Comparative sensitivity of five fish cell lines to wild type infectious haematopoietic necrosis virus from two Oregon sources

Abstract. Five fish cell lines (CHSE-214, STE-137, RTG-2, EPC and FHM) were compared for sensitivity to infectious haematopoietic necrosis virus (IHNV) from samples obtained from naturally-infected fish. Infectious ovarian fluids were obtained from steelhead trout, Salmo gairdneri Richardson, at the Round Butte Hatchery in central Oregon and tissue homogenates were prepared from chinook salmon, Oncorhynchus tshawytscha (Walbaum), alevins during an IHN virus epizootic at the Elk River Hatchery in coastal Oregon. The only lines to show characteristic viral cytopathology by plaque or end-point dilution assay for the steelhead trout virus isolate were the EPC and FHM cell lines. The chinook salmon isolates produced CPE in CHSE-214, STE-137, FHM and EPC cells. The titre of the salmon virus isolate was 10-50-fold higher on FHM and EPC cells by both assay methods. Neither by end-point nor plaque assay did the Round Butte or Elk River isolates produce CPE on RTG-2 cells. With both virus isolants both cell lines showed that greater sensitivity was obtained with plaque assay than with end-point titration. Pre-treatment of the cells with the polycation, polybrene, did not increase the virus titre in either assay. However, a transient enhancement in virus titre was observed in polybrene-treated STE-137 and CHSE-214 cells.     

Relationships between growth and disease resistance in rainbow trout,Oncorhynchus mykiss (Walbaum)

Rainbow trout from 23 families were evaluated for growth and resistance to the bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum and infectious haematopoietic necrosis (IHN) caused by IHN virus. Average family weights were between 161 and 263 g with an average of 225 g at 213 days post‐fertilization with specific growth rates ranging from 2.37 to 2.88. Per cent survival of fish challenged with F. psychrophilum was between 18% and 100%, while for those challenged with IHNV, the range was between 12% and 93%. Significant positive correlations were found for end body weight and resistance to IHN (P < 0.05) and for early body weight and resistance to BCWD (P < 0.1). However, no significant correlations were detected between resistance to both pathogens or disease resistance and overall genetic diversity or diversity within the major histocompatibility locus.     

Detection of infectious haematopoietic necrosis virus in river water and demonstration of waterborne transmission

Abstract. In a study of the possible role of waterborne infectious haematopoietic necrosis virus in transmission of the disease among spawning sockeye salmon, Oncorhynchus nerka (Walbaum), both infection rates and virus titres were higher in fish held at high density in a side channel than in fish in the adjacent river. Virus was never isolated from river water, but was found in water from the side channel at levels ranging from 32.5 to 1600 plaque-forming units (p.f.u.)/ml. Uninfected yearling sockeye salmon held in a box in the side channel developed localized gill infections with IHN virus. The disease did not progress to the viscera until a threshold titre of about 10⁵ p.f.u./g was reached in the gill. The effectiveness of the gill as a barrier limiting development of systemic infections means that waterborne IHN virus probably does not greatly increase the infection rate in a sockeye salmon population during spawning.     

A survey of viral diseases in farmed and feral salmonids in Switzerland

A field survey was carried out to study the occurrence and distribution of viruses causing diseases of major impact in fish farming, namely viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN) and infectious pancreatic necrosis (IPN) in farmed and wild fish in Switzerland. The presence of VHS virus (VHSV), IHN virus (IHNV) and IPN virus (IPNV) in the tissue samples was tested by virus isolation in cell cultures, and subsequent virus identification by immunofluorescence. The sera were screened for anti-VHSV antibodies (VHSV-AB) using a serum plaque neutralization test with complement addition. These data were then compared with results of a similar survey performed in 1984/85, and with data from routine diagnostic work completed at the Centre for Fish and Wildlife Health (FIWI) of the University of Bern from 1978 to 2001. Sampling sites included private and government fish farms as well as natural habitats from all major river catchments in Switzerland. In 2000/01, 522 tissue samples and 1910 sera were collected from 3400 fish. In 1984/85 1239 tissue samples and 694 sera were collected from 1628 fish. During the last 24 years of routine diagnostics at the FIWI, 1776 tissue samples were examined for presence of viruses. The results of the tissue analysis from the surveys in 1984/85 and 2000/01 showed low numbers of sites with virus-positive fish (five VHSV, three IPNV and three VHSV, one IPNV, respectively) in Swiss fish farms and rivers. The sites with virus-positive fish were located throughout the country. The decline in virus-positive cases observed between the two surveys agrees with data from the routine diagnostic work of the FIWI which show a decrease in total virus isolations from approximately 35 cases per year in the late 1970s, to approximately 10 cases per year during the last 10 years. However, in 1984/85 8.3% (58 of 694 serum samples) and in 2000/01 6.3% (121 of 1910 serum samples) proved to be positive for VHSV-AB. The 58 positive samples in 1984/85 originated from 40 of 175 sites (23%) and the 121 positive samples in 2000/01 were from 84 of 217 (29%) sites. These results are indicative of a wider distribution of VHSV than expected from the results of the virus isolations.     

Comparative stability of two salmonid viruses and poliovirus in fresh, estuarine and marine waters

Abstract. The inactivation rates in the aquatic environment of two fish pathogens, infectious pancieatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV), were compared with that of poliovirus type 1 as a representative of the human enterovirus group. The survival studies were performed using untreated fresh, estuarine and sea water samples held at 15 and 20°C. The results indicated that the salmonid viruses survived longer than poliovirus in the saline waters, whereas in fresh water poliovirus was the most stable of the three viruses. IPN virus proved to be most stable in estuarine water at 15°C, whereas the survival of IHN virus was favoured in fresh water. We also observed that at 20°C the inactivation rate for each virus was independent of salt concentration in estuarine and sea water. Although temperature exhibited a marked effect on virus stability in fresh water, the salmonid viruses presented similar survival patterns at both temperatures in sea water. In general the period of greatest viral inactivation correlated with higher bacterial numbers in the waters.     

传染性造血器官坏死病核酸疫苗的构建及其在虹鳟接种部位的消长规律

本研究将传染性造血器官坏死病病毒(infectious hematopoietic necrosis virus,IHNV)分离株SD-12糖蛋白(glycoprotein,G)基因克隆进商业化载体pc DNA3.1(+),构建了IHNV G的表达载体,即传染性造血器官坏死病(infectious hematopoietic necrosis,IHN)核酸疫苗,命名为p IHNsd-G。采用背鳍基部肌肉注射的方式,以2μg/尾的剂量免疫虹鳟(Oncorhynchus mykiss)鱼苗(5.0±0.5)g。于免疫后第4天及第7天,利用real-time PCR技术检测免疫虹鳟头肾及接种部位肌肉组织Mx-1基因表达情况;于免疫后第21天,以100倍半数组织培养感染剂量(tissue culture infective dose,TCID50)采取腹腔注射的方式进行攻毒实验,计算核酸疫苗相对保护率(relative percent survival,RPS);于免疫后第60天及150天检测免疫虹鳟血清IHNV中和抗体效价;最后,以p IHNsd-G的启动子序列和氨苄青霉素抗性基因序列为目标基因,利用PCR方法监测p IHNsd-G在免疫虹鳟接种部位的动态分布情况。结果显示:Mx-1基因在头肾和接种部位肌肉中均显著上调表达,并且在接种部位肌肉组织中明显高于同一时间点的头肾组织;攻毒实验中p IHNsd-G对虹鳟的相对保护率高达94.4%;而在免疫后第60天,所有免疫虹鳟血清中均存在中和抗体,其最高效价高达320,在免疫后第150天,最高抗体效价为80,自此,说明已成功获得有效的IHN核酸疫苗。p IHNsd-G在虹鳟接种部位的PCR监测结果显示:在免疫后的第1天即可在注射部位的肌肉中检测到全部p IHNsd-G目标片段,在第84天时已经无法从注射部位肌肉中扩增出全长氨苄青霉素抗性基因,而所有目标基因在第150天时均消失不见。本研究在成功构建IHN核酸疫苗并系统地验证了其有效性的基础上,开展了该疫苗在接种部位的动态分析研究,为IHN核酸疫苗的研发和安全性评价研究提供了基础数据。     

First evidence of infectious hematopoietic necrosis virus (IHNV) in the Netherlands

In spring 2008, infectious hematopoietic necrosis virus (IHNV) was detected for the first time in the Netherlands. The virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), from a put‐and‐take fishery with angling ponds. IHNV is the causative agent of a serious fish disease, infectious hematopoietic necrosis (IHN). From 2008 to 2011, we diagnosed eight IHNV infections in rainbow trout originating from six put‐and‐take fisheries (symptomatic and asymptomatic fish), and four IHNV infections from three rainbow trout farms (of which two were co‐infected by infectious pancreatic necrosis virus, IPNV), at water temperatures between 5 and 15 °C. At least one farm delivered trout to four of these eight IHNV‐positive farms. Mortalities related to IHNV were mostly <40%, but increased to nearly 100% in case of IHNV and IPNV co‐infection. Subsequent phylogenetic analysis revealed that these 12 isolates clustered into two different monophyletic groups within the European IHNV genogroup E. One of these two groups indicates a virus‐introduction event by a German trout import, whereas the second group indicates that IHNV was already (several years) in the Netherlands before its discovery in 2008.     

东北地区虹鳟IHN和IPN流行病学的初步研究

本文报道了虹鳟鱼病毒性疾病IHN和IPN的流行与危害情况，对这二种病毒性疾病的症状和病理变化作了初步的研究，并进行了病原学鉴定。文中建议严格地把住进口鱼卵或鱼苗的检疫工作，采取更严格的防疫措施，多层次地防止疫病的传播和蔓延。     

Inhibition of infectious pancreatic necrosis virus infectivity by extracts of rainbow trout, Salmo gairdneri Richardson, tissue

Abstract. Extracts of healthy rainbow trout liver, kidney, spleen and whole fry inhibited plaque production of infectious pancreatic necrosis virus (IPNV) in cell cultures. The mode of inhibition is not known, although it appears not to be manifest at the cellular level, as pre-treatment of the cell cultures with tissue extracts did not inhibit plaque production. Any effect on the virus itself was not permanent as the inhibition could be mitigated by treatment of virus/extract mixtures with 1,1,2-trichloro-1,1,2-trifluoroethane. The inhibition may be caused by prevention or reduction of virus attachment to the cell surface or, alternatively, the tissue extract may cause aggregation of the virus and thereby reduce the number of available infectious units. The inhibitory effect is also lost by dilution of the extract, reinforcing the claim that adequate dilutions of fish extracts prior to attempted virus isolation are necessary, especially for the detection of carrier fish.     

Factors controlling the early stages of viral haemorrhagic septicaemia epizootics: low exposure levels, virus amplification and fish-to-fish transmission

Viral haemorrhagic septicaemia virus, Genogroup IVa (VHSV), was highly infectious to Pacific herring, Clupea pallasii (Valenciennes), even at exposure doses occurring below the threshold of sensitivity for a standard viral plaque assay; however, further progression of the disease to a population‐level epizootic required viral amplification and effective fish‐to‐fish transmission. Among groups of herring injected with VHSV, the prevalence of infection was dose‐dependent, ranging from 100%, 75% and 38% after exposure to 19, 0.7 and 0.07 plaque‐forming units (PFU)/fish, respectively. Among Pacific herring exposed to waterborne VHSV (140 PFU mL^?1), the prevalence of infection, geometric mean viral tissue titre and cumulative mortality were greater among cohabitated herring than among cohorts that were held in individual aquaria, where fish‐to‐fish transmission was prevented. Fish‐to‐fish transmission among cohabitated herring probably occurred via exposure to shed virus which peaked at 680 PFU mL^?1; shed virus was not detected in the tank water from any isolated individuals. The results provide insights into mechanisms that initiate epizootic cascades in populations of wild herring and have implications for the design of VHSV surveys in wild fish populations.     

东北地区虹鳟鱼病流行情况初步调查报告

本文报导了1986—1987年东北地区虹鳟鱼常见病的调查结果。介绍了能引起经济损失的13种疾病的病原体、临诊症状及鱼病的鉴别诊断和防治。这些疾病分别为IHN、IPN、营养性肝病、真菌性病、小瓜虫病、三代虫病、孤菌病、尾柄病、旋转病(脑粘虫病)、白点病、复口吸虫病、气泡病、棘头虫病。其中IHN和旋转病属国内首次报导。     

An antigenic comparison of three strains of infectious pancreatic necrosis virus of salmonid fishes

Three strains of infectious pancreatic necrosis (IPN) virus of salmonid fishes were found to be closely related antigenically when compared by cross plaque neutralization. They differed significantly, however, in stability during freezing and storage at ?60°C. The American reference strain VR-299, which was the most stable, required consistently less antibody for a given degree of neutralization than the other two, regardless of whether the antiserum was specific for that strain or for either of the others. The CTT strain was the most unstable of the three, and required the greatest amount of antibody for a comparable degree of neutralization. The COHO strain was intermediate both in stability and in antibody requirement.The results seem to indicate that the CTT virus required the most antibody because it contained the largest amount of non-infectious virus (to chinook salmon embryo cells), which would still bind antibody and render it unavailable for neutralizing activity. The VR-299 virus contained the least amount of non-infectious virus, and consequently required the smallest amount of antibody.It is suggested that the relative stability of viral strains and possible variation in the content of non-infectious virus should be taken into account or controlled when quantitative neutralization data are used to differentiate or identify viral serotypes.     

Development of neutralizing antibody responses in muskellunge, Esox masquinongy (Mitchill), experimentally exposed to viral haemorrhagic septicaemia virus (genotype IVb)

A complement‐dependent 50% plaque neutralization test was used to assess the neutralizing antibody response in sera of muskellunge, Esox masquinongy, experimentally infected with viral haemorrhagic septicaemia virus (VHSV, genotype IVb) by immersion. Groups of muskellunge were challenged with varying concentrations of VHSV: Group 1 with 10² plaque‐forming units (pfu) mL^?1, Group 2 with 4 × 10³ pfu mL^?1, Group 3 with 10⁵ pfu mL^?1 and Group 4 with 0 pfu mL^?1. The fish were held at a temperature of 11 ± 1 °C and were sampled over a 20‐week period. Neutralizing antibodies were not detected in sera of any of the negative control fish throughout the study. Low neutralizing titres were detected in Groups 1–3 by 6 days post‐infection (p.i.). Neutralizing titres of <80 were not detected again until 3, 4 and 7 weeks p.i. for Groups 2, 3 and 1, respectively, with peak titres for those groups occurring 16, 11 and 17 weeks p.i., respectively. VHSV was detected in serum for up to 11 weeks p.i. Results of this study show that survivors can be detected by a serological technique, despite being virus negative. This may benefit the investigation of VHSV IVb distribution in the Great Lakes and the study of host immune responses to this emerging sublineage.     

Viral replication in excised fin tissues (VREFT) corresponds with prior exposure of Pacific herring, Clupea pallasii (Valenciennes), to viral haemorrhagic septicaemia virus (VHSV)

Procedures for a viral replication in excised fin tissue (VREFT) assay were adapted to Pacific herring, Clupea pallasii, and optimized both to reduce processing time and to provide the greatest resolution between naïve herring and those previously exposed to viral haemorrhagic septicaemia virus (VHSV), Genogroup IVa. The optimized procedures included removal of the left pectoral fin from a euthanized fish, inoculation of the fin with >10⁵ plaque‐forming units (PFU) mL^?1 VHSV for 1 h, rinsing the fin in fresh medium six times to remove unadsorbed virions, incubation of the fin in fresh medium for 4 days and enumeration of the viral titre in a sample of the incubation medium by plaque assay. The optimized VREFT assay was effective at identifying the prior exposure history of laboratory‐reared Pacific herring to VHSV. The geometric mean VREFT value was significantly greater (P < 0.01) among naïve herring (1.2 × 10³ PFU mL^?1) than among groups that survived exposure to VHSV (1.0–2.9 × 10² PFU mL^?1); additionally, the proportion of cultures with no detectable virus was significantly greater (P = 0.0002) among fish that survived exposure to VHSV (39–47%) than among naïve fish (3.3%). The optimized VREFT assay demonstrates promise for identifying VHSV exposure history and forecasting disease potential in populations of wild Pacific herring.     

Characterization of Herpes virus vitreum isolated from hyperplastic epidermal tissue of walleye,Stizostedion vitreum vitreum (Mitchill)*

Abstract. A new virus, provisionally named Herpesvirus vitreum, was isolated from hyperplastic epidermal tissue from a walleye, Stizostedion vitreum vitreum (Mitchill), taken in Saskatchewan, Canada. The virus, which was isolated in the walleye ovarian (WO) cell line, was identified as a herpesvirus on the basis of size (190–230 nm), morphology and apparent pattern of replication. The virus, which passes polycarbonate membranes of 200 nm mean pore diameter, was ether-labile. Virus replicated in WC-1 cells at 4 and 15°C, but not at 20°C. Although walleye cell lines (WO, WC-1, We-2) were susceptible to infection at 15°C, non-period cell lines were refractory. Syncytial formation and lysis occurred in susceptible cell lines. Virus was quantified by plaque assay at 13 to 15°C for two weeks. Replication was inhibited by 10^-3.0m phosphonoacetate and by 10^-5.0m 5-bromodeoxyuridine (BUDR), but addition of excess thymidine reversed the inhibition by BUDR. Viral replication in WO cells, but not in WC-1 cells, was inhibited by the antiherpetic drug acyclovir (10^-5.0m ). The relationship of the herpesvirus isolate and epithelial neoplasms was not determined.     

一株传染性造血器官坏死病病毒的致病性研究

为了对分离于山东某虹鳟养殖场的一株传染性造血器官坏死病毒株(IHNV-Sn1203)进行致病性检测与研究,将该IHNV-Sn1203毒株进行虹鳟鱼苗人工回接感染实验。结果显示,8d内人工感染实验鱼累计死亡率高达100%。收集大批濒死的病鱼样本,制备病理组织切片;利用鲤上皮细胞(EPC)进行细胞感染实验、病毒电镜观察、空斑实验、病毒滴度检测和聚类分析。病理组织切片显示,该病毒可造成虹鳟造血器官广泛性坏死;细胞感染实验结果显示,接种24 h后EPC细胞出现葡萄串状典型细胞病变(cytopathic effect,CPE),72 h后大部分细胞崩解脱落形成网状孔洞;电镜下清晰可见弹状病毒粒子大量存在于细胞质内,其在EPC细胞上的滴度为108.36TCID50/mL,并能形成2~4 mm空斑。对病毒核蛋白氨基酸序列的聚类分析结果显示,该病毒与标准毒株RB-1和WRAC的同源性分别为97%和93%,与国内报道的zyx株具有最高的同源性(99%)。研究表明,IHNV-Sn1203毒株能够在鱼体及敏感细胞中稳定繁殖,产生典型病变,具有较高的病毒滴度,对虹鳟鱼苗有很高的感染性和致死性。     

Comparative susceptibility among three stocks of yellow perch,Perca flavescens (Mitchill), to viral haemorrhagic septicaemia virus strain IVb from the Great Lakes

The Great Lakes strain of viral haemorrhagic septicaemia virus IVb (VHSV‐IVb) is capable of infecting a wide number of naive species and has been associated with large fish kills in the Midwestern United States since its discovery in 2005. The yellow perch, Perca flavescens (Mitchill), a freshwater species commonly found throughout inland waters of the United States and prized for its high value in sport and commercial fisheries, is a species documented in several fish kills affiliated with VHS. In the present study, differences in survival after infection with VHSV IVb were observed among juvenile fish from three yellow perch broodstocks that were originally derived from distinct wild populations, suggesting innate differences in susceptibility due to genetic variance. While all three stocks were susceptible upon waterborne exposure to VHS virus infection, fish derived from the Midwest (Lake Winnebago, WI) showed significantly lower cumulative % survival compared with two perch stocks derived from the East Coast (Perquimans River, NC and Choptank River, MD) of the United States. However, despite differences in apparent susceptibility, clinical signs did not vary between stocks and included moderate‐to‐severe haemorrhages at the pelvic and pectoral fin bases and exophthalmia. After the 28‐day challenge was complete, VHS virus was analysed in subsets of whole fish that had either survived or succumbed to the infection using both plaque assay and quantitative PCR methodologies. A direct correlation was identified between the two methods, suggesting the potential for both methods to be used to detect virus in a research setting.