Epizootic Haematopoietic Necrosis Virus Research Articles

Species which may act as vectors or reservoirs of diseases covered by the Animal Health Law: Listed pathogens of fish.

Vector or reservoir species of five fish diseases listed in the Animal Health Law were identified, based on evidence generated through an extensive literature review (ELR), to support a possible updating of Regulation (EU) 2018/1882. Fish species on or in which highly polymorphic region-deleted infectious salmon anaemia virus (HPR∆ ISAV), Koi herpes virus (KHV), epizootic haematopoietic necrosis virus (EHNV), infectious haematopoietic necrosis virus (IHNV) or viral haemorrhagic septicaemia virus (VHSV) were detected, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, the studied species was classified as a vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms or reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected fish was not found, these were defined as reservoirs. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors or reservoir fish species during transport was collected from scientific literature. For VHSV, IHNV or HPR∆ ISAV, it was concluded that under transport conditions at temperatures below 25°C, it is likely (66-90%) they will remain infective. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild, aquaculture establishments or through water supply can possibly transmit VHSV, IHNV or HPR∆ ISAV into a non-affected area when transported at a temperature below 25°C. The conclusion was the same for EHN and KHV; however, they are likely to remain infective under all transport temperatures.

Preparation and optimization of rapid and sensitive coagglutination test for detection of infectious pancreatic necrosis virus (IPNV)

The aim of this study is the development of a coagglutination test for rapid diagnosis of infectious pancreatic necrosis (IPN) virus which causes an acute infectious viral disease with high mortality in young salmonid fish. Serotypes Sp, Ab, WB of the IPNV were cultivated in BF-2 cells and purified by linear sucrose density gradient centrifugation. Hyperimmune sera against purified IPNV serotypes were collected from immunized guinea pigs. Coagglutination test conjugates were prepared by using sensitized S. aureus and hyperimmune sera. Cell culture derived salmonid pathogens such as infectious hematopoietic necrosis virus (IHNV), viral haemorrhagic septicaemia virus (VHSV) and epizootic hematopoietic necrosis virus (EHNV) were used to determine the specificity of the test as control. Upward comparison of the preferability for the rapid diagnosis was also carried out with ELISA and RT-PCR. The validation studies of newly developed coagglutination test were carried out in accordance with the Manual of Diagnostic Tests for Aquatic Animals. Consequently, determined detection limit of the test was approximately 105.25 TCID50/mL for the Sp and Ab serotypes and 107.75 TCID50/mL for the WB in laboratory conditions. The results indicated that newly developed coagglutination test was compatible with ELISA and RT-PCR in positive cell culture supernatants. It was concluded that this test would be an economic, rapid and reliable method for the diagnosis of IPN virus in cell culture.

Ranaviruses in captive and wild Australian lizards

Ranaviral infections have been associated with mass mortality events in captive and wild amphibian, fish, and reptile populations globally. In Australia, two distinct types of ranaviruses have been isolated: epizootic haematopoietic necrosis virus in fish and a Frog virus 3-like ranavirus in amphibians. Experimental studies and serum surveys have demonstrated that several Australian native fish, amphibian, and reptile species are susceptible to infection and supported the theory that ranavirus is naturally circulating in Australian herpetofauna. However, ranaviral infections have not been detected in captive or wild lizards in Australia. Oral-cloacal swabs were collected from 42 wild lizards from northern Queensland and 83 captive lizards from private collections held across three states/territories. Samples were tested for ranaviral DNA using a quantitative PCR assay. This assay detected ranaviral DNA in 30/83 (36.1%) captive and 33/42 (78.6%) wild lizard samples. This is the first time molecular evidence of ranavirus has been reported in Australian lizards.

Geographic Distribution of Epizootic haematopoietic necrosis virus (EHNV) in Freshwater Fish in South Eastern Australia: Lost Opportunity for a Notifiable Pathogen to Expand Its Geographic Range.

Epizootic haematopoietic necrosis virus (EHNV) was originally detected in Victoria, Australia in 1984. It spread rapidly over two decades with epidemic mortality events in wild redfin perch (Perca fluviatilis) and mild disease in farmed rainbow trout (Oncorhynchus mykiss) being documented across southeastern Australia in New South Wales (NSW), the Australian Capital Territory (ACT), Victoria, and South Australia. We conducted a survey for EHNV between July 2007 and June 2011. The disease occurred in juvenile redfin perch in ACT in December 2008, and in NSW in December 2009 and December 2010. Based on testing 3622 tissue and 492 blood samples collected from fish across southeastern Australia, it was concluded that EHNV was most likely absent from redfin perch outside the endemic area in the upper Murrumbidgee River catchment in the Murray–Darling Basin (MDB), and it was not detected in other fish species. The frequency of outbreaks in redfin perch has diminished over time, and there have been no reports since 2012. As the disease is notifiable and a range of fish species are known to be susceptible to EHNV, existing policies to reduce the likelihood of spreading out of the endemic area are justified.

An epidemiologic model of koi herpesvirus (KHV) biocontrol for carp in Australia

ABSTRACT In Australia, the common carp (Cyprinus carpio) is a significant pest species because it dominates fish communities in numerous catchment areas. In 2016, Australia launched a national control plan based on the use of Cyprinid herpesvirus 3 (CyHV3) to reduce carp densities. CyHV3 is exotic to Australia and is listed as by the World Organization for Animal Health (OIE) due to its substantial impact on global aquaculture production. Infection with CyHV3 causes koi herpesvirus disease (KHVD), a high mortality disease in common carp affecting all age classes of both wild and farmed fish. The objective of this review was to consider the current knowledge of CyHV3 transmission factors and discuss the potential for recurring epidemic-level mortality events in carp found in Australia. Case studies were presented comparing KHVD outbreaks in wild carp in Japan and Epizootic haematopoietic necrosis virus (EHNV) outbreaks in pest redfin perch (Perca fluviatilis L.) in Australia. The release of CyHV3 in therma...

Ausencia del virus de Necrosis Hematopoyética Epizoótica (EHNv) en truchas arcoíris (Oncorhynchus mykiss) enfermas en piscigranjas de la sierra del Perú

El objetivo del presente trabajo fue monitorear la detección del virus Necrosis Hematopoyética Epizoótica (EHNv) en truchas (Oncorhynchus mykiss) de piscigranjas de la Sierra del Perú. Se tomaron muestras de 111 peces enfermos (hígado, bazo, riñón anterior) de piscigranjas de las regiones de Áncash, Junín y Huancavelica, Perú. Las muestras de tejidos fueron procesados de inmediato para el aislamiento bacteriano y otro grupo de muestras fueron conservadas a -196 °C para el PCR. El ADN de cada tejido fue extraído mediante el método del Trizol y luego purificado con membranas de sílica (PureLinkGenomic DNA kit). El PCR se realizó utilizando los cebadores MCP-1 específicos para el EHNv siguiendo la metodología propuesta por la OIE y con el kit comercial VetPCRTM EHNV Detection para su confirmación. El aislamiento bacteriano se realizó con el sembrado en agar TSA y Anacker-Ordal (agar citófaga) de cada tejido y la identificación mediante tinción Gram y bioquímica bacteriana. No se detectó el ADN del EHNv en las muestras indicando que la prevalencia del virus es menor al 5% o no está presente en las piscigranjas muestreadas. Se aisló Yersinia ruckeri y Aeromonas spp en todas las muestras, indicando que la causa de la enfermedad y mortalidad presente en las piscigranjas se debían a la infección con estas bacterias.

Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses

Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ranaviruses. Here we report the development and partial validation of a TaqMan real-time qPCR assay. The primers and TaqMan probe targeted a conserved region of the major capsid protein (MCP) gene. A series of experiments using a 10-fold dilution series of Frog virus 3 (FV3) MCP plasmid DNA revealed linearity over a range of 7 orders of magnitude (107-101), a mean correlation coefficient (R2) of >0.99, and a mean efficiency of 96%. The coefficient of variation of intra- and inter-assay variability ranged from <0.1-3.5% and from 1.1-2.3%, respectively. The analytical sensitivity was determined to be 10 plasmid copies of FV3 DNA. The qPCR assay detected a panel of 33 different ranaviral isolates originating from fish, amphibian, and reptile hosts from all continents excluding Africa and Antarctica, thereby representing the global diversity of ranaviruses. The assay did not amplify highly divergent ranaviruses, members of other iridovirus genera, or members of the alloherpesvirus genus Cyprinivirus. DNA from fish tissue homogenates previously determined to be positive or negative for the ranavirus Epizootic hematopoietic necrosis virus by virus isolation demonstrated a diagnostic sensitivity of 95% and a diagnostic specificity of 100%. The reported qPCR assay provides an improved expedient diagnostic tool and can be used to elucidate important aspects of ranaviral pathogenesis and epidemiology in clinically and sublinically affected fish, amphibians, and reptiles.

DETECTION OF COPATHOGENS IN FREE-RANGING EASTERN BOX TURTLES ( TERRAPENE CAROLINA CAROLINA) IN ILLINOIS AND TENNESSEE.

Conservation efforts are investigating the impact of diseases within a species of interest, including prevalence and transmission and morbidity and mortality rates. However, the majority of these studies focus solely on the characteristics of a single pathogen. Recently, the role of copathogens has been reported to impact disease susceptibility and mortality. To that effect, a survey was conducted including 318 eastern box turtles ( Terrapene carolina carolina) from populations in Illinois and Tennessee in 2014 and 2015. Blood samples and oral swabs were collected for quantitative polymerase chain reaction (qPCR) of 15 different pathogens performed in a multiplex format using Fluidigm array technology. Four pathogens were found with varying qPCR prevalence: ranavirus (FV3; n = 2, 0.6%), Terrapene herpesvirus 1 (TerHV1; n = 129, 40.7%), box turtle Mycoplasma sp. (BT Myco; n = 14, 4.6%), and box turtle adenovirus (BT Adv1; n = 18, 11%). Thirteen pathogens were not identified in any sample, including Mycoplasma agassizii, M. testudineum, Salmonella enteriditis, S. typhmirium, Borrelia burgdorferi, Anaplasma phagocyophilum, tortoise intranuclear coccidia, Ambystoma tigrinum virus, Bohle iridovirus, Epizootic hematopoietic necrosis virus, and testudinid herpesvirus 2. Copathogen occurrence was rare but was observed in eight individuals with TerHV1-BT Myco detection and two animals with TerHV1-Adv1. Significant differences were observed in pathogen detection across season (TerHV1, BT Adv1, BT Myco, and TerHV1-Myco) and year (TerHV1, BT Adv1, and TerHV1-Myco). The results of this survey highlight that a single pathogen model may not adequately explain pathogen dynamics and that conservation efforts need to be aimed at detecting multiple pathogens in order to fully characterize population health.

Molecular epidemiology of Epizootic haematopoietic necrosis virus (EHNV)

Low genetic diversity of Epizootic haematopoietic necrosis virus (EHNV) was determined for the complete genome of 16 isolates spanning the natural range of hosts, geography and time since the first outbreaks of disease. Genomes ranged from 125,591–127,487 nucleotides with 97.47% pairwise identity and 106–109 genes. All isolates shared 101 core genes with 121 potential genes predicted within the pan-genome of this collection. There was high conservation within 90,181 nucleotides of the core genes with isolates separated by average genetic distance of 3.43 × 10−4 substitutions per site. Evolutionary analysis of the core genome strongly supported historical epidemiological evidence of iatrogenic spread of EHNV to naïve hosts and establishment of endemic status in discrete ecological niches. There was no evidence of structural genome reorganization, however, the complement of non-core genes and variation in repeat elements enabled fine scale molecular epidemiological investigation of this unpredictable pathogen of fish.

Risk of waterborne virus spread – review of survival of relevant fish and crustacean viruses in the aquatic environment and implications for control measures

AbstractAquatic animal diseases are a major constraint for increasing aquaculture production. Understanding the contribution of pathogen spread from infected aquaculture sites is critical in devising control measures in the event of an outbreak. We have reviewed the available literature on the persistence in the aquatic environment of several important viral pathogens of fish and crustaceans. These include infectious haematopoietic necrosis virus, viral haemorrhagic septicaemia virus, infectious salmon anaemia virus, koi herpes virus, epizootic haematopoietic necrosis virus and infectious pancreatic necrosis virus, white spot syndrome virus (WSSV), Taura syndrome virus and yellow head virus. Some trends were common to all viruses: (i) viability declined with increasing temperature (at temperatures above 0°C); (ii) higher biological loading in water correlated with reduction in detectable viable viruses; and (iii) virus decay in water is a function of time. Most aquatic animal viruses (AAVs) remained viable for several days or weeks. WSSV is particularly stable. Comparison of studies investigating survival parameters was sometimes difficult because of the different methods employed and different ways in which the data were presented. Data gaps are identified and experimental methods employed for testing critically assessed. The information presented in this review is directly relevant to design effective control measures for AAVs and to explore measures that reduce the economic impact of disease caused by these important pathogens.

Establishment and characterization of a heart-derived cell line from goldfish (Carassius auratus).

The goldfish Carassius auratus, a freshwater fish in the family Cyprinidae, was one of the earliest fish to be domesticated for ornamental purposes. A cell line was established from goldfish heart (GH) tissue to create a biological monitoring tool for viral diseases. The GH cell line was optimally maintained at 25°C in M199 medium supplemented with 10-20% fetal bovine serum. A chromosomal analysis indicated that the cell line remained diploid, with a mean chromosomal count of 100. In viral inoculation assays, significant cytopathic effects (CPEs) were caused by epizootic hematopoietic necrosis virus (EHNV), Andrias davidianus iridovirus (ADIV), and Bohle iridovirus (BIV) infections in the fish cells and the viral titers (average value) of EHNV, ADIV, and BIV in GH cells reached 105.0, 104.5, and 105.0TCID50/0.1mL, respectively, within 7days. However, no CPE was observed in the cells infected with viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), or grass carp reovirus (GCRV). These results suggest that the GH cell line is a valuable tool for studying viral pathogenesis.

IHNV与IPNV免疫层析试纸条的制备及应用

本文制备传染性造血器官坏死病毒(Infectious haematopoietic necrosis virus, IHNV)、传染性胰脏坏死病毒(Infectious pancreatic necrosis virus, IPNV)胶体金快速检测试纸，并对其性能进行检测。以柠檬酸三纳作为还原剂制备胶体金颗粒，标记IHNV、IPNV单克隆抗体，将羊抗鼠IgG、羊抗兔IgG、兔抗鼠和纯化后的2种单克隆抗体包被于NC膜上，作为质控线和检测线来检测样品中的IHNV和IPNV。结果表明所研制的试纸特异性良好，分别对鲤春血症病毒(SVCV)、病毒性出血性败血症病毒(VHSV)、流行性造血器官坏死病毒(EHNV)等常见病原菌进行测试，未出现交叉反应；敏感度达到105 CFU/mL，反应时间仅需5 min~10 min，可以用于感染上述2种病原的病鱼腹水现场检测。 In this paper, the colloidal gold strips of IHNV and IPNV for rapid detection was prepared, and their performance was tested. Citric acid three sodium was treated as deoxidizer to prepare gold particle, Label IHNV and IPNV monoclonal antibodies and coat on the NC membrane with the Goat anti mouse IgG, Goat anti rabbit IgG, Rabbit anti rat IgG, as the test line and control line to detect IHNV and IPNV. Test results showed that the specificity of Spring Viremia of Carp Virus (SVCV) was good. Viral Hemorrhagic Septicemia Virus (VHSV), Epizootic Haematopoietic Necrosis Virus (EHNV) and other common pathogens tested without cross reaction; The sensitivity was to 105CFU/mL and only the reaction time of 5 min to 10 min. They could be used to detect ascites in diseased fish by the above 2 pathogens on the spot.

Susceptibility of Australian Redfin Perch Perca fluviatilis Experimentally Challenged with Epizootic Hematopoietic Necrosis Virus (EHNV).

The ranavirus epizootic hematopoietic necrosis virus (EHNV) is endemic to Australia and is listed by the Office International des Epizooties. Clinical outbreaks have only been observed in wild populations of Redfin Perch Perca fluviatilis (also known as Eurasian Perch) and farmed populations of Rainbow Trout Oncorhynchus mykiss. The initial outbreaks of EHNV describe all age-classes of Redfin Perch as being susceptible and can lead to epidemic fish kills. Subsequently, experimental challenge studies using either cohabitation with the virus or injection exposures resulted in mixed susceptibilities across various age-groupings of Redfin Perch. We used an experimental bath challenge model to investigate the susceptibility of Redfin Perch collected from areas with and without a history of EHNV outbreaks. The median survival time for fish from Blowering Dam in New South Wales, a zone with a history of EHNV outbreaks, was 35 d, compared with fish from other areas, which had a median survival between 12 and 28 d postexposure. Redfin Perch from Blowering Dam demonstrated an increased mortality associated with epizootic hematopoietic necrosis up to approximately day 14 after exposure, and then there was a significantly reduced risk of mortality until the end of the trial compared with all other fish. Redfin Perch from Blowering Dam had markedly decreased susceptibility to EHNV, and less than 40% became infected following a bath challenge. In contrast, Redfin Perch from neighboring (e.g., Bethungra Dam and Tarcutta Creek) and distant water bodies (e.g., in Western Australia) with no previous history of EHNVdisplayed moderate to high susceptibility when given a bath challenge. Potential factors for the observed changes in the host-pathogen relationship include intense positive selection pressure for resistant fish following epizootic hematopoietic necrosis outbreaks and subsequent attenuation of the virulence of the virus in resistant fish. Received August 22, 2015; accepted February 13, 2016.

Differential response of the Senegalese sole (Solea senegalensis) Mx promoter to viral infections in two salmonid cell lines

Mx proteins are main effectors of the antiviral innate immune defence mediated by type I interferon (IFN I). The IFN I response is under a complex regulation; hence, one of the key issues in understanding virus-host interaction is the knowledge of the regulatory mechanisms governing this response. With this purpose, in this study Chinook salmon embryo cells (CHSE-214) and rainbow trout gonad cells (RTG-2) were transiently transfected with a vector containing the luciferase reporter gene under the control of the Senegalese sole Mx promoter. These transfected cells were infected with infectious pancreatic necrosis virus (IPNV), viral haemorrhagic septicaemia virus (VHSV) and epizootic haematopoietic necrosis virus (EHNV) at different doses in order to study the luciferase fold induction in response to viral infections. Transfected CHSE-214 cells infected with EHNV showed significant induction of the luciferase reporter gene, compared to control non-infected cells, at different times post infection (p.i.). The maximum expression was recorded at 24h p.i. in cells inoculated with 5×102TCID50/mL (2.17 folds compared to control cells). In these cells, the infection with IPNV and VHSV did not result in the luciferase expression at any time and doses tested. In transfected RTG-2 cells, VHSV stimulated luciferase expression, obtaining a maximum activity at 48h p.i. in cells infected with 5×102TCID50/mL (2.9 folds compared to control cells), whereas RTG-2 cells infected with IPNV and EHNV did not show significant luciferase activity at any time point. The different induction of the Senegalese sole Mx promoter in CHSE-214 and RTG-2 cells after infection with the same viruses indicates that cell-specific factors are significantly involved in the IFN-signalling response, and, probably, on the success of the strategies of these viruses to escape the IFN mechanisms. The use of these two different cellular systems might be an interesting approach to identify such cellular factors.

Diagnosis of nervous necrosis virus in orange-spotted grouper, Epinephelus coioides, by a rapid and convenient RT-PCR method

Viral nervous necrosis (VNN) causes high mortality in marine fish, especially in the grouper, worldwide and in China. Since there is no effective vaccine or drug to deal with VNN, early detection and prevention is important to block its outbreak. In this study, a reverse transcription-polymerase chain reaction (RT-PCR) was developed for the rapid, convenient, and sensitive detection of the VNN pathogen, nervous necrosis virus (NNV), in the grouper. The whole process was completed within 3.5 h from the RNA extraction to PCR product visualization. The detection limit of this method was 200 copies of NNV RNA standard, which corresponded to 200 copies of virus particles. This RT-PCR method was specific to the NNV detection with no cross-reactivity to other fish viral disease pathogens, such as infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), spring viraemia of carp virus (SVCV), epizootic haematopoietic necrosis virus (EHNV), and large yellow croaker iridovirus (LYCIV). With this method, the orange-spotted grouper (Epinephelus coioides) fry from hatcheries with or without incidence of the VNN epidemic in Fujian Province were detected. The results showed that all or 93% of the fry from the two hatcheries with incidence of the epidemic were diagnosed as positive, while 40% or 25% of fry from the two hatcheries without the VNN epidemic were also detected as NNV positive, indicating that this RT-PCR method can be used for rapid, sensitive detection of NNV infection and applied in the VNN epidemic alert.

A loop-mediated isothermal amplification method for the detection of members of the genus Ranavirus

A loop-mediated isothermal amplification (LAMP) method was developed for detection of members of the genus Ranavirus. The optimum reaction mixture contained 2.5 μL of each inner primer, RV-FIP (20pmol/μL) and RV-BIP (20pmol/μL), 0.5 μL of each outer primer, RV-F3 (10pmol/μL) and RV-B3 (10pmol/μL), 1.25 μL of each loop primer, RV-LF (20pmol/μL) and RV-LB (20pmol/μL), 3.5 μL dNTP mix (10mM each), 8 μL MgSO4 (25mM), 1 μL of Bst DNA polymerase (8 U/mL, large fragment; New England Biolabs Inc., Beverly, MA, USA), 2.5 μL 10×supplied buffer, and 1 μL of template DNA in a final volume of 25 μL. The optimum reaction conditions were 63 °C for 60min. This LAMP method could detect Andrias davidianus iridovirus (ADIV), soft-shelled turtle iridovirus (STIV), and epizootic hematopoietic necrosis virus (EHNV), all of which belong to the genus Ranavirus, but it could not detect other viruses such as koi herpes virus (KHV), channel catfish virus (CCV), infectious spleen and kidney necrosis virus (ISKNV) and white spot syndrome virus (WSSV). The detection limit of the LAMP method was 100 copies of STIV DNA segment, and the sensitivity was 10 times higher than that of the polymerase chain reaction (PCR) assay. The results could be estimated visually by eye when calcein was added.

Susceptibility of European sheatfish Silurus glanis to a panel of ranaviruses

Summary The current study was undertaken in order to assess the risk that different ranaviruses might impose on European sheatfish aquaculture. As the European sheatfish virus (ESV) is a known pathogen causing losses in European sheatfish aquaculture, it was assumed that closely related exotic ranaviruses might also be able to infect European sheatfish and probably cause disease and mortality in this species. The differential susceptibility of European sheatfish (Silurus glanis) to various ranavirus isolates was assessed at two different temperatures (15°C and 25°C) in a recirculation system. Fish were infected experimentally with a panel of ranavirus isolates including ESV, European catfish virus (ECV), European catfish virus isolate 24 (ECV-24), Epizootic haematopoietic necrosis virus (EHNV), Rana esculenta virus isolate Italy 282/ I02 (REV), short-finned eel virus (SERV), Bohle iridovirus (BIV), guppy virus 6 (GV6), doctor fish virus (DFV) and Frog virus 3 (FV3). Significant mortalities were observed, as expected, in fish infected with ESV at 15°C (100%) as well as at 25°C (86/83%). Fish infected with ECV at 15°C showed no clinical signs of disease (8% mortality), whereas those fish infected at 25°C exhibited a cumulative mortality of 54%. Fatal disease was also induced by Italian isolate ECV-24 at 25°C (81%). Virus isolates ESV, ECV and ECV-24, generally the most genetically closely related viruses, were successfully isolated from dead fish by cell culture with subsequent identification by polymerase chain reaction (PCR) and sequence analysis. However, no mortality or clinical signs of disease were observed in the groups of sheatfish infected with the other ranaviruses investigated in the study, and none of those viruses were re-isolated in cell culture or identified by PCR. It was concluded that European sheatfish are susceptible to infection with ESV, ECV and ECV-24 under laboratory conditions, but not to infection with EHNV, REV, SERV, BIV, GV6, DFV or FV3. For ESV, the incubation period was shorter at 25°C compared to 15°C water temperature, but whereas all fish died after ESV infection at 15°C, some fish survived the infection at 25°C. Futhermore, the very young sheatfish were susceptible to ECV and ECV-24 at 25°C, whereas there was no significant mortality in the group of older sheatfish challenged with ECV at 15°C. Therefore, the clinical characteristics of the disease seem to depend on the age of the fish as well as on the water temperature.

Susceptibility of the European common frog Rana temporaria to a panel of ranavirus isolates from fish and amphibian hosts

Ranaviruses are an emerging group of viruses and have been implicated in an increase of epidemics in susceptible species. They have a wide host range, infecting fish, amphibians and reptiles, with some isolates able to infect multiple species from different animal classes. Whilst some information exists on the pathogenicity of ranaviruses to novel hosts, there is none on the pathogenicity of fish ranaviruses to amphibians; this information is needed to develop measures to prevent the further spread of ranaviral disease in the aquatic environment. We undertook bath infection trials to assess the susceptibility of the European common frog Rana temporaria to 9 ranavirus isolates comprising doctor fish virus (DFV), European sheatfish virus (ESV), epizootic haematopoietic necrosis virus (EHNV), guppy virus 6 (GV6), pike-perch iridovirus (PPIV) and short-finned eel ranavirus (SERV) from fish hosts, and Bohle iridovirus (BIV), frog virus 3 (FV3) and Rana esculenta virus 282/I02 (REV) from amphibians. Animals were challenged as tadpoles at 15 and 20°C and as recent metamorphs at room temperature (20 ± 1°C) to investigate the effect of temperature and amphibian developmental stage on virus pathogenicity. Tadpoles were susceptible to FV3, PPIV and REV, but refractory to the other ranaviruses. Post-metamorphs were susceptible to FV3 and REV but refractory to BIV (the other ranaviruses were not tested). Significant mortality occurred in post-metamorphs and in tadpoles challenged at 20°C but was low in tadpoles challenged at 15°C. This study presents the first evidence of mortality in an amphibian species after challenge with ranavirus originally isolated from fish.

Experimental Infection of Australian Freshwater Fish with Epizootic Haematopoietic Necrosis Virus (EHNV)

The ranavirus, epizootic hematopoietic necrosis virus (EHNV), is endemic to southern Australia with natural outbreaks resulting in mass mortality events in wild Redfin Perch Perca fluviatilis (also known as Eurasian Perch) and less severe disease in farmed Rainbow Trout Oncorhynchus mykiss. To further investigate the host range for EHNV, 12 ecologically or economically important freshwater fish species from southeastern Australia were exposed experimentally to the virus. A bath-challenge model at 18 ± 3°C was employed with limited use of intraperitoneal inoculation to determine if a species was likely to be susceptible to EHNV. Of the species tested, Murray-Darling Rainbowfish Melanotaenia fluviatilis and Dewfish Tandanus tandanus (also known as Freshwater Catfish) were considered to be potentially susceptible species. EHNV was isolated from approximately 7% of surviving Eastern Mosquitofish Gambusia holbrooki, indicating this widespread alien fish species is a potential carrier. The infection of Silver Perch Bidyanus bidyanus and Macquarie Perch Macquaria australasica and the lack of infection in Murray Cod Maccullochella peelii peelii and Golden Perch Macquaria ambigua ambigua after exposure to EHNV via water confirmed earlier data from Langdon (1989). Five other species of native fish were potentially not susceptible to the virus or the fish were able to recover during the standard 35-d postchallenge observation period. Overall, it appeared that EHNV was less virulent in the present experimental model than in previous studies, but the reasons for this were not identified. Received May 21, 2012; accepted November 1, 2012.

A validated quantitative polymerase chain reaction assay for the detection of ranaviruses (Family Iridoviridae) in fish tissue and cell cultures, using EHNV as a model

Viruses in the genus Ranavirus (Family Iridoviridae) are important pathogens of fish, amphibians and reptiles and likely have a global distribution. Certification of freedom from infection, prevalence surveys, surveillance and routine diagnosis are hampered by lack of a validated rapid molecular test. In this study, a quantitative PCR (qPCR) assay for the detection of the major capsid protein gene of ranaviruses was developed and validated using Epizootic Haematopoietic Necrosis Virus (EHNV) as a model ranavirus. The assay had 100-fold greater analytical sensitivity compared to virus isolation in Bluegill fry (BF-2) cells, detected all viruses in a panel of 20 ranaviruses from Australia, America, Europe and the United Kingdom, did not detect two disputed members of the genus Ranavirus (doctorfish virus and guppy virus 6) and did not detect a megalocytivirus or two cyprinid herpesviruses. When applied to the detection of EHNV in fish tissue homogenates, 94.3% of samples which caused cytopathic effect (CPE) in BF-2 cells were positive in the qPCR, together with many others which did not display CPE but that arose from fish exposed to EHNV. Similarly, 98% of cell culture supernatants from cell monolayers with CPE were positive in the qPCR, together with other cultures of tissues from EHNV-exposed fish which did not display CPE. Overall a greater number of samples from exposed fish tested positive in qPCR compared to culture in BF-2 cells. The diagnostic specificity of the qPCR assay was 100% based on tests of tissue homogenates from 100 fish which were not exposed to EHNV. The assay appears to be suitable for a range of applications including surveillance and diagnosis, and for confirming the cause of CPE in BF-2 cells.