Title:

Aquatic Animal Health Subprogram: Characterisation of abalone herpes-like virus infections in abalone

Project Number:

2009-032

Organisation:

CSIRO Australian Animal Health Laboratory

Principal Investigator:

Mark S. Crane

Project Status:

Completed

FRDC Expenditure:

$401,292.00

Program(s):

Environment

Need

At the National Abalone Health Workplan Priority Setting Workshop held on 18 June 2008 in Melbourne, attended by representatives from DPI Victoria, SARDI, FRDC, VADA, TAC, EZAIA), AAGA, ACA, CSIRO, DPIW Tasmania, RecFish, DAFF and NSW DPI, it was agreed that to address the main epidemiological questions research should be focussed on 4 areas: 1) Development of diagnostics techniques with the highest priorities being: - Technique to isolate and concentrate the virus from abalone tissues (addressed in FRDC Project 2007/006) - A rapid and specific diagnostic test-development of a validated PCR-test for the AVG virus (addressed in FRDC Project 2007/006) - Secondary diagnostic test (ISH development addressed in FRDC Project 2007/006) - Validation of diagnostic tests (partly addressed in proposed extension of FRDC Project 2007/006 (PCR) and this project (ISH validation)) - Rollout of diagnostic tests across the States (addressed in proposed extension of FRDC Project 2007/006) - Distribution of infective dose in tissues (to be addressed by this proposal) 2) Disease aetiology and transmission with the highest priorities being: - Other bio-vectors and abiotic factors (to be addressed by this proposal) - Determination of the susceptibility of remnant populations following exposure to AVG (to be addressed by this proposal) 3) Virus inactivation with the highest priorities being: - Determine the viability of the AVG virus, including disinfection efficacy; efficacy of treatments (to be addressed by this proposal) - Survival of the virus in seawater (to be addressed by this proposal) - Survival of the virus on fomites (to be addressed by this proposal) 4) Disease surveillance and modelling with the highest priorities being: - National survey of stocks to determine current distribution of the virus, including latency, and resistance status of stocks (partly addressed by this proposal) - Determine the mode(s) of spread of the AVG (not addressed)

Objectives

1. Validate the developed in situ hybridisation diagnostic test including roll out to other States

2. Develop a quantitative assay (qPCR) for determining infectious dose for this virus

3. Determine the sensitivity of the virus to physico-chemical conditions including its stability in water/on fomites and its sensitivity to inactivation agents

4. Determine the role of mucus in viral transmission

5. Determine whether a latent stage exists in AVG

6. Determine the susceptibility of remnant populations of abalone previously exposed to AVG and known unexposed wild populations in South Australia

Final Report - 2009-032-DLD - Characterisation of abalone herpes-like virus infections in abalone

Final Report
ISBN:978-1-4863-0402-8
ISSN:
Author(s):
Date Published:August 2014
Principal Investigator: Mark Crane

Keywords: Abalone viral ganglioneuritis (AVG); abalone herpesvirus (AbHV); diagnostic methods; in situ hybridisation (ISH); polymerase chain reaction (PCR); diagnostic test validation; virus transmission; virus inactivation; virus stability

Summary

Scientists at CSIRO Australian Animal Health Laboratory, the Department of Environment and Primary Industries Victoria, South Australian Research and Development Institute, the Department of Primary Industries, Parks, Water and Environment Tasmania, and the University of Adelaide, collaborated on a major project investigating various aspects of the biology, detection and identification of abalone herpesvirus, the causative agent of abalone viral ganglioneuritis. The various aspects under investigation included determining the stability of the virus under various physico‐chemical conditions, providing new information on the current diagnostic tests for the detection and identification of the virus including subclinical infections, and determining the susceptibility of various abalone and other molluscan species to
infection and disease.

Much of the work built on research undertaken on previous FRDC‐funded projects and involved using diagnostic tests and the in vivo infectivity model developed previously. All in vivo research was undertaken at the CSIRO Australian Animal Health Laboratory, a high‐level bio‐security laboratory located in Geelong.


Aims/objectives
1. Validate the developed in situ hybridisation diagnostic test including roll‐out to other States.
2. Develop a quantitative assay (qPCR) for determining the infectious dose for this virus.
3. Determine the sensitivity of the virus to physico‐chemical conditions including its stability in water/on fomites and its sensitivity to inactivation agents.
4. Determine the role of mucus in viral transmission.
5. Determine whether or not a latent stage exists in AVG.
6. Determine the susceptibility of remnant populations of abalone previously exposed to AVG and known unexposed wild populations in South Australia.
7. Using all three available qPCR tests, determine their relative sensitivities and specificities by retesting previously collected samples from the abalone populations in Tasmania (additional objective added during the project).

Results/key findings
The project was successful in achieving all its objectives. Firstly, the developed in situ hybridisation diagnostic test was validated and made available to diagnostic laboratories in Australia and overseas. 

Secondly, it has been shown that a quantitative polymerase chain reaction (qPCR) can be used to determine the infectious dose for this virus. An analysis of the qPCR tests has determined that the ORF49 and ORF66 qPCR tests, when used in parallel, have a diagnostic sensitivity (DSe) of 86% and a diagnostic specificity (DSp) of >98% for the detection of sub‐clinical infections. 

Moreover, the sensitivity of the virus to various physico‐chemical conditions including its stability in water/on fomites and its sensitivity to inactivation agents has been determined. While the virus can be present in mucus secreted by infected abalone, mucus does not appear to play a significant role in prolonging viral infectivity in seawater. 

Available data indicate that abalone herpesvirus (AbHV) can exist as a sub‐clinical infection, and this is supported by investigations into disease events. 

In addition, it was shown that remnant populations of wild abalone (in Victoria) previously exposed to AbHV (studied here and in FRDC Project 2009/075: Determining the susceptibility of remnant populations of abalone previously exposed to AVG) remain susceptible to infection and disease.
Furthermore, it has been shown that known unexposed wild populations in South Australia are susceptible to infection and disease