A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

Rabies, resulting from infection by Rabies virus(RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabiesis essential for timely administration of post-exposure prophylaxisin humans and control of the disease in animals. Currently, only the direct fluorescent antibody(DFA) test is recommended for routine rabiesdiagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assayfor the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assaynamed LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirusgenus while maintaining sensitivity and specificity. The primers and probes of the LN34 assaytarget the highly conserved non-coding leader region and part of the nucleoprotein(N) coding sequence of the Lyssavirusgenome to maintain assayrobustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assaywas able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirusspecies. The LN34 assaywas successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assayrepresents a major improvement over previously published rabiesspecific RT-PCR and real-time RT-PCR assaysbecause of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabiesmolecular diagnostics. The LN34 assayprovides an alternative approach for rabiesdiagnostics, especially in rural areas and rabiesendemic regions that lack the conditions and broad experience required to run the standard DFA assay.