HCMV Glycoprotein Typing and Its Potential as a Biomarker to Determine the Outcome of Congenital Cytomegalovirus Infection

Abstract

Abstract Background: Human Cytomegalovirus (HCMV) is the most common congenital viral infection affecting 0.2-2.4% of all live births. Although most infected babies are asymptomatic at birth, congenital HCMV (cHCMV) is a major cause of sensorineural hearing loss, birth defects, and other severe neurological complications during childhood. The ability to predict outcomes of cHCMV infections is limited by a poor understanding of the pathogenesis of the infection. Viral and host factors likely contribute to the outcome, with variation in genes encoding essential envelope glycoproteins playing a major role. The virion carries 6 major surface glycoproteins within three complexes; gC-I (gB); gC-II (gM/gN); and gC-III (gH/gL/gO or gH/gL/UL128-131A). These glycoproteins are polymorphic, producing distinct genotypes and some are known to elicit neutralizing antibodies which makes them of interest for correlation with the severity and outcome of disease. However, previous studies linking individual genotypes with disease outcomes show contradictory results. Objectives: The current study aimed to (1) develop methods for the genetic characterization of the surface glycoproteins of HCMV strains and (2) evaluate the genomic variant distribution of HCMV in laboratory strains and clinical isolates from a range of HCMV-infected individuals including congenitally and postnatally infected infants, immunocompromised patients and immunocompetent individuals. In addition, (3) A systematic review of the current incidence, impact and birth prevalence of cHCMV infections among newborns in the African region was undertaken to determine whether the high maternal seroprevalence in this region resulted in better or poorer outcomes for cCMV-infected infants and whether glycoprotein typing could be clinically useful. Methodology: multiplex polymerase chain reaction (mPCR) in conjunction with MinION nanopore sequencing, analysed using a novel bioinformatics pipeline was applied to laboratory strains of the virus and a range of clinical samples and isolates from cytomegalovirus-infected patients. A systematic review of published studies was conducted using four scientific databases that assessed the rate of congenital HCMV infection in African countries using polymerase chain reaction (PCR) and/or culture-based methods for detection. Results: The protocol's capability to identify both single and mixed genotypes was assessed using five well-characterised laboratory strains of HCMV. The evaluation demonstrated accurate detection of all expected single and low-frequency minor genotypes within the five laboratory strains. The study also investigated genomic variants of glycoprotein genes in 13 HCMV isolates from three clinical groups. Among the patient groups, gB1 was the predominant single genotype (n=3, 23.1%) in gCI, followed by gM1 (n=8, 61.5%), gN4d (n=2, 15.4%) in gC-II, and gL4 (n=3, 23.1%), gH2 (n=2, 15.4%), and gO5 (n=2, 15.4%) for gC-III. Mixed infections involving multiple genotypes were detected in 72.2% (13/18) of samples tested. The UL128, UL130, and UL131A gene exhibited overall sequence divergence to be (0.0%-34.3%, 0.0%-8.0%, and 0.0%-5.4%) at the DNA level. Mixed genotypes were observed across all examined groups, but they were particularly frequent in the congenital samples and less common in the immunocompromised patients. Among the eleven studies that were included in the systematic review, a total of 8865 newborns were tested and the overall estimates of congenital HCMV birth incidence varied between 1.4% and 6.7%. Conclusion: The ability of the developed multiplex PCR and MinION nanopore sequencing method to simultaneously characterise all six genotypes allows the detection of genotype polymorphisms among the three glycoprotein complexes. Application of the method to clinical samples shows the high variability of the glycoprotein genes, particularly in congenitally infected infants. Although small sample numbers prevented any correlation between glycoprotein type and disease type or outcome, this method promises applicability in prospective studies involving a larger number of clinical samples. Data from the systematic review suggests it would be useful to conduct this in low-middle-income countries (LIMCs) to establish whether there is any correlation between specific HCMV glycoprotein genotypes and clinical outcomes and/or severity of the diseases. The understanding of the impact of genotype variations on replication within the host could facilitate the development of virologic prognostic markers for the outcomes of congenital and other HCMV-related diseases.

Date of Award	4 Jul 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Pamela Vallely (Main Supervisor) & Paul Klapper (Co Supervisor)