Characterization of Two Historic Smallpox Specimens from a Czech Museum

Abstract

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.

Re-Assembly and Analysis of an Ancient Variola Virus Genome

“…The most significant difference between the data presented in Figure 5 and the previous work is that our results push back the date of the tMRCA by about 100 years for all VARVs (from 1588–1645 to 1470–1563), as well as the split of the VARV major and minor clades (from 1734–1793 to 1579–1667). Interestingly, the dates for speciation of all VARV and the major/minor species are midway between those calculated by Duggan et al [ 1 ] and those recently calculated by Pajer et al [ 21 ], who sequenced the VARV-major genome from 1942 and the VARV-minor genome from 1850. The number of unique SNPs associated with particular viruses at nodes on the tree ( Figure 5 ) are consistent for all of the VARVs, suggesting that, after the speciation of VARV, there has been little to no recombination with other poxvirus species.…”

“…By using the improved assembly of the ancient VARV-VD21 genome together with TATV + CMLV genomes for phylogenetic analysis and the prediction of clade divergence dates, we determined a tMRCA for all VARVs within the range of 1470–1563. This is approximately midway between the latest two estimates from the publications describing the ancient VARV genomes [ 1 , 21 ]. The BEAST software predicted a split of the VARV and CMLV + TATV clades approximately 1250 years prior to the tMRCA for all VARVs.…”

“…We performed an evolutionary analysis of the VARV species using the Bayesian Markov chain Monte Carlo (MCMC) inference methods with BEAST 2 (Bayesian evolutionary analysis by sampling trees) software [ 20 ], with the rooted RAxML constructed maximum-likelihood tree as the starting tree. All of the viruses included in the evolutionary analysis had reliable collection dates, with the exception of VARV-VD21-uvic (our assembly), which was previously dated using radiocarbon dating techniques [ 1 ] , and VARV-V1588 and VARV-V563, which were previously dated by the degree of d - and l -aspartic acid racemization [ 21 ]. BEAST 2 was run using a fixed clock model, constant population size coalescent priors, and a 100,000,000 MCMC chain length.…”

Smallpox in the Post-Eradication Era

“…The authors conclude that the loss of gene function seen in modern VARVs compared to some other OPVs must have occurred before 1650 approximately. By calculating phylogenetic trees, the Lithuanian sequence fell basal to all VARV sequences [46,50]. Further analyses revealed that the time scale of VARV evolution is more recent than often supposed-the two major viral lineages diverged within the 18th and 19th centuries.…”

Galen and the Plague

“…Further genomic and historical work will certainly help shed light on the matter, even if no appropriate ancient DNA is forthcoming.93 87 Hawi 47 and 71 (TSM 113 and 121). 88 KJH 14 (TSM [71][72][73]. .…”