Characterizing intra-host influenza virus populations to predict emergence June 12, 2012 Forum on Microbial Threats Washington, DC Elodie Ghedin Center for Vaccine Research Dept. Computational & Systems Biology
Intra-host RNA virus diversity persistent HIV, HCV, (time) Replication errors, recombination HIGH DIVERSITY acute Influenza A?
Factors that affect viral diversity antivirals Immune pressure Transmission bottlenecks
Does natural selection occur within individual hosts? How big is the population bottleneck at transmission? What is the extent of mixed infection? What is the mutational spectrum within individual hosts? What is the fitness distribution of these mutations?
Influenza A Virus: 3 models to explain transmission and emergence A B C Host 1 Host 2 Host 1 Host 2 Host 1 Host 2 Immunocompromised Host 2 Persistent infection
What happens to viral diversity if influenza infection is persistent? persistent HIV, HCV, (time) HIGH DIVERSITY acute Influenza A? What happens in immunocompromised patients? (longer infection period)
Emergence of H275Y resistance in immunocompromised host Q1: Is the drug-resistant variant present at time 0 (sample 1) before oseltamivir treatment? Q2: What other variant positions are associated with H275Y? Q3: Is the drug-resistant virus a variant of the WT or due to a mixed infection?
NA Aligned short sequence reads on segments 1.4 Kb
Consensus assembly would read this as a CAC TAC leads to drug resistance
From alignment of sequence reads can generate summaries at each codon NA Position Total reads Dominant Minor 1 Minor 2 267 5260 V {GTC} 5212 99.09 A {GCC} 13 0.25 D {GAC} 8 0.15 268 5122 E {GAA} 5077 99.12 K {AAA} 14 0.27 E {GAG} 9 0.18 269 4895 M {ATG} 4848 99.04 V {GTG} 12 0.25 T {ACG} 10 0.20 270 4725 N {AAT} 4681 99.07 S {AGT} 13 0.28 D {GAT} 10 0.21 271 4738 A {GCC} 4699 99.18 T {ACC} 8 0.17 A {GCA} 7 0.15 272 4833 P {CCT} 4781 98.92 P {CCC} 10 0.21 P {CCA} 9 0.19 273 4816 N {AAT} 4782 99.29 S {AGT} 9 0.19 H {CAT} 6 0.12 274 4943 Y {TAT} 4899 99.11 Y {TAC} 9 0.18 C {TGT} 8 0.16 275 5006 H {CAC} 4796 95.81 Y {TAC} 157 3.14 R {CGC} 19 0.38 276 5569 Y {TAT} 5529 99.28 N {AAT} 12 0.22 Y {TAC} 7 0.13 277 5730 E {GAG} 5690 99.30 G {GGG} 13 0.23 E {GAA} 10 0.17 278 5746 E {GAA} 5710 99.37 E {GAG} 14 0.24 G {GGA} 7 0.12 279 5780 C {TGC} 5732 99.17 C {TGT} 10 0.17 Y {TAC} 9 0.16 280 5758 S {TCC} 5711 99.18 P {CCC} 13 0.23 S {TCA} 9 0.16 281 5774 C {TGT} 5703 98.77 S {AGT} 21 0.36 C {TGC} 11 0.19 282 5866 Y {TAT} 5818 99.18 Y {TAC} 17 0.29 H {CAT} 11 0.19 283 6089 P {CCT} 6017 98.82 P {CCC} 20 0.33 P {CCA} 16 0.26
Emergence of H275Y resistance in immunocompromised host Q1: Is the drug-resistant variant present at time 0 (sample 1) before oseltamivir treatment? Q2: What other variant positions are associated with H275Y? Q3: Is the drug-resistant virus a variant of the WT or due to a mixed infection?
Diversity of H1N1/2009: Many strains co-circulate (but antigenically similar) Wave 1 April 2009 Nelson et al. (2011) J. Virol. 85:828
Wave 2: mix across geographical regions but dominance of one strain Clade 7
Immunosuppressed patient infected during wave 2 of H1N1/2009 pandemic Sample 1 Sample 2 Consensus seq: H275 Deep seq: H275 Consensus seq: H275 Deep seq: H275 + 275Y tamiflu De novo mutations or mixed infection?
Reconstruction of variants? Representation of minor to major variants residue positions on the NA protein Frequency estimation for assembly?
Difficult to link positions on separate sequence reads Representation of minor to major variants Unique residue positions on the NA protein Sequence reads
The Assembly Phasing Problem Sample 1 Sample 2 Bansal et al (2008) Genome Res. 18:1336
R CLADE 2 Partial assembly of sequence reads demonstrates presence of co-infecting variants CLADE 7 Ghedin et al (2011) J. Inf. Dis. 203:168
Emergence of H275Y drug resistance mutation Q1: Is the drug-resistant variant present at time 0 (sample 1) before oseltamivir treatment? Q2: What other variant positions are associated with H275Y? Q3: Is the drug-resistant virus a variant of the WT or due to a mixed infection? Q4: Can the drug resistance mutation be transmitted?
Q1. Are drug-resistant viruses present as a subpopulation in the son? Q2. How is the bottleneck at transmission? Son Father t=i Tamiflu Rx RT-PCR: Drug sensitive? t=i t=i + 6 Tamiflu prophylaxis RT-PCR: Drug resistant
Son to Father Transmission Results for NA (codon 275) Q1: Is the drug-resistant strain present as a subpopulation in the individual carrying the oseltamivir-sensitive strain? YES
Log10 Sequence coverage of each codon on all proteins Son Father Major codons Most common minor variant codons
Account for all sources of stochastic variation to determine distribution of true variants Monte Carlo methods to generate empirical null distribution Simulation process: Assumptions: 5 days of infection/10 cycles of replication; Per site mutation rate: 1 in 10,000 Efficiency of replication: 0.8 20 simulated PCR cycles; Per site mutation rate: 1 in 10,000 Efficiency of replication: 0.8 Sequencing error: 0.0035 per site; Error profile used (accounting for HiSeq GC bias): Test statistic calculated for each position in simulated and experimental data; Report p-value calculated from rank
What is being transmitted? Son Father t=i t=i + 6 tamiflu
Summary 1. Multiple variants transmitted transmission bottleneck is not especially narrow. 2. Some variants are lost at transmission, and some attain a higher frequency in the recipient transmission plays a major role in shaping patterns of genetic diversity in influenza virus. 3. Pre-existing variation at 275 position in NA mutation not deleterious, compensatory mutations not necessary
Reconstructing chains of transmission for an epidemic? Consensus sequence R 0 =1.2 At the scale flu evolution happens, there are not enough changes to reconstruct transmission networks
Reconstructing transmission phylogeny Deep Sequencing significant changes in the distribution in the intra-host population
Acknowledgements Ghedin Lab: Jay DePasse Adam Fitch Eddie Holmes Jesse Papenburg Guy Boivin Marie-Eve Hamelin Lady Tatiana Pinilla Funding: NIH/NIAID