• Correlation between pre-treatment quasispecies complexity and treatment outcome in chronic HCV genotype 3a.

      Moreau, Isabelle; Levis, John; Crosbie, Orla; Kenny-Walsh, Elizabeth; Fanning, Liam J; Molecular Virology Diagnostic & Research Laboratory, Department of Medicine,, Clinical Sciences Building, Cork University Hospital, Cork, Ireland., i.moreau@ucc.ie (2012-02-03)
      Pre-treatment HCV quasispecies complexity and diversity may predict response to interferon based anti-viral therapy. The objective of this study was to retrospectively (1) examine temporal changes in quasispecies prior to the start of therapy and (2) investigate extensively quasispecies evolution in a group of 10 chronically infected patients with genotype 3a, treated with pegylated alpha2a-Interferon and ribavirin. The degree of sequence heterogeneity within the hypervariable region 1 was assessed by analyzing 20-30 individual clones in serial serum samples. Genetic parameters, including amino acid Shannon entropy, Hamming distance and genetic distance were calculated for each sample. Treatment outcome was divided into (1) sustained virological responders (SVR) and (2) treatment failure (TF). Our results indicate, (1) quasispecies complexity and diversity are lower in the SVR group, (2) quasispecies vary temporally and (3) genetic heterogeneity at baseline can be use to predict treatment outcome. We discuss the results from the perspective of replicative homeostasis.
    • The Irish paradigm on the natural progression of hepatitis C virus infection: an investigation in a homogeneous patient population infected with HCV 1b (review).

      Fanning, Liam J; Hepatitis C Unit, Department of Medicine, Clinical Sciences Building, National, University of Ireland, Cork, Cork University Hospital, Cork, Ireland., lfanning@ucc.ie (2012-02-03)
      The aetiological agent of chronic hepatitis C is the hepatitis C virus. The hepatitis C virus is spread by parenteral transmission of body fluids, primarily blood or blood products. In 1989, after more than a decade of research, HCV was isolated and characterised. The hepatitis C viral genome is a positive-sense, single-stranded RNA molecule approximately 9.4 kb in length, which encodes a polyprotein of about 3100 amino acids. There are 6 main genotypes of HCV, each further stratified by subtype. In 1994, a cohort of women was identified in Ireland as having been iatrogenically exposed to the hepatitis C virus. The women were all young and exposed as a consequence of the receipt of HCV 1b contaminated anti-D immunoglobulin. The source of the infection was identified as an acutely infected female. As part of a voluntary serological screening programme involving 62,667 people, 704 individuals were identified as seropositive for exposure to the hepatitis C virus; 55.4% were found to be positive for the viral genome 17 years after exposure. Of these women 98% had evidence of inflammation, but surprisingly, a remarkable 49% showed no evidence of fibrosis. Clinicopathology and virological analysis has identified associations between viral load and the histological activity index for inflammation, and, between inflammation and levels of the liver enzyme alanine aminotransferase. Infection at a younger age appears to protect individuals from progression to advanced liver disease. Molecular analyses of host immunogenetic elements shows that particular class II human leukocyte associated antigen alleles are associated with clearance of the hepatitis C virus. Additional class II alleles have been identified that are associated with stable viraemia over an extended period of patient follow-up. Although, investigation of large untreated homogeneous cohorts is likely to become more difficult, as the efficacy of anti-viral therapy improves, further investigation of host and viral factors that influence disease progression will help provide an evidence based approach were realistic expectations regarding patient prognosis can be ascertained.
    • Persistence of hepatitis C virus in a white population: associations with human leukocyte antigen class 1.

      Fanning, Liam J; Kenny-Walsh, Elizabeth; Shanahan, Fergus; Hepatitis C Unit, Department of Medicine, Clinical Sciences Building, Cork, University Hospital, National University of Ireland, Cork, Ireland., 1.fanning@ucc.ie (2012-02-03)
      The aim of this study was to define novel associations between human leukocyte antigen (HLA) class 1 alleles and persistence or clearance of the hepatitis C virus (HCV) in a white population. All individuals in the study were seropositive for anti-HCV antibodies. Viral status was determined by the Roche HCV Amplicor test. HLA-A, -B, -C allelic group profile was molecularly defined by reverse line probe hybridization. The strongest individual allelic group associations with persistent HCV infection were HLA A*11 (p = 0.044) and Cw*04 (p = 0.006). However, only the HLA C*04 association survived correction for multiple comparisons. Further analysis of alleles in linkage with HLA Cw*04 revealed that the haplotype HLA A*11, Cw*04 was present in 11 individuals, 10 of whom were viremic (p = 0.05). No gene dosage effect was observed. No association between HLA class 1 allelic groups and aviremia and virus load was evident in this white population. HLA B*44 is associated with low virus load in human immunodeficiency virus disease, but this association was not evident in this HCV-infected population. Novel HLA class 1 alleles associated with persistence of HCV have been identified.
    • Separation of Hepatitis C genotype 4a into IgG-depleted and IgG-enriched fractions reveals a unique quasispecies profile.

      Moreau, Isabelle; O'Sullivan, Hilary; Murray, Caroline; Levis, John; Crosbie, Orla; Kenny-Walsh, Elizabeth; Fanning, Liam J; Department of Medicine, Molecular Virology Diagnostic & Research Laboratory,, Clinical Sciences Building, Cork University Hospital, Cork, Ireland., i.moreau@ucc.ie (2012-02-03)
      BACKGROUND: Hepatitis C virus (HCV) circulates in an infected individual as a heterogeneous mixture of closely related viruses called quasispecies. The E1/E2 region of the HCV genome is hypervariable (HVR1) and is targeted by the humoral immune system. Hepatitis C virions are found in two forms: antibody associated or antibody free. The objective of this study was to investigate if separation of Hepatitis C virions into antibody enriched and antibody depleted fractions segregates quasispecies populations into distinctive swarms. RESULTS: A HCV genotype 4a specimen was fractionated into IgG-depleted and IgG-enriched fractions by use of Albumin/IgG depletion spin column. Clonal analysis of these two fractions was performed and then compared to an unfractionated sample. Following sequence analysis it was evident that the antibody depleted fraction was significantly more heterogeneous than the antibody enriched fraction, revealing a unique quasispecies profile. An in-frame 3 nt insertion was observed in 26% of clones in the unfractionated population and in 64% of clones in the IgG-depleted fraction. In addition, an in-frame 3 nt indel event was observed in 10% of clones in the unfractionated population and in 9% of clones in the IgG-depleted fraction. Neither of these latter events, which are rare occurrences in genotype 4a, was identified in the IgG-enriched fraction. CONCLUSION: In conclusion, the homogeneity of the IgG-enriched species is postulated to represent a sequence that was strongly recognised by the humoral immune system at the time the sample was obtained. The heterogeneous nature of the IgG-depleted fraction is discussed in the context of humoral escape.