Leen Vijgen

In Vitro Resistance Profile of the Hepatitis C Virus NS3/4A Protease Inhibitor TMC435

ABSTRACT TMC435 is a small-molecule inhibitor of the NS3/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC435 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS3 residue D168 was observed, with changes to V or A being the most frequent. Mutations at NS3 positions 43, 80, 155, and 156, alone or in combination, were also identified. A transient replicon assay confirmed the relevance of these positions for TMC435 inhibitory activity. The change in the 50% effective concentrations (EC50s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to ∼2,000-fold for those with the D168V or D168I mutation, compared to the EC50 for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC435 (<10-fold change in EC50s), while greater effects were observed for some replicons with mutations at positions 43, 155, and 156. TMC435 remained active against replicons with the specific mutations observed after in vitro or in vivo exposure to telaprevir or boceprevir, including most replicons with changes at positions 36, 54, and 170 (<3-fold change in EC50s). Replicons carrying mutations affecting the activity of TMC435 remained fully susceptible to alpha interferon and NS5A and NS5B inhibitors. Finally, combinations of TMC435 with alpha interferon and NS5B polymerase inhibitors prevented the formation of drug-resistant replicon colonies.

Complete Genomic Sequence of Human Coronavirus OC43: Molecular Clock Analysis Suggests a Relatively Recent Zoonotic Coronavirus Transmission Event

ABSTRACT Coronaviruses are enveloped, positive-stranded RNA viruses with a genome of approximately 30 kb. Based on genetic similarities, coronaviruses are classified into three groups. Two group 2 coronaviruses, human coronavirus OC43 (HCoV-OC43) and bovine coronavirus (BCoV), show remarkable antigenic and genetic similarities. In this study, we report the first complete genome sequence (30,738 nucleotides) of the prototype HCoV-OC43 strain (ATCC VR759). Complete genome and open reading frame (ORF) analyses were performed in comparison to the BCoV genome. In the region between the spike and membrane protein genes, a 290-nucleotide deletion is present, corresponding to the absence of BCoV ORFs ns4.9 and ns4.8. Nucleotide and amino acid similarity percentages were determined for the major HCoV-OC43 ORFs and for those of other group 2 coronaviruses. The highest degree of similarity is demonstrated between HCoV-OC43 and BCoV in all ORFs with the exception of the E gene. Molecular clock analysis of the spike gene sequences of BCoV and HCoV-OC43 suggests a relatively recent zoonotic transmission event and dates their most recent common ancestor to around 1890. An evolutionary rate in the order of 4 × 10−4 nucleotide changes per site per year was estimated. This is the first animal-human zoonotic pair of coronaviruses that can be analyzed in order to gain insights into the processes of adaptation of a nonhuman coronavirus to a human host, which is important for understanding the interspecies transmission events that led to the origin of the severe acute respiratory syndrome outbreak.

A novel pancoronavirus RT-PCR assay: frequent detection of human coronavirus NL63 in children hospitalized with respiratory tract infections in Belgium

BackgroundFour human coronaviruses are currently known to infect the respiratory tract: human coronaviruses OC43 (HCoV-OC43) and 229E (HCoV-229E), SARS associated coronavirus (SARS-CoV) and the recently identified human coronavirus NL63 (HCoV-NL63). In this study we explored the incidence of HCoV-NL63 infection in children diagnosed with respiratory tract infections in Belgium.MethodsSamples from children hospitalized with respiratory diseases during the winter seasons of 2003 and 2004 were evaluated for the presence of HCoV-NL63 using a optimized pancoronavirus RT-PCR assay.ResultsSeven HCoV-NL63 positive samples were identified, six were collected during January/February 2003 and one at the end of February 2004.ConclusionsOur results support the notation that HCoV-NL63 can cause serious respiratory symptoms in children. Sequence analysis of the S gene showed that our isolates could be classified into two subtypes corresponding to the two prototype HCoV-NL63 sequences isolated in The Netherlands in 1988 and 2003, indicating that these two subtypes may currently be cocirculating.

Subgroup Prevalence and Genotype Circulation Patterns of Human Respiratory Syncytial Virus in Belgium during Ten Successive Epidemic Seasons

ABSTRACT Human respiratory syncytial virus (HRSV) is the leading viral cause of severe respiratory illness for infants and young children worldwide. Two major antigenic groups (A and B) of HRSV exist, and viruses from both subgroups can cocirculate during epidemics; however, their frequencies might differ between seasons. The subgroup prevalence and genotype distribution patterns of HRSV strains were investigated in a community in Belgium during 10 successive epidemic seasons (1996 to 2006). A regular 3-year cyclic pattern of subgroup dominance was observed, consisting of two predominant HRSV-A seasons, followed by a single HRSV-B-dominant year. HRSV infections with both subgroups were more prevalent among children younger than 6 months and had a peak incidence in December. The most frequently detected genotypes were GA5 and GB13, the latter including strains with the 60-nucleotide duplication in the G gene. Furthermore, GA5 remained the dominant HRSV genotype in two consecutive epidemic seasons twice during the study period. Additional variability was detected among the GB13 isolates, due to the usage of a novel termination codon in the G gene. Dual infections with both HRSV subgroups were detected for 9 patients, and subsequent infections with the heterologous HRSV subgroup were documented for 15 patients. Among five patients with homologous reinfections, only one was caused by HRSV-B. Our results support the hypothesis that the overall prevalence of HRSV-A over HRSV-B could be due to a more-transient subgroup A-specific immune protection.

Evolutionary History of the Closely Related Group 2 Coronaviruses: Porcine Hemagglutinating Encephalomyelitis Virus, Bovine Coronavirus, and Human Coronavirus OC43

ABSTRACT The close genetic and antigenic relatedness among the group 2 coronaviruses human coronavirus OC43 (HCoV-OC43), bovine coronavirus (BCoV), and porcine hemagglutinating encephalomyelitis virus (PHEV) suggests that these three viruses with different host specificities diverged fairly recently. In this study, we determined the complete genomic sequence of PHEV (strain PHEV-VW572), revealing the presence of a truncated group 2-specific ns2 gene in PHEV in comparison to other group 2 coronaviruses. Using a relaxed molecular clock approach, we reconstructed the evolutionary relationships between PHEV, BCoV, and HCoV-OC43 in real-time units, which indicated relatively recent common ancestors for these species-specific coronaviruses.

Virology analyses of HCV isolates from genotype 1-infected patients treated with simeprevir plus peginterferon/ribavirin in Phase IIb/III studies

BACKGROUND & AIMS Simeprevir is an oral hepatitis C virus (HCV) NS3/4A protease inhibitor approved for treatment of chronic HCV infection. Baseline NS3 polymorphisms in all patients and emerging mutations in patients who failed to achieve sustained virologic response (SVR) with simeprevir plus peginterferon/ribavirin (PR) in Phase IIb/III studies are described. METHODS Baseline sequencing data were available for 2007 genotype 1 (GT1)-infected patients. Post-baseline data were available for 197/245 simeprevir-treated patients who did not achieve SVR. In vitro simeprevir susceptibility was assessed in a transient replicon assay as site-directed mutants or in chimeric replicons with patient-derived NS3 protease sequences. RESULTS Baseline NS3 polymorphisms at positions associated with reduced in vitro susceptibility to simeprevir (43, 80, 122, 155, 156, and/or 168; EC50 fold change >2.0) were uncommon (1.3% [26/2007]), with the exception of Q80K, which confers ∼10-fold reduction in simeprevir activity in vitro (13.7% [274/2007]; GT1a 29.5% [269/911], GT1b 0.5% [5/1096]). Baseline Q80K had minor effect on initial response to simeprevir/PR, but resulted in lower SVR rates. Overall, 91.4% of simeprevir-treated patients [180/197] without SVR had emerging mutations at NS3 positions 80, 122, 155, and/or 168 at failure (mainly R155K in GT1a with and without Q80K, and D168V in GT1b), conferring high-level resistance in vitro (EC50 fold change >50). Emerging mutations were no longer detectable by population sequencing at study end in 50% [90/180] of patients (median follow-up 28.4weeks). CONCLUSIONS Simeprevir treatment failure was usually associated with emerging high-level resistance mutations, which became undetectable over time in half of the patients.

In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine

Abstract We report on chloroquine, a 4-amino-quinoline, as an effective inhibitor of the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro. Chloroquine is a clinically approved drug effective against malaria. We tested chloroquine phosphate for its antiviral potential against SARS-CoV-induced cytopathicity in Vero E6 cell culture. Results indicate that the IC50 of chloroquine for antiviral activity (8.8±1.2μM) was significantly lower than its cytostatic activity; CC50 (261.3±14.5μM), yielding a selectivity index of 30. The IC50 of chloroquine for inhibition of SARS-CoV in vitro approximates the plasma concentrations of chloroquine reached during treatment of acute malaria. Addition of chloroquine to infected cultures could be delayed for up to 5h postinfection, without an important drop in antiviral activity. Chloroquine, an old antimalarial drug, may be considered for immediate use in the prevention and treatment of SARS-CoV infections.

Virologic response and characterisation of HCV genotype 2-6 in patients receiving TMC435 monotherapy (study TMC435-C202).

BACKGROUND & AIMS TMC435 is a potent, once-daily, investigational hepatitis C virus (HCV) NS3/4A protease inhibitor in phase III clinical development. In the phase II trial TMC435-C202 (NCT00812331), TMC435 displayed potent activity in genotype 4, 5 and 6 patients and in 3/6 genotype 2 patients, whereas no activity was observed with genotype 3. METHODS Thirty-seven patients received TMC435 monotherapy (200 mg once daily) for 7 days. HCV RNA, NS3 protease sequences and the corresponding phenotypes were evaluated. RESULTS Genotype and isolate-specific baseline polymorphisms at NS3 positions known to affect HCV protease inhibitor activity were present in all genotypes. Consistent with the antiviral activity observed in genotypes 4 and 6, TMC435 was active in vitro against all genotype 4 isolates, and against most genotype 6 polymorphisms when tested as single or double mutants. In contrast, in genotype 3 where no HCV RNA decline was observed, isolates displayed >700-fold increases in EC(50) attributed to the D168Q polymorphism. In genotypes 2 and 5, HCV RNA changes from baseline to Day 3 ranged between -0.3 to -3.6 and -1.5 to -4.0 log(10)IU/ml, respectively, and isolates or site-directed mutants displayed intermediate in vitro susceptibility to TMC435 with fold changes in EC(50) between 15 and 78. Viral breakthrough in genotypes 4-6 was associated with emerging mutations including Q80R, R155K and/or D168E/V. CONCLUSIONS Sequence and phenotypic analyses of baseline isolates identified polymorphisms which could explain the differences in antiviral activity between genotypes. Pathways of TMC435 resistance in genotypes 2-6 were similar to those identified in genotype 1.

Tracking the Evolution of Multiple In Vitro Hepatitis C Virus Replicon Variants under Protease Inhibitor Selection Pressure by 454 Deep Sequencing

ABSTRACT Resistance to hepatitis C virus (HCV) inhibitors targeting viral enzymes has been observed in in vitro replicon studies and during clinical trials. The factors determining the emergence of resistance and the changes in the viral quasispecies population under selective pressure are not fully understood. To assess the dynamics of variants emerging in vitro under various selective pressures with TMC380765, a potent macrocyclic HCV NS3/4A protease inhibitor, HCV genotype 1b replicon-containing cells were cultured in the presence of a low, high, or stepwise-increasing TMC380765 concentration(s). HCV replicon RNA from representative samples thus obtained was analyzed using (i) population, (ii) clonal, and (iii) 454 deep sequencing technologies. Depending on the concentration of TMC380765, distinct mutational patterns emerged. In particular, culturing with low concentrations resulted in the selection of low-level resistance mutations (F43S and A156G), whereas high concentrations resulted in the selection of high-level resistance mutations (A156V, D168V, and D168A). Clonal and 454 deep sequencing analysis of the replicon RNA allowed the identification of low-frequency preexisting mutations possibly contributing to the mutational pattern that emerged. Stepwise-increasing TMC380765 concentrations resulted in the emergence and disappearance of multiple replicon variants in response to the changing selection pressure. Moreover, two different codons for the wild-type amino acids were observed at certain NS3 positions within one population of replicons, which may contribute to the emerging mutational patterns. Deep sequencing technologies enabled the study of minority variants present in the HCV quasispecies population present at baseline and during antiviral drug pressure, giving new insights into the dynamics of resistance acquisition by HCV.

Plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle

Abstract We describe the antiviral activity of plant lectins with specificity for different glycan structures against the severe acute respiratory syndrome coronavirus (SARS-CoV) and the feline infectious peritonitis virus (FIPV) in vitro. The SARS-CoV emerged in 2002 as an important cause of severe lower respiratory tract infection in humans, and FIPV infection causes a chronic and often fatal peritonitis in cats. A unique collection of 33 plant lectins with different specificities were evaluated. The plant lectins possessed marked antiviral properties against both coronaviruses with EC50 values in the lower microgram/ml range (middle nanomolar range), being non-toxic (CC50) at 50–100μg/ml. The strongest anti-coronavirus activity was found predominantly among the mannose-binding lectins. In addition, a number of galactose-, N-acetylgalactosamine-, glucose-, and N-acetylglucosamine-specific plant agglutinines exhibited anti-coronaviral activity. A significant correlation (with an r-value of 0.70) between the EC50 values of the 10 mannose-specific plant lectins effective against the two coronaviruses was found. In contrast, little correlation was seen between the activity of other types of lectins. Two targets of possible antiviral intervention were identified in the replication cycle of SARS-CoV. The first target is located early in the replication cycle, most probably viral attachment, and the second target is located at the end of the infectious virus cycle.