Study of Quasispecies Complexity and Liver Damage Progression after Liver Transplantation in Hepatitis C Virus Infected Patients

Abstract

Cirrhosis derived from chronic hepatitis C virus (HCV) infection is still a common indication for liver transplantation (LT). Reinfection of the engrafted liver is universal in patients with detectable viral RNA at the time of transplant and causes fast progression of cirrhosis (within 5 years) in around one-third of these patients. To prevent damage to the liver graft, effective direct-acting antiviral (DAA) therapy is required as soon as possible. However, because of post-LT clinical instability, it is difficult to determine the optimal time to start DAAs with a low risk of complications. Evaluate changes in quasispecies complexity following LT and seek a predictive index of fast liver damage progression to determine the timing of DAA initiation. HCV genomes isolated from pre-LT and 15-day post-LT serum samples of ten patients, who underwent orthotopic LT, were quantified and sequenced using a next-generation sequencing platform. Sequence alignments, phylogenetic trees, quasispecies complexity measures, biostatistics analyses, adjusted R2 values, and analysis of variance (ANOVA) were carried out. Three different patterns of reinfection were observed (viral bottlenecking, conserved pre-LT population, and mixed populations), suggesting that bottlenecking or homogenization of the viral population is not a generalized effect after liver graft reinfection. None of the quasispecies complexity measures predicted the future degree of liver damage. Higher and more uniform viral load (VL) values were observed in all pre-LT samples, but values were more dispersed in post-LT samples. However, VL increased significantly from the pre-LT to 15-day post-LT samples in patients with advanced fibrosis at 1-year post-LT, suggesting that a VL increase on day 15 may be a predictor of fast liver fibrosis progression. HCV kinetics after LT differ between patients and are not fibrosis-dependent. Higher VL at day 15 post-LT versus pre-LT samples may predict fast liver fibrosis progression.

Keywords: complexity measures; fibrosis; hepatitis C virus; liver transplantation; variability; viral load.

Figure 1

Phylogenetic trees representing the liver quasispecies population before (pre-LT) and after (post-LT) liver transplantation. Haplotypes from the pre-LT quasispecies are represented in green, and haplotypes from the post-LT quasispecies are in orange, as examples of the three infection patterns found. (A) Patient 05; (B) Patient 08; (C) Patient 01. The nomenclature used was Pxx.y_z_vvvv, where xx is the patient identifier (P01, P02 etc), y is the sample identifier (0 for pre-LT, 1 for post-LT), z is the number of differences between the haplotype and the master sequence, and vvvv is an identifier for haplotypes with the same number of mutations in the quasispecies. The last percent number represents the frequency at which each haplotype is represented in the quasispecies.

Figure 1

Phylogenetic trees representing the liver quasispecies population before (pre-LT) and after (post-LT) liver transplantation. Haplotypes from the pre-LT quasispecies are represented in green, and haplotypes from the post-LT quasispecies are in orange, as examples of the three infection patterns found. (A) Patient 05; (B) Patient 08; (C) Patient 01. The nomenclature used was Pxx.y_z_vvvv, where xx is the patient identifier (P01, P02 etc), y is the sample identifier (0 for pre-LT, 1 for post-LT), z is the number of differences between the haplotype and the master sequence, and vvvv is an identifier for haplotypes with the same number of mutations in the quasispecies. The last percent number represents the frequency at which each haplotype is represented in the quasispecies.

Figure 1

Phylogenetic trees representing the liver quasispecies population before (pre-LT) and after (post-LT) liver transplantation. Haplotypes from the pre-LT quasispecies are represented in green, and haplotypes from the post-LT quasispecies are in orange, as examples of the three infection patterns found. (A) Patient 05; (B) Patient 08; (C) Patient 01. The nomenclature used was Pxx.y_z_vvvv, where xx is the patient identifier (P01, P02 etc), y is the sample identifier (0 for pre-LT, 1 for post-LT), z is the number of differences between the haplotype and the master sequence, and vvvv is an identifier for haplotypes with the same number of mutations in the quasispecies. The last percent number represents the frequency at which each haplotype is represented in the quasispecies.

Figure 2

Boxplots with diversity indices showing differences between the pre-LT and post-LT quasispecies. (A) D0: hill number of order 0, (B) D1: hill number of order 1, (C) D2: hill number of order 2, (D) Dinf: hill number of order infinity, (E) Mfmax: mutation frequency, (F) π: nucleotide diversity. Each patient is represented as *. The p-value resulting from the Mann-Whitney U-test is included.

Figure 3

Samples represented on the planes of the first three principal components. Each pair of samples is represented as an arrow, with pre-LT at the tail and post-LT at the head. (A) PC2 vs. PC1; (B) PC3 vs. PC1; (C) PC3 vs. PC2.

Figure 4

Viral load differences between pre and post-LT samples. (A) Arrows showing the evolution of log VL from pre-LT (arrow tails on the left) to 15-day post-LT (arrow heads on the right side of the box). At one year post-LT, patients with high fibrosis (F3–F4) in orange, patients with low fibrosis (F0–F2) in green. (B) Scatterplot with differences in log VL at 15 days post-LT minus pre-LT. The ANOVA p-value in the comparison of the two levels of liver damage is included. Each patient is represented as *.