Percorrer por autor "Chasqueira, Maria de Jesus"
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- Development of a multiplex PCR for tiled amplicon sequencing of highly variable Human Cytomegalovirus (HCMV) genomic regionsPublication . Carrasqueira, Patrícia; Ferreira, Rita; Lopo, Sílvia; Chasqueira, Maria de Jesus; Borges, Vítor; Paixão, Paulo; Gomes, João PauloHuman 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).
- Genetic and Epidemiological Insights Into Respiratory Syncytial Virus Infections: A Comparative Study of Hospitalized Versus Community Cases in Portugal (2021-2023)Publication . Lança, Miguel; Gaio, Vânia; Rodrigues, Ana Paula; Henriques, Camila; Gomes, Licínia; Dias, Daniela; Chasqueira, Maria de Jesus; Guiomar, Raquel; Melo, AryseBackground: Respiratory syncytial virus (RSV) is the leading cause of acute respiratory infection (ARI) in young children, but its genetic diversity requires ongoing surveillance. Methods: From 2021 to 2023, a total of 619 and 94 RSV-positive samples from the National Respiratory Syncytial Virus Surveillance Network (VigiRSV) and the Sentinel Influenza and other respiratory viruses surveillance network (Sentinel ISN), respectively, were analysed. The RSV A and RSV B typing was assessed by a multiplex real-time RT-PCR. Sanger sequencing was performed on a subset of samples (n = 495). Phylogenetic analysis was carried out on partial glycoprotein G sequences. Clinical and epidemiological data were compared through Pearson Chi-Square tests. Results: RSV Subgroup A was more prevalent (53.5%, 85/159) in the 2021/2022 season, whereas in the 2022/2023 season, it was RSV Subgroup B (82.1%, 435/530) in both networks. RSV A strains in VigiRSV clustered mainly to A.D.1 (39.0%, 39/100), whereas in Sentinel ISN, they clustered in A.D.5 (30.0%, 3/10). RSV Type B clustered mainly to B.D.E.1 (96.6%, 372/385) in both networks. All lineages cocirculated during the study period and in both surveillance networks. Regional clusters were identified for both subgroups. Conclusions: This study provides new insights into RSV genetic variability in Portugal, namely, the cocirculation of lineages and intravariability among lineages within both subgroups during the study period and in all Portuguese regions. However, our study is based on partial sequencing of the G gene, and because of this limitation, our results should be considered with great caution.