Infectious Salmon Anaemia Virus Strains Research Articles

Infectious Salmon Anemia Virus Infectivity Is Determined by Multiple Segments with an Important Contribution from Segment 5.

Infectious salmon anemia virus (ISAV) is the etiological agent of infectious salmon anemia. It belongs to the genus isavirus, one of the genera of the Orthomyxoviridae family, as does Influenzavirus A. The ISAV genome comprises eight negative-sense single-stranded RNA segments that code for at least 10 proteins. Although some ISAV strains can reach 100% mortality rates, the factors that determine isavirus infectivity remain unknown. However, some studies suggest that segments 5 and 6 are responsible for the different degrees of virulence and infectivity among ISAV subtypes, unlike the influenza A virus, where most segments are involved in the virus infectivity. In this work, synthetic reassortant viruses for the eight segments of ISAV were generated by reverse genetics, combining a highly virulent virus, ISAV 752_09 (HPR7b), and an avirulent strain, SK779/06 (HPR0). We characterized the rescued viruses and their capacity to replicate and infect different cell lines, produce plaques in ASK cells, and their ability to induce and modulate the cellular immune response in vitro. Our results show that the majority of ISAV segments are involved in at least one of the analyzed characteristics, segment 5 being one of the most important, allowing HPR0 viruses, among other things, to produce plaques and replicate in CHSE-214 cells. We determined that segments 5 and 6 participate in different stages of the viral cycle, and their compatibility is critical for viral infection. Additionally, we demonstrated that segment 2 can modulate the cellular immune response. Our results indicate a high degree of genetic compatibility between the genomic segments of HPR7b and HPR0, representing a latent risk of reassortant that would give rise to a new virus with an unknown phenotype.

Prevalence and genotypes of infectious salmon anaemia virus (ISAV) in returning wild Atlantic salmon (Salmo salar L.) in northern Norway.

Prevalence and genotypes of infectious salmon anaemia virus (ISAV) in returning wild Atlantic salmon (Salmo salar L.) in northern Norway.

An updated proposal for classification of infectious salmon anemia virus strains.

Biological databases contain a wealth of valuable information that can contribute to the enrichment of virtually any area. However, the exponential growth of information together with its dissemination through virtual networks has become a double-edged sword, promoting synonymy that leads to confusion and chaos. Organization of data is a big effort that must be accompanied by clarity, both in the deposited data and in the publications arising from them. In this report, an effort is made to organize the information related to infectious salmon anemia virus and its classification based on the variability of genomic segment 6.

RNA-Seq analysis in gills of atlantic salmon (Salmo salar) infected with newly identified ISAV strains in the atlantic Canada region

The Infectious salmon anemia virus (ISAV) is an important viral disease of farmed Atlantic salmon (Salmo salar) which has caused severe financial losses for salmon farmers around the world, including Atlantic Canada. It is listed as an OIE notifiable disease and to this day eradication of infected cages remains the current practice in many countries to mitigate the spread of the virus. All strains known to display any level of virulence are characterised by deletions in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) protein compared to ISAV-HPR0 strains and are designated ISAVHPR∆. In contrast, HPR0 strains are non-virulent and not detected by viral culture. In Atlantic Canada, improved management and husbandry practices resulted in no outbreaks caused by ISAV-HPR∆ strains from 2007 to 2012, with however ISAV-HPR0 strains (European genotype) still being detected. Starting in 2012, new ISAV-HPR∆ strains were responsible for disease outbreaks in fish farms in Nova Scotia, Newfoundland and New-Brunswick. This study was thus designed to characterize virulence associated to three of the newly identified ISAV-HPR∆ strains and investigate gene expression responses in gills of infected fish using RNA-Seq. All three ISAV-HPR∆ strains studied through the use of in vivo challenges resulted in cumulative mortalities below 40 percent and caused the differential expression of up to 1000 genes at the peak of infection compared to non-infected fish. Many genes linked to innate immunity were over-expressed; similar to what had been observed in the head-kidney through previous work. The use of RNA-Seq also enabled us to look at ISAV transcript abundance and showed that segment 8 had the highest abundance of all 8 segments of the genome. This information has given us great insight on how ISAV interacts with his host during the course of an infection.

Immersion challenge with low and highly virulent infectious salmon anaemia virus reveals different pathogenesis in Atlantic salmon, Salmo salar L.

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 mortality of 100% as compared to 20% in the LVI group by day 23 post-infection. Immersion challenge represented a more natural infection method and suggested that specific entry routes into the fish may be of key importance between ISAV strains. The results suggest that a difference in virulence is important for variations in virus dissemination and pathogenesis (disease development).

Infectious salmon anaemia virus (ISAV) in Chilean Atlantic salmon (Salmo salar) aquaculture: emergence of low pathogenic ISAV-HPR0 and re-emergence of virulent ISAV-HPR∆: HPR3 and HPR14.

AbstactInfectious 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-HPR∆). This study shows the historic dynamics of ISAV-HPR∆ and ISAV-HPR0 in Chile, the genetic relationship among ISAV-HPR0 reported worldwide and between ISAV-HPR0 and ISAV-HPR∆ 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-HPR∆ 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 (360PST362, 360PAN362 and 360PAT362) in HPR. The virus responsible for the 2013 ISAV-HPR∆ 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 360PAT362, which is the only type of ISAV-HPR0 variant found in Chile. This suggested to us that the 2013 ISAV-HPR∆ re-emerged from ISAV-HPR0 that is enzootic in Chilean salmon aquaculture and were not new introductions of virulent ISAV-HPR∆ 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-HPR∆ viruses and the possibility that it could mutate to virulent ISAV-HPR∆.

Molecular phylodynamics and protein modeling of infectious salmon anemia virus (ISAV)

BackgroundISAV is a member of the Orthomyxoviridae family that affects salmonids with disastrous results. It was first detected in 1984 in Norway and from then on it has been reported in Canada, United States, Scotland and the Faroe Islands. Recently, an outbreak was recorded in Chile with negative consequences for the local fishing industry. However, few studies have examined available data to test hypotheses associated with the phylogeographic partitioning of the infecting viral population, the population dynamics, or the evolutionary rates and demographic history of ISAV. To explore these issues, we collected relevant sequences of genes coding for both surface proteins from Chile, Canada, and Norway. We addressed questions regarding their phylogenetic relationships, evolutionary rates, and demographic history using modern phylogenetic methods.ResultsA recombination breakpoint was consistently detected in the Hemagglutinin-Esterase (he) gene at either side of the Highly Polymorphic Region (HPR), whereas no recombination breakpoints were detected in Fusion protein (f) gene. Evolutionary relationships of ISAV revealed the 2007 Chilean outbreak group as a monophyletic clade for f that has a sister relationship to the Norwegian isolates. Their tMRCA is consistent with epidemiological data and demographic history was successfully recovered showing a profound bottleneck with further population expansion. Finally, selection analyses detected ongoing diversifying selection in f and he codons associated with protease processing and the HPR region, respectively.ConclusionsOur results are consistent with the Norwegian origin hypothesis for the Chilean outbreak clade. In particular, ISAV HPR0 genotype is not the ancestor of all ISAV strains, although SK779/06 (HPR0) shares a common ancestor with the Chilean outbreak clade. Our analyses suggest that ISAV shows hallmarks typical of RNA viruses that can be exploited in epidemiological and surveillance settings. In addition, we hypothesized that genetic diversity of the HPR region is governed by recombination, probably due to template switching and that novel fusion gene proteolytic sites confer a selective advantage for the isolates that carry them. Additionally, protein modeling allowed us to relate the results of phylogenetic studies with the predicted structures. This study demonstrates that phylogenetic methods are important tools to predict future outbreaks of ISAV and other salmon pathogens.

Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences

BackgroundInfectious salmon anaemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon (Salmo salar); a disease first diagnosed in Norway in 1984. For over 25 years ISAV has caused major disease outbreaks in the Northern hemisphere, and remains an emerging fish pathogen because of the asymptomatic infections in marine wild fish and the potential for emergence of new epidemic strains. ISAV belongs to the family Orthomyxoviridae, together with influenza viruses but is sufficiently different to be assigned to its own genus, Isavirus. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; fusion (F) protein encoded on segment 5 and haemagglutinin-esterase (HE) protein encoded on segment 6. However, comparision between different ISAV isolates is complicated because there is presently no universally accepted nomenclature system for designation of genetic relatedness between ISAV isolates. The first outbreak of ISA in marine-farmed Atlantic salmon in the Southern hemisphere occurred in Chile starting in June 2007. In order to describe the molecular characteristics of the virus so as to understand its origins, how ISAV isolates are maintained and spread, and their virulence characteristics, we conducted a study where the viral sequences were directly amplified, cloned and sequenced from tissue samples collected from several ISA-affected fish on the different fish farms with confirmed or suspected ISA outbreaks in Chile. This paper describes the genetic characterization of a large number of ISAV strains associated with extensive outbreaks in Chile starting in June 2007, and their phylogenetic relationships with selected European and North American isolates that are representative of the genetic diversity of ISAV.ResultsRT-PCR for ISAV F and HE glycoprotein genes was performed directly on tissue samples collected from ISA-affected fish on different farms among 14 fish companies in Chile during the ISA outbreaks that started in June 2007. The genes of the F and HE glycoproteins were cloned and sequenced for 51 and 78 new isolates, respectively. An extensive comparative analysis of ISAV F and HE sequence data, including reference isolates sampled from Norway, Faroe Islands, Scotland, USA, and Canada was performed. Based on phylogenetic analysis of concatenated ISAV F and HE genes of 103 individual isolates, the isolates from the ISA outbreaks in Chile grouped in their own cluster of 7 distinct strains within Genotype I (European genotype) of ISAV, with the closest relatedness to Norwegian ISAVs isolated in 1997. The phylogenetic software program, BACKTRACK, estimated the Chile isolates diverged from Norway isolates about 1996 and, therefore, had been present in Chile for some time before the recent outbreaks. Analysis of the deduced F protein sequence showed 43 of 51 Chile isolates with an 11-amino acid insert between 265N and 266Q, with 100% sequence identity with Genotype I ISAV RNA segment 2. Twenty four different HE-HPRs, including HPR0, were detected, with HPR7b making up 79.7%. This is considered a manifestation of ISAV quasispecies HE protein sequence diversity.ConclusionTaken together, these findings suggest that the ISA outbreaks were caused by virus that was already present in Chile that mutated to new strains. This is the first comprehensive report tracing ISAV from Europe to South America.

In vivo correlates of infectious salmon anemia virus pathogenesis in fish

The phenotypic correlates of pathogenicity for Infectious salmon anemia virus (ISAV) in salmonid fishes have not been thoroughly studied to date. In this study, a comparison was made of 13 different strains of ISAV, isolated from different geographical regions between 1997 and 2004, for their infectivity in three fish species [Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch) and rainbow trout (Oncorhynchus mykiss)]. When the different virus isolates were used at an approximate inoculum dose of 10(6) TCID(50) in 0.2 ml per fish, it was found that the most virulent strains had an acute mortality phase in Atlantic salmon that started at 10-13 days post-inoculation and lasted for 9-15 days with a cumulative mortality of >/=90 %. These highly pathogenic strains also caused low mortality in rainbow trout, albeit later in infection. Viruses with a more delayed or protracted mortality phase resulting in cumulative mortalities of 50-89 % in Atlantic salmon were considered to be of intermediate pathogenicity and isolates with cumulative mortalities of </=49 % were considered to be of low pathogenicity. On this basis, three of the ISAV isolates showed a high-, eight an intermediate- and two a low-pathogenicity phenotype in Atlantic salmon. Coho salmon were resistant to all ISAV isolates. These results confirmed that there is variation in pathogenicity among ISAV strains for Atlantic salmon and rainbow trout, and that other salmonid species such as coho salmon can carry highly pathogenic strains of ISAV without showing signs of disease. The identified pathogenicity phenotypes may aid in the identification of molecular markers of ISAV virulence.

Isolation and identification of infectious salmon anaemia virus (ISAV) from Coho salmon in Chile.

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.