Abstract
Mosquito-borne viruses encompass a range of virus families, comprising a number of significant human pathogens (e.g., dengue viruses, West Nile virus, Chikungunya virus). Virulent strains of these viruses are continually evolving and expanding their geographic range, thus rapid and sensitive screening assays are required to detect emerging viruses and monitor their prevalence and spread in mosquito populations. Double-stranded RNA (dsRNA) is produced during the replication of many of these viruses as either an intermediate in RNA replication (e.g., flaviviruses, togaviruses) or the double-stranded RNA genome (e.g., reoviruses). Detection and discovery of novel viruses from field and clinical samples usually relies on recognition of antigens or nucleotide sequences conserved within a virus genus or family. However, due to the wide antigenic and genetic variation within and between viral families, many novel or divergent species can be overlooked by these approaches. We have developed two monoclonal antibodies (mAbs) which show co-localised staining with proteins involved in viral RNA replication in immunofluorescence assay (IFA), suggesting specific reactivity to viral dsRNA. By assessing binding against a panel of synthetic dsRNA molecules, we have shown that these mAbs recognise dsRNA greater than 30 base pairs in length in a sequence-independent manner. IFA and enzyme-linked immunosorbent assay (ELISA) were employed to demonstrate detection of a panel of RNA viruses from several families, in a range of cell types. These mAbs, termed monoclonal antibodies to viral RNA intermediates in cells (MAVRIC), have now been incorporated into a high-throughput, economical ELISA-based screening system for the detection and discovery of viruses from mosquito populations. Our results have demonstrated that this simple system enables the efficient detection and isolation of a range of known and novel viruses in cells inoculated with field-caught mosquito samples, and represents a rapid, sequence-independent, and cost-effective approach to virus discovery.
Highlights
Arthropod-borne viruses encompass a range of veterinary and medically significant viral pathogens belonging to five antigenically distinct families of RNA:DNA hybrid (RNA) viruses
The authors demonstrate the application of two antibodies to detect double-stranded RNA which is a common molecule produced in infection by a number of different viruses
The use of antibodies which react with double-stranded RNA independently of sequence allows for detection of a diverse range of viruses and has been instrumental in the detection of known arboviruses from three different families and the discovery of a number of previously unknown viruses from Australian mosquito populations
Summary
Arthropod-borne viruses (arboviruses) encompass a range of veterinary and medically significant viral pathogens belonging to five antigenically distinct families of RNA viruses. These families can be separated according to their genome type: those with positive-sense single-stranded RNA ((+)ssRNA) genomes, the Togaviridae, Flaviviridae; negative-sense RNA ((-) ssRNA) genomes, Rhabdoviridae and Bunyaviridae; and the double-stranded RNA (dsRNA) viruses of the Reoviridae family. These viruses cycle between haematophagous arthropod vectors and reservoir/amplifying vertebrate hosts. As a result many arthropod populations are routinely surveyed in an attempt to assess the risk of arboviruses and identify emerging pathogens
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