Changing of hepatitis C virus genotype patterns in France at the beginning of the third millenium: The GEMHEP GenoCII Study.

This cross-sectional study aimed to investigate, during a short period between 2000 and 2001, in a large population of patients with chronic hepatitis C, the epidemiological characteristics of hepatitis C virus (HCV) genotypes in France. Data from 26 referral centres, corresponding to 1769 patients with chronic hepatitis C were collected consecutively during a 6-month period. HCV genotyping in the 5'-non-coding region (NCR) was performed in each center using the line probe assay (LiPA, in 63% of cases), sequencing (25%) or primer-specific polymerase chain reaction (PCR) (12%). HCV genotypes 1a, 1b, 2, 3, 4, 5, non-subtyped 1 and mixed infection were found in 18, 27, 9, 21, 9, 3, 11 and 1% of our population, respectively. HCV genotype distribution was associated with gender, age, source and duration of infection, alanine aminotransferase (ALT) levels, cirrhosis, alcohol consumption, hepatitis B virus (HBV) and human immunodeficiency virus (HIV) coinfection. In multivariate analysis, only the source of infection was the independent factor significantly associated with genotype (P = 0.0001). In conclusion, this study shows a changing pattern of HCV genotypes in France, with i.v. drug abuse as the major risk factor, an increase of genotype 4, and to a lesser extent 1a and 5, and a decrease of genotypes 1b and 2. The modification of the HCV genotype pattern in France in the next 10 years may require new therapeutic strategies, and further survey studies.

[Assessment of the diagnostic value of RT-PCR on amniotic fluid for prenatal diagnosis of congenital rubella infection]. / L'infection rubéolique maternofoetale: apport de la biologie moléculaire.

AIM OF THE STUDY: To assess the diagnostic value of RT-PCR on amniotic fluid (AF) for prenatal diagnosis of congenital rubella infection. MATERIAL AND METHODS: RT-PCR on AF was compared to specific IgM antibody detection in foetuses and/or newborns in 45 pregnant women with confirmed primary infection. RESULTS: specificity of RT-PCR was 100% and sensitivity ranged between 83 and 95%. CONCLUSION: RT PCR may be considered as a valuable tool for prenatal diagnosis of foetal rubella infection.

Real-time PCR quantification of human cytomegalovirus DNA in amniotic fluid samples from mothers with primary infection.

A real-time PCR assay was developed to quantify human cytomegalovirus (HCMV) DNA in amniotic fluid (AF) samples collected from 30 pregnant women with primary HCMV infection as detected either from HCMV-immunoglobulin G (IgG) seroconversion or by the presence of HCMV-specific IgG and IgM associated with a low IgG avidity. Clinical information available for each case included ultrasonographic examination and fetal or newborn outcome. HCMV infection of fetuses or newborns was confirmed for the 30 studied cases. AF samples were subdivided into three groups. In group A (n = 13), fetuses presented major ultrasound abnormalities, and pregnancy was terminated. In group B (n = 13), fetuses had normal ultrasound findings, the pregnancy went to term, and the newborns were asymptomatic at birth. In group C (n = 4), fetuses had no or minor ultrasonographic signs, and pregnancy was terminated. The HCMV DNA load values in AF samples were significantly higher in group A (median, 2.8 x 10(5) genome equivalents [GE]/ml) than in group B (median, 8 x 10(3) GE/ml) (P = 0.014). Our findings suggest that HCMV load level in AF samples correlates with fetal clinical outcome but might also be dependent on other factors, such as the gestational age at the time of AF sampling and the time elapsed since maternal infection.

Diagnosis and prognostic markers of HCMV infection.

Human cytomegalovirus (HCMV) infections are frequently observed as well in immunocompetent as in immunocompromised patients. Diagnostic techniques for HCMV detection have greatly improved during the recent years. Detection of HCMV by culture has been bettered by centrifugating samples in a shell vial and by using monoclonal antibody to the immediate early antigen. Detection of antigens in leucocytes was facilitated by using whole blood instead of leucocytes separated by dextran sedimentation. Detection of HCMV recently sharpened by using molecular biology methods mainly based on the detection of the genome. Under certain circumstances, and especially in pregnant women, diagnosis of HCMV infection is essentially based on the detection of IgG and IgM antibodies. However, as IgM antibody is not a marker of primary infection, complementary tests are needed to help in the datation of the infection. Among them, the measurement of IgG avidity greatly improved the diagnosis of primary infections. In immunocompromised patients, very sensitive techniques are needed to diagnose HCMV infections. Detection of antigenemia and HCMV DNA are the methods of choice for an early detection of the infection. Diagnosis of HCMV-organ diseases largely depends on the infected organ and the presence of HCMV in the organ is, sometimes, difficult to interpret. Today, diagnosis of congenital infections is possible by detecting HCMV in the amniotic fluid. However, in order to reliably detect HCMV, amniocentesis must be performed after 21 week's gestation and at least 6 weeks after seroconversion. As all HCMV infections do not induce disease, prognostic markers are needed, as well in immunocompromised patients as in fetuses. Many studies were conducted in immunocompromised patients to find prognostic markers of HCMV infection in order to introduce preemptive therapy when needed. It seems that quantitative determination of HCMV DNA could be very useful to predict HCMV disease. Qualitative determination of mRNA could also be useful as a prognostic marker of HCMV disease. Determination of viral genotypes is more controversial. As for the infected fetuses, little was published about the prediction of sequelae. Some factors, such as viral load in amniotic fluid or in fetal blood or level of IgM antibody in fetal blood, may be predictive of sequelae, but these data require further studies.

Multicenter evaluation of a rapid and convenient method for determination of cytomegalovirus immunoglobulin G avidity.

An easy, rapid, and reproducible test to distinguish residual cytomegalovirus (CMV) immunoglobulin M (IgM) antibodies from antibodies produced in primary infection could be useful, especially for pregnant women. The CMV avidity of IgG antibodies with the VIDAS automated enzyme-linked fluorescent assay and 6 M urea was evaluated in a multicenter study to differentiate between primary CMV infections and past infections or reactivations. A total of 416 serum specimens were tested: 159 specimens were from follow-up of primary infections, and 257 were from past infections. All of the specimens from primary infections collected within 4 months (17 weeks) after the onset of the infection had an avidity index lower than 0.8. An avidity index higher than 0.8 excludes a recent primary infection of less than 4 months. However, an avidity index higher than 0.8 cannot confirm all past infections, since 48 specimens (18%) from past infections had an avidity index lower than 0.8 (between 0.5 and 0.8). The exclusion capacity could be improved (96.9%) by using a cutoff of 0.7, but this index would decrease the specificity of the technique, since the avidity index was found to be between 0.7 and 0.8 in two patients with recent primary infection. All specimens from primary infections obtained more than 4 months after the onset of infection had an avidity index more than 0.2. In this study, an avidity index less than 0.2 confirms the presence of a recent primary infection of less than 4 months. The VIDAS CMV IgG avidity test is a rapid, reproducible test with very good performance.

Congenital HCMV infection: a collaborative and comparative study of virus detection in amniotic fluid by culture and by PCR.

Cytomegalovirus (HCMV) infection is the leading cause of congenital virus infection in developed countries, affecting an estimated 1% of births. This antenatal infection can cause serious sequelae. Strategies for prevention and treatment must, therefore, be agreed upon, entailing a preliminary performance assessment of antenatal virus diagnosis techniques. Between 1992 and 1999, HCMV serology status was established for 19456 pregnant women in four French hospitals. Seronegative patients (55.4%) were given serology screening, and antenatal diagnosis was given to 152 women who had shown seroconversion during their pregnancies (1.4%). The detection of HCMV transmission from mother to fetus was finally established in 95 cases, using polymerase chain reaction (PCR) and viral culture methods for detecting HCMV in the amniotic fluid. These results were compared with viral culture of children's urine after birth, enabling us to distinguish between children really infected in utero (30%) and non-infected children (70%). The results of the virus culture and those of PCR were identical in 94 of the 95 cases, with one discrepancy (culture-/PCR+). The two diagnosis techniques had identical sensitivity (72%), with culture proving slightly more specific than PCR (98.4% as opposed to 96.9%). Positive prediction values for culture and for PCR were, respectively, 95.6 and 91.3%. Antenatal virus diagnosis on amniotic fluid was negative with both techniques in 8 out of 29 cases of children born with HCMV infection (VPN=89%). Over half of these wrongly negative results can be explained by amniocentesis carried out too early in the pregnancy or too early with respect to the mother's primary infection.

[The isolated presence of anti-HBc antibodies: prevalence and interpretation based on the results of viral DNA research and anti-HBs antibodies measurement after vaccination]. / Présence isolée de l'anticorps anti-HBc: prévalence et interprétation à l'aide des résultats de recherche d l'ADN viral et du dosage des anticorps anti-HBs après vaccination.

In order to study the significance of the isolated presence of anti-HBc antibodies, we have looked for the 3 classic serological markers of the hepatitis B (HBs antigen, anti-HBs and anti-HBc antibodies) in 1,586 hospital agents who are to vaccinate in the framework of a campaign of systematic vaccination of the hospital personnel of university hospitals of Sfax for a period of 18 months. We identified subjects who presented isolated anti-HBc antibodies (33 individuals = 2.08%). In these subjects'serum, we performed a research of the DNA of hepatitis B virus (HBV) with a PCR hybridization technique using a couple of primers. One week after administration of a vaccine dose, we also measured anti-HBs antibodies in their sera. Among the tested 18 personnel with anti-HBc isolated antibodies, 11.1% had low rates of anti-HBs antibodies indicating that there is presumably primary antibody response and therefore a false positivity of anti-HBc antibodies in pre-vaccinal serology; while 11.1% others had higher rates of anti-HBs antibodies corresponding to a secondary antibody response, which witness a previous HBV immunisation. The research of the HBV-DNA was positive in 11.1% of tested personnel, testifying a presumably chronic portage of the virus with low rates of the HBs antigen undetectable with the serological techniques. For the remainder subjects with isolated anti-HBc antibodies (66.7%), the interpretation remains ambiguous.

[Viral infections and pregnancy: contribution of amniotic fluid and blood samples]. / Infections virales et grossesse: apport des prélèvements amniotiques et sanguins.

The main viral infections prenatally detected in fetuses are: cytomegalovirus, parvovirus B19, rubella virus and varicellazoster virus infections. Prenatal diagnosis is based on the direct detection of the virus by culture (CMV), of its antigens or of its genome, essentially by PCR. This direct detection can be done either on fetal blood or on amniotic fluid. Prenatal diagnosis can also be performed by detection of specific IgM in fetal blood (rubella). Non specific markers of viral infection can also help in diagnosis. At the present time, prenatal diagnosis is essentially based on the detection of the viral genome in amniotic fluid. In order to better appreciate the severity of fetal infections, some groups have tried to identify prognostic markers of these infections. The viral load as well as the level of specific IgM could play a role in certain infections (CMV).

Unusual N-glycosylation of a recombinant human erythropoietin expressed in a human lymphoblastoid cell line does not alter its biological properties.

Erythropoietin (Epo) is a 166 amino acids protein containing three N-glycosylation sites (Asn-24, Asn-38, and Asn-83) and 1 O- glycosylation site (Ser-126) and involved in the regulation of the level of red blood cells. Today, only one recombinant human Epo (rHuEpo), produced in CHO cell line, is extensively used in therapy to cure severe anemia. The structure of the glycan chains of this rHuEpo slightly differ of those of the urinary human Epo (uHuEpo), considered as the natural Epo molecule. In an attempt to produce a rHuEpo as close as possible to the uHuEpo, Epo gene was expressed in a human lymphoblastoid cell line, named RPMI 1788. In order to fully characterize the Epo-RPMI, structural characterizations of the protein skeleton as well as glycan chains were undergone. As expected, the amino acid sequence of the Epo-RPMI conformed to that of uHuEpo. Surprisingly, the structure of some N-glycan chains, as mainly determined by ESI-MS, revealed some unusual characteristics. Thus, 80% of N-glycans possess a bisecting GlcNAc residue, 25% bear a second fucose residue which is present, in a large part, in a sialyl Le(x)motif, and 13% contain more than three LacNAc repeats (up to five per molecule). Despite these unusual structural characteristics, the data concerning the in vitro and in vivo biological activities were not impaired when compared to Epo-CHO and uHuEpo.

The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore.

Viral protein R (Vpr) encoded by HIV-1 is a facultative inducer of apoptosis. When added to intact cells or purified mitochondria, micromolar and submicromolar doses of synthetic Vpr cause a rapid dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), as well as the mitochondrial release of apoptogenic proteins such as cytochrome c or apoptosis inducing factor. The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr. Both mitochondrial and cytotoxic Vpr effects are prevented by Bcl-2, an inhibitor of the permeability transition pore complex (PTPC). Coincubation of purified organelles revealed that nuclear apoptosis is only induced by Vpr when mitochondria are present yet can be abolished by PTPC inhibitors. Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as the adenine nucleotide translocator (ANT) combined with Bax. Again, this effect is prevented by addition of recombinant Bcl-2. The Vpr COOH terminus binds purified ANT, as well as a molecular complex containing ANT and the voltage-dependent anion channel (VDAC), another PTPC component. Yeast strains lacking ANT or VDAC are less susceptible to Vpr-induced killing than control cells yet recover Vpr sensitivity when retransfected with yeast ANT or human VDAC. Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.

Determination of the sialylation level and of the ratio alpha-(2-->3)/alpha-(2-->6) sialyl linkages of N-glycans by methylation and GC/MS analysis.

A methodology for the determination of the sialylation pattern of N-glycans, extent of sialylation and the ratio between alpha-(2-->3) and alpha-(2-->6) sialyl linkages, is presented based on the labelling of the C-3 and C-6 hydroxyl groups of Gal residues obtained after permethylation, saponification, selective desialylation of sialylated oligosaccharides and methanolysis. Deuteromethylation and GC/MS analysis of Gal derivatives allow to determine the sialylation level of glycans. O-Ethyl ether labelling followed by GC analysis of the resulting Gal derivatives allows to obtain the ratio between alpha-(2-->3) and alpha-(2-->6) sialyl linkages. The method was applied to LNT (LcOse4: beta-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->3)-beta-D- Galp-(1-->4)-D-Glcp), LSTa (IV3NeuAcLcOse4: alpha-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D- GlcpNac-(1-->3-beta-D-Galp-(1-->4)-D-Glcp), LSTc (IV6NeuAcn LcOse4: alpha-Neup5Ac-(2-->6)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)- beta-D-Galp-(1-->4)-D-Glcp) and a bisialylated biantennary N-glycan in which sialic acid is bound to Gal residues via an alpha-(2-->6) linkage. Using this method, it was found that 92.8% of N-glycans in bovine fetuin is sialylated and that the ratio of alpha-(2-->6) versus alpha-(2-->3) sialyl linkages was 31:19.

Inhibition of HIV infection by pseudopeptides blocking viral envelope glycoprotein-mediated membrane fusion and cell death.

The RP dipeptide motif is highly conserved in the third hypervariable region (V3 loop) of the extracellular envelope glycoprotein of different types of HIV isolates. In view of this, we have designed and synthesized a construction referred to as "template assembled synthetic peptide" (TASP), in which a lysine-rich short polypeptide was used as a template to covalently anchor arrays of tripeptides, such as RPR, RPK, or KPR. The pentavalent presentation, 5(RPR)-, 5(RPK)-, or 5(KPR)-TASP, molecules manifested maximum inhibitory activity on HIV infection with a 50% inhibitory concentration value of 1-5 microM, respectively. Structure and inhibitory-activity relationship studies using analogs of 5(KPR)-TASP indicated that the positively charged side chains of the K and R residues in the tripeptide molecules are critical for the optimal inhibitory activity of the pentavalent construct. Interestingly, replacement of L-amino acid residues by D-amino acids or reduction of the peptide bond between the first two amino acids of the tripeptide generated peptide-TASP analogs active at sub-microM, concentrations. The anti-HIV action of the peptide-TASP constructs is specific, since they inhibit infection of several types of CD4-expressing cells by HIV-1 Lai and HIV-2 EHO but not by the simian SIV-mac isolate. Our results suggest that these inhibitors block three post-CD4 binding functions of the HIV envelope glycoproteins, mediation of viral entry, syncytium formation, and triggering cell death by apoptosis. As the peptide-TASP derivatives with unnatural amino acid sequences in the tripeptide moiety retain full inhibitory activity, they should provide potent protease-resistant peptide inhibitors as potential therapeutic agents for treatment of AIDS patients.