Cell-to-cell transmission is the main mechanism supporting bovine viral diarrhea virus spread in cell culture

After initiation of an infective cycle, spread of virus infection can occur fundamentally in two different ways: (i) viral particles can be released into the external environment and diffuse through the extracellular space until they interact with a new host cell, or (ii) virions can remain associated with infected cells promoting the direct passage between infected and uninfected cells that is referred to as direct cell-to-cell transmission. Although evidence for cell-associated transmission has accumulated for many different viruses, the ability of members of the genus pestivirus to use this mode of transmission has not been reported. Here we used a novel recombinant virus expressing the envelope glycoprotein E2 fused to mCherry fluorescent protein to monitor the spreading of bovine viral diarrhea virus (BVDV, the type member of pestiviuses) infection. To demonstrate direct cell-to-cell transmission of BVDV we developed a cell co-culture system that allowed us to prove the direct transmission from infected to uninfected cells in the presence of neutralizing antibodies. This mode of transmission requires cell-cell contacts and clathrin-mediated receptor-dependent endocytosis. Notably, it overcomes antibody blocking of the BVDV receptor CD46 indicating that cell-to-cell transmission of the virus involves the engagement of co-receptors on the target cell.Fil: Merwaiss, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Czibener, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Chikungunya virus replication rate determines the capacity of crossing tissue barriers in mosquitoes

Chikungunya virus (CHIKV) is a reemerging and rapidly spreading pathogen transmitted by mosquitoes. The emergence of new epidemic variants of the virus is associated with genetic evolutionary traits, including duplication of repeated RNA elements in the 3= untranslated region (UTR) that seemingly favor transmission by mosquitoes. The transmission potential of a given variant results from a complex interplay between virus populations and anatomical tissue barriers in the mosquito. Here, we used the wild-type CHIKV Caribbean strain and an engineered mutant harboring a deletion in the 3= UTR to dissect the interactions of virus variants with the anatomical barriers that impede transmission during the replication cycle of the virus in Aedes mosquitoes. Compared to the 3=-UTR mutant, we observed that the wild-type virus had a short extrinsic incubation period (EIP) after an infectious blood meal and was expectorated into mosquito saliva much more efficiently. We found that high viral titers in the midgut are not sufficient to escape the midgut escape barrier. Rather, viral replication kinetics play a crucial role in determining midgut escape and the transmission ability of CHIKV. Finally, competition tests in mosquitoes coinfected with wild-type and mutant viruses revealed that both viruses successfully colonized the midgut, but wild-type viruses effectively displaced mutant viruses during systemic infection due to their greater efficiency of escaping from the midgut into secondary tissues. Overall, our results uncover a link between CHIKV replication kinetics and the effect of bottlenecks on population diversity, as slowly replicating variants are less able to overcome the midgut escape barrier. IMPORTANCE It is well established that selective pressures in mosquito vectors impose population bottlenecks for arboviruses. Here, we used a CHIKV Caribbean lineage mutant carrying a deletion in the 3= UTR to study host-virus interactions in vivo in the epidemic mosquito vector Aedes aegypti. We found that the mutant virus had a delayed replication rate in mosquitoes, which lengthened the extrinsic incubation period (EIP) and reduced fitness relative to the wild-type virus. As a result, the mutant virus displayed a reduced capacity to cross anatomical barriers during the infection cycle in mosquitoes, thus reducing the virus transmission rate. Our findings show how selective pressures act on CHIKV noncoding regions to select variants with shorter EIPs that are preferentially transmitted by the mosquito vector.Fil: Merwaiss, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Centre National de la Recherche Scientifique; Francia. Institut Pasteur de Paris.; FranciaFil: Filomatori, Claudia Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Susuki, Yasutsugu. Centre National de la Recherche Scientifique; Francia. Institut Pasteur de Paris.; FranciaFil: Bardossy, Eugenia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Salehb, María Carla. Centre National de la Recherche Scientifique; Francia. Institut Pasteur de Paris.; Franci

Image-based Quantification of Direct Cell-to-cell Transmission of Bovine Viral Diarrhea Virus

Different viruses rely on direct cell-to-cell transmission to propagate infection within the infected host. Measuring this mode of transmission in cultured cells is often complicated by the contribution of cell free viruses to spread, and the difficulty to distinguish between primary infected cellsthat produce the virus and neighboring cells that are the target of spreading. Here, we present a protocol to quantify cell-to-cell transmission of the model pestivirus bovine viral diarrhea virus that is based on the co-culture of producer cells that are infected with a reporter virus expressing mCherry andtarget cells that stably express GFP. Spread of cell-free viruses is blocked by the presence of a neutralizing antibody in the cell culture medium, and cell-associated transmission is unequivocally quantified by numbering cells that are positive for both GFP and mCherry using automated analysis of fluorescence microscopy images.Fil: Merwaiss, Fernando. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Alvarez, Diego Ezequiel. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

Estudio de los mecanismos moleculares y celulares de la entrada y propagación del Virus de la Diarrea Viral Bovina

El virus de la diarrea viral bovina (BVDV) es un pestivirus patógeno del ganado vacuno cercanamente relacionado al virus de la hepatitis C (HCV) dentro de la familia Flaviviridae. La infección con BVDV reduce la productividad causando pérdidas económicas para la industria ganadera. La partícula viral comprende una membrana lipídica asociada a las glicoproteínas de envoltura Erns, E1 y E2, que rodean a la nucleocápside que contiene el genoma viral, el cual consiste en una única molécula de ARN simple cadena de polaridad positiva. E2 es el principal determinante antigénico en el animal infectado y produce una respuesta inmune con anticuerpos neutralizantes contra BVDV. A su vez cumple un rolfundamental para la entrada del virus a la célula huésped dado que interacciona con receptores en la superficie de la célula que median la endocitosis de la partícula viral, y forma un complejo con E1 que es esencial en la fusión de la envoltura del virus con la membrana del endosoma en medio ácido para liberar el genoma de ARN en el citoplasmade la célula infectada. La reciente determinación de la estructura cristalina de E2 ha abierto nuevas perspectivas en el estudio del mecanismo molecular de la entrada de BVDV. En este trabajo de tesis se desarrollaron nuevas herramientas genéticas y moleculares con elobjetivo de estudiar el mecanismo de entrada y propagación de BVDV, haciendo foco fundamentalmente en el rol protagónico de la glicoproteína de envoltura E2.En la primera parte de este trabajo se empleo genética invesa para producir una versión recombinante de BVDV que expresa GFP en el citoplasma de células infectadas. Utilizando este virus desarrollamos ensayos basados en la detección de GFP por microscopia de fluorescenica y citometría de flujo mediante los que pudimos demostrar que la expresión de dicha proteina funciona como reportero de la replicación de BVDV. Por otro lado se desarrolló un sistema de expresión y marcación sitio específica con biotina in vivo para la proteína de envoltura viral E2 que permitió producir una proteína recombinante marcada homogéneamente. La unión especifica de esta proteína recombinante a células susceptibles y su capacidad de bloquear la entrada de BVDV,sugieren que la misma mantiene un plegamiento y funcionalidad correctos.A partir de la bibliografía referida a las funciones de E2 y basándose en un análisis de conservación de secuencias se identificaron en la estructura de la proteína posibles motivos funcionales. En particular, analizamos el rol de un motivo de estructura tipo β-hairpin expuesto al solvente como posible responsable de la interacción con receptores. Sediseñaron mutaciones en dicha region, las cuales en el contexto de la proteína soluble resultaron en una disminucion de su capacidad de bloquear la entrada de BVDV. A continuación, se generó un virus recombinante portando las mutaciones en el β-hairpin y se observó que la infectividad de dicho virus está severamente disminuida con respecto alvirus, indicando que dicha región está involucrada en la interacción con receptores celulares.En la segunda parte de este trabajo se construyó una pareja de virus recombinantes citopático y no citopático que expresa una versión de E2 en fase con la proteína fluorescente mCherry en su extremo amino terminal. A través de la detección de mCherry se pudieron identificar células infectadas y seguir así la propagación de la infección en células vivas. Empleando esta herramienta nos propusimos estudiar si BVDV tiene la capacidad de propagar en forma directa de célula a célula. Para esto se diseñaron ensayos de co-cultivo, donde se pudo observar la propagación de la infección viral desde células productoras de virus hacia células blanco aun en presencia de suero neutralizante en el medio. De esta forma demostramos por primera vez la existencia de propagación célula a célula en pestivirus. Con estas mismas herramientas comenzamos a estudiar cuales son los factores virales y celulares que median este tipo de propagación, hallando que la misma depende de la interacción de E2 con receptores específicos.Fil: Merwaiss, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Universidad Nacional de San Martín; ArgentinaFil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Czibener, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Production of Potyvirus-Derived Nanoparticles Decorated with a Nanobody in Biofactory Plants

[EN] Viral nanoparticles (VNPs) have recently attracted attention for their use as building blocks for novel materials to support a range of functions of potential interest in nanotechnology and medicine. Viral capsids are ideal for presenting small epitopes by inserting them at an appropriate site on the selected coat protein (CP). VNPs presenting antibodies on their surfaces are considered highly promising tools for therapeutic and diagnostic purposes. Due to their size, nanobodies are an interesting alternative to classic antibodies for surface presentation. Nanobodies are the variable domains of heavy-chain (VHH) antibodies from animals belonging to the family Camelidae, which have several properties that make them attractive therapeutic molecules, such as their small size, simple structure, and high affinity and specificity. In this work, we have produced genetically encoded VNPs derived from two different potyviruses¿the largest group of RNA viruses that infect plants¿decorated with nanobodies. We have created a VNP derived from zucchini yellow mosaic virus (ZYMV) decorated with a nanobody against the green fluorescent protein (GFP) in zucchini (Cucurbita pepo) plants. As reported for other viruses, the expression of ZYMV-derived VNPs decorated with this nanobody was only made possible by including a picornavirus 2A splicing peptide between the fused proteins, which resulted in a mixed population of unmodified and decorated CPs. We have also produced tobacco etch virus (TEV)-derived VNPs in Nicotiana benthamiana plants decorated with the same nanobody against GFP. Strikingly, in this case, VNPs could be assembled by direct fusion of the nanobody to the viral CP with no 2A splicing involved, likely resulting in fully decorated VNPs. For both expression systems, correct assembly and purification of the recombinant VNPs was confirmed by transmission electron microscope; the functionality of the CP-fused nanobody was assessed by western blot and binding assays. In sum, here we report the production of genetically encoded plant-derived VNPs decorated with a nanobody. This system may be an attractive alternative for the sustainable production in plants of nanobody-containing nanomaterials for diagnostic and therapeutic purposes.This research was supported by grant PID 2020-114691RB-I00 from the Ministerio de Ciencia e Innovacion (Spain; co-financed by the European Region Development Fund) through the Agencia Estatal de Investigacion. MM is the recipient of a predoctoral contract (FPU16/05294) from the Ministerio de Educacion, Cultura y Deporte (Spain).Martí, MC.; Merwaiss, F.; Butkovic, A.; Daròs, J. (2022). Production of Potyvirus-Derived Nanoparticles Decorated with a Nanobody in Biofactory Plants. Frontiers in Bioengineering and Biotechnology. 10:1-13. https://doi.org/10.3389/fbioe.2022.8773631131

Impact of alphavirus 3’UTR plasticity on mosquito transmission

Alphaviruses such as chikungunya and western equine encephalitis viruses are important human pathogens transmitted by mosquitoes that have recently caused large epidemic and epizootic outbreaks. The epidemic potential of alphaviruses is often related to enhanced mosquito transmission. Tissue barriers and antiviral responses impose bottlenecks to viral populations in mosquitoes. Substitutions in the envelope proteins and the presence of repeated sequence elements (RSEs) in the 3’UTR of epidemic viruses were proposed to be specifically associated to efficient replication in mosquito vectors. Here, we discuss the molecular mechanisms that originated RSEs, the evolutionary forces that shape the 3’UTR of alphaviruses, and the significance of RSEs for mosquito transmission. Finally, the presence of RSEs in the 3’UTR of viral genomes appears as evolutionary trait associated to mosquito adaptation and emerges as a common feature among viruses from the alphavirus and flavivirus genera.Fil: Filomatori, Claudia Veronica. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Merwaiss, Fernando. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Bardossy, Eugenia Soledad. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Alvarez, Diego Ezequiel. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

BigA is a novel adhesin of Brucella that mediates adhesion to epithelial cells

Adhesion to cells is the initial step in the infectious cycle of basically all pathogenic bacteria, and to do so, microorganisms have evolved surface molecules that target different cellular receptors. Brucella is an intracellular pathogen that infects a wide range of mammals whose virulence is completely dependent on the capacity to replicate in phagocytes. Although much has been done to elucidate how Brucella multiplies in macrophages, we still do not understand how bacteria invade epithelial cells to perform a replicative cycle or what adhesion molecules are involved in the process. We report the identification in Brucella abortus of a novel adhesin that harbours a bacterial immunoglobulin-like domain and demonstrate that this protein is involved in the adhesion to polarized epithelial cells such as the Caco-2 and Madin-Darby canine kidney models targeting the bacteria to the cell-cell interaction membrane. While deletion of the gene significantly reduced adhesion, over-expression dramatically increased it. Addition of the recombinant protein to cells induced cytoskeleton rearrangements and showed that this adhesin targets proteins of the cell-cell interaction membrane in confluent cultures.Fil: Czibener, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Merwaiss, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Guaimas, Francisco Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: del Giudice, Mariela Giselda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Rey Serantes, Diego Armando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Spera, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ugalde, Juan Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

RNA recombination at Chikungunya virus 3'UTR as an evolutionary mechanism that provides adaptability.

The potential of RNA viruses to adapt to new environments relies on their ability to introduce changes in their genomes, which has resulted in the recent expansion of re-emergent viruses. Chikungunya virus is an important human pathogen transmitted by mosquitoes that, after 60 years of exclusive circulation in Asia and Africa, has rapidly spread in Europe and the Americas. Here, we examined the evolution of CHIKV in different hosts and uncovered host-specific requirements of the CHIKV 3'UTR. Sequence repeats are conserved at the CHIKV 3'UTR but vary in copy number among viral lineages. We found that these blocks of repeated sequences favor RNA recombination processes through copy-choice mechanism that acts concertedly with viral selection, determining the emergence of new viral variants. Functional analyses using a panel of mutant viruses indicated that opposite selective pressures in mosquito and mammalian cells impose a fitness cost during transmission that is alleviated by recombination guided by sequence repeats. Indeed, drastic changes in the frequency of viral variants with different numbers of repeats were detected during host switch. We propose that RNA recombination accelerates CHIKV adaptability, allowing the virus to overcome genetic bottlenecks within the mosquito host. These studies highlight the role of 3'UTR plasticity on CHIKV evolution, providing a new paradigm to explain the significance of sequence repetitions

A β-Hairpin motif in the envelope protein E2 mediates receptor binding of bovine viral diarrhea virus

Pestivirus envelope protein E2 is crucial to virus infection and accomplishes virus-receptor interaction during entry. However, mapping of E2 residues mediating these interactions has remained unexplored. In this study, to investigate the structure-function relationship for a β-hairpin motif exposed to the solvent in the crystal structure of bovine viral diarrhea virus (BVDV) E2, we designed two amino acidic substitutions that result in a change of electrostatic potential. First, using wild type and mutant E2 expressed as soluble recombinant proteins, we found that the mutant protein had reduced binding to susceptible cells compared to wild type and diminished ability to inhibit BVDV infection, suggesting a lower affinity for BVDV receptors. We then analyzed the effect of β-hairpin mutations in the context of recombinant viral particles. Mutant viruses recovered from cell culture supernatant after transfection of recombinant RNA had almost completely inhibited ability to re-infect susceptible cells, indicating an impact of mutations on BVDV infectivity. Finally, sequential passaging of the mutant virus resulted in the selection of a viral population in which β-hairpin mutations reverted to the wild type sequence to restore infectivity. Taken together, our results show that this conserved region of the E2 protein is critical for the interaction with host cell receptors.Fil: Merwaiss, Fernando. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Pascual, María José. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Pomilio, María Trinidad. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: López, María Gabriela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Alvarez, Diego Ezequiel. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

Structure-based drug design for envelope protein E2 uncovers a new class of bovine viral diarrhea inhibitors that block virus entry

Antiviral targeting of virus envelope proteins is an effective strategy for therapeutic intervention of viral infections. Here, we took a computer-guided approach with the aim of identifying new antivirals against the envelope protein E2 of bovine viral diarrhea virus (BVDV). BVDV is an enveloped virus with an RNA genome responsible for major economic losses of the cattle industry worldwide. Based on the crystal structure of the envelope protein E2, we defined a binding site at the interface of the two most distal domains from the virus membrane and pursued a hierarchical docking-based virtual screening search to identify small-molecule ligands of E2. Phenyl thiophene carboxamide derivative 12 (PTC12) emerged as a specific inhibitor of BVDV replication from in vitro antiviral activity screening of candidate molecules, displaying an IC50 of 0.30 μM against the reference NADL strain of the virus. Using reverse genetics we constructed a recombinant BVDV expressing GFP that served as a sensitive reporter for the study of the mechanism of action of antiviral compounds. Time of drug addition assays showed that PTC12 inhibited an early step of infection. The mechanism of action was further dissected to find that the compound specifically acted at the internalization step of virus entry. Interestingly, we demonstrated that similar to PTC12, the benzimidazole derivative 03 (BI03) selected in the virtual screen also inhibited internalization of BVDV. Furthermore, docking analysis of PTC12 and BI03 into the binding site revealed common interactions with amino acid residues in E2 suggesting that both compounds could share the same molecular target. In conclusion, starting from a targeted design strategy of antivirals against E2 we identified PTC12 as a potent inhibitor of BVDV entry. The compound can be valuable in the design of antiviral strategies in combination with already well-characterized polymerase inhibitors of BVDV.Instituto de VirologíaFil: Pascual, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Merwaiss, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Leal, Emilse Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias ; ArgentinaFil: Quintana, Maria Eugenia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Capozzo, Alejandra Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Tecnológicas; ArgentinaFil: Cavasotto, Claudio Norberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Bollini, Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias ; ArgentinaDil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin