Whole-genome characterization and resistance-associated substitutions in a new HCV genotype 1 subtype

HCV; Direct-acting antivirals; Genotype 1VHC; Antivirals d'acció directa; Genotip 1VHC; Antivirales de acción directa; Genotipo 1Hepatitis C virus (HCV) is a highly variable infectious agent, classified into 8 genotypes and 86 subtypes. Our laboratory has implemented an in-house developed high-resolution HCV subtyping method based on next-generation sequencing (NGS) for error-free classification of the virus using phylogenetic analysis and analysis of genetic distances in sequences from patient samples compared to reference sequences. During routine diagnostic, a sample from an Equatorial Guinea patient could not be classified into any of the existing subtypes. The whole genome was analyzed to confirm that the new isolate could be classified as a new HCV subtype. In addition, naturally occurring resistance-associated substitutions (RAS) were analyzed by NGS. Whole-genome analysis based on p-distances suggests that the sample belongs to a new HCV genotype 1 subtype. Several RAS in the NS3 (S122T, D168E and I170V) and NS5A protein (Q(1b)24K, R(1b)30Q and Y93L+Y93F) were found, which could limit the use of some inhibitors for treating this subtype. RAS studies of new subtypes are of great interest for tailoring treatment, as no data on treatment efficacy are reported. In our case, the patient has not yet been treated, and the RAS report will be used to design the most effective treatment

Deep-sequencing study of HCV G4a resistance-associated substitutions in Egyptian patients failing DAA treatment

Resistance-associated substitutions; RAS; Subtype 4aSustituciones asociadas a la resistencia; RAS; Subtipo 4aSubstitucions associades a la resistència; RAS; Subtipus 4aPurpose: To study resistance-associated substitutions using next-generation sequencing in Egyptian hepatitis C virus-infected patients failing direct-acting antiviral treatment. Methods: The current study describes three cases of treatment failure in patients referred to Zagazig Viral Hepatitis Treatment Center (ZVHTC), Sharkia Governorate, Egypt. RAS were identified and characterized using deep sequencing. The first patient had breakthrough while receiving a daclatasvir (DCV)+sofosbuvir (SOF) regimen, patient 2 relapsed after treatment with DCV+SOF+ribavirin (RBV), and patient 3 relapsed after DCV+SOF therapy. A serum sample was collected from each patient at failure and sent to Vall d’Hebron Research Institute at Hospital Universitari Vall d’Hebron in Barcelona (Spain) for deep-sequencing study to identify and characterize the RAS present in the samples. Results: The following were identified: L28M, L30S and L28M+L30S in patient 1, L30R in patient 2, and R155C, D168E, L28M, L30H, L30S, L28M+L30H, and L28M+L30S in patient 3. Conclusion: To the best of our knowledge, this is the first report from Egypt of patients failing DAA-based therapy, describing the associated RAS. This information will be of help to understand the natural history of HCV in Egyptian patients and guide the proper choice of retreatment protocols.This study was supported by the Spanish Ministry of Health, Consumer Affairs, and Social Welfare, grant name: Plan Estrategico Nacional contra la Hepatitis C. This study was also funded by Instituto de Salud Carlos III, PI15/00856 and PI16/00337, cofinanced by CIBERehd (Consorcio Centro de Investigacion en Red de Enfermedades Hepaticas y Digestivas), which is funded by Instituto de Salud Carlos III and Centro para el Desarrollo Tecnologico Industrial (CDTI) from the Spanish Ministry of Economy and Business, grant number, IDI-2015112

In-Host HEV Quasispecies Evolution Shows the Limits of Mutagenic Antiviral Treatments

Deep sequencing; Mutagens; QuasispeciesSeqüenciació profunda; Mutàgens; QuasiespècieSecuenciación profunda; Mutágenos; CuasiespecieHere, we report the in-host hepatitis E virus (HEV) quasispecies evolution in a chronically infected patient who was treated with three different regimens of ribavirin (RBV) for nearly 6 years. Sequential plasma samples were collected at different time points and subjected to RNA extraction and deep sequencing using the MiSeq Illumina platforms. Specifically, we RT-PCR amplified a single amplicon from the core region located in the open-reading frame 2 (ORF2). At the nucleotide level (genotype), our analysis showed an increase in the number of rare haplotypes and a drastic reduction in the frequency of the master (most represented) sequence during the period when the virus was found to be insensitive to RBV treatment. Contrarily, at the amino acid level (phenotype), our study revealed conservation of the amino acids, which is represented by a high prevalence of the master sequence. Our findings suggest that using mutagenic antivirals concomitant with high viral loads can lead to the selection and proliferation of a rich set of synonymous haplotypes that express the same phenotype. This can also lead to the selection and proliferation of conservative substitutions that express fitness-enhanced phenotypes. These results have important clinical implications, as they suggest that using mutagenic agents as a monotherapy treatment regimen in the absence of sufficiently effective viral inhibitors can result in diversification and proliferation of a highly diverse quasispecies resistant to further treatment. Therefore, such approaches should be avoided whenever possible.This study was partially supported by Plan Estratègic de Recerca i Innovació en Salut (PERIS)—Direcció General de Recerca i Innovació en Salut (DGRIS), Catalan Health Ministry, Generalitat de Catalunya; Centro para el Desarrollo Tecnológico Industrial (CDTI) from the Spanish Ministry of Economy and Business, grant number IDI-20200297, Grant PID2021-126447OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by ERDF A way of making Europe; Projects PI19/00301 and PI22/00258 funded by Instituto de Salud Carlos III (ISCIII) and cofounded by the European Union; and Gilead’s biomedical research project GLD21/00006. S.C.-C has received support from Spanish Ministry of Education, grant FPU21/04150. M.I.-L. received the support of a fellowship from the “la Caixa” Foundation (ID 100010434), whose code is “LCF/BQ/DR23/12000020”

Host-dependent editing of SARS-CoV-2 in COVID-19 patients

SARS-CoV-2; Mutacions; QuasiespècieSARS-CoV-2; Mutaciones; CuasiespecieSARS-CoV-2; Mutations; QuasispeciesA common trait among RNA viruses is their high capability to acquire genetic variability due to viral and host mechanisms. Next-generation sequencing (NGS) analysis enables the deep study of the viral quasispecies in samples from infected individuals. In this study, the viral quasispecies complexity and single nucleotide polymorphisms of the SARS-CoV-2 spike gene of coronavirus disease 2019 (COVID-19) patients with mild or severe disease were investigated using next-generation sequencing (Illumina platform). SARS-CoV-2 spike variability was higher in patients with long-lasting infection. Most substitutions found were present at frequencies lower than 1%, and had an A → G or T → C pattern, consistent with variants caused by adenosine deaminase acting on RNA-1 (ADAR1). ADAR1 affected a small fraction of replicating genomes, but produced multiple, mainly non-synonymous mutations. ADAR1 editing during replication rather than the RNA-dependent RNA polymerase (nsp12) was the predominant mechanism generating SARS-CoV-2 genetic variability. However, the mutations produced are not fixed in the infected human population, suggesting that ADAR1 may have an antiviral role, whereas nsp12-induced mutations occurring in patients with high viremia and persistent infection are the main source of new SARS-CoV-2 variants.This study was supported by the Direcció General de Recerca i Innovació en Salut (DGRIS) of the Catalan Health Ministry, Generalitat de Catalunya, through the Vall d’Hebron Institut de Recerca (VHIR); the European Regional Development Fund (ERDF) “A way to achieve Europe” by the Spanish Network for Research in Infectious Diseases [grant number REIPI RD16/0016/0003]; Instituto de Salud Carlos III [grant number FIS PI19/00301]; and Centro para el Desarrollo Tecnologico Industrial (CDTI) from the Ministry of Economic Affairs and Digital Transformation [grant number IDI-20200297]