Search

Trabajo presentado en el XVI Congreso Nacional de Virología, celebrado en Málaga (España) del 06 al 09 de septiembre de 2022., Despite the high sustained virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of patients do not achieve such a response. Identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultra-deep sequencing (UDS) methods for HCV characterization and patient management. By deep sequencing analysis of 220 subtyped HCV samples from infected patients who failed therapy, collected from 39 Spanish hospitals, we determined amino acid sequences of the DAA-target proteins NS3, NS5A and NS5B, by UDS of HCV patient samples, in search of resistanceassociated substitutions (RAS). Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide RAS. They were present frequently in basal and post-treatment virus of patients who failed therapy to different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Coherently, their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. Also, they have limited predicted disruptive effects on the threedimensional structures of the proteins harboring them. The information on HRSs that will be gathered during sequencing should be relevant not only to help predict treatment outcomes and disease progression but also to further understand HCV population dynamics, which appears much more complex than thought prior to the introduction of deep sequencing., The work at CBMSO was supported by grants SAF2014-52400-R from MINECO, SAF2017-87846-R and BFU2017-91384-EXP MICIU, PI18/00210 from ISCIII, S2013/ABI-2906 (PLATESA) and S2018/BAA-4370 (PLATESA2) from Comunidad de Madrid/FEDER. C.P. is supported by the Miguel Servet program of the ISCIII (CP14/00121 and CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd is funded by ISCIII. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). The work in Barcelona was supported by ISCIII, cofinanced by ERDF grant number PI19/00301 and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the MICIU, grant number IDI20151125. Work at CAB was supported by MINECO grant BIO2016-79618R and PID2019-104903RB-I00 (funded by the EU under the FEDER program) and by the Spanish State research agency (AEI) through project number MDM-2017-0737 Unidad de Excelencia “María de Maeztu”-Centro de Astrobiología (CSIC-INTA). Work at IBMB was supported by MICIN grant BIO2017-83906-P (funded by the EU under the FEDER program). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MICIU. B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo), cofinanced by Fondo Social Europeo (FSE).

Table S4. Subtype-specific oligonucleotides designed to sequence the NS3-coding region. (PDF 157 kb)

Table S3. Experimental samples and sequencing platforms used throughout this study. (PDF 181 kb)

Figure S8. Definition of the reference sequence for the ten subtypes under study. Nucleotide numbering at the beginning and at the end of NS3, NS5A and NS5B indicates the length of each coding region. Amino acid number of each coding region is indicated between the arrows. Positions at which resistance-associated substitutions (RAS) have been described are shown above the coding regions, and are included in our analyses. Alignments include a reference sequence for each subtype (1a, 1b, 2a, 2b, 2c, 2j, 3a, 4a, 4d, 4f); amino acid numbering above the boxes is indicated. Each reference sequence is defined as the most frequent amino acid at each position of the sequence ensemble for each subtype described in Table S1. Nucleotide and amino acid numbering are based on the HCV strain H77 (GenBank accession AF009606). (PDF 849 kb)

Table S5. Subtype-specific oligonucleotides designed to sequence the NS5A-coding region. (PDF 158 kb)

Table S2. Oligonucleotides used to perform the site-directed mutagenesis, qRT-PCR, control of basal amino acid sequencing error, and control of PCR recombination. (PDF 97 kb)

Figure S1. Subtype-specific oligonucleotides designed to sequence the NS3-coding region. Residue numbering is according to the reference strain AF009606. Positions in red are conserved among the different subtypes, and positions with different colors are discriminatory of a specific subtype (color codes given in the left column at each panel). Discriminatory positions for genotype are highlighted in pink. (PDF 1007 kb)

Figure S3. Subtype-specific oligonucleotides designed to sequence the NS5B-coding region. Residue numbering is according to the reference strain AF009606. Positions in red are conserved among the different subtypes, and positions with different colors are discriminatory of a specific subtype (color codes given in the left column at each panel). Discriminatory positions for genotype are highlighted in pink. (PDF 2762 kb)

Table S6. Subtype-specific oligonucleotides designed to sequence the NS5B-coding region. (PDF 250 kb)

Figure S2. Subtype-specific oligonucleotides designed to sequence the NS5A-coding region. Residue numbering is according to the reference strain AF009606. Positions in red are conserved among the different subtypes, and positions with different colors are discriminatory of a specific subtype (color codes given in the left column at each panel). Discriminatory positions for genotype are highlighted in pink. (PDF 1014 kb)

Background Since its first identification in the United Kingdom in late 2020, the highly transmissible B.1.1.7 variant of SARS-CoV-2, become dominant in several European countries raising great concern. Aim The aim of this study was to develop a duplex real-time RT-qPCR assay to detect, discriminate and quantitate SARS-CoV-2 variants containing one of its mutation signatures, the ΔHV69/70 deletion, to trace the community circulation of the B.1.1.7 variant in Spain through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19). Results B.1.1.7 variant was first detected in sewage from the Southern city of Málaga (Andalucía) in week 20_52, and multiple introductions during Christmas holidays were inferred in different parts of the country, earlier than clinical epidemiological reporting by the local authorities. Wastewater-based B.1.1.7 tracking showed a good correlation with clinical data and provided information at the local level. Data from WWTPs which reached B.1.1.7 prevalences higher than 90% for ≥ 2 consecutive weeks showed that 8.1±1.8 weeks were required for B.1.1.7 to become dominant. Conclusion The study highlights the applicability of RT-qPCR-based strategies to track specific mutations of variants of concern (VOCs) as soon as they are identified by clinical sequencing, and its integration into existing wastewater surveillance programs, as a cost-effective approach to complement clinical testing during the COVID-19 pandemic., This work was partially supported by the COVID-19 wastewater surveillance project (VATar COVID19), funded by the Spanish Ministry for the Ecological Transition and the Demographic Challenge of and the Spanish Ministry of Health; grants from CSIC (202070E101) and MICINN co-founded by AEI FEDER, UE (AGL2017-82909); grant ED431C 2018/18 from the Conselleria de Educacion, Universidade e Formacion Profesional, Xunta de Galicia (Spain); Direccio General de Recerca i Innovacio en Salut (DGRIS) Catalan Health Ministry Generalitat de Catalunya through Vall de Hebron Research Institute (VHIR), and Centro para el Desarrollo Tecnologico Industrial (CDTI) from the Spanish Ministry of Economy and Business, grant number IDI-20200297. Pilar Truchado is holding a Ramon y Cajal contract from the Ministerio de Ciencia e Innovacion. Adan Martinez is holding a predoctoral fellowship FI_SDUR from Generalitat de Catalunya. We gratefully acknowledge all the staff involved in the VATar COVID-19 project, working with sample collection and logistics. The authors are grateful to Promega Corporation (Madison, US) for technical advice, and thank Andrea Lopez de Mota for her technical support.