Novel polyomavirus detected in the feces of a chimpanzee by nested broad-spectrum PCR

Abstract

In order to screen for new polyomaviruses in samples derived from various animal species, degenerated PCR primer pairs were constructed. By using a nested PCR protocol, the sensitive detection of nine different polyomavirus genomes was demonstrated. The screening of field samples revealed the presence of a new polyomavirus, tentatively designated chimpanzee polyomavirus (ChPyV), in the feces of a juvenile chimpanzee (Pan troglodytes). Analysis of the region encoding the major capsid protein VP1 revealed a unique insertion in the EF loop of the protein and showed that ChPyV is a distinct virus related to the monkey polyomavirus B-lymphotropic polyomavirus and the human polyomavirus JC polyomavirus.

FIG. 1.

Testing the VP1-specific nested broad-spectrum PCR (A) and the ChPyV-specific PCR (B) with different polyomavirus genomes. Genomic DNA of SV40, BKPyV, JCPyV, LPyV, MPyV, MPtV, HaPyV, goose hemorrhagic polyomavirus (GHPV), and APV was excised from plasmids and circularized by use of T4 DNA ligase. For the ChPyV plasmid, DNA carrying the cloned secondary PCR product of sample 12 was used. A total of 1 ng of DNA was used as the template for each PCR. PCR products were separated on ethidium bromide-stained 2% agarose gels. (−) Ctrl, negative control; M, DNA ladder mix (Fermentas, Vilnius, Lithuania).

FIG. 2.

Detection of DNA sequences of a new polyomavirus in clinical sample 12 derived from a chimpanzee. (A) PCR products of seven field samples (#11 to #17), SV40-infected Vero cells (SV-40), and the negative control [(−) Ctrl] were separated on ethidium bromide-stained 2% agarose gels. (B) Analysis of products amplified with primers VP1-1f and VP1-1r (1st PCR) and primers VP1-2f and VP1-2r (2nd PCR) with single PCR protocols or with the first PCR followed by the second PCR in a nested PCR protocol by ethidium bromide-stained 2% agarose gel electrophoresis. The template DNA was derived from sample 12, SV40-infected Vero cells (SV-40), or the negative control [(−)-Ctrl]. M, DNA ladder mix (Fermentas). (C) Phylogenetic relationships of the new polyomavirus (ChPyV) with 10 other polyomaviruses, based on the nucleotide sequences of the whole VP1-encoding region, aligned by the ClustalW method.

FIG. 3.

Alignment of the deduced amino acid sequences of the VP1 of 11 polyomaviruses. Letters in the consensus sequence (in italics) indicate amino acids that are identical in all sequences; periods indicate positions where amino acids are not identical. Regions of VP1 forming outer loops as determined from the crystal structure of SV40 are boxed and designated BC, DE, EF, and HI loops according to reference .