Genetic Variation and Susceptibilities to Protease Inhibitors among Subtype B and F Isolates in Brazil

Abstract

The genetic variation of the human immunodeficiency virus type 1 (HIV-1) protease gene (prt) permits the classification of HIV-1 strains into five distinct protease subtypes, which follow thegag subtyping patterns. The susceptibilities of non-B-subtype strains to protease inhibitors (PIs) and other antiretroviral drugs remain largely unknown. Subtype F is the main non-B strain contributing to the Brazilian epidemic, accounting for 15 to 20% of these infections. In this work, we report the findings on 81 isolates from PI-naive Brazilian patients collected between 1993 and 1997. In addition, the relevant PI resistance mutations and their phenotypes were determined in vitro for 15 of these patients (B = 9 and F = 6). Among these, the subtype F samples evidenced high sensitivities in vitro to ritonavir and indinavir, with MICs at which 50 and 90% of the isolates are inhibited similar to those of both the Brazilian and the U.S. subtype B isolates. Analysis of the 81 Brazilianprt sequences demonstrated that the subtype F consensus sequence differs from the U.S. and Brazilian subtype B consensus in eight positions (I15V, E35D, M36I, R41K, R57K, Q61N, L63P, and L89M). The frequency of critical PI resistance substitutions (amino acid changes D30N, V82A/F/T, I84V, N88D, and L90M) among Brazilian isolates is very low (mean, 2.5%), and the associated secondary substitutions (amino acid positions 10L, 20K, 36M, 46M, 48G, 54I, 63P, 71A, and 77A) are infrequent. These observations document the relative rarity of resistance to PIs in the treatment of patients infected with HIV-1 subtype F in South America.

Impact of frequent natural polymorphisms at the protease gene on the in vitro susceptibility to protease inhibitors in HIV-1 non-B subtypes

“…This is the case for subtype C and reverse transcriptase inhibitors (Shafer et al, 1997), or for subtype G and PI . Controversial results have been published for subtype F Tanuri et al, 1999). However, in all these studies the total number of amino acid changes at the protease gene associated to PI resistance was not shown.…”

Low Accumulation of L90M in Protease from Subtype F HIV‐1 with Resistance to Protease Inhibitors Is Caused by the L89M Polymorphism

“…All 89L reversed clones were phenotyped for PI resistance (table [33]. Subtype F isolates are infrequently found to carry the L90M NFV and SQV-resistance mutation in HIV-1-infected individuals in whom therapy has failed [34,35,46].…”

“…Viral RNA was extracted with Trizol Reagent (Invitrogen), and the PR gene was amplified and sequenced as described elsewhere [33]. PR sequences were edited with DNAStar software and were submitted to the HIV-genotyping algorithm of the HIV RT and Protease Sequence Database (available at: http://hivdb.stanford.edu) [23].…”

“…Subtype F is the most prevalent (prevalence, 15%) non-B HIV-1 subtype found in Brazil [31][32][33]. Recently, we genotypically and phenotypically characterized Brazilian clinical isolates of HIV-1 of subtypes F and B infecting adults [34] and children [35] in whom antiretroviral therapy failed.…”

Genotypic resistance and HIV-1 subtype in Brazilian children on dual and triple combination therapy

“…Complementary DNA (cDNA) was synthesized using avian myeloblastosis virus RT (Stratagene, La Jolla, CA, USA) according to the manufacturer's protocol. Two regions were targeted in a 2-round nested PCR: the entire protease (PR) coding region (codons 1-99) and the first 235 codons of the reverse transcriptase (RT) coding region as described elsewhere (Caride et al, 2000;Tanuri et al, 1999b). PCR products were purified using QIAamp PCR purification kit (Qiagen).…”