Variola Virus Genomes Research Articles

An APOBEC3 Mutational Signature in the Genomes of Human-Infecting Orthopoxviruses.

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.

Phylogeographical and evolutionary history of variola major virus; a question of timescales?

Aim of this study was to reconstruct the phylogeography of variola virus (VARV) in the XX century, using 47 VARV whole genome sequences available in public databases, through two different methods for ancestral character reconstruction: a frequently used Bayesian framework and a fast maximum-likelihood (ML) based method. The substitution rate of the whole VARV genome was estimated to be between 6.7×10-6 and 1.1×10-5 substitutions/site/year. Both ML and Bayesian methods gave similar trees topology, showing two distinct monophyletic groups: one (known as P1) including the great part of variola major and the second (P2) including West African and American (variola minor) isolates and close evolutionary rate estimations, between 6.73×10-6 and 1.1×10-5 for the whole genome. The phylogeographical reconstruction of P1 suggested that the common ancestor of the variola major circulating in the Old World between the 1940s and the 1970s most probably originated in the Far East in the first decades of the XX century, and then spread to Indian subcontinent in the 1920s. India represented a center of further spread of VARV to eastern Africa in the 1940s and to the Middle East in the 1960s. The phylogeographic scenario obtained by the maximum-likelihood based method was congruent with that obtained by Bayesian framework, but the analysis was faster indicating the usefulness of this method in the analyses of large viral genomes. Our results may help to explain the controversial reconstructions of the history of VARV obtained using long or short timescale for calibration.

Variola virus genome sequenced from an eighteenth-century museum specimen supports the recent origin of smallpox.

Smallpox, caused by the variola virus (VARV), was a highly virulent disease with high mortality rates causing a major threat for global human health until its successful eradication in 1980. Despite previously published historic and modern VARV genomes, its past dissemination and diversity remain debated. To understand the evolutionary history of VARV with respect to historic and modern VARV genetic variation in Europe, we sequenced a VARV genome from a well-described eighteenth-century case from England (specimen P328). In our phylogenetic analysis, the new genome falls between the modern strains and another historic strain from Lithuania, supporting previous claims of larger diversity in early modern Europe compared to the twentieth century. Our analyses also resolve a previous controversy regarding the common ancestor between modern and historic strains by confirming a later date around the seventeenth century. Overall, our results point to the benefit of historic genomes for better resolution of past VARV diversity and highlight the value of such historic genomes from around the world to further understand the evolutionary history of smallpox as well as related diseases.This article is part of the theme issue ‘Insights into health and disease from ancient biomolecules’.

Diverse variola virus (smallpox) strains were widespread in northern Europe in the Viking Age.

Smallpox, one of the most devastating human diseases, killed between 300 million and 500 million people in the 20th century alone. We recovered viral sequences from 13 northern European individuals, including 11 dated to ~600-1050 CE, overlapping the Viking Age, and reconstructed near-complete variola virus genomes for four of them. The samples predate the earliest confirmed smallpox cases by ~1000 years, and the sequences reveal a now-extinct sister clade of the modern variola viruses that were in circulation before the eradication of smallpox. We date the most recent common ancestor of variola virus to ~1700 years ago. Distinct patterns of gene inactivation in the four near-complete sequences show that different evolutionary paths of genotypic host adaptation resulted in variola viruses that circulated widely among humans.

Comment: Characterization of Two Historic Smallpox Specimens from a Czech Museum.

The complete genome sequences of two strains of variola virus (VARV) sampled from human smallpox specimens present in the Czech National Museum, Prague, were recently determined, with one of the sequences estimated to date to the mid-19th century. Using molecular clock methods, the authors of this study go on to infer that the currently available strains of VARV share an older common ancestor, at around 1350 AD, than some recent estimates based on other archival human samples. Herein, we show that the two Czech strains exhibit anomalous branch lengths given their proposed age, and by assuming a constant rate of evolutionary change across the rest of the VARV phylogeny estimate that their true age in fact lies between 1918 and 1937. We therefore suggest that the age of the common ancestor of currently available VARV genomes most likely dates to late 16th and early 17th centuries and not ~1350 AD.

Re-Assembly and Analysis of an Ancient Variola Virus Genome.

We report a major improvement to the assembly of published short read sequencing data from an ancient variola virus (VARV) genome by the removal of contig-capping sequencing tags and manual searches for gap-spanning reads. The new assembly, together with camelpox and taterapox genomes, permitted new dates to be calculated for the last common ancestor of all VARV genomes. The analysis of recently sequenced VARV-like cowpox virus genomes showed that single nucleotide polymorphisms (SNPs) and amino acid changes in the vaccinia virus (VACV)-Cop-O1L ortholog, predicted to be associated with VARV host specificity and virulence, were introduced into the lineage before the divergence of these viruses. A comparison of the ancient and modern VARV genome sequences also revealed a measurable drift towards adenine + thymine (A + T) richness.

Characterization of Two Historic Smallpox Specimens from a Czech Museum.

Although smallpox has been known for centuries, the oldest available variola virus strains were isolated in the early 1940s. At that time, large regions of the world were already smallpox-free. Therefore, genetic information of these strains can represent only the very last fraction of a long evolutionary process. Based on the genomes of 48 strains, two clades are differentiated: Clade 1 includes variants of variola major, and clade 2 includes West African and variola minor (Alastrim) strains. Recently, the genome of an almost 400-year-old Lithuanian mummy was determined, which fell basal to all currently sequenced strains of variola virus on phylogenetic trees. Here, we determined two complete variola virus genomes from human tissues kept in a museum in Prague dating back 60 and 160 years, respectively. Moreover, mass spectrometry-based proteomic, chemical, and microscopic examinations were performed. The 60-year-old specimen was most likely an importation from India, a country with endemic smallpox at that time. The genome of the 160-year-old specimen is related to clade 2 West African and variola minor strains. This sequence likely represents a new endemic European variant of variola virus circulating in the midst of the 19th century in Europe.

The origin of the variola virus.

The question of the origin of smallpox, one of the major menaces to humankind, is a constant concern for the scientific community. Smallpox is caused by the agent referred to as the variola virus (VARV), which belongs to the genus Orthopoxvirus. In the last century, smallpox was declared eradicated from the human community; however, the mechanisms responsible for the emergence of new dangerous pathogens have yet to be unraveled. Evolutionary analyses of the molecular biological genomic data of various orthopoxviruses, involving a wide range of epidemiological and historical information about smallpox, have made it possible to date the emergence of VARV. Comparisons of the VARV genome to the genomes of the most closely related orthopoxviruses and the examination of the distribution their natural hosts’ ranges suggest that VARV emerged 3000 to 4000 years ago in the east of the African continent. The VARV evolution rate has been estimated to be approximately 2 × 10−6 substitutions/site/year for the central conserved genomic region and 4 × 10−6 substitutions/site/year for the synonymous substitutions in the genome. Presumably, the introduction of camels to Africa and the concurrent changes to the climate were the particular factors that triggered the divergent evolution of a cowpox-like ancestral virus and thereby led to the emergence of VARV.

EPIPOX: Immunoinformatic Characterization of the Shared T-Cell Epitome between Variola Virus and Related Pathogenic Orthopoxviruses.

Concerns that variola viruses might be used as bioweapons have renewed the interest in developing new and safer smallpox vaccines. Variola virus genomes are now widely available, allowing computational characterization of the entire T-cell epitome and the use of such information to develop safe and yet effective vaccines. To this end, we identified 124 proteins shared between various species of pathogenic orthopoxviruses including variola minor and major, monkeypox, cowpox, and vaccinia viruses, and we targeted them for T-cell epitope prediction. We recognized 8,106, and 8,483 unique class I and class II MHC-restricted T-cell epitopes that are shared by all mentioned orthopoxviruses. Subsequently, we developed an immunological resource, EPIPOX, upon the predicted T-cell epitome. EPIPOX is freely available online and it has been designed to facilitate reverse vaccinology. Thus, EPIPOX includes key epitope-focused protein annotations: time point expression, presence of leader and transmembrane signals, and known location on outer membrane structures of the infective viruses. These features can be used to select specific T-cell epitopes suitable for experimental validation restricted by single MHC alleles, as combinations thereof, or by MHC supertypes.

Identification of SNPs associated with variola virus virulence

BackgroundDecades after the eradication of smallpox, its etiological agent, variola virus (VARV), remains a threat as a potential bioweapon. Outbreaks of smallpox around the time of the global eradication effort exhibited variable case fatality rates (CFRs), likely attributable in part to complex viral genetic determinants of smallpox virulence. We aimed to identify genome-wide single nucleotide polymorphisms associated with CFR. We evaluated unadjusted and outbreak geographic location-adjusted models of single SNPs and two- and three-way interactions between SNPs.FindingsUsing the data mining approach multifactor dimensionality reduction (MDR), we identified five VARV SNPs in models significantly associated with CFR. The top performing unadjusted model and adjusted models both revealed the same two-way gene-gene interaction. We discuss the biological plausibility of the influence of the SNPs identified these and other significant models on the strain-specific virulence of VARV.ConclusionsWe have identified genetic loci in the VARV genome that are statistically associated with VARV virulence as measured by CFR. While our ability to infer a causal relationship between the specific SNPs identified in our analysis and VARV virulence is limited, our results suggest that smallpox severity is in part associated with VARV strain variation and that VARV virulence may be determined by multiple genetic loci. This study represents the first application of MDR to the identification of pathogen gene-gene interactions for predicting infectious disease outbreak severity.

Using time-structured data to estimate evolutionary rates of double-stranded DNA viruses.

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 × 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.

Comparative analysis of variable regions in the variola virus genome

Two segments of the variable terminal regions of the variola virus (VARV) genome were sequenced in 22 strains from the Russian collection, including about 13.5 kb of the left segment and about 10.5 kb of the right segment. The total length of the sequences was over 540 kb. Phylogenetic analysis of the new and published data determined the relationships among 70 VARV strains, the character of their clustering, and the intergroup and intragroup variation of the strain clusters. Loci with the highest polymorphism rate were identified and proved to map to noncoding regions or to regions of damaged open reading frames, characteristic of the ancestral virus. These loci offer attractive possibilities for developing a strategy of VARV strain genotyping. Recombination analysis by different methods did not detect, except for a single case, significant recombination events in the VARV strains examined.

Comparative Restriction Enzyme Analysis of the Genome in Variola Virus Strains from the Russian Collection

Comparative RFLP analysis was for the first time performed for 21 variola virus (VARV) strains of the Russian collection with 20 amplicons covering the total VARV genome. The amplicons were synthesized in the long polymerase chain reaction. A database useful as a reference for identifying VARV strains was generated. VARV strains isolated in different geographical regions were compared and proved to vary mostly in variable genome regions. Each of the dendrograms constructed included three clusters of African, Asian, and VARV-alastrim isolates. The VARV-alastrim isolates differed to the greatest extent from the other strains. VARV strains isolated during an ecdemic variola burst in Moscow (1960) grouped with Asian isolates. Polymorphism of VARV strains was for the first time observed for a single variola burst with a few affected patients.

Amplification refractory mutation system PCR assays for the detection of variola and Orthopoxvirus

PCR assays that can identify the presence of variola virus (VARV) sequences in an unknown DNA sample were developed using principles established for the amplification refractory mutation system (ARMS). The assay’s specificity utilised unique single nucleotide polymorphisms (SNP) identified among Orthopoxvirus (OPV) orthologs of the vaccinia virus Copenhagen strain A13L and A36R genes. When a variola virus specific primer was used with a consensus primer in an ARMS assay with different O rthopoxvirus genomes, a PCR product was only amplified from variola virus DNA. Incorporating a second consensus primer into the assay produced a multiplex PCR that provided Orthopoxvirus generic and variola-specific products with variola virus DNA. We tested two single nucleotide polymorphisms with a panel of 43 variola virus strains, collected over 40 years from countries across the world, and have shown that they provide reliable markers for variola virus identification. The variola virus specific primers did not produce amplicons with either assay format when tested with 50 other Orthopoxvirus DNA samples. Our analysis shows that these two polymorphisms were conserved in variola virus genomes and provide a reliable signature of Orthopoxvirus species identification.

Comparative analysis of the conserved region of the orthopoxvirus genome encoding the 36K and 12K proteins

Genes encoding virus-specific proteins with molecular masses of 36 kDa and 12 kDa were mapped in HindIII-P and HindIII-U DNA fragments of vaccinia strain LIVP and ectromelia strain K-1 viruses, respectively, by hybrid selection of RNA to cloned DNA fragments followed by in vitro translation. The 36K translation initiation codon was detected in the HindIII-J fragment. The nucleotide sequences of corresponding genes from vaccinia, ectromelia, cowpox and variola virus genomes were determined. The 12K protein has similarity to mammalian glutaredoxins. The derived amino acid sequence of the 36K polypeptide was compared with the protein bank PIR. No homology was found between the 36K protein and known structures of proteins. The 36K protein genes of vaccinia and ectromelia viruses were cloned in pUR290, which led to the production of E. coli chimeric proteins, consisting of the sequence of β-galactosidase and the viral protein on their C-ends. The chimeric proteins were shown to possess viral antigenic specificity. To identify the protein product of the 36K gene monospecific antisera to chimeric proteins were obtained. The late 36K protein is associated with virosomes but is not incorporated into the virions of orthopoxviruses.