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Human exploration of the solar system will expose crew members to galactic cosmic radiation (GCR), with a potential for adverse health effects. GCR particles (protons and ions) move at nearly the speed of light and easily penetrate space station walls, as well as the human body. Previously, we have shown reactivation of latent herpesviruses, including herpes simplex virus, Varicella zoster virus, Epstein-Barr virus, and cytomegalovirus (CMV), during stays at the International Space Station. Given the prevalence of latent CMV and the known propensity of space radiation to cause alterations in many cellular processes, we undertook this study to understand the role of GCR in reactivating latent CMV. Latently infected Kasumi cells with CMV were irradiated with 137 Cs gamma rays, 150 MeV protons, 600 MeV/n carbon ions, 600 MeV/n iron ions, proton ions, and simulated GCR. The CMV copy number increased significantly in the cells exposed to radiation as compared with the non-irradiated controls. Viral genome sequencing did not reveal significant nucleotide differences among the compared groups. However, transcriptome analysis showed the upregulation of transcription of the UL49 ORF, implicating it in the switch from latent to lytic replication. These findings support our hypothesis that GCR may be a strong contributor to the reactivation of CMV infection seen in ISS crew members.

Pasteurized donor human milk is provided by milk banks to very preterm babies where their maternal supply is insufficient or unavailable. Donor milk is currently processed by Holder pasteurization, producing a microbiologically safe product but significantly reducing immunoprotective components. Ultraviolet-C (UV-C) irradiation at 254 nm is being investigated as an alternative treatment method and has been shown to preserve components such as lactoferrin, lysozyme and secretory IgA considerably better than Holder pasteurization. We describe the inactivation of cytomegalovirus, a virus commonly excreted into breast milk, using UV-C irradiation. Full replication was ablated by various treatment doses. However, evidence of viral immediate early proteins within the cells was never completely eliminated indicating that some viral gene transcription was still occurring. In conclusion, UV-C may be a safe alternative to pasteurisation for the treatment of human donor milk that preserves the bioactivity. However, our data suggests that CMV inactivation will have to be carefully evaluated for each device designed to treat breast milk using UV-C irradiation.

Background: Cytomegalovirus (CMV)-infected human milk (HM) can lead to significant CMV morbidity and mortality in preterm very-low-birth weight infants. The eradication of CMV in HM while preserving its properties poses a major clinical challenge., Objective: We aimed to compare two methods used to neutralize the virus in HM, one recognized as partially effective (freezing) and another not tested to date (microwave exposure)., Materials and Methods: We sampled HM from 31 CMV-seropositive mothers whose infants were hospitalized at the Lis Maternity Hospital. Fifteen samples that were positive for CMV antigen were divided into five 5 mL aliquots: the first a control, the second was frozen at -20°C for 1 day, the third was frozen at -200°C for 3 days, and the fourth and fifth aliquots were exposed for 30 seconds to microwave radiation at a low-power setting (500 W) and high-power setting (750 W), respectively., Results: Only microwave radiation at a high-power setting led to complete neutralization of CMV in all samples. Low-power microwave irradiation had a 13% failure rate while 3-day freezing and 1-day freezing had failure rates of 7% and 20%, respectively., Conclusion: It is possible to eradicate CMV successfully in HM by using microwave radiation at a high-power setting. Further studies are needed to evaluate the effect of microwave heating on breast milk properties.

Nucleotide-binding oligomerization domain 2 (NOD2) is an important innate immune sensor of bacterial pathogens. Its induction results in activation of the classic NF-κB pathway and alternative pathways including type I IFN and autophagy. Although the importance of NOD2 in recognizing RNA viruses has recently been identified, its role in sensing DNA viruses has not been studied. We report that infection with human cytomegalovirus (HCMV) results in significant induction of NOD2 expression, beginning as early as 2 hours post infection and increasing steadily 24 hours post infection and afterwards. Infection with human herpesvirus 1 and 2 does not induce NOD2 expression. While the HCMV-encoded glycoprotein B is not required for NOD2 induction, a replication competent virion is necessary. Lentivirus-based NOD2 knockdown in human foreskin fibroblasts (HFFs) and U373 glioma cells leads to enhanced HCMV replication along with decreased levels of interferon beta (IFN-β) and the pro-inflammatory cytokine, IL8. NOD2 induction in HCMV-infected cells activates downstream NF-κB and interferon pathways supported by reduced nuclear localization of NF-κB and pIRF3 in NOD2 knockdown HFFs. Stable overexpression of NOD2 in HFFs restricts HCMV replication in association with increased levels of IFN-β and IL8. Similarly, transient overexpression of NOD2 in U373 cells or its downstream kinase, RIPK2, results in decreased HCMV replication and enhanced cytokine responses. However, overexpression of a mutant NOD2, 3020insC, associated with severe Crohn's disease, results in enhanced HCMV replication and decreased levels of IFN-β in U373 cells. These results show for the first time that NOD2 plays a significant role in HCMV replication and may provide a model for studies of HCMV recognition by the host cell and HCMV colitis in Crohn's disease.

Suitable host cell metabolic conditions are fundamental for the effective development of the human cytomegalovirus (HCMV) lytic cycle. Indeed, several studies have demonstrated the ability of this virus to interfere with cell cycle regulation, mainly by blocking proliferating cells in G1 or G1/S. In the present study, we demonstrate that HCMV deregulates the cell cycle of THP-1 macrophages (a cell line irreversibly arrested in G0) by pushing them into S and G2 phases. Moreover, we show that HCMV infection of THP-1 macrophages leads to Toll-like receptor 4 (TLR4) activation. Since various studies have indicated TLR4 to be involved in promoting cell proliferation, here we investigate the possible role of TLR4 in the observed HCMV-induced cell cycle perturbation. Our data strongly support TLR4 as a mediator of HCMV-triggered cell cycle activation in THP-1 macrophages favouring, in turn, the development of an efficient viral lytic cycle., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Although it has been suggested that cardiovascular disease incidence is increased among atomic bomb survivors, the existence of a causal relationship between radiation exposure and atherosclerosis is unclear. Microbial infections, including those caused by Chlamydia pneumoniae, Helicobacter pylori and cytomegalovirus, have recently been implicated in atherosclerosis. Since immune function is somewhat impaired among atomic bomb survivors, their immune defense against such infections might be diminished. To investigate this possibility, we measured antibody levels to the above microorganisms in the sera of survivors. We found that the levels of IgG and IgA antibodies to Chlamydia pneumoniae decreased significantly with radiation dose, whereas the levels of IgG antibodies to Helicobacter pylori or cytomegalovirus remained unchanged. The inflammation marker C-reactive protein was significantly and positively associated with level of antibodies to Chlamydia pneumoniae only in heavily exposed (>or=1000 mGy) survivors. These results may suggest that among atomic bomb survivors, immune response to Chlamydia pneumoniae is diminished and chronic inflammatory reactions related to Chlamydia pneumoniae infection are present.

The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway.

This study investigated the incidence of acquired cytomegalovirus (CMV) infection in very low birth weight infants (VLBWI) given CMV seropositive blood, and sought to determine whether filtering and irradiation of blood products could help prevent CMV infection and the time required to clear passively-derived anti-CMV IgG among 80 VLBWI transfused with filtered-irradiated blood, 20 VLBWI transfused with nonfiltered- nonirradiated blood and 26 nontransfused VLBWI. CMV IgG and IgM values were obtained from all blood products prior to transfusions, and from VLBWI at birth until the infants became seronegative. Urine was obtained for CMV culture at birth and every 3-4 weeks until 12 weeks after the final transfusion. The incidence of CMV IgG seropositivity among the 126 infants at birth and the blood products given were 96% and 95%, respectively. The incidence of acquired CMV infection was 4/100 (4%) in the transfused group: 2/80 (2.5%) and 2/20 (10%) in the filtered-irradiated and nonfiltered-nonirradiated transfusion groups, respectively. Approximately 9-10 months elapsed to clear passively acquired CMV IgG. The irradiation and filtering of the blood products did not seem to decrease the transfusion-related CMV infection rate in Korea among VLBWI, however, further validation is recommended in a larger cohort of infants.

One of the most important innate host defense mechanisms against viral infection is the induction of interferon (IFN)-stimulated genes (ISGs). Immediately upon entry, viruses activate interferon-regulatory factor 3 (IRF3), as well as nuclear factor kappaB (NF-kappaB), which transactivate a subset of ISGs, proinflammatory genes, as well as IFN genes. Most large DNA viruses exhibit countermeasures against induction of this response. However, whereas human cytomegalovirus (HCMV) inhibits IFN-dependent induction of ISGs, IFN-independent induction of ISGs is observed both in the presence and, even moreso, in the absence of viral gene expression. Rhesus CMV (RhCMV) is an emerging animal model for HCMV sharing important similarities in primary structure, epidemiology, and pathogenesis. To determine whether RhCMV would similarly induce ISGs, we performed DNA microarray and quantitative PCR analysis of ISG expression in rhesus fibroblasts infected with RhCMV or HCMV. In contrast to HCMV, however, RhCMV did not induce expression of ISGs or proinflammatory genes at any time after infection. Moreover, dimerization and nuclear accumulation of IRF3, readily observed in HCMV-infected cells, was absent from RhCMV-infected cells, whereas neither virus seemed to activate NFkappaB. RhCMV also blocked IRF3 activation by live or UV-inactivated HCMV, suggesting that RhCMV inhibits viral IRF3 activation and the resultant ISG induction with extraordinary efficiency. Since infection during inhibition of protein expression by cycloheximide or inactivation of viral gene expression by UV treatment did not trigger IRF3 activation or ISG expression by RhCMV, we conclude that RhCMV virions contain a novel inhibitor of IFN-independent viral induction of ISG expression by IRF3.

Background: A photochemical treatment (PCT) process utilizing amotosalen hydrochloride and long wavelength UVA light has been developed to inactivate pathogens in PLTs. This study investigated the effects of amotosalen/UVA treatment on free and latent murine CMV (MCMV) in PLT preparations using a murine model of transfusion-transmitted CMV (TT-CMV)., Study Design and Methods: In a model of latent MCMV infection, "donor" mice received 1 x 10(6) plaque-forming units (PFUs) MCMV and were rested 14 days. Subsequently harvested, pooled, and washed WBCs were PCR positive for MCMV. Murine WBC doses of 1 x 10(4), 1 x 10(5), and 1 x 10(6) were added to human apheresis PLTs in 35 percent autologous plasma and 65 percent PLT AS (PAS). The WBC-PLT products were treated with 150 micro mol/L amotosalen and 0.6 J per cm2 UVA and transfused via tail vein injection into recipient mice. Recipients were killed on Day 14. Blood and spleens were collected and assayed for MCMV by PCR. In a parallel model of active infection with free virus, human PLT in 35 percent autologous plasma and 65 percent PAS were dosed with 1 x 10(5) and 1 x 10(6) PFUs of MCMV. All other procedures were as described above., Results: In the absence of amotosalen/UVA-pretreatment, transfusion of PLT latently or actively infected with MCMV produced TT-CMV in a dose-dependent fashion. In contrast, all transfusion recipients of identical PLT preparations pretreated with amotosalen/UVA were uniformly PCR negative for MCMV (abrogation of TT-CMV; p < 0.05)., Conclusions: PCT of PLT preparations with the specified doses of amotosalen hydrochloride and UVA light prevents transfusion transmission of free and latent MCMV in a murine model. These results suggest that PCT of human PLTs with amotosalen/UVA should also effectively abrogate TT-CMV in the clinical setting.

Background and Objectives: Two different leucocyte-inactivation technologies--gamma irradiation and INACTINE PEN110--were evaluated for their effects on cell-associated human cytomegalovirus (CMV)., Materials and Methods: In vitro CMV-infected cells were spiked into leucoreduced red blood cell concentrates (RCC) or medium at a final concentration of 0.5 - 1 x 10(7) cells/ml to mimic non-leucoreduced levels of leucocytes. The spiked RCC/medium was divided into three equal units and treated with gamma irradiation at the US Food and Drug Administration (FDA)-approved dose of 25 Gy, with 0.1% v/v PEN110 at 22 degrees C for 24 h, or stored at 4 degrees C as a control. The treated and control cells were recovered and tested using infectivity, viability and polymerase chain reaction (PCR) assays., Results: Gamma-irradiated CMV-infected cells produced active virus, as shown by both infectivity assays and PCR quantification of viral DNA. PCR analysis demonstrated higher CMV DNA levels in gamma-irradiated, latently infected monocytic THP-1 cells than untreated control cells. The increased virus production in gamma-irradiated cells was paralleled by an increased metabolic rate and the development of enlarged multinuclear cells. In contrast, PEN110 treatment terminated virus replication and completely inactivated the infected cell., Conclusions: These results demonstrate that gamma irradiation, at levels currently used to treat RCC, has the capacity to induce expression of CMV, whereas PEN110 inhibits CMV replication and efficiently inactivates the infected cells.

The cellular response to viral infection often includes activation of pathways that shut off protein synthesis and thereby inhibit viral replication. In order to enable efficient replication, many viruses carry genes such as the E3L gene of vaccinia virus that counteract these host antiviral pathways. Vaccinia virus from which the E3L gene has been deleted (VVDeltaE3L) is highly sensitive to interferon and exhibits a restricted host range, replicating very inefficiently in many cell types, including human fibroblast and U373MG cells. To determine whether human cytomegalovirus (CMV) has a mechanism for preventing translational shutoff, we evaluated the ability of CMV to complement the deficiencies in replication and protein synthesis associated with VVDeltaE3L. CMV, but not UV-inactivated CMV, rescued VVDeltaE3L late gene expression and replication. Thus, complementation of the VVDeltaE3L defect appears to depend on de novo CMV gene expression and is not likely a result of CMV binding to the cell receptor or of a virion structural protein. CMV rescued VVDeltaE3L late gene expression even in the presence of ganciclovir, indicating that CMV late gene expression is not required for complementation of VVDeltaE3L. The striking decrease in overall translation after infection with VVDeltaE3L was prevented by prior infection with CMV. Finally, CMV blocked both the induction of eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation and activation of RNase L by VVDeltaE3L. These results suggest that CMV has one or more immediate-early or early genes that ensure maintenance of a high protein synthetic capacity during infection by preventing activation of the PKR/eIF2alpha phosphorylation and 2-5A oligoadenylate synthetase/RNase L pathways.

The effect of human cytomegalovirus (HCMV) infection on cellular mRNA accumulation was analyzed by gene chip technology. During a 48-h time course after infection of human diploid fibroblasts, 1,425 cellular mRNAs were found to be up-regulated or down-regulated by threefold or greater in at least two consecutive time points. Several classes of genes were prominently affected, including interferon response genes, cell cycle regulators, apoptosis regulators, inflammatory pathway genes, and immune regulators. The number of mRNAs that were up-regulated or down-regulated were roughly equal over the complete time course. However, for the first 8 h after infection, the number of up-regulated mRNAs was significantly less than the number of down-regulated mRNAs. By analyzing the mRNA expression profile of cells infected in the presence of cycloheximide, it was found that a minimum of 25 mRNAs were modulated by HCMV in the absence of protein synthesis. These included mRNAs encoded by a small number of interferon-responsive genes, as well as beta interferon itself. Cellular mRNA levels in cytomegalovirus-infected cells were compared to the levels in cells infected with UV-inactivated virus. The inactivated virus caused the up-regulation of a much greater number of mRNAs, many of which encoded proteins with antiviral roles, such as interferon-responsive genes and proinflammatory cytokines. These data argue that one or more newly synthesized viral gene products block the induction of antiviral pathways that are triggered by HCMV binding and entry.

Bone marrow suppression with leukopenia is frequently observed during human cytomegalovirus (HCMV) infection, and in vitro the cell colony formation of bone marrow progenitors is directly inhibited by HCMV. To better understand the mechanisms of HCMV's ability to directly inhibit the cell colony formation of hematopoietic cells, we examined the effect of HCMV infection on four hematopoietic cell lines, ML-3, HL-60, KG-1, and U-937. Similarly to the observed effect on hematopoietic progenitors, HCMV significantly inhibited the cell colony formation of KG-1 and U-937 cells, 40% and 30% respectively. Following HCMV infection, uptake of HCMV pp65 was detected in all cell lines. In contrast, no immediate early protein production could be observed. When the cell line KG-1 was challenged with UV-inactivated HCMV or with HCMV dense bodies, containing pp65 and other matrix proteins, a 20% to 25% inhibition of cell colony formation was found. In addition, a dose-dependent inhibition of proliferation of the KG-1 cells challenged with intact or UV-inactivated HCMV, was observed. Transfection of this cell line with vectors containing genes for the HCMV matrix protein pp65, revealed no inhibitory effect. In contrast, the transfection with pp71 resulted in a 20% growth inhibition. These results indicate that HCMV can induce inhibition of cell colony formation of hematopoietic cells without transcription of HCMV regulatory proteins, and that at least one HCMV matrix protein may play an important role in this inhibitory effect.

Effects of environmental levels of magnetic fields (MFs) on RNA synthesis have been investigated by using a cell-free system for in vitro transcription. Transcription reaction mixtures containing CMV immediate early promoter DNA plus HeLa cell nuclear extracts were exposed to each of three different MF field strengths, i.e., 10, 50, and 100 microT. Each MF exposed extract was paired with a simultaneous sham-exposure control. The present results show no significant differences in amounts of RNA synthesis in extracts of MF exposed compared with that in the sham controls. This finding is in contrast to results of prior studies of DNA synthesis in cell-free systems that showed MF exposure effects. The results of the present cell-free system studies suggest that the marked differences of MF exposure effects on DNA and on RNA synthesis direct attention to the complexity involved in confirming significant effects of exposures to environmental levels of MFs in biosystems in vivo and in vitro., (Copyright 1999 Wiley-Liss, Inc.)

Cytomegalovirus (CMV) infection induces the proinflammatory cytokine, interleukin (IL)-6, which may contribute to the pathology of the infection. In vitro CMV induction of IL-6 by human lung fibroblasts was studied. The quantity of cytokine in culture supernatants was maximal 20 h after infection and decreased thereafter. Transcription of the IL-6 gene and IL-6 protein expression were equally stimulated by infectious and UV-inactivated virus (CMV-UV). CMV-UV-stimulated IL-6 was inhibited by pyrrolidinedithiocarbamate (an inhibitor of the transcription factor, NF-kappaB) and by pertussis toxin (suggesting the involvement of a G protein) and occurred in the absence of CMV immediate-early antigen transcription. Neutralizing antibodies to IL-1beta or tumor necrosis factor-alpha did not affect CMV-UV-induced IL-6, but expression was inhibited by antibody to the CMV attachment glycoprotein. IL-6 production by fibroblasts occurs independently from productive infection but has characteristics that suggest a ligand receptor-mediated pathway. This function may be important in pathology or disease resolution.

Earlier studies have revealed that human cytomegalovirus rapidly inhibits the growth of fibroblasts, blocking cell cycle progression at multiple points, including the G1-to-S-phase transition. The present study demonstrates that the UL69 protein, a virus-encoded constituent of the virion, is able to arrest cell cycle progression when introduced into uninfected cells. Expression of the UL69 protein causes U2 OS cells and primary human fibroblasts to accumulate within the G1 compartment of the cell cycle, and serum fails to induce the progression of quiescent human fibroblasts into the S phase when the protein is present. Therefore, the UL69 protein is at least partially responsible for the cell cycle block that is instituted after infection of permissive cells with human cytomegalovirus.

Expression of many early viral genes during human cytomegalovirus (HCMV) infection is dependent on cellular transcription factors. Several immediate-early and early viral promoters contain DNA binding sites for cellular factors such as CREB, AP-1, serum response factor, and Elk-1, and these transcription factors can be activated by phosphorylation via the cellular mitogen-activated protein kinase (MAPK) signal transduction cascade. To determine if the extracellular signal-regulated MAPKs, ERK1 and ERK2, play a role in transcription factor activation during infection, we tested for ERK activity during viral infection. We found that HCMV infection resulted in the maintenance of previously activated ERK1 and ERK2 by a mechanism which appears to involve the inhibition of a cellular phosphatase activity. ERK phosphorylation and activity were sustained for at least 8 h after infection, whereas in mock-infected cells, ERK activity steadily declined by 1 h postinfection. The activity of at least one cellular substrate of the ERKs, the protein kinase RSK1, was also maintained during this period. UV inactivation experiments suggested that viral gene expression was required for sustained ERK activity. In turn, activation of the ERKs appeared to be important for viral gene expression, as evidenced by the observed decrease in the transcriptional activity of the HCMV UL112-113 promoter during infection in the presence of the MEK inhibitor PD98059. These data suggest that HCMV utilizes cellular signal transduction pathways to activate viral or cellular transcription factors involved in the control of early viral gene expression and DNA replication.

Premature newborns, who are at risk if infected with cytomegalovirus (CMV), has been recommended to receive blood from seronegative donors or leukocyte-reduced blood. The lower CMV infection rate seen in later studies is associated with the decreased use of fresh blood. One of the most significant risk factors of the infection is the use of fresh blood. CMV infection rate of filtered-irradiated blood newborn recipients in our prospective study did not differ from non-filtered and irradiated blood recipients. Gamma-irradiated blood is analog to leukodepleted blood in terms of abolished capability of immune response, even though the former contains adequate number of leukocyts. Mixed lymphocytes reaction of donor's lymphocyte plays a pivotal role in transmission of CMV from seropositive donors to recipients. It is likely that some newborns with post-transfusion graft-versus-host disease were misdiagnosed as transfusion-acquired CMV disease, as often overlap later CMV infection due to profound agranulocytosis. We hypothesize that donor lymphocytes abolished proliferating function by irradiation, storage or filtration are no more possible to evoke reaction against recipient's antigen and thus fail to transmit CMV from infected donor to recipient.

We used differential display analysis to identify mRNAs that accumulate to enhanced levels in human cytomegalovirus-infected cells as compared with mock-infected cells. RNAs were compared at 8 hr after infection of primary human fibroblasts. Fifty-seven partial cDNA clones were isolated, representing about 26 differentially expressed mRNAs. Eleven of the mRNAs were virus-coded, and 15 were of cellular origin. Six of the partial cDNA sequences have not been reported previously. All of the cellular mRNAs identified in the screen are induced by interferon alpha. The induction in virus-infected cells, however, does not involve the action of interferon or other small signaling molecules. Neutralizing antibodies that block virus infection also block the induction. These RNAs accumulate after infection with virus that has been inactivated by treatment with UV light, indicating that the inducer is present in virions. We conclude that human cytomegalovirus induces interferon-responsive mRNAs.

The human cytomegaloviruses (HCMVs) appear to have the potential to disrupt production of hematopoietic cytokines. We examined the production of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-8 by cultured and CMV-infected human umbilical vein endothelial cells (HUVECs) and compared this production with that of uninfected cells. Endothelial cells are, among other things, an integral component of human bone marrow stroma, and are responsible for production of factors that modulate the proliferation and differentiation of human hematopoietic progenitors. HCMV infection increased the production of GM-CSF in IL-1-primed HUVECs without altering GM-CSF levels in infected but unprimed HUVECs. However, this same virus was capable of causing increased production of the inhibitory cytokine IL-8. Both the viral pellet and the cleared viral supernatant appeared to contribute equally to the increased IL-8 and GM-CSF production, because each of these preparations alone was capable of exerting only half the effect seen with whole virus preparations. That both live virus and soluble protein factors within the viral stock contributed to the enhancement in GM-CSF and IL-8 production was further confirmed by inactivation with either ultraviolet or heat treatment of the viral stocks. Although the identity of the factor within the HCMV stock that contributes to this effect remains unknown, studies conducted in the presence of neutralizing antibodies or polymyxin B ruled out a role for tumor necrosis factor-alpha, IL-6, or endotoxin, all known inducers of GM-CSF. These studies indicate that HCMVs can exert both direct and indirect effects on the production of the hematopoietic factor GM-CSF and the inflammatory/inhibitory cytokine IL-8.

Viruses may be eliminated by the host immune system via complement-mediated lysis of infected cells. Previously, we have demonstrated that the synthesis of complement factor B by renal mesangial cells (MC) is enhanced by interferon-alpha (IFN-alpha), -beta and -gamma. In the present study we investigate the effect of human cytomegalovirus (HCMV) infection on the production of complement factors by MC. The production of factor B, C2, C4 and factor H by mock-infected MC was 0.2 +/- 0.4, 3.9 +/- 6.8, 1.7 +/- 0.8 and 149 +/- 36 ng/10(6) cells per 72 h, respectively. In HCMV-infected MC cultures an induction of both factor B and C2 protein synthesis was observed up to 2.2 +/- 1.1 and 156 +/- 74 ng/10(6) cells per 72 h, respectively. The synthesis of C4 and factor H of 2.9 +/- 2.0 and 146 +/- 31 ng/10(6) cells, respectively, was not altered significantly. By Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis it was demonstrated that factor B and C2 mRNA expression were up-regulated in HCMV-infected cell cultures, whereas the levels of C4 and factor H mRNA were not changed. When MC cultures were inoculated with heat- or UV-inactivated HCMV no enhancement of factor B mRNA expression was observed. The enhanced expression was not blocked by phosphono acetic acid (PAA), suggesting that expression of the HCMV immediate early or early genes is sufficient to induce complement synthesis. We conclude that infection of MC cultures with HCMV selectively induces complement C2 and factor B production, probably mediated by interferons.

The effect of cytomegalovirus (CMV) infection on the production of growth factors and negative regulators in unstimulated or lipopolysaccharide-stimulated human bone marrow stromal cells was assessed. After 5 days, the constitutive and lipopolysaccharide-stimulated production of growth factors was significantly decreased in CMV-infected compared with uninfected stromal cells. This decrease was noted as early as 72 h after infection and appeared to be strictly related to viral replication. On the other hand, the production of inhibitory factors was increased after CMV infection, and this increased release was detectable as early as 24 h after infection. No modulation in the production of interleukin-10 was observed after CMV infection. These data suggest that CMV disturbs the balanced cytokine network, which controls proliferation and differentiation of hematopoietic progenitors. CMV-induced myelosuppression results from this lack of production of growth factors and excess production of inhibitory factors.

PML is a protein associated with discrete spherical structures within the nucleus of normal cells. A defect in PML expression is observed in acute promyelocytic leukaemia as a consequence of a chromosomal translocation involving the genes encoding PML and the alpha retinoic acid receptor (RAR alpha). Disruption of PML bodies also occurs during herpes simplex virus infection after the immediate early protein Vmw110 has become associated with PML bodies. In this study, we followed the fate of PML bodies in human fibroblasts during the course of a human cytomegalovirus (CMV) infection. Disruption of PML bodies was observed to be dependent on de novo CMV gene expression and to occur within 4 h post-infection, concomitant with the onset of CMV IE gene expression. Although a transient increase in the number of PML bodies could be observed in some cells, PML exists predominantly as a diffuse nuclear protein during both the early and late stages of CMV infection. Although the function of PML bodies is still uncertain, their disruption may be important for efficient herpes virus replication.

Hypocrellin, a photodynamic perylene quinonoid isolated from the Chinese medicinal fungus Hypocrella bambuase, was evaluated for antiviral activity against the human immunodeficiency virus (HIV-1). Hypocrellin was phototoxic to HIV-1, almost as good as the structurally similar plant pigment hypericin, and like hypericin its activity required visible light. In contrast peroxyhypocrellin had little or no effect on the virus.

Persistent human cytomegalovirus (HCMV) infections are responsible for significant morbidity and mortality in immunocompromised individuals. One mechanism by which HCMV may develop persistence after primary infection is through inhibition of host cell human leukocyte antigen (HLA) class II expression with resultant escape from normal antiviral immune surveillance. Immunofluorescence flow cytometry of human endothelial cell (EC) cultures infected with HCMV AD169 and an EC propagated strain, VHL/E, showed a marked reduction in interferon-gamma (IFN-gamma)-induced surface expression of HLA-DR. This inhibition did not occur when EC were treated with ultraviolet-inactivated virus and IFN-gamma. HCMV, as determined by dual-labeling immunohistochemistry, inhibited induction of surface and cytoplasmic class II antigens specifically in infected cells. HCMV infection also inhibited IFN-gamma and tumor necrosis factor-alpha up-regulation of HLA class I expression. Northern blot analysis of infected, IFN-gamma-treated human umbilical vein endothelial cells revealed an absence of class II mRNA. Persistence of HCMV may result in part from its ability to inhibit HLA class II induction in infected cells.

Cytomegalovirus infection, a common complication in immunosuppressed graft recipients, bears an adverse impact on graft survival. Cytomegalovirus enhances the expression of the monotypic determinants of the class I major histocompatibility complex molecule by the endothelium, possibly rendering the endothelial cells more immunogenic and prone to attack by the allogeneic lymphocytes. In the present study, we focused on the effect of cytomegalovirus on the endothelial cell expression of different class I genes, on the relation between the extent of endothelial cell infection and the class I effect, and on the time course of the class I changes induced by the cytomegalovirus infection. Cytomegalovirus infection of primary cultures of human umbilical vein endothelial cells augmented the expression of the A2, A3, and B7 class I major histocompatibility complex genes when compared with uninfected cells. beta 2 microglobulin upregulation by the infected cells paralleled the changes in specific class I expression; this effect was significant only after 7 days after infection. Double immunocytochemical staining and fluorescence-activated cell sorter analysis revealed that the class I enhancement was uniform throughout the umbilical vein endothelial cell monolayer and not restricted to the cells that expressed cytomegalovirus early or late antigens. Ultraviolet-inactivated supernatants from infected umbilical vein endothelial cell did not increase class I expression on uninfected cells. In conclusion, cytomegalovirus might affect graft survival by amplifying the changes in class I expression beyond the sites of viral replication.

Clinically, there is an association between CMV infections and the occurrence of rejection after renal transplantation. Adhesion molecules such as intercellular adhesion molecules (ICAM) and MHC Ags are thought to be important in the induction and amplification of the rejection process. Therefore, we studied ICAM-1 and MHC expression after CMV infection or stimulation with cytokines. Cultured proximal tubular epithelial cells (PTEC) were stimulated with cytokines or infected with CMV. MHC class I, class II, and ICAM-1 expression were determined by radioimmunoassay. IFN-gamma induced class II and ICAM-1 expression. Small concentrations of IFN-alpha inhibited the IFN-gamma induced class II expression. CMV-infected PTEC displayed increased levels of ICAM-1 and class I expression. This enhancement is a direct effect of the virus on the infected cells and not mediated by soluble factors. Although MHC class II expression is not directly enhanced by CMV, infected PTEC display a normal increase of class II expression after stimulation with IFN-gamma.

Class I major histocompatibility complex (MHC) antigens of higher eukaryotes play a crucial role in the recognition of human cytomegalovirus (HCMV)-infected cells by cytotoxic T lymphocytes. In the present study, we have demonstrated that HCMV infection resulted in a marked reduction in the surface expression of class I MHC antigens on human embryonic lung fibroblasts (HEL). Even when HCMV-infected HEL were cultured in the presence of 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (DHPG), the reduction was observed to the same extent, indicating that HCMV DNA synthesis was not required for this phenomenon. However, immunoprecipitation studies have shown that there was no significant reduction in the synthesis of either the heavy chain or the light chain of class I MHC antigens after HCMV infection. In addition, Western blotting studies have revealed that the total amount of the antigens was almost unaltered after HCMV infection. These results suggest that the reduction in the cell surface expression of class I MHC antigens is due to some defect occurring post-translationally, most likely at the level of the correct complex formation and/or the intracellular transport of class I MHC antigens.

The photoactivated antiviral and cytotoxic activities of the naturally occurring thiophene, alpha-terthienyl (1), and 15 synthetic analogues were evaluated against murine cytomegalovirus and Sindbis virus, and murine mastocytoma cells. After irradiation with near UV light, alpha-terthienyl and most of its analogues had significant toxicity, with minimum inhibitory concentrations in the range of 0.02-40 microM. In the absence of near UV irradiation, only one analogue had antiviral activity and five were cytotoxic. The most active analogues were those containing carboxylic acid, hydroxyl, or cyano substituents. Quantitative structure-activity relationship analysis of thiophene phototoxicity suggested that the rate of singlet oxygen production is the primary determinant of antiviral and cytotoxic activities. For phototoxicity against murine cytomegalovirus, a significant role for hydrophobicity was also demonstrated. Tricyclic thiophenes show significant potential for photochemotherapy of viral infections and cancer, and further evaluation in animal models is recommended.

In an ethnopharmacological screening of medicinal plants used in Yunnan province of China, ethanol extracts from 31 plant species were assayed for inhibition of murine cytomegalovirus and Sindbis virus infections. Parallel assays were carried out with and without exposure to UVA radiation to test for photo-mediation of activity. Antiviral activity was observed with 16 of the plant extracts. Eight plant extracts have been selected for further studies, with the objective of characterizing the antiviral constituents.

The effect on cellular (c) oncogene RNA levels was investigated after infection of permissive cells with cell culture adapted strains (AD-169, C-87, Davis) and unadapted clinical isolates (82-1, 84-2, 85-1) of human cytomegalovirus (HCMV). The results indicate that both adapted and unadapted strains of HCMV induce substantial increases in c-oncogene RNA levels for fos, jun, and myc measured by Northern blot hybridization. Elimination of immediate early (IE) protein synthesis between 0 and 3 hrs or reduction of virus infectivity (99.99%) by UV-irradiation did not reduce the increase in c-oncogene RNA levels. Inhibition of viral and cellular protein synthesis by cycloheximide resulted in a high abundance (superinduction) of specific RNAs which hybridized to c-oncogene probes after infection with either adapted or unadapted strains of HCMV. These data suggest that IE viral gene expression is not essential for activation of c-oncogenes. Inhibition of DNA-dependent RNA synthesis by blocking RNA elongation with actinomycin-D or by inhibiting the activity of RNA polymerase II with alpha-amanitin significantly reduced the increase in c-oncogene RNA levels, suggesting that activation of cellular genes by HCMV is controlled at the level of transcription. Activation of c-oncogenes by HCMV may be particularly important because their protein products appear to be involved in initiation and regulation of viral and cellular gene expression.

The sensitivity of the polymerase chain reaction (PCR) can mean that even very low levels of contamination with the target DNA will result in a positive signal. At present this aspect is a major limitation in the use of PCR as a routine diagnostic method. By exposing PCR reagents to UV light, contaminating DNA can be inactivated, thus providing an opportunity to eradicate false positive reactions. UV irradiation was applied to PCR systems used for the detection of human cytomegalovirus (CMV) and human immunodeficiency virus (HIV) and shown to be effective in eradicating both laboratory encountered contamination and plasmid DNA (below 100 pg) added to PCR systems prior to UV exposure. The sensitivity of a PCR system to amplify the long terminal repeat (LTR) sequence of HIV-1 was not affected by the irradiation procedure; however, the ultimate sensitivity of a PCR system for the amplification of an early gene promotor sequence of the CMV genome was reduced 1000-fold. UV irradiation did not affect the size of the PCR product as determined by strand separating polyacrylamide gel electrophoresis of a 32P-labelled amplimer. Thus, a simple pre-exposure to UV light would seem a worthwhile step to incorporate into PCR protocols provided that the effects on sensitivity have been determined empirically for each PCR system.

The polycyclic compound hypericin, a known photodynamic agent, was investigated for antiviral activity in the presence and absence of light. The three viruses tested: murine cytomegalovirus; Sindbis virus; and human immunodeficiency virus type 1, were all susceptible to hypericin; but these antiviral activities were considerably enhanced, in a dose-dependent manner, by exposure to light.

Previous reports have demonstrated that the introduction of virus (MCMV, HSV-1) concurrent with a graft-versus-host reaction (GvHR) limited to a class I MHC disparity can result in the enhancement of GvHR-associated phenotypic (changes in CD4/CD8 ratio) and functional (inability to produce secondary antibody responses) alterations including the augmentation of in situ natural killer (NK) and donor anti-host cytotoxic T-cell activity. In the present study, we investigated whether immunogens other than infectious virus may be capable of enhancing GvHR. Mice receiving donor cells and the T-dependent antigen dinitrophenyl-bovine serum albumin (DNP-BSA) did not display exacerbated GvHR as evidenced by the absence of phenotypic alterations and the absence of elevated NK activity and the lack of donor anti-host cytotoxic activity. Furthermore, these recipients produced normal levels of IgM and IgG anti-DNP antibody. In addition, mice which received ultraviolet light (UV)-inactivated virus (even at 100 x dosage) together with donor cells also did not exhibit exacerbated GvHR. In total, these findings illustrate a novel ability of infectious virus to exacerbate GvH reactions and are consistent with the hypothesis that viral-induced immune responses may be important in the ability of a pathogen to induce the development of severe GvHR.

Exposure of human lung fibroblasts to human cytomegalovirus (HCMV) stimulated a rapid increase in the release of [3H] from cells prelabelled with radiolabelled arachidonic acid ([3H]AA). Maximum stimulation of [3H] release was observed at 20 min postinfection and was quantitatively similar to that induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA: 10 nM) or fetal calf serum (5%). The level of [3H] release was dependent on the multiplicity of infection, and appeared to be mediated by a component(s) of the virion, since the findings from three series of experiments suggested that neither infectious virus, nor HCMV-specific macromolecular synthesis was required for stimulation of [3H] release. (1) Inactivation of HCMV infectivity with ultra-violet (UV) light (approximately 254 nm, 4.80 x 10(4) ergs/mm2) did not diminish the stimulation of [3H] release. (2) Significant reduction in the level of [3H] release was not observed when infected cells were maintained in the presence of a protein synthesis inhibitor, cycloheximide (50 micrograms/ml), or an inhibitor of mRNA synthesis, 3'-deoxyadenosine (cordycepin, 50 micrograms/ml). (3) No correlation was established between the expression of HCMV immediate early (IE) antigens and the induction of [3H] release, since there was little, if any, synthesis of HCMV IE antigen detectable by anticomplement immunofluorescence through the first 30 min postinfection. These findings suggesting that the HCMV particle rapidly stimulates AA metabolism are consistent with the view that the interaction of a HCMV virion component(s) with the cell surface may initiate membrane-associated events similar to those induced by growth factors.

Enhanced survival of UV-irradiated human cytomegalovirus (HCMV) is demonstrated in normal human cells exposed to UV light prior to infection. The UV fluence that gave rise to maximum UV reactivation falls in the range of 15 J/m2. A large number of temperature-sensitive HCMV mutants were found under the peak of reactivation. These results confirm the existence of inducible SOS functions in human cells.

Heat-inactivated murine cytomegalovirus (MCMV) stimulates cellular DNA synthesis in WME, NMG, 3T3, WgIA, chick and NIK-8 cells, but active or u.v.-irradiated MCMV does not. The stimulation of DNA synthesis in NIL-8 and chick cells was studied in detail. We found that both the nuclear and the mitochondrial DNA synthesis were stimulated in these cells. There was no virus DNA synthesis during the period we studied (48 h). The stimulation of nuclear DNA synthesis was about threefold in NIL-8 and 2.5-fold in chick cells as measured by the rate of incorporation of 3H-thymidine (3H-dThd) in the CsCl fractions which banded at the density of cell DNA. The stimulation was about 9.5-fold in NIL-8 and 1.7-fold in chick cells as detected by autoradiography. There was a 3-fold and a 2.2-fold increase in the degree of incorporation of 3H-dThd into mitochondrial DNA of NIL-8 and chick cells, respectively. The amount of mitochondrial DNA obtained in infected cells of both kinds was about twice that in control cells. The synthesis of mitochondrial DNA was also stimulated by a factor of 2 in the thymidine kinaseless 3T3 cells which incorporate exogenous thymidine exclusively into mitochondrial DNA. There were no MCMV specific antigens detectable by immunofluorescence 5 h after infection, but diffuse nuclear fluorescence could be demonstrated 24 h after infection. Our results indicate that the heat-inactivated virus penetrates the cells, stimulates host DNA synthesis and induces synthesis of early MCMV antigens.

Over the last 6 years, our knowledge of the basic characteristics of human cytomegalovirus (HCMV) has been greatly enhanced, especially as concerns the microbiology of this virus. DNA relatedness studies show that there is 80% homology between different strains, regardless of their origin. Studies on the relationships between HCMV and host-cell metabolism reveal that it induces both a virus specific and a cellular DNA polymerase, stimulates ribosomal RNA, induces early protein synthesis and results in the liberation of "intrinsic" interferon. The sequence of appearance of virus-specific antigens, as well as the presence of virus-induced IgG-Fc receptors has been described. New morphological aspects of the virus itself, as well as of the lesions which it provokes, have been observed. Last, but perhaps most important, evidence is accumulating in favor of the oncogenic potential of this virus. These basic aspects of HCMV are presented and discussed herein, along with the effects of various drugs and supra-optimal temperature on the virus and the possibility of preparing a vaccine against it.

The results presented in this paper show that virus stocks enriched for defective particles are more effective in stimulating cellular DNA synthesis than are stocks relatively free of defective particles. Several lines of evidence support this conclusion: (1) Low levels of UV irradiation enhance the ability of standard virus stocks to stimulate cellular DNA synthesis. (2) Three different stocks derived from individual plaques (which presumably contain very few defective particles) and passaged only twice at low multiplicity, were found to be poor inducers of cellular DNA synthesis. (3) Stocks of virions obtained after serial undiluted passage which were shown to be enriched for defective particles were quite effective in stimulating cellular DNA synthesis.

The ultraviolet (UV-)sensitivity of the human cytomegalovirus (HCMV) genes coding for very early complement fixing and early antigens in human embryonic fibroblasts (HEF) and mouse embryonic fibroblasts (MEF) and the relation of these genes to the ability of the virus to stimulate host cell DNA synthesis were investigated. After 14 minutes of UV-irradiation of the virus inoculum only the very early complement fixing nuclear antigen (CMNA) developed in the HEF cells and only the early cytoplasmic antigen(s) was present in the MEF. In both HEF and MEF, host cell DNA synthesis was stimulated. We conclude that the ability of HCMV to stimulate host DNA synthesis is an early function of the viral genome and shows a high resistance to UV-irradiation. There is no direct correlation, however, between the ability of the virus to stimulate host cell DNA synthesis and the genes which code for the CMNA or for early cytoplasmic antigens.

Human cytomegalovirions and dense bodies labeled with [14C]glucosamine were analyzed by means of sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and autoradiography. The same glycopeptide composition was found in both cytomegalovirions and dense bodies. These consisted of at least 11 glycopeptides which ranged in molecular weight from 46,500 to over 170,000 daltons. Addition of purified human cytomegalovirus (CMV) at high multiplicity to confluent monolayers of human fibroblasts produced cell rounding and polykaryocytes containing 3 to 150 nuclei. The cell rounding was induced by the cytomegalovirions, but not by the cytomegalo dense bodies. Inhibition of protein synthesis, but not of DNA synthesis, prevented this effect, suggesting that cell rounding is protein mediated. In contrast, the formation of polykaryocytes by CMV was not affected by inhibitors of protein synthesis. UV irradiation of the virus, which abolishes infectivity, does not affect its fusion properties. CMV dense bodies, which contain very little or no DNA, also produce cell fusion although this effect is less pronounced than with virions. CMV-induced polykaryocytosis therefore appears to be a direct result of the interaction of cells with the input viral particles, a phenomenon usually referred to as early polykaryocytosis.

Ultraviolet (u.v.) light-irradiation of human cytomegalovirus (HCMV) resulted in differential inactivation of virus capacities, e.g. induction of cell rounding, early antigens (EA), nuclear inclusion, HCMV DNA synthesis, cellular DNA synthesis, HCMV-specific DNA polymerase, cellular DNA polymerases and plaque production, while the capacity of HCMV to penetrate cell nuclei was not critically impaired. These results indicated that the virus-coded functions expressed after infection were responsible for sll these events except for HCMV-induced stimulation of cellular RNA synthesis which was enhanced by irradiation of the virus at a low dose of u.v. light (6600 ergs/mm2). In these experiments phosphonoacetic acid was effectively utilized to detect EA formation by immunofluorescent staining and to differentiate cellular DNA synthesis from virus DNA synthesis.

The effect of human cytomegalovirus (CMV) on cell DNA synthesis and mitotic activity in hamster embryo fibroblasts was examined. The results indicated that CMV infected cells had increased rates of cell DNA replication and mitotic activity. Detection of the effect of CMV on these two parameters necessitated arrest of cells prior to infection with low serum concentrations. This lowered the background levels of DNA synthesis and cell division so that the effect of virus infection could be detected. The data indicate that cells arrested prior to infection demonstrate increased susceptibility to virus infection. It was also observed that the effect of CMV on both DNA replication and mitotic activity could be enhanced by irradiation with ultraviolet light of the virus prior to infection.