No abstract

Prospective molecular studies of HIV-1 pol region (2253–5250 in HXB2 genome) sequences from sequenced samples of 269 HIV-1-infected patients in Cyprus (2017–2021) revealed a transmission cluster of 14 unknown HIV-1 recombinants that were not classified as previously established CRFs. The earliest recombinant was collected in September 2017, and the transmission cluster continued to grow until November 2020. Near full-length HIV-1 genome sequences of the 11 of the 14 recombinants were successfully obtained (790–8795 in HXB2 genome) and aligned against a reference dataset of HIV-1 subtypes and CRFs. We employed MEGAX for maximum-likelihood tree construction (GTR model, 1000 bootstrap replicates), Cluster-Picker for phylogenetic clustering analysis (genetic distance ≤0.045, bootstrap support value ≥70%), and REGA-3.0 for subtype determination. Bootscan and similarity plot analyses (sliding window of 400 nucleotides overlapped by 40 nucleotides) were conducted using SimPlot-v3.5.1, and subregion confirmatory neighbour-joining tree analyses were conducted using MEGAX (Kimura two-parameter model, 1000 bootstrap replicates, ≥70% bootstrap-support value). Exclusive clustering of the HIV-1 recombinants revealed their uniqueness. The recombination analyses illustrated the same unique mosaic pattern with six putative intersubtype recombination breakpoints, seven fragments of subtypes CRF02_AG, G, J and an unclassified fragment. We conclusively characterized the mosaic structure of the novel HIV-1 CRF, named CRF91_cpx, by the Los Alamos HIV Sequence Database. Additionally, we identified a URF of CRF91_cpx with two additional recombination sites, generated by a recombination event between subtype B and CRF91_cpx. Since the identification of CRF91_cpx, two additional patient samples have been entered into the CRF91_cpx transmission cluster, demonstrating active growth.

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.

Emerging infectious viruses have led to global advances in the development of specific and sensitive detection techniques. Viruses have an inherent potential to easily mutate, presenting major hurdles for diagnostics and requiring methods capable of detecting genetically diverse viral strains. One such infectious agent is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged in December 2019 and has resulted in the global coronavirus disease 2019 (COVID-19) pandemic. This study presents a real-time reverse transcription PCR (RT-PCR) detection assay for SARS-CoV-2, taking into account its intrinsic polymorphic nature that arises due to genetic drift and recombination, as well as the possibility of continuous and multiple introductions of genetically nonidentical strains into the human population. This advance was achieved by using mismatch-tolerant molecular beacons designed to specifically detect the SARS-CoV-2 S, E, M, and N genes. These were applied to create a simple and reproducible real-time RT-PCR assay, which was validated using external quality control panels (QCMD: CVOP20, WHO: SARS-CoV-2-EQAP-01) and clinical samples. This assay was designed for high target detection accuracy and specificity and can also be readily adapted for the detection of other emerging and rapidly mutating pathogens.