TT viruses (TTV) of non-human primates and their relationship to the human TTV genotypes

Transfusion transmitted virus (TTV) is a recently discovered virus which was suspected to be a causative agent of non-Anon-F hepatitis. The virus was first identified in a Japanese patient who was hospitalized with acute hepatitis (Nishizawa et al., 1997 ). Initially, TTV was described as a non-enveloped, 3739 base long, single-stranded DNA virus. Based on these observations and the genome characteristics it was reported to be a parvovirus-like pathogen (Okamoto et al., 1998 ). Recently, extensive molecular and biophysical characterization of this virus has been performed (Mushahwar et al., 1999 ). It was confirmed that the virus is non-enveloped and has a single-stranded DNA genome, but that this comprises 3852 bases. Further analysis revealed that TTV possesses a negative-stranded genome which is circular. In addition, its buoyant density is significantly different from that of parvoviruses. The new data indicate that TTV is most closely related to the Circoviridae; however, the differences have led to the proposal that TTV is a member of a new virus family, tentatively named Circinoviridae.

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.

Serum samples.
Serum samples were collected from common chimpanzees (Pan troglodytes), rhesus macaques (Macaca mulatta) and long-tailed macaques (M. fascicularis), and from the New World monkey species cotton top tamarins (Saguinus oedipus), common marmosets (Callithrix jacchus) and common squirrel monkeys (Saimiri sciureus) housed at the Biomedical Primate Research Centre (BPRC). Serum samples from orangutans (Pongo pygmaeus) were obtained from animals kept at the Wanariset Orangutan Reintroduction Centre in East Kalimantan, Indonesia. Samples from pygmy chimpanzees (bonobo, Pan paniscus) were kindly supplied by A. Vandamme, Leuven, Belgium.

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.

Prevalence of TTV infections in non-human primates
Evidence for TTV infection in several non-human primate species was obtained by amplifying directly from serum a 228 bp fragment from the first open reading frame (ORF1) of the TTV genome (Okamoto et al., 1998 ). The non-human primate species examined originated from the Old World (Africa and Asia) as well as from the New World (South America). A total of 216 serum samples from eight different species was assayed. The results of the PCR analysis are revealed in Table 1. Individuals from two out of three great ape species tested (common chimpanzee, Pan troglodytes verus, and pygmy chimpanzee, Pan paniscus) showed evidence of infection with a TTV-like agent. A high incidence of TTV infection was found in a chimpanzee breeding colony, the founder animals of which originated from Sierra Leone in the 1960s. Of 123 animals tested 60 (48·8%) were positive for TTV in serum. Furthermore, we could amplify TTV sequences from 4/6 pygmy chimpanzee sera (66·7%). In all serum samples obtained from other species, including the only Asian great ape species, the orangutan (Pongo pygmaeus), no evidence of infection with TTV was found.

Table 1. Results of TTV PCR on non-human primate sera

Epidemiology of TTV in a captive, self-sustaining breeding colony of common chimpanzees
A detailed analysis of the incidence of TTV in a closed breeding colony of common chimpanzees was undertaken. TTV-positive and -negative animals were classified by sex and their year of birth (Table 2). No bias of TTV infection in males or females was found. When animals were divided into groups by age it was evident that a higher incidence of TTV infection was found in adult animals, and that the peak incidence (65·2%) was among animals between 10 and 20 years of age. A much lower prevalence (21·6%) was found in the newborn and juvenile chimpanzees. Based on the following observations we concluded that transmission from older animals in breeding groups likely plays a major role in the pattern transmission of TTV. Two groups of animals consisting of nine and seven individuals, respectively, had no evidence of TTV infection. One group consisted of 67-year-old animals, the other of 35-year-olds. Interestingly, at birth these animals had been separated from the colony because of rejection by their mothers; subsequently, they were hand-raised by animal caretakers and since that time housed together in age-matched social groups. In contrast, youngsters who were then housed in social groups together with infected animals had an incidence of TTV infection of 42%.

Table 2. Relationship between sex, age and the incidence of TTV infection in common chimpanzees

Nucleic and amino acid sequence analysis of ORF1
To investigate whether differences in sequence existed between the TT viruses from the chimpanzee populations studied the nucleotide sequences of the 228 bp ORF1 PCR products were determined. We performed sequence analysis on products obtained from sera of two pygmy chimpanzees (Bo_Ho and Bo_De) and seven common chimpanzees (Ch_Sy, Ch_Br, Ch_Ka, Ch_Pe, Ch_Bu, Ch_Ni and Ch_No). From each individual animal four to six clones were sequenced (except for Ch_Ni, Ch_No and Ch_Bu from which only a single sequence was obtained). In Fig. 1(A) an alignment of the nucleotide sequences of the different TT viruses is shown. In addition to the alignment, the percentage identity between each of the TTV sequences is listed in Table 3. Different TTV sequences were recovered from the common as well as the pygmy chimpanzees. Intraspecies sequence differences were found, as well as differences within infected individuals (Ch_Sy and Ch_Br) with several variants of TTV-like viruses. In total 14 distinct sequences were obtained, originating from nine individuals.

(68K): Fig. 1. Comparison of TTV ORF1 nucleotide sequences (A) and deduced amino acid sequences (B) obtained from pygmy chimpanzees and common chimpanzees. Sequence alignments were generated using ClustalW, as included in the MacVector sequence analysis software package. The Ch_Bu partial sequence was obtained from direct sequencing of the PCR fragment. Dots indicate nucleotides or amino acid residues identical to the Ch_Br2 sequence.

Table 3. Pairwise comparisons of TTV nucleotide sequences obtained from common and pygmy chimpanzees (expressed as percentage identity)

TT viruses from the pygmy chimpanzees, Bo_Ho and Bo_De, form one single sequence group with 99% pairwise identity (Table 3). Their DNA sequences possessed only 4562% nucleotide identity with the TT viruses detected in the common chimpanzees. In the group of TTV sequences recovered from the common chimpanzees, five types could be distinguished. One was represented by a single sequence, Ch_Sy2. The other groups were formed by Ch_Br2 and Ch_Pe (98% identity); Ch_Ka, Ch_Br1, Ch_Sy3 and Ch_Sy4 (8993% identity); Ch_Bu and Ch_Sy1 (85% identity); and Ch_Ni, Ch_No91 and Ch_No98 (9899% identity). Interestingly, the last group of TTV sequences was obtained from sera of two animals which belong to the chimpanzee subspecies Pan troglodytes schweinfurthii (P.t.s.), while all other sequences were isolated from the subspecies Pan troglodytes verus (P.t.v.).

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%).

(41K): Fig. 2. Relationship between TTV nucleotide sequences obtained from common and pygmy chimpanzees and DNA sequences from six human TTV genotypic variants. The tree represents an unrooted consensus tree, based on the nucleotide sequences, obtained with the neighbour-joining method. Bootstrap analysis was applied using 100 bootstrap values. The chimpanzee sequences are highlighted in shaded boxes. The six different TTV genotypes as described by Tanaka et al. (1998b ) are indicated. The human TTV isolates are identified by their GenBank database accession numbers followed by a two-letter code for their country of origin. Co, Colombia; Ja, Japan; Ge, Germany; It, Italy; Mo, Mongolia; Ca, Cameroon; Ga, Gabon; SL, Sierra Leone; UK, United Kingdom; In, Indonesia; Al, Albania; UZ, Uzbekistan; Sr, Sri Lanka; Za, Zanzibar; Ch, China. Sequence data of TTV described in this article are deposited in the EMBL and GenBank data libraries. The EMBL accession numbers are Y18906 to Y18918, and AJ38146.

Neighbour-joining analysis of the deduced amino acid sequences gave essentially identical results (Fig. 3), except that the P.t.v. TT viruses now clustered with genotype 5 and 6, whilst Ch_Sy1 and Ch_Bu formed a cluster that branches off before the human genotype 5 and 6 TTV. The latter are the most divergent viruses from the common chimpanzee TTV described here. Although not strongly supported by bootstrap analysis (66% at the DNA level, 23% at the protein level) they consistently branch off earlier than the other Pan troglodytes viruses.

(39K): Fig. 3. Phylogenetic relationship between TTV ORF1 amino acid sequences obtained from common and pygmy chimpanzees and analogous sequences from six human TTV genotypic variants. For further information see the legend to Fig. 2.

Infection with TTV is not restricted to humans, as has been described by several other research groups (Biagini et al., 1999 ; Charlton et al., 1998 ; Hohne et al., 1998 ; Mushahwar et al., 1999 ; Naoumov et al., 1998 ; Niel et al., 1999 ; Nishizawa et al., 1997 ; Okamoto et al., 1998 ; Simmonds et al., 1998 ; Tanaka et al., 1998a , b ). Here we report that TTV-related virus infections can also be found in at least two non-human primate species, the common chimpanzee and the pygmy chimpanzee. By using a human TTV-specific PCR assay we have discovered TTV sequences in the two chimpanzee species. No TTV-like viruses were found in six other non-human primate species. The latter does not necessarily exclude the possibility of TTV infections in these species, as primers based on sequences conserved among a limited number of human TT viruses were used in the assay (Okamoto et al., 1998 ). In future, with the availability of an increasing number of sequences of full-length genomes from different TTV genotypes, it may be possible to design improved PCR primers that will allow us to detect as-yet unknown TTV-related viruses.

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 ).

The support and technical assistance of Nel Otting and Natasja de Groot, Department of Immunobiology, Biomedical Primate Research Centre, is greatly appreciated.

Footnotes

The TTV sequence data described in this article have been deposited in the EMBL/GenBank data libraries, accession numbers Y18906 to Y18918, and AJ38146.