Investigation of Equus caballus papillomavirus type-2 (EcPV-2) in asymptomatic and symptomatic horses

Abstract

Papillomavirus (PV)-associated cancers are a well-recognized entity in humans as well as domestic and wild animals. Equus caballus papillomavirus type-2 (EcPV-2) has recently been recognized as a potential cause of genital squamous cell carcinomas (SCCs) in horses. However, little is currently understood regarding EcPV-2 in terms of exposure, transmission, persistent infection, clearance, interactions with host immune and cell proliferation pathways, nor its prognostic impact on clinical outcome – all critical information required for the development and application of meaningful diagnostic, therapeutic, prognostic and preventative measures. The current working theory of EcPV-2 in horses is that asymptomatic infection is rare, the virus shows strict genital tropism, transmission is via sexual contact, and EcPV-2 is a causal factor in genital SCCs, but not non-genital lesions. Here we review the current understanding of PV-associated cancer as it pertains to the better understood human papillomavirus (HPV) and bovine papillomavirus (BPV). We also highlight deficits in the literature as they relate specifically to EcPV-2 and horses. In Chapters 2 and 3, we use polymerase chain reaction (PCR) and RNA- in situ hybridization (R-ISH) to show that asymptomatic EcPV-2 infection, and genital SCC-associated EcPV-2 infections, are statistically similar amongst Western Canadian horses. Similarly, we show that EcPV-2 exposure, as measured by serology, is also not infrequent. We also identify in utero and aborted fetuses infected with EcPV-2. In Chapter 3, we measure clinical outcome (overall survival time) through the use of a retrospective survey, issued to Western Canadian veterinary practitioners. We show that the EcPV-2 status of genital SCCs is not a prognosticating factor. We identify treatment status and recurrence as major prognosticating factors, whereas histologic grade, treatment type, duration of lesions, and co-morbidity are not. In Chapter 4, we use a more extensive database to explore better the possibility of EcPV-2 infection in non-genital SCCs, such as those arising from the ocular and periocular tissues, something not identified in our Western Canadian population in Chapter 3. Here we again use PCR and R-ISH to identify viral nucleic acids in equine SCCs arising from a variety of anatomic locations, sampled from horses in the Colorado State University, Veterinary Diagnostic Laboratory service area. In doing so, we compile a collection of EcPV-2 positive non-genital SCCs that serve two purposes: 1) Provide additional data to compare to our asymptomatic tissues from Chapter 2, and 2) Act as the foundation for the materials needed in our Chapter 5 study. In Chapter 5, we use immunohistochemistry (IHC) to compare EcPV-2 status on cell cycle protein expression in both SCCs (genital and non-genital) and healthy tissues. We show that there is a difference in p53 expression of genital SCCs when compared by EcPV-2 status. We observe that p53 and Ki-67 expression is increased in an EcPV-2 positive genital SCC and is present in both the stratum basale and supra-basilar layers, whereas, the EcPV-2 negative genital SCC shows p53 and Ki-67 staining only within the stratum basale. A similar pattern was not readily evident in non-genital SCCs. In Chapter 5, we further attempt to infer p53 functionality by using IHC to detect changes in downstream effectors such as p21, Ki-67, and activated caspase-3. Finally, we strive to detect the presence of mutant p53 in equine SCCs. The combined conclusions of these works are 1) EcPV-2 exposure and asymptomatic infection are frequent in Western Canadian horses, 2) exposure likely occurs early in life, 3) transmission can occur in utero as well as through non-sexual routes, 4) the detection rate of EcPV-2 in SCCs is similar to that of healthy skin, but EcPV-2 infection in non-genital SCCs appears to be less 'intense' as compared to genital SCCs, 5) EcPV-2 status of genital SCCs does not impact overall survival times and, 6) EcPV-2 infection does influence p53 expression, although not in the anticipated manner of triggering p53 proteasomal degradation. We believe that all the above findings provide valuable contributions to the growing body of knowledge regarding EcPV-2 infection in horses and that further research is warranted.