Detection of the negative-strand hepatitis C virus RNA in tissues: implications for pathogenesis
Introduction
The hepatitis C virus (HCV) is an enveloped virus belonging to the Flaviviridae family. The virus genome is a linear, single-stranded RNA of ∼9600 nucleotides, which contains a single open reading frame encoding a polyprotein precursor of about 3000 aminoacids (Major and Feinstone, 1997). Since its genome functions as a messenger RNA for its polyprotein translation, it is also referred to as the positive-strand RNA. The replication of HCV RNA is believed to occur via its transcription into a complementary, genomic-length RNA, the so-called negative-strand HCV RNA. This is based on the comparison with the replication of other members of the Flaviviridae family that can be efficiently cultivated in vitro, thus allowing the characterization of the different steps of the viral replication (Chu and Westaway, 1985, Westaway, 1987, Gong et al., 1996). According to the model proposed for Flavi- (Chu and Westaway, 1985) and Pestivirus (Gong et al., 1996) replication, the viral genome is first transcribed into its negative-strand RNA. The double-stranded ‘replicative form’ (RF) RNA, consisting of the fully base-paired genomic- and negative-strand viral RNA's, functions as a recycling template for a semiconservative replication, where the progeny genomic-strand RNA is continuously transcribed and released for being incorporated in virions. Each nascent genomic-strand RNA molecule displace the previously synthesized genomic RNA in the 5′-3′ direction, and each RF may direct the synthesis of one or more progeny genomes at the same time (Gong et al., 1996). The complex of RF and its associated nascent genomic-strand RNA(s) is referred to as replication intermediate.
The steps of the HCV replication have not been defined. This is mostly due to the fact the development of in vitro replication systems for propagation of HCV has been difficult, with reliable systems for efficient long term virus replication being poorly reproducible (Shimizu et al., 1992, Fournier et al., 1998). Thus, the HCV replication has been so far studied essentially in in vivo models, i.e. the experimentally infected chimpanzee and the naturally infected humans. These studies have to cope with the very low level of replication of HCV (Major and Feinstone, 1997), hence the need to detect its RNA by amplification-based procedures. To achieve so, both the reverse transcription-polymerase chain reaction (RT-PCR) and signal amplification procedures, such as the branched DNA assay (Detmer et al., 1996), have been used.
The scope of the present paper is to discuss some technical aspects of the specific detection of the negative-strand HCV RNA by RT-PCR as well as our personal experience deriving from the application of this procedure to the study of HCV pathogenesis and cell tropism. Several review articles can be found in the literature on technical and virological aspects of the strand-specific detection of HCV RNA (Lanford and Chavez, 1998, Sangar and Carroll, 1998).
Section snippets
The technical issue of strand-specificity
Some authors have reported on the detection of intrahepatic HCV RNA of genomic polarity (Di Martino et al., 1997, Terrault et al., 1997). Although the ratio of liver to serum HCV RNA titers may be high (median 103, range 17.4–286) (Terrault et al., 1997), in some patients with high-level viremia the intrahepatic titer of genomic HCV RNA may be affected by the viral RNA of circulating virions, trapped in the liver tissue at the time of sampling. For this reason, we and others prefer to assess
Detection of negative-strand HCV RNA in chronic hepatitis C
One of the approaches used to study the pathogenesis of HCV-associated disease is to assess its replication in infected tissues and to establish anatomo-clinical correlations.
HCV RNA replication may be studied by in situ analysis techniques or by extraction-based procedures. The extraction-based techniques used to study the HCV replication in tissues include the Northern gel analysis and the strand-specific RT-PCR. Although extraction-based techniques do not allow to define the number and type
Negative-strand HCV RNA and recurrent hepatitis C after liver transplantation
We studied 23 patients who underwent an orthotopic liver transplantation (OLT) for end-stage, HCV-related cirrhosis (Negro et al., 1998). All of them had recurrent HCV infection early after OLT, since HCV RNA was detected in serum of all patients tested during the first week after OLT. Six patients, however, did not develop recurrent hepatitis: all six had an initial episode of rejection controlled by treatment but progressing to chronic rejection in two. Two further patients, among those
HCV and steatosis of the liver
A peculiar histopathological feature associated with chronic hepatitis C is the steatosis of the liver (Scheuer et al., 1992). The causes underlying the fat accumulation in chronic hepatitis C patients may encompass obesity, drugs, alcohol, diabetes and concomitant infections. However, even when all of these causes are carefully excluded, a significant proportion of patients persistently infected with HCV may still have a fatty liver. The proportion of patients infected with HCV who have a
Extrahepatic sites of HCV replication
In the context of the search for an extrahepatic reservoir of HCV replication, the strand-specific detection of the negative-strand HCV RNA has contributed to clarify which may be the major sites of HCV tropism. Peripheral blood mononuclear cells (PBMC) have been shown to harbor low-titer, HCV replication intermediate RNA (Lérat et al., 1996, Kao et al., 1997, Mellor et al., 1998, Bronowicki et al., 1998), and the HCV replication in these cells is further confirmed by the analysis of the viral
Acknowledgements
This work was supported by contract no. 32-52193.97 (to F.N.) from the Swiss National Science Foundation, a grant from the Fondation Cancer-Solidarité, Genève (to K.A.) and a Schering-Plough unrestricted educational grant. The authors thank Lelio Orci, Ferruccio Bonino and Jean-Michel Pawlotsky for advice and criticism and Joachim Brault and Colette Rossier for their skilled technical help.
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