Keith Way

Nucleotide sequence of the glycoprotein gene of viral haemorrhagic septicaemia (VHS) viruses from different geographical areas: a link between VHS in farmed fish species and viruses isolated from North Sea cod (Gadus morhua L.)

RT-PCR methods have been applied to the detection and sequencing of the glycoprotein gene of viral haemorrhagic septicaemia virus (VHSV), the rhabdovirus which causes viral haemorrhagic septicaemia (VHS) in farmed salmonid fish. Phylogenetic analysis of a 360 nt region of the glycoprotein gene from a range of marine and fresh water VHSV isolates identified three genogroups, I-III. Significantly, two virus isolates recovered from ulcerated North Sea cod caught off the Shetland Islands, and an isolate recovered from diseased turbot farmed on the island of Gigha, Scotland were assigned to the same genogroup. Moreover, a virus isolated from diseased turbot farmed on the Baltic Sea coast shared 99.4% nucleotide sequence similarity with a virus associated with a VHS outbreak in rainbow trout. This is the first time that a genetic link between a VHS outbreak and natural VHSV infections of marine fish species has been demonstrated.

First Report of Spring Viremia of Carp Virus (SVCV) in Wild Common Carp in North America

Abstract In spring 2002, an estimated 1,500 common carp Cyprinus carpio in Cedar Lake, northwestern Wisconsin, died over a 6-week period from late April through the first week in June. Three moribund carp were necropsied and had signs consistent with spring viremia of carp (SVC) disease, including petechiae and ecchymotic hemorrhages on the skin, ascites, and edematous kidney and spleen. A virus was isolated on fathead minnow cells and shown to be a rhabdovirus by electron microscopy. Immunoassay results indicated a close serological relationship with spring viremia of carp virus (SVCV). This was confirmed by a reverse transcriptase–polymerase chain reaction assay and subsequent analysis of a subsection glycoprotein gene. Immunocytochemistry and serum neutralization tests indicated that the Cedar Lake isolate did not share complete antigenic identity with the European reference SVCV. Also, the isolate showed an inhibition of cytopathic effect after repeated subculture in epithelioma papulosum cyprini and ...

Antibody response of two populations of common carp, Cyprinus carpio L., exposed to koi herpesvirus.

Common carp, Cyprinus carpio L., exposed to koi herpesvirus (KHV) may become persistently infected and populations containing such virus-infected individuals may transmit the virus to other fish when co-habited. Detection of virus-infected fish in a population is thus critical to surveillance and control programmes for KHV. A study was therefore designed to detect anti-KHV serum antibodies, with an enzyme-linked immunosorbent assay, in common carp following experimental exposures to KHV under varying environmental conditions. The study determined that a proportion of fish within a population experimentally exposed to KHV (at least 10-25%) develop high antibody titres (1/1600 or greater) to the virus, and this immunological response was detectable for several months (observed at the termination of the experiments at 65, 46 and 27 weeks post-exposure). Furthermore, this response was detected in one population of fish that did not succumb to a high level of mortality when maintained at water temperatures that were non-permissive for KHV. Elevating the water temperatures to permissive conditions for KHV resulted in recurrence of disease despite the presence of anti-virus antibodies, suggesting that serum antibodies alone are not protective under the conditions of our trials.

Emergence of cold water strawberry disease of rainbow trout Oncorynchus mykiss in England and Wales: outbreak investigations and transmission studies.

Cold water strawberry disease (CWSD), or red mark syndrome (RMS), is a severe dermatitis affecting the rainbow trout Oncorynchus mykiss. The condition, which presents as multifocal, raised lesions on the flanks of affected fish, was first diagnosed in Scotland in 2003 and has since spread to England and Wales. Results of field investigations indicated the condition had an infectious aetiology, with outbreaks in England linked to movements of live fish from affected sites in Scotland. Transmission trials confirmed these results, with 11 of 149 and 106 of 159 naive rainbow trout displaying CWSD-characteristic lesions 104 to 106 d after being cohabited with CWSD-affected fish from 2 farms (Farm B from England and Farm C from Wales, respectively). The condition apparently has a long latency, with the first characteristic lesions in the previously naive fish not definitively observed until 65 d (650 day-degrees) post-contact with affected fish. Affected fish from both outbreak investigations and the infection trial were examined for the presence of viruses, oomycetes, parasites and bacteria using a combination of techniques and methodologies (including culture-independent cloning of PCR-amplified bacterial 16S rRNA genes from lesions), with no potentially causative infectious agent consistently identified. The majority of the cloned phylotypes from both lesion and negative control skin samples were assigned to Acidovorax-like beta-Proteobacteria and Methylobacterium-like alpha-Proteobacteria.

Comparison of the resistance of selected families of common carp, Cyprinus carpio L., to koi herpesvirus: preliminary study.

Koi herpesvirus disease (KHD), caused by koi herpesvirus (KHV), is a highly infectious disease of carp, Cyprinus carpio L., usually causing high mortality (reviewed by Dixon 2008). The disease has a worldwide distribution and affects ornamental carp (koi carp) as well as common carp in both aquaculture and open waters (rivers, lakes, etc.). The disease is economically very important, having an impact on the trade in high value ornamental fish, sport fisheries and food production. Attempts have been made to reduce the economic impact of the disease by vaccination (Ronen, Perelberg, Abramowitz, Hutoran, Tinman, Bejerano, Steinitz & Kotler 2003; Perelberg, Ronen, Hutoran, Smith & Kotler 2005), but as yet no vaccine has been approved for use outside Israel. An alternative method of controlling the disease by use of resistant strains has been investigated (Shapira, Magen, Zak, Kotler, Hulata & Levavi-Sivan 2005; Zak, Perelberg, Magen, Milstein & Joseph 2007). The latter authors bred three hybrid and three pure crosses from two Hungarian and one Israeli strain of fish and reported survival rates after challenge with KHV of between 4% and 20%. Shapira et al. (2005) crossed two strains of domesticated carp with one strain of wild carp and exposed the progeny to KHV both in the laboratory and in the field. The mortalities ranged from 8.9% to 89.7% in the field trial and from 8.6% to 68.5% in the laboratory trial, and with one exception there was good comparability of survival rates of the different crosses in the two trials. To further explore the potential of producing strains of carp resistant to KHV, we have investigated the survival of a large number of carp families following exposure to the virus, and present the results here. This study is part of a larger study, which includes investigating production characteristics, the resistance of the families to Aeromonas hydrophila, post-infection changes at the genomic level (Williams, Li, Hughes, Gonzalez, Vernon, Vidal, Jeney, Jeney, Dixon, McAndrew, Bartfai, Orban, Trudeau, Rogers, Matthews, Fraser, Gracey & Cossins 2008) and proteomic level, heritability characteristics, the genetic correlation between different traits and gene linkage mapping. The production and other characteristics of the fish families will be described in full in a separate communication (Z. Jeney, unpublished data). Briefly, 96 families were produced by diallele crossing of four strains of carp (Duna, Amur, Tata and HAKI 15) maintained in a live gene bank at HAKI, Hungary. Five females and 10 males from each strain were used to produce six families per strain cross. The families were maintained in separate tanks until they were large enough to be injected with a passive integrated transponder (PIT) tag. Twenty fish from each of 94 families (there Journal of Fish Diseases 2009, 32, 1035–1039 doi:10.1111/j.1365-2761.2009.01081.x

Detection of novel strains of cyprinid herpesvirus closely related to koi herpesvirus

Cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus (KHV) is a devastating virus of carp. Using generic primers for the DNA polymerase and the major capsid protein genes of cyprinid herpesviruses, nucleotide sequences divergent from previously described CyHV-3 were obtained. At least 3 novel groups of putative CyHV-3-like viruses were identified, sharing 95 to 98% nucleotide identity with CyHV-3 strains. Carp carrying the CyHV-3 variants did not show clinical signs consistent with CyHV-3 infection and originated from locations with no actual CyHV-3 outbreaks. These strains might represent low- or non-pathogenic variants of CyHV-3.

Cyprinid Herpesvirus 3 : An Archetype of Fish Alloherpesviruses

The order Herpesvirales encompasses viruses that share structural, genetic, and biological properties. However, members of this order infect hosts ranging from molluscs to humans. It is currently divided into three phylogenetically related families. The Alloherpesviridae family contains viruses infecting fish and amphibians. There are 12 alloherpesviruses described to date, 10 of which infect fish. Over the last decade, cyprinid herpesvirus 3 (CyHV-3) infecting common and koi carp has emerged as the archetype of fish alloherpesviruses. Since its first description in the late 1990s, this virus has induced important economic losses in common and koi carp worldwide. It has also had negative environmental implications by affecting wild carp populations. These negative impacts and the importance of the host species have stimulated studies aimed at developing diagnostic and prophylactic tools. Unexpectedly, the data generated by these applied studies have stimulated interest in CyHV-3 as a model for fundamental research. This review intends to provide a complete overview of the knowledge currently available on CyHV-3.

Identification and Characterization of Cyprinid Herpesvirus-3 (CyHV-3) Encoded MicroRNAs

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in post-transcriptional gene regulation. Some viruses encode their own miRNAs and these are increasingly being recognized as important modulators of viral and host gene expression. Cyprinid herpesvirus 3 (CyHV-3) is a highly pathogenic agent that causes acute mass mortalities in carp (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) worldwide. Here, bioinformatic analyses of the CyHV-3 genome suggested the presence of non-conserved precursor miRNA (pre-miRNA) genes. Deep sequencing of small RNA fractions prepared from in vitro CyHV-3 infections led to the identification of potential miRNAs and miRNA–offset RNAs (moRNAs) derived from some bioinformatically predicted pre-miRNAs. DNA microarray hybridization analysis, Northern blotting and stem-loop RT-qPCR were then used to definitively confirm that CyHV-3 expresses two pre-miRNAs during infection in vitro. The evidence also suggested the presence of an additional four high-probability and two putative viral pre-miRNAs. MiRNAs from the two confirmed pre-miRNAs were also detected in gill tissue from CyHV-3-infected carp. We also present evidence that one confirmed miRNA can regulate the expression of a putative CyHV-3-encoded dUTPase. Candidate homologues of some CyHV-3 pre-miRNAs were identified in CyHV-1 and CyHV-2. This is the first report of miRNA and moRNA genes encoded by members of the Alloherpesviridae family, a group distantly related to the Herpesviridae family. The discovery of these novel CyHV-3 genes may help further our understanding of the biology of this economically important virus and their encoded miRNAs may have potential as biomarkers for the diagnosis of latent CyHV-3.

Spring viraemia of carp (SVC) in the UK: the road to freedom.

Spring viraemia of carp (SVC) is a disease of international importance that predominantly affects cyprinid fish and can cause significant mortality. In the United Kingdom (UK), SVC was first detected in 1977 with further cases occurring in fisheries, farms, wholesale and retail establishments throughout England and Wales (but not Scotland, where few cyprinid populations exist, nor Northern Ireland where SVC has never been detected) over the subsequent 30 years. Following a control and eradication programme for the disease initiated in 2005, the UK was recognised free of the disease in 2010. This study compiles historic records of SVC cases in England and Wales with a view to understanding its routes of introduction and spread, and assessing the effectiveness of the control and eradication programme in order to improve contingency plans to prevent and control future disease incursions in the cyprinid fish sectors. Between 1977 and 2010 the presence of SVC was confirmed on 108 occasions, with 65 of the cases occurring in sport fisheries and the majority of the remainder occurring in the ornamental fish sector. The study found that throughout the history of SVC in the UK, though cases were widely distributed, their occurrence was sporadic and the virus did not become endemic. All evidence indicates that SVC was not able to persist under UK environmental conditions, suggesting that the majority of cases were a result of new introductions to the UK as opposed to within-country spread. The control and eradication programme adopted in 2005 was highly effective and two years after its implementation cases of SVC ceased. Given the non-persistent nature of the pathogen the most important aspect of the control programme focused on preventing re-introduction of the virus to the UK. Despite the effectiveness of these controls against SVC, this approach is likely to be less effective against more persistent pathogens such as koi herpesvirus, which are likely to require more stringent measures to prevent within-country spread.

Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA)

Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme-linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs.