Development of PCR-based methods for detection of African lyssaviruses

Abstract

The etiological agent of rabies encephalitis belongs to the genus Lyssavirus in the Rhabdoviridae family. Lyssaviruses are negative sense, single stranded RNA viruses and cause an estimated 55 000 human deaths per year with 44% of these deaths occurring in Africa (WHO, 2005). With intense research effort and increased sequence information it is becoming evident that the Lyssavirus genus is much more diverse than initially thought and therefore diagnostic methods need to be modified accordingly. The African continent sustains a diverse variety of lyssaviruses, however, most countries in Africa do not have active surveillance or necessary diagnostic tools and therefore rabies-related lyssaviruses are underreported. Previous studies have indicated that real-time PCR has improved sensitivity and rapidity over conventional molecular diagnostic methods with the added advantage of allowing accurate estimations of viral load in a wide variety of samples. Several realtime PCR assays have been developed; however, none were specifically aimed at detection of lyssaviruses present on the African continent. This study was therefore aimed at evaluating certain molecular diagnostic methods for the detection of African lyssaviruses. Furthermore, the application of real-time PCR for various fields in lyssavirus research i.e. diagnostics, surveillance and pathogenicity studies were evaluated. This study revealed two different hemi-nested PCR assays capable of detecting representatives of African lyssaviruses. A real-time PCR was developed that was successful for the detection of African lyssaviruses. In addition, a quantitative assay and internal control was successfully employed for confirming ante-mortem human rabies diagnosis as well as post-mortem animal rabies diagnosis in formalin fixed brain material. As such the real-time PCR assay developed in this study could therefore be routinely used for ante-mortem diagnosis and as a confirmatory test for post-mortem diagnosis. The ability of this assay to detect and quantify all currently known African lyssaviruses not only offers improved surveillance capacity, but offers unique potential as a sensitive tool to track virus movement in pathogenicity studies. These aspects are important in our search for a better understanding of the complex epidemiological and viral characteristics of African lyssaviruses. Copyright