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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.

The factors that determine the characteristic seasonality of influenza remain enigmatic. Current models predict that occurrences of influenza outside the normal surveillance season within a temperate region largely reflect the importation of viruses from the alternate hemisphere or from equatorial regions in Asia. To help reveal the drivers of seasonality we investigated the origins and evolution of influenza viruses sampled during inter-seasonal periods in Australia. To this end we conducted an expansive phylogenetic analysis of 9912, 3804, and 3941 hemagglutinnin (HA) sequences from influenza A/H1N1pdm, A/H3N2, and B, respectively, collected globally during the period 2009-2014. Of the 1475 viruses sampled from Australia, 396 (26.8% of Australian, or 2.2% of global set) were sampled outside the monitored temperate influenza surveillance season (1 May - 31 October). Notably, rather than simply reflecting short-lived importations of virus from global localities with higher influenza prevalence, we documented a variety of more complex inter-seasonal transmission patterns including "stragglers" from the preceding season and "heralds" of the forthcoming season, and which included viruses sampled from clearly temperate regions within Australia. We also provide evidence for the persistence of influenza B virus between epidemic seasons, in which transmission of a viral lineage begins in one season and continues throughout the inter-seasonal period into the following season. Strikingly, a disproportionately high number of inter-seasonal influenza transmission events occurred in tropical and subtropical regions of Australia, providing further evidence that climate plays an important role in shaping patterns of influenza seasonality.