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.

Fig 1. Comparison of PCR success rates.

Partial pol genes were amplified for 47 group M viruses at six sites. The whole pol gene was amplified at the USA_FDA site.

Fig 2. Comparison of subtype results across all sites.

The subtype results from each site were compared to those determined by analyzing the 47 group M whole genome sequences at EQAPOL.

Fig 3. Recombination patterns in the pol gene and the near full length genome.

The recombination patterns of near full-length sequences determined at EQAPOL are shown and indicated on the far right. The pol gene region amplified at each site is indicated by the colored box. Subtypes are color coded (shown at the bottom).

Fig 4. Phylogenetic clusters of CRF sequences.

Partial pol gene sequences were aligned with all subtype references from the HIV sequence database. The phylogenetic tree was constructed using the neighbor-joining (NJ) method with Kimura two-parameter model. CRFs (06, 12, 14, 24 and 47) form distinct clusters and are depicted in different colors. The 50 sequences in the EQAPOL diversity panel are indicated by black dots. Asterisks indicate the EQAPOL sequences that are derived from the same strains used to define CRF24 and CRF47.

Fig 5. Phylogenetic analysis of partial pol gene sequences from all sites.

All group M virus sequences (47) from six sites were analyzed together with the EQAPOL reference sequences. The phylogenetic tree was constructed using the neighbor-joining (NJ) method with Kimura two-parameter model. Sequences from the USA_FDA site were not included due to divergent sequence fragments. Ambiguous bases in DEURF07ES002 (*) and DEURF07BR002 (#) from Canada and Brazil sites resulted in independent branches. The short sequences (⌘) of DEMD07UG002 and DEURF10US008 from the Malaysia site were excluded from the analysis.