Recovery of a recombinant salmonid alphavirus fully attenuated and protective for rainbow trout

Abstract

Sleeping disease virus (SDV) is a member of the new Salmonid alphavirus genus within the Togaviridae family. The single-stranded RNA genome of SDV is 11,894 nucleotides long, excluding the 3' poly(A) tail. A full-length cDNA has been generated; the cDNA was fused to a hammerhead ribozyme sequence at the 5' end and inserted into a transcription plasmid (pcDNA3) backbone, yielding pSDV. By transfection of pSDV into fish cells, recombinant SDV (rSDV) was successfully recovered. Demonstration of the recovery of rSDV was provided by immunofluorescence assay on rSDV-infected cells and by the presence of a genetic tag, a BlpI restriction enzyme site, introduced into the rSDV RNA genome. SDV infectious cDNA was used for two kinds of experiments (i) to evaluate the impact of various targeted mutations in nsP2 on viral replication and (ii) to study the virulence of rSDV in trout. For the latter aspect, when juvenile trout were infected by immersion in a water bath with the wild-type virus-like rSDV, no deaths or signs of disease appeared in fish, although they were readily infected. In contrast, cumulative mortality reached 80% in fish infected with the wild-type SDV (wtSDV). When rSDV-infected fish were challenged with wtSDV 3 and 5 months postinfection, a long-lasting protection was demonstrated. Interestingly, a variant rSDV (rSDV14) adapted to grow at a higher temperature, 14 degrees C instead of 10 degrees C, was shown to become pathogenic for trout. Comparison of the nucleotide sequences of wtSDV, rSDV, and rSDV14 genomes evidenced several amino acid changes, and some changes may be linked to the pathogenicity of SDV in trout.

FIG. 1.

Full-length SDV cDNA construct. Three cDNA fragments (fragments 1 to 3) covering the entire SDV genome were assembled by ligation into the multiple cloning site of the pBluescript plasmid using the EcoRI, SmaI, XbaI, and NotI restriction enzyme sites, yielding the pBS-SDV construct. An XbaI restriction enzyme site has been introduced into the junction region by changing 2 nucleotides (underlined) as indicated in the sequences in the box. The top and bottom sequences in the box are the wild-type and modified SDV sequences, respectively. Differences observed in comparison to the previously published sequences (18, 21) are indicated by letters (a to x) on the pBS-SDV construct. Nucleotide positions and amino acid changes are indicated in Table 2.

FIG. 2.

SDV replicons and infectious cDNA constructs. (A) Hammerhead ribozyme nucleotide sequence. The BamHI and NaeI restriction enzyme sites and the T7 promoter sequence are underlined. The ribozyme cleavage site and the beginning of the SDV genome are indicated by arrows. (B) The entire SDV cDNA was transferred from the pBluescript backbone into a pcDNA3 plasmid and fused to the hammerhead ribozyme sequence (HH) (see Materials and Methods). The gene encoding the structural protein was removed by BlpI/EcoRV restriction enzyme digestion and replaced by the reporter gene LUC (pnsP-Luc) or GFP (pnsP-GFP). (C) The LUC gene was removed from the pnsP-LUC construct and exchanged with structural (Struct.) genes by BlpI and EcoRV restriction enzyme digestion and ligation. A T7 terminator sequence (T7t) was added downstream of the poly(A) tail (pA), yielding the final construct, pSDV.

FIG. 3.

Expression of the reporter genes in cells by the SDV-derived replicons. BF-2 cells were transfected with either pnsP-LUC (A) or pnsP-GFP (B) construct and incubated at 10°C. (A) At different days posttransfection, cell lysates were incubated with the luciferase substrate. Emitted light was measured using a luminometer (Berthold) and quantified as light units. (B) Live cells were directly observed with a UV light microscope.

FIG. 4.

Recovery of rSDV. Supernatant from cells transfected with the pSDV construct was harvested and used to infect fresh BF-2 cells. (A) At 4 days postinfection, the cells were fixed and subjected to an indirect immunofluorescence antibody technique using a mixture of SDV MAbs. (B) Viral RNA was extracted from either wtSDV- or rSDV-infected cell supernatants and used as the template in RT-PCR using a pair of primers that span the additional BlpI restriction enzyme site created in the junction region upstream from the start codon of the structural (Struct.) genes. Both 1,326-nucleotide-long PCR products were either mock digested (−) or digested (+) with restriction enzyme BlpI, yielding the expected 990- and 336-nt DNA fragments for the rSDV PCR product, but leaving the wtSDV cDNA fragment intact. Lane M contains molecular size markers.

FIG. 5.

Effects of mutations in nsP2. The effects of three mutations in nsP2 were evaluated using either a replicon expressing the luciferase gene (white bars) or mutated infectious cDNA clone (black bars). Data are presented as log₁₀ of arbitrary emitted light units or PFU/ml for LUC activity and virus titer after one passage in cell culture. Amino acid mutations are indicated in the form of XnY, where X is the wild-type amino acid, n is the amino acid position in the nsP2 protein, and Y is the mutated amino acid. The wild-type nsP2 sequence (wt) is indicated. The results presented are the means of two individual experiments.

FIG. 6.

rSDV with an additional subgenomic promoter. (A) An additional transcriptional unit expressing the GFP as a reporter gene was inserted into the SDV genome either upstream or downstream of the structural genes (pGFP-SDV and pSDV-GFP), or three additional transcriptional GFP units were inserted upstream of the structural genes (pGFP₃-SDV). HH, hammerhead ribozyme sequence; pA, poly(A) tail. (B) GFP expression in either rSDV-GFP- or rGFP-SDV-infected BF-2 cells was monitored by UV light microscopy, and typical results for both rSDVs 7 days postinfection are presented. (C) Confirmation by RT-PCR of the three additional transcription units on RNA extracted from infected cells after one passage of the cell supernatant. Lanes: M, DNA molecular weight markers; rGFP-SDV, recombinant SDV containing one additional transcription unit; rGFP₃-SDV, recombinant SDV containing three additional transcription units. The sizes of the RT-PCR products (in nucleotides) are indicated to the left of the gel. The primers used for the RT-PCR are depicted in panel A.

FIG. 7.

Attenuation and protection of rSDV in trout. (A) Juvenile trout (n = 100; mean weight, 0.5 g) were infected by immersion in a water bath with 5 × 10⁴ PFU/ml of the wild-type SDV strain B or J (wtSDV-B or wtSDV-J) or with the recombinant SDV (rSDV) or mock infected. Mortalities were recorded every day and are expressed as a percentage of cumulative mortality. d.p.i, days postinfection. (B) Trout infected with rSDV 3 months (left) or 5 months (right) earlier were challenged with the wtSDV-B, and cumulative mortality was recorded 2 months later (months 5 and 7, respectively). Naïve trout are trout infected with the wtSDV-B (positive infection control). Mock-infected trout are trout treated under the same conditions except that cell culture medium was used.

FIG. 8.

Influence of temperature on the pathogenicity of SDV in trout. Juvenile trout (n = 100; mean weight, 0.5 g) were infected by immersion in a water bath with 5 × 10⁴ PFU/ml of each virus grown at 10°C or 14°C or mock infected. Mortalities were recorded every day and are expressed as a percentage of cumulative mortality. d.p.i, days postinfection.