Human cytomegalovirus in renal transplantation

Abstract

Human cytomegalovirus (HCMV) is a major cause of morbidity in renal transplant recipients due to its capacity for latency and reactivation under immunosuppression. Comprehensive genome characterisation is essential for advancing our understanding of HCMV diversity and pathogenesis; and hence, more effective clinical management. This thesis presents an integrated molecular workflow for the screening and whole-genome sequencing of HCMV in renal transplant settings. The work began with the development and optimisation of a multiplex long-range PCR (LR-PCR) protocol to enrich the full-length HCMV genome, targeting ~6.8 kb overlapping amplicons, to ensure genome-wide coverage and high amplification efficiency. Amplicons were sequenced using Oxford Nanopore Technologies (ONT) on the MinION Mk1B platform with Q10+ and Q20+ chemistries, generating high- fidelity assemblies with over 99% sequence identity. Bioinformatic processing via NanoGalaxy ensured accurate reconstruction of the viral genome. To address the challenges of non-specific amplification in multiplex PCR assays, NSA-IPI, a web-based tool, was developed to identify problematic primer interactions using a combinatorial pooling algorithm and mis-priming prediction via MegaBLAST. NSA-IPI reduced experimental burden and outperformed conventional primer- checking tools in detecting problematic primer interactions. Finally, a duplex TaqMan quantitative PCR (qPCR) assay was developed and validated for direct detection of HCMV DNA in unextracted plasma and serum samples from renal transplant recipients. The assay incorporated a heterologous internal control to detect PCR inhibition and demonstrated high sensitivity and robustness across 759 clinical samples. This extraction-free approach offers a rapid, cost-effective screening option for large-scale applications and retrospective studies. Collectively, the studies presented in this thesis establish a scalable and clinically relevant workflow for HCMV genomic surveillance, with broader implications for viral molecular pathogenesis and epidemiology.