African Swine Fever Virus Uses Macropinocytosis to Enter Host Cells

African swine fever (ASF) is caused by a large and highly pathogenic DNA virus, African swine fever virus (ASFV), which provokes severe economic losses and expansion threats. Presently, no specific protection or vaccine against ASF is available, despite the high hazard that the continued occurrence of the disease in sub-Saharan Africa, the recent outbreak in the Caucasus in 2007, and the potential dissemination to neighboring countries, represents. Although virus entry is a remarkable target for the development of protection tools, knowledge of the ASFV entry mechanism is still very limited. Whereas early studies have proposed that the virus enters cells through receptor-mediated endocytosis, the specific mechanism used by ASFV remains uncertain. Here we used the ASFV virulent isolate Ba71, adapted to grow in Vero cells (Ba71V), and the virulent strain E70 to demonstrate that entry and internalization of ASFV includes most of the features of macropinocytosis. By a combination of optical and electron microscopy, we show that the virus causes cytoplasm membrane perturbation, blebbing and ruffles. We have also found that internalization of the virions depends on actin reorganization, activity of Na+/H+ exchangers, and signaling events typical of the macropinocytic mechanism of endocytosis. The entry of virus into cells appears to directly stimulate dextran uptake, actin polarization and EGFR, PI3K-Akt, Pak1 and Rac1 activation. Inhibition of these key regulators of macropinocytosis, as well as treatment with the drug EIPA, results in a considerable decrease in ASFV entry and infection. In conclusion, this study identifies for the first time the whole pathway for ASFV entry, including the key cellular factors required for the uptake of the virus and the cell signaling involved

African swinw fever (ASF) virus: A missing link between poxviruses and iridoviruses

Trabajo presentado en el VII International Congress of Virology, celebrado en Edmonton (Canadá) del 09 al 14 de agosto de 1987

Erratun: G-force induced giant efficiency of nanoparticles internalization into living cells

The original version of this Article contained a typographical error in the spelling of the author Jose Luis F. Cuñado, which was incorrectly given as Jose Luis, F. Cuñad

The use of COS-1 cells for studies of field and laboratory African swine fever virus samples

Different naturally occurring, cell adapted or genetically manipulated stocks of African swine fever virus were able to infect directly cultures of COS-1 cells, producing extensive cytopathic effects and amounts from 106 to 107 of infective progeny virus per ml. The induction of late virus-specific proteins, demonstrated by RT-PCR and immunoblotting, and the development of lysis plaques by all the virus samples tested so far, allowed the optimization of both titration and diagnostic assays, as well as the proposal of a method for selection of virus clones during the generation of virus mutants with specific gene deletions.This work was supported by grants from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement KBBE-211691-ASFRISK and from Ministerio de Educación y Ciencia (BFU2007-63110/BMC), and by institutional grants from Fundación Ramón Areces and Banco Central Hispano. C. Hurtado was funded by Centro de Investigación en Sanidad Animal (CISA).Peer reviewe

Empleo de lauril galato en la prevención y tratamiento de infecciones causadas por el virus de la peste porcina (VPPA)

Empleo de lauril galato en la prevención y tratamiento de infecciones causadas por el virus de la peste porcina africana (VPPA). La presente invención se relaciona con el uso de lauril galato, o un derivado del mismo, en la elaboración de una composición farmacéutica antiviral para la prevención y/o el tratamiento de enfermedades provocadas por la infección causada por el virus de la peste porcina africana (VPPA).Peer reviewedConsejo Superior de Investigaciones Científicas (España), Fundación para la Investigación Biomédica del Hospital Universitario Ramón y CajalA1 Solicitud de patentes con informe sobre el estado de la técnic

Producción, valoración y diagnóstico de virus

23 páginas, 17 figuras.Los avances en el diagnóstico y valoración de los virus patógenos se han ido produciendo en paralelo con el refinamiento de las técnicas de cultivo de células eucariotas y de los ensayos para la detección de macromoléculas biológicas, cuya sensibilidad y especificidad ha experimentado un incremento muy importante en la última década. En este capítulo se revisan las técnicas y los requerimientos para el cultivo de células eucariotas, los métodos para la producción y purificación de preparaciones virales libres de contaminantes celulares, y los ensayos que posibilitan la detección y diagnóstico de las enfermedades producidas por la infección viral. Por razones de espacio esta revisión no puede ser muy exhaustiva, pero se abarcan en ella con cierta profundidad los diversos aspectos relacionados con la producción viral.Fundación BBVA.Peer reviewe

Empleo de lauril galato en la prevención y tratamiento de infecciones causadas por el virus de la peste porcina africana (VPPA)

The invention relates to the use of lauryl gallate, or a derivative thereof, in the production of a pharmaceutical antiviral composition for the prevention and/or treatment of diseases caused by infection resulting from African swine fever virus (ASFV).La invención relaciona al uso del galato del lauril, o a un derivado de eso, en la producción de una composición antiviral farmacéutica para la prevención y/o el tratamiento de enfermedades causados por la infección resultante del virus africano de la fiebre de los cerdos (ASFV).Peer reviewedConsejo Superior de Investigaciones Científicas (España)A1 Solicitud de patentes con informe sobre el estado de la técnic

Methods for Growing and Titrating African Swine Fever Virus Field and Laboratory Virus Samples

Growing African swine fever virus (ASFV) isolates obtained mainly from the field, but also engineered in the laboratory, is a critical step for diagnosis, titration, or virus infection studies. This unit describes a set of methods and protocols to produce and titrate any ASFV strain in cell cultures. The procedures include (1) basic techniques to prepare virus-sensitive target cells; (2) strategies for growth, concentration, and purification of virus stocks; and (3) the semi-quantitative (end dilution) and quantitative (plaque) assays for the determination of viral titers, and the use of different ASFV-sensitive cells as targets for virus production and titration.The work published by the author’s laboratory was supported by grants from the Spanish Ministerio de Ciencia e Innovación (BFU2007-63110/BMC and AGL2010-15199) and from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement KBBE- 211691- ASFRISK, and also by institutional grants from the Fundación Ramón Areces and Banco Santander Central Hispano.Peer reviewe

Inhibition of porcine viruses by different cell-targeted antiviral drugs

Antiviral compounds targeting cellular metabolism instead of virus components have become an interesting issue for preventing and controlling the spread of virus infection, either as sole treatment or as a complement of vaccination. Some of these compounds are involved in the control of lipid metabolism and/or membrane rearrangements. Here, we describe the effect of three of these cell-targeting antivirals: lauryl gallate (LG), valproic acid (VPA), and cerulenin (CRL) in the multiplication of viruses causing important porcine diseases. The results confirm the antiviral action in cultured cells of LG against African swine fever virus (ASFV), foot and mouth disease virus (FMDV), vesicular stomatitis virus (VSV), and swine vesicular disease virus (SVDV), as well as the inhibitory effect of VPA and CRL on ASFV infection. Other gallate esters have been also assayed for their inhibition of FMDV growth. The combined action of these antivirals has been also tested in ASFV infections, with some synergistic effects when LG and VPA were co-administered. Regarding the mode of action of the antivirals, experiments on the effect of the time of its addition in infected cell cultures indicated that the inhibition by VPA and CRL occurred at early times after ASFV infection, while LG inhibited a late step in FMDV infection. In all the cases, the presence of the antiviral reduced or abolished the induction of virus-specific proteins. Interestingly, LG also reduced mortality and FMDV load in a mouse model. The possible use of cell-targeted antivirals against porcine diseases is discussed.Spanish Ministerio de Ciencia e Innovación (2011-20E112), Ministerio de Economía y Competitividad (AGL2014-52395-C2-1-R and AGL2017-84097-C2-1-R), the European Community’s Seventh Framework Programme under grant agreement 311931-ASForce, Comunidad de Madrid co-financed with ECFEDER funds (S20149/ABI-2906-PLATESA; P2018/BAA-4370-PLATESA-CM), and by institutional grants from the Fundación Ramón Areces and Banco Santander Universidade

Cryo-electron tomography of isolated african swine fever virus (ASFV)

Resumen del trabajo presentado en el X Congreso Nacional de Virología, celebrado en Salamanca (España) del 21 al 24 de julio de 2009.African swine fever virus (ASFV) is a large nucleocytoplasmic DNA virus that shares the icosahedrical symmetry of iridoviruses and the complex genomic organization of the poxvirus, and it is actually the only member of the Asfaviridae family[1]. A common feature in these viral groups is that they are formed by several complex envelopes that have been investigated for decades producing uncertain results due to either the limited resolution or preparation artifacts [2,3]. The outbreak of the tomographic procedures, combined with cryo-preservation techniques, allows to follow structures and membranes in three dimensions through the whole reconstructed volume, thus revealing the continuity of structural elements without the interpretation problems arising by superposition derived from conventional two dimensional analyses of thin sections. We have obtained viral volumes of isolated AFSV particles revealing structural details of the outer envelopes, the icosahedral capsid shape as well as the internal arrangement of the core condensation. The analysis of the tomographic volumes from isolated ASFV particles indicates that, while the outer envelope seems to follow the icosahedrical symmetry, the order of the structural elements is not enough for typical symmetry superimposition for 3DEM reconstruction. This methodology combined with TOVIS (electron Tomography Of VItreus Sections), CEMOVIS (Cryo-Electron Microscopy Of VItreus Sections) and HPF-FS (High Pressure Freezing- Freeze Substitution) sections will allow us to study the formation and evolution of the ASFV envelopes inside the cell avoiding the purification damage of the viral particles