Identification and Characterization of a New and Distinct Molecular Subtype of Human T-Cell Lymphotropic Virus Type 2

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Identification and Characterization of a New and Distinct Molecular Subtype of Human T-Cell Lymphotropic Virus Type 2
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   1996, 70(3):1481. J. Virol. and W M HallGuerreiro, M P de Oliveira, P Loureiro, N Hammerschlak, S IjichiCosta Ferreira, S W Zhu, R Lorenco, M Ishak, V Azvedo, JN Eiraku, P Novoa, M da Costa Ferreira, C Monken, R Ishak, O da lymphotropic virus type 2.distinct molecular subtype of human T-cellIdentification and characterization of a new and information and services can be found at: These include:  CONTENT ALERTS  more»cite this article),Receive: RSS Feeds, eTOCs, free email alerts (when new articles Information about commercial reprint orders: To subscribe to to another ASM Journal go to:  on O c  t   o b  er 2  9  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  on O c  t   o b  er 2  9  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  on O c  t   o b  er 2  9  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  on O c  t   o b  er 2  9  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  on O c  t   o b  er 2  9  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  on O c  t   o b  er 2  9  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  on O c  t   o b  er 2  9  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  on O c  t   o b  er 2  9  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  on O c  t   o b  er 2  9  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  on O c  t   o b  er 2  9  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  on O c  t   o b  er 2  9  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  on O c  t   o b  er 2  9  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  on O c  t   o b  er 2  9  ,2  0 1  3  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   J OURNAL OF V IROLOGY , Mar. 1996, p. 1481–1492 Vol. 70, No. 30022-538X/96/$04.00 ϩ 0Copyright ᭧ 1996, American Society for Microbiology Identification and Characterization of a New and Distinct MolecularSubtype of Human T-Cell Lymphotropic Virus Type 2 NOBUTAKA EIRAKU, 1 PATRICIA NOVOA, 2 MARIZETE DA  COSTA FERREIRA, 2 CLAUDE MONKEN, 3 RICARDO ISHAK, 4 ORLANDO DA  COSTA FERREIRA, 2 SHI WEI ZHU, 1 ROSEMARIE LORENCO, 2 MARLUISA ISHAK, 4 VANIA AZVEDO, 4 JOAO GUERREIRO, 4 MARIA POMBO DE OLIVEIRA, 5 PAULA LOUREIRO, 6 NELSON HAMMERSCHLAK, 7 SHINJI IJICHI, 8  AND WILLIAM W. HALL  1 *  Laboratory of Medical Virology, The Rockefeller University, New York, New York 10021 1  ; Emilo Ribas Institute of  Infectious Diseases 2  and Hospital Israelita Albert Einstein, 7 Sao Paulo, Federal University of Para, Belem, 4  NationalCancer Institute, Rio de Janiero, 5  and Hemope, Recife, 6  Brazil; Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107  3  ; and Department of Internal Medicine, Kagoshima University, Kagoshima, Japan 8 Received 16 August 1995/Accepted 28 November 1995 Molecular studies have demonstrated the existence of at least two major subtypes of human T-cell lympho-tropic virus type 2 (HTLV-2), designated HTLV-2a and HTLV-2b. To further investigate the heterogeneity of this family of viruses, we have characterized the HTLV-2 subtypes present in several urban areas in Brazil.DNAs from peripheral blood mononuclear cells of a large number of infected individuals, the majority of whom were intravenous drug abusers, were analyzed by using PCR with restriction fragment length polymorphismand nucleotide sequencing analysis. Restriction fragment length polymorphism analysis of the env regionsuggested that all individuals were infected with the HTLV-2a subtype, and this was confirmed by nucleotidesequence analysis. In contrast, nucleotide sequence analysis of the long terminal repeat demonstrated thatalthough the viruses were more related to the HTLV-2a than to the HTLV-2b subtype, they clustered in adistinct phylogenetic group, suggesting that they may represent a new and distinct molecular subtype of HTLV-2. This conclusion was supported by nucleotide sequence analysis of the pX region, which demonstratedthat the Tax proteins of the Brazilian viruses differed from that of prototype HTLV-2a isolates but were moresimilar to that of HTLV-2b in that they would be expected to have an additional 25 amino acids at the carboxyterminus. In transient expression assays, the extended Tax proteins were found to be much more potenttransactivators of the virus long terminal repeat than the Tax protein of the prototype HTLV-2a subtype. Thestudies suggest that the Brazilian viruses analyzed in this study, while being phylogenetically related to theprototypic HTLV-2a seen in North America, are phenotypically more related to HTLV-2b and can be justifiablyclassified as a new molecular subtype, which has been tentatively designated HTLV-2c. Human T-cell lymphotropic virus type 1 (HTLV-1) and type2 (HTLV-2) are closely related mammalian retroviruses whichshare a number of biological properties and have a tropism forT lymphocytes (22, 27, 44, 54). HTLV-2 infection is endemic ina number of American Paleo-Indian populations (1, 2, 4, 6, 7,10, 14, 25, 26, 29, 33, 35, 37, 41, 43, 51, 56), and high rates of infection occur in intravenous drug abusers (IVDAs) in partsof North America, Europe, and Southeast Asia (13, 22, 44, 57). A number of studies of the viruses present in these populationshave demonstrated that there are two major molecular sub-types of HTLV-2, which have been designated HTLV-2a andHTLV-2b (5, 8, 22, 26, 44, 53). Whereas HTLV-2a has beenclearly shown to be the predominant infection in IVDAs inurban areas of North America (8, 44), HTLV-2b predominatesin the Paleo-Indian groups (5, 22, 41, 44, 52). To date,HTLV-2b has been shown to be the exclusive infection in theGuaymi of Panama (43), the Wayu of Colombia (29, 51), andthe Toba and Mataco groups of Argentina (10). In contrast,studies of Indian populations in North America have shownthat whereas HTLV-2b infection appears to predominate, anumber have infection with both subtypes (26). It seems pos-sible that these populations have endemic infection with onesubtype and that the second has only recently been introducedby interactions with individuals who had other risk factors forinfection.In contrast to the studies on the aforementioned Indiangroups, we have recently demonstrated that the Kayapo Indi-ans, an indigenous population of Amazon region of Brazil, areinfected with a distinct variant of the HTLV-2a subtype (30).Unfortunately, because of the small sample number and thelimited molecular analysis, it was impossible to determine if this virus should be considered a distinct and unique molecularsubtype. In the present study, we have attempted to character-ize HTLV-2 infections in urban areas of Brazil and to comparethese viruses with those present in the Kayapo Indian group.This effort has involved detailed nucleotide sequence analysisof three regions of the provirus, the env , long terminal repeat(LTR), and pX regions. The studies have demonstrated thatthe viruses in the urban areas are closely related to that presentin the Kayapo Indians but that they are phylogenetically dis-tinct from the HTLV-2a and HTLV-2b subtypes present inNorth America and Europe. This finding together with prelim-inary analysis of their phenotypic properties supports the viewthat these isolates represent and can be justifiably classified asa new and distinct molecular subtype of HTLV-2. MATERIALS AND METHODS Study population. Twenty-eight individuals from Sao Paulo (  n ϭ 24), Rio deJaniero (  n ϭ 2), and Recife (  n ϭ 2), 27 of whom were intravenous drug abusers(IVDAs), were identified as being seropositive for HTLV-2 by enzyme-linkedimmunosorbent assay (ELISA) and confirmatory Western blot (immunoblot) * Corresponding author. Mailing address: Laboratory of MedicalVirology, The Rockefeller University, 1230 York Ave., New York, NY10021. Phone: (212) 327-8581. Fax: (212) 327-8582. Electronic mailaddress:  analysis; 26 of the latter had concomitant human immunodeficiency virus infec-tion. Seropositive individuals were identified by initial screening for antibodies toHTLV-1 and/or HTLV-2 by ELISA (Coulter Immunology, Hialeah, Fla.). Re-active samples were analyzed by Western blotting (HTLV Blot 2.3; DiagnosticBiotechnology, Singapore) to confirm seropositivity and to differentiate HTLV-1and HTLV-2 infections. The srcins of the samples obtained from the KayapoIndian group have been previously described (30).Peripheral blood mononuclear cells from infected individuals were separatedby Ficoll-Hypaque centrifugation, and DNA was extracted by standard phenol-chloroform extraction methods. Amplification of the env region encoding thetransmembrane glycoprotein gp21 for restriction fragment length polymorphism(RFLP) and nucleotide sequence analysis was carried out by using nested PCR with methods previously described (23, 30). For amplification and nucleotidesequencing analysis of the LTR, nested PCR was carried out as follows. Reac-tions were performed in 100- ␮ l volumes containing 1 ␮ g of extracted DNA, 225 ␮ M each deoxynucleoside triphosphate, 100 pmol of each primer, 50 mM KCl,2.0 mM MgCl 2 , 10 mM Tris-HCl (pH 8.3), and 0.5 U of  Taq polymerase (Perkin-Elmer Cetus Corp., Norwalk, Conn.). Primer pairs used in the first round were5 Ј -CCTTACCCACTTCCCCTAGCGCTGA-3 Ј and 5 Ј -GGGAAAGCCCGTGGATTTGCCCCAT-3 Ј , corresponding to nucleotides (nt) 194 to 218 and 807 to831 of the HTLV-2-MoT isolate. The second round was carried out with thenested primers 5 Ј -AAAAGCGCAAGGACAGTTCAGGAGG-3 Ј and 5 Ј -ATCCCGGACGAGCCCCCACTTGTTT-3 Ј , corresponding to nt 290 to 314 and 759to 783. Five microliters of the initial amplified product was used in the nestedreaction. For RFLP analysis of the LTR, PCR was used to generate a 553-bpamplified product. After the first-round PCR as just described, a second primerpair, 5 Ј -TGACGATTACCCCCCTGCCCATAAA-3 Ј and 5 Ј -ATCCCGGACGAGCCCCCACTTGTTT-3 Ј , corresponding to nt 231 to 254 and 759 to 783 of the HTLV-2-MoT isolate, was used in the reaction to produce the nested prod-uct. The synthesis of this LTR product, which is larger than that used in thenucleotide sequence analysis, was necessary to achieve satisfactory resolution of all DNA fragments in the RFLP analysis of the HTLV-2a subtype (8). In eachexperiment, after an initial denaturation for 5 min at 94 Њ C, 35 cycles were carriedout in a DNA thermocycler (Perkin-Elmer Cetus). Each cycle consisted of denaturation for 40 s at 94 Њ C, annealing for 30 s at 57 Њ C, and extension for 1 minat 72 Њ C. The 35 cycles were followed by a 10-min extension at 72 Њ C. For ampli-fication and nucleotide sequencing of the entire pX region, nested PCR wascarried out to prepare two overlapping products (nt 7192 to 7791 and 7713 to8285). Primers in the first round for the first product were pX101 (nt 7153 to7173; 5 Ј -GGCAATCTCCTAAAATAGTCT-3 Ј ) and pX102 (nt 7815 to 7795;5 Ј -TGTGTGTAGGAACATTTTGTA-3 Ј ); primers in the nested reaction werepX103 (nt 7192 to 7212; 5 Ј -TTACAATCCTGTCTCCTCTCA-3 Ј ) and pX104 (nt7791 to 7771; 5 Ј -AAGTTCTTCTAATCGTTTTAG-3 Ј ). For the second overlap-ping product, the first-round primers were pX105 (nt 7683 to 7703; 5 Ј -GCTTTCCCCACCCATGACATG-3 Ј ) and pX106 (nt 8326 to 8306; 5 Ј -GGGCCGTGGTTTCAGTTCCTA-3 Ј ), and primers in the nested PCR were pX107 (nt 7713to 7733; 5 Ј -ACCCCATGTCATATTCTGCCA-3 Ј ) and pX108 (nt 8285 to 8265;5 Ј -AGCCTTTACTTGGGATTGTTT-3 Ј ). Reaction conditions were identicaland, following initial denaturation at 94 Њ C for 5 min, involved 35 cycles of denaturation at 94 Њ C for 40 s, annealing at 52.5 Њ C for 30 s, and chain elongationat 72 Њ C for 40 s. Upon completion of each PCR, an additional extension at 72 Њ Cfor 10 min was carried out.For amplification of the pX region which contains the expected stop codon of  tax , the following reaction conditions were used. The initial PCR was carried out with primers pX105 and pX106 as described above and was followed by nestedPCR using primers pX106 (see above) and LS1 (nt 8140 to 8160; 5 Ј -GAATA CACCAACATCCCTGTC-3 Ј ). Reaction conditions were as described above,and amplified products were analyzed by agarose gel electrophoresis withethidium bromide staining.In view of the large number of PCRs and particularly those involving the LTR,maximum precautions were taken to avoid contamination (32). Physically sepa-rate rooms were used for DNA isolation, preparation of reaction mixes, thePCR, and analysis of amplified products. All reactions were carried out withappropriate positive and negative controls. It should also be noted that contam-ination between the Kayapo Indian and IVDA samples would be highly unlikely,as studies of the former were carried out in different laboratories and at anearlier time. RFLP analysis. The amplified env gene product was directly digested with  Xho I as described previously (23). Digestion of the amplified product of HTLV-2a by this enzyme would be expected to generate two fragments (430 and180 bp), while the amplified product of HTLV-2b lacks the restriction site and isnot cleaved. Digestion products were visualized in agarose gels after staining withethidium bromide. RFLP analysis of the LTR was carried out as describedpreviously (8). PCR products were initially purified by using Wizard PCR Preps(Promega), and 10 ␮ l of the purified DNA was directly digested with 5 U of   Ava II, Ban II, Bgl I, Bsu 36I, Mse I, and Sma I (Boehringer Mannheim, Indianapo-lis, Ind.), either singly or in combination, using conditions recommended by themanufacturer. Products were electrophoresed on 3.0% agarose gels and visual-ized by ethidium bromide staining. Cloning and nucleotide sequence analysis of PCR-amplified products. Fol-lowing PCR and electrophoresis on low-melting-temperature agarose gels, theamplified products from the env , LTR, and pX regions were excised and clonedinto the pCR-Script SK( ϩ ) vector (Stratagene, La Jolla, Calif.), using conditionsrecommended by the manufacturer and as previously described (8). PlasmidDNA from recombinant clones was extracted by using the Promega miniprepsystem (Promega). Nucleotide sequence analysis was carried out on two to threeclones of each sample. DNAs were sequenced in both directions, using anautomatic sequencing apparatus (Applied Biosystems, Norwalk, Conn.). Phylogenetic analysis. Nucleotide sequences were aligned by using CLUSTAL W (version 1.5), obtained from the EMBL file server (12, 42). Phylogenetic trees were constructed by using programs from both the PHYLIP (version 3.5c)package (9) and the CLUSTAL W package (55). The nucleotide distancesamong the envelope clones were determined on nonidentical sequences, usingthe DNADIST program and correcting for evolutionary distance by using theKimura two-parameter model (31). A phylogenetic tree was constructed byleast-squares analysis using the FITCH program. To assess the reliability of thegroupings obtained with FITCH, 1,000 bootstrap replicates were generated byusing the SEQBOOT program, and the trees were analyzed by the CONSENSEprogram. Equivalent phylogenetic relationships were found among the envelopeclones by using the maximum-likelihood analysis program DNAML and by theneighbor-joining method (45), using the NEIGHBOR program. Phylogenetictrees for the LTR clones were constructed by using the neighbor-joining method(45) as implemented in CLUSTAL W. The reliability of these trees was statis-tically evaluated by using 10,000 bootstrap replicates. Equivalent phylogeneticrelationships among the LTR clones were found by using a least-squares analysis(FITCH) and a maximum-likelihood analysis (DNAML). The analysis includedthe 8 Brazilian IVDA samples and 25 other HTLV-2 LTR sequences, 18 se-quenced in our laboratory (8) and 7 from the published literature. Details of theformer have been previously published; these samples included 8 which had beenidentified as HTLV-2a and 10 identified as HTLV-2b (8). The latter included thetwo prototype subtypes of HTLV-2a and -2b, MoT (48) and NRA (34); G12, anisolate from a Guaymi Indian (43); two isolates from the Wayu Indians of Colombia (51); PygCam1, an isolate from a prostitute from Cameroon (38); andKT, an isolate from a prostitute from Ghana (28). Also included were two LTRsequences of the virus endemic in the Kayapo Indians of Brazil, one of which hasbeen recently published (30). Tax constructs. The expression vector pCG, used for the expression of Tax proteins, was kindly provided by M. Yoshida, Tokyo University, Tokyo, Japan.The pCG vector contains the cytomegalovirus promoter, the herpes simplex virus tk gene, 5 Ј untranslated leader, and initiation codon, rabbit ␤ -globin splicing andpolyadenylation signals, and the replication srcin of simian virus 40. To preparethe HTLV-2a Tax protein (Tax a), the extended Tax protein representative of the HTLV-2b isolates, and selected deletion mutants of these proteins, PCR wasused to amplify the corresponding gene regions with primers containing Xba Iand Bam HI restriction endonuclease sites in the 5 Ј and 3 Ј ends, respectively.Primers used in the PCR were as follows. An upstream primer 5 Ј -CGGGCTTCTAGACATGGCCCATTTCCCAGGATT-3 Ј (  Xba I site underlined) wasused in the preparation of both Tax a and the extended Tax proteins. Thedownstream primers for Tax a and the extended Tax protein were 5 Ј -GTAACTGGATCCCTAGTCGCCATTGTCATCCGA-3 Ј and 5 Ј -CGCCGCGGATCCTTACTTGGGATTGTTTGTGTG-3 Ј , respectively (  Bam HI sites underlined). Toprepare Tax a with a 55-amino-acid deletion in the carboxy terminus, the down-stream primer 5 Ј -CGCGGCGGATCCCTAGGAGGAGTATTGTATAAG-3 Ј (  Bam HI site underlined) was used. Thirty-five cycles of denaturation at 94 Њ C for30 s, annealing for a 1 min at 63 Њ C, and chain elongation at 72 Њ C for 2 min wereused in all reactions. Amplified products were initially cloned in the pCR-ScriptSK( ϩ ) vector as described above, and the purified plasmids were subsequentlydigested with Xba I and Bam HI. Inserts were separated by agarose gel electro-phoresis and directly ligated to the pCG vector which had been previouslydigested with Xba I and Bam HI. Transfections. Jurkat cells were transfected with the pCG Tax constructs andpHTLV-II-LTR-CAT (srcinally derived from pVS 2 CAT and kindly providedby K. Shimotohno, National Cancer Center Research Institute, Tokyo, Japan),using Lipofectin (Promega) as described by the manufacturer. One microgram of each plasmid was used to transfect 5 ϫ 10 6 cells which had been grown inantibiotic-free RPMI medium containing 10% fetal calf serum and subsequently washed twice with Opti-MEM (Gibco) without serum. Following transfection,cells were incubated for 3 h at 37 Њ C, at which time the Lipofectin-DNA com-plexes were removed. Cells were then incubated in RPMI containing 10% fetalcalf serum without antibiotics for an additional 48 h. CAT assays. Cells were collected, washed twice in ice-cold phosphate-bufferedsaline, resuspended in 300 ␮ l of Reporter lysis buffer (Promega), and incubatedat 60 Њ C for 15 min to inactivate endogenous deacetylase activity. After centrif-ugation, supernatants (100 ␮ l) were incubated with 3 ␮ l of [ 14 C]chloramphenicol(0.05 mCi/  ␮ l) and 5 ␮ l of  n -butyryl coenzyme A (5 ␮ g/  ␮ l), and reactions wereallowed to proceed at 37 Њ C for 2 h. Five hundred microliters of ethyl acetate wasadded to each sample, and after vortexing and centrifugation at 14,000 rpm for5 min, the upper organic phase was removed and evaporated to dryness on aSavant DNA SpeedVac apparatus. The residues were suspended in 30 ␮ l of ethylacetate, and 10- ␮ l samples were spotted onto thin-layer chromatography silicagel plates (J. Baker SG/IBZ plates) which had been preequilibrated for 1 h withchloroform-methanol (97:3). Chromatography was continued for 60 min, and theplates were then dried at room temperature and exposed to X-Omat X-ray filmfor 24 h with intensifying screens. After chromatography, the butyrylated prod- 1482 EIRAKU ET AL. J. V IROL  .  ucts were scraped from the plate and counted by liquid scintillation. Chloram-phenicol acetyltransferase (CAT) activity was averaged from a minimum of threeseparate assays.  Accession numbers. The GenBank accession numbers (first term) of theHTLV-2 env fragments included in the phylogenetic study (second term) are asfollows: U32905, 408N; U32901, MSA1bp; U32903, DOG (WH2); U32906,130P; U32904, 72969N; U32902, GAR (WH6); U32900, PAR (WH7); U19110,Kayapo 1 (KAY1); U19109, KAY2; U32899, Sao Paulo 1 (SP1); U32898, SP2;U32897, SP3; U32896, SP4; U32895, SP5; Z46837, PH230PCAM; PygCam1 (17). A number of additional sequences of the env region which were available foranalysis were not included, as these were found to have sequences identical tothose of the representative HTLV-2a (DOG) and HTLV-2b (130P) samplesincluded in the analysis (23, 26). The GenBank accession numbers of theHTLV-2 LTR fragments sequenced in our laboratory and included in the phy-logenetic study are as follows: L37129, WY; L37130, CAM; L37131, DOG;L37132, DSA; L37133, FH; L37134, FLN; L37135, FUC; L37136, GAR; L37137,JD; L37138, MIN; L37139, SAC; L371140, SC; L37141, VIN; L37142, AG;L37143, ASB; L37144, MER; L37145, PAR; L37146, WEN; U25135, KAY1;U32887, KAY2; U32888, SP1; U32889, SP2; U32890, SP3; U32891, SP4;U32892, SP5; U32893, SP6; U32894, SP7; U32886, Rio de Janeiro 1 (RJ1). Sevenpublished LTR sequences were also included in the analysis. In addition tosequences of the prototype (MoT, NRA, and G12) viruses (accession numbersare listed below), two sequences of HTLV-2b from two Wayu Indians (51),WYU-1 and WYU-2 (U12792 and U12794), and two of HTLV-2a from Africanprostitutes (28, 38) (PH230PCAM [GenBank accession number Z246838] andKT [28]) were included. The GenBank accession numbers for pX fragments areas follows: U32885, FLN; U32871, DOG; U32883, SAC; U32884, MIN; U32882,FUC; U32880, PAR; U32881, GAR; U32879, VIN; U32875, KAY1; U32874,KAY2; U32873, SP1; U32872, SP2; U32878, SP3; U32877, SP4; U32876, SP5.The GenBank accession numbers for the complete nucleotide sequences of theprototype HTLV-2a (MoT) (40) and for HTLV-2b (NRA) (30) and G12 (35) areM1D060, L20734, and L11456, respectively. RESULTSRFLP and nucleotide sequence analysis of the env region. RFLP analysis using Xho I digestion of the PCR product of the  env region encoding the transmembrane glycoprotein, gp21, was carried out to differentiate infections by the two knownHTLV-2 subtypes. Previous studies have shown that whereasall HTLV-2a isolates contain this enzyme site, it is absent inHTLV-2b isolates (23). It could be shown that the amplifiedproducts of all 28 seropositive samples were effectively cleaved, yielding two fragments with expected sizes of 450 and 180 ntand demonstrating that all were infected with a virus related tothe HTLV-2a subtype. To confirm infection by the HTLV-2asubtype and to determine the relationship of the viruses bothto prototype isolates and to the virus endemic in the KayapoIndians, the nucleotide sequence of the amplified env product was determined in five randomly chosen samples (SP1 to SP5).Phylogenetic analysis of a 589-nt region clearly demonstratedthat all of the Brazilian sequences segregated with prototypicHTLV-2a isolates from North America (Fig. 1). While theisolates did not cluster on a single branch, distinct phylogeneticgroups within the HTLV-2a subtype could not be clearly iden-tified. This reflects the marked conservation of nucleotide se-quence in this region of the proviruses (23, 26, 29). Examina-tion of the nucleotide sequences of the env clones revealed thatHTLV-2a (Mo) (40) and HTLV-2b (NRA) (30) differ by 24residues in the region examined. The Kayapo clones matchedHTLV-2a at 20 positions and HTLV-2b at 4 positions and hadone unique residue. The isolates from the urban areas, SP1 toSP5, matched HTLV-2a at 21 positions and HTLV-2b at 3positions and had two unique residues. Nucleotide sequence analysis of the LTR region. To furtherclarify the relationship of the Brazilian viruses to prototypeHTLV-2 isolates, the phylogenetic relationship of a 449-ntregion of the LTR was analyzed. Previous studies have shownthat analysis of the LTR region permitted the identification of distinct phylogenetic clusters within both virus subtypes (8, 43).Figures 2A and B show unrooted and rooted phylogenetictrees, respectively; in these analyses, 8 sequences (SP1 to SP7and RJ1) analyzed in the present study were compared withthe sequence of the virus present in two Kayapo Indians and anadditional 25 sequences representative of HTLV-2a and -2bisolates. It could be shown that all of the Brazilian viruses, while being more closely related to the HTLV-2a than to theHTLV-2b subtype, clearly clustered in a distinct phylogeneticgroup. The reliability of the phylogenetic trees was statisticallyevaluated by using 10,000 bootstrap replicates and provided aconfidence level of 97 to 98% in the branching of the Braziliansequences. Equivalent phylogenetic relationships were ob-tained with each method used. Examination of the nucleotidesequences of the LTR clones revealed that HTLV-2a andHTLV-2b differ at 20 positions in this region. The Braziliansequences matched HTLV-2a at 17 positions and HTLV-2b at3 positions and had unique residues at 4 positions. The resultsalso established the close relationship of the viruses present inthe urban areas of Brazil to that present in the Kayapo Indians. FIG. 1. Phylogenetic analysis of 589 nucleotides the env gene region encod-ing the transmembrane glycoprotein gp21 of HTLV-2 isolates from five IVDAsfrom Sao Paulo (SP1 to SP5) and two Kayapo Indians (KAY1 and KAY2) withrepresentative HTLV-2a and HTLV-2b isolates, including the prototypeHTLV-2a isolate (MoT) and the prototype HTLV-2b isolate (NRA), represen-tative isolates of HTLV-2a from a New York City IVDA (DOG) and North American Indians (130P and MSA1bp), and corresponding HTLV-2b isolatesfrom New York City IVDAs (GAR and PAR) and North American Indians(408N and 60405N). Other available sequences of both subtypes in these twopopulations (26) were intentionally not included in the analysis, as they werefound to have sequences identical to those of the representative isolates indi-cated above. Also included in the analysis were isolate G12, from a GuaymiIndian, and PH230CAM and PygCam1 from African prostitutes. The srcins of these isolates are detailed in Materials and Methods. The bootstrap statisticalanalysis was applied, using 1,000 bootstrap replicates with HTLV-1 (ATK) as theoutgroup. V OL  . 70, 1996 HTLV-2 SUBTYPES 1483  FIG. 2. (A) Unrooted phylogenetic analysis of a 449-nt region of the LTR. Included in the analysis were isolates from eight IVDAs from Brazil (SP1 to SP7 andRJ1) and two Kayapo Indians and 25 other viruses representative of HTLV-2a and HTLV-2b subtypes. Details of the latter viruses are outlined in Materials andMethods. The bootstrap statistical analysis was applied, using 10,000 bootstrap replicates. (B) Rooted phylogenetic analysis of the same 449 nt of the LTR with HTLV-1(ATK) as the outgroup. Sequences analyzed were as in panel A. 1484 EIRAKU ET AL. J. V IROL  .
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