Department of Virology, Biomedical Primate Research Centre (BPRC), PO Box 3306, 2280 GH Rijswijk, The Netherlands1
Division of Veterinary and Biomedical Sciences, Murdoch University, Western Australia2
Wanariset Orang-utan Reintroduction Centre, East Kalimantan, Indonesia3
Department of Animal Quarantine, East Kalimantan, Indonesia4
Author for correspondence: Ernst Verschoor. Fax +31 15 284 3986. e-mail verschoor{at}bprc.nl
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Abstract |
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Currently identified nonhuman primate hepadnaviruses are highly similar to the human HBVs in genome organization, and may cause acute or chronic hepatitis in their natural hosts (Lanford et al., 1998 ), or when passaged to other species (Norder et al., 1996
). Previously, we described hepadnaviruses that occur naturally in Bornean orang-utans (Pongo pygmaeus pygmaeus), now designated orang-utan hepadnavirus (OuHV), by sequence analysis of the S gene (Warren et al., 1998
, 1999
). Here we describe the molecular and evolutionary analysis of full-length genomes of two variants of OuHV present in the Bornean orang-utan population.
Phylogenetic analysis of the small S gene of OuHV isolates from seven chronic carriers suggested that OuHV could be classified in two genomic groups (Warren et al., 1999 ). Viruses obtained from the orang-utans Doel, Oon, Papa and Mojo, and those infecting Romeo, Somad and Lisa, were found on two different branches in the OuHV cluster. Both branches were supported with high bootstrap values of 84 and 100%, respectively.
The division of HBV into genomic groups (A to F) has been based on the definition that the difference between the complete genomes of viruses from different genotypes must be 8% or more (Okamoto et al., 1988 ). To solve the question whether Bornean orang-utans are infected by two distinct genotypes of OuHV or merely by different genomic variants, we determined the sequence of the complete genome of two representative viruses, one from each OuHV subcluster (Somad and Papa).
The isolation of viral genomic DNA, PCR amplification of subgenomic fragments and sequence analysis were performed essentially as described previously (Warren et al., 1999 ). The primers used for PCR and sequence analysis of the OuHV genome and their positions on the chimpanzee HBV genome are given in Table 1
. Sequencing of the cloned insert was performed using pUC/M13 forward and reverse sequencing primers, which bind on either side of the cloned insert, and with HBV-specific oligonucleotides. From each amplification reaction three clones were analysed.
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The Bornean orang-utan population consists of several geographically isolated and genetically different subpopulations (Groves et al., 1992 ; Rijksen & Meijaard, 1999
; Warren et al., 2001
; Zhi et al., 1996
). Co-evolution of OuHV with their hosts may have caused their divergence into at least two genomic variants. To test this hypothesis we examined the geographical distribution of both variants in the Indonesian part of Borneo (Kalimantan) by using a combined PCRrestriction fragment length polymorphism (PCRRFLP) assay to discriminate between infections with the genomic types (Fig. 1
). Sera from 25 viraemic orang-utans were screened. The majority of OuHV infections (n=19) could be classified as belonging to the Somad cluster, while the remaining infections were caused by viruses closely related to the Papa isolate. All OuHV-positive animals from East Kalimantan (n=12) were viruses closely related to Somad, while the remaining animals from other geographical origins (n=13) showed an almost equal distribution over both clusters (7/6). These findings suggest a correlation with geographical origin or subpopulation of the host and the virus variants. Such a correlation has also been described by Grethe et al. (2000)
for GiHBVs originating from Vietnam and Thailand. However, too few OuHV isolates from the various subpopulations have been examined to draw more definitive conclusions. Identification of additional viraemic orang-utans of known origin, together with development of a serological test capable of distinguishing between OuHV types, may be of assistance in further defining this issue in the future.
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Although the number of published genomes of ape hepadnaviruses is limited, certain conclusions may be drawn from our analysis. A distribution based on origin and species can be observed, consisting of the Asian and African ape hepadnaviruses. The former consist of two species-specific subclusters (orang-utan and gibbon viruses), both supported by high bootstrap values. The genomes of OuHV and GiHBV each differ by 8% or more from all other hepadnaviruses (data not shown); thus, based on the definition set by Okamoto et al. (1988) , OuHV and GiHBV form separate genotypes. The viruses isolated from gorilla and chimpanzees form one African genotype (bootstrap support 100%) comprising the African cluster (93 to 99% sequence similarity). Interestingly, the single GoHBV sequence falls within the chimpanzee viruses and, unlike OuHV and GiHBV, does not form a separate genotype. Accumulation of more gorilla and chimpanzee virus sequences is necessary to further investigate the true relationship between GoHBV and ChHBV.
The ape viruses are closely related to the human HBV genotypes A to E (92% bootstrap support), yet they belong to different subclades which branch off from a single branch. This is indicative of a common ancestor virus from which the ape hepadnaviruses and the HBV genotypes A to E (the Old World HBV) have evolved, as has also been suggested by others (Grethe et al., 2000 ; Hu et al., 2000
; MacDonald et al., 2000
; Takahashi et al., 2000
). A more recent cross-species transmission between humans and apes as proposed by Lanford et al. (1998
, 2000
) seems highly unlikely as this would have resulted in a mosaic branching pattern with human and ape viruses intermingled on the tree. In addition, the finding of closely related hepadnaviruses among ape species of Africa and Asia, as well as the cluster of species-specific hepadnaviruses within the Asian continent, strongly suggests an ancient hepadnavirus infection of the apes that may have been spread by multiple cross-species transmissions amongst these species. The evolution of different viral variants within one host species as described here for OuHV, and by Grethe et al. 2000
for GiHBV, also implies a much older infection event rather than a recent zoonotic event.
A question which remains is the relation of the HBV genotype F viruses and the virus carried by the South American woolly monkey with the other hepadnaviruses (Fig. 2). Several authors suggest that the F genotype viruses and the woolly monkey virus represent even older viruses (Arauz-Ruiz et al., 1997
; Lanford et al., 1998
; Norder et al., 1994
, 1996
). A more exhaustive search for hepadnavirus infections in nonhuman primates from all continents will be necessary to determine whether other primates harbour hepadnaviruses and at what prevalence, and how they differ from the ones already published. Only then it may be possible to test or verify our hypothesis and those put forward by others concerning the origin of primate hepadnaviruses and their evolutionary relationships.
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Acknowledgments |
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E.J.V. and K.S.W. contributed equally to the data described in this article.
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References |
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---|
Felsenstein, J. (1995). PHYLIP: Phylogeny Inference Package (version 3.572). Distributed by the author. University of Washington, Seattle, WA, USA.
Grethe, S., Heckel, J.-O., Rietschel, W. & Hufert, F. T. (2000). Molecular epidemiology of hepatitis B virus variants in non-human primates. Journal of Virology 74, 5377-5381.
Groves, C. P., Westwood, C. & Shea, B. T. (1992). Unfinished business: mahalanobis and a clockwork orang. Journal of Human Evolution 22, 327-340.
Hu, X., Margolis, H. S., Purcell, R. H., Ebert, J. & Robertson, B. H. (2000). Identification of hepatitis B virus indigenous to chimpanzees. Proceedings of the National Academy of Sciences, USA 97, 1661-1664.
Lanford, R. E., Chavez, D., Brasky, K. M., Burns, R. B. & Rico-Hesse, R. (1998). Isolation of a hepadnavirus from the woolly monkey, a New World primate. Proceedings of the National Academy of Sciences, USA 95, 5757-5761.
Lanford, R. E., Chavez, D., Rico-Hesse, R. & Mootnick, A. (2000). Hepadnavirus infection in captive gibbons. Journal of Virology 74, 2955-2959.
MacDonald, D. M., Holmes, E. C., Lewis, J. C. & Simmonds, P. (2000). Detection of hepatitis B virus infection in wild-born chimpanzees (Pan troglodytes verus): phylogenetic relationships with human and other primate genotypes. Journal of Virology 74, 4253-4257.
Marion, P. L., Oshiro, L. S., Regnery, D. C., Scullard, G. H. & Robinson, W. S. (1980). A virus in Beechey ground squirrels that is related to hepatitis B virus of humans. Proceedings of the National Academy of Sciences, USA 77, 2941-2945.[Abstract]
Norder, H., Courouce, A. M. & Magnius, L. O. (1994). Complete genomes, phylogenetic relatedness, and structural proteins of six strains of the hepatitis B virus, four of which represent two new genotypes. Virology 198, 489-503.[Medline]
Norder, H., Ebert, J. W., Fields, H. A., Mushahwar, I. K. & Magnius, L. O. (1996). Complete sequencing of a gibbon hepatitis B virus genome reveals a unique genotype distantly related to the chimpanzee hepatitis B virus. Virology 218, 214-223.[Medline]
Okamoto, H., Tsuda, F., Sakugawa, H., Sastrosoewignjo, R. I., Imai, M., Miyakawa, Y. & Mayumi, M. (1988). Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. Journal of General Virology 69, 2575-2583.[Abstract]
Perrière, G. & Gouy, M. (1996). WWW-Query: an on-line retrieval system for biological sequence banks. Biochemie 78, 364-369.[Medline]
Rambaut, A. (1995). SE-AL sequence alignment program V1.D1. Dept of Zoology, University of Oxford, Oxford, UK.
Rijksen, H. D. & Meijaard, E. (1999). Our Vanishing Relative. The Status of Wild Orang-utans at the Close of the Twentieth Century. Dordrecht: Kluwer Academic Publishers.
Summers, J., Smolec, J. M. & Snyder, R. (1978). A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proceedings of the National Academy of Sciences, USA 75, 4533-4537.[Abstract]
Swofford, D. L. (1999). PAUP*: Phylogenetic Analysis Using Parsimony (*and other methods), version 4.0b4, 4.0 edn. Sunderland, USA: Sinauer Associates.
Takahashi, K., Brotman, B., Usuda, S., Mishiro, S. & Prince, A. M. (2000). Full-genome sequence analyses of hepatitis B virus (HBV) strains recovered from chimpanzees infected in the wild: implications for an origin of HBV. Virology 267, 58-64.[Medline]
Vaudin, M., Wolstenholme, A. J., Tsiquaye, K. N., Zuckerman, A. J. & Harrison, T. J. (1988). The complete nucleotide sequence of the genome of a hepatitis B virus isolated from a naturally infected chimpanzee. Journal of General Virology 69, 1383-1389.[Abstract]
Warren, K. S., Niphuis, H., Heriyanto, Verschoor, E. J., Swan, R. A. & Heeney, J. L. (1998). Seroprevalence of specific viral infections in confiscated orangutans (Pongo pygmaeus). Journal of Medical Primatology 27, 33-37.[Medline]
Warren, K. S., Heeney, J. L., Swan, R. A., Heriyanto & Verschoor, E. J. (1999). A new group of hepadnaviruses naturally infecting orangutans (Pongo pygmaeus). Journal of Virology 73, 7860-7865.
Warren, K. S., Verschoor, E. J., Langenhuijzen, S., Heriyanto, Swan, R. A., Vigilant, L. & Heeney, J. L. (2001). Speciation and intra-subspecific variation of Bornean orangutans, Pongo pygmaeus pygmaeus. Molecular Biology and Evolution (in press).
Zhi, L., Karesh, W. B., Janczewski, D. N., Frazier-Taylor, H., Sajuthi, D., Gombek, F., Andau, M., Martenson, S. & OBrien, S. J. (1996). Genomic differentiation among natural populations of orang-utan (Pongo pygmaeus). Current Biology 6, 1326-1336.[Medline]
Received 7 September 2000;
accepted 22 November 2000.