A quantitative-PCR based method to estimate ranavirus viral load following normalisation by reference to an ultraconserved vertebrate target

- QPCR assay development for absolute quantification of amphibian-associated ranaviruses.
- Viral assay Exhibits 100% comparative specificity and sensitivity.
- A second qPCR for an ultra-conserved element of vertebrates quantifies host-cells.
- Permits robust viral load comparison between tissues, individuals, and species.
- Combined assays applied to track ranavirus growth in cell culture.

Ranaviruses are important pathogens of amphibians, reptiles and fish. To meet the need for an analytical method for generating normalised and comparable infection data for these diverse host species, two standard-curve based quantitative-PCR (qPCR) assays were developed enabling viral load estimation across these host groups. A viral qPCR targeting the major capsid protein (MCP) gene was developed which was specific to amphibian-associated ranaviruses with high analytical sensitivity (lower limit of detection: 4.23 plasmid standard copies per reaction) and high reproducibility across a wide dynamic range (coefficient of variation below 3.82% from 3 to 3 × 108 standard copies per reaction). The comparative sensitivity of the viral qPCR was 100% (n = 78) based on agreement with an established end-point PCR. Comparative specificity with the end-point PCR was also 100% (n = 94) using samples from sites with no history of ranavirus infection. To normalise viral quantities, a host qPCR was developed which targeted a single-copy, ultra-conserved non-coding element (UCNE) of vertebrates. Viral and host qPCRs were applied to track ranavirus growth in culture. The two assays offer a robust approach to viral load estimation and the host qPCR can be paired with assays targeting other pathogens to study infection burdens.