A genotypic resistance assay for the detection of drug resistance in the human immunodeficiency virus type 1 envelope gene

• Published in: Journal of virological methods Vol. 123; no. 1; pp. 25 - 34
• Main Authors: Van Laethem, Kristel, Schrooten, Yoeri, Lemey, Philippe, Wijngaerden, Eric Van, Wit, Stéphane De, Ranst, Marc Van, Vandamme, Anne-Mieke
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 2005; Amsterdam Elsevier; New York, NY
• Subjects: Biological and medical sciences; cDNA synthesis; Drug Resistance, Viral - genetics; Fundamental and applied biological sciences. Psychology; Genes, env - genetics; HIV Envelope Protein gp120 - genetics; HIV Envelope Protein gp41 - genetics; HIV-1 - classification; HIV-1 - drug effects; HIV-1 - genetics; HIV-1 subtypes; Human immunodeficiency virus 1; Humans; Microbial Sensitivity Tests; Microbiology; Molecular Sequence Data; PCR; Peptide Fragments - genetics; RNA extraction; Sensitivity; Sequence Analysis, DNA; Techniques used in virology; Virology; RNA extraction; HIV-1 subtypes; Sensitivity; PCR; cDNA synthesis; Microbiology; HIV-1 virus; Retroviridae; AIDS; Method; Lentivirus; Virology; Infection; Virus; Polymerase chain reaction; Resistance; Complementary DNA; Gene; Viral disease; Human immunodeficiency virus; Detection; Subtype
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2004.09.003

Since it is not clear yet whether enfuvirtide resistance is restricted to gp41, it was decided to develop a genotypic assay for the detection of drug resistance in the entire human immunodeficiency virus type 1 (HIV-1) env gene. Given the increasing prevalence of HIV-1 non-B subtypes in Europe, it is important to evaluate the performance of the assay on a panel of genetically divergent samples. A panel of 1 laboratory and 10 clinical isolates from 10 patients was tested, all enfuvirtide naïve and chosen according to the subtype as determined in the pol region (A, B, C, H, CRF01-AE, CRF02-AG, CRF05-DF, CRF11-cpx and U), while their env sequences belonged to subtypes A, B, C, H, A/G recombinant, B/H recombinant, CRF01-AE, CRF02-AG, CRF05-DF and CRF11-cpx. The detection limits of the gp120 and the gp41 PCRs ranged between 500 and 5000 RNA copies/ml plasma. The highest sensitivity was obtained for the laboratory strain, whereas the detection limit for all patient samples, except for the subtype C sample, was 1000 RNA copies/ml. The numerous insertions and deletions in the gp120 gene, that were often present as quasi-species, necessitated the sequencing of cloned PCR products. The gp41 gene displayed less diversity and less insertions/deletions. Especially, the heptad repeat 1 was highly conserved and none of the enfuvirtide naïve samples contained any of the already known enfuvirtide resistance mutations at amino acid positions 36–45. This study demonstrates that the assay is able to genotype genetically diverse HIV-1 strains with a good sensitivity.