Detection of Herpesvirus anguillae (AngHV‐1) in European eel Anguilla anguilla (L.) originating from northern Poland—assessment of suitability of selected diagnostic methods

The Community Action Plan requests EU member states to implement measures that ensure the recovery of the severely depleted European eel stocks. One of the main threats is posed by Anguillid herpesvirus 1 (AngHV‐1) leading to increased mortality in both wild and farmed eels. Following recommendations of the OIE to minimize the risk of obtaining false‐negative results, the main aim of the study was to optimize diagnostic methods for AngHV‐1 detection using conventional PCR, nested PCR and in situ hybridization assay. While 53.3% of the individual organ samples were tested positive for AngHV‐1 by PCR, the additional virus analysis via nested PCR revealed that the actual prevalence was 93.3%. In the cell cultivation passages, a cytopathic effect was hardly found in the first two rounds. In the third passage onto cell cultures, a lytic CPE was detected. The identification and confirmation of the viruses obtained from cell cultures as well as directly from the organ tissues were proceeded by PCR, nested PCR and sequencing of the PCR products. While no positive signal was detectable in the first round by PCR using samples from the third cell culture passages, the nested PCR provided weak but visible positive signals.     

Aquabirnaviruses isolated from marine organisms form a distinct genogroup from other aquabirnaviruses

A phylogenetic tree of aquabirnaviruses, including marine birnaviruses (MABV) and infectious pancreatic necrosis virus (IPNV), was developed based on the nucleotide sequences and deduced amino acid sequences of the polyprotein and VP5 genes of genomic segment A. In the polyprotein of MABV strains, the amino acid sequences were very similar, with identities of 98.3-99.7%. Twenty-one unique amino acid residues were found in the deduced amino acid sequences of the polyprotein gene of MABV strains. The phylogenetic tree based on the nucleotide sequence of genomic segment A and polyprotein sequences showed that 31 aquabirnavirus strains were clustered into seven genogroups. All MABV strains isolated in Japan and Korea were clustered into one genogroup which was distinct from other aquabirnaviruses. The seventh genogroup containing all MABV strains showed amino acid sequence similarities of 80.7-90.6% with other genogroups. In VP5, four unique residues were found in MABV strains when compared with IPNV strains. The MABV strains exhibited amino acid sequence similarities of 63.9-86.4% with IPNV strains. The amino acid sequences of VP5 were conserved among MABV strains, but differed from those of IPNV strains. The MABV strains isolated from different host species and different geographical areas were very similar to each other, suggesting that the MABV are distinct from the other genogroups.     

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.     

Genetic analysis of aquabirnaviruses isolated from wild fish reveals occurrence of natural reassortment of infectious pancreatic necrosis virus

In this study, we report the sequencing of the whole genome [including the 5' and 3' non-coding regions (NCR) of both segments A and B] of seven birnavirus strains isolated from wild fish from the Flemish Cap (FC) fishery at Newfoundland, Canada. From analysis and comparison of the sequences, most of the FC isolates clustered with the North American reference strains West Buxton (WB), Dry Mill and Jasper. One strain was included in the same genotype as the European strain Ab. In addition, at least in one case cohabitation of both type strains in an individual fish was demonstrated. These results clearly suggest the acquisition of the viruses from two different sources. The prevalence of the American type is easily explained by the close proximity of this fishing bank to the American coast whereas, although surprising, the presence of the European type strain could be because of migration of fish from European waters. In one strain, segment A and B sequences were typed differently (WB and Ab, respectively). These findings indicate natural reassortment between two strains of aquabirnaviruses in a host.     

Infections of nervous necrosis virus in wild and cage‐reared marine fish from South China Sea with unexpected wide host ranges

The concerns about the impact of the nervous necrosis virus (NNV) infections in wild fish have been raised. This paper presents the results of quarterly surveys of NNV in wild and cage‐reared marine fish from South China Sea. Samples of 892 wild fish belonging to 69 species and 381 cage‐reared fish belonging to 11 species were collected and were detected by seminested PCR and nested PCR. In the case of seminested PCR, the positive signal was detected in 3.0% and 3.1% samples of wild and cage‐reared fish, respectively. However, by nested RT‐PCR, the positive signal was observed in 42.3% and 63.0% samples of wild and cage‐reared fish, respectively. If the fish species were considered, the positive signal was detected in 21.7% and 72.7% species of wild and cage‐reared fish by seminested PCR assay, respectively. However, by nested RT‐PCR, the positive signal was observed in 65.2% and 100% species of wild and cage‐reared fish, respectively. The nucleotide sequences of the nested PCR products were determined. Phylogenetic tree showed that all the obtained viral isolates belonged to the red‐spotted grouper nervous necrosis virus (RGNNV) genotype. Thirty‐five species of the marine fish were the new hosts of NNV.     

鳗鲡疱疹病毒的分离与鉴定

为获知鳗鲡"脱粘败血病"与病毒的关系,实验用蔗糖密度梯度离心的方法从发病的欧洲鳗鲡内脏器官组织匀浆液中纯化了病毒粒子,负染后利用电镜观察;进一步用EO细胞对病毒进行了分离、培养,并对感染病毒的细胞进行超薄切片,电镜观察;然后,提取病毒DNA,利用鳗鲡疱疹病毒的PCR检测方法对其进行了鉴定。结果显示,接种匀浆上清液的EO细胞出现细胞融合的病变效应;分离病毒的病毒粒子具囊膜,大小约为200 nm;从感染病毒的细胞上清液DNA中扩增出特异性条带,序列测定与比对分析表明,该序列与鳗鲡疱疹病毒欧洲株(AngHV-1)的序列完全一致。研究表明,利用EO细胞分离了一株鳗鲡病毒,经形态观察和DNA分析,确认该病毒为鳗鲡疱疹病毒,命名为AngHV-FJ。该研究为深入开展鳗鲡疱疹病毒的致病机制及鳗鲡"脱粘败血病"的防控研究奠定了重要基础。     

First isolation of Flavobacterium psychrophilum associated with reports of moribund wild European eel (Anguilla anguilla) in Scotland

In late April 2015, the River Dee Trust informed Marine Scotland Science, Fish Health Inspectorate (FHI), that there had been observations of dead and moribund European eels on the River Dee. Later in May, the Spey Fishery Board also reported a number of moribund European eels in a rotary screw smolt trap on the River Spey. In total, 10 cases involving moribund eels were investigated in 2015 and one case in 2016. In addition, a health screen was conducted to investigate the potential presence of Flavobacterium psychrophilum in healthy eels and Atlantic salmon from the River Dee in 2015. Externally, the diseased eels demonstrated white patches in different locations of the body. In all cases, F. psychrophilum was detected by bacterial isolation and/or molecular methods. Three isolates were further characterized by whole‐genome sequencing (WGS) as belonging to sequence type 15 (ST15). Histological examination of diseased European eels revealed lesions at the level of the integument. The pathogen screen for F. psychrophilum in wild healthy fish tested negative by PCR. Further investigation is required to understand the pathogenicity of this bacterium on the health of eels and the potential impact on the wild salmonid population.     

Genogrouping of birnaviruses isolated from marine fish: a comparison of VP2/NS junction regions on genome segment A

Yellowtail ascites virus and related strains isolated from marine fish have been shown to be similar to infectious necrosis virus (IPNV) in terms of biological and serological characteristics. This paper explores the relationship of aquatic birnaviruses at the genetic level. The junction region on the genome segment A coding viral capsid protein VP2 and viral protease NS was amplified by PCR in six marine strains. Analysis of nucleotide and the deduced amino acid sequences revealed that the six marine strains have amino acid variations in the possible amino terminus of NS when compared to IPNV. The six marine strains form a new genogroup which is distinguished from three serotypes ofPNV.     

Anthropogenic spreading of anguillid herpesvirus 1 by stocking of infected farmed European eels,Anguilla anguilla (L.), in the Schlei fjord in northern Germany

The Schlei fjord in northern Germany is the recipient water of a comprehensive eel, Anguilla anguilla (L.), stocking programme. Since 2015, stocked eels become alizarin red S marked, but to date no control mechanism is implemented in this stock enhancement measure to prevent anthropogenic spreading of diseases. Consequentially, it was possible that farmed stocking cohorts of 2015 and 2016 (in total ca. 1040 kg) were subsequently tested positive for anguillid herpesvirus 1 (AngHV 1). For this study, 100 eels [total length (TL) 24.3–72.9 cm, age ca. 1–6 years] were caught in 2016 and investigated with regard to AngHV 1 infection, parasite load (Anguillicoloides crassus) and body conditions. 68% of the eels were found to be virus positive while larger specimens were more often infected. In addition, a fitted generalized linear model (area under the curve = 0.741) demonstrated that an increase in individual TL is accompanied with an increased risk of clinically relevant virus loads. Anguillicoloides crassus turned out to be an important stressor for eels, because parasite and virus load revealed a significant positive correlation. The results of this study evidently show the urgent need of a disease containment strategy for eel stocking programmes.     

Isolation and identification of a viral haemorrhagic septicaemia virus (VHSV) isolate from wild largemouth bass Micropterus salmoides in China

Fish rhabdoviruses are a family of viruses responsible for large‐scale fish die‐offs worldwide. Here, we reported the isolation and identification of a member of rhabdoviruses from wild largemouth bass (Micropterus salmoides) in the coastal area of the Pearl River Estuary, China. This virus isolate was identified as viral haemorrhagic septicaemia virus (VHSV) by specific RT‐PCR. Furthermore, the virus (VHSVLB2018) was isolated by cell culture using fathead minnow cells and confirmed by RT‐PCR. Electron microscopy showed the presence of bullet‐shaped viral particles in the cytoplasm of infected cells. The complete sequencing of VHSVLB2018 confirmed that it was genome configuration typical of rhabdoviruses. Phylogenetic analysis based on whole‐genome sequences and G gene nucleotides sequences revealed that VHSVLB2018 was assigned to VHSV genogroup Ⅳa. The pathogenicity of VHSVLB2018 was determined in infection experiments using specific pathogen‐free largemouth bass juveniles. VHSVLB2018‐infected fish showed typical clinical signs of VHSV disease, including darkened skin, petechial haemorrhages and pale enlarged livers, with the cumulative mortalities reached 63.3%–93.3% by 7 days post‐infection. VHSVLB2018 was re‐isolated from dead fish and confirmed by RT‐PCR. Together, this is the first report of isolation and identification of a VHSV isolate from wild largemouth bass in China.     

Carp oedema virus disease outbreaks in Czech and Slovak aquaculture

This work describes the first confirmed cases of carp oedema virus disease (CEVD) in Slovakia and the Czech Republic and the phylogenetic analysis of Czech and Slovak carp oedema virus (CEV) isolates. Four cases of disease outbreak in the Czech Republic are described, the oldest dating from mid-May 2013 and one case from Slovakia dating from May 2019. In all cases, virus presence was confirmed using nested PCR. PCR products were sequenced and compared with 357-bp nucleotide sequences encoding the CEV P4a protein in GenBank. In four cases of disease outbreak (three common carp breeding facilities and one koi garden pond), CEV detected belonged to genogroup I. In one case (koi garden pond), fish were confirmed as infected with CEV from genogroup II. This work complements data on CEV occurrence in European countries and contributes to a better understanding of the pathways leading to transmission of the virus throughout Europe.     

Mortality outbreak by perch rhabdovirus in European perch (Perca fluviatilis) farmed in Italy: Clinical presentation and phylogenetic analysis

This work reports a mortality outbreak, occurred in 2015 and affecting juveniles of European perch (Perca fluviatilis L.) farmed in Italy. Perch rhabdovirus (PRV) was detected by viral isolation and biomolecular investigations. Phylogenetic analysis clustered our isolate into genogroup B, which also includes PRV isolates from Perca fluviatilis identified in France (2004–2009); diagnostic investigations also revealed opportunistic bacteria (Aeromonas hydrophila) and parasites (Chilodonella piscicola). Since, occasionally, PRV has been reported in the natural environment, which is often a source of eggs and broodstock for farms, it could be possible that both similar France and Italian isolate were imported from a same place elsewhere and have a common origin. Improving biosecurity measures (batch control) and disinfection of egg strings with an iodine‐based solution helps prevent apparent vertical transmission of PRV.     

Virus infections of the European Eel in North Rhine Westphalian rivers

Viral infections have been suggested to play a role in the decline of the panmictic population of the European eel (Anguilla anguilla). However, despite the importance of knowledge about pathogenic eel viruses, little is known about their spread in the wild European eel population and only a few eel pathogenic viruses have been described so far. In this study, we aimed to investigate the health status of the A. anguilla stock in North Rhine Westphalia (NRW) State of Germany. For this purpose, we examined tissue samples of 16 elvers, 100 yellow eels and 6 silver eels, sampled from the rivers Rhine, Lippe and Ems. Virus detection was performed via a combination of cell culture and PCR. Next to the detection of frequently encountered pathogenic eel viruses (anguillid herpesvirus 1 and eel virus European X (EVEX)), we isolated the eel picornavirus 1 (EPV-1) from tissue of yellow eels and elvers and demonstrate the distribution of EPV-1 in wild eel population in NRW.     

Migration behaviour and escapement of European silver eels from a large lake and wetland system subject to water level management (Grand‐Lieu Lake,France): New insights from regulated acoustic telemetry data

Current knowledge about the downstream migration of sexually mature European eels (Anguilla anguilla) remains incomplete, particularly in still water habitats such as lakes and wetlands subject to water level management. However, for the management of this endangered species, it is important to understand migration dynamics, and contribution to the breeding stock. This study aimed to assess the parameters that trigger and guide the migration of silver eels in the largest floodplain lake and associated wetlands in France (the sluice regulated Grand‐Lieu Lake). A telemetry survey of 50 acoustic and PIT‐tagged female silver eels was performed during the 2015–2016 migration period. We deployed a novel telemetric approach, using receivers to delimit several restricted virtual boxes to determine the instantaneous location of individuals and to transform simple discrete telemetric data into presence/absence data. The low numbers leaving the lake centre are probably explained by the lack of orienting water flows or other environmental clues, but whilst the fate of 34% (17/50) of the tagged eels is unknown, 18% (9/50) were caught by commercial fishermen. Modelling showed that detections were not clearly associated with environmental factors typically involved in riverine migrations (e.g. current velocity, atmospheric pressure and temperature) but they were particularly associated with higher and increasing water levels and, for eels exiting the lake, a sharp increase when sluice gates were opened to an effective gap of >75 cm. It is concluded that management of water levels and sluice gate opening during the migration period might aid escapement of silver eels.     

First detection of spring viraemia of carp virus in common carp (Cyprinus carpio L.) affected by a septicaemic disease in Mexico

Spring viraemia of carp (SVC) is an infectious disease responsible for severe economic losses for various cyprinid species, particularly common carp (Cyprinus carpio carpio). The causative agent is the SVC virus (SVCV), a member of the Sprivivirus genus, Rhabdoviridae family, and a List 1 pathogen notifiable by the World Organization for Animal Health. This study describes the diagnosis of an SVCV pathogen isolated in October 2015 from wild common carp inhabiting a natural lagoon in central Mexico. While neither an epidemic nor fish mortalities were reported, the collected killed specimens exhibited clinical signs of disease (e.g., exopthalmia, moderate abdominal distension and haemorrhaging, as well as internal haemorrhages and adhesions). Histological results of injuries were consistent with the pathology caused by SVCV. This finding was supported by the isolation of a virus in EPC and BF‐2 cells and subsequent RT‐PCR confirmation of SVCV. The phylogenetic analyses of partial SVCV glycoprotein gene sequences classified the isolates into the Ia genogroup. These findings make this the first report of SVCV detection in Mexico, extending the southern geographical range of SVCV within North America. However, since this pathogen was detected in fish inhabiting a natural body of water without tributaries or effluents, it is difficult to estimate the risk of SVCV for other wild/feral cohabitating cyprinid species in the lagoon. The status of this virus is also unknown for other bodies of water within this region.     

Isolation of a novel polyomavirus,related to Japanese eel endothelial cell‐infecting virus,from marbled eels,Anguilla marmorata (Quoy & Gaimard)

Marbled eels, Anguilla marmorata (Quoy & Gaimard), cultured in Taiwan exhibited haemorrhage and mortality in January 2012. The severely diseased eels bled from the gills and showed congestion of the central venous sinus of the gill filaments and haemorrhage throughout the body similar to viral endothelial cell necrosis of eel. In this study, a novel polyomavirus (AmPyV) was isolated from the diseased eels using the AMPF cell line established from the pectoral fin of healthy marbled eels. AmPyV was found to encode a long T‐antigen orthologous gene. Phylogenetic analysis showed that AmPyV was closely related to Japanese eel endothelial cell‐infecting virus. PCR assays revealed AmPyV infection throughout the systemic organs. AmPyV proliferated in the AMPF, EK‐1 and EO‐2 cells at temperatures 25–30 °C, and the progeny virus yields were 10^7.0, 10^7.4 and 10^7.7 TCID₅₀ mL^?1, respectively. The purified virions were icosahedral particles, 70–80 nm in diameter. No clinical signs or mortality was observed among the eels injected with the virus; however, the virus was reisolated from the brain, eyes, kidneys, fins and gills of infected eels 2 month after injection. Our results suggest that AmPyV exhibits a latent infection. Pathogen of the disease needs to study further.     

鳗鲡疱疹病毒的生物学及理化特性

为了明确鳗鲡疱疹病毒(Anguillid herpesvirus, AngHV)的生物学及理化特性,本实验利用一株从欧洲鳗鲡"脱黏败血综合征"病料中分离的AngHV病毒株,研究了其增殖特性及其对主要鱼类细胞系的感染敏感性,进一步分析了其对热、酸碱、氯仿和乙醚等理化因子的耐受性。结果发现,AngHV感染的鳗鲡卵巢细胞系(eel ovary cell line, EO)内可见典型的疱疹病毒样颗粒,细胞出现时序性细胞病变;AngHV可在EO细胞系内稳定传代,较适宜扩繁温度为25～27°C,不能在鲤上皮瘤细胞系(epithelioma papilloma cyprinid cell line, EPC)、草鱼卵巢细胞系(grass carp ovary cell line, CO)、胖头逓肌肉细胞系(fathead minnow cell line, FHM)、大鳞大麻哈鱼胚胎细胞系(chinook salmon embryo cell line,CHSE-214)、虹鳟性腺细胞系(rainbow trout gonad cell line, RTG-2)及蓝鳃太阳鱼细胞系(bluegill ...     

An outbreak of Shewanella putrefaciens group in wild eels Anguilla anguilla L. favoured by hypoxic aquatic environments

Microbiological analyses were conducted on wild eels from the L′Albufera Lake (Spain). A total of 174 individuals were collected in two surveys (i.e. year 2008 and autumn–winter 2014) among those caught by local fishermen into the lagoon. The prevalence of Shewanella putrefaciens group was 1.7% in 2008 and rose above 32% in 2014. It was due to an outbreak of shewanellosis that presented a morbidity rate of 64%. S. putrefaciens group strains were isolated as pure cultures from the sick eels that showed white ulcers surrounded by a reddish inflammation, damage of the mouth, extensive skin discoloration, exophthalmia, ascites and bad odour. The S. putrefaciens group was recovered from freshwater samples taken at the L′Albufera system, along autumn–winter 2015. Its counts significantly increased in freshwater parallel to hypoxia and temperature rising. Shewanellae strains were identified as S. putrefaciens and S. xiamenensis by 16S rRNA gene sequencing. These isolates recovered from sick eels or freshwater were virulent for European eel by IP challenge (LD₅₀ 10⁶ CFU g^?1 body weight). They also caused 30–38% cumulative mortality, in European eels challenged by a 2‐h bath (10⁷ CFU mL^?1). These results suggest that shewanellosis could be transmitted through water highlighting the fact that hypoxic conditions increase this bacterium levels in water.