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Interpatient evolution of HCV in a country. Maximum Clade Credibility Tree constructed by Bayesian coalescent methodology from direct sequences of the first half of the E2 region in plasma samples from HCV genotype 1a isolations in Argentina. The tree is presented in time scale (calendar years). The overall process is depicted by the diversification of the current lineages from the 60's to the 80's independently of the actual time of sampling. The arrows indicate possible bottleneck effects. The dark red one indicates the possible introduction of HCV in the community, whereas the magenta ones may indicate the acquisition of the virus by different routes of transmission. Virtually, every transmission event is thought to be a bottleneck effect for virus diversity. The light blue shaded area represents the viral genetic diversity trough the time measured by the Bayesian Skyline methodology. 65
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Hepatitis C virus (HCV) has a short replication time, high mutation rates and large population sizes, all of which make it an excellent experimental model for evolution studies, because evolution can be visualized in real-time. In this review, we discuss the implications to study HCV evolution at the interpatient and intrapatient levels of infectio...
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Context 1
... In this context, it is known that HCV phylogenetic trees often span several decades so the time when two viral lineages coalesce is close (relative to the timescale of the phylogeny) to the actual transmission times. Accordingly, interpatient phylogenies of HCV sampled through time are not stepwise and show the persistence of multiple line- ages through time (Figure 1). The shapes of these trees are primarily determined by neutral demo- graphic and spatial dynamics in both immune-medi- ated positive selection and purifying selection for replication ability. ...
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Viral quasispecies (QS) has long been considered to affect the efficiency of hepatitis C virus (HCV) antiviral therapy. Yet a correlation between the QS diversity and treatment outcomes has not been established conclusively. We previously measured HCV QS diversity by genome-wide quantification of high-resolution mutation load in HCV 1a patients ach...
Background
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Citations
... Another possibility for the persistence of the C-A314G (K73R) mutation is the reduction in genetics of the viral population which reflects on the original members of a population which is mainly seen during the transmission of viruses from one host to another. Di Lello et al previously reported that during the transmission of hepatitis C virus from one individual to another, the genetic diversity of the virus was found to be decreased [61]. Another study also showed the decrease in genetic diversity of human-immunodeficiency virus (HIV) in HIVinfected individuals that shared needles during drug use [62]. ...
Dengue virus (DENV) causes approximately 390 million dengue infections worldwide every year. There were 22,777 reported DENV infections in Tainan, Taiwan in 2015. In this study, we sequenced the C-prM-E genes from 45 DENV 2015 strains, and phylogenetic analysis based on C-prM-E genes revealed that all strains were classified as DENV serotype 2 Cosmopolitan genotype. Sequence analysis comparing different DENV-2 genotypes and Cosmopolitan DENV-2 sequences prior to 2015 showed a clade replacement event in the DENV-2 Cosmopolitan genotype. Additionally, a major substitution C-A314G (K73R) was found in the capsid region which may have contributed to the clade replacement event. Reverse genetics virus rgC-A314G (K73R) showed slower replication in BHK-21 and C6/36 cells compared to wildtype virus, as well as a decrease in NS1 production in BHK-21-infected cells. After a series of passaging, the C-A314G (K73R) mutation reverted to wildtype and was thus considered to be unstable. Next generation sequencing (NGS) of three sera collected from a single DENV2-infected patient at 1-, 2-, and 5-days post-admission was employed to examine the genetic diversity over-time and mutations that may work in conjunction with C-A314G (K73R). Results showed that the number of haplotypes decreased with time in the DENV-infected patient. On the fifth day after admission, two new haplotypes emerged, and a single non-synonymous NS4A-L115I mutation was identified. Therefore, we have identified a persistent mutation C-A314G (K73R) in all of the DENV-2 isolates, and during the course of an infection, a single new non-synonymous mutation in the NS4A region appears in the virus population within a single host. The C-A314G (K73R) thus may have played a role in the DENV-2 2015 outbreak while the NS4A-L115I may be advantageous during DENV infection within the host.
... [17] As a result, we clarified for the first time that DAA-resistant HCV consisted of resistant virus haplotypes, quasispecies with various HASs, and changed over time during the treatment with different RASs. This indicates that resistance to DAA, [6] growth fitness in the host, [26] and the ability to escape from the immune reaction are different depending on the haplotype [27] and not on the simple individual signature mutations of HCV genomes as previously thought. The HCV haplotype may be involved in a pathological process, such as fibrosis or carcinogenesis, caused by persistent infection of HCV. ...
The method of analyzing individual resistant hepatitis C virus (HCV) by a combination of haplotyping and resistance-associated substitution (RAS) has not been fully elucidated because conventional sequencing has only yielded short and fragmented viral genomes. We performed haplotype analysis of HCV mutations in 12 asunaprevir/daclatasvir treatment-failure cases using the Oxford Nanopore sequencer. This enabled single-molecule long-read sequencing using rolling circle amplification (RCA) for correction of the sequencing error. RCA of the circularized reverse-transcription polymerase chain reaction products successfully produced DNA longer than 30 kilobase pairs (kb) containing multiple tandem repeats of a target 3 kb HCV genome. The long-read sequencing of these RCA products could determine the original sequence of the target single molecule as the consensus nucleotide sequence of the tandem repeats and revealed the presence of multiple viral haplotypes with the combination of various mutations in each host. In addition to already known signature RASs, such as NS3-D168 and NS5A-L31/Y93, there were various RASs specific to a different haplotype after treatment failure. The distribution of viral haplotype changed over time; some haplotypes disappeared without acquiring resistant mutations, and other haplotypes, which were not observed before treatment, appeared after treatment. Conclusion: The combination of various mutations other than the known signature RAS was suggested to influence the kinetics of individual HCV quasispecies in the direct-acting antiviral treatment. HCV haplotype dynamic analysis will provide novel information on the role of HCV diversity within the host, which will be useful for elucidating the pathological mechanism of HCV-related diseases.
... The evolvability of SARS-CoV-2 spike protein RBD in the presence of nAbs depends on both the mutation rate in the presence of selection pressure and the mutational tolerance of the spike protein. The mutation rate of SARS-CoV-2 is in line with that of other single-strand RNA viruses, [28], and is relatively high when compared against some members of this group (such as Hepatitis C) for which evolution has practical clinical consequences (S1 Table) [29,30]. Mutation rates themselves are evolvable and may increase over time due to natural selection [31]. ...
The spike protein receptor-binding domain (RBD) of SARS-CoV-2 is the molecular target for many vaccines and antibody-based prophylactics aimed at bringing COVID-19 under control. Such a narrow molecular focus raises the specter of viral immune evasion as a potential failure mode for these biomedical interventions. With the emergence of new strains of SARS-CoV-2 with altered transmissibility and immune evasion potential, a critical question is this: how easily can the virus escape neutralizing antibodies (nAbs) targeting the spike RBD? To answer this question, we combined an analysis of the RBD structure-function with an evolutionary modeling framework. Our structure-function analysis revealed that epitopes for RBD-targeting nAbs overlap one another substantially and can be evaded by escape mutants with ACE2 affinities comparable to the wild type, that are observed in sequence surveillance data and infect cells in vitro. This suggests that the fitness cost of nAb-evading mutations is low. We then used evolutionary modeling to predict the frequency of immune escape before and after the widespread presence of nAbs due to vaccines, passive immunization or natural immunity. Our modeling suggests that SARS-CoV-2 mutants with one or two mildly deleterious mutations are expected to exist in high numbers due to neutral genetic variation, and consequently resistance to vaccines or other prophylactics that rely on one or two antibodies for protection can develop quickly -and repeatedly- under positive selection. Predicted resistance timelines are comparable to those of the decay kinetics of nAbs raised against vaccinal or natural antigens, raising a second potential mechanism for loss of immunity in the population. Strategies for viral elimination should therefore be diversified across molecular targets and therapeutic modalities.
... Mutation rates for RNA viruses in general are among the highest known (on the order of 10 -6 per base per viral replication cycle). When compared against many other RNA viruses, such as Hepatitis C, for which evolution has practical clinical consequences, SARS-CoV-2 has a relatively high evolutionary rate (Table S1) (31,32). As mentioned earlier, the currently estimated mutation rate for SARS-CoV-2 is in the context of neutral genetic drift (19). ...
As many prophylactics targeting SARS-CoV-2 are aimed at the spike protein receptor-binding domain (RBD), we examined the risk of immune evasion from previously published RBD-targeting neutralizing antibodies (nAbs). Epitopes for RBD-targeting nAbs overlap one another substantially and can give rise to escape mutants with ACE2 affinities comparable to wild type that still infect cells in vitro . We used evolutionary modeling to predict the frequency of immune escape before and after the widespread presence of nAbs due to vaccines, passive immunization or natural immunity. Our modeling suggests that SARS-CoV-2 mutants with one or two mildly deleterious mutations are expected to exist in high numbers due to neutral genetic variation, and consequently resistance to single or double antibody combinations can develop quickly under positive selection.
One Sentence Summary
SARS-CoV-2 will evolve quickly to evade widely deployed spike RBD-targeting monoclonal antibodies, requiring combinations that rely on at least three antibodies targeting distinct epitopes to suppress viral immune evasion.
... Hepatitis C virus represents a major medical, social and economic problem worldwide, infecting about 3% of the world's population [1]. After a chronic, life-long disease [2]. In approximately 20% of patients, persistent viremia leads to fibrosis, cirrhosis and hepatocellular carcinoma [3]. ...
In chronically infected HCV patients emergence and evolution of fibrosis, as a consequence of virus persistence, can be considered as an indicator of disease advancement. Therefore the aim of this study was to correlate alterations of immune response in chronic HCV patients with liver histopathology. Sera cytokine levels and frequency of circulating and liver infiltrating cells were evaluated using 13plex Kit Flow Cytomix, flow cytometry and immunohistochemistry. We found that the number of circulating T lymphocytes (including CD4+, CD8+ and Treg) and B lymphocytes, as well as DCs, was higher in patients with no fibrosis than in healthy subjects. In patients with fibrosis frequency of these cells decreased, and contrarily, in the liver, number of T and B lymphocytes gradually increased with fibrosis. Importantly, in patients with advanced fibrosis, liver infiltrating regulatory T cells and DC-SIGN+ mononuclear cells with immunosuppressive and wound-healing effector functions were abundantly present. Cytokine profiling showed predominance of proinflammatory cytokines in patients with no fibrosis and a tendency of decline in level of all cytokines with severity of liver injury. Lower but sustained IL-4 production refers to Th2 predominance in higher stages of fibrosis. Altogether, our results reveal graduall alterations of immunological parameters during fibrosis evolution and illustrate the course of immunological events through disease progression.
... Viral molecular diversity is often significantly reduced upon virus transmission to a new host (bottleneck effect) [3][4][5][6]. The bottleneck phenomenon may be affected by the size of the inoculum, HCV genotype, viral load and complexity of the virus population in the donor (number and frequency of variants), as well as recipient host factors, such as IL28B genotype [7,8]. ...
Molecular characterization of early hepatitis C virus (HCV) infection remains rare. Ten out of 78 patients of a hematology/oncology center were found to be HCV RNA positive two to four months after hospitalization. Only two of the ten patients were anti-HCV positive. HCV hypervariable region 1 (HVR1) was amplified in seven patients (including one anti-HCV positive) and analyzed by next generation sequencing (NGS). Genetic variants were reconstructed by Shorah and an empirically established 0.5% variant frequency cut-off was implemented. These sequences were compared by phylogenetic and diversity analyses. Ten unrelated blood donors with newly acquired HCV infection detected at the time of donation (HCV RNA positive and anti-HCV negative) served as controls. One to seven HVR1 variants were found in each patient. Sequences intermixed phylogenetically with no evidence of clustering in individual patients. These sequences were more similar to each other (similarity 95.4% to 100.0%) than to those of controls (similarity 64.8% to 82.6%). An identical predominant variant was present in four patients, whereas other closely related variants dominated in the remaining three patients. In five patients the HCV population was limited to a single variant or one predominant variant and minor variants of less than 10% frequency. In conclusion, NGS analysis of a cluster of HCV infections acquired in the hospital setting revealed the presence of low diversity, very closely related variants in all patients, suggesting an early-stage infection with the same virus. NGS combined with phylogenetic analysis and classical epidemiological analysis could help in tracking of HCV outbreaks.
... It is defined as a marked reduction in the genetic diversity as a result of elimination of a significant proportion of viral variants (Bull et al. 2011). The bottleneck effect is observed during the ongoing infection and at the time of inter-host transmission (Di Lello et al. 2015). It is responsible for a lower viral genomic diversity in recipients soon after infection when compared with the respective donors even when transmission is caused by a large inoculum (e.g., blood transfusion) (D'Arienzo et al. 2013;Wang et al. 2010). ...
... With the development of the acute phase, the fittest variants evolve toward genetic diversification as a result of the immune system pressure. With time of infection, the environment becomes more stable which drives the HCV population toward homogeneity (Di Lello et al. 2015). The subsequent bottleneck effect is responsible for a decline in viral diversity and a decrease in the effective population size observed in subjects who subsequently develop chronic infection (Bull et al. 2011). ...
Hepatitis C virus (HCV) is the etiological agent of chronic hepatitis C and a major cause of liver cirrhosis and hepatocellular carcinoma. Only a minority of infected individuals can clear the virus spontaneously. The knowledge of the determinants of virus clearance would allow the development of effective methods preventing its further spread and optimizing treatment regimens. Viral factors associated with spontaneous virus clearance in the acute phase of infection, such as HCV genotype, virus heterogeneity, and the impact of viral proteins on the immune system have been characterized. Likewise, host genetic markers, such as the interleukin genotypes, HLA alleles, and factors affecting the T lymphocyte response appear to play an important role. Studies have revealed that natural clearance of HCV infection in the chronic phase is rare and its mechanisms are not well understood. In this review, we present the state-of-the art knowledge on the viral and host factors affecting the spontaneous elimination of HCV infection.
... HCV circulates in infected individuals as a complex mixture of genetically different, but closely related, viral variants [8], [9]. Constraints in the viral genome and protein structure prevent some variants from proliferating [10]. Rapid HCV evolution in a single host favors the emergence of mutants that can escape from specific immunity [11]. ...
Hepatitis C virus (HCV) evolves rapidly in a single host and circulates as a quasispecies wich is a complex mixture of genetically distinct virus’s but closely related namely variants. To identify intra-individual diversity and investigate their functional properties in vitro, it is necessary to define their quasispecies composition and isolate the HCV variants. This is possible using single genome amplification (SGA). This technique, based on serially diluted cDNA to amplify a single cDNA molecule (clonal amplicon), has already been used to determine individual HCV diversity. In these studies, positive PCR reactions from SGA were directly sequenced using Sanger technology. The detection of non-clonal amplicons is necessary for excluding them to facilitate further functional analysis. Here, we compared Next Generation Sequencing (NGS) with De Novo assembly and Sanger sequencing for their ability to distinguish clonal and non-clonal amplicons after SGA on one plasma specimen. All amplicons (n = 42) classified as clonal by NGS were also classified as clonal by Sanger sequencing. No double peaks were seen on electropherograms for non-clonal amplicons with position-specific nucleotide variation below 15% by NGS. Altogether, NGS circumvented many of the difficulties encountered when using Sanger sequencing after SGA and is an appropriate tool to reliability select clonal amplicons for further functional studies.
... HCV'nin ayırt edici özelliklerinden birisi, konak organizmada sıradışı bir genetik çeşitlilik oluşturabilme yeteneğidir 3 . Bu özellik, enfeksiyonun immün yanıt varlığında dahi kronikleşmesi ve antiviral ilaç direnci gibi önemli sonuçlar doğurmaktadır. ...
... Enfekte kişilerde HCV varyasyonları; hataya açık replikaz enzimi ve hızlı replikasyon döngülerine bağlı olarak, aynı olmayan ancak birbirine benzerlik gösteren bir genomlar topluluğu, yani türümsüler, olarak izlenir 3,8 . Türümsüler, konağın seçici baskısına karşı dinamik bir süreçte değişim gösteren bir mutant spektrumu oluşturur. ...
... Bu bulgular, klinik safhadan bağımsız olarak kontrol altına alınan HCV enfeksiyonlarında, virusa ait genetik çeşitlilikte azalma olduğunu göstermektedir. Yeni oluşan varyasyonlar ise sıklıkla HVR1 bölgesinde izlenmekte ve immün yanıta bağlı seçilimin etkisini vurgulamaktadır 3,4 . ...
Hepatitis C virus (HCV) is a globally-dispersed agent of chronic hepatitis with a significant public health threat, affecting over 110 million individuals throughout the world. The increased risk for chronicity after exposure and the lack of a protective vaccine make HCV is a leading infectious cause of cirrhosis, liver failure requiring transplantation and hepatocellular carcinoma. The replicative process and infection dynamics in the host enable HCV to generate an array of closely-related but non-identical genetic variants known as quasispecies in the infected individuals. Pathogenesis and outcome in HCV infections are directly affected by the virus genetic heterogeneity, reflected as the emergence of quasispecies in infected individuals. The evolution of these highly-diverse viral populations in the host directly influences the disease course, via providing a pool of variants capable of resuming viral replication under extrinsic and/or intrinsic selective pressures. Viral quasispecies go through several alterations during the course of the infection, and provide a background for the selection of escape mutants from the host humoral and cell-mediated immune responses and antiviral treatment. Supported by the robust next generation sequencing techniques, recent studies have provided significant insights on the genomic diversity and progression as well as on the origin and the epidemiology of HCV. This review provides an overview of the mechanisms of HCV genetic variability, and the interactions with the host, that affects clinical disease, covering viral and host determinants of humoral and cell-mediated immune responses, alterations during the early and late stages of the infection and disease progression leading to chronicity. In addition, current findings in virus evolution and epidemiology were briefly interpreted from the inter-species and population perspectives. The impact of viral genomic heterogeneity on antiviral treatment in the era of direct-acting agents is also discussed, along with an overview of current methods employed for the characterization of viral diversity.
Understanding within-host evolution is critical for predicting viral evolutionary outcomes, yet such studies are currently lacking due to difficulty involving human subjects. Hepatitis C virus (HCV) is an RNA virus with high mutation rates. Its complex evolutionary dynamics and extensive genetic diversity are demonstrated in over 67 known subtypes. In this study, we analyzed within-host mutation frequency patterns of three HCV subtypes, using a large number of samples obtained from treatment-naïve participants by next-generation sequencing. We report that overall mutation frequency patterns are similar among subtypes, yet subtype 3a consistently had lower mutation frequencies and nucleotide diversity, while subtype 1a had the highest. We found that about 50% of genomic sites are highly conserved across subtypes, which are likely under strong purifying selection. We also compared within-host and between-host selective pressures, which revealed that Hyper Variable Region 1 within hosts was under positive selection, but was under slightly negative selection between hosts, which indicates that many mutations created within hosts are removed during the transmission bottleneck. Examining the natural prevalence of known resistance-associated variants showed their consistent existence in the treatment-naïve participants. These results provide insights into the differences and similarities among HCV subtypes that may be used to develop and improve HCV therapies.
