Identification and characterization of an exogenous retrovirus from atlantic salmon swim bladder sarcomas

Abstract

A novel piscine retrovirus has been identified in association with an outbreak of leiomyosarcoma in the swim bladders of Atlantic salmon. The complete nucleotide sequence of the Atlantic salmon swim bladder sarcoma virus (SSSV) provirus is 10.9 kb in length and shares a structure and transcriptional profile similar to those of murine leukemia virus-like simple retroviruses. SSSV appears unique to simple retroviruses by not harboring sequences in the Atlantic salmon genome. Additionally, SSSV differs from other retroviruses in potentially utilizing a methionine tRNA primer binding site. SSSV-associated tumors contain high proviral copy numbers (greater than 30 per cell) and a polyclonal integration pattern. Phylogenetic analysis based on reverse transcriptase places SSSV with zebrafish endogenous retrovirus (ZFERV) between the Gammaretrovirus and Epsilonretrovirus genera. Large regions of continuous homology between SSSV and ZFERV Gag, Pol, and Env suggest that these viruses represent a new group of related piscine retroviruses.

FIG. 1.

(A) SSSV genome organization and splicing pattern. The numbers mark the boundaries of the ORFs and of the LTRs relative to the start of transcription (+1). The Gag-Pol polyprotein is predicted to be translated from a genomic RNA by suppression of a termination codon (TGA) at the junction of the gag and pol genes. LP, leader peptide. (B) Primers used for RT-PCR analysis of splice sites of SSSV. (C) Subgenomic viral transcripts. The positions of the major splice donor and acceptor sites relative to the start of transcription are indicated. The Env protein could be generated from either spliced subgenomic RNA.

FIG. 2.

Homology between SSSV and other retroviruses. Regions of homology between SSSV and ZFERV, MuLV, MPMV, and WDSV were determined by BLAST searches of putative SSSV Gag, Pol, and Env protein sequences. Regions of detectable protein identity between each virus and SSSV are delineated by overlapping bars. Percentages of identity between each protein in specified regions are noted above each bar with expected values in parentheses.

FIG. 3.

SSSV integration pattern in three salmon swim bladder sarcomas (T1 to T3) and control liver tissue (C). Genomic DNA cut with either PstI or KpnI and probed for SSSV pol sequences by Southern blotting. PstI sites are located upstream and downstream of SSSV pol and thus create a 2.5-kb fragment after PstI digestion. Enzyme digestion with KpnI produces a broad range of fragments greater than 6 kb due to the presence of a single KpnI site located upstream of pol and random sites in the Atlantic salmon genome.

FIG. 4.

Northern blot analysis of SSSV expression in swim bladder sarcoma tumors. Poly(A)⁺ RNA isolated from two individual tumors (T1 and T2) were probed with 552-bp DNA sequences from the U3 and R regions of SSSV LTR. The sizes of transcripts were determined by comparison to an RNA ladder marker run in parallel.

FIG. 5.

Unrooted phylogenetic tree of representative retroviruses based on an amino acid alignment of seven conserved domains in reverse transcriptase (52) totaling 178 residues. Retroviruses are designated as follows: MPMV, JSRV, MMTV (mouse mammary tumor virus), RSV (Rous sarcoma virus), EIAV (equine infectious anemia virus), FIV (feline immunodeficiency virus), Visna (visna virus), HIV-1, HIV-2, SIV-agm (simian immunodeficiency virus-agm), HTLV-1, HTLV-2, BLV (bovine leukemia virus), SSSV, SnRV, BFV (bovine foamy virus), HFV (human foamy virus), FeLV (feline leukemia virus), MuLV, GALV (gibbon ape leukemia virus), WDSV, WEHV-1, WEHV-2, ZFERV, and Xen-1. Phylogenetic and molecular evolutionary analyses were conducted using the neighbor-joining methods of MEGA version 2.1 software (24). Bootstrap values displayed at each branch point were determined from 100 replicates. Horizontal branch lengths are proportional to the degree of amino acid substitutions per site.