Identification of cytomegalovirus (CMV) infection by different laboratory methods in renal transplant recipients undergoing triple-drug immunosupressive treatment.

Early diagnosis of CMV infection is very important mainly in transplant recipients because CMV infection is a frequent complication after transplantation. In this work we compared different laboratory methods: ELISA (IgG, IgM), Western blot,shell vial, antigenemia assay (pp65), the immunofluorescent method with epithelial cells from urine (IF), DNA in leukocytes by PCR and DNA in leukocytes by hybridization (HCS) to estimate the most proper method for diagnosis of CMV in renal transplant recipients. This preliminary study showed that HCS, PCR and Western blot are sensitive methods for detecting CMV infection. Using HCS in quantitative variant we obtained a very good correlation between DNA load and clinical symptoms.     

Acute primary infection with cytomegalovirus (CMV) in kidney transplant recipients results in the appearance of a phenotypically aberrant CD8+ T cell population

Human cytomegalovirus (CMV) is a beta-herpesvirus that causes a chronic subclinical infection in healthy man. The immune system is unable to eliminate the virus completely, allowing virus to persist in a latent state. In the immunocompromised host, this equilibrium is disturbed, resulting in a clinical infection. In immunocompromised rats, clinical CMV infection is associated with an increase in NK cells and CD8+ T cells, including a phenotypically aberrant CD8+ T cell population. Using flow cytometry, we examined the effect of acute CMV infection on the composition of leukocyte subsets in immunocompromised patients. Therefore, we used peripheral blood of CMV seronegative patients receiving a kidney from a seronegative (control group) or a seropositive donor. Of the patients receiving a seropositive kidney, only the patients undergoing acute CMV infection were included (experimental group). Special attention was paid to the phenotype of the cytotoxic T cells. The development of acute CMV infection resulted in an increased NK cell number and an activation of both CD4+ and CD8+ T cells, as determined by HLA-DR expression. An aberrant CD8+ T cell subset with decreased expression of CD8 and TCR alphabeta appeared in the infected patients. Furthermore, the size of this subpopulation of CD8+ T cells is positively correlated with the viral load.     

Intracellular formation and processing of the heterotrimeric gH-gL-gO (gCIII) glycoprotein envelope complex of human cytomegalovirus

The human cytomegalovirus (HCMV) gCIII complex contains glycoprotein H (gH; gpUL75), glycoprotein L (gL; gpUL115), and glycoprotein O (gO; gpUL74). To examine how gH, gL, and gO interact within HCMV-infected cells to assemble the tripartite complex, pulse-chase experiments were performed. These analyses demonstrated that gH and gL associate by the end of the pulse period to form a disulfide dependent gH-gL complex. Subsequently, the gH-gL complex interacts with a 100-kDa precursor form of gO to form a 220-kDa precursor of the mature gH-gL-gO complex that contains a 125-kDa form of gO. The 220-kDa precursor complex (pgCIII) was sensitive to treatment with endoglycosidase H (endo H), while the mature gCIII complex was essentially resistant to digestion with this enzyme, suggesting that formation of pgCIII complex occurs in the endoplasmic reticulum (ER) and is processed to mature gH-gL-gO (gCIII) in a post-ER compartment. While the N-linked glycans on the 100-kDa form of gO were modified to endo H-resistant states as the 125-kDa gO formed, additional posttranslational modifications were detected on gO. These processing alterations were non-N-linked oligosaccharide modifications that could not be accounted for by phosphorylation or by O-glycosylation of the type sensitive to O-glycanase. Of gH, gL, gO, and the various complexes that they form, only the mature form of the complex was detectable at the infected cell membrane, as judged by surface biotinylation studies.     

The cytoplasmic tail of glycoprotein M (gpUL100) expresses trafficking signals required for human cytomegalovirus assembly and replication

The virion envelope of human cytomegalovirus (HCMV) is complex and consists of an incompletely defined number of glycoproteins. The gM/gN protein complex is the most abundant protein component of the envelope. Studies have indicated that deletion of the viral gene encoding either gM or gN is a lethal mutation. Analysis of the amino acid sequence of gM disclosed a C-terminal acidic cluster of amino acids and a tyrosine-containing trafficking motif, both of which are well-described trafficking/sorting signals in the cellular secretory pathway. To investigate the roles of these signals in the trafficking of the gM/gN complex during virus assembly, we made a series of gM (UL100 open reading frame) mutants in the AD169 strain of HCMV. Mutant viruses that lacked the entire C-terminal cytoplasmic tail of gM were not viable, suggesting that the cytoplasmic tail of gM is essential for virus replication. In addition, the gM mutant protein lacking the cytoplasmic domain exhibited decreased protein stability. Mutant viruses with a deletion of the acidic cluster or alanine substitutions in tyrosine-based motifs were viable but exhibited a replication-impaired phenotype suggestive of a defect in virion assembly. Analysis of these mutant gMs using static immunofluorescence and fluorescence recovery after photobleaching demonstrated delayed kinetics of intracellular localization of the gM/gN protein to the virus assembly compartment compared to the wild-type protein. These data suggest an important role of the glycoprotein gM during virus assembly, particularly in the dynamics of gM trafficking during viral-particle assembly.     

Effect of empathy trait on attention to various facial expressions: evidence from N170 and late positive potential (LPP)

Background

The present study sought to clarify the relationship between empathy trait and attention responses to happy, angry, surprised, afraid, and sad facial expressions. As indices of attention, we recorded event-related potentials (ERP) and focused on N170 and late positive potential (LPP) components.

Methods

Twenty-two participants (12 males, 10 females) discriminated facial expressions (happy, angry, surprised, afraid, and sad) from emotionally neutral faces under an oddball paradigm. The empathy trait of participants was measured using the Interpersonal Reactivity Index (IRI, J Pers Soc Psychol 44:113–126, 1983).

Results

Participants with higher IRI scores showed: 1) more negative amplitude of N170 (140 to 200 ms) in the right posterior temporal area elicited by happy, angry, surprised, and afraid faces; 2) more positive amplitude of early LPP (300 to 600 ms) in the parietal area elicited in response to angry and afraid faces; and 3) more positive amplitude of late LPP (600 to 800 ms) in the frontal area elicited in response to happy, angry, surprised, afraid, and sad faces, compared to participants with lower IRI scores.

Conclusions

These results suggest that individuals with high empathy pay attention to various facial expressions more than those with low empathy, from very-early stage (reflected in N170) to late-stage (reflected in LPP) processing of faces.     

Role of interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) in the control of the infection of monocyte-like cells with human cytomegalovirus (HCMV)

The role of cytokines in the control of HCMV infection has been studied in THP-1 cells, a macrophage-like cell model and in MRC-5 cells. HCMV replication was studied by immune detection of viral immediate-early antigens (IEA) and virus yield was evaluated in MRC-5 cells by immunoperoxidase staining. Pretreatment of MRC-5 and phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells with IFN-alpha or IFN-gamma for 24 hr prior to the infection reduced the number of infected cells and virus yield. A synergistic anti-CMV activity in synthesis of early proteins was obtained with these cytokines in combination with TNF-alpha in differentiated THP-1 cells only. Treatment of HCMV-infected differentiated THP-1 cells or MRC-5 cells with IFN-alpha or IFN-gamma alone had no inhibitory effect on virus replication, however the virus yield was reduced with ganciclovir. A synergistic anti-CMV activity in virus yield was obtained only when infected differentiated THP-1 cells were treated with ganciclovir in combination with IFN-gamma. The current study shows that IFN-alpha and IFN-gamma can play a role in the reduction of HCMV replication in macrophage-like cells and in the efficiency of therapies with ganciclovir in this cell type and that the anti-CMV effect of cytokines may be different in fibroblasts and in macrophage-like cells.     

Detection of hepatitis B virus infection in wild-born chimpanzees (Pan troglodytes verus): phylogenetic relationships with human and other primate genotypes

Infection with hepatitis B virus (HBV) was detected by serological testing for HBV surface antigen and by PCR assay for HBV DNA in serum samples from two common chimpanzees (Pan troglodytes subsp. verus) born in West Africa. The complete genome sequences obtained by nucleotide sequencing of overlapping DNA fragments amplified by PCR were compared with HBV variants recovered from other primates and with human genotypes A to F. Both chimpanzee sequences were 3, 182 nucleotides in length, and the surface gene sequence predicted the existence of a, d, and w serological determinants. Neither sequence contained stop codons in the precore region. On phylogenetic analysis, the HBV variants infecting the chimpanzees clustered together with a third chimpanzee HBV isolate independently obtained from an infected captive animal (A. J. Zuckerman, A. Thornton, C. R. Howard, K. N. Tsiquaye, D. M. Jones, and M. R. Brambell, Lancet ii:652-654, 1978), with an overall sequence similarity of >94%. This provides strong evidence for a chimpanzee-specific genotype of HBV which circulates in nature. These findings add to the recent evidence for infection in the wild of other Old and New World primates (gibbon, orangutan, and woolly monkey) with species-specific variants of HBV. There is no evidence for close phylogenetic clustering of variants found so far in primates with any of the established HBV genotypes from humans. With the new evidence for the widespread distribution of HBV in primates, hypotheses for the origins of human infection are reviewed.     

Evolutionary pattern of human respiratory syncytial virus (subgroup A): cocirculating lineages and correlation of genetic and antigenic changes in the G glycoprotein.

The genetic and antigenic variability of the G glycoproteins from 76 human respiratory syncytial (RS) viruses (subgroup A) isolated during six consecutive epidemics in either Montevideo, Uruguay, or Madrid, Spain, have been analyzed. Genetic diversity was evaluated for all viruses by the RNase A mismatch cleavage method and for selected strains by dideoxy sequencing. The sequences reported here were added to those published for six isolates from Birmingham, United Kingdom, and for two reference strains (A2 and Long), to derive a phylogenetic tree of subgroup A viruses that contained two main branches and several subbranches. During the same epidemic, viruses from different branches were isolated. In addition, closely related viruses were isolated in distant places and in different years. These results illustrate the capacity of the virus to spread worldwide, influencing its mode of evolution. The antigenic analysis of all isolates was carried out with a panel of anti-G monoclonal antibodies that recognized strain-specific (or variable) epitopes. A close correlation between genetic relatedness and antigenic relatedness in the G protein was observed. These results, together with an accumulation of amino acid changes in a major antigenic area of the G glycoprotein, suggest that immune selection may be a factor influencing the generation of RS virus diversity. The pattern of RS virus evolution is thus similar to that described for influenza type B viruses, expect that the level of genetic divergence among the G glycoproteins of RS virus isolates is the highest reported for an RNA virus gene product.     

Direct correlation between sialic acid binding and infection of cells by two human polyomaviruses (JC virus and BK virus)

For the human polyomaviruses JC virus (JCV) and BK virus (BKV), the first step to a successful infection involves binding to sialic acid moieties located on the surfaces of host cells. By stripping and then reconstituting specific sialic acid linkages on host cells, we show that JCV uses both α(2,3)-linked and α(2,6)-linked sialic acids on N-linked glycoproteins to infect cells. For both JCV and BKV, the sialic acid linkages required for cell surface binding directly correlate with the linkages required for infection. In addition to sialic acid linkage data, these data suggest that the third sugar from the carbohydrate chain terminus is important for virus binding and infection.     

Metal mediated protease inhibition: design and synthesis of inhibitors of the human cytomegalovirus (hCMV) protease

A versatile synthetic route to a novel series of bis-imidazolemethanes designed to inhibit the hCMV protease has been developed and a series of potential metal binding inhibitors has been identified. In selectivity assays, the compounds were highly specific for CMV protease and showed no inhibition (IC50 > 100 microM) of other prototypical serine proteases such as trypsin, elastase, and chymotrypsin. Although the presence of free zinc ions was found to be an absolute requirement for the in vitro biological activity of this class of inhibitor, the potency of the inhibitors could not be improved beyond the micromolar level.     

Nonprimate models of congenital cytomegalovirus (CMV) infection: gaining insight into pathogenesis and prevention of disease in newborns

Congenital and perinatal infections with cytomegalovirus (CMV) are responsible for considerable short- and long- term morbidity in infants. CMV is the most common congenital viral infection in the developed world, and is a common cause of neurodevelopmental injury, including mental retardation and sensorineural hearing loss (SNHL). Antiviral therapy has been shown to be valuable in ameliorating the severity of SNHL, but CMV disease control in newborns ultimately depends on successful development of a vaccine. Because CMVs are extremely species specific, preclinical evaluation of vaccines must be performed in animal models using the appropriate CMV of the animal being studied. Several small animal models available for CMV vaccine and pathogenesis research are described. The discussion focuses on the guinea pig model because guinea pig cytomegalovirus (GPCMV), which crosses the placenta and causes infection in utero, is uniquely useful. Examination of vaccines in the GPCMV and other nonprimate models should provide insights into the determinants of the host response that protect the fetus, and may help to prioritize potential vaccine strategies for use in human clinical trials related to this important public health problem.     

Interaction of human dynein light chain 1 (DYNLL1) with enterochelin esterase (Salmonella typhimurium) and protective antigen (Bacillus anthraci) might be the potential cause of human infection

The cytoplasmic dynein light chain 1 (DYNLL1) is an important constituent of motor proteins complex. In human it is encoded by DYNLL1 gene. It is involved in cargo transport functions and interacts with many viral proteins with the help of short linear consensus motif sequence (K/R) XTQT. Viral proteins bind to DYNLL1 through its consensus short linear motif (SLiM) sequence to reach the target site in the cell and cause different infections in the host. It is still unknown if bacterial proteins also contain the same conserved SLiMs sequence through which they bind to this motor protein and cause infections. So, it is important to investigate the role of DYNLL1 in human bacterial infections. The interaction partner proteins of DYNLL1 against conserved viral motif sequences were predicted through PDBSum. Pairwise sequence alignment, between viral motif sequence and that of predicted proteins, was performed to identify conserved region in predicted interaction partners. Docking between the DYNLL1 and new pathogenic interaction partners was performed, by using PatchDock, to explore the protein-protein binding quality. Interactions of docked complexes were visualized by DimPlot. Three pathogenic bacterial proteins i.e., enterochelin esterase (3MGA), protective antigen (3J9C) and putative lipoprotein (4KT3) were selected as candidate interaction partners of DYNLL1. The putative lipoprotein (4KT3) showed low quality binding with DYNLL1. So, enterochelin esterase (3MGA) and protective antigen (3J9C) were speculated to be involved in human bacterial infections by using DYNLL1 to reach their target sites.     

ABO(H) blood group antigens of the human erythrocyte membrane: Contribution of glycoprotein and glycolipid

Summary Formaldehyde-fixed human erythrocytes were extracted with sodium dodecyl sulfate and with three other solvent systems, at least two of which are known to remove glycolipids quantitatively. The extracted cells possessed the ability to absorb the ABO blood group-specific antibody at about onethird the level of unextracted cells. Treatment of fresh cells with pronase also reduced the ability of the cells to absorb the antibody, further supporting the presence of ABO blood group active glycoprotein in the membrane. Trypsinization of red cells, while removing PAS-1 and partly PAS-2, did not lead to any decrease in the activity. Papainization also did not diminish the activity, although PAS-1, PAS-2, and PAS-3 were removed from the cells. Thus, both glycolipid and glycoprotein contribute to ABO antigens of erythrocytes. Also, none of the three major glycoproteins of the membrane bears this activity.Presented at the Department of Atomic Energy Symposium on Receptors in Biological Systems held at Calcutta, India, during March 21–23, 1978.     

Complete DNA sequence analyses of the first two varicella-zoster virus glycoprotein E (D150N) mutant viruses found in North America: evolution of genotypes with an accelerated cell spread phenotype

Varicella-zoster virus (VZV) is considered to be one of the most genetically stable of all the herpesviruses. Yet two VZV strains with a D150N missense mutation within the gE glycoprotein were isolated in North America in 1998 and 2002. The mutant strains have an accelerated cell spread phenotype, which distinguishes them from all wild-type and laboratory viruses. Since the VZV genome contains 70 additional open reading frames (ORFs), the possibility existed that the phenotypic change was actually due to an as-yet-undiscovered mutation or deletion elsewhere in the genome. To exclude this hypothesis, the entire genomes of the two mutant viruses were sequenced and found to contain 124,883 (VZV-MSP) and 125,459 (VZV-BC) nucleotides. Coding single-nucleotide polymorphisms (SNPs) were identified in 14 ORFs. One missense mutation was discovered in gH, but none was found in gB, gI, gL, or gK. There were no coding SNPs in the major regulatory protein ORF 62. One polymorphism was discovered which could never have been anticipated based on current knowledge of herpesvirus genomics, namely, the origins of replication differed from those in the prototype strain but not in a manner expected to affect cell spread. When the two complete mutant VZV sequences were surveyed in their entirety, the most reasonable conclusion was that the increased cell spread phenotype was dependent substantially or solely on the single D150N polymorphism in glycoprotein gE. The genomic results also expanded the evolutionary database by identifying which VZV ORFs were more likely to mutate over time.     

Dendritic cell vaccination in an allogeneic stem cell recipient receiving a transplant from a human cytomegalovirus (HCMV)-seronegative donor: induction of a HCMV-specific Thelper cell response

Background aimsIn the absence of a protective immune response, human cytomegalovirus (HCMV) infection remains a life-threatening complication after allogeneic stem cell transplantation (SCT), especially in recipients of grafts from HCMV-seronegative donors. After allogeneic SCT from a seronegative donor, prolonged and severe immune deficiency often leads to infectious complications. Vaccination with antigen-loaded dendritic cells (DC) has been shown to be a potent approach for the induction of antigen-specific cytotoxic T-cell responses in vivo. For protection from subsequent HCMV reactivation, a sustained immune response is necessary, including antigen-specific CD4⁺ T cells.MethodsWe report the case of an 18-year-old girl with high-risk acute lymphoblastic leukemia that received an allogeneic SCT in CR2. After an HCMV infection, the graft was rejected and she received a second transplant from an HLA-mismatched, HCMV-seronegative family donor. She was treated with pp65-pulsed monocyte-derived DC at day 200 post-SCT, using a recombinant pp65 protein. Until day 200 post-SCT, HCMV reactivated six times with emerging viral resistance to antiviral chemotherapy.ResultsAfter vaccination with protein-pulsed DC, an induction and expansion of HCMV-specific T_helper cells and cytotoxic T lymphocytes was observed, associated with a sustained clearance of the HCMV viremia. Antiviral treatment could be tapered without recurrence of viremia within the first year post-SCT.Conclusionspp65-pulsed DC could induce antigen-specific T-cell responses even after a SCT from an HCMV-seronegative donor. After vaccination with pp65-pulsed DC, a sustained antigen-specific T-cell response prevented concurrent HCMV viremia. Emergence of antigen-specific T_helper cells may be essential for a sustained, functional T-cell response post-SCT.     

The rearrangement of the human alpha(1)-acid glycoprotein/orosomucoid gene: evidence for tandemly triplicated genes consisting of two AGP1 and one AGP2

The human alpha(1)-acid glycoprotein (AGP) or orosomucoid (ORM) is controlled by the two tandemly arranged genes, AGP1 and AGP2. The further duplication of the AGP1 gene has been suggested by a few duplicated ORM1 locus haplotypes including ORM1*F1. S and ORM1*B9. S, detected by isoelectric focusing. To clarify the triplication of the AGP gene, 39 DNA samples from Japanese subjects were studied by the long-range PCR of intergenic regions. The analysis of PCR products showed that the tandemly triplicated genes, AGP1A-AGP1B-AGP2, occurred on about 20% of chromosomes. These composites were divided into ORM1A*F1-ORM1B*S-ORM2*M and ORM1A*B9-ORM1B*S-ORM2*M by allelic variations. Furthermore, the former was classified into a few haplotypes by three synonymous sequence variations, which might have arisen through gene conversion-like events. The recombination breakpoints existed between the 5' flanking region and intron 2 of the AGP1B gene. Thus, it is likely that the rearrangement of the AGP gene has often occurred.