Quantitative real-time PCR detection of Rift Valley fever virus and its application to evaluation of antiviral compounds

Abstract

The Rift Valley fever virus (RVFV), a member of the genus Phlebovirus (family Bunyaviridae) is an enveloped negative-strand RNA virus with a tripartite genome. Until 2000, RVFV circulation was limited to the African continent, but the recent deadly outbreak in the Arabian Peninsula dramatically illustrated the need for rapid diagnostic methods, effective treatments, and prophylaxis. A method for quantifying the small RNA segment by a real-time detection reverse transcription (RT)-PCR using TaqMan technology and targeting the nonstructural protein-coding region was developed, and primers and a probe were designed. After optimization of the amplification reaction and establishment of a calibration curve with synthetic RNA transcribed in vitro from a plasmid containing the gene of interest, real-time RT-PCR was assessed with samples consisting of RVFV from infected Vero cells. The method was found to be specific for RVFV, and it was successfully applied to the detection of the RVFV genome in animal sera infected with RVFV as well as to the assessment of the efficiency of various drugs (ribavirin, alpha interferon, 6-azauridine, and glycyrrhizin) for antiviral activity. Altogether, the results indicated a strong correlation between the infectious virus titer and the amount of viral genome assayed by real time RT-PCR. This novel method could be of great interest for the rapid diagnosis and screening of new antiviral compounds, as it is sensitive and time saving and does not require manipulation of infectious material.

FIG. 1

Standard curve obtained with 10-fold serial dilutions of viral RVFV RNA. C_T values are plotted against different RNA amounts extracted from the infectious virus (titers running from 10⁶ to 10 TCID₅₀ · ml⁻¹).

FIG. 2

Effect of antiviral compounds on the RVFV (strain MP12) yield measured by either genomic (A) or virus (B) titer. Antiviral compound concentrations, increasing from C₁ to C₅, are given in Table 4. VT is the virus control (infected nontreated cells).