1 Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium
2 HIV and Retrovirology Branch, Division of AIDS, STD and TB Laboratory Research, National Center for HIV, STD and TB Prevention, Centers for Disease Control and Prevention, 1600 Clifton Rd, MS G-19, Atlanta, GA 30333, USA
3 HIV Immunology and Diagnostics Branch, Division of AIDS, STD and TB Laboratory Research, National Center for HIV, STD and TB Prevention, Centers for Disease Control and Prevention, 1600 Clifton Rd, MS G-19, Atlanta, GA 30333, USA
Correspondence
William M. Switzer
bis3{at}cdc.gov
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ABSTRACT |
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The complete genome of STLV-3/TGE-2117 has been assigned the GenBank accession number AY217650. The GenBank accession numbers for the STLV-3 tax and LTR and STLV-1 tax and env sequences are AY241678AY241693.
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INTRODUCTION |
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Despite these recent reports, the virological and immunological characteristics of STLV-3 infection of non-human primates (NHPs) remain poorly understood. STLV-3 has the highest amino acid sequence identity to the HTLV-1 and HTLV-2 p24 Gag (85 %) and envelope transmembrane proteins (7288 %, respectively) (Meertens et al., 2002; Van Brussel et al., 1997
). Thus, sera from STLV-3-infected primates show cross-reactivity to the p24 Gag and transmembrane (GD21) proteins in Western blot (WB) assays containing HTLV-1 antigens and conserved HTLV-1 and HTLV-2 recombinant GD21 proteins (Goubau et al., 1994
; Meertens et al., 2001
, 2002
; Meertens & Gessain 2003
; Takemura et al., 2002
; Van Dooren et al., 2001
). Sera from STLV-3-infected animals may also react in WB assays with the HTLV-2-type specific recombinant envelope (Env) protein, K55; thus, STLV-3 seroreactivity has been referred to as HTLV-2-like, though phylogenetic analysis clearly demonstrates the uniqueness of STLV-3 (Goubau et al., 1994
; Meertens et al., 2001
, 2002
; Meertens & Gessain 2003
; Takemura et al., 2002
; Van Dooren et al., 2001
). Therefore, the use of HTLV-1-based serological assays cannot reliably distinguish between STLV-3 and STLV-1/2 infections.
The recognition from recent data that STLV-3 is not a rare infection also highlights the importance of defining its prevalence among different primate species, understanding its genetic diversity and evolution and determining whether humans exposed to NHPs are infected with STLV-3-like variants. However, little is known about the epidemiology and genetic diversity of STLV-3. Full-length STLV-3 genomes have been obtained only for strains PH969, CTO-604, CTO-NG409 and PPA-F3. For all other STLV-3 strains, only short sequences in the pX and LTR regions are available (Takemura et al., 2002; Van Dooren et al., 2001
). Unlike STLV-1 and STLV-2, which are believed to be the simian equivalents of HTLV-1 and HTLV-2, it is not known whether STLV-3-like variants naturally infect humans. Limited testing of people with indeterminate HTLV serological results has found no evidence of STLV-3-like infections (Busch et al., 2000
; Vandamme et al., 1997
). However, phylogenetic studies of STLV-1 and HTLV-1 suggesting the occurrence of multiple interspecies transmissions of STLV (Gessain et al., 2002
; Mahieux et al., 1998
; Meertens et al., 2001
; Slattery et al., 1999
), combined with the ability of STLV-3 to grow in human cells in vitro (Goubau et al., 1994
), both suggest that humans may be at risk for cross-species infections with STLV-3. Zoonotic transmission of simian retroviruses to humans is not uncommon, since studies of workers occupationally exposed to NHPs have documented infection with a number of simian retroviruses, including simian immunodeficiency virus, simian foamy virus and simian type D retrovirus (Heneine et al., 1998
; Khabbaz et al., 1994
; Lerche et al., 2001
). Information on STLV-3 infection among captive primates may also help to assess the types of simian retroviruses that people may be occupationally exposed to. Thus, a better understanding of the genetic diversity of STLV-3 and its distribution among NHPs will help define the risks of transmitting these viruses to humans.
In this study we performed a serosurvey for STLV in gelada baboons (Theropithecus gelada) living in US zoos. We present here the serological and molecular characterization of the STLV found in these primates and describe the complete proviral sequence of a unique STLV-3 genome identified in one gelada baboon. In addition, we describe novel WB profiles in STLV-3-infected baboons and provide information regarding the genetic stability and transmission of STLV-3 among geladas housed at the same zoo.
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METHODS |
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Serological assays.
Initial screening for STLV antibodies in serum and plasma samples was performed by using the Vironostika HTLV-1/2 microelisa system (Organon-Teknika) following the manufacturer's instructions. Seroreactive samples were then tested in a WB test (HTLV Blot 2.4, Genelabs Diagnostics) containing disrupted HTLV-1 virions, a gp21 recombinant protein (GD21) common to both HTLV-1 and HTLV-2 and two HTLV-type-specific recombinant Env proteins, MTA-1 and K55, which allow serological differentiation of HTLV-1 and HTLV-2, respectively.
PCR analysis.
Generic nested PTLV tax and STLV-1 env or STLV-3-specific LTR PCR was performed on DNA lysates prepared from uncultured PBLs. The PTLV tax and STLV-3-specific LTR nested PCR was also performed on DNA extracted from frozen blood samples from seven other geladas. The primer sequences, expected product sizes and amplification conditions are given in Table 1. The generic PTLV tax primers used in the primary PCR amplification have been shown previously to amplify HTLV-1, HTLV-2 and divergent PTLVs, including STLV-2/Panp and STLV-3/PHA-PH969 (Busch et al., 2000
). In the current study, we also performed a semi-nested tax PCR using the primers PH2F and PH2R (Table 1
) to increase the amount of specific amplified product necessary for direct sequence analysis. The STLV-1 env and STLV-3 LTR primers were based on the HTLV-1/ATK and STLV-3/PHA-PH969 sequences with GenBank accession numbers J02029 and Y07616, respectively. To obtain the complete sequence of the unique gelada STLV-3, we PCR amplified sequential overlapping regions of the genome as previously described (Meertens et al., 2003
). Nested PCR products were electrophoresed in 1·8 % agarose gels and visualized by ethidium bromide staining.
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Neighbour-joining (NJ) and maximum-likelihood (ML) trees were constructed under the most appropriate evolutionary model determined with Modeltest 3-06 (Posada & Crandall, 1998). For the gag-pol-env-tax sequences, the general time-reversible model, allowing six different substitution rate categories, with gamma distributed rate heterogeneity and an estimated proportion of invariable sites, provided the best fit to the data. For the tax analysis, the transitional model allowing different rates among transitions including a gamma distributed rate heterogeneity seemed to be the best-fitting model. For the env sequences, the Tamura Nei evolutionary model was used with gamma distributed rate heterogeneity, taking into account a different substitution rate for transversions and purine and pyrimidine transitions. The NJ trees were constructed by iteratively optimizing the model parameters, followed by a bootstrap analysis of 1000 replicates. The ML trees were constructed starting from the NJ tree with optimized parameters using an heuristic search with the nearest neighbour interchange and the subtree pruningregrafting branch-swapping algorithm (Rogers & Swofford, 1999
). LTR sequences were aligned using the CLUSTAL W program (Thompson et al., 1994
) and distance-based LTR trees were generated using the Kimura two-parameter model in conjunction with the NJ method in the MEGA program (version 2.1) (Kumar et al., 2001
). Bootstrap replicates (1000) were used to test the reliability of the final topology of the LTR tree.
Molecular clock analysis and dating.
The molecular clock hypothesis was tested using the likelihood ratio test with the likelihoods for the ML and clock-like ML trees obtained in PAUP* (Swofford, 1998). The clock was tested with the best-fitting evolutionary model, estimated in Modeltest and with the NJ optimized parameters as described above. The evolutionary rate and subsequently the divergence times were estimated based on a known divergence time point and on the branch lengths of the ML clock tree according to the formula: evolutionary rate (r)=branch length (bl)/divergence time (t).
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RESULTS AND DISCUSSION |
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The tax and env sequences from the four remaining geladas with the HTLV-1-like WB profiles (TGE-598275, TGE-1884, TGE-2045 and TGE-5415) were identical and were found by BLAST analysis to be most similar to STLV-1 from mandrills and Celebes macaques (STLV-1/mnd and STLV-1/TE4, respectively). To characterize these STLV-1s further, we performed phylogenetic analysis of env sequences amplified from PBL DNA of these animals. This analysis showed that the gelada env sequences clustered with STLV-1 found naturally to infect Celebes macaques (Macaca tonkeana) (Fig. 2) (Ibrahim et al., 1995
). These four geladas were housed at the same zoo at one time on an island with Celebes crested and stump-tailed macaques (M. nigra and M. arctoides, respectively). Celebes macaques consist of seven primate species, including M. tonkeana and M. nigra, and thus the four gelada baboons were most likely infected via cross-species transmission from STLV-1-infected M. nigra. However, samples were not available from the M. nigra to investigate this hypothesis. None of the baboons in this study other than these four geladas were reported to have been in contact with other primate species, including other baboons.
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Phylogenetic analysis of concatenated gag-pol-env-tax sequences confirmed that TGE-2117 was infected with an STLV-3-like virus that clustered tightly with strain PH969 (Fig. 4a). A 180 bp tax sequence of all available STLV-3 sequences showed that TGE-2117 was closest to PH969 and Hyb2210 (Fig. 4b
). In both trees, the novel STLV-3/TGE-2117 was clearly related to the Ethiopian and Eritrean baboon strains, with strong bootstrap support of 100·0 in the gag-pol-env-tax trees (Fig. 4a
).
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Since STLV-3 is the most recently identified PTLV, very little is known about its transmission or genomic stability in infected animals. Sera archived in 1981 from five seropositive geladas from the same zoo included a captive-born mother (TGE-BK1), a wild-born father (TGE-BK3), their female offspring (TGE-2117) who was born in 1979 and two other females (TGE-BK2 and TGE-BK5). LTR sequences from the mother and TGE-2117 were identical and clustered together with 100 % bootstrap support by phylogenetic analysis (Fig. 5), suggesting a mother-to-offspring transmission of STLV-3 infection in this pair. The LTR sequences from two other STLV-3-infected female geladas (TGE-BK2 and TGE-BK5) were essentially identical to male gelada TGE-BK3, having >99 % nucleotide identity, suggesting a horizontal transmission. We also examined the genetic stability of STLV-3 over time by analysis of LTR sequences obtained from blood specimens from TGE-2117 and her parents that were collected 20 years apart. Interestingly, the LTR sequences for both parental baboons and the descendant TGE-2117 were identical, suggesting a high genetic stability of the STLV-3 genomes similar to that seen for other PTLVs (Slattery et al., 1999
). Viral persistence by oligoclonal expansion of infected lymphocytes within animals is believed to confer the high genetic stability of the PTLVs (Gabet et al., 2003
). This finding contrasts with the genetic mutability of other exogenous retroviruses, such as the human immunodeficiency virus, which has mutation rates 34 logs higher (Sharp et al., 1994
).
This study reports the serological and molecular characterization of a novel STLV-3 in Ethiopian T. gelada baboons and the full-length sequence analysis of this distinct STLV-3 found in animal TGE-2117. This report is the first to find and characterize STLV-3 and STLV-1 infection in this baboon species. The absence of STLV infection of geladas observed by others (Takemura et al., 2002) may be explained by differences in the sensitivities of the antibody screening assays used in each study and/or the sampling of gelada specimens originating from different Ethiopian locations (Takemura et al., 2002
). Interestingly, the STLV-3 LTR sequences obtained from five gelada baboons in the current study were more genetically related to an STLV-3 LTR sequence found in a wild-born Ethiopian P. hamadryas and P. anubis hybrid (Hyb2210) than to those found in either Eritrean or Ethiopian hamadryas baboons. These results suggest that the STLV-3s in the gelada and hybrid baboons are distinct from those found in hamadryas baboons and that the origin of the STLV-3s in the hybrid baboons may be from the gelada and not from infected hamadryas baboons as previously proposed (Takemura et al., 2002
).
Although fossil evidence suggests that the extant Theropithecus baboons once radiated across the African continent and exceeded the combined ranges of the Papio species, the gelada's current habitat is restricted to the high plateaus of Ethiopia (Pickford, 1993). In this region, gelada baboons are commonly sympatric with anubis and hamadryas baboons but occupy different ecological niches. In contrast, anubis and hamadryas baboons commonly interbreed, as reported elsewhere (Takemura et al., 2002
). None the less, these overlapping habitats may have provided opportunities for cross-species transmission of STLV-3 infections to or from each of these baboon species.
Questions about whether humans are infected with STLV-3-like viruses are raised by the widespread distribution of STLV-3 across Africa and its probable ancient existence (Goubau et al., 1994; Meertens et al., 2001
, 2002
; Meertens & Gessain, 2003
; Takemura et al., 2002
; Van Dooren et al., 2001
), the ability of STLV-3 to grow in human cells in vitro (Goubau et al., 1994
) and evidence for multiple interspecies transmissions that have occurred among the PTLV-1s (Gessain et al., 2002
; Slattery et al., 1999
). The presence of STLV-3-like viruses in primates such as geladas that are commonly hunted for their coats or shot while raiding crops may support the possibility of such infection in humans (Pickford, 1993
). In addition, the human population density on the Ethiopian plateau is among the highest in sub-Saharan Africa, thus placing the gelada in coexistence with humans living in this region and putting humans at risk for cross-species infections with STLV and other simian retroviruses. The finding by us and others of STLV-1 and STLV-3 infections in captive baboons also suggests that people working with these primates may be at risk for occupational exposure to these viruses (Goubau et al., 1994
, Meertens & Gessain, 2003
; Takemura et al., 2002
).
HTLV-1, HTLV-2 and HTLV-indeterminate WB profiles have all been observed in Ethiopian populations (Buckner et al., 1992; Vrielink et al., 1995
), but it is not known if any of these reactive samples are associated with STLV-3-like infections. While limited studies of 24 African and 325 non-African human specimens with HTLV-seroindeterminate WB results have detected no STLV-3-like infection (Busch et al., 2000
; Vandamme et al., 1997
), additional studies of larger numbers of human samples with not only indeterminate but also HTLV-1- and -2-like WB profiles will be necessary to assess fully the rate of infection with STLV-3-like viruses. Our finding of one STLV-3-infected gelada baboon with an HTLV-1-like WB profile and a previous report of an STLV-1-infected baboon with an HTLV-2-like serotype (Mahieux et al., 2000
) also raises questions about the accuracy of viral typing of PTLV infections by the currently used HTLV-1- and HTLV-2-type-specific Env peptides spiked onto HTLV-1 WB strips. Our results also highlight the need for better STLV-3-specific diagnostic assays to facilitate screening for STLV-3-like infections in both humans and NHPs.
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ACKNOWLEDGEMENTS |
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Use of trade names is for identification only and does not imply endorsement by the US Department of Health and Human Services, the Public Health Service, or the Centers for Disease Control and Prevention.
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Received 4 September 2003;
accepted 16 October 2003.