This study explored the occurrence of PV infections in NHP in northern Argentina and the possible scenarios for the evolution and dispersal of these viruses in the primate lineage. We identified two putative PV types. One was found in the oral mucosa of a wild Alouatta caraya female from Corrientes Province (called AcPV1), and the other was identified in a captive Sapajus sp. female (SPV1) from Misiones Province. The genetic and phylogenetic characterization of these PVs sequences allowed us to assign them to the Gamma-PV genus. Before this study, PV infections were reported for a number of other Platyrrhine species, including Saimiri sciureus, Alouatta guariba, Ateles geoffroyi Callicebus cupreus and Callithrix penicillata [7, 30, 32, 33, 34, 56]. Thus, our findings expand the range of described hosts for these viruses.
The diversity of cutaneous Gamma-PVs is known to be high. Several hundred partial PCR sequences and more than 100 complete reference genomes have been described in the last 10 years (6, 22, 42, 57, 58]. The evolutionary basis for this genetic diversity is unknown. However, it has been suggested that UV light-induced damage may contribute to a higher mutation rate of the sun-exposed PV in the skin of the Beta and Gamma genera .
Interestingly, the Gamma-PV genus was initially reported as belonging to the group of cutaneous PV because of being predominantly isolated in the cutaneous epithelium of the human skin [22, 42, 57, 59]. However, certain studies have shown that the oral cavity contains a broad spectrum of Gamma-PVs, which enlarges their proposed tropism [9, 58, 60]. In a recent publication, Chen et al. (2019) studied PV infection in different body parts of macaques, and found frequencies of 55.6% in genital swabs, 35.9% in oral swabs, and 29.9% in perianal swabs. Moreover, they found a significant difference in the distribution of PV genera at different body sites, with Alpha-PV infections being more frequent in genital samples (86.2%) and Gamma-PV infections more frequent in the oral cavity (90.3%) . Therefore, our identification of AcPV1 and SPV1 in samples of desquamated cells of the oral mucosa is consistent with these recent discoveries.
On the other hand, cross body site infection was also reported as being common among macaques . Thus, an alternative explanation for our finding is that primate skin-to-mouth contact could be responsible for the transmission of skin PV types to the oral cavity. In the case of NHP, grooming is a widespread activity that involves looking for parasites in the fur of peers and eating them [61, 62]. In this context, oral mucosa could represent a satellite niche produced by grooming. This is not a minor issue since tissue tropism has been indicated as one of the main determinants for the evolution of PV genera [18, 20, 30]. Therefore, future studies addressing the tropism of Gamma-PVs will be crucial for understanding virus niche adaptation.
Moreover, the Gamma-PV tree topology indicates the absence of a monophyletic pattern for viruses that infect the same species, with humans being the most striking example. Consequently, our statistical test rejected the host/hosted coevolution model. This finding is consistent with previous reports for Gamma-PV  and other PV-genera such as Alpha. In the later viruses from Papions (PCPV), Rhesus (RhPV-1) and Colobus (CCPV) are closely related to the human oncogenic types of the species Alpha-9, hence, do not possess a basal position in the phylogeny [13–15]. Increasing evidence supports the view that the first step in PV evolution was niche adaptation to tissue tropism. The explanation may explain the conflicts between the pathogen and host phylogenies [15, 17, 30].
Regarding the evolutionary history of PVs, the molecular dating of the Gamma-PV genus was estimated at 51 million years ago (MYA). Previous reports based on complete PV genomes generated dates ranging from 33 MYA to 45 MYA [9, 30, 56] and 50 to 60 MYA [21, 63]. This extent of variation can be attributed to the diversity (the number of species) and length (base pairs) of sequences involved in these different datasets. Nevertheless, all studies agree that the ancestral virus dates back to the Eocene (56 - 34 MYA). Current views on primate taxonomy agree that extant primate genera originated from a common ancestor during the Cretaceous/Paleocene boundary roughly 80 – 90 MYA, with the expansion of the major extant lineages Strepsirrhini, Tarsiiformes, and Simiiformes occurring during the Eocene . The importance of these data for our study concerns the Simiiformes Group, which comprises Platyrrhini (New World monkeys) and Catarrhini (Old World monkeys and humans) and has an estimated tMRCA of 43 (36 – 50) MYA . Therefore, molecular dating of the Gamma-PV types produces estimates that fall within the time frame of the evolution of the Primate Order.
Yet, the emergence of SPV1 during the last 11.9 MYA and of AcaPV at 46.6 MYA is not consistent with the evolutionary history of these primate host species. The biogeographic history of capuchins suggests a late Miocene geographic isolation of the gracile (Cebus) and robust (Sapajus) forms at 6.7 MYA . The divergence time between Alouatta species has also been estimated at 6.6 – 6.8 MYA . Therefore, it is clear that the evolution of the PVs predates the speciation of their respective hosts.
Finally, it is important to mention the potential role of cross-species transmission in our findings. For example, it is known that bovine Delta-PV infection causes tumors in horses, cape mountain zebras, giraffes, sable antelopes and buffaloes [67–70]. Unfortunately, there is relatively little information about humans as sources of PVs. Recent studies have revealed that a zookeeper temporarily tested positive for a chimpanzee PV , while a cat was infected with human HPV9 (possible by a cat owner, who was not tested) . These examples raise the possibility of viral transfer between human and non-human species. In our study, the Sapajus sp. sample came from a captive animal, although we were unable to include the zookeepers’ samples in this study.
The discovery of novel PVs, mainly in hosts in whom PV infection had not been previously reported, is significant, as it increases our knowledge about PV evolution and diversification. However, one of the study limitations is the use of a small DNA fragment for taxonomic, phylogenetic and molecular dating inferences. Unfortunately, we were unsuccessful in retrieving larger genes from our samples to expand this analysis. For this reason, other approaches such as enrichment of circular DNA by rolling circle amplification, and/or next generation sequencing may be needed for the characterization of these novel viruses in the future .