Development of a TaqMan® RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses

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Abstract

Chikungunya virus (CHIKV), a member of the alphavirus genus, is of considerable public health concern in Southeast Asian and African countries. However, despite serological evidence, the diagnosis of this arthropod-borne human disease is confirmed infrequently and needs to be improved. In fact, illness caused by CHIKV can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms, and laboratory confirmation of suspected cases is required to launch control measures during an epidemic. Moreover, no quantitative molecular tool is described to study CHIKV replication or detection in clinical samples and cell culture supernatants. In this study, a specific and sensitive CHIKV one-step TaqMan® RT-PCR assay was developed as a tool for the diagnosis of African CHIKV as well as a rapid indicator of active infection by quantifying viral load. This study also showed that a simple heat viral RNA release during the reverse transcription step constituted an alternative to the conventional RNA extraction method.

Introduction

Chikungunya virus (CHIKV) is an arthropod-borne alphavirus disseminated widely throughout Africa (Saluzzo et al., 1983, Muyembe-Tamfum et al., 2003), South-East Asia, Western Pacific and India (Lam et al., 2001). CHIKV is single strand positive RNA enveloped virus member of Togaviridae family and is transmitted from primates to humans generally by Aedes aegypti but also various aedine mosquitoes species (Yadav et al., 2003). Molecular epidemiological studies have shown that CHIKV clustered into three major distinct lineages, Asian, Central East and West African (Powers et al., 2000) and the virus originated probably in tropical Africa then imported into southern Asia. CHIKV illness has an important economic impact in many tropical countries, and due to the lack of specific symptoms, this infection cannot be differentiate from diseases such as dengue or yellow fever. CHIKV is classified serologically as a member of the Semliki Forest antigenic complex (Karabatsos, 1975) closely related to O’nyong-nyong virus (ONNV) because of its cross-reactivity. Detection of most alphaviruses is dependent currently on virus isolation from the blood of viremic patients, infected tissues or blood-feeding arthropods which process is time-consuming. The alphavirus species can be characterized by hemagglutination inhibition, complement fixation and neutralization of viral infectivity using reference sera (Calisher et al., 1988). For genus-specific detection of alphaviruses, an ELISA has been established (Greiser-Wilke et al., 1991) as well as sensitive RT-PCR or nested RT-PCR assays (Pfeffer et al., 1997, Powers et al., 2000, Paz Sanchez-Seco et al., 2001, Hasebe et al., 2002, Pfeffer et al., 2002, Bronzoni et al., 2004). Although CHIKV could be considered as a re-emerging threat, a few specific serological or molecular diagnosis tools are available. To date, only conventional RT-PCR methods have been suggested for the study of CHIKV replication in supernatants, clinical samples or for epidemiological survey. The aim of this study was to develop a rapid, sensitive and specific real-time RT-PCR method to detect and quantify African CHIKV in serum samples and infected cell supernatants. In a second step, this tool was optimised for direct viral RNA detection in small volumes without a previous RNA extraction or purification step (Thompson and Dietzgen, 1995, Klebe et al., 1996), in order to reduce sample processing, time and cost.

Section snippets

Virus preparation, titration and RNA extraction

Sindbis virus (SINV), Semliki Forest virus (SFV), Mayaro virus (MAYV), CHIKV strains 1720, 1728 and RCA (Central African genotypes with respective GenBank accession numbers AY549580, AY549582 and AY549584), Dengue virus (DENV), Yellow fever virus (YFV) were isolated in our laboratory. ONNV and CHIKV strain 37997 (West African genotype, GenBank accession number AF192892) were respectively kindly provided by Prof. H. Zeller (Institut Pasteur, Lyon, France) and the Institut Pasteur of Dakar.

All

Real-time RT-PCR detection

The quality of the synthetic CHIKV strain 1728 RNA transcript used for the development of the TaqMan® assay was estimated by different controls (data not shown): first the in vitro transcribed PCR product was sequenced; second the size and the purity of the RNA transcript was controlled by gel electrophoresis; third, direct amplification of the synthetic RNA with the Platinium Taq polymerase showed no DNA contamination. In pilot experiments, the Platinium Quantitative RT-PCR Thermoscript

Discussion

CHIKV, a member of the alphavirus genus, is of considerable public health importance in southeast Asian and African countries (Kit, 2002, Lam et al., 2001, Mourya et al., 2001). Although Chikungunya infections are rarely fatal and generally do not require admission to hospital, it is important to identify and quantify this infection for epidemiological studies. In fact, viral load should be a useful marker of disease progression and a measure of the antiviral compounds efficiency. Moreover, as

Acknowledgements

We thank H. Pugelli for primer synthesis and F. Tock for virus isolation. This work was supported by grants from the “service de santé des armées” and from the “délégation générale pour l’armement” (DGA).

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