鲤疱疹病毒Ⅱ型的理化及生物学特性和超微形态发生

为了查明鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2,Cy HV-2)的理化与生物学特性以及病毒在细胞内的超微形态发生过程,利用新建立的对Cy HV-2敏感的异育银鲫脑组织细胞系(Gi CB),对Cy HV-2的理化及生物学特性进行了详细研究,比较了不同来源鱼类细胞系对Cy HV-2感染的敏感性,并对体外培养细胞中Cy HV-2病毒粒子及其超微形态发生过程进行了电镜观察。结果显示,Cy HV-2对热、酸、碱、有机溶剂和冻融敏感;常用鱼类细胞系EPC、RTG-2、Koi-Fin、CIK、CCK、PF-Fin对Cy HV-2的感染不敏感,特异性巢式PCR检测盲传至第7代Cy HV-2细胞培养物,结果均为阴性;Cy HV-2在Gi CB细胞中的增殖动态研究结果表明:病毒感染细胞经过12 h的隐晦期,24 h开始进入对数生长期,96 h病毒滴度达到最高值(107.52±0.26 TCID50/m L),然后进入平台期;透射电子显微镜观察结果显示,Cy HV-2感染细胞可分为吸附与侵入、复制与装配、成熟与释放3个主要过程,病毒进入对数生长期后,被感染细胞内可见形态典型的疱疹病毒颗粒。     

鲤疱疹病毒2型微滴式数字PCR检测方法的建立及比较分析

本研究建立了定量检测鲤疱疹病毒2型(Cyprinid herpesvirus 2,CyHV-2)的微滴式数字PCR(Droplet digital PCR,ddPCR)检测方法,并与实时荧光定量PCR(Quantitative real-time PCR,qPCR)检测方法的灵敏性、重复性、特异性和临床样品检测做了比较分析.结果表明,与qPCR相比,ddPCR具有相同的特异性,其灵敏性比qPCR低20倍.在定量CyHV-2 DNA时,ddPCR (R2=0.994)和qPCR (R2=0.994)均表现出良好的线性关系,且2种检测方法间的定量值呈正相关(R2=0.989).在定量检测相同稀释度的CyHV-2 DNA时,qPCR的定量值始终比ddPCR高10倍.ddPCR的组内和组间重复变异系数(CV)分别为0.59％-11.26％和6.55％-23.21％,而qPCR为16.57％-27.56％和22.31％-56.73％,说明ddPCR具有更好的稳定性.在临床样品定量检测时,ddPCR的检出率稍高于qPCR.本研究建立的ddPCR能够准确定量检测CyHV-2,将为CyHV-2相关研究提供有益参考.     

鱼类疱疹病毒研究进展概述

疱疹病毒是双链DNA病毒家族成员之一,具有高度的传染性,严重危及人类及动物的健康;其中异疱疹病毒科各成员是引起水生动物病害的主要病原,严重制约全球水产养殖业的健康发展,尤其对我国鱼类养殖产业影响巨大.本文简要概述鱼类疱疹病毒研究发展历程、现状及进展,主要包括鲤疱疹病毒、(鮰)疱疹病毒、鲑疱疹病毒及其他鱼类疱疹病毒等,以...     

Distribution of the introduced cyprinid herpesvirus 3 in a wild population of common carp, Cyprinus carpio L.

Cyprinid herpesvirus 3 (CyHV-3), which causes a lethal disease in common carp, Cyprinus carpio L., and koi, C. carpio koi , first occurred in Lake Biwa, Japan in 2004. To elucidate distribution of CyHV-3 in a wild common carp population, we conducted a PCR survey of CyHV-3 among such fish in Lake Biwa in 2006. Only 6% (1/18) of the common carp smaller than 300 mm were positive with PCR, whereas 31% (18/58) of fish larger than 300 mm were positive. To evaluate their past exposure to CyHV-3 infection based on the presence of antibodies, we also measured the levels of serum anti-CyHV-3 antibodies in the carp, using an enzyme-linked immunosorbent assay. None (0/26) of the fish smaller than 300 mm was positive for the antibodies, whereas 54% (33/61) of fish larger than 300 mm were positive. Of the antibody-positive individuals, 44% (14/32) were also positive by PCR strongly suggesting that wild common carp that survived infection become CyHV-3 carriers. Five individuals were positive by PCR but negative for antibodies indicating that their infection with CyHV-3 had occurred recently. These results suggest that transmission of CyHV-3 from carriers to naïve common carp is still occurring in Lake Biwa.     

鲤疱疹病毒3型ORF136基因编码蛋白多克隆抗体的制备与鉴定

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。     

引起混养塘中异育银鲫和鲢发病死亡的病原及组织病理

混养的异育银鲫和鲢鱼种大批死亡,为明确发病死亡的病原和组织损伤并提供相关的疾病防控措施,进行了病鱼肉眼和显微镜检查、细菌学检测、病毒学检测、组织病理和药敏试验研究。结果发现,除在患病鱼体表偶然发现有少量不会引起充血等症状的杯体虫和车轮虫外,未在体内外发现其他寄生虫和真菌类病原;通过细菌分离、人工回感试验、生理生化特性和16S r RNA基因序列分析,从患病异育银鲫和鲢分离到的致病菌株均为嗜水气单胞菌;根据异育银鲫和鲢病毒性疾病的现状,使用鲤疱疹病毒2型(Cyprinid herpesvirus 2,Cy HV-2)DNA聚合酶基因的特异性引物分别对自然发病的异育银鲫和鲢进行PCR检测,只有异育银鲫检测到Cy HV-2,分别用它们的除菌组织上清液进行人工感染试验,只有异育银鲫出现充血症状和死亡现象;由此得出嗜水气单胞菌是异育银鲫和鲢发病死亡的主要病原,Cy HV-2是异育银鲫混合感染的次要病原。患病鲢与患病异育银鲫呈现出类似的组织病理现象,又有一些各自特有的组织病理表现,单纯细菌感染的鲢轻度病变以细胞颗粒变性为主,坏死细胞以核溶解为主,细菌和病毒混合感染的异育银鲫肝脏轻度病变以细胞滴状玻璃样变的变性为主,坏死组织细胞以核固缩和核碎裂为主,在肾脏和脾脏出现染色质边集于核膜的肿大细胞核,主要组织器官出现从变性到坏死的病理变化过程,最终失去应有的功能而死亡。依据药敏试验结果,建议内服诺氟沙星和氟苯尼考等抗生素防治本病的嗜水气单胞菌感染,混合感染Cy HV-2的异育银鲫可以通过注射Cy HV-2疫苗和生态养殖的方法控制和减少该病毒病感染和发展。     

Spatio-temporal analysis of cyprinid herpesvirus 3 genetic diversity at a local scale

Cyprinid herpesvirus 3 (CyHV-3), the causative agent of koi herpesvirus disease, is a major threat for carp populations in many countries worldwide, including Indonesia. It has been shown that many genotypes circulate worldwide, all highly related to one of the two known lineages U/I and J. In this study, we evaluated the spatial and temporal distribution of CyHV-3 strains in a small enzootic area, the lake of Cirata (West Java, Indonesia). Of the 365 samples analysed, from clinical or asymptomatic fish, 244 were found positive for CyHV-3, suggesting a high occurrence of the virus. Genotyping of these viral specimens with a range of molecular markers revealed the presence of numerous haplotypes in the host population, all related to the J lineage. In single individuals, mixed-genotype infections occurred at high frequency. The present results demonstrate that polymorphic molecular markers are suitable to monitor the genetic evolution of a viral population in an enzootic area.     

一种基于Ⅱ型鲤疱疹病毒衣壳蛋白72的免疫学检测方法

Ⅱ型鲤疱疹病毒(cyprinid herpesvirus 2,Cy HV-2)是引起养殖异育银鲫(Carassius auratus gibelio)造血器官坏死症的致病病原。在临床筛查中基于病毒核酸的PCR和real time PCR技术已经建立,但是稳定性更强的免疫学诊断技术国内外尚无报道。本研究目的是利用Cy HV-2编码的ORF72基因(Gen Bank登录号:AFJ20502.1)所编码的衣壳蛋白作为捕获抗原,通过识别感染病毒的鱼体中的相应抗体,从而对样本进行临床免疫学检测。首先采用PCR方法从纯化的Cy HV-2基因组中扩增ORF72基因,并把该基因克隆至原核表达载体PGEX-4T-3,并转化到大肠杆菌中诱导表达,诱导表达的产物通过SDS-PAGE进行鉴定,对表达的重组蛋白进行纯化。用已纯化的72重组蛋白对小鼠进行免疫,制得72重组蛋白的抗体。Western blot检测表明所制备的多克隆抗体既能识别原核表达的重组蛋白,也可以识别Cy HV-2病毒粒子上的衣壳蛋白72。在上述基础上建立了基于Western blot技术的Cy HV-2抗体检测技术:用纯化的72重组蛋白作为检测抗原,鲫鱼血清用作一抗,兔抗鲫Ig M多克隆抗体作为二抗,酶标羊抗兔作为三抗鉴定鲫鱼是否存在Cy HV-2特异性抗体。在对急性感染期的临床样本检测中,本方法能在所有样本中检测出ORF72特异性抗体存在,表明72重组蛋白作为相应抗体捕获原可以用于确诊鲫鱼是否感染Cy HV-2。本研究建立的实验室免疫学检测方法为商品化免疫学检测技术的开发奠定了基础,对Cy HV-2的检验检疫具有一定的临床应用价值。     

Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.     

Identification and detection of Pseudomonas plecoglossicida isolates with PCR primers targeting the gyrB region

Pseudomonas plecoglossicida is the agent of bacterial haemorrhagic ascites (BHA) in freshwater fish farming in Japan. To develop a rapid identification and detection method for P. plecoglossicida, a PCR amplification technique targeting the chromosomal DNA region coding the B subunit of the DNA gyrase (gyrB) was used. The nucleotide sequences of gyrB were determined in nine isolates of P. plecoglossicida and two other Pseudomonas species. On the basis of these determined sequences and the gyrB sequences of other Pseudomonas species or fish pathogenic bacteria deposited in international nucleotide sequence databases (GenBank/EMBL/DDBJ), PCR primers PL-G1F, PL-G1R, PL-G2F and PL-G2R were designed for specific amplification of the partial gyrB of P. plecoglossicida. The specificity of these primers in amplifying the gyrB of P. plecoglossicida was verified using selected strains of related bacterial species. The nested PCR technique was used to detect P. plecoglossicida from kidney and intestine of ayu. Primer pair PL-G1F and PL-G1R was used for the external PCR, and primer pair PL-G2F and PL-G2R for the internal PCR. Of 10 ayu juveniles, expected size PCR products were observed from intestine and kidney samples in one and two specimens, respectively. The PCR technique with primers based on the gyrB sequence is thus useful for the diagnosis of BHA.     

Development of a real‐time PCR assay for detection and quantification of Streptococcus iniae using the lactate permease gene

The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)‐based protocol for detection of zoonotic pathogen Streptococcus iniae in bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease‐encoding (lldY) gene was selected as a target for the design of S. iniae‐specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected with S. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted from S. iniae or tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 × 10¹ amplicon copies per assay (equivalent to 2 × 10^–9 ng/µl) using bacterial DNA and of 1.44 × 10¹ gene copies in tissues of fish infected with S. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification of S. iniae and its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories.     

Isolation of a cyprinid herpesvirus 2 from goldfish, Carassius auratus (L.), in the UK

Haematopoietic necrosis virus [cyprinid herpesvirus 2 (CyHV-2)] was isolated during disease outbreaks in goldfish, Carassius auratus, at an ornamental fish retail site in southern England in 2004. Signs of disease included lethargy and inappetence and were first seen after water temperatures increased from 14-15 to 19-21 degrees C. External gross pathology included pale patches on the gills and skin and internally the spleen was enlarged, often with distinctive white nodules. The most prominent histopathological changes observed were necrotic lesions in the spleen and kidney and focal patches of necrosis in the gill lamellae. Necrotic cells often contained nuclei with marginated chromatin and pale intranuclear inclusions. Ultrastructural examination of the spleen tissue revealed typical herpesvirus-like particles measuring 100 nm in diameter. The virus was isolated from extracts of gill tissue in KF-1 cells at 20 degrees C and oligonucleotide primer sets were designed based on conserved gene sequences and used to amplify viral DNA by polymerase chain reaction (PCR). The PCR assays were then used to detect the virus in DNA extracted from tissues sampled during earlier disease investigations at the retail site owner's holding facility in 2002 and 2003 and stored at -70 degrees C since then. Polymerase gene-specific PCR amplification products obtained from tissue samples and from the virus isolated in cell culture shared 100% nucleotide sequence identity with the published sequence for CyHV-2.     

应用RAPD标记对东方鲀属进行种类鉴别及其聚类分析

利用RAPD技术对红鳍东方鲀、假晴东方鲀、暗纹东方鲀和从日本引进的红鳍东方鲀4个种进行了遗传标记鉴别研究。在事先优化的条件下，20个随机引物共扩增出134条谱带清晰、重复性高的DNA片段，其中多态性片段77条。结果证实每一种均有其特异性扩增图谱，可作为种鉴定的依据；用UPGMA和NJ等方法进行聚类，构建的系统树与借助形态学和生化特征进行的传统分类结果一致。研究表明，RAPD作为一种新的分子标记，在海洋动物遗传鉴定方面具有巨大的潜力。     

应用RAPD标记对东方(鱼屯)属进行种类鉴别及其聚类分析

利用RAPD技术对红鳍东方、假睛东方、暗纹东方和从日本引进的红鳍东方4个种进行了遗传标记鉴别研究.在事先优化的条件下,20个随机引物共扩增出134条谱带清晰、重复性高的DNA片段,其中多态性片段77条.结果证实每一种均有其特异性扩增图谱,可作为种鉴定的依据;用UPGMA和NJ等方法进行聚类,构建的系统树与借助形态学和生化特征进行的传统分类结果一致.研究表明,RAPD作为一种新的分子标记,在海洋动物遗传鉴定方面具有巨大的潜力.     

Detection of Flexibacter maritimus in fish tissue using nested PCR amplification

A nested polymerase chain reaction (PCR) system was developed for the detection of Flexibacter maritimus from fish tissue. The total procedure for the diagnosis of marine flexibacteriosis, from the point of DNA extraction to the electrophoretic analysis, can be performed in < 4 h. This was achieved by the combination of a short thermal cycling programme with a rapid DNA extraction procedure. The assay was extremely sensitive, capable of detecting as few as 75 cfu mg(-1) fish tissue. The accuracy of the nested PCR was confirmed under field conditions using tissue samples recovered during 1993-2002 from fish suffering marine flexibacteriosis. The nested PCR method proved to be efficient for the rapid and sensitive detection of F. maritimus from fish tissues and can be used for routine diagnosis of the disease caused by this pathogen.     

The use of a real-time PCR primer/probe set to observe infectivity of Yersinia ruckeri in Chinook salmon, Oncorhynchus tshawytscha (Walbaum), and steelhead trout, Oncorhynchus mykiss (Walbaum)

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), a common pathogen affecting aquaculture facilities and implicated in large losses of cultured fish. Fisheries scientists continue to gain a greater understanding of the disease and the pathogen by investigating methods of identification and pre- and post-infection treatment. In this study, a real-time PCR probe set for Y. ruckeri was developed to detect daily changes in the bacterial load during pathogen challenges. Two species of fish, Chinook salmon, Oncorhynchus tshawytscha, and steelhead trout, Oncorhynchus mykiss, were exposed to two strains of Y. ruckeri (Hag and SC) during bath challenges. A subset of fish was killed daily for 14 days, and the kidney tissue was biopsied to enumerate copies of pathogen DNA per gram of tissue. While Chinook exposed to either the Hag or SC strains exhibited similar pathogen loads, those exposed to the Hag strain displayed higher mortality (～66%) than fish exposed to the SC strain (～24% mortality). Steelhead exposed to the Hag strain exhibited a greater pathogen load and higher mortality (～42%) than those exposed to the SC strain (<1% mortality). Steelhead challenged with either strain showed lower pathogen loads than Chinook. The study illustrates the efficacy of the probe set to enumerate Y. ruckeri bacterial growth in the kidneys of fish. Also, strains of Y. ruckeri display species-specific growth patterns that result in differential mortality and pathogen load.     

鲤疱疹病毒Ⅱ型主要免疫原性蛋白的鉴定

为了鉴定CyHV-2的主要免疫原性蛋白,本研究用分离的CyHV-2-YC-1分离株(下简称CyHV-2)感染异育银鲫尾鳍细胞系(GiCF),用蔗糖密度梯度超速离心法对细胞感染液中的CyHV-2进行纯化,纯化后的CyHV-2病毒免疫小鼠制备抗CyHV-2多克隆抗体。纯化的病毒颗粒经变性聚丙烯酰胺凝胶电泳(SDS-PAGE)和考马斯亮蓝染色后,用抗CyHV-2多克隆抗体进行Western blotting分析和质谱鉴定。结果显示,透射电镜下观察发现50%～66%的蔗糖梯度多见完整囊膜包裹的CyHV-2病毒颗粒,也有少量囊膜破损的病毒颗粒。Western blotting结果显示,抗CyHV-2多克隆抗体与多种病毒蛋白具有特异性免疫反应,质谱鉴定显示,其中8种主要免疫原性蛋白分别是ORF92、ORF115、ORF25、ORF57、ORF66、ORF72、ORF131和ORF132。研究表明,通过蔗糖密度梯度超速离心法提纯CyHV-2病毒颗粒后,本研究制备的抗CyHV-2多克隆抗体能够特异性识别CyHV-2病毒的主要免疫原性蛋白。本研究将为CyHV-2免疫学检测方法的建立以及疫苗的研制提供更多的候选抗原。     

Comparison of the pathogenicity of Francisella orientalis in Nile tilapia (Oreochromis niloticus), Asian seabass (Lates calcarifer) and largemouth bass (Micropterus salmoides) through experimental intraperitoneal infection

Francisella orientalis is a highly virulent, emerging bacterium that causes mass mortalities in tilapia. This pathogen also affects numerous other warm-water fish species, including three-line grunt, hybrid striped bass and various ornamental fish. This study sheds light on two new species of fish that are susceptible to F. orientalis. Asian seabass and largemouth bass showed variable levels of susceptibility in a bacterial challenge experiment. After intraperitoneally injected with a dose of 10⁶ CFU/fish, a total of 64.28% and 21.42% mortalities were obtained in Asian seabass and largemouth bass, respectively. Meanwhile, Nile tilapia showed acute mortality of 100%. All fish showed typical lesions of francisellosis, including multifocal granulomas in the spleen and head kidney. Immunohistochemical analysis revealed strong positive signals inside the granulomas of all fish. The bacterial recovery in solid media from infected fish was highest in Nile tilapia (85.71%), followed by Asian seabass (35.71%) and largemouth bass (21.42%). PCR results tested 100% positive for Nile tilapia, and 78.57% and 21.42% for Asian seabass and largemouth bass, respectively. In conclusion, Asian seabass and largemouth bass are susceptible to this pathogen, which warrants new management strategies when employing predation polyculture systems of these species with tilapia.     

抗鲤疱疹Ⅱ型病毒卵黄抗体制备及其功能鉴定

为制备抗鲤疱疹Ⅱ型病毒(CyHV-2)的卵黄抗体,探索防治异育银鲫(Carassius auratus gibelio)鳃出血病的新方法和途径,本研究利用原核表达系统产生具免疫原性的重组CyHV-2-ORF72衣壳蛋白,纯化后免疫蛋鸡;二次免疫后采用间接ELISA法抽检免疫蛋的特异性卵黄抗体(IgY)含量,收集抗体效价达到1∶8000的免疫蛋。将免疫蛋分成全蛋组与蛋黄组,采用微包膜和冷冻干燥技术,分别制成全蛋粉或蛋黄粉,利用Western blot和ELISA技术检测全蛋粉和蛋黄粉中的IgY特异性及效价。将蛋黄粉稀释液与CyHV-2混合后孵育鲤鱼上皮瘤细胞,进行抗体中和试验。以0.15%和0.30%的添加量分别将抗CyHV-2-ORF72的免疫全蛋粉拌入商品饲料制成功能性饲料,投喂已发病的两个池塘的异育银鲫进行生产性治疗试验。结果显示,全蛋粉和蛋黄粉中的IgY均能特异性地结合CyHV-2-ORF72衣壳蛋白,效价达到1∶8000~1∶16000;抗CyHV-2-ORF72的IgY与CyHV-2具有较好的亲和力,中和活性高于鼠抗ORF72血清;两个池塘异育银鲫成活率分别达到74.3%和87.6%,远高于对照池的28.3%和31.5%。综上所述,本研究制备的抗CyHV-2-ORF72卵黄抗体可特异性地中和CyHV-2病毒,具较显著的免疫保护作用。     

Herpesviral haematopoietic necrosis virus (CyHV-2) infection: case studies from commercial goldfish farms

Herpesviral haematopoietic necrosis is a disease of goldfish, Carassius auratus , caused by Cyprinid herpesvirus-2 (CyHV-2) infection. Quantitative PCR was carried out on tissue homogenates from healthy goldfish fingerlings, broodfish, eggs and fry directly sampled from commercial farms, from moribund fish submitted to our laboratory for disease diagnosis, and on naturally-infected CyHV-2 carriers subjected to experimental stress treatments. Healthy fish from 14 of 18 farms were positive with copy numbers ranging from tens to 10⁷ copies μg⁻¹ DNA extracted from infected fish. Of 118 pools of broodfish tested, 42 were positive. The CyHV-2 was detected in one lot of fry produced from disinfected eggs. Testing of moribund goldfish, in which we could not detect any other pathogens, produced 12 of 30 cases with 10⁶–10⁸ copies of CyHV-2 μg⁻¹ DNA extracted. Subjecting healthy CyHV-2 carriers to cold shock (22–10 °C) but not heat, ammonia or high pH, increased viral copy numbers from mean copy number (±SE) of 7.3 ± 11 to 394 ± 55 μg⁻¹ DNA extracted after 24 h. CyHV-2 is widespread on commercial goldfish farms and outbreaks apparently occur when healthy carriers are subjected to a sharp temperature drop followed by holding at the permissive temperature for the disease.