Development and Validation of a Bioinformatic Workflow for the Rapid Detection of Viruses in Biosecurity.

The field of biosecurity has greatly benefited from the widespread adoption of high-throughput sequencing technologies, for its ability to deeply query plant and animal samples for pathogens for which no tests exist. However, the bioinformatics analysis tools designed for rapid analysis of these sequencing datasets are not developed with this application in mind, limiting the ability of diagnosticians to standardise their workflows using published tool kits. We sought to assess previously published bioinformatic tools for their ability to identify plant- and animal-infecting viruses while distinguishing from the host genetic material. We discovered that many of the current generation of virus-detection pipelines are not adequate for this task, being outperformed by more generic classification tools. We created synthetic MinION and HiSeq libraries simulating plant and animal infections of economically important viruses and assessed a series of tools for their suitability for rapid and accurate detection of infection, and further tested the top performing tools against the VIROMOCK Challenge dataset to ensure that our findings were reproducible when compared with international standards. Our work demonstrated that several methods provide sensitive and specific detection of agriculturally important viruses in a timely manner and provides a key piece of ground truthing for method development in this space.

Expression of Renal Vitamin D and Phosphatonin-Related Genes in a Sheep Model of Osteoporosis.

Osteoporosis is a significant public health issue around the world, with post-menopausal osteoporosis due to estrogen deficiency resulting in approximately ¾ of cases. In this study, 18 aged Merino ewes were ovariectomized, and 10 were controls. Three of the ovariectomized ewes were treated weekly with 400 mg of methylprednisolone for 5 months and three were treated weekly for 2 months, followed by a 3-month recovery period. At 2 months, five control animals and six ovariectomized animals were euthanized. At 5 months, all the remaining ewes were euthanized. Kidney samples were collected postmortem for qPCR analysis of NPT1, PTH1R, NPT2a, NPT2c, Klotho, FGFR1IIIc, VDR, CYP24A1, CYP27B1, TRPV5, TRPV6, CalD9k, CalD28k, PMCA and NCX1. Ovariectomized sheep had significantly greater VDR expression compared with other groups. Ovariectomized sheep treated with glucocorticoids for 2 months followed by euthanasia at 5 months showed significant differences in TRPV5, CYP24A1 and klotho gene expression compared to other groups. Differences in klotho expression were most marked after adjustment for repeated measures (p = 0.1). Klotho is known as the "anti-aging" hormone and is involved in calcium and phosphorus metabolism. Klotho may be involved in the recovery of bone mineral density in ovariectomized sheep treated with glucocorticoids for 2 months followed by euthanasia at 5 months. Further research on the role of klotho is recommended.

Uncoupling Molecular Testing for SARS-CoV-2 From International Supply Chains.

The rapid global rise of COVID-19 from late 2019 caught major manufacturers of RT-qPCR reagents by surprise and threw into sharp focus the heavy reliance of molecular diagnostic providers on a handful of reagent suppliers. In addition, lockdown and transport bans, necessarily imposed to contain disease spread, put pressure on global supply lines with freight volumes severely restricted. These issues were acutely felt in New Zealand, an island nation located at the end of most supply lines. This led New Zealand scientists to pose the hypothetical question: in a doomsday scenario where access to COVID-19 RT-qPCR reagents became unavailable, would New Zealand possess the expertise and infrastructure to make its own reagents onshore? In this work we describe a review of New Zealand's COVID-19 test requirements, bring together local experts and resources to make all reagents for the RT-qPCR process, and create a COVID-19 diagnostic assay referred to as HomeBrew (HB) RT-qPCR from onshore synthesized components. This one-step RT-qPCR assay was evaluated using clinical samples and shown to be comparable to a commercial COVID-19 assay. Through this work we show New Zealand has both the expertise and, with sufficient lead time and forward planning, infrastructure capacity to meet reagent supply challenges if they were ever to emerge.

Differential recognition of peptides within feline coronavirus polyprotein 1 ab by sera from healthy cats and cats with feline infectious peritonitis.

The aim of the study was to identify peptides within the polyprotein (Pp) 1 ab that are differentially recognised by cats with either enteric or systemic disease following infection with feline coronavirus. Overlapping 12-mer peptides (n = 28,426) across the entire Pp1ab were arrayed on peptide chips and reacted with pooled sera from coronavirus seropositive cats and from one seronegative cat. Eleven peptides were further tested in ELISA with individual serum samples, and three were selected for further screening. Two peptides (16433 and 4934) in the nsp3 region encoding the papain 1 and 2 proteases were identified for final testing. Peptide 4934 reacted equally with positive sera from healthy cats and cats with feline infectious peritonitis (FIP), while peptide 16433 was recognized predominantly by FIP-affected cats. The value of antibody tests based on these peptides in differentiating between the enteric and FIP forms of feline coronavirus infection remains to be determined.

ß-Mannosidosis in German Shepherd Dogs.

A neurological disease was investigated in 3 German Shepherd pups from the same litter that failed to grow normally, appeared stiff, were reluctant to move, and were deaf. They developed intermittent seizures and ataxia and had proprioceptive defects. Histopathology showed severe vacuolation of neurons, astrocytes in nervous tissue, renal tubular epithelial cells, and macrophages in nervous tissue, spleen, and liver. Vacuoles appeared empty with no storage material stained by periodic acid-Schiff (PAS) or Sudan black stains, leading to a diagnosis of a lysosomal storage disease and in particular an oligosaccharidosis. Biochemical and genomic studies showed that this was ß-mannosidosis, not previously diagnosed in dogs. A c.560T>A transition in exon 4 of the MANBA gene was found, which segregated in these and other family members in a manner consistent with it being the causative mutation of an autosomal recessive disease. This mutation led to substitution of isoleucine to asparagine at position 187 of the 885 amino acid enzyme, a change expected to have functional significance.