Zoology/Biology session

Amphibian populations are drastically shrinking or disappearing worldwide. In addition to the well-known environmental factors, infectious diseases, including viruses, play a significant role in this process. Among them, ranaviruses (RV), which can cause severe local mortality events, have already been detected in Hungary by the sensitive qPCR method, yet no genetic characterisation has been done. In the last decade, two new frog herpesviruses have been described from several Western European countries, and signs resembling these infections have been reported in our country as well. Still, the causative agents have not been detected and characterised. In amphibians, only a few species have been described with partial circovirus (CV) sequences, in two cases these were proven to be endogenous viruses, residues incorporated into the host genome. Yet, amphibians remain a class without a complete genome of an infectious (exogenous) circovirus described. Our study aimed to detect representatives of the above-listed virus groups in Hungary, from freshly collected swab samples by polymerase chain reaction (PCR) and to characterise them genetically. Our samples were collected in 14 habitats near Budapest, from a total of 56 common toads (Bufo bufo) and 48 agile frogs (Rana dalmatina). During the screening of viruses, we targeted the major capsid protein gene of RV, the DNA polymerase gene of herpesviruses and the rep gene of circoviruses. Additional primers were designed to reveal the full length of a gene (ORF97) of the detected ranid herpesvirus, which was presumed to be more variable, and to obtain the full genome of the circovirus (or to confirm its endogeneity). We detected RV in two agile frogs but their genetic characterisation is still ongoing. The presence of ranid herpesvirus-3 was confirmed in 12 agile frogs from four different habitats. Based on partial DNA polymerase gene (1200bp) and full ORF 97 gene (3750bp) sequences, this Hungarian strain differs from the Western European prototype by 3-4%. Bufonid herpesvirus was detected from one toad in each of two habitats, with short sequences identical to the Swiss prototype. When screening the common toad samples, using the highly degenerate CV primers, we could verify the presence of a partial rep gene in 10 individuals. Additionally, 13 individuals provided a specific size amplicon without reliable sequence, while the remaining individuals yielded no PCR product at all. Using primers designed by us, we confirmed that the toad circovirus is endogenous, and we identified its site of integration into the host chromosome. In the case of agile frog samples, the CV monitoring PCR resulted in the description of a novel cyclovirus sequence and as a side finding the detection of a pigeon circovirus contaminant.