Quantitative nested PCR for human cytomegalovirus DNA: analysis using NucleoScan 2001

A quantitative nested DNA PCR for human cytomegalovirus (CMV) was developed using PCR mimic DNA as an internal standard. A combination of ultra-thin gel and enhanced detection of ethidium bromide-stained PCR bands allowed us to achieve a 25-fold increase in sensitivity over the conventional gel electrophoreses, down to 0.1 ng of DNA per band.When the technique was applied to measuring CMV load in CMV positive leucocyte DNA preparations its sensitivity spanned the range from single to hundreds of CMV DNA copies in 0.1 microg of total white blood cell DNA.     

Reactivation of latent human cytomegalovirus in CD14(+) monocytes is differentiation dependent.

We have previously demonstrated reactivation of latent human cytomegalovirus (HCMV) in myeloid lineage cells obtained from healthy donors. Virus was obtained from allogenically stimulated monocyte-derived macrophages (Allo-MDM), but not from macrophages differentiated by mitogenic stimulation (ConA-MDM). In the present study, the cellular and cytokine components essential for HCMV replication and reactivation were examined in Allo-MDM. The importance of both CD4(+) and CD8(+) T cells in the generation of HCMV-permissive Allo-MDM was demonstrated by negative selection or blocking experiments using antibodies directed against both HLA class I and HLA class II molecules. Interestingly, contact of monocytes with CD4 or CD8 T cells was not essential for reactivation of HCMV, since virus was observed in macrophages derived from CD14(+) monocytes stimulated by supernatants produced by allogeneic stimulation of peripheral blood mononuclear cells. Examination of the cytokines produced in Allo-MDM and ConA-MDM cultures indicated a significant difference in the kinetics of production and quantity of these factors. Further examination of the cytokines essential for the generation of HCMV-permissive Allo-MDM identified gamma interferon (IFN-gamma) but not interleukin-1 or -2, tumor necrosis factor alpha, or granulocyte-macrophage colony-stimulating factor as critical components in the generation of these macrophages. In addition, although IFN-gamma was crucial for reactivation of latent HCMV, addition of IFN-gamma to unstimulated macrophage cultures was insufficient to reactivate virus. Thus, this study characterizes two distinct monocyte-derived cell types which can be distinguished by their ability to reactivate and support HCMV replication and identifies the critical importance of IFN-gamma in the reactivation of HCMV.     

Quantitative analysis of human emotions]

Experimental models describing the relationship between emotional stress, information, and needs are considered. The approach used is based on mental reproduction of emotional events such as joy, anger, fear, and distress. Experimental verification of the models revealed the presence of a linear component in the formula that relates the parameters of emotions, needs, and information.     

In silico pattern-based analysis of the human cytomegalovirus genome

More than 200 open reading frames (ORFs) from the human cytomegalovirus genome have been reported as potentially coding for proteins. We have used two pattern-based in silico approaches to analyze this set of putative viral genes. With the help of an objective annotation method that is based on the Bio-Dictionary, a comprehensive collection of amino acid patterns that describes the currently known natural sequence space of proteins, we have reannotated all of the previously reported putative genes of the human cytomegalovirus. Also, with the help of MUSCA, a pattern-based multiple sequence alignment algorithm, we have reexamined the original human cytomegalovirus gene family definitions. Our analysis of the genome shows that many of the coded proteins comprise amino acid combinations that are unique to either the human cytomegalovirus or the larger group of herpesviruses. We have confirmed that a surprisingly large portion of the analyzed ORFs encode membrane proteins, and we have discovered a significant number of previously uncharacterized proteins that are predicted to be G-protein-coupled receptor homologues. The analysis also indicates that many of the encoded proteins undergo posttranslational modifications such as hydroxylation, phosphorylation, and glycosylation. ORFs encoding proteins with similar functional behavior appear in neighboring regions of the human cytomegalovirus genome. All of the results of the present study can be found and interactively explored online (http://cbcsrv.watson.ibm.com/virus/).     

Quantitative behavior analysis and human values

Many scientists believe that among the virtues of quantitative science are that its facts are free from personal, social, political, economic, and other cultural influences, or at least, if they are not, they should be. Radical behaviorism suggests, however, that a science of behavior must apply to peoples' everyday professional behaviors, including those of quantitative behavior analysts. The behaviors of quantitative behavior analysts, however, like the behaviors of everyone else, depend on the cultures to which they belong. A quantitative science of behavior must therefore describe and explain the cultural and human values of quantitative behavior analysts. In this sense, a quantitative science of behavior must apply to itself. No such "reflexive behavior analysis" currently exists and its development might shed considerable light on the basic nature of behavior analysis.     

Structural and functional analysis of human cytomegalovirus US3 protein

Human cytomegalovirus (HCMV) unique short region 3 (US3) protein, a type I membrane protein, prevents maturation of class I major histocompatibility complex (MHC) molecules by retaining them in the endoplasmic reticulum (ER) and thus helps inhibit antigen presentation to cytotoxic T cells. US3 molecules bind to class I MHC molecules in a transient fashion but retain them very efficiently in the ER nonetheless. The US3 luminal domain is responsible for ER retention of US3 itself, while both the US3 luminal and transmembrane domains are necessary for retaining class I MHC in the ER. We have expressed the luminal domain of US3 molecule in Escherichia coli and analyzed its secondary structure by using nuclear magnetic resonance. We then predicted the US3 tertiary structure by modeling it based on the US2 structure. Unlike the luminal domain of US2, the US3 luminal domain does not obviously interact with class I MHC molecules. The luminal domain of US3 dynamically oligomerizes in vitro and full-length US3 molecules associate with each other in vivo. We present a model depicting how dynamic oligomerization of US3 may enhance its ability to retain class I molecules within the ER.     

Thermoinactivation of human cytomegalovirus

Vonka, Vladimir (Baylor University College of Medicine, Houston, Tex.), and Matilda Benyesh-Melnick. Thermoinactivation of human cytomegalovirus. J. Bacteriol. 91:221-226. 1966.-The inactivation at 4 and 37 C of several strains of human cytomegalovirus was studied. The preliminary findings that freshly harvested cytomegalovirus was inactivated more rapidly at 4 C than at higher temperatures was confirmed. Intracellular virus still within infected cells was found to be more stable at 4 C than virus released by sonic treatment just before incubation at 4 C. The composition of the diluent played an important role. In tris(hydroxymethyl)-aminomethane buffer, virus was unstable at both 4 and 37 C, with the rate of inactivation faster at 4 than at 37 C. Similar results were obtained when bicarbonate-phosphate buffer or Eagle's medium when bicarbonate was used as virus diluent. Calf serum stabilized the virus at 37 C, but not at 4 C. The deletion of bicarbonate from Eagle's medium had a stabilizing effect at both temperatures. An even greater stabilizing effect at both 4 and 37 C was obtained when distilled water was used as virus diluent. Inactivation rates varied from one strain to the next at 4 C but not at 37 C. Differences were found also with virus progeny derived from a single strain, but harvested at different stages during virus multiplication. Virus harvested early was more labile at 4 than at 37 C, whereas the late virus was more labile at the higher temperature. Intracellular and extracellular virus preparations were inactivated at the same rates at either 4 or 37 C.     

Antigens of human cytomegalovirus

In considering HCMV antigens one must take into consideration not only structural proteins of virus particles but also HCMV specific proteins associated with the infected cell, for all of these proteins may play a part in eliciting an humoral and/or cell-mediated immune response in the infected individual. The virion is composed of some 35 polypeptides ranging in molecular weight from 12 000 to more than 200 000 daltons (Table I). Viral polypeptide synthesis at the level of the infected cell occurs in three waves, the immediate early, the early and the late (Table II). During the immediate early and early phases a dozen polypeptides appear. Two glycoproteins appear during the early period but these are poorly represented in the virion. Many antigens have been described both in the cytoplasm and nucleus during these periods (Table II). Viral DNA synthesis marks the beginning of the late phase of virus replication. Many new proteins and glycoproteins appear but not all of them will become part of the virus particle (Table II). It is interesting to compare the kinetics of appearance of antibodies as detected by different serodiagnostic techniques, at the time of primary infection, with the location of the antigens which these antibodies detect in the infected cells (Table III). CMV-IgM, the first antibodies to be detected, react with late appearing intracellular nuclear inclusion antigens. This contrasts with the relatively long time required for the development of neutralizing antibodies which react with antigens accessible not only on the viral envelope and at the infected cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of human tissue-specific differences in methylation

Tissue-specific differentially methylated regions (tDMRs) have been identified and implicated for their indispensable involvement in mammalian development and tissue differentiation. In this report, a quantitative DNA methylation analysis was performed for 13 human orthologous regions of recently confirmed mouse tDMRs by using Sequenom Mass Array, by which bisulfite-treated fragments are quantitatively detected using time of flight mass spectroscopy analysis. Eight regions were shown as tDMRs in various tissues from three independent individuals. Testis DNA samples from eight individuals were also analyzed for methylation. Interestingly, there is evidence that the DNA methylation level is divergent among individuals. DNA methylation levels of five testis-specific DMRs were significantly inversely correlated with the number of spermatocytes. However, a positive correlation was seen at tDMRs located near the TRIM38 and CASZ1 genes. Our results indicate that tDMRs are conserved between mouse and human and may have an important role in regulating tissue function, differentiation, and aging.     

Viral interleukin-10 expressed by human cytomegalovirus during the latent phase of infection modulates latently infected myeloid cell differentiation

The human cytomegalovirus UL111A gene is expressed during latent and productive infections, and it codes for homologs of interleukin-10 (IL-10). We examined whether viral IL-10 expressed during latency altered differentiation of latently infected myeloid progenitors. In comparison to infection with parental virus or mock infection, latent infection with a virus in which the gene encoding viral IL-10 has been deleted upregulated cytokines associated with dendritic cell (DC) formation and increased the proportion of myeloid DCs. These data demonstrate that viral IL-10 restricts the ability of latently infected myeloid progenitors to differentiate into DCs and identifies an immunomodulatory role for viral IL-10 which may limit the host's ability to clear latent virus.     

Studies of latent cytomegalovirus infection: the macrophage as a virus-harboring cell

During chronic infection of mice with mouse cytomegalovirus (MCMV), the virus was isolated from various tissues by cocultivation with allogeneic mouse embryonic fibroblasts (MEF). Infectious virus was recovered from over 15% of the pancreases, salivary glands, kidneys, lacrimal glands, and spleens. When activated macrophages were obtained by intraperitoneal injection of peptone into mice infected 3 months earlier, they harbored MCMV. Macrophages or lymphocytes were infected with MCMV in vitro and injected into normal mice intravenously. The peritoneal cavities of these mice were then stimulated by peptone injection 3 months after the transfer, and peritoneal or splenic macrophages and lymphocytes were cocultured with allogeneic MEF. MCMV was recovered from the peritoneal and splenic macrophages and not from the lymphocytes.     

Design and analysis of rhesus cytomegalovirus IL-10 mutants as a model for novel vaccines against human cytomegalovirus

Background

Human cytomegalovirus (HCMV) expresses a viral ortholog (CMVIL-10) of human cellular interleukin-10 (cIL-10). Despite only ∼26% amino acid sequence identity, CMVIL-10 exhibits comparable immunosuppressive activity with cIL-10, attenuates HCMV antiviral immune responses, and contributes to lifelong persistence within infected hosts. The low sequence identity between CMVIL-10 and cIL-10 suggests vaccination with CMVIL-10 may generate antibodies that specifically neutralize CMVIL-10 biological activity, but not the cellular cytokine, cIL-10. However, immunization with functional CMVIL-10 might be detrimental to the host because of its immunosuppressive properties.

Methods and Findings

Structural biology was used to engineer biologically inactive mutants of CMVIL-10 that would, upon vaccination, elicit a potent immune response to the wild-type viral cytokine. To test the designed proteins, the mutations were incorporated into the rhesus cytomegalovirus (RhCMV) ortholog of CMVIL-10 (RhCMVIL-10) and used to vaccinate RhCMV-infected rhesus macaques. Immunization with the inactive RhCMVIL-10 mutants stimulated antibodies against wild-type RhCMVIL-10 that neutralized its biological activity, but did not cross-react with rhesus cellular IL-10.

Conclusion

This study demonstrates an immunization strategy to neutralize RhCMVIL-10 biological activity using non-functional RhCMVIL-10 antigens. The results provide the methodology for targeting CMVIL-10 in vaccine, and therapeutic strategies, to nullify HCMV''s ability to (1) skew innate and adaptive immunity, (2) disseminate from the site of primary mucosal infection, and (3) establish a lifelong persistent infection.