Evolution and diversity of nucleotide and dinucleotide composition in poxviruses.

Poxviruses (family Poxviridae) have long dsDNA genomes and infect a wide range of hosts, including insects, birds, reptiles and mammals. These viruses have substantial incidence, prevalence and disease burden in humans and in other animals. Nucleotide and dinucleotide composition, mostly CpG and TpA, have been largely studied in viral genomes because of their evolutionary and functional implications. We analysed here the nucleotide and dinucleotide composition, as well as codon usage bias, of a set of representative poxvirus genomes, with a very diverse host spectrum. After correcting for overall nucleotide composition, entomopoxviruses displayed low overall GC content, no enrichment in TpA and large variation in CpG enrichment, while chordopoxviruses showed large variation in nucleotide composition, no obvious depletion in CpG and a weak trend for TpA depletion in GC-rich genomes. Overall, intergenome variation in dinucleotide composition in poxviruses is largely accounted for by variation in overall genomic GC levels. Nonetheless, using vaccinia virus as a model, we found that genes expressed at the earliest times in infection are more CpG-depleted than genes expressed at later stages. This observation has parallels in betahepesviruses (also large dsDNA viruses) and suggests an antiviral role for the innate immune system (e.g. via the zinc-finger antiviral protein ZAP) in the early phases of poxvirus infection. We also analysed codon usage bias in poxviruses and we observed that it is mostly determined by genomic GC content, and that stratification after host taxonomy does not contribute to explaining codon usage bias diversity. By analysis of within-species diversity, we show that genomic GC content is the result of mutational biases. Poxvirus genomes that encode a DNA ligase are significantly AT-richer than those that do not, suggesting that DNA repair systems shape mutation biases. Our data shed light on the evolution of poxviruses and inform strategies for their genetic manipulation for therapeutic purposes.

Selective events at individual sites underlie the evolution of monkeypox virus clades.

In endemic regions (West Africa and the Congo Basin), the genetic diversity of monkeypox virus (MPXV) is geographically structured into two major clades (Clades I and II) that differ in virulence and host associations. Clade IIb is closely related to the B.1 lineage, which is dominating a worldwide outbreak initiated in 2022. Lineage B.1 has however accumulated mutations of unknown significance that most likely result from apolipoprotein B mRNA editing catalytic polypeptide-like 3 (APOBEC3) editing. We applied a population genetics-phylogenetics approach to investigate the evolution of MPXV during historical viral spread in Africa and to infer the distribution of fitness effects. We observed a high preponderance of codons evolving under strong purifying selection, particularly in viral genes involved in morphogenesis and replication or transcription. However, signals of positive selection were also detected and were enriched in genes involved in immunomodulation and/or virulence. In particular, several genes showing evidence of positive selection were found to hijack different steps of the cellular pathway that senses cytosolic DNA. Also, a few selected sites in genes that are not directly involved in immunomodulation are suggestive of antibody escape or other immune-mediated pressures. Because orthopoxvirus host range is primarily determined by the interaction with the host immune system, we suggest that the positive selection signals represent signatures of host adaptation and contribute to the different virulence of Clade I and II MPXVs. We also used the calculated selection coefficients to infer the effects of mutations that define the predominant human MPXV1 (hMPXV1) lineage B.1, as well as the changes that have been accumulating during the worldwide outbreak. Results indicated that a proportion of deleterious mutations were purged from the predominant outbreak lineage, whose spread was not driven by the presence of beneficial changes. Polymorphic mutations with a predicted beneficial effect on fitness are few and have a low frequency. It remains to be determined whether they have any significance for ongoing virus evolution.

Analysis of variola virus molecular evolution suggests an old origin of the virus consistent with historical records.

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.

Geographic Structuring and Divergence Time Frame of Monkeypox Virus in the Endemic Region.

BACKGROUND: Monkeypox is an emerging zoonosis endemic to Central and West Africa. Monkeypox virus (MPXV) is genetically structured in 2 major clades (clades 1 and 2/3), but its evolution is poorly explored. METHODS: We retrieved MPXV genomes from public repositories and we analyzed geographic patterns using STRUCTURE. Molecular dating was performed using a using a Bayesian approach. RESULTS: We show that the population transmitted in West Africa (clades 2/3) experienced limited drift. Conversely, clade 1 (transmitted in the Congo Basin) possibly underwent a bottleneck or founder effect. Depending on the model used, we estimated that the 2 clades separated ∼560-860 (highest posterior density: 450-960) years ago, a period characterized by expansions and contractions of rainforest areas, possibly creating the ecological conditions for the MPXV reservoir(s) to migrate. In the Congo Basin, MPXV diversity is characterized by 4 subpopulations that show no geographic structuring. Conversely, clades 2/3 are spatially structured with 2 populations located West and East of the Dahomey Gap. CONCLUSIONS: The distinct histories of the 2 clades may derive from differences in MPXV ecology in West and Central Africa.

Evolution of the orthopoxvirus core genome.

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.

Monkeypox virus: The changing facets of a zoonotic pathogen.

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.

Genetic ancestry and population structure of vaccinia virus.

Vaccinia virus (VACV) was used for smallpox eradication, but its ultimate origin remains unknown. The genetic relationships among vaccine stocks are also poorly understood. We analyzed 63 vaccine strains with different origin, as well horsepox virus (HPXV). Results indicated the genetic diversity of VACV is intermediate between variola and cowpox viruses, and that mutation contributed more than recombination to VACV evolution. STRUCTURE identified 9 contributing subpopulations and showed that the lowest drift was experienced by the ancestry components of Tian Tan and HPXV/Mütter/Mulford genomes. Subpopulations that experienced very strong drift include those that contributed the ancestry of MVA and IHD-W, in good agreement with the very long passage history of these vaccines. Another highly drifted population contributed the full ancestry of viruses sampled from human/cattle infections in Brazil and, partially, to IOC clones, strongly suggesting that the recurrent infections in Brazil derive from the spillback of IOC to the feral state.

Homology-based classification of accessory proteins in coronavirus genomes uncovers extremely dynamic evolution of gene content.

Coronaviruses (CoVs) have complex genomes that encode a fixed array of structural and nonstructural components, as well as a variety of accessory proteins that differ even among closely related viruses. Accessory proteins often play a role in the suppression of immune responses and may represent virulence factors. Despite their relevance for CoV phenotypic variability, information on accessory proteins is fragmentary. We applied a systematic approach based on homology detection to create a comprehensive catalogue of accessory proteins encoded by CoVs. Our analyses grouped accessory proteins into 379 orthogroups and 12 super-groups. No orthogroup was shared by the four CoV genera and very few were present in all or most viruses in the same genus, reflecting the dynamic evolution of CoV genomes. We observed differences in the distribution of accessory proteins in CoV genera. Alphacoronaviruses harboured the largest diversity of accessory open reading frames (ORFs), deltacoronaviruses the smallest. However, the average number of accessory proteins per genome was highest in betacoronaviruses. Analysis of the evolutionary history of some orthogroups indicated that the different CoV genera adopted similar evolutionary strategies. Thus, alphacoronaviruses and betacoronaviruses acquired phosphodiesterases and spike-like accessory proteins independently, whereas horizontal gene transfer from reoviruses endowed betacoronaviruses and deltacoronaviruses with fusion-associated small transmembrane (FAST) proteins. Finally, analysis of accessory ORFs in annotated CoV genomes indicated ambiguity in their naming. This complicates cross-communication among researchers and hinders automated searches of large data sets (e.g., PubMed, GenBank). We suggest that orthogroup membership is used together with a naming system to provide information on protein function.