Emergence of fatal European genotype CyHV-3/KHV in mainland China

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is a highly infectious causative agent to common carp and koi worldwide. The virus is mainly consisted of European and Asian genotype isolates. To date, no European genotype CyHV-3 has been found emerging in the East and Southeast Asian regions. In late March 2011, an outbreak of CyHV-3 disease occurred in Guangzhou City, Guangdong Province, China, resulting in the deaths of approximately 200 large-sized adult koi within four weeks. One moribund koi was sampled for CyHV-3 isolation. Thus, a CyHV-3 was isolated in KCF-1 cells and designated as KHV-GZ11. Abundant mature or immature virions in infected KCF-1 cells were observed under a transmission electron micrograph. In addition, intra-nuclear inclusion body-like structures with masses of virions were also observed. Based on the TK and ORF136H genes, the sequence analyses revealed that KHV-GZ11 is a distinct European genotype of CyHV-3. Moreover, the infectivity experiment showed that KHV-GZ11 was highly virulent to koi. In summary, we are the first to confirm the emergence of fatal European genotype CyHV-3/KHV in East and Southeast Asia. Our study will provide new insight to explore the virus origin and epidemiology, as well as its pathogenicity.     

锦鲤摩氏摩根氏菌的分离鉴定与药敏性研究

为鉴定2009年山东某养殖场锦鲤病病原,本研究从病鱼体内分离到3株优势菌,经回归感染实验证实编号为A400502的分离株为该病的致病菌.该菌为革兰氏阴性杆菌,周生鞭毛,大小为0.5 μm～0.7 μm×1.0 μm～1.5 μm.其生化特征为鸟氨酸脱羧酶、尿素酶、酚红反应阳性,赖氨酸脱羧酶和精氨酸双水解酶阴性.利用16S rDNA序列分析和比对结果构建的系统发育树表明,该菌与登录号为EF455493的摩氏摩根氏菌(Morganella morganii)的同源性高达99％,因此将该菌鉴定为摩氏摩根氏菌.26种药物的敏感实验结果表明,该分离株对复达欣、庆大霉素、新霉素等11种药物高度敏感.     

Antibody screening identifies 78 putative host proteins involved in Cyprinid herpesvirus 3 infection or propagation in common carp,Cyprinus carpio L

Cyprinid herpesvirus 3 (CyHV‐3) is the aetiological agent of a serious and notifiable disease afflicting common and koi carp, Cyprinus carpio L., termed koi herpesvirus disease (KHVD). Significant progress has been achieved in the last 15 years, since the initial reports surfaced from Germany, USA and Israel of the CyHV‐3 virus, in terms of pathology and detection. However, relatively few studies have been carried out in understanding viral replication and propagation. Antibody‐based affinity has been used for detection of CyHV‐3 in enzyme‐linked immunosorbent assay and PCR‐based techniques, and immunohistological assays have been used to describe a CyHV‐3 membrane protein, termed ORF81. In this study, monoclonal antibodies linked to N‐hydroxysuccinimide (NHS)‐activated spin columns were used to purify CyHV‐3 and host proteins from tissue samples originating in either CyHV‐3 symptomatic or asymptomatic fish. The samples were next analysed either by polyacrylamide gel electrophoresis (PAGE) and subsequently by electrospray ionization coupled to mass spectrometry (ESI‐MS) or by ESI‐MS analysis directly after purification. A total of 78 host proteins and five CyHV‐3 proteins were identified in the two analyses. These data can be used to develop novel control methods for CyHV‐3, based on pathways or proteins identified in this study.     

淡水鱼病毒性疾病的研究进展

为了更好地预防和治疗淡水鱼病毒病。综述了淡水鱼病毒病的研究情况。主要介绍了锦鲤疱疹病毒病、鲤春病毒血症、传染性胰腺坏死病毒病、传染性造血器官坏死症、病毒性出血性败血症等疾病的流行情况、国内外研究进展、病原学的研究、疾病的诊断与防治、并对淡水鱼病毒病研究中存在的问题进行了分析和探讨。旨在为淡水鱼病毒病的深入研究提供理论基础和科学依据。     

Detection of Koi herpesvirus: impact of extraction method,primer set and DNA polymerase on the sensitivity of polymerase chain reaction examinations

Since virus isolation is seldom successful, KHV infection is commonly detected by PCR examination. A number of different PCR assays have been described in recent years. However, at present no commonly accepted PCR method is used amongst different laboratories. The aim of this study was to check if the examination of infected fish by different PCR methods yielded comparable results. We used tissue samples of three KHV‐infected koi, one KHV‐infected common carp, one KHV‐infected goldfish and one non‐infected common carp. DNA was extracted with DNAzol Reagent, High Pure PCR Template DNA Preparation Kit and QIAamp DNA Mini Kit. The DNA was tested by PCR with different combinations of published primer sets –KHV‐F and ‐R, KHV‐Gray‐2F and ‐2R and KHV‐TKf and ‐TKr – plus different DNA polymerases – a standard Taq DNA polymerase, a Platinum (hotstart) Taq DNA polymerase and a Platinum (hotstart) Pfx DNA polymerase with proofreading activity. The different extraction methods produced DNA solutions with different yields of DNA and different degrees of homogeneity. Also, the sensitivity of the PCR depended on the choice of the primer set and polymerase. Not all infected fish could be identified with all methods; there were large differences in the sensitivity between methods.     

Effect of ploidy on the mortality of Crassostrea gigas spat caused by OsHV‐1 in France using unselected and selected OsHV‐1 resistant oysters

The effect of ploidy on the mortality of Crassostrea gigas spat caused by the ostreid herpesvirus (OsHV‐1) genotype μVar was investigated at five sites along the Atlantic coast in France in 2011. Sibling diploids and triploids were produced using either unselected or selected OsHV‐1‐resistant oysters. No significant interactions were found between the factors of environment, genotype and ploidy at the endpoint dates. The mean mortality rates at the sites were 62% and 59% for diploids and triploids, respectively, and the two rates were not significantly different. The mean mortality rates were 33% and 32% for sibling diploids and triploids, respectively, when OsHV‐1‐resistant parents were used and 91% and 85%, respectively, when unselected parents were used. The results were confirmed through other broodstocks tested in 2013. Our study is the first to clearly show that mortality related to OsHV‐1 is similar between diploids and triploids in C. gigas when the same germplasm is used for both ploidy. Furthermore, OsHV‐1 resistance was not substantially altered by triploidization, indicating that the achieved selective breeding of diploid oysters for OsHV‐1 resistance can be translated into improved survival in triploids.     

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。