We characterized the complete genome of a novel dog circovirus (DogCV) from the liver of a dog with severe hemorrhagic gastroenteritis, vasculitis, and granulomatous lymphadenitis. DogCV was detected by PCR in fecal samples from 19/168 (11.3%) dogs with diarrhea and 14/204 (6.9%) healthy dogs and in blood from 19/409 (3.3%) of dogs with thrombocytopenia and neutropenia, fever of unknown origin, or past tick bite. Co-infection with other canine pathogens was detected for 13/19 (68%) DogCV-positive dogs with diarrhea. DogCV capsid proteins from different dogs varied by up to 8%. In situ hybridization and transmission electron microscopy detected DogCV in the lymph nodes and spleens of 4 dogs with vascular compromise and histiocytic inflammation. The detection of a circovirus in tissues of dogs expands the known tropism of these viruses to a second mammalian host. Our results indicate that circovirus, alone or in co-infection with other pathogens, might contribute to illness and death in dogs.

Bovine papillomaviruses (BPV1/BPV2) have long been associated with equine sarcoids; deciphering their contribution has been difficult due to their ubiquitous presence on skin and in the environment, as well as the lack of decent techniques to interrogate their role in pathogenesis. We have developed and characterized an in situ hybridization (ISH) assay that uses a pool of probes complementary to portions of the E5, E6, and E7 genes. This assay is highly sensitive for direct visualization of viral transcript and nucleic acid in routinely processed histopathologic samples. We demonstrate here the visualization of BPV nucleic acid in 18 of 18 equine sarcoids, whereas no detectable viral DNA was present in 15 of 15 nonsarcoid controls by this technique. In nearly 90% (16/18) of the sarcoids, 50% or more of the fibroblastic cell nuclei distributed throughout the neoplasm had detectable hybridization. In the remaining 2 cases, fewer than half of the fibroblastic cells contained detectable hybridization, but viral nucleic acid was also detected in epithelial cells of the sebaceous glands, hair follicles and epidermis. A sensitive ISH assay is an indispensable addition to the molecular methods used to detect viral nucleic acid in tissue. We have used this technique to determine the specific cellular localization and distribution of BPV in a subset of equine sarcoids.

Tumors of any type are exceedingly rare in raccoons. High-grade brain tumors, consistently located in the frontal lobes and olfactory tracts, were detected in 10 raccoons during March 2010–May 2012 in California and Oregon, suggesting an emerging, infectious origin. We have identified a candidate etiologic agent, dubbed raccoon polyomavirus, that was present in the tumor tissue of all affected animals but not in tissues from 20 unaffected animals. Southern blot hybridization and rolling circle amplification showed the episomal viral genome in the tumors. The multifunctional nuclear protein large T-antigen was detectable by immunohistochemical analyses in a subset of neoplastic cells. Raccoon polyomavirus may contribute to the development of malignant brain tumors of raccoons.

Raccoon polyomavirus (RacPyV) is associated with 100% of neuroglial tumors in free-ranging raccoons. Other tumor-associated polyomaviruses (PyVs), including simian virus 40 (SV40), murine PyV, and Merkel cell PyV, are found integrated in the host genome in neoplastic cells, where they constitutively express splice variants of the tumor antigen (TAg) gene. We have previously reported that RacPyV exists only as an episome (nonintegrated) in neuroglial tumors. Here, we have investigated TAg transcription in primary tumor tissue by transcriptome analysis, and we identified the alternatively spliced TAg transcripts for RacPyV. We also determined that TAg was highly transcribed relative to host cellular genes. We further colocalized TAg DNA and mRNA by in situ hybridization and found that the majority of tumor cells showed positive staining. Lastly, we examined the stability of the viral genome and TAg transcription by quantitative reverse transcriptase PCR in cultured tumor cells in vitro and in a mouse xenograft model. When tumor cells were cultured in vitro, TAg transcription increased nearly 2 log-fold over that of parental tumor tissue by passage 17. Both episomal viral genome and TAg transcription were faithfully maintained in culture and in tumors arising from xenotransplantation of cultured cells in mice. This study represents a minimal criterion for RacPyV's association with neuroglial tumors and a novel mechanism of stability for a polyomavirus in cancer. IMPORTANCEThe natural cycle of polyomaviruses in mammals is to persist in the host without causing disease, but they can cause cancer in humans or in other animals. Because this is an unpredictable and rare event, the oncogenic potential of polyomavirus is primarily evaluated in laboratory animal models. Recently, raccoon polyomavirus (RacPyV) was identified in neuroglial tumors of freeranging raccoons. Viral copy number was consistently high in these tumors but was low or undetectable in nontumor tissue or in unaffected raccoons. Unlike other oncogenic polyomaviruses, RacPyV was episomal, not integrated, in these tumors. To determine the stability of the viral genome and sustained transcription of the oncogenic tumor antigen genes, we cultured primary raccoon tumor cells and passaged them in mice, confirming the nonintegrated state of the virus and the maintenance of viral gene transcription throughout. RacPyV provides a naturally occurring and tractable model for a novel mechanism of polyomavirus-mediated oncogenesis.

Equus caballus papillomavirus 2 (EcPV2) has been proposed as an etiologic agent for genital squamous cell carcinoma (SCC), the most common malignant tumor of the horse penis. EcPV2 is commonly detected by polymerase chain reaction (PCR) on normal horse genitalia; therefore, unraveling the virus' role in oncogenic transformation requires other methods of detection. In this study, a highly sensitive multiple-probe chromogenic in situ hybridization (ISH) technique was designed to recognize the E6/E7 oncogenes of EcPV2. ISH demonstrated abundant virus within 6 of 13 penile and preputial SCCs, whereas evidence of solar damage was found in 6 cases that were negative for EcPV2 by ISH. The ISH technique is valuable for studies of pathogenesis, since it demonstrates for the first time that the vast majority of neoplastic cells contain virus. Moreover, hybridization was present in all metastases examined, implying stability of E6/E7 expression in these clonal populations of neoplastic cells. This study contributes to the accumulating evidence for a causal role of EcPV2 in a subset of genital SCCs in horses.

We report the identification of a novel papillomavirus, Fulmarus glacialis papillomavirus 1 (FgPV1), present within an interdigital foot mass of a Northern Fulmar (Fulmarus glacialis). The mass of interest was composed of normal stratified and keratinized epithelium and dense mesenchymal cells with central cartilaginous islands. Within the nuclei of many chondrocytes were loose aggregates or paracrystalline arrays of virions approximately 50 nm in size. Degenerate polymerase chain reaction was used to identify the virus as a putative papillomavirus, and the entire viral genome of 8132 base pairs was subsequently amplified and sequenced. Analysis revealed canonical papillomavirus architecture, including the early open reading frames E6, E7, E1, and E2 and the 2 late proteins L1 and L2. FgPV1 is most closely related to a cluster of avian and reptilian papillomaviruses as visualized by phylogenetic trees. This observation suggests that papillomavirus virion production can occur in mesenchymal cells. Keywords birds, histocytochemistry, chondrocytes, papillomavirus infections, inclusion bodies-viral, transmission electron microscopy, genome-viralThe Papillomaviridae family continues to expand with the ongoing discovery of previously unidentified species occupying over 30 different genera. 46 Avian papillomaviruses have been described in more than 12 avian species and 6 orders 27 since the 1970s. 28,33 In that time, only 3 have been fully sequenced: Francolinus leucoscepus papillomavirus 1 (FlPV1), Psittacus erithacus papillomavirus (PePV), and Fringilla coelebs papillomavirus (FcPV).31,41,44 Among species described so far, avian papillomaviruses and mammalian papillomaviruses share epitheliotropism and similar tissue distribution. In psittacines, virus has been detected in intestinal mucosal cells, 13,16,19,23,27,30,40 nasolacrimal epithelium, 13,16,22 and oral mucosa at the commissure of beaks. 22There is also a report of papillomavirus-associated growths on the legs and feet of two related finch species, Fringilla. coelebs and Fringilla montifringilla. 31 To date, there have been no reports of papillomaviruses or any neoplastic lesions in free-ranging seabirds despite the identification of papillomaviruses in numerous marine mammals, 5,35 including dolphins, 36,37 porpoises, 43 and sea turtles. 18Regardless of host species, productive papillomavirus infection (formation of infectious particles) has been invariably tied to the terminal differentiation of epithelial cells. A small number of papillomaviruses are capable of infecting and transforming fibroblasts, but these infections are considered nonproductive. The members of the Deltapapillomavirus genus such as bovine (Bos taurus) papillomaviruses 1 and 2 (BPV1, BPV2), for example, can infect fibroblasts in cattle, horses, and cats and is associated with transformation in these cells, causing the formation of fibropapillomas, also known as sarcoids in nonbovid species. 20 In these cases, virion production is considered limited to infected cells within the o...

There is evidence that raccoon polyomavirus is causative for neuroglial brain tumors in the western United States. It is unknown if infection is limited to geographic locales where tumors have been reported or is widespread, like human polyomaviruses. We demonstrate raccoons in western, eastern and midwestern states have been exposed to RacPyV by detection of antibodies to capsid protein, VP1. While raccoons in eastern and midwestern states are seropositive, exposure is lower than in the western states. Additionally, across geographic areas seropositivity is higher in older compared to younger raccoons, similar to polyomavirus exposure in humans. Serum titers are significantly higher in raccoons with tumors compared to raccoons without. Unlike polyomavirus-associated diseases in humans, we did not detect significant sequence variation between tumor and non-tumor tissue in raccoons with tumors compared to those without tumors. This warrants further investigation into co-morbid diseases or genetic susceptibility studies of the host.

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