Abstract
Over the past 30 years, there has been a consistently high prevalence of urogenital carcinoma in adult California sea lions (Zalophus californianus) stranding along the west coast of the US.1-3 Primary tumors form in the vagina, cervix or penis, and aggressively metastasize throughout the body, resulting in stranding or death.2 Contaminants, genetic predisposition and a virus, otarine herpesvirus 1 (OtHV1), have all been associated with this cancer, however the pathogenesis is not fully understood.4-8 OtHV1 has been detected in genital swabs from animals with and without urogenital carcinoma, making it unclear if this virus is causative or a consequence of this cancer.7 Here we explore the role of OtHV1 by examining the viral genome and gene expression patterns in cervix tissue from sea lions with and without urogenital carcinoma.
Next generation sequencing (MiSeq) was used to generate the OtHV1 genome from a sea lion cervical tumor. The viral genome was annotated and compared to those of herpesviruses that cause cancer in humans (Kaposi’s Sarcoma Herpesvirus and Epstein Barr Herpesvirus). Within the OtHV1 genome, several herpesvirus gene homologues known to induce cancer were identified. Next, RNA in situ hybridization probes (Basescope) were designed to bind to suspected viral oncogene mRNA to determine whether they were actively expressed in cancer tissue and/or in healthy sea lion cervix. Five OtHV1 genes of interest were assessed in nine normal cervices and 16 cervices with urogenital carcinoma.
In sea lions without cancer, no expression of OtHV1 genes was seen in normal cervical epithelium. However, in sea lions with urogenital carcinoma, there was very high expression of all OtHV1 genes examined within the carcinoma cells of the cervical epithelium. This indicates that OtHV1 expression is associated with tumor tissue and not normal cervical epithelium.
The homologous viral oncogenes, and localization of viral expression in cervical urogenital carcinoma cells, strongly supports the hypothesis that OtHV1 plays a significant role in the development of this persistently common cancer observed in California sea lions. Like herpesvirus-induced cancer in humans, it is likely that a variety of influences are affecting the pathogenicity of herpesvirus during the development of urogenital carcinoma. Future research on the effects of contaminant exposure and genetic predisposition on the behavior of OtHV1 may provide important insight into how virally induced cancer are perpetuated in sea lions as well as other mammalian species.
Acknowledgements
The study was funded by the Geoffrey Hughes Research Fellowship and The Marine Mammal Center. We thank the dedicated staff and volunteers at The Marine Mammal Center for the care provided to all animals. Particular thanks to Cara Field, Barbie Halaska, Christine Fontaine, Tenaya Norris, Lauren Rust, Erin Brodie, and Liz Wheeler for assisting with necropsies and tissue sampling. This study was permitted by the National Marine Fisheries Service Marine Mammal Protection Act permit number 18786 and the Institutional Animal Care and Use Committees of the University of Florida (no. 201608188) and the Marine Mammal Center (2014-1).
*Presenting author
Literature Cited
1. Gulland FMD, Trupkiewicz JG, Spraker TR, Lowenstine LJ. 1996. Metastatic carcinoma of probable transitional cell origin in 66 free-living California sea lions (Zalophus californianus), 1979 to 1994. J Wildl Dis 32:250–258.
2. Greig DJ, Gulland FMD, Kreuder C. 2005. A Decade of Live California Sea Lion (Zalophus californianus) Strandings Along the Central California Coast: Causes and Trends, 1991–2000. Aquatic Mammals 31:11–22.
3. Deming AC, Colegrove KM, Duignan PJ, Hall AJ, Wellehan JFX, Gulland FMD. 2018. Prevalence of Urogenital Carcinoma in Stranded California Sea Lions (Zalophus californianus) from 2005–15. J Wildl Dis 54:581–586.
4. Randhawa N, Gulland FMD, Ylitalo GM, DeLong R, Mazet JAK. 2015. Sentinel California sea lions provide insight into legacy organochlorine exposure trends and their association with cancer and infectious disease. One Health 1:37–43.
5. Acevedo-Whitehouse K, Gulland FMD, Greig D, Amos W. 2003. Inbreeding: Disease susceptibility in California sea lions. Nature 422:35.
6. King DP, Hure MC, Goldstein T, Aldridge BM, Gulland FMD, Saliki JT, Buckles EL, Lowenstine LJ, Stott JL. 2002. Otarine herpesvirus-1: a novel gammaherpesvirus associated with urogenital carcinoma in California sea lions (Zalophus californianus). Vet Microbiol 86:131–137.
7. Buckles EL, Lowenstine LJ, DeLong RL, Melin SR, Vittore RK, Wong HN, Ross GL, St Leger JA, Greig DJ, Duerr RS, Gulland FMD, Stott JL. 2007. Age-prevalence of Otarine Herpesvirus-1, a tumor-associated virus, and possibility of its sexual transmission in California sea lions. Vet Microbiol 120:1–8.
8. Gulland FMD, Hall AJ, Ylitalo GM, Colegrove KM, Norris T, Duignan PJ, Halaska B, Acevedo Whitehouse K, Lowenstine LJ, Deming AC and Rowles TK (2020) Persistent Contaminants and Herpesvirus OtHV1 Are Positively Associated with Cancer in Wild California Sea Lions (Zalophus californianus). Front. Mar. Sci. 7:602565.