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Alpha interferon and not gamma interferon inhibits salmonid alphavirus subtype 3 replication in vitro.

ABSTRACT: Salmonid alphavirus (SAV) is an emerging virus in salmonid aquaculture, with SAV-3 being the only subtype found in Norway. Until now, there has been little focus on the alpha interferon (IFN-alpha)-induced antiviral responses during virus infection in vivo or in vitro in fish. The possible involvement of IFN-gamma in the response to SAV-3 is also not known. In this study, the two IFNs were cloned and expressed as recombinant proteins (recombinant IFN-alpha [rIFN-alpha] and rIFN-gamma) and used for in vitro studies. SAV-3 infection in a permissive salmon cell line (TO cells) results in IFN-alpha and IFN-stimulated gene (ISG) mRNA upregulation. Preinfection treatment (4 to 24 h prior to infection) with salmon rIFN-alpha induces an antiviral state that inhibits the replication of SAV-3 and protects the cells against virus-induced cytopathic effects (CPE). The antiviral state coincides with a strong expression of Mx and ISG15 mRNA and Mx protein expression. When rIFN-alpha is administered at the time of infection and up to 24 h postinfection, virus replication is not inhibited, and cells are not protected against virus-induced CPE. By 40 h postinfection, the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) is phosphorylated concomitant with the expression of the E2 protein as assessed by Western blotting. Postinfection treatment with rIFN-alpha results in a moderate reduction in E2 expression levels in accordance with a moderate downregulation of cellular protein synthesis, an approximately 65% reduction by 60 h postinfection. rIFN-gamma has only a minor inhibitory effect on SAV-3 replication in vitro. SAV-3 is sensitive to the preinfection antiviral state induced by rIFN-alpha, while postinfection antiviral responses or postinfection treatment with rIFN-alpha is not able to limit viral replication.

SUBMITTER: Xu C

PROVIDER: S-EPMC2919011 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Alpha interferon and not gamma interferon inhibits salmonid alphavirus subtype 3 replication in vitro.

Xu Cheng C Guo Tz-Chun TC Mutoloki Stephen S Haugland Øyvind Ø Marjara Inderjit S IS Evensen Øystein Ø

Journal of virology 20100623 17

Salmonid alphavirus (SAV) is an emerging virus in salmonid aquaculture, with SAV-3 being the only subtype found in Norway. Until now, there has been little focus on the alpha interferon (IFN-alpha)-induced antiviral responses during virus infection in vivo or in vitro in fish. The possible involvement of IFN-gamma in the response to SAV-3 is also not known. In this study, the two IFNs were cloned and expressed as recombinant proteins (recombinant IFN-alpha [rIFN-alpha] and rIFN-gamma) and used f ...[more]

PMID: 20573808

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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.

Project description:The Janus kinase (Jak) and signaling transducer activator of transcription (Stat) pathway mediates the signaling of genes required for cellular development and homeostasis. To elucidate the effect of type I IFN on the Jak/stat pathway in salmonid alphavirus subtype 3 (SAV3) infected macrophage/dendritic like TO-cells derived from Atlantic salmon (Salmo salar L) headkidney leukocytes, we used a differential transcriptome analysis by RNA-seq and the Kyoto encyclopedia of genes and genomes (KEGGs) pathway analysis to generate a repertoire of de novo assembled genes from type I IFN treated and non-treated TO-cells infected with SAV3.Concurrent SAV3 infection with type I IFN treatment of TO-cells suppressed SAV3 structural protein (SP) expression by 2log10 at 2 days post infection compared to SAV3 infection without IFN treatment which paved way to evaluating the impact of type I IFN on expression of Jak/stat pathway genes in SAV3 infected TO-cells. In the absence of type I IFN treatment, SAV3 downregulated several Jak/stat pathway genes that included type I and II receptor genes, Jak2, tyrosine kinase 2 (Tyk2), Stat3 and Stat5 pointing to possible failure to activate the Jak/stat signaling pathway and inhibition of signal transducers caused by SAV3 infection. Although the suppressor of cytokine signaling (SOCS) genes 1 and 3 were upregulated in the IFN treated cells, only SOCS3 was downregulated in the SAV3 infected cells which points to inhibition of SOCS3 by SAV3 infection in TO-cells.Data presented in this study shows that SAV3 infection downregulates several genes of the Jak/stat pathway, which could be an immune evasion strategy, used to block the transcription of antiviral genes that would interfere with SAV3 replication in TO-cells. Overall, we have shown that combining de novo assembly with pathway based transcriptome analyses provides a contextual approach to understanding the molecular networks of genes that form the Jak/stat pathway in TO-cells infected by SAV3.

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Alpha interferon and not gamma interferon inhibits salmonid alphavirus subtype 3 replication in vitro.

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Alpha interferon and not gamma interferon inhibits salmonid alphavirus subtype 3 replication in vitro.

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