Human CD8+ T cell cross-reactivity across influenza A, B and C viruses.

Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8+ T cells confer cross-protection against IAV strains, however the responses of CD8+ T cells to IBV and ICV are understudied. We investigated the breadth of CD8+ T cell cross-recognition and provide evidence of CD8+ T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8+ T cell epitopes from IBVs that were protective in mice and found memory CD8+ T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8+ T cells displayed tissue-resident memory phenotypes. Notably, CD38+Ki67+CD8+ effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8+ T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines.

B-cell characteristics define HCV reinfection outcome.

BACKGROUND & AIMS: In individuals highly exposed to HCV, reinfection is common, suggesting that natural development of sterilising immunity is difficult. In those that are reinfected, some will develop a persistent infection, while a small proportion repeatedly clear the virus, suggesting natural protection is possible. The aim of this study was to characterise immune responses associated with rapid natural clearance of HCV reinfection. METHODS: Broad neutralising antibodies (nAbs) and Envelope 2 (E2)-specific memory B cell (MBC) responses were examined longitudinally in 15 individuals with varied reinfection outcomes. RESULTS: Broad nAb responses were associated with MBC recall, but not with clearance of reinfection. Strong evidence of antigen imprinting was found, and the B-cell receptor repertoire showed a high level of clonality with ongoing somatic hypermutation of many clones over subsequent reinfection events. Single-cell transcriptomic analyses showed that cleared reinfections featured an activated transcriptomic profile in HCV-specific B cells that rapidly expanded upon reinfection. CONCLUSIONS: MBC quality, but not necessarily breadth of nAb responses, is important for protection against antigenically diverse variants, which is encouraging for HCV vaccine development. IMPACT AND IMPLICATIONS: HCV continues to have a major health burden globally. Limitations in the health infrastructure for diagnosis and treatment, as well as high rates of reinfection, indicate that a vaccine that can protect against chronic HCV infection will greatly complement current efforts to eliminate HCV-related disease. With alternative approaches to testing vaccines, such as controlled human inoculation trials under consideration, we desperately need to identify the correlates of immune protection. In this study, in a small but rare cohort of high-risk injecting drug users who were reinfected multiple times, breadth of neutralisation was not associated with ultimate clearance of the reinfection event. Alternatively, characteristics of the HCV-specific B-cell response associated with B-cell proliferation were. This study indicates that humoral responses are important for protection and suggests that for genetically very diverse viruses, such as HCV, it may be beneficial to look beyond just antibodies as correlates of protection.

B cell immunodominance in primary hepatitis C virus infection.

BACKGROUND & AIMS: Neutralising antibodies (NAbs) play a key role in clearance of HCV. NAbs have been isolated and mapped to several domains on the HCV envelope proteins. However, the immunodominance of these epitopes in HCV infection remains unknown, hindering efforts to elicit optimal epitope-specific responses. Furthermore, it remains unclear which epitope-specific responses are associated with broad NAb (bNAb) activity in primary HCV infection. The aim of this study was to define B cell immunodominance in primary HCV, and its implications on neutralisation breadth and clearance. METHODS: Using samples from 168 patients with primary HCV infection, the antibody responses targeted 2 immunodominant domains, termed domains B and C. Genotype 1 and 3 infections were associated with responses targeted towards different bNAb domains. RESULTS: No epitopes were uniquely targeted by clearers compared to those who developed chronic infection. Samples with bNAb activity were enriched for multi-specific responses directed towards the epitopes antigenic region 3, antigenic region 4, and domain D, and did not target non-neutralising domains. CONCLUSIONS: This study outlines for the first time a clear NAb immunodominance profile in primary HCV infection, and indicates that it is influenced by the infecting virus. It also highlights the need for a vaccination strategy to induce multi-specific responses that do not target non-neutralising domains. LAY SUMMARY: Neutralising antibodies will likely form a key component of a protective hepatitis C virus vaccine. In this work we characterise the predominant neutralising and non-neutralising antibody (epitope) targets in acute hepatitis C virus infection. We have defined the natural hierarchy of epitope immunodominance, and demonstrated that viral genotype can impact on this hierarchy. Our findings highlight key epitopes that are associated with broadly neutralising antibodies, and the deleterious impact of mounting a response towards some of these domains on neutralising breadth. These findings should guide future efforts to design immunogens aimed at generating neutralising antibodies with a vaccine candidate.

B-cell receptor reconstruction from single-cell RNA-seq with VDJPuzzle.

Motivation: The B-cell receptor (BCR) performs essential functions for the adaptive immune system including recognition of pathogen-derived antigens. The vast repertoire and adaptive variation of BCR sequences due to V(D)J recombination and somatic hypermutation necessitates single-cell characterization of BCR sequences. Single-cell RNA sequencing presents the opportunity for simultaneous capture of paired BCR heavy and light chains and the transcriptomic signature. Results: We developed VDJPuzzle, a novel bioinformatic tool that reconstructs productive, full-length B-cell receptor sequences of both heavy and light chains and extract somatic mutations on the VDJ region. VDJPuzzle successfully reconstructed BCRs from 100% (n=117) human and 96.5% (n=200) murine B cells. The reconstructed BCRs were successfully validated with single-cell Sanger sequencing. Availability and implementation: VDJPuzzle is available at https://bitbucket.org/kirbyvisp/vdjpuzzle2. Supplementary information: Supplementary data are available at Bioinformatics online.

Genomic variability of within-host hepatitis C variants in acute infection.

Interactions between the host immune system and the viral variants determine persistence of hepatitis C virus (HCV) infection after the acute phase of infection. This study describes the genetic variability of within-host HCV viral variants in acute infection and correlates it with host- and virus-related traits and infection outcome. Next generation sequence data (Illumina, MiSeq platform) of viral genomes from 116 incident acute infections (within 180 days of infection) were analysed to determine all the single nucleotide polymorphism (SNP) frequencies above a threshold of 0.1%. The variability of the SNPs for the full open reading frame of the genome as well as for each protein coding region were compared using mean standardized Shannon entropy (SE) values calculated separately for synonymous and nonsynonymous mutations. The envelope glycoproteins regions (E1 and E2) had the highest SE values (indicating greater variability) followed by the NS5B region. Nonsynonymous mutations rather than synonymous mutations were the main contributors to genomic variability in acute infection. The mean difference of Shannon entropy was also compared between subjects after categorizing the samples according to host and virus-related traits. Host IFNL3 allele CC polymorphism at rs12979860 (vs others) and viral genotype 1a (vs 3a) were associated with higher genomic variability across the viral open reading frame. Time since infection, host gender or continent of origin was not associated with the viral genomic variability. Viral genomic variability did not predict spontaneous clearance.

Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses.

Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3' untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and ß-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as "vesicle packets" (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.

Historical Trends in the Hepatitis C Virus Epidemics in North America and Australia.

BACKGROUND: Bayesian evolutionary analysis (coalescent analysis) based on genetic sequences has been used to describe the origins and spread of rapidly mutating RNA viruses, such as influenza, Ebola, human immunodeficiency virus (HIV), and hepatitis C virus (HCV). METHODS: Full-length subtype 1a and 3a sequences from early HCV infections from the International Collaborative of Incident HIV and Hepatitis C in Injecting Cohorts (InC3), as well as from public databases from a time window of 1977-2012, were used in a coalescent analysis with BEAST software to estimate the origin and progression of the HCV epidemics in Australia and North America. Convergent temporal trends were sought via independent epidemiological modeling. RESULTS: The epidemic of subtype 3a had more recent origins (around 1950) than subtype 1a (around 1920) in both continents. In both modeling approaches and in both continents, the epidemics underwent exponential growth between 1955 and 1975, which then stabilized in the late 20th century. CONCLUSIONS: Historical events that fuelled the emergence and spread of injecting drug use, such as the advent of intravenous medical therapies and devices, and growth in the heroin trade, as well as population mixing during armed conflicts, were likely drivers for the cross-continental spread of the HCV epidemics.

A method for near full-length amplification and sequencing for six hepatitis C virus genotypes.

BACKGROUND: Hepatitis C virus (HCV) is a rapidly evolving RNA virus that has been classified into seven genotypes. All HCV genotypes cause chronic hepatitis, which ultimately leads to liver diseases such as cirrhosis. The genotypes are unevenly distributed across the globe, with genotypes 1 and 3 being the most prevalent. Until recently, molecular epidemiological studies of HCV evolution within the host and at the population level have been limited to the analyses of partial viral genome segments, as it has been technically challenging to amplify and sequence the full-length of the 9.6 kb HCV genome. Although recent improvements have been made in full genome sequencing methodologies, these protocols are still either limited to a specific genotype or cost-inefficient. RESULTS: In this study we describe a genotype-specific protocol for the amplification and sequencing of the near-full length genome of all six major HCV genotypes. We applied this protocol to 122 HCV positive clinical samples, and had a successful genome amplification rate of 90%, when the viral load was greater than 15,000 IU/ml. The assay was shown to have a detection limit of 1-3 cDNA copies per reaction. The method was tested with both Illumina and PacBio single molecule, real-time (SMRT) sequencing technologies. Illumina sequencing resulted in deep coverage and allowed detection of rare variants as well as HCV co-infection with multiple genotypes. The application of the method with PacBio RS resulted in sequence reads greater than 9 kb that covered the near full-length HCV amplicon in a single read and enabled analysis of the near full-length quasispecies. CONCLUSIONS: The protocol described herein can be utilised for rapid amplification and sequencing of the near-full length HCV genome in a cost efficient manner suitable for a wide range of applications.

Linking the T cell receptor to the single cell transcriptome in antigen-specific human T cells.

Heterogeneity of T cells is a hallmark of a successful adaptive immune response, harnessing the vast diversity of antigen-specific T cells into a coordinated evolution of effector and memory outcomes. The T cell receptor (TCR) repertoire is highly diverse to account for the highly heterogeneous antigenic world. During the response to a virus multiple individual clones of antigen specific CD8+ (Ag-specific) T cells can be identified against a single epitope and multiple epitopes are recognised. Advances in single-cell technologies have provided the potential to study Ag-specific T cell heterogeneity at both surface phenotype and transcriptome levels, thereby allowing investigation of the diversity within the same apparent sub-population. We propose a new method (VDJPuzzle) to reconstruct the native TCRαß from single cell RNA-seq data of Ag-specific T cells and then to link these with the gene expression profile of individual cells. We applied this method using rare Ag-specific T cells isolated from peripheral blood of a subject who cleared hepatitis C virus infection. We successfully reconstructed productive TCRαß in 56 of a total of 63 cells (89%), with double α and double ß in 18, and 7% respectively, and double TCRαß in 2 cells. The method was validated via standard single cell PCR sequencing of the TCR. We demonstrate that single-cell transcriptome analysis can successfully distinguish Ag-specific T cell populations sorted directly from resting memory cells in peripheral blood and sorted after ex vivo stimulation. This approach allows a detailed analysis of the TCR diversity and its relationship with the transcriptional profile of different clones.

Cross-genotypic examination of hepatitis C virus polymerase inhibitors reveals a novel mechanism of action for thumb binders.

Direct-acting antivirals (DAAs) targeting proteins encoded by the hepatitis C virus (HCV) genome have great potential for the treatment of HCV infections. However, the efficacy of DAAs designed to target genotype 1 (G1) HCV against non-G1 viruses has not been characterized fully. In this study, we investigated the inhibitory activities of nonnucleoside inhibitors (NNIs) against the HCV RNA-dependent RNA polymerase (RdRp). We examined the ability of six NNIs to inhibit G1b, G2a, and G3a subgenomic replicons in cell culture, as well as in vitro transcription by G1b and G3a recombinant RdRps. Of the six G1 NNIs, only the palm II binder nesbuvir demonstrated activity against G1, G2, and G3 HCV, in both replicon and recombinant enzyme models. The thumb I binder JTK-109 also inhibited G1b and G3a replicons and recombinant enzymes but was 41-fold less active against the G2a replicon. The four other NNIs, which included a palm I binder (setrobuvir), two thumb II binders (lomibuvir and filibuvir), and a palm ß-hairpin binder (tegobuvir), all showed at least 40-fold decreases in potency against G2a and G3a replicons and the G3a enzyme. This antiviral resistance was largely conferred by naturally occurring amino acid residues in the G2a and G3a RdRps that are associated with G1 resistance. Lomibuvir and filibuvir (thumb II binders) inhibited primer-dependent but not de novo activity of the G1b polymerase. Surprisingly, these compounds instead specifically enhanced the de novo activity at concentrations of ≥ 100 nM. These findings highlight a potential differential mode of RdRp inhibition for HCV NNIs, depending on their prospective binding pockets, and also demonstrate a surprising enhancement of de novo activity for thumb RdRp binders. These results also provide a better understanding of the antiviral coverage for these polymerase inhibitors, which will likely be used in future combinational interferon-free therapies.

Nonnucleoside inhibitors of norovirus RNA polymerase: scaffolds for rational drug design.

Norovirus (NoV) is the leading cause of acute gastroenteritis worldwide, causing over 200,000 deaths a year. NoV is nonenveloped, with a single-stranded RNA genome, and is primarily transmitted person to person. The viral RNA-dependent RNA polymerase (RdRp) is critical for the production of genomic and subgenomic RNA and is therefore a prime target for antiviral therapies. Using high-throughput screening, nearly 20,000 "lead-like" compounds were tested for inhibitory activity against the NoV genogroup II, genotype 4 (GII.4) RdRp. The four most potent hits demonstrated half-maximal inhibitory concentrations (IC50s) between 5.0 µM and 9.8 µM against the target RdRp. Compounds NIC02 and NIC04 revealed a mixed mode of inhibition, while NIC10 and NIC12 were uncompetitive RdRp inhibitors. When examined using enzymes from related viruses, NIC02 demonstrated broad inhibitory activity while NIC04 was the most specific GII.4 RdRp inhibitor. The antiviral activity was examined using available NoV cell culture models; the GI.1 replicon and the infectious GV.1 murine norovirus (MNV). NIC02 and NIC04 inhibited the replication of the GI.1 replicon, with 50% effective concentrations (EC50s) of 30.1 µM and 71.1 µM, respectively, while NIC10 and NIC12 had no observable effect on the NoV GI.1 replicon. In the MNV model, NIC02 reduced plaque numbers, size, and viral RNA levels in a dose-dependent manner (EC50s between 2.3 µM and 4.8 µM). The remaining three compounds also reduced MNV replication, although with higher EC50s, ranging from 32 µM to 38 µM. In summary, we have identified novel nonnucleoside inhibitor scaffolds that will provide a starting framework for the development and future optimization of targeted antivirals against NoV.

Envelope-Specific IgG3 and IgG1 Responses Are Associated with Clearance of Acute Hepatitis C Virus Infection.

Hepatitis C virus (HCV) can be cleared naturally in a subset of individuals. However, the asymptomatic nature of acute HCV infection makes the study of the early immune response and defining the correlates of protection challenging. Despite this, there is now strong evidence implicating the humoral immune response, specifically neutralising antibodies, in determining the clearance or chronicity outcomes of primary HCV infection. In general, immunoglobulin G (IgG) plays the major role in viral neutralisation. However, there are limited investigations of anti-HCV envelope protein 2 (E2) isotypes (IgM, IgG, IgA) and IgG subclasses (IgG1-4) in early HCV infection. In this study, using a rare cohort of 14 very recently HCV-infected individuals (4-45 days) with varying disease outcome (n = 7 clearers), the timing and potency of anti-HCV E2 isotypes and IgG subclasses were examined longitudinally, in relation to neutralising antibody activity. Clearance was associated with anti-E2 IgG, specifically IgG1 and IgG3, and appeared essential to prevent the emergence of new HCV variants and the chronic infection outcome. Interestingly, these IgG responses were accompanied by IgM antibodies and were associated with neutralising antibody activity in the subjects who cleared infection. These findings provide novel insights into the early humoral immune response characteristics associated with HCV disease outcome.

Single-Cell Transcriptome Analysis of T Cells.

Single-cell RNA-seq (scRNA-seq) has provided novel routes to investigate the heterogeneous populations of T cells and is rapidly becoming a common tool for molecular profiling and identification of novel subsets and functions. This chapter offers an experimental and computational workflow for scRNA-seq analysis of T cells. We focus on the analyses of scRNA-seq data derived from plate-based sorted T cells using flow cytometry and full-length transcriptome protocols such as Smart-Seq2. However, the proposed pipeline can be applied to other high-throughput approaches such as UMI-based methods. We describe a detailed bioinformatics pipeline that can be easily reproduced and discuss future directions and current limitations of these methods in the context of T cell biology.

Clearance of hepatitis C virus is associated with early and potent but narrowly-directed, Envelope-specific antibodies.

Hepatitis C virus (HCV) is one of very few viruses that are either naturally cleared, or alternatively persist to cause chronic disease. Viral diversity and escape, as well as host adaptive immune factors, are believed to control the outcome. To date, there is limited understanding of the critical, early host-pathogen interactions. The asymptomatic nature of early HCV infection generally prevents identification of the transmitted/founder (T/F) virus, and thus the study of host responses directed against the autologous T/F strain. In this study, 14 rare subjects identified from very early in infection (4-45 days) with varied disease outcomes (n = 7 clearers) were examined in regard to the timing, breadth, and magnitude of the neutralizing antibody (nAb) response, as well as evolution of the T/F strain. Clearance was associated with earlier onset and more potent nAb responses appearing at a mean of 71 days post-infection (DPI), but these responses were narrowly directed against the autologous T/F virus or closely related variants. In contrast, a delayed onset of nAbs (mean 425 DPI) was observed in chronic progressors that appear to have targeted longitudinal variants rather than the T/F strain. The nAb responses in the chronic progressors mapped to known CD81 binding epitopes, and were associated with rapid emergence of new viral variants with reduced CD81 binding. We propose that the prolonged period of viremia in the absence of nAbs in these subjects was associated with an increase in viral diversity, affording the virus greater options to escape nAb pressure once it emerged. These findings indicate that timing of the nAb response is essential for clearance. Further investigation of the specificities of the early nAbs and the factors regulating early induction of protective nAbs is needed.

A method for detecting hepatitis C envelope specific memory B cells from multiple genotypes using cocktail E2 tetramers.

Hepatitis C (HCV) is a rapidly mutating RNA virus, with a strong propensity to cause chronic infection and progressive liver disease. Recent evidence has shown that early appearance of neutralizing antibodies in primary infection is associated with clearance. Little is known about the characteristics of HCV-specific B cells and their correlation with outcomes in primary infection, as there is a lack of sensitive tools for HCV-specific B cells which are present at very low frequency. We describe the development and optimisation of tetramer staining for flow cytometric detection of HCV-specific B cells using a cocktail of two recombinant HCV Envelope-2 (rE2) glycoproteins (from genotype 1a and 3a; Gt1a and Gt3a) and streptavidin dyes. The optimal weight to weight (w/w) ratio of streptavidin-phycoerythrin (PE) and rE2 proteins were determined for sensitive detection using HCV E2-specific hybridoma cell lines and peripheral blood mononuclear cells (PBMC) from HCV-infected individuals. In a cross-sectional set of PBMC samples collected from 33 subjects with either chronic infection or previous clearance, HCV E2-specific B cells (CD19+CD20+CD10-IgD-tetramer+) were detected in 29 subjects (87.8%), with a mean frequency of 0.45% (0.012-2.20%). To validate the specificity of tetramer staining, 367 HCV E2-specific B cells were single cell sorted from 9 PBMC samples before monoclonal antibodies (mAbs) were synthesised, with 87.5% being reactive to E2 via ELISA. Of these mAbs, 284 and 246 clones were reactive to either Gt1a or Gt3a E2 proteins, respectively. This is a sensitive and robust method for future studies investigating B cell responses against the HCV Envelope protein.

Understanding the Determinants of BnAb Induction in Acute HCV Infection.

Despite recent advances in curative therapy, hepatitis C virus (HCV) still remains a global threat. In order to achieve global elimination, a prophylactic vaccine should be considered high priority. Previous immunogens used to induce broad neutralising antibodies (BnAbs) have been met with limited success. To improve immunogen design, factors associated with the early development of BnAbs in natural infection must first be understood. In this study, 43 subjects identified with acute HCV were analysed longitudinally using a panel of heterogeneous HCV pseudoparticles (HCVpp), to understand the emergence of BnAbs. Compared to those infected with a single genotype, early BnAb development was associated with subjects co-infected with at least 2 HCV subtypes during acute infection. In those that were mono-infected, BnAbs were seen to emerge with increasing viral persistence. If subjects acquired a secondary infection, nAb breadth was seen to boost upon viral re-exposure. Importantly, this data highlights the potential for multivalent and prime-boost vaccine strategies to induce BnAbs against HCV in humans. However, the data also indicate that the infecting genotype may influence the development of BnAbs. Therefore, the choice of antigen will need to be carefully considered in future vaccine trials.

Clonally diverse CD38+HLA-DR+CD8+ T cells persist during fatal H7N9 disease.

Severe influenza A virus (IAV) infection is associated with immune dysfunction. Here, we show circulating CD8+ T-cell profiles from patients hospitalized with avian H7N9, seasonal IAV, and influenza vaccinees. Patient survival reflects an early, transient prevalence of highly activated CD38+HLA-DR+PD-1+ CD8+ T cells, whereas the prolonged persistence of this set is found in ultimately fatal cases. Single-cell T cell receptor (TCR)-αß analyses of activated CD38+HLA-DR+CD8+ T cells show similar TCRαß diversity but differential clonal expansion kinetics in surviving and fatal H7N9 patients. Delayed clonal expansion associated with an early dichotomy at a transcriptome level (as detected by single-cell RNAseq) is found in CD38+HLA-DR+CD8+ T cells from patients who succumbed to the disease, suggesting a divergent differentiation pathway of CD38+HLA-DR+CD8+ T cells from the outset during fatal disease. Our study proposes that effective expansion of cross-reactive influenza-specific TCRαß clonotypes with appropriate transcriptome signatures is needed for early protection against severe influenza disease.

Understanding the complex evolution of rapidly mutating viruses with deep sequencing: Beyond the analysis of viral diversity.

With the advent of affordable deep sequencing technologies, detection of low frequency variants within genetically diverse viral populations can now be achieved with unprecedented depth and efficiency. The high-resolution data provided by next generation sequencing technologies is currently recognised as the gold standard in estimation of viral diversity. In the analysis of rapidly mutating viruses, longitudinal deep sequencing datasets from viral genomes during individual infection episodes, as well as at the epidemiological level during outbreaks, now allow for more sophisticated analyses such as statistical estimates of the impact of complex mutation patterns on the evolution of the viral populations both within and between hosts. These analyses are revealing more accurate descriptions of the evolutionary dynamics that underpin the rapid adaptation of these viruses to the host response, and to drug therapies. This review assesses recent developments in methods and provide informative research examples using deep sequencing data generated from rapidly mutating viruses infecting humans, particularly hepatitis C virus (HCV), human immunodeficiency virus (HIV), Ebola virus and influenza virus, to understand the evolution of viral genomes and to explore the relationship between viral mutations and the host adaptive immune response. Finally, we discuss limitations in current technologies, and future directions that take advantage of publically available large deep sequencing datasets.

Broad-spectrum non-nucleoside inhibitors for caliciviruses.

Viruses of the Caliciviridae cause significant and sometimes lethal diseases, however despite substantial research efforts, specific antivirals are lacking. Broad-spectrum antivirals could combat multiple viral pathogens, offering a rapid solution when no therapies exist. The RNA-dependent RNA polymerase (RdRp) is an attractive antiviral target as it is essential for viral replication and lacks mammalian homologs. To focus the search for pan-Caliciviridae antivirals, the RdRp was probed with non-nucleoside inhibitors (NNIs) developed against hepatitis C virus (HCV) to reveal both allosteric ligands for structure-activity relationship enhancement, and highly-conserved RdRp pockets for antiviral targeting. The ability of HCV NNIs to inhibit calicivirus RdRp activities was assessed using in vitro enzyme and murine norovirus cell culture assays. Results revealed that three NNIs which bound the HCV RdRp Thumb I (TI) site also inhibited transcriptional activities of six RdRps spanning the Norovirus, Sapovirus and Lagovirus genera of the Caliciviridae. These NNIs included JTK-109 (RdRp inhibition range: IC50 4.3-16.6 µM), TMC-647055 (IC50 range: 18.8-45.4 µM) and Beclabuvir (IC50 range: 23.8->100 µM). In silico studies and site-directed mutagenesis indicated the JTK-109 binding site was within the calicivirus RdRp thumb domain, in a pocket termed Site-B, which is highly-conserved within all calicivirus RdRps. Additionally, RdRp inhibition assays revealed that JTK-109 was antagonistic with the previously reported RdRp inhibitor pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) tetrasodium salt (PPNDS), that also binds to Site-B. Moreover, like JTK-109, PPNDS was also a potent inhibitor of polymerases from six viruses spanning the three Caliciviridae genera tested (IC50 range: 0.1-2.3 µM). Together, this study demonstrates the potential for de novo development of broad-spectrum antivirals that target the highly-conserved RdRp thumb pocket, Site-B. We also revealed three broad-spectrum HCV NNIs that could be used as antiviral scaffolds for further development against caliciviruses and other viruses.

Dynamic evolution of hepatitis C virus resistance-associated substitutions in the absence of antiviral treatment.

Resistance against new hepatitis C virus (HCV) antivirals is an area of increasing interest. Resistance-associated substitutions (RASs) have been identified in treatment-naïve individuals, but pressures driving treatment-independent RAS emergence are poorly understood. We analysed the longitudinal evolution of RASs in twelve participants with early acute HCV infections. Full-genome deep sequences were analysed for changes in RAS frequency within NS3, NS5A and NS5B-coding regions over the course of the infection. Emergence of RASs relevant only to the polymerase non-nucleoside inhibitors (NNI) was detected, and these lay within CD8+ T-cell epitopes. Conversely, the loss of NNI RASs over time appeared likely to be driven by viral fitness constraints. These results highlight the importance of monitoring CD8+ T cell epitope-associated RASs in populations with dominant HLA types.