HIV Type 1 Diversity from Newly Diagnosed Patients in Santos Metropolitan Area=Brazil
Dercy Jose de Sa-Filho, Rafael Favero Ambar, Natalia Brenneken Duarte, ´ Rafael Braganca Rodrigues Matias, Valeria Candido, Luiz Henrique Gagliani, and Marcos Montani Caseiro ¸´
HIV-1 from infected subjects has been characterized in order to provide a more accurate view of the strains that are currently found in a given region. In this report, we focused on characterizing the pol gene diversity obtained from newly diagnosed patients in Santos metropolitan area, Brazil. This region is composed of nine cities and an international port. Analysis of the 33samples revealed that 22 strains belonged to subtype B, 4 to subtype F, and 2 to subtype C; 5 strains were B=F recombinants. Our results demonstrated that 18.2% of samples were primary antiretroviral resistance genotypic mutations, with high-level resistance to reverse transcriptase inhibitors in both subtypes B and F and in recombinant forms B=F. Our data revealed that the primary antiretroviralresistance genotypic mutations should be carefully investigated in developing countries with widespread access to antiretrovirals, such as Brazil.
he Brazilian epidemic of human immunodeﬁciency virus type 1 (HIV-1) is complex with the presence of subtypes B, F, and C, and a multitude of recombinant genomes emerging from these subtypes.1–8 We previously described two HIV-1 circulating recombinantforms in Santos, Brazil classiﬁeds as CRF28_BF and CRF29_BF.5 The Santos metropolitan area includes an international port and nine cities. It was the ﬁrst region of Brazil to include HIV-1 antiretroviral therapy in HIV=AIDS programs in 1996. In this article we describe sequence analyses of the pol (protease and reverse transcriptase) gene in HIV-1 newly diagnosed individuals from the Santosmetropolitan area. We report the genetic diversity and primary drug resistance mutations found in subtypes B and F and recombinants B=F. We studied 33 samples from newly diagnosed patients enrolled in Brazilian HIV=AIDS programs. Samples were collected in 2006–2008, after informed consent was obtained. The population group studied included 33 unrelated individuals, 18 male and 15 female, with a mean ageof 35 years and a mean viral load of 4.8 log10 copies=ml (b-DNA, Siemens). The DNA was extracted from the buffy coat using the QIAamp DNA Blood Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer’s procedure. Ampliﬁcation of the HIV-1 pol gene was carried out using nested polymerase chain reaction (PCR). Reagents and thermocycling proﬁles have
been previously described.3 PuriﬁedPCR products were sequenced bidirectionally with an ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase (Applied Biosystems, Foster City, CA). The reaction products were sequenced and analyzed using a model ABI 3100 automatic sequencer (Applied Biosystems). Sequences were corrected and assembled using the Sequencer 4.0 program (Genecodes, Ann Arbor, MI).Phylogenetic analysis was conducted in the 1200-bp protease and reverse transcriptase regions of pol. Alignments were generated using Clustal X. Phylogenetic relationships were established using the neighbor-joining methods present in the PAUP evolutionary package (PAUP version 4.0b10). Trees were constructed in accordance with the HKY85 evolutionary method and the consistency of tree topologieswas evaluated by bootstrapping. The GenBank accession numbers used in the comparative phylogenetic analysis are as follows: subtype A: AB098330 and AF004885; subtype B: M93258 and M38431; subtype C: AF286228 and U52953; subtype D: AF133821 and AF457090; subtype F: AY173957 and AF005494; subtype G: AF061640 and AY371121; subtype H: AF005496 and AF190128; subtype J: AF082394 and AF082395; subtype...