1. The Outcome of Acute Hepatitis C Predicted by the Evolution of the Viral Quasispecies Farci et al. (2000) Science 288, 339-344. Georg Gerber HST.120 December 15, 2006

2. HCV overview HCV sequenced in 1989 – major cause of non-A, non-B viral hepatitis Approximately 170 million people infected worldwide > 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

3. Envelope Core EnvelopeGlycoproteins Viral RNA (9400 nucleotides) 55-65 nm Hepatitis C Virus

5. HCV Genotypes Viral polymerase is highly error-prone (≈1 nucleotide change per replication cycle) At least 6 viral genotypes and >90 subtypes Genotypes determined by highly conserved 5’NC 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)

6. HCV Genotype Distribution 1a, 1b 2a, 2b, 3a 1b 2a, 2b, 2c, 3a 4 5a 1b 1b, 6 1b, 3a 3b 2a 4

7. 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 couldn’t cross BBB complemented by “opposite” phenotype)

8. Host Response Clearance of acute infection doesn’t protect against re- infection, but does protect against chronic state Role of naturally acquired antibodies unclear, because don’t 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

9. “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

10. 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

12. Viral Population Analysis For patients w/o FH, 3-4 viral infection time-point samples: 1.The first HCV PCR-positive sample (2-5 weeks post- transfusion) 2.Prior to antibody seroconversion (before or at the time of ALT peak) 3.One to two add’l 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)

13. 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

15. 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

18. 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.)

20. 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

21. Criticism Small patient population → limited statistical power Limited statistical analysis: –Correction for multiple hypothesis testing? –Significance of phylogenetic findings? Lack of explicit biological mechanism doesn’t allow inference of causality Thus, conclusions really only suggestive of associations, patterns or trends

22. Criticism (cont.) Are these findings counterintuitive? Shouldn’t 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

23. 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

24. 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 >6 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