Lynette M. Williams

Development and validation of a TaqMan ® PCR assay for the Australian abalone herpes-like virus

The recent emergence of a herpes-like virus in both farmed and wild populations of abalone in Victoria, Australia, has been associated with high mortality rates in animals of all ages. Based on viral genome sequence information, a virus-specific real-time TaqMan assay was developed for detection and identification of the abalone herpes-like virus (AbHV). The assay was shown to be specific as it did not detect other viruses from either the Herpesvirales or the Iridovirales orders which have genome sequence similarities. However, the TaqMan assay was able to detect DNA from the Taiwanese abalone herpes-like virus, suggesting a relationship between the Taiwanese and Australian viruses. In addition, the assay detected < 300 copies of recombinant plasmid DNA per reaction. Performance characteristics for the AbHV TaqMan assay were established using 1673 samples from different abalone populations in Victoria and Tasmania. The highest diagnostic sensitivity and specificity were 96.7 (95% CI: 82.7 to 99.4) and 99.7 (95% CI: 99.3 to 99.9), respectively, at a threshold cycle (C(T)) value of 35.8. The results from 2 separate laboratories indicated good repeatability and reproducibility. This molecular assay has already proven useful in confirming presumptive diagnosis (based on the presence of ganglioneuritis) of diseased abalone in Victorian waters as well as being a tool for surveillance of wild abalone stocks in other parts of Australia.

New yellow head virus genotype (YHV7) in giant tiger shrimp Penaeus monodon indigenous to northern Australia

In 2012, giant tiger shrimp Penaeus monodon originally sourced from Joseph Bonaparte Gulf in northern Australia were examined in an attempt to identify the cause of elevated mortalities among broodstock at a Queensland hatchery. Nucleic acid extracted from ethanol-fixed gills of 3 individual shrimp tested positive using the OIE YHV Protocol 2 RT-PCR designed to differentiate yellow head virus (YHV1) from gill-associated virus (GAV, synonymous with YHV2) and the OIE YHV Protocol 3 RT-nested PCR designed for consensus detection of YHV genotypes. Sequence analysis of the 794 bp (Protocol 2) and 359 bp (Protocol 3) amplicons from 2 distinct regions of ORF1b showed that the yellow-head-complex virus detected was novel when compared with Genotypes 1 to 6. Nucleotide identity on the Protocol 2 and Protocol 3 ORF1b sequences was highest with the highly pathogenic YHV1 genotype (81 and 87%, respectively) that emerged in P. monodon in Thailand and lower with GAV (78 and 82%, respectively) that is enzootic to P. monodon inhabiting eastern Australia. Comparison of a longer (725 bp) ORF1b sequence, spanning the Protocol 3 region and amplified using a modified YH30/31 RT-nPCR, provided further phylogenetic evidence for the virus being distinct from the 6 described YHV genotypes. The virus represents a unique seventh YHV genotype (YHV7). Despite the mortalities observed, the role of YHV7 remains unknown.

EVALUATION OF ABALONE VIRAL GANGLIONEURITIS RESISTANCE AMONG WILD ABALONE POPULATIONS ALONG THE VICTORIAN COAST OF AUSTRALIA

ABSTRACT Between May 2006 and February 2010, abalone viral ganglioneuritis caused by abalone herpes virus (AbHV) spread along the coast of Victoria, Australia, and devastated wild abalone populations, causing high mortality (up to 90% in some areas). However, some abalone from the affected populations survived the epizootic and thus may be naturally resistant to the disease. To test this hypothesis, abalone (Haliotis rubra) from 5 reefs within the geographical range for AVG were collected and tested for resistance to infection and disease. Thus, mature survivors (abalone ∼160 mm in length) and juvenile “new recruits” (abalone ∼70 mm in length) were exposed to the virus using an experimental infectivity model to determine the presence of any potential resistance to the virus. Exposure to AbHV was performed by immersion using 3 serial viral dilutions to ensure that abalone were exposed to at least 1 viral concentration that would provide a morbidity dose of intermediate level. Results indicated that morbidity curves for the wild abalone groups (both mature and juvenile) were similar to those of the susceptible, naive, farmed hybrid (Haliotis laevigata × Haliotis rubra) abalone (positive control) groups. Histological lesions typical of abalone viral ganglioneuritis, and viral DNA, were detected in moribund, challenged abalone, confirming AbHV as the causative disease agent. Results suggest that the surviving wild abalone are not resistant to AbHV and were probably not exposed to pathogenic doses of the virus during the initial outbreak that commenced in 2006.

Abalone viral ganglioneuritis: Establishment and use of an experimental immersion challenge system for the study of abalone herpes virus infections in Australian abalone

In late 2005, acute mortalities occurred in abalone on farms located in Victoria, Australia. Disease was associated with infection by an abalone herpes virus (AbHV). Subsequently, starting in 2006, the disease (abalone viral ganglioneuritis; AVG) was discovered in wild abalone in Victorian open waters. Currently, it continues to spread, albeit at a slow rate, along the Victorian coast-line. Here, we report on experimental transmission trials that were carried out by immersion using water into which diseased abalone had shed infectious viral particles. At various time points following exposure, naïve abalone were assessed by an AbHV-specific real-time PCR and histological analyses including in situ hybridization (ISH). Results demonstrated that while exposed abalone began displaying clinical signs of the disease from 60 hours post exposure (hpe), they tested positive for the presence of viral DNA at 36 hpe. Of further interest, the AbHV DNA probe used in the ISH assay detected the virus as early as 48 hpe.

Australian abalone (Haliotis laevigata, H. rubra and H. conicopora) are susceptible to infection by multiple abalone herpesvirus genotypes

From 2006 to 2012, acute mortalities occurred in farmed and wild abalone (Haliotis spp.) along the coast of Victoria, Australia. The disease (abalone viral ganglioneuritis; AVG) is associated with infection by an abalone herpesvirus (AbHV). The relative pathogenicity of 5 known variants of AbHV was evaluated on abalone stocks from different states in Australia. Results indicated that all virus variants (Vic1, Tas1, Tas2, Tas3 and Tas4) cause disease and mortality in all abalone stocks tested (greenlip, blacklip and brownlip). In order to avoid further AVG outbreaks in Australian wild abalone, strict regulations on the transfer of abalone stocks must be implemented.

Immunological changes in response to herpesvirus infection in abalone Haliotis laevigata and Haliotis rubra hybrids

Australian abalone production has been affected by outbreaks of abalone viral ganglioneuritis (AVG) caused by a herpesvirus (AbHV). In this study, we undertook experimental transmission trials by immersion to study the abalone immune response to infection with AbHV. Representative cellular and humoural immune parameters of abalone, including total haemocyte count (THC), superoxide anion (SO) and antiviral activity against herpes simplex virus type 1 (HSV-1), were examined in apparently healthy (sub-clinical) and moribund abalone after challenge. In the early infection, sub-clinical stage (days 1-3), THC was found to increase significantly in infected abalone. TaqMan qPCR confirmed 20.5% higher viral load in moribund abalone compared to apparently healthy abalone, indicating that the abundance of AbHV within abalone is linked to their clinical signs. At the clinical stage of infection, THC was significantly lower in moribund abalone, but increased in AbHV-exposed but apparently healthy abalone, in comparison to non-infected controls. SO was reduced in all abalone that were PCR-positive for AbHV. THC and SO level were found to be negatively correlated with the presence of AbHV in abalone, but no effect of AbVH exposure was observed on the haemolymph antiviral activity. These results suggest that abalone mount an initial cellular immune response to AbHV infection, but this response cannot be sustained under high viral loads, leading to mortality.

Molecular characterisation of Australasian isolates of aquatic birnaviruses.

An aquatic birnavirus, first isolated in Australia from farmed Atlantic salmon in Tasmania in 1998, has continued to be re-isolated on an infrequent but regular basis. Due to its low pathogenicity, there has been little urgency to undertake a comprehensive characterisation of this aquatic birnavirus. However, faced with possible incursions of any new aquatic birnaviruses, specific identification and differentiation of this virus from other, pathogenic, aquatic birnaviruses such as infectious pancreatic necrosis virus (IPNV) are becoming increasingly important. The present study determined the nucleic acid sequence of the aquatic birnavirus originally isolated in 1998, as well as a subsequent isolate from 2002. The sequences of the VP2 and VP5 genes were compared to that of other aquatic birnaviruses, including non-pathogenic aquatic birnavirus isolates from New Zealand and pathogenic infectious pancreatic necrosis virus isolates from North America and Europe. The deduced amino acid (aa) sequences indicate that the Australian and New Zealand isolates fall within Genogroup 5 together with IPNV strains Sp, DPL, Fr10 and N1. Thus, Genogroup 5 appears to contain aquatic birnavirus isolates from quite diverse host and geographical ranges. Using the sequence information derived from this study, a simple diagnostic test has been developed that differentiates the current Australian isolates from all other aquatic birnaviruses, including the closely related isolates from New Zealand.

Development and application of molecular methods (PCR) for detection of Tasmanian Atlantic salmon reovirus

Molecular (PCR) diagnostic tests for the detection and identification of aquareovirus in general, and Tasmanian Atlantic salmon reovirus (TSRV) specifically, were developed, and their diagnostic sensitivity and specificity were determined and compared with virus isolation in cell culture. Intralaboratory and interlaboratory comparison of PCR (conventional hemi-nested RT-PCR & RT-qPCR) and virus isolation in cell culture using finfish cell lines, CHSE-214 and EPC, was carried out for the detection and identification of TSRV using field samples of farmed Atlantic salmon Salmo salar, L. from various aquaculture sites around Tasmania. The interlaboratory comparison of diagnostic methods was carried out between two laboratories, AAHL-CSIRO and DPIPWE-Tasmania. A total of 144 fish from nine sites (12-33 fish per site) were sampled from two regions of Tasmania (Tamar River estuary in the north and Huon River estuary in the south-east) during late spring to early summer of 2009, and the data were analysed using different statistical approaches. The prevalence of TSRV ranged from 6% to 22% in both regions. All the diagnostic methods (data from both laboratories) had high specificity, while the estimated sensitivity varied between tests with RT-qPCR being the most sensitive (95.2%) method followed by virus isolation and then conventional hemi-nested RT-PCR.

Molecular confirmation of infectious spleen and kidney necrosis virus (ISKNV) in farmed and imported ornamental fish in Australia.

Viruses of the genus Megalocytivirus have not been detected in wild populations of fish in Australia but circulate in imported ornamental fish. In 2012, detection of a megalocytivirus in healthy platys Xiphophorus maculatus was reported from a farm in Australia during surveillance testing as part of a research project undertaken at the University of Sydney. Confirmatory testing of the original samples at the AAHL Fish Diseases Laboratory verified the presence of an infectious spleen and kidney necrosis virus (ISKNV)-like virus. Additional sampling at the positive farm confirmed the persistence of the virus in the platys, with 39 of 265 (14.7%) samples testing positive. Comparison of 3 separate gene regions of the virus with those of ISKNV confirmed the detection of a virus indistinguishable from ISKNV. Subsequently, ISKNV was also detected in a range of imported ornamental fish from several countries between 2013 and 2014, by screening with real-time PCR and confirmation by conventional PCR and sequence analysis. Accordingly, the current importation of live ornamental fish acts as a potential perpetual source for the establishment of ISKNV viruses within Australia. The testing of the farmed and imported ornamental fish verified the utility of the probe-based real-time PCR assay for screening of ornamental fish for Megalocytivirus.

Molecular characterization of Tasmanian aquabirnaviruses from 1998 to 2013.

Tasmanian aquabirnaviruses (TABVs) have been isolated intermittently since 1998 from healthy Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss farmed in Macquarie Harbour, Tasmania, Australia. However, beginning in 2011, TABVs have been isolated from rainbow trout in association with mortality events. To determine if recent molecular changes in TABV were contributing to increased mortalities, next generation sequencing was undertaken on 14 TABVs isolated from 1998 to 2013. Sequencing of both genome segments and analysis of the 5 viral proteins they encode revealed that minimal changes had occurred in the past 15 yr. Of the amino acid changes detected only 1, alanine to aspartic acid at position 139 of the minor structural VP3 protein, was unique to the recent disease events. The most dramatic changes observed were in the length of the non-structural VP5 protein varying from 43 to 133 amino acids. However, the amino acid substitution in VP3 and variable VP5 length were unlikely to have resulted in increased TABV pathogenicity. The genome of a novel Australian aquabirnavirus, Victorian trout aquabirnavirus (VTAB) was also sequenced and compared to TABV isolates.