The effort to more fully characterize the subtypes in circulation around the Caribbean was initiated by an examination of the viruses from HIV-positive patients from four countries: Dominican Republic (DO), Haiti (HT), Jamaica (JM) and Trinidad and Tobago (TT). The nearly full-length genomes of HIV-1 were amplified from the viral RNA and sequenced and represent the first nearly full-length sequences of HIV-1 from the Dominican Republic and Jamaica and the first in over a decade from Haiti and Trinidad and Tobago. To evaluate the genetic distribution pattern of HIV-1 in the Caribbean region, we analyzed phylogenetic relationships and genetic variability among HIV-1 strains isolated from patient samples collected between 2000 and 2005 in seroprevalence studies in Trinidad and Tobago, Jamaica, Haiti, and the Dominican Republic. Materials and Methods Study Subjects Blood samples were drawn from patients attending the following centers: GHESKIO in Port-au-Prince, Haiti; Ministry of Health, Kingston, Jamaica; IDCP, Santo Domingo, Dominican Republic; Medical Research Foundation of Trinidad and Tobago, Port of Spain, Trinidad and Tobago. Patients were newly diagnosed as HIV(+) and had been infected for an unknown period of time. Serum was collected, frozen and sent to IHV, Baltimore, MD for analysis. All patients gave written informed consent and the study, including the notification of the subjects of their HIV status, was approved by the IRB of the University of Maryland, Baltimore, and the local participating institutions. Reverse Transcription and Amplification The methods for RNA extraction, reverse transcription (RT), polymerase chain reaction (PCR) and the primers used for the nearly full-length PCR amplification have been previously described [10,11]. Briefly, RNA was extracted from 140 ml of serum concentrated from 500 ml, using QIAamp Viral RNA Mini Assay (Qiagen, Valencia, CA). Reverse transcription of RNA was performed using SuperScriptTM III RNase H2 Reverse Tran- scriptase (Invitrogen, Carlsbad, CA) with RT3474R and UN- INEF79 primers for the partial pol region and the nearly full-length genome amplification, respectively. cDNA synthesized with UNINEF79 as primer was used as template for the nearly full- length PCR amplification with limiting-dilution methods [11]. This method utilizes one, two or three overlapping amplicons to cover most of the HIV genome, from 795 to 9180 on HXB-2 (Genbank Accession No. K03455) and is routinely successful with well preserved serum having a viral load of 105 or greater. The amplicon does not, however, include the U5, R or U3 components of the viral RNA. Specifically, reverse transcription of the RNA was performed by priming with UNINEF79 (59-GCACTCAAGGCAAGCTT- TATTGAGGCTT-39) close to the 39 end of the viral RNA or by VIF-VPUoutR1 (59-GGTACCCCATAATAGACTGTRA- CCCACAA-39) in vpu [11]. The extracted RNA (3 ml) was reverse transcribed in a total volume of 20 ml with 500 mM dNTP, 2.5 mM primer, 16 RT buffer, 5 mM MgCl2, 10 mM DTT, 40 U RnaseOUT, and 400 U SuperScriptTM III RNase H2 RT (Invitrogen, Carlsbad, CA). The RNA, primer and dNTPs were first incubated at 65uC for 5 minutes, then the remaining reagents were added for cDNA synthesis at 50uC for 2 hours, followed by 85uC for 5 minutes. Then 2 U E. coli RNase H (Invitrogen, Carlsbad, CA) was added, and the reaction tubes were incubated at 37uC for 20 minutes followed by 70uC for 15 minutes. HIV Subtypes in Caribbean Two or three regions of the viral genome were independently amplified from the cDNA to contain a nearly full-length genome of HIV-1. The two-amplicon strategy consisted of one amplicon of about 2.6-kb consisting of gag and part of pol (nts 769-3338 HXB-2 (Genbank Acc No: K03455)) and another amplicon of approxi- mately 7.0-kb stretching from the 59 end of pol to the middle of nef (nts 2143–9181 HXB-2). A three-amplicon strategy was used as needed. The first amplicon was the gag-pol amplicon described above, the second spanned pol to vpu (nts 2483–6231 HXB-2) and the third env to nef (nts 5861–9181 HXB-2). The reaction volume was 50 ml, containing 16 PCR buffer, 350 mM dNTP mixture, 0.4 mM of each primer and 5 U Expand Long Template PCR enzyme mixture (Roche Diagnostics, Indianapolis, IN). Cycling conditions for the first round were: 94uC for 2 minutes and then 10 cycles of (94uC 10 s, 60uC 30 s, 68uC 3 min), then 20 cycles of (94uC 10 s, 55uC 30 s, 68uC 3 min) followed by 68uC for 10 minutes. For the second round, cycling conditions were the same, except that the annealing temperature for the first 10 cycles was 65uC and the incubation at 68uC was for 8 minutes. The products of reverse transcription with the primer RT3474R were employed for amplification of the partial pol region. The first- round amplification was done with 2 primers: Pro5F (59- AGAAATTGCAGGGCCCCTAGGAA) and RT3474R (59- GAATCTCTCTGTTTTCTGCCAG), using AmpliTaq Gold (Applied Biosystems, Foster City, CA) and 2 mM of MgCl2 in a total volume of 50 mL. The second-round amplification was completed using the following 2 primers: Pro3F (59-AGANCA- GAGCCAACAGCCCCACCA) and ProRT (59- TTTCCCCAC- TAACTTCTGTATGTCATTGACA). The first-round amplifi- cation reactions began with a ‘‘hot start’’ at 95uC for 10 minutes to activate the polymerase, followed by 30 cycles for 30 seconds at 94uC, 30 seconds at 55uC, and 1.5 minutes at 72uC, then a step at 7 minutes at 72uC was followed to end the cycles. For the second- round amplification, the cycling conditions were the same except for an annealing temperature of 58uC and the use of 40 cycles instead of 30. The partial pol amplicon contained the coding sequences for protease and part of reverse transcriptase (RT), corresponding to nts 2167 through 3307 on HXB-2. Amplified DNA for the partial pol and nearly full-length regions were purified and sequenced in the Applied Biosystems 3130xl automated sequencer using Big Dye terminators (Applied Biosys- tems), and the sequences were assembled with Sequencher v4.6 (Gene Codes Corporation, Ann Arbor, MI). When multiple amplicons were used, they were not merged unless the overlapping sequences were within 2% of each other. Nearly full genome and partial pol sequences were deposited in Genbank under accession numbers EU839596– EU839610 and EU439709–EU439774, respectively. Analysis. A multiple alignment of the newly derived protease/RT sequences and nearly full-length genome sequences with selected reference sequences was constructed, consisting of 1069 nts and 8364 nts, respectively. Phylogenetic trees were generated, and the consistency of branching order was evaluated using SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE modules of the Phylogeny Inference Package (V3.52c). Recombinant analysis was performed with Simplot, version 3.4, and alignment examination was used to determine precise breakpoints [12]. After breakpoint identification, each segment was extracted and analyzed phylogenetically to confirm the assignment of subtype; breakpoint locations were designed relative to HXB-2. The Kimura 2-parameter method was used to calculate pairwise genetic distances. Analysis of the protease-RT sequences for mutations that might lead to resistance to antiretroviral drugs was performed using the Stanford University Database (http://hivdb.stanford.edu/hiv). PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4814