Avihepadnavirus Diversity in Parrots Is Comparable to That Found Amongst All Other Avian Species

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Virology 438 (2013) 98–105

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Virology

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Avihepadnavirus diversity in parrots is comparable to that found amongst all other avian species

Tomasz Piasecki a, Gordon W. Harkins b, Klaudia Chrza˛stek a, Laurel Julian c, Darren P. Martin d, Arvind Varsani c,e,f,n

a Department of Epizootiology with Clinic of Birds and Exotic Animals, Faculty of Veterinary Medicine, Wroc!aw University of Environmental and Life Sciences, 50-360 Wroc!aw, Poland b South African National Bioinformatics Institute, University of the Western Cape, Cape Town, South Africa c School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand d Institute of Infectious Diseases and Molecular Medicine, Computational Biology Group, University of Cape Town, South Africa e Division of Medical Biochemistry, Department of Clinical Laboratory Sciences, University of Cape Town, Observatory, Western Cape 7700, South Africa f Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand

article info abstract

Article history: Avihepadnaviruses have previously been isolated from various species of duck, goose, stork, heron and Received 9 November 2012 crane. Recently the ﬁrst parrot avihepadnavirus was isolated from a Ring-necked Parakeet in Poland. In Returned to author for revisions this study, 41 psittacine liver samples archived in Poland over the last nine years were tested for 12 January 2013 presence of Parrot hepatitis B virus (PHBV). We cloned and sequenced PHBV isolates from 18 birds Accepted 17 January 2013 including a Crimson Rosella, an African grey parrot and sixteen Ring-necked Parakeets. PHBV isolates Available online 12 February 2013 display a degree of diversity (478% genome wide pairwise identity) that is comparable to that found Keywords: amongst all other avihepadnaviruses (479% genome wide pairwise identity). The PHBV viruses can be Avihepadnaviruses subdivided into seven genetically distinct groups (tentatively named A-G) of which the two isolated of Psittacines PHBV-G are the most divergent sharing 79% genome wide pairwise identity with all their PHBVs. All Hepatitis B virus PHBV isolates display classical avihepadnavirus genome architecture. Parrot hepatitis B virus & 2013 Elsevier Inc. All rights reserved.

Introduction a natural infection in mammals is unknown. Although not for in vitro replication (Blum et al., 1992) the X protein is apparently The family Hepadnaviridae contains enveloped DNA viruses with required for in vivo replication (Zoulim et al., 1994). While both partially double-stranded relaxed circular DNA (rcDNA) genomes of orthohepadnaviruses and avihepadnaviruses are hepatotropic and approximately 3 kb. These viruses have an unusual replication can cause transient or chronic liver infections, only orthohepadna- strategy involving reverse transcription of an RNA intermediate viruses are known to cause liver disease and cancer (Funk et al., (Seeger and Mason, 2000). There are two currently defined hepad- 2007). navirus genera: The genus Orthohepadnavirus, which contains As with orthohepadnaviruses, avihepadnaviruses have very species that infect mammals, and the genus Avihepadnavirus, which narrow host ranges. It is however, likely that host range switches contains species that infect birds. Although species in these two do occur in that the relatedness of viruses infecting different hosts genera share very little sequence similarity they have similar is not always reflective of the relationships of those hosts (Funk genome organisations, with species in both groups containing three et al., 2007). Host specificity is thought to be determined by the homologous open reading frames (ORFs), encoding surface proteins preS domain of the L-envelope protein (Ishikawa and Ganem, (preS/S), the nucleocapsid protein and the e-antigen (pre C/C), and 1995). Evidence of recombination amongst Duck hepatitis B virus the viral polymerase (P) (Seeger and Mason, 2000). A fourth ORF, (DHBV) isolates (Liu et al., 2010; Piasecki et al., 2012) suggests the encoding a protein called X, is found in all orthohepadnaviruses, possibility that strains with either expanded or larger host ranges but is apparently only present in some avihepadnaviruses (Chang might arise through recombinational transfers of this genome et al., 2001; Guo et al., 2005). The exact role of the X protein during region between distantly related viruses. To date avihepadnaviruses have been found infecting various species of duck (Duck hepatitis B virus, DHBV), geese (Snow goose n Corresponding author at: School of Biological Sciences, University of Canter- hepatitis B virus, SGHBV, and Ross goose hepatitis B virus, bury, Private Bag 4800, Christchurch 8140, New Zealand. Fax: þ64 3 364 2590. E-mail addresses: [email protected], RGHBV), cranes (Crane hepatitis B virus, CHBV), herons (Heron [email protected] (A. Varsani). hepatitis B virus, DHBV), and storks (Stork hepatitis B virus, SHBV;

(Chang et al., 1999; Guo et al., 2005; Prassolov et al., 2003; Pult avihepadnaviruses (i.e. excluding the divergent parrot infecting et al., 2001; Sprengel et al., 1988). Most recently an avihepadna- group) is less that 6000 years ago. In light of this, and given the virus was discovered for the ﬁrst time in a Ring-necked Parakeet diversity of the PHBV genomes that we have sequenced, we addi- (Psittacula krameri) in Poland (Piasecki et al., 2012). In the light of tionally decided to re-estimate the nucleotide substitution rates and the discovery of this Parrot hepatitis B virus (PHBV), we decided associated TMRCA’s using a larger sample of 77 avihepadnaviruses to test a library of archived parrot liver samples collected in including 18 new sequences of PHBV isolates sampled in Poland. Poland between 2003 and 2011 for similar avihepadnaviruses in order to determine the prevalence of this virus, its genomic diversity and the extent of its psittacine species host range. Results and discussion Endogenous hepadnaviruses have also been discovered in various avian species including ﬁnches, olive sunbird, dark-eyed junco Parrot avihepanavirus infections and disease pathology (Gilbert and Feschotte, 2010; Katzourakis and Gifford, 2010)and more recently in the budgerigar (Cui and Holmes, 2012; Liu et al., DNA was extracted from liver samples (n¼41) representing 12 2012). Analysis of the endogenous viruses indicates that viruses species (see Table 1 for details) from birds that had died from distantly related to extant avihepadnaviruses likely became inte- unknown diseases. Of the 41 samples screened, we found 18 to be grated into avian genomes at least as long ago as 19 million years PHBV positive. These comprised 15 samples from Ring-necked (Gilbert and Feschotte, 2010; Katzourakis and Gifford, 2010). Cui and Parakeets and one sample each from an African Grey Parrot Holmes, (2012) suggest that despite the fact that endogenous (Psittacus erithacus), an Alexandrine Parakeet (Psittacula eupatria) avihepadnaviruses have been inherited between certain bird species, and a Crimson Rosella (Platycercus elegans). We determined the the circulating exogenous avihepadnaviruses do not show obvious full genomic sequences of one cloned genome from each of these evidence of virus-host co-divergence and the fact that there has been 18 birds. some cross species transmission, will complicate attempts to accu- A review of the pathology of the PHBV positive birds revealed rately date the origin of the avihepadnaviruses based on the diver- that infection was mainly observed in young birds (2–5 weeks: gence times of their host species. Nonetheless, earlier estimates by n¼9; 2–5 months: n¼4; 6–9 months: n¼6) with a high incidence Zhou and Holmes (2007) suggest that, based on hepadnavirus amongst Ring-necked Parakeets (85% of analysed birds; pathology substitution rates of between 104–106 substitutions/site/year observations are summarised in Tables 1 and 2). Interestingly, the time to the most recent ancestor (TMRCA) of all then known these birds succumbed to sudden deaths without any physical

Table 1 Details of PHBV infected parrots, full genome accession number and speciﬁc pathology related to individual birds.

PHBV Lab GenBank Common Sampling Age of Liver spleen others PHBV APV BFDV strains ID accession # name date birds

PHBV-A 328 JN565944 Ring necked 16.03.2007 unknown none – – þþ parakeet PHBV-A 337 JX274026 Ring necked 29.03.2007 6 months Swollen marble-mottled Splenomegaly – þþ parakeet haemorrhage congestion PHBV-B 360 JX274025 Ring necked 18.05.2007 6 months Small hepatomegaly Splenomegaly – þ parakeet congestion PHBV-B 410 JX274024 African grey 22.08.2007 – – – – þþ parrot PHBV-C 755 JX274023 Ring necked 12.01.2009 o1 year – – – þ parakeet PHBV-D 830 JX274022 Alexandrine 26.03.2009 3–4 weeks Swollen, marble congestion Splenomegaly Subcutaneous þþþ parakeet petechiae, pale muscles PHBV-G 902 JX274018 Crimson 23.06.2009 6 months – – – þþ rosella PHBV-E 904 JX274021 Ring necked 23.06.2009 o1 year – – – þþ parakeet PHBV-F 1032 JX274020 Ring necked 12.03.2010 3 weeks Hepatomegaly marble Subcutaneous þþ parakeet -mottled haemorrhage petechiae, Pale congestion muscles PHBV-A 1035 JX274035 Ring necked 12.03.2010 2–4 weeks Swollen, pallor Muscle þþ parakeet petechiae PHBV-A 1036 JX274034 Ring necked 12.03.2010 2–4 weeks Swollen, pallor Muscle þþ parakeet petechiae PHBV-A 1037 JX274033 Ring necked 12.03.2010 2–4 weeks Swollen, pallor Muscle þ parakeet petechiae PHBV-A 1194 JX274032 Ring necked 4.03.2011 3–6 weeks Swollen, pallor Muscle þ parakeet petechiae PHBV-A 1195 JX274031 Ring necked 4.03.2011 3–6 weeks Swollen, pallor Muscle þ parakeet petechiae PHBV-A 1220 JX274030 Ring necked 7.09.2011 o 1 year Hepatomegaly marble Splenomegaly þþ parakeet - mottled haemorrhage PHBV-A 1232 JX274029 Ring necked 07.06.2011 3–5 weeks Hepatomegaly congestion Splenomegaly Muscle þþ parakeet petechiae PHBV-G 1233 JX274019 Ring necked 07.06.2011 3–5 weeks Hepatomegaly congestion Splenomegaly Muscle þþþ parakeet petechiae PHBV-A 1248 JX274028 Ring necked 09.09.2011 o 1 year Swollen þþ parakeet PHBV-A 1288 JX274027 Ring necked 24.11.2011 6 months Swollen marble-mottled Splenomegaly Marble Subcutaneous þþ parakeet haemorrhage Pallor appearance petechiae 100 T. Piasecki et al. / Virology 438 (2013) 98–105 symptoms. In summary, gross lesions were frequently observed in organisations to all other avihepadnaviruses, with three main the livers and spleens of these birds, while in some cases spleno- open reading frames encoding the PreC/C, PreS/S and poly- megaly and marble spleens were also noted. Muscles and subcuta- merase polyproteins. These genome sequences also have neous petechiae were observed more frequently than petechiae on many previously identiﬁed sequence motifs know to be essential the heart (Table 1). These symptoms were not observed in birds that for nucleic acid synthesis (such as the epsilon motif, the DR1 and were found to be negative for PHBV. However, it must be noted that DR2 motifs, TATA cis regulatory elements, transcription factor we are unable to verify that the observed pathology is linked binding sites; Fig. 1, Supplementary Fig. 1)(Mueller-Hill and directly to pathological PHBV infections. Loeb, 2002). Therefore the overall disease pathology resulting in psittacines Various domains within the predicted PHBV proteome such from PHBV infection is currently unknown and there is an urgent as the terminal domain, the reverse transcriptase domain need to address this gap in our knowledge. Nonetheless, we noted (RNA-dependent DNA polymerase), the RNase H domain, the that at least 13/18 of the PHBV infected birds were coinfected with putative myristylation sites, the host range determination either Avian polyomavirus (APV) or Beak and feather disease virus region, the gp120 binding site, the p120 binding region, the (BFDV; Table 1). BFDV is the main causative agent of Psittacine transmembrane domain, the conserved avian insertion domain, beak and feather disease (PBFD), and frequent coinfections invol- the hhr domain and a X-like domain were identiﬁed within the ving APV and PHBV, might indicate that these viruses together polyprotein, preS/S and the PreC/C ORFs of the PHBVs (Lilienbaum constiute an important hitherto unappreciated disease complex. et al., 1993; Liu et al., 1994; Zoulim and Seeger, 1994) (supplementary Fig. 2). As is the case with HHBV and STHBV, some of the PHBV isolates (labelled A, B, C, D and E in Figs. 2 and 3) have Avihepadnavirus genome organisation and conserved domains an X-like ORF. Similarly, PHBV-G has a homologous X-like element (as do DHBV, SHBV, CHBV and RGHBV) with a potential As with the PHBV isolate previously described, the 18 PHBV alternative start codon (supplementary Fig. 3). In the case of full genome sequences determined here have similar genome woodchuck hepatitis B virus (WHV) the X-like ORF, through in vivo experiments, has been shown to be required for the Table 2 establishment of chronic infections and could contribute to Summary of pathological conditions found to be potentially associated hepatocarcinogenesis (Chang et al., 2001). The balance of evi- with PHBV. dence, however, suggests that by itself this gene does not cause orthohepadnavirus related carcinogenesis (reviewed by (Seeger Gross lesion Percentage PHBV infected parrots (%) and Mason, 2000).

Swollen Liver 60.0 Maximum likelihood phylogenetic and pairwise identity analyses of Marble-mottled haemorrhage avihepadnavirus isolates Liver 33.3 Spleen 6.7 Haemorrhage A maximum likelihood (ML) phylogenetic tree of the full Myocardial 26.7 genomes of avihepadnavirus (Fig. 2) indicated that the 18 Subcutaneous 40.0 new PHBV isolates (Table 1; Genbank accession # JX274018– Muscle 20.0 Liver congestion 40.0 JX274035; supplementary Fig. 1) could be subdivided into two Hepatomegaly 33.3 major clades (Fig. 2); one with isolates sharing 495% pairwise Pallor identities (1p-distances with pairwise deletion of gaps; isolates Liver 33.3 labelled A–F in Figs. 2 and 3) with the PHBV isolate described by Muscle 20.0 Piasecki et al., (2012), whereas the second is more distantly related Splenomegaly 46.7 Hydropericardium 6.7 (79% pairwise identity) with the two isolates from Ring-necked Parakeet and Crimson Rosella sharing 99.4% pairwise identity

TATA box TATA box HNF3 C/EBP HNF1 TATA box Epsilon PAS

HNF2 DR2 DR1 hhr domain NLS Avian insertion domain

PreC/C Core protein

thr/pro kinase motif Terminal protein Spacer RNA-dependent DNA polymerase

Polymerase polyprotein p120 binding site gp120 binding site RNaseH PreS/S Host range S protein determinaton region Transmembrane domain HNF1:GTTAATGRTTAAACC HNF2:CAGTGTTTRCTTTY HNF3:GTGCAGRCAG (PHBV-A- F) /ATCGACGCCA (PHBV-G) Epsilon motif:GCCAAGGTATYTTTACGTCTTGCTTGCTGTTGYCTGCATTGRCTGTACCTTYGG PAS:AATAAA DR1 / DR2:TACACCCCTCTC C/EBP: RCATGGCGCAATAT

Fig. 1. Illustration of the genome organisation of PHBV isolates with various domains and motifs highlighted. T. Piasecki et al. / Virology 438 (2013) 98–105 101

(isolates P902 and P1233 labelled G in Figs. 2 and 3; Table 1; SGHBV [AF110998] GenBank accession # JX274018 and JX274019). 100 SGHBV [AF110997] SGHBV [AF110999] A comparative analysis of pairwise genetic distances of the full 93 SGHBV [AF110996] genomes of avihepadnaviruses revealed that the pairwise identity 74 SGHBV [AF111000] DHBV-K [M21953] of the two most distantly related PHBV isolates analysed here DHBV-J [AF404406] 100 (22%; based on genome wide pairwise distance calculated with 99 DHBV-I [AJ006350] pairwise deletion of gaps) is similar to that of the two most 88 DHBV-I [M32991] DHBV-A [X60213] 97 divergent non-PHBV avihepadnaviruses (i.e. DHBV, SGHBV, SHBV, DHBV-A [M32990] CHBV, RGHBV, HHBV and STHBV; 21%; Fig. 3). Similarly, pairwise 99 DHBV-A [AY521227] comparisons (2927 pairwise comparisons) of 77 avihepadnavirus DHBV-A [AY433937] 100 DHBV-A [AY521226] full genome sequences indicated that they all share 475% DHBV-A [HM043822] pairwise identities (Fig. 3). The diversity amongst the 39 DHBV DHBV 99 DHBV-B [HM590411] 100 DHBV-B [DQ276978] isolates (Fig. 3) is relatively low (p-distanceso10%), as is the case SGHBV 87 DHBV-B [HQ328779] for sparsely sampled isolates of CHBV (p-distanceso2%), SGHBV SHBV DHBV-B [AY536371] 100 (p-distanceso1%) and STHBV (p-distanceso1%). CHBV 99 DHBV-B [AY392760] DHBV-L [EU429325] RGHBV The distribution of pairwise identities clearly indicates troughs DHBV-C [X58568] at 80–81% and 96–95% which could easily be used for strain and STHBV 100 DHBV-C [X58569] genotype demarcations. This can easily be expanded beyond the HHBV DHBV-D [EU429324] PHBV 87 DHBV-E [AY294029] current criteria for species demarcation set by ICTV for avihepad- DHBV-E [AF047045] naviruses based on DHBV and HHBV isolates, which state that DHBV-E [AF505512] DHBV-E [AY294028] isolates with 421.6% pairwise distance should be classiﬁed as 99 DHBV-E [AY250904] new species. Based on the information available and the distribu- 99 81 DHBV-E [AY250903] 85 tion of pairwise identities (Fig. 3), we propose that avihepadna- DHBV-E [AY250902] 93 DHBV-E [AY250901] virus sequences with o80% pairwise identity should perhaps be DHBV-E [HQ214130] considered as new species. Finally, we can easily further classify 98 DHBV-E [AF493986] these sequences based on a 95% genome-wide sequence identity 99 DHBV-E [X74623] 100 DHBV-E [X12798] clustering rule, i.e. isolates sharing 495% pairwise identity 98 94 DHBV-E [M60677] belong to the variant grouping. For descriptive purposes we have DHBV-E [AY294656] DHBV-E [K01834] tentatively assigned the names A–G to the various PHBV variant 93 DHBV-E [EU429326] groupings and A–L to the DHBV variant groupings (Figs. 2–4, 100 DHBV-F [X58567] supplementary Figs. 1–3, Table 1). 100 DHBV-F [HQ132730] 99 DHBV-F [DQ195079] DHBV-G [AY494851] Polyprotein, PreC/C and PreS/S maximum likelihood 99 DHBV-H [AY494850] phylogenetic analyses 99 SHBV-A [AY494853] SHBV-B [AY494852] 96 CHBV [AJ441113] ML phylogenetic trees of the polymerase polyprotein, Prec/C CHBV [AJ441112] 100 CHBV [AJ441111] and Pres/S amino acid sequences show that while the overall tree 100 RGHBV-A [M95589] topology inferred from these different proteins is similar to that of RGHBV-A [AY494848] 100 the full genome trees, there are some noteworthy subtle differ- RHBV-B [AY494849] HHBV [M22056] ences (Fig. 4, supplementary Fig. 2). For example, the PreC/C and 100 STHBV [AJ251934] PreS/S proteins of SHBV isolates cluster in-amongst those of 100 STHBV [AJ251936] DHBV, whereas the polyproteins of the SHBV isolates cluster 73 STHBV [AJ251935] STHBV [AJ251937] basal to those of the DHBV and SGHBV isolates. Most of the other PHBV-A [JX274031] 73 PHBV-A [JX274032] differences between the trees are in the patterns of branching 84 PHBV-A [JX274033] within the DHBV (A–L) and PHBV (A–F) clusters (Fig. 4, Supple- 77 PHBV-A [JX274027] mentary Fig. 2). The PreC/C, PreS/S and polymerase polyprotein 100 PHBV-A [JN565944] sequences of the PHBV-A, -B, -C, -D, -E, -F isolates share 498.5% 100 PHBV-A [JX274026] PHBV-A [JX274028] amino acid similarity, which is similar to that shared by the two 99 PHBV-A [JX274035] PHBV-G isolates. However, when PHBV-A, -B, -C, -D, -E, -F are PHBV-A [JX274029] 96 collectively compared to PHBV-G, the PreC/C, PreS/S and poly- PHBV-A [JX274034] 61 PHBV-A [JX274030] merase polyproteins of these share only 82%, 65% and 70% PHBV-F [JX274020] similarity respectively. A comparative analysis of PHBV with all PHBV-D [JX274022] 100 91 PHBV-C [JX274023] other avihepadnaviruses indicates that they respectively share 90 PHBV-B [JX274025] o76%, 65% and 65% similarity in the PreC/C, PreS/S and poly- 100 PHBV-B [JX274024] merase polyproteins. The similarities in comparisons of proteins PHBV-E [JX274021] 100 PHBV-G [JX274018] 0.1 nucleotide substitutions per site of PHBV-A, -B, -C, -D, -E, -F and -G between themselves and with PHBV-G [JX274019] all other avihepadnaviruses is striking but not unexpected given that similar degrees of diversity were observed with their full Fig. 2. Maximum likelihood phylogenetic tree based on full genomes illustrating the evolutionary relationships between 77 avihepadnaviruses (rooted with ortho- genome nucleotide sequences. hepadnavirus isolate Hepatitis B virus subtype ayr, GenBank accession # X04615). Branch supports based on 1000 bootstrap replicates are provided next to each Recombination analysis branch.

Other than the recombination events reported by Piasecki et al. (2012) we found no new events of recombination amongst the world will assist in identifying new recombinants, recombi- the avihepadnaviruses. However, large scale screening and deter- nation hotspots and over-all recombination patterns that can be mination of PHBV genomes from infected parrots from all around compared to those of orthohepadnaviruses (Bollyky et al., 1996; 102 T. Piasecki et al. / Virology 438 (2013) 98–105

Proportion of pairwise identities (2927 pairwise comparisons) 100 HHBV [M22056] 0 0.10 0.18 0.20 0.02 0.04 0.06 0.08 0.12 0.14 STHBV [AJ251934] 0.16 Percentage nucleotide pairwise identities

STHBV [AJ251936] CHBV DHBV-A DHBV- B DHBV-C DHBV-D DHBV-E DHBV-F DHBV-G DHBV-H DHBV-I DHBV-J DHBV-K DHBV-L HHBV RGHBV-A RGHBV-B SHBV-A SHBV-B PHBV-A PHBV-B PHBV-C PHBV-D PHBV-E PHBV-F PHBV-G SGHBV STHBV RGHBV SHBV DHBV PHBV All non PHBV aviheapdnaviruses 100 STHBV [AJ251935] 1 11 11 1 1121 1 1111 5 4 2 2 99 STHBV [AJ251937] 95 PHBV-A [JX274031] 16 98 3 PHBV-A [JX274032] 97 6 2 2 PHBV-A [JX274033] 3 11 96 PHBV-A [JX274027] 5 95 PHBV-A [JN565944] 90 94 PHBV-A [JX274026] 93 3 PHBV-A [JX274028] 92 Variants PHBV-A [JX274035] 91 PHBV-A [JX274029] 90 85 40 PHBV-A [JX274034] 89 PHBV-A [JX274030] 2 88 PHBV-F [JX274020]

Percentage pairwise nucleotide identity 87 PHBV-D [JX274022] 86 PHBV-C [JX274023] 80 PHBV-B [JX274025] 85 PHBV-B [JX274024] 84 PHBV-E [JX274021] 83 PHBV-G [JX274018] 82 PHBV-G [JX274019] 81 75 ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] 80 Species 58 79 19 78 JX274019 JX274018 JX274031 JX274032 JX274033 JX274027 JX274026 JX274028 JX274035 JX274029 JX274034 JX274030 JX274020 JX274022 JX274023 JX274025 JX274024 JX274021 77 76 HHBV [M22056]

STHBV [AJ251934] STHBV [AJ251936] STHBV [AJ251935] STHBV [AJ251937] 75 PHBV-F [ PHBV-F PHBV-A [ PHBV-A [ PHBV-A [ PHBV-A [ PHBV-A [ PHBV-A [ PHBV-A [ PHBV-A [ PHBV-A [ PHBV-B [ PHBV-B [ PHBV-E PHBV-A [ PHBV-A [ PHBV-A PHBV-A [JN565944] [ PHBV-D [ PHBV-C PHBV-G [ PHBV-G PHBV-G [ PHBV-G

Fig. 3. (A) Two-dimensional graphical illustration of genome-wide percentage pairwise nucleotide sequence identities (calculated with pairwise deletion of gaps) of the PHBV isolates and those of the divergent HHBV and STHBV isolates. (B) Distribution of percentage pairwise identities of avihepadnaviruses (right) and comparison of diversity amongst various avihepadnaviruses species (left; number at the bottom of the bars denote number of isolates in each pairwise comparison).

Fallot et al., 2012; Liu et al., 2010; Piasecki et al., 2012; Simmonds groups with the DHBV clade rendering the RGHBV sequences para- and Midgley, 2005; Zhou and Holmes, 2007). Further, this will phyletic on the tree. The posterior probabilities of this split were, also assist in establishing the global presence and movement of however, poorly supported (posterior probability¼0.53). In contrast, these viruses amongst parrots, especially in the light of the ever the recombination-free C gene exponential relaxed-clock MCC expanding trade in these birds as pets. tree (not shown) the STHBV and the HHBV sequences form a monophyletic clade at the base of the tree that is reminiscent of the tree presented by Zhou and Holmes (2007). The statistical support Estimation of nucleotide substitution rates for this arrangement was however, extremely weak (posterior probability¼0.33). We attempted to infer the nucleotide substitution rates of The estimated mean substitution rates with 95% highest prob- avihepadnaviruses using our expanded dataset of full genome ability density (HPD) intervals for the full genome and C gene datasets sequences (all of which had known sampling dates) using the ranged between 1.01 104 (95% HPD, 4.22 1051.77 104) molecular clock analysis methods implemented in the computer and 9.38 105 (95% HPD, 4.19 1051.61 104)(supplemen- programme BEAST v1.7.3 (Drummond and Rambaut, 2007). We tary Table 1). These nucleotide substitution rates overlap with the tested a range of plausible demographic and molecular clock estimated mean rates of 7.32 104 and 4.85 104 subs/site/year models and attempted to identify which of these ﬁtted the data reported previously by Zhou and Holmes (2007) for the low- and high best using Bayes factor tests (Kass and Raftery, 1995). These tests recombinant regions of the genome respectively from 35 avihepad- indicated that for both the full genome dataset and a C gene naviruses sampled between 1981 and 2001. dataset containing no evidence of recombination, a relaxed-clock Under the constant population size, exponential growth and provided a better ﬁt to the data than a strict-clock model expansion models and a relaxed-clock, the estimated median irrespective of the demographic model employed (full genome time to the most recent common ancestor (TMRCA) of all the

BFlog 10 ¼70.13, capsid 12.89), but was equivocal between differ- analysed avihepadnaviruses ranged between 1485 (95% HPD 468- ent demographic models under the relaxed clock (full genome 3587) years (observed with the C gene dataset under an expo-

BFlog 10 ¼0.21). nential growth model) and 4753 (95% HPD 1935-8535) years For the sake of brevity, the maximum clade credibility (MCC) (observed with the full genome dataset under a constant popula- trees for the full genome and C gene datasets under a constant tion size model) (supplementary Table 1). population size relaxed-clock model are shown in supplementary It should be emphasised however, thatthedegreeofclock-like Figs. 4 and 5. The full genome trees had identical topologies but evolution evident in the data is low. Using a regression approach differed from the ML tree in Fig. 2 in that the rooting achieved whereby the root-to-tip genetic distances inferred from neighbor- with the MCC trees (supplementary Fig. 4) indicates reasonable joining phylogenetic trees are regressed against sampling times support (posterior probability0.83) for all the PHBV sequences using the programme PATH-O-GEN v1.3 (http://tree.bio.ed.ac.uk/ branching from the root of the tree (as is indicated in the preS/S software/pathogen/), indicated that there is no strong evidence for a tree of Fig. 4). signiﬁcant temporal signal in either the C gene (r2¼0.08916, For the recombination free C gene dataset the constant (supple- residual mean squared¼9.32 105)orfullgenome(r2¼5.1324 mentary Fig. 5) and expansion model relaxed-clock MCC trees had 103, residual mean squared¼2.41 105) datasets. Thus any identical topologies to their full genome counterparts with the attempt to date the origin of these isolated would be invalid with exception that sequence, RGHBV-B [AY494849_USA_2001], which the current available data. T. Piasecki et al. / Virology 438 (2013) 98–105 103

DHBV DHBV-E [M60677] DHBV-E [X12798] DHBV-E [K01834] DHBV-E [K01834] PrecC/C DHBV-E [M60677] PrecS/S DHBV-E [AF505512] Polyproteins SGHBV DHBV-E [AY294029] DHBV-E [HQ214130] DHBV-E [AY294029] DHBV-E [AY294028] DHBV-E [AY294028] 72 DHBV-E [HQ214130] SHBV DHBV-E [AY250902] DHBV-E [AY294029] DHBV-E [AY294656] 99 87 DHBV-E [X74623] DHBV-E [K01834] DHBV-E [AF493986] CHBV DHBV-E [X12798] DHBV-E [AY250903] DHBV-E [AF047045] 95 DHBV-E [HQ214130] DHBV-E [AY250901] 99 DHBV-E [X74623] RGHBV 63 81 DHBV-E [AY294656] DHBV-E [AF493986] 90 DHBV-E [X12798] STHBV 85 DHBV-E [AY250904] DHBV-E [X74623] DHBV-E [M60677] DHBV-E [AY250903] DHBV-E [AF505512] 99 DHBV-E [AY294028] HHBV DHBV-E [AF047045] DHBV-E [AY250904] 95 DHBV-E [AY250904] DHBV-E [AY250901] DHBV-E [AY294656] 81 DHBV-E [AY250903] PHBV 89 75 DHBV-E [AF505512] DHBV-E [AY250902] 86 DHBV-E [AY250901] DHBV-E [AF493986] DHBV-E [AF047045] DHBV-E [AY250902] DHBV-E [EU429326] DHBV-F [X58567] 89 DHBV-E [EU429326] DHBV-F [X58567] 92 DHBV-F [HQ132730] DHBV-D [EU429324] 73 DHBV-F [DQ195079] 95 89 DHBV-F [DQ195079] 100 DHBV-F [X58567] DHBV-F [HQ132730] DHBV-E [EU429326] DHBV-F [HQ132730] 96 SHBV-A [AY494853] DHBV-D [EU429324] 97 DHBV-F [DQ195079] SHBV-B [AY494852] 99 SHBV-A [AY494853] DHBV-A [X60213] 74 DHBV-A [X60213] SHBV-B [AY494852] DHBV-A [HM043822] DHBV-I [M32991] 87 DHBV-G [AY494851] 96 DHBV-A [AY521226] 99 DHBV-A [AY521227] 99 92 DHBV-H [AY494850] DHBV-A [AY521227] 96 DHBV-J [AF404406] 97 DHBV-C [X5869] DHBV-A [AY433937] 77 DHBV-A [AY433937] DHBV-C [X58568] DHBV-A [M32990] DHBV-A [AY521226] 85 DHBV-B [HQ328779] 77 DHBV-I [M32991] DHBV-A [HM043822] 72 DHBV-B [HM590411] 91 98 99 DHBV-I [AJ006350] DHBV-K [M21953] 75 DHBV-B [AY536371] DHBV-J [AF404406] DHBV-A [M32990] 95 DHBV-B [AY392760] DHBV-K [M21953] 91 86 DHBV-L [EU429325] 91 DHBV-B [DQ276978] 99 DHBV-C [X58569] 71 DHBV-B [HQ328779] 68 DHBV-L [EU429325] DHBV-C [X58568] 84 DHBV-B [HM590411] 76 DHBV-A [X60213] 97 DHBV-B [HM590411] 93 77 DHBV-B [DQ276978] DHBV-A [AY433937] 8890 DHBV-B [DQ276978] DHBV-D [EU429324] 83 DHBV-A [HM043822] DHBV-B [HQ328779] DHBV-I [AJ006350] DHBV-A [AY521227] 94 99 DHBV-B [AY536371] 75 97 DHBV-C [X58569] 95 DHBV-A [AY521226] 10073 DHBV-B [AY392760] 97 DHBV-C [X58568] 98 DHBV-A [M32990] DHBV-L [EU429325] 83 DHBV-B [AY392760] DHBV-I [M32991] SGHBV [AF111000] DHBV-B [AY536371] DHBV-I [AJ006350] 100 SGHBV [AF110999] SGHBV [AF111000] 94 DHBV-J [AF404406] 99 SGHBV [AF110996] 96 99 SGHBV [AF110996] DHBV-K [M21953] SGHBV [AF110998] SGHBV [AF110997] 77 SGHBV [AF111000] SGHBV [AF110997] SGHBV [AF110999] SGHBV [AF110996] DHBV-G [AY494851] SGHBV [AF110998] SGHBV [AF110999] 95 96 DHBV-H [AY494850] DHBV-G [AY494851] 94 SGHBV [AF110998] 99 SHBV-A [AY494853] 88 DHBV-H [AY494850] SGHBV [AF110997] SHBV-B [AY494852] 100 CHBV [AJ441111] 99 RGHBV-B [AY494849] 98 95 CHBV [AJ441113] 100 97 CHBV [AJ441112] RGHBV-A [AY494848] CHBV [AJ441112] 88 CHBV [AJ441113] 78 RGHBV-A [M95589] CHBV [AJ441111] 91 RGHBV-A [AY494848] 92 91 CHBV [AJ441111] 99 RGHBV-A [M95589] 90 RGHBV-A [M95589] CHBV [AJ441112] 100 RGHBV-A [AY494848] RGHBV-B [AY494849] 82 CHBV [AJ441113] RGHBV-B [AY494849] STHBV [AJ251934] STHBV [AJ251934] 76 STHBV [AJ251934] 99 75 STHBV [AJ251935] 98 STHBV [AJ251936] 99 STHBV [AJ251935] 99 STHBV [AJ251936] 100 STHBV [AJ251935] 100 STHBV [AJ251936] STHBV [AJ251937] STHBV [AJ251937] STHBV [AJ251937] HHBV [M22056] HHBV [M22056] HHBV [M22056] PHBV-A [JX274026] PHBV-C [JX274023] PHBV-D [JX274022] PHBV-A [JX274027] 77 PHBV-A [JX274026] PHBV-C [JX274023] 99 PHBV-A [JN565944] 69 PHBV-A [JX274029] 100 PHBV-B [JX274025] PHBV-A [JX274033] PHBV-A [JX274034] PHBV-B [JX274024] 79 PHBV-A [JX274032] PHBV-A [JX274032] PHBV-E [JX274021] 92 83 PHBV-A [JX274031] PHBV-A [JX274027] 66 PHBV-F [JX274020] PHBV-A [JX274029] 85 PHBV-A [JN565944] PHBV-A [JX274035] PHBV-A [JX274034] 86 PHBV-A [JX274035] PHBV-A [JX274031] PHBV-A [JX274035] 99 PHBV-A [JX274033] 100 PHBV-A [JX274027] 74 98 PHBV-A [JX274028] PHBV-A [JX274031] PHBV-A [JX274029] 90 PHBV-D [JX274022] PHBV-A [JX274028] 98 PHBV-A [JN565944] PHBV-B [JX274024] 75 PHBV-A [JX274030] PHBV-A [JX274028] PHBV-B [JX274025] 75 PHBV-E [JX274021] PHBV-A [JX274034] 77 PHBV-F [JX274020] 87 PHBV-D [JX274022] PHBV-A [JX274026] 99 99 79 PHBV-A [JX274030] PHBV-F [JX274020] 85 PHBV-A [JX274033] 99 PHBV-C [JX274023] PHBV-B [JX274024] PHBV-A [JX274032] PHBV-E [JX274021] 79 PHBV-B [JX274025] PHBV-A [JX274030] PHBV-G [JX274018] 100 PHBV-G [JX274018] PHBV-G [JX274018] 99 PHBV-G [JX274019] PHBV-G [JX274019] 100 PHBV-G [JX274019]

0.05 amino acid substitutions per site 0.1 amino acid substitutions per site 0.1 amino acid substitutions per site

Fig. 4. Maximum likelihood phylogenetic trees of the PreC/C, PreS/S and polyproteins of 77 avihepadnaviruses (rooted with orthohepadnavirus isolate GenBank accession # X04615). Branch supports based on aLRT are provided next to each branch.

Conclusions Material and methods

This study provides verification of the initial discovery DNA extraction, amplification, cloning and sequencing of parrot of PHBV infections in parrots. While the only cases of infection hepadnavirus isolates so far have been found in Poland, further testing of psittacine species in other countries will determine how widespread DNA from liver samples was extracted using commercially avail- PHBV infection is among these birds. Such testing will hopefully able Genomic Mini kit (A&A Biotechnology, Gdynia, Poland), accord- provide information on the potential global distribution of these ing to the manufacturer’s instructions. Total DNA was extracted from viruses, and facilitate the detection of any new recombinants samples and subjected to rolling-circle amplification (RCA) using amongst them. Perhaps testing for HBV should be routinely TempliPhiTM (GE Healthcare, USA), to non-specifically amplify any carried out in aviaries, especially when psittacine birds die circular DNA (such as circular viral genomes) as described in Piasecki unexpectedly and where the presence of BFDV or APV infections et al. (2012). The RCA concatemers were digested with BamHI, HindIII are also suspected or detected. Although we have attempted to and XmnI restriction enzymes. The resulting fragments ranging in size estimate the TMRCAs of all the currently sampled avihepadna- from 1–3 kb were cloned into plasmid vectors (BamHI and HindIII virus full genomes, the analysis of more avihepadnavirus restricted pGEM3ZF and pJET1.2 for XmnI restricted products) and sequence data, particularly from archived samples, will be sequenced. Based on the resulting sequence information we designed required to obtain credible estimates of the substitution rates of back-to-back primers to amplify full PHBV genomes. One micro litre these viruses. Without such data it will not be possible to of the RCA product was used for PCR amplification using primers properly reconcile the apparently high evolution rates of avihe- PHBV-F 50 - GGAYTTCTCTCAGTTYTCCAAAGG - 30 and PHBV-R 50 - padnaviruses with the fact that close relatives of these viruses ACCACRARTCTAGCYTCCYCAGT - 30 with Kapa HiFi HotStart DNA likely became integrated into bird genomes at least as long ago as polymerase (Kapa Biosystems, USA) using the following protocol: 19 million years. initial denaturation at 94 1C for 2 min, 25 cycles of 98 1Cfor20s, 104 T. Piasecki et al. / Virology 438 (2013) 98–105

52 1C for 30 s, 72 1C for 3 min, followed by a ﬁnal extension at 72 1C was supported by the Polish Ministry of Education and Science for 5 min and a ﬁnal renaturation step at 4 1Cfor10min.The (grant no. N308 036 32/3369). amplicons were resolved on a 0.7% agarose gel, 3 kb bands excised, cleaned with Mega-spin agarose gel extraction kit (Intron, Korea) and cloned into pJET1.2 vector (Fermentas, USA). The plasmid isolated Appendix A. Supporting information from a single E. coli colony was sequenced by Macrogen Inc (Korea) by primer walking. Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2013.01.009. DNA sequence manipulation and dataset preparations

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