Genome-wide gene expression analysis of anguillid herpesvirus 1

<p>Background: Whereas temporal gene expression in mammalian herpesviruses has been studied extensively, little is known about gene expression in fish herpesviruses. Here we report a genome-wide transcription analysis of a fish herpesvirus, anguillid herpesvirus 1, in cell culture, studied during the first 6 hours of infection using reverse transcription quantitative PCR.</p> <p>Results: Four immediate-early genes – open reading frames 1, 6A, 127 and 131 – were identified on the basis of expression in the presence of a protein synthesis inhibitor and unique expression profiles during infection in the absence of inhibitor. All of these genes are located within or near the terminal direct repeats. The remaining 122 open reading frames were clustered into groups on the basis of transcription profiles during infection. Expression of these genes was also studied in the presence of a viral DNA polymerase inhibitor, enabling classification into early, early-late and late genes. In general, clustering by expression profile and classification by inhibitor studies corresponded well. Most early genes encode enzymes and proteins involved in DNA replication, most late genes encode structural proteins, and early-late genes encode non-structural as well as structural proteins.</p> <p>Conclusions: Overall, anguillid herpesvirus 1 gene expression was shown to be regulated in a temporal fashion, comparable to that of mammalian herpesviruses.</p&gt

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

Molecular characterization of the alloherpesvirus anguillid herpesvirus 1

All herpesviruses belong to the order Herpesvirales, which consists of the families Herpesviridae, Alloherpesviridae and Malacoherpesviridae. Although herpesviruses share unique morphological characteristics, only the gene encoding the ATPase subunit of terminase is detectably conserved throughout the order. The family Herpesviridae, which comprises mammalian, avian and reptilian herpesviruses, has been studied extensively, but much less knowledge is available for members of the families Alloherpesviridae and Malacoherpesviridae, which respectively comprise amphibian and fish, and invertebrate herpesviruses. Anguillid herpesvirus 1 (AngHV1) frequently causes disease in wild and cultured European eel, a traditionally important fish species in the Netherlands. Hence, in this study AngHV1 was chosen as a model for the family Alloherpesviridae. The aim of the study was to characterize AngHV1 at the molecular level, and to determine its similarities and differences as compared with other herpesviruses. AngHV1 has a genome of close to 250 kbp, including an 11 kbp terminal direct repeat. The genome contains a total of 129 protein-coding genes, five of which are duplicated in the terminal repeat. Since only a dozen genes are detectably conserved among fish and amphibian herpesviruses, the family Alloherpesviridae appears to be more divergent than the family Herpesviridae, among which more than 40 genes are conserved. Taxonomically, AngHV1 is most closely related to the cyprinid herpesviruses. High-resolution transcriptome analysis based on deep sequencing revealed that RNA splicing is much more abundant than had been assumed. A total of 58 functional splice junctions were identified. Eleven genes contain integral, spliced protein-coding exons, and nine contain 5’-untranslated exons or, in instances of alternative splicing, 5’-untranslated or -translated exons. In contrast to mammalian herpesviruses, overall levels of antisense transcription in AngHV1 were low, and no abundant, non-overlapping non-coding RNAs were identified. A genome-wide expression study using qPCR showed that gene expression is regulated in a temporal fashion, similar to mammalian herpesviruses. The putative regulatory immediate-early genes of AngHV1 were identified, and appeared to be located within and near the terminal repeats. The remaining open reading frames were classified into early, early-late and late genes. Most early genes encode enzymes and proteins involved in DNA replication, and most late genes encode structural proteins. The structural proteins of AngHV1 were identified using a combination of classical virus purification and fractionation techniques and modern mass spectrometry analyses. A total of 40 different structural proteins were identified, of which 7 could be assigned to the capsid, 11 to the envelope, and 22 to the tegument. Although no convincing sequence homology is apparent between the herpesvirus families for any of the structural proteins, virion composition shows many similarities. AngHV1 encodes several viral homologs of components of the host immune system, including an interleukin-10-like open reading frame. Although amino acid sequence homology between the European eel interleukin-10 and the AngHV1 interleukin-10 homolog is low, the three-dimensional structures as predicted by modelling are highly similar, suggesting functionality. Overall, despite the virtual absence of detectable genetic similarities, AngHV1 and the other alloherpesviruses resemble members of the family Herpesviridae in many fundamental aspects

Zoönosen : uit de ziekenboeg

Zoönosen zijn infectieziekten die overdraagbaar van dier op mens, zowel vanuit koudbloedige als warmbloedige dieren Ook bij contacten met aquacultuur- en visserijdieren is er een klein risico voor de mens op het oplopen van een ziekte. In de Nederlandse aquacultuur komen af en toe zoönotische bacteriën voor, die contactzoönose kunnen veroorzaken. Met voldoende hygiëne op aquacultuurbedrijven ontwikkelen deze bacteriën zich meestal niet tot een zoönose. Toch is het belangrijk, dat men op de hoogte is van risico's en mee helpt aan een goede diagnose van een aquatische zoönose, omdat deze in eerste instantie door de huisarts vaak niet als zodanig herkend wordt

Ziekten van vissen, schaal- en schelpdieren, van belang voor de Nederlandse aquacultuur

In de Nederlandse aquacultuur wordt een groot aantal vis- en schelpdiersoorten gekweekt en geteeld, daarnaast is er een zeer beperkte houderij van schaaldieren. Net als in andere dierlijke productiesectoren komen in de aquacultuursector verschillende ziekten voor die een belemmering kunnen vormen voor de productie. Dit boek geeft een overzicht van de belangrijkste vis-, schaaldier- en schelpdierziekten in Nederland

Diagnostic yield of repeat sampling with immunoassay, real-time PCR, and toxigenic culture for the detection of toxigenic Clostridium difficile in an epidemic and a non-epidemic setting

Current international guidelines lack definite conclusions regarding repeat stool sampling for the detection of toxigenic Clostridium difficile. We assessed the value of repeat sampling and compared the diagnostic yield in an epidemic to a non-epidemic setting. Consecutive fecal samples obtained during two time frames were analyzed using direct stool immunoassay toxin testing (enzyme immunoassay [EIA]), direct stool real-time PCR toxin gene testing, and toxigenic culture. Samples collected within 7 days of the initial sample were considered repeat tests. In the epidemic setting 989 patients were analyzed, and in the non-epidemic setting 1,015. In the epidemic setting 204 patients had two or more specimens included for analysis and in the non-epidemic setting 287 patients. In the epidemic setting 136 samples yielded a positive results, either by EIA or toxigenic culture; of these, 108 were positive according to EIA and 123 according to toxigenic culture. In the first test round 98 (90.7 %, 95 % CI 85.3 to 96.2), 114 (92.7 %, 88.1 to 97.3), and 126 (92.6 %, 88.3 to 97.0) positives were detected. Subsequent test rounds yielded 10 (9.3 %, 3.8 to 14.7), 9 (7.3 %, 2.7 to 11.9), and 10 (7.4 %, 3.0 to 11.7) extra positives. In the non-epidemic setting EIA, toxigenic culture and PCR detected 33, 66, and 83 positives. The three tests combined 93 detected positives. In the first test round 30 (90.9 %, 81.1 to 100.7), 63 (95.5 %, 90.4 to 110.5), 76 (91.6 %, 85.6 to 97.5), and 87 (93.5 %, 88.6 to 98.5) positives were detected. Subsequent test rounds yielded 3 (9.1 %, −0.7 to 18.9), 3 (4.5 %, −0.5 to 9.6), 7 (8.4 %, 2.5 to 14.4), and 6 (6.5 %, 1.5 to 11.4) extra positives. In conclusion, repeat testing resulted in 4.5 % to 9.3 % extra positives. No significant difference between the settings studied could be demonstrated. Repeat sampling and multimodality testing may be chosen in an outbreak situation to detect all cases, effectively controlling nosocomial spread

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 pathogenesi

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 r2-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

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

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 A. anguilla yellow and silver eels 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