Comparative HIV-1 Phylogenies Characterized by PR/RT, Pol and Near-Full-Length Genome Sequences

Abstract

In an effort to evaluate the accuracy of HIV-1 phylogenies based on genomes of increasing length, we developed a comprehensive near-full-length HIV-1 genome RT–PCR assay and performed a comparative evaluation via phylogenetic analyses. To this end, we conducted comparative analyses of HIV-1 phylogenies derived based on HIV-1 PR/RT (2253–3359 in the HXB2 genome) and pol region (2253–5250 in the HXB2 genome) sequences isolated from 134 HIV-1-infected patients in Cyprus (2017–2019). The HIV-1 genotypic subtypes determined using six subtyping tools (REGA 3.0, COMET 2.3, jpHMM, SCUEAL, Stanford, and Geno2pheno) were compared to investigate the discrepancies generated among different tools. To evaluate the accuracy of defined HIV-1 phylogenies, the samples exhibiting at least one discrepant subtyping result among different subtyping tools in both PR/RT and pol regions or only in the pol region (n = 38) were selected for near-full-length HIV-1 genome (790–8795 in HXB2 genome) sequencing using a newly developed RT–PCR/sequencing assay. The obtained sequences were employed for HIV-1 genotypic subtype determination and subjected to comparative phylogenetic-based analyses. It was observed that 39.6% of the 134 samples presented discrepancies in the PR/RT region, while 28.4% presented discrepancies in the pol region. REGA 3.0 produced the fewest discrepancies collectively in both regions and was selected for subsequent subtyping and comparative phylogenetic analyses of near-full-length HIV-1 genome sequences. The analyses of near-full-length HIV-1 genome sequences identified 68.4% of the 38 ‘discrepant samples’ (n = 26) as belonging to uncharacterized recombinant HIV-1 strains, while 21.1% were circulating recombinant forms (CRFs) (n = 8) and 10.5% belonged to pure group M subtypes (n = 4). The findings demonstrated a significant reduction of 11.2% in discrepancies when pol region sequences were used compared to PR/RT region sequences, indicating that increased nucleotide sequence lengths are directly correlated with more consistent subtype classification. The results also revealed that if the discrepancy in pol region subtyping results persists, then there is a high likelihood (89.5%) that the query sequence is a recombinant HIV-1 strain, 68.4% of which belong to uncharacterized recombinant HIV-1 strains. The results of this study showed that REGA 3.0 presented the best performance in subtyping recombinant HIV-1 strains, while Stanford performed better in defining phylogenies of pure group M subtypes. The study highlights that, especially in populations with polyphyletic HIV-1 epidemics resulting in a high prevalence of recombinant HIV-1 strains, neither PR/RT nor pol region sequences are reliable for the determination of HIV-1 genotypic subtypes in samples showing discrepancies among different subtyping tools, and only near-full-length or full-length HIV-1 genome sequences are sufficiently accurate.

How to report and make sense of a new HIV-1 circulating recombinant form?

“…Second, Bayesian phylogenetic analysis is suggested to infer the origin and population history of a new CRF ( Suchard et al, 2018 ). Third, recent study showed that only near-full-length HIV-1 genomic sequences, rather than partial pol sequences, are sufficiently accurate for the determination of HIV-1 genotypic subtypes, especially CRFs ( Topcu et al, 2022b ). In spite of the advances in NGS, however, HIV-1 full-length genomic sequencing is still not practical for HIV-1 molecular epidemiological studies.…”

Reconstructing the phylodynamic history and geographic spread of the CRF01_AE-predominant HIV-1 epidemic in the Philippines from PR/RT sequences sampled from 2008-2018

“…The trend of higher CRF01_AE R e in our study is consistent with the hypothesis of faster transmissibility over subtype B, although further analysis should be done to confirm these findings. Use of whole genome sequences instead of subgenomic sequences may lead to more precise estimates of these epidemiological parameters for each subtype [18] as well as allow for more accurate classification of subtypes and recombinants forms [17]. The same study by Salvaña et al also suggested higher rates of transmitted drug resistance (TDR) among CRF01_AE infections compared to other HIV subtypes, which if confirmed may be a mechanism by which the current antiretroviral treatment regimens in the country that were tailored for non-CRF01_AE epidemics selectively favor the survival and transmission of CRF01_AE [9].…”

“…Although whole genome sequences would be able to more accurately distinguish recombinant sequences [17] and lead to more precise estimates [18], partial pol sequences from drug resistance genotyping have been shown sufficient to reconstruct transmission histories phylogenetically and are highly available data from routine testing [19]. The Research Institute for Tropical Medicine (RITM) DRG database has archived pol protease/reverse transcriptase (PR/RT) sequences from routine testing for over a decade from referred samples spanning multiple Philippine regions, compiling a suitable and available dataset for reconstructing subtype-specific phylodynamics.…”

Validation of an HIV whole genome sequencing method for HIV drug resistance testing in an Australian clinical microbiology laboratory

“…10 Utilizing the whole genome to determine subtypes provides greater accuracy, especially for recombinant subtypes. 24 However, discrepancies in subtype determination have been shown to arise based on the region and proportion of the genome used as well as with a choice of subtyping tool. 24 Therefore, despite having the whole genome available to us, we did not use it to determine subtype as part of this validation.…”