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.

Complete Genome Sequence of African Swine Fever Virus Isolated from a Domestic Pig in Timor-Leste, 2019.

Here, we report the complete genome sequence of the African swine fever virus (ASFV) isolate ASFV/Timor-Leste/2019/1, isolated from a domestic pig during the first outbreak of ASF in Timor-Leste in 2019. Using target enrichment short-read Illumina data combined with long-read Oxford Nanopore data, we assembled a full-length genome sequence of 192,237 bp.

Risk factors for intellectual disability in children with spastic cerebral palsy.

BACKGROUND: Cerebral palsy (CP) is a non-progressive disorder of posture and movement caused by prenatal or perinatal lesions of the brain. Children with CP are also at increased risk of other disabilities, for example, intellectual disability. Previous studies suggest the risk of intellectual disability varies in complex ways according to the type of motor impairment and perinatal factors such as gestational age. OBJECTIVE: To determine the patterns of risk of intellectual disability in children with spastic CP. DESIGN: Cross-sectional, population-based study using the Northern Ireland Cerebral Palsy Register. PARTICIPANTS: Persons born in 1981-2008 with congenital bilateral or unilateral spastic CP (N=1452). OUTCOME MEASURE: The outcome measure was severe intellectual disability (IQ <50), as reported by clinicians known to the child. Data pertaining to CP subtype, sex, gestational age, birth weight and functional level were included in analyses. RESULTS: Severe intellectual disability was significantly more prevalent in children with bilateral spastic CP (BSCP) compared with children with unilateral spastic CP (χ² (2)=162.60, p<0.001). Compared with very preterm infants with BSCP, the risk of intellectual disability increased in moderately preterm (OR=3.97, 95% CI 1.04 to 15.23) and at-term (OR=2.51, 95% CI 1.16 to 5.44) children with BSCP. CONCLUSIONS: Children with BSCP are at increased risk of intellectual disability, with those born at term at the highest risk. The findings highlight the importance of early screening, particularly for children with BSCP born at term.

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.

Midsagittal tissue bridges are associated with walking ability in incomplete spinal cord injury: A magnetic resonance imaging case series.

Context: Following spinal cord injury (SCI), early prediction of future walking ability is difficult, due to factors such as spinal shock, sedation, impending surgery, and secondary long bone fracture. Accurate, objective biomarkers used in the acute stage of SCI would inform individualized patient management and enhance both patient/family expectations and treatment outcomes. Using magnetic resonance imaging (MRI) and specifically a midsagittal T2-weighted image, the amount of tissue bridging (measured as spared spinal cord tissue) shows potential to serve as such a biomarker. Ten participants with incomplete SCI received MRI of the spinal cord. Using the midsagittal T2-weighted image, anterior and posterior tissue bridges were calculated as the distance from cerebrospinal fluid to the damage. Then, the midsagittal tissue bridge ratio was calculated as the sum of anterior and posterior tissue bridges divided by the spinal cord diameter. Each participant also performed a 6-minute walk test, where the total distance walked was measured within six minutes.Findings: The midsagittal tissue bridge ratio measure demonstrated a high level of inter-rater reliability (ICC = 0.90). Midsagittal tissue bridge ratios were significantly related to distance walked in six minutes (R = 0.68, P = 0.03).Conclusion/clinical relevance: We uniquely demonstrated that midsagittal tissue bridge ratios were correlated walking ability. These preliminary findings suggest potential for this measure to be considered a prognostic biomarker of residual walking ability following SCI.

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.

Establishing the inter-rater reliability of spinal cord damage manual measurement using magnetic resonance imaging.

Study design: Retrospective study. Objectives: To establish the inter-rater reliability in the quantitative evaluation of spinal cord damage following cervical incomplete spinal cord injury (SCI) utilizing magnetic resonance imaging (MRI). MRI was used to perform manual measurements of the cranial and caudal boundaries of edema, edema length, midsagittal tissue bridge ratio, axial damage ratio, and edema volume in 10 participants with cervical incomplete SCI. Setting: Academic university setting. Methods: Structural MRIs of 10 participants with SCI were collected from Northwestern University's Neuromuscular Imaging and Research Lab. All manual measures were performed using OsiriX (Pixmeo Sarl, Geneva, Switzerland). Intraclass correlation coefficients (ICC) were used to determine inter-rater reliability across seven raters of varying experience. Results: High-to-excellent inter-rater reliability was found for all measures. ICC values for cranial/caudal levels of involvement, edema length, midsagittal tissue bridge ratio, axial damage ratio, and edema volume were 0.99, 0.98, 0.90, 0.84, and 0.93, respectively. Conclusions: Manual MRI measures of spinal cord damage are reliable between raters. Researchers and clinicians may confidently utilize manual MRI measures to quantify cord damage. Future research to predict functional recovery following SCI and better inform clinical management is warranted.

Investigating the Behavior of Published PAINS Alerts Using a Pharmaceutical Company Data Set.

Biochemical assay interference is becoming increasingly recognized as a significant waste of resource in drug discovery, both in industry and academia. A seminal publication from Baell and Holloway raised the awareness of this issue, and they published a set of alerts to identify what they described as PAINS (pan-assay interference compounds). These alerts have been taken up by drug discovery groups, even though the original paper had a somewhat limited data set. Here, we have taken Lilly's far larger internal data set to assess the PAINS alerts on four criteria: promiscuity (over six assay formats including AlphaScreen), compound stability, cytotoxicity, and presence of a high Hill slope as a surrogate for non-1:1 protein-ligand binding. It was found that only three of the alerts show pan-assay promiscuity, and the alerts appear to encode primarily AlphaScreen promiscuous molecules. Although not enriching for pan-assay promiscuity, many of the alerts do encode molecules that are unstable, show cytotoxicity, and increase the prevalence of high Hill slopes.

Two-Site Evaluation of the Repeatability and Precision of an Automated Dual-Column Hydrogen/Deuterium Exchange Mass Spectrometry Platform.

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein-ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test-retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error.

Integrating Everything: The Molecule Selection Toolkit, a System for Compound Prioritization in Drug Discovery.

In recent years there have been numerous papers on the topic of multiattribute optimization in pharmaceutical discovery chemistry, applied to compound prioritization. Many solutions proposed are static in nature; fixed functions are proposed for general purpose use. As needs change, these are modified and proposed as the latest enhancement. Rather than producing one more set of static functions, this work proposes a flexible approach to prioritizing compounds. Most published approaches also lack a design component. This work describes a comprehensive implementation that includes predictive modeling, multiattribute optimization, and modern statistical design. This gives a complete package for effectively prioritizing compounds for lead generation and lead optimization. The approach described has been used at our company in various stages of discovery since 2001. An adaptable system alleviates the need for different static solutions, each of which inevitably must be updated as the needs of a project change.

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.

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.

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.

Selectivity data: assessment, predictions, concordance, and implications.

Could high-quality in silico predictions in drug discovery eventually replace part or most of experimental testing? To evaluate the agreement of selectivity data from different experimental or predictive sources, we introduce the new metric concordance minimum significant ratio (cMSR). Empowered by cMSR, we find the overall level of agreement between predicted and experimental data to be comparable to that found between experimental results from different sources. However, for molecules that are either highly selective or potent, the concordance between different experimental sources is significantly higher than the concordance between experimental and predicted values. We also show that computational models built from one data set are less predictive for other data sources and highlight the importance of bias correction for assessing selectivity data. Finally, we show that small-molecule target space relationships derived from different data sources and predictive models share overall similarity but can significantly differ in details.

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.

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.

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.