Antibodies enhance the infection of phorbol-ester-differentiated human monocyte-like cells with coxsackievirus B4.

Coxsackievirus B4 (CV-B4), in presence of antibodies and through a specific viral receptor CAR and Fcγ receptors II and III, can infect monocytes which results in interferon-α synthesis. The antibody-dependent enhancement of CV-B4 infection in the human monocytic-like THP-1 cell line has been investigated. The preincubation of CV-B4 with human plasma or human polyvalent immunoglobulins enhanced the infection of phorbol-myristate-acetate (PMA)-activated THP-1 cell cultures. CV-B4 replicated in these cells as demonstrated by the intracellular detection of infectious particles, viral protein VP1 (immunofluorescence), positive and negative viral RNA (RT-PCR). The viability of infected and control cell cultures was not different up to 20 days post-infection. Activated cell cultures inoculated with CV-B4 harbored intracellular RNA up to 14 days post-infection and produced IFNα that was detected by intracellular immunofluorescence staining as soon as 4 h post-infection with a maximum at 48 h post-infection and by RT-PCR all along the experiment. Together, these data demonstrate that PMA-activated THP-1 cells can be infected with CV-B4, can produce IFNα as a result of interactions between the virus, antibodies and specific receptors. This cellular model can be used to investigate further the mechanism and the result of the antibody-dependent enhancement of CV-B4 infection.

A respiratory syncytial virus isolate enables the testing of virucidal products.

The respiratory syncytial virus (RSV) is known as a major cause of respiratory infections and nosocomial diseases. Testing this virus is rather difficult due to the problems encountered in producing it at a high titer without using any purification method. A RSV isolate which replicates to high level on a Hep-2 cell line with an infectious titer of at least 10(7)TCID(50)mL(-1) in culture supernatant fluids has been identified. Thanks to this isolate, the virucidal effects of two products, a hand rub solution and a surface disinfectant, were conveniently tested according to the EN 14476:2007-02 procedure.

[Emergent viruses: SARS-associate coronavirus and H5N1 influenza virus]. / Virus respiratoires émergents : virus du Sras et virus influenza A/H5N1 hautement pathogène.

Two viral agents with RNA genome are responsible for emerging illnesses: influenza virus A/H5N1 and Severe Acute Respiratory Syndrome virus (SARS). For the diagnosis of SARS virus infection, an epidemiological investigation is necessary to know whether the patient has been exposed to a risk in a country where the SARS virus is circulating or whether the patient had worked in a laboratory handling SARS virus. The detection of SARS virus is possible in various clinical samples (including urine) by viral culture or RT-PCR. The handling of those samples and RNA extraction must be performed in a BSL3 laboratory. The SARS virus RT-PCR is poorly sensitive, therefore the test should be performed on samples collected consecutively for several days. In front of a suspicion of A/H5N1, similar procedures are recommended. An epidemiologic investigation is necessary to specify whether the patient stayed in a country where A/H5N1 virus was circulating. Clinical samples needed for a specific diagnosis are: nasopharyngeal, throat-swab or fecal samples, cerebrospinal fluid and blood. The presence of A/H5N1 virus is confirmed by viral isolation or RNA detection by RT-PCR. RNA extraction must be performed in a BSL3 laboratory. For diagnosis of A/H5N1 virus infection, RT-PCR test amplifies specifically a fragment of H5 gene (Hemagglutinin). In french laboratories of medical virology, procedures are ready to diagnose the first case of A/H5N1 virus infection and cases of reemerging SARS virus infection.

[Various approaches of therapeutic vaccination for the treatment of HIV type 1 infection]. / Différentes approches de vaccination thérapeutique dans le traitement de l'infection par le VIH-1.

HIV-1 infection is a major pandemic situation. With the advent of highly active antiretroviral therapy (HAART), morbidity and mortality associated with HIV-1 infection have been dramatically reduced. However, HAART does not enable eradication of the virus. The efficacy of these new regimens is limited by problems over long-term use such as toxicity and resistance. Therapeutic vaccination is an alternative approach to HIV-1 infection. The main aim is to boost and reinforce virus-specific host immune responses. Several immunogens and schedules of immunization have been tested. In this review, various strategies designed for therapeutic vaccines for HIV-1 infection are presented.

A coding RNA sequence acts as a replication signal in cardioviruses.

Theiler's virus and Mengo virus are representatives of the Cardiovirus genus within the picornavirus family. Their genome is an 8-kilobase long positive strand RNA molecule. This RNA molecule plays three roles in infected cells: It serves as a messenger RNA, acts as a template for genome replication, and is encapsidated to form progeny virions. We observed that a cis-acting signal required for replication of Theiler's virus was contained within a 130-nt stretch of the region encoding the capsid protein VP2. This RNA sequence does not influence internal ribosome entry site-mediated translation initiation and thus likely acts directly as a signal for the replication complex. We found a similar signal in the VP2-coding sequence of Mengo virus, and both signals could be functionally exchanged. Within the replication element, a 9-nt sequence that is highly conserved among cardioviruses was shown to be essential for replication. This conserved sequence was contained in mostly unpaired regions of the RNA secondary structure predicted for the replication elements of the various cardioviruses. Interestingly, a similar replication element has been reported to occur in the distantly related human rhinovirus type 14, suggesting that such elements could be conserved throughout the picornavirus family. However, the different location of the replication elements in rhinovirus and cardioviruses, and the fact that they were not functionally exchangeable, is raising intriguing questions about the evolution of such signals in picornaviruses.

Rapid detection of enterovirus in clinical specimens using PCR and microwell capture hybridization assay.

A rapid detection method of enteroviral RNA in clinical samples using PCR and a microwell capture hybridization assay is described. PCR products were labelled directly by digoxigenin-dUTP during the amplification step. The labelled amplicons were hybridized with a biotinylated oligo-probe and captured on commercially available test microwells coated with streptavidin. The hybridized amplicons labelled with digoxigenin were detected using anti-digoxigenin Fab fragments conjugated to peroxidase and colorimetric reaction automatically measured. This method detected as few as 0.01 PFU/100 microl of biological sample with a result obtained within 8 h. Using this method, we were able to detect enteroviral RNA in 23 of 35 clinical specimens from 16 of 17 patients with suspected acute or chronic enteroviral infection. The samples included cerebrospinal fluid, broncho-pulmonary lavage, pericardial effusion, throat swabs, stools, sera, muscular and myocardial biopsies. In contrast, virus was isolated in cell culture in only 8 of 28 clinical specimens from 6 of the 17 patients. This easy-to-perform assay has useful potential in the rapid detection of enterovirus in acute or chronic infection. This methodology could be used for a rapid qualitative detection of other RNA viruses.

[Polyvalent hyperimmune immunoglobulins prevent the infection of monocytic type cells by human cytomegalovirus]. / Les immunoglobulines polyvalentes hyperimmunes préviennent l'infection des cellules de type monocytaire par le cytomégalovirus humain.

Monocyte/macrophage cell types can be infected with Human Cytomegalovirus (HCMV) could be a reservoir and a vehicle for virus spread in infected patients. We developed a model to study the effects of antiviral molecules on these cells. The monocytic-like cell line THP-1 and the human diploïd cells MRC-5 were used. THP-1 cells were cultivated with a phorbol 12-myristate 13-acetate (PMA) for 24h prior to the infection. We studied infection of these cells with reference strain (AD-169). A cell free virus suspension of HCMV was preincubated with hyperimmune polyvalent immunoglobulins. The infection of the cells was studied on the basis of immune detection of viral immediate early antigens (IEA) in nucleus 24h after culture. Our results showed that hyperimmune polyvalent immunoglobulins have been able to neutralize fibroblasts and THP-1 cells infection, whereas control antibodies did not inhibit the infection of the cells. This is the first report of the use of monocytic-like cells for testing the efficiency of anti-CMV molecules.

Detection of enteroviral RNA by polymerase chain reaction in endomyocardial tissue of patients with chronic cardiac diseases.

Enteroviruses are suspected to be etiologic agents in myocarditis and cardiomyopathy. The prevalence of enteroviral (EV) heart infection in patients with chronic cardiomyopathy was determined through detection of specific EV genomic sequences using reverse transcription and polymerase chain reaction (RT-PCR) followed by slot blotting. Endomyocardial biopsies from the explanted hearts of 19 patients with dilated cardiomyopathy (DCM) and 14 patients with chronic coronary disease (CCD) were examined. EV genome was detected in 11 of 19 patients with DCM and in 8 of 14 patients with CCD. Ventricular biopsies from the control group, which included 35 healthy heart patients and 33 patients with myocardial infarction, were negative by EV RT-PCR. The percentage of patients showing presence of EV-RNA was almost similar in the DCM (57.9%) and CCD (57.1%) groups. The present study demonstrates that enterovirus RNA sequences persist in the myocardium in a significant proportion of patients suffering from end-stage ischaemic and dilated cardiac diseases and supports the hypothesis of a possible direct link between EV infection and the pathogenesis of chronic heart disease.

Evidence that neomycin inhibits human cytomegalovirus infection of fibroblasts.

The effect of phosphoinositide-binding aminoglycosides, such as neomycin, gentamicin and streptomycin, on human cytomegalovirus (HCMV) infection of human fibroblasts MRC-5 was studied. The inhibition of HCMV infection was obtained with all of these molecules but neomycin was more effective than the others. We showed that the inoculation of the cells with cell-free viral suspension in presence of neomycin concentrations above 5 mM at 37 degrees C, inhibited more than 98% the HCMV infection. However, the preincubation of the fibroblasts with neomycin at 4 degrees C, before the removal of the drug and the inoculation of the cells, induced only a 30% decrease in the number of infected cells. Addition of neomycin after the HCMV-binding at 4 degrees C or the infection of the cells was less efficient to inhibit HCMV infection than the standard incubation of neomycin during inoculation of the fibroblasts. Indeed, 1 hour after the inoculation of the cells at 37 degrees C, neomycin still inhibited HCMV infection, but 4 hours after the inoculation, this drug had no effect on HCMV infection. Our findings demonstrated that neomycin must be present at the time of infection in order to exert a full inhibiting effect. The effect of neomycin on the HCMV infection was almost immediate upon the addition of the drug (binding and/or internalization) and after the virus internalization (inhibition of immediate-early events). We suggest that neomycin and other aminoglycoside antibiotics may interact with HCMV glycoproteins for binding to similar structural features of cell surface heparan sulfate proteoglycans and may inhibit HCMV infection in fibroblasts by disrupting phosphoinositide-mediated events in the cells.

Detection of enterovirus-specific RNA sequences in explanted myocardium biopsy specimens from patients with dilated or ischemic cardiomyopathy.

Enteroviral RNA (EV-RNA) was detected in endomyocardial tissue by means of retrotranscription and polymerase chain reaction (RT-PCR) followed by slot-blot hybridization. The myocardial biopsy specimens studied were taken at the time of heart transplantation from 15 patients with dilated cardiomyopathy (DCM) and from 10 patients with ischemic cardiomyopathy (ICM). Specimens from 18 (72%) of the 25 patients were positive for EV-RNA, whereas no control specimens (myocardial specimens from 29 healthy organ donors and atrial specimens from 15 patients with acute myocardial infarction treated by anatomic bypass) yielded evidence of EV-RNA. The rates of EV-RNA detection for the two groups requiring heart transplantation did not differ significantly (66.7% vs. 80.0%; chi 2 test). Our findings support a link between enteroviral infection in both DCM and ICM and suggest a pathogenic role for the enteroviruses.

Cell membrane bound N-acetylneuraminic acid is involved in the infection of fibroblasts and phorbol-ester differentiated monocyte-like cells with human cytomegalovirus (HCMV).

We focused on the role of membrane bound sugar residues in the infection of fibroblasts and monocyte-like cells with human cytomegalovirus (HCMV). Treatment of phorbol 12-myristate 13-acetate (PMA) differentiated monocyte-like cells THP-1 or human fibroblasts MRC-5 with lectins specific for N-acetylneuraminic acid (NeuAc) blocked infection with HCMV. HCMV failed to infect sialidase-treated differentiated THP-1 cells or MRC-5 cells. By using NeuAc, N-glycolylneuraminic acid (NeuGl) and alpha 2-3, but not alpha 2-6, sialyl-oligosaccharide, the infection of cells was less efficient. NeuAc was more potent inhibitor than NeuGl. These observations suggest that the sialic acid specificity and the nature of the interglycosidic linkage at the end of the complex carbohydrates may play an important role. Analogous experiments indicated that HCMV binds to N-acetylglucosamine (GlcNAc) in addition to NeuAc. Human cytomegalovirus infection in differentiated THP-1 cells and in human fibroblasts was inhibited by incubation of the virus with 20 micrograms/ml of heparin before and during the adsorption period. Treatment of the cells with heparinase or heparitinase inhibited infection with HCMV. We emphasized the role of NeuAc and GlcNAc and heparan sulfate proteoglycans at the surface of the cells, in the early steps of infection of both human fibroblasts and PMA differentiated monocyte-like cells with HCMV.

[Current status of human cytomegalovirus disease]. / Actualités sur la pathologie du cytomégalovirus humain.

Human cytomegalovirus (HCMV) can establish lifelong persistence after primary infection with reactivation occurring as a result of immunosuppression. There is much evidence that molecular interactions between the immune system and the HCMV are responsible for immune escape. HCMV in many cells especially in mononuclear blood cells during latency are frequently the source of transmission and spreading and results in a variety of disorders. In this review some data about acute infection in immunocompetent host (mononucleosis, hepatitis), about intrauterine HCMV infection, about infection and endogenous reinfection in bone marrow and solid organ transplant recipients (pneumonitis) and about HCMV disease in AIDS patients (encephalitis, neuropathy, retinitis, colitis) are investigated. Moreover, HCMV associated vasculitis is described in patients with myocarditis, rheumatoid arthritis or polyradiculopathy. HCMV could play an important role in atherosclerosis. Several types of human malignancy have been linked to HCMV and it has been shown that HCMV ie genes upregulate expression of cellular oncogenes. The diagnosis of HCMV infection is carried out by viremia in cell culture using immediate early antigen staining, by antigenaemia which appears to be an early quantitative and predictive tool, by HCMV DNA detection using hybridization and PCR, and by IgM and IgG antibody evaluation. Two antiviral drugs are used for treatment: ganciclovir and phosphonoformic acid; few resistant clinical isolates have been reported. Specific gammaglobulin activity is discussed. HCMV vaccine is not available.

Significance and clinical relevance of the detection of herpes simplex virus DNA by the polymerase chain reaction in cerebrospinal fluid from patients with presumed encephalitis.

The polymerase chain reaction (PCR) was used to detect DNA of herpes simplex virus (HSV) in 38 samples of cerebrospinal fluid (CSF) obtained from 22 patients (7 children and 15 adults) 1-20 days after the onset of encephalitis. The results were best with amplification on the pellet of the CSF-extracted DNA and with analysis of the amplified products by dot-blotting (sensitivity, 100%). A highly significant difference was evident in the chi 2 test when PCR was compared with specific antigen detection or antibody evaluation (n = 19; chi 2 = 7; sensitivity = 100% vs. 63%) or with interferon alpha determination (n = 20; chi 2 = 11; sensitivity = 95% vs. 42%). PCR was positive as early as 1 day after onset of disease and was often the first test to become positive. The detection of HSV DNA by PCR is the most specific, rapid, and sensitive tool for early diagnosis and therapeutic management of acute HSV encephalitis.

Soluble CD23 in human immunodeficiency virus type 1 infected patients.

The level of sCD23 produced in the course of human immunodeficiency virus (HIV) infection was measured in patients grouped according to the Centers for Disease Control by using an immunoradiometric assay. Soluble CD23 was evaluated in supernatants of peripheral blood mononuclear cell (PBMC) (10(6) cells/ml) stimulated by phytohemagglutinin (PHA). Compared with healthy controls (m +/- S.D. = 1.0 +/- 0.34 U/ml, n = 7), higher values were observed in some of the patients of group II (asymptomatic) (m +/- S.D. = 2 +/- 1.33, n = 9) and some of the patients of group IV (AIDS) (m +/- S.D. = 1.3 +/- 1.40, n = 8). Those results prompted us to compare the plasma levels of sCD23 in group II and group IV HIV-infected patients and in healthy individuals. Soluble CD23 plasma levels in healthy patients (n = 42) ranged from 0 to 1.5 U/ml (m +/- S.D. = 0.9 +/- 0.33), in group II patients (n = 17) from 0 to 3 U/ml (m +/- S.D. = 0.92 +/- 0.83) and in group IV patients (n = 73) from 0 to 2.9 U/ml (m +/- S.D. = 1.15 +/- 0.71). The differences between the patients and the healthy individuals were not statistically significant but individual sCD23 values higher than 2 U/ml were obtained in 6% of the group II patients and 16.7% of the group IV patients. Increased values of sCD23 were obtained in plasma from patients with secondary infectious diseases (groups IV-Cl and IV-C2) and from patients without secondary infectious diseases (group II, group IV-A and group IV-B).(ABSTRACT TRUNCATED AT 250 WORDS)

[Production, purification and evaluation of human cytomegalovirus 66 kD structural antigen for antibody detection by ELISA]. / Production, purification et évaluation de l'antigène de structure 66 kD du cytomégalovirus humain pour la détection des anticorps par ELISA.

An ELISA method using human CMV major polypeptide (66 kD) purified by electroelution was used to detect IgG and IgM antibodies and was compared with a conventional ELISA using the whole virus. The ELISA-66 kD showed a good sensitivity (98.2%) and had a better specificity than the ELISA-whole virus (97.5% versus 80.3%). Cross reactions observed, particularly with sera containing anti-EBV antibodies, were ruled out.