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Smallpox Evolution, History Explored With Genome Sequence From Ancient Remains

NEW YORK (GenomeWeb) – An ancient DNA analysis suggests a new or altered form of variola virus (VARV) may have contributed to the smallpox outbreaks that began mushrooming in Europe and other parts of the world in the 16th century.

Researchers from McMaster University and elsewhere used in-solution capture and whole-genome sequencing to assess an ancient VARV isolate that was uncovered by chance in present-day Lithuania in the naturally mummified, partial remains of a child who died in the mid-1600s. The cause of his or her death is unknown, though historical records suggest there was a smallpox outbreak in Lithuania at around the same time.

As reported in Current Biology today, the team's analysis of the new VARV genome and existing sequences from 20th century VARVs pointed to a shared common ancestor in the late 1500s or mid-1600s, perhaps reflecting the advent of a strain capable of causing more widespread smallpox outbreaks at around the same time that Europeans were actively exploring and colonizing new locations around the world.

"It seems like this is a time when something is happening with smallpox in Europe. Even though there are older bills of mortality, when historians look at them they don't really see this epidemic outbreak of smallpox the way it seems to start cropping up in the 17th century," first author Ana Duggan, a post-doctoral researcher in Hendrik Poinar's anthropology lab at the McMaster Ancient DNA Centre, told GenomeWeb. "That corresponds nicely to not only the time that we've dated our sample, but also to the time that we dated the coalescence of our sample and the 20th century variola [virus] strains." 

While some prior studies suggest VARV evolution primarily occurred over the past few centuries, there are examples of mummified remains going back thousands of years with marks that some have attributed to smallpox, including 3,000-year-old Egyptian mummies with skin lesions, as well as ancient texts from India and China describing diseases with symptoms that resemble smallpox.

Still, Duggan cautioned that such retrospective diagnoses are difficult. Since conditions such as chickenpox or measles involve some similar disease features, "there's a lingering question there," she said.

Naturally occurring smallpox was successfully eradicated by vaccination and has not been documented since the late 1970s — a major public health victory that has, fortunately, made it tricky to find new VARV isolates to study genetically.

For their new analysis, Duggan, Poinar, and their colleagues focused on samples from the mummy of a two- to four-year-old child who died between 1643 and 1665, prior to the advent of the smallpox vaccination.

"There was no reason to think the sample had smallpox. It was a surprise to us," Duggan noted. "We had, originally, access to many different mummies … And, in this one particular mummy, when we were looking through our original sequencing results and we compared the sequence to a BLAST database, we saw a small section of reads — about 200 — that kept coming back as variola virus."

Although there was no obvious evidence of smallpox infection, such as a rash, scarring, or lesions visible with the naked eye, the team explored the potential VARV infection further, using MYcroarray MYbaits in-solution capture with custom variola virus RNA baits to capture VARV genetic material from the young child's remains.

The captured VARV material was then sequenced on Illumina HiSeq 1500 instruments, producing reads that were assembled into a 187,565 base-pair assembly. The genome was covered to an average depth of 18-fold and contained sequences representing all known VARV genes. The researchers also generated sufficient host reads to put together the mitochondrial genome for the infected child, who belonged to a common European haplogroup.

The team's subsequent analyses of the ancient smallpox-causing virus — alongside camelpox, taterapox, and dozens of VARV genome sequences — placed it at the base of the VARV phylogenetic tree.

Based on subtle differences between the sequenced VARV isolates, the researchers untangled evolutionary patterns in the smallpox-causing virus over time. For example, their data suggested that a previously documented split between major and minor VARV clades took place in the mid- to late-18th century, not long after smallpox vaccination or other forms of inoculation became more widespread.

"The split between the major and minor [clades] correlating with that general time suggests that maybe a practice of inoculation or vaccination was something that provided an evolutionary pressure to cause this split between the major and the minor groups," Duggan said.

From molecular clock data, the team concluded that smallpox culprits from both clades shared a common ancestor going back to around 1588 to 1645. That may reflect a moment in history when VARV was undergoing some sort of change, Duggan explained, though there is not enough functional information available for VARV to pinpoint the nature of that metamorphosis, if it did take place.

"That late 16th century coalescence is really interesting. The reports that come back from historians, and bills of mortality indicating that there seems to be some kind of shift there is really interesting," Duggan said. "Whether it's just a switch, like there's something that changes with virulence, or it's a different strain that moves in and takes over another one, we can't tell."

Overall, VARV appears to have a long phylogenetic branch length relative to other orthopoxviruses that have been studied, Duggan noted, though it is unclear whether this reflects extended evolution in a human system or in a yet-unidentified animal reservoir of the virus.