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Summary: Treatment with the direct‐acting antiviral agent (DAA) sofosbuvir (SOF), an NS5B inhibitor, and velpatasvir (VEL), an NS5A inhibitor, demonstrates viral cure rates of ≥95% in hepatitis C virus (HCV) genotypes (GT) 1‐6. Here, we investigated intrapatient HCV diversity in NS5A and NS5B using Shannon entropy to examine the relationship between viral diversity and treatment outcome. At baseline, HCV diversity was lowest in patients infected with HCV GT3 as compared to the other GTs, and viral diversity was greater in NS5A than NS5B (P < .0001). Treatment outcome with SOF/VEL or the comparator regimen of SOF with ribavirin (RBV) was not correlated with baseline diversity. However, among persons treated with SOF/VEL, a decrease in diversity from baseline was observed at relapse in the majority virologic failures, consistent with a viral bottleneck event at relapse. In contrast, an increase in diversity was observed in 27% of SOF+RBV virologic failures. We investigated whether the increase in diversity was due to an increase in the transition rate, one mode of potential RBV‐mediated mutagenesis; however, we found no evidence of this mechanism. Overall, we did not observe that viral diversity at baseline influenced treatment outcome, but the diversity changes observed at relapse can improve our understanding of RBV viral suppression in vivo. [ABSTRACT FROM AUTHOR]

Antiviral nucleos(t)ide analogue therapies inhibit HBV replication and suppress the HBV DNA levels in patients with chronic HBV infection. Since HBV RNAs are expressed from cccDNA or HBV integrated sequences, independently of viral genome replication, levels of HBV RNAs in plasma may remain high following treatment with nucleos(t)ide analogue. Thus, HBV RNAs have been proposed to be used as a viral biomarker for treatment outcome and disease progression. Recent investigations of plasma HBV RNAs described the presence of full length as well as subgenomic forms of RNA. To support the usage of plasma HBV RNAs as a viral biomarker, further understanding of HBV RNA composition in clinical samples is needed. Here, sequence of extracellular HBV RNAs was characterized in plasma samples of patients with chronic HBV infection using two independent RNA amplification methods that do not use HBV-specific primers for amplification: total RNA (NuGEN RNAseq) and mRNA (TruSeq RNAseq). Sequencing coverage was obtained across the full length of HBV genome for both methods, confirming the presence of full-length HBV RNA in plasma. The sequence of HBV RNA was nearly identical to plasma HBV DNA sequence in each sample with only 0-14 (median 4) mismatches over 3 kb. Thus, sequence of HBV RNA plasma reflects the intrahepatic viral reservoir and can be used for monitoring of sequence variants such as resistance in clinical trials. Additionally, RNA splice forms, different polyA tails start positions and presence of HBV-human chimeric transcript were identified., (© 2022 John Wiley & Sons Ltd.)

More systematic analysis of hepatitis B virus (HBV) genome diversity, linked with tenofovir disoproxil fumarate (TDF) treatment and HBeAg seroconversion, are needed. GS-US-174-0115 was a double-blind, placebo-controlled, Phase 3, 192-week clinical trial that evaluated TDF in adolescents with chronic hepatitis B (CHB). HBV full-genome deep sequencing was performed using Illumina MiSeq at baseline (BL; n = 85), Week 8 (W8; n = 80), Week 72 (W72; PBO only, n = 42), and treatment-free follow-up (TDF only, n = 25). The viral diversity was calculated using Shannon entropy and population nucleotide diversity with a 2% variant cutoff. Our data showed (i) a higher viral diversity in the X region at baseline than the core/polymerase/surface regions, (ii) higher core/surface viral diversity at baseline for patients with seroconversion, (iii) an expected reduction in viral diversity after 8 weeks of TDF treatment, and (iv) a drop in viral diversity at W72 for patients receiving placebo with a seroconversion (n = 7). The higher viral diversity in X was associated with higher baseline alanine aminotransferase (ALT) levels (p < .001). Patients with greater reduction of diversity at W8 of TDF treatment had higher baseline ALT levels. For placebo patients who seroconverted, the drop in viral diversity at W72 (p = .04) coincided with reduction of serum HBV DNA (average change from baseline = -4.10 log10 copies/ml) and unique combinations of variants were enriched in a patient's viral population post seroconversion. The basal core promoter (BCP) variants, A1762T and G1764A, and the pC variant, G1896A, were most often enriched at or after seroconversion., (© 2021 John Wiley & Sons Ltd.)

Both the A194T and a quadruple mutation CYEI (S106C, H126Y, D134E and L269I) in hepatitis B virus (HBV) polymerase reverse transcriptase domain (pol/RT) are suggested to be associated with treatment failure with tenofovir disoproxil fumarate (TDF). To further evaluate this assertion, the prevalence of these mutations at baseline as well as their development and/or loss during TDF and tenofovir alafenamide (TAF) treatment was analysed in 3886 patients enrolled in Gilead HBV clinical studies. In total, six out of 3886 (0.2%) patients carried the rtA194T mutation, while only 1 patient carried a triple CYE and 2 patients carried a quadruple CYEI mutation at baseline. All the patients harbouring rtA194T or CYE/CYEI at baseline achieved viral suppression by week 96 after TDF or TAF treatment. No patients developed an rtA194T mutation or > 1 substitution of CYEI, and the number of patients losing any substitutions of CYEI (n = 17) was similar to the number who developed a single substitution of CYEI (n = 32) during treatment. Phenotypic evaluation of the site-directed mutant (SDM) panel containing these mutations with or without other resistance mutations did not demonstrate a significant shift in TFV and TAF potency in vitro. No evidence of rtA194T and CYEI conferring resistance to TDF or TAF was observed based on the treatment responses to TDF or TAF in patients with mutations at baseline, the lack of selection of mutations after starting TDF or TAF treatment and no change in susceptibility to TFV or TAF in vitro., (© 2020 John Wiley & Sons Ltd.)

Velpatasvir is a pan-genotypic hepatitis C virus (HCV) NS5A inhibitor, which is used with sofosbuvir for treatment of infection with HCV genotypes 1-6. In vitro resistance studies were performed to characterize NS5A changes that might confer reduced velpatasvir susceptibility in vivo. Resistance selection studies using HCV replicon cells for subtypes 1a, 1b, 2a, 2b, 3a, 4a, 5a and 6a identified NS5A resistance-associated substitutions (RASs) at nine positions, most often 28M/S/T, 31F/I/M/P/V and 93D/H/N/S. In subtype 1a, RASs were selected at positions 31 and/or 93, while in subtype 1b, replicons with two or more RASs at positions 31, 54 or 93 were selected. Y93H was selected in subtypes 1a, 1b, 2a, 3a and 4a. In subtype 5a or 6a, L31P or P32L/Q was selected, respectively. Velpatasvir susceptibility of 358 replicons from genotypes 1 to 6 containing one or more NS5A RASs was also evaluated. The majority (63%) of subtypes 1a and 1b single RAS-containing replicons retained susceptibility to velpatasvir (<2.5-fold change in EC 50 ). High levels of resistance to velpatasvir were observed for six single mutants in subtype 1a, including M28G, A92K, Y93H/N/R/W and for one mutant, A92K, in subtype 1b. Most single mutants in subtypes 2a, 2b, 3a, 4a and 5a displayed low levels of reduced velpatasvir susceptibility. High-level resistance was observed for C92T and Y93H/N in subtype 2b, Y93H/S in 3a, and L31V and P32A/L/Q/R in 6a, and several double mutants in these subtypes. Overall, velpatasvir maintained activity against most common RASs that are known to confer resistance to first-generation NS5A inhibitors., (© 2019 John Wiley & Sons Ltd.)