Extreme-halophiles: their role in the arsenic biogeochemical cycle

Biofilms, mats and microbialites dwell under extreme environmental conditions (high salinity, extreme aridity, pH and arsenic concentration) in the Argentinean Puna and the Atacama Desert. Microbial communities inhabiting those ecosystems are poorly known. Arsenic metabolism is proposed to be an ancient mechanism in microbial life. Besides, some bacteria and archaea are not only able to use detoxification processes to grow under high arsenic concentration, but also, some of them are able to exploit arsenic as a bioenergetic substrate in either anaerobic arsenate respiration or chemolithotrophic growth on arsenite. Only four aioAB coding for arsenite oxidase and two arrA coding for arsenate reductase sequences from haloarchaea were previously deposited in the NCBI Database, but have not been reported in the literature. The arrA arsenate reductases are reliable indicators of anaerobic As (V) respiration and catalyze the electron transfer to the As (V) terminal acceptor in dissimilatory arsenatereducing prokaryotes (DARPs). In this work, we are presenting our first steps in the study of the arsenic biogeochemical cycle in these ecosystems. Thus, the aim of this study was to isolate and to study the arsenic metabolism genes of the isolated extreme halophile microorganisms as well as to test the growth in minimal medium using different carbon sources. Mats and microbialites samples were taken from the water’s edge of Laguna Tebenquiche, Laguna Brava (Salar de Atacama, Chile) during December 2012 and from gaylusite crystals (Laguna Diamante) in August 2014. Samples were enriched and plated in WS medium supplemented with arsenic (AsIII 0.5mM and AsV 20mM). Arsenite oxidase (aioB) and Arsenate reductase (arrA) primers specific for haloarchaea were designed using PrimerProspector software. Selected primers were aioB-1190F (5’-GCTCMTSACCGGCAGCGTCG-3’), aioB-1507R (5’-YGATCTCGTCGATGTCGGCG-3’), arrA-417F (5’CCCGAGTTCGAGCCSATCTC-3’) and arrA-614R (5’GCRCAGATCGMGCTGTGGGA-3’). In order to identify the isolates we used Archaea-specific primers for 16S rDNA gene amplification: 344F (5´- ACG GGG YGC AGC AGG CGC GA-3´) and 915R (5´- GTG CTC CCC CGC CAA TTC CT -3´). Fragments of 577 bp, 317pb and 197pb were obtained from 16S rDNA, aioB and arrA genes respectively. Universal primers 27F and 1492R were used to amplify 16S rDNA in bacterial isolates. 25 isolates belonging to Archaea and Bacteria Domain were obtained; they are related to the Phylum Euryarchaeota, Firmicutes and Proteobacteria. AioB and arrA genes were found in most of the isolates and DNA from the samples (mats, microbialites and biofilm). The best carbon source tested was pyruvate and acetate, being pyruvate better in all cases. Promising results were obtained in the search of organisms able to use arsenic in their bioenergetic metabolism. More studies are underway to try to better understand these very interesting systems.Fil: Rasuk, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Ordoñez, Omar Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Soria, Mariana Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaXI Congreso Argentino de Microbiología GeneralCordobaArgentinaSociedad Argentina de Microbiología Genera

Long˗term obsidian circulation in quebrada del toro (Salta), Northwestern Argentina

Este trabajo describe los resultados de los análisis de fluorescencia de rayos X (XRF) realizados sobre 30 muestras de obsidiana de sitios de Quebrada del Toro (Salta, Argentina), que cubren un lapso de alrededor de 2000 años de ocupación (ca. 2500˗500 AP). Establecida la procedencia de las muestras se discuten los patrones de distribución de esta materia prima observados entre los diferentes sitios y períodos considerados. Los resultados alcanzados permiten argumentar el uso relativamente sostenido en el tiempo de las canteras de Alto Tocomar, Laguna Blanca˗Zapaleri y Ona˗Las Cuevas (aunque esta última está ausente durante un intervalo de alrededor de 500 años), y algunas variaciones temporales en el uso de fuentes menores. Estos resultados proveen información para discutir el acceso y consumo de obsidianas desde una perspectiva de larga duración. Estas prácticas son evaluadas en el marco de los procesos de cambio ocurridos en el rango cronológico abordado, caracterizados por importantes transformaciones en los sistemas sociopolíticos y económicos.Fil: de Feo, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. División Arqueología; ArgentinaFil: Soria, Silvia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones en Ciencias Sociales y Humanidades. Universidad Nacional de Salta. Facultad de Humanidades. Instituto de Investigaciones en Ciencias Sociales y Humanidades; ArgentinaFil: Macoritto Torcivia, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones en Ciencias Sociales y Humanidades. Universidad Nacional de Salta. Facultad de Humanidades. Instituto de Investigaciones en Ciencias Sociales y Humanidades; Argentin

Bacteriorodhopsin or arsenite as energy source in the growth of haloarchaea

Laguna Diamante (S 26°1’50’’ O 67°2’32’’) ubicada en el cráter del volcán Galán en Catamarca, a 4750 m. s.n.m., presenta condiciones extremas como: elevado pH, salinidad, alta radiación ultravioleta (UV) y alto contenido de metales pesados y metaloides, principalmente arsénico (As) [1], el cuál es un compuesto altamente tóxico y muy distribuido en la corteza terrestre [2]. En este ambiente, han sido reportadas unas biopelículas rojas (BD) formadas en la parte inferior de microbialitos de tipo leiolitos, y los estudios metagenómicos demostraron que están constituidas por un 94% de haloarqueas [1]. Algunas haloarqueas presentan un mecanismo fotosintético que les permite producir ATP de una forma similar al realizado en la cadena transportadora, pero a diferencia de ésta, los protones expulsados no provienen de complejos proteicos sino de una bomba de protones fotoexitable, la “bacteriorrodopsina” (BR) [3]. Mecanismo que les permite enfrentar las bajas concentraciones de oxígeno de ambientes hipersalinos. Objetivo: estudiar la influencia de la luz en el crecimiento microbiano de haloarqueas aisladas de BD en presencia y ausencia de arsenito (As III). Para ello, se utilizaron cinco haloarqueas aisladas de BD del género Halorubrum (DM1, DM2, DM3, DM4 y DM5) para evaluar su crecimiento en medio mínimo (CDM con piruvato como fuente de carbono [5]) con y sin adición de AsIII (1 mM de concentración final); usando el medio de aislamiento WJK [4] como control. Los cultivos fueron incubados a 37 °C y agitados a 120 r.p.m. entre 7 – 10 días (dependiendo del tiempo de generación de la cepa) midiendo la densidad óptica (Do) a 600 nm cada 12 h. Se utilizaron dos condiciones: luz blanca fría (400 – 700 nm) y oscuridad (cubriendo los frascos con láminas de aluminio). Se observó que de las cepas estudiadas, DM2 presentó un mayor crecimiento en CDM con As III en oscuridad, mientras que en luz el mejor crecimiento fue observado en CDM sin la adición de As III, en el resto de las condiciones el crecimiento fue escaso. A partir de estos resultados obtenidos se sugiere que en presencia de luz DM2 utilizaría el sistema de la bacteriorodopsina para obtener energía y sostener su crecimiento, mientras que en oscuridad la obtención de energía sería a través del uso del As III como donador de electrones.Fil: Soria, Mariana Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Rasuk, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Ordoñez, Omar Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaIII Reunión de Fotobiólogos Moleculares ArgentinosSan Miguel de TucumánArgentinaGrupo Argentino de Fotobiologí

Los oncoides de las quínoas: Un nuevo depósito de microbialitos en el salar de antofalla (Catamarca, Argentina).

The Salar de Antofalla (salt flat) is located in the Puna region of Catamarca, in northern Argentina. In this paper we report and provide the first descriptive data of Las Quínoas, a modern system of oncoids located in the western margin of the salt flat. Oncoids were studied by insitu logging, polished and thin sections analysis. In addition, the 16s rRNA genes of microbial mats associated with these oncoids were amplified and sequenced to characterize the microbial biodiversity. Oncoids present discoidal to subrounded morphologies and sizes up to 15 cm in diameter. They are scattered along channels, which originate from the groundwater springs of a wetland and enter the salt flat. Its macrostructure is concentric and composed by three zones: 1. A nucleus zone built by clastic material in a carbonate matrix. 2. A well-laminated zone around the nucleus that shows two types of mesostructures: concentrically stacked spheroids (SS-C) and randomly stacked hemispheroids (SS-R), both showing an alternation of dense and dark micritic laminae with light micritic to microsparitic laminae. 3. A poorly-laminated zone, in the outermost sector of oncoids, with two types of mesostructures too: a laminated mesostructure composed also of an alternation of dense and dark micritic laminae with light micritic to microsparitic laminae, and a non-laminated mesostructure composed of agglomerated and cemented clastic material within a calcareous matrix (wackestones-packstones). Regarding the microbial diversity, the analyzed oncoids in this work are mainly inhabited by Proteobacteria (ca. 37.5%), Bacteroidetes (ca. 25.0%), and in less proportion Planctomycetes, Actinobacteria and Cyanobacteria.El salar de Antofalla se encuentra en la región de la Puna de Catamarca, en el norte de Argentina. En este artículo informamos y proporcionamos los primeros datos descriptivos de Las Quínoas, un sistema moderno de oncoides ubicado en el margen occidental de este salar. Ellos se estudiaron mediante registro in situ, análisis de secciones pulidas y delgadas. Además, los genes de ARNr 16s de tapices microbianos asociados con estos oncoides se amplificaron y secuenciaron para caracterizar la biodiversidad microbiana. Los oncoides presentan morfologías discoidales a subredondeadas y tamaños de hasta 15 cm de diámetro. Se encuentran dispersos a lo largo de canales, que se originan de las surgentes de agua de una vega y entran al salar. Su macroestructura es concéntrica y está compuesta por tres zonas: 1. Una zona de núcleo construida por material clástico en una matriz de carbonato. 2. Una zona bien laminada alrededor del núcleo que muestra dos tipos de mesoestructuras: esferoides concéntricamente apilados (SS-C) y hemisferoides apilados aleatoriamente (SS-R), ambos con una alternancia de láminas micríticas densas y oscuras con micríticas claras a microesparíticas. 3. Una zona pobremente laminada, en el sector más externo de los oncoides, también con dos tipos de mesoestructuras: una mesoestructura laminada compuesta por la alternancia de láminas micríticas densas y oscuras con láminas micríticas claras a microesparíticas, y una mesoestructura no laminada compuesta de material clástico aglomerado y cementado dentro de una matriz calcárea (wackestones-packstones). En cuanto a la diversidad microbiana, los oncoides analizados en este trabajo están habitados principalmente por Proteobacterias (ca. 37,5%), Bacteroidetes (ca. 25,0%), y en menor proporción Planctomicetes, Actinobacterias y Cianobacterias.Fil: Villafañe, Patricio Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Saona Acuña, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Gómez, Fernando Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lencina, Agustina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Catamarca. Universidad Nacional de Catamarca. Centro de Investigaciones y Transferencia de Catamarca; ArgentinaFil: Soria, Mariana Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentin

First Report on the Plasmidome From a High-Altitude Lake of the Andean Puna

Mobile genetic elements, including plasmids, drive the evolution of prokaryotic genomes through the horizontal transfer of genes allowing genetic exchange between bacteria. Moreover, plasmids carry accessory genes, which encode functions that may offer an advantage to the host. Thus, it is expected that in a certain ecological niche, plasmids are enriched in accessory functions, which are important for their hosts to proliferate in that niche. Puquio de Campo Naranja is a high-altitude lake from the Andean Puna exposed to multiple extreme conditions, including high UV radiation, alkalinity, high concentrations of arsenic, heavy metals, dissolved salts, high thermal amplitude and low O2 pressure. Microorganisms living in this lake need to develop efficient mechanisms and strategies to cope under these conditions. The aim of this study was to characterize the plasmidome of microbialites from Puquio de Campo Naranja, and identify potential hosts and encoded functions using a deep-sequencing approach. The potential ecological impact of the plasmidome, including plasmids from cultivable and non-cultivable microorganisms, is described for the first time in a lake representing an extreme environment of the Puna. This study showed that the recovered genetic information for the plasmidome was novel in comparison to the metagenome derived from the same environment. The study of the total plasmid population allowed the identification of genetic features typically encoded by plasmids, such as resistance and virulence factors. The resistance genes comprised resistances to heavy metals, antibiotics and stress factors. These results highlight the key role of plasmids for their hosts and impact of extrachromosomal elements to thrive in a certain ecological niche.Fil: Perez, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Kurth, Daniel German. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Soria, Mariana Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Castillo Villamizar, Genis Andrés. Universität Göttingen; AlemaniaFil: Poehlein, Anja. Universität Göttingen; AlemaniaFil: Daniel, Rolf. Universität Göttingen; AlemaniaFil: Dib, Julian Rafael. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentin

Krüppel-like factor 6 Is required for oxidative and oncogene-iduced cellular senescence

Krüppel-like factor 6 (KLF6) is a transcription factor involved in the regulation of several cellular processes. Regarding its role in tumorigenesis, KLF6 is considered a tumor suppressor. Numerous reports demonstrate its frequent genomic loss or down-regulation, implying a functional inactivation in a broad range of human cancers. Previous work from our laboratory showed that the down-regulation of KLF6 expression in normal fibroblasts leads to cellular transformation, while its ectopic expression interferes with the oncogenic transformation triggered by activated Ras through a cell cycle arrest. We hypothesize that the growth suppressor activity of KLF6 may involve the induction of cellular senescence thereby helping to prevent the proliferation of cells at risk of neoplastic transformation. Here, we explored the association of KLF6 up-regulation in two different cellular senescence scenarios. We found that KLF6 silencing bypasses both oxidative and oncogene-induced senescence. In this context, KLF6 expression per se was capable to trigger cellular senescence in both normal and tumoral contexts. As such, the findings presented in this report provide insights into a potential mechanism by which KLF6 may play a suppressing role of uncontrolled or damaged cell proliferation.Fil: Sabatino, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Castellaro, Andrés Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Racca, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Carbajosa González, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Pansa, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Soria, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Bocco, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

Population Disequilibrium as Promoter of Adaptive Explorations in Hepatitis C Virus

Coronavirus SARS-CoV-2; COVID-19; 2019-nCoV; Virus de l'hepatitis C; Vacunes universalsCoronavirus SARS-CoV-2; COVID-19; 2019-nCoV; Virus de la hepatitis C; Vacunas universalesCoronavirus SARS-CoV-2; COVID-19; 2019-nCoV; Hepatitis C virus; Universal vaccinesReplication 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)

Baseline hepatitis C virus resistance-associated substitutions present at frequencies lower than 15% may be clinically significant

Background: Controversy is ongoing about whether a minority mutant present at frequencies below 15% may be clinically relevant and should be considered to guide treatment. Methods: Resistance-associated substitution (RAS) studies were performed in patients before and at failure of antiviral treatments using Next-generation hepatitis C virus (HCV) sequencing (NGS). Results: We have found two patients with genotype 1a infection having RAS in 3.5%-7.1% of the viral population at baseline that were selected during ledipasvir + sofosbuvir treatment. Coselection of RAS located in a region not directly affected by the antiviral treatment also occurred. This observation calls into question, the recommendations to guide RAS-based direct-acting antiviral (DAA) treatment only when RAS are present in > 15% of the sequences generated. Conclusion: Our results suggests that RAS study should include all three HCV DAA target proteins and minority mutants should be considered as clinically relevant

Partial restoration of immune response in Hepatitis C patients after viral clearance by direct-acting antiviral therapy

Fibrosi hepàtica; Cèl·lules T; Teràpia de citocinesFibrosis hepática; Células T; Terapia de citocinasLiver fibrosis; T cells; Cytokine therapyBackground & aims HCV CD4+ and CD8+ specific T cells responses are functionally impaired during chronic hepatitis C infection. DAAs therapies eradicate HCV infection in more than 95% of treated patients. However, the impact of HCV elimination on immune responses remain controversial. Here, we aimed to investigate whether HCV cure by DAAs could reverse the impaired immune response to HCV. Methods We analyzed 27 chronic HCV infected patients undergoing DAA treatment in tertiary care hospital, and we determined the phenotypical and functional changes in both HCV CD8+ and CD4+ specific T-cells before and after viral clearance. PD-1, TIM-3 and LAG-3 cell-surface expression was assessed by flow cytometry to determine CD4+ T cell exhaustion. Functional responses to HCV were analyzed by IFN-Ɣ ELISPOT, intracellular cytokine staining (IL-2 and IFN-Ɣ) and CFSE-based proliferation assays. Results We observed a significant decrease in the expression of PD-1 in CD4+ T-cells after 12 weeks of viral clearance in non-cirrhotic patients (p = 0.033) and in treatment-naive patients (p = 0.010), indicating a partial CD4 phenotype restoration. IFN-Ɣ and IL-2 cytokines production by HCV-specific CD4+ and CD8+ T cells remained impaired upon HCV eradication. Finally, a significant increase of the proliferation capacity of both HCV CD4+ and CD8+ specific T-cells was observed after HCV elimination by DAAs therapies. Conclusions Our results show that in chronically infected patients HCV elimination by DAA treatment lead to partial reversion of CD4+ T cell exhaustion. Moreover, proliferative capacity of HCV-specific CD4+ and CD8+ T cells is recovered after DAA’s therapies.This study was funded by Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF): grant numbers PI16/00337, PI18/00210 and PI19/00301. 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)

Study of Quasispecies Complexity and Liver Damage Progression after Liver Transplantation in Hepatitis C Virus Infected Patients

Fibrosis; Hepatitis C virus; Viral loadFibrosis; Virus de la hepatitis C; Carga viralFibrosi; Virus de l'hepatitis C; Càrrega viralCirrhosis derived from chronic hepatitis C virus (HCV) infection is still a common indication for liver transplantation (LT). Reinfection of the engrafted liver is universal in patients with detectable viral RNA at the time of transplant and causes fast progression of cirrhosis (within 5 years) in around one-third of these patients. To prevent damage to the liver graft, effective direct-acting antiviral (DAA) therapy is required as soon as possible. However, because of post-LT clinical instability, it is difficult to determine the optimal time to start DAAs with a low risk of complications. Evaluate changes in quasispecies complexity following LT and seek a predictive index of fast liver damage progression to determine the timing of DAA initiation. HCV genomes isolated from pre-LT and 15-day post-LT serum samples of ten patients, who underwent orthotopic LT, were quantified and sequenced using a next-generation sequencing platform. Sequence alignments, phylogenetic trees, quasispecies complexity measures, biostatistics analyses, adjusted R2 values, and analysis of variance (ANOVA) were carried out. Three different patterns of reinfection were observed (viral bottlenecking, conserved pre-LT population, and mixed populations), suggesting that bottlenecking or homogenization of the viral population is not a generalized effect after liver graft reinfection. None of the quasispecies complexity measures predicted the future degree of liver damage. Higher and more uniform viral load (VL) values were observed in all pre-LT samples, but values were more dispersed in post-LT samples. However, VL increased significantly from the pre-LT to 15-day post-LT samples in patients with advanced fibrosis at 1-year post-LT, suggesting that a VL increase on day 15 may be a predictor of fast liver fibrosis progression. HCV kinetics after LT differ between patients and are not fibrosis-dependent. Higher VL at day 15 post-LT versus pre-LT samples may predict fast liver fibrosis progression.This study was supported by grants from Instituto de Salud Carlos III cofinanced by the European Regional Development Fund (ERDF) with grant numbers PI19/00533, PI19/00301, Clinical Trial Gov. Identifier: NCT01707849, and from Centro para el Desarrollo Tecnológico Industrial-CDTI of the Spanish Ministry of Economics and Competitiveness (MINECO) grant number, IDI-20200297. C.P. is supported by the Miguel Servet program of Instituto de Salud Carlos III, grant CP14/00121, cofinanced by the ERDF. Astellas Pharma Inc and Novartis Pharma also provided funding for the study, but these companies had had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript