Genome-wide patterns of intrahuman dengue virus diversity reveal associations with viral phylogenetic clade and interhost diversity

Analogous to observations in RNA viruses such as human immunodeficiency virus, genetic variation associated with intrahost dengue virus (DENV) populations has been postulated to influence viral fitness and disease pathogenesis. Previous attempts to investigate intrahost genetic variation in DENV characterized only a few viral genes or a limited number of full-length genomes. We developed a whole-genome amplification approach coupled with deep sequencing to capture intrahost diversity across the entire coding region of DENV-2. Using this approach, we sequenced DENV-2 genomes from the serum of 22 Nicaraguan individuals with secondary DENV infection and captured ～75% of the DENV genome in each sample (range, 40 to 98%). We identified and quantified variants using a highly sensitive and specific method and determined that the extent of diversity was considerably lower than previous estimates. Significant differences in intrahost diversity were detected between genes and also between antigenically distinct domains of the Envelope gene. Interestingly, a strong association was discerned between the extent of intrahost diversity in a few genes and viral clade identity. Additionally, the abundance of viral variants within a host, as well as the impact of viral mutations on amino acid encoding and predicted protein function, determined whether intrahost variants were observed at the interhost level in circulating Nicaraguan DENV-2 populations, strongly suggestive of purifying selection across transmission events. Our data illustrate the value of high-coverage genome-wide analysis of intrahost diversity for high-resolution mapping of the relationship between intrahost diversity and clinical, epidemiological, and virological parameters of viral infection.     

High-resolution analysis of intrahost genetic diversity in dengue virus serotype 1 infection identifies mixed infections

Little is known about the rate at which genetic variation is generated within intrahost populations of dengue virus (DENV) and what implications this diversity has for dengue pathogenesis, disease severity, and host immunity. Previous studies of intrahost DENV variation have used a low frequency of sampling and/or experimental methods that do not fully account for errors generated through amplification and sequencing of viral RNAs. We investigated the extent and pattern of genetic diversity in sequence data in domain III (DIII) of the envelope (E) gene in serial plasma samples (n = 49) taken from 17 patients infected with DENV type 1 (DENV-1), totaling some 8,458 clones. Statistically rigorous approaches were employed to account for artifactual variants resulting from amplification and sequencing, which we suggest have played a major role in previous studies of intrahost genetic variation. Accordingly, nucleotide sequence diversities of viral populations were very low, with conservative estimates of the average levels of genetic diversity ranging from 0 to 0.0013. Despite such sequence conservation, we observed clear evidence for mixed infection, with the presence of multiple phylogenetically distinct lineages present within the same host, while the presence of stop codon mutations in some samples suggests the action of complementation. In contrast to some previous studies we observed no relationship between the extent and pattern of DENV-1 genetic diversity and disease severity, immune status, or level of viremia.     

Temporal variations in the hepatitis C virus intrahost population during chronic infection

The intrahost evolution of hepatitis C virus (HCV) holds keys to understanding mechanisms responsible for the establishment of chronic infections and to development of a vaccine and therapeutics. In this study, intrahost variants of two variable HCV genomic regions, HVR1 and NS5A, were sequenced from four treatment-naïve chronically infected patients who were followed up from the acute stage of infection for 9 to 18 years. Median-joining network analysis indicated that the majority of the HCV intrahost variants were observed only at certain time points, but some variants were detectable at more than one time point. In all patients, these variants were found organized into communities or subpopulations. We hypothesize that HCV intrahost evolution is defined by two processes: incremental changes within communities through random mutation and alternations between coexisting communities. The HCV population was observed to incrementally evolve within a single community during approximately the first 3 years of infection, followed by dispersion into several subpopulations. Two patients demonstrated this pattern of dispersion for the rest of the observation period, while HCV variants in the other two patients converged into another single subpopulation after ∼9 to 12 years of dispersion. The final subpopulation in these two patients was under purifying selection. Intrahost HCV evolution in all four patients was characterized by a consistent increase in negative selection over time, suggesting the increasing HCV adaptation to the host late in infection. The data suggest specific staging of HCV intrahost evolution.     

Evolution of Hypervariable Region 1 of Hepatitis C Virus in Primary Infection

The hypervariable region 1 (HVR-1) of the putative envelope encoding E2 region of hepatitis C virus (HCV) RNA was analyzed in sequential samples from three patients with acute type C hepatitis infected from different sources to address (i) the dynamics of intrahost HCV variability during the primary infection and (ii) the role of host selective pressure in driving viral genetic evolution. HVR-1 sequences from 20 clones per each point in time were analyzed after amplification, cloning, and purification of plasmid DNA from single colonies of transformed cells. The intrasample evolutionary analysis (nonsynonymous mutations per nonsynonymous site [K_a], synonymous mutations per synonymous site [K_s], K_a/K_s ratio, and genetic distances [gd]) documented low gd in early samples (ranging from 2.11 to 7.79%) and a further decrease after seroconversion (from 0 to 4.80%), suggesting that primary HCV infection is an oligoclonal event, and found different levels and dynamics of host pressure in the three cases. The intersample analysis (pairwise comparisons of intrapatient sequences; rK_a, rK_s, rK_a/rK_s ratio, and gd) confirmed the individual features of HCV genetic evolution in the three subjects and pointed to the relative contribution of either neutral evolution or selective forces in driving viral variability, documenting that adaptation of HCV for persistence in vivo follows different routes, probably representing the molecular counterpart of the viral fitness for individual environments.     

A unique, predominant hepatitis C virus variant found in an infant born to a mother with multiple variants.

To demonstrate vertical transmission of hepatitis C virus (HCV) from an HCV-infected, non-human immunodeficiency virus type 1-infected mother to her infant and to assess the distribution of viral species in the mother and infant, the hypervariable region of the gene encoding the putative envelope glycoprotein E2 (E2HV) was sequenced in three mothers and one mother-infant pair. The data indicate that (i) quasi-species distributions of HCV E2HV variants were found in all four mothers, (ii) a single predominant HCV E2HV variant was found in the infant of a mother shown to have nine predominant E2HV variants, and (iii) the infant's E2HV variant was highly related to, but not identical with, the nine variants identified in the mother at the time of birth. These findings indicate that HCV is transmitted from mother to infant and raise the possibility that the transmission occurs in utero.     

Impact of CCR5delta32 host genetic background and disease progression on HIV-1 intrahost evolutionary processes: efficient hypothesis testing through hierarchical phylogenetic models

The interplay between C-C chemokine receptor type 5 (CCR5) host genetic background, disease progression, and intrahost HIV-1 evolutionary dynamics remains unclear because differences in viral evolution between hosts limit the ability to draw conclusions across hosts stratified into clinically relevant populations. Similar inference problems are proliferating across many measurably evolving pathogens for which intrahost sequence samples are readily available. To this end, we propose novel hierarchical phylogenetic models (HPMs) that incorporate fixed effects to test for differences in dynamics across host populations in a formal statistical framework employing stochastic search variable selection and model averaging. To clarify the role of CCR5 host genetic background and disease progression on viral evolutionary patterns, we obtain gp120 envelope sequences from clonal HIV-1 variants isolated at multiple time points in the course of infection from populations of HIV-1-infected individuals who only harbored CCR5-using HIV-1 variants at all time points. Presence or absence of a CCR5 wt/Δ32 genotype and progressive or long-term nonprogressive course of infection stratify the clinical populations in a two-way design. As compared with the standard approach of analyzing sequences from each patient independently, the HPM provides more efficient estimation of evolutionary parameters such as nucleotide substitution rates and d(N)/d(S) rate ratios, as shown by significant shrinkage of the estimator variance. The fixed effects also correct for nonindependence of data between populations and results in even further shrinkage of individual patient estimates. Model selection suggests an association between nucleotide substitution rate and disease progression, but a role for CCR5 genotype remains elusive. Given the absence of clear d(N)/d(S) differences between patient groups, delayed onset of AIDS symptoms appears to be solely associated with lower viral replication rates rather than with differences in selection on amino acid fixation.     

Hepatitis C Virus Variants with Decreased Sensitivity to Direct-Acting Antivirals (DAAs) Were Rarely Observed in DAA-Naive Patients prior to Treatment

The prevalence of naturally occurring hepatitis C virus (HCV) variants that are less sensitive to direct-acting antiviral (DAA) inhibitors has not been fully characterized. We used population sequence analysis to assess the frequency of such variants in plasma samples from 3,447 DAA-naive patients with genotype 1 HCV. In general, HCV variants with lower-level resistance (3- to 25-fold increased 50% inhibitor concentration [IC₅₀]) to telaprevir were observed as the dominant species in 0 to 3% of patients, depending on the specific variant, whereas higher-level resistant variants (>25-fold-increased IC₅₀) were not observed. Specific variants resistant to NS5A inhibitors were predominant in up to 6% of patients. Most variants resistant to nucleo(s/t)ide active-site NS5B polymerase inhibitors were not observed, whereas variants resistant to non-nucleoside allosteric inhibitors were observed in up to 18% of patients. The presence of DAA-resistant variants in NS5A, NS5B, or NS3 (including telaprevir-resistant variants), in baseline samples of treatment-naive patients receiving a telaprevir-based regimen in phase 3 studies did not affect the sustained viral response (SVR). Treatment-naive patients with viral populations containing the telaprevir-resistant variants NS3 V36M, T54S, or R155K at baseline achieved a 74% SVR rate, whereas patients with no resistant variants detected prior to treatment achieved a 76% SVR rate. The effect of specific resistant variant frequency on response to various DAA treatments in different patient populations, including interferon nonresponders, should be further studied.     

Temporal dynamics of SARS-CoV-2 mutation accumulation within and across infected hosts

Analysis of SARS-CoV-2 genetic diversity within infected hosts can provide insight into the generation and spread of new viral variants and may enable high resolution inference of transmission chains. However, little is known about temporal aspects of SARS-CoV-2 intrahost diversity and the extent to which shared diversity reflects convergent evolution as opposed to transmission linkage. Here we use high depth of coverage sequencing to identify within-host genetic variants in 325 specimens from hospitalized COVID-19 patients and infected employees at a single medical center. We validated our variant calling by sequencing defined RNA mixtures and identified viral load as a critical factor in variant identification. By leveraging clinical metadata, we found that intrahost diversity is low and does not vary by time from symptom onset. This suggests that variants will only rarely rise to appreciable frequency prior to transmission. Although there was generally little shared variation across the sequenced cohort, we identified intrahost variants shared across individuals who were unlikely to be related by transmission. These variants did not precede a rise in frequency in global consensus genomes, suggesting that intrahost variants may have limited utility for predicting future lineages. These results provide important context for sequence-based inference in SARS-CoV-2 evolution and epidemiology.     

Persistence of multiple genetic lineages within intrahost populations of Ross River virus

We examined the structure and extent of genetic diversity in intrahost populations of Ross River virus (RRV) in samples from six human patients, focusing on the nonstructural (nsP3) and structural (E2) protein genes. Strikingly, although the samples were collected from contrasting ecological settings 3,000 kilometers apart in Australia, we observed multiple viral lineages in four of the six individuals, which is indicative of widespread mixed infections. In addition, a comparison with previously published RRV sequences revealed that these distinct lineages have been in circulation for at least 5 years, and we were able to document their long-term persistence over extensive geographical distances.     

Influence of increased CD4 cell counts on the genetic variability of hepatitis C virus in patients co-infected with human immunodeficiency virus I.

In order to study the effect of increased CD4 cell counts on the biology of hepatitis C virus (HCV), we analyzed the genetic variability of HCV generated over 8 y in eight human immunodeficiency virus-1 (HIV-1) and HCV co-infected patients. This was a retrospective study in which HIV patients were selected who had profound immune impairment evident over four years and were co-infected with HCV genotype 1 and who then went on highly active antiretroviral therapy (HAART). These patients achieved different degrees of immune reconstitution, measured as increased CD4 cell counts during a 4- to 8-y period, following initiation of HAART. HCV genetic variability was determined by measuring the genetic diversity (Hamming distance, HD), and complexity (number of viral variants) in plasma samples collected at yearly intervals just before and after the initiation of HAART. The parameters were assessed by molecular cloning and sequencing of a 575-bp fragment including the HCV envelope 1 and envelope 2 genes (E1/E2), containing the hypervariable region 1 (HVR1). significantly increased HVR1 genetic diversity was observed in analyzed samples where the patients' CD4 cell counts were > or =100 compared with CD4 cell counts <100. A significant increase in genetic diversity in HVR1 was detected in co-infected patients whose CD4 cell counts increased from <100 to >400 over a period of more than 4 y of HAART therapy. This was in contrast to a minimal increase in HCV genetic diversity of HVR1 occurring in patients whose CD4 cell counts failed to rise much over 200 over 7 y of follow up. Insertion and deletion of HCV genomic fragments in the E1/E2 region was documented in one patient who developed fulminant hepatitis C.     

Analysis of envelope sequence variants suggests multiple mechanisms of mother-to-child transmission of human immunodeficiency virus type 1.

In order to elucidate the molecular mechanisms involved in human immunodeficiency virus type 1 (HIV-1) mother-to-child transmission, we have analyzed the genetic variation within the V3 hypervariable domain and flanking regions of the HIV-1 envelope gene in four mother-child transmission pairs. Phylogenetic analysis and amino acid sequence comparison were performed on cell-associated viral sequences derived from maternal samples collected at different time points during pregnancy, after delivery, and from child samples collected from the time of birth until the child was approximately 1 year of age. Heterogeneous sequence populations were observed to be present in all maternal samples collected during pregnancy and postdelivery. In three newborns, viral sequence populations obtained within 2 weeks after birth revealed a high level of V3 sequence variability. In contrast, V3 sequences obtained from the fourth child (diagnosed at the age of 1 month) displayed a more restricted heterogeneity. The phylogenetic analysis performed for each mother-child sequence set suggested that several mechanisms may potentially be involved in HIV-1 vertical transmission. For one pair, child sequences were homogeneous and clustered in a single branch within the phylogenetic tree, consistent with selective transmission of a single maternal variant. For the other three pairs, the child sequences were more heterogeneous and clustered in several separate branches within the tree. In these cases, it appeared likely that more than one maternal variant was responsible for infection of the child. In conclusion, no single mechanism can account for mother-to-child HIV-1 transmission; both the selective transmission of a single maternal variant and multiple transmission events may occur.     

Oxidative stress in small-for-gestational age (SGA) term newborns and their mothers

This study used malondialdehyde (MDA) determination by HPLC and enzymatic assays for total serum peroxides and antioxidant capacity to evaluate oxidative stress in 47 healthy full-term small-for-gestational age (SGA) newborns vs 67 appropriate-for-gestational age (AGA) newborns. Blood samples were collected at delivery from umbilical cord artery and vein and from peripheral blood of the babies on the third day after birth. Blood samples of mothers were also collected and compared with blood of 29 normal non-pregnant women (NPW). Serum peroxide values were significantly higher in both groups of mothers than in NPW, decreasing towards the third day in AGA mothers, while persisting in SGA mothers. Antioxidant capacity of sera of both groups of mothers was lower than NPW. Both SGA mothers and babies had increased MDA at delivery, unlike AGA counterparts. MDA levels in umbilical vein were higher than in umbilical arteries, while immunohistochemistry revealed abundant presence of 4-hydroxynonenal (HNE)-protein adducts only in stroma of the SGA placenta. These results show that both mothers and babies are exposed to oxidative stress during and after delivery, which is more pronounced and persistent in the perinatal period of the SGA group, while lipid peroxidation in placenta could play a role in SGA pathophysiology.     

Extensive genome-wide variability of human cytomegalovirus in congenitally infected infants

Research has shown that RNA virus populations are highly variable, most likely due to low fidelity replication of RNA genomes. It is generally assumed that populations of DNA viruses will be less complex and show reduced variability when compared to RNA viruses. Here, we describe the use of high throughput sequencing for a genome wide study of viral populations from urine samples of neonates with congenital human cytomegalovirus (HCMV) infections. We show that HCMV intrahost genomic variability, both at the nucleotide and amino acid level, is comparable to many RNA viruses, including HIV. Within intrahost populations, we find evidence of selective sweeps that may have resulted from immune-mediated mechanisms. Similarly, genome wide, population genetic analyses suggest that positive selection has contributed to the divergence of the HCMV species from its most recent ancestor. These data provide evidence that HCMV, a virus with a large dsDNA genome, exists as a complex mixture of genome types in humans and offer insights into the evolution of the virus.     

Highly sensitive and specific detection of rare variants in mixed viral populations from massively parallel sequence data

Viruses diversify over time within hosts, often undercutting the effectiveness of host defenses and therapeutic interventions. To design successful vaccines and therapeutics, it is critical to better understand viral diversification, including comprehensively characterizing the genetic variants in viral intra-host populations and modeling changes from transmission through the course of infection. Massively parallel sequencing technologies can overcome the cost constraints of older sequencing methods and obtain the high sequence coverage needed to detect rare genetic variants (< 1%) within an infected host, and to assay variants without prior knowledge. Critical to interpreting deep sequence data sets is the ability to distinguish biological variants from process errors with high sensitivity and specificity. To address this challenge, we describe V-Phaser, an algorithm able to recognize rare biological variants in mixed populations. V-Phaser uses covariation (i.e. phasing) between observed variants to increase sensitivity and an expectation maximization algorithm that iteratively recalibrates base quality scores to increase specificity. Overall, V-Phaser achieved > 97% sensitivity and > 97% specificity on control read sets. On data derived from a patient after four years of HIV-1 infection, V-Phaser detected 2,015 variants across the -10 kb genome, including 603 rare variants (< 1% frequency) detected only using phase information. V-Phaser identified variants at frequencies down to 0.2%, comparable to the detection threshold of allele-specific PCR, a method that requires prior knowledge of the variants. The high sensitivity and specificity of V-Phaser enables identifying and tracking changes in low frequency variants in mixed populations such as RNA viruses.     

Molecular Evidence for Mother-to-Child Transmission of Multiple Variants by Analysis of RNA and DNA Sequences of Human Immunodeficiency Virus Type 1

We have examined the viral selection that may occur during transmission by studying the env gene sequences from four cases of mother-to-child transmission of human immunodeficiency virus type 1. The V3 region sequences were directly amplified from both plasma viral RNA and peripheral blood mononuclear cells containing proviral DNA from mothers at delivery and at the time of diagnosis for children. Transmission occurred perinatally in three cases. The similarity of the viral sequences in each infant sample contrasted with the heterogeneous viral populations in the mothers. Phylogenetic analysis indicated the transmission of one or a few closely related maternal minor virus variants. In contrast, the child virus population in the fourth case was as heterogeneous as that of his mother, and phylogenetic analysis strongly suggested the transmission of multiple maternal variants. This case of multiple transmission was confirmed by analyzing sequences obtained at three times after delivery. Strains with sequences corresponding to the syncytium-inducing phenotype were also transmitted in this fourth case, and this was associated with the rapid development of disease in the child. There was no evidence for transmission of particular viral variants from mother to infant. We have thus described a particular case of vertical human immunodeficiency virus type 1 transmission with the transmission of multiple maternal variants to the infant and a rapid, fatal outcome in the child.     

Distinct viral populations differentiate and evolve independently in a single perennial host plant

The complex structure of virus populations has been the object of intensive study in bacteria, animals, and plants for over a decade. While it is clear that tremendous genetic diversity is rapidly generated during viral replication, the distribution of this diversity within a single host remains an obscure area in this field of science. Among animal viruses, only Human immunodeficiency virus and Hepatitis C virus populations have recently been thoroughly investigated at an intrahost level, where they are structured as metapopulations, demonstrating that the host cannot be considered simply as a "bag" containing a homogeneous or unstructured swarm of mutant viral genomes. In plants, a few reports suggested a possible heterogeneous distribution of virus variants at different locations within the host but provided no clues as to how this heterogeneity is structured. Here, we report the most exhaustive study of the structure and evolution of a virus population ever reported at the intrahost level through the analysis of a Prunus tree infected by Plum pox virus for over 13 years following a single inoculation event and by using analysis of molecular variance at different hierarchical levels combined with nested clade analysis. We demonstrate that, following systemic invasion of the host, the virus population differentiates into several distinct populations that are isolated in different branches, where they evolve independently through contiguous range expansion while colonizing newly formed organs. Moreover, we present and discuss evidence that the tree harbors a huge "bank" of viral clones, each isolated in one of the myriad leaves.     

Assessing the effects of human mixing patterns on human immunodeficiency virus-1 interhost phylogenetics through social network simulation

Geneticists seeking to understand HIV-1 evolution among human hosts generally assume that hosts represent a panmictic population. Social science research demonstrates that the network patterns over which HIV-1 spreads are highly nonrandom, but the effect of these patterns on the genetic diversity of HIV-1 and other sexually transmitted pathogens has yet to be thoroughly examined. In addition, interhost phylogenetic models rarely account explicitly for genetic diversity arising from intrahost dynamics. This study outlines a graph-theoretic framework (exponential random graph modeling, ERGM) for the estimation, inference, and simulation of dynamic partnership networks. This approach is used to simulate HIV-1 transmission and evolution under eight mixing patterns resembling those observed in empirical human populations, while simultaneously incorporating intrahost viral diversity. Models of parametric growth fit panmictic populations well, yielding estimates of total viral effective population on the order of the product of infected host size and intrahost effective viral population size. Populations exhibiting patterns of nonrandom mixing differ more widely in estimates of effective population size they yield, however, and reconstructions of population dynamics can exhibit severe errors if panmixis is assumed. I discuss implications for HIV-1 phylogenetics and the potential for ERGM to provide a general framework for addressing these issues.     

Contribution of intra- and interhost dynamics to norovirus evolution

Norovirus (NoV) is an emerging RNA virus that has been associated with global epidemics of gastroenteritis. Each global epidemic arises with the emergence of novel antigenic variants. While the majority of NoV infections are mild and self-limiting, in the young, elderly, and immunocompromised, severe and prolonged illness can result. As yet, there is no vaccine or therapeutic treatment to prevent or control infection. In order to design effective control strategies, it is important to understand the mechanisms and source of the new antigenic variants. In this study, we used next-generation sequencing (NGS) technology to investigate genetic diversification in three contexts: the impact of a NoV transmission event on viral diversity and the contribution to diversity of intrahost evolution over both a short period of time (10 days), in accordance with a typical acute NoV infection, and a prolonged period of time (288 days), as observed for NoV chronic infections of immunocompromised individuals. Investigations of the transmission event revealed that minor variants at frequencies as low as 0.01% were successfully transmitted, indicating that transmission is an important source of diversity at the interhost level of NoV evolution. Our results also suggest that chronically infected immunocompromised subjects represent a potential reservoir for the emergence of new viral variants. In contrast, in a typical acute NoV infection, the viral population was highly homogenous and relatively stable. These results indicate that the evolution of NoV occurs through multiple mechanisms.     

Sequential bottlenecks drive viral evolution in early acute hepatitis C virus infection

Hepatitis C is a pandemic human RNA virus, which commonly causes chronic infection and liver disease. The characterization of viral populations that successfully initiate infection, and also those that drive progression to chronicity is instrumental for understanding pathogenesis and vaccine design. A comprehensive and longitudinal analysis of the viral population was conducted in four subjects followed from very early acute infection to resolution of disease outcome. By means of next generation sequencing (NGS) and standard cloning/Sanger sequencing, genetic diversity and viral variants were quantified over the course of the infection at frequencies as low as 0.1%. Phylogenetic analysis of reassembled viral variants revealed acute infection was dominated by two sequential bottleneck events, irrespective of subsequent chronicity or clearance. The first bottleneck was associated with transmission, with one to two viral variants successfully establishing infection. The second occurred approximately 100 days post-infection, and was characterized by a decline in viral diversity. In the two subjects who developed chronic infection, this second bottleneck was followed by the emergence of a new viral population, which evolved from the founder variants via a selective sweep with fixation in a small number of mutated sites. The diversity at sites with non-synonymous mutation was higher in predicted cytotoxic T cell epitopes, suggesting immune-driven evolution. These results provide the first detailed analysis of early within-host evolution of HCV, indicating strong selective forces limit viral evolution in the acute phase of infection.     

Viral evolution and interferon resistance of hepatitis C virus RNA replication in a cell culture model

Hepatitis C virus (HCV) replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy. We evaluated HCV-IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV type 1b subgenomic RNA replicon that differed at only two sites within the NS5A-encoding region. A replicon with a K insertion at HCV codon 2040 replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure. In contrast, a replicon with an L2198S point mutation replicated poorly and triggered a cellular response characterized by IFN-beta production and low-level IFN-stimulated gene (ISG) expression. When maintained in long term-culture, the L2198S RNA evolved into a stable high-passage (HP) variant with six additional point mutations throughout the HCV protein-encoding region that enhanced viral replication. The HP RNA transduced Huh7 cells with more than 1,000-fold greater efficiency than its L2198S progenitor or the K2040 sequence. Replication of the HP RNA resisted suppression by IFN-alpha treatment and was associated with virus-directed reduction in host cell expression of ISG56, an antagonist of HCV RNA translation. Accordingly, the HP RNA was retained within polyribosome complexes in vivo that were refractory to IFN-induced disassembly. These results identify ISG56 as a translational control effector of the host response to HCV and provide direct evidence to link this response to viral sequence evolution, ISG regulation, and selection of the IFN-resistant viral phenotype.