Development and genomic characterization of a diverse HIV-1 variant reference panel for nucleic acid-based testing

The high worldwide genetic diversity of HIV poses significant challenges for its detection and diagnosis by nucleic acid testing (NAT). Well-characterized reference panels are important for evaluating the analytical performance of HIV tests.

To develop a reference panel for HIV NAT that reflects the genetic diversity of circulating strains.

HIV was cultured from blood specimens collected from blood donor and clinical sites in Cameroon. Metagenomic next-generation sequencing in combination with spiked primer enrichment along with Sanger sequencing were used to sequence 101 cultured HIV-1 samples representing 59 strains. To establish an HIV-1 variant reference panel, a diverse subset of cultured viruses was analyzed in multiple laboratories with different assays to determine consensus viral loads.

Near full-length HIV-1 genomes, with an average of 9589 base pairs (bp), were recovered from 37 (62.7%) of the 59 strains. The whole genome sequences of 28 strains exhibited more than 95% similarity to our previously reported genomes obtained by Sanger sequencing. An HIV variant reference panel for NAT comprising 18 diverse HIV-1 strains was developed. The panel included four subtypes, four circulating recombinant forms, and eight unique recombinant forms. Strains were prepared at low (n = 18, 2.53 log10 copies/mL), medium (n = 18, 3.61 log10 copies/mL), and high viral loads (n = 15, 4.66 log10 copies/mL), yielding 51 panel members in total.

This diverse HIV reference panel can be used to evaluate the performance of HIV NAT and is available upon request to developers and manufacturers of HIV tests.