Abstract
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.
Highlights
Ranaviruses are large double stranded DNA viruses with broad host ranges which can cause systemic disease in ectothermic vertebrates (Chinchar, 2002)
This study describes the development of two quantitative PCR (qPCR) assays: one to detect associated ranaviruses (AARVs) by targeting a conserved region of the major capsid protein (MCP) gene, and a second qPCR assay targeting a single-copy ultra-conserved non-coding element (UCNE) of vertebrates, which serves to quantify host cells
Candidate UCNEs were retained if the following conditions were met: 1) single hits were returned for more than 90% of vertebrates suggesting that a specific qPCR assay could be designed and the sequence was present as a single copy in the haploid genome; 2) no hits to invertebrate species were returned
Summary
Ranaviruses (genus Ranavirus; family Iridoviridae) are large double stranded DNA viruses with broad host ranges which can cause systemic disease in ectothermic vertebrates (Chinchar, 2002). The genus has been divided into amphibian-associated ranaviruses (AARVs) − previously, frequently referred to as amphibian-like ranaviruses (Jancovich et al, 2010; Price, 2016) − and the fish associated SanteeCooper ranaviruses and grouper iridoviruses (GIV-like) based on phylogenetics (Jancovich et al, 2015). AARVs have been repeatedly associated with disease in amphibian hosts but can infect reptiles and fish (Stöhr et al, 2015). Ranavirus infection of amphibians and epizootic haematopoietic necrosis disease in fish are both listed as notifiable diseases by the World Organisation for Animal Health (OIE). Despite not being included on this list, quantitative PCR (qPCR)-based methods are commonly used as a screening tool for ranavirus (Black et al, 2017)
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