Analysis of the Complexity of a Biological Clone from a Diverse HCV Population

Hepatitis C virus (HCV) infects the liver and affects over 180 million people worldwide. Approximately 25% of patients infected by HCV overcome the infection, but the remaining 75% evolve towards chronicity. Chronic infection will cause damage to the liver and can lead to cirrhosis and a higher risk of developing hepatocarcinoma. HCV is an enveloped RNA virus with a single stranded RNA of positive polarity as its genome. The mutation rate of the virus classifies it as a quasispecies, a model that describes a cloud of viruses with related genomes. Researchers experience difficulty when finding treatments since it is so challenging to isolate a single strain suitable to represent the whole; the high genetic variability of HCV has made it nearly impossible to prepare a preventive vaccine. The main goal of this study was to estimate the mutant spectrum complexity of a biological clone isolated from a multiply-passaged HCV population. The general objective of the laboratory is to distinguish viral features that depend on mutant spectrum complexity from those that depend on passage history. To carry out the project, an RNA clone was isolated from a virus that was previously passaged 200 times in human hepatoma cells in culture. Reverse transcription was used with primers designed to target a specific protein (NS5A) in the HCV genome in order to create the complementary DNA, which was then ligated into a plasmid vector. The vector was transformed into viable DH5α bacteria, and colonies that harbored the relevant viral sequences were analyzed. Templiphi protocols were used to amplify the viral DNA within each molecular clone, and the amplified DNA was then sent to Macrogen for sequencing. The final sequences were assembled using Lasergene software. Previous studies have given two possibilities for the complexity of the mutant spectrum: it can either resemble the population it was cloned from or it can resemble the original unpassaged virus. Results indicate the latter is occurring; both the minimum and maximum mutation frequencies for the molecular clones more closely resemble the original unpassaged HCV population than the passage 200 population. Furthermore, while the estimated average number of point mutations per genome within the biological clone of passage 200 and a biological clone of the original unpassaged population is similar (3.3 and 5.5, respectively), the number of insertions and deletions is fourfold higher (3.3 and 0.82, respectively). These results, together with biological assays currently ongoing in the laboratory, represent a relevant contribution to distinguish HCV properties that may depend on mutant spectrum complexity and extended replication.