Green qPCR assay in this study has been developed to demonstrate that the universal primer pair, JW12 and N165-146 targets the N gene successfully for each of the known Lyssavirus
species. We developed this to address the problems associated with the diagnosis of divergent Lyssavirus
infections. There are currently numerous assays available for rabies and Lyssavirus
detection and these have recently been reviewed (Fooks et al., 2009
). Of the several real-time PCR assays published, however, none has been validated against for the detection of all published Lyssavirus
species. Despite the need for further assessment of the sensitivity of this assay, including as a possible one-step technique, we have demonstrated that this primer pair in a real-time application is more sensitive for the detection of all Lyssavirus
cDNA that we tested, when compared with a known pan-Lyssavirus
hnRT-PCR. For some genetically similar viruses, e.g. RV2 and RV611 from LBV lineage C, there appeared to be substantial differences in limits of detection by qPCR. Whilst standardising for total RNA, the true quantity of virus RNA is unknown in these samples. Using plasmid clones addresses this somewhat (see below), however, the substantial differences in detection between the qPCR and hnRT-PCR for LBV lineage C viruses may reflect poor primer binding of the hnRT-PCR for LBV viruses RV2 and RV3, compared to the qPCR primer pair. This has been discussed previously by another group (Coertse et al., 2010
). Further evidence of the increased primer binding efficiency for this universal primer pair has been previously demonstrated on a panel of over 40 RABV and a range of EBLV-1 and -2 viruses (Wakeley et al., 2005
), which demonstrated a similarly greater sensitivity over the hnRT-PCR to that found in this study. This new assay overcomes restrictions in the current hnRT-PCR assay which requires multiple transfers of material and substantial time (both in required man-hours for preparation and thermal cycling) (Heaton et al., 1997
). Despite this, the hnRT-PCR has been sufficient to detect virus from each virus-positive brain sample and therefore still offers a useful tool for rabies diagnosis where conventional PCR technology exists. Importantly, hnRT-PCR produces a PCR product that is readily sequenced.
The use of only two universal primers (JW12 and N165-146) plus SYBR® Green (Applied Biosystems, Foster City, CA, USA) dsDNA dye is an easy to use assay which enabled each of the viruses archived in the WHO and OIE rabies reference laboratory at VLA to be detected in infected brain material. The PCR assay was shown to be highly sensitive, detecting between 5 and 50 copies of N-gene target sequences when cDNA from MOKV, DUVV and LBV (lineage A, B and C) were cloned into plasmid vectors and 25–195 copies of LBV RNA generated by in vitro transcription. Sensitivity for MOKV and DUVV RNA was lower, with 190–1430 copies detected. In each case, when used on infected material, the assay was more sensitive than the previously published hnRT-PCR, however future studies should assess the RNA detection limit for each of the viruses known. Additional attempts to further optimise the RT step, which leads to a loss of sensitivity, should also be made.
The use of synthetic DNA for the Lyssaviruses
isolated on single occasions from bats is novel and demonstrates that this JW12/N165-146 primer pair is able to detect cDNA from all currently known species. The detection limit was good; with the JW12/N165-146 SYBR®
Green assay able to detect 5 DNA copies of DNA Ultramers. This JW12/N165-146 primer set has previously been described for use with RABV, EBLV-1 and EBLV-2 TaqMan®
probes (Wakeley et al., 2005
) and in this study was shown to detect ABLV cDNA when used in conjunction with the dsDNA dye, SYBR®
Green (Applied Biosystems, Foster City, CA, USA). This assay, therefore, has the potential to be expanded to incorporate an RT step to make this assay a single tube test. Further testing, however, will be required to validate this assay for use as an OIE/WHO prescribed test, and it is likely that additional advances in technology will be required if the ultimate aim of having a sensitive diagnostic assay which differentiates between viruses is achieved (Fooks et al., 2009
). Despite the lack of ability of the SYBR®
Green (Applied Biosystems, Foster City, CA, USA) assay to differentiate species, the simplicity of this assay makes this an attractive option for laboratory use as a screening surveillance tool, enabling further analysis by hnRT-PCR or examination of the real-time PCR product by cloning and sequencing.
The current WHO “gold standard” test is the fluorescent antibody test (FAT), which uses a conjugated monoclonal antibody against the RABV nucleoprotein, but is believed to detect all viruses. Developing a sensitive real-time PCR assay capable of detecting all bat and phylogroup II Lyssaviruses
is particularly important, given that there is a considerable lack of understanding regarding the ecology of most bat Lyssaviruses
and that of MOKV. Indeed, the reservoir of MOKV is still unknown, despite it being a zoonotic infection leading to human deaths. The assay described should allow the detection of low levels of viral nucleic acid for which current vaccines offer no protection and may be used as a tool for the surveillance of phylogroup I, II and III Lyssaviruses
in a range of hosts where active infection is suspected. The FAT, whilst very useful and able to detect nucleoprotein, it is not as sensitive as the nucleic-acid detecting hnRT-PCR described by Heaton et al. (1997)
. Therefore, the development of this assay, approximately 200-fold more sensitive than the hnRT-PCR, may allow better estimation of the true number of cases of MOKV, DUVV and LBV infection. Further studies are required to assess the sensitivity and specificity of this assay, however, this assay already may be useful, where real-time technology exists. For example, the universal primer pair has already been used with species specific probes (Wakeley et al., 2005
) for use in an OIE/WHO reference laboratory.