The aim of this work was to evaluate transplacental transmission of hepatitis C virus (HCV) in HIV-negative pregnant women who were HCV-PCR-positive, and also to determine the serotypes of the virus in these cases. Therefore, 767 pregnant women were screened for anti-HCV antibodies, hepatitis B surface antigen (HBsAg) and anti-HIV antibodies. HCV PCR was performed for HCV-positive women. Those who were PCR-positive were tested for anti-HCV IgM. Neonates of PCR-positive mothers were tested for virus transmission by the PCR test. Virus serotyping was done for mothers and neonates. Anti-HCV antibodies were detected in 105 out of 767 (13.7%) pregnant women. PCR was positive in 18 out of 67 HCV-positive women (26.9%). Transplacental transmission occurred in 11.1% of HIV-negative pregnant women. HCV type 4 predominates in Egypt (83.3%). Mothers who are PCR-positive and have high aspartate aminotransferase and positive anti-HCV IgM are most likely to transmit HCV to their babies.
We have selected a human immunodeficiency virus type 1 (HIV1) using the technique of in vitro selection to generate variants that are resistant to didanosine and/or stavudine. After serial passages of the Lai strain of HIV1 in MT-2 cells in increased concentrations of didanosine-stavudine association, 2 novel mutations in reverse transcriptase at codon 57 (Asp->His) and at codon 98 (Ala-->Val) were observed. These mutations were associated with an 11.5-fold increase in the didanosine and a 4.5-fold increase in the stavudine 50% inhibitory concentration.
Thousands of cases of dengue fever (DF) and several cases of dengue haemorrhagic fever were recorded in French Guiana during the recent outbreak of dengue-2 virus (1991-1992) and in subsequent years. One case with clinical signs typical of classical DF with neurological complications is reported in this study. The neurological features (encephalitis) appeared during the acute phase, 2 days after the onset of fever. Dengue-2 virus was detected in both the cerebrospinal fluid and blood sample. This case was fatal. This first reported case of classical DF with encephalitis in French Guiana is a new demonstration of the potential neurovirulence of dengue viruses.
A herpes simplex virus type 1 (HSV1) productive, persistently infected murine macrophage-like culture was obtained by infection at low multiplicity (0.001). Persistent cultures were characterized by spontaneously ceasing to produce infectious viruses after initial HSV1 production. A similar pattern of virus production was observed in cultures obtained by reinfection of the persistent macrophages with the original virus. Nonetheless, in persistent and reinfected cultures (17/18) which did not produce infectious viruses, viral proteins were detected by immunoblot, although their molecular weight, number and pattern differed among the cultures.
In this study, we analysed by transmission electron microscopy (TEM), sequential details of morphological modifications that accompanied viral morphogenesis in the lymphoblastoid cell line (LCL) TOFE infected in vitro with hepatitis C virus (HCV). As previously reported, we observed virus-like particles (VLPs) in cytoplasmic vesicles mainly located in the perinuclear region of infected cells. In this area, the Golgi apparatus and the endoplasmic reticulum (ER) appeared hyperplastic, remarkably enriched in vesicles and lysosomal structures. Furthermore, only in this perinuclear region, cytopathic-effect(CPE)-like changes seemed to originate, consisting in enlarged cytoplasmic vacuoles filled with degenerative amorphous material containing VLPs. Finally, the complete filling-up of the cytoplasm with these degenerative vacuoles, in addition to cellular lysis displayed by some cells, appeared as the possible terminal pattern of the infectious process. Our data suggest that in vitro HCV-infected TOFE cells undergo typical CPE-like changes that may be connected with virus replication.
West African sheep appear to play a central role as virus hosts in the maintenance cycle of Crimean-Congo haemorrhagic fever (CCHF) virus in endemic areas and also because of their role as a principal host of the CCHF virus tick vector. In an effort to clarify CCHF epidemiological significance in sheep, we studied the biological and clinical aspects of sheep experimentally infected with CCHF virus. West African sheep breeds were infected either by intraperitoneal inoculation or by infestation with experimentally CCHF-virus-infected ticks (Hyalomma truncatum). A total of 17 sheep including controls as well as 5 lambs from their progeny were monitored. A moderate but constant fever was observed (39.7 degrees C +/- 0.3) which correlates with the viraemia. Virus was reisolated from blood samples taken from day 3 to day 9 postinfection (p.i.) at a mean titre of 3.3 log LD50/ml. The virus was detected for a period of time of 7 days in non-immune sheep and for less than 4 days in previously immunized sheep. In non-immune sheep, antibody detected by ELISA showed an IgM response on day 7 p.i., followed by an IgG response one day later. Five infected sheep, surveyed for liver and kidney biological markers, showed hepatic dysfunction with a moderate serum aspartate transferase rise to 210 U/l. Out of four sheep tested for blood markers, two showed an abnormal blood cell count, with marked neutrophilia of up to 63% lasting for two weeks. Infected pregnant ewes produced antibodies in their milk at a significant titre (1:1,000), and antibodies were recovered in the sera of nursing lambs from their first meal to 50 days after birth. These findings are discussed; they demonstrate that, in spite of a high turnover of local sheep herds (median age of 3 years) and long-term CCHF antibody persistence (> 3 years), sheep can be infected and efficiently transmit the virus at least once in a lifetime.
We report the structure and antigenicity of the third variable region (V3) of the HIV2 envelope glycoprotein by the use of linear and cyclic peptides. To this end, a peptide mimicking this region was synthesized and purified, both as an iodoacetamidated linear peptide and a disulphide-bridged cyclic peptide. The cross-reactivity of three monoclonal antibodies (mAbs) produced against the envelope glycoprotein gp140 with the linear and cyclic peptides was tested with ELISA. The results showed that the cyclic peptide is a better ligand for the 3 mAbs 125-F, 125-J and 125-K. The avidity of the mAb/peptide interaction was further analysed by determining the concentration of linear or cyclic peptide leading to 50% inhibition of mAb-peptide complex formation (K-0.5). The K-0.5 value of mAb 125-F, which displayed the best reactivity with gp140, was estimated to be 5 times higher for the linear (K-0.5 = 1.5 x 10(-6) M) than for the cyclic peptide (K-0.5 = 3 x 10(-7) M). This indicates a higher affinity of mAb 125-F for the cyclic peptide. mAb 125-J, which exhibited a lower avidity for the gp140 compared to mAb 125-F, had a similar affinity for the cyclic and the linear peptides (K-0.5 = 3 x 10(-7) M). mAb 125-K had the lowest reactivity with gp140 and its binding to adsorbed peptide could not be inhibited by the soluble linear or cyclic peptide used up to 10(-5) M. These results suggest that cyclic peptides may have a higher propensity for adopting a native-like structure for the peptide/antibody interaction. Nuclear magnetic resonance experiments at 25 degrees C in phosphate buffer pH 5.4, however, showed that neither peptide displayed a well-defined structure.
In this preliminary report, we provide evidence that the human B-lymphoblastoid cell line (LCL) CE, bone-marrow-derived, previously reported to be permissive to hepatitis C virus, is also permissive to HIV1 infection. HIV1 genomes were detectable in cell supernatants, virus RNA transcripts and proviral DNAs in cell extracts at different times postinfection. Therefore, we propose this LCL cell line as a tool for exploring the mutual intereactions of the two viruses in double-infected cells.
Many recent investigations have shown that both HBV and anti-HBs antibodies coexist in the same patient, and HBV can be found in individuals with anti-HBc antibodies in the absence of immunologically detectable HBsAg. In most cases, mutant forms of HBV affecting the region of the envelope gene coding for the group a determinant recognized by human antibodies have been found.The nature of the group a determinant was revisited with an ELISA involving dissociated, but not alkylated, envelope subunits. No antibody recognizing a continuous epitope of the major S envelope protein could be found in humans; the full activity of human anti-HBs antibodies appeared to be focused on the discontinuous group a determinant. The immunological human repertoire against the HBsAg group a determinant was analysed by competitive inhibition of three mouse monoclonal antibodies (mAbs) selected as recognizing three distinct specificities on the group a determinant. Antibodies to specificities #1 and 3 were found in 52/70 anti-HBs human sera and generally predominated over specificity #2, which was lacking in some sera. The heterogeneity of the group a determinant suggested by these data argue for the use of more than one type of anti-HBs mAb for seroprevention of recurrence after liver transplantation and HBs serodiagnosis. Provided all three types of mAbs characterized here recognize HBV variants with mutations in the a determinant and are virus-neutralizing, it may be helpful, after association of such mAbs, to use them for diagnosis and to devise new immunotherapeutic strategies to prevent emergence of HBsAg escape mutants.
We previously demonstrated that the human lymphoblastoid B cell line (LCL) TOFE, derived from normal human bone marrow, is permissive to HCV infection. In this report we developed an in vitro HCV adsorption-inhibition assay based on TOFE cells, to reveal the presence of neutralizing antibodies in sera from acutely infected patients.
Genetic variation in glycoprotein B (gB) may play a role in human cytomegalovirus (HCMV) pathogenesis. Using restriction endonuclease digestion and DNA sequencing, a unique gB genotype was identified in eight HCMV strains isolated from five patients with the acquired immune deficiency syndrome. Nucleic acid homology to the four previously described gB genotypes ranged from 79 to 91% for the two major variable regions of gB. Studies of the role of gB in HCMV pathogenesis should recognize the existence of live gB genotypes.
Sandfly fever, a vector-borne disease endemic in the Mediterranean region, is caused by Toscana virus (TOS). The disease is increasingly important as a travel-related infection. Serological diagnosis is currently dependent on viral antigens derived from TOS-infected cell cultures. In this study, we report the cloning and expression of the TOS nucleoprotein (N) in Escherichia coli and evaluation of the recombinant (r) TOS N protein as an antigen for immunoblot assays. The TOS N gene was amplified by reverse-transcriptase polymerase chain reaction and cloned into the bacterial expression vector pTrcHis-A. Sera with known TOS antibody status were used to evaluate the immunoblot assay. The expressed rTOS N protein was purified and used as antigen for immunoblots. By recombinant immunoblot, the TOS antibody status (IgM and/or IgG) of the test panel was correctly identified. No cross-reactivity was detected. The rTOS N protein is useful as an antigen for immunoblot assays, and will enable more laboratories to perform TOS antibody diagnosis.
Based on the analysis of HDV genomes from different areas of the world, three genotypes of HDV have been identified. Genotype I is the most prevalent and widespread. Genotype II is represented by two isolates from Japan and Taiwan. Genotype III has been found only in the Amazonian basin where it is associated with a history of severe disease, fulminant hepatitis with microvesicular steatosis (spongiocytosis). We report here the cloning and the analysis of the complete viral genome from woodchuck serumderived HDV RNA after transmission from Central African Republic (RCA) patients with fulminant spongiocytic delta hepatitis. Two overlapping cDNA fragments, covering the entire HDV genome, were generated by RT-PCR and cloned. Three clones obtained from each fragment were fully sequenced. A complete consensus RCA HDV genome was reconstituted. The individual and the consensus nucleotide sequences were compared with those of 16 other fully sequenced isolates belonging to the three genotypes. Phylogenetic trees generated by the neighbour joining method firmly place our isolate in genotype I, and show that this RCA isolate differs significantly from the east African isolates previously analysed. Transfection experiments showed that the isolate is replication-competent, but less so than the control “wild-type” strain. Two novel mutations encountered in this work, one in the antigenomic ribozyme sequence and one affecting delta antigen, were studied.
We isolated three monoclonal antibodies (mAbs), H3e, H11b and H16h, which were capable of inhibiting syncytium formation induced in a human T-cell line MOLT-4 or a human glioma line U251 MG by coculture with human T-cell leukaemia virus type I (HTLV-I)-positive human T-cell lines. The mAbs partially inhibited the plating of pseudotypes of vesicular stomatitis virus (VSV) bearing envelope antigens of HTLV-I. Formation of proviral DNA was also inhibited when indicator cells were treated with the mAbs before adsorption of HTLV-I, but not after its adsorption. They did not inhibit syncytium formation induced by human immunodeficiency virus type 1. Flow cytometry revealed that H16h hardly reacted with various HTLV-I-positive T cells, while H3e and H11b reacted with HTLV-I-positive human cells as well as HTLV-I-negative human cells. H11b and H16h immunoprecipitated the membrane antigen with a molecular weight of 20 and 110-130 kDa, respectively. Western blot analysis showed that H3e, H11b and H16h bound to the protein of 20, 20 and 110-130 kDa, respectively. Thus, these findings suggest that the 20- and 110-130-kDa cell surface proteins may play a role at the early stage of HTLV-I infection.
The purpose of our investigation was to ascertain the presence of viral replication in subjects positive for antibodies to hepatitis C virus (anti-HCV) with persistently normal values of liver tests, to define their natural history, and to determine whether the immunoglobulin Nt (IgM) response could be a useful parameter to distinguish viraemic from non-viraemic patients.Twenty-seven subjects were selected based on their anti-HCV positivity and sustained normality (for at least 18 months) of alanine-aminotransferase (ALT) values. They were enrolled into the study and observed for another 1-4 years (mean 2.6). Fifteen out of 27 subjects were positive for hepatitis C virus ribonucleic acid (HCV RNA) and 12/15 were also positive for IgM. The remaining 12/27 patients were negative for both assessments. ALT levels remained in the norm throughout the investigation for all 27 subjects studied; the 15 viraemic patients showed persistent positivity for HCV RNA and the 12 positive for IgM anti-core also maintained their positivity. Patients shown to be negative for HCV RNA and IgM sustained their negativity throughout the study. Our results indicate that some patients remain viraemic while not having and/or developing clinical and biochemical signs of liver damage. IgM anti-HCV seems to be a specific index of viraemia in HCV-positives and could be useful for monitoring these patients.
Hepatitis G virus (HGV) is a recently described hepatotropic parenterally transmitted flavivirus. The presence of HGV was tested in 61 patients with a request for confirmation of HCV active infection. Thirty-two patients were in haemodialysis and 29 were referred from wards other than nephrology. Active HCV and HGV infections were determined by detection of their viral RNA in serum. Evaluation of previous HGV infection was carried out by detection of antibodies to E2 antigen. HCV prevalence was 62.29% (38/61). HGV-active infection was found in 11.47% (7/61) of the population studied: in 18.7% (6/32) of the haemodialysed patients and in 3.4% (1/29) of patients belonging to the other group. HGV prevalence increased two-fold when previous infection was also considered. HGV clearance was prospectively detected in 5 out of the 7 patients with active infection, and at an earlier stage for those patients coinfected with HCV. Anti-E2 seropositivity was associated with HGV clearance in only two patients.
Recombinant Mengo viruses expressing heterologous genes have proven to be safe and immunogenic in both mice and primates, and to be able to induce both humoral and cellular immune responses (Altmeyer et al., 1995, 1996). Several recombinant Mengo viruses expressing either a large region (aa 65-206) of the HIV1 nef gene product, or cytotoxic T lymphocyte (CTL) epitopic regions from the SIV Gag (aa 182-190), Nef (aa 155-178) and Pol (aa 587-601) gene products were engineered. The heterologous antigens were expressed either as fusion proteins with the Mengo virus leader (L) protein, or in cleaved form through autocatalytic cleavage by the foot-and-mouth disease virus 2A protein. Rhesus macaques and BALB/c mice inoculated with the Mengo virus SIV recombinants failed to develop CTL responses against the SIV gene products, while one of the HIV-Nef recombinants induced a weak CTL response in mice directed to an HIV1 Nef peptide spanning positions 182-198. In contrast, BALB/c mice immunized with vaccinia virus recombinants expressing HIV1 Nef developed a strong CTL response to the 182-198 peptide and also responded to a second peptide spanning positions 73-81. These results indicate that Mengo virus recombinants expressing HIV1 Nef and SIV CTL epitopes are weak immunogens. One of the fusion recombinants expressing SIV CTL epitopes failed to infect macaques even when used at high doses, while the recombinant expressing HIV1 Nef as a fusion protein failed to infect BALB/c mice. These results demonstrate that the expression of certain heterologous sequences as fusion proteins with L can result in the loss of the ability of the recombinant to infect normally susceptible animals.
The capacity of Moloney murine leukaemia virus (MoMLV) to infect neonatal hepatocytes and to accelerate liver carcinogenesis was examined in a transgenic mouse model. WHV/c-myc mice which are highly susceptible to the development of liver tumours were infected with MoMLV shortly after birth, when expression of the murine ecotropic retroviral receptor gene was still detectable in the neonatal liver. All MoMLV-infected transgenic mice and non-transgenic littermates succumbed to T-cell lymphomas within 2-9 months; during this period of time, three infected transgenic animals developed primary hepatocellular carcinomas. Remarkably, one of these liver tumours arose significantly faster than tumours from uninfected WHV/c-myc controls, and it harboured a unique MoMLV provirus. The provirus integration site was located 5.5 kb upstream of the first exon of the syndecan-4 gene, which encodes a heparan sulphate proteoglycan implicated in growth factor activation and protein kinase C distribution in focal adhesions. Our data provide evidence for clonal MoMLV provirus integration in a hepatocellular carcinoma, and indicate that parenchymal liver cells may be susceptible to MoMLV infection following neonatal inoculation.
