Consensus PCR protocols for the detection of amphibian herpesviruses (Batrachovirus)

Amphibians have been disappearing at an unprecedented rate worldwide. Among the proposed contributing factors are infectious diseases. Investigations have focused mainly on ranavirus and chytrids; however, additional agents may be relevant stressors. Two novel batrachoviruses have been discovered (ranid herpesvirus 3 [RaHV-3] and bufonid herpesvirus 1 [BfHV-1]). Their clinical role is still to be clarified; however, both have been associated with obvious skin lesions in their respective hosts. Herein we present 2 consensus PCR protocols that can be used to detect all of the known and, possibly, yet to be discovered batrachoviruses. We targeted a 200 nt long, highly conserved region of the DNA terminase gene. We established a sensitive protocol, which can detect both European batrachoviruses (European batrachovirus PCR protocol; RaHV-3 and BfHV-1) and a panbatrachovirus PCR protocol detecting all known batrachoviruses, including ranid herpesvirus 1 and 2 (RaHV-1, -2). The limit of detection (LOD) for the European batrachovirus protocol was 10(1)copies of RaHV-3 and 10(2)copies of BfHV-1 per reaction. The panbatrachovirus protocol could detect all known batrachoviruses with LODs of 10(3)(RaHV-3, BfHV-1, RaHV-1) to 10(4)copies (RaHV-2) per reaction. These novel detection tools can be used as a first line of detection when herpesviral infection in amphibians is suspected, followed by additional PCRs with herpesvirus-specific primers in the case of known viral species, or sequencing as in the case of novel batrachoviruses.