scholarly journals Asparagine deprivation causes a reversible inhibition of Human Cytomegalovirus acute virus replication

2019 ◽  
Author(s):  
Chen-Hsuin Lee ◽  
Samantha Griffiths ◽  
Paul Digard ◽  
Nhan T. Pham ◽  
Manfred Auer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Virus Replication ◽  
Human Cytomegalovirus ◽  
Late Onset ◽  
Host Factors ◽  
Prolonged Treatment ◽  
Solid Organ ◽  
Hcmv Disease ◽  
Wide Range ◽  
Hcmv Replication

AbstractAs obligate intracellular pathogens, viruses rely on the host cell machinery to replicate efficiently, with the host metabolism extensively manipulated for this purpose. High throughput siRNA screens provide a systematic approach for the identification of novel host-virus interactions. Here, we report a large-scale screen for host factors important for human cytomegalovirus (HCMV), consisting of 6,881 siRNAs. We identified 47 proviral factors and 68 antiviral factors involved in a wide range of cellular processes including the mediator complex, proteasome function and mRNA splicing. Focused characterisation of one of the hits, asparagine synthetase (ASNS), demonstrated a strict requirement for asparagine for HCMV replication which leads to an early block in virus replication before the onset of DNA amplification. This effect is specific to HCMV, as knockdown of ASNS had little effect on herpes simplex virus-1 or influenza A virus replication, suggesting the restriction is not simply due to a failure in protein production. Remarkably, virus replication could be completely rescued seven days post-infection with addition of exogenous asparagine, indicating that while virus replication is restricted at an early stage, it maintains the capacity for full replication days after initial infection. This study represents the most comprehensive siRNA screen for the identification of host factors involved in HCMV replication and identifies the non-essential amino acid, asparagine as a critical factor in regulating HCMV virus replication. These results have implications for control of viral latency and the clinical treatment of HCMV in patients.ImportanceHCMV accounts for more than 60% of complications associated with solid organ transplant patients. Prophylactic or preventative treatment with antivirals, such as ganciclovir, reduces the occurrence of early onset HCMV disease. However, late onset disease remains a significant problem and prolonged treatment, especially in patients with suppressed immune systems, greatly increases the risk of antiviral resistance. Very few antivirals have been developed for use against HCMV since the licensing of ganciclovir, and of these, the same viral genes are often targeted, reducing the usefulness of these drugs against resistant strains. An alternative approach is to target host genes essential for virus replication. Here we demonstrate that HCMV replication is highly dependent on levels of the amino acid asparagine and knockdown of a critical enzyme involved in asparagine synthesis results in severe attenuation of virus replication. These results suggest that reducing asparagine levels through dietary restriction or chemotherapeutic treatment could limit HCMV replication in patients.

scholarly journals Asparagine Deprivation Causes a Reversible Inhibition of Human Cytomegalovirus Acute Virus Replication

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Chen-Hsuin Lee ◽  
Samantha Griffiths ◽  
Paul Digard ◽  
Nhan Pham ◽  
Manfred Auer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Virus Replication ◽  
Human Cytomegalovirus ◽  
Late Onset ◽  
Host Factors ◽  
Prolonged Treatment ◽  
Solid Organ ◽  
Hcmv Disease ◽  
Wide Range ◽  
Hcmv Replication

ABSTRACT As obligate intracellular pathogens, viruses rely on the host cell machinery to replicate efficiently, with the host metabolism extensively manipulated for this purpose. High-throughput small interfering RNA (siRNA) screens provide a systematic approach for the identification of novel host-virus interactions. Here, we report a large-scale screen for host factors important for human cytomegalovirus (HCMV), consisting of 6,881 siRNAs. We identified 47 proviral factors and 68 antiviral factors involved in a wide range of cellular processes, including the mediator complex, proteasome function, and mRNA splicing. Focused characterization of one of the hits, asparagine synthetase (ASNS), demonstrated a strict requirement for asparagine for HCMV replication which leads to an early block in virus replication before the onset of DNA amplification. This effect is specific to HCMV, as knockdown of ASNS had little effect on herpes simplex virus 1 or influenza A virus replication, suggesting that the restriction is not simply due to a failure in protein production. Remarkably, virus replication could be completely rescued 7 days postinfection with the addition of exogenous asparagine, indicating that while virus replication is restricted at an early stage, it maintains the capacity for full replication days after initial infection. This study represents the most comprehensive siRNA screen for the identification of host factors involved in HCMV replication and identifies the nonessential amino acid asparagine as a critical factor in regulating HCMV virus replication. These results have implications for control of viral latency and the clinical treatment of HCMV in patients. IMPORTANCE HCMV accounts for more than 60% of complications associated with solid organ transplant patients. Prophylactic or preventative treatment with antivirals, such as ganciclovir, reduces the occurrence of early onset HCMV disease. However, late onset disease remains a significant problem, and prolonged treatment, especially in patients with suppressed immune systems, greatly increases the risk of antiviral resistance. Very few antivirals have been developed for use against HCMV since the licensing of ganciclovir, and of these, the same viral genes are often targeted, reducing the usefulness of these drugs against resistant strains. An alternative approach is to target host genes essential for virus replication. Here we demonstrate that HCMV replication is highly dependent on levels of the amino acid asparagine and that knockdown of a critical enzyme involved in asparagine synthesis results in severe attenuation of virus replication. These results suggest that reducing asparagine levels through dietary restriction or chemotherapeutic treatment could limit HCMV replication in patients.

scholarly journals Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Dominique McCormick ◽  
Yao-Tang Lin ◽  
Finn Grey
Keyword(s):  
Virus Replication ◽  
Human Cytomegalovirus ◽  
High Throughput ◽  
Small Interfering Rna ◽  
Virus Production ◽  
Host Factors ◽  
Solid Organ ◽  
Combining Data ◽  
Interfering Rna ◽  
Host Genes

ABSTRACT As obligate intracellular parasites, viruses are completely dependent on host factors for replication. Assembly and egress of complex virus particles, such as human cytomegalovirus (HCMV), are likely to require many host factors. Despite this, relatively few have been identified and characterized. This study describes a novel high-throughput, two-step small interfering RNA (siRNA) screen, which independently measures virus replication and virus production. By combining data from replication and virus production, multiple candidate genes were identified in which knockdown resulted in substantial loss of virus production with limited effect on primary replication, suggesting roles in later stages such as virus assembly and egress. Knockdown of the top candidates, ERC1, RAB4B, COPA, and COPB2, caused profound loss of virus production. Despite COPA and COPB2 being reported to function in the same complex, knockdown of these genes produced distinct phenotypes. Furthermore, knockdown of COPA caused increased expression of viral late genes despite substantial inhibition of viral DNA replication. This suggests that efficient viral genome replication is not required for late gene expression. Finally, we show that RAB4B relocates to the viral assembly compartment following infection with HCMV and knockdown of RAB4B reduces the release of intact virion particles, suggesting that it plays a role in virion assembly and egress. This study demonstrates a powerful high-throughput screen for identification of host-virus interactions, identifies multiple host genes associated with HCMV assembly and egress, and uncovers potentially independent functions for coatomer components COPA and COPB2 during infection. IMPORTANCE Human cytomegalovirus infection is a significant cause of disease in immunocompromised populations, individuals with heart disease, and recipients of solid organ and bone marrow transplants. HCMV is also the leading cause of infectious congenital birth defects. The majority of antivirals in clinical use target components of the virus to specifically inhibit replication. However, a major drawback of this approach is the emergence of resistance. An alternative approach is to target host factors that the virus requires for successful infection. In this study, multiple host factors were identified that were found to be essential for the production of newly infectious human cytomegalovirus. Identifying which host genes are necessary for virus replication extends our understanding of how viruses replicate and how cells function and provides potential targets for novel antivirals.

scholarly journals Strategy of Human Cytomegalovirus To Escape Interferon Beta-Induced APOBEC3G Editing Activity

2018 ◽  
Vol 92 (19) ◽  
Author(s):  
Sara Pautasso ◽  
Ganna Galitska ◽  
Valentina Dell'Oste ◽  
Matteo Biolatti ◽  
Rachele Cagliani ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Hot Spots ◽  
Interferon Beta ◽  
Hcmv Infection ◽  
Viral Infections ◽  
Innate Immune ◽  
Mutational Robustness ◽  
Wide Range ◽  
Hcmv Replication ◽  
Apobec3 Family

ABSTRACTThe apolipoprotein B editing enzyme catalytic subunit 3 (APOBEC3) is a family of DNA cytosine deaminases that mutate and inactivate viral genomes by single-strand DNA editing, thus providing an innate immune response against a wide range of DNA and RNA viruses. In particular, APOBEC3A (A3A), a member of the APOBEC3 family, is induced by human cytomegalovirus (HCMV) in decidual tissues where it efficiently restricts HCMV replication, thereby acting as an intrinsic innate immune effector at the maternal-fetal interface. However, the widespread incidence of congenital HCMV infection implies that HCMV has evolved to counteract APOBEC3-induced mutagenesis through mechanisms that still remain to be fully established. Here, we have assessed gene expression and deaminase activity of various APOBEC3 gene family members in HCMV-infected primary human foreskin fibroblasts (HFFs). Specifically, we show that APOBEC3G (A3G) gene products and, to a lesser degree, those of A3F but not of A3A, are upregulated in HCMV-infected HFFs. We also show that HCMV-mediated induction of A3G expression is mediated by interferon beta (IFN-β), which is produced early during HCMV infection. However, knockout or overexpression of A3G does not affect HCMV replication, indicating that A3G is not a restriction factor for HCMV. Finally, through a bioinformatics approach, we show that HCMV has evolved mutational robustness against IFN-β by limiting the presence of A3G hot spots in essential open reading frames (ORFs) of its genome. Overall, our findings uncover a novel immune evasion strategy by HCMV with profound implications for HCMV infections.IMPORTANCEAPOBEC3 family of proteins plays a pivotal role in intrinsic immunity defense mechanisms against multiple viral infections, including retroviruses, through the deamination activity. However, the currently available data on APOBEC3 editing mechanisms upon HCMV infection remain unclear. In the present study, we show that particularly the APOBEC3G (A3G) member of the deaminase family is strongly induced upon infection with HCMV in fibroblasts and that its upregulation is mediated by IFN-β. Furthermore, we were able to demonstrate that neither A3G knockout nor A3G overexpression appears to modulate HCMV replication, indicating that A3G does not inhibit HCMV replication. This may be explained by HCMV escape strategy from A3G activity through depletion of the preferred nucleotide motifs (hot spots) from its genome. The results may shed light on antiviral potential of APOBEC3 activity during HCMV infection, as well as the viral counteracting mechanisms under A3G-mediated selective pressure.

scholarly journals Improved Detection of Mutated Human Cytomegalovirus UL97 by Pyrosequencing

2010 ◽  
Vol 54 (12) ◽  
pp. 5234-5241 ◽  
Author(s):  
Birgit Schindele ◽  
Luise Apelt ◽  
Jörg Hofmann ◽  
Andreas Nitsche ◽  
Detlef Michel ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Prolonged Treatment ◽  
Solid Organ ◽  
Immune Functions ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Reading Frame ◽  
Mutant Sequence ◽  
Cell Transplants

ABSTRACT Ganciclovir (GCV) resistance frequently occurs upon prolonged treatment of ongoing active human cytomegalovirus (HCMV) infection in individuals with immature or compromised immune functions (e.g., recipients of solid-organ and hematopoietic stem cell transplants). Using pyrosequencing (PSQ), we established fast and sensitive detection of GCV resistance-associated mutations occurring in the HCMV open reading frame UL97. These mutations have been repeatedly associated with clinical treatment failure. We designed four PSQ assays and evaluated them by analyzing mixtures of plasmids or bacterial artificial chromosome-derived viruses containing UL97 wild-type and mutant sequences. A minimum level of 6% mutant sequence variants could be detected in these mixtures. In order to further evaluate the novel PSQ assays, we tested clinical specimens from patients with active HCMV infections. The results were compared with those obtained by conventional dideoxy chain terminator sequencing. As the PSQ method was more sensitive in detecting minor HCMV mutant fractions in a wild-type population, it is suggested that pyrosequencing is a useful tool for the early detection of emerging GCV-resistant HCMV in GCV-treated patients.

scholarly journals The Thrombopoietin Receptor Agonist Eltrombopag Inhibits Human Cytomegalovirus Replication Via Iron Chelation

Cells ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 31 ◽  
Author(s):  
Jens-Uwe Vogel ◽  
Sophie Schmidt ◽  
Daniel Schmidt ◽  
Florian Rothweiler ◽  
Benjamin Koch ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Receptor Agonist ◽  
Iron Chelation ◽  
Drug Resistant ◽  
Hematopoietic Stem ◽  
Thrombopoietin Receptor ◽  
Hcmv Disease ◽  
Adult Mesenchymal Stem Cells ◽  
Thrombopoietin Receptor Agonist ◽  
Hcmv Replication

The thrombopoietin receptor agonist eltrombopag was successfully used against human cytomegalovirus (HCMV)-associated thrombocytopenia refractory to immunomodulatory and antiviral drugs. These effects were ascribed to the effects of eltrombopag on megakaryocytes. Here, we tested whether eltrombopag may also exert direct antiviral effects. Therapeutic eltrombopag concentrations inhibited HCMV replication in human fibroblasts and adult mesenchymal stem cells infected with six different virus strains and drug-resistant clinical isolates. Eltrombopag also synergistically increased the anti-HCMV activity of the mainstay drug ganciclovir. Time-of-addition experiments suggested that eltrombopag interfered with HCMV replication after virus entry. Eltrombopag was effective in thrombopoietin receptor-negative cells, and the addition of Fe3+ prevented the anti-HCMV effects, indicating that it inhibits HCMV replication via iron chelation. This may be of particular interest for the treatment of cytopenias after hematopoietic stem cell transplantation, as HCMV reactivation is a major reason for transplantation failure. Since therapeutic eltrombopag concentrations are effective against drug-resistant viruses, and synergistically increase the effects of ganciclovir, eltrombopag is also a drug-repurposing candidate for the treatment of therapy-refractory HCMV disease.

scholarly journals Human Cytomegalovirus-Encoded Human Interleukin-10 (IL-10) Homolog Amplifies Its Immunomodulatory Potential by Upregulating Human IL-10 in Monocytes

2016 ◽  
Vol 90 (8) ◽  
pp. 3819-3827 ◽  
Author(s):  
Selmir Avdic ◽  
Brian P. McSharry ◽  
Megan Steain ◽  
Emma Poole ◽  
John Sinclair ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Feedback Loop ◽  
Interleukin 10 ◽  
Negative Regulator ◽  
Viral Gene ◽  
Heme Oxygenase 1 ◽  
Solid Organ ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Wide Range

ABSTRACTThe human cytomegalovirus (HCMV) gene UL111A encodes cytomegalovirus-encoded human interleukin-10 (cmvIL-10), a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). This viral homolog exhibits a range of immunomodulatory functions, including suppression of proinflammatory cytokine production and dendritic cell (DC) maturation, as well as inhibition of major histocompatibility complex (MHC) class I and class II. Here, we present data showing that cmvIL-10 upregulates hIL-10, and we identify CD14+monocytes and monocyte-derived macrophages and DCs as major sources of hIL-10 secretion in response to cmvIL-10. Monocyte activation was not a prerequisite for cmvIL-10-mediated upregulation of hIL-10, which was dose dependent and controlled at the transcriptional level. Furthermore, cmvIL-10 upregulated expression of tumor progression locus 2 (TPL2), which is a regulator of the positive hIL-10 feedback loop, whereas expression of a negative regulator of the hIL-10 feedback loop, dual-specificity phosphatase 1 (DUSP1), remained unchanged. Engagement of the hIL-10 receptor (hIL-10R) by cmvIL-10 led to upregulation of heme oxygenase 1 (HO-1), an enzyme linked with suppression of inflammatory responses, and this upregulation was required for cmvIL-10-mediated upregulation of hIL-10. We also demonstrate an important role for both phosphatidylinositol 3-kinase (PI3K) and STAT3 in the upregulation of HO-1 and hIL-10 by cmvIL-10. In addition to upregulating hIL-10, cmvIL-10 could exert a direct immunomodulatory function, as demonstrated by its capacity to upregulate expression of cell surface CD163 when hIL-10 was neutralized. This study identifies a mechanistic basis for cmvIL-10 function, including the capacity of this viral cytokine to potentially amplify its immunosuppressive impact by upregulating hIL-10 expression.IMPORTANCEHuman cytomegalovirus (HCMV) is a large, double-stranded DNA virus that causes significant human disease, particularly in the congenital setting and in solid-organ and hematopoietic stem cell transplant patients. A prominent feature of HCMV is the wide range of viral gene products that it encodes which function to modulate host defenses. One of these is cmvIL-10, which is a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). In this study, we report that, in addition to exerting a direct biological impact, cmvIL-10 upregulates the expression of hIL-10 by primary blood-derived monocytes and that it does so by modulating existing cellular pathways. This capacity of cmvIL-10 to upregulate hIL-10 represents a mechanism by which HCMV may amplify its immunomodulatory impact during infection.

scholarly journals Human cytomegalovirus infection among treatment-naive HIV-1 infected patients in Ethiopia

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247264
Author(s):  
Mulugeta Kiros ◽  
Alene Geteneh ◽  
Henok Andualem ◽  
Derbie Alemu ◽  
Abebech Tesfaye ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Addis Ababa ◽  
Laboratory Diagnosis ◽  
Conventional Pcr ◽  
Hcmv Disease ◽  
Treatment Naïve ◽  
Hcmv Replication ◽  
Human Cytomegalovirus Infection ◽  
Hiv 1

Subclinical human cytomegalovirus (HCMV) replication is associated with immune dysfunction in immuno-suppressed antiretroviral therapy (ART) naive HIV infected individuals. No data is documented in Ethiopia so far concerning HCMV co-infection among HIV infected individuals. Hence, this study was aimed at generating data regarding the prevalence of active HCMV infection among treatment-naive HIV-infected individuals from Ethiopia. For this purpose, we enrolled 97 treatment-naive HIV infected study subjects in Addis Ababa from June to December 2018. ELISA and conventional PCR were performed consecutively to detect HCMV specific IgM antibody and HCMV DNA respectively. Of the 97 study subjects, 12 (12.4%) were positive for anti-CMV IgM antibodies but were not confirmed by PCR. With regard to the PCR positivity, 4/97 (4.1%) samples were positive for HCMV DNA. No statically significant associations were found between the dependent and independent variables. The presence of HCMV DNA in the current study highlights the need for a routine laboratory diagnosis for preventing HCMV disease among HIV-infected individuals early. Besides, the use of anti-CMV therapy for these CMV viremic individuals is also recommended as this can reduce the burden of CMV complications and consecutively prolonging the life of HIV infected individuals.

scholarly journals Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Lisa M. Wise ◽  
Yuecheng Xi ◽  
John G. Purdy
Keyword(s):  
Aryl Hydrocarbon Receptor ◽  
Virus Replication ◽  
Human Cytomegalovirus ◽  
Hypoxia Inducible Factor ◽  
Hypoxia Inducible Factor 1Α ◽  
Indoleamine 2,3 Dioxygenase ◽  
Aryl Hydrocarbon ◽  
Infected Cells ◽  
Hcmv Replication ◽  
Rate Limiting

ABSTRACT Human cytomegalovirus (HCMV) replication depends on the activities of several host regulators of metabolism. Hypoxia-inducible factor 1α (HIF1α) was previously proposed to support virus replication through its metabolic regulatory function. HIF1α protein levels rise in response to HCMV infection in nonhypoxic conditions, but its effect on HCMV replication was not investigated. We addressed the role of HIF1α in HCMV replication by generating primary human cells with HIF1α knocked out using CRISPR/Cas9. When HIF1α was absent, we found that HCMV replication was enhanced, showing that HIF1α suppresses viral replication. We used untargeted metabolomics to determine if HIF1α regulates metabolite concentrations in HCMV-infected cells. We discovered that in HCMV-infected cells, HIF1α suppresses intracellular and extracellular concentrations of kynurenine. HIF1α also suppressed the expression of indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting enzyme in kynurenine synthesis. In addition to its role in tryptophan metabolism, kynurenine acts as a signaling messenger by activating aryl hydrocarbon receptor (AhR). Inhibiting AhR reduces HCMV replication, while activating AhR with an exogenous ligand increases virus replication. Moreover, we found that feeding kynurenine to cells promotes HCMV replication. Overall, our findings indicate that HIF1α reduces HCMV replication by regulating metabolism and metabolite signaling. IMPORTANCE Viruses, including human cytomegalovirus (HCMV), reprogram cellular metabolism using host metabolic regulators to support virus replication. Alternatively, in response to infection, the host can use metabolism to limit virus replication. Here, our findings show that the host uses hypoxia-inducible factor 1α (HIF1α) as a metabolic regulator to reduce HCMV replication. Further, we found that HIF1α suppresses kynurenine synthesis, a metabolite that can promote HCMV replication by signaling through the aryl hydrocarbon receptor (AhR). In infected cells, the rate-limiting enzyme in kynurenine synthesis, indoleamine 2,3-dioxygenase 1 (IDO1), is suppressed by a HIF1α-dependent mechanism. Our findings describe a functional connection between HIF1α, IDO1, and AhR that allows HIF1α to limit HCMV replication through metabolic regulation, advancing our understanding of virus-host interactions.

scholarly journals The thrombopoietin receptor agonist eltrombopag inhibits human cytomegalovirus replication via iron chelation

2019 ◽  
Author(s):  
Jens-Uwe Vogel ◽  
Sophie Schmidt ◽  
Daniel Schmidt ◽  
Florian Rothweiler ◽  
Benjamin Koch ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Receptor Agonist ◽  
Iron Chelation ◽  
Haematopoietic Stem Cell ◽  
Drug Resistant ◽  
Thrombopoietin Receptor ◽  
Hcmv Disease ◽  
Adult Mesenchymal Stem Cells ◽  
Thrombopoietin Receptor Agonist ◽  
Hcmv Replication

AbstractThe thrombopoietin receptor agonist eltrombopag was successfully used against human cytomegalovirus (HCMV)-associated thrombocytopenia refractory to immunomodulatory and antiviral drugs. These effects were ascribed to effects of eltrombopag on megakaryocytes. Here, we tested whether eltrombopag may also exert direct antiviral effects. Therapeutic eltrombopag concentrations inhibited HCMV replication in human fibroblasts and adult mesenchymal stem cells infected with six different virus strains and drug-resistant clinical isolates. Eltrombopag also synergistically increased the anti-HCMV activity of the mainstay drug ganciclovir. Time-of-addition experiments suggested that eltrombopag interferes with HCMV replication after virus entry. Eltrombopag was effective in thrombopoietin receptor-negative cells, and addition of Fe3+ prevented the anti-HCMV effects, indicating that it inhibits HCMV replication via iron chelation. This may be of particular interest for the treatment of cytopenias after haematopoietic stem cell transplantation, as HCMV reactivation is a major reason for transplantation failure. Since therapeutic eltrombopag concentrations are effective against drug-resistant viruses and synergistically increase the effects of ganciclovir, eltrombopag is also a drug repurposing candidate for the treatment of therapy-refractory HCMV disease.

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