Constitutive expression of Atlantic salmon Mx1 protein in CHSE-214 cells confers resistance to Infectious Salmon Anaemia virus
© Kibenge et al; licensee BioMed Central Ltd. 2005
Received: 08 July 2005
Accepted: 26 August 2005
Published: 26 August 2005
Infectious salmon anaemia (ISA) is a highly fatal viral disease affecting marine-farmed Atlantic salmon which is caused by ISA virus (ISAV), a fish orthomyxovirus that has recently been assigned to the new genus Isavirus within the family Orthomyxoviridae. Mx proteins are among the interferon (IFN)-induced proteins responsible for the development of an antiviral state in vertebrate cells. We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and Chinook salmon embryo (CHSE-214) cells constitutively expressing Atlantic salmon Mx1 protein (ASMx1) to examine the antiviral properties of ASMx1 against two ISAV strains, NBISA01 and HKS-36, having phenotypically different growth properties (cytopathic vs non-cytopathic) in the CHSE-214 cell line. We present evidence that ISAV is sensitive to ASMx1. CHSE-214 cells constitutively expressing ASMx1 showed increased resistance to infection with the cytopathic ISAV strain NBISA01, manifested as delayed development of cytopathic effects (CPE) and significant reduction in the severity of CPE, as well as a 10-fold reduction in virus yield. However, by real-time RT-PCR we observed no significant difference in the mean threshold cycle (Ct) values of ISAV RNA levels, suggesting that the ASMx1 activity on ISAV occurs at the post-transcription steps of virus replication, possibly in the cytoplasm.
Infectious salmon anaemia (ISA) virus (ISAV), the causative agent of a highly fatal disease of marine-farmed Atlantic salmon, is a fish orthomyxovirus that has recently been assigned to the new genus Isavirus within the family Orthomyxoviridae . The disease has caused severe economic losses to the salmon-farming industry in several countries in the northern hemisphere, and confirmed positive diagnostic results for ISA are reportable to the "Office International des Epizooties", OIE . Viruses in the genus Isavirus are enveloped particles of 90–140 nm diameter with surface projections consisting of a combined haemagglutinin-esterase (HE) protein  and a separate putative fusion (F) protein . The genome is composed of eight segments of linear, single-stranded negative sense RNA ranging in length from 1.0 to 2.4 kb with a total molecular size of approximately 14.3 kb . Sequence analysis of several ISAV isolates on the eight segments consistently reveals two genotypes, one European and one North American. The gene-coding assignments of the ISAV genome differ from those of other orthomyxoviruses [2, 4].
Some strains of ISAV can replicate and cause CPE in the CHSE-214 cell line while others do not replicate in this cell line at all [6, 7]. The molecular basis for this phenotypic difference is not clearly known. Although there is a strong geographical correlation in that practically all ISAV isolates of the European genotype do not cause CPE in the CHSE cell line, this phenotype is not related to sequence variation in the HE protein .
In general, fish viral diseases are difficult to control due to the high susceptibility of fish at an early age, and insufficient knowledge of pathogenesis of virus infections. In this context, we are studying the mechanisms by which ISAV interacts with its host, including the identification of markers for ISAV virulence. Vertebrates , including fish , mount an early strong innate immune response against virus infections, characterized by the induction and secretion of cytokines, such as type I interferons (IFN-α/β) that mediate an antiviral state. Secreted IFNs signal through a common receptor activating a JAK/STAT signaling pathway which leads to the transcriptional upregulation of numerous IFN-stimulated genes (ISGs), a number of which encode antiviral proteins [10–12]. The induced antiviral proteins include dsRNA-dependent protein kinase R (PKR), 2',5'-oligoadenylate synthetase (OAS), and the Mx proteins [10, 12]. Viruses have evolved mechanisms to subvert the host IFN response [12, 13]. Previous in-vitro studies revealed that ASMx1 inhibited the replication of infectious pancreatic necrosis virus (IPNV), a dsRNA virus belonging to the Birnaviridae family, but it did not appear to inhibit replication of ISAV [14, 15]. It was concluded from those results that ISAV and IPNV have developed different strategies to avoid the IFN-system of Atlantic salmon. Thus, we were interested in analyzing ISAV strains having phenotypically different growth properties in the CHSE-214 cell line to gain further insight in ISAV virulence. We present evidence that ISAV is sensitive to the antiviral activity of ASMx1.
Inhibition of ISAV NBISA01 by AsMx1 in CHSE-214 cells.
In an attempt to assess the effects of ASMx1 on the viral mRNA levels, we used real-time RT-PCR in the LightCycler with RNA Amplification Kit SYBR Green I (Roche Applied Science) and PCR primers FA-3/RA-3 targeting a 220-bp product on ISAV segment 8 . This assay has been shown to be 100 times more sensitive than the conventional one-tube RT-PCR assay, and to differentiate ISAV isolates into three CHSE phenotypes (replicating cytopathic, replicating non-cytopathic, and non-replicating) based on their ability to replicate and cause CPE in CHSE-214 cells . Total RNA was extracted from 300 μl of cell culture supernatants of the uninfected and ISAV-infected tissue cultures harvested at 14 dpi by using TRIZOL reagent (Invitrogen Life Technologies). The RNA pellet was dissolved in 10 μl of RNase free water and 1 μl was used in real-time RT-PCR. The assay was performed on three replicates of each virus sample. The thermal conditions were one cycle of reverse transcription at 55°C for 30 min, pre-denaturation at 95°C for 30 s followed by 50 cycles of 95°C for 5s, 59°C for 10s, 72°C for 10s, and data acquisition at 80°C for 2s. Melting curve analysis was performed from 70°C to 95°C in 0.1°C/s increments to assess the specificity of the RT-PCR products. The quantitative (Ct values) and melting curve data were analyzed using LightCycler software version 3.5 (Roche Applied Science). The real-time amplification reaction products were also resolved by 1% agarose gel electrophoresis in 0.5 × TBE buffer and visualized by staining with ethidium bromide and photographed under 304 nm UV light.
To our knowledge these are the first studies demonstrating that ASMx1 interferes with ISAV replication and suggest that this interference occurs after viral mRNA synthesis. Orthomyxoviruses are known to be sensitive to the antiviral action of type I IFN, and have evolved viral IFN antagonists to suppress IFN induction [17–19]. Influenza A virus NS1 protein binds dsRNA  and prevents activation of PKR  and OAS . Additionally, the NS1 protein is able to prevent activation of NFκ-B  and IRF-3  which are necessary for IFN-β synthesis in virus infected cells. Influenza B virus NS1 protein also inhibits activation of IRF-3 , and the activity of ISG15, one of the major type I IFN inducible proteins . Thogoto virus protein ML suppresses IRF-3 function . It is therefore expected that ISAV also encodes protein(s) with IFN antagonistic properties, giving the virus an advantage in its fight against the antiviral host response to infection. The complete ISAV protein profile has not yet been definitively established. ISAV segment 8, like its influenza A virus counterpart, has been shown to encode two proteins; one open reading frame (ORF) was shown to encode a major structural protein corresponding to the virus matrix protein , and the other ORF is considered to encode the non-structural protein  albeit with no sequence similarity to the influenza NS1 proteins . It would be interesting to see if IFN and ISGs knockdowns would enhance the growth and/or cytopathogenicity of ISAV. This would provide further insight into the exact mechanisms by which ISAV interacts with the Atlantic salmon IFN system with implications for ISAV virulence.
List of abbreviations
- Infectious salmon anaemia virus (ISAV):
Atlantic salmon Mx1 protein (ASMx1), cytopathic effect (CPE), Tris borate EDTA (TBE).
We kindly thank Dr. Børre Robertsen for the CHSE-214 cells constitutively expressing ASMx1. This work was supported by Natural Sciences and Engineering Research Council (NSERC) of Canada Strategic Grants and an NSERC Discovery Grant.
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