A tale of two strands: Reverse‐transcriptase polymerase chain reaction detection of hepatitis C virus replication

‘‘It was the best of times, it was the worst of times . ’’ This phrase from Charles Dickens aptly describes the impact of the polymerase chain reaction (PCR) on hepatitis C research. Clearly, the combination of reverse-transcriptase polymerase chain reaction (RT-PCR) and PCR amplification of viral complementary DNA (cDNA) has had a pivotal role in the identification of the virus responsible for the majority of non-A non-B hepatitis cases. However, although it has allowed the molecular characterization of this virus, the unique sensitivity of RT-PCR has also created a number of problems. For example, a cell culture system for the propagation of hepatitis C virus (HCV) would be of great benefit. However, formal proof of replication in such systems has not been easy to obtain. The use of ‘‘strand-specific’’ RT-PCR to detect the replicative intermediates (minus-strand RNA) of HCV has been taken as evidence for viral replication. However, recent developments have shown that strandspecific RT-PCR is fraught with problems, and many early studies claiming low-level viral replication based on the detection of minus-strand RNA may be flawed. Here, we review the steps that must be taken if RT-PCR detection of minus-strand RNA is to be used to indicate the replication of virus in cell cultures or to identify those cells that may be productively infected in vivo. The identification of HCV was a triumph for modern molecular biology techniques,1 and its role as an agent in serious liver disease is now established beyond reasonable doubt.2 Another putative hepatitis virus has been identified recently, GBV-C or ‘‘hepatitis G virus’’ (GBV-C/HGV). This virus has distinct similarities to HCV.3,4 Sequence analysis shows that both are related to the flaviviruses, indicating that they are positive-strand RNA viruses with a genome containing a single open reading frame encoding a single polyprotein.5 This large polyprotein is cleaved to several structural and nonstructural proteins by cellular and viral proteases during replication of the virus. The nonstructural proteins include a protein (NS5B) that has the characteristics of an RNA-dependent RNA polymerase.6 Progress with HCV research has now reached such a stage that several well-defined molecular clones of the viral genome have been constructed. Messenger RNAs made from these clones can generate an infection when inoculated directly into the liver of chimpanzees.7,8 Other positive-strand RNA viruses, including the flaviviruses, have been shown to undergo replication via a negative sense intermediate.9 The similarity of hepatitis C with these viruses makes it difficult to envisage any other mechanism for replication of HCV: Thus, the demonstration of a negative-sense molecule would be indicative of viral replication. Because most of the information concerning the use of strand-specific RT-PCR detection of viral replication concerns HCV, the following discussion will concentrate on that virus. How these results pertain to GBV-C/HGV will be discussed at the end of this article. The very low levels of HCV found in infected individuals and the presumed low-level replication of the virus in hosts and tissue culture mean that RT-PCR is often the only suitable method for detecting viral RNA. However, the potential for contamination of the reaction mix and the strand-specificity of the reaction are issues that require careful consideration. Contamination is always a concern with RT-PCR techniques. However, there are several excellent articles on how these concerns can be minimized.10 Early work invariably used nested RT-PCR (in which the products of a first round of PCR were reamplified using a second, nested set of primers). Although this method gave extreme sensitivity and increased specificity, it was fraught with difficulties. After a single cycle of amplification, even a weak positive frequently contained many picograms of product. The opening of this reaction tube, to add reagents for a second cycle of amplification, could easily lead to contamination of other samples. We realize that some investigators have solved the problem of nested PCR contamination, but this is no trivial matter, and systems are now available that can readily detect 200 copies/mL of target RNA in a single round of PCR amplification. Furthermore the reverse-transcription and PCR steps may be done in a single tube with no requirements for opening the tube between these steps.11 This makes control of contamination a much simpler matter. We firmly believe that this level of sensitivity should be sufficient for most, perhaps all applications. There may still be rare instances, although we stress that they will be rare, when use of a nested reaction will be required. However, this should only be done on samples that have already been shown to be negative in a single-round RT-PCR reaction, and the transfer of reagents must be done in a room that is different from the room used to prepare the reaction mix for first-round RT-PCR. An alternative to nesting is to perform a Southern blot on the product. This will increase the sensitivity as well as show specificity without the risk of cross-contamination. Early attempts to detect negative-strand RNA in vivo and in vitro used a primer specific for the negative strand during the initial reverse-transcriptase step. The reverse transcriptase was then inactivated, and the synthesized DNA was amplified by PCR. Using this technique, negative-strand RNA was found in many tissues and in vitro infected cells. However, Abbreviations: PCR, polymerase chain reaction; RT-PCR, reverse-transcriptase polymerase chain reaction; cDNA, complementary DNA; HCV, hepatitis C virus; PBMC, peripheral blood mononuclear cell. From the Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, TX, and Glaxo Wellcome Research and Development, Medicines Research Center, Hertfordshire, UK. Received June 15, 1998; accepted August 14, 1998. Address reprint requests to: David V. Sangar, Ph.D., Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, TX 775551019. Fax: (409) 772-3757; e-mail: dvsangar@utmb.edu. Copyright r 1998 by the American Association for the Study of Liver Diseases. 0270-9139/98/2805-0001$3.00/0