Accelerated ISAV replication detection by cell culture methods combined with time-monitoring RT-qPCR (original) (raw)
Isolation and identification of infectious salmon anaemia virus (ISAV) from Coho salmon in Chile
Diseases of Aquatic Organisms, 2001
The isolation of infectious salmon anaemia virus (ISAV) from asymptomatic wild fish species including wild salmon, sea trout and eel established that wild fish can be a reservoir of ISAV for farmed Atlantic salmon. This report characterizes the biological properties of ISAV isolated from a disease outbreak in farmed Coho salmon in Chile and compares it with ISAV isolated from farmed Atlantic salmon in Canada and Europe. The virus that was isolated from Coho salmon tissues was initially detected with ISAV-specific RT-PCR (reverse transcription-polymerase chain reaction). The ability of the virus to grow in cell culture was poor, as cytopathology was not always conspicuous and isolation required passage in the presence of trypsin. Virus replication in cell culture was detected by RT-PCR and IFAT (indirect fluorescent antibody test), and the virus morphology was confirmed by positive staining electron microscopy. Further analysis of the Chilean virus revealed similarities to Canadian ISAV isolates in their ability to grow in the CHSE-214 cell line and in viral protein profile. Sequence analysis of genome segment 2, which encodes the viral RNA polymerase PB1, and segment 8, which encodes the nonstructural proteins NS1 and NS2, showed the Chilean virus to be very similar to Canadian strains of ISAV. This high sequence similarity of ISAV strains of geographically distinct origins illustrates the highly conserved nature of ISAV proteins PB1, NS1 and NS2 of ISAV. It is noteworthy that ISAV was associated with disease outbreaks in farmed Coho salmon in Chile without corresponding clinical disease in farmed Atlantic salmon. This outbreak, which produced high mortality in Coho salmon due to ISAV, is unique and may represent the introduction of the virus to a native wild fish population or a new strain of ISAV.
Virology Journal, 2016
Background: Infectious salmon anaemia (ISA) virus (ISAV) belongs to the genus Isavirus, family Orthomyxoviridae. ISAV occurs in two basic genotypes, North American and European. The European genotype is more widespread and shows greater genetic variation and greater virulence variation than the North American genotype. To date, all of the ISAV isolates from the clinical disease, ISA, have had deletions in the highly polymorphic region (HPR) on ISAV segment 6 (ISAV-HPRΔ) relative to ISAV-HPR0, named numerically from ISAV-HPR1 to over ISAV-HPR30. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish. It is recognized that asymptomatically ISAV-infected fish exist. There is no universally accepted ISAV RT-qPCR TaqMan® assay. Most diagnostic laboratories use the primer-probe set targeting a 104 bp-fragment on ISAV segment 8. Some laboratories and researchers have found a primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive. Methods: In this study, we tested 1,106 fish tissue samples collected from (i) market-bought farmed salmonids and (ii) wild salmon from throughout British Columbia (BC), Canada, for ISAV using real time RT-qPCR targeting segment 8 and/or conventional RT-PCR with segment 8 primers and segment 6 HPR primers, and by virus isolation attempts using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. The sequences from the conventional PCR products were compared by multiple alignment and phylogenetic analyses. Results: Seventy-nine samples were "non-negative" with at least one of these tests in one or more replicates. The ISAV segment 6 HPR sequences from the PCR products matched ISAV variants, HPR5 on 29 samples, one sample had both HPR5 and HPR7b and one matched HPR0. All sequences were of European genotype. In addition, alignment of sequences of the conventional PCR product segment 8 showed they had a single nucleotide mutation in the region of the probe sequence and a 9-nucleotide overlap with the reverse primer sequence of the real time RT-qPCR assay. None of the classical ISAV segment 8 sequences in the GenBank have this mutation in the probe-binding site of the assay, suggesting the presence of a novel ISAV variant in BC. A phylogenetic tree of these sequences showed that some ISAV sequences diverted early from the classical European genotype sequences, while others have evolved separately. All virus isolation attempts on the samples were negative, and thus the samples were considered "negative" in terms of the threshold trigger set for Canadian federal regulatory action; i.e., successful virus isolation in cell culture.
Diseases of Aquatic Organisms, 2010
The objective of this study was to evaluate the application of a TaqMan ® real-time reverse transcriptase PCR (RT-PCR) assay for the detection of infectious salmon anaemia virus (ISAV) in formalin-fixed paraffin-embedded (FFPE) fish tissues from Atlantic salmon Salmo salar with and without clinical signs of infection, and to compare it with histological and immunohistochemical (IHC) techniques. Sixteen fish samples obtained in 2007 and 2008 from 4 different farms in Chile were examined. The real-time RT-PCR allowed the detection of ISAV in FFPE samples from 9 of 16 fish, regardless of the organs analyzed, whereas 4 of the real-time RT-PCR negative fish were positive as indicated by histological examination and 3 of the real-time RT-PCR positive fish were negative as indicated by immunohistochemistry evaluation. The presence of ISAV in RT-PCR positive samples was confirmed by amplicon sequencing. This work constitutes the first report on the use of real-time RT-PCR for the detection of ISAV in FFPE sections. The assay is very useful for the examination of archival wax-embedded tissues, and allows for both prospective and retrospective evaluation of tissue samples for the presence of ISAV. However, the method only confirms the presence of the pathogen and should be used in combination with histopathology, which is a more precise tool. The combination of both techniques would be invaluable for confirmatory diagnosis of infectious salmon anaemia (ISA), which is essential for solving salmon farm problems.
Assessment of the in vitro survival of the Infectious Salmon Anaemia Virus (ISAV)
Bulletin- European Association of Fish Pathologists
caused by ISA virus (ISAV). A deeper understanding of the biophysical properties of ISAV is needed in vitro survival of ISAV under varying water types and temperature. Multiple aliquots of four ISAV strains were -observed when the temperature increased. ISAV could survive for extended periods in freshwater at low temperature but the survival of ISAV was greatly decreased in seawater notwithstanding water temperature. This study has shown that temperature and water type play an important role in the in vitro ISAV survival.
Journal of General Virology, 2011
Infectious salmon anemia virus (ISAV) is an orthomyxovirus responsible for a significant disease of farmed Atlantic salmon. Fallowing and re-establishment of the Atlantic salmon farming industry in the Faroes following a recent devastating infectious salmon anaemia (ISA) disease epidemic provided a unique opportunity to study the risk of re-emergence of disease. Over 53 months, 2787 of 34 573 (8.1 %) apparently healthy Atlantic salmon analysed tested positive for ISAV by RT-PCR. Sequence analysis revealed the putative low-pathogenic ISAV-HPR0 subtype in all cases. Results demonstrated that ISAV-HPR0 appeared as a seasonal and transient infection without detectable ISA mortality or pathology. This finding, coupled to an apparent gill tropism of ISAV-HPR0, suggests ISAV-HPR0 causes a subclinical respiratory infection more like seasonal influenza, as opposed to the systemic infection and serious disease caused by highly pathogenic ISAV. The mean time before marine sites became infected was 7.7 months after transfer to seawater of the fish, suggesting a potentially unknown marine reservoir of infection. Sequence analysis identified two main subtypes of ISAV-HPR0 sequences, one of which showed close genetic association with ISAV isolates responsible for the disease outbreak in the Faroes. Thus ISAV-HPR0 might represent an ancestor of pathogenic variants and thus be a potential risk factor in the emergence of new strains of disease-causing ISAV. Our data, however, suggest that the risk of emergence of pathogenic ISAV variants from a reservoir of ISAV-HPR0 is low. This risk is probably being further reduced by practical management strategies adopted in the Faroes and aimed at reducing the potential for maintenance and adaptation of ISAV-HPR0.
Virology Journal, 2013
Infectious salmon anaemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), which belongs to the genus Isavirus, family Orthomyxoviridae. ISA is caused by virulent ISAV strains with deletions in a highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) protein (designated virulent ISAV-HPRΔ). This study shows the historic dynamics of ISAV-HPRΔ and ISAV-HPR0 in Chile, the genetic relationship among ISAV-HPR0 reported worldwide and between ISAV-HPR0 and ISAV-HPRΔ in Chile, and reports the 2013 ISA outbreak in Chile. The first ISA outbreak in Chile occurred from mid-June 2007 to 2010 and involved the virulent ISAV-HPR7b, which was then replaced by a low pathogenic ISAV-HPR0 variant. We analyzed this variant in 66 laboratory-confirmed ISAV-HPR0 cases in Chile in comparison to virulent ISAV-HPRΔ that caused two new ISA outbreaks in April 2013. Multiple alignment and phylogenetic analysis of HE sequences from all ISAV-HPR0 viruses allowed us to identify three genomic clusters, which correlated with three residue patterns of ISAV-HPR0 ( 360 PST 362 , 360 PAN 362 and 360 PAT 362 ) in HPR. The virus responsible for the 2013 ISAV-HPRΔ cases in Chile belonged to ISAV-HPR3 and ISAV-HPR14, and in phylogenetic analyses, both clustered with the ISAV-HPR0 found in Chile. The ISAV-HPR14 had the ISAV-HPR0 residue pattern 360 PAT 362 , which is the only type of ISAV-HPR0 variant found in Chile. This suggested to us that the 2013 ISAV-HPRΔ re-emerged from ISAV-HPR0 that is enzootic in Chilean salmon aquaculture and were not new introductions of virulent ISAV-HPRΔ to Chile. The clinical presentations and diagnostic evidence of the 2013 ISA cases indicated a mixed infection of ISAV with the ectoparasite Caligus rogercresseyi and the bacterium Piscirickettsia salmonis, which underscores the need for active ISAV surveillance in areas where ISAV-HPR0 is enzootic, to ensure early detection and control of new ISA outbreaks, as it is considered a risk factor. This is the first report of ISA linked directly to the presence of ISAV-HPR0, and provides strong evidence supporting the contention that ISAV-HPR0 shows a strong relationship to virulent ISAV-HPRΔ viruses and the possibility that it could mutate to virulent ISAV-HPRΔ.
Diseases of Aquatic Organisms, 2012
Infectious salmon anaemia (ISA) is a severe disease in farmed Atlantic salmon Salmo salar that has caused epidemic outbreaks in most salmon-producing countries worldwide. The disease is caused by virulent ISA virus (ISAV). Low virulent variants of the virus, characterised by a full-length sequence in the highly polymorphic region of segment 6 in the virus genome, have been reported with increasing frequencies. These variants of the virus, termed HPR0, have been proposed to be ancestors of virulent ISAV. We examined this idea through studies of the phylogeographic and environmental distribution of ISAV-HPR0, as well as phylogeographic associations between virulent ISAV and ISAV-HPR0. Samples from 232 fish groups were screened for ISAV. Real-time RT-PCR was used for detection of ISAV, and the ISAV haemagglutinin esterase (HE) gene was characterised for positive samples. A Mantel test was used to test phylogeographic associations between pairs of ISAV-HPR0 HE gene sequences. A rank test was used to test associations between HE gene sequences from virulent ISAV and ISAV-HPR0. ISAV-HPR0 was detected in fish groups both in freshwater and marine environments, and in juveniles, on-grown marine salmon and broodstock salmon. Genetic and geographic distances between pairs of ISAV-HPR0 HE gene sequences were positively correlated, suggesting that the population of ISAV-HPR0 is geographically structured. Finally, we found a spatial association between fish groups with virulent ISAV (n = 21) and fish groups with ISAV-HPR0 (n = 27), supporting the hypothesis that ISAV-HPR0 may undergo a transition to virulent ISAV.
Diseases of Aquatic Organisms, 1999
Atlantic salmon Salmo salar L. were injected intrapentoneally with infectious salmon anaemia virus (1SAV)-infective tissue homogenate to clarify the tissue distribution of ISAV in a time course study. Fish were sampled at 11 different intervals between 1 and 40 d post-infection (p.i.) and mid-kidney, head kidney, liver, spleen, intestine, gills, muscle and heart were tested for the presence of ISAV by reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that during a disease outbreak, ISAV is present in most organs. It was possible to detect ISAV at all sampling times in at least 1 of the fish examined. However, for the first 8 d p.i. positive RT-PCR results were predominantly found in samples from the head hdney and mid-kidney. Fish giving positive samples after Day 13 p.i. were RT-PCR positive in most organs. These results indicated that between Days 8 to 13 p.i. considerable replication of the virus occurred, combined with wide tissue dissemination. KEY WORDS: Infectious salmon anaemia virus (ISAV) . ISAV tissue distribution . RT-PCR detection of ISAV 0 Inter-Research 1999 Resale of fuU article not permittea
Journal of fish diseases, 2015
The salmonid orthomyxovirus infectious salmon anaemia virus (ISAV) causes disease of varying severity in farmed Atlantic salmon, Salmo salar L. Field observations suggest that host factors, the environment and differences between ISAV strains attribute to the large variation in disease progression. Variation in host mortality and dissemination of ISAV isolates with high and low virulence (based on a previously published injection challenge) were investigated using immersion challenge. Virus dissemination was determined using real-time PCR and immunohistochemistry in several organs, including blood. Surprisingly, the low virulent virus (LVI) replicated and produced nucleoprotein at earlier time points post-infection compared to the virus of high virulence (HVI). This was particularly noticeable in the gills as indicated by different viral load profiles. However, the HVI reached a higher maximum viral load in all tested organs and full blood. This was associated with a higher mortalit...