# Next-Generation Sequencing Reveals Continued Circulation of Rare HIV-1 Subtypes in the Democratic Republic of the Congo and Refines the Estimate of the Emergence Dates of Three Subtypes

## TL;DR

This study uses next-generation sequencing to reveal that rare HIV-1 subtypes still circulate in the Democratic Republic of the Congo and updates estimates of when these subtypes first emerged.

## Contribution

The study provides new insights into the emergence dates of rare HIV-1 subtypes and highlights ongoing diversity in the DRC.

## Key findings

- NGS identified a wide range of HIV-1 subtypes and recombinant forms circulating in the DRC.
- Bayesian analysis pushed back the estimated emergence dates of subtypes G, H, and J by 3 to 7 years.
- Rare HIV-1 subtypes in the DRC may challenge diagnostics and vaccine development.

## Abstract

HIV-1 diversified for decades within the Democratic Republic of the Congo (DRC) before spreading globally in the early 1980s. Thus, the DRC is home to some of the most ancestral and diverse HIV-1 strains. Recent serosurveys conducted from 2017 to 2019 in Kinshasa, DRC, indicated high prevalence of HIV-1, yet sequence data is lacking from this period. Given the history of circulating rare HIV-1 subtypes in the DRC, a viral whole-genome sequencing study was conducted to determine current diversity in the greater Kinshasa area. Next-generation sequencing (NGS) through metagenomic and target enrichment methods was conducted on 197 specimens collected from 2017 to 2019. A large array of HIV subtypes (A, B, C, D, F1, G, H, J, and K), circulating recombinant forms (CRF01_AE, CRF02_AG, CRF05_DF, CRF11_cpx, CRF13_cpx, CRF25_cpx, CRF 45_cpx, and CRF92_C2U), unique recombinant forms, and unclassifiable sequences were observed, with many branching in basal positions within, or outside of, many subtypes on phylogenetic trees. Incorporating these new sequences into Bayesian inference of phylogeny pushes back the dates of the most recent common ancestors of HIV-1 group M and the rare subtypes G, H, and J by between 3 and 7 years each. The DRC continues to harbor diverse and rare HIV-1 subtypes that could challenge diagnostic tests, treatments, and vaccines. In addition to shifting subtype emergence dates, the sequences from our study are evidence that rare strains continue to circulate and should be regularly monitored.