锦鲤疱疹病毒-CJ株ORF81基因的克隆及生物信息学分析

为了解锦鲤疱疹病毒中国吉林株(KHV-CJ) ORF81蛋白的结构特征和进化关系,用框镜鲤尾鳍原代细胞增殖KHV-CJ,提取其DNA,经PCR扩增,获得ORF81基因,将其克隆到pMD18-T载体中,构建重组质粒.应用生物信息学方法初步分析KHV-CJ ORF81基因的结构和功能,并与GenBank上已公布的3株KHV构建系统进化树.结果显示,获得了长771 bp的ORF81基因,编码256个氨基酸;预测ORF81基因的理论分子量为28 246.50 u,等电点为8.404;疏水性大于亲水性;信号肽切割部位最可能位于29位的S(丝氨酸);有4个跨膜区;抗原表位预测显示抗原性良好;结构预测显示,不存在N-糖基化位点、存在6个O-糖基化位点和11个磷酸化位点;系统进化树分析显示,与锦鲤疱疹病毒以色列株属同一分支.     

锦鲤疱疹病毒3型ORF126基因克隆及生物信息学分析

为研究锦鲤疱疹病毒3型(KHV-3)ORF126基因编码蛋白的功能,我们对该蛋白的结构特征进行研究分析。本实验采用PCR扩增技术获得KHV ORF126基因的完整序列,构建pMD19-T-ORF 126重组质粒,应用生物信息学方法分析ORF126基因编码蛋白的理化特征。结果显示:KHV ORF126基因翻译合成273个氨基酸;ExPasy预测该蛋白的理论分子量为29.9kDa,等电点为5.11;信号肽的切割部位最可能位于第19~20位氨基酸之间;跨膜区位于第145~168位氨基酸;抗原表位预测显示抗原性良好;编码蛋白不含N-糖基化位点,含有4个O-糖基化位点和17个磷酸化位点。     

拟态弧菌安徽分离株外膜蛋白基因OmpU的克隆测序与生物信息学分析

测序结果显示4株拟态弧菌OmpU基因片段长为849~876bp,编码283~292个氨基酸;拟态弧菌HX4分离株OmpU基因ORF长1 038 bp,编码346个氨基酸。生物信息学分析结果显示,OmpU基因在拟态弧菌安徽分离株之间及其与霍乱弧菌参考株间均具有较高保守性,彼此间核苷酸同源性和氨基酸同源性分别介于82.8%~99.6%和83.3%~100%;拟态弧菌OmpU蛋白N端前22个氨基酸组成信号肽,N末端有1个明显疏水区,并含有多个蛋白激酶磷酸化位点和糖基化位点;OmpU蛋白含有2个明显跨膜域,二级结构中富含β-折叠结构,在蛋白的53~66 aa、185~192 aa、215~219 aa和275~281 aa之间可能存在抗原表位。     

锦鲤疱疹病毒CJ株ORF27基因克隆及生物信息学分析

为了解锦鲤疱疹病毒中国吉林株（KHV-CJ）ORF27基因编码蛋白的结构特征和进化关系,采用镜鲤尾鳍原代细胞增殖KHV-CJ,提取其DNA,经PCR扩增,获得ORF27基因,将其克隆在pMD18-T载体中,构建重组质粒。应用生物信息学方法初步分析KHV-CJ ORF27基因的结构和功能,并与GenBank上公布的三株KHV构建系统进化树。结果显示：获得207bp的ORF27基因,编码69个氨基酸;预测ORF27编码蛋白的理论分子质量为7366.62Da,等电点为4.487;抗原表位预测显示抗原性良好;编码蛋白存在1个N-糖基化位点、4个O糖基化位点和5个磷酸化位点;系统进化树分析表明与锦鲤疱疹病毒美国株（KHV-U）属同一分支。     

锦鲤疱疹病春主要囊膜蛋白基因的PCR扩增与序列分析

为了进一步研究锦鲤疱疹病毒主要囊膜蛋白(KHV-MEP)的功能及锦鲤疱疹病毒(KHV)的感染机制,根据KHV-MEP基因序列设计并合成1对引物,从自然感染KHV发病的锦鲤(Cyprinus carpio Koi)肝组织总DNA中扩增获得特异性基因片段.将所得基因片段克隆到pMD18-T Simple Vector载体中,获得重组质粒T-KMEP;酶切鉴定后进行序列测定,并采用氨基酸亲水性分析软件Tmpred对其编码氨基酸序列进行分析;在对该片段所编码氨基酸可能抗原位点分析的基础上,进行PCR改造构建原核表达载体,获得重组表达载体pBV-KMEP1和pBV-KMEP2.所获得的基因片段大小为771 bp,该基因片段与GenBank中已登录的KHV-MEP基因(AB178537)的同源性为100%,是一个完整的开放阅读框,所编码的蛋白由256个氨基酸组成,分子量为28.2kD,等电点(PI)为8.65.该序列含有4个跨膜区,可构成主要抗原决定簇.结果显示所获得的目的基因片段就是锦鲤主要囊膜蛋白全基因.     

锦鲤疱疹病毒主要囊膜蛋白基因的PCR扩增与序列分析

为了进一步研究锦鲤疱疹病毒主要囊膜蛋白（KHV-MEP）的功能及锦鲤疱疹病毒（KHV）的感染机制,根据KHV-MEP基因序列设计并合成1对引物,从自然感染KHV发病的锦鲤（Cyprinus carpio Koi）肝组织总DNA中扩增获得特异性基因片段。将所得基因片段克隆到pMD18-T Simple Vector载体中,获得重组质粒T-KMEP;酶切鉴定后进行序列测定,并采用氨基酸亲水性分析软件TMpred对其编码氨基酸序列进行分析;在对该片段所编码氨基酸可能抗原位点分析的基础上,进行PCR改造构建原核表达载体,获得重组表达载体pBV-KMEP1和pBV-KMEP2。所获得的基因片段大小为771bp,该基因片段与GenBank中已登录的KHV-MEP基因（AB178537）的同源性为100%,是一个完整的开放阅读框,所编码的蛋白由256个氨基酸组成,分子量为28.2kD,等电点（PI）为8.65。该序列含有4个跨膜区,可构成主要抗原决定簇。结果显示所获得的目的基因片段就是锦鲤主要囊膜蛋白全基因。     

猪繁殖与呼吸综合征病毒云南株的分离鉴定

<正>云南农业大学动物科学技术学院李富祥等用Marc145细胞从云南某猪场分离到两株猪繁殖与呼吸综合征病毒。PCR方法对NSP2基因进行扩增结果表明,分离株缺失了90个核苷酸。对分离株ORF5基因序列进行扩增和测序,进行同源性及遗传特性分析,结果表明,分离到的两株PRRSV位于进化树的同一个小分支上,其核苷酸同源性为     

传染性皮下及造血组织坏死病毒NJ株ORF3基因的原核表达及生物信息学分析

为探究本实验室分离的传染性皮下及造血组织坏死病毒NJ株(IHHNV-NJ)ORF3基因编码蛋白的结构特征,本实验根据ORF3基因序列设计引物,利用PCR方法克隆ORF3基因序列,并构建至原核表达载体p ET-32a(+)中。对成功构建的p ET32a-ORF3重组表达载体进行原核表达,获得49 ku的融合蛋白,符合预期大小。通过生物信息学软件对ORF3基因编码蛋白序列进行分析,结果显示,ORF3基因序列长度为990 bp,编码329个氨基酸;ORF3基因编码蛋白理论分子质量为37 385.2 u,等电点为7.22,为亲水性蛋白;该编码蛋白序列不存在跨膜区、信号肽切割位点;二级结构含有55.9%的α-螺旋、52.0%的β-折叠以及13.4%的β-转角;抗原表位分布较广泛,抗原性强;该编码蛋白序列不存在潜在的N-糖基化位点,存在13个潜在的O-糖基化位点和17个潜在的磷酸化位点。进化树结果表明,NJ株的ORF3基因编码蛋白序列与6株IHHNV序列同源性均高于96%,与厄瓜多尔株同源性最高,为99.7%。研究表明,ORF3基因编码IHHNV衣壳蛋白;O-糖基化位点可参与衣壳蛋白的组装过程及细胞侵染过程,磷酸化位点可参与病毒在对虾细胞内的增殖过程;ORF3编码蛋白序列保守性强,不影响病毒毒力和个体间感染能力。     

锦鲤疱疹病毒GZ1301株的分离与鉴定

2013年4月,广东省一锦鲤养殖场暴发不明病因疾病,濒死锦鲤在塘边游动缓慢直至死亡,死亡率高达100%。现场采样发现,发病锦鲤体长25 cm,眼球凹陷,胸鳍及腹鳍出现出血斑点,解剖发现内脏器官包括肝、脾、肾肿大。细菌分离结果显示,内脏器官肝脏和肾脏中未分离到细菌。提取自然发病鱼的肝、脾、肾、鳃组织DNA作为模板,采用世界动物卫生组织(OIE)推荐的锦鲤疱疹病毒(KHV)检测引物进行PCR扩增,均能扩增出预期大小的特异性产物。NCBI的Blast搜索结果显示,扩增序列与KHV胸苷激酶(thymidine kinase,TK)基因核苷酸序列同源性为99%。病鱼内脏组织研磨过滤除菌后,腹腔注射20尾锦鲤,可复制出与自然发病相似的症状,并于7 d内全部死亡。取病鱼的鳃和肾脏研磨过滤除菌后进行细胞感染实验,结果显示,组织滤液感染CCB细胞后,盲传5代可以观察到典型的细胞病变效应(CPE)。将出现典型CPE的CCB细胞进行超薄切片制备和电镜观察,电镜下病毒呈对称20面体,直径约100 nm。将出现典型CPE的细胞进行间接免疫荧光实验,可以观察到特异性荧光。根据TK基因全长序列建立系统进化树,证实该毒株为KHV亚洲型毒株,暂命名为KHV-GZ1301株。研究结果可为KHV起源进化、分类以及疾病防控提供重要材料。     

克氏原螯虾病原弗氏柠檬酸杆菌的分离鉴定及其药敏与黏附特性

为了明确安徽省当涂县淡水养殖克氏原螯虾暴发性疾病的病原,取濒临死亡的病虾分离病原。从肝胰腺中分离到一株优势细菌(命名为XLX1分离株),人工感染试验证实其具有较强的致病性;采用形态学检查、生理生化特性测定和细菌16S r RNA基因序列分析,确定XLX1分离株为弗氏柠檬酸杆菌。进一步对XLX1分离株进行药物敏感性、携带黏附素基因情况和细胞黏附性进行检测。结果显示:该分离株对头孢噻肟、庆大霉素、阿米卡星、硫酸新霉素、恩诺沙星、诺氟沙星、替考拉宁和米诺环素敏感或中敏,对其他7种测试药物呈现耐药。该分离株携带黏附素基因cfa和ure基因簇;序列分析发现ure ABC结构基因和ure D关键辅助基因高度保守,在虾源分离株与人源参考株间前者的核苷酸和氨基酸序列同源性分别在93.2%~98.3%和91.7%~97.4%,后者的核苷酸和氨基酸序列同源性分别在90.8%~98.3%和94.7%~98.7%;与人源参考株相比,虾源分离株Ure ABC结构蛋白中第294、600和608位氨基酸,以及Ure D蛋白中的第62和122位氨基酸发生了有意突变。XLX1分离株可以聚集方式黏附于EPC细胞周围,平均黏附菌数为29.8±5.3,但随黏附时间延长,EPC细胞出现病变。上述研究结果可为防控弗氏柠檬酸杆菌引起的水产动物疾病提供理论依据。     

黑龙江地区鲤春病毒血症病毒的分离与基因型分析

对2015—2016年黑龙江不同地区的40个养殖场送检的鲤(Cyprinus carpio)进行鲤春病毒血症病毒(spring viremia of carp virus,SVCV)的细胞培养分离、PCR鉴定、病毒滴度测定、病毒表面糖蛋白(glycoprotein,G)氨基酸序列聚类分析及基因分型研究。细胞培养结果显示,来自4个不同养殖场的鲤组织样本能够感染鲤上皮细胞(epithelioma papulosum cyprini,EPC)产生典型细胞病变(cytopathic effect,CPE),收集病毒悬液分别称为Shlj1~Shlj4。PCR鉴定结果表明,该4株病毒均为SVCV。病毒滴度实验测算出SVCV Shlj 1~Shlj 4的滴度分别为10~(6.28)、10~(6.88)、10~(7.57)和106.38 TCID50/mL。Shlj的糖蛋白基因核苷酸序列的聚类分析和遗传进化分析结果显示,Shlj 1~Shlj 4与Gen Bank收录的中国参考株A2、BJ0505-2和美国参考株USA、212364聚为一簇,同源性为98.4%~99.8%;Shlj 1~Shlj 4毒株之间的糖蛋白核苷酸序列相似性在98.6%~99.8%,其中Shlj 3与美国SCVC毒株USA、212364具有最高的核苷酸相似性(99.8%),Shlj 2与英国参考毒株880163具有最低的相似性(88.0%)。糖蛋白氨基酸序列比对结果显示,Shlj4中氨基酸突变最多,与另3个毒株差异较大。基因型分析结果显示,Shlj 1~Shlj 4均为基因Ia型。本研究结果表明,黑龙江地区2015—2016年间的SVCV检出率约为10%,并且来源于不同养殖场的病毒分离株的核酸序列呈现不同程度的差异,该结果进一步证明SVCV毒株在中国不同的鲤养殖环境中正在不断地进化。     

锦鲤疱疹病毒囊膜蛋白ORF59的克隆、分析及其主要B细胞表位区的原核表达

从感染锦鲤疱疹病毒（Koi herpesvirus,KHV）的锦鲤（Cyprinus carpiokoi）肾脏组织中提取DNA,通过PCR扩增了KHVORF59基因。该基因全长411bp,所编码的蛋白包含136个氨基酸,分子量14.3kDa,等电点（PI）6.91,有12个潜在的O糖基化位点。此研究克隆的KHVORF59基因第130位碱基由G突变为A,使其编码的第44位氨基酸由Ala突变为Thr。采用DNAStar程序,在综合分析二级结构柔性区、蛋白的亲水性、表面可能性和抗原性指数的基础上,预测了KHVORF59蛋白主要B细胞表位,并将其区段的编码序列与KHVORF59完整编码序列分别克隆入原核表达载体pET-32a（＋）,构建重组质粒pET32a-ORF59S和pET32a-ORF59C,转入大肠杆菌Rosetta菌株,IPTG诱导表达。SDS-PAGE及WesternBlot分析显示,pET32a-ORF59S可以高效表达,表达的截短KHVORF59蛋白主要以可溶性形式存在,采用HisBindResin填料,层析纯化了该截短蛋白。     

Characterization of the monoclonal antibody specific to the ORF72 protein of koi herpesvirus and cellular distribution analysis of the viral protein

Koi herpesvirus (KHV) is an emerging pathogen of koi and common carp that causes a severe disease and mass mortality of infected fish. The KHV ORF72 protein is an important capsid protein that has been suggested to be a candidate for the development of diagnostic reagents and KHV vaccines. The purpose of this study was to clone and express the KHV ORF72 gene for further preparation of a specific monoclonal antibody (mAb) and to analyse cellular distribution of the viral protein. The mAb 3E1 could specifically recognize the expressed ORF72 protein of transfected cells by indirect immunofluorescence, and the antigenic site recognized by the mAb 3E1 was mapped to the region of N-terminal 124 residues of KHV ORF72. This mAb was further demonstrated to specifically detect the KHV-infected fish tissue by immunohistochemistry, thereby suggesting its high diagnostic potential. In addition, the cellular distribution analysis of the KHV ORF72 protein revealed that the region of amino acid residues 125–247 was related to mitochondrial localization and proliferation. Furthermore, a putative nuclear export signal (NES) of ORF72 at the residues 201–212 was confirmed on the basis of its function associated with NES activity.     

Koi herpesvirus epizootic in cultured carp and koi, Cyprinus carpio L., in Taiwan

Koi herpesvirus (KHV) poses a significant threat to cultured koi and common carp, both Cyprinus carpio L. Since the first reported case in Israel in 1998, KHV has rapidly spread worldwide. This study investigates the spread of KHV to Taiwan by collecting 49 cases of suspected common carp and koi infections from 2003 to 2005 for analysis. Clinical signs included lethargy, anorexia, increased respiratory movements and uncoordinated swimming. Hyperaemia, haemorrhage on body surface and necrotic gill filaments were recorded. Gill epithelial hyperplasia, necrosis and eosinophilic intranuclear inclusion bodies were observed by histological examination, while virions were detected using transmission electron microscopy. By detecting the presence of the KHV thymidine kinase (TK) gene and the KHV 9/5 gene using polymerase chain reaction (PCR), 37 cases were identified as KHV-positive, and the cumulative mortality of infected fish was 70-100%. Positive cases showed identical sequences for the genes analysed, implying that they were of the same origin. For the KHV 9/5 gene sequence, these cases exhibited 100% identity with the Japanese strain (TUMST1, accession number AP008984) and 99% identity with the Israeli (KHV-I, DQ177346) and US (KHV-U, DQ657948) strains. Additionally, a loop-mediated isothermal amplification (LAMP) assay was performed and found to be more sensitive than PCR tests, suggesting its potential use as a rapid diagnostic method for KHV. This is the first epidemiological study of KHV infection in cultured common carp and koi in Taiwan.     

Inactivation of koi‐herpesvirus in water using bacteria isolated from carp intestines and carp habitats

Since its first outbreak in Japan in 2003, koi‐herpesvirus (KHV) remains a challenge to the carp Cyprinus carpio L. breeding industry. In this study, inactivation of KHV in water from carp habitats (carp habitat water) was investigated with the aim of developing a model for rapidly inactivating the pathogen in aquaculture effluent. Experiments with live fish showed that, in carp habitat water, KHV lost its infectivity within 3 days. Indications were that inactivation of KHV was caused by the antagonistic activity of bacteria (anti‐KHV bacteria) in the water from carp habitats. Carp habitat water and the intestinal contents of carp were therefore screened for anti‐KHV bacteria. Of 581 bacterial isolates, 23 showed anti‐KHV activity. An effluent treatment model for the disinfection of KHV in aquaculture effluent water using anti‐KHV bacteria was developed and evaluated. The model showed a decrease in cumulative mortality and in the number of KHV genome copies in kidney tissue of fish injected with treated effluent compared with a positive control. It is thought that anti‐KHV bacteria isolated from the intestinal contents of carp and from carp habitat water can be used to control KHV outbreaks.     

First detection of koi herpesvirus from koi,Cyprinus carpio L. experiencing mass mortalities in Iran: clinical,histopathological and molecular study

Koi herpesvirus (KHV) is the aetiological agent of an emerging disease (KHVD) associated with mass mortalities in koi and common carp and reported from at least 30 countries. We report the first detection of KHV from koi in Iran using clinical, histopathological and molecular studies. KHV‐infected fish showed reduced swimming activity, sunken eyes and increased mucus production on skin and fins. On post‐mortem examination, gill necrosis was observed in the majority of fish. Histopathologically, the gill showed diffuse necrosis of the branchial epithelial cells. Margination of chromatin was detected in gills, kidney, heart, spleen, intestine and brain. In addition, sequence analyses of the TK gene, ORF 136 and marker I and II, demonstrates that Iranian KHV isolates were identical and classified as variant A1 of TUSMT1 (J strain) and displayed the I⁺⁺II⁺ allele of this Asian genotype.     

Carp edema virus in Polish aquaculture – evidence of significant sequence divergence and a new lineage in common carp Cyprinus carpio (L.)

Fish samples initially collected by local veterinarians on the common and koi carp farms in Poland between 2013 and 2015 as part of a KHV surveillance programme, when the water temperature was between 16 and 26 °C, and were also tested for CEV by qPCR. A partial 478 nucleotide fragment of the 4a gene was subsequently generated from 17 qPCR‐positive common carp Cyprinus carpio samples from 36 farm sites tested during the period. Sequence alignments and analysis revealed the presence of CEV in Poland both in common carp as well as in koi carp farms, and phylogenetic analysis assigned the Polish CEV sequences into three distinct genogroups. A lineage which includes the original sequences obtained from koi carp in Japan (genogroup II) included sequences from both koi carp and common carp, and the second lineage (genogroup I) contained sequences from common carp only. A third lineage (genogroup III) which was more closely related to the genogroup II also consisted of sequences from common carp only. The latter represents a lineage of CEV not previously described in the literature.     

Detection of koi herpesvirus in common carp, Cyprinus carpio L., by loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a protocol for koi herpes virus (KHV) detection in common carp was designed. A set of four primers, two inner and two outer, were designed based on the sequence of the thymidine kinase (tk) gene of KHV. Time and temperature conditions for detection of KHV were optimized for 60 min at 65 degrees C. The detection limit using LAMP was found to be similar to that by polymerase chain reaction. In this study, we have developed a highly sensitive and rapid diagnostic procedure for detection of KHV infection in common carp.     

我国五大湖日本沼虾线粒体COI基因部分片段序列比较

对我国五大湖日本沼虾100个野生个体的线粒体细胞色素氧化酶亚基I（COI）部分序列进行了测定和分析，经比对获得578bp核苷酸片段，发现49个变异位点，得到35个单倍型，包括7个共享单倍型，各群体都具有较好的单倍型多态性和核苷酸多态性，其中鄱阳湖群体遗传多样性相对最高。AMOVA分析表明，五群体间总遗传分化系数F_st＝0.31873 （P<0.05）， 群体间具有较高的遗传分化。MEGA3.1软件计算五群体的Kimura 2-paramter遗传距离，洞庭湖群体和巢湖群体之间的遗传距离最远为0.0191，巢湖群体和洪泽湖群体之间的遗传距离最近为0.0051。以同属胖掌沼虾（Macrobrachium inflatum）为外群分别构建了NJ和UPGMA系统树，结果显示洞庭湖和鄱阳湖为一族群，太湖、巢湖和洪泽湖为一族群。