Differential Expression of PDGF Receptor-α in Human Placental Trophoblasts Leads to Different Entry Pathways by Human Cytomegalovirus Strains.

Human cytomegalovirus (CMV) is the leading non-genetic cause of fetal malformation in developed countries. CMV placental infection is a pre-requisite for materno-fetal transmission of virus, and fetal infection. We investigated the roles of the viral pentameric complex gH/gL/pUL128-pUL131A, and cellular platelet-derived growth factor receptor-α (PDGFRα) for CMV infection in first trimester extravillous-derived (SGHPL-4) and villous-derived (HTR-8/SVneo) trophoblast cells. Infection with four CMV clinical and laboratory strains (Merlin, TB40E, Towne, AD169), and Merlin deletion mutants of UL128-, UL130-, and UL131A-genes, showed a cell type-dependent requirement of the viral pentameric complex for infection of trophoblast cells. The viral pentameric complex was essential for infection of villous trophoblasts, but non-essential for extravillous trophoblasts. Blocking of PDGFRα in extravillous trophoblasts, which naturally express PDGFRα, inhibited entry of pentameric complex-deficient CMV strains, but not the entry of pentameric positive CMV strains. Transient expression of PDGFRα in villous trophoblasts, which are naturally deficient in PDGFRα, promoted the entry of CMV strains lacking gH/gL/pUL128-pUL131A, but had no effect on entry of pentameric positive CMV strains. These results suggest PDGFRα is an important cell receptor for entry of CMV mutant strains lacking gH/gL/pUL128-pUL131A complexes in some placental cells, suggesting these entry pathways could be potential antiviral targets.

New insight into the phosphorylation-regulated intranuclear localization of human cytomegalovirus pUL69 mediated by cyclin-dependent kinases (CDKs) and viral CDK orthologue pUL97.

Cyclin-dependent kinases (CDKs) are multifaceted regulators involved in the replication of human cytomegalovirus. Recently, we demonstrated an interaction of CDK9-cyclin T1 as well as viral CDK orthologue pUL97 with the viral regulator pUL69, thereby leading to pUL69-activating phosphorylation. Here, we demonstrate that colocalization and direct pUL69-cyclin T1 interaction is independent of viral strains and host cell types. In vitro phosphorylation of pUL69 by CDK9 or pUL97 did not occur in a single site-specific manner, but at multiple sites. The previously described fine-speckled nuclear aggregation of pUL69 was assigned to the late phase of viral replication. CDK inhibitors, including a novel inhibitor of the CDK-activating kinase CDK7, massively intensified this fine-speckled accumulation. Interestingly, we also observed spontaneous pUL69 accumulation in the absence of inhibitors at a lower frequency. These findings provide new insight into pUL69 kinase interregulation and emphasize the importance of pUL69 phosphorylation for correct intranuclear localization.

Congenital cytomegalovirus infection in pregnancy: a review of prevalence, clinical features, diagnosis and prevention.

Human cytomegalovirus (CMV) is under-recognised, despite being the leading infectious cause of congenital malformation, affecting ~0.3% of Australian live births. Approximately 11% of infants born with congenital CMV infection are symptomatic, resulting in clinical manifestations, including jaundice, hepatosplenomegaly, petechiae, microcephaly, intrauterine growth restriction and death. Congenital CMV infection may cause severe long-term sequelae, including progressive sensorineural hearing loss and developmental delay in 40-58% of symptomatic neonates, and ~14% of initially asymptomatic infected neonates. Up to 50% of maternal CMV infections have nonspecific clinical manifestations, and most remain undetected unless specific serological testing is undertaken. The combination of serology tests for CMV-specific IgM, IgG and IgG avidity provide improved distinction between primary and secondary maternal infections. In pregnancies with confirmed primary maternal CMV infection, amniocentesis with CMV-PCR performed on amniotic fluid, undertaken after 21-22 weeks gestation, may determine whether maternofetal virus transmission has occurred. Ultrasound and, to a lesser extent, magnetic resonance imaging are valuable tools to assess fetal structural and growth abnormalities, although the absence of fetal abnormalities does not exclude fetal damage. Diagnosis of congenital CMV infection at birth or in the first 3 weeks of an infant's life is crucial, as this should prompt interventions for prevention of delayed-onset hearing loss and neurodevelopmental delay in affected infants. Prevention strategies should also target mothers because increased awareness and hygiene measures may reduce maternal infection. Recognition of the importance of CMV in pregnancy and in neonates is increasingly needed, particularly as therapeutic and preventive interventions expand for this serious problem.

Prevention of congenital cytomegalovirus complications by maternal and neonatal treatments: a systematic review.

Human cytomegalovirus is the leading non-genetic cause of congenital malformation in developed countries. Congenital CMV may result in fetal and neonatal death or development of serious clinical sequelae. In this review, we identified evidence-based interventions for prevention of congenital CMV at the primary level (prevention of maternal infection), secondary level (risk reduction of fetal infection and disease) and tertiary level (risk reduction of infected neonates being affected by CMV). A systematic review of existing literature revealed 24 eligible studies that met the inclusion criteria. Prevention of maternal infection using hygiene and behavioural interventions reduced maternal seroconversion rates during pregnancy. However, evidence suggested maternal adherence to education on preventative behaviours was a limiting factor. Treatment of maternal CMV infection with hyperimmune globulin (HIG) showed some evidence for efficacy in prevention of fetal infection and fetal/neonatal morbidity with a reasonable safety profile. However, more robust clinical evidence is required before HIG therapy can be routinely recommended. Limited evidence also existed for the safety and efficacy of established CMV antivirals (valaciclovir, ganciclovir and valganciclovir) to treat neonatal consequences of CMV infection, but toxicity and lack of randomised clinical trial data remain major issues. In the absence of a licensed CMV vaccine or robust clinical evidence for anti-CMV therapeutics, patient education and behavioural interventions that emphasise adherence remain the best preventative strategies for congenital CMV. There is a strong need for further data on the use of HIG and other antivirals in pregnancy, as well as the development of less toxic, novel, antiviral agents.

Human cytomegalovirus directly modulates expression of chemokine CCL2 (MCP-1) during viral replication.

Human cytomegalovirus (CMV) infects monocytes and other haematopoietic progenitor cells which then act as reservoirs for latency and virus dissemination. The chemokine CCL2 (monocyte chemotactic protein-1 or MCP-1) exhibits potent chemotactic activity for monocytes and is a likely target for CMV-induced immunomodulation. In this study, we demonstrate CMV modulates CCL2 expression in MRC-5 fibroblasts with multiplicity-dependent kinetics, where CCL2 is upregulated during early stage infection, followed by CCL2 inhibition at late stage infection. This CMV-induced CCL2 modulation was dependent upon virus replication, as UV-inactivated virus did not elicit any changes in CCL2 levels. Dual immunofluorescence staining showed CMV strains AD169, purified AD169, Merlin, FIX WT (FLAG-US28/WT) and pUS28-deficient FIX (FIX-ΔUS28) all induced upregulation of CCL2 primarily within infected cells. Focal upregulation of CCL2 within FIX-ΔUS28-infected cells demonstrated intracellular CCL2 accumulation was independent of CCL2 sequestration by the CMV-encoded chemokine receptor US28. Infection with purified virus confirmed CMV-induced CCL2 upregulation was not due to any CCL2-inducing factors contained within non-purified virus stocks. The CMV-induced CCL2 expression kinetics occurred concurrently with modulation of the CCL2 transcriptional activators NF-κB, interferon regulatory factor 3 and cytokine IFN-ß, independent of virus strain, and with the establishment of viral replication compartments within infected cell nuclei. This is the first report to our knowledge to demonstrate CMV modulation of CCL2 expression within infected cells during viral replication. This immune modulation may facilitate virus dissemination, establishment of latency and pathogenesis of CMV-induced host disease.

Prevalence of viruses in stool of premature neonates at a neonatal intensive care unit.

AIM: Premature neonates represent a population highly vulnerable to infection. This study aims to profile viral colonisation of gut and the associated clinical manifestations among premature neonates admitted to a neonatal intensive care unit (NICU) in Australia. METHODS: In a cohort of 75 premature neonates born at less than 32 weeks gestation, who were followed for 4 weeks following admission to a NICU in Sydney, Australia, multiplex polymerase chain reaction assays were used to determine viral presence in stool, and clinical data were examined. RESULTS: Overall, viral RNA or DNA was detected in 24/419 (5.7%) of specimens, including norovirus genogroup 2 (1.9%), enterovirus (1.2%), herpes simplex virus-2 (1.2%), cytomegalovirus (0.7%), Epstein-Barr virus (0.5%) and rotavirus (0.2%). Viral infection was detected in 13/75 (17%) of premature neonates at some time point, including five (7%) neonates shedding more than one type of virus in stool. A higher rate of infection was observed among premature neonates with intrauterine growth restriction (56%) compared with those infants born appropriate for gestational age (12%. P = 0.006). CONCLUSION: The overall viral detection rate in stool of 5.7% (affecting 17% of neonates) indicates viral infections are an important health risk for premature infants in NICU.

Cytomegalovirus infection during pregnancy with maternofetal transmission induces a proinflammatory cytokine bias in placenta and amniotic fluid.

Congenital infection with cytomegalovirus (CMV) can induce immune responses and placental damage. By use of immunoassay panels, 27 cytokines were assessed in midtrimester amniotic fluid from 8 patients with congenital CMV, in midtrimester sera from 12 pregnant women with primary CMV infection, and in amniotic fluid and serum from uninfected maternal controls. Levels of the cytokines tumor necrosis factor α, interleukin 1ß, interleukin 12, and interleukin 17; the chemokines CCL2, CCL4, and CXCL10; and the growth factors granulocyte-macrophage colony-stimulating factor and platelet-derived growth factor bb were significantly elevated in amniotic fluid from congenital CMV patients (P < .01). Only CXCL10 was significantly higher in sera from CMV-infected pregnant women. CMV infection during pregnancy is associated with a shift in cytokine expression toward a proinflammatory state.

Successful corneal autograft after clearance of anterior chamber cytomegalovirus with oral valganciclovir in a patient with multiple failed corneal allografts.

PURPOSE: To report a case of recurrent corneal graft failure because of cytomegalovirus (CMV) infection and to demonstrate successful clearance of the virus with oral valganciclovir, confirmed by polymerase chain reaction (PCR) assay. This allowed for a successful corneal autograft to be performed. METHODS: Interventional case report. RESULTS: A 90-year-old white man with 4 previous corneal graft failures in his right eye is presented. His visual acuity was no light perception in the left eye subsequent to ocular trauma. His initial penetrating keratoplasty for pseudophakic bullous keratopathy was from a human leukocyte antigen-matched multiorgan donor who was CMV-seropositive. An anterior chamber paracentesis was performed to exclude an infective etiology. CMV was detected on PCR of aqueous humor. After a 12-week course of oral valganciclovir, a repeat aqueous PCR test confirmed the clearance of CMV. A corneal autograft from his left eye was subsequently performed with good outcome. CONCLUSIONS: We present a case of successful corneal autograft after clearance of CMV from the anterior chamber (PCR confirmed) in a patient treated with oral valganciclovir.

Cyclin-dependent Kinases Phosphorylate the Cytomegalovirus RNA Export Protein pUL69 and Modulate Its Nuclear Localization and Activity.

Replication of human cytomegalovirus (HCMV) is subject to regulation by cellular protein kinases. Recently, we and others reported that inhibition of cyclin-dependent protein kinases (CDKs) or the viral CDK ortholog pUL97 can induce intranuclear speckled aggregation of the viral mRNA export factor, pUL69. Here we provide the first evidence for a direct regulatory role of CDKs on pUL69 functionality. Although replication of all HCMV strains was dependent on CDK activity, we found strain-specific differences in the amount of CDK inhibitor-induced pUL69 aggregate formation. In all cases analyzed, the inhibitor-induced pUL69 aggregates were clearly localized within viral replication centers but not subnuclear splicing, pore complex, or aggresome structures. The CDK9 and cyclin T1 proteins colocalized with these pUL69 aggregates, whereas other CDKs behaved differently. Phosphorylation analyses in vivo and in vitro demonstrated pUL69 was strongly phosphorylated in HCMV-infected fibroblasts and that CDKs represent a novel class of pUL69-phosphorylating kinases. Moreover, the analysis of CDK inhibitors in a pUL69-dependent nuclear mRNA export assay provided evidence for functional impairment of pUL69 under suppression of CDK activity. Thus, our data underline the crucial importance of CDKs for HCMV replication, and indicate a direct impact of CDK9-cyclin T1 on the nuclear localization and activity of the viral regulator pUL69.

Successful valganciclovir treatment of post-transplant cytomegalovirus infection in the presence of UL97 mutation N597D.

Mutations in the human cytomegalovirus (CMV) UL97 protein kinase are the most common mechanism of ganciclovir (GCV) resistance in the clinical setting. A CMV strain with a previously unrecognized UL97 mutation N597D was identified in the blood of a heart transplant recipient who experienced a persistent CMV infection with high viral loads accompanying pain and fever while receiving valganciclovir (valGCV) therapy. The N597D mutation was transferred by mutagenesis to an antiviral sensitive CMV strain for analysis of antiviral susceptibility by standardized phenotypic assay. Recombinant phenotyping showed N597D conferred a less than twofold increase in GCV IC(50) compared to the sensitive control strain. Despite the presence of this mutation, valGCV eventually resolved the infection after 6 weeks of therapy. A subsequent CMV reactivation was also responsive to valganciclovir. This case illustrates the diversity of UL97 mutations in the codon segment 590-607 usually associated with GCV resistance, with some mutations producing minimal levels of resistance that do not preclude a therapeutic response to the drug. Accurate interpretation of genotypic test results ultimately requires experimental determination of the level of resistance conferred by newly discovered UL97 mutations.

Identification of putative functional motifs in viral proteins essential for human cytomegalovirus DNA replication.

Six of the eleven genes essential for Human cytomegalovirus (HCMV) DNA synthesis have been analyzed for putative structural motifs that may have a significant functional role in DNA replication. The genes studied encode for the DNA polymerase accessory protein (UL44), single-stranded DNA binding protein (UL57), primase-helicase complex (UL70, UL102, and UL105), and the putative initiator protein (UL84). The full-length open reading frames of these genes were highly conserved between ten isolates with amino acid sequence identity of >97% for all genes. Using ScanProsite software from the Expert Protein Analysis System (ExPASy) proteomics server, we have mapped putative motifs throughout these HCMV replication genes. Interesting motifs identified include casein kinase-2 (CKII) phosphorylation sites, a microbodies signal motif in UL57, and an ATP binding site in the putative UL105 helicase. Our investigations have also elucidated motif-rich regions of the UL44 DNA polymerase accessory protein and identified cysteine motifs that have potential implications for UL57 and UL70 primase. Taken together, these findings provide insights to regions of these HCMV replication proteins that are important for post-translation modification, activation, and overall function, and this information can be utilized to target further research into these proteins and advance the development of novel antiviral agents that target these processes.

Genotypic analysis of two hypervariable human cytomegalovirus genes.

Most human cytomegalovirus (HCMV) genes are highly conserved in sequence among strains, but some exhibit a substantial degree of variation. Two of these genes are UL146, which encodes a CXC chemokine, and UL139, which is predicted to encode a membrane glycoprotein. The sequences of these genes were determined from a collection of 184 HCMV samples obtained from Africa, Australia, Asia, Europe, and North America. UL146 is hypervariable throughout, whereas variation in UL139 is concentrated in a sequence encoding a potentially highly glycosylated region. The UL146 sequences fell into 14 genotypes, as did all previously reported sequences. The UL139 sequences grouped into 8 genotypes, and all previously reported sequences fell into a subset of these. There were minor differences among continents in genotypic frequencies for UL146 and UL139, but no clear geographical separation, and identical nucleotide sequences were represented among communities distant from each other. The frequent detection of multiple genotypes indicated that mixed infections are common. For both genes, the degree of divergence was sufficient to preclude reliable sequence alignments between genotypes in the most variable regions, and the mode of evolution involved in generating the genotypes could not be discerned. Within genotypes, constraint appears to have been the predominant mode, and positive selection was detected marginally at best. No evidence was found for linkage disequilibrium. The emerging scenario is that the HCMV genotypes developed in early human populations (or even earlier), becoming established via founder or bottleneck effects, and have spread, recombined and mixed worldwide in more recent times.

Emergence and persistence of multiple antiviral-resistant CMV strains in a highly immunocompromised child.

BACKGROUND: The emergence of human cytomegalovirus (CMV) antiviral resistance plays a significant role in disease progression in immunocompromised patients who have received antiviral therapy. OBJECTIVES: To determine the pattern of antiviral-resistant CMV strains in a highly immunocompromised child. STUDY DESIGN: Retrospective specimens of blood and urine were analysed using PCR-sequencing to identify antiviral-resistant CMV strains containing UL97 or UL54 mutations. RESULTS: CMV strains resistant to antiviral agents contributed to disease in a bone marrow transplant recipient with X-linked severe combined immunodeficiency (SCID) treated with ganciclovir (GCV) and foscarnet (FOS). Retrospective analyses detected GCV-resistant CMV (L595S) in a specimen taken after disease progression. This GCV-resistant CMV strain persisted for 1 year, after which time it was no longer detected even though the patient continued to receive GCV. A FOS-resistant strain (T700A) then emerged even though no FOS had been administered in the preceding year. CONCLUSION: The detection of antiviral-resistant CMV did not follow the patterns found in other patients tested for antiviral resistance, including emergence of a FOS-resistant strain in the absence of antiviral-selective pressure. These findings indicate the patient's underlying immunosuppressive condition should be considered for diagnosis and management of resistant CMV.

Multidrug resistance conferred by novel DNA polymerase mutations in human cytomegalovirus isolates.

The emergence of antiviral-resistant cytomegalovirus (CMV) strains is a continuing clinical problem, with increased numbers of immunocompromised patients given longer-duration antiviral prophylaxis. Two previously unrecognized CMV DNA polymerase mutations (N408K and A834P) identified separately and together in at-risk lung and kidney transplant recipients and a third mutation (L737M) identified in a liver transplant recipient were characterized by marker transfer to antiviral-sensitive laboratory strains AD169 and Towne. Subsequent phenotypic analyses of recombinant strains demonstrated the ability of mutation N408K to confer ganciclovir (GCV) and cidofovir (CDV) resistance and of mutation A834P to confer GCV, foscarnet, and CDV resistance. Mutation L737M did not confer resistance to any of the antiviral agents tested. A recombinant strain containing both N408K and A834P demonstrated increased GCV and CDV resistance compared to the levels of resistance of the virus containing only the A834P mutation. The addition of mutation N408K in combination with A834P also partially reconstituted the replication impairment of recombinant virus containing only A834P. This suggests that perturbation of both DNA polymerization (A834P) and exonuclease (N408K) activities contributes to antiviral resistance and altered replication kinetics in these mutant strains. The identification of these multidrug-resistant CMV strains in at-risk seronegative recipients of organs from seropositive donors suggests that improved prophylactic and treatment strategies are required. The additive effect of multiple mutations on antiviral susceptibility suggests that increasing antiviral-resistant phenotypes can result from different virus-antiviral interactions.

The interplay between host and viral factors in shaping the outcome of cytomegalovirus infection.

Cytomegalovirus (CMV) remains a major human pathogen causing significant morbidity and mortality in immunosuppressed or immunoimmature individuals. Although significant advances have been made in dissecting out certain features of the host response to human CMV (HCMV) infection, the strict species specificity of CMVs means that most aspects of antiviral immunity are best assessed in animal models. The mouse model of murine CMV (MCMV) infection is an important tool for analysis of in vivo features of host-virus interactions and responses to antiviral drugs that are difficult to assess in humans. Important studies of the contribution of host resistance genes to infection outcome, interplays between innate and adaptive host immune responses, the contribution of virus immune evasion genes and genetic variation in these genes to the establishment of persistence and in vivo studies of resistance to antiviral drugs have benefited from the well-developed MCMV model. In this review, we discuss recent advances in the immunobiology of host-CMV interactions that provide intriguing insights into the complex interplay between host and virus that ultimately facilitates viral persistence. We also discuss recent studies of genetic responses to antiviral therapy, particularly changes in DNA polymerase and protein kinase genes of MCMV and HCMV.

Characterisation of transcripts from the human cytomegalovirus genes TRL7, UL20a, UL36, UL65, UL94, US3 and US34.

The genome of human cytomegalovirus (HCMV) has been studied extensively in some regions, but not others. In this study, transcripts of the genome were further characterised for open reading frames (ORFs) TRL7, UL36, UL65, UL94, US3 and US34, and for the previously unrecognised ORF, UL20a. Reverse transcription-PCR demonstrated the presence of spliced transcripts from the putative glycoprotein gene, UL20a, at early and late times post-infection. US3 full-length and spliced transcripts, including a previously unidentified transcript (US3ii), were described at immediate early times. Sequencing of the complete ORFs of UL20a and US3 from 21 clinical isolates showed that US3 is well conserved in all isolates (97-100% identity), whereas UL20a shows more variation at the nucleotide level, with 90-100% identity. The limits of transcription, and splice donor and acceptor sequences for UL20a and US3 were conserved in all isolates, indicating likely conservation of mRNA splicing patterns. Sequencing a late cDNA library identified the limits of transcription for ORFs TRL7, UL94 and US34 and transcription from the TRL7 ORF was confirmed by northern blotting. Transcripts were found that were congruent with UL36 and UL65, but these differed in the limits previously predicted for these ORFs. These findings show the variation between predicted and actual transcription and indicate the complex nature of transcription from HCMV ORFs.