Archaeovirology efforts provided a rich portrait of the evolutionary history of variola virus (VARV, the cause of smallpox), which was characterized by lineage extinctions and a relatively recent origin of the virus as a human pathogen (~1700 years ago, ya). This contrasts with historical records suggesting the presence of smallpox as early as 3500 ya. By performing an analysis of ancestry components in modern, historic, and ancient genomes, we unveil the progressive drifting of VARV lineages from a common ancestral population and we show that a small proportion of Viking Age ancestry persisted until the 18th century. After the split of the P-I and P-II lineages, the former experienced a severe bottleneck. With respect to the emergence of VARV as a human pathogen, we revise time estimates by accounting for the time-dependent rate phenomenon. We thus estimate that VARV emerged earlier than 3800 ya, supporting its presence in ancient societies, as pockmarked Egyptian mummies suggest.

In the last five years, the prevalence of monkeypox has been increasing both in the regions considered endemic for the disease (West and Central Africa) and worldwide. Indeed, in July 2022, the World Health Organization declared the ongoing global outbreak of monkeypox a public health emergency of international concern. The disease is caused by monkeypox virus (MPXV), a member of the Orthopoxvirus genus, which also includes variola virus (the causative agent of smallpox) and vaccinia virus (used in the smallpox eradication campaign). Here, we review aspects of MPXV genetic diversity and epidemiology, with an emphasis on its genome structure, host range, and relationship with other orthopoxviruses. We also summarize the most recent findings deriving from the sequencing of outbreak MPXV genomes, and we discuss the apparent changing of MPXV evolutionary trajectory, which is characterized by the accumulation of point mutations rather than by gene gains/losses. Whereas the availability of a vaccine, the relatively mild presentation of the disease, and its relatively low transmissibility speak in favor of an efficient control of the global outbreak, the wide host range of MPXV raises concerns about the possible establishment of novel reservoirs. We also call for the deployment of field surveys and genomic surveillance programs to identify and control the MPXV reservoirs in West and Central Africa.

The ongoing worldwide monkeypox outbreak is caused by viral lineages (globally referred to as hMPXV1) that are related to but distinct from clade IIb MPXV viruses transmitted within Nigeria. Analysis of the genetic differences has indicated that APOBEC-mediated editing might be responsible for the unexpectedly high number of mutations observed in hMPXV1 genomes. Here, using 1,624 publicly available hMPXV1 sequences, we analyzed the mutations that accrued between 2017 and the emergence of the current predominant variant (B.1), as well as those that that have been accumulating during the 2022 outbreak. We confirmed an overwhelming prevalence of C-to-T and G-to-A mutations, with a sequence context (5'-TC-3') consistent with the preferences of several human APOBEC3 enzymes. We also found that mutations preferentially occur in highly expressed viral genes, although no transcriptional asymmetry was observed. A comparison of the mutation spectrum and context was also performed against the human-specific variola virus (VARV) and the zoonotic cowpox virus (CPXV), as well as fowlpox virus (FWPV). The results indicated that in VARV genomes, C-to-T and G-to-A changes were more common than the opposite substitutions, although the effect was less marked than for hMPXV1. Conversely, no preference toward C-to-T and G-to-A changes was observed in CPXV and FWPV. Consistently, the sequence context of C-to-T changes confirmed a preference for a T in the -1 position for VARV, but not for CPXV or FWPV. Overall, our results strongly support the view that, irrespective of the transmission route, orthopoxviruses infecting humans are edited by the host APOBEC3 enzymes. IMPORTANCE Analysis of the viral lineages responsible for the 2022 monkeypox outbreak suggested that APOBEC enzymes are driving hMPXV1 evolution. Using 1,624 public sequences, we analyzed the mutations that accumulated between 2017 and the emergence of the predominant variant and those that characterize the last outbreak. We found that the mutation spectrum of hMPXV1 has been dominated by TC-to-TT and GA-to-AA changes, consistent with the editing activity of human APOBEC3 proteins. We also found that mutations preferentially affect highly expressed viral genes, possibly because transcription exposes single-stranded DNA (ssDNA), a target of APOBEC3 editing. Notably, analysis of the human-specific variola virus (VARV) and the zoonotic cowpox virus (CPXV) indicated that in VARV genomes, TC-to-TT and GA-to-AA changes are likewise extremely frequent. Conversely, no preference toward TC-to-TT and GA-to-AA changes is observed in CPXV. These results suggest that APOBEC3 proteins have an impact on the evolution of different human-infecting orthopoxviruses.

Orthopoxviruses comprise several relevant pathogens, including the causative agent of smallpox and monkeypox virus. Analysis of orthopoxvirus genome evolution mainly focused on gene gains/losses. We instead analyzed core genes, which are conserved in all orthopoxviruses. We show that, despite their strong constraint, some genes involved in viral morphogenesis and transcription/replication were targets of pervasive positive selection, which was relatively uncommon in immunomodulatory genes. However at least three of the positively selected genes, E3L, A24R, and H3L, might have evolved in response to immune selection. Episodic positive selection was particularly common on the internal branches of the orthopox phylogeny and on the monkeypox virus lineage. The latter showed evidence of episodic positive selection at the D14L gene, which encodes a modulator of complement activation (MOPICE). Notably, two genes (B1R and A33R) targeted by episodic selection on more than one branch are involved in forms of intra-genomic conflict. Finally, we found that, in orthopoxvirus proteomes, intrinsically disordered regions (IDRs) tend to be less constrained and are common targets of positive selection. Extension of our analysis to all poxviruses showed no evidence that the IDR fraction differs with host range. Conversely, we found a strong effect of base composition, which was however not sufficient to explain IDR fraction. We thus suggest that, in poxviruses, the IDR fraction is maintained by modulating GC content to accommodate disorder-promoting codons. Overall, our data provide novel insight in orthopoxvirus evolution and provide a list of genes and sites that are expected to modulate viral phenotypes.