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Desenvolvimento de uma metodologia para sequenciação de regiões altamente polimórficas de HCMV para avaliação dos perfis mutacionais da população viral
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O citomegalovírus humano (HCMV, do inglês human cytomegalovirus) possui um genoma de ~236 kb sendo o herpesvírus que causa infeções no Homem com maior diversidade genética conhecida. Apesar dos inúmeros esforços para associar os polimorfismos virais e a diversidade da população intra- paciente com o aumento da aptidão viral, com os resultados clínicos e com a latência, as correlações permanecem inconclusivas. Neste estudo, foi desenvolvido um ensaio de tilling PCR multiplex para a sequenciação simultânea de múltiplas regiões genómicas hipervariáveis do HCMV, com o objetivo de caracterizar o polimorfismo deste vírus. Para tirar partido do potencial da sequenciação direcionada de última geração (tNGS, do inglês targeted next-generation sequencing), foi concebida uma tilling PCR multiplex para amplificação de ~ 8% do genoma de HCMV em 7 regiões discretas abrangendo 14 loci polimórficos de interesse (UL33, UL55, UL73, UL74, UL75, UL100, UL115, UL128, UL130, UL131A, UL144, UL146, UL147 e UL147A). Esta abordagem teve por base o desenho de 145 primers diferentes que em conjunto originam 51 amplicões. A otimização de todo o workflow foi realizada tanto ao nível técnico como ao nível da análise bioinformática, com resultados muito promissores. A metodologia foi aplicada a um conjunto de 30 amostras clínicas, tendo-se verificado que cinco apresentavam resultados compatíveis com infeções mistas, sendo que nenhuma destas provinha de infeções congénitas. Da sequenciação das 7 regiões das restantes 25 amostras resultaram sequências consensus de elevada qualidade para a maior parte delas correspondendo a uma elevada taxa de sucesso de 92.6% (162/175). De notar que embora o sucesso da metodologia tenda a diminuir com menores cargas virais foi total mesmo para amostras clínicas com carga viral mais baixa (Ct>25). Verificou-se ainda que, tanto ao nível das sequências concatenadas como gene-a-gene, as amostras clínicas portuguesas não só apresentavam variabilidade genética idêntica à já identificada no conjunto global das sequências retiradas do NCBI (n = 344) como apresentavam diferentes níveis de diversidade entre si. Por fim, focando a análise de polimorfismo em pares de amostras de 3 hospedeiros diferentes verificou-se que estas não apresentavam quaisquer diferenças entre si. Em conclusão a metodologia desenvolvida abre perspetivas promissoras para investigações futuras, que permitam estudar a relação entre polimorfismos do HCMV, a sua aptidão viral e correlacioná-los com os desfechos clínicos.
Human cytomegalovirus (HCMV) has a genome of ~236 kb and is the herpesvirus that causes infections in humans with the greatest known genetic diversity. Despite numerous efforts to associate viral polymorphisms and intra-patient population diversity with increased viral fitness, clinical outcomes, and latency, the correlations remain inconclusive. In this study, a multiplex PCR tilling assay was developed for the simultaneous sequencing of multiple hypervariable genomic regions of HCMV, with the aim of characterizing the polymorphism of this virus. To take advantage of the potential of targeted next-generation sequencing (tNGS), a multiplex tiling PCR was designed to amplify ~8% of the HCMV genome in 7 discrete regions covering 14 polymorphic loci of interest (UL33, UL55, UL73, UL74, UL75, UL100, UL115, UL128, UL130, UL131A, UL144, UL146, UL147, and UL147A). The entire workflow was optimized both technically and in terms of bioinformatic analysis, with very promising results. That said, the methodology was applied to a set of clinical samples (n = 30), where it was found that 5 of the samples had results compatible with mixed infections and that none of them were congenital infections. From the sequencing of the 7 regions of the remaining 25 samples, high-quality consensus sequences were obtained for most of them, corresponding to a high success rate of 92.6% (162/175). It should be noted that although the success of the methodology tends to decrease with lower viral loads, it was total even for clinical samples with lower viral loads (Ct>25). It was also found that, both in terms of concatenated sequences and gene-togene, the Portuguese clinical samples not only showed genetic variability identical to that already identified in the overall set of samples (n = 344) but also showed different levels of diversity among themselves. Finally, focusing on the analysis of polymorphism in pairs of samples from three different hosts, it was found that these did not show any differences between them. This methodology opens up promising prospects for future research to further investigate the relationship between HCMV polymorphisms, their viral fitness, and clinical outcomes.
Human cytomegalovirus (HCMV) has a genome of ~236 kb and is the herpesvirus that causes infections in humans with the greatest known genetic diversity. Despite numerous efforts to associate viral polymorphisms and intra-patient population diversity with increased viral fitness, clinical outcomes, and latency, the correlations remain inconclusive. In this study, a multiplex PCR tilling assay was developed for the simultaneous sequencing of multiple hypervariable genomic regions of HCMV, with the aim of characterizing the polymorphism of this virus. To take advantage of the potential of targeted next-generation sequencing (tNGS), a multiplex tiling PCR was designed to amplify ~8% of the HCMV genome in 7 discrete regions covering 14 polymorphic loci of interest (UL33, UL55, UL73, UL74, UL75, UL100, UL115, UL128, UL130, UL131A, UL144, UL146, UL147, and UL147A). The entire workflow was optimized both technically and in terms of bioinformatic analysis, with very promising results. That said, the methodology was applied to a set of clinical samples (n = 30), where it was found that 5 of the samples had results compatible with mixed infections and that none of them were congenital infections. From the sequencing of the 7 regions of the remaining 25 samples, high-quality consensus sequences were obtained for most of them, corresponding to a high success rate of 92.6% (162/175). It should be noted that although the success of the methodology tends to decrease with lower viral loads, it was total even for clinical samples with lower viral loads (Ct>25). It was also found that, both in terms of concatenated sequences and gene-togene, the Portuguese clinical samples not only showed genetic variability identical to that already identified in the overall set of samples (n = 344) but also showed different levels of diversity among themselves. Finally, focusing on the analysis of polymorphism in pairs of samples from three different hosts, it was found that these did not show any differences between them. This methodology opens up promising prospects for future research to further investigate the relationship between HCMV polymorphisms, their viral fitness, and clinical outcomes.
Descrição
Dissertação de Mestrado apresentada à Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2025. http://hdl.handle.net/10362/191656
Palavras-chave
Citomegalovírus Humano (HCMV) Diversidade Genética Tilling PCR Multiplex Tiling Sequenciação de Nova Geração (NGS) Genes Estruturais Genes Evasão Imune Vigilância Molecular Infecções Sistémicas e Zoonoses
Contexto Educativo
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Licença CC
Sem licença CC