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Studies of Ancient Hepatitis B Reveal Previously Unknown Lineages in Europe

NEW YORK (GenomeWeb) – Mutations, recombination events, and apparent lineage extinctions may have obscured some of the genetic clues needed to clearly untangle the origins of the hepatitis B virus (HBV), two new studies suggest. 

"The estimates people have been dealing with before, in terms of [HBV] mutation rate, for example, have been problematic because they've only been using contemporary strains, which are just a subset of what has been around," Eske Willerslev, a researcher affiliated with the University of Copenhagen's Centre for GeoGenetics, the University of Cambridge, and the Wellcome Trust Sanger Institute, said in a conference call this week.

Willerslev and colleagues from Denmark, the UK, and elsewhere published a paper on ancient HBV in Nature this week. For that study, they tapped into HBV sequences generated as byproducts of ancient human genome sequencing studies on Bronze and Iron Age individuals, generating full or partial genome sequences for a dozen 800- to 4,500-year-old HBV samples from Eurasia.

"After we sequenced the [ancient human] genomes by shotgun sequencing, the vast majority of the DNA we were getting out was actually non-human," Willerslev explained. "Originally, this was nothing we paid much attention to. It was just expensive and kind of a waste product. But now, we have started investigating this waste product for possible pathogens."

By sifting through sequence data for 167 Bronze Age individuals and 137 individuals from a study centered on the Iron Age, the researchers narrowed in on 25 samples containing sequences that lined up with the HBV genome. From there, they used HBV-targeted enrichment and Illumina HiSeq2500 shotgun sequencing to generate 0.4-fold to more than 89-fold average coverage of each ancient HBV genome, subsequently focusing on the 12 most completely sequenced samples.

The team's phylogenetic analysis, which included more than 100 modern-day HBV isolates from humans and non-human primates, suggests that the HBV tree stretches between 8,600 and 20,900 years. It also uncovered previously unknown HBV genotypes and sequence recombinations, along with ancient European genotypes that are now better known for causing infections in other parts of the world, such as Africa or Asia.

"Based on the observation that [HBV] 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," the authors wrote. "These data support a scenario in which all present-day HBV diversity arose only after the split of the Old World and New World genotypes."

Willerslev said the availability of ancient HBV sequences "allows you to address very fundamental questions about the evolution and development of this disease." More broadly, he noted, the new data "provides a catalog" of past mutations in the HBV genome.

Because it is conceivable that some of those mutations may return at some point, he added, and the sequences serve as "not a full catalog, but at least a start of a catalog of possible variances that we may see in the future."

In an accompanying Nature paper that primarily focused on human migrations in and around the Eurasian steppe, members of the same team provided insights into the spread of another pathogen identified through ancient genome sequencing: Yersinia pestis, the pathogen behind the Justinian plague.

Sequences for two ancient Y. pestis isolates, the authors wrote, provide "provisional support for the hypothesis that the pandemic was brought to Europe toward the end of the Hunnic period through the Silk Road along the southern fringes of the steppes."

The strategy used for the ancient HBV and Y. pestis analyses will only pick up DNA-based pathogens, particularly those reaching sufficient blood concentrations to be retained in the ancient tooth or bone material, first author Barbara Mühlemann, a pathogen evolution researcher at the University of Cambridge, said during this week's call.

For a paper published online today in eLife, meanwhile, another team, led by Johannes Krause at the Max Planck Institute for the Science of Human History, presented its own analysis of three ancient HBV samples that went back up to around 7,000 years.

There, researchers considered sequences from two Neolithic HBV isolates and one medieval isolate, first identified through shotgun metagenomic sequencing and screening of dozens of human remains from archeological sites in Germany.

Again, they found evidence that versions of HBV once circulating in Europe are no longer detected today. They noted that the Neolithic samples in particular did not group well with known human HBV strains in their phylogenetic analysis, which included the three ancient isolates from Germany and nearly 500 modern HBV strains. Instead, they appeared to fall between the known human and non-human primate strains.

The medieval strain appeared to fall into its own distinct lineage, albeit somewhat closer to the HBV strains in circulation today, the team reported. And the overall genomic structure of the ancient strains "closely resembled that of modern hepatitis B viruses."

"Although the ancient forms show a relationship to modern isolates," Krause and his colleagues wrote, "they appear to represent distinct lineages that have no close modern relatives and are possibly extinct today."

In a statement, co-first author Ben Krause-Kyora, a clinical molecular biology researcher affiliated with Kiel University and the Max Planck institute, noted that "[m]ore ancient precursors, intermediates, and modern strains of both human and non-human primate HBV strains need to be sequenced to disentangle the complex evolution of this virus."