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

Abstract

A B S T R A C TRanaviruses 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 amphibianassociated 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 × 10 8 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.

Temperature is a key driver of a wildlife epidemic and future warming will increase impacts

“…Temperature was not retained as a significant predictor of in vivo viral load when logarithms of viral loads were modelled as a function of exposure and temperature treatments, survival outcome, and the interactions. However, the minimum adequate model did retain exposure (F2, 51 = 277, p < 0.001), survival outcome (F1, 51 = 197, p < 0.001), and their interaction (F2, 51 = 25.7, p = 1.88 × 10 -4 ; Fig. S4).…”

“…DNA was eluted in 100µL final volumes. Viral loads were estimated using quantitative PCRs against both host and viral targets (method, reaction mix and settings followed Leung et al 51 ).…”

“…Cells were grown on 96 well plates until more than 90% confluent and then inoculated with virus in a ten-fold dilution series ranging from a multiplicity of infection of approximately 2 x 10 -6 to 2 x 10 3 (five wells per dilution with an additional one well per dilution receiving a sham exposure of cell culture media only as a negative control). Titres of viral isolate stocks were equalised by reference to qPCR scores (following Leung et al51 ). Plates were incubated at six temperatures(10, 14, 18, 22, 26, and 30°C) and monitored daily for cytopathic effect (plaques in the cell layer).…”

Effects of historic and projected climate change on the range and impacts of an emerging wildlife disease

“…Cells were grown on 96-well plates until more than 90% confluent and then inoculated with virus in a 10-fold dilution series ranging from an estimated multiplicity of infection of approximately 2 × 10 −6 to 2 × 10 3 (five wells per dilution with an additional one well per dilution receiving a sham exposure of cell culture media only as a negative control). Titers of viral isolate stocks were equalized by reference to qPCR scores [following Leung et al (2017)]. Plates were incubated at six temperatures (10, 14, 18, 22, 26, and 30°C) and monitored daily for cytopathic effect (plaques in the cell layer).…”

Sublethal Effects of Wild-Type and a vIF-2&alpha;-Knockout <em>Frog virus 3</em> on Post-Metamorphic Wood Frogs (<em>Rana sylvatica</em>)

“…Tissue samples were assessed for Ranavirus following the protocol for quantitative PCR (qPCR) described by Leung et al [40], and viral loads were quantified as described by Hoverman et al [14].…”