Long Terminal Repeat Sequence Analysis of HTLV-2 Molecular Variants Identified in Southern Brazil

H uman T-lymphotropic virus type 2 (HTLV-2) appears to be an endemic infection of ancient people in both Africa and the Americas, but epidemic among intravenous drug users (IDUs) in urban areas of North America, Europe, and Southeast Asia. ^1,2 In Brazil, HTLV-2 has long been considered endemic among indigenous population from the Amazon region (North) ^3,4 and recently in the South region. ⁵ HTLV-2 had been detected in low frequencies in the general population as blood donors, ^{6 –8} and in high frequencies in HIV/AIDS and IDUs, mostly of the Southeast and South of Brazil. ^{9 –11} Thus, in Brazil, HTLV-2 infection is endemic in the North and epidemic in the South.

The routes of HTLV-2 transmission depends on the population analyzed, where sexual intercourse and breast-feeding are the main forms among Amerindians and the general population of Brazil, ^4,7 while blood-born transfusion by sharing HTLV-infected needles account for transmission/acquisition among IDUs. ^{6,9 –11}

Molecular characterization of env, long terminal repeat (LTR), and tax genomic regions of HTLV-2 has recognized three HTLV-2 subtypes: HTLV-2a, HTLV-2b, and HTLV-2d. In Brazil, a unique HTLV-2 subtype was detected among IDUs and Amerindians; this subtype possesses a long transactivating protein Tax similar to HTLV-2b and the env, and LTR genomic regions similar to HTLV-2a. ⁴ Therefore, this HTLV-2 subtype was tentatively named HTLV-2c, ⁴ albeit complete nucleotide sequences of several genomes had unequivocally demonstrated that the Brazilian HTLV-2 strains are molecular variants of the HTLV-2a subtype. ^{12 –14} Interestingly, molecular analysis of Brazilian HTLV-2a isolates disclosed that they have originated from two sources: Brazilian Amerindians and European/North American IDUs. ¹⁵ HTLV-2b subtype, although endemic in Amerindians from Central America, Colombia, Venezuela, Chile, and Argentina, was only recently detected in Brazil among blood donors from the South, ¹⁶ and in IDUs infected with HIV from the Southeast. ¹⁷

Several years ago, we conducted a study on the prevalence of HTLV-1 and HTLV-2 in HIV/AIDS patients from Londrina and vicinities, South of Brazil, and disclosed high frequency of HIV/HTLV-2 co-infection in these patients. ¹¹ Using restriction fragment length polymorphism (RFLP) analysis of LTR and env genomic regions, we identified HTLV-2a circulating in this geographic region. ¹⁸ At present, we decided to extend this data searching for HTLV-2a subgroups correlating with risk factor for HTLV transmission/acquisition. Using a classification system that accommodate restriction sites described by Switzer ¹⁹ and Eiraku, ²⁰ we employed seven enzymes in RFLP LTR genomic analysis (i.e., Dra II, Ban II, Ava II, Sau I, Bgl I, SacII, and Sma I). Nested polymerase chain reaction was used to amplify a 666-bp LTR region (nt. 64 to 729), ¹⁸ and the products obtained were submitted to RFLP and sequencing analysis. Predicted endonuclease maps were constructed using Sequencher 4.7 software and NEBcutter V2.0 (http://tools.neb.com/NEBcutter2/index.php) tools and compared with RFLP gel patterns.

LTR-amplified products were sequenced in a ABI 3100 Genetic Analyser (Applied Biosystems, Foster City, CA). All sequencing chromatograms obtained were assembled and manually edited with Sequencher 4.7 software. Multiple alignments were performed using ClustalW multiple-sequence alignment software, with a reference set available in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank). HTLV-2 LTR subtype was screened at NCBI (http://www.ncbi.nlm.nih.gov) using Genotyping and BLAST tools. Neighbor Joining (NJ) and Maximum Likelihood (ML) trees were constructed based in appropriate nucleotide substitution models determined by Modeltest v3.7 (HKY+i+G model), using PAUP (v4b10). Bootstrapping was performed using the stepwise addition algorithm for 1000 replicates.

Phylogenetic analysis of a segment of 458 bp of 88 HTLV-2 LTR sequences including the fourteen new sequences of Londrina, Southern Brazil (GenBank accession number GU573730 to GU573743) were used to construct the phylogenetic tree (Fig. 1).

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FIG. 1.

Representative Neighbor Joining phylogenetic tree of partial HTLV-2 LTR region (458 bp) generated with the HKY+i+G model, using Paup 4b10. Bootstrap values above 70% in key branches are depicted. Sequences from Londrina, Southern Brazil are in bold.

Seventy sequences were obtained in GenBank: 21 sequences of Brazilian Amerindians, blood donors, and IDUs (19 of subtype 2a, and two of subtype 2b), 49 sequences of endemic and epidemic populations of other countries [15 of subtype 2a, 33 of subtype 2b, and one of subtype 2d (considered as the outgroup)] along with four sequences from Sao Paulo (Southeast Brazil) not published yet (SP17, SP281, SP305, SP510), were included in the analysis.

As shown in Figure 1, all HTLV-2 sequences from Londrina clustered into subtype 2a beside other Brazilian sequences. Figure 2 shows in detail HTLV-2a cluster, grouping the new sequences into three main subgroups named a4, a5, and a6, according to RFLP. Table 1 shows the predicted fragments obtained by RFLP maps, confirming the results obtained by phylogenetic trees (Figs. 1 and 2) and gel profiles (not shown). Interestingly, one of sequences (BRLO23-02) although classified as subgroup a4 by RFLP, clustered apart of this subgroup on the phylogenetic tree, but near to other Brazilian sequences; RP329 and K96 that belong to a blood donor from Ribeirão Preto and a Kayapo Indian from Amazonia. ¹⁴ Of note, one branch of HTLV-2a subgroup a6 was almost exclusively composed of sequences from Londrina. Additionally, sequences from Sao Paulo, not yet in the GenBank, clustered in the same three groups of HTLV-2a from Londrina. Although the limited number of samples analyzed, we could speculate on the route of virus transmission/acquisition. In fact, when we searched for risk factors of such patients, sequences of subgroup a4 (including the BRLO23-02) were of individuals of sexual risk factor, while sequences of subgroups a5 and a6 were of IDUs.

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FIG. 2.

Representative Maximum Likelihood phylogenetic tree of partial HTLV-2a LTR region (458 bp) generated with the HKY+i+G model, using Paup v4b10. The analyzed samples are showed in bold.

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Table 1.

Predicted Fragments Obtained by Sequencing and Restriction Fragment Length Polymorphism Analysis of a Product of 458-bp (nt 181 to 638) of HTLV-2 LTR Region of Sequences from Londrina, Southern Brazil

Enzymes	Subgroup a4 fragment size	Subgroup a5 fragment size	Subgroup a6 fragment size	Subgroup a6′ fragment size
AvaII	225/233	225/233	225/233	225/233
DraII	458	458	458	458
BanII	293/165	135/158/165	137/156/165	293/165
Bsu36I	303/155	303/155	303/155	303/155
SacII	458	458	458	458
BgII	450/8	450/8	450/8	450/8
SmaI	340/118	340/118	458	458

Sequences belonging to each subgroup according to Switzer et al., 1995 classification system:¹⁹ HTLV-2 a4: BRLO18, BRLO19, BRLO23, BRLO24, BRLO27, BRLO29, and SP305; HTLV-2 a5: BRLO7, SP17, and SP510; HTLV-2 a6: BRLO9, BRLO12, BRLO21, BRLO22, BRLO28, BRLO31, and SP281; HTLV-2 a6’: BRLO26.

These results corroborate the results obtained in previous studies that clustered Brazilian viruses in a single HTLV-2a clade, although with considerable diversity among them. ^{2,12 –14,17,19,20} Additionally, several studies conducted with Brazilian HTLV-2 sequences pointed virus transmission from Indians to IDUs of urban areas, ^{1 –4,12 –14,17,19,20} but the finding of a group almost exclusively of IDUs (subgroup a6) could represent different route of virus transmission or different evolutionary rate. In accordance of the last hypotheses, higher evolutionary rate and genetic diversity of HTLV-2 are detected in European IDUs than in Indians endemic populations. ²¹ Therefore, we could speculate these events to occur among IDUs of Londrina, Southern Brazil.

Moreover, the present study confirms RFLP as an alternative method for performing HTLV-2 epidemiological studies in countries with poor resources setting. In conclusion, this is the first study on molecular characterization of HTLV-2 sequences from Londrina, Southern Brazil, providing insights in HTLV-2 dynamic epidemic in Brazil.