Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples

Objective To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. Design A published hemi‐nested reverse transcriptase polymerase chain reaction (RT‐PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT‐PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi‐nested RT‐PCR assays. The sequences of RT‐PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. Results The nested RT‐PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi‐nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT‐PCR assay, with a detection level of approximately 10 genome equivalents per µl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. Conclusion The nested RT‐PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT‐PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis.