Background: With the advent of Next Generation Sequencing (NGS) technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field. Most RNA viruses have relatively small genomes in comparison to other organisms and as such, would appear to be an obvious success story for the use of NGS technologies. However, due to the relatively low abundance of viral RNA in relation to host RNA, RNA viruses have proved relatively difficult to sequence using NGS technologies. Here we detail a simple, robust methodology, without the use of ultra-centrifugation, filtration or viral enrichment protocols, to prepare RNA from diagnostic clinical tissue samples, cell monolayers and tissue culture supernatant, for subsequent sequencing on the Roche 454 platform. Results: As representative RNA viruses, full genome sequence was successfully obtained from known lyssaviruses belonging to recognized species and a novel lyssavirus species using these protocols and assembling the reads using de novo algorithms. Furthermore, genome sequences were generated from considerably less than 200 ng RNA, indicating that manufacturers’ minimum template guidance is conservative. In addition to obtaining genome consensus sequence, a high proportion of SNPs (Single Nucleotide Polymorphisms) were identified in the majority of samples analyzed. Conclusions: The approaches reported clearly facilitate successful full genome lyssavirus sequencing and can be universally applied to discovering and obtaining consensus genome sequences of RNA viruses from a variety of sources.

Several reverse transcription-PCR (RT-PCR) methods have been reported for the detection of rabies and rabies-related viruses. These methods invariably involve multiple transfers of nucleic acids between different tubes, with the risk of contamination leading to the production of false-positive results. Here we describe a single, closed-tube, nonnested RT-PCR with TaqMan technology that distinguishes between classical rabies virus (genotype 1) and European bat lyssaviruses 1 and 2 (genotypes 5 and 6) in real time. The TaqMan assay is rapid, sensitive, and specific and allows for the genotyping of unknown isolates concomitant with the RT-PCR. The assay can be applied quantitatively and the use of an internal control enables the quality of the isolated template to be assessed. Despite sequence heterogeneity in the N gene between the different genotypes, a universal forward and reverse primer set has been designed, allowing for the simplification of previously described assays. We propose that within a geographically constrained area, this assay will be a useful tool for the detection and differentiation of members of the Lyssavirus genus.

Fifty-five rabies virus isolates originating from different regions of the former Soviet Union (FSU) were compared with isolates originating from Eurasia, Africa, and North America according to com...

In Europe, two bat lyssaviruses referred to as European bat lyssaviruses (EBLVs) types 1 and 2 (genotypes 5 and 6 respectively) which are closely related to classical rabies virus are responsible for an emerging zoonosis. EBLVs are host restricted to bats, and have been known to infect not only their primary hosts but also in rare circumstances, induce spillover infections to terrestrial mammals including domestic livestock, wildlife and man. Although spillover infections have occurred, there has been no evidence that the virus adapted to a new host. Since 1977, four human deaths from EBLVs have been reported. None of them had a record of prophylactic rabies immunization. Only fragmentary data exist about the effectiveness of current vaccines in cross-protection against EBLVs. It is clear that EBLV in bats cannot be eliminated using conventional strategies similar to the control programmes based on vaccine baits used for fox rabies in Europe during the 1980s. Due to the protected status of bats in Europe, our knowledge of EBLV prevalence and epidemiology is limited. It is possible that EBLV is under-reported and that the recorded cases of EBLV represent only a small proportion of the actual number of infected bats. For this reason, any interaction between man and bats in Europe must be considered as a possible exposure. Human exposure through biting incidents, especially unprovoked attacks, should be treated immediately with rabies post-exposure treatment and the bat, where possible, retained for laboratory analysis. Preventative measures include educating all bat handlers of the risks posed by rabies-infected animals and advising them to be immunized. This review provides a brief history of EBLVs, their distribution in host species and the public health risks.