Clinical Findings and Tumor Pathology
During March 2010–May 2012, 52 raccoons were submitted to the California Animal Health and Food Safety Laboratory at the University of California, Davis, for full necropsy, with rabies screening performed as part of state diagnostic protocol. Ten (19%) raccoons had brain tumors within the cranial portion of their frontal lobe(s), most of which spanned the cribiform plate of the ethmoid bone and extended into, or from, the olfactory tract (). Nine of the affected animals represented separate collection events from 3 adjacent counties in California, and 1 affected animal was shipped from southern Oregon. In all instances, the raccoons were exhibiting neurologic signs, including wandering in the daylight, approaching humans, or exhibiting lack of consciousness. Overall, other tissues, including lymphoid and hematopoietic tissues such as bone marrow, thymus, lymph nodes, and spleen, were generally within normal limits.
Figure 1 Pathology of raccoon polyomavirus–associated tumors. A) Normal anatomy, head of unaffected raccoon, midsagittal section. An intact cribriform plate separates the ethmoid turbinates from the olfactory tract. B) Gross pathology, head of raccoon (more ...)
In one of the affected raccoons (Rac7), a presumed early (grossly undetectable) tumor was exclusively localized within the olfactory tract and segments of the axonal bundles of olfactory nerves within the ethmoid turbinates, which makes this the likely site of tumor origin. Most of the tumors were histologically pleomorphic, and each of the 10 tumors have been alternately and preliminarily classified as MPNSTs or glioblastomas on the basis of criteria such as anatomic location, histopathology (), and a panel of histochemical and immunohistochemical stains, including reticulin stain, glial fibrillary acidic protein, vimentin, pancytokeratin, neuron-specific nuclear protein, Olig2, synaptophysin, and laminin. Among consistent findings in the glial tumors was expression of glial fibrillary acidic protein, variably sized regions of necrosis (), and high mitotic activity. Tumors classified as MPNSTs also were positive for glial fibrillary acidic protein; however, neoplastic cells were individually surrounded by a reticulin-positive framework, which was immunoreactive with laminin. Viral inclusions were not detectable histologically, and no viral particles were visualized by transmission electron microscopy.
RacPyV Discovery by Consensus PCR
JCV is a human PyV that has been associated with brain tumors, including a broad range of glial-origin tumors. In experimental studies, transgenic mice expressing JCV LT-Ag under the control of the Mad-4 promoter develop MPNSTs and under the regulation of the CY (archetype) promoter develop medulloblastomas and glioblastomas (19
). Because LT-Ag is a principal PyV protein that orchestrates oncogenesis (13
), we chose it as the target for consensus PCR detection. Using an alignment of 21 PyVs, we designed 4 sets of degenerate primers to amplify conserved regions within LT-Ag (Technical Appendix Figure 3
). Amplification was successful by using primers PLMA_F3 and PLMA_R1, yielding a 270-bp product from tumor tissue of the 3 raccoons originally tested. Thus far, the virus has not been detected in brain tissue or multiple other tissues (kidney, spleen, feces, salivary gland, lung, tonsil, nasal swab) tested in 20 unaffected animals collected from the same geographic region. Full length viral genomes (6/10) or partial genomes (4/10, ranging from 2,998 bp to 4,667 bp) were generated by using a combination of primer walking and specific RacPyV primers.
RacPyV Genome Analysis
The novel virus was most closely related, phylogenetically, to a clade of viruses that includes MCPyV and was named raccoon polyomavirus (RacPyV). PyVs are small, circular, double-stranded DNA viruses, and analysis of the RacPyV genome showed characteristic organization for Polyomaviridae
). RacPyV encodes putative open reading frames (ORFs) with homology to PyV LT-Ag, sT-Ag, the structural VPs 1–3, and a single bidirectional noncoding regulatory region (NCRR). Because of sequence variation among the raccoon isolates, the designations 1–10 indicate the order of animal submission and which raccoon harbored which respective RacPyV; that is, RacPyV1 was found in Rac1 and was the first tumor and RacPyV discovered.
Figure 2 Genome organization of RacPyV. The entire dsDNA viral genome for RacPyV10 comprises 5,015 bp. The viral genome has a noncoding regulatory region and putative open reading frames for the late proteins VP1, VP2, and VP3 and the early proteins LT-Ag and (more ...)
Phylogenetic trees of the deduced PyV proteins constructed by using the neighbor-joining method show that the putative early and late proteins of RacPyV are most closely related to those of MCPyV and chimpanzee PyV (). The 10 RacPyV genomes had a total of 30 single nucleotide polymorphisms (SNPs) (). Most (29/30) of the SNPs were synonymous substitutions or in noncoding regions, as seen with strain variants of other PyVs. Half (15/30) of the SNPs were unique to the single Oregon case (Rac6), perhaps reflecting its geographic distance from the California raccoons. Three distinct nucleotide deletion events occurred across RacPyV5 and RacPyV10. RacPyV5 had a 26-bp deletion within the putative LT-Ag intron and a 12-bp deletion within the putative NCRR; RacPyV10 had a 1-bp deletion within the putative LT-Ag intron. The genome of RacPyV3 is deposited in GenBank (accession no. JQ178241).
Figure 3 Phylogenetic relationship of RacPyV with representative polyomavirus species. Phylogenetic trees were individually generated on the basis of amino acid sequences of LT-Ag (A), sT-Ag (B), VP1, and VP2 (C, D) by using the neighbor-joining method with p-distance (more ...)
Figure 4 Partial genome sequences (ranging from 2,998 bp in raccoon polyomavirus 6 [RacPyV6] to 4,667 bp in RacPyV9) were obtained from 4 raccoons that either had undergone prolonged storage (RacPyV9) or were available only as formalin-fixed, paraffin-embedded (more ...)
The NCRR of RacPyV encodes multiple consensus pentanucleotide LT-Ag binding sites (GAGGC) on both strands as seen in other PyVs. RacPyV LT-Ag has 46% homology to MCPyV LT-Ag and has features conserved among PyV LT-Ags, including the LXCXE motif necessary for LT-Ag association with the retinoblastoma protein and the Walker A box GPXXXGKT, which suggests that RacPyV LT-Ag is an ATPase that binds to p53 (26
). In experimental models, these sites are crucial components of virus-mediated transformation (20
The putative ORF encoding LT-Ag in RacPyV encodes putative splice sites surrounding a 387-bp intron. In other PyVs, sT-Ag, which shares a start site with LT-Ag, promotes cell transformation through negative regulation of the protein phosphatase 2A (PP2A) family of serine-threonine phosphatases (26
). In transcription and expression studies, sT-Ag terminates at a stop codon within the LT-Ag intron. Aside from chimpanzee PyV (Azzie isolate) (28
), all PyVs discovered thus far contain this canonical intronic stop codon that defines the ORF for sT-Ag. However, among the 7 RacPyVs for which the intron of LT-Ag was completely sequenced, only 2 (RacPyV5 and RacPyV10) contain intronic in-frame stop codons that would define an independent sT-Ag. In the 5 other RacPyVs, the entire T-antigen coding region is in-frame as a putative single ORF. Although expression studies are needed, among possible explanations are that a single T-Ag contains both LT-Ag and sT-Ag domains, that sT-Ag could be expressed as an alternately spliced form of the T-Ag coding region, that sT-Ag is generated proteolytically, or that it is not expressed. Of the 2 viruses that have a stop codon in-frame within the LT-Ag intron, RacPyV10 has a stop codon that terminates sT-Ag after the PP2A domains, as is seen in other PyVs, whereas RacPyV5 encodes a stop codon before the PP2A domains. However, the sT-Ag of MCPyV constructs altered to lack a functional PP2A binding domain still initiates cell transformation (29
Southern blot hybridizations and RCA were performed to ascertain whether RacPyV existed in episomal, integrated, or a combination of these forms. Viral DNA of sufficient quantity and quality was present in 5/10 tumors for these assays. In all cases, an ≈5-kb band for the KpnI-digested samples was absent or present faintly in undigested conditions (, panel A, closed arrow). The band variably present at ≈9 kb is expected to be a circular nicked form of the genome (, panel A, open arrow). RCA, by using random hexamers, was performed on DNA extracted from the same set of tumor tissues. RCA products of the tumor tissues produced the expected ≈5-kb band after KpnI digestion, confirming the Southern blot result that RacPyV is not detectably integrated in the host genome and demonstrating that it exists in circular form in tumor cells (, panel B). As expected, Southern blot and RCA analyses performed on DNA extracted from normal, unaffected raccoon brain tissue produced no bands (, panels A, B).
Figure 5 Raccoon polyomavirus (RacPyV) is episomal in raccoon brain tumors as detected by Southern blot hybridization and rolling circle amplification. A unique KpnI site in viral protein 1 (Technical Appendix Figure 2) conserved across the viral genomes was predicted (more ...)
Expression of RacPyV LT-Ag in the Nuclei of Tumor Cells
To determine the expression of LT-Ag, we performed immunohistochemical analyses with a monoclonal antibody that recognizes a peptide within exon 2 of MCPyV LT-Ag. The LT-Ag was absent () in unaffected regions of the brain and absent in unaffected raccoon tissues but was detected within the nuclei of a subset of neoplastic cells in 4 tested raccoon tumors (). Immunohistochemical analyses for wild-type p53 also was positive in a subset of neoplastic cells () but was negative in the adjacent normal brain. Furthermore, double-labeling immunofluorescence showed the co-localization of LT-Ag with wild-type p53 in the nuclei of neoplastic cells ().
Figure 6 Expression of large T-antigen (LT-Ag) and p53 within a subset of tumors. A) Control, immunohistochemical analysis of frontal lobe of normal raccoon brain tissue. Astrocytes in this image and throughout the section were not immunoreactive for LT-Ag. Original (more ...)