Carrasqueira, PatríciaFerreira, RitaLopo, SílviaChasqueira, Maria de JesusBorges, VítorPaixão, PauloGomes, João Paulo2026-03-242026-03-242025-09-19http://hdl.handle.net/10400.18/11297Human cytomegalovirus (HCMV) possesses a ~236 kb genome and exhibits genetic diversity mostly concentrated in discrete hypervariable regions, such as envelope glycoprotein genes like UL55 (gB), UL73 (gN), and UL75 (gH), and recombination occurs frequently. Despite numerous efforts to associate viral polymorphisms and intra-patient population diversity with enhanced viral fitness, clinical outcomes, and latency, correlations remains inconclusive. Therefore, in this study a multiplex tiling PCR assay was developed, for the simultaneous sequencing of multiple hypervariable HCMV genomic regions, aiming for a comprehensive characterization of viral population variability and dynamics. To take advantage of the vast potential of targeted next-generation sequencing (tNGS), a 2-pool primer scheme (51 amplicons) for multiplex tiling PCR was designed, taking into account the genetic diversity of 358 HCMV genome sequences available at NCBI Virus (https://www.ncbi.nlm.nih.gov/labs/virus). The novel multiplex targets seven independent genomic regions, encompassing 14 polymorphic loci of interest (UL33, UL55, UL73–UL75, UL100, UL115, UL128–UL131A, and UL144–UL147A). Protocol optimization and performance assays were conducted using Illumina NGS and subsequent bioinformatics analysis with the online platform INSaFLU-TELEVIR (https://insaflu.insa.pt/). Preliminary results of the application of the novel multiplex tNGS assay to clinical samples (n = 11) from different matrices (urine, BALF, plasma, placenta) collected between 2020 and 2024 demonstrated its high potential, with successful amplification and sequencing of all amplicons in all samples with a Ct value ≤25. The assay also proved effective in sequencing highly polymorphic regions and detecting mixed infections. We anticipate that this innovative approach will open new avenues for characterizing circulating HCMV diversity and investigating whether viral population diversity and specific genetic profiles correlate with clinical outcomes, ultimately supporting earlier interventions and improved management of HCMV infections (e.g. congenital infections).engHuman Cytomegalovirus (HCMV)Next Generation SequencingTiling MultiplexPCRTestingInfecções Sistémicas e Zoonosesconference object