Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection

Abstract

The definitive diagnosis of lyssavirus infection (including rabies) in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR) based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR) method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan) RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR) and a second reaction using an intercalating dye (SYBR Green) to detect other lyssavirus species (pan-lyssa RT-qPCR). The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135) including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5%) and saliva (54%) samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco). This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for lyssavirus surveillance.

Fig 1. Multiple alignment of the 102 partial polymerase sequences of RABV species, with nucleotide sequences and positions for the Taq3long and Taq17revlong primers and TaqMan hybridization probes RABV4 and RABV5 of the pan-RABV RT-qPCR assay.

The oligonucleotide sequence of each primer and probe is indicated in bold, together with its name and an arrow indicating the sense direction. Identity to primer and probe sequences is highlighted in gray. Dots indicate identity to the reference sequence 9106MAR. Positions are indicated according to the reference sequence PV (GenBank accession number M13215). Asterisks indicated partial polymerase sequences obtained in this study. A description of the RABV isolates included in this multiple alignment is provided in S1 Table.

Fig 2. Multiple alignment of 45 partial polymerase sequences from lyssaviruses other than RABV species, with nucleotide sequences and the positions of the Taq5long and Taq16revlong primers (pan-lyssa RT-qPCR).

The oligonucleotide sequence of each primer is indicated in bold, together with its name and an arrow indicating the sense direction. Identity to primer sequences is highlighted in gray. Dots indicate identity to the reference sequence 04006SEN. Positions are indicated according to the reference sequence PV (GenBank accession number M13215). Asterisks indicated partial polymerase sequences obtained in this study. Lyssavirus species are indicated on the left side of the figure, and a description of the lyssavirus isolates included in this multiple alignment is provided in S1 Table.