Specific Detection of Rinderpest Virus by Real-Time Reverse Transcription-PCR in Preclinical and Clinical Samples from Experimentally Infected Cattle

Abstract

A highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (rRT-PCR)system, was developed. Five different RPV genomic targets were examined, and one was selected and optimized to detect viral RNA in infected tissue culture fluid with a level of detection ranging from 0.59 to 87.5 50% tissue culture infectious doses (TCID 50 ) per reaction depending on the viral isolate. The strain sensitivity of the test was validated on 16 RPV strains belonging to all three phylogenetic branches described for RPV. No cross-reactivity was detected with closely related peste des petit ruminants or with symptomatically similar viruses, including all seven serotypes of foot-and-mouth disease virus, two serotypes of vesicular stomatitis virus, bluetongue virus, and bovine herpes virus type 2. In samples from experimentally infected cattle, our real-time RT-PCR test was significantly more sensitive than the gold standard test of virus isolation, allowing the detection of the disease 2 to 4 days prior to the appearance of clinical signs. The comparison of clinical samples with putative diagnostic value from live animals showed that conjunctival swabs and blood buffy coat were the samples of choice for epidemiological surveillance, while lymph nodes performed the best as postmortem specimens. This portable and rapid real-time RT-PCR has the capability of the preclinical detection of RPV and provides differential diagnosis from look-alike diseases of cattle. As RPV is declared globally eradicated, this test provides an important rapid virus detection tool that does not require the use of infectious virus and allows the processing of a large number of samples.

Full genome sequencing of archived wild type and vaccine rinderpest virus isolates prior to their destruction

“…Sequencing libraries. All samples were first screened by reverse transcription real time PCR (RT-qPCR) specific for RPV 18 . RPV-positive RNA samples were processed to create sequencing libraries which were sequenced using an Illumina MiSeq.…”

“…The only published RT-qPCR assay for RPV 18 was developed after the virus was declared eradicated, and so was not subject to the kind of global testing undergone by the simple RT-PCR assays in use during the control and eradication programme 6 . It was therefore useful to assess this assay against the large set of RPV isolates in this study.…”

Protection of Cattle against Rinderpest by Vaccination with Wild-Type but Not Attenuated Strains of Peste des Petits Ruminants Virus

“…Total RNA was extracted using an LSI MagVet Universal Isolation kit (Thermo Fisher) on a MagMax Express-96 processor. RPV RNA was assayed by quantitative reverse transcription-PCR (RT-qPCR) in 5 μl of the extracted RNA using AgPath-ID one-step RT-PCR reagents and a modification of the primer/probe set L10 described by Carrillo and colleagues ( 21 ), in which the probe had a 3′ quencher/minor groove binder (Thermo Fisher) in place of the 3′ 6-carboxytetramethylrhodamine (TAMRA) quencher used in the original study, as this was required to allow the probe to work at 60°C. Results are expressed as the mean 40- C T value, where C T is the threshold cycle.…”

Long-term functional alterations in sports concussion

“…7,30,31,33 Specifically, N-acetylaspartate is consistently diminished in concussed athletes relative to nonconcussed athletes. Other neurometabolic alterations include diminished levels of both glutamate, which seem to normalize by 6 months postinjury, and myoinositol, which may be elevated in the chronic postinjury phase.…”