The Outcome of Acute Hepatitis C Predicted by the Evolution of the Viral Quasispecies Farci et al. (2000) Science 288, 339-344.

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The Outcome of Acute Hepatitis C Predicted by the
Evolution of the Viral QuasispeciesFarci et al.
(2000) Science 288, 339-344.

• Georg Gerber
• HST.120
• December 15, 2006

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HCV overview

• HCV sequenced in 1989 major cause of non-A,
  non-B viral hepatitis
• Approximately 170 million people infected
  worldwide
• gt 38,000 new cases in U.S. annually
• Chronic infection in 70 untreated people (10
  if treated)
• Cirrhosis in 20 and hepatocellular carcinoma in
  2.5 of chronically infected
• No vaccine
• Treatment effective in only 50-60 of patients
  and has significant toxicity

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Hepatitis C Virus
Envelope
Core
Viral RNA (9400 nucleotides)
Envelope Glycoproteins
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HCV Genotypes

• Viral polymerase is highly error-prone (1
  nucleotide change per replication cycle)
• At least 6 viral genotypes and gt90 subtypes
• Genotypes determined by highly conserved 5NC
  sequence
• In the U.S.
• 75 genotype 1
• 15 genotype 2
• 10 genotype 3
• Difference in sustained virological response
  (SVR) w/ Rx
• genotype 1 - 42-46 SVR
• genotypes 2 and 3 76-80 (shorter Rx effective)

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HCV Genotype Distribution
1b 2a, 2b, 2c, 3a
1b
1a, 1b 2a, 2b, 3a
2a
4
1b, 6
1b, 3a
4
3b
1b, 3a
5a
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Concept of Viral Quasispecies

• Low-fidelity viral polymerase produces an
  ensemble of related sequences
• View evolutionary pressure as acting on the viral
  population, not individual variants
• Extremely high mutations rates bad for the virus,
  but too low rates make non-adaptive
• Recent work by Vignuzzi et al. (Nature 2006)
• Poliovirus mutant with high fidelity polymerase
• Achieves WT replication rates, but loses
  neurotropism and pathogenicity
• Chemically induced mutagenesis ? restored
  neurotropism and pathogenicity
• Demonstrate strains that complement each other
  (strain that was pathogenic but couldnt cross
  BBB complemented by opposite phenotype)

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Host Response

• Clearance of acute infection doesnt protect
  against re-infection, but does protect against
  chronic state
• Role of naturally acquired antibodies unclear,
  because dont protect against re-infection and
  viral clearance can occur w/o anti-HCV antibodies
  in both humans and chimps
• Unclear why some become chronically infected
  many proposed mechanisms for viral modulation of
  host defenses

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The Outcome of Acute Hepatitis C Predicted by
the Evolution of the Viral Quasispecies

• Investigated relationship between viral genetic
  diversity in patients and infection outcome
  (e.g., resolving, chronic, fulminant)
• Opportunity to study natural history of HCV
  infection because
• Post-transfusion infections (before universal
  blood screen) can pinpoint date of infection
• Prior to current treatment protocols

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Patients

• Twelve patients categorized as
• Fulminant hepatitis (FH)
• Resolving hepatitis
• Chronic, slowly progressors (mild and stable for
  more than 20 years)
• Chronic, rapid progressors (liver-related death
  within 5 years of the onset of infection)
• Three patients in each group

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Viral Population Analysis

• For patients w/o FH, 3-4 viral infection
  time-point samples
• The first HCV PCR-positive sample (2-5 weeks
  post-transfusion)
• Prior to antibody seroconversion (before or at
  the time of ALT peak)
• One to two addl after seroconversion
• FH patients 2 time-points (one before and one
  after seroconversion)
• DNA amplified from E1/E2 genes (558 nucleotides)
  and cloned
• Mean of 10.6 clones sequenced from each sample
  (total of 414 sequences)

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Viral Genetic Diversity and Number of Viral
Variants

• Genetic diversity mean Hamming distance between
  a.a. sequences w/in or outside 31 a.a.s
  hypervariable region 1 (HVR1) of E2
• Number of viral variants number of unique a.a.
  sequences w/in or outside 31 a.a.s HVR1 of E2

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Viral Genetic Diversity and Number of Viral
Variants Analysis Conclusions

• After seroconversion, patients w/ chronic
  infection had large increase in viral genetic
  diversity w/in HVR1 patients w/ resolving
  disease showed a marked decrease in the same
  measure (both changes statistically significant)
• Genetic diversity and the of viral variants
  outside the HVR1 region was consistently lower
  than w/in HVR1 and showed little temporal change
• Patients w/ FH had the lowest levels of viral
  genetic diversity

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Analysis for positive selection

• Analyzed the of synonymous (silent) versus
  non-synonymous (a.a. replacements) changes
• For each patient, derived time-point 1 consensus
  sequence and compared to all sequences from the
  last time-point
• Mean of non-syn. substitutions per site per
  week in HVR1 higher in patients w/ progressing
  hepatitis than in patients w/ either resolving or
  FH (statistically significant)
• Non-syn. substitutions consistently lower outside
  HVR1 w/ no significant differences among patient
  populations
• No significant differences in syn. substitutions
  either inside or outside HVR1 among patient
  populations
• Syn. substitutions slightly higher both inside
  and outside the HVR1 in patients w/ FH (not
  statistically signif.)

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Study conclusions

• Provides evidence that outcome of HCV infection
  may be related to viral genetic diversity that
  emerges w/in first few months of infection
• Patients w/ chronic infection had large increase
  in viral genetic diversity w/in HVR1 and
  significantly more non-synonymous substitutions
  than in other patient populations
• Patients w/ resolving disease had decrease in
  viral genetic diversity w/in HVR1
• Genetic diversity outside HVR1 was consistently
  lower than w/in HVR1, showed little temporal
  change, and low rates of both syn. and non-syn.
  substitutions across all patient populations

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Criticism

• Small patient population ? limited statistical
  power
• Limited statistical analysis
• Correction for multiple hypothesis testing?
• Significance of phylogenetic findings?
• Lack of explicit biological mechanism doesnt
  allow inference of causality
• Thus, conclusions really only suggestive of
  associations, patterns or trends

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Criticism (cont.)

• Are these findings counterintuitive? Shouldnt
  an effective immune response exert selective
  pressure ? viral diversification?
• Authors speculate that patients who clear HCV are
  mounting an effective response that eliminates
  many viral variants, and note a similar trend in
  patients responding well to interferon therapy
• 2006 study by same authors (similar methodology)
  of children perinatally infected w/ HCV
• Children w/ highest ALT levels (worst liver
  damage) harbored the most homogeneous viral
  populations
• Authors speculate due to differences in strength
  of the cytotoxic T-cell response versus B-cell
  mediated response

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Viral tropism

• All necessary host receptors not yet elucidated
• E2 binds to CD81 (present on many cells including
  hepatocytes) necessary but not sufficient
• Many other receptors identified low-density
  lipoprotein receptor, scavenger receptor class-B
  type-I (SR-BI), L-SIGN and DC-SIGN
• Some evidence can infect non-hepatocytes

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Treatment and prophylaxis

• Pegylated IFN-alpha and ribavirin for 24 or 48
  weeks
• Sustained virological response (SVR) no
  detectable HCV RNA during treatment and for gt6
  months after stopping therapy
• genotype 1 - 42-46 SVR
• genotypes 2 and 3 76-80 (shorter Rx effective)
• Concerns about drug resistance and relatively low
  SVRs, so much work on developing new drugs and a
  vaccine