Species-level identification of trypanosomes infecting Australian wildlife by High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR).

Conventional nested PCR and Sanger sequencing methods are currently the gold standards for detecting trypanosomes in wildlife. However, these techniques are time-consuming and can often overlook mixed infections. True trypanosome prevalence can thus be underrepresented. Here, we designed an 18S rDNA-based real-time quantitative PCR (qPCR) assay coupled with High-Resolution Melting Analysis (HRMA) to detect and discriminate three Trypanosoma species (T. copemani, T. noyesi, and T. vegrandis) commonly infecting Australian marsupials. A total of 68 genetically characterised samples from blood and tissue were used to validate the High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR) assay. A further 87 marsupial samples consisting of blood, tissue and in vitro cultures derived from wildlife blood samples, were screened for the first time using this assay, and species identity confirmed using conventional PCR and Sanger sequencing. All three Trypanosoma species were successfully detected in pure cultures using the HRM-qPCR assay, and in samples containing mixed trypanosome infections. Of the 87 marsupial samples screened using the HRM-qPCR assay, 93.1% were positive for trypanosomes, and 8.0% contained more than one trypanosome species. In addition to the three targeted Trypanosoma species, this assay was also able to detect and identify other native and exotic trypanosomes. The turnaround time for this assay, from sample preparation to obtaining results, was less than 2 h, with a detection limit of 10 copies of the amplicon in a reaction for each of the targeted trypanosome species. This more rapid and sensitive diagnostic tool provides a high throughput platform for the detection, identification and quantification of trypanosome infections. It will also improve understanding of host diversity and parasite relationships and facilitate conservation management decisions.

The complete nucleotide sequence of Subterranean clover mottle virus.

The complete nucleotide sequence of Subterranean clover mottle virus (SCMoV) genomic RNA has been determined. The SCMoV genome is 4,258 nucleotides in length. It shares most nucleotide and amino acid sequence identity with the genome of Lucerne transient streak virus (LTSV). SCMoV RNA encodes four overlapping open reading frames and has a genome organisation similar to that of Cocksfoot mottle virus (CfMV). ORF1 and ORF4 are predicted to encode single proteins. ORF2 is predicted to encode two proteins that are derived from a -1 translational frameshift between two overlapping reading frames (ORF2a and ORF2b). A search of amino acid databases did not find a significant match for ORF1 and the function of this protein remains unclear. ORF2a contains a motif typical of chymotrypsin-like serine proteases and ORF2b has motifs characteristically present in positive-stranded RNA-dependent RNA polymerases. ORF4 is likely to be expressed from a subgenomic RNA and encodes the viral coat protein. The ORF2a/ORF2b overlapping gene expression strategy used by SCMoV and CfMV is similar to that of the poleroviruses and differ from that of other published sobemoviruses. These results suggest that the sobemoviruses could now be divided into two distinct subgroups based on those that express the RNA-dependent RNA polymerase from a single, in-frame polyprotein, and those that express it via a -1 translational frameshifting mechanism.