Development of a Rift Valley fever real-time RT-PCR assay that can detect all three genome segments

Abstract

Outbreaks of Rift Valley fever in Kenya, Madagascar, Mauritania, and South Africa had devastating effects on livestock and human health. In addition, this disease is a food security issue for endemic countries. There is growing concern for the potential introduction of RVF into non-endemic countries. A number of single-gene target amplification assays have been developed for the rapid detection of RVF viral RNA. This paper describes the development of an improved amplification assay that includes two confirmatory target RNA segments (L and M) and a third target gene, NSs, which is deleted in the Clone 13 commercial vaccine and other candidate vaccines. The assay also contains an exogenous RNA control added during the PCR setup for detection of amplification inhibitors. The assay was evaluated initially with samples from experimentally infected animals, after which clinical veterinary and human samples from endemic countries were tested for further evaluation. The assay has a sensitivity range of 66.7-100% and a specificity of 92.0-100% depending on the comparison. The assay has an overall sensitivity of 92.5%, specificity of 95% and a positive predictive value of 98.7%. The single-tube assay provides confirmation of the presence of RVFV RNA for improved confidence in diagnostic results and a "differentiate infected from vaccinated animals" (DIVA)--compatible marker for RVFV NSs--deleted vaccines, which is useful for RVF endemic countries, but especially important in non-endemic countries.

Absence of Evidence of Rift Valley Fever Infection in Eulemur fulvus (Brown Lemur) in Mayotte During an Interepidemic Period

“…Blood samples were centrifuged and sera were kept at −80°C and shipped to La Reunion (France) for laboratory testing. To look for acute infections, detection of RVF virus genome was performed using the RT-PCR technique based on the L segment with a detection limit of 0.5 TCID 50 /mL (Wilson et al 2013). Evidence of past infections was examined by detecting RVF-specific IgG antibodies in serum (ID Screen RVF Competition multispecies ELISA kit; IDVet, Grabels, France) with a diagnostic sensitivity of 98% and specificity of 100% (Kortekaas et al 2013).…”

Rift Valley Fever Virus, Japanese Encephalitis Virus, and African Swine Fever Virus: Three Transboundary, Vector-Borne, Veterinary Biothreats With Diverse Surveillance, and Response Capacity Needs

“…This assay was successfully validated on human RVF clinical samples, and has clinical diagnostic capabilities out to at least 10 days post-symptomonset. The assay developed by Wilson et al amplifies the L and M segments as confirmatory targets, and includes a third target gene, NSs, which is deleted in multiple vaccine candidates (64). This approach will be tremendously advantageous for simultaneously monitoring vaccine coverage vs. virus spread.…”

Artificial Synthesis of Conserved Segment S Gene Fragment of Rift Valley Fever Virus and Preliminary Study of Its Reverse Transcription Loop-Mediated Isothermal Amplification Detection Method

“…As there is no ideal vaccine, developing a rapid detection and diagnosis technology has important agricultural and public health significance for RVFV control in both epidemic and non-epidemic areas [14]. Presently, apart from traditional methods such as virus isolation, serology diagnosis, virus neutralization testing, and hemagglutination inhibition tests, there are also RT-PCR [15], ELISA [2], nested RT-PCR [16], fluorescence RT-PCR [3], and RT-LAMP [17] technologies available for detecting and diagnosing RVFV. As there is no RVFV pathogen in China, because of the pathogen's characteristics (such as being harmful to both human and animals, and its high transmissibility, pathogenicity, and perniciousness), research on RVFV is seldom reported in China [18].…”

“…When infected by RVFV, pregnant dams show an abortion rate of 90 -100 %. The mortality rate for adult animals of different species varies in the range 10 to 60 %, and that of newborn lambs reaches 90 -100 % [2][3][4]. Humans infected by the virus have moderate symptoms such as chills, headache, and pain in the extremities (that is, influenza-like symptoms) or there may be other unapparent symptoms.…”

“…Humans infected by the virus have moderate symptoms such as chills, headache, and pain in the extremities (that is, influenza-like symptoms) or there may be other unapparent symptoms. Infection usually lasts for several days and may lead to retinal injury, temporary or permanent blindness, encephalitis, hemorrhagic hepatitis, and, in serious cases, even death [2][3][4][5]. Heavy economic losses have been inflicted on countries and areas as a result of RVF epidemics and the threat to human health posed by RVF is serious as well.…”