Infectivity of an ancient hepatitis B virus cloned after retrieval from archaeological human remains

Background HBV evolution, diversity and phylogeographic history remain unclear. The successful retrieval of HBV DNA from archaeological human remains allows genotypic and phenotypic studies of HBV lineages over thousands of years. Aim of this study was to generate, clone and characterize the infectivity of a 1,160-years-old HBV isolate of genotype D (GT-D, DA222) in vitro and in vivo.

Methods The ancient HBV strain (DA222) was cloned based on shotgun sequencing from archaeological human remains (Mühlemann, Nature 2018). DA222-HBsAg-pseudotyped-(ps)HDV was used to infect HepG2-NTCP cells. HBV generated from 1.5mer plasmids was used to infect primary human hepatocytes (PHH) and humanized mice (USG). Viral markers were measured by RT-PCR, Abbott Architect system and immunohistochemistry.

Results Infection of HepG2-NTCP cells with DA222-HBsAg-psHDV corroborated the NTCP-conserved mode of infection, while genuine DA222 showed productive infection in PHHs. DA222 and a modern GT-D control displayed similar kinetics of HBV DNA viremia development (median 2E9 vs. 7E9, respectively; week 15) in humanized mice. pgRNA in serum (3E7 vs. 2E8 copies/ml) and liver (23 vs. 41 rel. exp.), as well as intrahepatic rcDNA (294 vs. 1479 copies/PHH) were slightly lower in DA222-infected mice, whereas cccDNA loads (1.5 vs. 2 copies/PHH) and human ISGs (MXA; ISG15; IP10; STAT1; OAS1) appeared comparable. Immunofluorescence indicated a typical HBcAg staining pattern.

Conclusion We established a productive infection of an ancient HBV strain. The in-vivo-replication capabilities of DA222 were nearly as good as modern HBV. Our approach opens new possibilities for phenotypic characterization of infection and pathogenic potential of ancient HBV lineages.

Publikationsverlauf

Artikel online veröffentlicht:
18. Januar 2023