Nanopore sequencing provides snapshots of the genetic variation within salmonid alphavirus-3 (SAV3) during an ongoing infection in Atlantic salmon (Salmo salar) and brown trout (Salmo trutta).

Frequent RNA virus mutations raise concerns about evolving virulent variants. The purpose of this study was to investigate genetic variation in salmonid alphavirus-3 (SAV3) over the course of an experimental infection in Atlantic salmon and brown trout. Atlantic salmon and brown trout parr were infected using a cohabitation challenge, and heart samples were collected for analysis of the SAV3 genome at 2-, 4- and 8-weeks post-challenge. PCR was used to amplify eight overlapping amplicons covering 98.8% of the SAV3 genome. The amplicons were subsequently sequenced using the Nanopore platform. Nanopore sequencing identified a multitude of single nucleotide variants (SNVs) and deletions. The variation was widespread across the SAV3 genome in samples from both species. Mostly, specific SNVs were observed in single fish at some sampling time points, but two relatively frequent (i.e., major) SNVs were observed in two out of four fish within the same experimental group. Two other, less frequent (i.e., minor) SNVs only showed an increase in frequency in brown trout. Nanopore reads were de novo clustered using a 99% sequence identity threshold. For each amplicon, a number of variant clusters were observed that were defined by relatively large deletions. Nonmetric multidimensional scaling analysis integrating the cluster data for eight amplicons indicated that late in infection, SAV3 genomes isolated from brown trout had greater variation than those from Atlantic salmon. The sequencing methods and bioinformatics pipeline presented in this study provide an approach to investigate the composition of genetic diversity during viral infections.

Disease Progression in Lake Trout (Salvelinus namaycush) Experimentally Infected With Epizootic Epitheliotropic Disease Virus (Salmonid Herpesvirus-3).

Epizootic epitheliotropic disease virus (salmonid herpesvirus-3; EEDV) is responsible for the death of millions of hatchery-raised lake trout (Salvelinus namaycush) in the Laurentian Great Lakes Basin. However, little is known about its biology, pathology, tropism, and host interactions. In this study, the presence and disease progression of EEDV were evaluated following exposure of naïve juvenile lake trout to EEDV via bath immersion under controlled laboratory conditions (n = 84 infected; n = 44 control). Individual tissues (n = 10 per fish), collected over 6 weeks, were analyzed for viral load by quantitative polymerase chain reaction, gross and histopathologic changes, and virus cellular targets using in situ hybridization. Skin, fin, and ocular tissues were the earliest viral targets and yielded the highest viral loads throughout the course of infection. Early gross lesions included exophthalmia, ocular hemorrhage, fin congestion, and hyperemia of visceral blood vessels. Advanced disease was characterized by multifocal to coalescing erosions and ulcerations of the skin, and congestion of visceral organs. Microscopically, there was cellular degeneration and necrosis in the epidermis and spleen, and lymphohistiocytic perivasculitis of the dermis, omentum, and the epicardium. EEDV DNA was first detected by in situ hybridization in epithelial cells of the epidermis, with subsequent labeling in the epithelial lining of primary and secondary gill lamellae. During advanced disease, EEDV was detected in endothelial and dendritic cells as well as blood monocytes. This study characterized EEDV tissue tropism and associated pathologic features, to guide research aimed at understanding EEDV disease ecology and improving strategies for disease control.

Single-step genomic evaluation improves accuracy of breeding value predictions for resistance to infectious pancreatic necrosis virus in rainbow trout.

The aim of this study was to compare the accuracy of breeding values (EBVs) predicted using the traditional pedigree based Best Linear Unbiased Prediction (PBLUP) and the single-step genomic Best Linear Unbiased Prediction (ssGBLUP) for resistance against infectious pancreatic necrosis virus (IPNV) in rainbow trout. A total of 2278 animals were challenged against IPNV and 768 individuals were genotyped using a 57 K single nucleotide polymorphism array for rainbow trout. Accuracies for both methods were assessed using five-fold cross-validation. The heritabilities were higher for PBLUP compared to ssGBLUP. The ssGBLUP accuracies outperformed PBLUP in 7 and 11% for days to death and binary survival, respectively. The ssGBLUP could be an alternative approach to improve the accuracy of breeding values for resistance against infectious pancreatic necrosis virus in rainbow trout, using information from genotyped and non-genotyped animals.

A survey of salmon gill poxvirus (SGPV) in wild salmonids in Norway.

In 2016, the Norwegian health monitoring programme for wild salmonids conducted a real-time PCR-based screening for salmon gill poxvirus (SGPV) in anadromous Arctic char (Salvelinus alpinus L.), anadromous and non-anadromous Atlantic salmon (Salmo salar L.) and trout (Salmo trutta L.). SGPV was widely distributed in wild Atlantic salmon returning from marine migration. In addition, characteristic gill lesions, including apoptosis, were detected in this species. A low amount of SGPV DNA, as indicated by high Ct-values, was detected in anadromous trout, but only in fish cohabiting with SGPV-positive salmon. SGPV was not detected in trout and salmon from non-anadromous water courses, and thus seems to be primarily linked to the marine environment. This could indicate that trout are not a natural host for the virus. SGPV was not detected in Arctic char but, due to a low sample size, these results are inconclusive. The use of freshwater from anadromous water sources may constitute a risk of introducing SGPV to aquaculture facilities. Moreover, SGPV-infected Atlantic salmon farms will hold considerable potential for virus propagation and spillback to wild populations. This interaction should therefore be further investigated.

Development and use of an Arctic charr cell line to study antiviral responses at extremely low temperatures.

Arctic charr (Salvelinus alpinus) are the northernmost distributed freshwater fish and can grow at water temperatures as low as 0.2 °C. Other teleost species have impaired immune function at temperatures that Arctic charr thrive in, and thus, charr may maintain immune function at these temperatures. In this study, a fibroblastic cell line, named ACBA, derived from the bulbus arteriosus (BA) of Arctic charr was developed for use in immune studies at various temperatures. ACBA has undergone more than forty passages at 18 °C over 3 years, while showing no signs of senescence-associated ß-galactosidase activity and producing nitric oxide. Remarkably, ACBA cells survived and maintained some mitotic activity even at 1 °C for over 3 months. At these low temperatures, ACBA also continued to produce MH class I proteins. After challenge with poly I:C, only antiviral Mx proteins were induced while MH proteins remained constant. When exposed to live viruses, ACBA was shown to permit viral infection and replication of IPNV, VHSV IVa and CSV at 14 °C. Yet at the preferred temperature of 4 °C, only VHSV IVa was shown to replicate within ACBA. This study provides evidence that Arctic charr cells can maintain immune function while also resisting infection with intracellular pathogens at low temperatures.

[IDENTIFICATION OF THE INFECTIOUS PANCREATIC NECROSIS VIRUS (IPNV) USING THE ENZYME IMMUNOASSAY].

The infectious pancreatic necrosis (IPN) caused by a non-enveloped virus of the Birnaviridae family is one of the most important loss factors in the salmonid aquaculture. Virus isolation in the sensitive cell cultures has been approved in the Russian Federation as the diagnostic method for determination of IPNV antigen. This work gives the results of the development of the diagnostic test to reveal IPNV using the antigen-bound ELISA (sandwich ELISA). The developed test supplements a new diagnostic method and verifies some disputable results obtained with classical methods.

Mutation of N-glycosylation Sites in Salmonid Alphavirus (SAV) Envelope Proteins Attenuate the Virus in Cell Culture.

Salmonid alphavirus (SAV) is the cause of pancreas disease and sleeping disease in farmed salmonid fish in Europe. The spread of these diseases has been difficult to control with biosecurity and current vaccination strategies, and increased understanding of the viral pathogenesis could be beneficial for the development of novel vaccine strategies. N-glycosylation of viral envelope proteins may be crucial for viral virulence and a possible target for its purposed attenuation. In this study, we mutated the N-glycosylation consensus motifs of the E1 and E2 glycoproteins of a SAV3 infectious clone using site-directed mutagenesis. Mutation of the glycosylation motif in E1 gave a complete inactivation of the virus as no viral replication could be detected in cell culture and infectious particles could not be rescued. In contrast, infectious virus particles could be recovered from the SAV3 E2 mutants (E2319Q, E2319A), but not if they were accompanied by lack of N-glycosylation in E1. Compared to the non-mutated infectious clone, the SAV3-E2319Q and SAV3-E2319A recombinant viruses produced less cytopathic effects in cell culture and lower amounts of infectious viral particles. In conclusion, the substitution in the N-linked glycosylation site in E2 attenuated SAV3 in cell culture. The findings could be useful for immunization strategies using live attenuated vaccines and testing in fish will be desirable to study the clone's properties in vivo.

PCR assay for improved diagnostics of epitheliotropic disease virus (EEDV) in lake trout Salvelinus namaycush.

Epizootic epitheliotropic disease virus (EEDV) has caused catastrophic losses among hatchery-reared juvenile lake trout Salvelinus namaycush since the early 1980s and remains a major impediment to lake trout restoration in the Great Lakes basin of the USA. Although EEDV has been tentatively designated as a herpesvirus based upon morphological criteria, further characterization of the virus and development of improved detection methods have been hampered by the inability to propagate the virus in cell culture. Recently obtained sequence data for a region of the putative terminase gene from EEDV as well as the related Salmonid herpesvirus 1 and 2 have permitted the development of a polymerase chain reaction (PCR) assay for specific detection of EEDV. The new EEDV PCR demonstrated both an excellent analytic sensitivity and specificity and detected viral DNA as present in the skin of lake trout during periods of active viral outbreaks. In addition, EEDV DNA was detected among healthy appearing juveniles and in the ovarian fluids of spawning adults. Here we describe the development and initial validation steps of the EEDV PCR as a replacement for current diagnostic methods that require virus extraction from the skin, partial purification by isopycnic centrifugation, and visualization of negatively-stained virions by electron microscopy.

Shedding of the Salmonid Herpesvirus-3 by Infected Lake Trout (Salvelinus namaycush).

Salmonid Herpesvirus-3, commonly known as the Epizootic Epitheliotropic Disease virus (EEDV), causes a disease of lake trout (Salvelinus namaycush) that has killed millions of fish over the past several decades. Currently, most aspects of EEDV disease ecology remain unknown. In this study, we investigated EEDV shedding in experimentally challenged (intracoelomic injection) lake trout that were individually microchipped. In order to assess viral shedding, each infected fish was placed in individual static, aerated aquaria for a period of 8 h, after which the water was assessed for the presence of EEDV DNA using quantitative PCR. Water sampling was conducted every seven days for 93 days post-infection (pi), followed by additional sampling after one year. Results demonstrated that lake trout began shedding EEDV into the water as early as 9 days pi. Shedding peaked approximately three weeks pi and ceased after nine weeks pi. In contrast, mortalities did not occur until 40 days pi. Although mortality reached 73.9%, surviving fish ceased shedding and continued to grow. However, additional shedding was detected 58 weeks after infection in 66% of surviving fish. Findings of this study demonstrate that EEDV is shed into the water by infected lake trout hosts for extended periods of time, a mechanism that favors virus dissemination.

Piscine Orthoreovirus 3 Is Not the Causative Pathogen of Proliferative Darkening Syndrome (PDS) of Brown Trout (Salmo trutta fario).

The proliferative darkening syndrome (PDS) is a lethal disease of brown trout (Salmo trutta fario) which occurs in several alpine Bavarian limestone rivers. Because mortality can reach 100%, PDS is a serious threat for affected fish populations. Recently, Kuehn and colleagues reported that a high throughput RNA sequencing approach identified a piscine orthoreovirus (PRV) as a causative agent of PDS. We investigated samples from PDS-affected fish obtained from two exposure experiments performed at the river Iller in 2008 and 2009. Using a RT-qPCR and a well-established next-generation RNA sequencing pipeline for pathogen detection, PRV-specific RNA was not detectable in PDS fish from 2009. In contrast, PRV RNA was readily detectable in several organs from diseased fish in 2008. However, similar virus loads were detectable in the control fish which were not exposed to Iller water and did not show any signs of the disease. Therefore, we conclude that PRV is not the causative agent of PDS of brown trout in the rhithral region of alpine Bavarian limestone rivers. The abovementioned study by Kuehn used only samples from the exposure experiment from 2008 and detected a subclinical PRV bystander infection. Work is ongoing to identify the causative agent of PDS.

Development of a loop-mediated isothermal amplification assay for the detection and quantification of epizootic epitheliotropic disease virus (salmonid herpesvirus-3).

Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus-3) causes a serious disease hatchery-reared lake trout (Salvelinus namaycush), threatening restoration efforts of this species in North America. The current inability to replicate EEDV in vitro necessitates the search for a reproducible, sensitive, and specific assay that allows for its detection and quantitation in a time- and cost-effective manner. Herein, we describe a loop-mediated isothermal amplification (LAMP) assay that was developed for the quantitative detection of EEDV in infected fish tissues. The newly developed LAMP reaction was optimized in the presence of calcein, and the best results were produced using 2 mM MgCl2, 1.8 mM dNTPs and at an incubation temperature of 67.1 °C. This method was highly specific to EEDV, as it showed no cross-reactivity with several fish viruses, including Salmonid Herpesvirus-1, -2, -4, and -5, Infectious Pancreatic Necrosis Virus, Spring Viremia of Carp Virus, Infectious Hematopoietic Necrosis Virus, Golden Shiner Reovirus, Fathead Minnow Nidovirus, and Viral Hemorrhagic Septicemia Virus. The analytical sensitivity of the EEDV-LAMP method was estimated to be as low as 16 copies of plasmid per reaction. When infected fish tissue was used, a positive reaction could be obtained when an infected gill tissue sample that contained 430 viral copies/µg was diluted up to five orders of magnitude. The sensitivity and specificity of the newly developed LAMP assay compared to the SYBR Green qPCR assay were 84.3% and 93.3%, respectively. The quantitative LAMP for EEDV had a correlation coefficient (R2 = 0.980), and did not differ significantly from the SYBR Green quantitative PCR assay (p > 0.05). Given its cost- and time-effectiveness, this quantitative LAMP assay is suitable for screening lake trout populations and for the initial diagnosis of clinical cases.

Identification of a piscine reovirus-related pathogen in proliferative darkening syndrome (PDS) infected brown trout (Salmo trutta fario) using a next-generation technology detection pipeline.

The proliferative darkening syndrome (PDS) is an annually recurring disease that causes species-specific die-off of brown trout (Salmo trutta fario) with a mortality rate of near 100% in pre-alpine rivers of central Europe. So far the etiology and causation of this disease is still unclear. The objective of this study was to identify the cause of PDS using a next-generation technology detection pipeline. Following the hypothesis that PDS is caused by an infectious agent, brown trout specimens were exposed to water from a heavily affected pre-alpine river with annual occurrence of the disease. Specimens were sampled over the entire time period from potential infection through death. Transcriptomic analysis (microarray) and RT-qPCR of brown trout liver tissue evidenced strong gene expression response of immune-associated genes. Messenger RNA of specimens with synchronous immune expression profiles were ultra-deep sequenced using next-generation sequencing technology (NGS). Bioinformatic processing of generated reads and gap-filling Sanger re-sequencing of the identified pathogen genome revealed strong evidence that a piscine-related reovirus is the causative organism of PDS. The identified pathogen is phylogenetically closely related to the family of piscine reoviruses (PRV) which are considered as the causation of different fish diseases in Atlantic and Pacific salmonid species such as Salmo salar and Onchorhynchus kisutch. This study also highlights that the approach of first screening immune responses along a timeline in order to identify synchronously affected stages in different specimens which subsequently were ultra-deep sequenced is an effective approach in pathogen detection. In particular, the identification of specimens with synchronous molecular immune response patterns combined with NGS sequencing and gap-filling re-sequencing resulted in the successful pathogen detection of PDS.

Development of a rapid, sensitive and non-lethal diagnostic assay for the detection of viral haemorrhagic septicaemia virus.

A non-lethal diagnostic procedure based on polymerase chain reaction (PCR) technology was developed to detect viral haemorrhagic septicaemia virus (VHSV). Sensitivity of the assay was tested using purified viral RNA and seeded tissues. Detection limits of the reverse transcriptase-polymerase chain reaction (RT-PCR) assay were estimated to be 10 fg of purified RNA and 0.97 x 10(3) or 10(0) TCID(50)/g of seeded tissue, depending on the experimental approach employed (viral adsorption allowed for 1 or 24h). Addition of nested PCR increased sensitivity up to 100-fold when cDNA excised from the agarose gel was used as template. Both, RT-PCR and nested RT-PCR, as well as Southern blot were applied to RNA extracted from blood of experimentally infected brown trout and the results were compared with those obtained by applying the same techniques to tissues and also with those of conventional viral isolation in cell culture. The superiority of the nested RT-PCR applied to blood samples has been clearly demonstrated in terms of sensitivity, obtaining positive results in 85% of fish tested, as against 40% obtained by RT-PCR and Southern blot, and only 5% viral isolations in cell culture. This procedure could turn into an important tool for screening of wild stocks as well as valuable individuals in commercial fish farms, since it makes to kill the fish unnecessary.

Initial Detection and Molecular Characterization of Namaycush Herpesvirus (Salmonid Herpesvirus 5) in Lake Trout.

A novel herpesvirus was found by molecular methods in samples of Lake Trout Salvelinus namaycush from Lake Erie, Pennsylvania, and Lake Ontario, Keuka Lake, and Lake Otsego, New York. Based on PCR amplification and partial sequencing of polymerase, terminase, and glycoprotein genes, a number of isolates were identified as a novel virus, which we have named Namaycush herpesvirus (NamHV) salmonid herpesvirus 5 (SalHV5). Phylogenetic analyses of three NamHV genes indicated strong clustering with other members of the genus Salmonivirus, placing these isolates into family Alloherpesviridae. The NamHV isolates were identical in the three partially sequenced genes; however, they varied from other salmonid herpesviruses in nucleotide sequence identity. In all three of the genes sequenced, NamHV shared the highest sequence identity with Atlantic Salmon papillomatosis virus (ASPV; SalHV4) isolated from Atlantic Salmon Salmo salar in northern Europe, including northwestern Russia. These results lead one to believe that NamHV and ASPV have a common ancestor that may have made a relatively recent host jump from Atlantic Salmon to Lake Trout or vice versa. Partial nucleotide sequence comparisons between NamHV and ASPV for the polymerase and glycoprotein genes differ by >5% and >10%, respectively. Additional nucleotide sequence comparisons between NamHV and epizootic epitheliotropic disease virus (EEDV/SalHV3) in the terminase, glycoprotein, and polymerase genes differ by >5%, >20%, and >10%, respectively. Thus, NamHV and EEDV may be occupying discrete ecological niches in Lake Trout. Even though NamHV shared the highest genetic identity with ASPV, each of these viruses has a separate host species, which also implies speciation. Additionally, NamHV has been detected over the last 4 years in four separate water bodies across two states, which suggests that NamHV is a distinct, naturally replicating lineage. This, in combination with a divergence in nucleotide sequence from EEDV, indicates that NamHV is a new species in the genus Salmonivirus. Received April 20, 2015; accepted October 11, 2015.

A Quantitative Polymerase Chain Reaction Assay for the Detection and Quantification of Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus 3).

Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R(2) = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR. Received April 20, 2015; accepted November 10, 2015.

The genetic diversity and epizootiology of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which causes a serious disease in salmonid fish. The T1 ribonuclease fingerprinting method was used to compare the RNA genomes of 26 isolates of IHNV recovered from sockeye salmon (Oncorhynchus nerka), chinook salmon (O. tshawytscha), and steelhead trout (O. mykiss) throughout the enzootic portion of western North America. Most of the isolates analyzed in this study were from a single year (1987) to limit time of isolation as a source of genetic variation. In addition, isolates from different years collected at three sites were analyzed to investigate genetic drift or evolution of IHNV within specific locations. All of the isolates examined by T1 fingerprint analysis contained less than a 50% variation in spot location and were represented by a single fingerprint group. The observed variation was estimated to correspond to less than 5% variation in the nucleic acid sequence. However, sufficient variation was detected to separate the isolates into four subgroups which appeared to correlate to different geographic regions. Host species appeared not to be a significant source of variation. The evolutionary and epizootiologic significance of these findings and their relationship to other evidence of genetic variation in IHNV isolates are discussed.

Molecular epizootiology and evolution of the glycoprotein and non-virion protein genes of infectious hematopoietic necrosis virus, a fish rhabdovirus.

Infectious hematopoietic necrosis virus (IHNV) causes a highly lethal, economically important disease of salmon and trout. The virus is enzootic throughout western North America, and has been spread to Asia and Europe. The nucleotide sequences of the glycoprotein (G) and non-virion (NV) genes of 12 diverse IHNV isolates were determined in order to examine the molecular epizootiology of IHN, the primary structure and conservation of NV, and the evolution of the virus. The G and NV genes and their encoded proteins were highly conserved, with a maximum pairwise nucleotide divergence of 3.6 and 4.4%, and amino acid divergence of 3.7 and 6.2%, respectively. Conservation of NV protein sequence (111 amino acids in length) confirms that the protein is functional and plays an important role in virus replication. The phylogenetic relationship of viruses was found to correlate with the geographic origin of virus isolates rather than with host species or time of isolation. These data are consistent with stable maintenance of virus in enzootic foci. Two main IHNV genetic lineages were identified; one in the Columbia River Basin (Oregon, Washington and Idaho), the other in the Sacramento River Basin (California). The first major IHNV outbreak in chinook salmon in 1973 in the Columbia River was genetically linked to importation of virus-infected fish eggs from the Sacramento River where outbreaks in chinook salmon are common. However, the introduced virus apparently did not persist, subsequent virus outbreaks in Columbia River chinook salmon being associated with Columbia River genetic lineages. In general, virus monoclonal antibody reactivity profiles and phylogenetic relationships correlated well.

Fast neutralization/immunoperoxidase assay for viral haemorrhagic septicaemia with anti-nucleoprotein monoclonal antibody.

An enzyme-immunohistochemical procedure was employed to facilitate neutralization/diagnostic tests for viral haemorrhagic septicaemia virus (VHSV), a significant pathogen in trout farms throughout Europe. The method described can be used for trout or mice antibodies; increases speed (1 day), simplicity, and minimizes the use of reagents compared to other neutralization assays. Furthermore, the test requires a minimum handling of the cell cultures under sterile conditions, decreasing frequent contamination due to the non-sterile conditions of the fish pathological samples. Foci of 5-20 infected epithelioma papillosum carp (EPC) cells are detected and counted with an inverted microscope in under 16 h after infection of EPC monolayers using a high titre anti-N VHSV monoclonal antibody (MAb) 2C9. MAb 2C9 recognizes different viral haemorrhagic septicaemia virus serotypes and VHSV isolates from different host species (trout, salmon and barbel) and Spanish geographical locations. The high titre and specificity of MAb 2C9 favour its conjugation to peroxidase and also make it possible to use in direct immunoperoxidase staining of the VHSV infected EPC monolayers. This neutralization/immunoperoxidase assay should improve diagnostics that use currently agarose or methylcellulose plaque reduction neutralization assays.

Differential diagnosis of fish pathogenic rhabdoviruses by reverse transcriptase-dependent polymerase chain reaction.

Reverse transcriptase-dependent polymerase chain reaction (RT-PCR) was applied to the detection and differentiation of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) using primer pairs designed for the amplification of glycoprotein G-specific gene fragments of the two viruses. The products of 443 bp (VHS) and 548 bp (IHN), respectively, were amplified from the total RNA extracts of RTG-2 cells infected with a total of 9 different strains of either VHS virus or IHN virus. Restriction analysis using FokI, and DNA sequencing of the PCR products demonstrated specificity of the amplification. The RT-PCR amplification of VHSV or IHNV G-genes was found to be a simple, highly specific and sensitive method allowing differential diagnosis of VHS and IHN within 8 h.

Use of RT-PCR for diagnosis of infectious salmon anaemia virus (ISAV) in carrier sea trout Salmo trutta after experimental infection.

The emergence of infectious salmon anaemia virus (ISAV) in Canada and Scotland and frequent new outbreaks of the disease in Norway strongly suggest that there are natural reservoirs for the virus. The main host for the ISA virus is probably a fish occurring in the coastal area, most likely a salmonid fish. Since sea trout is an abundant species along the Norwegian coast, common in areas where ISA outbreaks occur, and possibly a life-long carrier of the ISA virus, a study was initiated to evaluate reverse transcriptase polymerase chain reaction (RT-PCR) for diagnosis of the virus in experimentally infected trout. Several tissues (kidney, spleen, heart and skin) were collected from the trout during a 135 d period. The following diagnostic methods for detection of the ISA virus were compared: cell culture (Atlantic Salmon Kidney, ASK cells), challenge of disease-free salmon with blood from the infected trout, and RT-PCR. The RT-PCR was the most sensitive of these methods. With the help of this technique it was possible to pick out positive individuals throughout the experimental period of 135 d. Challenge of disease-free salmon were more sensitive than cell culture (ASK cells). These 2 latter methods require use of the immunofluorescent antibody test (IFAT) or RT-PCR for verification of presence of ISA virus.