1 Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, PO Box 201652, 20206 Hamburg, Germany
2 Florida Keys Wild Bird Centre, Tavernier, FL 33070, USA
3 Institute for Zoo and Wildlife Research, Alfred-Kowalke-Str. 17, 10315 Berlin, Germany
4 School of Medicine, University of North Carolina, Department of Microbiology and Immunology, Chapel Hill, NC 27599-7290, USA
Correspondence
Hüseyin Sirma
sirma{at}hpi.uni-hamburg.de
Falko Steinbach
steinbach{at}izw-berlin.de
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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Hepadnaviruses are generally known for their rather narrow host range. For instance, human HBV infects only human, chimpanzee (Pan troglodytes), chacma baboon (Papio ursinus orientalis) and, to some extent, tree shrew (Tupaia belangeri) hepatocytes (Yan et al., 1996; Guidotti et al., 1999
; Kedda et al., 2000
; Takahashi et al., 2000
). Woodchuck hepatitis B virus (WHV), another example of an orthohepadnavirus, has only been detected in Eastern woodchucks (Marmota monax) and does not infect the related alpine marmosets (Marmota marmota) (Tyler et al., 1981
; Chomel et al., 1984
). Ground squirrel hepatitis B virus (GSHV) infects only some species of squirrel (Spermophilus beecheyi and Spermophilus richardsonii) and the phylogenetically closely related chipmunks (Eutamias spp.) (Trueba et al., 1985
). A closely related virus (arctic squirrel hepatitis virus; ASHV) was identified in arctic ground squirrels (Testut et al., 1996
), but its host range has not been studied so far.
Avihepadnaviruses have been detected in various duck species [Anas spp.; duck hepatitis B virus (DHBV); reviewed by Schödel et al., 1991; Triyatni et al., 2001
], snow geese [Anser caerulescens; snow goose hepatitis B virus (SGHBV); Chang et al., 1999
], a Ross' goose [Anser rossii; Ross' goose hepatitis B virus (RGHBV); GenBank accession no. M95589], white storks [Ciconia ciconia; stork hepatitis B virus (STHBV); Pult et al., 2001
], demoiselle and grey crowned cranes [Anthropoides virgo and Balearica regulorum, respectively; crane hepatitis B virus (CHBV); Prassolov et al., 2003
], and grey herons [Ardea cinerea; heron hepatitis B virus (HHBV); Sprengel et al., 1988
; Netter et al., 1997
]. Like their mammalian counterparts, avihepadnaviruses have a rather narrow host range. For instance, DHBV infects only certain duck and goose species, but does not infect Muscovy ducks or chickens (Schödel et al., 1991
; Sprengel et al., 1991
; Pugh & Simmons, 1994
). Little is known about the host range of HHBV or STHBV. Despite its substantial sequence similarity to DHBV, HHBV does not infect ducks and only infects primary duck hepatocytes very inefficiently (Sprengel et al., 1988
). Recently, it has been reported that cranes are naturally infected with a hepadnavirus, designated CHBV (Prassolov et al., 2003
). Cranes are phylogenetically very distant from ducks and are more closely related to herons and storks. Interestingly, however, CHBV infects primary duck hepatocytes with an efficiency similar to that of DHBV. Collectively, these and related data suggest that the host range of hepadnaviruses cannot be simply predicted based on the evolutionary relatedness of their respective hosts.
Naturally occurring DHBV infections have been reported in Pekin ducks (Anas domesticus) and related species from China, USA, Canada, Europe, India and South Africa (Schödel et al., 1991; Munshi et al., 1994
; Triyatni et al., 2001
; Mangisa et al., 2004
). Phylogenetic analysis of the various isolates demonstrated a rather high variability among DHBV strains, whereas genomes from other avihepadnaviruses identified so far, such as STHBV and CHBV, appear less variable (Sprengel et al., 1988
; Netter et al., 1997
; Triyatni et al., 2001
; Pult et al., 2001
; Prassolov et al., 2003
).
Species specificity of hepadnaviruses seems to be, at least to some extent, determined at the level of virus entry, involving the preS part of the large envelope protein L. Substitution of the N-terminal region (aa 2290) of the HHBV-specific preS domain with the corresponding sequence from DHBV renders HHBV infectious for ducks and is therefore sufficient to overcome the species barrier of HHBV in primary duck hepatocytes (Ishikawa & Ganem, 1995). Accordingly, comparative genomic and subgenomic sequence alignment from different avihepadnaviruses facilitates prediction of the specific properties of each virus and helps to gain insight into the mechanisms controlling species specificity and host adaptation.
DNA sequences from full-length genomes of the few HHBV isolates reported diverge from those of DHBV by >20 %. The preS region of the HHBV L protein is hypervariable. It has the lowest sequence similarity to all hepadnaviral proteins, diverging by about 50 % from the DHBV protein (Sprengel et al., 1988, 1991
; Netter et al., 1997
). However, the biological significance of this divergence is not yet clear. This is partly because only a few cloned HHBV full-length genome sequences are available, which have all been derived from captive herons in German zoos. Based on the available data, it is not clear whether HHBV infection occurs at all in free-living herons, as cross-species virus transmission in zoos cannot be excluded as a possible source of these reported infections.
Here, it is shown that HHBV infection occurs not only in captive grey herons, but also with high prevalence in free-living birds. HHBV has been detected in another heron species, the great blue heron (Ardea herodias), as well as in two of its subspecies (great white heron and Würdemann's heron). Thus, the data shown here demonstrate for the first time the occurrence of this virus in free-living birds and show that HHBV is an endogenous virus of several heron species.
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METHODS |
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PCR amplification and sequencing of viral DNA from serum.
Sera were screened for viral genomes without prior DNA extraction. One microlitre of native serum was diluted in 199 µl lysis buffer [50 mM KCl; 0·45 % (v/v) Tween; 0·5 % (v/v) NP-40; 10 mM Tris/HCl (pH 8·3)] and subjected to proteinase K (Roche) digestion at 56 °C for 2 h. Thereafter, proteinase K was inactivated by heating at 95 °C for 10 min. For amplification of the complete HHBV genome, 5 µl digested sample was subjected to PCR using HHBV-4-specific full-length primers, which anneal to the so-called nick region of the viral DNA. Primer F1 aligns to nt 25392561 (5'-GAAGATCTGCTCTTCATTACACCCCTCTCCATTCGGAGC-3') and the reverse primer R1 to nt 25192541 (5'-GAAGATCTGCTCTTCTAATCTTAGAGACCACATAGCCT-3'). PCR was carried out by using the Expand High Fidelity PCR system (Roche) as described previously (Netter et al., 1997; Prassolov et al., 2003
). Amplified PCR products were analysed on an ethidium bromide-stained 1 % agarose gel, purified by using a QIAquick Gel Extraction kit (Qiagen) according to the manufacturer's instructions and sequenced directly.
The subgenomic sequence containing the preS/S ORF was amplified by using purified full-length genome as template and primers F2 annealing to nt 772789 (5'-GCGGCGGCCGCGCACCTTGTCCCGCAACA-3') and R2 annealing to nt 18011821 (5'-GCGGCCGCGTCATTTTGTCAAAGTTGATC-3') of HHBV 4, containing a heterologous restriction site for NotI. PCR products were digested with NotI and subcloned into the vector pcDNA3 (Invitrogen). Sequencing of cloned pcDNA3-HHBV-preS/S was carried out by using an ABI BigDye Terminator Cycle Sequencing kit. Sequencing PCR was performed by using primers T7 (5'-TAATACGACTCACTATAGGG-3') and SP6 (5'-ATTTAGGTGACACTATAGAA-3'). Sequence analysis was performed by using a 3100 Genetic Analyser (ABI) and MACVECTOR software.
To calculate sequence divergence values among the different viruses, the software MEGA 2.1 (Kumar et al., 2001) was used. Phylogenetic relationships and genetic distances of the virus genomes were estimated by using neighbour-joining (NJ) algorithms implemented by MEGA. Bootstrap analyses were based on 1000 replications. The significance of the branch lengths in the NJ tree was examined by a standard error test using the confidence probability. The phylogenetic network was constructed by using the median-joining (MJ) algorithm of NETWORK 4.0 (Bandelt et al., 1999
). MJ networks include all most-parsimonious trees supported by the data and are particularly appropriate for the detailed resolution encountered in closely related intra- or interspecific datasets.
SDS-PAGE and immunoblotting.
Immunoblot analysis was performed for detection of viral L antigen in serum samples by using antisera, as described previously (Pult et al., 2001; Prassolov et al., 2003
). An aliquot of each serum sample (1 µl) was diluted in 49 µl PBS (pH 7). Samples were denatured in 50 µl 2x Laemmli buffer and boiled at 99 °C for 5 min. Each sample (20 µl) was separated by 15 % denaturing SDS-PAGE and the proteins were transferred to nitrocellulose membranes. The membranes were then blocked for 1 h at room temperature in 5 % dry milk dissolved in TBST (Tris-buffered saline, 50 mM Tris/HCl, 150 mM NaCl, 0·1 % Tween 20) and incubated overnight at 4 °C with an HHBV preS-specific rabbit antiserum at a 1 : 2000 dilution. After several washings with TBST, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (Dianova) at a dilution of 1 : 20 000. Proteins were visualized by chemiluminescence (SuperSignal West Dura; Pierce).
Electron microscopic analysis of virus particles.
Electron microscopy was performed to obtain ultrastructural evidence for hepadnavirus particles in the respective bird sera. The great blue heron serum analysed was positive, as assayed by immunoblotting and PCR, whereas the control serum was negative in both assays. Heron sera were incubated with micro-carriers pre-coated with HHBV preS antiserum. After several washings, the carriers were pelleted by low-speed centrifugation, washed, resuspended in PBS and subsequently transferred into capillary tubes, as described previously (Hohenberg et al., 1994; Pult et al., 2001
). For electron microscopic analysis, samples were fixed with 2·5 % glutaraldehyde in PBS for 1 h at room temperature, washed and post-fixed for 30 min with 1 % OsO4 in PBS. For ultrathin sectioning, the samples were gradually dehydrated with ethanol and embedded in ERL resin. Ultrathin sections were counterstained with 2 % uranyl acetate and lead citrate. All electron micrographs were obtained with a Philips CM120 transmission electron microscope at 60 kV.
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RESULTS |
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DISCUSSION |
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HHBV is clearly distinct from avihepadnaviruses isolated from snow geese (SGHBV), Ross' goose (RGHBV), white storks (STHBV), cranes (CHBV) and ducks (DHBV). The host range-determining sequence motif at the N-terminal end of the L protein of all HHBV isolates is very similar. As the WTP motif in the DHBV preS region, which was shown previously to be critical for hepatocyte infection (Sunyach et al., 1999), is changed to WTE in all of the HHBV isolates (Netter et al., 1997
; this study), it seems likely that HHBV infects only heron and possibly closely related species. In our study, however, naturally occurring HHBV or related viruses were not detected in egrets of the same geographical area, suggesting species barriers to infection. Alternatively, this may be due to the low number of samples available and further analysis may be required.
DHBV isolates cluster in at least two groups (Asian isolates, including Australia, versus isolates from the USA and Europe). The majority of DHBV strains from Asia were isolated from closely related duck species and are less conserved than the few USA/European strains isolated from distinct species. Genomes from HHBV, SGHBV, STHBV and CHBV were conserved and not divided into subclusters (Triyatni et al., 2001; Prassolov et al., 2003
). Accordingly, the higher diversity of DHBV strains was considered to be unusual in avihepadnaviral evolution, but data regarding other avian HBV-like viruses were limited and restricted the extent of comparative analysis. All isolates of HHBV analysed thus far were from captive grey herons from northern Germany (Sprengel et al., 1988
; Netter et al., 1997
). Likewise, STHBV was isolated and sequenced from captive white storks from Germany (Pult et al., 2001
) and the isolates from snow geese were all collected from one zoo in Germany (Chang et al., 1999
). Interestingly, the CHBV isolates were obtained from two different species (Anthropoides virgo vs Balearica regulorum) and subfamilies (Gruinae vs Balearicinae), but again were all derived from German zoos. Therefore, lateral transmission of CHBV in captivity cannot be excluded so far.
As hepadnaviruses replicate via an RNA intermediate and error-prone reverse transcription, the low variability in most avihepadnaviral genomes was unexpected. The highly conserved nature of HHBV, even among viruses isolated from different heron species (Fig. 4), suggests that host specificity and/or geographical barriers have a strong impact on the evolution of new viruses. Phylogenetic analysis of the HHBV L gene demonstrates that the HHBV strains from grey (Germany, Europe) and blue (Florida, USA) herons form separate clusters, suggesting that regional aspects may also be important for understanding the evolution of HHBV (Fig. 5
). Thus, our study adds important information and a new perspective on avihepadnaviral divergence, spreading and evolution. In nature, however, DHBV spread occurs by vertical transmission; horizontal transmission of avian hepadnaviruses has not been demonstrated conclusively so far.
Our data demonstrate that the previously isolated HHBV strains from captive grey herons (from German zoos) (Netter et al., 1997) are related closely to the HHBV strain infecting the free-ranging birds from North America. Phylogenetic analysis of all HHBV isolates (Fig. 6a
) shows clearly that the North American and European isolates of HHBV are related sister clades that share a common ancestor. The close sequence identity (the DNA sequences coding for the L protein of HHBV 4 and the new isolate HHBV 43 are approx. 95 % identical), the ancestral position of the US isolates (Fig. 6a
) and the high rate of infection found in herons from Florida suggest that the captive European grey herons were infected by natural, horizontal or iatrogenic transmission with an ancestral virus of these US heron species that has evolved since by continued transmissions in the zoo setting. Taking into account the short history of zoological gardens and the animal trade, it is assumed that formation of the two clusters is less than 150 years old. At present, it cannot be formally excluded that HHBV 4 or closely related strains are prevalent in European wild grey herons and that these strains may resemble intermediate strains in the evolution of HHBV. To clarify this possibility, analysis of viruses isolated from wild herons from Europe must be performed to see whether these animals are an alternative source of infection for zoo animals.
Although the grey and great blue heron evolved a long time ago and today exist allopatrically as geographically isolated populations, some ornithologists consider them as conspecific or allied together as a superspecies. Such a close relationship would be compatible with the high sequence identity of the viruses from the two sister clades. From this point of view, an alternative explanation arises in that the HHBV genome sequences may be constrained and do not change as readily as DHBV in domesticated ducks. The two lineages of HHBV would then still have a common ancestor, but the node would have to date back more than 100 000 years before the last ice age, which seems rather unlikely according to our current knowledge of the virus life cycle and evolution dynamics.
In conclusion, the existence of HHBV in a free-living heron species has been shown, thereby expanding the current knowledge of naturally occurring avihepadnavirus infections. The present data argue against a monophyletic origin of avihepadnaviruses, as well as strict co-evolution of the viruses and their hosts. To determine the importance of the hostspecies interaction, geographical restriction and adaptation in avihepadnaviral evolution, further studies on closely related heron species or other Ardea species in different geographical regions need to be performed.
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ACKNOWLEDGEMENTS |
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Received 26 November 2004;
accepted 18 January 2005.
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