Effect of Simulated Cosmic Radiation on Cytomegalovirus Reactivation and Lytic Replication.

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 137Cs 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.

Levels of antibodies to microorganisms implicated in atherosclerosis and of C-reactive protein among atomic bomb survivors.

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.

Inactivation of Cytomegalovirus in Breast Milk Using Ultraviolet-C Irradiation: Opportunities for a New Treatment Option in Breast Milk Banking.

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.

Eradication of Cytomegalovirus from Human Milk by Microwave Irradiation: A Pilot Study.

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.

Altered production of GM-CSF and IL-8 in cytomegalovirus-infected, IL-1-primed umbilical cord endothelial cells.

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.

Cytomegalovirus directly enhances MHC class I and intercellular adhesion molecule-1 expression on cultured proximal tubular epithelial cells.

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.

Cytomegalovirus infection amplifies class I major histocompatibility complex expression on cultured human endothelial 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.

Eliminating PCR contamination: is UV irradiation the answer?

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.

Induction of chromosome aberrations and mitotic arrest by cytomegalovirus in human cells.

Human cytomegalovirus (CMV) is potentially an effective but often overlooked genotoxic agent in humans. We report here evidence that indicates that infection by CMV can induce chromosome alterations and mitotic inhibition. The frequency of chromosome aberrations induced was dependent on the input multiplicity of infection (m.o.i.) for human lung fibroblasts (LU), but not for human peripheral blood lymphocytes (PBLs) when both cell types were infected at the GO phase of the cell cycle. The aberrations induced by CMV were mostly chromatid breaks and chromosome pulverizations that resembled prematurely condensed S-phase chromatin. Pulverized chromosomes were not observed in LU cells infected with virus stocks that had been rendered nonlytic by UV-irradiation at 24,000 ergs/mm2 or from infection of human lymphocytes. In LU cells infected with UV-irradiated CMV, the frequency of aberrations induced was inversely dependent on the extent of the exposure of the CMV stock to the UV-light. In permissive CMV infection of proliferating LU cells at 24 hr after subculture, a high percentage (greater than 40%) of the metaphase cells were arrested at their first metaphase and displayed severely condensed chromosomes when harvested 48 hr later. A significant increase (p less than 0.05) in the chromosome aberration frequency was also observed. Our study shows that CMV infection is genotoxic to host cells. The types and extent of damage are dependent on the viral genome expression and on the cell cycle stage of the cells at the time of infection. The possible mechanisms for induction of chromosome damage by CMV are discussed.

Antiviral activity of the photoactive plant pigment hypericin.

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.

Hypocrellin, from Hypocrella bambuase, is phototoxic to human immunodeficiency virus.

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.

Structure-activity studies of photoactivated antiviral and cytotoxic tricyclic thiophenes.

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.

Enhanced reactivation of ultraviolet-irradiated human cytomegalovirus in normal host cells.

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.

Screening of medicinal plants from Yunnan Province in southwest China for antiviral activity.

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.

Human cytomegalovirus-induced inhibition of exogenous thymidine uptake into cell DNA in HEL cells stimulated to proliferate with serum.

In contrast with the previous findings by us and others, human cytomegalovirus (HCMV) infection caused inhibition of exogenous 3H-dThd uptake into cell DNA in human embryonic lung (HEL) cells which were stimulated to proliferate with serum at the time of infection. The response of HEL cells to HCMV infection varied depending on the HEL cell population, and the inhibition of 3H-dThd incorporation was observed in a particular HEL cell population prepared in our laboratory. That the inhibition was caused by HCMV virion was shown by: (i) antiserum against HCMV prevented the virus inoculum from inhibiting 3H-dThd uptake, and (ii) removal of virions from an infecting virus stock by centrifugation resulted in no inhibition. The virus-induced inhibition was enhanced by uv-irradiation of infecting virus, suggesting that the expression of viral gene functions was not necessarily required for the inhibition. comparison between autoradiography of 3H-dThd-labeled infected cells and the total amounts of 3H incorporated into DNA indicated that HCMV reduced the overall rate of 3H incorporation into DNA in infected cells and the number of cells entering the S phase of the cell cycle.

[Cytomegalovirus transmission through blood transfusion to newborn recipients].

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.

Activation of nucleotide oligomerization domain 2 (NOD2) by human cytomegalovirus initiates innate immune responses and restricts virus replication.

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-ß) 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-ß 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-ß 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.

Toll-like receptor 4 is involved in the cell cycle modulation and required for effective human cytomegalovirus infection in THP-1 macrophages.

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.