The advent of next generation sequencing technologies has greatly improved our capacity for detection and identification of new viruses in mosquitoes and thereby facilitated the proposal of numerous novel virus taxons. However, this approach is expensive and often does not allow for viral isolation.
We have developed a broad-spectrum virus detection assay based on monoclonal antibodies to viral RNA intermediates in cells (MAVRIC). This assay uses two monoclonal antibodies (mAbs) which target double-stranded RNA (dsRNA) intermediates produced in cells infected with single-stranded positive-sense RNA viruses and double-stranded RNA viruses. This approach couples isolation of viruses from mosquito samples by cell culture inoculation, with fixed-cell ELISA to detect viral replication in a sequence-independent manner. This alleviates the requirement for virus-specific antibodies or production of overt cytopathic effects to identify virus-positive cultures. The non-sequence specific affinity of the MAVRIC antibodies also allows for non-biased enrichment of long double-stranded viral RNA from infected cell lysates for identification of viral sequence and removal of contaminating host sequence.
To date, this system has been instrumental in the discovery of a number of novel viruses in mosquitoes, including a new species in the Mesoniviridae family, a novel orbivirus, negevirus, and three new flavivirus species. The MAVRIC system represents a streamlined and economical approach for the detection and discovery of viruses in mosquito populations and is currently being trialled in a number of laboratories in Australia to assess its potential for integration into routine arbovirus surveillance programs.