Department of Virology, Biomedical Primate Research Centre, PO Box 3306, 2280 GH Rijswijk, The Netherlands1
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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Introduction |
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Since the discovery of TTV, studies have been published describing the prevalence of TTV infection in people with acute or chronic hepatitis, as well as in blood donors and drug users, but also in healthy persons (Biagini et al., 1998 ; Charlton et al., 1998
; Höhne et al., 1998
; MacDonald et al., 1999
; Naoumov et al., 1998
; Niel et al., 1999
; Simmonds et al., 1998
; Tanaka et al., 1998a
). From these studies it has become apparent that it is currently impossible to ascribe TTV to any specific disease. TTV can be transmitted parenterally and has been found in plasma and peripheral blood mononuclear cells (Okamoto et al., 1999
). However, non-parenteral transmission is also possible as TTV can be excreted in faeces (Okamoto et al., 1999
). Molecular and phylogenetic analysis of PCR fragments revealed that TTV could be divided into several genotypes that are found worldwide without any direct correlation with geographical distribution or disease (Biagini et al., 1999
; Höhne et al., 1998
; Mushahwar et al., 1999
; Okamoto et al., 1999
; Tanaka et al., 1998b
; Viazov et al., 1998
).
This study investigated whether non-human primates are infected with TTV. We also determined whether these viruses are related to the known human TTV genotypes and if they could be a possible reservoir for human TTV infections. We analysed serum samples from eight different non-human primate species originating from the Old and the New World. TTV could be detected only in the two chimpanzee species studied. Subsequent molecular and phylogenetic analysis revealed that chimpanzee TTV consists of heterogeneous group of viruses closely related to, but distinct from, human TTV.
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Methods |
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PCR and nucleic acid sequencing.
Viral DNA was isolated from 100 µl of serum using a modified procedure previously used for isolating viral RNA (Ten Haaft et al., 1998 ). TTV sequences were amplified in a semi-nested PCR using the primers NG059, NG061 and NG063 (Okamoto et al., 1998
). The first PCR reaction was performed in a 50 µl volume using 10 µl of viral DNA sample, 50 pmol of primers NG059 and NG063, 0·2 mM of each dNTP, 2·6 mM MgCl2 and 2·5 U AmpliTaqGold (Perkin-Elmer). Samples were pre-heated for 15 min at 94 °C to activate the enzyme, and then cycled for 30 s at 94 °C, 20 s at 55 °C and 30 s at 72 °C for 35 rounds of amplification in a PTC-2000 thermal cycler (MJ Research). A 2 µl volume from the first PCR was then transferred to the second reaction mixture, which contained primers NG061 and NG063 but with MgCl2 at a concentration of 2·7 mM, and amplified as described above.
The PCR products were analysed on a 1·8% agarose gel, isolated from this gel using a Qiagen QIAquick gel extraction kit, and cloned in a pGEM-T vector (Promega). Sequence reactions were performed with the ABI PRISM dRhodamine terminator cycle sequencing kit and the nucleotide sequence was determined using an ABI PRISM 310 Genetic Analyser (PE Applied Biosystems).
Sequence analysis.
Analysis of the TTV sequences was performed using MacVector 6.0 and AssemblyLIGN software packages (Oxford Molecular Ltd). Phylogenetic analysis of nucleotide and deduced amino acid sequences was performed using the PHYLIP software package, version 3.572 (Felsenstein, 1995 ). The PHYLIP program SEQBOOT was used to bootstrap data in which 100 data sets were analysed. Pairwise distances were calculated with DNADIST using the Kimura two-parameter method and the NEIGHBOR program (neighbour-joining method) was used to create dendrograms. CONSENSE was used to create consensus trees that were visualized using the program NJplot.
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Results |
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Within individuals different sequence types could also be detected. From individual Ch_Sy four sequences were isolated, belonging to three different groups with only limited sequence identity ( 50%), while two types of TTV sequence (49% identity) were recovered from Ch_Br. One animal, Ch_No, was positive for TTV in serum samples taken at two different time-points spaced 7 years apart. These sequences (Ch_No91 and Ch_No98) differed at only two nucleotides. This implies a relatively low mutation rate for TTV and suggests that the above-mentioned cases of infection of an individual with multiple TTV types are more likely the result of separate infection events than from evolution of a single progenitor TTV within the animal. In addition to the DNA alignment we also show (Fig. 1 B) the deduced amino acid sequences of ORF1. This protein alignment fully confirms the division into different types described above.
Phylogenetic relationship of chimpanzee and human TT viruses
To examine the relationship of the viruses detected in the two chimpanzee species with the six human TTV genotypes described by Tanaka et al. (1998b ) we generated unrooted phylogenetic trees of the nucleotide and amino acid sequences. The neighbour-joining tree of nucleotide sequences is depicted in Fig. 2
. The phylogenetic analysis fully confirms the description of the different TTV types in chimpanzees, but in addition reveals that the pygmy chimpanzee TT viruses and the common chimpanzee TT viruses are found on separate branches of the tree. Pygmy chimpanzee TT viruses cluster with human genotype 3 as well as two West African genotype 2 TT viruses, with a strong bootstrap support of 96%. Viruses detected in the two subspecies of the common chimpanzee (P.t.v. and P.t.s.) all form one branch of the tree together with human TT viruses belonging to genotype 5 (bootstrap support 66%).
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Discussion |
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Transmission of TTV within chimpanzee populations was not investigated in detail; however, the majority of infections seemed to have occurred during adolescence. The highest incidence was found in older animals, 1020 years of age. Importantly, group housing of hand-reared animals directly after birth seemed to break the cycle of virus transmission. The latter excludes in utero transmission or peripartal transmission as a major mode of transmission in these animals. Circumstantial evidence suggests that in this chimpanzee colony the most likely of mode of infection is by the oralfaecal route. Chimpanzees which are housed together with older TTV-positive animals soon become infected, with an incidence of 42%. No relationship between TTV infection of offspring and the TTV status of parents could be identified (data not shown). As fighting (parenteral transmission) between these animals is not common, and in utero or perinatal transmission is not observed, the oralfaecal route is the most likely. Indeed, in humans TTV has been found in faeces of TTV-infected individuals (Okamoto et al., 1999 ), also suggesting that transmission of TTV may take place in situations where strict hygiene is not practised. The question remains, as is also the case in human TTV infection (Cossart, 1998
), whether TTV causes disease in chimpanzees. No overt clinical signs were found which corresponded to TTV infection in these animals and certainly there was no evidence of hepatitis. Our observations to date suggest that in chimpanzees, as in humans, the disease-causing potential of TTV-related viruses remains undefined.
As has also been reported for humans (Biagini et al., 1999 ; Mushahwar et al., 1999
), chimpanzees can be infected with multiple (sub)types of TTV. One animal described in this article (Ch_Sy) was infected with at least three types of TTV. All types found in Ch_Sy belonged to the cluster encompassing all of the Pan troglodytes viruses and the human TTV genotypes 5 and 6. Many chimpanzees of the subspecies P.t.v. described in this article have a history of being used for non-A, non-B hepatitis research. It is thus conceivable that (some) TT viruses described here are of recent human origin. However, the fact that all the P.t.v. TT viruses described here cluster with a relatively high bootstrap support makes this unlikely. In the case of infection with human TTV one would not expect clustering, but rather a distribution of TTV sequences along all branches of the tree representing different human genotypes. Also, clustering with TT viruses isolated from animals without an experimental history (Ch_Ni and Ch_No) makes infection with the human viruses unlikely.
We also found, analogous to the human situation (Biagini et al., 1999 ), that TTV in chimpanzees causes a chronic and persistent asymptomatic infection. One animal, Ch_No, was documented to be persistently infected with TFV for more than 7 years. Circumstantial evidence points to the possibility that TTV relatively frequently establishes persistent infections in chimpanzees. Such a high incidence level of active infections observed in a single cross-sectional sample of this chimpanzee population suggests that persistent infections are common.
The absence of apparent disease caused by TTV infection in chimpanzees may point at an ancient hostpathogen relationship between these great apes and TT viruses (May, 1995 ). An indication of a long-standing relationship between chimpanzees and TTV-like viruses is provided by the phylogenetic analysis of TTV nucleotide and amino acid sequences. The two subspecies of the common chimpanzee, P.t.v. and P.t.s., have geographically widely separated origins. P.t.v., the western subspecies, is found in Western Africa from Guinea to Ghana, while P.t.s. inhabits areas of Central Africa (Gonder et al., 1997
; Morin et al., 1994
). The viruses from these two common chimpanzee subspecies, however, form one cluster with very high bootstrap support (DNA 99%, amino acid 92%). This suggests that the common chimpanzee viruses originate from the same progenitor TT virus. As subspeciation/geographical separation of P.t.v. and P.t.s. may have taken place as long as 1·6 million years ago (Morin et al., 1994
), TTV infection of common chimpanzees may have occurred before that time. On the other hand, TT viruses from the two different African chimpanzee species, the common chimpanzee and the pygmy chimpanzee, are found in distinct clusters together with different human genotypes that are located on two separate branches of the phylogenetic tree. This implies that two unrelated zoonotic events in history may have been associated with cross-species transmission from chimpanzee to human or vice versa. If so, such events must have taken place after both species had diverged from each other 2·57 million years ago (Arnason et al., 1996
; Begun, 1992
; Ruvolo et al., 1993
).
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Acknowledgments |
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Footnotes |
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References |
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Received 6 May 1999;
accepted 15 June 1999.