Scientists Identify Candidate New HIV-1 Subtype in Patient from the Democratic Republic of the Congo

A team of French researchers has reported the discovery of a viral genome that may represent an entirely new subtype of HIV-1, the virus responsible for the global AIDS pandemic. The near-complete genome was obtained from a woman in her sixties originally from the Democratic Republic of the Congo (DRC), who was diagnosed with HIV-1 in 1989, with a blood transfusion in the DRC considered the likely mode of transmission. The strain, provisionally designated as a candidate new subtype tentatively termed M, has been described in the journal New Microbes and New Infections by Philippe Colson of Aix-Marseille University and colleagues, based at the Marseille Public and University Hospitals.

HIV-1 currently comprises four lineages: group M, for major; group N, for non-M non-O; group O, for outlier; and group P, named to continue in alphabetical order. Group M is by far the most prevalent lineage and the only pandemic strain, believed to have originated in Kinshasa, the capital of the DRC, with the emergence of HIV-1 estimated to have occurred around the 1920s in the DRC. Within group M, ten subtypes have been delineated, labeled A through D, F through H, and K through L, along with nine sub-subtypes such as A1 through A8 and F1 and F2. These subtypes are thought to have diverged from one or a few ancestral HIV-1 strains after a single chimpanzee-to-human transmission, some of them around the 1950s. The most recently recognized subtype, L, was formally established in 2020 on the basis of complete genome sequences identified in the DRC between 1983 and 2001.

The designation of a new HIV-1 subtype is governed by strict criteria. Researchers must identify at least three viral strains or complete genomes from distinct, epidemiologically unlinked individuals. Phylogenetically, each subtype should form a distinct monophyletic clade, with approximately equal genetic distances separating it from the other subtypes, producing a characteristic star-like phylogeny. Established group M subtypes differ by a mean nucleotide divergence of approximately 15 percent at the genome level, with considerable variation between genes. In addition, genomes classified within a given subtype must be non-mosaic, distinguishing them from recombinant genomes that arise when viruses of different subtypes exchange genetic material inside coinfected cells.

The new report describes an HIV-1 genome recovered from residues of peripheral blood mononuclear cells from a single blood sample collected during the summer of 2023. The team extracted viral DNA using a commercial kit and amplified the genome with conventional polymerase chain reaction primers as well as a whole-genome HIV-1 genotyping assay. Sequencing was performed using three complementary platforms: Sanger sequencing, Oxford Nanopore technology on a GridION instrument, and Illumina technology on a MiSeq instrument. The resulting near-full-length consensus genome, deposited in GenBank under accession number PZ307881, was 8,950 nucleotides long and covered 92 percent of the subtype B reference genome, with the shortfall mostly due to sequencing defects at the 5-prime and 3-prime ends.

Comparative genomic analysis revealed that the ten best matches in the GenBank database exhibited nucleotide similarities of only 83.29 to 83.60 percent to the new sequence. These closest genomes had been collected from patients in the DRC, the United States, the Netherlands, and Gabon between 1987 and 2018. When aligned against representative genomes of the ten established subtypes, the new genome showed a mean nucleotide divergence of 12.69 percent, ranging between 11.87 and 13.28 percent, while the ten established subtypes diverged from one another by a mean of 12.64 percent, ranging between 10.01 and 13.73 percent. In other words, the candidate subtype is as distant from every known subtype as those subtypes are from each other, and pairwise comparisons showed no statistically significant differences in divergence. Similar results held when the genomes were cut into eight fragments corresponding to different genomic regions, with regional divergences for the candidate genome ranging from 8.55 to 20.23 percent, closely mirroring the 8.49 to 20.25 percent range observed among established subtypes.

Phylogenetic analyses reinforced this picture. Using IQ-TREE and Nextclade software, with a simian immunodeficiency virus genome from Gabon as an outgroup, the researchers found that the candidate genome formed a distinct branch within an overall star-like tree, the hallmark topology of genuine subtypes. The closest established subtype was J, supported by a bootstrap value of 99 percent. Independent trees built from each of the eight genome fragments likewise showed star-like topologies with the candidate sequence on its own branch. Bootscan analysis, which uses a sliding window of 200 nucleotides to detect recombination, found no recombination events, satisfying another key criterion for subtype status. Nextclade classified the genome as an other clade, and a tree incorporating 1,020 genomes from the Los Alamos National Laboratory HIV Sequence Database placed it on a new node. Taken together, the researchers concluded that the genome belongs to a candidate new subtype, which they named M following subtype L in alphabetical order, while acknowledging the potential nomenclatural ambiguity of reusing the letter that already designates the pandemic group.

The clinical course of the patient offers an intriguing backdrop to the genomic findings. At the start of antiretroviral treatment, her CD4-positive T lymphocyte count was 351 cells per cubic millimeter with a CD4-to-CD8 ratio of 0.23, and plasma HIV-1 RNA stood at 5.03 log10 copies per milliliter. Subsequently, her CD4 count fluctuated between 406 cells per cubic millimeter in 1999 and 2,394 cells per cubic millimeter in 2008, and has remained above 1,000 since 2007. Plasma viral load has been undetectable during regular monitoring from 2008 onward, except for a reading of 2.2 log10 copies per milliliter in 2024. Notably, a Western blot performed in 2023 showed antibody reactivities only to envelope and capsid proteins, with no response to Pol proteins. The authors suggest this unusual profile may partly reflect reduced antibody affinity for antigenically divergent proteins, or an overall weak antibody response to HIV-1 antigens, which particularly affects Pol-encoded proteins such as reverse transcriptase, protease, and integrase, especially after more than three decades of infection and long-term antiretroviral therapy.

Genotypic resistance analysis using algorithms from the French National Agency for AIDS Research and the Stanford University HIV drug resistance database found no naturally present amino acid mutations conferring resistance to any currently available antiretroviral drug, apart from two substitutions acquired during treatment failures: Y181C in reverse transcriptase, detected transiently in 2003 during a virological failure episode on nevirapine, and I84V in protease, detected in 2007 during failure on atazanavir and ritonavir. Viral tropism was predicted to be R5, indicating use of the CCR5 co-receptor. The researchers cautioned that the absence of natural resistance mutations should be interpreted carefully given the markedly different genetic background of this genome, though the finding is congruent with observations for both rare and prevalent subtypes.

The team also turned to molecular modeling to explore how the roughly 40 amino acid differences in the candidate subtype’s reverse transcriptase, the central enzyme of HIV replication and a prime drug target, might alter its structure. Mapping the mutations onto the reference enzyme structure revealed changes predicted to reshape several surface loops, including a closure of the F130 to P150 buckle and a flattening of the K281 to P294 loop. Alterations in the N57 to D76 region, corresponding to the functionally important beta3-beta4 loop within the fingers subdomain, were associated with a greater space between the fingers and palm subdomains. The surface electrostatic potential was modified but remained globally electropositive, particularly in the ligand-interacting area. The modeled enzyme-ligand interaction energy decreased slightly, from minus 646 to minus 512 kilojoules per mole, because the number of amino acids involved in ligand interaction fell from 15 to 7. The authors speculate that such changes could modulate enzyme processivity and, by promoting dissociation and re-association of the enzyme-substrate complex, potentially increase template switching and genetic recombination, though they stress these hypotheses derive entirely from in silico modeling and require in vitro biochemical validation.

The discovery underscores that HIV-1 diversity remains far from fully charted and reinforces the status of the DRC as the epicenter of the group M pandemic and a region where rare subtypes continue to circulate. Rare established subtypes, including F, H, J, K, and L, each account for less than 1 percent of global HIV-1 infections and circulate primarily in the DRC, Angola, and Cameroon, with limited data on their transmissibility, virulence, and pathogenicity. The researchers also observed notable intra-sample diversity, with mean intra-host nucleotide divergence of 1.61 percent and a maximum of 5.52 percent, suggesting intra-patient viral evolution or, less likely given the candidate subtype’s apparent rarity, superinfection with multiple variants during the patient’s decades of stays in the DRC. Formal recognition of the new subtype will require detecting it in at least two more epidemiologically unlinked individuals, and the authors call for further studies, primarily in the DRC, to establish its prevalence and genotypic and phenotypic characteristics.

Subject of Research: Discovery of a candidate new HIV-1 group M subtype in a patient from the Democratic Republic of the Congo

Article Title: A case of infection with a candidate new subtype, tentatively termed M, of human immunodeficiency virus type 1 of group M

Article References: A case of infection with a candidate new subtype, tentatively termed M, of human immunodeficiency virus type 1 of group M. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: HIV-1, subtype M, Democratic Republic of the Congo, viral phylogenetics, whole-genome sequencing, reverse transcriptase, group M, subtype L, molecular modeling, antiretroviral resistance, HIV diversity, New Microbes and New Infections