Smallpox has left an unmistakable mark on human history, killing at least 300 million people in the 20th century alone. In spite of its notoriety, the virus's origins still remain elusive.

Now, a team of scientists in Italy has pushed our best estimates for the emergence of smallpox back by another 2,000 years, verifying historical sources that suggested the disease has plagued human societies since ancient times, back when pharaohs ruled.

Advances in genetic sequencing technologies have enabled researchers to undertake increasingly detailed analyses on fragments of ancient viral DNA in recent years, inching them closer to knowing where and when smallpox emerged.

Thanks to a lucky find in Lithuania in 2016, scientists traced smallpox as far back as the 1500s using viral DNA lifted from the remains of a young boy. In 2020, viral DNA from Viking Age skeletons pushed genetic evidence for the latest emergence of smallpox back another few years, to some time before 1050 CE.

However, historical records have suggested something like smallpox plagued ancient societies even earlier than this. Descriptions of symptoms resembling those of the disease have been found in 4th-century texts from China, and Egyptian mummies with pockmark scarring also suggest smallpox was circulating some 3,000 to 4,000 years ago.

But definitive genetic evidence – akin to the molecular fingerprint of a virus – to support this theory has been hard to find.

Scientists can still infer a lot about a virus's past and its evolutionary history when they have enough samples to compare. They can see how a virus has changed over time and work out how fast or slow a virus picks up genetic mutations. From there, scientists can wind the 'molecular clock' back to estimate when an ancestral version of the virus likely existed.

In the case of smallpox, the disease is caused by the variola virus or VARV. In this new study, bioinformatician Diego Forni of the Scientific Institute for Research, Hospitalization and Healthcare (IRCCS) in Italy led a team into taking another look at the genetic sequences of 54 VARV samples, retrieved from previously published works or a research database.

This included four ancient VARV genomes from the Viking Age and two historic VARV genomes from the 17th and 18th centuries, along with 48 modern VARV sequences from before smallpox was eradicated in 1980.

Using this set of viral sequences, the researchers reconstructed the evolutionary history of the smallpox virus, showing how it branched out from a single common ancestor into different strains that either spread around the world, or petered out.

In their models, they adjusted for the way the rate of viral evolution appears to slow down when looking at longer time spans and speed up over shorter time periods. The most recent common ancestor of all the VARV genomes, they found, dates back to around 3,800 years ago or earlier.

Comparing the VARV sequences to those of two related orthopoxviruses – taterapox (which infects gerbils) and camelpox – the analysis also showed that the smallpox virus ancestor split off from its relatives around 7,700 years ago.

That still leaves a pretty wide window for when smallpox may have spilled over into humans, somewhere roughly between 8,000 and 4,000 years ago, the researchers say. But even so, it adds to evidence suggesting smallpox has been with us for millennia longer than previous analyses of viral DNA samples had suggested.

"Variola virus may be much, much older than we thought," says Forni. "This is important because it confirms the historical hypothesis that smallpox existed in ancient societies."

While these new dating estimates put smallpox in the right timeframe to match historical accounts of Egyptian pharaohs bearing smallpox scars, some skepticism remains as to whether the disease was widespread back then, as contemporary written documents contain few mentions of smallpox-like symptoms.

"A number of other infectious diseases cause a rash similar to smallpox and only the sequencing of archaeological specimens will provide information on which ancient societies were affected by the disease," Forni and colleagues conclude in their paper.

The study was published in Microbial Genomics.