The paradox of HBV evolution as revealed from a 16th century mummy.

Zoe Patterson Ross,Sebastian Duchêne,Gino Fornaciari,Valentina Giuffra,John-Sebastian Eden,Mark W Douglas,Hendrik N Poinar,Debi Poinar,Ana T Duggan,Jennifer Klunk,Edward C Holmes,Siobain Duffy

doi:10.1371/journal.ppat.1006750

Abstract

Hepatitis B virus (HBV) is a ubiquitous viral pathogen associated with large-scale morbidity and mortality in humans. However, there is considerable uncertainty over the time-scale of its origin and evolution. Initial shotgun data from a mid-16th century Italian child mummy, that was previously paleopathologically identified as having been infected with Variola virus (VARV, the agent of smallpox), showed no DNA reads for VARV yet did for hepatitis B virus (HBV). Previously, electron microscopy provided evidence for the presence of VARV in this sample, although similar analyses conducted here did not reveal any VARV particles. We attempted to enrich and sequence for both VARV and HBV DNA. Although we did not recover any reads identified as VARV, we were successful in reconstructing an HBV genome at 163.8X coverage. Strikingly, both the HBV sequence and that of the associated host mitochondrial DNA displayed a nearly identical cytosine deamination pattern near the termini of DNA fragments, characteristic of an ancient origin. In contrast, phylogenetic analyses revealed a close relationship between the putative ancient virus and contemporary HBV strains (of genotype D), at first suggesting contamination. In addressing this paradox we demonstrate that HBV evolution is characterized by a marked lack of temporal structure. This confounds attempts to use molecular clock-based methods to date the origin of this virus over the time-frame sampled so far, and means that phylogenetic measures alone cannot yet be used to determine HBV sequence authenticity. If genuine, this phylogenetic pattern indicates that the genotypes of HBV diversified long before the 16th century, and enables comparison of potential pathogenic similarities between modern and ancient HBV. These results have important implications for our understanding of the emergence and evolution of this common viral pathogen.

Highlights

The comparative analysis of viral genomes provides a wide and informative view of evolutionary patterns and processes
We used ancient DNA techniques to recover the complete genome sequence of an Hepatitis B virus (HBV) from the mummified remains of a child discovered in the 16th century from Naples, Italy
Our analysis of this specimen resulted in two contrasting findings: while the damage patterns lend credence to this HBV sequence being authentically 16th century, phylogenetic analysis revealed a close relationship to recently sampled viruses as expected if the sequence were a modern contaminant

Summary

Introduction

The comparative analysis of viral genomes provides a wide and informative view of evolutionary patterns and processes. ADNA techniques have been used in studies of major 20th century epidemics of influenza virus and human immunodeficiency virus [14, 15] Taken together, these studies have helped to clarify the causative agents of specific outbreaks, whether ancient strains differ markedly from recent ones, and the evolutionary and epidemiological processes that have likely shaped virus diversity. These studies have helped to clarify the causative agents of specific outbreaks, whether ancient strains differ markedly from recent ones, and the evolutionary and epidemiological processes that have likely shaped virus diversity They have provided key information on the dynamics of evolutionary change, including the ‘timedependent’ nature of viral evolution in which estimates of evolutionary rates are routinely elevated toward the present and decline toward the past due to a combination of unpurged transient deleterious mutations in the short-term and site saturation in the long-term [16]

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Discussion

Conclusion