E E evolution at transmission
E1E2 evolution at transmission has been explored in multiple previous studies. Vertical transmission selects for E1E2 variants with increased replication fitness due of CTL escape mutations (Honegger et al., 2013). Experimental infection of chimeric liver mice selects for E1E2 variants with increased capacity for cellular entry (Brown et al., 2012) while in orthotopic liver transplantation, preferential transmission of neutralization resistant E1E2s with enhanced entry capacity occurs (Fafi-Kremer et al., 2010, Felmlee et al., 2016, Fofana et al., 2012). In our study focusing on the HCV protease, no evidence for T-cell reversion was observed upon transmission. However, while no selection of advantageous amino acids was observed, multiple silent transitional substitutions, which were present at >1% in the initial patient serum or donor inoculum, became dominant in the population post-transmission. Whether these fixations confer a replication or translation advantage into viral populations in a new host liver remains untested and requires further investigation. However as an identical pattern was detected in independent transmissions, this observation is unlikely due to random genetic drift. Despite high-titer inoculations, sites exhibiting variation >1% in the population were reduced in all recipients, with differential levels of viral diversity transmitted: most detectable variation was due to synonymous transitions. NS3 was surprisingly stable upon transmission, with an identical amino lactacystin consensus in the donor and all recipients. These data indicate a combination of strong purifying selection and a genetic bottleneck shape viral protease evolution at transmission. The majority of nucleotide positions exhibit only low-frequency SNVs in the patient sera, DM and recipient animal, indicating that selective constraints limit the frequencies of SNVs that can reach high frequencies in the viral population.
While single genome amplification (SGA) has advantages over clonal sequencing to assess viral population diversity (Salazar-Gonzalez et al., 2008), sampling depth remains highly restricted and represents only a fraction of the total population. NGS allows viral populations to be scanned at unprecedented depth, orders of magnitude greater than previous approaches. Our analyses reveal the presence of a low frequency mutational cloud in all populations analyzed. The extent and diversity of this cloud, or the extreme RAS heterogeneity observed prior to virological breakthrough would not be accurately represented using clonal/SGA approaches. The virally encoded RdRp is responsible for genome replication via a negative stranded intermediate and has low fidelity. The resultant mutational spectrum generated facilitates the rapid emergence of escape mutants in response to selective pressures. Due to the limitations of current technologies, a proportion of the low frequency SNVs detected are likely to represent assay noise (Whitfield and Andino, 2016). However, the low frequency SNV cloud observed also contains a combination of defective or fitness-impaired viral genomes and represents the raw variation on which natural selection can act in viral populations, evidenced by the rapid emergence of RAS upon therapy initiation. In conclusion, these analyses provide novel insights into the dynamics of HCV protease populations at transmission and under therapeutic intervention, quantifying the evolutionary processes shaping NS3 sequence divergence and governing RAS emergence in vivo.
Acknowledgements TFB acknowledges grant support from the European Union (ERC-AdG-2014-671231-HEPCIR, FP7 HepaMab, H2020-2015-667273-HEP-CAR), the NIH (NIAID U19AI123862) and LabEx HEPSYS (ANR-10-LABX-0028_ HEPSYS). PM was supported by Ghent University (Concerted Action Grant 01G01712), the Agency for Innovation by Science and Technology (IWT SBO project HLIM-3D), the Belgian Science Policy Office (BELSPO; IUAP P7/47-HEPRO-2) and the European Union (FP7, HepaMab). TP was supported the German Centre for Infection Research (DZIF). Ciluprevir BILN 2061 (purity 99%) was kindly provided by Gilead (Foster City, CA).