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The discrepant results available in the literature about the presence of hepatitis C virus (HCV) RNA in seminal plasma of men chronically infected by this agent are related, at least in part, to the molecular techniques used and particularly to the wide range of protocols dedicated to RNA extraction. In order to evaluate these protocols and to standardize the method of detection of HCV RNA in this fluid, a panel of coded specimens was tested blindly in 12 French laboratories; it included 14 seminal plasma specimens and four water controls spiked with HCV RNA ranging from 10 to 20000 IU/ml and two HCV-negative seminal plasma specimens. The extraction step was performed according to methods using either silica beads (NucliSens [Organon Teknika S.A., Fresnes, France]; RNA viral kit [Qiagen, Courtaboeuf, France]) or guanidinium thiocyanate (Amplicor HCV assay; Roche Diagnostics, Meylan, France), preceded or not by a centrifugation of the seminal plasma. For the amplification step, all the laboratories performed the same reverse transcription-PCR technique (Amplicor HCV Cobas assay). The percentage of correct results ranged from 53.3 to 100, the poorest results being obtained when no centrifugation step preceded the Amplicor extraction protocol. The rate of correct results was significantly higher in laboratories using a preliminary centrifugation of the specimen (P = 0.034 by chi-square test). By contrast, the overall number of correct results was not correlated to the initial volume of sample used for the test. These results allowed us to validate standardized techniques adapted to the performance of this test on a routine basis, especially in men infected with HCV and involved in programs of medically assisted reproduction.

Lamivudine (3TC) is a nucleoside analogue which inhibits replication of HIV and HBV and which is used in the treatment of chronic hepatitis B-infected patients with safety and efficacy. The activity of lamivudine was evaluated by the measurement of DNA-HBV concentration in plasma using a very sensitive assay (1,000 copies/mL) (Amplicor VHB Monitor. Roche). Ten patients chronically infected with hepatitis B (group A) and 24 patients with HIV-1 co-infection (group B) were enrolled. In 9 patients of group A, HBVDNA load was undetectable a median of 3.5 months after the beginning of treatment and remained negative for 2 years with hepatitis Be antigen disappearing and normal alanine aminotransferase concentration. In the last immunodeficient patient, the virus which had been resistant to three interferon treatments, was also resistant to lamivudine. In five patients of group B, HBV DNA load remained undetectable after 18 months with HBe antigen disappearing and baseline concentration of alanine aminotransferase. In the remaining 19 patients after a transient decrease of HBV DNA concentration for one year, HBV DNA load increased again without disappearing of HBe antigen and without decrease of alanine aminotransferase concentration showing lamivudine resistant hepatitis B virus. Mutations in the YMDD motif of the DNA polymerase gene were identified in 11 patients (3 with M550V/I mutation; 7 with M550V/I and L256M mutations; 1 with M550V/I, L526M and V519L mutations). In 6 of these patients, was found a M184V mutation in the VIH polymerase. No correlation could be observed between the mutations detected in the two viruses. Using a sensitive HBV-DNA assay, efficacy of lamivudine for a long time in HBV infected patients was proved. However, the prevalence of lamivudine resistance is related to duration of treatment and it may be necessary to use a multitherapy.

Increased levels of IFN-alpha have been found in patients with type 1 diabetes who have detectable levels of coxsackievirus B4 (CVB4) RNA in their blood. The IFN-alpha-inducing activity of CVB4 in vitro is weak but can be enhanced by human IgGs. Therefore, it was investigated in vitro whether a preferential IFN-alpha response of peripheral blood mononuclear cells (PBMCs) to CVB4 exists in patients with type 1 diabetes (n=56) compared with healthy subjects (n=20) and whether antibodies play a role. In patients, the levels of IFN-alpha obtained after stimulation by PBMCs with CVB4 were higher (P=0.008), an individual IFN-alpha response by PBMCs to CVB4 was more frequent (P=0.0004) and increased levels of IFN-alpha were observed in CVB4-infected whole blood cultures. The IFN-alpha-inducing activity of patients plasma and IgGs mixed with CVB4 and then added to PBMCs was high in comparison with healthy subjects (P<0.001) and was inhibited by preincubating the cells with anti-FcgammaRII, anti-FcgammaRIII and anti-CAR (coxsackievirus and adenovirus receptor) antibodies. The strong IFN-alpha responsiveness of PBMCs to CVB4 suggested that IgGs bound to the cell surface might play a role. A short 56 degrees C incubation of PBMCs from patients responsive to CVB4 generated supernatants, which, when added to cells, exhibited IFN-alpha-enhancing activity in combination with CVB4, whereas those of controls did not. Specific antibodies for FcgammaRI, FcgammaRII and CAR inhibited this activity. These studies demonstrate that CVB4, through interactions with circulating and/or cell-bound IgGs, can strongly induce the production of IFN-alpha by PBMCs from patients with type 1 diabetes.

A quantitative PCR assay (RS Elosa CMV, Lambdatech) was used to quantitate HCMV DNA in maternal amniotic fluid of 12 fetuses with congenital infection (group 1) and of 10 fetuses without congenital infection (group 2). HCMV detection was performed for both groups using culture and qualitative PCR. Histologic examinations of fetal tissues and placenta were carried out for 9 patients from group 1. The amniotic fluid viral loads were negative in all patients of group 2. In group 1, all viral loads were high (from 1.105 to > 107 cop/mL) and no difference was observed between symptomatic and asymptomatic foetuses. Further evaluation on larger samples is needed to define more precisely the pronostic value of HCMV DNA quantification in amniotic fluid.

IgG devoid of neutralizing activity and isolated from donor plasma by chromatography formed immune complexes with coxsackievirus B4 (CVB4) and significantly increased the infection of peripheral blood mononuclear cells with CVB4. The major host cells for CVB4 infection enhanced with IgG are monocytic CD14+ cells. The roles of CVB and adenovirus receptor and Fcgamma receptor II and III have been shown. Increased viral replication and the release of infectious particles were demonstrated when interferon (IFN)-alpha produced by infected cells was first neutralized by use of antibodies. The CVB4 IgG-induced synthesis of IFN-alpha by monocytes reflected entry and uncoating of CVB4 but not of viral replication and required the presence of CVB4 RNA inside the cells. Thus, CVB4 can infect monocytes by an antibody-dependent mechanism through interactions between the virus, antiviral antibodies, and specific receptors that result in IFN-alpha production.

Coxsackievirus B4 (CVB4) can be found in circulating blood of patients; however, the interaction of CVB4 with peripheral blood mononuclear cells (PBMCs) is poorly understood. CVB4 induced low levels of IFN-alpha synthesis in PBMCs from healthy donors. In contrast, preincubation of infectious CVB4 with plasma from these donors containing anti-CVB4 antibodies strongly enhanced the synthesis of IFN-alpha. IgG obtained from plasma by chromatography formed immune complexes with CVB4 and increased significantly the CVB4-induced production of IFN-alpha by PBMCs. These antibodies did not have a neutralizing effect on CVB4 infection of Hep-2 cells. The role of CVB and adenovirus receptor (CAR), FcgammaRII and FcgammaRIII in the increased synthesis of IFN-alpha induced by CVB4 preincubated with IgG was shown by inhibition with specific antibodies. The major interferon-alpha-producing cells in response to CVB4-IgG complexes were CD14(+) cells and monocyte-enriched PBMCs. With the latter, detection of IFN-alpha by immunostaining was positive whereas in monocyte-depleted PBMCs it was not. This study shows that CVB4-induced synthesis of IFN-alpha by PBMCs can be enhanced by an antibody-dependent mechanism through interactions between the virus, non-neutralizing antivirus antibodies, FcgammaRII and III and CAR.

The interactions of coxsackievirus B3 (CVB3), CVB4E2 (diabetogenic), and CVB4JBV (nondiabetogenic) strains with human pancreatic islets from eight adult brain-dead donors were investigated. Persistent replication of viruses in human islets was proved by detection of viral RNA by in situ hybridization, VP1 capsid protein by immunofluorescence (IF) staining, negative-strand viral RNA by reverse transcription-PCR in extracted RNA from islets, and release of infectious particles up to 30 days after infection without obvious cytolysis. By double IF staining, glucagon-containing alpha cells and insulin-containing beta cells were shown to be susceptible to CVB. The persistence of CVB3 and CVB4 in islet cells was associated with the chronic synthesis of alpha interferon (IFN-alpha), as evidenced by the detection of IFN-alpha mRNA and immunoreactive IFN-alpha with antiviral activity. By double IF staining, IFN-alpha was detected in insulin-producing beta cells only. Experiments with neutralizing anti-coxsackievirus and adenovirus receptor (CAR) antibodies provided evidence that CAR was expressed by alpha and beta cells and that it played a role in the infection of these cells with CVB and the consecutive IFN-alpha expression in beta cells. The viral replication and the expression of IFN-alpha in islets were not restricted to the CVB4E2 diabetogenic strain and did not depend on the genetic background of the host. The neutralization of endogenous IFN-alpha significantly enhanced the CVB replication in islet cells and resulted in rapid destruction of islets. Thus, human beta cells can harbor a persistent CVB infection, and CVB-induced IFN-alpha plays a role in the initiation and/or maintenance of chronic CVB infection in human islets.

To investigate enterovirus replication versus persistence in end-stage cardiac diseases, endomyocardial biopsies from explanted hearts of 70 patients with idiopathic dilated cardiomyopathy (IDCM), 64 patients with chronic coronary disease (CCD), and 45 donors of healthy hearts (controls) were examined by reverse transcriptase-polymerase chain reaction for genomic and antigenomic enterovirus RNA and by VP1 antigen immunohistochemistry. Enterovirus genome was detected in 25 of 70 patients with IDCM and in 21 of 64 patients with CCDs (35.7 vs. 32.8%, respectively; P=.12). Of the 46 patients positive for genomic RNA, only 3 exhibited antigenomic RNA and VP1 antigen that demonstrated active viral replication, whereas 43 had latent infection characterized by the absence of antigenomic RNA associated with or not with VP1 antigen expression. No viral component was detected in control subjects. The findings demonstrate that a small percentage of patients with end-stage chronic cardiac diseases had active enterovirus replication in their myocardium.

The activation of the interferon (IFN)-alpha system and its relationship with coxsackievirus B (CVB) infection has been analyzed in 56 patients with insulin-dependent diabetes mellitus (IDDM; 25 children and 31 adults). Elevated levels of IFN-alpha were found in plasma of 70% of patients (39/56), and a positive detection of IFN-alpha mRNA in blood cells by reverse transcriptase-polymerase chain reaction (RT-PCR) was observed in 75% of patients (42/56). Enterovirus (EV) RNA assayed by seminested RT-PCR was detected in the blood of 50% of IFN-alpha-positive patients but not in any IFN-alpha-negative patients. The results of genotype analysis of amplified EV RNA sequences (5 CVB2, 8 CVB3, and 8 CVB4) were concordant with the results of CVB-neutralization tests. The comparison between IFN-alpha, EV RNA, and serology suggested that the proportion of CVB infection associated with IFN-alpha positivity might be higher than is predicted from the investigation of EV RNA. Together, the results suggest that, in a majority of cases, a CVB infection is associated with clinical IDDM.

The pattern of human immunodeficiency virus (HIV)-1 antigen-activated production of interferon (IFN)-gamma by immunocompetent cells of HIV-1 infected patients has been studied using a simplified assay combining a small volume (25 microliter) of whole blood stimulation with various HIV-1 antigens, and cytokine measurement in the same wells of microtitre plates (enzyme-linked immunotrapping assay, ELITA). The levels of IFN-gamma were higher using this assay than in the supernatant from stimulated whole blood cultures, therefore ELITA was used in the rest of the study. Specific immune responses to HIV-1 proteins (gp120, p24) and synthetic peptides derived from these proteins and from gp41 were detected in patients, but not in healthy controls. Decreased levels of IFN-gamma were observed in CDC class B (n = 5) and C (n = 4), compared with CDC class A (n = 5), following HIV-1 antigen-specific challenge. The positive response of cells from different patients to overlapping peptides of p25 (amino acids 329-344 and 335-351) was suggestive of a new epitope of HIV-1 gag recognized by T cells in the overlap region. In conclusion, the difference in in vitro antigen-specific T-cell responses of HIV-1-infected patients was shown using the ELITA method. Our results raise the possibility of using this method in screening specific antigens in HIV-1 infection.

Type I interferon system is an important part of host's innate defense mechanisms against viral infections. The type I interferons mediate in part their antiviral effect via induction of various proteins. Among them the most widely known are 2'-5' oligoadenylate synthetase (2'-5' OAS) and a protein kinase (PKR). MxA, an other antiviral protein, is specifically induced by the type I interferons. The MxA protein contains the dynamin signature, which is implicated in transport processes. The MxA protein appears to block the replication of certain viruses at poorly defined steps. There are substantial differences in the antiviral activity of MxA between virus types. Indeed, the replication of vesicular stomatitis virus and influenza virus is inhibited by MxA, but not the one of type I herpes simplex virus. Measurements of interferon alpha and MxA levels may be of high value in clinical practice. Interferon alpha can be detected by using a bioassay based on the interferon alpha ability to protect cultured cells from the cytopathic effect caused by a selected challenged virus, or by using immunological techniques. The current bioassays are the most sensitive methods but they are cumbersome and lengthy, even though simplifications have been proposed. Immunological techniques are easier, however they do not explore the biological activity of the circulating interferon. The presence of type I interferon in biological samples (serum, plasma, cerebro-spinal fluid, cultured cell supernatants) can be indirectly assessed by capability of interferon alpha to induce in vitro the synthesis of MxA in a dose dependent manner in cultured cells. Following to the lysis of the cells, the induced MxA can be quantitated and hence the type I-interferon concentration can be determinated in samples. The quantitation of MxA protein in peripheral blood lysates can be useful as a specific marker of acute viral infections. A minute amount of whole blood (15 mul) is sufficient which facilitates its use in pediatrics. The specifically type-I-interferons inducible MxA protein is also a potential useful marker in the management of interferon alpha-treatment. Moreover, the detection of interferon alpha and antiviral proteins constitute an indirect approach for investigating the hypothesis of the role of viruses in chronic diseases with suspected infectious aetiology.

The presence of human papillomavirus (HPV) DNA in 79 cervical specimens obtained from 70 patients was studied by using a molecular hybridization technique performed in tube. The results were compared to those of the cytological and histological studies. The molecular hybridization technique in tube (Hybrid Capture I) detects two groups of HPV types. One group is highly associated with the development of cancer (types 16, 18, 31, 33, 35, 45, 51, 52, 56) whereas the second group (types 6, 11, 42, 43, 44) is not. Among 42 patients with cervical lesions before any treatment, high risk DNA of HPV was found in 50% of those with low grade cytology and 90% with high grade cytology. In total, 32 out of the 42 patients (76%) who presented histological lesions, were actually infected by HPV. Samples were obtained before and after treatment from 9 patients. Seven out of 9 presented high grade cervical intraepithelial neoplasia (CIN) and 2 other patients had low grade CIN. HPV DNA was not detected in any of the patients after treatment. Detection of HPV DNA by molecular hybridization in tube is simple, sensitive, standardized, inexpensive and is well adapted to screening programs. It can be used in complement of the cytological diagnosis, in the surveillance of equivocal cytological abnormalities, and in the follow-up of treated patients.

Background: Human polyomavirus JC (JCV) induces human progressive multifocal leukoencephalopathy (PML) in patients with AIDS. Peripheral blood mononuclear cells (PBMC) of HIV-1-positive immunocompetent and immunocompromised patients can harbour JCV genome, but their precise role in JCV latency or reactivation status before the onset of PML remains hypothetical., Objectives: To assess JCV latency or reactivation status in PBMC of HIV-1-positive immunocompromised patients without PML., Design: A group of 82 HIV-1-positive immunocompromised patients who did not have PML were compared with 10 patients with AIDS and PML and with 69 HIV-1-positive immunocompetent patients without PML., Methods: DNA and total RNA were extracted from PBMC. The presence of JCV DNA was demonstrated by a semi-nested polymerase chain reaction (PCR). By using primer pairs specific for an early gene,T, and a late gene, VP1, the expression of both early and late gene mRNA in PBMC could be identified using reverse transcriptase (RT) PCR., Results: JCV DNA was detected by PCR in 17.4% of 69 HIV-1-positive immunocompetent patients, in 23.2% of 82 HIV-1-positive immunocompromised patients, and in 60% of 10 patients with AIDS and PML. No correlation could be drawn between the detection of JCV DNA in the PBMC and the clinical or biological status of the HIV-1-positive patients. By using RT-PCR procedures, no expression of JCV early and late mRNA in PBMC was found in any patients., Conclusions: JCV DNA is detectable in the PBMC of 20.5% of 151 HIV-1-infected patients independently of the CDC (Centers for Disease Control and Prevention) stages of the infection. Moreover, our results suggest that active replication of JCV in PBMC appears to be absent or at least a very rare event in HIV-1-positive immunocompromised patients with and without PML.

We studied the in vitro HIV-1 antigen-stimulated production of IFN-gamma and IL-4 in HIV-1-infected patients and its relationship with viral replication as assessed through the plasma level of HIV-1 RNA. The levels of IFN-gamma and IL-4 were higher in supernatants of stimulated whole blood cultures than in stimulated peripheral blood mononuclear cell cultures, therefore whole blood cultures were used in the rest of the study. Specific IFN-gamma and IL-4 responses to HIV-1 p24 antigen were observed in HIV-1-infected patients but not in healthy controls (n = 23). A lower proportion of individuals with a positive IFN-gamma response to HIV-1 p24 was observed in patients at a declining clinical stage: 62% in asymptomatic patients (CDC group A, n = 16) versus 19% in symptomatic patients (CDC groups B and C, n = 21; P = 0.007, chi2 testing), whereas the proportion of individuals with a positive IL-4 response to HIV-1 p24 was almost similar in both groups of patients (25% versus 23.8%). Increased IL-4 production by HIV-1 p24-activated immunocompetent cells of patients and a predominant IL-4 response to HIV-1 p24 (with IL-4/IFN-gamma > 1) were positively correlated with an increased viral load. In contrast, there was no correlation between the mitogen-stimulated production of IL-4 and IFN-gamma and the viral load in plasma. The CD8 T cells from whole blood of patients, but not from controls played a significant role in the HIV-1 p24-activated production of IFN-gamma and IL-4. In conclusion, HIV-1-antigen-stimulated whole blood appears to be a valuable tool to study the production of IL-4 in HIV-1-infected patients. The cytokine profile pattern in response to epitopes of HIV-1 gag p24 may play an important role in the host immune response to HIV-1.

We investigated the biological properties of interferon (IFN)-alpha produced by Sendai virus (SV)-activated whole blood cultures in 20 patients infected with human immunodeficiency virus (HIV)-1 and 24 healthy controls. Supernatants of cultures were assayed for IFN-alpha by using an immunological method (DELFIA), biological methods and an in-vitro MxA induction assay. The levels of intracellular MxA protein were detected by an immunochemiluminescence assay. The levels of IFN-alpha in patients measured by DELFIA were significantly lower than those in healthy controls (P < 0.0001), but the antiviral activity of IFN-alpha in patients infected with HIV-1 was lower than predicted from DELFIA. The IFN-alpha produced by cells of patients infected with HIV-1 was able to induce MxA protein in human amnions WISH cells but was unable to protect these cells against Vesicular Stomatitis Virus (VSV)-induced cytopathic effects. A relative increased capability to induce the production of MxA protein in vitro was observed with the IFN-alpha contained in culture supernatant of virus-activated whole blood of HIV-1-infected patients with increased levels of MxA in their peripheral blood. These data suggest that biological properties of IFN-alpha produced in the course of HIV-1 infection are different from those observed with IFN-alpha of healthy subjects.

The value of soluble receptor for tumor necrosis factor type II (sTNFRII) as a strong and early predictor of HIV disease progression was suggested. Recently it has been reported that sTNFRII may provide an indication of the HIV load. In this work we focused on the relationship between sTNFRII and HIV burden in 95 HIV-1+ patients without AIDS grouped according to the 1993 classification of the CDC as group A, n = 55, and group B, n = 40. Compared with healthy controls, higher values of sTNFRII were obtained in all groups of HIV-1 infected patients (P < 0.001), but we found no inverse correlation between sTNFRII and CD4+ lymphocyte counts in CDC group A and B of the disease, and no correlation with log RNA copy number in patients with CD4 T-cell counts > 499/microl. A correlation was obtained between sTNFRII and the viral load in patients with CD4 T-cell counts ranging from 200 to 499/microl, but only in CDC group B patients (P < 0.01, n = 26). There was no correlation between the variations of sTNFRII and HIV-1 RNA levels in 19 CDC group A and 15 CDC group B clinically stable patients in the course of a short follow up. The plasma level of sTNFRII do not appear as a valuable surrogate marker of the plasma level of HIV-1 RNA in patients. Further investigations are needed to define the mechanism of the raised level of sTNFRII in HIV-1 infected patients.

Singlet oxygen (1O2, 1delta(g)) selectively oxidizes many biological targets, some of which, such as viruses, are located intracellularly under in vivo conditions. Considering the short lifetime of 1O2 in aqueous media, it is essential to generate this species in close proximity to the targets. Therefore, a water-soluble and non-ionic carrier of 1O2, DHPNO2, has been designed to convey 1O2 through lipid membranes. In contrast to the known anionic carrier NDPO2, which inactivates only extracellular enveloped viruses, the new compound exhibits virucidal activity on all types of viruses, enveloped (HIV) and non-enveloped (Poliovirus), extracellular and intracellular. HIV inactivation can also be achieved in the presence of red blood cells, suggesting the possible use of DHPNO2 in the decontamination of cellular blood products.

It has been reported that in vitro biological properties of human immunodeficiency virus type 1 (HIV-1) isolates from patients are correlated with the prognosis of HIV-1 infection. A rapid assay was developed to study the phenotype of HIV-1 isolates. The P4 cell line is a HIV-1 infectible Hela CD4 cell carrying the bacterial LacZ gene under the control of the HIV-1 LTR (long terminal repeat). Conventional peripheral blood mononuclear cells (PBMCs) co-culture and heparinized whole blood (HWB) co-culture with normal PBMCs were used for HIV-1 isolated strains from 17 HIV-1-infected patients. The sensitivity of P4 cells was higher than that of MT-2 cells for detecting syncytia induced by HIV-1LAI (lymphadenopathy-associated virus). Like MT-2 cells, P4 cells enable the detection of syncytium inducing strains isolated in peripheral blood mononuclear cells (PBMCs) and HWB cultures. HIV-1 isolates with both culture methods from certain patients induced cytolysis without syncytium in P4 cells but had no cytopathic effect on MT-2 cells. The experiments are in favour of the direct effect of HIV-1 isolates of these patients in the lysis of P4 cells but its mechanism has not been elucidated. It was shown that the combination of whole blood culture for HIV-1 isolation and phenotype study with P4 cell assay is rapid and sensitive and could be used to monitor HIV-1-infected patients.

A PCR assay for detection of enterovirus RNA in multiple specimen types from patients with neurological infections was evaluated. Combined PCR assay of cerebrospinal fluid and serum (systemic specimens) was more sensitive than assaying either specimen alone in children but not in adults. Compared with PCR in systemic specimens, detection of enterovirus RNA in throat swabs showed a sensitivity of 62.5% and a specificity of 75.6%.

Human parvovirus B19 primary infection during pregnancy is responsible for 27% of non autoimmune hydrops fetalis. Parvovirus B19 antigen detection and parvovirus B19 IgM and IgG antibody determination using enzyme immunoassays are not reliable for diagnostic purposes and lack of specificity. Parvovirus B19 DNA detection in amniotic fluid, fetal blood, ascitic fluid, and fetal biopsies or placenta specimens seems to be the best method for the diagnosis. Ninety-seven samples from 70 cases of spontaneous abortions after fetal death or hydrops fetalis were examined using PCR. A 270-bp length fragment of the NSI gene was amplified using PCR followed by electrophoresis, by Dot-blot hybridization assay using a biotinylated probe and by Southern-blot hybridization assay using a horseradish peroxidase-labelled probe followed by chemiluminescent assay. The Southern-blot hybridization assay was the longest test but the most sensitive. The parvovirus B19 genome was identified in 10 cases. In two cases, intrauterine blood transfusions led to the cessation of symptoms and to the birth of normal babies.

Molecular biology techniques are applied for the diagnosis of meningoencephalitis due to herpesviruses, enteroviruses or polyomaviruses, for the diagnosis of human cytomegalovirus, human parvovirus B19, varicella-zoster virus and rubella virus infections occurring during pregnancy, for the diagnosis and the management of retrovirus infections (HIV and HTLV) and of hepatitis (HBV and HCV), for papillomavirus typing and to detect a link between virus and clinical manifestations (cardiomyopathy or insulinodependent diabetes with coxsackievirus B: Kaposi's sarcoma with HHV 8) or to investigate an environmental contamination with viruses. These new molecular markers which are both qualitative and quantitative represent an important advance in the field of viral diagnosis research, in the monitoring of viral load during the course of infection, in the therapy control of viral disease and in the epidemiology of virus spread. Standardization and automatization are obtained using available commercial reagents and kits.

Molecular biology techniques which can rapidly detect a few copy number of viral genome allow to obtain new parameters for the diagnosis, the prognosis, and the monitoring of viral diseases in addition to or in substitution for cell culture and antigen and antibody detection. The methods are based on the virus structural organization and on their replication into cells. They use the tools which have been developed to analyse nucleic acids. Molecular hybridization (dot-blot, southern-blot, liquid phase hybridization, in situ hybridization), enzymatic amplification (PCR, nested-PCR, RT-PCR, NASBA, bDNA, multiplex PCR), DNA and RNA quantification (quantitative PCR or RT-PCR, NASBA, bDNA) and genotyping (virus types, mutations, variant virus) are used. Nucleic acid extracts native or amplified with a reporter molecule (dUTP-biotin or dUTP-digoxigenin) are identified using gel electrophoresis followed by hybridization or using microwell capture hybridization assay and colorimetric immunoenzymatic or bioluminescence assays.

The studies indicating the importance of TNF alpha in dengue virus infection have led us to determine whether monocyte-like cells produce TNF alpha exposure after dengue virus. The supernatant fluids of mosquito cells (AP61) infected with dengue virus (DV) type 1 and DV type 3 were harvested 7 days post-infection and clarified. DV inactivation was performed in the presence of betapropiolactone that preserves antigenicity of viruses. We used the monocytic-like cell line THP-1 that is a model system of TNF alpha production. Polymyxin B (50 micrograms/ml) was added to block untoward effects resulting from possible LPS contamination of media or cultures. THP-1 cells were primed with a phorbol ester (PMA) for 24 h, then they were cultured for 4 and 24 h in the presence of inactivated culture supernatant of dengue infected AP61 cells or control preparations. The concentrations of TNF alpha in the culture supernatants were measured by using an immunoenzymatic assay. PMA-treated THP-1 cells rapidly secreted TNF alpha in response to inactivated culture supernatant of DV-infected cells. We found high levels of TNF alpha with cells exposed to DV1 and DV3 preparations compared with controls (mean values; 465 and 829 vs. 70 pg/ml, respectively, at 24 h post exposure, n = 4). We obtained a substantial inhibition of the enhancing activity of DV1 and DV3 infected supernatants in the presence of dengue hyperimmune mouse ascitic fluids. Our results demonstrate that exposure of monocytes/macrophages to DV particles or virus proteins derived from DV may be responsible for the enhanced production of TNF alpha in DV-infected patients.

We determined the degree of activation of the TNF alpha system and the levels of IL-8 in HIV-1 infection. TNF alpha is one of the most potent agents for the induction of IL-8. TNF alpha may be cleared rapidly from the circulation, however soluble tumor necrosis factor receptor (sTNFR) p75 which is more stable may reflect the degree of activation of the TNF alpha system. We measured concentrations of sTNFR p75 and IL-8 with immunoassays in the plasma of 99 HIV-1-infected patients at different stages of disease (Centers for Disease Control classification) and in 20 healthy control subjects. Plasma levels of sTNFR p75 were elevated in most of the asymptomatic seropositive patients without CD4+ T cell depletion (Stage IIA) and in most of patients of all clinical groups compared with controls. However, in the majority of those patients plasma levels of IL-8 were not higher than those of controls. Our results suggest that sTNFR p75 levels but not IL-8 levels in circulating blood are high in the course of HIV-1 infection.

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.

Production of cytokines by immunocompetent cells in vitro may be assessed after stimulation with polyclonal activators. Because it mimics the natural environment, diluted whole blood (WB) culture may be the most appropriate milieu in which to study cytokine production in vitro. We tested TNF alpha production by small volume of whole blood (25 microliters) from HIV-1 positive patients by using a one-step procedure that combines WB stimulation with LPS and PHA and cytokine measurement. We studied 30 patients without secondary infection or at distance of secondary infection staged according to the classification proposed by the CDC and 12 healthy seronegative subjects. The mean values of TNF alpha from patients were not statistically different from those from normal controls however in certain patients at different stages of the disease higher values than the mean +2 SD of controls and lower values than the mean -2 SD of controls were obtained. Heparinized blood from 5 control subjects had been collected sequentially during a period of 5 months. The individual variation of TNF alpha production were limited for all these individuals. For each of 6 HIV-1 patients, whole blood samples were collected sequentially during a period of 5 months and in most of patients large variations of levels of TNF alpha were observed from one sample to another one. Our method can detect abnormal cytokine production in HIV-1 positive individuals and can become a useful tool to investigate the role of cytokines in the outcome of HIV-1 infection.

The pathogenesis of CVB3-induced chronic myocarditis remains unknown. Activated monocytes and macrophages may maintain ongoing inflammation during a persistent CVB3 infection and possibly represent the major mechanism leading to chronic myocarditis. We decided to study the activation status of cells by studying TNF alpha secretion in vitro using whole blood culture in CVB3-induced murine chronic myocarditis. Seven DBA/2 +/+ mice and 18 NMRI nu/nu mice were inoculated intraperitoneally with 5x10(5) pfu of CVB3, and mice were mock-infected. Thirty-one days post-infection, all mice were sacrificed, blood samples were obtained from the heart, and the heart was removed. Enteroviral genomic detection by RT-PCR, virus isolation and histological analysis of heart samples were performed. Heparinized whole blood (25 microliters) was cultured for 4 hr and 24 hr in sterile 96 well-plate containing 225 microliters RPMI in the presence or the absence of activators (LPS + PHA). The TNF alpha levels in the whole blood from mock-infected DBA/2 (n = 4) and NMRI nu/nu mice (n = 5) were not different. A moderate increase of TNF alpha was observed in three out of five DBA/2 mice with negative CVB3 that had no histological abnormalities in myocardium. An increased level of TNF alpha was found in the sole DBA/2 mouse with positive CVB3 detection and chronic myocarditis. An increased level of TNF alpha was found in one out of nine NMRI nu/nu mice with positive CVB3 detection and chronic myocarditis and in one out of seven mice with positive CVB3 detection exempt of lesions in myocardium. In other infected mice, the level of TNF alpha was normal. Enteroviral genome was not detected in the blood from infected mice at 31 days post-infection. The increased TNF alpha level in some mice may be designed for a beneficial inflammatory and immune response, however, an exaggerated release may be associated with an adverse effect. The normal TNF alpha level in whole blood cultures from mice with chronic myocarditis does not exclude enhanced cytokine production at infected loci such as myocardial tissue. This is the first report to use whole blood cultures to study the production of cytokines in virus-induced disease in a small animal model.

Soluble forms of the two molecular species of the cell surface TNF receptors (sTNFR p55 and sTNFR p75) can reduce the activity of TNF alpha but they may also enhance its function by stabilizing the active TNF alpha oligomer. Considering the pathophysiological importance of sTNFR p75 for the regulation of the bioavailability of TNF alpha in the body, we determined the serum levels of sTNFR p75 and TNF alpha in 45 children and 28 adults with laboratory-confirmed dengue infection by using immunoassays. The serum samples were obtained from day 1 to day 15 after the onset of the disease during the 1989-1990 outbreak of dengue-3 in Tahiti, French Polynesia. The patients were clinically classified as having dengue hemorrhagic fever (DHF) and graded according to the criteria of the World Health Organization (WHO) into four grades from less severe (grade I) to severe (grade IV). The sera of both children and adult patients of all severity grades contained higher levels of sTNFR p75 than the sera of control subjects. Although high levels of TNF alpha were also detected in children and adults among grade I, II, III and IV patients, we found no correlation between sTNFR p75 and TNF alpha. We observed in adults a moderate elevation of sTNFR p75 and TNF alpha in sera compared with that observed in children. The raised levels of immunoreactive sTNFR p75 and TNF alpha in all clinical groups of dengue-infected patients strongly indicate activation of the TNF alpha system during dengue infection. The balance between sTNFR p75 and TNF alpha may be altered in dengue infection. Further investigations are needed to understand the role of sTNFR p75 and TNF alpha in the pathogenesis of DHF and to improve the management of dengue infection.

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.

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.

Hepatitis E, a faecal-oral waterborne acute viral hepatitis, occurs most frequently in epidemic outbreaks in developing countries. Hepatitis E virus (HEV) is spherical, nonenveloped and varied in size from 27 to 34 nm with a spiked surface. Experimental HEV infection in primates, molecular cloning involving cDNA libraries and recombinant protein technology indicate that the genome can be assigned to a 7.6 kb single stranded positive polyadenylated RNA. In a 5'NS-S poly A 3' molecular structure, three partially overlapping ORF have been identified. Two strains (Burma and Mexico) have been studied and although they related to caliciviruses, the genetic organization indicates that it represents different agents ranged in a new subgroup: the alpha-like viruses. HEV is an important cause of morbidity and mortality in developing countries in the 15 to 40 year age-group and although the mortality rate is low in the general population (0.5 to 3%), it averages 17 to 20% among third-trimester pregnant women. Several sporadic cases have been recently identified among children. The prevalence of anti-HEV antibodies in blood donors ranges from 2 to 3% in North Europe and USA and from 6.8% in Spain to 70% in Thailand. The average incubation period is 6 weeks. Chronic liver disease, persistent viraemia and oncogenicity have not been observed. HEV particles are identified in stool or bile by immune electron microscopy, capture immunoassay or RT-PCR.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of commercial multichannel air-cooled laser flow cytometers which allow measurement of one or more characteristic parameters of particles or cells has been furthered due to advances in probes and fluorochromes. FITC-conjugated antibodies specific for selected cell surface molecules or for viral antigen expression in infected cells, fluorescein diacetate for labeling cells as a parameter of viability and propidium iodide as a DNA stain are generally used. Flow cytometric assays have been developed in virology to study the interactions of viruses with the immune system (Hantan Virus), to evaluate the diagnosis of secondary immunodeficiency syndromes (HIV), to analyze the protein and DNA content of virally infected cells during the cell cycle (SV40), to detect immediate early, early or late antigens in HCMV-infected cells, to assess the HCMV persistence in mononuclear cells, for use as a model for studying virus attachment to cellular receptors (EBV-echovirus), to screen and evaluate antiviral agents (Influenza C, HCMV, HSV, HIV) and to quantify antigens or antibodies simultaneously against various viruses (HCMV and HSV) or against different antigenic glycoproteins (HIV). This technology has thus become an important tool for the clinical virology and microbial serology laboratories.

Quantitative determination of alpha interferon (IFN) is used as an early marker in viral encephalitis. IFN is detected during 10 days following the onset of clinical symptoms. In 26 patients (11 children from 1 day to 6 year old and 15 adults from 17 to 70 year old) with central nervous system disorders (15 meningo-encephalitis, 5 meningitis, 1 myelitis, 1 polyradiculoneuritis, 1 dementia, 1 epilepsy and 2 other), alpha IFN is quantified using a cytopathic effect inhibition assay of VSV on MDBK cells. The mean value of alpha IFN is 80 UI/ml (range from 0 to 512 UI/ml). Virus involved are herpes virus in 38.5% of cases (10/26) and 66% of viral meningoencephalitis (8/12), H.I.V. in 3 cases, VZV in 2 and measles virus in 1 case. Viral aetiology is suspected in six other patients. The results show the importance of early determination of alpha IFN (immediately after the first symptoms and on the first admission to the hospital) in sera and cerebrospinal fluids (CSF) simultaneously with viral culture and antibody research. The presence of alpha IFN only in CSF and a higher titre of alpha IFN in CSF than in serum are important data to distinguish primitive acute necrotizing encephalitis from post eruptive or post infectious perivenous encephalitis. In herpes virus infections with specific treatment all the patients recover. However to prevent brain damage in survivors the treatment should be established as soon as possible.

About six observations of toxocariasis (visceral larva migrans syndrome). We relate six observations of toxocariasis among children. In one case, an ocular localization is probable. For other five patients, they are inapparent forms. The allergologist pediatrician may be consulted because of a major hypereosinophilia (greater than 10,000/mm3) and an elevation of total IgE (greater than 2,000 UI/ml). Allergic and current parasitologic assays are negative and diagnostic key is given by toxocara serology. We insist on interest and reliability of passive hemagglutination test with a purified antigen (titer greater than or equal to 1/320). Treatment now is preferably flubendazole (50 mg/kg/day for six days) eventually renewed.

Coxsackie B virus infections are common and frequently asymptomatic. However in young people, they can cause primary congestive cardiomyopathy and complicate previous cardiovascular illnesses. Virus diagnosis is difficult and based mainly on the detection of significant rising or stating high neutralizing antibody titers. A clinical and epidemiological five years study investigated 3,856 sera. 30.2% of the patients had evidence of a significant antibody titer (greater than or equal to 64) to one of the group B Coxsackie viruses; this percentage reaches 34.6% in cardiology and fluctuates from year to year; Coxsackie B2 is predominant (55.9%) in cardiology; coxsackie B1 and B2 antibody responses were detected more frequently than B3, B4 and B5. Coxsackie B6 appears to be uncommon. From these, 66 patients have evidence of coxsackie B virus infections with 60.2% for Coxsackie B2; they include pericarditis (30.3%), acute and chronic congestive cardiomyopathies (19.7%). Moreover it has been suggested that Coxsackie B virus might be responsible for electrocardiographic abnormalities (9.1%), ischemic heart disease and myocardial infarction. Enzyme linked immunosorbent assay (ELISA) and radioimmunoassay with purified antigens (VP1 and VP4) did not appear better than microneutralisation for evaluation of IgM and IgG antibodies. To elucidate the mechanisms of cardiac injury it is refer to viral replication, virus specific and auto-reactive T cells cytotoxic activities.

A rapid, simple and preparative method is described for the recovery of the seven highest molecular weight proteins (HMWP) from Mycoplasma pneumoniae membrane. The yield of proteins obtained was approximately 90%. The method involved the separation of M. pneumoniae proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroelution of HMWP. These eluted antigens were used in an ELISA to measure IgG antibodies in sera from 9 blood donors and 9 patients with M. pneumoniae infection. The specificity of M. pneumoniae HMWP was examined by competition ELISA and immunoblotting with different mycoplasma species encountered in the respiratory tract.

During a 4-years period 122 patients admitted to the cardiologic hospital in Lille presented a significative positivity of serological reaction against Coxsackievirus B. Among these patients 19 suffered from acute pericarditis, 4 from cardiac arrhythmias, 3 from Bornholm syndrome and 14 from apparently primary congestive cardiomyopathy. In most of these 14 patients representing 18.4% of congestive cardiomyopathies investigated at the same time, hemodynamic studies and heart scanning with radioactive gallium were performed. 7 of these patients had a chronic (group A) and 7 an acute evolution (group B). In group A the scans were negative 4 times out of 5, whereas in group B they were positive in 5 out of 5 examinations. The control group including 3247 patients coming from the Institut Pasteur exhibited serological positivity in 4.6% of cases. Comparison of this group with that of cardiomyopathies shows a significant difference (p less than 0.001). The authors draw the attention to the frequency of positive serodiagnosis in congestive cardiomyopathy and stress the importance of gallium scintiscanning.

In patients infected by Fasciola hepatica, total IgE and specific IgE antibodies have been determined by radioimmunoassays, and IgG, IgA, IgM levels by radial immunodiffusion test (Mancini, 1965). Moreover, total and specific IgE levels have been related to parasite egg burden, age, clinical features and eosinophilia. Elevated total IgE and specific IgE antibodies levels have been found respectively in 76% and 48% of the patients whereas there was no significant variations in other immunoglobulins levels. However, though the amount of total and specific IgE was lower than in other helminthic diseases, it appears to be a significant data of the immune response to parasites as it has been reported and discussed previously. It has been shown a significant relationship between total and specific IgE levels, the number of lines by immunoelectrophoresis, and the results of the indirect haemagglutination and indirect fluorescent antibody techniques; each method appeared to be in equal value to perform the early diagnosis of human Fasciola hepatica. In addition, specific IgE antibodies levels were correlated with eosinophilia specially when it exceeds 15%. This results demonstrate the availability of their measurement in the diagnosis of fascioliasis versus other diseases with marked eosinophilia.