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African swine fever virus (ASFV) is an extremely genetically and phenotypically heterogeneous pathogen. Previously, we have demonstrated that experimental inoculation of pigs with an attenuated strain, Katanga-350 (genotype I, seroimmunotype I) (ASFV-Katanga-350), can induce protective immunity in 80% of European domestic pigs against the homologous virulent European strain Lisbon-57. At least 50% of the surviving pigs received protection from subsequent intramuscular infection with a heterologous virulent strain, Stavropol 01/08 (genotype II, seroimmunotype VIII) (ASFV-Stavropol 01/08). In this study, we assessed clinical signs, the levels of viremia, viral DNA, anti-ASFV antibodies and post-mortem changes caused by subsequent intramuscular injection with ASFV-Katanga-350 and heterologous ASFV-Stavropol 01/08. Inoculation of pigs with the ASFV-Katanga-350 did not protect animals from the disease in the case of the subsequent challenged ASFV-Stavropol 01/08. However, 40% of pigs were protected from death. Moreover, the surviving animals showed no pathomorphological changes or the presence of an infectious virus in the organs after euthanasia at 35 days post challenging. The ability/inability of attenuated strains to form a certain level of protection against heterologous isolates needs a theoretical background and experimental confirmation.

ilustraciones, fotografías, graficas, tablas La tuberculosis (TB) es una enfermedad infecciosa causada por el bacilo ácido alcohol resistente Mycobacterium tuberculosis (Mtb). La TB es una amenaza para la salud pública, debido a su alta incidencia, la aparición de cepas multi-fármaco-resistentes (MDR) y extremadamente-fármaco-resistentes (XDR), la coinfección con VIH y la eficacia limitada de la vacuna BCG. El diseño de nuevas estrategias de control requiere una mejor comprensión de los mecanismos moleculares utilizados por Mtb para ser un patógeno intracelular tan exitoso. En este sentido, algunos estudios han sugerido la importancia de las ATPasas tipo P en la fisiología y la supervivencia intracelular de las micobacterias. Un meta-análisis del perfil transcripcional de las ATPasas tipo P de Mtb bajo condiciones de estrés como hipoxia, estrés oxidativo, inanición, intoxicación por agentes químicos y procesos de infección in vitro e in vivo, evidenció la expresión diferencial de estos transportadores frente a estas condiciones. De las 12 ATPasas tipo P presentes en el genoma de Mtb, CtpF, que codifica para un transportador de Ca2+, es la ATPasa que muestra mayores niveles de transcripción en las diferentes condiciones de estrés. Particularmente, varias ATPasas tipo P (ctpF, ctpG, ctpC, ctpH y ctpV) exhibieron un aumento en los niveles de expresión durante la infección de macrófagos humanos, sugiriendo la importancia de dichas proteínas en los procesos de infección. Considerando la relevancia funcional de las ATPasas tipo P, el objetivo principal de este trabajo fue evaluar el efecto de la deleción de ATPasas tipo P transportadoras de metales alcalino/alcalinotérreos en la viabilidad y virulencia de Mtb. Para lograr este objetivo, mediante técnicas de recombinería se construyeron los mutantes defectivos en los genes ctpF y ctpH de Mtb, y a partir de distintos análisis funcionales con las cepas mutantes, se demostró que ambos transportadores están implicados en el eflujo de Ca2+. Adicionalmente, las cepas mutantes (MtbΔctpF y MtbΔctpH) mostraron hipersensibilidad frente agentes oxidantes en comparación con la cepa tipo silvestre (MtbWT), indicando un vinculo entre el transporte de Ca2+ y los mecanismos que utiliza el bacilo para neutralizar especies reactivas del ambiente intrafagosomal. Por otro lado, se evaluó en un modelo celular y animal el efecto de la deleción del gen ctpF en la virulencia Mtb. Así, se evidenció que dicha mutación genera una disminución significativa en la capacidad replicativa de Mtb en macrófagos alveolares murinos de la línea celular MH-S. Asimismo, se comparó la virulencia de las cepas MtbΔctpF y MtbWT en un ensayo de sobrevida en ratones BALB/c, encontrando que los ratones infectados con la cepa mutante mostraban mayor tiempo medio de supervivencia, sugiriendo la atenuación de la cepa mutante. Finalmente, se comprobó la existencia de estrategias complementarias que permiten contrarrestar deficiencias en el transporte iónico mediado por las ATPasa tipo P en Mtb. En efecto, se encontró que la cepa MtbΔctpF sobreexpresa el gen ctpH frente a concentraciones tóxicas de Ca2+ y durante los procesos de infección in vitro. De manera similar, el mutante MtbΔctpH sobreexpresó el gen ctpF bajo condiciones tóxicas de Ca2+, sugiriendo una posible actividad compensatoria entre CtpF y CtpH en Mtb. En general, los resultados obtenidos en este trabajo demuestran que las Ca+2-ATPasas están involucradas en la respuesta frente sustancias tóxicas, siendo fundamentales para la supervivencia celular de Mtb. Además, CtpF es relevante para la proliferación intracelular, y su deleción genera atenuación del bacilo tuberculoso en un modelo experimental de TB pulmonar. De esta manera, CtpF es fundamental para la virulencia de Mtb, por lo que podría considerarse un interesante blanco de atenuación. (texto tomado de la fuente) Tuberculosis (TB) is an infectious disease caused by the acid and alcohol-resistant bacillus Mycobacterium tuberculosis (Mtb). TB is considered a public health threat due to its high incidence, the emergence of drug-resistant strains (MDR and XDR), coinfection with HIV, and the limited efficacy of the BCG vaccine. Consequently, the design of new TB control strategies relies on a better comprehension of the molecular mechanisms used by Mtb to be a successful intracellular pathogen. In this sense, some studies have suggested that P-type ATPases are relevant to the physiology and intracellular survival of mycobacteria. Specifically, a meta-analysis of the transcriptional levels of Mtb P-type ATPases under conditions of hypoxia, oxidative stress, starvation, intoxication by chemical agents, and in vitro and in vivo infection processes indicated that these transporters are differentially expressed in these situations. Among the 12 P-type ATPases encoded in the Mtb genome, CtpF encodes a Ca2+ transporter and is the most activated against the conditions studied. Furthermore, several P-type ATPases (ctpF, ctpG, ctpC, ctpH, and ctpV) show over-expression during infection of human macrophages, suggesting the relevance of these proteins in the infection process. Therefore, the main objective of this study was to evaluate the effect of the deletion of alkali/alkaline earth metal-transporting P-type ATPases on the viability and virulence of Mtb. For this, recombination techniques were applied to construct mutants defective in the Mtb ctpF and ctpH genes that encode alkali/alkaline earth metal-transporting ATPases. Diverse functional analyses of these mutants demonstrated that both transporters are involved in Ca2+ efflux. Additionally, the mutant strains (MtbΔctpF and MtbΔctpH) showed hypersensitivity to oxidizing agents compared to the wild type strain (MtbWT), indicating a link between Ca2+ transport and the mechanism used by the bacillus to neutralize reactive species in the intraphagosomal environment. Moreover, the effect ctpF gene deletion on Mtb virulence was evaluated in cellular and animal infection models. Accordingly, this deletion generated a significant decrease in the replicative capacity of Mtb in murine alveolar macrophages of the MH-S cell line. Furthermore, a comparison of the virulence of MtbΔctpF and MtbWT strains through survival tests in BALB/c mice demonstrated that mice infected with the mutant strain showed a longer mean survival time, suggesting the attenuation of the deleted strain. Finally, the existence of a possible compensatory mechanism to counteract deficiencies in ion transport mediated by P-type ATPases in Mtb was evaluated. Indeed, the MtbΔctpF strain over-expresses the ctpH gene against toxic concentrations of Ca2+ and during in vitro infection processes. Similarly, the MtbΔctpH mutant over-expresses the ctpF gene under toxic concentrations of Ca2+, suggesting a possible compensatory activity between ctpF and ctpH in Mtb. This study demonstrates that Ca+2-ATPases are involved in the response to toxic substances, being essential for Mtb survival. Furthermore, CtpF is required for the intracellular proliferation of the mycobacteria, whereas its deletion attenuates the bacillus in an experimental model of pulmonary TB. Overall, CtpF is critical for Mtb virulence and can be an interesting attenuation target. Convocatoria nacional para el apoyo a proyectos de investigación y creación artística de la Universidad Nacional de Colombia 2017-2018 (Código 2010100-29088). Convocatoria nacional para el fomento de alianzas interdisciplinarias que articulen investigación, creación, extensión y formación en la Universidad Nacional de Colombia 2019-2021 (Código 2010100-29665). Doctorado Doctor en Ciencias - Bioquímica Para el desarrollo de los objetivos propuestos en el presente trabajo, se estableció una estrategia experimental que incluyó herramientas bioquímicas, microbiológicas y de biología molecular. Hospedero-Patógeno

The African swine fever virus (ASFV) is the cause of a recent pandemic that is threatening the global pig industry. The virus infects domestic and wild pigs and manifests with a variety of clinical symptoms, depending on the strain. No commercial vaccine is currently available to protect animals from this virus, but some attenuated and recombinant live vaccine candidates might be effective against the disease. This article describes the immunobiological characteristics of one such candidate—the laboratory-attenuated ASFV strain, Katanga-350—which belongs to genotype I. In this study, we assessed clinical signs and post-mortem changes, the levels of viremia and the presence of viral DNA caused by injection of ASF virus strains Katanga-350, Lisbon-57, and Stavropol 08/01. Intramuscular injection of this strain protected 80% of pigs from a virulent strain of the same genotype and seroimmunotype (Lisbon-57). At least 50% of the surviving pigs received protection from subsequent intramuscular infection with a heterologous (genotype II, seroimmunotype VIII) virulent strain (Stavropol 08/01). Virus-specific antibodies were detectable in serum and saliva samples between 8–78 days after the first inoculation of the Katanga-350 strain (the observational period). The results suggested that this strain could serve as a basis for the development of a recombinant vaccine against ASF viruses belonging to seroimmunotype I.

Genotype-matched vaccines provide ideal protection against infection caused by new Newcastle disease virus (NDV) genotypes or variants even in the vaccinated chickens. In this study, we report a protocol for attenuation and rapid development of a velogenic NDV isolate as an effective vaccine candidate, using a simple and reliable infectious cloning platform. Based on DHN3, a genotype VII velogenic NDV isolate, recombinant rDHN3 was rescued by co-transfection of plasmids expressing the genomic RNA, NDV proteins NP, P and L, and the T7 polymerase without using a helper virus. Subsequently, an attenuated strain rDHN3-mF was produced by substitution of residues from amino acids 112 to 117 in the DHN3 F protein with the corresponding sequence from the LaSota strain. Both rDHN3 and rDHN3-mF are genetically stable during propagation in cell culture and chicken embryos. Further characterization through determination of EID50, MDT and clinical assessments confirmed that rDHN3 is velogenic and rDHN3-mF lentogenic. Vaccination of one-week-old SPF chicks with inactivated rDHN3-mF produced much higher anti-DHN3 antibody response and better protection against live DHN3 challenge than did the commercial LaSota vaccine, providing 100% protection and much earlier viral clearance. This attenuated NDV isolate would merit further development into a vaccine product.

This article summarizes the study results on the generation of attenuated strains of African swine fever virus (ASFV) of seroimmunotypes I–VIII and the creation of live vaccines for temporary protection of pigs during a period of epizootics in the surveillance zone (a zone adjacent to the area of outbreak). These studies were initiated at the Federal Research Center for Virology and Microbiology (FRCVM, formerly VNIIVViM) at the time of introduction of the pathogen to the Iberian Peninsula in the middle of the 20th century. The developed experimental vaccines against ASFV seroimmunotypes I–V provided protection against virulent strains of homologous seroimmunotypes by day 14 after vaccination, lasting at least four months.

The threat of cholera spread beyond the borders of endemic countries and the realness of the emergence of introduced epidemic foci remain the actual problems and neccessitate continuous development of specific prophylaxis of this disease. The review is dedicated to the analysis of the effectiveness of licensed cholera vaccines as well as to possible perspectives of the advancement of cholera specific prophylaxis.

The major Mycobacterium tuberculosis virulence factor ESAT-6 exported by the ESX-1 secretion system has been described as a pro-apoptotic factor by several independent groups in recent years, sustaining a role for apoptosis in M. tuberculosis pathogenesis. This role has been supported by independent studies in which apoptosis has been shown as a hallmark feature in human and mouse lungs infected with virulent strains. Nevertheless, the role of apoptosis during mycobacterial infection is subject to an intense debate. Several works maintain that apoptosis is more evident with attenuated strains, whereas virulent mycobacteria tend to inhibit this process, suggesting that apoptosis induction may be a host mechanism to control infection. In this review, we summarize the evidences that support the involvement of ESX-1-induced apoptosis in virulence, intending to provide a rational treatise for the role of programmed cell death during M. tuberculosis infection.