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Using Time-Structured Data to Estimate Evolutionary Rates of Double-Stranded DNA Viruses
Abstract and Figures
Double-stranded (ds) DNA viruses are often described as evolving through long-term codivergent associations with their hosts, a pattern that is expected to be associated with low rates of nucleotide substitution. However, the hypothesis of codivergence between dsDNA viruses and their hosts has rarely been rigorously tested, even though the vast majority of nucleotide substitution rate estimates for dsDNA viruses are based upon this assumption. It is therefore important to estimate the evolutionary rates of dsDNA viruses independent of the assumption of host-virus codivergence. Here, we explore the use of temporally structured sequence data within a Bayesian framework to estimate the evolutionary rates for seven human dsDNA viruses, including variola virus (VARV) (the causative agent of smallpox) and herpes simplex virus-1. Our analyses reveal that although the VARV genome is likely to evolve at a rate of approximately 1 x 10(-5) substitutions/site/year and hence approaching that of many RNA viruses, the evolutionary rates of many other dsDNA viruses remain problematic to estimate. Synthetic data sets were constructed to inform our interpretation of the substitution rates estimated for these dsDNA viruses and the analysis of these demonstrated that given a sequence data set of appropriate length and sampling depth, it is possible to use time-structured analyses to estimate the substitution rates of many dsDNA viruses independently from the assumption of host-virus codivergence. Finally, the discovery that some dsDNA viruses may evolve at rates approaching those of RNA viruses has important implications for our understanding of the long-term evolutionary history and emergence potential of this major group of viruses.
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... Por su tamaño extenso, se le dificulta el ingreso al huésped y la replicación propia, ya que alerta rápidamente al sistema inmunológico, lo que activa señales de evasión por parte del huésped (Kaler et al., 2022;Petersen et al., 2019). El virus Mpox tiene una baja frecuencia de mutaciones genómicas, dado su estructura bicatenaria del ADN y la actividad exonucleasa 3'-5' de su ADN polimerasa (Elde et al., 2012;Firth et al., 2010). No obstante, el virus Mpox de 2022 difiere de los virus documentados entre 2018-2019 en una media de 50 polimorfismos de un solo nucleótido; considerando estimaciones anteriores de la tasa de sustitución de orthopoxvirus, lo cual es más alto de lo esperado (Firth et al., 2010;Huang et al., 2022). ...
... El virus Mpox tiene una baja frecuencia de mutaciones genómicas, dado su estructura bicatenaria del ADN y la actividad exonucleasa 3'-5' de su ADN polimerasa (Elde et al., 2012;Firth et al., 2010). No obstante, el virus Mpox de 2022 difiere de los virus documentados entre 2018-2019 en una media de 50 polimorfismos de un solo nucleótido; considerando estimaciones anteriores de la tasa de sustitución de orthopoxvirus, lo cual es más alto de lo esperado (Firth et al., 2010;Huang et al., 2022). ...
Mpox (viruela símica) es una enfermedad viral que puede producir síntomas como erupción cutánea, adenomegalias y fiebre. Pese a su baja tasa de mortalidad, se relaciona con complicaciones que pueden comprometer la calidad de vida de las personas. Fue identificada por primera vez en humanos en 1970, desde entonces se mantenía una distribución geográfica y una incidencia controladas. No obstante, desde el año 2022 su incidencia ha aumentado al igual que su distribución geográfica alcanzando un total de 89.596 casos confirmados por laboratorio y 663 casos probables, incluidas 157 muertes, reportados al 28 de agosto de 2023; llegando incluso a países no endémicos. A continuación, se presenta una revisión narrativa con base en información recolectada en bases de datos como UpToDate, Google Scholar, Medline, ClinicalKey, Scopus, Embase, Pubmed; con el objetivo de describir los aspectos más relevantes de Mpox en humanos desde una perspectiva epidemiológica actual, además de presentar datos fisiopatológicos, de diagnóstico, tratamiento, pronóstico y rehabilitación.
... The monkeypox virus has few genetic changes because of the DNA double-stranded structure and 3′-5′ exonuclease activity of its DNA polymerase (Elde et al. 2012). However, based on prior estimations of the substitution rate for Orthopoxviruses, the 2022 monkeypox virus differs from the associated 2018-2019 viruses by an average of 50 single-nucleotide polymorphisms (SNPs), which is notably greater than expected (Firth et al. 2010). Such a split in mostly mice and non-human animals, and found that they can get sick from the virus Reynolds et al. 2019). ...
Although the smallpox virus has been eradicated worldwide, the World Health Organization (WHO) has issued a warning about the virus’s potential to propagate globally. The WHO labeled monkeypox a world public health emergency in July 2022, requiring urgent prevention and treatment. The monkeypox virus is a part of the Poxviridae family, Orthopoxvirus genus, and is accountable for smallpox, which has killed over a million people in the past. Natural hosts of the virus include squirrels, Gambian rodents, chimpanzees, and other monkeys. The monkeypox virus has transmitted to humans through primary vectors (various animal species) and secondary vectors, including direct touch with lesions, breathing particles from body fluids, and infected bedding. The viral particles are ovoid or brick-shaped, 200–250 nm in diameter, contain a single double-stranded DNA molecule, and reproduce only in the cytoplasm of infected cells. Monkeypox causes fever, cold, muscle pains, headache, fatigue, and backache. The phylogenetic investigation distinguished between two genetic clades of monkeypox: the more pathogenic Congo Basin clade and the West Africa clade. In recent years, the geographical spread of the human monkeypox virus has accelerated despite a paucity of information regarding the disease’s emergence, ecology, and epidemiology. Using lesion samples and polymerase chain reaction (PCR), the monkeypox virus was diagnosed. In the USA, the improved Ankara vaccine can now be used to protect people who are at a higher risk of getting monkeypox. Antivirals that we have now work well against smallpox and may stop the spread of monkeypox, but there is no particular therapy for monkeypox.
... Overall, 129 viral genomes, sharing 50 SNVs (single nucleotide variants) and differing from the sequences of the 2018-2019 MPXV, have been shared through GISAID. As reported in the literature [12], the number of SNVs in the sequences from the 2022 outbreak appears to be higher than the estimated substitution rate for Orthopoxviruses (i.e., 1-2 substitutions per site per year) [13]. Most changes involved extensive G-to-A/C-to-T replacements, suggesting that the intrinsic host antiviral mechanism, based on APOBEC3 enzymes, may have driven MPXV evolution since 2017 [8,14,15]. ...
In 2022, an unprecedented outbreak of mpox raged in several nations. Sequences from the 2022 outbreak reveal a higher nucleotide substitution if compared with the estimated rate for orthopoxviruses. Recently, intra-lesion SNVs (single nucleotide variants) have been described, and these have been suggested as possible sources of genetic variation. Until now, it has not been clear if the presence of several SNVs could represents the result of local mutagenesis or a possible co-infection. We investigated the significance of SNVs through whole-genome sequencing analysis of four unrelated mpox cases. In addition to the known mutations harboured by the circulating strains of virus (MPXV), 7 novel mutations were identified, including SNVs located in genes that are involved in immune evasion mechanisms and/or viral fitness, six of these appeared to be APOBEC3-driven. Interestingly, three patients exhibited the coexistence of mutated and wild-type alleles for five non-synonymous variants. In addition, two patients, apparently unrelated, showed an analogous pattern for two novel mutations, albeit with divergent frequencies. The coexistence of mixed viral populations, harbouring non-synonymous mutations in patients, supports the hypothesis of possible co-infection. Additional investigations of larger clinical cohorts are essential to validating intra-patient viral genome heterogeneity and determining the possibility of co-presence events of slightly divergent MPXV strains.
... MPXV of clade IIb most probably originated from clade II in a 2017 outbreak in Nigeria.2 Phylogenetically, these two clades differ by a mean of 50 SNPs, which is roughly 6-12-fold more than previous estimations of the substitution rate for Orthopoxviruses (1-2 substitutions per genome per year).2,25 In the present study, we can confirm that the underlying MPXV strain belongs to clade IIb and thus confirm the already known genome sequence differences between clade II and IIb.2 Clade IIb exhibits molecular signatures that can been associated with the F I G U R E 5 Realigned reads from SRR20698670 against their assembly ON563414.3 ...
In 2022, a series of human monkeypox cases in multiple countries led to the largest and most widespread outbreak outside the known endemic areas. Setup of proper genomic surveillance is of utmost importance to control such outbreaks. To this end, we performed Nanopore (PromethION P24) and Illumina (NextSeq. 2000) Whole Genome Sequencing (WGS) of a monkeypox sample. Adaptive sampling was applied for in silico depletion of the human host genome, allowing for the enrichment of low abundance viral DNA without a priori knowledge of sample composition. Nanopore sequencing allowed for high viral genome coverage, tracking of sample composition during sequencing, strain determination, and preliminary assessment of mutational pattern. In addition to that, only Nanopore data allowed us to resolve the entire monkeypox virus genome, with respect to two structural variants belonging to the genes OPG015 and OPG208. These SVs in important host range genes seem stable throughout the outbreak and are frequently misassembled and/or misanno-tated due to the prevalence of short read sequencing or short read first assembly. Ideally, standalone standard Illumina sequencing should not be used for Monkeypox WGS and de novo assembly, since it will obfuscate the structure of the genome, which has an impact on the quality and completeness of the genomes deposited in public databases and thus possibly on the ability to evaluate the complete genetic reason for the host range change of monkeypox in the current pandemic. K E Y W O R D S engineering and technology, evolution, genetic mapping, genetics, mutation/mutation rate, poxvirus, research and analysis methods, virus classification J Med Virol. 2024;96:e29610. wileyonlinelibrary.com/journal/jmv |
... BEAST analyses were conducted for these randomized data sets and the mean 591 rates were compared between runs. The data had sufficient temporal signal if 592 the 95% credible interval of mean rates of the date-randomized datasets did not 593 overlap with that of the original sampling dataset [78,79]. ...
Antimicrobial resistance (AMR) poses a serious threat to the clinical management of typhoid fever. AMR in Salmonella Typhi ( S . Typhi) is commonly associated with the H58 lineage, a lineage that arose comparatively recently before becoming globally disseminated. To better understand when and how H58 emerged and became dominant, we performed detailed phylogenetic analyses on contemporary genome sequences from S . Typhi isolated in the period spanning the emergence. Our dataset, which contains the earliest described H58 S . Typhi organism, indicates that ancestral H58 organisms were already multi-drug resistant (MDR). These organisms emerged spontaneously in India in 1987 and became radially distributed throughout South Asia and then globally in the ensuing years. These early organisms were associated with a single long branch, possessing mutations associated with increased bile tolerance, suggesting that the first H58 organism was generated during chronic carriage. The subsequent use of fluoroquinolones led to several independent mutations in gyrA . The ability of H58 to acquire and maintain AMR genes continues to pose a threat, as extensively drug-resistant (XDR; MDR plus resistance to ciprofloxacin and third generation cephalosporins) variants, have emerged recently in this lineage. Understanding where and how H58 S . Typhi originated and became successful is key to understand how AMR drives successful lineages of bacterial pathogens. Additionally, these data can inform optimal targeting of typhoid conjugate vaccines (TCVs) for reducing the potential for emergence and the impact of new drug-resistant variants. Emphasis should also be placed upon the prospective identification and treatment of chronic carriers to prevent the emergence of new drug resistant variants with the ability to spread efficiently.
... The relaxed (uncorrelated lognormal) clock model, that allows for evolutionary rate variations among branches of the tree and the constant-coalescent model were found to best fit our data. To assess the temporal signal of these Bayesian estimates, we conducted a date-randomisation test where sampling times were assigned randomly to the sequences, and the analysis re-run 20 times with the best fitting models (constant-coalescent demographic and uncorrelated lognormal clock) Duchene et al., 2016;Firth et al., 2010. The date-randomisation test revealed that these data displayed 'strong' temporal structure (meeting the criterion CR2 of Duchene et al., 2016). ...
Five years before the 2022 global mpox outbreak Nigeria reported its first cases in nearly 40 years, with the ongoing epidemic since driven by sustained human-to-human transmission. However, limited genomic data has left questions about the timing and origin of the mpox virus (MPXV) emergence. Here we generated 112 MPXV genomes from Nigeria from 2021 to 2023. We identify the closest zoonotic outgroup to the human epidemic in southern Nigeria, and estimate that the lineage transmitting from human-to-human emerged around July 2014, circulating cryptically until detected in September 2017. The epidemic originated in Southern Nigeria, particularly Rivers State, which also acted as a persistent and dominant source of viral dissemination to other states. We show that APOBEC3 activity increased MPXV's evolutionary rate twenty-fold during human-to-human transmission. We also show how Delphy, a tool for near-real-time Bayesian phylogenetics, can aid rapid outbreak analytics. Our study sheds light on MPXV's establishment in West Africa before the 2022 global outbreak and highlights the need for improved pathogen surveillance and response.
Historically, monkeypox (mpox) was a zoonotic disease endemic in Africa. However, in 2022, a global outbreak occurred following a substantial increase in cases in Africa, coupled with spread by international travellers to other continents. Between January 2022 and October 2023, about 91,000 confirmed cases from 115 countries were reported, leading the World Health Organization to declare a public health emergency. The basic biology of monkeypox virus (MPXV) can be inferred from other poxviruses, such as vaccinia virus, and confirmed by genome sequencing. Here the biology of MPXV is reviewed, together with a discussion of adaptive changes during MPXV evolution and implications for transmission. Studying MPXV biology is important to inform specific host interactions, to aid in ongoing outbreaks and to predict those in the future.
Modified vaccinia virus Ankara (MVA) has been widely tested in clinical trials as recombinant vector vaccine against infectious diseases and cancers in humans and animals. However, one biosafety concern about the use of MVA vectored vaccine is the potential for MVA to recombine with naturally occurring orthopoxviruses in cells and hosts in which it multiplies poorly and, therefore, producing viruses with mosaic genomes with altered genetic and phenotypic properties. We previously conducted co-infection and superinfection experiments with MVA vectored influenza vaccine (MVA-HANP) and a feline Cowpox virus (CPXV-No-F1) in Vero cells (that were semi-permissive to MVA infection) and showed that recombination occurred in both co-infected and superinfected cells. In this study, we selected the putative recombinant viruses and performed genomic characterization of these viruses. Some putative recombinant viruses displayed plaque morphology distinct of that of the parental viruses. Our analysis demonstrated that they had mosaic genomes of different lengths. The recombinant viruses, with a genome more similar to MVA-HANP (>50%), rescued deleted and/or fragmented genes in MVA and gained new host ranges genes. Our analysis also revealed that some MVA-HANP contained a partially deleted transgene expression cassette and one recombinant virus contained part of the transgene expression cassette similar to that incomplete MVA-HANP. The recombination in co-infected and superinfected Vero cells resulted in recombinant viruses with unpredictable biological and genetic properties as well as recovery of delete/fragmented genes in MVA and transfer of the transgene into replication competent CPXV. These results are relevant to hazard characterization and risk assessment of MVA vectored biologicals.
Introduction: The global headlines have been dominated by the sudden and widespread outbreak of monkeypox, a rare and endemic zoonotic disease caused by the monkeypox virus (MPXV). Genomic composition based machine learning (ML) methods have recently shown promise in identifying host adaptability and evolutionary patterns of virus. Our study aimed to analyze the genomic characteristics and evolutionary patterns of MPXV using ML methods.
Methods: The open reading frame (ORF) regions of full-length MPXV genomes were filtered and 165 ORFs were selected as clusters with the highest homology. Unsupervised machine learning methods of t-distributed stochastic neighbor embedding (t-SNE), Principal Component Analysis (PCA), and hierarchical clustering were performed to observe the DCR characteristics of the selected ORF clusters.
Results: The results showed that MPXV sequences post-2022 showed an obvious linear adaptive evolution, indicating that it has become more adapted to the human host after accumulating mutations. For further accurate analysis, the ORF regions with larger variations were filtered out based on the ranking of homology difference to narrow down the key ORF clusters, which drew the same conclusion of linear adaptability. Then key differential protein structures were predicted by AlphaFold 2, which meant that difference in main domains might be one of the internal reasons for linear adaptive evolution.
Discussion: Understanding the process of linear adaptation is critical in the constant evolutionary struggle between viruses and their hosts, playing a significant role in crafting effective measures to tackle viral diseases. Therefore, the present study provides valuable insights into the evolutionary patterns of the MPXV in 2022 from the perspective of genomic composition characteristics analysis through ML methods.
The evolutionary interaction between influenza A virus and the human immune system, manifest as 'antigenic drift' of the viral haemagglutinin, is one of the best described patterns in molecular evolution. However, little is known about the genome-scale evolutionary dynamics of this pathogen. Similarly, how genomic processes relate to global influenza epidemiology, in which the A/H3N2 and A/H1N1 subtypes co-circulate, is poorly understood. Here through an analysis of 1,302 complete viral genomes sampled from temperate populations in both hemispheres, we show that the genomic evolution of influenza A virus is characterized by a complex interplay between frequent reassortment and periodic selective sweeps. The A/H3N2 and A/H1N1 subtypes exhibit different evolutionary dynamics, with diverse lineages circulating in A/H1N1, indicative of weaker antigenic drift. These results suggest a sink–source model of viral ecology in which new lineages are seeded from a persistent influenza reservoir, which we hypothesize to be located in the tropics, to sink populations in temperate regions.
Previous restriction fragment length polymorphism analysis divided variola virus (VARV) strains into two subtypes, one of
which included West African and South American isolates. This allowed a dating to be introduced for the first time in estimation
of the VARV evolution rate. The results were used to analyze the molecular evolution of the total family Poxviridae. Comparisons
of the known nucleotide sequences were performed for the extended conserved central genome region in 42 orthopoxvirus strains
and for the eight genes of multisubunit RNA polymerase in 65 viruses belonging to various genera of the family Poxviridae.
Using the Bayesian dating method, the mutation accumulation rate of poxviruses was estimated at (1.7–8.8) × 10−6 nucleotide substitutions per site per year. Computations showed that the modern poxvirus genera started diverging from an
ancestral virus more than 200 thousand years ago and that an ancestor of the genus Orthopoxvirus emerged 131 ± 45 thousand years ago. The other genera of mammalian poxviruses with a low GC content diverged approximately
110–90 thousand years ago. The independent evolution of VARV started 3.4 ± 0.8 thousand years ago. It was shown with the example
of VARV and the monkeypox virus (MPXV) that divergent evolution of these orthopoxviruses started and the West African subtypes
of VARV and MPXV were formed as geographical conditions changed to allow isolation of West African animals from other African
regions.
A heteroduplex mobility assay was used to identify variants of varicella-zoster virus circulating in the United Kingdom and elsewhere. Within the United Kingdom, 58 segregating sites were found out of the 23,266 examined (0.25%), and nucleotide diversity was estimated to be 0.00063. These are an order of magnitude smaller than comparable estimates from herpes simplex virus type 1. Sixteen substitutions were nonsynonymous, the majority of which were clustered within surface-expressed proteins. Extensive genetic correlation between widely spaced sites indicated that recombination has been rare. Phylogenetic analysis of varicella-zoster viruses from four continents distinguished at least three major genetic clades. Most geographical regions contained only one of these three strains, apart from the United Kingdom and Brazil, where two or more strains were found. There was minimal genetic differentiation (one or fewer substitutions in 1,895 bases surveyed) between the samples collected from Africa (Guinea Bissau, Zambia) and the Indian subcontinent (Bangladesh, South India), suggesting recent rapid spread and/or low mutation rates. The geographic pattern of strain distribution would favor a major influence of the former. The genetic uniformity of most virus populations makes recombination difficult to detect. However, at least one probable recombinant between two of the major strains was found among the samples originating from Brazil, where mixtures of genotypes co-occur.
Porcine circovirus 2 (PCV2) is the primary etiological agent of postweaning multisystemic wasting syndrome (PMWS), one of the most economically important emerging swine diseases worldwide. Virulent PCV2 was first identified following nearly simultaneous outbreaks of PMWS in North America and Europe in the 1990s and has since achieved global distribution. However, the processes responsible for the emergence and spread of PCV2 remain poorly understood. Here, phylogenetic and cophylogenetic inferences were utilized to address key questions on the time scale, processes, and geographic diffusion of emerging PCV2. The results of these analyses suggest that the two genotypes of PCV2 (PCV2a and PCV2b) are likely to have emerged from a common ancestor approximately 100 years ago and have been on independent evolutionary trajectories since that time, despite cocirculating in the same host species and geographic regions. The patterns of geographic movement of PCV2 that we recovered appear to mimic those of the global pig trade and suggest that the movement of asymptomatic animals is likely to have facilitated the rapid spread of virulent PCV2 around the globe. We further estimated the rate of nucleotide substitution for PCV2 to be on the order of 1.2 x 10(-3) substitutions/site/year, the highest yet recorded for a single-stranded DNA virus. This high rate of evolution may allow PCV2 to maintain evolutionary dynamics closer to those of single-stranded RNA viruses than to those of double-stranded DNA viruses, further facilitating the rapid emergence of PCV2 worldwide.
— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.
Many evolutionary studies of birds rely on the estimation of molecular divergence times and substitution rates. In order to perform such analyses, it is necessary to incorporate some form of calibration information: a known substitution rate, radiometric ages of heterochronous sequences, or inferred ages of lineage splitting events. All three of these techniques have been employed in avian molecular studies, but their usage has not been entirely satisfactory. For example, the ‘traditional’ avian mitochondrial substitution rate of 2% per million years is frequently adopted without acknowledgement of the associated uncertainty. Similarly, fossil and biogeographic information is almost always converted into an errorless calibration point. In both cases, the resulting estimates of divergence times and substitution rates will be artificially precise, which has a considerable impact on hypothesis testing. In addition, using such a simplistic approach to calibration discards much of the information offered by the fossil record. A number of more sophisticated calibration methods have recently been introduced, culminating in the development of probability distribution-based calibrations. In this article, I discuss the use of this new class of methods and offer guidelines for choosing a calibration technique.
Unlike vertebrates, for which paleontological data are available, and RNA viruses, which display a high rate of genetic variation, an objective estimate of time parameters for the molecular evolution of DNA viruses, which display a low rate of accumulation of mutations, is a complex problem. Genomic studies of a set of smallpox (variola) virus (VARV) isolates demonstrated the patterns of phylogenetic relationships between geographic variants of this virus. Using archival data on smallpox outbreaks and the results of phylogenetic analyses of poxvirus genomes, different research teams have obtained contradictory data on the possible time point of VARV origin. I discuss the approaches used for dating of VARV evolution and adduce the arguments favoring its historically recent origin.
Picornaviruses are responsible for some of the most common and debilitating illnesses affecting humans and animals worldwide. To extend our knowledge of the evolution of picornaviruses and their molecular epidemiology, phylogenetic relationships among 11 genera and the unassigned seal picornavirus type 1 were estimated from the conserved proteins 2C, 3C(pro), and 3D(pol). Each gene was analyzed separately and as a combined dataset. Different tree topologies were recovered from each gene. However, their sequences were determined to be combinable based on our finding of no recombination among genera and failing to reject the hypothesis of homogeneity among datasets using ILD tests. The combined data tree topology was identical to the 3D(pol) gene tree; a topology largely consistent with previous phylogenetic hypotheses based on 3D(pol) and the coding genome. Phylogenetic trees estimated from six phenotypic characters were not congruent with those recovered from molecular datasets; further supporting the hypothesis that viral phenotypes are highly plastic. Finally, we tested the hypothesis of host-virus cophylogeny. Both global and individual tests of the relationships between host and virus trees failed to detect a significant association. These results emphasize the importance of horizontal transmission among host species for picornavirus diversification rather than vertical transmission accompanying speciation.
The poxviruses (family Poxviridae) are a family of double-stranded viruses including several species that infect humans and their domestic animals, most notably Variola virus (VARV), the causative agent of smallpox. The evolutionary biology of these viruses poses numerous questions, for which we have only partial answers at present. Here we review evidence regarding the origin of poxviruses, the frequency of host transfer in poxvirus history, horizontal transfer of host genes to poxviruses, and the population processes accounting for patterns of nucleotide sequence polymorphism.