Epidemiology and pathogenesis of Nervous Necrosis Virus

Abstract

Viral Nervous Necrosis (VNN) is a globally distributed disease that affects a large number of finfish species, causing significant economic losses on affected farms. The causative agent is a small single stranded RNA virus called Nervous Necrosis Virus (NNV) from the genus Betanodavirus. NNV is neurotropic; clinical signs involve abnormal behaviour and high mortality associated with histopathological findings of vacuolating necrosis in the central nervous system and retina. In Australia, NNV has been isolated from Australian bass (Macquaria Novemaculeata) and barramundi (Lates calcarifer) populations recurrently for the past 10 and 25 years, respectively. However, the prognosis of NNV infection is highly variable. Although NNV became notorious for mass mortalities in marine fish hatcheries, it is often detected in apparently healthy individuals in the absence of clinical signs or histopathological lesions. Current knowledge on NNV epidemiology and pathogenesis is fragmentary. It is still unclear how the virus is transmitted between hosts and why some individuals are susceptible to VNN while others are not. The aim of this work was to study the epidemiology and pathogenesis of NNV in Australian native species with a focus on transmission and disease determinants to provide a basis for the development of prevention and control strategies. In Chapter 3, a partially retrospective study was conducted on the occurrence of NNV at the Darwin Aquaculture Centre (DAC), a barramundi hatchery. Observations on NNV detection frequency and distribution provided clues to the possible transmission pathways of the virus, including the potential role of broodstock as reservoirs, and the age-dependency of the disease. To assess the NNV exposure distribution among populations of adult fish, an indirect antibody detection ELISA was developed (Chapter 4). The assay was optimized and compared with a competitive ELISA format to provide the best discriminatory power between sera from immunized and non-immunized populations. After defining the best antibody detection protocol (the indirect ELISA), the diagnostic accuracy of the assay was assessed in naturally exposed subjects using a Bayesian approach in the absence of a gold standard (Chapter 5). After validation of the ELISA, a single point and repeated cross sectional analysis of NNV seroprevalence was conducted on native Australian adult fish populations (barramundi, Australian bass and groupers Epinephelus sp.) (Chapter 6). Survey results discredited the role of broodstock as NNV reservoirs based on the lack of correspondence between NNV seroprevalence and the occurrence of NNV outbreaks at the hatchery level. Results also suggested that the exposure of adult fish to NNV antigens must be progressive as seroconversion was often observed and the seroprevalence tended to be higher in older fish populations. From this and previous accumulated epidemiological evidence, horizontal transmission of NNV was considered most likely. An environmental reservoir outside the hatcheries has yet to be investigated. In Chapter 7, the factors influencing the pathogenesis of NNV in barramundi were explored. Juveniles of different ages were challenged by immersion to analyse the influence of the age of the host on VNN disease expression. Additionally, to test the influence of the virus isolate, juveniles were challenged with two inoculums obtained from NNV outbreaks in barramundi populations with different disease presentation (clinical and subclinical). Results showed that fish from all the age groups tested (range 20 to 63 days post hatch) were susceptible to NNV infection. However, the survival of the fish following NNV challenge was highly influenced by the age of the host. Juveniles of 5 weeks of age and older showed no clinical signs and their survival odds were the same as the non-challenged controls, whereas younger fish developed clinical disease. No significant effect on disease severity was noted between different NNV isolates. In Chapter 8, the factors influencing the pathogenesis of NNV in Australian bass were explored. In addition to the age of the host and the isolate factor, the influence of the dose of the virus and the water temperature on VNN expression was examined. As with barramundi, the disease expression in Australian bass was age dependent. The severity of the disease was affected by the water temperature in younger fish but it did not affect the outcome in fish above 5 weeks of age. The dose of the virus influenced the incidence of infection but not the severity of the disease expression. Again, no significant effect on disease severity was noted between the two isolates tested. From the experimental challenge of barramundi and Australian bass, further observations on NNV pathogenesis were provided: incubation period, minimum infectious dose, tissue distribution, shedding and humoral immune response. The results from this study narrow the knowledge gap on NNV transmission mechanisms and provide important insights into the virus pathogenesis in barramundi and Australian bass. The virus is most likely being transmitted horizontally and VNN disease expression as distinct from infection with NNV is highly age dependent. From this evidence, recommendations are made on the direction of efforts to control VNN at the farm level.