甘油缓冲液保存;4):乙醇保存;5):异丙醇保存.结果表明5种方法保存的患病锦鲤组织利用PCR检测都为阳性;组织匀浆后感染健康锦鲤,保存在50％磷酸甘油缓冲液、乙醇和异丙醇的患病组织失去了感染能力,而保存在-80°C超低温冰箱和液氮中的组织均可以使健康锦鲤感染KHV;将5种保存方法保存的组织匀浆后感染CCB细胞,只有保存在50％磷酸甘油缓冲液中的患病鱼组织可以感染CCB细胞.此外,将细胞病毒液存放于4°C冰箱、20°C冰箱中病毒感染力(TCID50/0.1mL)显著下降(P＜0.05),而将病毒液存放于-80°C超低温冰箱、液氮中病毒的感染力无明显差异(P＞0.05).可以利用-80°C超低温、液氮超低温长期保存患病组织病料和细胞病毒液,而保存在50％磷酸甘油缓冲液的组织病料有利于进行病毒的细胞分离.

摘要： 本发明公开了一种鲤浮肿病毒和锦鲤疱疹病毒双重实时荧光定量PCR检测方法，该方法包括以下步骤：第一、合成引物与TaqMan探针：根据CEV P4a基因序列，对引物序列进行优化，上游引物第1个碱基改为简并碱基W，下游引物第1个碱基改为简并碱基R；根据KHV ORF7基因保守序列，设计1对特异性引物和1条TaqMan探针；分别使用FAM和VIC作为探针报告基团，BHQ1作为探针淬灭基团；第二、确定反应体系及条件：采用20μL反应体系，2×Probe PCR Master Mix 10μL，上、下游引物和探针引物终浓度在0.2μmol/L～0.8μmol/L之间，QN ROX参考染料0.1μL，模板2.5μL，DEPC水补足20μL；反应程序为：95°C预变性2min，1个循环；95°C5s，50～60°C退火31s，40个循环。

摘要： 本发明提供一种检测锦鲤疱疹病毒的试剂盒，适用于聚合酶连锁反应，试剂盒包含引物对，引物对包含如序列辨识编号1所示的正向引物和序列辨识编号2所示的反向引物。由此，可以快速且特异性地自待测样本中检测锦鲤疱疹病毒。

摘要： 本发明公开了一种锦鲤疱疹病毒RPA引物组、探针和检测试剂盒及其应用，所述RPA引物组序列如SEQ ID NO.4～19所示，所述探针序列如SEQ ID NO.2～3所示。本发明中提供的探针和引物灵敏度高，检测效果稳定，特异性强。基于此构建的检测试剂及检测试剂盒均具有很高的检测灵敏度，最低检测限为100copy/μL，检测时间仅需4～18min，相比KHV检测金标准PCR及real‑time PCR均具有时间和检测精度上的优势，具有极高的应用价值。

摘要： 本发明公开了抗锦鲤疱疹病毒的单克隆抗体制备方法及其应用，本发明制备的抗锦鲤疱疹病毒的单克隆抗体是由保藏编号为CCTCC NO：C201887的小鼠杂交瘤细胞株分泌产生的，单克隆抗体的效价为1：1.28×10

摘要： 本发明涉及：重组锦鲤疱疹病毒(KHV)或鲤疱疹病毒3(CyHV-3)，其在下列物种中具有免疫原性：鱼，优选鲤类，更优选鲤鱼(Cyprinus carpio)；疫苗，用于预防和/或治疗由重组锦鲤疱疹病毒(KHV)或CyHV-3引发的疾病。将5种重组疱疹病毒用于给予下列物种抵抗由锦鲤疱疹病毒(KHV)或鲤疱疹病毒3(CyHV-3)引发的疾病的免疫力：鱼，优选鲤类，更优选鲤鱼(Cyprinus carpio)。

摘要： 本发明公开了一种鲤浮肿病毒和锦鲤疱疹病毒三重PCR检测方法，该方法包括以下步骤：第一、选择与合成引物：选择扩增CEV P4a基因548bp片段，KHV TK基因409bp片段和KHV Sph基因292bp片段的3对特异性引物；第二、制备标准品：提取鲤浮肿病毒和锦鲤疱疹病毒的DNA，合成CEV P4a基因序列548bp片段，克隆到pEASY‑T1载体；合成KHV TK基因序列409bp片段和KHV Sph基因序列292bp片段，克隆到PUC57‑Kan载体上，构建CEV‑P4a、KHV‑TK和KHV‑Sph标准质粒，根据质粒浓度计算拷贝数；第三、确定三重PCR退火温度及优化引物浓度。

摘要： 本发明公开了一种鲤浮肿病毒和锦鲤疱疹病毒双重实时荧光定量PCR检测方法，该方法包括以下步骤：第一、合成引物与TaqMan探针：根据CEV P4a基因序列，对引物序列进行优化，上游引物第1个碱基改为简并碱基W，下游引物第1个碱基改为简并碱基R；根据KHV ORF7基因保守序列，设计1对特异性引物和1条TaqMan探针；分别使用FAM和VIC作为探针报告基团，BHQ1作为探针淬灭基团；第二、确定反应体系及条件：采用20μL反应体系，2×Probe PCR Master Mix 10μL，上、下游引物和探针引物终浓度在0.2μmol/L～0.8μmol/L之间，QN ROX参考染料0.1μL，模板2.5μL，DEPC水补足20μL；反应程序为：95°C预变性2min，1个循环；95°C5s，50～60°C退火31s，40个循环。

摘要： 本发明公开了一种锦鲤疱疹病毒RPA引物组、探针和检测试剂盒及其应用，所述RPA引物组序列如SEQ ID NO.4～19所示，所述探针序列如SEQ ID NO.2～3所示。本发明中提供的探针和引物灵敏度高，检测效果稳定，特异性强。基于此构建的检测试剂及检测试剂盒均具有很高的检测灵敏度，最低检测限为100copy/μL，检测时间仅需4～18min，相比KHV检测金标准PCR及real‑time PCR均具有时间和检测精度上的优势，具有极高的应用价值。

摘要： 本发明涉及一种锦鲤疱疹病毒的检测试剂盒和检测方法，所述试剂盒包括有引物，所述引物为：序列如SEQ ID NO.1和SEQ ID NO.2的一对外引物，序列如SEQ ID NO.3和SEQ ID NO.4所示的一对探针引物，以及序列如SEQ ID NO.5所示的一条交叉引物。本发明通过到设计到一组鲤疱疹病毒的检测引物，并且，通过优化调整该引物的用量比例，采用交叉引物等温扩增技术，取得了很好的扩增效果。而且，通过优化扩增体系中的其他参数，配合所设计的引物，实现了对鲤疱疹病毒的检测高特异性和高灵敏度的检测。

摘要： 本发明公开了一种基于锦鲤疱疹病毒ORF149的碳纳米管载核酸疫苗及应用，本发明成功构建了单壁碳纳米管载pcDNA‑ORF149核酸疫苗系统，提高鱼体抗体水平，其相对免疫保护率为81.9％，免疫水平远远高于其他免疫系统，具有在水产养殖和病原研究的应用潜质。