Analysis of the 5'UTR of HCV genotype 3 grown in vitro in human B cells, T cells, and macrophages.

BACKGROUND: Previously, we have reported the isolation and molecular characterization of human Hepatitis C virus genotype 1 (HCV-1) from infected patients. We are now reporting an analysis of HCV obtained from patients infected with HCV genotype 3 (HCV-3) as diagnosed by clinical laboratories. RESULTS: HCV was cultured in vitro using our system. HCV RNA was isolated from patients' blood and from HCV cultured in various cell types for up to three months. The 5'UTR of these isolates were used for comparisons. Results revealed a number of sequence changes as compared to the serum RNA. The HCV RNA produced efficiently by infected macrophages, B-cells, and T-cells had sequences similar to HCV-1, which suggests that selection of the variants was performed at the level of macrophages. Virus with sequences similar to HCV-1 replicated better in macrophages than HCV having a 5'UTR similar to HCV-3. CONCLUSIONS: Although HCV-3 replicates in cell types such as B-cells, T-cells, and macrophages, it may require a different primary cell type for the same purpose. Therefore, in our opinion, HCV-3 does not replicate efficiently in macrophages, and patients infected with HCV-3 may contain a population of HCV-1 in their blood.

The simultaneous presence and expression of human hepatitis C virus (HCV), human herpesvirus-6 (HHV-6), and human immunodeficiency virus-1 (HIV-1) in a single human T-cell.

We have developed a system that isolates and replicates HCV in vitro. These isolates are called CIMM-HCV. This system has made it possible to analyze the biology, nature, and extent of HCV variability, among other things. Individuals that are infected with HIV-1 are often also infected with HCV and HHV-6. In addition to HCV, our lab has systems for replicating HIV-1 and HHV-6. We asked whether all these viruses could infect the same cells. We report here the successful infection of a T-cell (CEM) by CIMM-HCV, HHV-6, and HIV-1. PCR analyses demonstrated that the CEM cells were productively infected by HHV-6A. RT-PCR showed that the same cell culture was positive for HCV and HIV-1. Co-infection of a T-cell by all three viruses was confirmed by transmission electron microscopy (TEM). All these viruses are highly cytolytic; therefore, triply-infected cells were short lived. However, HIV-1 and HCV co-infected cells unexpectedly lasted for several weeks. Viral replication was unhindered and the phenomenon of 'dominance' was not observed in our experiments. In addition, CIMM-HCV was present in the perinuclear space, suggesting their possible synthesis in the nucleus. This report is based entirely on viruses produced in vitro in our laboratories. As part of the determinations of host ranges of these viruses, studies were designed to demonstrate the infection of a single cell by these viruses and to study the consequences of this phenomenon. All measurements were made on cultured cells and cell culture supernatants.

Transmission of human hepatitis C virus from patients in secondary cells for long term culture.

Infection by human hepatitis C virus (HCV) is the principal cause of post-transfusion hepatitis and chronic liver diseases worldwide. A reliable in vitro culture system for the isolation and analysis of this virus is not currently available, and, as a consequence, HCV pathogenesis is poorly understood. We report here the first robust in vitro system for the isolation and propagation of HCV from infected donor blood. This system involves infecting freshly prepared macrophages with HCV and then transmission of macrophage-adapted virus into freshly immortalized B-cells from human fetal cord blood. Using this system, newly isolated HCV have been replicated in vitro in continuous cultures for over 130 weeks. These isolates were also transmitted by cell-free methods into different cell types, including B-cells, T-cells and neuronal precursor cells. These secondarily infected cells also produced in vitro transmissible infectious virus. Replication of HCV-RNA was validated by RT-PCR analysis and by in situ hybridization. Although nucleic acid sequencing of the HCV isolate reported here indicates that the isolate is probably of type 1a, other HCV types have also been isolated using this system. Western blot analysis shows the synthesis of major HCV structural proteins. We present here, for the first time, a method for productively growing HCV in vitro for prolonged periods of time. This method allows studies related to understanding the replication process, viral pathogenesis, and the development of anti-HCV drugs and vaccines.