Gene duplication, gene loss, and recombination events with variola virus shaped the complex evolutionary path of historical American horsepox-based smallpox vaccines.

Vaccinia virus is the active component of all modern smallpox vaccines after the mid-20th century, but it is uncertain to what extent cowpox, vaccinia, and horsepox viruses were used to produce vaccines before then. Genome sequences of six smallpox vaccines used in the United States between 1850 and 1902, namely VK01, VK02, VK05, VK08, VK12, and Mulford_1902 vaccines, revealed >99.5% similarity with a 1976 strain of horsepox in the genome core. However, how these historical vaccines relate to horsepox and vaccinia viruses is still unknown. Here, we present a detailed investigation of the gene content and genomic structure of these historical smallpox vaccines. Except for VK05, all historical vaccines differ from horsepox in the genomic architecture of the flanking variable regions showing complex patterns of gene duplication/transposition, gene fragmentation, and gene loss. The Mulford_1902 vaccine is the closest virus to contemporary vaccinia viruses and the VK02 vaccine is the most different, with several stretches of variola virus genes recombined in its genome. Our data suggest that in the late 19th and early 20th centuries, different horsepox-based vaccines and probably related unsampled progenitors of modern vaccinia virus coexisted. A better understanding of the evolutionary path of the now extinct horsepox-based vaccines will increase our knowledge of the origins of contemporary vaccinia viruses and the pathways that led to the consolidation of current smallpox vaccines. This is particularly important now that the resumption of production of smallpox vaccines for use against mpox is widely discussed, as is the improvement of available vaccines. IMPORTANCE Modern smallpox vaccines, such as those used against mpox, are made from vaccinia viruses, but it is still unknown whether cowpox, horsepox, or vaccinia viruses were used in the early 20th century or earlier. The mystery began to be solved when the genomes of six historical smallpox vaccines used in the United States from 1850 to 1902 were determined. Our work analyzed in detail the genomes of these six historical vaccines, revealing a complex genomic structure. Historical vaccines are highly similar to horsepox in the core of their genomes, but some are closer to the structure of vaccinia virus at the ends of the genome. One of the vaccines is a recombinant virus with parts of variola virus recombined into its genome. Our data add valuable information for understanding the evolutionary path of current smallpox vaccines and the genetic makeup of the potentially extinct group of horsepox viruses.