Exposure dynamics of Ross River virus in horses - Horses as potential sentinels (a One Health approach).

Ross River virus (RRV), the most medically and economically important arbovirus in Australia, has been the most prevalent arbovirus infections in humans for many years. Infected humans and horses often suffer similar clinical symptoms. We conducted a prospective longitudinal study over a 3.5-year period to investigate the exposure dynamics of RRV in three foal cohorts (n = 32) born in a subtropical region of South East Queensland, Australia, between 2020 and 2022. RRV-specific seroconversion was detected in 56% (n = 18) of foals with a median time to seroconversion, after waning of maternal antibodies, of 429 days (95% CI: 294-582). The median age at seroconversion was 69 weeks (95% CI: 53-57). Seroconversion events were only detected between December and March (Southern Hemisphere summer) over the entire study period. Cox proportion hazards regression analyses revealed that seroconversions were significantly (p < 0.05) associated with air temperature in the month of seroconversion. Time-lags in meteorological variables were not significantly (p > 0.05) associated with seroconversion, except for relative humidity (p = 0.036 at 2-month time-lag). This is in contrast to research results of RRV infection in humans, which peaked between March and May (Autumn) and with a 0-3 month time-lag for various meteorological risk factors. Therefore, horses may be suitable sentinels for monitoring active arbovirus circulation and could be used for early arbovirus outbreak detection in human populations.

On the prediction of SAV transmission among Norwegian aquaculture sites.

Pancreas Disease (PD) is a viral disease that affects Atlantic salmon (Salmo salar) in Norwegian, Scottish and Irish aquaculture. It is caused by salmonid alphavirus (SAV) and represents a significant problem in salmonid farming. Infection with SAV leads to reduced growth, mortality, product downgrading, and has a significant financial impact for the farms. The overall aim of this study is to evaluate the effect of various factors on the transmission of SAV and to create a predictive model capable of providing an early warning system for salmon farms within the Norwegian waters. Using a combination of publicly available databases, specifically BarentsWatch, and privately held PCR analyses a feature set consisting of 11 unique features was created based on the input parameters of the databases. An ensemble model was developed based on this feature set using XG-Boost, Ada-Boost, Random Forest and a Multilayer Perceptron. It was possible to successfully predict SAV transmission with 94.4% accuracy. Moreover, it was possible to predict SAV transmission 8 weeks in advance of a 'PD registration' at individual aquaculture salmon farming sites. Important predictors included well boat movement, environmental factors, proximity to sites with a 'PD registration' and seasonality.

Seroconversion of anti-Getah virus antibody among Japanese native Noma horses around 2012.

Getah virus (GETV), an arthropod-borne virus transmitted by mosquitoes, has been isolated from several animals. GETV infection in horses shows clinical signs such as fever, rash, and edema in the leg. Noma horses are one of the eight Japanese native horses. The present study aimed to clarify the occurrence of GETV infection in Noma horses. Serum samples collected from Noma horses were analyzed using a virus neutralization test and enzyme-linked immunosorbent assay and showed that the anti-GETV antibody titers in the samples collected in 2017 were significantly higher than those collected in 2012. We concluded that a seroconversion of anti-GETV antibodies was occurred in the Noma horse population around 2012, providing evidence of the GETV epidemic in Japan circa 2012.

Salmonid alphavirus non-structural protein 2 is a key protein that activates the NF-κB signaling pathway to mediate inflammatory responses.

Salmonid alphavirus (SAV) infection of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) causes pancreas disease (PD) with typical inflammatory responses, such as necrosis of the exocrine pancreas, cardiomyopathy and skeletal myopathy. However, the pathogenic mechanism underlying SAV infection is still unclear. Inflammation may cause damage to the body, but it is a defense response against infection by pathogenic microorganisms, of which nuclear factor-kappa B (NF-κB) is the main regulator. This study revealed that SAV can activate NF-κB, of which the viral nonstructural protein Nsp2 is the major activating protein. SAV activates the NF-κB signaling pathway by simultaneously up-regulating TLR3, 7, 8 and then the expression of the signaling molecule myeloid differentiation factor 88 (Myd88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). We found that Nsp2 can induce IκB degradation and p65 phosphorylation and transnucleation, and activate NF-κB downstream inflammatory cytokines. Nsp2 may simultaneously activate NF-κB through TLR3,7,8-dependent signaling pathways. Overexpression of Nsp2 can up-regulate mitochondrial antiviral signaling protein (MAVS) and then promote the expression of IFNa1 and antiviral protein Mx, which inhibits viral replication. This study shows that Nsp2 acts as a key activator protein for the NF-κB signaling pathway, which induces inflammation post-SAV infection. This study systematically analyzes the molecular mechanism of SAV activation of the NF-κB signaling pathway, and provides a theoretical basis for revealing the mechanism of innate immune response and inflammatory injury caused by SAV.

Epidemiological Study of Multiple Zoonotic Mosquito-Borne Alphaviruses in Horses in Queensland, Australia (2018-2020).

The increased frequency of extreme weather events due to climate change has complicated the epidemiological pattern of mosquito-borne diseases, as the host and vector dynamics shift to adapt. However, little is known about the seroprevalence of common mosquito-borne virus infections in horses in Australia. In this study, serological surveys for multiple alphaviruses were performed on samples taken from 622 horses across two horse populations (racehorses and horses residing on The University of Queensland (UQ) campus) in Queensland using the gold standard virus neutralization test. As is the case in humans across Australia, Ross River virus (RRV) is the most common arbovirus infection in horses, followed by Barmah Forest virus, with an overall apparent seroprevalence of 48.6% (302/622) and 4.3% (26/607), respectively. Horses aged over 6 years old (OR 1.86, p = 0.01) and residing at UQ (OR 5.8, p < 0.001) were significantly associated with seroconversion to RRV. A significant medium correlation (r = 0.626, p < 0.001) between RRV and Getah virus (GETV) neutralizing antibody titers was identified. Collectively, these results advance the current epidemiological knowledge of arbovirus exposure in a susceptible host in Australia. The potential use of horses as sentinels for arbovirus monitoring should be considered. Furthermore, since GETV is currently exotic to Australia, antibodies cross-reactivity between RRV and GETV should be further investigated for cross-protection, which may also help to inform vaccine developments.

Epidemiological and Genomic Characterisation of Middelburg and Sindbis Alphaviruses Identified in Horses with Febrile and Neurological Infections, South Africa (2014-2018).

Although Old World alphaviruses, Middelburg- (MIDV) and Sindbis virus (SINV), have previously been detected in horses and wildlife with neurologic disease in South Africa, the pathogenesis and clinical presentation of MIDV and SINV infections in animals are not well documented. Clinical samples from horses across South Africa with acute or fatal neurologic and febrile infections submitted between 2014-2018 were investigated. In total, 69/1084 (6.36%) and 11/1084 (1.01%) horses tested positive for MIDV and SINV, respectively, by real-time reverse transcription (RT) PCR. Main signs/outcomes for MIDV (n = 69): 73.91% neurological, 75.36% fever, 28.99% icterus and anorexia, respectively, 8.70% fatalities; SINV (n = 11): 54.54% neurological, 72.73% fever, 36.36% anorexia and 18.18% fatalities. MIDV cases peaked in the late summer/autumn across most South African provinces while SINV cases did not show a clear seasonality and were detected in fewer South African provinces. MIDV could still be detected in blood samples via RT-PCR for up to 71,417 and 21 days after onset of signs in 4 horses respectively, suggesting prolonged replication relative to SINV which could only be detected in the initial sample. Phylogenetic analyses based on partial sequences of the nsP4 (MIDV n = 59 and SINV n = 7) and E1 (MIDV n = 45) genes, as well as full genome sequences (MIDV n = 6), clustered the MIDV and SINV strains from the present study with previously detected strains. MIDV infection appears to be more prevalent in horses than SINV infection based on RT-PCR results, however, prevalence estimates might be different when also considering serological surveillance data.

First confirmation of salmonid alphavirus infection in rainbow trout in Bosnia and Herzegovina and Serbia.

Sleeping disease is a highly infectious viral disease caused by salmonid alphavirus subtype 2 (SAV2 FW), affecting mainly rainbow trout Oncorhynchus mykiss farmed in freshwater. During March to May 2014, disease episodes with clinical signs of sleeping disease in rainbow trout fingerlings occurred almost simultaneously in 2 trout farms located in Bosnia and Herzegovina (BiH) and Serbia. The infection of rainbow trout with SAV2 FW in 2 farms was confirmed by virus isolation and molecular methods. This is the first isolation and molecular characterization of SAV2 FW in BiH and Serbia.

Alphavirus Identification in Neotropical Bats.

Alphaviruses (Togaviridae) are arthropod-borne viruses responsible for several emerging diseases, maintained in nature through transmission between hematophagous arthropod vectors and susceptible vertebrate hosts. Although bats harbor many species of viruses, their role as reservoir hosts in emergent zoonoses has been verified only in a few cases. With bats being the second most diverse order of mammals, their implication in arbovirus infections needs to be elucidated. Reports on arbovirus infections in bats are scarce, especially in South American indigenous species. In this work, we report the genomic detection and identification of two different alphaviruses in oral swabs from bats captured in Northern Uruguay. Phylogenetic analysis identified Río Negro virus (RNV) in two different species: Tadarida brasiliensis (n = 6) and Myotis spp. (n = 1) and eastern equine encephalitis virus (EEEV) in Myotis spp. (n = 2). Previous studies of our group identified RNV and EEEV in mosquitoes and horse serology, suggesting that they may be circulating in enzootic cycles in our country. Our findings reveal that bats can be infected by these arboviruses and that chiropterans could participate in the viral natural cycle as virus amplifiers or dead-end hosts. Further studies are warranted to elucidate the role of these mammals in the biological cycle of these alphaviruses in Uruguay.

Structural Insights into Alphavirus Assembly Revealed by the Cryo-EM Structure of Getah Virus.

Getah virus (GETV) is a member of the alphavirus genus, and it infects a variety of animal species, including horses, pigs, cattle, and foxes. Human infection with this virus has also been reported. The structure of GETV has not yet been determined. In this study, we report the cryo-EM structure of GETV at a resolution of 3.5 Å. This structure reveals conformational polymorphism of the envelope glycoproteins E1 and E2 at icosahedral 3-fold and quasi-3-fold axes, which is believed to be a necessary organization in forming a curvature surface of virions. In our density map, three extra densities are identified, one of which is believed a "pocket factor"; the other two are located by domain D of E2, and they may maintain the stability of E1/E2 heterodimers. We also identify three N-glycosylations at E1 N141, E2 N200, and E2 N262, which might be associated with receptor binding and membrane fusion. The resolving of the structure of GETV provides new insights into the structure and assembly of alphaviruses and lays a basis for studying the differences of biology and pathogenicity between arthritogenic and encephalitic alphaviruses.

Serological Positivity against Selected Flaviviruses and Alphaviruses in Free-Ranging Bats and Birds from Costa Rica Evidence Exposure to Arboviruses Seldom Reported Locally in Humans.

Arboviruses have two ecological transmission cycles: sylvatic and urban. For some, the sylvatic cycle has not been thoroughly described in America. To study the role of wildlife in a putative sylvatic cycle, we sampled free-ranging bats and birds in two arbovirus endemic locations and analyzed them using molecular, serological, and histological methods. No current infection was detected, and no significant arbovirus-associated histological changes were observed. Neutralizing antibodies were detected against selected arboviruses. In bats, positivity in 34.95% for DENV-1, 16.26% for DENV-2, 5.69% for DENV-3, 4.87% for DENV-4, 2.43% for WNV, 4.87% for SLEV, 0.81% for YFV, 7.31% for EEEV, and 0.81% for VEEV was found. Antibodies against ZIKV were not detected. In birds, PRNT results were positive against WNV in 0.80%, SLEV in 5.64%, EEEV in 8.4%, and VEEV in 5.63%. An additional retrospective PRNT analysis was performed using bat samples from three additional DENV endemic sites resulting in a 3.27% prevalence for WNV and 1.63% for SLEV. Interestingly, one sample resulted unequivocally WNV positive confirmed by serum titration. These results suggest that free-ranging bats and birds are exposed to not currently reported hyperendemic-human infecting Flavivirus and Alphavirus; however, their role as reservoirs or hosts is still undetermined.

Development and Evaluation of DNA Vaccine Against Salmonid Alphavirus.

Despite vaccination, pancreas disease (PD) caused by salmonid alphavirus (SAV) has been the economically most important virus disease in salmon farming in Ireland, Scotland, and Norway. A vaccine based on DNA plasmid has been authorized to be used in Norwegian aquaculture since 2018. DNA vaccination of plasmids expressed subcellular viral proteins have been shown its particular protective effect against SAV3 that surface expression of the E2 protein with the whole viral protein construct, yielding a more effective vaccine. The chapter describes methods to design and test the sublocalization of expressed viral protein and the performance evaluation of vaccines against SAV3 infection in Atlantic salmon.

A systematic review and meta-analysis of the potential non-human animal reservoirs and arthropod vectors of the Mayaro virus.

Improving our understanding of Mayaro virus (MAYV) ecology is critical to guide surveillance and risk assessment. We conducted a PRISMA-adherent systematic review of the published and grey literature to identify potential arthropod vectors and non-human animal reservoirs of MAYV. We searched PubMed/MEDLINE, Embase, Web of Science, SciELO and grey-literature sources including PAHO databases and dissertation repositories. Studies were included if they assessed MAYV virological/immunological measured occurrence in field-caught, domestic, or sentinel animals or in field-caught arthropods. We conducted an animal seroprevalence meta-analysis using a random effects model. We compiled granular georeferenced maps of non-human MAYV occurrence and graded the quality of the studies using a customized framework. Overall, 57 studies were eligible out of 1523 screened, published between the years 1961 and 2020. Seventeen studies reported MAYV positivity in wild mammals, birds, or reptiles and five studies reported MAYV positivity in domestic animals. MAYV positivity was reported in 12 orders of wild-caught vertebrates, most frequently in the orders Charadriiformes and Primate. Sixteen studies detected MAYV in wild-caught mosquito genera including Haemagogus, Aedes, Culex, Psorophora, Coquillettidia, and Sabethes. Vertebrate animals or arthropods with MAYV were detected in Brazil, Panama, Peru, French Guiana, Colombia, Trinidad, Venezuela, Argentina, and Paraguay. Among non-human vertebrates, the Primate order had the highest pooled seroprevalence at 13.1% (95% CI: 4.3-25.1%). From the three most studied primate genera we found the highest seroprevalence was in Alouatta (32.2%, 95% CI: 0.0-79.2%), followed by Callithrix (17.8%, 95% CI: 8.6-28.5%), and Cebus/Sapajus (3.7%, 95% CI: 0.0-11.1%). We further found that MAYV occurs in a wide range of vectors beyond Haemagogus spp. The quality of evidence behind these findings was variable and prompts calls for standardization of reporting of arbovirus occurrence. These findings support further risk emergence prediction, guide field surveillance efforts, and prompt further in-vivo studies to better define the ecological drivers of MAYV maintenance and potential for emergence.

Genomic Epidemiology of Salmonid Alphavirus in Norwegian Aquaculture Reveals Recent Subtype-2 Transmission Dynamics and Novel Subtype-3 Lineages.

Viral disease poses a major barrier to sustainable aquaculture, with outbreaks causing large economic losses and growing concerns for fish welfare. Genomic epidemiology can support disease control by providing rapid inferences on viral evolution and disease transmission. In this study, genomic epidemiology was used to investigate salmonid alphavirus (SAV), the causative agent of pancreas disease (PD) in Atlantic salmon. Our aim was to reconstruct SAV subtype-2 (SAV2) diversity and transmission dynamics in recent Norwegian aquaculture, including the origin of SAV2 in regions where this subtype is not tolerated under current legislation. Using nanopore sequencing, we captured ~90% of the SAV2 genome for n = 68 field isolates from 10 aquaculture production regions sampled between 2018 and 2020. Using time-calibrated phylogenetics, we infer that, following its introduction to Norway around 2010, SAV2 split into two clades (SAV2a and 2b) around 2013. While co-present at the same sites near the boundary of Møre og Romsdal and Trøndelag, SAV2a and 2b were generally detected in non-overlapping locations at more Southern and Northern latitudes, respectively. We provide evidence for recent SAV2 transmission over large distances, revealing a strong connection between Møre og Romsdal and SAV2 detected in 2019/20 in Rogaland. We also demonstrate separate introductions of SAV2a and 2b outside the SAV2 zone in Sognefjorden (Vestland), connected to samples from Møre og Romsdal and Trøndelag, respectively, and a likely 100 km Northward transmission of SAV2b within Trøndelag. Finally, we recovered genomes of SAV2a and SAV3 co-infecting single fish in Rogaland, involving novel SAV3 lineages that diverged from previously characterized strains >25 years ago. Overall, this study demonstrates useful applications of genomic epidemiology for tracking viral disease spread in aquaculture.

Realtime case study simulations of transmission of Pancreas Disease (PD) in Norwegian salmonid farming for disease control purposes.

Pancreas Disease (PD) is a viral disease caused by Salmonid Alphavirus (SAV). It affects farmed salmonids in the North Atlantic, and leads to reduced feed intake and increased mortality with reduced production and welfare as a consequence. In 2013, the estimated cost of an outbreak on an average salmon farm was about 6.6 mil €. In Norway, PD has been notifiable since 2008, and regulations to mitigate disease spread are in place. However, despite the regulations, 140-170 farms are affected by PD every year. The aquaculture industry is growing continuously, introducing farms in new geographical areas, and fish are moved between hydrographically separated zones for trade and slaughter. All such movements and relocations need to be approved by the competent authorities. Thus, there is a demand for support to farmers and competent authorities when making decisions on disease management and especially on the effect of moving infected fish. We have used a disease-transmission model for outbreak-simulation in real time for assessing the probability of disease transmission from a farm that gets infected with PD. We have also simulated the effects of three different control-regimes: no stamping-out, delayed stamping-out or immediate stamping-out, on the transmission of PD to surrounding farms. Simulations showed that the immediate stamping out of an infected farm led to effective containment of an outbreak. No stamping out led to up to 32.1% of farms within 100 km of the index farm to become effected. We have used real production data for the model building and the scenario simulations, and the results illustrate that a risk assessment of horizontal disease transmission must be undertaken on a case-by-case basis, because the time and place of the outbreak has a large influence on the risk of transmission.

Development of a reverse transcription recombinase-aided amplification assay for detection of Getah virus.

GETV, an arbo-borne zoonotic virus of the genus Alphavirus, which causes diarrhea and reproduction disorders in swine, lead to serious economic losses to the swine industry in China. At present, the existing methods for GETV detection are time-consuming and low sensitivity, so, a rapid, accurate and sensitive GETV detection method is urgently needed. In this study, a fluorescent reverse transcription recombinase-assisted amplification method (RT-RAA) was successfully established for the rapid detection of GETV. The sensitivity of this method to GETV was 8 copies/reaction and 20 TCID50/reaction. No cross-reaction with other viruses. A total of 118 samples were prepared for GETV detection using fluorescent RT-RAA and SYBR Green I RT-qPCR, the coincidence rate of the two methods was 100%. The results suggest that the RT-RAA method is rapid, sensitive and specific for GETV detection and can be applied in the clinical.

Emergence of Salmonid Alphavirus Genotype 2 in Norway-Molecular Characterization of Viral Strains Circulating in Norway and Scotland.

Pancreas disease (PD) and sleeping disease (SD), caused by an alphavirus, are endemic in European salmonid aquaculture, causing significant mortality, reduced growth and poor flesh quality. In 2010, a new variant of salmonid alphavirus emerged in Norway, marine salmonid alphavirus genotype 2 (SAV2). As this genotype is highly prevalent in Scotland, transmission through well boat traffic was hypothesized as one possible source of infection. In this study, we performed full-length genome sequencing of SAV2 sampled between 2006 and 2012 in Norway and Scotland, and present the first comprehensive full-length characterization of Norwegian marine SAV2 strains. We analyze their relationship with selected Scottish SAV2 strains and explore the genetic diversity of SAV. Our results show that all Norwegian marine SAV2 share a recent last common ancestor with marine SAV2 circulating in Scotland and a higher level of genomic diversity among the Scottish marine SAV2 strains compared to strains from Norway. These findings support the hypothesis of a single introduction of SAV2 to Norway sometime from 2006-2010, followed by horizontal spread along the coast.

Effect of vaccines against pancreas disease in farmed Atlantic salmon.

Pancreas disease (PD) caused by salmonid alphavirus (SAV) continues to negatively impact salmon farming. To assess the effect on growth and mortality of three vaccines against PD, two controlled field designs were employed: one controlled field study with individual marked fish (PIT tag) assessing three PD vaccines and three controls groups, and a second controlled field study with group marked fish (Maxilla) comparing two PD vaccines against controls. In addition, a descriptive study using whole cages compared fish immunized with two different PD vaccines against controls. The target populations experienced a natural PD outbreak where both SAV 2 and SAV 3 were identified. Only one of the PD vaccines provided statistically significant improvements in harvest weight of 0.43 kg (CI: 0.29-0.57) and 0.51 kg (CI: 0.36-0.65) compared with the control in the PIT tag and the Maxilla study, respectively. In the latter, a significant reduction in mortality of 1.31 (CI:0.8-1.8) per cent points was registered for the same vaccine compared with controls. These results aligned with the growth and PD-specific mortality registered in the descriptive Cage study. The data in this study show a difference in the efficacy of PD vaccines in farmed Atlantic salmon.

Ultrastructural insights into the replication cycle of salmon pancreas disease virus (SPDV) using salmon cardiac primary cultures (SCPCs).

Salmon pancreas disease virus (SPDV) has been affecting the salmon farming industry for over 30 years, but despite the substantial amount of studies, there are still a number of recognized knowledge gaps, for example in the transmission of the virus. In this work, an ultrastructural morphological approach was used to describe observations after infection by SPDV of an ex vivo cardiac model generated from Atlantic salmon embryos. The observations in this study and those available on previous ultrastructural work on SPDV are compared and contrasted with the current knowledge on terrestrial mammalian and insect alphaviral replication cycles, which is deeper than that of SPDV both morphologically and mechanistically. Despite their limitations, morphological descriptions remain an excellent way to generate novel hypotheses, and this has been the aim of this work. This study has used a target host, ex vivo model and resulted in some previously undescribed features, including filopodial membrane projections, cytoplasmic stress granules or putative intracytoplasmic budding. The latter suggests a new hypothesis that warrants further mechanistic research: SPDV in salmon may have retained the capacity for non-cytolytic (persistent) infections by intracellular budding, similar to that noted in arthropod vectors of other alphaviruses. In the notable absence of a known intermediate host for SPDV, the presence of this pattern suggests that both cytopathic and persistent infections may coexist in the same host. It is our hope that the ultrastructural comparison presented here stimulates new research that brings the knowledge on SPDV replication cycle up to a similar level to that of terrestrial alphaviruses.

Effect of two constant light regimens on antibody profiles and immune gene expression in Atlantic salmon following vaccination and experimental challenge with salmonid alphavirus.

Before seawater transfer, farmed Atlantic salmon are subjected to treatments that may affect the immune system and susceptibility to pathogens. E.g., exposure to constant light (CL) stimulates smoltification, which prepares salmon to life in sea water, but endocrine changes in this period are associated with suppression of immune genes. Salmon are vaccinated towards end of the freshwater period to safeguard that adequate vaccine efficacy is achieved by the time the fish is transferred to sea. In the present study, we investigated how the responses to vaccination and viral infection varied depending on the time of CL onset relative to vaccination. The salmon were either exposed to CL two weeks prior to vaccination (2-PRI) or exposed to CL at the time of vaccination (0-PRI). A cohabitant challenge with salmonid alphavirus, the causative agent of pancreatic disease, was performed 9 weeks post vaccination. The immunological effects of the different light manipulation were examined at 0- and 6-weeks post vaccination, and 6 weeks post challenge. Antibody levels in serum were measured using a serological bead-based multiplex panel as well as ELISA, and 92 immune genes in heart and spleen were measured using an integrated fluidic circuit-based qPCR array for multiple gene expression. The 2-PRI group showed a moderate transcript down-regulation of genes in the heart at the time of vaccination, which were restored 6 weeks after vaccination (WPV). Conversely, at 6WPV a down-regulation was seen for the 0-PRI fish. Moreover, the 2-PRI group had significantly higher levels of antibodies binding to three of the vaccine components at 6WPV, compared to 0-PRI. In response to SAV challenge, transcription of immune genes between 2-PRI and 0-PRI was markedly dissimilar in the heart and spleen of control fish, but no difference was found between vaccinated salmon from the two CL regimens. Thus, by using labor-saving high throughput detection methods, we demonstrated that light regimens affected antibody production and transcription of immune genes in non-vaccinated and virus challenged salmon, but the differences between the light treatment groups appeared eliminated by vaccination.

Filtration, concentration and detection of salmonid alphavirus in seawater during a post-smolt salmon (Salmo salar) cohabitant challenge.

Currently, the prevalence of salmonid alphavirus (SAV) in Norwegian Atlantic salmon farms is largely surveyed via sacrificing fish and sampling of organ tissue on a monthly basis. However, a more cost-efficient, straightforward, rapid, reliable, reproducible and animal welfare friendly method based on the detection of SAV in water could be considered as an alternative method. In the present study, such a method was developed and optimized through a 6 wk cohabitant challenge trial, using post-smolt Atlantic salmon Salmo salar L challenged with high or low doses of SAV subtype 3 (SAV3). Tank water and tissue samples from cohabitant fish were collected at 16 time points. SAV3 was concentrated from the water by filtration, using either electronegative or electropositive membrane filters, which were subsequently rinsed with one of 4 different buffer solutions. SAV3 was detected first in tank water (7 d post-challenge, DPC), and later in cohabitant fish organ tissue samples (12 DPC). The electronegative filter (MF-Millipore™) and rinsing with NucliSENS® easyMAG® Lysis Buffer presented the best SAV3 recovery. A significant positive correlation was found between SAV3 in the tank water concentrates and the mid-kidney samples. Based on these results, detection of SAV3 in filtrated seawater is believed to have the potential to serve as an alternative method for surveillance of SAV in Atlantic salmon farms.