A fully attenuated recombinant Salmonid alphavirus becomes pathogenic through a single amino acid change in the E2 glycoprotein

Abstract

A recombinant sleeping disease virus (rSDV) was previously shown to be totally attenuated and provide long-term protection in trout (C. Moriette, M. Leberre, A. Lamoureux, T. L. Lai, M. Brémont, J. Virol. 80:4088-4098, 2006). Sequence comparison of the rSDV to wild-type genomes exhibited a number of nucleotide changes. In the current study, we demonstrate that the virulent phenotype of SDV was essentially associated with two amino acid changes, V8A and M136T, in the E2 glycoprotein, with the V8A change mostly being involved in the acquisition of the virulent phenotype.

Fig 1

Schematic representation of the pSDV infectious cDNA. Restriction enzyme sites used in the study are indicated, as are the nucleotides and amino acid changes in the E2 glycoprotein.

Fig 2

Pathogenicity of rSDVStruct_wt in trout. Fifty juvenile trout (mean weight, 4.4 g) were infected by bath immersion with 5 × 10⁴ PFU/ml of wtSDV, rSDV, or rSDVStruct_wt or were mock infected. Mortalities were recorded every day postinfection over a 2-month period of time. A comparison of survival between groups was performed with the log rank χ² test on the Kaplan-Meier survival data using GraphPad Prism (GraphPad, San Diego, CA).

Fig 3

(A) Multiple-sequence alignment of alphavirus E2 glycoprotein amino acid sequences. E2 glycoprotein amino acid sequences from prototype alphaviruses are aligned with that of salmonid alphavirus (SAV), SDV, and SPDV. E2 SAV amino acids A8 and T136 are highlighted in red, as is T12 of E2 from chikungunya virus (CHIKV). The extra E2 domain for SAV is highlighted in yellow. Amino acids in gray shading are the residues conserved in all of the presented E2 alphavirus sequences. SFV, Semliki Forest virus; RRV, Ross River virus; MAYA, Mayaro virus; WEEV, Western equine encephalitis virus; AURA, Aura virus; VEEV, Venezuelan equine encephalitis virus. (B) Schematic representation of the genome of the various rSDV. Amino acid changes in the E2 glycoprotein are indicated at positions 8 and 136.

Fig 4

Growth curve kinetics of the various rSDV. BF2 cells plated in 24-well plates were infected at a multiplicity of infection of 0.1 with each of the rSDV. At various days postinfection, aliquots of viral supernatant were taken and clarified. Viral titers were determined by plaque assay using serial 10-fold dilutions to infect (in duplicate) BF-2 cells plated in 96-well plates. Plaques were visualized 6 days later by indirect immunofluorescence assay with a monoclonal antibody, 17H23, directed against E2. The titers were expressed in PFU/ml. Viral titers were compared by one-way analysis of variance (ANOVA) with Tukey's multiple-comparison post test using GraphPad Prism (GraphPad, SanDiego, CA). Viruses that were assigned to statistically similar groups (and, thus, share a letter) are not significantly different from each other (P > 0.05), whereas those that were not assigned to statistically similar groups are significantly different (P < 0.05).

Fig 5

Cumulative mortalities induced in trout by the various SDV. Twenty-five juvenile trout (mean weight, 3 g) were infected by intraperitoneal injection with 10⁵ PFU/fish of rSDV, rSDVStruct_wt, rSDV-E2_A8, or rSDV-E2_T136 or were mock infected. Mortalities were recorded each day postinjection over a 2-month period. For statistical grouping, a comparison of survival between groups was performed with the log rank χ² test on the Kaplan-Meier survival data using GraphPad Prism (GraphPad, San Diego, CA). Viruses that were assigned to statistically similar groups (and, thus, share a letter) are not significantly different from each other (P > 0.05), whereas those that were not assigned to statistically similar groups are significantly different (P < 0.01).