* Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium
Department of Zoology, University of Oxford, Oxford, U.K.
Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan
Université Catholique de Louvain, Unité de Virologie, Bruxelles, Belgium
| Hospital San Roque, San Salvador de Jujuy, Argentina
¶ Laboratoire de Rétrovirologie, Institut Pasteur de la Guyane, Cayenne, French Guiana
# Instituto de Medicina Tropical Alexander Von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru
** Gonçalo Moniz Research Center, Oswaldo Cruz Foundation, Bahia School of Medicine and Public Health, Salvador, Bahia, Brazil
Correspondence: E-mail: Sonia.VanDooren{at}uz.kuleuven.ac.be.
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Abstract |
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Key Words: HTLV-1 vertical transmission evolutionary rate molecular clock
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Introduction |
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The genetic stability of HTLV has been indicated by several estimates of the evolutionary rate of the virus. The investigation of intrafamilial HTLV infections has demonstrated the presence of almost identical HTLV-1 sequences in several family members sampled over several generations (Nerurkar et al. 1993; Liu et al. 1994). In a more precise manner, the evolutionary rate of HTLV-1, measured as the number of nucleotide substitutions per site per year, has been estimated by phylogenetic analysis using two different methods. The conventional approach, which involves dividing the difference in branch lengths in a phylogenetic tree by the difference in isolation time (Li, Tanimura, and Sharp 1988), although straightforward, is not ideally suited for HTLV strains, as the observed sequence divergence is usually too small for reliable estimates to be obtained. This method has only been applied to HTLV-2 strains from intravenous drug users (Salemi et al. 1999). The second approach assumes a known time for a particular node in the HTLV phylogeny. All current calculations of the HTLV-1 evolutionary rate are based on an assumed time point in the migration history of the human host population (Yanagihara et al. 1995; Salemi et al. 1998; Van Dooren et al. 1998; Salemi,. Desmyter, and. Vandamme 2000; Van Dooren, Salemi, and Vandamme 2001). This time point is obtained from anthropological studies, so the estimated rates are dependent on the accuracy of the anthropological date and rely heavily on the assumption that the phylogenetic node in question coincides with the anthropological event.
Herein, we report an alternative approach that uses HTLV-1 familial transmission data that is independent of anthropological dates. Familial HTLV-1 is predominantly transmitted vertically from mother to offspring or horizontally through sexual contact. However, only HTLV-1 sequences from vertically infected family members can contribute to the estimation of evolutionary rates, as data on the timing of horizontal transmissions are almost impossible to collect, forcing us to exclude these events from our calculations.
To obtain an estimate of the molecular evolutionary rate of HTLV-1 that is independent of anthropological data, we have sequenced the complete LTR region and a 522 bp fragment of the gp21 env region from HTLV-1 strains in eight families. First, the phylogenetic relationships of the strains were studied. Subsequently, we estimated the evolutionary rate of the sequenced genome regions from the total number of mutations accumulated during the combined period of vertical infection that was represented by the pedigrees of the infected families.
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Materials and Methods |
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The accession numbers of the new Latin American sequences are AY324777 to AY324788 (LTR) and AY324789 to AY324800 (env). For the African family members, the accession numbers are Z31659 (MOMJ LTR) and X88884 (MOMS env).
Phylogeny
Only the phylogeny of the LTR region was studied, as closely related strains are best investigated using a highly variable genome region. A Blast search (www.ncbi.nml.gov/blast) was conducted on the Latin American isolates as an aid to HTLV-1 subtype identification. A detailed phylogenetic analysis was then performed. ClustalX (Jeanmougin et al. 1998) was used to align the nine nonidentical familial sequences with 59 HTLV-1 reference sequences from GenBank that belonged to the same subtype. Reference strains from the same geographic area were preferentially chosen, and three strains from other subtypes were included as outgroups. Minor editing of the alignment was performed manually in MacClade version 3.04 (Maddison and Maddison 1992). Phylogenetic trees were estimated with PAUP* version 4.0b10 (Swofford 1998). The Tamura-Nei substitution model with gamma-distributed rate heterogeneity among sites was chosen as best model for PTLV-1 strains (Salemi, Desmyter, and Vandamme 2000) and was thus used to construct neighbor-joining (NJ) and maximum-likelihood (ML) trees. Using empirical base frequencies, the NJ tree was constructed by optimizing the substitution rate matrix and gamma shape parameter three times; this was followed by a bootstrap analysis (1,000 replicates). The ML tree was estimated using the substitution model and parameters obtained above. A heuristic ML search was performed using the subtree-pruning-regrafting branch-swapping algorithm and an NJ starting tree. Statistical support for the ML tree branches was calculated in PAUP* using a likelihood ratio test that compared the likelihood of the estimated branch length with that of a zero branch length.
HTLV-1 Evolutionary Rate Estimation
The sequences from each family were aligned and the position and number of observed mutations in the LTR and env regions were scored (see fig. 1).
The HTLV evolutionary rate was calculated using a homogenous Poisson process model. Taken together, the pedigrees represent a total amount of time (t years), during which n mutations were observed in the combined LTR-env region. Therefore, the likelihood of the evolutionary rate () in this region is
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Results |
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Phylogeny
Figure 2 shows the results of the phylogenetic analysis. Both NJ and ML trees showed similar topologies and demonstrated that HTLV-1b, HTLV-1c, and HTLV-1d were appropriate outgroups (NJ bootstrap, 83%; ML, P < 0.01). Within the HTLV-1a part of the tree, different subgroups could be identified (as previously noted by Yamashita et al. [1996] and Van Dooren et al. [1998]). These subgroups were strongly supported in the ML analysis (P < 0.01) but only moderately in the NJ bootstrap analysis. The nine nonidentical familial LTR sequences belonged to the cosmopolitan subtype HTLV-1a. All the Peruvian and Argentinean familial HTLV-1 strains cluster within the transcontinental subgroup A of subtype HTLV-1a. More specifically, they group within the previously described Latin American clade (NJ, 83%; ML, P < 0.01). The strains of family AA (Ar55, Ar56, Ar57, Ar58, and Ar63) were an exception, as they seemed to belong to a different Latin American clade of subgroup A (NJ, 88%; ML, P < 0.01). Nonidentical HTLV-1 strains from the same family cluster closely together, with the exception of Ar11 from family E, which is more distantly related to other strains from the same family (Ar12, Ar15, Ar16, and Ar64). The family from French Guyana cluster within the West African/Caribbean subgroup C of subtype HTLV-1a (NJ, 60%; ML, P < 0.01), together with a strain (NM1626) from the same geographic area. The strains sampled from the Brazilian family are very closely related to Bl3.Peru and were all obtained from individuals of black origin. The NJ analysis clustered the Brazilian and Peruvian strains and positioned them as the most divergent lineage within the Japanese subgroup B, although this was not supported by the bootstrap analysis. However, the ML analysis supported a branch that clustered Br4 and Br9 with Bl3.Peru, separate from any other known HTLV-1a subgroup.
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Among the eight investigated families, 16 vertical transmission chains were available for study (see fig. 3), containing two mutations in the LTR region and three mutations in the env region. Two of the proposed positions were ambiguous (one in the LTR sequence from MODI that was not fixed in the next generation and one in the env sequence from Ar47), making it impossible to draw conclusions regarding mutation fixation. Mutations in the LTR sequences occurred only in the R and U5 regions, and mutations in the env region occurred only at third codon positions (two synonymous mutations in family U and one nonsynonymous mutation in the Peruvian family).
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Discussion |
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Calculation of evolutionary rates usually requires the assumption of a molecular clock (Zuckerkandl and Pauling 1962). However, the validity of the clock assumption for viruses is still a matter of controversy (Holmes 2003). Although the hypothesis of a strict molecular clock is rejected for many viral data sets, most likely because evolutionary rates vary among lineages, the resulting effect on estimated dates may be small if the rate variation is not large (Jenkins et al. 2002). Previous studies of PTLV have shown that the clock assumption may be applied but only under certain conditions. Either lineages (or complete clades) deviating from the clock must be removed from the analysis (Van Dooren et al. 1998), or sites affecting the molecular clock must be eliminated (Salemi et al. 2000; Van Dooren et al. 2001), resulting in the loss of molecular information. Alternatively, statistical methods that explicitly incorporate rate variation could be used (e.g., Thorne et al. 1998). HTLV-1 sequences from vertical transmission chains might be expected to evolve in a more clocklike manner, as the heterogeneity in the evolutionary rate is probably smaller when closely related sequences are considered.
The estimation of the HTLV-1 evolutionary rate using a molecular clock requires an accurate calibration date for at least one node in the phylogeny. Previous analyses (Yanagihara et al. 1995; Salemi et al. 1998; Salemi, Desmyter, and Vandamme 2000; Van Dooren et al. 1998; Van Dooren, Salemi, and Vandamme 2001) have used time points based on anthropological events in the history of the viral host. In the analysis presented here, the calibration dates represent a well-known time frame, corresponding to the total number of years during which mutations in the vertical transmission chains occurred. More specifically, this time frame was expressed as the highest and lowest possible times for the accumulation of mutations among the different pedigrees. The reasoning behind the highest possible time is that, theoretically, one cannot know whether a mutation observed in the HTLV strain of the offspring was already present in the HTLV population of the mother before she gave birth (e.g., as a minor variant among the major HTLV clones). The lowest possible time assumes that the mutation really arose within the offspring. Thus for each mother-offspring pair, the highest or lowest possible times represent the minimum and maximum amount of shared common ancestry.
The inclusion of ambiguous positions in the calculations led to several different rate estimates. The estimates presented in table 1 revealed that the LTR and env regions evolve at approximately equivalent rates, with a slightly higher rate for the envelope gene compared with the LTR noncoding region. The combined LTR-env region resulted in estimates with smaller confidence intervals, thanks to an increased total amount of mutations.
It is important to note that the rate estimates provided in table 1 reflect the evolution of HTLV-1 within vertically infected family members. As vertical HTLV-1 transmission is one of the main transmission routes in endemically infected areas, our estimate should reflect the true HTLV-1 evolutionary rate in such populations. All previously published estimates lie within the confidence limits of our new estimates. Our estimates for the LTR correspond to those of a previous study that investigated the cosmopolitan HTLV-1a subtype and its introduction into Latin America. (Van Dooren et al. 1998). The hypothesis concerning the dissemination of HTLV-1a in the New World is still a matter of controversy. Some analyses suggest the virus was first introduced on the American continent by an ancient migration of mongoloids across the Bering Strait 40,000 to 10,000 years ago (Miura et al. 1994, 1997; Yamashita et al. 1998; Ohkura et al. 1999; Li et al. 1999, Ramirez et al. 2002). Other molecular epidemiological studies indicate that HTLV was first introduced along with the post-Columbian African migration, which started approximately 400 years ago (Gessain et al. 1992, Gessain, Gallo, and Franchini 2000; Vandamme et al. 1994, 2000). Assuming either an ancient introduction or a post-Columbian introduction, the HTLV-1 LTR rate was estimated to be around 1.25 to 5x10-7 or 1.25x10-5 subs./site/year, respectively. These estimates correspond to the upper and lower 95% confidence limits of the estimates for the LTR provided here. However, this comparison is complicated by the worldwide distribution of HTLV-1a strains included in the previous study; these strains are not necessarily restricted to the endemically infected populations. If horizontal transmission is greater among these nonendemic HTLV-1a isolates, then a rate that only takes into account vertical transmission will be an underestimate. In that case, the recent introduction theory would be more plausible. We have previously published an estimated rate for the combined LTR+ env third codon position (Van Dooren, Salemi, and Vandamme 2001), using a data set containing African HTLV-1 subtypes and some STLV-1 strains that cluster with the human strains. In that study, the earliest human migrations to Melanesia and Australia 60,000 to 40,000 years ago were used as an anthropological calibration point, as these dates could be correlated with the isolated presence of HTLV-1 subtype c in Australia and Melanesia. This study reported a rate of 1.56 ± 0.43x10-6 subs./site/year, similar to the range calculated here for four mutations. The analysis of Van Dooren, Salemi, and Vandamme (2001) contained simian strains, and the presence of this cross-species transmission event could have led to a difference between the estimated rates. Other published estimates of the evolutionary rate in PTLV, based on larger concatenated gene regions, resulted in rate estimates of 2.5 to 6.8x10-7 subs./site/year (Yanagihara et al. 1995) and 1.67 ± 0.17x10-6 subs./site/year at the third codon position, using the Melanesian migration calibration (Salemi, Desmyter, and Vandamme 2000). These data cannot be compared directly with the vertical transmission chain estimates presented here, because different coding regions and/or different PTLV types were investigated.
Using 16 HTLV-1 vertical transmission chains from eight HTLV-1 infected families, we have confirmed the genetic stability of the virus, even in genomic regions such as LTR and env that are expected to be relatively variable. Our estimates are based on a new approach that is independent of anthropological calibrations. In the future, more precise rate estimates could be calculated if samples and medical histories from patients were gathered over more than three generations, illustrating the importance of long-term sample collection, curation, and storage.
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Acknowledgements |
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Footnotes |
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