A. Kirn

High hepatitis C viraemia and impaired antibody response in patients coinfected with HIV

ObjectiveTo compare hepatitis C virus (HCV) load in patients infected with HCV alone and those coinfected with HIV, and to evaluate the antibody response to HCV in the case of HIV infection. DesignPatients coinfected with both HCV and HIV have been shown to develop hepatic changes more rapidly, which may be due to an interaction between HCV and HIV. In a prospective study, serum samples were taken from 150 patients. MethodsUsing reverse transcription followed by polymerase chain reaction and the branched DNA assay, we detected HCV RNA in 75 patients coinfected with HIV and HCV and in 75 patients infected with HCV alone. The HIV RNA was also quantified by the branched DNA assay and the p24 antigenaemia was determined by enzyme-linked immunosorbent assay. The immune response to HCV was studied in the 150 patients by the use of third generation recombinant immunoblot assay (RIBA). ResultsAlthough a comparable number of patients had detectable HCV viraemia in both groups, HCV RNA was quantifiable in 79% of HIV-positive patients and in only 43% of HIV-negative patients (P < 10-5), and the mean HCV RNA level was much higher in the HIV-positive group than in the HIV-negative group (P < 10-7). The quantity of HCV RNA did not correlate with the CD4 count, p24 antigenaemia or HIV RNA level. The analysis of RIBA showed 14.7% indeterminate or negative results in the HIV-positive group and only 4% indeterminate results in the HIV-negative group. HIV-positive patients had reactivity to less antigen bands than HIV-negative patients (P < 10-3), and they had a weaker reactivity to c100, c33c and NS5 antigen bands than HIV-negative patients. ConclusionOur results show that in the case of HIV infection, the HCV RNA levels are strongly increased, but HCV load is not linked to the immunosuppression induced by HIV; therefore, the present data do not support the hypothesis of a direct interaction between HIV and HCV.

IN VITRO INFECTION OF PERIPHERAL BLOOD MONONUCLEAR CELLS BY HEPATITIS C VIRUS

To study the in vitro susceptibility of peripheral blood mononuclear cells (PBMC) to hepatitis C virus (HCV), we incubated cells from healthy donors with HCV-positive sera. Using RT-PCR and in situ hybridization, the genomic viral RNA was detected in PBMC and in their supernatants until 25 days post-incubation. The PBMC of the different donors were not all permissive to HCV, but results were more constantly positive when cells from four donors were pooled. Quantification of the genomic viral RNA by the branched-DNA assay showed a decrease in the HCV RNA concentration during the first week of culture followed by a peak during the second or third week, and also an increase in the total amount of viral RNA in the inoculated cells. Although HCV RNA could be detected in the supernatants by RT-PCR, the concentration was very low. Using a sense-specific RT-PCR method, the HCV negative-strand was also detected in the cells but not in the supernatants. In two experiments PBMC were successfully infected using HCV-positive culture supernatants, therefore suggesting that infectious particles can be produced in this system. Our findings demonstrate that PBMC are permissive for HCV replication in vitro but the replication level is very low. The HCV RNA concentration measured in PBMC of 10 chronically infected patients was not significantly higher than the maximal concentration obtained in PBMC infected in vitro.

Intracellular delivery of nucleoside monophosphates through a reductase-mediated activation process

On the basis of three different models (namely: ddU, AZT and PMEA), mononucleotide phosphotriester derivatives were designed to be able to liberate the corresponding monophosphate (or phosphonate) inside the cell through a reductase-mediated activation process. It was demonstrated that the use of bis[S-(2-hydroxyethylsulfidyl)-2-thioethyl] esters of ddUMP (11), AZTMP (12) and PMEA (17) resulted in intracellular delivery of the parent monophosphate (or phosphonate). This point was corroborated by observation of an anti-HIV effect of, 11 in various cell lines, for 12 in CEM TK- cells and by the enhanced activity observed for 17. Furthermore, the reported decomposition data in cell extracts fully confirm the validity of this approach and show unambiguously the potential for intracellular reductase-mediated activation of the starting drug.

Equal inhibition of the replication of human immunodeficiency virus in human T-cell culture by ddA BIS(SATE)phosphotriester and 3′-azido-2′,3′-dideoxythymidine

It is shown that ddA bis(SATE)phosphotriester is one of the most potent anti-HIV agents in cell culture. Compared with the parent nucleoside, ddA, an increase of 3 orders of magnitude was observed in the EC50, which makes this compound as active as AZT. This can be tentatively explained if one considers that direct ddAMP intracellular delivery shunts the well established ddA/ddI metabolism pathway.

Bicyclic imidazo derivatives, a new class of highly selective inhibitors for the human immunodeficiency virus type 1.

In the search for new antiviral agents against human immunodeficiency virus, different members of two imidazoheterocycle families (imidazothiazoles, imidazopyridines) have been found to display potent inhibitory effects on the replication of HIV-1. Three of these derivatives, which show significant anti-HIV-1 activity, have been chosen for further studies. The analysis of these compounds and their comparison to AZT and TIBO revealed that these bicyclic imidazo derivatives represent a class of highly specific inhibitors of HIV-1, but not of HIV-2 or simian immunodeficiency virus (SIV). Their inhibition of HIV-1 is mediated through interaction with the reverse transcriptase (RT). The mechanism of action of these bicyclic imidazo derivatives may be similar to that of the other non-nucleoside RT inhibitors (NNRTIs).

Host range deletion mutant of vaccinia virus defective in human cells

Abstract A vaccinia virus host range (hr) mutant unable to multiply in most human cell lines assayed has been isolated after nitrous acid mutagenesis. This mutant also displayed various alterations in plaque morphology and cytopathic effect on permissive cell lines. The block in multiplication in human cells was at an early stage of infection. Only early cytoplasmic RNA and early viral-induced polypeptides could be detected and there was no evidence of morphological events within viroplasms. Protein synthesis constantly declined as infection proceeded suggesting either that early viral mRNA was unstable or that the mutant virus was defective in a step necessary for the maintenance of translation. Restriction enzyme digestion of DNA from the hr mutant revealed that it was deleted of about 12.6 × 10 6 daltons in the left-hand end of the genome leaving intact a fragment containing the terminal crosslink. In both permissive and nonpermissive cells the mutant failed to induce the synthesis of an early 42K polypeptide which could thus be encoded within a region of the deleted sequence. The failure to segregate the various phenotypic and biochemical properties of the hr mutant from one another through recombination with a temperature sensitive mutant indicated that the pleiotropic characteristics of the mutant virus were due to the deletion.

Differing Infection Patterns of Dengue and Yellow Fever Viruses in a Human Hepatoma Cell Line

Dengue (DEN) and yellow fever (YF) viruses are responsible for human diseases with symptoms ranging from mild fever to hepatitis and/or hemorrhages. Whereas DEN virus typically induces only limited foci of necrosis in the liver, YF virus infection is characterized by devastating lesions. In a human hepatoma cell line (HepG2), the kinetics of DEN and YF virus replication and release from the cells and the nature of host cell response to viral infection were compared. DEN virus infection was characterized by the early appearance of intracellular viral antigens, major ultrastructural cytopathic changes as early as 32 h after infection, extensive apoptotic cell death, and a low production of infectious particles. In contrast, YF virus grew exponentially to high titers and induced cytopathic changes only 72 h after infection. Differences between the infection processes of the two viruses observed in the hepatoma cell line may explain the different liver pathologies.

Effects of corticosteroids on HCV infection.

The risk factors for clinical recurrent hepatitis C in liver transplant recipients are not clearly defined. It has been suggested that the corticosteroids included in the treatments of patients undergoing allograft rejection might induce acute hepatitis by increasing HCV replication. In this study we investigated the effects of corticosteroid boluses on HCV viremia in liver allograft recipients treated for acute rejection. Since we had previously developed a model of HCV replication in peripheral blood mononuclear cells (PBMC) in vitro, we also studied the effects of corticosteroids on HCV replication in vitro. A transient peak of HCV viremia was observed in patients treated with corticosteroid boluses for an acute allograft rejection. In the cell cultures, corticosteroids induced an increase of the total amount of viral RNA detectable. Our results demonstrate that corticosteroids induce an increase of hepatitis C virus replication in vivo and in vitro.

Human immunodeficiency virus type 1 infection of human CD4-transgenic rabbits

Investigations of human immunodeficiency virus (HIV) infection of man have benefited from the study of relevant animal models of the infection and disease. However, the ultimate models use primate species which are either endangered, not generally available, or expensive to maintain. A transgenic rabbit specifically and stably expressing human CD4 protein on T lymphocytes was assessed as a new laboratory animal model for HIV-1 infection. In vitro studies demonstrate that lymphocytes derived from the transgenic rabbits are more susceptible to HIV-1IIIB infection than those from normal rabbits. In vivo infection of huCD4-transgenic rabbits using HIV-1IIIB-infected autologous lymphocytes was demonstrated by virus isolation, detection of HIV-1-specific DNA in peripheral blood lymphocytes and seroconversion to various HIV-1 proteins. Viral DNA was detected in the tissues of one rabbit sacrificed 7 weeks post-infection and virus was isolated from lymph node. Although these transgenic rabbits are less sensitive to HIV-1 infection than man, such a small and inexpensive animal model may be a useful tool.

Increase of the anti-HIV activity of D4T in human T-cell culture by the use of the sate pronucleotide approach

Abstract The bis( S -acetyl-2-thioethyl) phosphotriester derivative of 2′,3′-didehydro-2′,3′-dideoxythymidine has been synthesized and evaluated for its inhibitory effects on the replication of HIV-1 in several cell culture systems. This pronucleotide showed potent anti-HIV activity and proved to be significantly superior to the parent nucleoside with regard to the antiviral efficiency.