Characterization of the Drug Resistance Profiles of Patients Infected with CRF07_BC Using Phenotypic Assay and Ultra-Deep Pyrosequencing

Abstract

The usefulness of ultra-deep pyrosequencing (UDPS) for the diagnosis of HIV-1 drug resistance (DR) remains to be determined. Previously, we reported an explosive outbreak of HIV-1 circulating recombinant form (CRF) 07_BC among injection drug users (IDUs) in Taiwan in 2004. The goal of this study was to characterize the DR of CRF07_BC strains using different assays including UDPS. Seven CRF07_BC isolates including 4 from early epidemic (collected in 2004–2005) and 3 from late epidemic (collected in 2008) were obtained from treatment-naïve patient’s peripheral blood mononuclear cells. Viral RNA was extracted directly from patient’s plasma or from cultural supernatant and the pol sequences were determined using RT-PCR sequencing or UDPS. For comparison, phenotypic drug susceptibility assay using MAGIC-5 cells (in-house phenotypic assay) and Antivirogram were performed. In-house phenotypic assay showed that all the early epidemic and none of the late epidemic CRF07_BC isolates were resistant to most protease inhibitors (PIs) (4.4–47.3 fold). Neither genotypic assay nor Antivirogram detected any DR mutations. UDPS showed that early epidemic isolates contained 0.01–0.08% of PI DR major mutations. Furthermore, the combinations of major and accessory PI DR mutations significantly correlated with the phenotypic DR. The in-house phenotypic assay is superior to other conventional phenotypic assays in the detection of DR variants with a frequency as low as 0.01%.

Amino Acid Deletions in p6Gag Domain of HIV-1 CRF07_BC Ameliorate Galectin-3 Mediated Enhancement in Viral Budding

“…Unfortunately, there were a lack of studies available to address this question. More recently, Hung et al [39] reported that clinical CRF07_BC-7d isolates with the mutations in the protease (PR) region had obtained the resistance to protease inhibitors (PIs), including ritonavir, saquinavir, indinavir, nelfinavir, and amprenavir (resistant fold range 4.4-47.3). Some studies from mainland China indicate that potential drug-resistant mutants of Chinese CRF07_BC viruses (most isolates were non-amino acid deletion strains) were resistant to Nevirapine (NVP), which is the most frequently used antiretroviral drug in China [40].…”