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

William C Wilson,Marco Romito,Dane C Jasperson,Hana Weingartl,Yatinder S Binepal,Moabi R Maluleke,David B Wallace,Petrus Jansen Van Vuren,Janusz T Paweska

doi:10.1016/j.jviromet.2013.07.006

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.

Highlights

Recent outbreaks of Rift Valley fever (RVF) in Kenya (Nguku et al, 2010), Madagascar (Carroll et al, 2011), South Africa (Archer et al, 2011) and Mauritania (El Mamy et al, 2011) have highlighted the need for rapid and reliable diagnostic tools for detecting this zoonotic disease of significant public health, veterinary and socio-economic importance
The possible unintentional or intentional introduction of Rift Valley fever virus (RVFV) into a non-endemic country is of significant concern and it is considered a high priority zoonotic disease (Chevalier et al, 2010; Hartley et al, 2011)
The current reverse transcriptasepolymerase chain reaction (rRT-PCR) assays all detect a single region on the RVF viral genome

Summary

Introduction

Recent outbreaks of Rift Valley fever (RVF) in Kenya (Nguku et al, 2010), Madagascar (Carroll et al, 2011), South Africa (Archer et al, 2011) and Mauritania (El Mamy et al, 2011) have highlighted the need for rapid and reliable diagnostic tools for detecting this zoonotic disease of significant public health, veterinary and socio-economic importance. Disease outbreaks vary significantly in the percentage of susceptible animals affected, ranging from 15% to 90% (EMPRES, 2005). Those which succumb display severe disease, coupled with abortion in pregnant cattle, sheep, and goats and 70–100% mortality in young animals (Gerdes, 2004). Endemic prevalence of antibody in humans ranges from 5 to 40% (EMPRES, 2005), but increases with risk factors such as livestock handling, exposure to aerosols during the slaughter of infected animals and consumption of raw milk (Gerdes, 2004). Infection of humans can result in hepatitis, hemorrhagic fever, encephalitis, ocular degeneration, and death (Al-Hazmi et al, 2003; Archer et al, 2011; Gerdes, 2004)

Methods

Results

Conclusion