Characterization of two novel polyomaviruses of birds by using multiply primed rolling-circle amplification of their genomes

Abstract

Polyomaviruses are small nonenveloped particles with a circular double-stranded genome, approximately 5 kbp in size. The mammalian polyomaviruses mainly cause persistent subclinical infections in their natural nonimmunocompromised hosts. In contrast, the polyomaviruses of birds--avian polyomavirus (APV) and goose hemorrhagic polyomavirus (GHPV)--are the primary agents of acute and chronic disease with high mortality rates in young birds. Screening of field samples of diseased birds by consensus PCR revealed the presence of two novel polyomaviruses in the liver of an Eurasian bullfinch (Pyrrhula pyrrhula griseiventris) and in the spleen of a Eurasian jackdaw (Corvus monedula), tentatively designated as finch polyomavirus (FPyV) and crow polyomavirus (CPyV), respectively. The genomes of the viruses were amplified by using multiply primed rolling-circle amplification and cloned. Analysis of the FPyV and CPyV genome sequences revealed a close relationship to APV and GHPV, indicating the existence of a distinct avian group among the polyomaviruses. The main characteristics of this group are (i) involvement in fatal disease, (ii) the existence of an additional open reading frame in the 5' region of the late mRNAs, and (iii) a different manner of DNA binding of the large tumor antigen compared to that of the mammalian polyomaviruses.

FIG. 1.

Detection of novel polyomaviruses by nested broad-spectrum PCR, amplification of their genomes by RCA and long-range PCR, and attempts to rescue infectious virus from the cloned genome of FPyV. The products were analyzed on ethidium bromide-stained agarose gels. The positions of the specific products are indicated by arrows; the sizes of molecular mass markers are specified next to the panels. (A) Secondary PCR products of the VP1-specific nested broad-spectrum PCR obtained with samples derived from Eurasian bullfinches (Bullfinches 1 to 3), a Eurasian jackdaw, a goose, or a negative control (−). (B) RCA products of the sample of Eurasian bullfinch 2 (Bullfinch), the goose, and the Eurasian jackdaw after digestion with EcoRI or PstI. (C) RCA products after pretreatment of the sample of Eurasian bullfinch 2 with BamHI (lin.) or without pretreatment (circ.) and analysis of the RCA products using BamHI. (D) PCR products obtained after long-range PCR with the sample of the Eurasian jackdaw either with (RCA+) or without (RCA−) preamplification by RCA, as well as a negative control (−). (E) Immunoblot analysis of CE cells transfected (T) with the excised and circularized genome of FPyV from plasmid 1 (FPyV1), plasmid 2 (FPyV2), a plasmid containing the APV genome (APV), or without DNA (−). P1 and P2, CE cells after one or two consecutive passages, respectively, of the supernatants of the transfected cells. A rabbit serum directed against APV particles and cross-reacting with other polyomavirus VP1 molecules was used.

FIG. 2.

Genome organization of FPyV and CPyV. Coding regions for large T-Ag, small T-Ag, VP1, VP2, VP3, and ORF-X are marked by arrows. Intron sequences inferred from the DNA sequences as well as the restriction sites of the enzymes used in RCA analysis are indicated. NCR, noncoding regulatory region.

FIG. 3.

Phylogenetic relationship of 12 polyomaviruses. The phylogenetic tree was established using the nucleotide sequences of the whole genomes (A) or the amino acid sequences of the large T-Ag (B) with the Clustal W method. Clusters of avian viruses (APV, CPyV, FPyV, and GHPV), primate viruses (BKPyV, JCPyV, and SV40), and rodent viruses (HaPyV and MPyV) are indicated. CPyV and FPyV are marked by arrows.

FIG. 4.

Comparison of the DNA binding domain of large T-Ags of polyomaviruses and the corresponding target sequences within the noncoding regulatory region. (A) Alignment of the DNA-binding regions of 12 polyomavirus large T-Ags. The NLS and the DNA-binding domains A and B2 in SV40 are indicated by dotted lines. Basic amino acid residues within the NLS region are shaded gray. Sequences that are conserved in the avian viruses but not in the mammalian viruses are boxed. (B) Nucleotide sequence of an 80-bp fragment of the noncoding regulatory region of six polyomaviruses. The (putative) binding sequences for the large T-Ag are shaded gray; the orientation of the binding sequences is indicated by arrows. For MPyV, the complementary strand (c) is shown due to the inverse nucleotide numbering of its genome.

FIG. 5.

Comparison of the amino acid sequences encoded by the additional ORFs in the 5′ region of the late mRNA of the avian polyomaviruses. (A) Phylogenetic tree based on an alignment of the amino acid sequences of VP4 of APV and the ORF-X sequences of FPyV, CPyV, and GHPV. (B) Alignment of the respective amino acid sequences. The coiled-coil motifs in the APV VP4 and FPyV ORF-X proteins as well as a region of high similarity between the ORF-X sequences of CPyV and GHPV are indicated; conserved amino acid residues are shaded gray.