Speckled with lesions across its dried, ghastly face, the nearly 500-year-old child mummy buried in a church in Naples, Italy, was long thought to be the earliest evidence of smallpox during the Italian Renaissance period.
But decades after researchers first examined it, a group of scientists has taken another look at the toddler's remains and discovered that it most likely suffered from a different scourge: the hepatitis B virus.
The finding, published Thursday in the journal PLOS Pathogens, provides further evidence that the hepatitis B virus has afflicted humans for centuries. Today more than 250 million people live with the virus worldwide, and it killed nearly 900,000 people in 2015, mostly through liver disease, according to the World Health Organization.
"This very common and nasty bug has been with us at least 500 years, and I suspect it may have been here many, many thousands of years," said Edward Holmes, an evolutionary biologist from the University of Sydney and an author on the paper.
The mummified child, who died at 2 years old, was buried in the Basilica of Saint Domenico Maggiore in Naples during the 16th century. The researchers who have worked there acknowledge some of the emotional challenges to studying the toddler's remains.
"There's this hollowness yet this ghostlike pain still there which is fascinating from a scientific perspective but horrific from a parental perspective," said Hendrik Poinar, an evolutionary geneticist at McMaster University in Ontario and an author of the recent paper.
In the crypt, the child's coffin rests near dozens of other wooden burial boxes, some of which held the bodies of Aragonese princes and Neapolitan nobles. Their corpses were clothed in decorated woven fabrics and precious silk. Many were embalmed but some were naturally mummified by the dry conditions of the basilica.
"The mummies of San Domenico Maggiore are unique in Italy not only for the antiquity and excellent state of preservation of the bodies," Gino Fornaciari, a paleopathologist at the University of Pisa and an author of the paper, said in an email, "but also for the fame of the personages, whose lives and causes of death are well known."
Fornaciari first studied the child mummy in the 1980s. Using an electron microscope, he found what he thought were traces of smallpox, also known as variola virus, in its remains. Since then the child has served as a quintessential example of early European smallpox. That made it a prime target for Holmes and his colleagues who have tried to map out the timeline and diversity of the smallpox virus.
In 2016, Holmes and his team had found traces of smallpox in a 17th-century Lithuanian mummy. By re-examining the Naples mummy with molecular tools, they expected to push back the timeline of smallpox in Europe another hundred years.
"That was the hope, that this would be a slam dunk," Poinar said. "But it's never a slam dunk when you think it's a slam dunk."
Instead, when they sequenced the genome of the child mummy and performed their molecular analysis they found no trace of the smallpox virus. But they did find evidence of the hepatitis B virus.
At first they did not pay much attention to the finding. Then they realized that a symptom of hepatitis B infection in children is a facial rash called Gianotti-Crosti syndrome. They thought perhaps the hepatitis B virus caused the dots on the child's mummified face.
They performed another molecular analysis and found that the genetic information they the extracted from the ancient hepatitis B virus resembled that of a modern-day hepatitis B virus genome. To the researchers, that was a red flag for contamination.
They spent the next year and a half trying to figure out whether they had actually found traces of ancient hepatitis B virus, or whether instead the virus they found was a contaminant from one of the researchers who uncovered the mummy in the 1980s. As hepatitis B infections were common in Italy at that time, Poinar said it was possible that the virus signals they saw were from contamination.
Further analysis showed that the genetic material from the hepatitis B virus they found on the mummy was damaged in much the same way that genetic information from the mummy itself was damaged. That indicated that it was similarly as old. But to further corroborate their hunch, the team performed an evolutionary analysis using several other old strains of hepatitis B virus, and found that the virus itself evolves very slowly. That suggests that in a period of about 500 years, the hepatitis B virus has not changed much.
Poinar and Holmes said they are confident that they found perhaps the earliest evidence of hepatitis B infection in Italy, but would not completely rule out the possibility of contamination.
"I'm 80-20 at this point, or maybe 90-10, that it's not contamination," Poinar said.