Next time you picture a Mongol horde sweeping across the Asian steppes on horseback, imagine that about two-thirds of them have liver disease. Hepatitis B is a virus that attacks the liver, causing scarring, organ failure, and sometimes cancer. Its origins and evolutionary history are still a bit of an enigma, but viral DNA left behind in the bones and teeth of ancient people from the Asian steppe may help reconstruct part of our long history with the disease.

The virus showed up in what have been considered extraneous sequences of DNA that are associated with DNA samples but not part of the human genome. Typically, software gets rid of these sequences and uses what's left to assemble the human genome.

Viruses and genomes

While DNA sequencing has focused on the human portion of human genome data, that's starting to change. “Originally, this was nothing we paid much attention to. It was just expensive and kind of a waste product, but now we've started investigating this waste product for possible positives,” said Copenhagen University evolutionary geneticist Eske Willerslev. It's how his team found evidence that Mongol warriors from the steppe carried an early form of the pathogen that would later become the 541-542 CE Justinian Plague.

Viruses show up during DNA sequencing for two reasons. About eight percent of the human genome is made up of fragments of viral DNA that we’ve acquired over the last 1.8 million years or so. One category of this viral DNA, the so-called endogenous retroviruses, have actually worked themselves into our chromosomes, and they're passed from parent to child with all the rest of our genetic makeup.

But most viruses don't integrate into the genome as part of their life cycle. The hepatitis B DNA that Cambridge University pathogen evolution researcher Barbara Mühlemann and her colleagues have extracted from Bronze Age and Iron Age samples is like this; it remains separate from the host's genome. But that doesn't mean there's no hepatitis B DNA in the cells of infected individuals. As a result, it's picked up during genome sequencing and can be identified as viral DNA that happened to be in a person’s cells when they died but wasn’t part of their chromosomes. Researchers call this exogenous viral DNA.

A 4,500-year-old sample from the group is the oldest exogenous viral DNA ever recovered so far, beating out a 450-year-old hepatitis B sample found in an earlier study.

The unromantic past

When Mühlemann and her colleagues sequenced 304 genomes from the skeletons of people who lived on the steppe during the Iron and Bronze Ages (from about 4,500 years ago up until about 800 years ago) they checked the waste sequences for possible evidence of ancient infections. Twenty-five people out of 304 had DNA from the hepatitis B virus in their bones.

“The samples that are sequenced are obviously either teeth or bones, and we think that the reason why we find viruses in those samples is because those are tissues that have blood flow going through them, and the virus gets to a high concentration in the blood, which is why we find them in those tissues,” said Mühlemann. “That essentially means that we're only able to find viruses that cause infections with high titers in the blood, and the individual has to die in the state like that.”

That means that HBV on the ancient steppes may have been as prevalent as it is in some of the most heavily impacted areas of the world today—or more so. In South Sudan, for instance, about 22.38 percent of people are infected. And in areas where prevalence is over about 8 percent, a surprisingly high majority of adults, 70 to 90 percent, show signs of having been infected at some point in their lives.

“About 10 percent or so of the samples of the individuals we are studying have nasty pathogens in them, and when we use teeth, you also find that 50 percent of the individuals have all kinds of oral infections that you might not die of but it's not nice that you have. So the picture that emerged from this line of work is that a lot of people were running around with diseases in the past,” said Willerslev. “It certainly kind of cracked my romantic picture of the Bronze Age and Iron Age.”

The more you know

They sequenced 12 of the best-preserved ancient viral genomes and combined them with modern genomes to analyze the evolutionary relationships between different strains of the virus. What they found is that hepatitis B was part of life on the Eurasian steppes for thousands of years, and its evolutionary story is more complicated than anyone thought. Mühlemann and her colleagues found at least one strain of hepatitis B that’s now extinct, having apparently faded out of existence sometime in the last 4,500 years. And according to a phylogenetic tree mapping the evolutionary relationship between the genomes, one of the nine major genotypes circulating today was the product of an ancient recombination between two strains of the hepatitis B virus.

“Based on the observation that genotypes go extinct and can be created by recombination, the ancient sequence data show that the diversity that we observe today is only a subset of the diversity that has ever existed,” Mühlemann and her colleagues wrote. Most of the genetic diversity in hepatitis B viruses today probably arose sometime between 25,000 and 13,400 years ago, when the genetic lineages of Old World hepatitis B strains and New World hepatitis B strains split, according to Mühlemann and her colleagues.

Understanding some of that long-lost genetic diversity and how the virus evolved into the strains we see today may equip us to fight it more effectively in the future. By looking at genetic variants of the virus in the past, researchers can get a preview of the kinds of mutations that are likely to arise in the future. The World Health Organization has set a goal of significantly reducing the worldwide burden of hepatitis B by 2030. The best hope for that, at the moment, rests on a combination of vaccines to protect the uninfected and antiviral drugs to treat the infected, but the wrong mutation in the virus’ genome could render those weapons useless. Knowing which mutations may be coming could give us an advantage.

“It's good to have a very in-depth look at those sequences, also potentially in the lab, to see if the variation that we see can tell us anything about the changes that the virus could make in the future, and if such changes occur, whether our vaccines and our antivirals and our diagnostic tests still work,” said Mühlemann. “In case of the arising of that variant of the virus in the future, that might help us to know how we have to adapt or change our diagnostic tests or our vaccines.”

People and pathogens

The 12 hepatitis B genomes may also help tell us where the virus came from. We still don’t know where hepatitis B first evolved, for instance, and while it’s going to take more viral genomes and a lot more analysis to answer that question, Mühlemann and her colleagues say there’s already reason to think that some current hypotheses about where hepatitis B arose and how it spread around the world may need some revision.

“Some ideas that have been around over the years, [such as] that hepatitis B came from America and very recently entered Europe some 500 years ago, are certainly wrong, because of course these are going much further back in time,” said Willerslev.

One modern strain, genotype A, was previously thought to have emerged in Africa and spread to the Americas and India within the last few centuries via the slave trade. But Mühlemann and her colleagues found some ancestral strains of type A in people living on the steppe as early as 4,300 years ago: some from the Sintasha culture in what is now southwest Russia, and one in a person from the Scythian culture in what is now Hungary.

That, according to Natural History Museum of Denmark paleogeneticist Peter de Barros Damgaard, “suggests that it's kind of the other way around, that it came from Europe, but it was introduced to South Asian populations historically recently, without having a massive human population admixture.”

And Damgaard says that’s an important point about tracking the movement of diseases through human history. Paleogenomicists use changes in human genomes to track large-scale population movements and interactions between cultures, but DNA can only capture the kind of intercultural relations that leave a genetic signature, which means interbreeding on a fairly large scale for a period of time. The exchange of diseases can provide an independent line of evidence for paleogenomicists to check their work against, but diseases spread between populations much more easily than genes do.

“While it's super exciting to try to correlate the human dispersals with the dispersal of pathogens, there for sure are limitations to doing that, because the pathogen can also spread without you having massive population admixture,” he said. “Once the pathogen is introduced, it can also spread horizontally without you having to have a human population admixture that's so considerable that you actually see it.”

But that may mean that some pathogens can help track other kinds of interactions between groups of people.

“There’s now a number of cases emerging where you can say we don't see much of that sexual contact [between populations],” said Willerslev. Bronze Age Scythian culture, for example, was pretty uniform across a wide swath of the steppe, but the Scythians themselves actually consisted of several genetically distinct populations.

“So how did that kind of similar culture come about? What kind of processes? And there I think maybe some of the pathogens can be one way to address some of these questions,” said Willerslev.

Searching for ancient diseases

Answering some of those remaining questions will require more ancient viral genomes, not just from hepatitis B but from other pathogens. Hepatitis B is particularly easy to find in ancient DNA samples because infected people carry lots of the virus in their blood for several years, increasing the chances its DNA will be preserved in bone cells. The virus particle itself is very stable over time. But other viruses, including influenza and variola (pox) have been found in archaeological human remains as much as 400 years old, and Mühlemann says that others like herpes viruses, parvoviruses, and adenoviruses might be good candidates.

The team has set up dedicated systems to scan what was once considered waste DNA for sequences that match known viruses and bacteria (except for RNA viruses like influenza, whose genes won’t be preserved in human DNA), and they’re still working through the sequences from samples they’ve already taken from ancient bones.

“We haven't even completed that yet, but obviously also for future samples we will be screening for all known viruses and all known bacteria,” said Willerslev.

And it’s possible that in one of those future samples, they may find even older traces of hepatitis B or another virus. “In a number of cases, you will find that it has survived, in time,” said Willerslev.

Nature, 2018. DOI: 10.1038/s41586-018-0097-z (About DOIs).