COMMENTARY Do Taq-generated RT-PCR products from RNA viruses accurately reflect viral genetic heterogeneity? B. Mullan, 1 P. Sheehy, 1,2 F. Shanahan 1 and L. Fanning 1 1 Hepatitis C Unit, Department of Medicine; and 2 Department of Microbiology, University College Cork, Cork, Ireland Received February 2003; accepted for publication March 2003 SUMMARY. Since the first report of genetically heterogene- ous, or quasispecies, populations of RNA viruses, the genetic heterogeneity of the RNA genomes of major viral pathogens has been extensively studied. These studies aim to provide 1 insights into the evolutionary pressures that act upon vir- uses, in order to define windows where anti-viral therapies will be most effective, to take prognostic values from viral genetic distributions at a given time, and to aid the devel- opment of novel therapeutic compounds that may tilt viral replication towards information loss. Many methodologies are employed to analyse genetic distributions of a virus in a given sample, but all involve the generation, and subsequent analysis, of the sequence infor- mation contained in a reverse-transcription-polymerase chain reaction (RT-PCR) product. Despite the fact that the aim of these RT-PCRs is to obtain sequence information from viral genomes, their application to this task is approached without adequate consideration of this end-goal. The estab- lishment of an RT-PCR for a specific viral target genome generally proceeds in the same fashion as one would apply to establishing a PCR to determine the presence or absence of a specific target sequence in a given sample. However, it is becoming increasingly apparent that RT-PCR products generated by amplification with the ubiquitous thermostable DNA polymerase Taq, coupled with standard cloning and sequencing methodologies, has the potential to yield inac- curate and misleading data as pertains to the information content of populations of RNA viral genomes. This review discusses varying approaches employed to analyse hetero- geneous populations of hepatitis C virus RNA genomes. Keywords: error rate, hepatitis C virus, mismatch, quasispe- cies, reverse-transcribed polymerase chain reaction. INTRODUCTION The concept of heterogeneous populations of RNA molecules was first proposed by Eigen and colleagues who defined the genetic organization of simple replicons at the population level. They proposed a model to explain the adaptability and rapid evolution of simple replicons that probably populated the earth at the onset of life [1,2]. As part of this theory, Eigen proposed the concept of a quasispecies, a group of self- replicating RNA molecules, which are different, but closely related to each other, and evolve as a single unit when adapting to changes in the environment [3]. RNA viral quasispecies were first reported for the phage Qb [2,4,5] and, similar to the proposal of Eigen, are defined as a master sequence with an associated mutant spectrum. The master sequence is the dominant nucleotide sequence in the viral genomic distribution. At the most general level, the number of individual viral variants in a given sample is termed the quasispecies complexity, and the genetic relatedness of these variants is termed the quasispecies diversity. Collectively, both the quasispecies complexity and diversity are com- monly termed the quasispecies distribution, population or spectrum. It is important when considering the properties endowed upon an RNA virus by its genome, that a given RNA viral isolate is not considered as a wild type or mutant type virus. An RNA virus is not a virus per se, but a rapidly evolving quasispecies population of RNA genomes, some of which have greater or lesser virulence. The quasispecies must be considered as a whole, rather than by its indi- vidual components, and the quasispecies acts as the unit of selection [2,6]. In RNA viral populations, the origin of quasispecies is due to an error-prone viral RNA polymerase, and the absence of an editing, or proofreading, function during polymerization. It is also a function of the virus generation time (number of replication events in a given time period) [7,8]. The basis for the error-prone nature of viral RNA Abbreviations: HCV, hepatitis C virus; VSV, vesicular stomatis virus. Correspondence: Brian Mullan, Laboratoire de Vectorologie et Transfert de Genes, CNRS UMR 8121/Aventis Pharma, Institut Gustave Roussy, 39, rue Camille Desmoulins, Villejuif cedex, F94805, France. E-mail: bmjmullan@yahoo.co.uk Journal of Viral Hepatitis, 2004, 11, 108–114 Ó 2004 Blackwell Publishing Ltd