Genetic Characterization of a Panel of Diverse HIV-1 Isolates at Seven International Sites

Abstract

HIV-1 subtypes and drug resistance are routinely tested by many international surveillance groups. However, results from different sites often vary. A systematic comparison of results from multiple sites is needed to determine whether a standardized protocol is required for consistent and accurate data analysis. A panel of well-characterized HIV-1 isolates (N = 50) from the External Quality Assurance Program Oversight Laboratory (EQAPOL) was assembled for evaluation at seven international sites. This virus panel included seven subtypes, six circulating recombinant forms (CRFs), nine unique recombinant forms (URFs) and three group O viruses. Seven viruses contained 10 major drug resistance mutations (DRMs). HIV-1 isolates were prepared at a concentration of 107 copies/ml and compiled into blinded panels. Subtypes and DRMs were determined with partial or full pol gene sequences by conventional Sanger sequencing and/or Next Generation Sequencing (NGS). Subtype and DRM results were reported and decoded for comparison with full-length genome sequences generated by EQAPOL. The partial pol gene was amplified by RT-PCR and sequenced for 89.4%-100% of group M viruses at six sites. Subtyping results of majority of the viruses (83%-97.9%) were correctly determined for the partial pol sequences. All 10 major DRMs in seven isolates were detected at these six sites. The complete pol gene sequence was also obtained by NGS at one site. However, this method missed six group M viruses and sequences contained host chromosome fragments. Three group O viruses were only characterized with additional group O-specific RT-PCR primers employed by one site. These results indicate that PCR protocols and subtyping tools should be standardized to efficiently amplify diverse viruses and more consistently assign virus genotypes, which is critical for accurate global subtype and drug resistance surveillance. Targeted NGS analysis of partial pol sequences can serve as an alternative approach, especially for detection of low-abundance DRMs.

Distinctive variation in the U3R region of the 5' Long Terminal Repeat from diverse HIV-1 strains

“…Thus, we retrieved a total of 80 contigs that were subtyped as follows: clade B (n = 20), CRF12_BF (n = 5), clade C (n = 8), clade D (n = 10), clade F2 (n = 5), clade G (n = 3), clade A1 (n = 10), CRF02_AG (n = 9) and CRF01_AE (n = 10). For majority of the sequences analyzed, the subtype assigned to the 5’LTR of the HIV-1 isolate was concordant with its previously reported primary genotype [ 44 – 46 , 52 ]. However, we found that additional contigs from 5 clade B specimens were subtyped as CRF12_BF (BF), one of the two contigs from a CRF02_AG (AG) specimen was assigned to be F2, and all the LTR sequences retrieved from the CRF22_01A1 (01A1) specimens were subtyped as CRF01_AE (AE) Table 2 .…”

“…Sixty five high titer virus derived from de-identified plasma specimens obtained from DUKE EQAPOL viral diversity program [ 44 ], and five CRF02_AG virus isolated from stored and de-identified plasma specimens [ 45 – 47 ] as previously described [ 48 ] were selected for this study. The study was approved by the Duke University Institutional Review Board for clinical investigation (Pro0029507) and the Review Board of the US HHS/Food and Drug Administration Research in Human Subjects Committee (exempt reference number 01-044B).…”

Comparison of Detection Limits of Fourth- and Fifth-Generation Combination HIV Antigen-Antibody, p24 Antigen, and Viral Load Assays on Diverse HIV Isolates

“…To address this, well-characterized genetically diverse HIV clinical isolates assembled by the External Quality Assurance Program Oversight Laboratory (EQAPOL) were used to develop limit-of-detection (LOD) panels for assessment of the analytic sensitivities across platforms using p24 Ag-capture components, i.e., HIV fourth-generation diagnostic assays, stand-alone p24 Ag enzyme immunoassays (EIAs), novel p24 Ag digital detection technologies, and rapid point-of-care (POC) p24 Ag and Ab assays, relative to the analytic sensitivities of VL assays. Twenty isolates were selected from the EQAPOL Genotype Diversity Panel (19) to represent globally relevant subtypes and circulating recombinant forms (CRFs) and further characterized to confirm VL and p24 concentrations to ensure that the Ag/RNA ratios were consistent with the expected values (see Table S1 in the supplemental material). Both manufacturers and clinical and research labs participated in the comparison study ( Table 1).…”

“…Samples were selected as being representative from geographically diverse origins as a subset of the EQAPOL Genetic Diversity Panel and characterized by nearly full-length genome sequencing for subtype and recombination pattern by single-genome amplification (SGA) at the EQAPOL core laboratory and phylogenetic analysis, as previously described (see Fig. S1 in the supplemental material) (19). This genetic characterization confirmed that gag region sequences were consistent with the genotype assignment (see the GenBank accession number assignments in reference 19).…”

“…This genetic characterization confirmed that gag region sequences were consistent with the genotype assignment (see the GenBank accession number assignments in reference 19). Isolates with a ratio of VL/p24 within the expected range of 20 ϫ 10 3 to 60 ϫ 10 3 copies/pg were selected to ensure that the p24 Ag reactivity was attributable to virion-associated p24 Ag (Table S1) (19,20). Additional spiking experiments were conducted to establish that lysed cell debris from infected culture cells would have made an insignificant contribution to supernatant VL and p24 levels (see Fig.…”