Rapid Detection of SARS-CoV-2 Variants of Concern by Genomic Surveillance Techniques.

This chapter describes the application of genomic, transcriptomic, proteomic, and metabolomic methods in the study of SARS-CoV-2 variants of concern. We also describe the important role of machine learning tools to identify the most significant biomarker signatures and discuss the latest point-of-care devices that can be used to translate these findings to the physician's office or to bedside care. The main emphasis is placed on increasing our diagnostic capacity and predictability of disease outcomes to guide the most appropriate treatment strategies.

The architecture of clonal expansions in morphologically normal tissue from cancerous and non-cancerous prostates.

Background: Up to 80% of cases of prostate cancer present with multifocal independent tumour lesions leading to the concept of a field effect present in the normal prostate predisposing to cancer development. In the present study we applied Whole Genome DNA Sequencing (WGS) to a group of morphologically normal tissue (n = 51), including benign prostatic hyperplasia (BPH) and non-BPH samples, from men with and men without prostate cancer. We assess whether the observed genetic changes in morphologically normal tissue are linked to the development of cancer in the prostate.

Multiplex Quantitative Polymerase Chain Reaction Test to Identify SARS-CoV-2 Variants.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for detection and quantitation of gene expression in real time. This is achieved through the incorporation and measurement of fluorescent reporter probes in the amplified cDNA strands, since the fluorescent signals increase as the reaction progresses. The availability of multiple probes which fluoresce at different wavelengths allows for multiplexing as this gives rise to amplicons with unique fluorescent signatures.

Multiplex Quantitative Polymerase Chain Reaction Diagnostic Test for SARS-CoV-2 and Influenza A/B Viruses.

COVID-19 disease caused by the novel SARS-CoV-2 virus represents a new challenge for healthcare systems. The molecular confirmation of infection is crucial to guide public health decision-making. This task could be made more difficult during the next influenza season.

Rapid recontamination of a farm building occurs after attempted prion removal.

The transmissible spongiform encephalopathy scrapie of sheep/goats and chronic wasting disease of cervids are associated with environmental reservoirs of infectivity. Preventing environmental prions acting as a source of infectivity to healthy animals is of major concern to farms that have had outbreaks of scrapie and also to the health management of wild and farmed cervids. Here, an efficient scrapie decontamination protocol was applied to a farm with high levels of environmental contamination with the scrapie agent.

Sequencing of prostate cancers identifies new cancer genes, routes of progression and drug targets.

Prostate cancer represents a substantial clinical challenge because it is difficult to predict outcome and advanced disease is often fatal. We sequenced the whole genomes of 112 primary and metastatic prostate cancer samples. From joint analysis of these cancers with those from previous studies (930 cancers in total), we found evidence for 22 previously unidentified putative driver genes harboring coding mutations, as well as evidence for NEAT1 and FOXA1 acting as drivers through noncoding mutations.

Rapid and Easy Protocol for Quantification of Next-Generation Sequencing Libraries.

The emergence of next-generation sequencing (NGS) over the last 10 years has increased the efficiency of DNA sequencing in terms of speed, ease, and price. However, the exact quantification of a NGS library is crucial in order to obtain good data on sequencing platforms developed by the current market leader Illumina. Different approaches for DNA quantification are available currently and the most commonly used are based on analysis of the physical properties of the DNA through spectrophotometric or fluorometric methods.

Appraising the relevance of DNA copy number loss and gain in prostate cancer using whole genome DNA sequence data.

A variety of models have been proposed to explain regions of recurrent somatic copy number alteration (SCNA) in human cancer. Our study employs Whole Genome DNA Sequence (WGS) data from tumor samples (n = 103) to comprehensively assess the role of the Knudson two hit genetic model in SCNA generation in prostate cancer. 64 recurrent regions of loss and gain were detected, of which 28 were novel, including regions of loss with more than 15% frequency at Chr4p15.

Multiplex Single Nucleotide Polymorphism Analyses.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for detection and quantitation of gene expression in real time. This is achieved through the incorporation and measurement of fluorescent reporter probes in the amplified cDNA strands, since the fluorescent signals increase as the reaction progresses. The availability of multiple probes that fluoresce at different wavelengths allows for multiplexing as this gives rise to amplicons with unique fluorescent signatures.

Multiplex Analysis Using cDNA Transcriptomic Profiling.

DNA microarrays contain microscopic DNA spots attached to a solid surface. Each spot contains picomolar levels of a specific DNA probe sequence and hybridization to the corresponding gene products can be detected and quantitated through the use of fluorescently labeled target DNA. In this format, DNA microarrays can be used to measure the expression level of thousands of genes in a single experiment.

Multiplex Analyses Using Real-Time Quantitative PCR.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for the detection and quantitation of gene expression in real time. Multiplex qPCR requires the use of probe-based assays, in which each probe is labeled with a unique fluorescent dye, resulting in different observed colors for each assay. The signal from each dye is used to quantitate the amount of each target separately in the same tube or well.

Evidence of scrapie transmission to sheep via goat milk.

Background: Previous studies confirmed that classical scrapie can be transmitted via milk in sheep. The current study aimed to investigate whether scrapie can also be transmitted via goat milk using in vivo (new-born lambs fed milk from scrapie-affected goats due to the unavailability of goat kids from guaranteed scrapie-free herds) and in vitro methods (serial protein misfolding cyclic amplification [sPMCA] on milk samples).

Results: In an initial pilot study, new-born lambs of two different prion protein gene (PRNP) genotypes (six VRQ/VRQ and five ARQ/ARQ) were orally challenged with 5 g brain homogenate from two scrapie-affected goats to determine susceptibility of sheep to goat scrapie.

Minimum Effective Dose of Cattle and Sheep BSE for Oral Sheep Infection.

The minimum dose required to cause infection of Romney and Suffolk sheep of the ARQ/ARQ or ARQ/ARR prion protein gene genotypes following oral inoculation with Romney or Suffolk a sheep Bovine spongiform encephalopathy (BSE)-derived or cattle BSE-derived agent was investigated using doses ranging from 0.0005g to 5g. ARQ/ARQ sheep which were methionine (M) / threonine (T) heterozygous or T/T homozygous at codon 112 of the Prnp gene, dosed ARQ/ARR sheep and undosed controls did not show any evidence of infection.

Dynamics of the natural transmission of bovine spongiform encephalopathy within an intensively managed sheep flock.

Sheep are susceptible to the bovine spongiform encephalopathy (BSE) agent and in the UK they may have been exposed to BSE via contaminated meat and bone meal. An experimental sheep flock was established to determine whether ovine BSE could be naturally transmitted under conditions of intensive husbandry. The flock consisted of 113 sheep of different breeds and susceptible PRNP genotypes orally dosed with BSE, 159 sheep subsequently born to them and 125 unchallenged sentinel controls.

Further characterisation of transmissible spongiform encephalopathy phenotypes after inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain.

Background: The infectious agent responsible for the bovine spongiform encephalopathy (BSE) epidemic in Great Britain is a transmissible spongiform encephalopathy (TSE) strain with uniform properties but the origin of this strain remains unknown. Based on the hypothesis that classical BSE may have been caused by a TSE strain present in sheep, cattle were inoculated intracerebrally with two different pools of brains from scrapie-affected sheep sourced prior to and during the BSE epidemic to investigate resulting disease phenotypes and characterise their causal agents by transmission to rodents.

Results: As reported in 2006, intracerebral inoculation of cattle with pre-1975 and post-1990 scrapie brain pools produced two distinct disease phenotypes, which were unlike classical BSE.

Circulation of prions within dust on a scrapie affected farm.

Prion diseases are fatal neurological disorders that affect humans and animals. Scrapie of sheep/goats and Chronic Wasting Disease (CWD) of deer/elk are contagious prion diseases where environmental reservoirs have a direct link to the transmission of disease. Using protein misfolding cyclic amplification we demonstrate that scrapie PrP(Sc) can be detected within circulating dusts that are present on a farm that is naturally contaminated with sheep scrapie.

Persistence of ovine scrapie infectivity in a farm environment following cleaning and decontamination.

Scrapie of sheep/goats and chronic wasting disease of deer/elk are contagious prion diseases where environmental reservoirs are directly implicated in the transmission of disease. In this study, the effectiveness of recommended scrapie farm decontamination regimens was evaluated by a sheep bioassay using buildings naturally contaminated with scrapie. Pens within a farm building were treated with either 20,000 parts per million free chorine solution for one hour or were treated with the same but were followed by painting and full re-galvanisation or replacement of metalwork within the pen.

Elements modulating the prion species barrier and its passage consequences.

The specific characteristics of Transmissible Spongiform Encephalopathy (TSE) strains may be altered during passage across a species barrier. In this study we investigated the biochemical and biological characteristics of Bovine Spongiform Encephalopathy (BSE) after transmission in both natural host species (cattle, sheep, pigs and mice) and in transgenic mice overexpressing the corresponding cellular prion protein (PrPC) in comparison with other non-BSE related prions from the same species. After these passages, most features of the BSE agent remained unchanged.

Bovine spongiform encephalopathy: the effect of oral exposure dose on attack rate and incubation period in cattle - an update.

Background: To provide information on dose-response and aid in modelling the exposure dynamics of the BSE epidemic in the United Kingdom groups of cattle were exposed orally to a range of different doses of brainstem homogenate of known infectious titre from clinical cases of classical bovine spongiform encephalopathy (BSE). Interim data from this study was published in 2007. This communication documents additional BSE cases, which occurred subsequently, examines possible influence of the bovine prion protein gene on disease incidence and revises estimates of effective oral exposure.

Ultrastructural findings in pigs experimentally infected with bovine spongiform encephalopathy agent.

We report here an electron microscopic study of selected nervous system tissues from pigs infected experimentally with the agent of bovine spongiform encephalopathy (BSE). Generally, the ultrastructural neuropathology of BSE-affected pig brain resembled that of BSE-affected cattle brain. Spongiform change, in the form of membrane-bound vacuoles separated by septae into secondary chambers, dominated the pathology.

Relationship between clinical signs and postmortem test status in cattle experimentally infected with the bovine spongiform encephalopathy agent.

Background: Various clinical protocols have been developed to aid in the clinical diagnosis of classical bovine spongiform encephalopathy (BSE), which is confirmed by postmortem examinations based on vacuolation and accumulation of disease-associated prion protein (PrPd) in the brain. The present study investigated the occurrence and progression of sixty selected clinical signs and behaviour combinations in 513 experimentally exposed cattle subsequently categorised postmortem as confirmed or unconfirmed BSE cases. Appropriate undosed or saline inoculated controls were examined similarly and the data analysed to explore the possible occurrence of BSE-specific clinical expression in animals unconfirmed by postmortem examinations.

Monitoring of clinical signs in goats with transmissible spongiform encephalopathies.

Background: As there is limited information about the clinical signs of BSE and scrapie in goats, studies were conducted to describe the clinical progression of scrapie and BSE in goats and to evaluate a short clinical protocol for its use in detecting scrapie-affected goats in two herds with previously confirmed scrapie cases. Clinical assessments were carried out in five goats intracerebrally infected with the BSE agent as well as five reported scrapie suspects and 346 goats subject to cull from the two herds, 24 of which were retained for further monitoring. The brain and selected lymphoid tissue were examined by postmortem tests for disease confirmation.

Transmissibility studies of vacuolar changes in the rostral colliculus of pigs.

Background: Histopathological examinations of brains from healthy pigs have revealed localised vacuolar changes, predominantly in the rostral colliculus, that are similar to the neuropil vacuolation featured in the transmissible spongiform encephalopathies and have been described in pigs challenged parenterally with the agent causing bovine spongiform encephalopathy (BSE). Feedstuff containing BSE-contaminated meat and bone meal (MBM) may have been fed to pigs prior to the ban of mammalian MBM in feed of farmed livestock in the United Kingdom in 1996, but there is no evidence of the natural occurrence of a transmissible spongiform encephalopathy (TSE) in the domestic pig. Furthermore, experimental transmission of BSE to pigs by the oral route has been unsuccessful.

Transcriptional changes in the brains of cattle orally infected with the bovine spongiform encephalopathy agent precede detection of infectivity.

Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. BSE can be transmitted experimentally between cattle through the oral route, and in this study, brain tissue samples from animals at different time points postinoculation were analyzed for changes in gene expression. The aims of this study were to identify differentially regulated genes during the progression of BSE using microarray-based gene expression profiling and to understand the effect of prion pathogenesis on gene expression.