## 4 Molecular Analyses
### Viruses in Parchments  
Until recently, viruses have not been investigated as an integral part of the microbiome of ancient parchment manuscripts. This is simply because they had previously been overlooked due to the use of technologies that did not allow for their detection, i.e., only cultivation or sequencing of DNA target regions. However, the few recent studies that have employed the shotgun NGS approach have revealed that viruses can account for a significant part of the parchment microbiome [2,3], with proportions of them that are as high as 50% of the sequences that have been obtained [8] (see Figure 5). Although these data are still very preliminary and require more advanced bioinformatics analyses, they indicate a new future direction in the emerging field of biocodicology. [PERSON] et al. [2] already showed the presence of viruses in the samples from the York Gospels, but they did not analyze these sequences in depth, showing only that some of them belonged to the _Siphoviridae_ group. This observation has been confirmed in our studies on the ancient Slavonic parchment manuscripts, where the _Siphoviridae_ group was widely represented in some of the manuscripts, such as the _L. Sinaiticum_ S5, _C. Assemanianus_ P and the _Glagolita Clozianus_, specifically with sequences being related to the _Propionibacterium_-phage (see Figure 9). This indicates a direct relationship with the bacteria that were found in these manuscripts, where _Propionibacterium_ was detected in a relatively high proportion in their microbiomes [3,8] (and in _Glagolita Clozianus_ in this study, see Figure 7), and this indicates that part of the viruses that were found are bacteriophages, i.e., they prey on the bacteria that colonize the parchment. Figure 9 shows that diversity and proportion of the viruses can be very different in the investigated Slavonic manuscripts, which can also reveal the differences in their history of use. Besides the bacteriophages, a considerable proportion of the viruses that were found are partially members of the \"vivobiota\", i.e., viruses that are commonly found in healthy humans [89]. This is the case of the Merkel cell polyomavirus (MCV), which was widely distributed in some of the parchments, especially in the Kiev folia [8]. This virus causes most of the cases of Merkel cell carcinoma, a type of rare skin cancer [90]. However, it is also found on healthy skin [91]. The presence of the MCV on the surface of some of the parchments is most likely due to its close contact with human skin, considering its widespread manipulation throughout the centuries. However, can viruses reveal any other information about the parchments beyond this? Our previous and current results suggest that they can, in fact, reveal or support important information about this material. Some of the viruses that were detected in our shotgun NGS analyses on the ancient Slavonic manuscripts provide fascinating information that confirms our conclusions about the source of the skins and even unveils the possible origins of some of the inks that were used for the elaboration of the manuscripts [3]. This was the case for the detection of the Jaagsiekte sheep retrovirus (JSRV) [92], which causes a contagious lung cancer in sheep, known as ovine pulmonary adenocarcinoma (OPA). JSRV has an endogenous counterpart, which is inserted in all of the sheep genomes, with about 27 copies of endogenous retroviruses (en)SRVs) per genome. Endogenous JSRVs have several functions in the development of domestic sheep, as they help in blocking the replication cycle of JSRV and have a key involvement in the maturation of the sheep embryo [93]. This virus was found in the _L. Sinaiticum_ S5, the _C. Assemanianus_ P [3] and the _Glagolita Clozianus_ (this study, see Figure 9), all having been confirmed to be manufactured with sheepskin (see Table 1).