Pathogenesis of experimental salmonid alphavirus infection in vivo: an ultrastructural insight.
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ABSTRACT:
SUBMITTER: Herath TK
PROVIDER: S-EPMC4705579 | biostudies-literature | 2016 Jan
REPOSITORIES: biostudies-literature
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Project description:MicroRNAs (miRNAs) control multiple biological processes including the innate immune responses by negative post-transcriptional regulation of gene expression. As there were no studies on the role(s) of miRNAs in viral diseases in Atlantic salmon, we aimed to identify miRNAs responding to salmonid alphavirus (SAV) infection. Their expression were studied at different time points post infection with SAV isolates associated with different mortalities. Furthermore, the genome sequences of the identified miRNAs were analysed to reveal putative cis-regulatory elements, and, finally, their putative target genes were predicted.Twenty differentially expressed miRNAs (DE miRNAs) were identified. The expression of the majority of these increased post infection with maximum levels reached after the viral load were stabilized or decreasing. On the other hand, some miRNAs (e.g. the miRNA-21 family) showed decreased expression at the early time points post infection. There were significant differences in the temporal expression of individual miRNA associated with different SAV isolates. Target gene prediction in SAV responsive immune network genes showed that seventeen of the DE miRNAs could target 24 genes (e.g. IRF3, IRF7). Applying the Atlantic salmon transcriptome as input 28 more immune network genes were revealed as putative targets (e.g. IRF5, IRF4). The majority of the predicted target genes promote inflammatory response. The upstream sequences of the miRNA genes revealed a high density of cis-regulatory sequences known as binding sites for immune network transcription factors (TFs). A high expression in the late phase could therefore be due to increased transcription promoted by immune response activated TFs. Based on the in silico target predictions, we discuss their putative roles as early promotors or late inhibitors of inflammation. We propose that the differences in expressions associated with different SAV isolates could contribute to their differences in mortality rates.This study represents the first steps in exploring miRNAs important in viral-host interaction in Atlantic salmon. We identified several miRNAs responding to SAV infection. Some likely to prohibit harmful inflammation while other may promote an early immune response. Their predicted functions need to be validated and further studied in functional assays to fully understand their roles in immune homeostasis.
| S-EPMC5418855 | biostudies-literature
Project description:Interactions among host, microbiota and viral pathogens are complex and poorly understood. The goal of the present study is to assess the changes in the skin microbial community of Atlantic salmon (Salmo salar L.) in response to experimental infection with salmonid alphavirus (SAV). The salmon skin microbial community was determined using 16S rDNA pyrosequencing in five different experimental groups: control, 7 days after infection with low-dose SAV, 14 days after infection with low-dose SAV, 7 days after infection with high-dose SAV, and 14 days after infection with high-dose SAV. Both infection treatment and time after infection were strong predictors of the skin microbial community composition. Skin samples from SAV3 infected fish showed an unbalanced microbiota characterized by a decreased abundance of Proteobacteria such as Oleispira sp. and increased abundances of opportunistic taxa including Flavobacteriaceae, Streptococcaceae and Tenacibaculum sp. These results demonstrate that viral infections can result in skin dysbiosis likely rendering the host more susceptible to secondary bacterial infections.
| S-EPMC5338768 | biostudies-literature
Project description:Salmonid alphavirus subtype-3 (SAV-3) infection in Atlantic salmon is exclusively found in Norway. The salmonid alphaviruses have been well characterized at the genome level but there is limited information about the host-pathogen interaction phenomena. This study was undertaken to characterize the replication and spread of SAV-3 in internal organs of experimentally infected Atlantic salmon and the subsequent innate and adaptive immune responses. In addition, suitability of a cohabitation challenge model for this virus was also examined. Groups of fish were infected by intramuscular injection (IM), cohabited (CO) or kept uninfected in a separate tank. Samples of pancreas, kidney, spleen, heart and skeletal muscles were collected at 2, 4 and 8 weeks post infection (wpi). Pathological changes were assessed by histology concurrently with viral loads and mRNA expression of immune genes by real time RT-PCR. Pathological changes were only observed in the pancreas and heart (target organs) of both IM and CO groups, with changes appearing first in the pancreas (2 wpi) in the former. Lesions with increasing severity over time coincided with high viral loads despite significant induction of IFN-α, Mx and ISG15. IFN-γ and MHC-I were expressed in all tissues examined and their induction appeared in parallel with that of IL-10. Inflammatory genes TNF-α, IL-12 and IL-8 were only induced in the heart during pathology while T cell-related genes CD3ε, CD4, CD8, TCR-α and MHC-II were expressed in target organs at 8 wpi. These findings suggest that the onset of innate responses came too late to limit virus replication. Furthermore, SAV-3 infections in Atlantic salmon induce Th1/cytotoxic responses in common with other alphaviruses infecting higher vertebrates. Our findings demonstrate that SAV-3 can be transmitted via the water making it suitable for a cohabitation challenge model.
| S-EPMC3505155 | biostudies-literature
Project description:With increasing interest in the use of triploid salmon in commercial aquaculture, gaining an understanding of how economically important pathogens affect triploid stocks is important. To compare the susceptibility of diploid and triploid Atlantic salmon (Salmo salar L.) to viral pathogens, fry were experimentally infected with Salmonid alphavirus sub-type 1 (SAV1), the aetiological agent of pancreas disease (PD) affecting Atlantic salmon aquaculture in Europe. Three groups of fry were exposed to the virus via different routes of infection: intraperitoneal injection (IP), bath immersion, or cohabitation (co-hab) and untreated fry were used as a control group. Mortalities commenced in the co-hab challenged diploid and triploid fish from 11 days post infection (dpi), and the experiment was terminated at 17 dpi. Both diploid and triploid IP challenged groups had similar levels of cumulative mortality at the end of the experimental period (41.1% and 38.9% respectively), and these were significantly higher (p < 0.01) than for the other challenge routes. A TaqMan-based quantitative PCR was used to assess SAV load in the heart, a main target organ of the virus, and also liver, which does not normally display any pathological changes during clinical infections, but exhibited severe degenerative lesions in the present study. The median viral RNA copy number was higher in diploid fish compared to triploid fish in both the heart and the liver of all three challenged groups. However, a significant statistical difference (p < 0.05) was only apparent in the liver of the co-hab groups. Diploid fry also displayed significantly higher levels of pancreatic and myocardial degeneration than triploids. This study showed that both diploid and triploid fry are susceptible to experimental SAV1 infection. The lower virus load seen in the triploids compared to the diploids may possibly be related to differences in cell metabolism between the two groups, however, further investigation is necessary to confirm this and also to assess the outcome of PD outbreaks in other developmental stages of the fish when maintained in commercial production systems.
| S-EPMC5614425 | biostudies-literature
Project description:For more than three decades, diseases caused by salmonid alphaviruses (SAV) have become a major problem of increasing economic importance in the European fish-farming industry. However, experimental infection trials with SAV result in low or no mortality i.e very different from most field outbreaks of pancreas disease (PD). This probably reflects the difficulties in reproducing complex biotic and abiotic field conditions in the laboratory. In this study we looked at the relationship between SAV-infection in salmon and sub-lethal environmental hypoxia as a result of reduced flow-through in tank systems.The experiment demonstrated that constant reduced oxygen levels (60-65% oxygen saturation: 6.5-7.0 mg/L) did not significantly increase the severity or the progress of pancreas disease (PD). These conclusions are based upon assessments of a semi-quantitative histopathological lesion score system, morbidities/mortalities, and levels of SAV RNA in tissues and water (measured by 1 MDS electropositive virus filters and downstream real-time RT-PCR). Furthermore, we demonstrate that the fish population shed detectable levels of the virus into the surrounding water during viraemia; 4-13 days after i.p. infection, and prior to appearance of severe lesions in heart (21-35 dpi). After this period, viral RNA from SAV could not be detected in water samples although still present in tissues (gills and hearts) at lasting low levels. Lesions could be seen in exocrine pancreas at 7-21 days post infection, but no muscle lesions were seen.In our study, experimentally induced hypoxia failed to explain the discrepancy between the severities reported from field outbreaks of SAV-disease and experimental infections. Reduction of oxygen levels to constant suboptimal levels had no effect on the severity of lesions caused by SAV-infection or the progress of the disease. Furthermore, we present a modified VIRADEL method which can be used to detect virus in water and to supplement experimental infection trials with information related to viral shedding. By using this method, we were able to demonstrate for the first time that shedding of SAV from the fish population into the surrounding water coincides with viraemia.
| S-EPMC2936311 | biostudies-literature
Project description:BackgroundPancreas disease (PD) is a viral fish disease which in recent years has significantly affected Norwegian salmonid aquaculture. In Norway, the aetiological agent salmonid alphavirus (SAV) has been found to be represented by the subtype 3 only. SAV subtype 3 has in previous analyses been found to show a lower genetic divergence than the subtypes found to cause PD in Ireland and Scotland. The aim of this study was to evaluate the nucleotide (nt) and amino acid divergence and the phylogenetic relationship of 33 recent SAV subtype 3 sequences. The samples from which the sequences were obtained originated from both PD endemic and non-endemic regions in an attempt to investigate agent origin/spread. Multiple samples throughout the seawater production phase from several salmonid populations were included to investigate genetic variation during an outbreak. The analyses were mainly based on partial sequences from the E2 gene. For some samples, additional partial 6 K and nsP3 gene sequences were available.ResultsThe nucleotide divergence for all gene fragments ranged from total identity (0.0% divergence) to 0.45% (1103 nt fragment of E2), 1.11% (451 nt fragment of E2), 0.94% (6 K) and 0.28% (nsP3). This low nucleotide divergence corresponded well to previous reports on SAV 3 sequences; however the observed divergence for the short E2 fragment was higher than that previously reported. When compared to SAVH20/03 (AY604235), amino acid substitutions were detected in all assessed gene fragments however the in vivo significance of these on for example disease outbreak mortality could not be concluded on. The phylogenetic tree based on the 451 nt E2 fragment showed that the sequences divided into two clusters with low genetic divergence, representing only a single SAV subtype.ConclusionsThe analysed sequences represented two clusters of a single SAV subtype; however some of the observed sequence divergence was higher than that previously reported by other researchers. Larger scale, full length sequence analyses should be instigated to allow further phylogenetic and molecular epidemiology investigations of SAV subtype 3.
| S-EPMC2925375 | biostudies-literature
Project description:BackgroundSalmonid alphavirus (SAV) is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed.ResultsInfectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF) encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2197, nsP3263 and nsP3323 severely reduced infectivity, a serine to proline mutation in E2206 appeared to enhance the virus titer production.ConclusionWe have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.
| S-EPMC2949663 | biostudies-literature
Project description:The microbial communities that live in symbiosis with the mucosal surfaces of animals provide the host with defense strategies against pathogens. These microbial communities are largely shaped by the environment and the host genetics. Triploid Atlantic salmon (Salmo salar) are being considered for aquaculture as they are reproductively sterile and thus cannot contaminate the natural gene pool. It has not been previously investigated how the microbiome of triploid salmon compares to that of their diploid counterparts. In this study, we compare the steady-state skin and gill microbiome of both diploid and triploid salmon, and determine the effects of salmonid alphavirus 3 experimental infection on their microbial composition. Our results show limited differences in the skin-associated microbiome between triploid and diploid salmon, irrespective of infection. In the gills, we observed a high incidence of the bacterial pathogen Candidatus Branchiomonas, with higher abundance in diploid compared to triploid control fish. Diploid salmon infected with SAV3 showed greater histopathological signs of epitheliocystis compared to controls, a phenomenon not observed in triploid fish. Our results indicate that ploidy can affect the alpha diversity of the gills but not the skin-associated microbial community. Importantly, during a natural outbreak of Branchiomonas sp. the gill microbiome of diploid Atlantic salmon became significantly more dominated by this pathogen than in triploid animals. Thus, our results suggest that ploidy may play a role on Atlantic salmon gill health and provide insights into co-infection with SAV3 and C. Branchiomonas in Atlantic salmon.
| S-EPMC7894865 | biostudies-literature
Project description:Alphaviruses, members of the positive-sense, single-stranded RNA virus family Togaviridae, represent a re-emerging public health concern worldwide as mosquito vectors expand into new geographic ranges. Members of the alphavirus genus tend to induce clinical disease characterized by rash, arthralgia, and arthritis (chikungunya virus, Ross River virus, and Semliki Forest virus) or encephalomyelitis (eastern equine encephalitis virus, western equine encephalitis virus, and Venezuelan equine encephalitis virus), though some patients who recover from the initial acute illness may develop long-term sequelae, regardless of the specific infecting virus. Studies examining the natural disease course in humans and experimental infection in cell culture and animal models reveal that host genetics play a major role in influencing susceptibility to infection and severity of clinical disease. Genome-wide genetic screens, including loss of function screens, microarrays, RNA-sequencing, and candidate gene studies, have further elucidated the role host genetics play in the response to virus infection, with the immune response being found in particular to majorly influence the outcome. This review describes the current knowledge of the mechanisms by which host genetic factors influence alphavirus pathogenesis and discusses emerging technologies that are poised to increase our understanding of the complex interplay between viral and host genetics on disease susceptibility and clinical outcome.
| S-EPMC6488303 | biostudies-literature
Project description:B cell responses are a crucial part of the adaptive immune response to viral infection. Infection by salmonid alphavirus subtype 3 (SAV3) causes pancreas disease (PD) in Atlantic salmon (Salmo salar) and is a serious concern to the aquaculture industry. In this study, we have used intraperitoneal (IP) infection with SAV3 as a model to characterize local B cell responses in the peritoneal cavity (PerC) and systemic immune tissues (head kidney/spleen). Intraperitoneal administration of vaccines is common in Atlantic salmon and understanding more about the local PerC B cell response is fundamental. Intraperitoneal SAV3 infection clearly induced PerC B cell responses as assessed by increased frequency of IgM+ B cells and total IgM secreting cells (ASC). These PerC responses were prolonged up to nine weeks post-infection and positively correlated to the anti-SAV3 E2 and to neutralizing antibody responses in serum. For the systemic immune sites, virus-induced changes in B cell responses were more modest or decreased compared to controls in the same period. Collectively, data reported herein indicated that PerC could serve as a peripheral immunological site by providing a niche for prolonged maintenance of the ASC response in Atlantic salmon.
| S-EPMC7511533 | biostudies-literature