Highly Sensitive and Broadly Reactive Quantitative Reverse Transcription-PCR Assay for High-Throughput Detection of Rift Valley Fever Virus

Abstract

Rift Valley fever (RVF) virus is a mosquito-borne virus associated with large-scale epizootics/epidemics throughout Africa and the Arabian peninsula. Virus infection can result in economically disastrous "abortion storms" and high newborn mortality in livestock. Human infections result in a flu-like illness, with 1 to 2% of patients developing severe complications, including encephalitis or hemorrhagic fever with high fatality rates. There is a critical need for a highly sensitive and specific molecular diagnostic assay capable of detecting the natural genetic spectrum of RVF viruses. We report here the establishment of a pan-RVF virus quantitative real-time reverse transcription-PCR assay with high analytical sensitivity (ϳ5 RNA copies of in vitro-transcribed RNA/reaction or ϳ0.1 PFU of infectious virus/reaction) and efficiency (standard curve slope ‫؍‬ ؊3.66).

Elevated Antibodies Against Rift Valley Fever Virus Among Humans with Exposure to Ruminants in Saudi Arabia

“…However, although viremia and IgM antibody persistence for RVFV have been well documented, less is known about the long-term persistence of human IgG antibodies against RVFV; therefore, post-outbreak exposure cannot be completely ruled out. 15 Furthermore, the detection of IgM antibody in one of the PRNT-positive samples could suggest the possibility of recent exposure to RVFV. More evidence would be necessary, including the collection of prospective exposure data, to better ascertain whether a recent exposure had occurred.…”

“…A one-step qRT-PCR protocol was performed using the iScript cDNA synthesis kit (BioRad, Hercules, CA) using primers, probes, and cycling protocol previously described. 15 A positive control of extracted RNA from the RVFV MP-12 strain was prepared for each run, and a no template and negative control. All qRT-PCR samples were tested in triplicate.…”

Rift Valley Fever Virus Lacking the NSs and NSm Genes Is Highly Attenuated, Confers Protective Immunity from Virulent Virus Challenge, and Allows for Differential Identification of Infected and Vaccinated Animals

“…Whole rat blood was assayed for the presence and quantity of RVF virus-specific RNA following protocols described previously (8). Quantification of total serum RVF virus RNA was calculated directly via interpolation from a standard curve generated from serial dilutions of stock RVF virus strain ZH501, of a known titer, in whole rat blood extracted and processed in a manner identical to that of each experimental replicate q-RT-PCR run.…”

“…Briefly, 25 l of whole rat blood (either from vaccinated/challenged animals or from stock virus serial dilutions) was added to 300 l of 2ϫ noncellular lysis buffer (Applied Biosystems), and total RNA was extracted (ABI 6100 nucleic acid workstation; Applied Biosystems). After RNA was extracted, cDNA was generated by random hexamer priming (high-capacity cDNA kit; Applied Biosystems), followed by RVF virus-specific q-PCR (Universal q-PCR master mixture; Applied Biosystems), using the protocol, the primer/probe combination, and the thermocycling conditions outlined previously (8). Results are reported as RVF virus PFU equivalents (eq)/ml of rat blood.…”

Rift Valley Fever Outbreak in Livestock, Mozambique, 2014

“…A quantitative real-time reverse transcription PCR was performed as described (10). Positive samples were subsequently subjected to a conventional RT-PCR to amplify a 490-nt region of the medium segment as described (11).…”