Immunization with Neospora caninum profilin induces limited protection and a regulatory T-cell response in mice.

Profilins are actin-binding proteins that regulate the polymerization of actin filaments. In apicomplexan parasites, they are essential for invasion. Profilins also trigger the immune response of the host by activating TLRs on dendritic cells (DCs), inducing the production of pro-inflammatory cytokines. In this study we characterized for the first time the immune response and protection elicited by a vaccine based on Neospora caninum profilin in mice. Groups of eight BALB/c mice received either two doses of a recombinant N. caninum profilin expressed in Escherichia coli. (rNcPRO) or PBS, both formulated with an aqueous soy-based adjuvant enriched in TLR-agonists. Specific anti-profilin antibodies were detected in rNcPRO-vaccinated animals, mainly IgM and IgG3, which were consumed after infection. Splenocytes from rNcPRO-immunized animals proliferated after an in vitro stimulation with rNcPRO before and after challenge. An impairment of the cellular response was observed in NcPRO vaccinated and infected mice following an in vitro stimulation with native antigens of N. caninum, related to an increase in the percentage of CD4+CD25+FoxP3+. Two out of five rNcPRO-vaccinated challenged mice were protected; they were negative for parasite DNA in the brain and showed no histopathological lesions, which were found in all PBS-vaccinated animals. As a whole, our results provide evidence of a regulatory response elicited by immunization with rNcPRO, and suggest a role of profilin in the modulation and/or evasion of immune responses against N. caninum.

In-vivo Activity of IFN-λ and IFN-α Against Bovine-Viral-Diarrhea Virus in a Mouse Model.

Bovine-viral-diarrhea virus (BVDV) can cause significant economic losses in livestock. The disease is controlled with vaccination and bovines are susceptible until vaccine immunity develops and may remain vulnerable if a persistently infected animal is left on the farm; therefore, an antiviral agent that reduces virus infectivity can be a useful tool in control programs. Although many compounds with promising in-vitro efficacy have been identified, the lack of laboratory-animal models limited their potential for further clinical development. Recently, we described the activity of type I and III interferons, IFN-α and IFN-λ respectively, against several BVDV strains in-vitro. In this study, we analyzed the in-vivo efficacy of both IFNs using a BALB/c-mouse model. Mice infected with two type-2 BVDV field strains developed a viremia with different kinetics, depending on the infecting strain's virulence, that persisted for 56 days post-infection (dpi). Mice infected with the low-virulence strain elicited high systemic TNF-α levels at 2 dpi. IFNs were first applied subcutaneously 1 day before or after infection. The two IFNs reduced viremia with different kinetics, depending on whether either one was applied before or after infection. In a second experiment, we increased the number of applications of both IFNs. All the treatments reduced viremia compared to untreated mice. The application of IFN-λ pre- and post-infection reduced viremia over time. This study is the first proof of the concept of the antiviral potency of IFN-λ against BVDV in-vivo, thus encouraging further trails for a potential use of this cytokine in cattle.

Interferon lambda protects cattle against bovine viral diarrhea virus infection.

Interferon lambda (IFN-λ) plays an important role in inducing an antiviral state in mucosal surfaces and has been used as an effective biotherapeutic against several viral diseases. Here we performed a proof of concept study on the activity of a biologically active recombinant bovine IFN-λ (rIFN-λ) produced in eukaryotic cells against Bovine Viral Diarrhea Virus (BVDV) in cattle. We first confirmed the lack of toxicity of different concentrations of rIFN-λ in bovine peripheral blood cells and the safety of its subcutaneous application in calves in doses up to 12 IU/kg. The antiviral activity of the rIFN-λ against BVDV was assessed in calves that were inoculated with 6 IU/kg of rIFN-λ (n = 4) or mock-treated (n = 2) two days before and after challenge with a BVDV type-2 non-cytopathic strain. Mock-treated animals developed respiratory disease, shedded the virus from 4 to 7 days post-infection (dpi) and had viremia between 4 and 14 dpi. Conversely, calves treated with rIFN-λ did not develop clinical symptoms. The virus was not found in nasal secretions or sera. Only one animal had a positive viral RNA detection in serum at 7 dpi. All infected animals treated with rIFN-λ increased systemic type-I IFNs levels at 4 dpi. The antiviral treatment induced an earlier onset of the anti-BVDV neutralizing antibodies. Altogether, these results constitute the proof-of-principle of bovine IFN-λ as an antiviral biotherapeutic to protect cattle against the clinical disease caused by BVDV.

A direct high-throughput in Cell-ELISA for measuring infectivity of cytopathic and non-cytopathic bovine viral diarrhoea virus strains applied to the assessment of antiviral activity.

Low-cost high-throughput methods applicable to any virus strain are required for screening antiviral compounds against multiple field strains. Colorimetric cell-viability assays are used for this purpose as long as the viruses are cytopathic (CP) in cell culture. However, bovine viral diarrhoea virus (BVDV) strains circulating in the field are mostly non-cytopathic (NCP). An In Cell-ELISA aimed to measure viral infectivity by detecting a conserved protein produced during viral replication (non-structural protein 3, "NS3") was developed. The ELISA is performed without harvesting the cells, directly on the 96-wells culture plate. NS3 In Cell-ELISA was tested for its ability to assess BVDV-specific antiviral activity of recombinant bovine type I and III IFNs. Results correlated to those measured by qRT-PCR and virus titration. NS3 In Cell-ELISA was also efficient in estimating the IC50 of two compounds with different antiviral activity. Estimation of the 50% inhibition dose of each IFN using six BVDV strains of different biotype and genotype showed that CP strains were more susceptible to both IFNs than NCP, while type 2 NCP viruses were more sensitive to IFN-I. The In Cell-ELISA format using a detector antibody against a conserved non-structural protein can be potentially applied to accurately measure infectivity of any viral strain.

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.

Bovine Viral Diarrhea Virus Infects Monocyte-Derived Bovine Dendritic Cells by an E2-Glycoprotein-Mediated Mechanism and Transiently Impairs Antigen Presentation.

Infection of professional antigen presenting cells by viruses can have a marked effect on these cells and important consequences for the generation of subsequent immune responses. In this study, we demonstrate that different strains of bovine viral diarrhea virus (BVDV) infect bovine dendritic cells differentiated from nonadherent peripheral monocytes (moDCs). BVDV did not cause apoptosis in these cells. Infection of moDC was prevented by incubating the virus with anti-E2 antibodies or by pretreating the cells with recombinant E2 protein before BVDV contact, suggesting that BVDV infects moDC through an E2-mediated mechanism. Virus entry was not reduced by incubating moDC with Mannan or ethylenediaminetetraacetic acid (EDTA) before infection, suggesting that Ca(2+) and mannose receptor-dependent pathways are not mediating BVDV entry to moDC. Infected moDC did not completely upregulate maturation surface markers. Infection, but not treatment with inactivated virus, prevented moDC to present a third-party antigen to primed CD4(+) T cells within the first 24 hours postinfection (hpi). Antigen-presenting capacity was recovered when viral replication diminished at 48 hpi, suggesting that active infection may interfere with moDC maturation. Altogether, our results suggest an important role of infected DCs in BVDV-induced immunopathogenesis.

Mesotelioma y ocupación: revisión de casos en Uruguay: 2002-2014 / Mesothelioma and occupation: review of cases in Uruguay: 2002-2014 / Mesotelioma e ocupação: revisão de casos no Uruguai: 2002-2014

El mesotelioma es considerado en el mundo industrializado a consecuencia de la exposición ocupacional a fibras de asbesto. A nivel país se considera una enfermedad profesional. El objetivo del presente trabajo fue conocer y describir casos de mesotelioma notificados en Uruguay entre los años 2002 y 2014, con énfasis en los aspectos de la exposición ocupacional. El presente trabajo corresponde a un estudio descriptivo retrospectivo, a partir de los casos notificados se recrearon historias médicas enlazando con datos de servicios asistenciales. Se identificaron fuentes de exposición al asbesto en diferentes ocupaciones e industrias en el país. Resultados: fueron notificados 122 casos. Se accedió a la historia clínica en un tercio (47/122). El dato ocupación estaba consignado solo en 27/47, en 3/47 se explicitaba la exposición al asbesto/amianto. Los sectores productivos identificados mayoritariamente correspondieron a transporte, metalúrgico, construcción y limpieza. Se evidenció un registro insuficiente del dato ocupación y de los antecedentes laborales. Ésta información laboral es fundamental para establecer el nexo causal de la exposición en estudio y la condición de enfermedad profesional. La gravedad de la enfermedad y el conocimiento del riesgo derivado de la exposición, laboral, justifica el desarrollo de políticas en salud ocupacional. Es necesario fortalecer la formación de los profesionales de la salud sobre la importancia del trabajo como determinante del proceso salud - enfermedad.

Mesothelioma is considered in the industrialized world as a consequence of occupational exposure to asbestos fibers - asbestos. At the country level it is considered an occupational disease. The objective was to know and describe cases of mesothelioma notified in Uruguay between the years 2002 and 2014, with emphasis on aspects of occupational exposure. The present work corresponds to a retrospective descriptive study, from the reported cases medical records were recreated linking with data from healthcare services. Sources of asbestos exposure were identified in different occupations and industries in the country. Results: 122 cases were notified. The medical history was accessed in one third (47/122). The occupation data was only in 27/47, in 3/47 the exposure to asbestos / asbestos was specified. The productive sectors identified mainly corresponded to transportation, metallurgy, construction and cleaning. Insufficient registration of occupation and employment history was evidenced. This work information is essential to establish the causal link between the exposure under study and the occupational disease condition. The severity of the disease and knowledge of the risk derived from exposure occupational, justify the development of occupation health policies. It is necessary to strengthen the training of health professionals on the importance of work as a determinant of the health - disease process.

O mesotelioma é considerado no mundo industrializado como consequência da exposição ocupacional às fibras de amianto - o asbesto. Em nível nacional, é considerada uma doença ocupacional. O objetivo foi conhecer e descrever os casos de mesotelioma notificados no Uruguai entre os anos de 2002 a 2014, com ênfase nos aspectos de exposição ocupacional. O presente trabalho corresponde a um estudo descritivo retrospectivo, a partir dos casos relatados, prontuários médicos foram recriados vinculando-os a dados de serviços de saúde. Fontes de exposição ao amianto foram identificadas em diferentes ocupações e indústrias no país. Resultados: foram notificados 122 casos. O histórico médico foi acessado em um terço (47/122). Os dados de ocupação foram apenas em 27/47, em 3/47 foi especificada a exposição ao amianto / amianto. Os setores produtivos identificados corresponderam principalmente a transportes, metalurgia, construção e limpeza. Foi evidenciado registro insuficiente de ocupação e histórico de empregos. Essas informações de trabalho são essenciais para estabelecer o nexo causal entre a exposição em estudo e a condição de doença ocupacional. A gravidade da doença e o conhecimento do risco decorrente da exposição ocupacional, justificam o desenvolvimento de políticas de saúde ocupacional. É preciso fortalecer a formação dos profissionais de saúde sobre a importância do trabalho como determinante do processo saúde - doença.

Safety and immunogenicity of a soluble native Neospora caninum tachyzoite-extract vaccine formulated with a soy lecithin/ß-glucan adjuvant in pregnant cattle.

The global economic impact of Neospora caninum infection in cattle herds has promoted the development of vaccines that can be safely used during pregnancy. The aim of this study was to evaluate the safety and immunogenicity of a vaccine formulated with the soluble fraction of tachyzoite's lysate and a soy-based aqueous adjuvant (sNcAg/AVEC), which was protective in the mouse model and induced strong IFN-γ responses and high avidity antibodies in non-pregnant cattle. Ten pregnant heifers were vaccinated twice during the first trimester of gestation and 8 remained unvaccinated. Anti-N. caninum immune responses were efficiently primed by vaccination, evidenced by a quick induction of IgM serum titers (7dpv) and a prompt switch to high avidity IgG shortly after infection (performed at 78 or 225 days of gestation; n=5 each); while naïve cattle elicited lower IgG titers, with a delayed kinetics. High systemic IFN-γ levels were induced after infection which did not interfere with pregnancy. No local or systemic adverse effects were recorded along the study. Calves were born in term and in good health conditions, showing that the sNcAg/AVEC vaccine was safe when applied to healthy heifers during the first trimester of gestation.