Date Published: October 24, 2018
Publisher: Public Library of Science
Author(s): Jing Zhang, Deborah S. Finlaison, Melinda J. Frost, Sarah Gestier, Xingnian Gu, Jane Hall, Cheryl Jenkins, Kate Parrish, Andrew J. Read, Mukesh Srivastava, Karrie Rose, Peter D. Kirkland, Luis Menéndez-Arias.
In mid-February 2015, a large number of deaths were observed in the sole extant population of an endangered species of freshwater snapping turtle, Myuchelys georgesi, in a coastal river in New South Wales, Australia. Mortalities continued for approximately 7 weeks and affected mostly adult animals. More than 400 dead or dying animals were observed and population surveys conducted after the outbreak had ceased indicated that only a very small proportion of the population had survived, severely threatening the viability of the wild population. At necropsy, animals were in poor body condition, had bilateral swollen eyelids and some animals had tan foci on the skin of the ventral thighs. Histological examination revealed peri-orbital, splenic and nephric inflammation and necrosis. A virus was isolated in cell culture from a range of tissues. Nucleic acid sequencing of the virus isolate has identified the entire genome and indicates that this is a novel nidovirus that has a low level of nucleotide similarity to recognised nidoviruses. Its closest relatives are nidoviruses that have recently been described in pythons and lizards, usually in association with respiratory disease. In contrast, in the affected turtles, the most significant pathological changes were in the kidneys. Real time PCR assays developed to detect this virus demonstrated very high virus loads in affected tissues. In situ hybridisation studies confirmed the presence of viral nucleic acid in tissues in association with pathological changes. Collectively these data suggest that this virus is the likely cause of the mortalities that now threaten the survival of this species. Bellinger River Virus is the name proposed for this new virus.
The Bellinger River snapping turtle, Myuchelys georgesi, is a species of freshwater turtle that, prior to this outbreak, was rare and has a very restricted habitat. It is confined solely to a 60 kilometre section of the Bellinger River, and a short section of the adjacent Kalang River in northern coastal New South Wales (NSW), Australia. While the turtle had been described as “locally abundant” it was also described as “meets the criteria for being listed as a vulnerable species under the Threatened Species Conservation Act of NSW” . In 2014 it was estimated that there were approximately 2500 of this species in the wild . Commencing in mid-February 2015, a number of people using and managing the environment surrounding the Bellinger River observed a large number of deaths in M. georgesi. A multi-agency investigation was undertaken to establish the cause and extent of the outbreak. Mortalities continued for 7 weeks and involved mostly adult animals. More than 400 dead or dying animals were observed and population surveys conducted after the outbreak had ceased indicated that only a small proportion of the total population had survived with very few adults remaining. No other species, including the sympatric Murray River turtle (Emydura macquarii), appeared to be affected. Full details of the prevailing environmental conditions and the extent of the investigation have been described by others . A number of moribund and dead M. georgesi were collected for post mortem examination at the Australian Registry of Wildlife Health, Taronga Conservation Society Australia Mosman, NSW. Tissue samples were referred to a number of different laboratories to test for a wide range of potential pathogens, toxins and water quality assessment. No consistent results were obtained from bacterial cultures and tests for mycoplasma, trichomonas, chlamydia and toxins gave negative results. On the basis of the histological changes and a failure to identify any other causative agent, a viral aetiology was suspected. Testing was undertaken at several other laboratories and ranaviruses, adenoviruses, paramyxoviruses (ferlavirus) and herpesviruses were excluded . Samples were also referred to the Elizabeth Macarthur Agriculture Institute, Menangle NSW. Here we report the isolation and characterisation of a novel nidovirus and provide evidence for its involvement as the principal pathogen in this disease outbreak.
The virus described in this study, for which the name “Bellinger River virus” (BRV) is proposed, is believed to have had a profound impact on the wild Myuchelys georgesi population. The data presented provide strong, indirect evidence that this virus is the principal aetiological agent involved in the deaths of these M. georgesi. Unfortunately, as this is a now a critically endangered species of turtle , it is not possible to undertake experimental transmission studies to fulfil Koch’s postulates. Nevertheless, the criteria for disease causation defined by Fredericks and Relman  have been met. BRV, as a novel nidovirus, was isolated from tissues of diseased animals, very high levels of viral RNA were detected in tissues with marked pathological changes and in situ hybridisation assays demonstrated the presence of specific viral RNA in lesions in kidneys and eye tissue–two of the main affected organs. No or very low levels of viral RNA were detected in normal animals tested at the time of the outbreak. Collectively these data suggest that this virus is the likely cause of these mortalities. The high levels of viral RNA in several different organ systems would suggest that this virus is actively replicating in these organs and these detections are not an incidental finding or due to contamination as a result of either ingestion or inhalation of virus from the environment. Nevertheless, although we believe that BRV is the principal pathogen, it is inevitable that other factors are likely to have contributed to the onset and severity of disease. The turtles were probably already in a stressed and potentially immunosuppressed state as they had lost considerable body condition . The higher water temperatures may have supported and perhaps enhanced virus replication, a phenomenon that is well known for a number of aquatic viruses [32, 33] and, although other pathogens have not been identified, it is likely that other microbes, even commensals, may have contributed to disease severity and ultimately the death of these turtles.