Equus caballus Papillomavirus Type-9 (EcPV9): First Detection in Asymptomatic Italian Horses

Abstract

Papillomavirus (PV) infections may be related to anogenital lesions and cancer development in humans and several other animal species. To date, 11 different PVs have been reported in horses. Among them, a newly described PV named Equus caballus Papillomavirus Type9 (EcPV9) was thus far only reported in the semen of a stallion with penile lesions in Australia. This study reports for the first time the presence of EcPV9 in asymptomatic Italian horses. From July 2020 to January 2022, genital brush samples were collected from 209 horses with no apparent signs of neoplastic disease and no PV-associated lesions, clinically examined at the Didactic Veterinary University Hospital (OVUD) of Perugia and at the Veterinary University Hospital (OVU) of Turin. Brushes were submitted to real-time PCR targeting the EcPV9-L1 region. The first amplification targeted a region of ~116 bp, followed by the amplification and sequencing of ~533 bp of the positive samples. EcPV9-L1 DNA was found in eleven horses (5.3%), all female and mainly English Thoroughbred. Co-infection with EcPV2-L1 was found in 7 out of the 11 EcPV9-L1 positive horses (63.6%). This study contributes to the description of the prevalence of exposure or infection of EcPVs in the horse population in Italy, for which data are still limited. In this regard, here we provide a phylogenetic analysis and the completely reconstructed viral genomes of two Italian EcPV type 9 isolates, as well as four EcPV type 2 obtained from co-infected animals.

Genetic Characterization of a Novel Equus caballus Papillomavirus Isolated from a Thoroughbred Mare

“…Out of the 216 examined horses using the EcPV10-specific Real Time assay, only 7 mares and 1 stallion tested positive. Despite EcPV10 belonging to Dyoiota 1, the genus that includes the most prevalent PVs within the Italian horse population, i.e., EcPV2 and EcPV9 [ 3 , 13 ], our results indicate a limited circulation of EcPV10 in Italy, to date.…”

“…Next, we performed aa pairwise alignments using BLASTp of the six viral ORFs against most closely related PVs ( Figure 3 ). EcPV10 L1, used to classify EcPV [ 3 , 16 ], shared 64.4%, 64.3%, 58.37%, and 60.5% aa similarity with EcPV2, EcPV9, EcPV4, and EcPV5, respectively ( Table 2 ). EcPV10 E1 shared 57.7%, 51.4%, 50.2%, and 48.2% aa similarity with EcPV2, EcPV9, EcPV4, and EcPV5, respectively.…”

“…Upon infection, the viral genome can be either integrated into the host DNA or maintained as multiple episomes replicate concomitantly with the host cells. Usually, the integration is correlated with higher disease severity [ 3 , 4 ].…”

“…PVs’ genome sizes range from 6953 bp ( Chelonia mydas papillomavirus type 1, CmPV1) to 8607 bp ( Canine papillomavirus type 1, CPV1) [ 1 ]. Regardless of host species and PV type, all the PV genomes encode for at least five genes, divided into early (E) and late (L) genes according to their expression during the infection cycle, and an upstream regulatory region (URR) contains enhancers and promoters [ 3 ]. E1 is a helicase involved in replication, while E2 is a transcription factor that enhances E1 and E2 expression, both expressed during the early stage of the infection [ 14 ].…”

“…Three additional genes (E5, E6, E7), involved in cell growth and immune response, are considered accessory genes acquired during the PV evolution, and their presence vary among the different phylogenetic groups [ 1 ]. Among the PV genes, L1 is the most conserved one and, for this reason, used for PV detection and identification [ 3 , 16 ]. Hence, according to their relative clinical history and conditions, horses admitted to the veterinary hospital are routinely tested for the presence of PVs using specific detection and quantification methods targeting the L1 gene.…”