Characterization of untranslated regions of the salmonid alphavirus 3 (SAV3) genome and construction of a SAV3 based replicon

Salmonid alphavirus (SAV) causes disease in farmed salmonid fish and is divided into different genetic subtypes (SAV1-6). Here we report the cloning and characterization of the 5'- and 3'- untranslated regions (UTR) of a SAV3 isolated from Atlantic salmon in Norway. The sequences of the UTRs are very similar to those of SAV1 and SAV2, but single nucleotide polymorphisms are present, also in the 3' - conserved sequence element (3'-CSE). Prediction of the RNA secondary structure suggested putative stem-loop structures in both the 5'- and 3'-ends, similar to those of alphaviruses from the terrestrial environment, indicating that the general genome replication initiation strategy for alphaviruses is also utilized by SAV. A DNA replicon vector, pmSAV3, based upon a pVAX1 backbone and the SAV3 genome was constructed, and the SAV3 non-structural proteins were used to express a reporter gene controlled by the SAV3 subgenomic promoter. Transfection of pmSAV3 into CHSE and BF2 cell lines resulted in expression of the reporter protein, confirming that the cloned SAV3 replication apparatus and UTRs are functional in fish cells

Development of infectious cDNA clones of Salmonid alphavirus subtype 3

<p>Abstract</p> <p>Background</p> <p>Salmonid 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.</p> <p>Results</p> <p>Infectious 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, nsP3₂₆₃and nsP3₃₂₃severely reduced infectivity, a serine to proline mutation in E2₂₀₆appeared to enhance the virus titer production.</p> <p>Conclusion</p> <p>We have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.</p

Low Temperature-Dependent Salmonid Alphavirus Glycoprotein Processing and Recombinant Virus-Like Particle Formation

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ∼17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production

LE VIRUS RESPONSABLE DE LA MALADIE DU SOMMEIL CHEZ LES SALMONIDES (UN NOUVEAU TYPE D'ALPHAVIRUS)

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Genetically modified attenuated salmonid alphavirus: A potential strategy for immunization of Atlantic salmon

Pancreas disease (PD) is a serious challenge in European salmonid aquaculture caused by salmonid alphavirus (SAV). In this study, we report the effect of immunization of Atlantic salmon with three attenuated infectious SAV3 strains with targeted mutations in a glycosylation site of the envelope E2 protein and/or in a nuclear localization signal in the capsid protein. In a pilot experiment, it was shown that the mutated viral strains replicated in fish, transmitted to naïve cohabitants and that the transmission had not altered the sequences. In the main experiment, the fish were immunized with the strains and challenged with SAV3 eight weeks after immunization. Immunization resulted in infection both in injected fish and 2 weeks later in the cohabitant fish, followed by a persistent but declining load of the mutated virus variants in the hearts. The immunized fish developed clinical signs and pathology consistent with PD prior to challenge. However, fish injected with the virus mutated in both E2 and capsid showed little clinical signs and had higher average weight gain than the groups immunized with the single mutated variants. The SAV strain used for challenge was not detected in the immunized fish indicating that these fish were protected against superinfection with SAV during the 12 weeks of the experiment

Genetically modified attenuated salmonid alphavirus: A potential strategy for immunization of Atlantic salmon

Pancreas disease (PD) is a serious challenge in European salmonid aquaculture caused by salmonid alphavirus (SAV). In this study, we report the effect of immunization of Atlantic salmon with three attenuated infectious SAV3 strains with targeted mutations in a glycosylation site of the envelope E2 protein and/or in a nuclear localization signal in the capsid protein. In a pilot experiment, it was shown that the mutated viral strains replicated in fish, transmitted to naïve cohabitants and that the transmission had not altered the sequences. In the main experiment, the fish were immunized with the strains and challenged with SAV3 eight weeks after immunization. Immunization resulted in infection both in injected fish and 2 weeks later in the cohabitant fish, followed by a persistent but declining load of the mutated virus variants in the hearts. The immunized fish developed clinical signs and pathology consistent with PD prior to challenge. However, fish injected with the virus mutated in both E2 and capsid showed little clinical signs and had higher average weight gain than the groups immunized with the single mutated variants. The SAV strain used for challenge was not detected in the immunized fish indicating that these fish were protected against superinfection with SAV during the 12 weeks of the experiment

SAV3-E2 detection on the surface of <i>Sf</i>9 cells after recombinant baculovirus expression.

<p>Cells were infected with Ac-SAV3 at 12°C, 15°C, 18°C and 27°C. Cells were fixed with 4% paraformaldehyde and subjected to immunostaining with α-E2 mabs. Cells were analyzed by confocal microscopy and positive staining indicates the presence of E2 at the surface of infected cells.</p

SAV3 structural cassette expression and VLP formation by temperature-shift in <i>Sf</i>9 insect cells.

<p><i>Sf</i>9 cells were infected with Ac-SAV3, incubated for 2 days at 27°C and subsequently transferred to 12°C for 3 days. A) Cell cultures were treated with/without PNGase F and analyzed by WB using α-E2 mabs. B) Infected cells were treated with 4% paraformaldehyde and subjected to surface immunostaining with α-E2 mabs. C) The medium fraction the infected cell culture was analyzed by TEM for the presence of VLPs.</p

SAV3 structural cassette expression, using recombinant baculoviruses.

<p>A) Schematic representation of the SAV3 structural cassette as it is expressed by recombinant baculoviruses. The molecular mass of the proteins are indicated and shaded areas represent transmembrane domains or signal sequences (ss). Autocatalytic (A), furin (F) and signalase (S) cleavage sites are indicated, asterisks represent N-linked glycosylation sites. B) Protein expression in <i>Sf</i>9 cells was analyzed by CBB and WB using SAV α-E1 and SAV α-E2 mabs. C) Whole cell lysates were treated with/without PNGase F and analyzed with SAV α-E2 mabs. D) Cells infected with Ac-GFP and Ac-SAV3 and stained with Hoechst. CPE was evaluated by brightfield and fluorescence microscopy. Arrows indicate dense nuclear bodies in Ac-SAV3-infected insect cells.</p