Genomes of hepatitis E virus (HEV), rubivirus and cutthroat virus (CTV) contain a region of high proline density and low amino acid (aa) complexity, named the polyproline region (PPR). In HEV genotypes 1, 3 and 4, it is the only region within the non-structural open reading frame (ORF1) with positive selection (4–10 codons with dN/dS>1). This region has the highest density of sites with homoplasy values >0.5. Genotypes 3 and 4 show ∼3-fold increase in homoplastic density (HD) in the PPR compared to any other region in ORF1, genotype 1 does not exhibit significant HD (p<0.0001). PPR sequence divergence was found to be 2-fold greater for HEV genotypes 3 and 4 than for genotype 1. The data suggest the PPR plays an important role in host-range adaptation. Although the PPR appears to be hypervariable and homoplastic, it retains as much phylogenetic signal as any other similar sized region in the ORF1, indicating that convergent evolution operates within the major HEV phylogenetic lineages. Analyses of sequence-based secondary structure and the tertiary structure identify PPR as an intrinsically disordered region (IDR), implicating its role in regulation of replication. The identified propensity for the disorder-to-order state transitions indicates the PPR is involved in protein-protein interactions. Furthermore, the PPR of all four HEV genotypes contains seven putative linear binding motifs for ligands involved in the regulation of a wide number of cellular signaling processes. Structure-based analysis of possible molecular functions of these motifs showed the PPR is prone to bind a wide variety of ligands. Collectively, these data suggest a role for the PPR in HEV adaptation. Particularly as an IDR, the PPR likely contributes to fine tuning of viral replication through protein-protein interactions and should be considered as a target for development of novel anti-viral drugs.

Hepatitis E virus (HEV) is a human pathogen that causes acute hepatitis. When an HEV capsid protein containing a 52-amino-acid deletion at the C terminus and a 111-amino-acid deletion at the N terminus is expressed in insect cells, the recombinant HEV capsid protein can self-assemble into a T‫1؍‬ virus-like particle (VLP) that retains the antigenicity of the native HEV virion. In this study, we used cryoelectron microscopy and image reconstruction to show that anti-HEV monoclonal antibodies bind to the protruding domain of the capsid protein at the lateral side of the spikes. Molecular docking of the HEV VLP crystal structure revealed that Fab224 covered three surface loops of the recombinant truncated second open reading frame (ORF2) protein (PORF2) at the top part of the spike. We also determined the structure of a chimeric HEV VLP and located the inserted B-cell tag, an epitope of 11 amino acids coupled to the C-terminal end of the recombinant ORF2 protein. The binding site of Fab224 appeared to be distinct from the location of the inserted B-cell tag, suggesting that the chimeric VLP could elicit immunity against both HEV and an inserted foreign epitope. Therefore, the T‫1؍‬ HEV VLP is a novel delivery system for displaying foreign epitopes at the VLP surface in order to induce antibodies against both HEV and the inserted epitope.Hepatitis E virus (HEV) is a causative agent of acute hepatitis in humans and is primarily transmitted via the fecal-oral route. HEV is thus resistant to the low pH and digestive enzymes associated with the stomach and gastrointestinal tract. HEV regularly causes epidemics in many tropical and subtropical countries. In India, 101 outbreaks were confirmed by serological analysis in the state of Maharashtra in the last 5 years (6), and the lifetime risk of HEV infection exceeds 60% (28). Sporadic cases have also been reported in regions where HEV is endemic, as well as in areas where it is not endemic. Although some of these cases were associated with travel, many cases involved patients without a history of travel to regions where HEV is endemic. Accumulating evidence suggests that sporadic infection occurs through a zoonotic route and is not limited to developing countries. Seroprevalence suggests hepatitis E infection may also be prevalent in high-income countries (21), such as the United States (17), the United Kingdom (3), and Japan (18). The overall mortality rate of HEV infection during an outbreak generally ranges from 1 to 15%, and the highest mortality occurs in pregnant women, with fatality rates of up to 30% (19).The HEV virion is composed of a 7.2-kb single-stranded RNA molecule and a 32-to 34-nm icosahedral capsid. The HEV genome contains three open reading frames (ORFs).The capsid protein, encoded by the second open reading frame (ORF2), located at the 3Ј terminus of the genome, comprises 660 amino acids and is responsible for most capsid-related functions, such as assembly, host interaction, and immunogenicity. Recombinant ORF2 proteins can induce antibodies that block...

h GB virus B (GBV-B; family Flaviviridae, genus Hepacivirus) has been studied in New World primates as a model for human hepatitis C virus infection, but the distribution of GBV-B and its relatives in nature has remained obscure. Here, we report the discovery of a novel and highly divergent GBV-B-like virus in an Old World monkey, the black-and-white colobus (Colobus guereza), in Uganda. The new virus, guereza hepacivirus (GHV), clusters phylogenetically with GBV-B and recently described hepaciviruses infecting African bats and North American rodents, and it shows evidence of ancient recombination with these other hepaciviruses. Direct sequencing of reverse-transcribed RNA from blood plasma from three of nine colobus monkeys yielded near-complete GHV genomes, comprising two distinct viral variants. The viruses contain an exceptionally long nonstructural 5A (NS5A) gene, approximately half of which codes for a protein with no discernible homology to known proteins. Computational structure-based analyses indicate that the amino terminus of the GHV NS5A protein may serve a zinc-binding function, similar to the NS5A of other viruses within the family Flaviviridae. However, the 521-amino-acid carboxy terminus is intrinsically disordered, reflecting an unusual degree of structural plasticity and polyfunctionality. These findings shed new light on the natural history and evolution of the hepaciviruses and on the extent of structural variation within the Flaviviridae.

Hepatitis C virus is a genetically heterogeneous RNA virus that is a major cause of liver disease worldwide. Here, we show that, despite its extensive heterogeneity, the evolution of hepatitis C virus is primarily shaped by negative selection and that numerous coordinated substitutions in the polyprotein can be organized into a scale-free network whose degree of connections between sites follows a power-law distribution. This network shares all major properties with many complex biological and technological networks. The topological structure and hierarchical organization of this network suggest that a small number of amino acid sites exert extensive impact on hepatitis C virus evolution. Nonstructural proteins are enriched for negatively selected sites of high centrality, whereas structural proteins are enriched for positively selected sites located in the periphery of the network. The complex network of coordinated substitutions is an emergent property of genetic systems with implications for evolution, vaccine research, and drug development. In addition to such properties as polymorphism or strength of selection, the epistatic connectivity mapped in the network is important for typing individual sites, proteins, or entire genetic systems. The network topology may help devise molecular intervention strategies for disrupting viral functions or impeding compensatory changes for vaccine escape or drug resistance mutations. Also, it may be used to find new therapeutic targets, as suggested in this study for the NS4A protein, which plays an important role in the network.complex systems ͉ scale-free network ͉ covariation ͉ natural selection ͉ epistasis H epatitis C virus (HCV) is a major cause of liver disease worldwide. The global prevalence of HCV infection is estimated to be 2.2%, representing 130 million people (1). HCV causes chronic infection in 70-85% of infected adults (2). There is no vaccine against HCV and current antiviral therapy is relatively toxic, being effective in 50-60% of patients treated (3). HCV is a single-stranded RNA virus of Ϸ9.4 kb belonging to the Flaviviridae family (4). The positive-sense genome of HCV contains one large ORF that encodes a polyprotein that can undergo proteolytic cleavage into 10 mature proteins (C-E1-E2-P7-NS2-NS3-NS4A-NS4B-NS5A-NS5B). The structural proteins, the core (C) and envelope glycoproteins E1 and E2, are present in the N-terminal part of the polyprotein and presumably self-assemble to form the virion. The nonstructural (NS) proteins have various functions and form the replication complex (5).The HCV genome continually mutates during virus replication. Although a high rate of mutation significantly contributes to the enormous adaptability of RNA viruses, it also limits the size of viral genomes by causing error catastrophe (6). The small size of viral genomes imposes strong evolutionary constraints on their organization, as a result of which each genomic region may encode multiple and often conflicting functions. Such genomic organization requires a tight coor...

Treatment with lamivudine of patients infected with hepatitis B virus (HBV) results in a high rate of drug resistance, which is primarily associated with the rtM204I/V substitution in the HBV reverse transcriptase domain. Here we show that the rtM204I/V substitution, although essential, is insufficient for establishing resistance against lamivudine. The analysis of 639 HBV whole-genome sequences obtained from 11 patients shows that rtM204I/V is independently acquired by more than one intra-host HBV variant, indicating the convergent nature of lamivudine resistance. The differential capacity of HBV variants to develop drug resistance suggests that fitness effects of drug-resistance mutations depend on the genetic structure of the HBV genome. An analysis of Bayesian networks that connect rtM204I/V to many sites of HBV proteins confirms that lamivudine resistance is a complex trait encoded by the entire HBV genome rather than by a single mutation. These findings have implications for public health and offer a more general framework for understanding drug resistance.

Genotype-specific sensitivity of the hepatitis C virus (HCV) to interferon-ribavirin (IFN-RBV) combination therapy and reduced HCV response to IFN-RBV as infection progresses from acute to chronic infection suggest that HCV genetic factors and intrahost HCV evolution play important roles in therapy outcomes. HCV polyprotein sequences (n ‫؍‬ 40) from 10 patients with unsustainable response (UR) (breakthrough and relapse) and 10 patients with no response (NR) following therapy were identified through the Virahep-C study. Bayesian networks (BNs) were constructed to relate interrelationships among HCV polymorphic sites to UR/NR outcomes. All models showed an extensive interdependence of HCV sites and strong connections (P < 0.003) to therapy response. Although all HCV proteins contributed to the networks, the topological properties of sites differed among proteins. E2 and NS5A together contributed ϳ40% of all sites and ϳ62% of all links to the polyprotein BN. The NS5A BN and E2 BN predicted UR/NR outcomes with 85% and 97.5% accuracy, respectively, in 10-fold cross-validation experiments. The NS5A model constructed using physicochemical properties of only five sites was shown to predict the UR/NR outcomes with 83.3% accuracy for 6 UR and 12 NR cases of the HALT-C study. Thus, HCV adaptation to IFN-RBV is a complex trait encoded in the interrelationships among many sites along the entire HCV polyprotein. E2 and NS5A generate broad epistatic connectivity across the HCV polyprotein and essentially shape intrahost HCV evolution toward the IFN-RBV resistance. Both proteins can be used to accurately predict the outcomes of IFN-RBV therapy.Hepatitis C virus (HCV) is the major etiologic agent of blood-borne non-A, non-B hepatitis (25). Chronic HCV infection is an established risk factor for the development of liver diseases, such as fibrosis, cirrhosis, and hepatocellular carcinoma (33,124,125). Approximately 70% to 80% of HCVinfected patients fail to clear the virus and progress to chronicity (89a). At present, there are no preventive vaccines against HCV. The current, accepted therapeutic approach to treating chronic hepatitis C infection involves a 24-or 48-week course of pegylated alpha interferon (IFN-␣) combined with ribavirin (RBV) (i.e. IFN-RBV therapy) (48, 52). Because only 50% to 70% of chronically infected patients develop a sustained virologic response (SVR) to this treatment (48,52,55,80) and because patient intolerance to such therapy is common (61, 68, 120), the development and application of other therapeutic approaches using antiviral compounds that act against HCV more efficaciously and yet generate lower rates of adverse effects are major clinical management and public health objectives. Therapeutic failure presents in two forms: (i) complete resistance to treatment (no response [NR]) and (ii) unsustainable response (UR), which is characterized by an increase in HCV load observed during therapy after an initial period of decline in viral load (breakthrough) or observed after cessation of therapy (relapse)...

BackgroundIdentification of acute or recent hepatitis C virus (HCV) infections is important for detecting outbreaks and devising timely public health interventions for interruption of transmission. Epidemiological investigations and chemistry-based laboratory tests are 2 main approaches that are available for identification of acute HCV infection. However, owing to complexity, both approaches are not efficient. Here, we describe a new sequence alignment-free method to discriminate between recent (R) and chronic (C) HCV infection using next-generation sequencing (NGS) data derived from the HCV hypervariable region 1 (HVR1).ResultsUsing dinucleotide auto correlation (DAC), we identified physical-chemical (PhyChem) features of HVR1 variants. Significant (p < 9.58 × 10−4) differences in the means and frequency distributions of PhyChem features were found between HVR1 variants sampled from patients with recent vs chronic (R/C) infection. Moreover, the R-associated variants were found to occupy distinct and discrete PhyChem spaces. A radial basis function neural network classifier trained on the PhyChem features of intra-host HVR1 variants accurately classified R/C-HVR1 variants (classification accuracy (CA) = 94.85%; area under the ROC curve, AUROC = 0.979), in 10-fold cross-validation). The classifier was accurate in assigning individual HVR1 variants to R/C-classes in the testing set (CA = 84.15%; AUROC = 0.912) and in detection of infection duration (R/C-class) in patients (CA = 88.45%). Statistical tests and evaluation of the classifier on randomly-labeled datasets indicate that classifiers’ CA is robust (p < 0.001) and unlikely due to random correlations (CA = 59.04% and AUROC = 0.50).ConclusionsThe PhyChem features of intra-host HVR1 variants are strongly associated with the duration of HCV infection. Application of the PhyChem biomarkers to models for detection of the R/C-state of HCV infection in patients offers a new opportunity for detection of outbreaks and for molecular surveillance. The method will be available at https://webappx.cdc.gov/GHOST/ to the authenticated users of Global Hepatitis Outbreak and Surveillance Technology (GHOST) for further testing and validation.

BackgroundChronic infection with hepatitis C virus (HCV) is a risk factor for liver diseases such as fibrosis, cirrhosis and hepatocellular carcinoma. HCV genetic heterogeneity was hypothesized to be associated with severity of liver disease. However, no reliable viral markers predicting disease severity have been identified. Here, we report the utility of sequences from 3 HCV 1b genomic regions, Core, NS3 and NS5b, to identify viral genetic markers associated with fast and slow rate of fibrosis progression (RFP) among patients with and without liver transplantation (n = 42).MethodsA correlation-based feature selection (CFS) method was used to detect and identify RFP-relevant viral markers. Machine-learning techniques, linear projection (LP) and Bayesian Networks (BN), were used to assess and identify associations between the HCV sequences and RFP.ResultsBoth clustering of HCV sequences in LP graphs using physicochemical properties of nucleotides and BN analysis using polymorphic sites showed similarities among HCV variants sampled from patients with a similar RFP, while distinct HCV genetic properties were found associated with fast or slow RFP. Several RFP-relevant HCV sites were identified. Computational models parameterized using the identified sites accurately associated HCV strains with RFP in 70/30 split cross-validation (90-95% accuracy) and in validation tests (85-90% accuracy). Validation tests of the models constructed for patients with or without liver transplantation suggest that the RFP-relevant genetic markers identified in the HCV Core, NS3 and NS5b genomic regions may be useful for the prediction of RFP regardless of transplant status of patients.ConclusionsThe apparent strong genetic association to RFP suggests that HCV genetic heterogeneity has a quantifiable effect on severity of liver disease, thus presenting opportunity for developing genetic assays for measuring virulence of HCV strains in clinical and public health settings.