Elizabeth Kenny-Walsh

Divergent and convergent evolution after a common-source outbreak of hepatitis C virus

The genomic sequences of viruses that are highly mutable and cause chronic infection tend to diverge over time. We report that these changes represent both immune-driven selection and, in the absence of immune pressure, reversion toward an ancestral consensus. Sequence changes in hepatitis C virus (HCV) structural and nonstructural genes were studied in a cohort of women accidentally infected with HCV in a rare common-source outbreak. We compared sequences present in serum obtained 18–22 yr after infection to sequences present in the shared inoculum and found that HCV evolved along a distinct path in each woman. Amino acid substitutions in known epitopes were directed away from consensus in persons having the HLA allele associated with that epitope (immune selection), and toward consensus in those lacking the allele (reversion). These data suggest that vaccines for genetically diverse viruses may be more effective if they represent consensus sequence, rather than a human isolate.

Tissue viral load variability in chronic hepatitis C.

OBJECTIVE:Liver biopsy is regarded as the gold standard for assessing disease activity in chronic hepatitis C, but sampling error is a potential limitation. Whether sampling variability applies equally to viral load assessment as it does to histology is uncertain. To examine this, we compared viral load between right- and left-lobe biopsy specimens from patients infected with hepatitis C virus (HCV).METHODS:Bilobe biopsies were taken from 16 patients who were serum positive for HCV RNA by reverse transcription-polymerase chain reaction. Genotype was identified by reverse line probe hybridization. There was an absence of competing risk factors for infectious and other liver diseases in this patient group. Histology and hepatic viral load were assessed blindly. None of the patients had received antiviral therapy at the time of study.RESULTS:Detection of HCV in right and left lobes was concordant with serum positivity in all cases. The viral load between lobes was highly correlated (p = 0.0003, r = 0.79). In contrast, the histological activity indices of inflammation and fibrosis/cirrhosis were poorly correlated between lobes (p = 0.038, r = 0.60, and p = 0.098, r = 0.50, respectively).CONCLUSION:Hepatic viral load variability does not suffer from the same degree of heterogeneity of sampling variability as does histology.

THE NATURAL HISTORY OF HEPATITIS C VIRUS INFECTION

The natural history of HCV infection remains ill-defined. The knowledge accumulated on the progression of HCV to date is important, however. It is now abundantly clear that the progression of disease is generally slow, and the development of cirrhosis and its complications is a possibility, not a probability as hitherto thought. Predicting the outcome remains a quandary for clinicians. Ultimately it will be possible to define the natural history of hepatitis C infection through a combination of research in the fields of virology, immunology, and molecular biology and by monitoring the biochemical and histologic progress of the disease. Only then will it be possible to intervene appropriately and develop new therapies to prevent the progression to cirrhosis and hepatocellular carcinoma.

In vitro replication models for the hepatitis C virus

Summary.  Soon after the discovery of the hepatitis C virus (HCV), attention turned to the development of models whereby replication of the virus could be investigated. Among the HCV replication models developed, the HCV RNA replicon model and the newly discovered infectious cell culture systems have had an immediate impact on the study of HCV replication, and will continue to lead to important advances in our understanding of HCV replication. The aim of this study is to deal with developments in HCV replication models in a chronological order from the early 1990s to the recent infectious HCV cell culture systems.

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

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 α2a-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.

Insertion and recombination events at hypervariable region 1 over 9.6 years of hepatitis C virus chronic infection.

Hepatitis C virus (HCV) exists as a quasispecies within an infected individual. We have previously reported an in-frame 3 bp insertion event at the N-terminal region of the E2 glycoprotein from a genotype 4a HCV isolate giving rise to an atypical 28 aa hypervariable region (HVR) 1. To further explore quasispecies evolution at the HVR1, serum samples collected over 9.6 years from the same chronically infected, treatment naïve individual were subjected to retrospective clonal analysis. Uniquely, we observed that isolates containing this atypical HVR1 not only persisted for 7.6 years, but dominated the quasispecies swarm. Just as striking was the collapse of this population of variants towards the end of the sampling period in synchrony with variants containing a classical HVR1 from the same lineage. The replication space was subsequently occupied by a second minor lineage, which itself was only intermittently detectable at earlier sampling points. In conjunction with the observed genetic shift, the coexistence of two distinct HVR1 populations facilitated the detection of putative intra-subtype recombinants, which included the identification of the likely ancestral parental donors. Juxtaposed to the considerable plasticity of the HVR1, we also document a degree of mutational inflexibility as each of the HVR1 subpopulations within our dataset exhibited overall genetic conservation and convergence. Finally, we raise the issue of genetic analysis in the context of mixed lineage infections.

Analysis of the Evolution and Structure of a Complex Intrahost Viral Population in Chronic Hepatitis C Virus Mapped by Ultradeep Pyrosequencing

ABSTRACT Hepatitis C virus (HCV) causes chronic infection in up to 50% to 80% of infected individuals. Hypervariable region 1 (HVR1) variability is frequently studied to gain an insight into the mechanisms of HCV adaptation during chronic infection, but the changes to and persistence of HCV subpopulations during intrahost evolution are poorly understood. In this study, we used ultradeep pyrosequencing (UDPS) to map the viral heterogeneity of a single patient over 9.6 years of chronic HCV genotype 4a infection. Informed error correction of the raw UDPS data was performed using a temporally matched clonal data set. The resultant data set reported the detection of low-frequency recombinants throughout the study period, implying that recombination is an active mechanism through which HCV can explore novel sequence space. The data indicate that polyvirus infection of hepatocytes has occurred but that the fitness quotients of recombinant daughter virions are too low for the daughter virions to compete against the parental genomes. The subpopulations of parental genomes contributing to the recombination events highlighted a dynamic virome where subpopulations of variants are in competition. In addition, we provide direct evidence that demonstrates the growth of subdominant populations to dominance in the absence of a detectable humoral response. IMPORTANCE Analysis of ultradeep pyrosequencing data sets derived from virus amplicons frequently relies on software tools that are not optimized for amplicon analysis, assume random incorporation of sequencing errors, and are focused on achieving higher specificity at the expense of sensitivity. Such analysis is further complicated by the presence of hypervariable regions. In this study, we made use of a temporally matched reference sequence data set to inform error correction algorithms. Using this methodology, we were able to (i) detect multiple instances of hepatitis C virus intrasubtype recombination at the E1/E2 junction (a phenomenon rarely reported in the literature) and (ii) interrogate the longitudinal quasispecies complexity of the virome. Parallel to the UDPS, isolation of IgG-bound virions was found to coincide with the collapse of specific viral subpopulations.

Constraints on Viral Evolution during Chronic Hepatitis C Virus Infection Arising from a Common-Source Exposure

ABSTRACT Extraordinary viral sequence diversity and rapid viral genetic evolution are hallmarks of hepatitis C virus (HCV) infection. Viral sequence evolution has previously been shown to mediate escape from cytotoxic T-lymphocyte (CTL) and neutralizing antibody responses in acute HCV infection. HCV evolution continues during chronic infection, but the pressures driving these changes are poorly defined. We analyzed plasma virus sequence evolution in 5.2-kb hemigenomes from multiple longitudinal time points isolated from individuals in the Irish anti-D cohort, who were infected with HCV from a common source in 1977 to 1978. We found phylogenetically distinct quasispecies populations at different plasma time points isolated late in chronic infection, suggesting ongoing viral evolution and quasispecies replacement over time. We saw evidence of early pressure driving net evolution away from a computationally reconstructed common ancestor, known as Bole1b, in predicted CTL epitopes and E1E2, with balanced evolution toward and away from the Bole1b amino acid sequence in the remainder of the genome. Late in chronic infection, the rate of evolution toward the Bole1b sequence increased, resulting in net neutral evolution relative to Bole1b across the entire 5.2-kb hemigenome. Surprisingly, even late in chronic infection, net amino acid evolution away from the infecting inoculum sequence still could be observed. These data suggest that, late in chronic infection, ongoing HCV evolution is not random genetic drift but rather the product of strong pressure toward a common ancestor and concurrent net ongoing evolution away from the inoculum virus sequence, likely balancing replicative fitness and ongoing immune escape.

Hepatitis C virus liver disease in women infected with contaminated anti-D immunoglobulin

Screening for hepatitis C virus (HCV) infection is carried out by detection of antibodies to the virus (enzyme‐linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA)) with confirmation by identification of HCV RNA genome in serum (polymerase chain reaction (PCR)). We describe the histological features on liver biopsy in 88 women with chronic HCV infection (serum positive on ELISA, RIBA and PCR) acquired from virus contaminated anti‐D immunoglobulin. For the majority of these patients the time interval from virus infection to presentation was between 17 and 18 years. We separately assessed necroinflammatory disease activity and architectural features on liver biopsy and applied a scoring system which permitted semi‐quantitative documentation of abnormal features. Only three women showed liver biopsies within normal limits (± focal steatosis). The remaining 85 cases showed a predominantly mild or moderate degree of disease activity with interface hepatitis (56.8% of cases), spotty necrosis, apoptosis and focal inflammation (88.6% of cases) and portal inflammation (90.9% of cases). Confluent necrosis was an uncommon finding (2.3% of cases).

Viral load change and sequential evolution of entire hepatitis C virus genome in Irish recipients of single source-contaminated anti-D immunoglobulin*

Summary.  In hepatitis C virus (HCV) infection, serum viral load is important in the prediction of therapeutic efficacy. However, factors that affect the viral load remain poorly understood. To identify viral genomic elements responsible for the viral load, we investigated samples from a population of Irish women who were iatrogenically infected from a single HCV source by administration of HCV 1b‐contaminated anti‐D immune globulin between 1977 and 1978 (Kenny‐Walsh, N Engl J Med 1999; 340: 1228). About 15 patients were divided into two groups, viral load increasing group (11 patients) and decreasing group (4 patients). Pairs of sera were collected from each patient at interval between 1.1 and 5.8 years. Full‐length sequences of HCV genome were determined, and analyzed for changes in each patient. Sliding window analysis showed that the decreasing group had significantly higher mutation rates in a short segment of NS5B region that may affect the activity of RNA‐dependent RNA polymerase. By comparing each coding regions, significantly higher mutation numbers were accumulated in NS5A region in the increasing group than the decreasing group (0.92 vs 0.16 nucleotides/site/year, P = 0.021). The mutation in certain positions of the HCV genome may be determinant factors of the viral load in a relatively homogeneous patient population.