Convallatoxin-Induced Reduction of Methionine Import Effectively Inhibits Human Cytomegalovirus Infection and Replication.

Cytomegalovirus (CMV) is a ubiquitous human pathogen that increases the morbidity and mortality of immunocompromised individuals. The current FDA-approved treatments for CMV infection are intended to be virus specific, yet they have significant adverse side effects, including nephrotoxicity and hematological toxicity. Thus, there is a medical need for safer and more effective CMV therapeutics. Using a high-content screen, we identified the cardiac glycoside convallatoxin as an effective compound that inhibits CMV infection. Using a panel of cardiac glycoside variants, we assessed the structural elements critical for anti-CMV activity by both experimental and in silico methods. Analysis of the antiviral effects, toxicities, and pharmacodynamics of different variants of cardiac glycosides identified the mechanism of inhibition as reduction of methionine import, leading to decreased immediate-early gene translation without significant toxicity. Also, convallatoxin was found to dramatically reduce the proliferation of clinical CMV strains, implying that its mechanism of action is an effective strategy to block CMV dissemination. Our study has uncovered the mechanism and structural elements of convallatoxin, which are important for effectively inhibiting CMV infection by targeting the expression of immediate-early genes. IMPORTANCE: Cytomegalovirus is a highly prevalent virus capable of causing severe disease in certain populations. The current FDA-approved therapeutics all target the same stage of the viral life cycle and induce toxicity and viral resistance. We identified convallatoxin, a novel cell-targeting antiviral that inhibits CMV infection by decreasing the synthesis of viral proteins. At doses low enough for cells to tolerate, convallatoxin was able to inhibit primary isolates of CMV, including those resistant to the anti-CMV drug ganciclovir. In addition to identifying convallatoxin as a novel antiviral, limiting mRNA translation has a dramatic impact on CMV infection and proliferation.

CD160 activation by herpesvirus entry mediator augments inflammatory cytokine production and cytolytic function by NK cells.

Lymphocyte activation is regulated by costimulatory and inhibitory receptors, of which both B and T lymphocyte attenuator (BTLA) and CD160 engage herpesvirus entry mediator (HVEM). Notably, it remains unclear how HVEM functions with each of its ligands during immune responses. In this study, we show that HVEM specifically activates CD160 on effector NK cells challenged with virus-infected cells. Human CD56(dim) NK cells were costimulated specifically by HVEM but not by other receptors that share the HVEM ligands LIGHT, Lymphotoxin-α, or BTLA. HVEM enhanced human NK cell activation by type I IFN and IL-2, resulting in increased IFN-γ and TNF-α secretion, and tumor cell-expressed HVEM activated CD160 in a human NK cell line, causing rapid hyperphosphorylation of serine kinases ERK1/2 and AKT and enhanced cytolysis of target cells. In contrast, HVEM activation of BTLA reduced cytolysis of target cells. Together, our results demonstrate that HVEM functions as a regulator of immune function that activates NK cells via CD160 and limits lymphocyte-induced inflammation via association with BTLA.

Virological and immunological characteristics of human cytomegalovirus infection associated with Alzheimer disease.

Serum, cerebrospinal fluid (CSF), and cryopreserved lymphocytes from subjects in the Rush Alzheimer's Disease Center Religious Orders Study were analyzed for associations between cytomegalovirus (CMV) infection and clinical and pathological markers of Alzheimer disease. CMV antibody levels were associated with neurofibrillary tangles (NFTs). CSF interferon γ was only detected in seropositive subjects and was significantly associated with NFTs. The percentage of senescent T cells (CD4+ or CD8+CD28-CD57+) was significantly higher for CMV-seropositive as compared to CMV-seronegative subjects and was marginally associated with the pathologic diagnosis of Alzheimer disease (CD4+) or amyloid-ß (CD8+). Immunocytochemical analysis showed induction of amyloid-ß in human foreskin fibroblasts (HFFs) infected with each of 3 clinical CMV strains. In the same subjects, there was no association of herpes simplex virus type 1 (HSV-1) antibody levels with CMV antibody levels or clinical or pathological markers of Alzheimer disease. HSV-1 infection of HFFs did not induce amyloid-ß. These data support an association between CMV and the development of Alzheimer disease.

Modeling of human cytomegalovirus maternal-fetal transmission in a novel decidual organ culture.

Human cytomegalovirus (HCMV) is the leading cause of congenital infection, associated with severe birth defects and intrauterine growth retardation. The mechanism of HCMV transmission via the maternal-fetal interface is largely unknown, and there are no animal models for HCMV. The initial stages of infection are believed to occur in the maternal decidua. Here we employed a novel decidual organ culture, using both clinically derived and laboratory-derived viral strains, for the ex vivo modeling of HCMV transmission in the maternal-fetal interface. Viral spread in the tissue was demonstrated by the progression of infected-cell foci, with a 1.3- to 2-log increase in HCMV DNA and RNA levels between days 2 and 9 postinfection, the expression of immediate-early and late proteins, the appearance of typical histopathological features of natural infection, and dose-dependent inhibition of infection by ganciclovir and acyclovir. HCMV infected a wide range of cells in the decidua, including invasive cytotrophoblasts, macrophages, and endothelial, decidual, and dendritic cells. Cell-to-cell viral spread was revealed by focal extension of infected-cell clusters, inability to recover infectious extracellular virus, and high relative proportions (88 to 93%) of cell-associated viral DNA. Intriguingly, neutralizing HCMV hyperimmune globulins exhibited inhibitory activity against viral spread in the decidua even when added at 24 h postinfection-providing a mechanistic basis for their clinical use in prenatal prevention. The ex vivo-infected decidual cultures offer unique insight into patterns of viral tropism and spread, defining initial stages of congenital HCMV transmission, and can facilitate evaluation of the effects of new antiviral interventions within the maternal-fetal interface milieu.

Antiviral drug resistance of human cytomegalovirus.

The study of human cytomegalovirus (HCMV) antiviral drug resistance has enhanced knowledge of the virological targets and the mechanisms of antiviral activity. The currently approved drugs, ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV), target the viral DNA polymerase. GCV anabolism also requires phosphorylation by the virus-encoded UL97 kinase. GCV resistance mutations have been identified in both genes, while FOS and CDV mutations occur only in the DNA polymerase gene. Confirmation of resistance mutations requires phenotypic analysis; however, phenotypic assays are too time-consuming for diagnostic purposes. Genotypic assays based on sequencing provide more rapid results but are dependent on prior validation by phenotypic methods. Reports from many laboratories have produced an evolving list of confirmed resistance mutations, although differences in interpretation have led to some confusion. Recombinant phenotyping methods performed in a few research laboratories have resolved some of the conflicting results. Treatment options for drug-resistant HCMV infections are complex and have not been subjected to controlled clinical trials, although consensus guidelines have been proposed. This review summarizes the virological and clinical data pertaining to HCMV antiviral drug resistance.

Multilaboratory evaluation of real-time PCR tests for hepatitis B virus DNA quantification.

The performance characteristics of four different assays for hepatitis B virus (HBV) quantification were assessed: the Abbott RealTime HBV IUO, the Roche Cobas AmpliPrep/Cobas TaqMan HBV test, the Roche Cobas TaqMan HBV test with HighPure system, and the Qiagen artus HBV TM ASR. Limit of detection (LOD), linear range, reproducibility, and agreement were determined using a serially diluted plasma sample from a single chronically infected subject. Each assay was tested by at least three laboratories. The LOD of the RealTime and two TaqMan assays was approximately 1.0 log(10) IU/ml; for artus HBV (which used the lowest volume of extracted DNA), it was approximately 1.5 log(10) IU/ml. The linear range spanned 1.0 to at least 7.0 log(10) IU/ml for all assays. Median values were consistently lowest for artus HBV and highest for Cobas AmpliPrep/Cobas TaqMan HBV. Assays incorporating automated nucleic acid extraction were the most reproducible; however, the overall variability was minor since the standard deviations for the means of all tested concentrations were ≤0.32 log(10) IU/ml for all assays. False-positive results were observed with all assays; the highest rates occurred with tests using manual nucleic acid extraction. The performance characteristics of these assays suggest that they are useful for management and therapeutic monitoring of chronic HBV infection.

Author Correction: Ginkgolic acid inhibits fusion of enveloped viruses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ginkgolic acid inhibits fusion of enveloped viruses.

Ginkgolic acids (GA) are alkylphenol constituents of the leaves and fruits of Ginkgo biloba. GA has shown pleiotropic effects in vitro, including: antitumor effects through inhibition of lipogenesis; decreased expression of invasion associated proteins through AMPK activation; and potential rescue of amyloid-ß (Aß) induced synaptic impairment. GA was also reported to have activity against Escherichia coli and Staphylococcus aureus. Several mechanisms for this activity have been suggested including: SUMOylation inhibition; blocking formation of the E1-SUMO intermediate; inhibition of fatty acid synthase; non-specific SIRT inhibition; and activation of protein phosphatase type-2C. Here we report that GA inhibits Herpes simplex virus type 1 (HSV-1) by inhibition of both fusion and viral protein synthesis. Additionally, we report that GA inhibits human cytomegalovirus (HCMV) genome replication and Zika virus (ZIKV) infection of normal human astrocytes (NHA). We show a broad spectrum of fusion inhibition by GA of all three classes of fusion proteins including HIV, Ebola virus (EBOV), influenza A virus (IAV) and Epstein Barr virus (EBV). In addition, we show inhibition of a non-enveloped adenovirus. Our experiments suggest that GA inhibits virion entry by blocking the initial fusion event. Data showing inhibition of HSV-1 and CMV replication, when GA is administered post-infection, suggest a possible secondary mechanism targeting protein and DNA synthesis. Thus, in light of the strong effect of GA on viral infection, even after the infection begins, it may potentially be used to treat acute infections (e.g. Coronavirus, EBOV, ZIKV, IAV and measles), and also topically for the successful treatment of active lesions (e.g. HSV-1, HSV-2 and varicella-zoster virus (VZV)).

High-throughput sequence analysis of variants of human cytomegalovirus strains Towne and AD169.

The genomes of commonly used variants of human cytomegalovirus (HCMV) strains Towne and AD169 each contain a substantial mutation in which a region (U(L)/b') at the right end of the long unique region has been replaced by an inverted duplication of a region from the left end of the genome. Using high-throughput technology, we have sequenced HCMV strain Towne (ATCC VR-977) and confirmed the presence of two variants, one exhibiting the replacement in U(L)/b' and the other intact in this region. Both variants are mutated in genes RL13, UL1, UL40, UL130, US1 and US9. We have also sequenced a novel AD169 variant (varUC) that is intact in U(L)/b' except for a small deletion that affects genes UL144, UL142, UL141 and UL140. Like other AD169 variants, varUC is mutated in genes RL5A, RL13, UL36 and UL131A. A subpopulation of varUC contains an additional deletion affecting genes IRS1, US1 and US2.

Multisite comparison of anti-human immunodeficiency virus microbicide activity in explant assays using a novel endpoint analysis.

Microbicide candidates with promising in vitro activity are often advanced for evaluations using human primary tissue explants relevant to the in vivo mucosal transmission of human immunodeficiency virus type 1 (HIV-1), such as tonsil, cervical, or rectal tissue. To compare virus growth or the anti-HIV-1 efficacies of candidate microbicides in tissue explants, a novel soft-endpoint method was evaluated to provide a single, objective measurement of virus growth. The applicability of the soft endpoint is shown across several different ex vivo tissue types, with the method performed in different laboratories, and for a candidate microbicide (PRO 2000). The soft-endpoint method was compared to several other endpoint methods, including (i) the growth of virus on specific days after infection, (ii) the area under the virus growth curve, and (iii) the slope of the virus growth curve. Virus growth at the assay soft endpoint was compared between laboratories, methods, and experimental conditions, using nonparametric statistical analyses. Intra-assay variability determinations using the coefficient of variation demonstrated higher variability for virus growth in rectal explants. Significant virus inhibition by PRO 2000 and significant differences in the growth of certain primary HIV-1 isolates were observed by the majority of laboratories. These studies indicate that different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy when (i) the soft endpoint or another standardized endpoint is used, (ii) drugs and/or virus reagents are centrally sourced, and (iii) the same explant tissue type and method are used. Application of the soft-endpoint method reduces the inherent variability in comparisons of preclinical assays used for microbicide development.

Increased CMV IgG Antibody Titer is Associated with Non-AIDS Events among Virologically Suppressed HIV-Positive Persons.

BACKGROUND: Among HIV-positive individuals, increased levels of inflammation and immune activation persist even in the setting of effective antiretroviral therapy (ART) and are associated with greater rates of non-AIDS events. The etiology of this persistent inflammation is incompletely understood. METHODS: Using a well-characterized cohort of 322 HIV-infected individuals on suppressive ART, we conducted a case-control study. Cytomegalovirus (CMV) immunoglobulin G (IgG) levels, plasma biomarkers, and T-cell phenotypes were measured/characterized from samples collected 1 year after ART initiation. Conditional logistic regression for matched case-control studies analyzed the associations of year 1 CMV-specific IgG level with the subsequent occurrence of any non-AIDS event. Correlations between continuous CMV IgG antibody levels and soluble and cellular markers were assessed. RESULTS: We found that higher levels of CMV IgG were associated with increased risk of non-AIDS events (OR = 1.58 per IQR [95% CI: 1.12, 2.24], P = 0.01) and with elevated soluble and cellular markers of inflammation. CONCLUSIONS: The magnitude of the host immune response to CMV may play a role in the persistent inflammation and resultant morbid events observed in the HIV-positive population.

Artesunate as a potent antiviral agent in a patient with late drug-resistant cytomegalovirus infection after hematopoietic stem cell transplantation.

This is the first report of treatment of cytomegalovirus infection with artesunate, for a stem cell transplant recipient with a newly identified foscarnet-resistant and ganciclovir-resistant DNA polymerase L776M mutation. Artesunate treatment resulted in a 1.7-2.1-log reduction in viral load by treatment day 7, with a viral half-life of 0.9-1.9 days, indicating a highly effective block in viral replication.

Upregulation of human cytomegalovirus by HIV type 1 in human lymphoid tissue ex vivo.

HIV-1 copathogens are believed to play a critical role in progression to AIDS. Human cytomegalovirus (HCMV) has a high prevalence in the general population and is a common copathogen in HIV-1-infected individuals. Important events in copathogen interactions with HIV-1 take place in lymphoid tissue where critical events in HIV-1 disease occur. Here, we used an experimental system of human lymphoid tissue ex vivo to investigate interactions of HCMV with HIV-1. We inoculated ex vivo blocks of human lymphoid tissue with a recombinant strain of HCMV, expressing the green fluorescent protein, and HIV-1 and monitored viral replication and the phenotype of productively infected cells. HCMV readily replicated in tissue blocks as revealed by the release of HCMV viral DNA and an increasing number of viral-positive cells. Immunophenotyping of HCMV-infected cells showed a preferential infection of activated lymphocytes. The number of these cells significantly increased in HIV-1-coinfected tissues. Accordingly, HCMV replication was enhanced 2- to-3 fold. This upregulation occurred in tissues infected with either CXCR4- or CCR5-utilizing HIV-1. Thus, HIV-1 creates new targets for HCMV, which may explain the strong association of HCMV with HIV-1 infection in vivo. Ex vivo-infected human lymphoid tissue constitutes a model to study the mechanisms of HCMV tissue pathogenesis and its interactions with HIV-1 and this model may provide new targets for anti-HIV-1 therapy.

Differential inhibition of human cytomegalovirus (HCMV) by toll-like receptor ligands mediated by interferon-beta in human foreskin fibroblasts and cervical tissue.

Human cytomegalovirus (HCMV) can be acquired sexually and is shed from the genital tract. Cross-sectional studies in women show that changes in genital tract microbial flora affect HCMV infection and/or shedding. Since genital microbial flora may affect HCMV infection or replication by stimulating cells through Toll-like receptors (TLR), we assessed the effects of defined TLR-ligands on HCMV replication in foreskin fibroblasts and ectocervical tissue. Poly I:C (a TLR3-ligand) and lipopolysaccharide (LPS, a TLR4-ligand) inhibited HCMV and induced secretion of IL-8 and Interferon-beta (IFNbeta) in both foreskin fibroblasts and ectocervical tissue. The anti-HCMV effect was reversed by antibody to IFNbeta. CpG (TLR9 ligand) and lipoteichoic acid (LTA, TLR2 ligand) also inhibited HCMV infection in ectocervical tissue and this anti-HCMV effect was also reversed by anti-IFNbeta antibody. In contrast, LTA and CpG did not inhibit HCMV infection in foreskin fibroblasts. This study shows that TLR ligands induce an HCMV-antiviral effect that is mediated by IFNbeta suggesting that changes in genital tract flora may affect HCMV infection or shedding by stimulating TLR. This study also contrasts the utility of two models that can be used for assessing the interaction of microbial flora with HCMV in the genital tract. Clear differences in the response to different TLR ligands suggests the explant model more closely reflects in vivo responses to genital infections.

Analysis of the human cytomegalovirus genomic region from UL146 through UL147A reveals sequence hypervariability, genotypic stability, and overlapping transcripts.

BACKGROUND: Although the sequence of the human cytomegalovirus (HCMV) genome is generally conserved among unrelated clinical strains, some open reading frames (ORFs) are highly variable. UL146 and UL147, which encode CXC chemokine homologues are among these variable ORFs. RESULTS: The region of the HCMV genome from UL146 through UL147A was analyzed in clinical strains for sequence variability, genotypic stability, and transcriptional expression. The UL146 sequences in clinical strains from two geographically distant sites were assigned to 12 sequence groups that differ by over 60% at the amino acid level. The same groups were generated by sequences from the UL146-UL147 intergenic region and the UL147 ORF. In contrast to the high level of sequence variability among unrelated clinical strains, the sequences of UL146 through UL147A from isolates of the same strain were highly stable after repeated passage both in vitro and in vivo. Riboprobes homologous to these ORFs detected multiple overlapping transcripts differing in temporal expression. UL146 sequences are present only on the largest transcript, which also contains all of the downstream ORFs including UL148 and UL132. The sizes and hybridization patterns of the transcripts are consistent with a common 3'-terminus downstream of the UL132 ORF. Early-late expression of the transcripts associated with UL146 and UL147 is compatible with the potential role of CXC chemokines in pathogenesis associated with viral replication. CONCLUSION: Clinical isolates from two different geographic sites cluster in the same groups based on the hypervariability of the UL146, UL147, or the intergenic sequences, which provides strong evidence for linkage and no evidence for interstrain recombination within this region. The sequence of individual strains was absolutely stable in vitro and in vivo, which indicates that sequence drift is not a mechanism for the observed sequence hypervariability. There is also no evidence of transcriptional splicing, although multiple overlapping transcripts extending into the adjacent UL148 and UL132 open reading frames were detected using gene-specific probes.

The Association of Human Cytomegalovirus with Biomarkers of Inflammation and Immune Activation in HIV-1-Infected Women.

Three groups of cytomegalovirus (CMV)-seropositive women (total n = 164) were selected from the Chicago Women's Interagency HIV-1 Study to investigate the association between CMV coinfection and immune activation: (1) HIV-1 viremic, (2) HIV-1 aviremic, and (3) HIV-1 uninfected. Quantitative measures of CMV serum IgG, CMV DNA, and serum biomarkers interleukin (IL)-6, soluble CD163 (sCD163), soluble CD14 (sCD14), and interferon gamma-induced protein (IP10) were obtained. Levels of CMV IgG and the serum biomarkers were significantly higher in the HIV-1 viremic group compared to the aviremic and uninfected groups (p < 0.001). No significant associations with CMV IgG levels were found for HIV-uninfected women. When each of the HIV-infected groups was analyzed, sCD14 levels in the viremic women were significantly associated with CMV IgG levels with p < 0.02 when adjusted for age, CD4 count, and HIV viral load. There was also a modest association (p = 0.036) with IL-6 from plasma and cervical vaginal lavage specimens both unadjusted and adjusted for CD4 count and HIV viral load. The association of CMV IgG level with sCD14 implicates the monocyte as a potential site for interaction of the two viruses, which eventually may lead to non-AIDS-defining pathological conditions.

Emergence of ganciclovir-resistant cytomegalovirus in lung transplant recipients.

BACKGROUND: Since ganciclovir-resistant cytomegalovirus (CMV) disease was initially described in a patient with acquired immunodeficiency syndrome (AIDS) in 1986, the incidence of ganciclovir-resistant CMV disease appears to be increasing in immunocompromised patients. More recently, there have been sporadic reports of ganciclovir-resistant CMV disease in solid organ transplantation. METHODS: We retrospectively assessed the incidence of ganciclovir-resistant CMV disease in all lung transplant recipients transplanted between 6/93 and 6/01 at Loyola University Medical Center. All patients underwent routine CMV blood culture, shell vial assay as well as phenotypic and genotypic anti-viral susceptibility testing according to a pre-determined schedule. The number of CMV episodes, intravenous ganciclovir use, acute and chronic rejection and survival data were documented for all patients. RESULTS: Twelve of 212 (6%) transplant recipients developed ganciclovir-resistant CMV disease. Ganciclovir resistance was associated with a higher number of CMV episodes (3.4 +/- 2.3 episodes/patient vs 1.7 +/- 0.7 episodes/patient [p < 0.05]) and an increased exposure to cumulative intravenous ganciclovir in the primary CMV-mismatched (D(+)R(-)) population (22 +/- 10 vs 13 +/- 7 days [p < 0.05]) compared with patients who did not develop ganciclovir resistance. In addition, the use of daclizumab therapy was associated with a 7-fold greater likelihood of developing ganciclovir resistance (p < 0.0001). The presence of ganciclovir-resistant CMV disease in our population was associated with a decreased survival that could be attributed to CMV disease itself (p < 0.05). CONCLUSIONS: By screening all lung transplant recipients with CMV disease for ganciclovir resistance, we were able to detect a higher incidence of ganciclovir-resistant CMV disease (6%) than previously seen in solid organ transplantation. High-risk patients (D(+)R(-) CMV serostatus) who receive anti-lymphocytic therapy should be monitored aggressively and treated to prevent the development of ganciclovir resistance and avert a negative outcome.

Evaluation of real-time PCR laboratory-developed tests using analyte-specific reagents for cytomegalovirus quantification.

Viral load testing for cytomegalovirus (CMV) has become the standard for the diagnosis of infection and monitoring of therapy at many transplant centers. However, no viral load test has been approved by the FDA. Therefore, many laboratories rely on laboratory-developed assays. This study evaluated the performance characteristics of two real-time PCR tests developed using the artus CMV analyte-specific reagents (ASRs). One version is distributed by Abbott Molecular and the other by QIAGEN. For plasma specimens, the Abbott test had a limit of detection of 2.3 log10 copies/ml and a linear range up to at least 6.0 log10 copies/ml. Comparison of plasma viral loads using the Abbott test and the Roche Amplicor Monitor test showed a mean difference of -0.012 log10 copies/ml. In addition, the Abbott test viral loads correlated with the Digene Hybrid Capture assay ratios. Viral loads obtained from plasma specimens tested by the Abbott and QIAGEN tests were in very close agreement (mean difference, 0.144 log10 copies/ml). When the QIAGEN test was evaluated with the QIAGEN, MagNA Pure, and easyMAG extraction methods, the viral loads for all three methods were within 0.370 log10 copies/ml. Thus, there is good agreement between viral loads obtained by the different tests using the same extraction method or by the same test using different extraction methods. The availability of real-time PCR ASRs provides additional reagents that can be used for CMV viral load testing.

Human cytomegalovirus and human immunodeficiency virus type-1 co-infection in human cervical tissue.

Human cytomegalovirus (HCMV) and human immunodeficiency virus type-1 (HIV-1) infect the female genital tract. A human cervical explant model was developed to study single and dual infection by these viruses in the genital compartment. An HCMV strain expressing green fluorescent protein, and two clinical HCMV strains produced peak viral DNA copies at 14 to 21 days post-infection. Peak levels of HIV-1(Ba-L) p24 antigen occurred at 7 days post-infection. HIV-1(Ba-L) appeared to enhance HCMV in co-infected tissues. Singly and dually infected explants produced increased levels of cytokines IL-6, IL-8, and GRO-alpha in culture supernatants. Immunohistochemical and flow cytometric analysis showed HCMV infection of leukocytes with the phenotype CD45+/CD1a+/CD14+/HLA-DR+ but not stromal or endothelial cells. Cells expressing both GFP and HIV-1 p24 antigen were detected in co-infected tissues. The cervical explants provide an ex vivo human model for examining mechanisms of virus-virus interaction and pathogenesis in clinically relevant tissue.