Superinfection and cure of infected cells as mechanisms for hepatitis C virus adaptation and persistence. Proceedings of the National Academy of Sciences of the United States of America RNA viruses exist as a genetically diverse quasispecies with extraordinary ability to adapt to abrupt changes in the host environment. However, the molecular mechanisms that contribute to their rapid adaptation and persistence in vivo are not well studied. Here, we probe hepatitis C virus (HCV) persistence by analyzing clinical samples taken from subjects who were treated with a second-generation HCV protease inhibitor. Frequent longitudinal viral load determinations and large-scale single-genome sequence analyses revealed rapid antiviral resistance development, and surprisingly, dynamic turnover of dominant drug-resistant mutant populations long after treatment cessation. We fitted mathematical models to both the viral load and the viral sequencing data, and the results provided strong support for the critical roles that superinfection and cure of infected cells play in facilitating the rapid turnover and persistence of viral populations. More broadly, our results highlight the importance of considering viral dynamics and competition at the intracellular level in understanding rapid viral adaptation. Thus, we propose a theoretical framework integrating viral and molecular mechanisms to explain rapid viral evolution, resistance, and persistence despite antiviral treatment and host immune responses. 10.1073/pnas.1805267115

Machine-learning based patient classification using Hepatitis B virus full-length genome quasispecies from Asian and European cohorts. Mueller-Breckenridge Alan J,Garcia-Alcalde Fernando,Wildum Steffen,Smits Saskia L,de Man Robert A,van Campenhout Margo J H,Brouwer Willem P,Niu Jianjun,Young John A T,Najera Isabel,Zhu Lina,Wu Daitze,Racek Tomas,Hundie Gadissa Bedada,Lin Yong,Boucher Charles A,van de Vijver David,Haagmans Bart L Scientific reports Chronic infection with Hepatitis B virus (HBV) is a major risk factor for the development of advanced liver disease including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The relative contribution of virological factors to disease progression has not been fully defined and tools aiding the deconvolution of complex patient virus profiles is an unmet clinical need. Variable viral mutant signatures develop within individual patients due to the low-fidelity replication of the viral polymerase creating 'quasispecies' populations. Here we present the first comprehensive survey of the diversity of HBV quasispecies through ultra-deep sequencing of the complete HBV genome across two distinct European and Asian patient populations. Seroconversion to the HBV e antigen (HBeAg) represents a critical clinical waymark in infected individuals. Using a machine learning approach, a model was developed to determine the viral variants that accurately classify HBeAg status. Serial surveys of patient quasispecies populations and advanced analytics will facilitate clinical decision support for chronic HBV infection and direct therapeutic strategies through improved patient stratification. 10.1038/s41598-019-55445-8

Deep sequencing analysis of quasispecies in the HBV pre-S region and its association with hepatocellular carcinoma. Zhang An-Ye,Lai Ching-Lung,Huang Fung-Yu,Seto Wai-Kay,Fung James,Wong Danny Ka-Ho,Yuen Man-Fung Journal of gastroenterology BACKGROUND:The association between the evolution of hepatitis B virus (HBV) quasispecies and the development of hepatocellular carcinoma (HCC) is unknown. METHODS:We used deep sequencing to examine the dynamics of HBV quasispecies and their relationship to HCC development. Thirty-two chronic hepatitis B (CHB) patients with HCC (HCC group) and 32 matched CHB patients without HCC (controls) were recruited. Fourteen patients from each group had serial sera available up to 9 years before the time of the present study. Deep sequencing of the HBV pre-S regions was performed. HBV quasispecies complexity, diversity, and intrapatient prevalence of pre-S deletions/mutations were analyzed. RESULTS:Compared with control patients, HCC patients had a significant greater quasispecies complexity (p = 0.04 at the nucleotide level), greater diversity (p = 0.004 and 0.009 at the nucleotide level and the amino acid level respectively), and a trend of greater complexity at the amino acid level (p = 0.065). HCC patients had a higher intrapatient prevalence of pre-S deletions and point mutations (at codons 4, 27, and 167) compared with the control patients (all p < 0.05). Longitudinal observation in the sera of 14 HCC patients showed that quasispecies complexity (p = 0.027 and 0.024 at the nucleotide level and the amino acid level respectively) and diversity (p = 0.035 and 0.031 at the nucleotide level and the amino acid level respectively) increased as the disease progressed to HCC. CONCLUSIONS:Increased HBV quasispecies complexity and diversity in the pre-S region, probably reflecting enhanced virus-host interplay, was associated with disease progression from CHB to HCC. 10.1007/s00535-017-1334-1

Quasispecies Diversity Is a Major Risk Factor for Vertical Hepatitis C Virus Transmission. Larouche Ariane,Milton McSween Kimberly-Ann,Calderon Virginie,Fauteux-Daniel Sébastien,Boulais Jonathan,Ransy Doris G,Boucher Marc,Lamarre Valérie,Lapointe Normand,Boucoiran Isabelle,Money Deborah M,Krajden Mel,Le Campion Armelle,Soudeyns Hugo The Journal of infectious diseases BACKGROUND:Vertical transmission is the major cause of pediatric hepatitis C virus (HCV) infection. The objective of this study was to better understand HCV pathogenesis in pregnant women and provide insights into risk factors and mechanisms involved in vertical transmission. METHODS:Evolutionary dynamics of HCV variant spectra and HCV-specific neutralizing antibody responses were examined using high-throughput sequencing and pseudoparticle-based assays in pregnant women monoinfected with HCV (n = 17) or coinfected with HCV and human immunodeficiency virus (HIV)-1 (n = 15). RESULTS:Overall, statistically significant associations were found between HCV quasispecies diversity, selective pressure exerted on the HCV E2 envelope protein, and neutralizing activity of maternal immunoglobulins. Women with low quasispecies diversity displayed significantly higher mean aspartate aminotransferase and alanine aminotransferase levels throughout pregnancy, but this difference was restricted to monoinfected participants. Low quasispecies diversity and inefficient neutralizing activity were also significantly associated with vertical transmission, but only in the monoinfected group. CONCLUSIONS:These results indicate that maternal neutralizing antibody responses play a role in the prevention of vertical HCV transmission, but not in presence of HIV-1 coinfection, and suggest that the mechanism of vertical transmission may be different between monoinfected and coinfected women. These findings could inform management strategies for the prevention of vertical HCV transmission. 10.1093/infdis/jiy581

Kinetics of hepatitis B surface antigen quasispecies during REP 2139-Ca therapy in HBeAg-positive chronic HBV infection. Usman Zainab,Mijočević Hrvoje,Karimzadeh Hadi,Däumer Martin,Al-Mathab Mamun,Bazinet Michel,Frishman Dmitrij,Vaillant Andrew,Roggendorf Michael Journal of viral hepatitis Chronic HBV infection results in various clinical manifestations due to different levels of immune response. In recent years, hepatitis B treatment has improved by long-term administration of nucleos(t)ide analogues (NUCs) and peg-interferon. Nucleic acid polymers (NAPs; REP 2139-Ca and REP 2139-Mg) are new antiviral drugs that block the assembly of subviral particles, thus preventing the release of HBsAg and allowing its clearance and restoration of functional control of infection when combined with various immunotherapies. In the REP 102 study (NCT02646189), 9 of 12 patients showed substantial reduction of HBsAg and seroconversion to anti-HBs in response to REP 2139-Ca, whereas 3 of 12 patients did not show responses (>1 log reduction of HBsAg and HBV DNA from baseline). We characterized the dynamic changes of HBV quasispecies (QS) within the major hydrophilic region (MHR) of the 'pre-S/S' open reading frame including the 'a' determinant in responders and nonresponders of the REP 102 study and four untreated matched controls. HBV QS complexity at baseline varied slightly between responders and nonresponders (P = .28). However, these responders showed significant decline in viral complexity (P = .001) as REP 2139-Ca therapy progressed but no significant change in complexity was observed among the nonresponders (P = .99). The MHR mutations were more frequently observed in responders than in nonresponders and matched controls. No mutations were observed in 'a' determinant of major QS population which may interfere with the detection of HBsAg by diagnostic assays. No specific mutations were found within the MHR which could explain patients' poor HBsAg response during REP 2139-Ca therapy. 10.1111/jvh.13180