Genetic Characterization of a Novel HIV-1 Circulating Recombinant Form (CRF91_cpx) and Identification of Three Additional Putative CRFs: Increased Prevalence of URFs and CRFs in the Polyphyletic HIV-1 Infection in Cyprus.

Abstract

HIV is characterized by a high degree of genetic variation, as it is evident from numerous genetically distinct subtypes (within the major group M) and circulating recombinant forms (CRFs). The genetic diversity of HIV is attributed to its nature as a fast-replicating virus, coupled with a high mutation rate and the lack of proof-reading ability of reverse transcriptase. The HIV-1 epidemic in Cyprus is characterized as highly polyphyletic due to the influx of various HIV-1 group M subtypes from Africa, Europe, and Asia. The coexistence of numerous subtypes in the same geographic area raises the potential for the generation of new recombinant strains. Thus, the investigation of the viral HIV-1 nucleotide sequences uncovered the existence of a novel circulating recombinant form (CRF91_cpx) in Cyprus and raised evidence for the existence of additional three CRFs. The four transmission clusters (clusters 4,5,9 and 16) were identified through phylogenetic analyses of HIV-1 pol region (2253-5250 on HXB2 genome) sequences, derived from consenting HIV 1 positive patients in Cyprus (2017-2021) collected as part of routine surveillance and antiretroviral resistance analyses. The HIV-1 genotypic subtypes were determined using REGA-3.0. Subsequently, a multiple sequence alignment was created using CLUSTALW algorithm, followed by phylogenetic analyses performed on the MEGA-X software for the construction of maximum-likelihood trees (GTR model, 1000 bootstrap replicates). The identification of the transmission clusters was performed on Cluster-Picker software (genetic distance ≤0.045, bootstrap support value ≥70%). Later, near full-length HIV-1 genome (790-8795 on HXB2 genome) sequences were obtained, which were aligned against a reference dataset of all known HIV-1 subtypes and CRFs (RIP alignment 2017 and 2020), and phylogenetic analyses were repeated. Next, similarity plot and bootscan analyses (sliding window of 400 nucleotides overlapped by 40 nucleotides) were performed against a reference set of all HIV-1 group M pure subtypes, CRF02_AG and CRF56_cpx using SimPlot v3.5.1 software for each of the HIV-1 recombinant query sequences belonging to the four new CRF strains. Consequently, subregion confirmatory neighbor-joining tree analyses were performed using MEGA-X software for confirmation of the recombination breakpoints and subtype origin of each fragment (Kimura two-parameter model, 1000 bootstrap replicates, ≥70% bootstrap-support value was considered definitive). In the maximum-likelihood trees of near full-length genome sequences, the HIV-1 recombinant sequences of each cluster exclusively clustered together, revealing their high genetic similarity and uniqueness. The subtypes of the near full-length genome sequences for cluster 9 were determined as “Rec. of 02_AG, G, J”, cluster 4 as “Rec. of 56_cpx, G” and cluster 5 and 16 as “Rec. of A1, B”. The similarity plot and the bootscan analyses illustrated the same unique mosaic pattern for each of the HIV-1 recombinant clusters, revealing six putative inter-subtype recombination breakpoints for the cluster 9 “Rec. of 02_AG, G, J”, two inter-subtype recombination breakpoints for the cluster 4 “Rec of 56_cpx, G”, and seven inter-subtype recombination breakpoints for the two recombinant clusters 5 and 16 “Rec of A1, B”. The neighbor-joining subregion confirmatory analyses confirmed the subtype origin of each fragment. Thus, it was concluded that the corresponding query sequences of each of the HIV-1 recombinant clusters have the same and unique mosaic pattern, which consists of fragments of subtypes CRF02_AG, G, J, and a fragment of unknown subtype origin for the cluster 9 the subtypes CRF56_cpx and G for the cluster 4, and the subtypes A1 and B for the clusters 5 and 16. In conclusion, we have characterized the mosaic structure of the new HIV-1 circulating recombinant form, named CRF91_cpx (cluster 9) by the Los Alamos HIV Database according to the standards of HIV nomenclature. Since the identification of the CRF91_cpx, 2 more patient samples have been introduced into the CRF91_cpx transmission cluster, demonstrating an active growth. For the cluster 4, “Rec. of 56_cpx, G” and clusters 5 and 16 “Rec of A1, B”, all the evidence indicate that these recombinant strains are novel CRFs, and as such approval is pending from the Los Alamos HIV Database to name them according to the standards of HIV nomenclature.