Popular Science: How can we analyse Hepatitis C virus variants?
The Hepatitis C virus (HCV) infects mainly liver cells and can cause serious liver disease. Chronic HCV infection has a long asymptomatic initial phase that can ultimately result in liver cancer.
HCV has a single-molecule RNA genome. Based on differences in the RNA sequence, scientists distinguish seven HCV genotypes and more than 70 subtypes. Variants of viral RNA emerge during replication of the virus and synthesis of new RNA. Sometimes, mistakes are made when genomes are copied, resulting in synthesis of viral particles with differences in their RNA. Even in a single patient we can find a mixture of HCV variants.
New variants of viral RNA emerge at a high rate and can affect viral properties, such as its ability to cause disease or resist medication. This presents a serious problem in the treatment of Hepatitis C. In the past few years, new drugs to treat HCV have been developed. However, new viral variants able to resist them appeared almost immediately.
Scientists from Charles University decided to study the differences in the IRES sequence in the HCV genome. The IRES sequence is a part of viral RNA necessary for synthesising viral proteins and replicating viral RNA. This regulatory sequence therefore plays an important role in the viral replication cycle.
Over the past few years, several studies have investigated the possible correlation between mutations in HCV IRES and patient response to antiviral therapy. The results obtained were contradictory, however. The team of scientists therefore decided to once again analyse the previously published data. They conducted a bioinformatics analysis, which showed no association between HCV IRES mutations and therapeutic responsiveness.
The researchers from Charles University then stopped examining specific mutations in IRES sequences. Instead, they wanted to understand how to assess the properties of IRES sequences present in a patient. This led to the research team developing a new method that enabled the activity of different IRES sequences to be compared.
The authors of the study isolated RNA from the blood samples of three HCV infected patients and prepared a library of IRES sequences. The scientists used the fact that the IRES sequence controls the synthesis of viral proteins. They inserted IRES sequences isolated from patients into DNA vectors (called plasmids) containing a gene that codes fluorescent protein EGFP. The synthesis of fluorescent protein therefore became controlled by IRES sequences.
A method called transfection was used to insert prepared DNA constructs into cells. The scientists then observed the synthesis of fluorescent protein EGFP using flow cytometry, which is a method enabling fluorescence in individual cells to be measured. The activity of IRES sequences based on the efficiency of EGFP synthesis could therefore be compared.
The analysis revealed that there are indeed differences in IRES activity in patients. For example, the scientists observed that IRES activity was lower in patients who underwent several rounds of unsuccessful treatment than in patients who had not yet begun treatment. One possible explanation is that unsuccessful treatment might develop a selective pressure that could result in higher fractions of less active viral variants. It is also possible that lower IRES activity could serve as a diagnostic tool for predicting patient responsiveness to treatment. However, more experiments must be conducted before any conclusions are drawn.
The authors thus developed a method allowing us to compare diversity in IRES sequences in a patient as well as the activity of individual IRES sequences. The researchers hope that an analysis of all the viral variants present in patients can help us better understand the disease.
Vopálenský V, Khawaja A, Rožnovský L, Mrázek J, Mašek T, et al. Characterization of Hepatitis C Virus IRES Quasispecies - From the Individual to the Pool. Front Microbiol 2018;9:731.