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Friday, 21st of December 2012

• VARIOLA VIRUS IN A 300-YEAR-OLD SIBERIAN MUMMY

N Engl J Med 2012; 367:2057-2059, November 22, 2012

Full text, without figure, is below.

Also at http://www.nejm.org/doi/full/10.1056/NEJMc1208124?query=infectious-disease

To the Editor:

Smallpox, which is caused by the variola virus of the Poxviridae family and the orthopoxvirus genus, is among the most devastating human diseases. It may have originated and spread from Egypt, the Near East, or the Indus Valley 3000 to 4000 years ago, and historical reports indicate epidemics in China as early as the first century A.D. and in Europe during the 6th century. By the mid-18th century, smallpox was a worldwide endemic disease. It was eradicated after vaccination campaigns began more than 200 years ago.1

Variola DNA is about 186 kbp, with genes distributed across conserved (central) or variable (termini) regions. Sequence analysis has revealed two main clusters: clade 1 includes variants of variola major, and clade 2 includes West Africa strains and variola minor (alastrim).2 The oldest sequences that have been characterized originate from biologic samples obtained from patients during the past five to six decades.

In 2004, a French and Russian team identified several archeological sites in northeastern Siberia (in Sakha Republic [Yakutia], Russian Federation). Each site consisted of frozen wooden graves buried in the permafrost and dating from the late 17th to early 18th century.3 One of these graves contained five frozen mummies (Figure 1AFigure 1 Grave Containing Five Mummies and Phylogenetic Analysis of Virus Detected in Tissue Samples Obtained from Mummy 2.; and see the Supplementary Appendix, available with the full text of this letter at NEJM.org). This discovery was very unusual, since burial of bodies individually was the standard practice in Yakutia at that time. Analysis of the grave also suggested that the corpses were buried shortly after death.4

Biologic samples from mummy 2 were obtained for histologic and molecular investigations. Microscopical examination of pulmonary tissue showed iron inclusions suggestive of the presence of blood after a possible hemorrhagic episode (Fig. S1 in the Supplementary Appendix). On the basis of these observations, the hypothesis of a sudden and lethal infection was considered, one of which was variola infection.

We confirmed this hypothesis by performing successful polymerase-chain-reaction (PCR) amplification of three short fragments (B7R/hemagglutinin, A30L/14-kD protein, and E9L/DNA polymerase) of the variola genome (PoxSib strain, 718 bp of sequence information) (Fig. S2 in the Supplementary Appendix). To rule out the persistence of intact viral particles, long-distance PCR analyses (E9L assay, approximately 2 kb) were performed. No positive results were obtained, suggesting an extensive fragmentation of the viral genome. Phylogenetic analyses confirmed that PoxSib was variola-related, clustering together with 18 representative variola human sequences, but distinct from contemporary clades 1 and 2 (Figure 1B). Bayesian analysis that included PoxSib extended the origin of smallpox viral strains as far back as A.D. 120 (geometric mean, A.D. 928). Thus, PoxSib could be a direct progenitor of modern viral strains or a member of an ancient lineage that did not cause outbreaks in the 20th century. It could be linked to the epidemic of 1714, which was described in studies conducted during the 18th century. The disease may have been imported to Yakutia during Russian conquest.5

These data show that mummified bodies frozen in the Siberian permafrost are a reservoir of DNA fragments from ancient pathogens. This genetic information could provide clues to past epidemics.

Philippe Biagini, Ph.D.
Viral Emergence and Co-Evolution Unit, Marseille, France 
philippe.biagini@efs.sante.fr

Catherine Thèves, Ph.D.
Patricia Balaresque, Ph.D.
Molecular Anthropology and Image Synthesis Laboratory, Toulouse, France

Annie Géraut, M.D.
Catherine Cannet, M.Sc.
Institute of Legal Medicine, Strasbourg, France

Christine Keyser, Ph.D.
Molecular Anthropology and Image Synthesis Laboratory, Strasbourg, France

Dariya Nikolaeva, M.Sc.
North-Eastern Federal University, Yakutsk, Russian Federation

Patrice Gérard, M.Sc.
Sylvie Duchesne, M.Sc.
Molecular Anthropology and Image Synthesis Laboratory, Toulouse, France

Ludovic Orlando, Ph.D.
Eske Willerslev, Ph.D.
Center for GeoGenetics, Copenhagen, Denmark

Anatoly N. Alekseev, Ph.D.
North-Eastern Federal University, Yakutsk, Russian Federation

Philippe de Micco, M.D., Ph.D.
Viral Emergence and Co-Evolution Unit, Marseille, France

Bertrand Ludes, M.D., Ph.D.
Eric Crubézy, M.D., Ph.D.
Molecular Anthropology and Image Synthesis Laboratory, Toulouse, France

Supported by the French Archaeological Mission in Oriental Siberia (Ministry of Foreign and European Affairs, France), North-Eastern Federal University (Yakutsk, Sakha Republic), and the Human Adaptation program of the French Polar Institute Paul Émile Victor.

Disclosure forms provided by the authors are available with the full text of this letter at NEJM.org.

 References

1Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and its eradication. Geneva: World Health Organization, 1988.

2Lefkowitz EJ, Wang C, Upton C. Poxviruses: past, present and future. Virus Res 2006;117:105-118
CrossRef | Web of Science

3Crubezy E, Amory S, Keyser C, et al. Human evolution in Siberia: from frozen bodies to ancient DNA. BMC Evol Biol 2010;10:25-25
CrossRef | Web of Science

4Crubézy E, Alexeev A. Chamane, Kyss, jeune fille des glaces. Paris: Editions Errance, 2007.

5Zinner EP. Sibir v izvestiakh zapadnoevropeiskikh pouteshestvennikov i outchenykh XVIII v. Vostoshno-Sibirskoe Knijnoe Izdatelstvo Ed, 1968.