Human Cytomegalovirus Disruption of Calcium Signaling in Neural Progenitor Cells and Organoids

ABSTRACTThe herpesvirus human cytomegalovirus (HCMV) is a leading cause of congenital birth defects. Infection can result in infants born with a variety of symptoms, including hepatosplenomegaly, microcephaly, and developmental disabilities. Microcephaly is associated with disruptions in the neural progenitor cell (NPC) population. Here, we defined the impact of HCMV infection on neural tissue development and calcium regulation, a critical activity in neural development. Regulation of intracellular calcium involves purinergic receptors and voltage-gated calcium channels (VGCC). HCMV infection compromised the ability of both pathways in NPCs as well as fibroblasts to respond to stimulation. We observed significant drops in basal calcium levels in infected NPCs which were accompanied by loss in VGCC activity and purinergic receptor responses. However, uninfected cells in the population retained responsiveness. Addition of the HCMV inhibitor maribavir reduced viral spread but failed to restore activity in infected cells. To study neural development, we infected three-dimensional cortical organoids with HCMV. Infection spread to a subset of cells over time and disrupted organoid structure, with alterations in developmental and neural layering markers. Organoid-derived infected neurons and astrocytes were unable to respond to stimulation whereas uninfected cells retained nearly normal responses. Maribavir partially restored structural features, including neural rosette formation, and dampened the impact of infection on neural cellular function. Using a tissue model system, we have demonstrated that HCMV alters cortical neural layering and disrupts calcium regulation in infected cells.IMPORTANCEHuman cytomegalovirus (HCMV) replicates in several cell types throughout the body, causing disease in the absence of an effective immune response. Studies on HCMV require cultured human cells and tissues due to species specificity. In these studies, we investigated the impact of infection on developing three-dimensional cortical organoid tissues, with specific emphasis on cell-type-dependent calcium signaling. Calcium signaling is an essential function during neural differentiation and cortical development. We observed that HCMV infects and spreads within these tissues, ultimately disrupting cortical structure. Infected cells exhibited depleted calcium stores and loss of ATP- and KCl-stimulated calcium signaling while uninfected cells in the population maintained nearly normal responses. Some protection was provided by the viral inhibitor maribavir. Overall, our studies provide new insights into the impact of HCMV on cortical tissue development and function.

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The Carboxy-Terminal Tail of Human Cytomegalovirus (HCMV) US28 Regulates both Chemokine-Independent and Chemokine-Dependent Signaling in HCMV-Infected Cells

Journal of Virology ◽

10.1128/jvi.00354-09 ◽

2009 ◽

Vol 83 (19) ◽

pp. 10016-10027 ◽

Cited By ~ 24

Author(s):

Melissa P. Stropes ◽

Olivia D. Schneider ◽

William A. Zagorski ◽

Jeanette L. C. Miller ◽

William E. Miller

Keyword(s):

Human Cytomegalovirus ◽

Calcium Release ◽

Hcmv Infection ◽

Calcium Signal ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Domain Truncation ◽

Infected Cells ◽

Heterologous Expression Systems ◽

Carboxy Terminal

ABSTRACT The human cytomegalovirus (HCMV)-encoded G-protein-coupled receptor (GPCR) US28 is a potent activator of a number of signaling pathways in HCMV-infected cells. The intracellular carboxy-terminal domain of US28 contains residues critical for the regulation of US28 signaling in heterologous expression systems; however, the role that this domain plays during HCMV infection remains unknown. For this study, we constructed an HCMV recombinant virus encoding a carboxy-terminal domain truncation mutant of US28, FLAG-US28/1-314, to investigate the role that this domain plays in US28 signaling. We demonstrate that US28/1-314 exhibits a more potent phospholipase C-β (PLC-β) signal than does wild-type US28, indicating that the carboxy-terminal domain plays an important role in regulating agonist-independent signaling in infected cells. Moreover, HMCV-infected cells expressing the US28/1-314 mutant exhibit a prolonged calcium signal in response to CCL5, indicating that the US28 carboxy-terminal domain also regulates agonist-dependent signaling. Finally, while the chemokine CX3CL1 behaves as an inverse agonist or inhibitor of constitutive US28 signaling to PLC-β, we demonstrate that CX3CL1 functions as an agonist with regard to US28-stimulated calcium release. This study is the first to demonstrate that the carboxy terminus of US28 controls US28 signaling in the context of HCMV infection and indicates that chemokines such as CX3CL1 can decrease constitutive US28 signals and yet simultaneously promote nonconstitutive US28 signals.

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Intrahost Dynamics of Human Cytomegalovirus Variants Acquired by Seronegative Glycoprotein B Vaccinees

Journal of Virology ◽

10.1128/jvi.01695-18 ◽

2018 ◽

Vol 93 (5) ◽

Cited By ~ 9

Author(s):

Cody S. Nelson ◽

Diana Vera Cruz ◽

Melody Su ◽

Guanhua Xie ◽

Nathan Vandergrift ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Rational Design ◽

Hcmv Infection ◽

Vaccine Trial ◽

The United States ◽

Glycoprotein B ◽

Viral Population ◽

Vaginal Fluid ◽

The Impact ◽

Placebo Recipients

ABSTRACTHuman cytomegalovirus (HCMV) is the most common congenital infection worldwide and a frequent cause of hearing loss and debilitating neurologic disease in newborn infants. Thus, a vaccine to prevent HCMV-associated congenital disease is a public health priority. One potential strategy is vaccination of women of child bearing age to prevent maternal HCMV acquisition during pregnancy. The glycoprotein B (gB) plus MF59 adjuvant subunit vaccine is the most efficacious tested clinically to date, demonstrating 50% protection against primary HCMV infection in a phase 2 clinical trial. Yet, the impact of gB/MF59-elicited immune responses on the population of viruses acquired by trial participants has not been assessed. In this analysis, we employed quantitative PCR as well as multiple sequencing methodologies to interrogate the magnitude and genetic composition of HCMV populations infecting gB/MF59 vaccinees and placebo recipients. We identified several differences between the viral dynamics in acutely infected vaccinees and placebo recipients. First, viral load was reduced in the saliva of gB vaccinees, though not in whole blood, vaginal fluid, or urine. Additionally, we observed possible anatomic compartmentalization of gB variants in the majority of vaccinees compared to only a single placebo recipient. Finally, we observed reduced acquisition of genetically related gB1, gB2, and gB4 genotype “supergroup” HCMV variants among vaccine recipients, suggesting that the gB1 genotype vaccine construct may have elicited partial protection against HCMV viruses with antigenically similar gB sequences. These findings suggest that gB immunization had a measurable impact on viral intrahost population dynamics and support future analysis of a larger cohort.IMPORTANCEThough not a household name like Zika virus, human cytomegalovirus (HCMV) causes permanent neurologic disability in one newborn child every hour in the United States, which is more than that for Down syndrome, fetal alcohol syndrome, and neural tube defects combined. There are currently no established effective measures to prevent viral transmission to the infant following HCMV infection of a pregnant mother. However, the glycoprotein B (gB)/MF59 vaccine, which aims to prevent pregnant women from acquiring HCMV, is the most successful HCMV vaccine tested clinically to date. Here, we used viral DNA isolated from patients enrolled in a gB vaccine trial who acquired HCMV and identified several impacts that this vaccine had on the size, distribution, and composition of thein vivoviral population. These results have increased our understanding of why the gB/MF59 vaccine was partially efficacious, and such investigations will inform future rational design of a vaccine to prevent congenital HCMV.

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Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01641-05 ◽

2006 ◽

Vol 50 (8) ◽

pp. 2806-2813 ◽

Cited By ~ 10

Author(s):

T. Ueno ◽

Y. Eizuru ◽

H. Katano ◽

T. Kurata ◽

T. Sata ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Cell Line ◽

Real Time ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Fluorescent Protein ◽

Clinical Isolates ◽

Pml Bodies ◽

Infected Cells ◽

Green Fluorescent

ABSTRACT Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for “immediate-early 1”) protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

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Human Cytomegalovirus Infection Dysregulates the Localization and Stability of NICD1 and Jag1 in Neural Progenitor Cells

Journal of Virology ◽

10.1128/jvi.00351-15 ◽

2015 ◽

Vol 89 (13) ◽

pp. 6792-6804 ◽

Cited By ~ 19

Author(s):

Xiao-Jun Li ◽

Xi-Juan Liu ◽

Bo Yang ◽

Ya-Ru Fu ◽

Fei Zhao ◽

...

Keyword(s):

Stem Cell ◽

Notch Signaling ◽

Progenitor Cells ◽

Human Cytomegalovirus ◽

Neural Progenitor Cells ◽

Hcmv Infection ◽

Fetal Brain ◽

Neural Progenitor ◽

Notch Signaling Pathway ◽

Protein Levels

ABSTRACTHuman cytomegalovirus (HCMV) infection of the developing fetus frequently results in major neural developmental damage. In previous studies, HCMV was shown to downregulate neural progenitor/stem cell (NPC) markers and induce abnormal differentiation. As Notch signaling plays a vital role in the maintenance of stem cell status and is a switch that governs NPC differentiation, the effect of HCMV infection on the Notch signaling pathway in NPCs was investigated. HCMV downregulated mRNA levels of Notch1 and its ligand, Jag1, and reduced protein levels and altered the intracellular localization of Jag1 and the intracellular effector form of Notch1, NICD1. These effects required HCMV gene expression and appeared to be mediated through enhanced proteasomal degradation. Transient expression of the viral tegument proteins of pp71 and UL26 reduced NICD1 and Jag1 protein levels endogenously and exogenously. Given the critical role of Notch signaling in NPC growth and differentiation, these findings reveal important mechanisms by which HCMV disturbs neural cell developmentin vitro. Similar eventsin vivomay be associated with HCMV-mediated neuropathogenesis during congenital infection in the fetal brain.IMPORTANCECongenital human cytomegalovirus (HCMV) infection is the leading cause of birth defects that primarily manifest as neurological disabilities. Neural progenitor cells (NPCs), key players in fetal brain development, are the most susceptible cell type for HCMV infection in the fetal brain. Studies have shown that NPCs are fully permissive for HCMV infection, which causes neural cell loss and premature differentiation, thereby perturbing NPC fate. Elucidation of virus-host interactions that govern NPC proliferation and differentiation is critical to understanding neuropathogenesis. The Notch signaling pathway is critical for maintaining stem cell status and functions as a switch for differentiation of NPCs. Our investigation into the impact of HCMV infection on this pathway revealed that HCMV dysregulates Notch signaling by altering expression of the Notch ligand Jag1, Notch1, and its active effector in NPCs. These results suggest a mechanism for the neuropathogenesis induced by HCMV infection that includes altered NPC differentiation and proliferation.

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P2Y2 purinergic receptor is induced following human cytomegalovirus infection and its activity is required for efficient viral replication

10.1101/051391 ◽

2016 ◽

Author(s):

Saisai Chen ◽

Thomas Shenk ◽

Maciej T. Nogalski

Keyword(s):

Viral Replication ◽

Host Cell ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Purinergic Receptor ◽

Purinergic Signaling ◽

Receptor Expression ◽

Receptor Activity ◽

P2y2 Receptor ◽

Infected Cells

AbstractHuman cytomegalovirus (HCMV) manipulates many aspects of host cell biology to create an intracellular milieu optimally supportive of its replication and spread. The current study reveals a role for purinergic signaling in HCMV infection. The levels of several components of the purinergic signaling system, including the P2Y2 receptor, were altered in HCMV-infected fibroblasts. P2Y2 receptor RNA and protein are strongly induced following infection. Pharmacological inhibition of receptor activity or knockdown of receptor expression markedly reduced the production of infectious HCMV progeny. When P2Y2 activity was inhibited, the accumulation of most viral RNAs tested and viral DNA was reduced. In addition, the level of cytosolic calcium within infected cells was reduced when P2Y2 signaling was blocked. The HCMV-coded UL37x1 protein was previously shown to induce calcium flux from the smooth endoplasmic reticulum to the cytosol, and the present study demonstrates that P2Y2 function is required for this mobilization. We conclude that P2Y2 supports the production of HCMV progeny, possibly at multiple points within the viral replication cycle that interface with signaling pathways induced by the purinergic receptor.ImportanceHCMV infection is ubiquitous and can cause life-threatening disease in immunocompromised patients, debilitating birth defects in newborns, and has been increasingly associated with a wide range of chronic conditions. Such broad clinical implications result from the modulation of multiple host cell processes. This study documents that cellular purinergic signaling is usurped in HCMV-infected cells and that the function of this signaling axis is critical for efficient HCMV infection. Therefore, we speculate that blocking P2Y2 receptor activity has the potential to become an attractive novel treatment option for HCMV infection.

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Diagnosis and Management of Human Cytomegalovirus Infection in the Mother, Fetus, and Newborn Infant

Clinical Microbiology Reviews ◽

10.1128/cmr.15.4.680-715.2002 ◽

2002 ◽

Vol 15 (4) ◽

pp. 680-715 ◽

Cited By ~ 353

Author(s):

Maria Grazia Revello ◽

Giuseppe Gerna

Keyword(s):

Prenatal Diagnosis ◽

Human Cytomegalovirus ◽

Primary Infection ◽

Hcmv Infection ◽

Prognostic Markers ◽

Recurrent Infection ◽

Congenital Infection ◽

Immunoglobulin M ◽

Hcmv Disease ◽

The Impact

SUMMARY Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection and mental retardation. HCMV infection, while causing asymptomatic infections in most immunocompetent subjects, can be transmitted during pregnancy from the mother with primary (and also recurrent) infection to the fetus. Hence, careful diagnosis of primary infection is required in the pregnant woman based on the most sensitive serologic assays (immunoglobulin M [IgM] and IgG avidity assays) and conventional virologic and molecular procedures for virus detection in blood. Maternal prognostic markers of fetal infection are still under investigation. If primary infection is diagnosed in a timely manner, prenatal diagnosis can be offered, including the search for virus and virus components in fetal blood and amniotic fluid, with fetal prognostic markers of HCMV disease still to be defined. However, the final step for definite diagnosis of congenital HCMV infection is detection of virus in the blood or urine in the first 1 to 2 weeks of life. To date, treatment of congenital infection with antiviral drugs is only palliative both prior to and after birth, whereas the only efficacious preventive measure seems to be the development of a safe and immunogenic vaccine, including recombinant, subunit, DNA, and peptide-based vaccines now under investigation. The following controversial issues are discussed in the light of the most recent advances in the field: the actual perception of the problem; universal serologic screening before pregnancy; the impact of correct counseling on decision making by the couple involved; the role of prenatal diagnosis in ascertaining transmission of virus to the fetus; the impact of preconceptional and periconceptional infections on the prevalence of congenital infection; and the prevalence of congenitally infected babies born to mothers who were immune prior to pregnancy compared to the number born to mothers undergoing primary infection during pregnancy.

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Activation of the NF-κB Pathway in Human Cytomegalovirus-Infected Cells Is Necessary for Efficient Transactivation of the Major Immediate-Early Promoter

Journal of Virology ◽

10.1128/jvi.78.9.4498-4507.2004 ◽

2004 ◽

Vol 78 (9) ◽

pp. 4498-4507 ◽

Cited By ~ 98

Author(s):

Ian B. DeMeritt ◽

Liesl E. Milford ◽

Andrew D. Yurochko

Keyword(s):

Human Cytomegalovirus ◽

Time Course ◽

Hcmv Infection ◽

Dominant Negative ◽

Immediate Early ◽

Primary Role ◽

Early Promoter ◽

Ikk Complex ◽

Infected Cells ◽

Major Immediate Early Promoter

ABSTRACT We previously demonstrated that human cytomegalovirus (HCMV) infection induced the activation of the cellular transcription factor NF-κB. Here, we investigate the mechanism for the HCMV-induced NF-κB activation and the role that the induced NF-κB plays in transactivation of the major immediate-early promoter (MIEP) and production of immediate-early (IE) proteins. Using a dominant-negative inhibitor of NF-κB, the IκB-superrepressor, we demonstrated that active NF-κB is critical for transactivation of the HCMV MIEP. Investigation of the mechanisms of NF-κB activation following HCMV infection showed a rapid and sustained decrease in the inhibitors of NF-κB, IκBα and IκBβ. Because the IκB kinases (IKKs) regulate the degradation of the IκBs, virus-mediated changes in the IKKs were examined next. Using dominant-negative forms of the IKKs, we showed significant decreases in transactivation of the MIEP in the presence of these mutants. In addition, protein levels of members of the IKK complex and IKK kinase activity were upregulated throughout the time course of infection. Lastly, the role NF-κB plays in HCMV IE mRNA and protein production during infection was examined. Using aspirin and MG-132, we demonstrated that production of IE protein and mRNA was significantly decreased and delayed in infected cells treated with these drugs. Together, the results of these studies suggest that virus-mediated NF-κB activation, through the dysregulation of the IKK complex, plays a primary role in the initiation of the HCMV gene cascade in fibroblasts and may provide new targets for therapeutic intervention.

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CD8+ T-cell recognition of human cytomegalovirus latency-associated determinant pUL138

Journal of General Virology ◽

10.1099/vir.0.020982-0 ◽

2010 ◽

Vol 91 (8) ◽

pp. 2040-2048 ◽

Cited By ~ 18

Author(s):

Siok-Keen Tey ◽

Felicia Goodrum ◽

Rajiv Khanna

Keyword(s):

T Cells ◽

T Cell ◽

Human Cytomegalovirus ◽

Cell Response ◽

Hcmv Infection ◽

Mononuclear Cells ◽

T Cell Response ◽

Cell Recognition ◽

T Cell Recognition ◽

Infected Cells

Recent studies have shown that long-term persistence of human cytomegalovirus (HCMV) in mononuclear cells of myeloid lineage is dependent on the UL138 open reading frame, which promotes latent infection. Although T-cell recognition of protein antigens from all stages of lytic HCMV infection is well established, it is not clear whether proteins expressed during latent HCMV infection can also be recognized. This study conducted an analysis of T-cell response towards proteins associated with HCMV latency. Ex vivo analysis of T cells from healthy virus carriers revealed a dominant CD8+ T-cell response to the latency-associated pUL138 protein, which recognized a non-canonical 13 aa epitope in association with HLA-B*3501. These pUL138-specific T cells displayed a range of memory phenotypes that were in general less differentiated than that previously described in T cells specific for HCMV lytic antigens. Antigen-presentation assays revealed that endogenous pUL138 could be presented efficiently by HCMV-infected cells. However, T-cell recognition of pUL138 was dependent on newly synthesized protein, with little presentation from stable, long-lived protein. These data demonstrate that T cells targeting latency-associated protein products exist, although HCMV may limit the presentation of latent proteins, thereby restricting T-cell recognition of latently infected cells.

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Checks and balances between human cytomegalovirus replication and indoleamine-2,3-dioxygenase

Journal of General Virology ◽

10.1099/vir.0.061994-0 ◽

2014 ◽

Vol 95 (3) ◽

pp. 659-670 ◽

Cited By ~ 9

Author(s):

Albert Zimmermann ◽

Sebastian Hauka ◽

Marco Maywald ◽

Vu Thuy Khanh Le ◽

Silvia K. Schmidt ◽

...

Keyword(s):

Epithelial Cells ◽

Human Cytomegalovirus ◽

Cell Types ◽

Interferon Γ ◽

Counter Measures ◽

Indoleamine 2,3 Dioxygenase ◽

Infected Cells ◽

Hcmv Replication ◽

Ifn Γ ◽

The Impact

Despite a rigorous blockade of interferon-γ (IFN-γ) signalling in infected fibroblasts as a mechanism of immune evasion by human cytomegalovirus (HCMV), IFN-γ induced indoleamine-2,3-dioxygenase (IDO) has been proposed to represent the major antiviral restriction factor limiting HCMV replication in epithelial cells. Here we show that HCMV efficiently blocks transcription of IFN-γ-induced IDO mRNA both in infected fibroblasts and epithelial cells even in the presence of a preexisting IFN-induced antiviral state. This interference results in severe suppression of IDO bioactivity in HCMV-infected cells and restoration of vigorous HCMV replication. Depletion of IDO expression nonetheless substantially alleviated the antiviral impact of IFN-γ treatment in both cell types. These findings highlight the effectiveness of this IFN-γ induced effector gene in restricting HCMV productivity, but also the impact of viral counter-measures.

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Genetic signatures of human cytomegalovirus variants acquired by seronegative glycoprotein B vaccinees

10.1101/432922 ◽

2018 ◽

Cited By ~ 1

Author(s):

Cody S. Nelson ◽

Diana Vera Cruz ◽

Melody Su ◽

Guanhua Xie ◽

Nathan Vandergrift ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Rational Design ◽

Hcmv Infection ◽

Vaccine Trial ◽

The United States ◽

Glycoprotein B ◽

Viral Population ◽

The Impact ◽

Placebo Recipients

AbstractHuman cytomegalovirus (HCMV) is the most common congenital infection worldwide, and a frequent cause of hearing loss or debilitating neurologic disease in newborn infants. Thus, a vaccine to prevent HCMV-associated congenital disease is a public health priority. One potential strategy is vaccination of women of child-bearing age to prevent maternal HCMV acquisition during pregnancy. The glycoprotein B (gB) + MF59 adjuvant subunit vaccine is the most efficacious tested clinically to date, demonstrating approximately 50% protection against HCMV infection of seronegative women in multiple phase 2 trials. Yet, the impact of gB/MF59-elicited immune responses on the population of viruses acquired by trial participants has not been assessed. In this analysis, we employed quantitative PCR as well as multiple sequencing methodologies to interrogate the magnitude and genetic composition of HCMV populations infecting gB/MF59 vaccinees and placebo recipients. We identified several differences between the viral dynamics of acutely-infected vaccinees and placebo recipients. First, there was reduced magnitude viral shedding in the saliva of gB vaccinees. Additionally, employing a panel of tests for genetic compartmentalization, we noted tissue-specific gB haplotypes in the majority of vaccinees though only in a single placebo recipient. Finally, we observed reduced acquisition of genetically-related gB1, gB2, and gB4 genotype “supergroup” HCMV variants among vaccine recipients, suggesting that the gB1 genotype vaccine construct may have elicited partial protection against HCMV viruses with antigenically-similar gB sequences. These findings indicate that gB immunization may have had a measurable impact on viral intrahost population dynamics and support future analysis of a larger cohort.Author SummaryThough not a household name like Zika virus, human cytomegalovirus (HCMV) causes permanent neurologic disability in one newborn child every hour in the United States - more than Down syndrome, fetal alcohol syndrome, and neural tube defects combined. There are currently no established effective preventative measures to inhibit congenital HCMV transmission following acute or chronic HCMV infection of a pregnant mother. However, the glycoprotein B (gB) vaccine is the most effective HCMV vaccine tried clinically to date. Here, we utilized high-throughput, next-generation sequencing of viral DNA isolated from patients enrolled in a gB vaccine trial, and identified several impacts that this vaccine had on the size, distribution, and composition of thein vivoviral population. These results have increased our understanding of why the gB/MF59 vaccine was partially efficacious and will inform future rational design of a vaccine to prevent congenital HCMV.

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