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, 26 (24), 3407-3412

17 th Century Variola Virus Reveals the Recent History of Smallpox


17 th Century Variola Virus Reveals the Recent History of Smallpox

Ana T Duggan et al. Curr Biol.


Smallpox holds a unique position in the history of medicine. It was the first disease for which a vaccine was developed and remains the only human disease eradicated by vaccination. Although there have been claims of smallpox in Egypt, India, and China dating back millennia [1-4], the timescale of emergence of the causative agent, variola virus (VARV), and how it evolved in the context of increasingly widespread immunization, have proven controversial [4-9]. In particular, some molecular-clock-based studies have suggested that key events in VARV evolution only occurred during the last two centuries [4-6] and hence in apparent conflict with anecdotal historical reports, although it is difficult to distinguish smallpox from other pustular rashes by description alone. To address these issues, we captured, sequenced, and reconstructed a draft genome of an ancient strain of VARV, sampled from a Lithuanian child mummy dating between 1643 and 1665 and close to the time of several documented European epidemics [1, 2, 10]. When compared to vaccinia virus, this archival strain contained the same pattern of gene degradation as 20th century VARVs, indicating that such loss of gene function had occurred before ca. 1650. Strikingly, the mummy sequence fell basal to all currently sequenced strains of VARV on phylogenetic trees. Molecular-clock analyses revealed a strong clock-like structure and that the timescale of smallpox evolution is more recent than often supposed, with the diversification of major viral lineages only occurring within the 18th and 19th centuries, concomitant with the development of modern vaccination.

Keywords: Lithuanian Mummy Project; ancient DNA; evolution; molecular clock; phylogeny; smallpox; variola virus.


Figure 1
Figure 1
Dominican Church of the Holy Spirit, Vilnius Left: Lithuania is shaded in yellow, with the red star indicating the city of Vilnius, the location of the Dominican Church of the Holy Spirit where the VD21 specimen was found and dated to approximately 1654. Dates in black indicate known smallpox outbreaks in nearby countries during the 17th century [1, 2]. Top right: the Dominican Church of the Holy Spirit in Vilnius, Lithuania. Bottom right: the crypt where the child mummy was located. See also Figure S1 and Table S1.
Figure 2
Figure 2
VARV Genome Reconstructed from VD21 (A) Coverage of reference NC_001611.1 variola (major) virus genome. The inner-most circle (light purple) indicates the full 185,578 bp length of the reference, and the inner yellow circle depicts GC content across the reference genome with the genomic average of 32.7% indicated by the thin dark ring. The dark-purple ring indicates the location of annotated genes in the reference. The outer-most ring (dark orange) represents the coverage depth of reads from sample VD21 mapped to the NC_001611.1 reference sequence averaged across 25 bp windows. Average coverage was 18× (minimum 0× to maximum 60×). The concentric gray lines represent intervals of 10× coverage. The plot was constructed with Circos [14]. (B) Conservation of genomic sequence between VD21 and the VARV reference genome NC_001611.1. The plot was constructed with Dotter [15]. See also Figure S2.
Figure 3
Figure 3
Evolutionary History of VARV (A) Maximum-likelihood phylogeny of 43 strains of VARV rooted using homologous sequences from camels (camelpox) and African gerbils (taterapox), which are the orthopoxviruses most closely related to VARV. All horizontal branch lengths are drawn to a scale of nucleotide substitutions per site, and bootstrap values are shown for key nodes, with year of sampling shown for the VARV strains. The VD21 strain is shown in red, and the major clades of VARV (P-I and P-II, with the later the containing alastrim minor strains) are marked. (B) Posterior probability densities of mean evolutionary rate estimates for VARV under different molecular-clock and coalescent models, possible 14C dates for VD21 (i.e., “1789” assumes VD21 is from 1789 rather than 1654), and with VD21 excluded (i.e., “−VD21”) from the analysis (see Table S3 for full results). (C) Maximum clade credibility tree showing the timescale of VARV evolution inferred under a strict molecular clock and a constant population size, with the VD21 lineage shown in red and the 95% credible intervals for a number of other key divergence events also shown. The date (1796) of the introduction of the cowpox-based smallpox vaccine by Jenner, which we include as an historical reference point only, is marked by a hatched line, and it is important to note that a process of smallpox variolation (inoculation) had most likely been in existence for many centuries prior to this. See also Figure S3 and Tables S2 and S3.

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