Estudio in vitro e in vivo de una cepa recombinante de Herpesvirus bovino 1 (BoHV-1deltagEbetagal)

Bovine herpesvirus 1 is an alfaherpesvirus that infects cattle producing respiratory, genital and reproductive disease. It is a neurotropic virus that establishes latency in sensory ganglia, and\nviral reexcretion after reactivation of latent infections is responsible for the perpetuation of the\nagent in the field. In our country, seroprevalence can reach 80% and vaccination is the strategy used to control this disease. A recombinant virus was developed in our laboratory to be used as a marker vaccine, from BoHV-1.1 strain Los Angeles (LA) through allelic replacement of the gene encoding glycoprotein E by the lac z gene, named BoHV-1?gE?gal. Genomic characterization of BoHV-1?gE?gal strain verified the absence of changes in the\ngE gene flanking regions, and specifically identified the sites where allelic exchange occurred. The RFLP study showed that this strain has a different pattern than expected, indicating changes in certain restriction sites. The study of the in vitro behavior established that this strain is less\nefficient in producing infectious viral particles as well as in viral dispersion when compared to the\nparental or other gE gene deleted strains, both in the mechanism of cell-to-cell spread and in the\nrelease of infectious viral particles into the extracellular medium. BoHV-1?gE?gal virus is\nattenuated in the natural host, and this attenuation is greater than that described for other gE gene deleted strains, albeit immunogenic, it is not excreted nor produces clinical disease after intranasal infection, and does not establish a latent infection or is reactivated experimentally. The experience of BoHV-1 viral infection and the methodology developed in this work applied to the analysis of samples from infected animals are a valuable tool for studying the\npathogenesis of genetically modified viruses, recombinant virus and field isolates. On the other\nhand, it is a contribution to the knowledge of parental BoHV-1 LA strain which is used in viral\nchallenge experiences. The results of this thesis work, taken together with the history of immunogenicity, protection against viral challenge and safety of BoHV-1?gE?gal strain and the increasing global trend towards the use of live marker vaccines in controlling diseases caused by BoHV-1, indicate that BoHV-1?gE?gal strain could be incorporated as a live immunogen in vaccination schemes of future BoHV-1 control and eradication plans in our country without risk of\ncausing illness, latency or virus spread to other animals, therefore reducing the incidence of bovine respiratory disease.\nFil: Kotsias, Fiorella. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Buenos Aires, ArgentinaFil: Kotsias, Fiorella. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones en Ciencias Veterinarias y Agronómicas. Instituto de Virología. Castelar, ArgentinaEl herpesvirus bovino 1 es un alfaherpesvirus que infecta al ganado bovino produciendo cuadros clínicos respiratorios, genitales y/o reproductivos. Es un virus neurotrópico que establece latencia en ganglios sensoriales, y la reexcreción viral luego de la reactivación de la infección\nlatente es responsable de la perpetuación del agente en los rodeos. En nuestro país la seroprevalencia puede alcanzar el 80% y la vacunación es la estrategia utilizada para el control de\nla enfermedad. En nuestro laboratorio se desarrolló, a partir de la cepa BoHV-1.1 Los Angeles (LA), un virus recombinante mediante el reemplazo alélico del gen que codifica para la\nglicoproteina E por el gen lac z, denominada BoHV-1?gE?gal, para su utilización como vacuna\nmarcadora. La caracterización genómica de la cepa BoHV-1?gE?gal verificó la ausencia de cambios en las regiones flanqueantes al gen gE y permitió identificar específicamente los sitios donde ocurrió\nel intercambio alélico. El estudio de RFLP mostró que esta cepa posee un patrón diferente al esperado, indicando modificaciones en ciertos sitios de restricción. El estudio del comportamiento\nin vitro mostró que esta cepa es menos eficiente en la producción de partículas virales infectivas\nasí como en la dispersión viral respecto a la cepa parental BoHV-1 LA y a otras cepas gE-, tanto en\nel mecanismo de pasaje célula a célula como en el de liberación de partículas virales infectivas al\nmedio extracelular. El virus BoHV-1?gE?gal se encuentra atenuado en su virulencia en el\nhospedador natural, y esta atenuación es mayor que la descripta para otras cepas gE-, ya que si bien induce una respuesta inmune humoral sérica y de mucosas, no se excreta ni produce enfermedad clínica luego de la infección intranasal, así como tampoco establece una infección latente ni se reactiva experimentalmente.\nLa experiencia de infección viral con BoHV-1 y la metodología desarrollada en este trabajo aplicada al análisis de muestras provenientes de animales infectados constituyen una valiosa herramienta para el estudio de la patogenia de virus genéticamente modificados, virus\nrecombinantes y aislamientos de campo. Por otro lado, resulta una contribución al conocimiento de la cepa parental BoHV-1 LA que se utiliza en las experiencias de desafío viral. Los resultados de\neste trabajo de Tesis, conjuntamente con los antecedentes de inmunogenicidad, protección frente al desafío viral e inocuidad de la cepa BoHV-1?gE?gal, sumado a la creciente tendencia mundial en el uso de vacunas marcadoras vivas en el control de la enfermedad causada por BoHV-1, indican que la cepa BoHV-1?gE?gal podría incorporarse como inmunógeno vivo en los esquemas de\nvacunación de futuros planes de control y erradicación de BoHV-1 en nuestro país sin riesgos de\nproducir enfermedad, latencia o diseminarse a otros animales, reduciendo la incidencia de\nenfermedad respiratoria bovina

Genomic characterization of canine circovirus associated with fatal disease in dogs in South America

Canine circovirus (CanineCV) was detected, together with canine parvovirus (CPV), in samples from an outbreak of fatal gastroenteritis in dogs in Argentina. We obtained the full-length genome of this recently discovered virus by overlapping PCR, designated strain UBA-Baires. Sequence analysis revealed a highly conserved genome but also showed several unique mutations in amino acids from the capsid protein that have not been previously reported. Phylogenetic analysis shows that this strain is more closely related to European strains than to viruses detected in North America or Asia. Although the pathogenic role of CanineCV in dogs is still unclear, this study highlights the importance of CanineCV as a coinfecting virus in disease development. To our knowledge, this is the first report of the involvement of CanineCV in severe clinical disease in dogs in South America. Our results expand our information on the geographical extent of this virus and contribute to the understanding of its role in disease.Fil: Kotsias, Fiorella. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Gastronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Bucafusco, Danilo. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; ArgentinaFil: Nuñez, Denise Anabel. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; ArgentinaFil: Lago Borisovsky, Lucía Antonella. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; ArgentinaFil: Rodriguez, Mariana. No especifíca;Fil: Bratanich, Ana Cristina. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentin

Comparative study on the in vitro and in vivo properties of two bovine herpesvirus-5 reference strains

<p>Abstract</p> <p>Background</p> <p>Bovine herpesvirus 5 (BoHV-5) is an alphaherpesvirus responsible for meningoencephalitis in young cattle and it is antigenically and genetically related to bovine herpesvirus 1. BoHV-5 outbreaks are sporadic and restricted in their geographical distribution, being mostly detected in the Southern hemisphere. The N569 and A663 strains are prototypes of the "a" and "b" subtypes of BoHV-5, however, scarce information about their <it>in vitro </it>and <it>in vivo </it>properties is currently available.</p> <p>Methods</p> <p>For the <it>in vitro </it>comparison between BoHV-5 A663 and N569 strains, viral growth kinetics, lysis and infection plaque size assays were performed. Additionally, an experimental infection of cattle with BoHV-5 A663 and N569 strains was carried out. Viral excretion, development of neurological signs, presence of specific antibodies in serum and nasal swabs and presence of latent BoHV-5 DNA in trigeminal ganglion, were analyzed. Histopathological examination of samples belonging to inoculated animals was also performed.</p> <p>Results</p> <p>The lytic capacity and the cell-to-cell spread was lower for the A663 strain compared to the N569 strain, however, the production of total infectious viral particles was similar between both strains. Concerning the <it>in vivo </it>properties, the A663 and N569 strains are able to induce similar degrees of pathogenicity in cattle.</p> <p>Conclusions</p> <p>Our results show that the A663 strain used in this study is less adapted to <it>in vitro </it>replication in MDBK cells than the N569 strain and, although slight differences were observed, both strains are able to induce a similar degree of virulence in the natural host.</p

In vitro-generated interspecific recombinants between bovine herpesviruses 1 and 5 show attenuated replication characteristics and establish latency in the natural host

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Dendritic cell maturation and cross-presentation: timing matters!

As a population, dendritic cells (DCs) appear to be the best cross-presenters of internalized antigens on major histocompatibility complex class I molecules in the mouse. To do this, DCs have developed a number of unique and dedicated means to control their endocytic and phagocytic pathways: among them, the capacity to limit acidification of their phagosomes, to prevent proteolytic degradation, to delay fusion of phagosomes to lysosomes, to recruit ER proteins to phagosomes, and to export phagocytosed antigens to the cytosol. The regulation of phagocytic functions, and thereby of antigen processing and presentation by innate signaling, represents a critical level of integration of adaptive and innate immune responses. Understanding how innate signals control antigen cross-presentation is critical to define effective vaccination strategies for CD8+ T-cell responses.Fil: Alloatti, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Inserm; FranciaFil: Kotsias, Fiorella. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Inserm; Francia. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento Virologia; ArgentinaFil: Magalhaes, Joao Gamelas. Inserm; FranciaFil: Amigorena, Sebastian. Inserm; Franci

Evaluation of the effect of Saccharomyces cerevisiae on E coli of interest in farming poultry

Objetivos. Verificar la acción aglutinante de Saccharomyces cerevisiae sobre distintos géneros bacterianos y sobre E coli aisladas de materia fecal de animales alimentados con y sin aditivos de Saccharomyces cerevisiae in vitro. Material y métodos. Se estudio la propiedad de aglutinación de E. coli y Pseudomonas aeruginosa y de E coli aisladas de pollos alimentados con y sin aditivos, de Saccharomyces cerevisiae con la técnica de aglutinación en portaobjetos y observación microscópica. Resultados. Las E coli aisladas fueron positivas a la aglutinación in vitro y se observó la reducción en el número de bacterias entéricas totales en el grupo de animales tratados con aditivo con respecto al grupo no tratado. Conclusiones. En este estudio se demostró la capacidad de Saccharomyces cerevisiae de aglutinar cepas bacterianas in vitro. La reducción en el número de bacterias entéricas totales en el grupo de animales tratados con respecto al grupo control, sugiere que un fenómeno similar se estaría produciendo in vivo, evitando probablemente la unión de las bacterias a la pared intestinal.Objectives. To verify binding action of Saccharomyces cerevisiae on different bacterial genera, and E coli isolated from feces of animals fed with and without additives, in vitro. Material and methods. The binding property of E. coli and Pseudomonas aeruginosa and E. coli isolated with slide agglutination technique microscopic observation and study. Results. The E coli isolates were positive for agglutination in vitro and reduction was observed in the total number of enteric bacteria in the group of treated animals compared to untreated group. Conclusions. In this study it was demonstrated the ability of Saccharomyces cerevisiae to bind bacterial strains in vitro. The reduction in the total number of enteric bacteria in the group of animals treated with the control group, suggesting that a similar phenomenon will be produced in vivo, probably preventing binding of bacteria to the intestinal wall .Fil: Guida, Nora. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Mesplet, Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Kotsias, Fiorella. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Gónzalez, Solange. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Bustos, Carla. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Laiño, Mariano Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Franco, Pablo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Picos, Jorge Alberto. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Mascolo, Marcela. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; Argentin

<it>In vitro</it>-generated interspecific recombinants between bovine herpesviruses 1 and 5 show attenuated replication characteristics and establish latency in the natural host

Abstract Background Interspecific recombinant viruses R1ΔgC and R2ΔgI were isolated after in vitro co-infection with BoHV-1 and BoHV-5, two closely related alphaherpesviruses that infect cattle. The genetic characterization of R1ΔgC and R2ΔgI showed that they are composed of different sections of the parental genomes. The aim of this study was the characterization of the in vivo behavior of these recombinants in the natural host. Results Four groups of four 3-month-old calves of both genders were intranasally inoculated with either the recombinant or parental viruses. A control group of two animals was also included. Viral excretion and clinical signs were monitored after infection. Histopathological examination of the central nervous system (CNS) was performed and the establishment of latency in trigeminal ganglia was analyzed by PCR. The humoral response was also evaluated using ELISA tests. Three out of four animals from the BoHV-5 infected group excreted virus for 4-10 days. Two calves shed R1ΔgC virus for one day. In R2ΔgI and BoHV-1.2ΔgCΔgI groups, infectious virus was isolated only after two or three blind passages. None of the infected animals developed neurological signs, although those infected with BoHV-5 showed histopathological evidence of viral infection. Latent viral DNA was detected in at least one calf from each infected group. Serum and/or mucosal antibodies were detected in all groups. Conclusion Both BoHV-1/-5 recombinants and the BoHV-1 parental strain are attenuated in calves, although they are able to replicate in animals at low rates and to establish latent infections.</p