Accelerated ISAV replication detection by cell culture methods combined with time-monitoring RT-qPCR (original) (raw)
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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.
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.
Detection of infectious salmon anaemia virus by real-time RT–PCR
Journal of Virological Methods, 2004
A one-tube real-time reverse transcription-polymerase chain reaction (RT-PCR) using LightCycler technology and SYBR Green chemistry that quantitatively detects infectious salmon anaemia virus (ISAV) in biological samples is described. The assay utilized primers targeting ISAV RNA segment 8, with ISAV isolate U5575-1 as template. The entire optimized assay, including one cycle of reverse transcription, 50 cycles of complementary DNA amplification, and data acquisition and analysis took only 80 min. The melting curve and gel electrophoresis analyses of real-time RT-PCR showed harmony with each other as a virus-specific single melting peak and a product of the expected size of 211 bp were obtained. A regression line between the mean threshold cycle (Ct) values and viral template concentrations over a 1:10 5 dilution range with an r 2 value of 0.962 and a slope of −3.771 indicated that the assay was highly reproducible. This assay was 100 times more sensitive than the conventional one-tube RT-PCR assay when compared on the same sample. Analysis of different tissues from fish that survived an ISAV experimental infection further confirmed that real-time RT-PCR was more sensitive than regular RT-PCR for detection of ISAV nucleic acids. Temporal analysis of ISAV-infected TO cell cultures showed that the amount of the specific viral RNA increased more than 100-fold within 32 h post-inoculation (p.i.) and over 1200-fold by 144 h p.i. The melting curve analysis throughout the duration of the infection sampled had a single melting peak suggesting that the virus population was uniform in the targeted region. Quantitative analysis of CHSE-214 cell cultures infected with different ISAV isolates indicated that ISAV isolates, based on their ability to replicate and cause cytopathic effects in CHSE-214 cells, may be differentiated into three CHSE phenotypes: replicating cytopathic, replicating non-cytopathic, and non-replicating. Thus, the SYBR Green real-time RT-PCR is a sensitive, rapid, and highly reproducible assay that can be used to quantitate ISAV in biological samples.
Diseases of Aquatic Organisms, 2001
A reverse transcription polymerase chain reaction (RT-PCR) was used to study the early phase of infectious salmon anaemia virus (ISAV) infection in Atlantic salmon Salmo salar L. The detection threshold for the RT-PCR was estimated to be 0.01 to 0.1 TCID 50 . A protocol that closely mimics the conditions in populations of farmed salmon was used. The major port of ISAV entry was most likely the gills, but oral entry could not be excluded. The gills and heart were RT-PCR positive 5 d post infection and there was a rapid viraemic spread of the virus after entry. Ten or more days post infection, most organs yielded RT-PCR positive samples. The viral load of the fish followed a 2-phase curve with the first maximum at approximately 15 d and a minimum around 25 d. After 25 d, there was a steady increase in viral load until all sampled organs eventually became positive. In an experiment in which the transportation of material from field to diagnostic laboratory was simulated, the transportation of whole fish was found to be optimal for the performance of RT-PCR.
Virology Journal, 2008
Background: Infectious salmon anaemia (ISA) virus (ISAV), which causes ISA in marine-farmed Atlantic salmon, is an orthomyxovirus belonging to the genus Isavirus, family Orthomyxoviridae. ISAV agglutinates erythrocytes of several fish species and it is generally accepted that the ISAV receptor destroying enzyme dissolves this haemagglutination except for Atlantic salmon erythrocytes. Recent work indicates that ISAV isolates that are able to elute from Atlantic salmon erythrocytes cause low mortality in challenge experiments using Atlantic salmon. Previous work on ISAVinduced haemagglutination using the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-0851, showed endocytosis of NBISA01 but not RPC/NB-04-0851. Realtime RT-PCR was used to assess the viral RNA levels in the ISAV-induced haemagglutination reaction samples, and we observed a slight increase in viral RNA transcripts by 36 hours in the haemagglutination reaction with NBISA01 virus when the experiment was terminated. However, a longer sampling interval was considered necessary to confirm ISAV replication in fish erythrocytes and to determine if the infected cells mounted any innate immune response. This study examined the possible ISAV replication and Type I interferon (IFN) system gene induction in Atlantic salmon erythrocytes following ISAV haemagglutination.
Journal of Fish Diseases, 2009
Real-time PCR assays are being increasingly applied to the detection of fish pathogens due to their sensitivity, specificity and potential for high throughput sample processing. Such assays allow for the ready and efficient inclusion of appropriate quality controls which are fundamental to scientific integrity and to satisfying the demands of diagnostic test accreditation. In this article, we report development of a universal positive control strategy for real-time PCR assays, which has been used to support and improve a previously published method for detection of infectious salmon anaemia virus (ISAV). The strategy employed uses an RNA mimic template, which is based on the ISAV segment 8 target sequence but includes an artificial universal positive control sequence. Inclusion of this sequence, which is targeted by a second specific probe carrying a different fluorophore to the primary assay, allows for convenient screening of all real-time PCR reactions for the presence of contaminating positive control material. The development of readily distinguishable artificial positive control material offers distinct advantages to real-time PCR assays over using control material derived from clinical material.
Prevalence of infectious salmon anaemia virus (ISAV) in wild salmonids in western Norway
Diseases of Aquatic Organisms, 2005
Studies of infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Ireland, Canada, the USA and Chile, suggest that natural reservoirs for this virus can be found on both sides of the North Atlantic. Based on existing information about ISAV it is believed to be maintained in wild populations of trout and salmon in Europe. It has further been suggested that ISAV is transmitted between wild hosts, mainly during their freshwater spawning phase in rivers, and that wild salmonids, mainly trout, are possible carriers of benign wild-type variants of ISAV. Change in virulence is probably a result of deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates after transmission to farmed salmon. Hence, it has been suggested that the frequency of new outbreaks of ISA in farmed salmon could partly reflect natural variation in the prevalence of ISAV in wild populations of salmonids. The aims of the present study were to screen for ISAV in wild salmonids during spawning in rivers and to determine the pathogenicity of resultant isolates from wild fish. Tissues from wild salmonids were screened by RT-PCR and real-time PCR. The prevalence of ISAV in wild trout Salmo trutta varied from 62 to 100% between tested rivers in 2001. The prevalence dropped in 2002, ranging from 13 to 36% in the same rivers and to only 6% in 2003. All ISAV were nonpathogenic when injected into disease-free Atlantic salmon, but were capable of propagation, as indicated by subsequent viral recovery. However, non-pathogenic ISAV has also been found in farmed salmon, where a prevalence as high as 60% has been registered, but with no mortalities occurring. Based on the results of the present and other studies, it must be concluded that vital information about the importance of wild and man-made reservoirs for the emergence of ISA in salmon farming is still lacking. This information can only be gained by further screening of possible reservoirs, combined with the development of a molecular tool for typing virulence and the geographical origin of the virus isolates.