Transduction of enteric Escherichia coli isolates with a derivative of Shiga toxin 2-encoding bacteriophage phi3538 isolated from Escherichia coli O157:H7.
(25/2392)
We investigated the ability of a detoxified derivative of a Shiga toxin 2 (Stx2)-encoding bacteriophage to infect and lysogenize enteric Escherichia coli strains and to develop infectious progeny from such lysogenized strains. The stx(2) gene of the patient E. coli O157:H7 isolate 3538/95 was replaced by the chloramphenicol acetyltransferase (cat) gene from plasmid pACYC184. Phage phi3538(Deltastx(2)::cat) was isolated after induction of E. coli O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Stx-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and E. coli from the physiological stool microflora were infected with phi3538(Deltastx(2)::cat), and plaque formation and lysogenic conversion of wild-type E. coli strains were investigated. With the exception of one EIEC strain, none of the E. coli strains supported the formation of plaques when used as indicators for phi3538(Deltastx(2)::cat). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC, and 1 of 6 E. coli isolates from the stool microflora of healthy individuals integrated the phage in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of phi3538(Deltastx(2)::cat) that formed plaques on a lawn of E. coli laboratory strain C600. The results of our study demonstrate that phi3538(Deltastx(2)::cat) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic E. coli and that the lysogens produced infectious phage progeny. Stx-encoding bacteriophages are able to spread stx genes among enteric E. coli strains. (+info)
Functional antibody response to human cytomegalovirus in immunocompetent and HIV-1 infected individuals with antibodies that inhibit virus penetration into cells and intercellular transmission of viral infection.
(26/2392)
Antibodies mediating post-attachment virus neutralisation (PN), inhibition of human cytomegalovirus (HCMV)-induced cell fusion in the glioblastoma cell line U373 (IF) and global neutralising activity (NA) were quantified in sera from healthy immunocompetent individuals, asymptomatic HIV-1-infected subjects and AIDS patients to further characterise the neutralising antibody response to HCMV in these population groups and to assess whether HIV-1-infected individuals exhibited an abnormal functional antibody profile. PN and IF antibodies accounted for a minor fraction of the NA activity of sera from all population groups. Sera from HIV-1-infected individuals (particularly AIDS patients) displayed higher levels of PN and IF antibodies than those from the healthy control group; however, the relative contribution of these antibodies to the global serum NA activity appeared to be lower in the former individuals than in immunocompetent controls. Serum antibodies preventing HCMV cell-to-cell spread (IP) were then measured to determine whether a specific deficiency could be detected in the HIV-1-infected group population. Serum IP antibody titres were significantly higher in HIV-1-infected individuals (particularly in AIDS patients) than in controls. The potential implications of the data for explaining the pathogenesis of HCMV infection are discussed. (+info)
The role of B lymphocytes in coxsackievirus B3 infection.
(27/2392)
Coxsackieviruses are important human pathogens, frequently causing myocarditis, pancreatitis, and a variety of less severe diseases. B lymphocytes appear central to the interaction between these viruses and their mammalian hosts, because agammaglobulinemic humans, genetically incapable of antibody production, are susceptible to chronic infections by coxsackieviruses and related enteroviruses, such as poliovirus and echovirus. However, recent studies show that Type B coxsackievirus (CVB) infects B lymphocytes soon after infection, suggesting the possibility that these cells may play some role in virus dissemination and/or that the virus may be able to modulate the host immune response. We analyzed the role of B lymphocytes in CVB infection and confirmed that CVB infects B lymphocytes, and extended these findings to show that this is a productive infection involving approximately 1 to 10% of the cells; however, infectious center assays show that other splenocytes are infected at approximately the same frequency. Virus is readily detectable by in situ hybridization in the spleen of immunocompetent mice but is difficult to detect in mice deficient in B cells (BcKO mice), consistent with much of the splenic signal being the result of B cell infection. Surprisingly, given the extent of their infection, B cells express barely detectable levels of the murine coxsackievirus-adenovirus receptor (mCAR), suggesting that another means of cell entry may be used. We found no evidence of B cell depletion following CVB infection, indicating that this is not the explanation for the transient immunosuppression previously reported. Virus replication and dissemination are slightly delayed in BcKO mice, consistent with B cells' playing a role as an important early target of infection and/or a means to distribute the virus to many tissues. In addition, we show that BcKO mice recapitulate a central feature of human agammaglobulinemia: CVB establishes chronic infection in a variety of organs (heart, liver, brain, kidney, lung, pancreas, spleen). In most of these tissues the viral titers remain high (10(5)-10(8) plaque forming units (pfu) per gram of tissue) for the life of the mouse, and in several there is severe pathology, particularly severe myocardial fibrosis with ventricular dilation, reminiscent of the dilated cardiomyopathy seen in humans with chronic enteroviral myocarditis. Transfer of B and/or T cells from non-immune mice had no discernible effect, whereas equivalent transfers from immune mice often resulted in transient or permanent disappearance of detectable CVB. (+info)
Effects of antigen dose and immunization regimens on antibody responses to a cytomegalovirus glycoprotein B subunit vaccine.
(28/2392)
The purpose of this phase I study was to evaluate the safety and immunogenicity of 2 doses of cytomegalovirus glycoprotein B (CMV gB)/MF59 vaccine at 3 different immunization schedules. Ninety-five volunteers were randomized to 6 groups. Antibodies to gB represent the majority of the CMV-specific neutralizing response. Three groups received 5 microgram of gB antigen combined with MF59 (a proprietary adjuvant) and 3 groups received a 30-microgram dose at 0, 1, and 2 months; 0, 1, and 4 months; or 0, 1, and 6 months. The vaccine was well tolerated, and there was no significant difference in antibody production between the 2 doses. The vaccine induced highest antibody titers when given at 0, 1, and 6 months. A low dose of CMV gB/MF59 may be the preferred dose for future studies. (+info)
Identification of functional domains in the 14-kilodalton envelope protein (A27L) of vaccinia virus.
(29/2392)
The mechanism of entry of vaccinia virus (VV) into cells is still a poorly understood process. A 14-kDa protein (encoded by the A27L gene) in the envelope of intracellular mature virus (IMV) has been implicated in virus-cell attachment, virus-cell fusion, and virus release from cells. We have previously described the structural organization of the VV 14-kDa protein, consisting of a triple-stranded coiled-coil region responsible for oligomer formation and a predicted Leu zipper-like third alpha helix with an important role in the interaction with a 21-kDa membrane protein (encoded by the A17L gene) thought to anchor the 14-kDa protein to the envelope of IMV (M.-I. Vazquez, G. Rivas, D. Cregut, L. Serrano, and M. Esteban, J. Virol. 72:10126-10137, 1998). To identify the functional domains important for virus entry and release, we have generated VV recombinants containing a copy of the A27L gene regulated by the lacI operator-repressor system of Escherichia coli (VVIndA27L) in the thymidine kinase locus and a mutant form of the A27L gene in the hemagglutinin locus but expressed constitutively under the control of an early-late VV promoter. Cells infected with a VV recombinant that expresses a mutant 14-kDa form lacking the first 29 amino acids at the N terminus failed to form extracellular enveloped virus (EEV). Fusion-from-without assays with purified virus confirmed that the fusion process was mediated by the 14-kDa protein and the fusion domain to be contained within amino acids 29 to 43 of the N-terminal region. Competitive inhibition of the infection process with soluble heparin and synthetic peptides and in vitro experiments with purified mutant proteins identified the heparin binding domain within amino acids 21 to 33, suggesting that this domain is involved in virus-cell binding via heparan sulfate. Thus, the N terminus of the 14-kDa protein contains a heparin binding domain, a fusion domain, and a domain responsible for interacting with proteins or lipids in the Golgi stacks for EEV formation and virus spread. (+info)
Inhibition of cell-free human T-cell leukemia virus type 1 infection at a postbinding step by the synthetic peptide derived from an ectodomain of the gp21 transmembrane glycoprotein.
(30/2392)
To investigate the roles of human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) proteins gp46 and gp21 in the early steps of infection, the effects of the 23 synthetic peptides covering the entire Env proteins on transmission of cell-free HTLV-1 were examined by PCR and by the plaque assay using a pseudotype of vesicular stomatis virus (VSV) bearing the Env of HTLV-1 [VSV(HTLV-1)]. The synthetic peptide corresponding to amino acids 400 to 429 of the gp21 Env protein (gp21 peptide 400-429, Cys-Arg-Phe-Pro-Asn-Ile-Thr-Asn-Ser-His-Val-Pro-Ile-Leu-Gln-Glu-Arg-P ro-Pro-Leu-Glu-Asn-Arg-Val-Leu-Thr-Gly-Trp-Gly-Leu) strongly inhibited infection of cell-free HTLV-1. By using the mutant peptide, Asn407, Ser408, and Leu413, -419, -424, and -429 were confirmed to be important amino acids for neutralizing activity of the gp21 peptide 400-429. Addition of this peptide before or during adsorption of HTLV-1 at 4 degrees C did not affect its entry. However, HTLV-1 infection was inhibited about 60% when the gp21 peptide 400-429 was added even 30 min after adsorption of HTLV-1 to cells, indicating that the amino acid sequence 400 to 429 on the gp21 Env protein plays an important role at the postbinding step of HTLV-1 infection. In contrast, a monoclonal antibody reported to recognize the gp46 191-196 peptide inhibited the infection of HTLV-1 at the binding step. (+info)
Cytopathic effect inhibition assay for determining the in-vitro susceptibility of herpes simplex virus to antiviral agents.
(31/2392)
We compare a rapid dilution method for the determination of antiviral susceptibility of herpes simplex virus (HSV) with the plaque reduction assay. A total of 84 HSV clinical isolates were studied by both methods to detect in-vitro resistance to acyclovir and foscarnet. The rapid method showed for the detection of HSV isolates resistant to acyclovir and foscarnet, a sensitivity of 96. 8% and 100% and specificity of 100% and 100%, respectively. This method provides an easy and accurate screening procedure for the susceptibility testing of HSV to antiviral agents. (+info)
Truncation of the C-terminal acidic transcriptional activation domain of herpes simplex virus VP16 renders expression of the immediate-early genes almost entirely dependent on ICP0.
(32/2392)
The herpes simplex virus (HSV) proteins VP16 and ICP0 play key roles in stimulating the onset of the viral lytic cycle. We sought to explore the regulatory links between these proteins by studying the phenotypes of viral mutants in which the activation functions of both were simultaneously inactivated. This analysis unexpectedly revealed that truncation of the C-terminal transcriptional activation domain of VP16 (allele V422) in an ICP0-deficient background almost completely eliminated immediate-early gene expression and virus replication in Vero and HEL cells. The doubly mutant viral genome persisted in a quiescent state for at least 10 days in HEL cells infected at high multiplicity and could be reactivated by superinfection with wild-type HSV. In contrast, the in1814 VP16 mutation produced a markedly less severe phenotype in the same ICP0-deficient background. These data demonstrate that expression of the immediate-early genes requires ICP0 when the C-terminal activation domain of VP16 is deleted and raise the possibility that the in1814 form of VP16 retains a residual ability to stimulate gene expression during virus infection. (+info)