Performance characteristics of two real-time TaqMan polymerase chain reaction assays for the detection of WSSV in clinically diseased and apparently healthy prawns.

This study aimed to generate data on performance characteristics for 2 real-time TaqMan PCR assays (CSIRO and WOAH WSSV qPCRs) for the purposes of (1) detection of white spot syndrome virus (WSSV) in clinically diseased prawns and (2) detection of WSSV in apparently healthy prawns. Analytical sensitivity of both assays was 2 to 20 genome copies per reaction, and analytical specificity was 100% after testing nucleic acid from 9 heterologous prawn pathogens and 4 prawn species. Results obtained after testing more than 20 000 samples in up to 559 runs with the CSIRO WSSV qPCR and up to 293 runs with the WOAH WSSV qPCR demonstrated satisfactory repeatability for both assays. Both assays demonstrated median diagnostic sensitivity (DSe) 100% (95% CI: 94.9-100%) when testing clinically diseased prawns. When 1591 test results from apparently healthy prawns were analysed by Bayesian latent class analysis, median DSe and diagnostic specificity (DSp) were 82.9% (95% probability interval [PI]: 75.0-90.2%) and 99.7% (95% PI: 98.6-99.99%) for the CSIRO WSSV qPCR and 76.8% (95% PI: 68.9-84.9%) and 99.7% (95% PI: 98.7-99.99%) for the WOAH WSSV qPCR. When both assays were interpreted in parallel, median DSe increased to 98.3 (95% PI: 91.6-99.99%), and median DSp decreased slightly to 99.4% (95% PI: 97.9-99.99%). Routine testing of quantified positive controls by laboratories in the Australian laboratory network demonstrated satisfactory reproducibility of the CSIRO WSSV qPCR assay. Both assays demonstrated comparable performance characteristics, and the results contribute to the validation data required in the WOAH validation pathway for the purposes of detection of WSSV in clinically diseased and apparently healthy prawns.

Isolation and characterisation of an ISKNV-genotype megalocytivirus from imported angelfish Pterophyllum scalare.

Using cultures of the SKF-9 cell line, megalocytivirus AFIV-16 was isolated from imported angelfish Pterophyllum scalare held in quarantine at the Australian border. The cytopathic effect caused by isolate AFIV-16 presented as cell rounding and enlargement, but complete destruction of the infected cell cultures did not occur. The infected cells demonstrated immunocytochemical reactivity with monoclonal antibody M10, which is used for diagnosis of OIE-listed red sea bream iridoviral disease. Using electron microscopy, the virus particles, consisting of hexagonal nucleocapsids, were observed in the cytoplasm of SKF-9 cells. The replication of AFIV-16 in cultured SKF-9 cells was significantly greater at 28°C incubation than at 22 and 25°C incubation, whereas no difference in growth characteristics was observed for red sea bream iridovirus (RSIV) isolate KagYT-96 across this temperature range. Whole genome sequencing demonstrated that AFIV-16 has a 99.96% similarity to infectious spleen and kidney necrosis virus (ISKNV), the type species in the genus Megalocytivirus. AFIV-16 was classified into ISKNV genotype Clade 1 by phylogenetic analysis of the major capsid protein gene nucleotide sequence. This is the first report of whole genome sequencing of an ISKNV genotype megalocytivirus isolated from ornamental fish.

Pilchard orthomyxovirus (POMV). I. Characterisation of an emerging virus isolated from pilchards Sardinops sagax and Atlantic salmon Salmo salar.

An orthomyxo-like virus was first isolated in 1998 as an incidental discovery from pilchards Sardinops sagax collected from waters off the South Australian coast. In the following 2 decades, orthomyxo-like viruses have been isolated from healthy pilchards in South Australia and Tasmania. In 2006, an orthomyxo-like virus was also isolated from farmed Atlantic salmon Salmo salar in Tasmania during routine surveillance and, again, from 2012 onwards from diseased Atlantic salmon. Using transmission electron microscopy, these viruses were identified as belonging to the family Orthomyxoviridae. To further characterise the viruses, the genomes of 11 viral isolates were sequenced. The open reading frames (ORFs) that encode 10 putative proteins from 8 viral genome segments were assembled from Illumina MiSeq next generation sequencing (NGS) data. The complete genome of a 2014 isolate was also assembled from NGS, RNA-sequencing (RNA-seq) data, that included conserved motifs that shared commonalities with infectious salmon anaemia virus, rainbow trout orthomyxovirus and Influenzavirus A. The presence of 8 viral proteins translated from genome segments was confirmed by mass spectrometric analysis including 2 novel proteins with no known orthologs. Sequence analysis of the ORFs, non-coding regions and proteins indicated that the viruses had minimal diversity and hence were named pilchard orthomyxovirus (POMV), based on the fish host species of its first isolation. The low homology of POMV proteins with previously characterised orthomyxoviruses suggests that POMV is the first virus to be characterised from a new genus within the Orthomyxoviridae. To facilitate more rapid detection and subsequent diagnostic confirmation of POMV infections, TaqMan and conventional nested PCRs were designed.

Diagnostic test accuracy when screening for Haliotid herpesvirus 1 (AbHV) in apparently healthy populations of Australian abalone Haliotis spp.

The accuracy of 3 real-time PCR assays (ORF49, ORF66 and ORF77) and histopathology was evaluated for the purpose of demonstrating or certifying abalone free from Haliotid herpesvirus 1 (AbHV), the causative agent of abalone viral ganglioneuritis. Analytically, all 3 qPCRs showed equivalent limit of detection (20 copies per reaction); however, ORF49 could not detect 2 of the AbHV genotypes. A selection of 1452 archive specimens sourced from apparently healthy abalone populations was screened using all 4 tests. In the absence of a perfect reference standard, a Bayesian latent class analysis was built to estimate diagnostic sensitivity (DSe), diagnostic specificity (DSp) and likelihood ratios of a positive (LR+) and negative test result (LR-) for each individual test and for all possible combinations of test pairs interpreted either in series or in parallel. The pair ORF49/ORF66 interpreted in parallel performed the best both analytically and diagnostically to demonstrate freedom from AbHV in an established population of abalone and to certify individual abalone free from AbHV for trade or movement purposes (DSe = 96.0%, 95% posterior credibility interval [PCI]: 82.6 to 99.9; DSp = 97.7%, 95% PCI: 96.4 to 99.4; LR+ = 41.4, 95% PCI: 27.4 to 148.7; LR- = 0.041, 95% PCI: 0.001 to 0.176). Histopathology showed very poor DSe (DSe = 6.3%, 95% PCI: 2.4 to 13.1) as expected since most infected abalone in the study were likely sub-clinical with limited pathological change. Nevertheless, we recommend histopathology when clinically investigating outbreaks to find potential, new, emerging AbHV genotype(s) that may not be detectable by either ORF49 or ORF66.

Preliminary characterization of Tasmanian aquareovirus (TSRV) isolates.

In an attempt to determine whether or not genetic variants of the Tasmanian strain of Atlantic salmon aquareovirus (TSRV) exist, 14 isolates of TSRV, originating from various locations in Tasmania, covering a 20-year period (1990-2010), obtained from various host species and tissues, and isolated on different cell lines, were selected for this study. Two categories, termed "typical" and "atypical", of variants of TSRV were identified based on preliminary genotypic and phenotypic characterization carried out on these 14 different isolates. In addition, electron microscopic examination indicated the existence of at least three variants based on viral particle size. Finally, this study demonstrated the existence of at least one new variant of TSRV isolates, other than the more commonly isolated typical TSRV isolates, in farmed Tasmanian Atlantic salmon.

Innate resistance of New Zealand paua to abalone viral ganglioneuritis.

The susceptibility of New Zealand paua (Haliotis iris) to infection by abalone herpesvirus (Haliotid herpesvirus 1; HaHV) and to the disease abalone viral ganglioneuritis (AVG) was determined. Infection challenges performed by intra-muscular injection and by immersion in infectious water containing HaHV demonstrated that New Zealand paua were highly resistant to infection by Haliotid herpesvirus 1 and were fully resistant to the disease AVG.

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.

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.

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.

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.

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 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.

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.

Development and characterisation of pilchard (Sardinops sagax neopilchardus) cell lines derived from liver and heart tissues.

Two cell lines have been established from juvenile pilchards (Sardinops sagax neopilchardus) caught in waters off the Victorian coast of Australia. Following establishment of primary cultures derived from different pilchard tissues, using various cell culture media, a pilchard liver (PL) cell line and a pilchard heart (PH) cell line have been maintained in Eagle's minimal essential medium supplemented with 10% foetal bovine serum for over four years. The cell lines have been cryopreserved in liquid nitrogen and can be recovered from storage with good cell viability. Stock cell cultures have been maintained at 20-22 degrees C on a continuous basis in normal atmosphere (100% air), with weekly subculture at a split ratio of 3:1. The origin of the cell cultures was confirmed by PCR analysis using primers designed to be specific for pilchard mitochondrial DNA. In addition, the liver cell line was cloned and both the parental cell line and clones thereof were shown to be susceptible to a broad range of marine and freshwater viral pathogens of fish.