Stella A. Atim

Small ruminants (sheep and goats) are one of the few mammals in which an exogenous retrovirus (XRV) and closely related endogenous retroviral elements (ERV) coexist within the same host genome. The betaretroviruses Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) cause pulmonary and nasal adenocarcinomas, respectively, and share extensive sequence similarity with their endogenous counterparts. Consequently, molecular surveillance must rely on assays that can unequivocally distinguish true exogenous infection from ERV-derived templates; failure to do so compromises diagnosis, phylogenetic inference, and epidemiological conclusions. We retrieved all complete JSRV, ENTV-1/2, and related ERV genomes deposited in public repositories and performed a comprehensive alignment. Only a limited number of genomic segments were capable of distinguishing exogenous from endogenous sequences. We refer to these as discriminating regions (DRs). Phylogenies built using DRs revealed that several entries annotated as XRV are, in fact, ERV-derived or chimeric artefacts generated by short-amplicon reconstruction. A systematic literature review of over 100 articles identified 286 distinct primers and probes used for the XRV amplification. In-silico mapping of each oligonucleotide onto the full alignment showed that only 28 % reliably differentiate XRV from ERV. We experimentally validated the predictive power of this approach for 17 primer/probe sets, confirming that non-discriminating assays produce false-positive signals from endogenous templates. The misannotation of ERV sequences as exogenous viruses has resulting in the population of databases with dubious entries, fostering erroneous hypotheses such as vector-borne transmission of JSRV and ENTV. To address this issue, we propose a concise set of criteria for assay design, validation, and database annotation emphasizing DR targeting, specificity testing against endogenous templates, and transparent reporting. Although this framework was developed for small ruminants, it is readily applicable to any host–virus system in which exogenous viruses coexist with endogenous viral elements. This will strengthen viral surveillance, phylogenetics, and the One Health initiatives.

Small ruminants (sheep and goats) are one of the few mammals in which an exogenous retrovirus (XRV) and closely related endogenous retroviral (ERV) elements coexist within the same host genome. The betaretroviruses Jaagsiekte sheep retrovirus (JSRV) and Enzootic Nasal Tumour Virus (ENTV) cause pulmonary and nasal adenocarcinomas, respectively, and share extensive sequence similarity with their endogenous counterparts. Consequently, molecular surveillance must rely on assays that can unequivocally distinguish true exogenous infection from ERV-derived templates; failure to do so compromises diagnosis, phylogenetic inference, and epidemiological conclusions. We retrieved all complete JSRV, ENTV-1/2, and related ERV genomes deposited in public repositories and performed a comprehensive alignment. Only a limited number of genomic segments were capable of distinguishing exogenous from endogenous sequences. We refer to these as discriminating regions (DRs). Phylogenies built using DRs revealed that several entries annotated as XRV are, in fact, ERV-derived or chimeric artefacts generated by short-amplicon reconstruction. A systematic literature review of over 100 articles identified 286 distinct primers and probes used for the XRV amplification. In-silico mapping of each oligonucleotide onto the full alignment showed that only 28% reliably differentiate XRV from ERV. We experimentally validated the predictive power of this approach for 17 primer/probe sets, confirming that non-discriminating assays produce false-positive signals from endogenous templates. The misannotation of ERV sequences as exogenous viruses has resulted in the population of databases with dubious entries, fostering erroneous hypotheses such as vector-borne transmission of JSRV and ENTV. To address this issue, we propose a concise set of criteria for assay design, validation, and database annotation, emphasizing DR targeting, specificity testing against endogenous templates, and transparent reporting. Although this framework was developed for small ruminants, it is readily applicable to any host–virus system in which exogenous viruses coexist with endogenous viral elements. This will strengthen viral surveillance, phylogenetics, and the One Health initiatives.

African swine fever (ASF) is a devastating disease of pigs. Without a vaccine, early detection and rapid diagnosis of ASF is a crucial step towards effective disease control. In many countries where ASF is endemic, laboratory infrastructure including sampling and sample shipment is inadequate, and a rapid laboratory confirmation would require that the diagnosis is performed at regional laboratories close to the pig farms of concern, or even at the farm‐side. This study intended to evaluate measures including sample preparation methods, dry‐down assay, and a portable, battery‐powered real‐time PCR instrument, to improve molecular diagnosis under field conditions. A simple dilution of blood samples, either in Phosphate‐buffered saline or a commercial buffer, worked similarly to beads‐based nucleic acid extraction using a magnet as the core equipment; the latter method did work as well for those samples with low viral load or high Ct values. The real‐time PCR assay using a Universal ProbeLibrary (UPL) probe tolerated suspected inhibitory substances present in the prepared samples better, whereas a dry‐down assay had a higher diagnostic sensitivity. Additionally, an inhibition control assay proved to be helpful in avoiding false negative results when interpreting negative results of samples that might be of low quality or with inadequate reduction of inhibitory substances. When tested with synthetic DNA standards, the portable instrument performed at a level approaching the stationary thermocycler. In summary, the developments of suitable sample preparation, robust and thermal‐stable real‐time PCR assays with inhibition control, and battery‐powered portable thermocyclers with middle‐throughput offer one way forward to provide rapid, reliable molecular diagnosis under challenging field conditions.