Paul Hick

Identification and characterisation of an ostreid herpesvirus-1 microvariant (OsHV-1 µ-var) in Crassostrea gigas (Pacific oysters) in Australia.

Between November 2010 and January 2011, triploid Crassostrea gigas (Pacific oysters) cultivated in the Georges River, New South Wales, experienced >95% mortality. Mortalities also occurred in wild diploid C. gigas in the Georges River and shortly thereafter in the adjacent Parramatta River estuary upstream from Sydney Harbour. Neighbouring Saccostrea glomerata (Sydney rock oysters) did not experience mortalities in either estuary. Surviving oysters were collected to investigate the cause of mortalities. Histologically all oysters displayed significant pathology, and molecular testing revealed a high prevalence of ostreid herpesvirus-1 (OsHV-1). Quantitative PCR indicated that many C. gigas were carrying a high viral load at the time of sampling, while the load in S. glomerata was significantly lower (p < 0.001). Subsequent in situ hybridisation experiments confirmed the presence of a herpesvirus in C. gigas but not S. glomerata tissues, suggesting that S. glomerata is not susceptible to infection with OsHV-1. Naïve sentinel triploid C. gigas placed in the Georges River estuary in January 2011 quickly became infected and experienced nearly 100% mortality within 2 wk of exposure, indicating the persistence of the virus in the environment. Phylogenetic analysis of sequences derived from the C2/C6 region of the virus revealed that the Australian strain of OsHV-1 belongs to the microvariant (µ-var) cluster, which has been associated with severe mortalities in C. gigas in other countries since 2008. Environmental data revealed that the Woolooware Bay outbreaks occurred during a time of considerable environmental disturbance, with increased water temperatures, heavy rainfall, a toxic phytoplankton bloom and the presence of a pathogenic Vibrio sp. all potentially contributing to oyster stress. This is the first confirmed report of OsHV-1 µ-var related C. gigas mortalities in Australia.

Characterization of Virulent West Nile Virus Kunjin Strain, Australia, 2011

An encephalitis outbreak among horses was caused by a pathogenic variant of Kunjin virus.

Distribution and Host Range of Ranaviruses

Ranaviruses are globally distributed pathogens in amphibian, fish, and reptile communities that appear to be emerging. Cases of ranavirus infection or disease have been confirmed in at least 105 amphibian species (18 families), 41 fish species (22 families), and 29 reptile species (12 families). Ranaviruses have been documented on all continents except Antarctica, and are frequently associated with mass die-offs. Host susceptibility differs among species, with some species harboring subclinical infections and likely serving as reservoirs for the virus, and other highly susceptible species amplifying the virus. Currently, there are six recognized species of ranavirus, and all are not equally pathogenic among hosts. Frog virus 3 (FV3) is the type species of the genus Ranavirus, and appears to be the most globally distributed species infecting ectothermic taxonomic across three vertebrate classes. International commerce involving subclinically infected ectothermic vertebrates undoubtedly has contributed to the global distribution and emergence of ranaviruses. Herein, we describe the global distributed species infecting ectothermic vertebrates across three taxonomic classes.

Optimisation and validation of a real-time reverse transcriptase-polymerase chain reaction assay for detection of betanodavirus.

A RT-qPCR assay that was developed and optimised for detection of betanodaviruses was validated for use as a diagnostic test for viral nervous necrosis disease of fish. Four betanodavirus genotypes were detected but the sensitivity was greatest for redspotted grouper nervous necrosis virus (RGNNV). The analytical sensitivity was 10-1000-fold greater than that of a nested RT-PCR assay and the limit of detection was <0.4 TCID(50) units per reaction. The assay was highly repeatable (standard deviation of estimated log(10)(viral copies) 0.10+/-0.08) and reproducible (standard deviation of estimated log(10)(viral copies) 0.08+/-0.06). Diagnostic accuracy was assessed in 2193 samples comprising tissue homogenates from Australian bass (Macquaria novemaculeata) and barramundi (Lates calcarifer), and also in SSN-1 tissue culture supernatants, using virus isolation in striped snake head (SSN-1) cell culture as the gold standard. Diagnostic sensitivity and specificity were 100% when the assay was applied to Australian bass tissue and SSN-1 tissue culture supernatants, but for barramundi tissue were 99.1% and 92.8%, respectively. The apparent imperfect specificity was shown by specific amplification of alternate regions of the betanodavirus genome to be due to the lower sensitivity of virus isolation. This is the first study to report the diagnostic performance of a RT-qPCR assay for detection of betanodavirus.

A SIMPLE CENTRIFUGATION METHOD FOR IMPROVING THE DETECTION OF OSTREID HERPESVIRUS-1 (OsHV-1) IN NATURAL SEAWATER SAMPLES WITH AN ASSESSMENT OF THE POTENTIAL FOR PARTICULATE ATTACHMENT

Ostreid herpesvirus-1 (OsHV-1) is responsible for massive mortality events in commercially farmed Pacific oysters (Crassostrea gigas) in Australia, New Zealand, Europe and the USA. Economic losses have been severe in many countries since 2008, associated with a strain known as OsHV-1μ-var. Despite intensive studies of the virus itself, there is almost no information on its detection in natural seawater, how it is spread over wide geographic distance in water or on how it is transmitted from oyster to oyster via seawater. The aim of the current work was to (1) assess and compare several centrifugation methods in order to detect OsHV-1 in natural seawater samples using real-time quantitative PCR, in such a way that large numbers of samples could be processed efficiently and (2) assess the potential for particulate attachment of OsHV-1 using filtration. Compared to testing unprocessed seawater samples, centrifugation of seawater at 1000×g for 20 min with testing of the pellet improved OsHV-1 detection rates by two fold. Results suggest that OsHV-1 may be attached to particles large enough to be pelleted at low g-force, as well as in the form of small particles, free virus or free viral DNA. Filtration of seawater using low protein binding filters could not be used to assess OsHV-1 particle attachment, due to interactions between particles, free virus or free viral DNA and the membranes.

ICTV Virus Taxonomy Profile: Iridoviridae

The Iridoviridae is a family of large, icosahedral viruses with double-stranded DNA genomes ranging in size from 103 to 220 kbp. Members of the subfamily Alphairidovirinae infect ectothermic vertebrates (bony fish, amphibians and reptiles), whereas members of the subfamily Betairidovirinae mainly infect insects and crustaceans. Infections can be either covert or patent, and in vertebrates they can lead to high levels of mortality among commercially and ecologically important fish and amphibians. This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Iridoviridae, which is available at www.ictv.global/report/iridoviridae.

Comparative Pathology of Ranaviruses and Diagnostic Techniques

Recognizing the pathological changes caused by ranaviruses, understanding how to properly collect test samples, and knowing what diagnostic tools to choose are key to detecting ranaviruses and in determining whether they are a factor in morbidity and mortality events. Whether infection occurs in fish, reptiles, or amphibians, clinical disease is typically acute and can affect a high proportion of the population. Among ectothermic vertebrates, affected individuals can present with hemorrhages, edema, and necrosis. Generally, microscopic examination reveals intracytoplasmic inclusion bodies and necrosis of hematopoietic tissues, vascular endothelium, and epithelial cells. Ultimately, the type and severity of the lesions that develop vary depending upon the host species, type of ranavirus, or environmental factors. Our ability to identify lesions caused by ranaviruses is improving because of the knowledge gained from laboratory experiments and the improvement of existing, or development of new diagnostic tests. There is no single Gold Standard test for ranavirus detection, rather the diagnostic test chosen depends on the question asked. For example, a surveillance study may use quantitative real-time PCR (qPCR) to detect ranaviruses, but an investigation of a mortality event may use virus isolation, qPCR, histopathology, electron microscopy, and bioassay. To date, a treatment for ranavirus infections has not been found; however, vaccine development against iridoviruses is showing promise for both DNA and live vaccines within the aquaculture industry.

Optimization of Betanodavirus culture and enumeration in striped snakehead fish cells

An optimized culture method for detection of infection of fish with the Red spotted grouper nervous necrosis virus (RGNNV) genotype of betanodavirus in striped snakehead (SSN-1, Channa striatus) cells is described. Inoculation of fish tissue homogenates at the same time or within 4 hr of seeding the SSN-1 cells was as sensitive as the method recommended by the World Organization for Animal Health, where homogenates were adsorbed onto an established cell monolayer. Such modification halved the time required and the costs of consumables, and reduced the potential for error when processing large numbers of samples. Positive culture results were obtained from 88.3% of 392 fish tissue homogenates in which RGNNV was detected using a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay; 99.7% of 943 tissue homogenates, which were qRT-PCR negative, were cell culture negative. Cytopathic effect (CPE) was characterized by large intracytoplasmic vacuoles in 0.1–60% of cells. Detachment of affected cells from the culture surface resulting in progressive disruption of the monolayer occurred in 46.4% of primary cultures and 96.0% of subcultures of positive samples. Identification of CPE that did not disrupt the cell monolayer increased estimates of the 50% tissue culture infective dose (TCID50) by 1.07–2.79 logs (95% confidence interval). The predicted mean TCID50/ml was 3.3 logs higher when cells were inoculated less than 36 hr after subculture at less than 80% confluence compared to cells inoculated at greater than 80% confluence and more than 36 hr after subculture (P < 0.05).

Hendra Virus Infection in Dog, Australia, 2013.

Hendra virus occasionally causes severe disease in horses and humans. In Australia in 2013, infection was detected in a dog that had been in contact with an infected horse. Abnormalities and viral RNA were found in the dog’s kidney, brain, lymph nodes, spleen, and liver. Dogs should be kept away from infected horses.

Bayesian estimation of diagnostic sensitivity and specificity of a nervous necrosis virus antibody ELISA

Diagnosis of nervous necrosis virus (NNV) infection in susceptible fish species is mostly performed post-mortem due to the neurotropism of the causative agent and the only validated diagnostic assays require samples from brain and retinal tissue. However, a non-lethal alternative to test for exposure of fish to NNV is needed. An indirect ELISA for the detection of anti-NNV antibodies in was recently developed and evaluated to detect responses in the sera from immunized fish. For this study, we assessed the accuracy of the assay at detecting specific antibodies from naturally exposed fish using field samples from populations with differing infection status. We applied a Bayesian model, using RTqPCR as a second test. Median estimates of the diagnostic sensitivity and specificity of the VNN ELISA were 81.8% and 86.7%, respectively. We concluded that the assay was fit for the purpose of identifying animals in naturally exposed populations. With further evaluation in larger populations the test might be used to inform implementation of control measures, and for estimating infection prevalence to facilitate risk analysis. To our knowledge this is the first report on the diagnostic accuracy of an antibody ELISA for an infectious disease in finfish.