The genome of a Mesozoic paleovirus reveals the evolution of hepatitis B viruses

Abstract

Paleovirology involves the identification of ancient endogenous viral elements within eukaryotic genomes. The evolutionary origins of the reverse-transcribing hepatitis B viruses, however, remain elusive, due to the small number of endogenized sequences present in host genomes. Here we report a comprehensively dated genomic record of hepatitis B virus endogenizations that spans bird evolution from >82 to <12.1 million years ago. The oldest virus relic extends over a 99% complete hepatitis B virus genome sequence and constitutes the first discovery of a Mesozoic paleovirus genome. We show that Hepadnaviridae are >63 million years older than previously known and provide direct evidence for coexistence of hepatitis B viruses and birds during the Mesozoic and Cenozoic Eras. Finally, phylogenetic analyses and distribution of hepatitis B virus relics suggest that birds potentially are the ancestral hosts of Hepadnaviridae and mammalian hepatitis B viruses probably emerged after a bird-mammal host switch. Our study reveals previously undiscovered and multi-faceted insights into prehistoric hepatitis B virus evolution and provides valuable resources for future studies, such as in-vitro resurrection of Mesozoic hepadnaviruses.

ARTICLE

Received 21 Sep 2012 | Accepted 26 Mar 2013 | Published 30 Apr 2013

DOI: 10.1038/ncomms2798

The genome of a Mesozoic paleovirus reveals the evolution of hepatitis B viruses

Alexander Suh1,w, Jrgen Brosius1, Jrgen Schmitz1,* & Jan Ole Kriegs1,2,*

Paleovirology involves the identication of ancient endogenous viral elements within eukaryotic genomes. The evolutionary origins of the reverse-transcribing hepatitis B viruses, however, remain elusive, due to the small number of endogenized sequences present in host genomes. Here we report a comprehensively dated genomic record of hepatitis B virus endogenizations that spans bird evolution from 482 to o12.1 million years ago. The oldest virus relic extends over a 99% complete hepatitis B virus genome sequence and constitutes the rst discovery of a Mesozoic paleovirus genome. We show that Hepadnaviridae are 463 million years older than previously known and provide direct evidence for coexistence of hepatitis B viruses and birds during the Mesozoic and Cenozoic Eras. Finally, phylogenetic analyses and distribution of hepatitis B virus relics suggest that birds potentially are the ancestral hosts of Hepadnaviridae and mammalian hepatitis B viruses probably emerged after a birdmammal host switch. Our study reveals previously undiscovered and multi-faceted insights into prehistoric hepatitis B virus evolution and provides valuable resources for future studies, such as in-vitro resurrection of Mesozoic hepadnaviruses.

1 Institute of Experimental Pathology (ZMBE), University of Mnster, Von-Esmarch-Stra 56, D-48149 Mnster, Germany. 2 LWL-Museum fr Naturkunde, Westfalisches Landesmuseum mit Planetarium, Sentruper Stra 285, D-48161 Mnster, Germany. * These authors contributed equally to this work.w Present address: Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, Norbyvagen 18D, SE-752 36 Uppsala, Sweden.

Correspondence and requests for materials should be addressed to A.S. (email: mailto:[email protected]

Web End [email protected] ).

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The emerging eld of paleovirology1 has discovered a wealth of endogenous viral elements (EVEs)2 scattered within eukaryotic genomes. Surprisingly, in addition to the

retroviral origin of many EVEs, all other major groups of eukaryotic viruses exhibit a more or less pronounced genomic record of endogenizations, too3. Such genomic relics arise as a consequence of viral inltration of the hosts germline, followed by insertion and xation of virus-derived DNA fragments within the hosts genome4,5. As each virus relic is present at a specic genomic location, the presence/absence patterns of orthologous EVE insertions among closely and less closely related species permit the reconstruction of the lower and upper age boundary of endogenization events3.

Hepatitis B viruses (HBVs) belong to the Hepadnaviridae family of reverse-transcribing DNA viruses and constitute a major global health issue by infecting more than a third of the human population6. Until very recently2,7, these viruses were presumed to completely lack a record of genomic endogenizations, as none of the sequenced genomes of their extant avian, rodent and primate hosts8 (that is, human, chimp, gorilla, orangutan, gibbon) exhibit HBV-derived sequences. Consequently, insights into the evolutionary origin of Hepadnaviridae were based solely on sequence analyses of extant HBVs, dating the last common ancestor of Orthohepadnaviridae and Avihepadnaviridae to 30,000 years9 or 125,000 years10 ago. It was rather the recently published11 genome of the zebra nch, a bird species that is not documented to be an extant HBV host8, that revealed the rst evidence for HBV-derived EVEs in BLAST12-based analyses2,7. Gilbert and Feschotte7 identied about a dozen HBV-derived fragments, each with a length extending over B4 to B40% of the extant duck HBV (DHBV) genome, and revealed compelling evidence that at least one of these EVEs was inserted as early as 419 million years ago (MYA).

Here, we report a dated record of HBV germline inltrations in the lineage leading to the zebra nch that discloses even more far-reaching surprises. We conducted comprehensive presence/ absence analyses (Supplementary Table S1) of previously2,7 and newly detected HBV-derived EVEs in the zebra nch genome (Supplementary Table S2) using a dense taxon sampling, permitting the reconstruction of HBV endogenization events during bird evolution and revealing the 99% complete genome sequence of a paleovirus nested within an intron of the FRY (that is, Drosophila Furry homolog) gene.

ResultsHepadnavirus endogenizations and bird evolution. From the 12 endogenous zebra nch HBV-derived EVEs (eZHBVs) recognizable via tBLASTx (see Methods), we were able to amplify eight insertion loci in all those bird species that are necessary for a precise reconstruction of the respective insertion event. The resultant eZHBV presence/absence patterns (Supplementary Table S1) revealed HBV endogenizations during bird evolution in different geological epochs (Fig. 1). The youngest HBV-derived EVE (eZHBV_M) is present in the zebra nch and absent in the orthologous position in all other sampled taxa; thus, its insertion occurred o12.1 MYA during the Miocene or even more recently.

Six of our eight dated EVEs are either of Oligocene (eZHBV_O1-O5; 26.6-34.2 MYA) or Eocene (eZHBV_E; 34.2-35 MYA) origin, as they are present in passeroid songbirds to the exclusion of either leafbirds and sunbirds or sunbirds alone. Strikingly, we identied an even older eZHBV insertion (eZHBV_C) that is shared among all major neoavian clades sensu ref. 13 and absent in non-neoavian taxa (for example, chicken), strongly suggesting that this HBV endogenization occurred in the common ancestor of Neoaves. As both the most conservative14 and the most

comprehensive15 molecular analyses date the existence of the neoavian ancestor to 474 MYA14 or 8294 MYA15, the neoavian-wide presence (Fig. 2) of eZHBV_C provides direct evidence for the existence and endogenization of Hepadnaviridae during the Upper Cretaceous. The hepadnaviral genomic record of germline inltrations is thus considerably older than previously assumed7. Even more surprisingly, the eZHBV_C EVE constitutes a continuous and 99% complete paleovirus genome sequence (Fig. 3a) that has been buried in neoavian genomes since the Mesozoic Era.

Long-term substitution rates of Hepadnaviridae. On the basis of the orthologous eZHBV sequences obtained in our presence/ absence screenings of each of the eight EVEs, we derived (see Methods) hypothetical ancestral sequences (HASs) for further sequence analyses, as we expect that these HASs approximate the respective sequence condition of each HBV fragment at the time of its endogenization. By combining pairwise HAS divergences (Supplementary Table S3) with the dating of the upper and lower boundaries of each HBV endogenization event, we calculated long-term nucleotide substitution rates (Supplementary Table S3) during the different prehistoric chapters of HBV evolution (Fig. 1). Notably, the lowest substitution rates are found when comparing Mesozoic versus Eocene eZHBVs (that is, 1.17 10 8

substitutions per site per year) as well as Oligocene versus Miocene eZHBVs (that is, 1.54 10 8 substitutions per site per

year), whereas Eocene versus Oligocene and Oligocene versus Oligocene eZHBV comparisons resulted in slightly higher values(3.46 10 8 and 5.23 10 8 substitutions per site per year). All

these eZHBV substitution rates are similar to previous estimations7 (based on pairwise comparisons of eZHBVs versus extant avian HBVs) and suggest that the Mesozoic and Cenozoic long-term substitution rates of Hepadnaviridae are more than 1,000-fold slower7 than the extant short-term substitution rates of human HBVs16, even during their e-antigen positive phase17.

Genome evolution of Hepadnaviridae. Our study provides direct evidence that the compact genomic organisation of Hepadnaviridae has remained largely unchanged for the last 482 MYA of hepadnaviral evolution. This assumption is based on the presence of partially overlapping polymerase (pol), pre-surface/surface (preS/S) and precore/core (preC/C) open reading frames (ORFs)8 in the HAS of the 99% complete Mesozoic paleovirus genome (eZHBV_C). Furthermore, our analyses of extant HBV and paleoviral eZHBV_C protein sequences unravel an unexpected degree of amino-acid sequence conservation in certain regions of HBV genomes (Fig. 3bd). Most strikingly, the reverse transcriptase region of the pol ORF exhibits 27.1% perfectly conserved amino-acid positions (Fig. 3b), but also other genomic regions (for example, N-terminal protein, RNase H, S ORF, C ORF) are thoroughly conserved at 12.218.5% of their amino-acid sites (Fig. 3bd). Merely the spacer region of the pol ORF, the preC-specic region, and the preS-specic region show a relatively low proportion (that is, o5%) of unvaried amino-acid positions (Fig. 3bd).

Despite their genomic similarities, the most striking difference between Orthohepadnaviridae and Avihepadnaviridae is the presence of an X protein in the genomes of human and other mammalian HBVs8, a regulatory protein with multiple protein-interacting functions1820 and a tumor-promoting effect18. When we thus performed tBLASTn searches of mammalian HBV X proteins in the eZHBV_C genome, we found no evidence for the presence of an intact or at least a degenerated X ORF in this Mesozoic paleovirus. Additionally, nucleotide sequence alignments of eZHBV_C and extant avian HBVs revealed that

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2798 ARTICLE

1.17 x108

3.46 x108

5.23 x108

1.54 x108

Taeniopygia (zebra finch)

eZHBV_O1

Chloebia (Gouldian finch)

eZHBV_O2

Ploceidae

eZHBV_O3

Ploceus (weaver)

Carpodacus (rosefinch)

eZHBV_O4

Passeroidea

Anthus (pipit)

eZHBV_O5

eZHBV_M

eZHBV_E

Radiation of

Passeriformes

Prunella (accentor)

Chloropsis (leafbird)

Cinnyris (sunbird)

Other passerine lineages

Other neoavian lineages

Gallus (chicken)

Radiation of

Neoaves

eZHBV_C

Dendrocygna (whistling duck)

Struthio (ostrich)

110

100

90

80

70

60

50

40

30

20

10

Lower

Cretaceous

Upper

Cretaceous

Paleocene

Eocene

Oligocene

Quaternary

Pliocene

Miocene

Figure 1 | Hepatitis B virus endogenization events during bird evolution. Presence/absence analyses of zebra nch HBV EVE insertion loci (Supplementary Table S1; for full alignments, see Supplementary Data 1) revealed eZHBV endogenization events in the ancestor of Neoaves, during early passeroid evolution and during recent ploceid evolution. The avian tree of life13,34 is shown as a simplied chronogram based on molecular dates of interordinal avian relationships15, as well as relationships within Passeriformes32 and Ploceidae (Supplementary Fig. S1). Based on HASs of the dated EVEs, nucleotide substitution rates (in substitutions/site/year) were calculated (Supplementary Table S3) for the respective parts of eZHBV evolution and denoted above the tree. Endogenization events are depicted as icosahedrons and temporal ranges of insertion events are shown as coloured rectangles (colours correspond to the respective geological epoch in the International Stratigraphic Chart; http://www.stratigraphy.org/ics%20chart/09_2010/ StratChart2010.pdf). Although eZHBV_O1 (grey letters) was previously dated7, we sampled two missing taxa (Prunella, Chloropsis) that permit a more precise dating.

eZHBV_C lacks the hypothetical start codon of an X-like ORF that has been annotated in some Avihepadnaviridae based on the structural prediction sensu ref. 21 (Fig. 3e) or the ORF prediction sensu ref. 22 (Fig. 3f). Thus, our paleovirological indication for the absence of the X ORF in a Mesozoic paleovirus suggests that, in contrast to previous assumptions10,21, the oncogenic X protein of Orthohepadnaviridae might not have been present in the common ancestor of Hepadnaviridae. Instead, it probably emerged de novo via overprinting23 of pol and precore ORF parts in the common ancestor of mammalian HBVs.

Phylogeny of Hepadnaviridae. Our phylogenetic analyses of the pol protein from eZHBV HASs and extant HBVs reveal three ancient lineages of zebra nch eZHBV EVEs (also when including very recently published24,25 budgerigar HBV EVEs, Supplementary Fig. S3) and further insights into early hepadnaviral evolution (Fig. 4). The Eocene/Oligocene EVEs (eZHBV_E

eZHBV_O1-O5) group as sister to extant avian HBVs, whereas the youngest (eZHBV_M) and the oldest (eZHBV_C) HBV-derived EVEs each appear to be independent lineages that are rather distantly related to extant avian HBVs. The exact branching order of eZHBV_M and eZHBV_C in relation to extant HBVs depends on the position of the root in the HBV phylogeny. When the tree is rooted to mammalian HBVs (Fig. 4a), the EVE record and tree topology imply that Orthohepadnaviridae and Avihepadnaviridae separated before the endogenization of eZHBV_C, that is, 494 to 482 MYA.

Accordingly, the ancestor of Hepadnaviridae probably infected the amniote ancestor that lived 4324 MYA26 and subsequently diverged into avian and mammalian HBVs with the emergence of the bird and mammal lineages, respectively. As an alternative to this codivergence scenario, the rooting of the tree to the oldest HBV EVE (eZHBV_C) suggests the paraphyly of HBV EVEs (Fig. 4b) with a potential relationship of eZHBV_M and extant

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213 1107

| 2401

| 5177

| 5855

|

|

G

AGG

eZHBV_C (HAS)

TCCCAAGCTTGT CAATT TTAAAACTGCCATA

Taeniopygia guttata Cinnyris asiaticus Parus palustris Acanthisitta chloris Nestor notabilis Falco sparverius Cariama cristata Asio otusAlcedo atthisPicus viridis Trogon viridis Buteo lagopus Cathartes aura

Balearica pavonina

Gallus gallusDendrocygna viduataStruthio camelus (103 MY)

(94 MY)

GG..GG.GG..GG

A

AAG

TATG GTCCTCCAAAAGCGGAGTTCTCAAGCTTAT TGATT TTACAACTGCCACAGAAACAG-GAAACTGC CCTGATAAG ATTCTCCAAAAGTTGAGTTCTCAAACTTAT T---- TTACAACTGCCACAGAAACAG-AAAACTGC ......... .............................. TGATT TTTCAACTGCCAGAGAAACAG-AAAACTGC CCTGA.... .............................. ..... TTAAAACTGCCACAGAAGGAG-AAAACTGC CCTGATATG ------------------------------ CAATT TTAAAACTGCC------ACAG-AAAACTGC CCTGATATA GTTCTCCAAAAGCTGAATTCCCAAGCTTGT CAATT TTAAAACTGCCACAGAAACAG-AACACTGC CCTGA.... .............................. ..... TTAAAACCACCACAGAAACAGAAAAACCAC CCTGACATG ------------------------------ CAGTT .............................. .....CAGG ------------------------------ ----- ------------------------------ CCTGA.... .............................. ..... TTAGGACTGCCACAGAAACAC-TAAACTGC CTTGA.... .............................. ..... TTAAAACTGCTACAGAAACAG-AAAACTGT CTTGCTATG GTTCTCCAAAGGCTGAATTCCCAAGCTTCT TAATT TTAAAGCTGCCATAGAAACAA-AAAACTAC CCTGATATA GTTCTCCAAAAGTTGAATTGCTAAGCTTGT CAGTT TTAAAACTGCCACAGAAACAG-AAACCTGC CCTGA

TATG GTTCTCCAAAAGTGGAATTCCCAAGCTTGT ..... .............................. CCTGA

TTAT GTTAACTGGATGTTG--------------- ----- ---------------AAATGG-AAACCTGC TTT--TATT GTTAACCAAAGGTCA--------------- ----- ---------------AAACGG-AAAACTGC GCTGATGTG GTTATCCAAAGTTTG--------------- ----- ---------------AAACAG-AAAGCTGC CTTGA

(35 MY)

(41 MY)

(60 MY)

(70 MY)

(75 MY)

(76 MY)

(78 MY)

(78 MY)

(78 MY)

(78 MY)

(78 MY)

(78 MY)

Larus ridibundus (79 MY)

Columba palumbusTachybaptus ruficollis (82 MY)

(82 MY)

Ciconia ciconia (80 MY)

Opisthocomus hoazinApus apusChrysolampis mosquitus (82 MY)

(82 MY)

G

AG

TATG CTTCTCAAAAAATTGAATTCCCAAGCTTGT CAATT TTAAAACTGCCAATGAAACAG-CAAACTGC TCTGA

TATG GTTCTCCGAAAGTTCAATTCCCAAGCTTAT CAATT CTAAAACTACCACAGAAACAG-AAATCTGC CCTGATATG -TTCTCCAAAAGTTTAATTCTCAAGCTTGT ..... .............................. CCTRA

TATG GTTCTCCAAAAGTTGAATTACCAAGCTTGT CAATT TTAAAACTGCCACAGAAACAG-AAAACTGC CCTGA

TATG GTTCTCCAGAAGTTGA--------GCTTGT CAATT ------------------------------ .....TATG ------------------------------ ----- ------------------------------ CCTGATATG ------------------------------ ----- ------------------------------ CCTGA

(80 MY)

(80 MY)

(94 MY)

Figure 2 | Presence/absence pattern of the Mesozoic eZHBV_C paleovirus within avian genomes. Non-neoavian representatives (Gallus, Dendrocygna, Struthio) exhibit the ancestral absence situation, whereas neoavian representatives exhibit the unambiguous presence of the eZHBV_C fragment. In Alcedo, Apus and Chrysolampis, birds that are each phylogenetically nested34 within Neoaves, large and unspecic deletions have led to the secondary loss of almost the complete intron (spanning the paleovirus anking sequences). Approximate dates of species divergence15,32 to the zebra nch (Taeniopygia)

are shown in italicized letters. The eZHBV_C presence/absence region is boxed and aligned to the HAS of eZHBV_C. Missing sequence information is denoted as grey dots and numbers denote positions in the full alignment of the eZHBV_C locus (Supplementary Data 1).

mammalian HBVs that is moderately supported by a bootstrap value of 69% and a Bayesian posterior probability of 0.82. Considering this topology and the apparent absence of HBV EVEs in mammalian genomes, we hypothesize that the common ancestor of Hepadnaviridae probably infected birds and later, following a host switch, one of the hepadnaviral lineages infected mammals. If the assumption of a birdmammal host switch is correct, Orthohepadnaviridae would constitute a virus taxon that is younger than the Mesozoic eZHBV_C EVE (that is, o94 to o82 MYA) and probably even as young as o12.1 MYA when including the putative phylogenetic position of the Miocene eZHBV_M EVE.

DiscussionOur paleovirological results comprise direct evidence for virushost coexistence of hepadnaviruses in the Upper Cretaceous, a nding that was so far restricted to bornaviruses and circoviruses (based on shared ancient EVE insertions2), as well as foamy viruses (based on their codivergence with placental mammals27). Above all, this study is the rst to present the genome sequence of a Mesozoic paleovirus. Our insights into long-term sequence evolution, genome evolution and hypothetical ancestral hosts change the understanding of the prehistoric evolution of Hepadnaviridae, including the hypothetical origin of mammalian HBVs. We anticipate that this will be of signicance to a variety of disciplines involved in studying HBVs or their hosts, including medical research and palaeontology. Additionally, our results emphasize the importance of conducting comprehensive presence/absence analyses in addition to the computational extraction24,25 of paleoviral sequences. With the foreseeable sequencing of a wealth of animal genomes28, we expect that our Mesozoic paleovirus genome is just the tip of the iceberg of prehistoric virus genomes.

Methods

General approach. We conducted tBLASTx searches using the DHBV genome (accession code AY494851) as query and the zebra nch genome as database (genomic blast server at the National Center for Biotechnology and Information;

http://blast.ncbi.nlm.nih.gov/

Web End =http://blast.ncbi.nlm.nih.gov/ ). We complemented these tBLASTx searches by conducting additional searches using the eZHBV fragments of Katzourakis and Gifford2 and Gilbert and Feschotte7 as queries. Their eZHBV sequences2,7 had been detected likewise (that is, using tBLASTx searches of DHBV against the zebra nch genome). After we extracted BLAST hits (E o10 5) including anking sequences of 45 kb from the taeGut1 assembly in Genome Browser29 (http://genome.ucsc.edu/cgi-bin/hgBlat

Web End =http:// http://genome.ucsc.edu/cgi-bin/hgBlat

Web End =genome.ucsc.edu/cgi-bin/hgBlat ), we aligned these to the respective orthologous regions of the chicken genome (galGal3 assembly) using MAFFT30 (FFT-NS-2, version 6, http://mafft.cbrc.jp/alignment/server/index.html

Web End =http://mafft.cbrc.jp/alignment/server/index.html ). Based on the insertion sites identied in these sequence alignments, we reconstructed that the total of 16 eZHBV fragments stem from 12 germline inltration events, as some of these fragments arose via post-insertional duplication of the genomic locus (that is, eZHBVf g7; eZHBVl l*7) and others appear to be the result of a big deletion

within a beforehand larger eZHBV fragment (that is, eZHBVb k7; eZHBVi j7).

Eight of these insertion loci exhibited anking sequences that were suitable for primer design and PCR amplication, that is, well-conserved coding or noncoding regions near the respective eZHBV insertion. Note that we did not consider the previously studied7 eZHBVj and eZHBVl EVEs, as for these, no absence situation could be amplied7, prohibiting the precise dating of these insertions.

Subsequent to our comprehensive presence/absence screening of eight eZHBV insertion loci in vitro (see below), we aligned (Supplementary Data 1) all sequences of each orthologous locus using MAFFT (E-INS-i, version 6) and ascertained presence/absence character states (Supplementary Table S1). Our dense taxon sampling (see below) permitted the maximum likelihood-based reconstruction of HASs in MEGA531 (Tamura-Nei model of nucleotide substitution, gamma-distributed rates among sites, ve discrete gamma categories, tree topology from Fig. 1 and ref. 13) for each orthologous eZHBV insertion shared by more than two species. The resultant ancestral sequences exhibit only a few frameshift mutations and stop codons (Supplementary Table S2).

Taxon sampling. Our taxon sampling comprises representatives of all major passeroid taxa32 (including the major ploceid33 taxa within Passeroidea) on the lineage leading to the zebra nch and thus permits an accurate detection of the upper and lower age boundary of eZHBV endogenization events. For eZHBV_O1-O5 and eZHBV_E, we sampled Anthus gustavi, Carpodacus erythrinus, Prunella modularis (LWL00359), Chloropsis aurifrons, Cinnyris asiaticus and Turdus merula. In the case of eZHBV_M, we additionally sampled Chloebia gouldiae and Ploceus castaneiceps, whereas for eZHBV_C, we included Parus palustris and representatives of the major neoavian lineages13,34 (see Supplementary Table S1 for more information and ref. 13 for species names). Sequence-based verication of species identity was conducted as previously described13. In the case of eZHBV_O1, we completed the taxon sampling by using previously published sequences7 of Chloebia gouldiae, Junco hyemalis and Cyanomitra olivaceus from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/

Web End =http://www.ncbi.nlm.nih.gov/Genbank/; accession codes HQ116567, HQ116565, and HQ116564).

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a

Extant

DHBV

pol vX * *

preC C

* preS S *

1,332

Miocene

eZHBV_M

1,781

2,774

eZHBV_O1

eZHBV_E

418

eZHBV_O2

556

474

Oligocene

Eocene

eZHBV_O3

764

518

eZHBV_O4 eZHBV_O5

681 1,565

1,756

298

747

Upper

Cretaceous

2,551

2,514

eZHBV_C

b

N-terminal protein (16.8%) Spacer (0.7%) Reverse transcriptase (27.1%) RNase H (15.3%)

100%

67%

33%

100 200 300 400 500 600 700 800 900

c d

preS (3.8%)

18

S (18.5%)

100%

67%

33%

100 200 300 400

e f

eZHBV_C

DHBV

CHBV

HHBV

ACATTCAAGAATTAT TCCTACTGTTAGAATATGTTCAGGAATTGC TATCAATGCTTCTAGATGTGCAGGAACTTC TGTTAACGCTTCAAGACATACAGGAATTGC TGTCAATGCTTCAAG

preC (0%) C (12.2%) 100%

67%

33%

100 200 300

eZHBV_C GGACTCCTTCAAAGT TGAAACAATTTGTAT

DHBV TTGTTCCAAGCAAAT TACCATTGTTTATGA

CHBV TTGTCCCCAGTAAAT TACCATTGTTTATGA

HHBV ATGTACCCAGTAAAT TACCATTGTTTATGA

Figure 3 | Genome organisation and sequence conservation of the Mesozoic hepatitis B paleovirus (eZHBV_C). (a) The HAS of the eZHBV_C insertion covers B99% of the circular 3,024-bp extant DHBV genome (AY494851), whereas the other seven dated EVEs are HBV genome fragments that range from 4.6% (eZHBV_O2) to 14.7% (eZHBV_E and eZHBV_M) coverage. For the DHBV genome, the start (arrow) and stop (asterisk) codon positions for the ORFs of the polymerase protein22 (pol; red), the presurface/surface protein22 (preS/S; blue), and the precore/core protein22 (preC/C; green) are shown, as well as the putative location of the structural vestige of an X protein21 (vX; orange). (bd) The pol (b), preS/S (c) and preC/C (d) proteins of the eZHBV_C HAS exhibit amino-acid positions (for full alignments, see Supplementary Data 2) that are both conserved among extant avian HBVs (blue graph) and among extant mammalian HBVs (orange graph). The proportions of perfectly conserved sites (that is, 100% conservation) within the respective regions of each ORF are denoted as italicized percentages and alignment gaps in the eZHBV_C sequence are shown as grey background. (e,f) The eZHBV_C HAS lacks an intact start codon for the structural prediction of a vestigial X protein21 (e) of DHBV and crane HBV (CHBV), as well as an intact ORF for the X-like protein22 (f) of heron HBV (HHBV). Intact start (ATG) and stop (TAA/TGA) codons are boxed.

In vitro screening. We conducted a comprehensive in vitro screening for our eight eZHBV insertion loci that is consistent with the procedures described by Suhet al.13 in their study of retroposon presence/absence patterns. Briey, eZHBV insertion loci were amplied via touchdown PCR, followed by purication and cloning of the PCR amplicons, and subsequent sequencing of the cloned fragments13. In the case of the large eZHBV_C insertion locus, a modied touchdown PCR protocol was used (5 min elongation time instead of the 80 s in the normal touchdown PCR protocol13), as well as various primers (Supplementary Table S4) for amplication and/or direct sequencing of the resultant PCR amplicons.

Sequence analyses. Owing to the large size of many eZHBV_C PCR amplicons(3.64.3 kb), we sequenced the full eZHBV_C locus sequence in four distantly related13,34 neoavians (that is, Nestor notabilis, Buteo lagopus, Larus ridibundus and Columba palumbus) and used these sequences for HAS reconstruction of eZHBV_C. The other complete neoavian sequences (that is, Taeniopygia guttata,

Cinnyris asiaticus, Alcedo atthis, Apus apus and Chrysolampis mosquitus) were not included due to the presence of large tandem duplications or large secondary deletions within the HBV-derived sequence.

We calculated pairwise distances of eZHBV HASs using MEGA5 (maximum composite likelihood, inclusion of transitions transversions, uniform rates

among sites, homogeneous patterns among lineages, complete deletion of alignment gaps). Dividing these by the mean time interval between the endogenization of the respective older and younger paleovirus, we estimated nucleotide substitution rates of the different prehistoric chapters of HBV evolution (Supplementary Table S3).

For further in-depth sequence analyses, amino-acid sequences of eZHBV paleoviruses were estimated after removal of frameshifts (that is, exclusion of frameshifting insertions, insertion of alignment gaps in frameshifting deletions) and internal stop codons (that is, replacement by X).

Sequence conservation graphs were compiled on the basis of amino-acid sequence alignments of pol, preS/S, preC/C ORFs (Supplementary Data 2) and by plotting extant HBV sequences (DHBV, CHBV, HHBV, HBV, WMHBV, WHV)

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a b

eZHBV_C

eZHBV_C

DHBV

CHBV

HHBV

DHBV

CHBV

HHBV

Avihepadnaviridae

69/0.82

95/1.00

95/1.00

94/1.00

70/0.68

100/1.00

70/0.68

100/1.00

eZHBV_O

eZHBV_E

eZHBV_O

eZHBV_E

eZHBV_M

Avihepadnaviridae

eZHBV_M

Quaternary

Pliocene

Miocene

Ancestral amniote codivergence

94/1.00

HBV

WMHBV

WHV

69/0.82

100/1.00

100/1.00

Orthohepadnaviridae

100/1.00

100/1.00

HBV

WMHBV

WHV

Orthohepadnaviridae

Birdmammal host switch

90

80

70

60

50

40

30

20

10

90

80

70

60

50

40

30

20

10

Upper

Cretaceous

Paleocene

Eocene

Oligocene

Quaternary

Pliocene

Miocene

Upper

Cretaceous

Paleocene

Eocene

Oligocene

Figure 4 | Phylogeny of hepatitis B viruses and two scenarios on the origin of mammalian HBVs. (a,b) Consensus tree (grey branches not to scale) of maximum likelihood and Bayesian phylogenetic analyses (Supplementary Fig. S2a,b; for full alignment, see Supplementary Data 3) of the polymerase protein from extinct eZHBV HASs and extant HBVs. Note that this tree topology is corroborated by our whole-genome nucleotide sequence analysis (Supplementary Fig. S2c). Rooting the tree to mammalian HBVs (a) places the origin of Orthohepadnaviridae before the endogenization of eZHBV_C and implies ancient codivergence of avian and mammalian HBVs. Alternatively, the same tree rooted to the Mesozoic eZHBV_C EVE (b) implies the emergence of Orthohepadnaviridae after a host switch from birds to mammals at some point after the endogenization of eZHBV_C. The representatives of Orthohepadnaviridae are extant HBVs from human (HBV), woolly monkey (WMHBV), and woodchuck (WHV), complemented by representatives of Avihepadnaviridae from duck (DHBV), crane (CHBV) and heron (HHBV). Rectangles are coloured according to the respective geological epoch in the International Stratigraphic Chart and denote the temporal ranges of insertion events. Nodes are labelled with maximum likelihood bootstrap values (in %) and Bayesian posterior probabilities (italicized).

to the Mesozoic eZHBV_C paleovirus. We coded sequence identities with the value 1, whereas non-identical alignment positions (including gaps) received a value of 0. Perfect (that is, 100%) conservation of an alignment position thus corresponds to a cumulative value of 6. Note that alignment gaps in the reference sequence (eZHBV_C) were treated as missing data and therefore marked with grey background in Fig. 3 (panels bd).

Finally, we performed phylogenetic sequence analyses of the pol protein of HBV EVEs and extant HBVs, as all but one (eZHBV_O1) of the eZHBV EVEs map to at least a part of this hepadnaviral ORF. To obviate problems arising from too short sequences of the four Oligocene EVEs overlapping with the pol ORF (eZHBV_O2-O5), we concatenated these into a chimaeric sequence, as we assume that these EVEs are likely to be closely related and of similar age. We compiled an alignment of complete and partial pol protein sequences (Supplementary Data 3) using MAFFT (E-INS-i, version 6) alignment and manual rechecking. Following model testing in MEGA5 that revealed WAG as most and JTT as second-most appropriate model, various phylogenetic analyses were performed using the CIPRES Science Gateway35 (http://www.phylo.org/portal2/

Web End =http://www.phylo.org/portal2/) and TOPALi36 (version 2.5). Irrespective of the model used (WAG or JTT), maximum likelihood (RAxML37) and Bayesian (MrBayes38) inferences of phylogeny constantly yielded the same topology (two examples are shown in Supplementary Fig. S2a,b). To evaluate the putative distant relationship of the youngest paleovirus (eZHBV_M) and the other eZHBV EVEs, we furthermore conducted a Bayesian sequence analysis (with GTR as best-t model) of aligned nucleotides of whole-genome sequences (extant HBVs and eZHBV_C) plus eZHBV genome fragments. The resultant tree (Supplementary Fig. S2c) exhibits the same tree topology, except for a basal polytomy involving eZHBV_M. Thus, eZHBV_M forms a distinct hepadnaviral lineage that, if it is indeed related to mammalian HBVs, implies a relatively recent birdmammal host switch.

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Acknowledgements

We thank Werner Beckmann (LWL-DNA- und Gewebearchiv), Hans-Heiner Bergmann, Sharon Birks (Burke Museum), Andrew Fidler, Franziska Anni Franke, Stephanie Hodges, Ommo Hppop, Harald Jockusch, Gerald Mayr (Senckenberg Museum), Simone Schehka (Allwetterzoo Mnster), Dirk Schneider, Sandra Silinski (Allwetterzoo Mnster), Timm Spretke (Zoo Halle), Mark Byung-Moon Suh, Michael Wink, and Anne-C. Zakrzewski for providing feather, blood and tissue samples. A.S. thanks Harald Hausen and Daniel Chourrout for hospitality and scientic discussions in the writing phase of this study during his guest reseach stay at the Sars International Centre for Marine Molecular Biology (Bergen, Norway). Regine Jahn helped with editing, Jn Baldur Hljberg provided the bird paintings. This research was funded by the Deutsche Forschungsgemeinschaft (KR3639 to J.O.K. and J.S.).

Author contributions

A.S. conceived the study, performed in silico and in vitro experiments, analysed the data and wrote the manuscript. J.O.K. and J.S. provided funding. J.B., J.O.K. andJ.S. contributed reagents, materials and analysis tools. J.B., J.O.K. and J.S. discussed and commented on the data and the manuscript.

Additional information

Accession codes: All nucleotide sequences generated in this study have been deposited in at DDBJ/EMBL/GenBank under the accession codes KC750086 to KC750148. To provide a resource for future hepadnaviral studies, we deposited a frameshift-free FASTA sequence of the eZHBV_C genome sequence as Supplementary Information (Supplementary Data 4).

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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Competing nancial interests: The authors declare no competing nancial interests.

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How to cite this article: Suh, A. et al. The genome of a Mesozoic paleovirus reveals the evolution of hepatitis B viruses. Nat. Commun. 4:1791 doi: 10.1038/ncomms2798 (2013).

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