O.L.M. Haenen

Complete genome sequence and taxonomic position of anguillid herpesvirus 1.

Eel herpesvirus or anguillid herpesvirus 1 (AngHV1) frequently causes disease in freshwater eels. The complete genome sequence of AngHV1 and its taxonomic position within the family Alloherpesviridae were determined. Shotgun sequencing revealed a 249 kbp genome including an 11 kbp terminal direct repeat that contains 7 of the 136 predicted protein-coding open reading frames. Twelve of these genes are conserved among other members of the family Alloherpesviridae and another 28 genes have clear homologues in cyprinid herpesvirus 3. Phylogenetic analyses based on amino acid sequences of five conserved genes, including the ATPase subunit of the terminase, confirm the position of AngHV1 within the family Alloherpesviridae, where it is most closely related to the cyprinid herpesviruses. Our analyses support a recent proposal to subdivide the family Alloherpesviridae into two sister clades, one containing AngHV1 and the cyprinid herpesviruses and the other containing Ictalurid herpesvirus 1 and the ranid herpesviruses.

The alloherpesviral counterparts of interleukin 10 in European eel and common carp.

Viral interleukin 10 (IL-10) like open reading frames have been identified in several pox- and herpesviruses, including the fish herpesviruses Anguillid herpesvirus 1 (AngHV-1) and Cyprinid herpesvirus 3 (CyHV-3). European eel (Anguilla anguilla) IL-10 was sequenced, in order to compare European eel and common carp (Cyprinus carpio) IL-10 with their alloherpesviral counterparts. Homology between the virus and host IL-10 amino acid sequences is low, which is confirmed by phylogenetic analysis. However, the three dimensional structures of the fish and alloherpesviral IL-10 proteins as predicted by modeling are highly similar to human IL-10. Closely related AngHV-1 and CyHV-3 are expected to have obtained their viral IL-10 genes independently in the course of coexistence with their respective hosts. The presence and structural conservation of these alloherpesviral IL-10 genes suggest that they might play an important role in the evolution of pathogenesis.

Viral diseases of wild and farmed European eel Anguilla anguilla with particular reference to the Netherlands.

Diseases are an important cause of losses and decreased production rates in freshwater eel farming, and have been suggested to play a contributory role in the worldwide decline in wild freshwater eel stocks. Three commonly detected pathogenic viruses of European eel Anguilla anguilla are the aquabirnavirus eel virus European (EVE), the rhabdovirus eel virus European X (EVEX), and the alloherpesvirus anguillid herpesvirus 1 (AngHV1). In general, all 3 viruses cause a nonspecific haemorrhagic disease with increased mortality rates. This review provides an overview of the current knowledge on the aetiology, prevalence, clinical signs and gross pathology of these 3 viruses. Reported experimental infections showed the temperature dependency and potential pathogenicity of these viruses for eels and other fish species. In addition to the published literature, an overview of the isolation of pathogenic viruses from wild and farmed A. anguilla in the Netherlands during the past 2 decades is given. A total of 249 wild A. anguilla, 39 batches of glass eels intended for farming purposes, and 239 batches of farmed European eels were necropsied and examined virologically. AngHV1 was isolated from wild yellow and silver A. anguilla from the Netherlands from 1998 until the present, while EVEX was only found sporadically, and EVE was never isolated. In farmed A. anguilla AngHV1 was also the most commonly isolated virus, followed by EVE and EVEX.

Identification and localization of the structural proteins of anguillid herpesvirus 1

Many of the known fish herpesviruses have important aquaculture species as their natural host, and may cause serious disease and mortality. Anguillid herpesvirus 1 (AngHV-1) causes a hemorrhagic disease in European eel, Anguilla anguilla. Despite their importance, fundamental molecular knowledge on fish herpesviruses is still limited. In this study we describe the identification and localization of the structural proteins of AngHV-1. Purified virions were fractionated into a capsid-tegument and an envelope fraction, and premature capsids were isolated from infected cells. Proteins were extracted by different methods and identified by mass spectrometry. A total of 40 structural proteins were identified, of which 7 could be assigned to the capsid, 11 to the envelope, and 22 to the tegument. The identification and localization of these proteins allowed functional predictions. Our findings include the identification of the putative capsid triplex protein 1, the predominant tegument protein, and the major antigenic envelope proteins. Eighteen of the 40 AngHV-1 structural proteins had sequence homologues in related Cyprinid herpesvirus 3 (CyHV-3). Conservation of fish herpesvirus structural genes seemed to be high for the capsid proteins, limited for the tegument proteins, and low for the envelope proteins. The identification and localization of the structural proteins of AngHV-1 in this study adds to the fundamental knowledge of members of the Alloherpesviridae family, especially of the Cyprinivirus genus.

Thermal preference of juvenile Dover sole (Solea solea) in relation to thermal acclimation and optimal growth temperature.

Dover sole (Solea solea) is an obligate ectotherm with a natural thermal habitat ranging from approximately 5 to 27°C. Thermal optima for growth lie in the range of 20 to 25°C. More precise information on thermal optima for growth is needed for cost-effective Dover sole aquaculture. The main objective of this study was to determine the optimal growth temperature of juvenile Dover sole (Solea solea) and in addition to test the hypothesis that the final preferendum equals the optimal growth temperature. Temperature preference was measured in a circular preference chamber for Dover sole acclimated to 18, 22 and 28°C. Optimal growth temperature was measured by rearing Dover sole at 19, 22, 25 and 28°C. The optimal growth temperature resulting from this growth experiment was 22.7°C for Dover sole with a size between 30 to 50 g. The temperature preferred by juvenile Dover sole increases with acclimation temperature and exceeds the optimal temperature for growth. A final preferendum could not be detected. Although a confounding effect of behavioural fever on temperature preference could not be entirely excluded, thermal preference and thermal optima for physiological processes seem to be unrelated in Dover sole.

Impact of Eel Viruses on Recruitment of European Eel

Eels have an uncommon catadromic life cycle with exceptional migratory patterns to their spawning grounds several thousand kilometres away: the European eel (Anguilla anguilla) travels over 5,500 km to the Sargasso Sea (Schmidt 1923; McCleave and Kleckner 1987; Tesch 1982; Tesch and Wegner 1990); the American eel (A. rostrata) migrates over 4,000 km also to the Sargasso Sea (Castonguay and McCleave 1987; McCleave and Kleckner 1987; Tesch and Wegner 1990); the Australian eel (A. aus-tralis) travels over 5,000 km into the Pacific Ocean to spawn (Jellyman 1987); and the Japanese eel (A. japonica) travels over 4,000 km to an area near the Marianna Islands in the Philippines to spawn (Tsukamoto 1992). Evidently such long distance swimming will place those fishes under extra stress caused by the long starvation period, the high energy cost of the journey, and the many changes in the environment such as salt water, darkness, high pressure, and low temperatures, among other stress factors. Stress is often a basis for disease in eel, especially in intensive eel culture (Haenen and Engelsma, 2005 unpublished finding). Nowadays, global transport of live fishes for aquaculture has facilitated the global spread of pathogens from diseased to healthy stocks. Within the last few decades, aquaculture has become an important production branch in our society. Its global production has more than doubled between 1986 and 1996 in tonnage and value, and over one quarter of human fish consumption at world scale is now produced in aquaculture (Naylor et al. 2000). The Netherlands is one of the leading eel producing & trading countries (Heinsbroek and Kamstra 1995). Blanc (1997) showed that nearly 100 pathogens have been introduced into European water bodies since the introduction of aquaculture. Worldwide many diseases are known in both wild and cultured eel. Parasites, for example trematodes, Anguillicola crassus(nematode), and Myxidium giardi (myxosporean)occur naturally in wild eel populations, mostly in low numbers, without causing mortality (Koie 1988; Van Banning and Haenen 1990; Borgsteede et al. 1999). However, under culture conditions, with eels kept in high densities, they may be harmful. Eel pathogenic bacteria like Vibrio vulnificus, Vibrio anguillarum, Pseudomonas anguillisepticaand Edwardsiella tardamay also cause disease, especially when a stress factor is involved or when the eel is injured (Veenstra et al. 1993; Austin and Austin 1999; Haenen and Davidse 2001). As far as we know, the clinical signs are often more severe under culture conditions compared to in the wild.

Development and validation of a two-step real-time RT-PCR for the detection of eel virus European X in European eel, Anguilla anguilla

Eel virus European X (EVEX) is one of the most common pathogenic viruses in farmed and wild European eel (Anguilla anguilla) in the Netherlands. The virus causes a hemorrhagic disease resulting in increased mortality rates. Cell culture and antibody-based detection of EVEX are laborious and time consuming. Therefore, a two-step real-time reverse transcriptase (RT-)PCR assay was developed for rapid detection of EVEX. Primers and probe for the assay were designed based on a sequence of the RNA polymerase or L gene of EVEX. The real-time RT-PCR assay was validated both for use with SYBR Green chemistry and for use with a TaqMan probe. The assay is sensitive, specific, repeatable, efficient and has a high r²-value. The real-time RT-PCR assay was further evaluated by testing field samples of European eels from the Netherlands, which were positive or negative for EVEX by virus isolation followed by an indirect fluorescent antibody test. The real-time RT-PCR assay allows rapid, sensitive and specific laboratory detection of EVEX in RNA extracts from 10% eel organ suspensions and cell cultures with cytopathic effects, and is a valuable contribution to the diagnosis of viral diseases of eel.

Complete genome sequence of a common midwife toad virus-like ranavirus associated with mass mortalities in wild amphibians in the Netherlands

ABSTRACT A ranavirus associated with mass mortalities in wild water frogs (Pelophylax spp.) and other amphibians in the Netherlands since 2010 was isolated, and its complete genome sequence was determined. The virus has a genome of 107,772 bp and shows 96.5% sequence identity with the common midwife toad virus from Spain.

Development and validation of a real-time PCR assay for the detection of anguillid herpesvirus 1

Anguillid herpesvirus 1 (AngHV1) causes a haemorrhagic disease with increased mortality in wild and farmed European eel, Anguilla anguilla (L.) and Japanese eel Anguilla japonica, Temminck & Schlegel). Detection of AngHV1 is currently based on virus isolation in cell culture, antibody-based typing assays or conventional PCR. We developed, optimized and concisely validated a diagnostic TaqMan probe based real-time PCR assay for the detection of AngHV1. The primers and probe target AngHV1 open reading frame 57, encoding the capsid protease and scaffold protein. Compared to conventional PCR, the developed real-time PCR is faster, less labour-intensive and has a reduced risk of cross-contamination. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability, efficiency and r(2) -value. The diagnostic performance of the assay was determined by testing 10% w/v organ suspensions and virus cultures from wild and farmed European eels from the Netherlands by conventional and real-time PCR. The developed real-time PCR assay is a useful tool for the rapid and sensitive detection of AngHV1 in 10% w/v organ suspensions from wild and farmed European eels.

Vibrio vulnificus outbreaks in Dutch eel farms since 1996: strain diversity and impact

Vibrio vulnificus is a potentially zoonotic bacterial pathogen of fish, which can infect humans (causing necrotic fasciitis). We analysed 24 V. vulnificus isolates (from 23 severe eel disease outbreaks in 8 Dutch eel farms during 1996 to 2009, and 1 clinical strain from an eel farmer) for genetic correlation and zoonotic potential. Strains were typed using biotyping and molecular typing by high-throughput multilocus sequence typing (hiMLST) and REP-PCR (Diversilab®). We identified 19 strains of biotype 1 and 5 of biotype 2 (4 from eels, 1 from the eel farmer), that were subdivided into 8 MLST types (ST) according to the international standard method. This is the first report of V. vulnificus biotype 1 outbreaks in Dutch eel farms. Seven of the 8 STs, of unknown zoonotic potential, were newly identified and were deposited in the MLST database. The REP-PCR and the MLST were highly concordant, indicating that the REP-PCR is a useful alternative for MLST. The strains isolated from the farmer and his eels were ST 112, a known potential zoonotic strain. Antimicrobial resistance to cefoxitin was found in most of the V. vulnificus strains, and an increasing resistance to quinolones, trimethoprim + sulphonamide and tetracycline was found over time in strain ST 140. Virulence testing of isolates from diseased eels is recommended, and medical practitioners should be informed about the potential risk of zoonotic infections by V. vulnificus from eels for the prevention of infection especially among high-risk individuals. Additional use of molecular typing methods such as hiMLST and Diversilab® is recommended for epidemiological purposes during V. vulnificus outbreaks.