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.

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.

The avian Z-linked gene DMRT1 is required for male sex determination in the chicken.

Sex in birds is chromosomally based, as in mammals, but the sex chromosomes are different and the mechanism of avian sex determination has been a long-standing mystery. In the chicken and all other birds, the homogametic sex is male (ZZ) and the heterogametic sex is female (ZW). Two hypotheses have been proposed for the mechanism of avian sex determination. The W (female) chromosome may carry a dominant-acting ovary determinant. Alternatively, the dosage of a Z-linked gene may mediate sex determination, two doses being required for male development (ZZ). A strong candidate avian sex-determinant under the dosage hypothesis is the conserved Z-linked gene, DMRT1 (doublesex and mab-3-related transcription factor 1). Here we used RNA interference (RNAi) to knock down DMRT1 in early chicken embryos. Reduction of DMRT1 protein expression in ovo leads to feminization of the embryonic gonads in genetically male (ZZ) embryos. Affected males show partial sex reversal, characterized by feminization of the gonads. The feminized left gonad shows female-like histology, disorganized testis cords and a decline in the testicular marker, SOX9. The ovarian marker, aromatase, is ectopically activated. The feminized right gonad shows a more variable loss of DMRT1 and ectopic aromatase activation, suggesting differential sensitivity to DMRT1 between left and right gonads. Germ cells also show a female pattern of distribution in the feminized male gonads. These results indicate that DMRT1 is required for testis determination in the chicken. Our data support the Z dosage hypothesis for avian sex determination.

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.

Characterisation and comparison of the chicken H1 RNA polymerase III promoter for short hairpin RNA expression.

The U6 and 7SK RNA polymerase III promoters are widely used in RNAi research for the expression of shRNAs. However, with their increasing use in vitro and in vivo, issues associated with cytotoxicity have become apparent with their use. Therefore, alternative promoters such as the weaker H1 promoter are becoming a popular choice. With interest in the chicken as a model organism, we aimed to identify and characterise the chicken H1 promoter for the expression of shRNAs for the purpose of RNAi. The chicken H1 promoter was isolated and sequence analysis identified conserved RNA polymerase III promoter elements. A shRNA expression cassette containing the chicken H1 promoter and shRNA targeting enhanced green fluorescent protein (EGFP) was developed. An RNAse protection assay confirmed activity of the promoter determined by the detection of expressed shRNAs. Comparison of the H1 promoter to the chicken RNA polymerase III 7SK and U6 promoters demonstrated that expressed shRNAs from the H1 promoter induced gene specific silencing, albeit to lower levels in comparison to both 7SK and U6 promoters. Here we have identified a new tool for RNAi research with specific applications to the chicken. The availability of a RNA polymerase III promoter that drives shRNA expression to reduced levels will greatly benefit in ovo/in vivo applications where there are concerns of cytotoxicity resulting from overexpression of an shRNA.

Transgenic Chickens Overexpressing Aromatase Have High Estrogen Levels but Maintain a Predominantly Male Phenotype.

Estrogens play a key role in sexual differentiation of both the gonads and external traits in birds. The production of estrogen occurs via a well-characterized steroidogenic pathway, which is a multistep process involving several enzymes, including cytochrome P450 aromatase. In chicken embryos, the aromatase gene (CYP19A1) is expressed female-specifically from the time of gonadal sex differentiation. Ectopic overexpression of aromatase in male chicken embryos induces gonadal sex reversal, and male embryos treated with estradiol become feminized; however, this is not permanent. To test whether a continuous supply of estrogen in adult chickens could induce stable male to female sex reversal, 2 transgenic male chickens overexpressing aromatase were generated using the Tol2/transposase system. These birds had robust ectopic aromatase expression, which resulted in the production of high serum levels of estradiol. Transgenic males had female-like wattle and comb growth and feathering, but they retained male weights, displayed leg spurs, and developed testes. Despite the small sample size, this data strongly suggests that high levels of circulating estrogen are insufficient to maintain a female gonadal phenotype in adult birds. Previous observations of gynandromorph birds and embryos with mixed sex chimeric gonads have highlighted the role of cell autonomous sex identity in chickens. This might imply that in the study described here, direct genetic effects of the male chromosomes largely prevailed over the hormonal profile of the aromatase transgenic birds. This data therefore support the emerging view of at least partial cell autonomous sex development in birds. However, a larger study will confirm this intriguing observation.

Development of retroviral vectors for tissue-restricted expression in chicken embryonic gonads.

The chicken embryo has long been a useful model organism for studying development, including sex determination and gonadal differentiation. However, manipulating gene expression specifically in the embryonic avian gonad has been difficult. The viral vector RCASBP can be readily used for embryo-wide transgene expression; however global mis-expression using this method can cause deleterious off-target effects and embryo-lethality. In an attempt to develop vectors for the over-expression of sequences in chicken embryonic urogenital tissues, the viral vector RCANBP was engineered to contain predicted promoter sequences of gonadal-expressed genes. Several promoters were analysed and it was found that although the SF1 promoter produced a tissue-restricted expression pattern that was highest in the mesonephros and liver, it was also higher in the gonads compared to the rest of the body. The location of EGFP expression from the SF1 promoter overlapped with several key gonad-expressed sex development genes; however expression was generally low-level and was not seen in all gonadal cells. To further validate this sequence the key testis determinant DMRT1 was over-expressed in female embryos, which due to insufficient levels had no effect on gonad development. The female gene aromatase was then over-expressed in male embryos, which disrupted the testis pathway as demonstrated by a reduction in AMH protein. Taken together, although these data showed that the SF1 promoter can be used for functional studies in ovo, a stronger promoter sequence would likely be required for the functional analysis of gonad genes that require high-level expression.

Characterization of zebrafish polymerase III promoters for the expression of short-hairpin RNA interference molecules.

RNA interference (RNAi) is a powerful, sequence specific, and long-lasting method of gene knockdown, and can be elicited by the expression of short-hairpin RNA (shRNA) molecules driven via polymerase III type 3 promoters from a DNA vector or transgene. To further develop RNAi as a tool in zebrafish, we have characterized the zebrafish U6 and H1 snRNA promoters and compared the efficiency of each of the promoters to express an shRNA and silence a reporter gene, relative to previously characterized U6 promoters from pufferfish, chicken, and mouse. Our results show that the zebrafish polymerase III promoters were capable of effective gene silencing in the zebrafish ZF4 cell line, but were ineffective in mammalian Vero cells. In contrast, mouse and chicken promoters were active in Vero but not ZF4 cells, highlighting the importance of homologous promoters to achieve effective silencing.

Overexpression of aromatase alone is sufficient for ovarian development in genetically male chicken embryos.

Estrogens play a key role in sexual differentiation of both the gonads and external traits in birds. The production of estrogen occurs via a well-characterised steroidogenic pathway, which is a multi-step process involving several enzymes, including cytochrome P450 aromatase. In chicken embryos, the aromatase gene (CYP19A1) is expressed female-specifically from the time of gonadal sex differentiation. To further explore the role of aromatase in sex determination, we ectopically delivered this enzyme using the retroviral vector RCASBP in ovo. Aromatase overexpression in male chicken embryos induced gonadal sex-reversal characterised by an enlargement of the left gonad and development of ovarian structures such as a thickened outer cortex and medulla with lacunae. In addition, the expression of key male gonad developmental genes (DMRT1, SOX9 and Anti-Müllerian hormone (AMH)) was suppressed, and the distribution of germ cells in sex-reversed males followed the female pattern. The detection of SCP3 protein in late stage sex-reversed male embryonic gonads indicated that these genetically male germ cells had entered meiosis, a process that normally only occurs in female embryonic germ cells. This work shows for the first time that the addition of aromatase into a developing male embryo is sufficient to direct ovarian development, suggesting that male gonads have the complete capacity to develop as ovaries if provided with aromatase.