Screening for simian type-D retrovirus infection in macaques, using nested polymerase chain reaction

Abstract

A nested polymerase chain reaction (PCR) assay was developed to detect proviral DNA in peripheral blood mononuclear cells (PBMC) of macaques infected with simian type-D retrovirus (SRV/D). Primers were designed to amplify gag gene sequences of SRV/D serotype 1, 2, and 3 viral genomes and were used in a single assay for simultaneous detection of infection with SRV/D-1, SRV/D-2, or SRV/D-3. Results of plasmid dilution studies indicate sensitivity of nested PCR in the range of 1 to 10 genomic copies. The PBMC samples from 395 macaques of unknown SRV/D status, obtained from several primate facilities, were tested in parallel by Western blot (immunoblot) analysis, virus isolation, and nested PCR. Infection was detected in 60 (15.2%) animals by nested PCR, in 40 (10.1%) animals by virus isolation, and in 28 (7.1%) animals by immunoblot. All 40 culture-positive samples were positive by nested PCR. In addition, 11 of 23 immunoblot-positive/virus isolation-negative samples, 2 of 20 immunoblot-indeterminate/virus isolation-negative samples, and 7 of 312 immunoblot-negative/virus isolation-negative samples were identified as positive by nested PCR. Nested PCR is a sensitive and specific assay for simultaneous screening for infection with serotypes 1, 2, and 3 of simian type D retrovirus, and is a powerful tool for rapid screening and surveillance in macaque colonies.