To the Editor:

Smallpox was declared eradicated in 19801 thanks to the use of the vaccine initially developed by Edward Jenner in 1798.2 Since that time, it has generally been assumed that the smallpox vaccine is based on cowpox virus, even though it has been known since the late 1930s that the virus that is used to immunize against smallpox, now referred to as vaccinia, differs from cowpox virus. The true origin of vaccinia virus is unknown, and it is usually described as a laboratory virus without a known natural host. Smallpox vaccines from many different sources were used until 1967, when the World Health Organization standardized four vaccinia strains that were widely used in the global smallpox eradication effort, although other vaccines were also used.1

Figure 1. Figure 1. Mulford 1902 Smallpox Vaccine. Panel A shows the original wooden and glass containers that held the capillaries with the Mulford 1902 smallpox vaccine. Panel B shows a sequence-similarity plot for the Mulford 1902 vaccine virus as compared with its closest relative, horsepox virus. The gray line is a plot of the percentage similarity between the genomes of the Mulford 1902 smallpox vaccine virus and horsepox virus (GenBank accession number, DQ792504.1), with shading beneath the line used to highlight the degree of similarity. We selected horsepox virus as the closest relative on the basis of the core genome, defined as the region between and including the genes F9L and A24R (vaccinia virus Copenhagen genome annotation, GenBank accession number, M35027.1) and indicated with darker gray shading. The unique vaccinia deletions of 10.7 kb on the left and 5.5 kb on the right in the Mulford 1902 vaccine, which are not seen in horsepox virus sequence, are shown in red.

We analyzed a smallpox vaccine manufactured for use by the end of 1902 by the Philadelphia company H.K. Mulford, which merged with Sharpe and Dohme in 1929 (Figure 1A). (Because the vaccines of this time had a maximum shelf life of 6 months, we assume that the vaccine was also produced in 1902.) DNA extracted from the glycerinated vaccine was subjected to whole-genome amplification with the use of the TruSeq Nano DNA library preparation kit and was subsequently sequenced on an Illumina MiSeq instrument; the sequence was then assembled into a genome (GenBank accession number, MF477237; 196,464 bp) with the use of the bioinformatics tool SPAdes Genome Assembler. Phylogenetic analyses were based on the alignment of 65 relevant full-genome orthopoxvirus sequences with the use of MAFFT software, version 7.205.

Genomic and phylogenetic analysis revealed that the core genome of the virus in this vial of the Mulford 1902 vaccine has the highest degree of similarity (99.7%) to horsepox virus (Figure 1B). Curiously, the deletions found at each end of the sequence of this Mulford 1902 smallpox vaccine strain are also found in current vaccinia virus but not in cowpox or horsepox virus. The Mulford 1902 vaccine clustered with horsepox virus regardless of the algorithm used to determine phylogenetic relationships.3

The results of our analysis of this 1902 smallpox vaccine provide evidence of the suspected role of horsepox in the origin of the smallpox vaccine,4 a role that was suspected even by Jenner himself.2 In fact, during the 19th century, inocula derived from cowpox or horsepox were used interchangeably to immunize against smallpox. The origin of the Mulford 1902 vaccine stock is unknown, but it was probably obtained from Europe, because horsepox was absent from the Americas.5 The identification of a link between 19th-century and modern smallpox vaccines strengthens the hypothesis that the horsepox virus may be the ancestor of the vaccinia lineage.4

Livia Schrick, Ph.D.

Simon H. Tausch, M.Sc.

P. Wojciech Dabrowski, Ph.D.

Robert Koch Institute, Berlin, Germany

Clarissa R. Damaso, Ph.D.

Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

José Esparza, M.D., Ph.D.

University of Maryland School of Medicine, Baltimore, MD

Andreas Nitsche, Ph.D.

Robert Koch Institute, Berlin, Germany

[email protected]

Supported by the German Ministry of Health and by research fellowships (to Dr. Damaso) from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). Disclosure forms provided by the authors are available with the full text of this letter at NEJM.org.