Unité des Virus Emergents, EA 871, Laboratoire de Virologie Moléculaire, Établissement Français du Sang Alpes-Méditerranée, 149 Boulevard Baille, 13005 Marseille, France1
Unité des Virus Emergents, EA 871, Laboratoire de Virologie Moléculaire, Tropicale et Transfusionnelle, Faculté de Médecine, 27 Boulevard Jean Moulin, 13005 Marseille, France2
Author for correspondence: Xavier de Lamballerie. Fax +33 491 32 44 95. e-mail virophdm{at}gulliver.fr
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
To date, the complete sequence of ten Japanese TLMV isolates has been determined by Takahashi et al. (2000) and deposited in databases. However, there have been no reports of complete sequence data from non-Japanese isolates, phylogenetic analysis of genomes or epidemiological investigations.
In this study, we report the first complete sequence of a European isolate (PB4TL) of TLMV. Viral DNA was extracted from the peripheral blood mononuclear cells (PBMCs) of a French haemodialysis patient using a commercial kit (High Pure viral nucleic acid kit, Roche), according to the manufacturers instructions. Primers specific for the non-coding region (NCR) of the viral genome were designed according to alignment analysis of the available full-length Japanese TLMV sequences. These NCR-specific primers (TLMS, sense 5' ATTWRMATTGCCGACCACAAAC 3', and TLMS2INV, antisense 5' GTTTCTTGCCCRKTCCGCYAG 3') amplified a 288 nt product using standard PCR conditions with an annealing temperature of 55°C. Determination of the sequence of this 288 nt PCR product allowed the design of two new sets of specific primers that could be used for two successive steps of inverted PCR around the circular genome of TLMV (Fig. 1a). Primers used for first-round inverted PCR were SF1 (sense 5' TGGCTGAGTTTATGCCGCTAGACG 3') and RF1 (antisense 5' TCCCCGCCTAGTTATGACGGTGTG 3'). For the nested inverted PCR, primers SF2 (sense 5' GAAGACGGACAACGACTTCGGCTG 3') and RF2 (antisense 5' CATGAATAATAATGAGCAAAAGAG 3') were used.
|
Sequence analysis revealed that the genome of the PB4TL isolate was 2910 nt in length and contained two large ORFs, corresponding to ORF1 (nt 5292556; 676 aa) and ORF2 (nt 341640; 99 aa), similar to those present in the genomes of the Japanese isolates described by Takahashi et al. (2000) . In contrast, no initiation codon was found for the proposed ORF3 reported by the same authors. It is noteworthy that this is also the case for the Japanese isolate CLC-156. To further investigate the probable functionality of the different ORFs, full-length TLMV sequences were aligned using the CLUSTAL W program, version 1.74 (Thompson et al., 1994
). As expected, it was observed that the two first codon positions were less variable than the third position along the sequences of ORFs 1 and 2. In the region where the two genes overlap, the coding constraint applies to all codon positions, resulting in a lower global variability (Fig. 1b
). In this region, we also detected signature sequences of overlapping genes (Pavesi, 2000
): the end of the ORF2 sequence includes a large number of acidic amino acid residues, while the beginning of ORF1 includes a large number of basic amino acid residues. This situation is similar to that observed in the genome of another circovirus, CAV, in the region where the genes of the 24 and 52 kDa proteins overlap (Pavesi, 2000
). Altogether, these observations indicate that both ORFs 1 and 2 of TLMV are functional. In contrast, we did not identify a significant drop in nucleotide variability at the end of ORF1, which is the region corresponding to the putative ORF3. Moreover, no signature sequence of overlapping genes could be detected in that part of the genome. Together with the absence of an initiation codon in that region for at least two TLMV isolates, these data suggest that the probability of ORF3 being functional is low.
In the NCR of the viral genomes analysed, the alignment of full-length sequences allowed the identification of two highly conserved patterns located between nucleotides 183 and 332 (positions are related to strain CBD279), downstream of the GC-rich zone. Interestingly, the nucleotide patterns spanning positions 183198 and 247298 correspond to sequences conserved among the most highly divergent TTV isolates. Another conserved pattern, common to both TTV and TLMV, has been previously identified in the NCR upstream of the GC-rich zone by Takahashi et al. (2000) . Other circovirus-related motifs are present in the genome of TLMV: in particular, a CAV-like VP2 amino acid motif (Wx7Hx3Cx1Cx5H) is present in the TLMV ORF2, a motif which is also observed in TTV sequences (Hijikata et al., 1999
; Biagini et al., 2000b
; Takahashi et al., 2000
). Finally, since circoviruses replicate using a rolling circle mechanism, we investigated the presence of motifs related to the Rep protein, which possesses up to four sequence motifs and is conserved among many plant and animal circoviruses (Niagro et al., 1998
). Conserved motifs 1 (FTL/FxTL), 2 (HxH) and 3 (YxxK) were identified in ORF1 of numerous TLMV isolates, while motif 4 (GxxxxGKS) and the P-loop (putative ATP/GTP-binding motif) could not be found, as previously reported in the case of TTV and CAV.
Pairwise comparison of complete nucleotide sequences was performed using the software program MEGA (Kumar et al., 1993 ) to assess the genetic relationship between the 11 TLMV isolates tested. The p-distance algorithm for distance determination, the neighbour-joining method for tree-drawing and a bootstrap resampling of 500 replications were used. An unrooted phylogenetic tree was constructed in which three main groups, supported by a 100% bootstrap confidence level, could be distinguished (Fig. 2a
). Group 1 includes isolates PB4TL, NLC030, CLC062, CLC156 and CBD203. Group 2 includes isolates CBD231, CBD279, CLC138 and CLC205 and group 3 includes isolates NLC023 and NLC026. The topology of this phylogram was compared with those observed using independent alignments of subgenomic sequences. Similar groupings were found for ORFs 1 and 2. A tree corresponding to the ORF1 region is shown in Fig. 2(b)
. Phylogenetic analysis of amino acid sequences corresponding to ORFs 1 and 2 provided similar groupings to those identified by nucleotide sequence analysis (data not shown). The untranslated region of the genome was also submitted to phylogenetic analysis. The region of the NCR located upstream of the GC-rich zone produced phylogenetic groupings similar to those obtained from complete or coding sequences. This was not the case for the region of the NCR located downstream of the GC-rich zone. The topology of group 2 was identical to that observed when using complete sequences, but the positions of some isolates (PB4TL, NLC026 and CLC156) belonging to groups 1 and 3 within the tree showed important variations (Fig. 2c
). One of the hypotheses that could take this observation into account is the existence of recombination events between TLMV isolates. The location of such recombinations within the NCR could be favoured by the existence of several highly conserved nucleotide patterns. This hypothesis was tested by submitting the alignments of full-length sequences to analysis by the Recombination Detection Program (RDP) (Martin & Rybicki, 2000
). Despite the low number of sequences available, RDP determined a high probability for recombination breakpoints, all of them located within the NCR. These findings are in accordance with recent results concerning TTV (Worobey, 2000
). In particular, TTV isolate NLC026 was identified as a possible recombinant (P=2x10-8) between a minor parent belonging to the group 1 PB4TL lineage (region spanning nt 2852330) and a major parent belonging to the group 3 NLC023 lineage. This would explain the phylogenetic assignment of NLC026 to group 3 when complete or coding sequences are analysed and its apparent genetic relatedness to the PB4TL isolate when non-coding sequences are analysed. Similarly, within group 1, isolate CLC156 was identified as a possible recombinant between one parent belonging to the PB4TL/NLC026 lineage for the NCR (nt 14213) and another parent belonging to the CLC062 lineage for the rest of the genome. The hypothesis that some of the complete sequences used for analysis are recombinant sequences derived from different viruses present in the original samples cannot be formally ruled out. In the case of isolate PB4TL, however, the fact that identical sequences were obtained using specific or degenerated/direct or reverse PCR systems renders this hypothesis improbable.
|
|
Finally, our analysis of TLMV sequences confirms that the general organization of the genome and the presence of conserved nucleotide or amino acid motifs show a clear relatedness with other circoviruses such as TTV or CAV. However, these virus species are only distantly related to each other as shown by genetic distances (>75%) or by analysis of their G+C content, which revealed a high degree of discrepancy (the calculated values from full-length sequences are 37·7, 51·7 and 56·4% for TLMV, TTV and CAV, respectively). Preliminary observations suggest that, as previously observed for TTV, the infection of humans by TLMV is extremely frequent and generally non-symptomatic. This reinforces the hypothesis of Simmonds et al. (1999) that circoviruses could be a part of the normal human flora and constitutes a new and important physiological concept, since the active replication of viruses that chronically infect humans (such as Herpesviridae) is generally considered to be associated with pathology. The presence of TTV or TLMV virus particles in the blood of a large proportion of healthy individuals demonstrates that viraemia (and thus active virus replication) is not always associated with disease. However, the example of the saprophytic bacterial flora of humans reminds us that this kind of co-habitation is the result of a subtle balance between the multiplication of micro-organisms and the host defences. Consequently, the high prevalence of TTV or TLMV infections should not be interpreted as proof that these viruses are never implicated in human pathologies.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Biagini, P., Gallian, P., Touinssi, M., Cantaloube, J. F., Zapitelli, J., de Lamballerie, X. & de Micco, P.(2000b). High prevalence of TT virus infection in French blood donors revealed by the use of three PCR systems. Transfusion 40, 590-595.[Medline]
Gallian, P., Biagini, P., Zhong, S., Touinssi, M., Yeo, W., Cantaloube, J. F., Attoui, H., de Micco, P., Johnson, P. J. & de Lamballerie, X.(2000). TT virus: a study of molecular epidemiology and transmission of genotypes 1, 2 and 3. Journal of Clinical Virology 17, 43-49.[Medline]
Hijikata, M., Takahashi, K. & Mishiro, S.(1999). Complete circular DNA genome of a TT virus variant (isolate name SANBAN) and 44 partial ORF2 sequences implicating a great degree of diversity beyond genotypes. Virology 260, 17-22.[Medline]
Kumar, S., Tamura, K. & Nei, M. (1993). MEGA: Molecular evolutionary genetics analysis, version 1.02. Pensylvania State University, PA, USA.
Martin, D. P. & Rybicki, E. P.(2000). RDP: detection of recombination amongst aligned sequences. Bioinformatics 16, 562-563.[Abstract]
Miyata, H., Tsunoda, H., Kazi, A., Yamada, A., Khan, M. A., Murakami, J., Kamahora, T., Shiraki, K. & Hino, S.(1999). Identification of a novel GC-rich 113-nucleotide region to complete the circular, single-stranded DNA genome of TT virus, the first human circovirus. Journal of Virology 73, 3582-3586.
Niagro, F. D., Forsthoefel, A. N., Lawther, R. P., Kamalanathan, L., Ritchie, B. W., Latimer, K. S. & Lukert, P. D.(1998). Beak and feather disease virus and porcine circovirus genomes: intermediates between the geminiviruses and plant circoviruses. Archives of Virology 143, 1723-1744.[Medline]
Nishizawa, T., Okamoto, H., Konishi, K., Yoshizawa, H., Miyakawa, Y. & Mayumi, M.(1997). A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology. Biochemical and Biophysical Research Communications 241, 92-97.[Medline]
Pavesi, A.(2000). Detection of signature sequences in overlapping genes and prediction of a novel overlapping gene in hepatitis G virus. Journal of Molecular Evolution 50, 284-295.[Medline]
Simmonds, P., Prescott, L. E., Logue, C., Davidson, F., Thomas, A. E. & Ludlam, C. A.(1999). TT virus part of the normal human flora? Journal of Infectious Diseases 180, 1748-1750.[Medline]
Takahashi, K., Iwasa, Y., Hijikata, M. & Mishiro, S.(2000). Identification of a new human DNA virus (TTV-like mini virus, TLMV) intermediately related to TT virus and chicken anemia virus. Archives of Virology 145, 979-993.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J.(1994). CLUSTAL W: improving the sensitivity of progressive multiple alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673-4680.[Abstract]
Worobey, M.(2000). Extensive homologous recombination among widely divergent TT viruses. Journal of Virology 74, 7666-7670.
Yuasa, N., Taniguchi, T. & Yoshida, I.(1979). Isolation and characteristics of an agent inducing anemia in chicks. Avian Diseases 23, 366-385.
Received 9 August 2000;
accepted 4 October 2000.