Development of a consensus reverse transcription PCR assay for the specific detection of tortoise picornaviruses

Abstract

Picornaviruses (PVs) of different terrestrial tortoise species, previously designated as Virus "X," have been frequently detected from various tissues by virus isolation in Terrapene heart cell culture as the preferred laboratory method for diagnosis. Here, we describe the development of 2 diagnostic reverse transcription (RT)-PCR-based assays for the identification and characterization of tortoise PVs belonging to the tentative genus Topivirus To test the novel diagnostic systems, PVs were isolated from swab and tissue samples collected in Germany, Italy, and Hungary between 2000 and 2013. All 25 tested isolates gave positive results with both novel consensus primer sets. Sequencing of the amplified products confirmed that all studied viruses were members of the new proposed genus Topivirus Phylogenetic analyses clearly distinguished 2 lineages within the genus. Based on sequence analysis, no association was observed between the geographic distribution and genetic relatedness. Furthermore, no strict host specificity was indicated. The PCR-based diagnosis may provide a time-saving and sensitive method to detect tortoise PVs, and evaluation of PV presence in these animals may help control virus spread.

Investigations on the prevalence of tortoise picorna-virus in captive tortoises in Germany

“…Breeders in Germany have contact to other breeders, and animals are bred and sold, so the distribution of potentially sick/virus positive animals is likely to encompass the whole of Europe. Additionally, we tested several animals in each collection, other than in the investigations in Europe, where either samples in routine diagnostics of various owners, or animals in field studies were tested (10,13). We also support the potential role of the virus in shell softening in juvenile T. graeca, as all positive juveniles from a positive breeding pair showed clinical signs of the disease.…”

“…Antibody titres may also change in different stages of infection, which could also explain the negative animals. By now two more PCR protocols have been established, different strains have been detected and a higher number of samples have been tested positive using more than one protocol (13). It might be possible to retest our samples to include genetic variation and to get an even higher or more acurate prevalence in Germany.…”

“…Sequence analysis of isolate 1243/37 Zu, taken from a juvenile tortoise deceased after shell softening, revealed a picornavirus with a genome of approximately 8 kb in length (9). Consequently, RT-PCR for virus detection was developed by two different authors (9,13). At this point, the names tortoise testivirus 1 and tortoise picor navirus were proposed (6,9).…”

“…ToPV was thought to be a causative agent in the clinical pattern of soft carapace and plastron, but was also isolated in healthy tortoises in previous studies, for routine survey on chelonian viruses (10,(12)(13)(14)(15). Heuser et al (6) isolated the virus from different species but only T. graeca and one Geochelone elegans individual demonstrated clinical signs, whereas other species showed normal development but also showed seroconversion.…”

“…In August 2017, ToPV was classified as Torchivirus by the International Committee on Taxonomy of Viruses (ICTV) (8). Recent investigations consider genetic variability in detected ToPV (10,13). The present study focuses on the prevalence of ToPV in tortoise collections in Germany, since previous work focused on only one collection.…”