Neutralizing antibody and CD8+ T cell responses following BA.4/5 bivalent COVID-19 booster vaccination in adults with and without prior exposure to SARS-CoV-2.

As severe acute respiratory coronavirus 2 (SARS-CoV-2) variants continue to emerge, it is important to characterize immune responses against variants which can inform on protection efficacies following booster vaccination. In this study, neutralizing breadth and antigen-specific CD8+ T cell responses were analyzed in both infection-naïve and infection-experienced individuals following administration of a booster bivalent Wuhan-Hu-1+BA.4/5 Comirnaty® mRNA vaccine. Significantly higher neutralizing titers were found after this vaccination compared to the pre-third booster vaccination time point. Further, neutralizing breadth to omicron variants, including BA.1, BA.2, BA.5, BQ.1 and XBB.1, was found to be boosted following bivalent vaccination. SARS-CoV-2-specific CD8+ T cells were identified, but with no evidence that frequencies were increased following booster vaccinations. Spike protein-specific CD8+ T cells were the only responses detected after vaccination and non-spike-specific CD8+ T cells were only detected after infection. Both spike-specific and non-spike-specific CD8+ T cells were found at much lower frequencies than CD8+ T cells specific to cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza (Flu). Taken together, these results show that the bivalent Wuhan-Hu-1+BA.4/5 Comirnaty® mRNA vaccine boosted the breadth of neutralization to newer SARS-CoV-2 variants and that vaccination is able to induce spike protein-specific CD8+ T cell responses, which are maintained longitudinally.

Characterization of multi-DAA resistance using a novel hepatitis C virus genotype 3a infectious culture system.

BACKGROUND AND AIMS: The high HCV infection cure rates achieved with direct-acting antiviral (DAA) treatments could be compromised in the future by the emergence of antiviral resistance. Thus, it is essential to understand the viral determinants that influence DAA resistance, which is most prevalent in genotype 3. We aimed at studying how resistance to protease-, NS5A-, and NS5B-inhibitors influences the activities of glecaprevir/pibrentasvir, sofosbuvir/velpatasvir, and sofosbuvir/velpatasvir/voxilaprevir in cell culture, and how the HCV genome adapts to selective pressure by successive rounds of treatment failure. APPROACH AND RESULTS: A previously developed in vivo infectious cDNA clone of strain S52 (genotype 3a) was adapted to efficiently replicate and propagate in human hepatoma cells (Huh7.5) using 31 adaptive substitutions. DAA escape experiments resulted in the selection of S52 variants with decreased drug susceptibility (resistance), which was linked to the emergence of known resistance-associated substitutions (RASs). NS5A-inhibitor resistance was sufficient to promote treatment failure with double-DAA but not triple-DAA regimens. Enhanced viral fitness associated with the selection of sofosbuvir resistance accelerated escape from DAAs. After serial DAA treatment failure, HCV genetic evolution led to a complex genome-wide network of substitutions, some of which coevolved with known RASs. CONCLUSIONS: Baseline NS5A-RAS can compromise the efficacy of double-DAA pangenotypic regimens for HCV genotype 3, and enhanced viral fitness can accelerate treatment failure. Persistence of RASs after successive treatment failure is facilitated by the remarkable evolutionary capacity and plasticity of the HCV genome. Proof-of-concept for the potential development of multi-DAA resistance is shown.

Molecular Determinants of Mouse Adaptation of Rat Hepacivirus.

The lack of robust immunocompetent animal models for hepatitis C virus (HCV) impedes vaccine development and studies of immune responses. Norway rat hepacivirus (NrHV) infection in rats shares HCV-defining characteristics, including hepatotropism, chronicity, immune responses, and aspects of liver pathology. To exploit genetic variants and research tools, we previously adapted NrHV to prolonged infection in laboratory mice. Through intrahepatic RNA inoculation of molecular clones of the identified variants, we here characterized four mutations in the envelope proteins responsible for mouse adaptation, including one disrupting a glycosylation site. These mutations led to high-titer viremia, similar to that observed in rats. In 4-week-old mice, infection was cleared after around 5 weeks compared to 2 to 3 weeks for nonadapted virus. In contrast, the mutations led to persistent but attenuated infection in rats, and they partially reverted, accompanied by an increase in viremia. Attenuated infection in rat but not mouse hepatoma cells demonstrated that the characterized mutations were indeed mouse adaptive rather than generally adaptive across species and that species determinants and not immune interactions were responsible for attenuation in rats. Unlike persistent NrHV infection in rats, acute resolving infection in mice was not associated with the development of neutralizing antibodies. Finally, infection of scavenger receptor B-I (SR-BI) knockout mice suggested that adaptation to mouse SR-BI was not a primary function of the identified mutations. Rather, the virus may have adapted to lower dependency on SR-BI, thereby potentially surpassing species-specific differences. In conclusion, we identified specific determinants of NrHV mouse adaptation, suggesting species-specific interactions during entry. IMPORTANCE A prophylactic vaccine is required to achieve the World Health Organization's objective for hepatitis C virus elimination as a serious public health threat. However, the lack of robust immunocompetent animal models supporting hepatitis C virus infection impedes vaccine development as well as studies of immune responses and viral evasion. Hepatitis C virus-related hepaciviruses were discovered in a number of animal species and provide useful surrogate infection models. Norway rat hepacivirus is of particular interest, as it enables studies in rats, an immunocompetent and widely used small laboratory animal model. Its adaptation to robust infection also in laboratory mice provides access to a broader set of mouse genetic lines and comprehensive research tools. The presented mouse-adapted infectious clones will be of utility for reverse genetic studies, and the Norway rat hepacivirus mouse model will facilitate studies of hepacivirus infection for in-depth characterization of virus-host interactions, immune responses, and liver pathology.

Identification of novel neutralizing determinants for protection against HCV.

BACKGROUND AND AIMS: HCV evasion of neutralizing antibodies (nAb) results in viral persistence and poses challenges to the development of an urgently needed vaccine. N-linked glycosylation of viral envelope proteins is a key mechanism for such evasion. To facilitate rational vaccine design, we aimed to identify determinants of protection of conserved neutralizing epitopes. APPROACH AND RESULTS: Using a reverse evolutionary approach, we passaged genotype 1a, 1b, 2a, 3a, and 4a HCV with envelope proteins (E1 and E2) derived from chronically infected patients without selective pressure by nAb in cell culture. Compared with the original viruses, HCV recombinants, engineered to harbor substitutions identified in polyclonal cell culture-passaged viruses, showed highly increased fitness and exposure of conserved neutralizing epitopes in antigenic regions 3 and 4, associated with protection from chronic infection. Further reverse genetic studies of acquired E1/E2 substitutions identified positions 418 and 532 in the N1 and N6 glycosylation motifs, localizing to adjacent E2 areas, as key regulators of changes of the E1/E2 conformational state, which governed viral sensitivity to nAb. These effects were independent of predicted glycan occupancy. CONCLUSIONS: We show how N-linked glycosylation motifs can trigger dramatic changes in HCV sensitivity to nAb, independent of glycan occupancy. These findings aid in the understanding of HCV nAb evasion and rational vaccine design, as they can be exploited to stabilize the structurally flexible envelope proteins in an open conformation, exposing important neutralizing epitopes. Finally, this work resulted in a panel of highly fit cell culture infectious HCV recombinants.

Inactivated genotype 1a, 2a and 3a HCV vaccine candidates induced broadly neutralising antibodies in mice.

OBJECTIVE: A prophylactic vaccine is needed to control the HCV epidemic, with genotypes 1-3 causing >80% of worldwide infections. Vaccine development is hampered by HCV heterogeneity, viral escape including protection of conserved neutralising epitopes and suboptimal efficacy of HCV cell culture systems. We developed cell culture-based inactivated genotype 1-3 HCV vaccine candidates to present natively folded envelope proteins to elicit neutralising antibodies. DESIGN: High-yield genotype 1a, 2a and 3a HCV were developed by serial passage of TNcc, J6cc and DBN3acc in Huh7.5 cells and engineering of acquired mutations detected by next-generation sequencing. Neutralising epitope exposure was determined in cell-based neutralisation assays using human monoclonal antibodies AR3A and AR4A, and polyclonal antibody C211. BALB/c mice were immunised with processed and inactivated genotype 1a, 2a or 3a viruses using AddaVax, a homologue of the licenced adjuvant MF-59. Purified mouse and patient serum IgG were assayed for neutralisation capacity; mouse IgG and immune-sera were assayed for E1/E2 binding. RESULTS: Compared with the original viruses, high-yield viruses had up to ~1000 fold increased infectivity titres (peak titres: 6-7 log10 focus-forming units (FFU)/mL) and up to ~2470 fold increased exposure of conserved neutralising epitopes. Vaccine-induced IgG broadly neutralised genotype 1-6 HCV (EC50: 30-193 µg/mL; mean 71 µg/mL), compared favourably with IgG from chronically infected patients, and bound genotype 1-3 E1/E2; immune-sera endpoint titres reached up to 32 000. CONCLUSION: High-yield genotype 1-3 HCV could be developed as basis for inactivated vaccine candidates inducing broadly neutralising antibodies in mice supporting further preclinical development.

Neutralization and receptor use of infectious culture-derived rat hepacivirus as a model for HCV.

BACKGROUND AND AIMS: Lack of tractable immunocompetent animal models amenable to robust experimental challenge impedes vaccine efforts for HCV. Infection with rodent hepacivirus from Rattus norvegicus (RHV-rn1) in rats shares HCV-defining characteristics, including liver tropism, chronicity, and pathology. RHV in vitro cultivation would facilitate genetic studies on particle production, host factor interactions, and evaluation of antibody neutralization guiding HCV vaccine approaches. APPROACH AND RESULTS: We report an infectious reverse genetic cell culture system for RHV-rn1 using highly permissive rat hepatoma cells and adaptive mutations in the E2, NS4B, and NS5A viral proteins. Cell culture-derived RHV-rn1 particles (RHVcc) share hallmark biophysical characteristics of HCV and are infectious in mice and rats. Culture adaptive mutations attenuated RHVcc in immunocompetent rats, and the mutations reverted following prolonged infection, but not in severe combined immunodeficiency (SCID) mice, suggesting that adaptive immune pressure is a primary driver of reversion. Accordingly, sera from RHVcc-infected SCID mice or the early acute phase of immunocompetent mice and rats were infectious in culture. We further established an in vitro RHVcc neutralization assay, and observed neutralizing activity of rat sera specifically from the chronic phase of infection. Finally, we found that scavenger receptor class B type I promoted RHV-rn1 entry in vitro and in vivo. CONCLUSIONS: The RHV-rn1 infectious cell culture system enables studies of humoral immune responses against hepacivirus infection. Moreover, recapitulation of the entire RHV-rn1 infectious cycle in cell culture will facilitate reverse genetic studies and the exploration of tropism and virus-host interactions.

Efficacy of Ion-Channel Inhibitors Amantadine, Memantine and Rimantadine for the Treatment of SARS-CoV-2 In Vitro.

We report the in vitro efficacy of ion-channel inhibitors amantadine, memantine and rimantadine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In VeroE6 cells, rimantadine was most potent followed by memantine and amantadine (50% effective concentrations: 36, 80 and 116 µM, respectively). Rimantadine also showed the highest selectivity index, followed by amantadine and memantine (17.3, 12.2 and 7.6, respectively). Similar results were observed in human hepatoma Huh7.5 and lung carcinoma A549-hACE2 cells. Inhibitors interacted in a similar antagonistic manner with remdesivir and had a similar barrier to viral escape. Rimantadine acted mainly at the viral post-entry level and partially at the viral entry level. Based on these results, rimantadine showed the most promise for treatment of SARS-CoV-2.

In vitro efficacy of artemisinin-based treatments against SARS-CoV-2.

Effective and affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are needed. We report in vitro efficacy of Artemisia annua extracts as well as artemisinin, artesunate, and artemether against SARS-CoV-2. The latter two are approved active pharmaceutical ingredients of anti-malarial drugs. Concentration-response antiviral treatment assays, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that treatment with all studied extracts and compounds inhibited SARS-CoV-2 infection of VeroE6 cells, human hepatoma Huh7.5 cells and human lung cancer A549-hACE2 cells, without obvious influence of the cell type on antiviral efficacy. In treatment assays, artesunate proved most potent (range of 50% effective concentrations (EC50) in different cell types: 7-12 µg/mL), followed by artemether (53-98 µg/mL), A. annua extracts (83-260 µg/mL) and artemisinin (151 to at least 208 µg/mL). The selectivity indices (SI), calculated based on treatment and cell viability assays, were mostly below 10 (range 2 to 54), suggesting a small therapeutic window. Time-of-addition experiments in A549-hACE2 cells revealed that artesunate targeted SARS-CoV-2 at the post-entry level. Peak plasma concentrations of artesunate exceeding EC50 values can be achieved. Clinical studies are required to further evaluate the utility of these compounds as COVID-19 treatment.

Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 In Vitro.

Antivirals targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could improve treatment of COVID-19. We evaluated the efficacy of clinically relevant hepatitis C virus (HCV) NS3 protease inhibitors (PIs) against SARS-CoV-2 and their interactions with remdesivir, the only direct-acting antiviral approved for COVID-19 treatment. HCV PIs showed differential potency in short-term treatment assays based on the detection of SARS-CoV-2 spike protein in Vero E6 cells. Linear PIs boceprevir, telaprevir, and narlaprevir had 50% effective concentrations (EC50) of ∼40 µM. Among the macrocyclic PIs, simeprevir had the highest (EC50, 15 µM) and glecaprevir the lowest (EC50, >178 µM) potency, with paritaprevir, grazoprevir, voxilaprevir, vaniprevir, danoprevir, and deldeprevir in between. Acyclic PIs asunaprevir and faldaprevir had EC50s of 72 and 23 µM, respectively. ACH-806, inhibiting the HCV NS4A protease cofactor, had an EC50 of 46 µM. Similar and slightly increased PI potencies were found in human hepatoma Huh7.5 cells and human lung carcinoma A549-hACE2 cells, respectively. Selectivity indexes based on antiviral and cell viability assays were highest for linear PIs. In short-term treatments, combination of macrocyclic but not linear PIs with remdesivir showed synergism in Vero E6 and A549-hACE2 cells. Longer-term treatment of infected Vero E6 and A549-hACE2 cells with 1-fold EC50 PI revealed minor differences in the barrier to SARS-CoV-2 escape. Viral suppression was achieved with 3- to 8-fold EC50 boceprevir or 1-fold EC50 simeprevir or grazoprevir, but not boceprevir, in combination with 0.4- to 0.8-fold EC50 remdesivir; these concentrations did not lead to viral suppression in single treatments. This study could inform the development and application of protease inhibitors for optimized antiviral treatments of COVID-19.

Overcoming Culture Restriction for SARS-CoV-2 in Human Cells Facilitates the Screening of Compounds Inhibiting Viral Replication.

Efforts to mitigate the coronavirus disease 2019 (COVID-19) pandemic include the screening of existing antiviral molecules that could be repurposed to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Although SARS-CoV-2 replicates and propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (NUCs) often show decreased activity in these cells due to inefficient metabolization. SARS-CoV-2 exhibits low viability in human cells in culture. Here, serial passages of a SARS-CoV-2 isolate (original-SARS2) in the human hepatoma cell clone Huh7.5 led to the selection of a variant (adapted-SARS2) with significantly improved infectivity in human liver (Huh7 and Huh7.5) and lung cancer (unmodified Calu-1 and A549) cells. The adapted virus exhibited mutations in the spike protein, including a 9-amino-acid deletion and 3 amino acid changes (E484D, P812R, and Q954H). E484D also emerged in Vero E6-cultured viruses that became viable in A549 cells. Original and adapted viruses were susceptible to scavenger receptor class B type 1 (SR-B1) receptor blocking, and adapted-SARS2 exhibited significantly less dependence on ACE2. Both variants were similarly neutralized by COVID-19 convalescent-phase plasma, but adapted-SARS2 exhibited increased susceptibility to exogenous type I interferon. Remdesivir inhibited original- and adapted-SARS2 similarly, demonstrating the utility of the system for the screening of NUCs. Among the tested NUCs, only remdesivir, molnupiravir, and, to a limited extent, galidesivir showed antiviral effects across human cell lines, whereas sofosbuvir, ribavirin, and favipiravir had no apparent activity. Analogously to the emergence of spike mutations in vivo, the spike protein is under intense adaptive selection pressure in cell culture. Our results indicate that the emergence of spike mutations will most likely not affect the activity of remdesivir.

Identification of specific amino acid residues in the border disease virus glycoprotein E2 that modify virus growth in pig cells but not in sheep cells.

Border disease virus (BDV) envelope glycoprotein E2 is required for entry into cells and is a determinant of host tropism for sheep and pig cells. Here, we describe adaptive changes in the BDV E2 protein that modify virus replication in pig cells. To achieve this, two BDV isolates, initially collected from a pig and a sheep on the same farm, were passaged in primary sheep and pig cells in parallel with a rescued variant of the pig virus derived from a cloned full-length BDV cDNA. The pig isolate and the rescued virus shared the same amino acid sequence, but the sheep isolate differed at ten residues, including two substitutions in E2 (K771E and Y925H). During serial passage in cells, the viruses displayed clear selectivity for growth in sheep cells; only the cDNA-derived virus adapted to grow in pig cells. Sequencing revealed an amino acid substitution (Q739R) in the E2 domain DA of this rescued virus. Adaptation at the same residue (Q739K/Q739R) was also observed after passaging of the pig isolate in sheep cells. Use of reverse genetics confirmed that changing residue Q739 to R or K (each positively charged) was sufficient to achieve adaptation to pig cells. Furthermore, this change in host tropism was suppressed if Q739R was combined with K771E. Another substitution (Q728R), conferring an additional positive charge, acquired during passaging, restored the growth of the Q739R/K771E variant. Overall, this study provided evidence that specific, positively charged, residues in the E2 domain DA are crucial for pig-cell tropism of BDV.

Equine pegiviruses cause persistent infection of bone marrow and are not associated with hepatitis.

Pegiviruses frequently cause persistent infection (as defined by >6 months), but unlike most other Flaviviridae members, no apparent clinical disease. Human pegivirus (HPgV, previously GBV-C) is detectable in 1-4% of healthy individuals and another 5-13% are seropositive. Some evidence for infection of bone marrow and spleen exists. Equine pegivirus 1 (EPgV-1) is not linked to disease, whereas another pegivirus, Theiler's disease-associated virus (TDAV), was identified in an outbreak of acute serum hepatitis (Theiler's disease) in horses. Although no subsequent reports link TDAV to disease, any association with hepatitis has not been formally examined. Here, we characterized EPgV-1 and TDAV tropism, sequence diversity, persistence and association with liver disease in horses. Among more than 20 tissue types, we consistently detected high viral loads only in serum, bone marrow and spleen, and viral RNA replication was consistently identified in bone marrow. PBMCs and lymph nodes, but not liver, were sporadically positive. To exclude potential effects of co-infecting agents in experimental infections, we constructed full-length consensus cDNA clones; this was enabled by determination of the complete viral genomes, including a novel TDAV 3' terminus. Clone derived RNA transcripts were used for direct intrasplenic inoculation of healthy horses. This led to productive infection detectable from week 2-3 and persisting beyond the 28 weeks of study. We did not observe any clinical signs of illness or elevation of circulating liver enzymes. The polyprotein consensus sequences did not change, suggesting that both clones were fully functional. To our knowledge, this is the first successful extrahepatic viral RNA launch and the first robust reverse genetics system for a pegivirus. In conclusion, equine pegiviruses are bone marrow tropic, cause persistent infection in horses, and are not associated with hepatitis. Based on these findings, it may be appropriate to rename the group of TDAV and related viruses as EPgV-2.

Analysis of Virus Population Profiles within Pigs Infected with Virulent Classical Swine Fever Viruses: Evidence for Bottlenecks in Transmission but Absence of Tissue-Specific Virus Variants.

Classical swine fever virus (CSFV) contains a specific motif within the E2 glycoprotein that differs between strains of different virulence. In the highly virulent CSFV strain Koslov, this motif comprises residues S763/L764 in the polyprotein. However, L763/P764 represent the predominant alleles in published CSFV genomes. In this study, changes were introduced into the CSFV strain Koslov (here called vKos_SL) to generate modified CSFVs with substitutions at residues 763 and/or 764 (vKos_LL, vKos_SP, and vKos_LP). The properties of these mutant viruses, in comparison to those of vKos_SL, were determined in pigs. Each of the viruses was virulent and induced typical clinical signs of CSF, but the vKos_LP strain produced them significantly earlier. Full-length CSFV cDNA amplicons (12.3 kb) derived from sera of infected pigs were deep sequenced and cloned to reveal the individual haplotypes that contributed to the single-nucleotide polymorphism (SNP) profiles observed in the virus population. The SNP profiles for vKos_SL and vKos_LL displayed low-level heterogeneity across the entire genome, whereas vKos_SP and vKos_LP displayed limited diversity with a few high-frequency SNPs. This indicated that vKos_SL and vKos_LL exhibited a higher level of fitness in the host and more stability at the consensus level, whereas several consensus changes were observed in the vKos_SP and vKos_LP sequences, pointing to adaptation. For each virus, only a subset of the variants present within the virus inoculums were maintained in the infected pigs. No clear tissue-dependent quasispecies differentiation occurred within inoculated pigs; however, clear evidence for transmission bottlenecks to contact animals was observed, with subsequent loss of sequence diversity.IMPORTANCE The surface-exposed E2 protein of classical swine fever virus is required for its interaction with host cells. A short motif within this protein varies between strains of different virulence. The importance of two particular amino acid residues in determining the properties of a highly virulent strain of the virus has been analyzed. Each of the different viruses tested proved highly virulent, but one of them produced earlier, but not more severe, disease. By analyzing the virus genomes present within infected pigs, it was found that the viruses which replicated within inoculated animals were only a subset of those within the virus inoculum. Furthermore, following contact transmission, it was shown that a very restricted set of viruses had transferred between animals. There were no significant differences in the virus populations present in various tissues of the infected animals. These results indicate mechanisms of virus population change during transmission between animals.

High density Huh7.5 cell hollow fiber bioreactor culture for high-yield production of hepatitis C virus and studies of antivirals.

Chronic hepatitis C virus (HCV) infection poses a serious global public health burden. Despite the recent development of effective treatments there is a large unmet need for a prophylactic vaccine. Further, antiviral resistance might compromise treatment efficiency in the future. HCV cell culture systems are typically based on Huh7 and derived hepatoma cell lines cultured in monolayers. However, efficient high cell density culture systems for high-yield HCV production and studies of antivirals are lacking. We established a system based on Huh7.5 cells cultured in a hollow fiber bioreactor in the presence or absence of bovine serum. Using an adapted chimeric genotype 5a virus, we achieved peak HCV infectivity and RNA titers of 7.6 log10 FFU/mL and 10.4 log10 IU/mL, respectively. Bioreactor derived HCV showed high genetic stability, as well as buoyant density, sensitivity to neutralizing antibodies AR3A and AR4A, and dependency on HCV co-receptors CD81 and SR-BI comparable to that of HCV produced in monolayer cell cultures. Using the bioreactor platform, treatment with the NS5A inhibitor daclatasvir resulted in HCV escape mediated by the NS5A resistance substitution Y93H. In conclusion, we established an efficient high cell density HCV culture system with implications for studies of antivirals and vaccine development.

Direct acting antiviral treatment of chronic hepatitis C in Denmark: factors associated with and barriers to treatment initiation.

OBJECTIVES: We describe factors associated with and barriers to initiation of Direct Acting Antiviral (DAA) treatment in patients with chronic hepatitis C, who fulfill national fibrosis treatment guidelines in Denmark. MATERIALS AND METHODS: In this nationwide cohort study, we included patients with chronic hepatitis C from The Danish Database for Hepatitis B and C (DANHEP) who fulfilled fibrosis treatment criteria. Factors associated with treatment initiation and treatment failure were determined by logistic regression analyses. Medical records were reviewed from patients who fulfilled fibrosis treatment criteria, but did not initiate DAA treatment to determine the cause. RESULTS: In 344 (49%) of 700 patients, who fulfilled treatment criteria, factors associated with DAA treatment initiation were transmission by other routes than injecting drug use odds ratio (OR) 2.13 (CI: 1.38-3.28), previous treatment failure OR 2.58 (CI: 1.84-3.61) and ALT above upper limit of normal OR 1.60 (CI: 1.18-2.17). The most frequent reasons for not starting treatment among 356 (51%) patients were non-adherence to medical appointments (n = 107/30%) and ongoing substance use (n = 61/17%). Treatment failure with viral relapse occurred in 19 (5.5%) patients, who were more likely to have failed previous treatment OR 4.53 (CI: 1.59-12.91). CONCLUSIONS: In this nationwide cohort study, we found non-adherence to medical appointments and active substance use to be major obstacles for DAA treatment initiation. Our findings highlight the need for interventions that can overcome these barriers and increase the number of patients who can initiate and benefit from curative DAA treatment.

HCV Genotype 6a Escape From and Resistance to Velpatasvir, Pibrentasvir, and Sofosbuvir in Robust Infectious Cell Culture Models.

BACKGROUND & AIMS: Chronic liver diseases caused by hepatitis C virus (HCV) genotype 6 are prevalent in Asia, and millions of people require treatment with direct-acting antiviral regimens, such as NS5A inhibitor velpatasvir combined with the NS5B polymerase inhibitor sofosbuvir. We developed infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance. METHODS: The consensus sequences of strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined by Sanger sequencing of genomes amplified by reverse-transcription polymerase chain reaction. In vitro noninfectious full-length clones of these 6a strains were subsequently adapted in Huh7.5 cells, primarily by using substitutions identified in JFH1-based Core-NS5A and Core-NS5B genotype 6a recombinants. We studied the efficacy of NS5A and NS5B inhibitors in concentration-response assays. We examined the effects of long-term culture of Huh7.5 cells incubated with velpatasvir and sofosbuvir singly or combined following infection with passaged full-length HK2 or HK6a recombinant viruses. Resistance-associated substitutions (RAS) were identified by Sanger and next-generation sequencing, and their effects on viral fitness and in drug susceptibility were determined in reverse-genetic experiments. RESULTS: Adapted full-length HCV genotype 6a recombinants HK2cc and HK6acc had fast propagation kinetics and high infectivity titers. Compared with an HCV genotype 1a recombinant, HCV genotype 6a recombinants of strains HK2 and HK6a were equally sensitive to daclatasvir, elbasvir, velpatasvir, pibrentasvir, and sofosbuvir, but less sensitive to ledipasvir, ombitasvir, and dasabuvir. Long-term exposure of HCV genotype 6a-infected Huh7.5 cells with a combination of velpatasvir and sofosbuvir resulted in clearance of the virus, but the virus escaped the effects of single inhibitors via emergence of the RAS L31V in NS5A (conferring resistance to velpatasvir) and S282T in NS5B (conferring resistance to sofosbuvir). Engineered recombinant genotype 6a viruses with single RAS mediated resistance to velpatasvir or sofosbuvir. HCV genotype 6a viruses with RAS NS5A-L31V or NS5B-S282T were however, able to propagate and escape in Huh7.5 cells exposed to the combination of velpatasvir and sofosbuvir. Further, HCV genotype 6a with NS5A-L31V was able to propagate and escape in the presence of pibrentasvir with emergence of NS5A-L28S, conferring a high level of resistance to this inhibitor. CONCLUSIONS: Strains of HCV genotype 6a isolated from patients can be adapted to propagate in cultured cells, permitting studies of the complete life cycle for this important genotype. The combination of velpatasvir and sofosbuvir is required to block propagation of original HCV genotype 6a, which quickly becomes resistant to single inhibitors via the rapid emergence and persistence of RAS. These features of HCV genotype 6a could compromise treatment.

Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

Spd2 assists Spd1 in the modulation of ribonucleotide reductase architecture but does not regulate deoxynucleotide pools.

In yeasts, small intrinsically disordered proteins (IDPs) modulate ribonucleotide reductase (RNR) activity to ensure an optimal supply of dNTPs for DNA synthesis. The Schizosaccharomyces pombe Spd1 protein can directly inhibit the large RNR subunit (R1), import the small subunit (R2) into the nucleus and induce an architectural change in the R1-R2 holocomplex. Here, we report the characterization of Spd2, a protein with sequence similarity to Spd1. We show that Spd2 is a CRL4(Cdt2)-controlled IDP that functions together with Spd1 in the DNA damage response and in modulation of RNR architecture. However, Spd2 does not regulate dNTP pools and R2 nuclear import. Furthermore, deletion of spd2 only weakly suppresses the Rad3(ATR) checkpoint dependency of CRL4(Cdt2) mutants. However, when we raised intracellular dNTP pools by inactivation of RNR feedback inhibition, deletion of spd2 could suppress the checkpoint dependency of CRL4(Cdt2) mutant cells to the same extent as deletion of spd1. Collectively, these observations suggest that Spd1 on its own regulates dNTP pools, whereas in combination with Spd2 it modulates RNR architecture and sensitizes cells to DNA damage.