Abstract

Cetacean morbillivirus (CeMV) is considered one of the most important viral pathogens in cetaceans. In infected cetaceans, the virus causes serious respiratory, central nervous system disease and immunosuppression which lead to serious secondary bacterial and fungal infections. CeMV was shown to be associated with high-mortality events of the population of several cetacean species along the coast of the United States, Spain, and Italy in the past three decades. Stranded cetacean events resulting from CeMV infection in other areas including Asia, Oceania, and South America also were sporadically reported. The information of CeMV infection is still lacking in cetaceans around these areas. In addition, in previous studies there were some cases that were positive in RT-PCR testing, but there were no typical pathological findings resulting from CeMV infection. It revealed that it could have been misdiagnosis of CeMV infection if researchers made a diagnosis only relying on the results of pathological examination on retrospective study. It also may bring risks of false-negative results in several conditions such as applying less sensitive detection method and the cases with a persistent infection with low copies of virus. To clarify the real distribution of CeMV and possible carriers, the retrospective study of CeMV is essential. However, fresh tissues as nucleic acid sources on retrospective study are more limited than formalin-fixed paraffin-embedded (FFPE) samples on the acquisition and preservation.

In this study, a high-efficient, sensitive and throughput real-time RT-PCR (qRT-PCR) combining with high-resolution melting (HRM) for genotyping using FFPE samples was developed. The result showed the 19-year stored FFPE sample still included sufficient amount and quality of RNA, indicating the feasibility of this study. The designed specific primers in qRT-PCR target on the highly conserved sequence of CeMV phosphoprotein gene. The Morbillivirus-positive FFPE samples from several countries (Spain, Brazil, Hawaii) and different virus strains (DMV, PWMV and novel CeMVs) were used. The result showed that this novel technique successfully amplified nucleic acid from every sample and the following HRM genotyping could distinguish different virus strains. This efficient method for diagnosing and genotyping of CeMV strains could be applied to the future retrospective studies using FFPE samples. Through investigation of the relationship of virus variation and responsiveness to CeMV in cetaceans, we will have a better understanding of not only the different prevalence and outbreak conditions between Atlantic and Pacific, but also the mechanism of variable host response to pathogens. This unique opportunity would help us in the future to elucidate the role of virus strains in disease susceptibility, resistance, and progression.

* Presenting author