2022 taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

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.

To pool or not to pool? Guidelines for pooling samples for use in surveillance testing of infectious diseases in aquatic animals.

Samples from multiple animals may be pooled and tested to reduce costs of surveillance for infectious agents in aquatic animal populations. The primary advantage of pooling is increased population-level coverage when prevalence is low (<10%) and the number of tests is fixed, because of increased likelihood of including target analyte from at least one infected animal in a tested pool. Important questions and a priori design considerations need to be addressed. Unfortunately, pooling recommendations in disease-specific chapters of the 2018 OIE Aquatic Manual are incomplete and, except for amphibian chytrid fungus, are not supported by peer-reviewed research. A systematic review identified only 12 peer-reviewed aquatic diagnostic accuracy and surveillance studies using pooled samples. No clear patterns for pooling methods and characteristics were evident across reviewed studies, although most authors agreed there is a negative effect on detection. Therefore, our purpose was to review pooling procedures used in published aquatic infectious disease research, present evidence-based guidelines, and provide simulated data examples for white spot syndrome virus in shrimp. A decision tree of pooling guidelines was developed for use by peer-reviewed journals and research institutions for the design, statistical analysis and reporting of comparative accuracy studies of individual and pooled tests for surveillance purposes.

TaqMan real-time and conventional nested PCR tests specific to yellow head virus genotype 7 (YHV7) identified in giant tiger shrimp in Australia.

In 2013, a unique seventh yellow head virus genotype (YHV7) was detected in Black Tiger shrimp (Penaeus monodon) broodstock that suffered high mortality following their capture from Joseph Bonaparte Gulf (JBG) in northern Australia. To assist with its diagnosis and assessment of its distribution, prevalence and pathogenicity, YHV7-specific TaqMan real-time qPCR and conventional nested PCR primer sets were designed to ORF1b gene sequences divergent from the other YHV genotypes. Using high (≥108) copies of plasmid (p)DNA controls containing ORF1b gene inserts of representative strains of YHV genotypes 1-7, both PCR tests displayed specificity for YHV7. Amplifications of serial 10-fold dilutions of quantified YHV7 pDNA and synthetic ssRNA showed that both tests could reliably detect 10 genome copies. Pleopods/gills from wild P. monodon sourced from locations in geographically disparate regions across northern Australia as well as 96 juveniles (48 either appearing normal or displaying signs of morbidity) from a commercial pond experiencing mortalities were screened to partially validate the diagnostic capacity of the qPCR test. Based on these data and PCR primer/probe sequence mismatches with other newly identified YHV genotypes, both YHV7-specific PCR tests should prove useful in the sensitive detection and discrimination of this genotype from YHV 2 (gill-associated virus) and YHV6 that can occur in Australian P. monodon, as well as from YHV genotypes currently listed as exotic to Australia.

Control performance of fish short term reproduction assays with fathead minnow (Pimephales promelas).

The fish short-term reproduction assay (FSTRA) is an in vivo screen to assess potential interactions with the fish endocrine system. After a 21-day exposure period vitellogenin (VTG) and secondary sexual characteristics are measured in males and females. Egg production and fertility are also monitored daily throughout the test. This paper presents data from 49 studies performed to satisfy test orders from the United States Environmental Protection Agency's Endocrine Disruptor Screening Program. Data Evaluation Records were used to collate the typical control variability and performance of test parameters in FSTRAs conducted in different laboratories with fathead minnow (Pimephales promelas). We also examine the statistical power of FSTRA endpoints and assess whether available historical control data (HCD) assist evidence-based interpretation of the endpoints. Statistically significant inter-laboratory differences were found for all endpoints except survival. HCD could therefore be usefully developed on a laboratory-by-laboratory basis to aid interpretation of new study data. Reliable HCD ranges could be developed for survival, body weight/length, gonadal somatic index, fertilisation success, and male tubercle score, and used in association with stated test acceptability criteria to interpret FSTRA data. In contrast, high intra- and inter-laboratory control variability for VTG and fecundity means that HCD for these endpoints are of limited use during study interpretation.

Investigating the natural resistance of blackfoot p-a%%KERN_ERR%%ua Haliotis iris to abalone viral ganglioneuritis using whole transcriptome analysis.

The natural resistance of New Zealand blackfoot p-a%%%%%%%%%%%%%%KERN_ERR%%KERN_ERR%%KERN_ERR%%KERN_ERR%%KERN_ERR%%KERN_ERR%%KERN_ERR%%ua Haliotis iris to infection by haliotid herpesvirus-1 (HaHV-1) and to the disease abalone viral ganglioneuritis was investigated in experimentally challenged p-aua using high throughput RNA-sequencing. HaHV-1-challenged p-aua up-regulated broad classes of genes that contained chitin-binding peritrophin-A domains, which seem to play diverse roles in the p-aua immune response. The p-aua also up-regulated vascular adhesion protein-1 (VAP-1), an important adhesion molecule for lymphocytes, and chitotriosidase-1 (CHIT-1), an immunologically important gene in mammalian immune systems. Moreover, several blood coagulation pathways were dysregulated in the p-aua, possibly indicating viral modulation. We also saw several indications that neurological tissues were specifically affected by HaHV-1, including the dysregulation of beta-1,4-N-acetylgalactosaminyltransferase (B4GALNT), GM2 ganglioside, neuroligin-4 and the Notch signalling pathway. This research may support the development of molecular therapeutics useful to control and/or manage viral outbreaks in abalone culture.

Assessing the population relevance of endocrine-disrupting effects for nontarget vertebrates exposed to plant protection products.

The European Commission intends to protect vertebrate wildlife populations by regulating plant protection product (PPP) active substances that have endocrine-disrupting properties with a hazard-based approach. In this paper we consider how the Commission's hazard-based regulation and accompanying guidance can be operationalized to ensure that a technically robust process is used to distinguish between substances with adverse population-level effects and those for which it can be demonstrated that adverse effects observed (typically in the laboratory) do not translate into adverse effects at the population level. Our approach is to use population models within the adverse outcome pathway framework to link the nonlinear relationship between adverse effects at the individual and population levels in the following way: (1) use specific protection goals for focal wildlife populations within an ecosystem services framework; (2) model the effects of changes in population-related inputs on focal species populations with individual-based population models to determine thresholds between negligible and nonnegligible (i.e., adverse) population-level effects; (3) compare these thresholds with the relevant endpoints from laboratory toxicity tests to determine whether they are likely to be exceeded at hazard-based limits or the maximum tolerated dose/concentration from the experimental studies. If the population threshold is not exceeded, then the substance should not be classified as an endocrine disruptor with population-relevant adversity unless there are other lines of evidence within a weight-of-evidence approach to challenge this. We believe this approach is scientifically robust and still addresses the political and legal requirement for a hazard-based assessment. Integr Environ Assess Manag 2019;15:278-291. © 2018 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

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.

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.

Acute oral toxicity of chemicals in terrestrial life stages of amphibians: Comparisons to birds and mammals.

Amphibians are currently the most threatened and rapidly declining group of vertebrates and this has raised concerns about their potential sensitivity and exposure to plant protection products and other chemicals. Current environmental risk assessment procedures rely on surrogate species (e.g. fish and birds) to cover the risk to aquatic and terrestrial life stages of amphibians, respectively. Whilst a recent meta-analysis has shown that in most cases amphibian aquatic life stages are less sensitive to chemicals than fish, little research has been conducted on the comparative sensitivity of terrestrial amphibian life stages. Therefore, in this paper we address the questions "What is the relative sensitivity of terrestrial amphibian life stages to acute chemical oral exposure when compared with mammals and birds?" and "Are there correlations between oral toxicity data for amphibians and data for mammals or birds?" Identifying a relationship between these data may help to avoid additional vertebrate testing. Acute oral amphibian toxicity data collected from the scientific literature and ecotoxicological databases were compared with toxicity data for mammals and birds. Toxicity data for terrestrial amphibian life stages are generally sparse, as noted in previous reviews. Single-dose oral toxicity data for terrestrial amphibian life stages were available for 26 chemicals and these were positively correlated with LD50 values for mammals, while no correlation was found for birds. Further, the data suggest that oral toxicity to terrestrial amphibian life stages is similar to or lower than that for mammals and birds, with a few exceptions. Thus, mammals or birds are considered adequate toxicity surrogates for use in the assessment of the oral exposure route in amphibians. However, there is a need for further data on a wider range of chemicals to explore the wider applicability of the current analyses and recommendations.

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.

Recommended reporting standards for test accuracy studies of infectious diseases of finfish, amphibians, molluscs and crustaceans: the STRADAS-aquatic checklist.

Complete and transparent reporting of key elements of diagnostic accuracy studies for infectious diseases in cultured and wild aquatic animals benefits end-users of these tests, enabling the rational design of surveillance programs, the assessment of test results from clinical cases and comparisons of diagnostic test performance. Based on deficiencies in the Standards for Reporting of Diagnostic Accuracy (STARD) guidelines identified in a prior finfish study (Gardner et al. 2014), we adapted the Standards for Reporting of Animal Diagnostic Accuracy Studies-paratuberculosis (STRADAS-paraTB) checklist of 25 reporting items to increase their relevance to finfish, amphibians, molluscs, and crustaceans and provided examples and explanations for each item. The checklist, known as STRADAS-aquatic, was developed and refined by an expert group of 14 transdisciplinary scientists with experience in test evaluation studies using field and experimental samples, in operation of reference laboratories for aquatic animal pathogens, and in development of international aquatic animal health policy. The main changes to the STRADAS-paraTB checklist were to nomenclature related to the species, the addition of guidelines for experimental challenge studies, and the designation of some items as relevant only to experimental studies and ante-mortem tests. We believe that adoption of these guidelines will improve reporting of primary studies of test accuracy for aquatic animal diseases and facilitate assessment of their fitness-for-purpose. Given the importance of diagnostic tests to underpin the Sanitary and Phytosanitary agreement of the World Trade Organization, the principles outlined in this paper should be applied to other World Organisation for Animal Health (OIE)-relevant species.

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.