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Aspergillus fumigatus is a filamentous fungus that can infect the lungs of patients with immunosuppression and/or underlying lung diseases. The mortality associated with chronic and invasive aspergillosis infections remain very high, despite availability of antifungal treatments. In the last decade, there has been a worrisome emergence and spread of resistance to the first-line antifungals, the azoles. The mortality caused by resistant isolates is even higher, and patient management is complicated as the therapeutic options are reduced. Nevertheless, treatment failure is also common in patients infected with azole-susceptible isolates, which can be due to several non-mutually exclusive reasons, such as poor drug absorption. In addition, the phenomena of tolerance or persistence, where susceptible pathogens can survive the action of an antimicrobial for extended periods, have been associated with treatment failure in bacterial infections, and their occurrence in fungal infections already proposed. Here, we demonstrate that some isolates of A. fumigatus display persistence to voriconazole. A subpopulation of the persister isolates can survive for extended periods and even grow at low rates in the presence of supra-MIC of voriconazole and seemingly other azoles. Persistence cannot be eradicated with adjuvant drugs or antifungal combinations and seemed to reduce the efficacy of treatment for certain individuals in a Galleria mellonella model of infection. Furthermore, persistence implies a distinct transcriptional profile, demonstrating that it is an active response. We propose that azole persistence might be a relevant and underestimated factor that could influence the outcome of infection in human aspergillosis. Importance: The phenomena of antibacterial tolerance and persistence, where pathogenic microbes can survive for extended periods in the presence of cidal drug concentrations, have received significant attention in the last decade. Several mechanisms of action have been elucidated, and their relevance for treatment failure in bacterial infections demonstrated. In contrast, our knowledge of antifungal tolerance and, in particular, persistence is still very limited. In this study, we have characterized the response of the prominent fungal pathogen Aspergillus fumigatus to the first-line therapy antifungal voriconazole. We comprehensively show that some isolates display persistence to this fungicidal antifungal and propose various potential mechanisms of action. In addition, using an alternative model of infection, we provide initial evidence to suggest that persistence may cause treatment failure in some individuals. Therefore, we propose that azole persistence is an important factor to consider and further investigate in A. fumigatus. J.A. is funded by an Atracción de Talento Modalidad 1 (020-T1/BMD-200) contract of the Madrid Regional Government. J.S. has been funded by a BSAC Scholarship (bsac-2016-0049). C.V. was funded by FAPESP (2108/00715-3 and 2020/01131-5). G.H.G. hasbeen funded by FAPESP (2016/07870-9 and 2021/04977-5), CNPq (301058/2019-9 and404735/2018-5) and by the NIH/NIAID (grant R01AI153356). S.G. was cofunded by the NIHR Manchester Research Centre and the Fungal Infection Trust. Sí

Aspergillus fumigatusis a filamentous fungus that can infect the lungs of patients with immunosuppression and/or underlying lung-diseases. The mortality associated with chronic and invasive aspergillosis infections remain very high, despite availability of antifungal treatments. In the last decade, there has been a worrisome emergence and spread of resistance to the first line antifungals, the azoles. The mortality caused by resistant isolates is even higher, and patient management is complicated as the therapeutic options are reduced. Nevertheless, treatment failure is also common in patients infected with azole-susceptible isolates, which can be due to several non- mutually exclusive reasons, such as poor drug absorption. In addition, the phenomena of tolerance or persistence, where susceptible pathogens can survive the action of an antimicrobial for extended periods, have been associated with treatment failure in bacterial infections, and their occurrence in fungal infections already proposed. Here, we demonstrate that some isolates ofA. fumigatusdisplay persistence to voriconazole. A sub-population of the persister isolates can survive for extended periods and even grow at slow rates in the presence of supra-MIC (Minimum Inhibitory Concentration) of voriconazole and seemingly other azoles. Persistence cannot be eradicated with adjuvant drugs or antifungal combinations and seems to reduce the efficacy of treatment for certain individuals in aGalleria mellonellamodel of infection. Furthermore, persistence implies a distinct transcriptional profile, demonstrating that it is an active response. We propose that azole persistence might be a relevant and underestimated factor that could influence the outcome of infection in human aspergillosis.IMPORTANCEThe phenomena of antibacterial tolerance and persistence, where pathogenic microbes can survive for extended periods in the presence of cidal drug concentrations, have received significant attention in the last decade. Several mechanisms of action have been elucidated, and their relevance for treatment failure in bacterial infections demonstrated. In contrast, our knowledge about antifungal tolerance and, particularly, persistence are still very scarce. In this study, we have characterised the response of the prominent fungal pathogenAspergillus fumigatusto the first line therapy antifungal voriconazole. We comprehensively show that some isolates display persistence to this fungicidal antifungal and identify various potential mechanisms of action. In addition, using an alternative model of infection, we provide initial evidence to suggest that persistence may cause treatment failure in some individuals. Therefore, we propose that azole persistence is an important factor to consider and further investigate inA. fumigatus.

The error rate displayed during template copying to produce viral RNA progeny is a biologically relevant parameter of the replication complexes of viruses. It has consequences for virus–host interactions, and it represents the first step in the diversification of viruses in nature. Measurements during infections and with purified viral polymerases indicate that mutation rates for RNA viruses are in the range of 10 to 10 copying errors per nucleotide incorporated into the nascent RNA product. Although viruses are thought to exploit high error rates for adaptation to changing environments, some of them possess misincorporation correcting activities. One of them is a proofreading-repair 3 to 5 exonuclease present in coronaviruses that may decrease the error rate during replication. Here we review experimental evidence and models of information maintenance that explain why elevated mutation rates have been preserved during the evolution of RNA (and some DNA) viruses. The models also offer an interpretation of why error correction mechanisms have evolved to maintain the stability of genetic information carried out by large viral RNA genomes such as the coronaviruses., Ministerio de Economía y Competitividad (MINECO), grant number SAF2014-52400-R, SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER), and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged

Replication of RNA viruses is characterized by exploration of sequence space which facilitates their adaptation to changing environments. It is generally accepted that such exploration takes place mainly in response to positive selection, and that further diversification is boosted by modifications of virus population size, particularly bottleneck events. Our recent results with hepatitis C virus (HCV) have shown that the expansion in sequence space of a viral clone continues despite prolonged replication in a stable cell culture environment. Diagnosis of the expansion was based on the quantification of diversity indices, the occurrence of intra-population mutational waves (variations in mutant frequencies), and greater individual residue variations in mutant spectra than those anticipated from sequence alignments in data banks. In the present report, we review our previous results, and show additionally that mutational waves in amplicons from the NS5A-NS5B-coding region are equally prominent during HCV passage in the absence or presence of the mutagenic nucleotide analogues favipiravir or ribavirin. In addition, by extending our previous analysis to amplicons of the NS3- and NS5A-coding region, we provide further evidence of the incongruence between amino acid conservation scores in mutant spectra from infected patients and in the Los Alamos National Laboratory HCV data banks. We hypothesize that these observations have as a common origin a permanent state of HCV population disequilibrium even upon extensive viral replication in the absence of external selective constraints or changes in population size. Such a persistent disequilibrium¿revealed by the changing composition of the mutant spectrum¿may facilitate finding alternative mutational pathways for HCV antiviral resistance. The possible significance of our model for other genetically variable viruses is discussed., The work at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO), SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER), and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. 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 Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF) Grant No. PI19/00301 and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the MICIU, Grant No. IDI-20200297. 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). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MCIU. 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).

Publisher's version disponible en: http://hdl.handle.net/10261/257116, COVID-19 severity and progression are determined by several host and virological factors that may influence the final outcome of SARS-CoV-2-infected patients. The objective of this work is to determine a possible association between the viral load, obtained from nasopharyngeal swabs, and the severity of the infection in a cohort of 448 SARS-CoV-2-infected patients from a hospital in Madrid during the first outbreak of the pandemic in Spain. To perform this, we have clinically classified patients as mild, moderate and severe COVID-19 according to a number of clinical parameters such as hospitalization requirement, need of oxygen therapy, admission to intensive care units and/or exitus. Here we report a statistically significant correlation between viral load and disease severity, being high viral load associated with worse clinical prognosis, independently of several previously identified risk factors such as age, sex, hypertension, cardiovascular disease, diabetes, obesity, and lung disease (asthma and chronic obstructive pulmonary disease). The data presented here reinforce the viral load as a potential biomarker for predicting disease severity in SARS-CoV-2-infected patients. It is also an important parameter in viral evolution since it relates to the numbers and types of variant genomes present in a viral population, a potential determinant of disease progression., This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 Research Call COV20/00181) co-financed by European Development Regional Fund (A way to achieve Europe). The work was also supported by grants CSIC-COV19-014 from Consejo Superior de Investigaciones Cientificas (CSIC), BFU2017-91384-EXP from Ministerio de Ciencia, Innovacion y Universidades (MICIU), PI18/00210 from Instituto de Salud Carlos III. C.P., M.C. and P.M. are supported by the Miguel Servet program of the Instituto de Salud Carlos III (CPII19/00001, CPII17/00006 and CP16/00116, respectively) cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigacion en Red de Enfermedades Hepaticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundacion Ramon Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). 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). R. L.-V. is supported by predoctoral contract PEJD-2019-PRE/BMD-16414 from Comunidad de Madrid. R L-R is sponsored by the IIS-Fundacion Jimenez Diaz-UAM Genomic Medicine Chair.

Despite the high virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral 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. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV from 220 patients who failed therapy, which are not bona fide resistance-associated substitutions (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 three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value of treatment response are discussed.

Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. 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. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed., The work at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO), SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MICIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER), and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121 and CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. 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 Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF) grant number PI19/00301 and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the MICIU, grant number IDI-20151125. 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).