Hepatitis Viruses and Molecular Hepatitis Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 7, Room 201, 7 Center Dr MSC 0740, Bethesda, MD 20892-0740, USA1
Department of Internal Medicine, University of Milan, IRCCS Maggiore Hospital, Milan, Italy2
Author for correspondence: Jens Bukh. Fax +1 301 402 0524. e-mail jbukh{at}niaid.nih.gov
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Abstract |
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TTV transmission can occur by the parenteral route (Nishizawa et al., 1997 ; Simmonds et al., 1998
) and the relatively high prevalence of TTV among drug addicts, haemodialysis patients and haemophilia patients suggests that this is an important transmission route (Desai et al., 1999
; Forns et al., 1999
; Leary et al., 1999
). Shedding of TTV in bile and faeces has been demonstrated, suggesting that faecaloral transmission may also occur (Okamoto et al., 1998a
; Ukita et al., 1999
).
TTV has a negative-sense, single-stranded circular DNA genome of approximately 3·8 kb (Hijikata et al., 1999 ; Miyata et al., 1999
; Mushahwar et al., 1999
; Okamoto et al., 1998b
, 1999a
). Because of the lack of significant sequence similarity between TTV and circoviruses, it has been proposed that TTV belongs to a new virus family tentatively designated Circinoviridae (Mushahwar et al., 1999
). Three putative ORFs were recognized (Erker et al., 1999
). The sequence between the end of ORF3 and the beginning of ORF2 is defined as the UTR.
TTV is characterized by extreme genetic heterogeneity: currently >15 major genetic groups have been identified in humans (Okamoto et al., 1998b , 1999a
, b
; Prescott et al., 1999
; Tanaka et al., 1998
). The primary aim of the present study was to determine whether naive chimpanzees bred in captivity were infected with TTV. The genetic heterogeneity of TTV in chimpanzees as well as in hepatitis C virus (HCV)-infected humans was studied by analysis of two independent genome regions.
For TTV DNA detection, 100 µl serum was diluted 1:5 with 50 mM TrisHCl (pH 8), 1 mM EDTA, 100 mM NaCl and 0·5% SDS and digested with 1 mg/ml proteinase K, at 65 °C for 1 h. DNA was extracted by 500 µl phenol:chloroform:isoamyl alcohol (25:24:1) (Gibco/BRL) and precipitated overnight in 1/50 vol. 3 M sodium acetate, 2 vol. ice-cold ethanol and 1 µl glycogen (Boehringer Mannheim). After centrifugation at 4 °C for 20 min, the pellet was washed with 1 ml 70% ethanol. DNA amplification was performed by PCR as described previously, using two different sets of primers located within ORF1 (Forns et al., 1999 ) and the UTR (Takahashi et al., 1998a
), respectively. The positive control was 100 µl of a 10-2 dilution of a serum sample from a TTV-positive patient with a titre of 103 genome equivalents (GE)/ml, as determined by hemi-nested PCR (Okamoto et al., 1998 b
). This sample had a titre of 103104 GE/ml in both ORF1-PCR and UTR-PCR. At least three negative controls were included in each experiment.
A 10% (w/v) faecal suspension in PBS (pH 7·4) (10 ml) was centrifuged in an IEC Centra-8R refrigerated centrifuge at 3000 r.p.m. for 30 min. Supernatant was recovered and centrifuged in an Eppendorf Refrigerated Microcentrifuge at 7500 r.p.m. for 10 min. DNA was extracted from 200 µl aliquots of supernatant as described above. TTV DNA was amplified by UTR-PCR.
PCR products corresponding to positions 26184 (159 nt) and 19392163 (225 nt) of isolate TA278 (GenBank accession number AB008394) (Okamoto et al., 1998b ), excluding primer sequences, were purified by the Wizard PCR purification kit (Promega). When necessary, PCR products were cloned into vector pCR2.1 with the original TA cloning kit (Invitrogen). Both DNA strands were sequenced. Relevant TTV sequences published up to August 1999 were analysed in the present study. Sequences were analysed with GeneWorks and MacVector (Oxford Molecular Group), as well as CLUSTALW (version 1.6) and PAUP (version 4.0), both from the GCG Sequence Analysis Package (version 10).
Serum samples from nine Italian patients with chronic hepatitis C and a plasma sample from patient H obtained during the acute phase of post-transfusion hepatitis C (Feinstone et al., 1981 ) were studied. Eight patients (80%) were positive for TTV DNA by ORF1-PCR. The ORF1-PCR product, which was sequenced directly, was 225 nt in each case (Fig. 1
). Phylogenetic relationships of these ORF1 sequences to those of published TTV strains representing recognized genotypes were determined (Fig. 2a
). Genotypes 1, 2, 3 and 6 were recovered from Italian patients, whereas genotype 4 was found in the US patient.
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To genotype the chimpanzee strains, we tried to amplify the ORF1 region used to genotype the human strains. However, ORF1 DNA was amplified from only two (CH1304 and CH1545) of eight naive chimpanzees that tested TTV DNA-positive by UTR-PCR (three positive animals were not tested by ORF1-PCR). The ORF1 sequence recovered from chimpanzee 1545 in nested PCR was 222 nt long with a single amino acid deletion compared with the prototype (Fig. 1). It shared approximately 4055% nucleotide and 2550% amino acid identity with the corresponding sequences from humans. The corresponding ORF1 sequence from chimpanzee CH1304, which was positive after single-round PCR, but negative in nested PCR, was 231 nt with two amino acid insertions compared with the TTV prototype sequence (Fig. 1
). It shared approximately 4055% nucleotide and 2550% amino acid identity with the corresponding sequences from humans. Phylogenetic analysis of the ORF1 nucleotide consensus sequences suggested that TTV recovered from these two chimpanzees represented two new genotypes (Fig. 2a
).
Sera from a chimpanzee (CH1410) that had been inoculated with plasma containing both TTV and HCV were retrospectively analysed to determine if the chimpanzee became infected with TTV. Chimpanzee CH1410 was inoculated intravenously with 1 ml plasma HC-J4/91 from a chimpanzee chronically infected with HCV (Okamoto et al., 1992 ) after inoculation with plasma pooled from several chimpanzees infected with a Japanese blood donor-derived agent. ORF1-PCR products amplified from the inoculum and from chimpanzee CH1410 at week 12 post-inoculation had 100% sequence similarity, indicating that TTV in the inoculum had infected the chimpanzee (Fig. 1
; Fig. 2a
). This sequence belonged to the same TTV genotype as the sequence recovered from naive chimpanzee CH1545 (see above). The ORF1 sequence recovered during late follow-up in this experimentally infected chimpanzee belonged to a third new TTV genotype (Fig. 1
; Fig. 2a
); it had approximately 4560% nucleotide and 3050% amino acid identity to the corresponding human sequences.
Since TTV has been reported in human faeces, we examined faeces from infected chimpanzees for TTV. TTV DNA was detected by UTR-PCR in faeces from two of five animals tested. Both animals were repeatedly positive for TTV DNA in faecal samples taken during the chronic phase of infection. In each case, TTV sequences recovered from faeces had a high similarity (>98%) with sequences recovered from serum of the same animal.
We also tested non-human primates other than chimpanzees for TTV. Serum collected from 40 tamarins (Saguinus mystax), four owl monkeys (Aotus trivirgatus) and six rhesus monkeys (Macacca mulatta) were all TTV DNA-negative by UTR-PCR.
In the present study, we found a high prevalence of TTV in HCV-infected humans and in naive chimpanzees bred in captivity. All TTV sequences recovered from patients belonged to genotypes already recognized among humans. In contrast, TTV sequences recovered from chimpanzees could not be classified within the genotypes currently recognized.
Clonal analysis demonstrated that eight of the ten TTV-infected patients were co-infected with multiple TTV variants; two patients were each infected with at least seven variants. The high degree of genetic heterogeneity suggested that most patients were co-infected with different TTV genotypes, although it must be acknowledged that the region analysed could not accurately predict TTV genotype distribution. In addition, we demonstrated that naive chimpanzees also had mixed TTV infections; this was previously reported for humans (Ball et al., 1999 ; Forns et al., 1999
; Mushahwar et al., 1999
; Takayama et al., 1999
). Our data suggest mixed TTV infection is also common in chimpanzees. Therefore, infection with one TTV variant does not seem to generate protection against infection with other variants.
Based on our analysis of published ORF1 TTV sequences, at least 18 genotypes of TTV circulate among humans (Fig. 2a). We found genotypes 1, 2, 3 and 6 among Italian patients and genotype 4 in the US patient. Genotypes 14 have worldwide distribution, whereas genotype 6 has been identified only in Asia (Forns et al., 1999
; Mushahwar et al., 1999
; Tanaka et al., 1998
). Unfortunately, different designations have been assigned by different groups to isolates of the same genotype. The genotype designations used in the present study are those used in the first published report of that genotype (Okamoto et al., 1998b
, 1999a
, 1999b
; Simmonds et al., 1998
; Tanaka et al., 1998
).
We found that 65% of naive chimpanzees bred in captivity in the USA were infected with TTV. While we cannot rule out that these chimpanzees were infected by contact with humans, the more likely explanation for this high prevalence is that TTV was spread among the animals, most likely by faecaloral transmission. TTV DNA was detected in faeces from two infected chimpanzees. In addition, the TTV genomic sequences recovered from the chimpanzees were significantly different from those recovered from humans; the ORF1 sequences recovered from a subset of the infected chimpanzees belonged to genotypes not previously recognized among humans and all but one of the UTR sequences recovered from these chimpanzees formed a separate cluster with 79% bootstrap support in the phylogenetic analysis. It is noteworthy, however, that we identified a subset of UTR sequences from three humans that were more closely related to these chimpanzee sequences (89% bootstrap support) than to the other TTV sequences analysed.
Recently, a similarly high TTV prevalence was reported for chimpanzees kept in captivity in the USA (Leary et al., 1999 ) and the Netherlands (Verschoor et al., 1999
). Leary et al. (1999)
reported that TTV sequences (corresponding to a different UTR region from the one we analysed) recovered from chimpanzees were similar to human sequences; however, this region might be too conserved to reveal species-specific differences. Verschoor et al. (1999)
reported ORF1 sequences from chimpanzees; most of these formed a separate cluster from human sequences although there was some support for classifying these sequences with human genotype 5 isolates (bootstrap support 66%; in general, a bootstrap support of >70% is considered significant). These sequences were published following completion of our data analysis and therefore are not included in Fig. 2(a)
. However, subsequent phylogenetic analysis of selected isolates (data not shown) indicated that one chimpanzee isolate (Ch_Sy2) identified by Verschoor et al. (1999)
belongs to the same genotype as that of naive chimpanzee 1545, as well as that of a Japanese chimpanzee (J4-91); identities among these three isolates were 8591% and 9396% at the nucleotide and deduced amino acid levels, respectively. In our analysis, we found no support for classifying our two isolates with isolates of genotype 5 or other human TTV isolates. We therefore believe that this genotype, infecting chimpanzees in three different animal facilities, represents a chimpanzee TTV. The two additional TTV variants identified in chimpanzees (CH1304 and CH1410) were not found by Verschoor et al. (1999)
. ORF1 sequences from CH1304 or CH1410 determined in the present study did not cluster with any human isolates but the CH1304 sequence clustered with a sequence determined for isolate Ch_Sy1 by Verschoor et al. (1999)
; however, the ORF1 identities between these two isolates were only 71% and 74% at the nucleotide and deduced amino acid levels, respectively.
TTV was not detected in other non-human primate species tested (tamarins, owl monkeys and rhesus monkeys). Leary et al. (1999) recently reported TTV in tamarins and owl monkeys, but not in common marmosets or rhesus monkeys, whereas Verschoor et al. (1999)
did not detect TTV in tamarins, common marmosets or rhesus monkeys. Several studies have recently reported great variation in the detection of TTV DNA depending on the PCR primers used (Leary et al., 1999
; Okamoto et al., 1999b
; Takahashi et al., 1998a
). Further studies using the most conserved primers in PCR assays and testing a larger number of naive animals are warranted to determine the prevalence of TTV in non-human primates other than chimpanzees. Interestingly, TTV was recently detected with a relatively high prevalence in chickens, pigs, cows and sheep, so TTV infection is apparently widespread (Leary et al., 1999
).
In conclusion, our data indicate that TTV was present in chimpanzees, as in humans, with a high prevalence. Chimpanzees were naturally infected with TTV variants that have not been identified among humans.
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Acknowledgments |
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Footnotes |
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References |
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Received 8 October 1999;
accepted 7 January 2000.