A single nucleotide polymorphism in IL28B affects viral evolution of hepatitis C quasispecies after pegylated interferon and ribavirin therapy

Abstract

Interleukin-28B (IL28B) polymorphisms are associated with viral response to peginterferon and ribavirin (RBV) in chronic hepatitis C (HCV). Their recognition represents a breakthrough in the understanding of the role of the host in viral eradication. How these polymorphisms determine viral eradication is unknown. The IL-28B variants are hypothesized to have a differential impact on HCV quasispecies evolution during treatment with pegylated interferon (PEG-IFN) and RBV. In this study, HCV RNA levels were measured at early time points in 33 naïve genotype 1 hepatitis C patients and clonal analysis of the entire NS5A region was performed on sera from baseline and Day 7. Site rs12979860 polymorphisms were determined by direct sequencing of PCR products and classified into CC, CT, and TT and were identified in 13, 11, and 9 patients, respectively. The CC polymorphism more commonly was seen in Whites versus Blacks [12/21 (57%) vs. 1/12 (8%), P = 0.009] and HIV-infected versus mono-infected [13/25 (52%) vs. 0/8 (0%), P = 0.009]. Patients with CC and non-CC had similar baseline viral loads. More patients with the CC polymorphism had amino acid substitutions in NS5A compared to non-CC patients. Despite similar baseline viral diversity, by Day 7, significantly more patients with CC had higher non-synonymous substitution values compared to non-CC (P = 0.02). Chronic hepatitis C patients with the CC IL28B polymorphism have a higher number of amino acid substitutions in the NS5A region and early viral evolution due to greater interferon induced selective pressure during this critical period of treatment.

Figure 1

Figure. Evolutionary relationships of HCV NS5A consensus sequences from patients with CC, CT and TT polymorphism. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 1.03173567 is shown. The percentage of replicate trees in which the associated sequence clustered together in the bootstrap test (2000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Phylogenetic analyses were conducted in MEGA4. The red circle represent the HCV genotype 1a reference sequence (H77); the red square represent HCV 1b reference sequence (HCV-J); the black circle represent patients with CC polymorphism, grey circle represent patients with CT polymorphism and white circle represent patients with TT polymorphism.

Figure 2

Sequence diversity at baseline and day 7 in patients with CC and non-CC polymorphisms. Black box represents patients with CC and white box represent patients with non-CC. SGD, genetic distance within quasispecies; dS, synonymous substitutions, dN, non-synonymous substitutions.

Figure 3

Sequence diversity at baseline and day 7 in Black and White patients: Black box represents Black patients and white box represents White patients. SGD, genetic distance within quasispecies; dS, synonymous substitutions, dN, non-synonymous substitutions.

Figure 4

dN value in Whites with CC, Whites with non-CC and Blacks with CC polymorphisms, dN, non-synonymous substitutions.