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During their lifespan, high-voltage (HV) electrical systems are subjected to operating conditions in which electrical, mechanical, thermal and environmental-related stresses occur. These conditions over time lead to unforeseen failures caused by various types of defects. For this reason, there are several technologies for measuring and monitoring the electrical systems, with the aim of minimizing the number of faults. The early detection of defects, preferably in their incipient state, will enable the necessary corrective actions to be taken in order to avoid unforeseen failures. These failures generally lead to human risks and material damage, lack of power supply and significant economic losses. An efficient maintenance technique for the early detection of defects consists of the supervision of the dielectrics status in the installations by means of on-line partial discharge (PD) measurement. Nowadays, there are numerous systems in the market for the measurement of PD in HV installations. The most efficient with a reasonable cost will be those that offer greater security guarantees and the best positioned in the market. Currently, technology developers and users of PD measuring systems face difficulties related to the lack of reference procedures for their complete characterization and to the technical and economic drawback of performing the characterization tests on site or in laboratory installations. To deal with the previous difficulties, in this paper a novel method for the complete and standardized characterization of PD measuring systems is presented. The applicability of this method is mainly adapted for the characterization of systems operating in on-line applications using high-frequency current transformer (HFCT) sensors. For the appropriate application of the method, an associated and necessary scale modular test platform is used. In the test platform, the real on-site measuring conditions of an HV insulated distribution line are simulated in a controlled way. Practical characterizations, showing the convenience and advantages of applying the method using the modular test platform, are also presented.
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In general, a high voltage (HV) substation can be made up of multiple insulation subsystems: an air insulation subsystem (AIS), gas insulation subsystem (GIS), liquid insulation subsystem (power transformers), and solid insulation subsystem (power cables), all of them with their grounding structures interconnected and linked to the substation earth. Partial discharge (PD) pulses, which are generated in a HV apparatus belonging to a subsystem, travel through the grounding structures of the others. PD analyzers using high-frequency current transformer (HFCT) sensors, which are installed at the connections between the grounding structures, are sensitive to these traveling pulses. In a substation made up of an AIS, several non-critical PD sources can be detected, such as possible corona, air surface, or floating discharges. To perform the correct diagnosis, non-critical PD sources must be separated from critical PD sources related to insulation defects, such as a cavity in a solid dielectric material, mobile particles in SF6, or surface discharges in oil. Powerful diagnostic tools using PD clustering and phase-resolved PD (PRPD) pattern recognition have been developed to check the insulation condition of HV substations. However, a common issue is how to determine the subsystem in which a critical PD source is located when there are several PD sources, and a critical one is near the boundary between two HV subsystems, e.g., a cavity defect located between a cable end and a GIS. The traveling direction of the detected PD is valuable information to determine the subsystem in which the insulation defect is located. However, incorrect diagnostics are usually due to the constraints of PD measuring systems and inadequate PD diagnostic procedures. This paper presents a diagnostic procedure using an appropriate PD analyzer with multiple HFCT sensors to carry out efficient insulation condition diagnoses. This PD procedure has been developed on the basis of laboratory tests, transient signal modeling, and validation tests. The validation tests were carried out in a special test bench developed for the characterization of PD analyzers. To demonstrate the effectiveness of the procedure, a real case is also presented, where satisfactory results are shown.
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African horse sickness (AHS) is a highly severe disease caused by a viral etiological agent, African horse sickness virus (AHSV). It is endemic in sub-Saharan Africa, while sporadic outbreaks have occurred in North Africa, Asia, and Europe, with the most recent cases in Thailand. AHSV transmission between equines occurs primarily by biting midges of the genus Culicoides, especially C. imicola, with a wide distribution globally. As research in horses is highly restricted due to a variety of factors, small laboratory animal models that reproduce clinical signs and pathology observed in natural infection of AHSV are highly needed. Here, we investigated the expression profile of several pro-inflammatory cytokines in target organs and serum of IFNAR (-/-) mice, to continue characterizing this established animal model and to go deep into the innate immune responses that are still needed.
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Vírus da Doença Equina Africana , Doença Equina Africana , Receptor de Interferon alfa e beta , Animais , Camundongos , África Subsaariana , Doença Equina Africana/genética , Vírus da Doença Equina Africana/metabolismo , Vírus da Doença Equina Africana/patogenicidade , Ceratopogonidae , Europa (Continente) , Cavalos/genética , RNA Mensageiro/genética , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologiaRESUMO
PA-X is a nonstructural protein of influenza A virus (IAV), which is encoded by the polymerase acidic (PA) N-terminal region that contains a C-terminal +1 frameshifted sequence. IAV PA-X protein modulates virus-induced host innate immune responses and viral pathogenicity via suppression of host gene expression or cellular shutoff, through cellular mRNA cleavage. Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype naturally infect different avian species, they have an enormous economic impact in the poultry farming, and they also have zoonotic and pandemic potential, representing a risk to human public health. In the present study, we describe a novel bacterium-based approach to identify amino acid residues in the PA-X protein of the HPAIV A/Viet Nam/1203/2004 H5N1 that are important for its ability to inhibit host protein expression or cellular shutoff activity. Identified PA-X mutants displayed a reduced shutoff activity compared to that of the wild-type A/Viet Nam/1203/2004 H5N1 PA-X protein. Notably, this new bacterium-based screening allowed us to identify amino acid residues widely distributed over the entire N-terminal region of PA-X. Furthermore, we found that some of the residues affecting A/Viet Nam/1203/2004 H5N1 PA-X host shutoff activity also affect PA polymerase activity in a minigenome assay. This information could be used for the rational design of new and more effective compounds with antiviral activity against IAV. Moreover, our results demonstrate the feasibility of using this bacterium-based approach to identify amino acid residues important for the activity of viral proteins to inhibit host gene expression. IMPORTANCE Highly pathogenic avian influenza viruses continue to pose a huge threat to global animal and human health. Despite of the limited genome size of Influenza A virus (IAV), the virus encodes eight main viral structural proteins and multiple accessory nonstructural proteins, depending on the IAV type, subtype, or strain. One of the IAV accessory proteins, PA-X, is encoded by the polymerase acidic (PA) protein and is involved in pathogenicity through the modulation of IAV-induced host inflammatory and innate immune responses. However, the molecular mechanism(s) of IAV PA-X regulation of the host immune response is not well understood. Here, we used, for the first time, a bacterium-based approach for the identification of amino acids important for the ability of IAV PA-X to induce host shutoff activity and describe novel residues relevant for its ability to inhibit host gene expression, and their contribution in PA polymerase activity.
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Aminoácidos , Expressão Gênica , Interações Hospedeiro-Patógeno , Virus da Influenza A Subtipo H5N1 , Proteínas Repressoras , Proteínas não Estruturais Virais , Aminoácidos/genética , Aminoácidos/imunologia , Animais , Bactérias/virologia , Aves/imunologia , Expressão Gênica/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Aviária/imunologia , Influenza Aviária/virologia , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Vietnã , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologiaRESUMO
Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report modified vaccinia virus Ankara (MVA) and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS21-180). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt, or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD8+ T-cell responses against NS1, NS2-Nt, or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4, and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1-NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. IMPORTANCE Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved nonstructural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines.
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Vírus Bluetongue/imunologia , Bluetongue/prevenção & controle , Vetores Genéticos/genética , Vaccinia virus/genética , Proteínas não Estruturais Virais/genética , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus Bluetongue/classificação , Vetores Genéticos/imunologia , Imunidade Celular , Imunização , Imunogenicidade da Vacina , Sorogrupo , Ovinos , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vaccinia virus/imunologia , Proteínas não Estruturais Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Bluetongue virus (BTV) and African horse sickness virus (AHSV) are vector-borne viruses belonging to the Orbivirus genus, which are transmitted between hosts primarily by biting midges of the genus Culicoides. With recent BTV and AHSV outbreaks causing epidemics and important economy losses, there is a pressing need for efficacious drugs to treat and control the spread of these infections. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antiviral activity. Here, we evaluated ATA as a potential antiviral compound against Orbivirus infections in both mammalian and insect cells. Notably, ATA was able to prevent the replication of BTV and AHSV in both cell types in a time- and concentration-dependent manner. In addition, we evaluated the effect of ATA in vivo using a mouse model of infection. ATA did not protect mice against a lethal challenge with BTV or AHSV, most probably due to the in vivo effect of ATA on immune system regulation. Overall, these results demonstrate that ATA has inhibitory activity against Orbivirus replication in vitro, but further in vivo analysis will be required before considering it as a potential therapy for future clinical evaluation.
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Vírus da Doença Equina Africana/efeitos dos fármacos , Ácido Aurintricarboxílico/farmacocinética , Vírus Bluetongue/efeitos dos fármacos , Viroses/tratamento farmacológico , Doença Equina Africana/tratamento farmacológico , Doença Equina Africana/genética , Doença Equina Africana/virologia , Vírus da Doença Equina Africana/genética , Vírus da Doença Equina Africana/patogenicidade , Animais , Vírus Bluetongue/genética , Vírus Bluetongue/patogenicidade , Ceratopogonidae/patogenicidade , Ceratopogonidae/virologia , Cavalos/virologia , Ovinos/virologia , Viroses/genética , Viroses/virologia , Replicação Viral/efeitos dos fármacosRESUMO
The development of vaccines against bluetongue, a prevalent livestock disease, has been focused on surface antigens that induce strong neutralizing antibody responses. Because of their antigenic variability, these vaccines are usually serotype restricted. We now show that a single highly conserved nonstructural protein, NS1, expressed in a modified vaccinia Ankara virus (MVA) vector can provide multiserotype protection in IFNAR-/- 129 mice against bluetongue virus (BTV) that is largely dependent on CD8 T cell responses. We found that the protective antigenic capacity of NS1 resides within the N terminus of the protein and is provided in the absence of neutralizing antibodies. The protective CD8 T cell response requires the presence of a specific peptide within the N terminus of NS1, since its deletion ablates the efficacy of the vaccine formulation. These data reveal the importance of the nonstructural protein NS1 in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine.IMPORTANCE Conventional vaccines have controlled or limited BTV expansion in the past, but they cannot address the need for cross-protection among serotypes and do not allow distinguishing between infected and vaccinated animals (DIVA strategy). There is a need to develop universal vaccines that induce effective protection against multiple BTV serotypes. In this work we have shown the importance of the nonstructural protein NS1, conserved among all the BTV serotypes, in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine.
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Vírus Bluetongue/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos Imunodominantes/imunologia , Receptor de Interferon alfa e beta/deficiência , Proteínas não Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Bluetongue/prevenção & controle , Células Cultivadas , Modelos Animais de Doenças , Portadores de Fármacos , ELISPOT , Vetores Genéticos , Interferon gama/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Knockout , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vaccinia virus/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
The aim of this work was to evaluate the immunogenicity and efficacy of DNA and MVA vaccines encoding the RVFV glycoproteins Gn and Gc in an ovine model of RVFV infection. Adult sheep of both sexes were challenged 12 weeks after the last immunization and clinical, virological, biochemical and immunological consequences, were analyzed. Strategies based on immunization with homologous DNA or heterologous DNA/MVA prime-boost were able to induce a rapid in vitro neutralizing antibody response as well as IFNγ production after in vitro virus specific re-stimulation. In these animals we observed reduced viremia levels and less clinical signs when compared with mock-immunized controls. In contrast, sheep inoculated with a homologous MVA prime-boost showed increased viremia correlating with the absence of detectable neutralizing antibody responses, despite of inducing cellular responses after the last immunization. However, faster induction of neutralizing antibodies and IFNγ production after challenge were found in this group when compared to the mock vaccinated group, indicative of a primed immune response. In conclusion, these results suggest that vaccination strategies based on DNA priming were able to mount and maintain specific anti-RVFV glycoprotein immune responses upon homologous or heterologous booster doses, warranting further optimization in large animal models of infection.
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Febre do Vale de Rift/prevenção & controle , Vírus da Febre do Vale do Rift/imunologia , Doenças dos Ovinos/prevenção & controle , Vacinação/veterinária , Vacinas de DNA/farmacologia , Vacinas Virais/farmacologia , Animais , Feminino , Masculino , Febre do Vale de Rift/virologia , Ovinos , Doenças dos Ovinos/virologiaRESUMO
Partial discharge (PD) measurements provide valuable information for assessing the condition of high voltage (HV) insulation systems, contributing to their quality assurance. Different PD measuring techniques have been developed in the last years specially designed to perform on-line measurements. Non-conventional PD methods operating in high frequency bands are usually used when this type of tests are carried out. In PD measurements the signal acquisition, the subsequent signal processing and the capability to obtain an accurate diagnosis are conditioned by the selection of a suitable detection technique and by the implementation of effective signal processing tools. This paper proposes an optimized electromagnetic detection method based on the combined use of wideband PD sensors for measurements performed in the HF and UHF frequency ranges, together with the implementation of powerful processing tools. The effectiveness of the measuring techniques proposed is demonstrated through an example, where several PD sources are measured simultaneously in a HV installation consisting of a cable system connected by a plug-in terminal to a gas insulated substation (GIS) compartment.
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Epizootic hemorrhagic disease (EHD), caused by Epizootic hemorrhagic disease virus (EHDV), is an emerging and severe livestock disease. Recent incursion and distribution of EHDV in Europe have outlined the emerging character of EHD. Despite its worldwide impact, numerous knowledge gaps exist. A range of inconveniences restricts utilization of natural hosts of EHDV. Here, we show that adult mice deficient in type I IFN receptor (IFNAR(-/-)) are highly susceptible to EHDV-6 and EHDV-8 infection when the virus is administered subcutaneously. Disease was characterized by ruffled hair, reluctance to move, dehydration and conjunctivitis, with viraemia detected from day 5 post-infection. A deeper characterization of EHDV-8 infection showed viral replication in the lung, liver, spleen, kidney, testis and ovaries. Importantly, increased expression levels of pro-inflammatory cytokines IL-1ß, IL-6 and CXCL2 were observed in spleen after EHDV-8 infection. Furthermore, IFNAR(-/-) adult mice immunized with a EHDV-8 inactivated vaccine elicited neutralizing antibodies specific of EHDV-8 and full protection against challenge with a lethal dose of this virus. This study also explores the possibilities of this animal model for study of BTV and EHDV coinfection. In summary, the IFNAR(-/-) mouse model faithfully recapitulates EHD and can be applied for vaccine testing, which can facilitate progress in addressing the animal health challenge posed by this virus.
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Modelos Animais de Doenças , Vírus da Doença Hemorrágica Epizoótica , Receptor de Interferon alfa e beta , Vacinas Virais , Animais , Camundongos , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Vírus da Doença Hemorrágica Epizoótica/imunologia , Vírus da Doença Hemorrágica Epizoótica/genética , Vacinas Virais/imunologia , Infecções por Reoviridae/imunologia , Feminino , Camundongos Knockout , Anticorpos Neutralizantes/imunologia , MasculinoRESUMO
Introduction: Bluetongue (BT), caused by bluetongue virus (BTV), is an important arthropod-borne livestock disease listed by the World Organization for Animal Health. Live-attenuated and inactivated vaccines have permitted to control BT but they do not simultaneously protect against the myriad of BTV serotypes. Recently, we identified the highly conserved BTV nonstructural protein NS1 and the N-terminal region of NS2 as antigens capable of conferring multiserotype protection against BTV. Methods: Here, we designed Modified Vaccinia Ankara (MVA) viral vectors that expressed BTV-4 proteins VP2 or VP7 along with NS1 and NS2-Nt as well as MVAs that expressed proteins VP2, VP7 or NS1 and NS2-Nt. Results: Immunization of IFNAR(-/-) mice with two doses of MVA-NS1-2A-NS2-Nt protected mice from BTV-4M infection by the induction of an antigen-specific T cell immune response. Despite rMVA expressing VP7 alone were not protective in the IFNAR(-/-) mouse model, inclusion of VP7 in the vaccine formulation amplified the cell-mediated response induced by NS1 and NS2-Nt. Expression of VP2 elicited protective non-cross-reactive neutralizing antibodies (nAbs) in immunized animals and improved the protection observed in the MVA-NS1-2A-NS2-Nt immunized mice when these three BTV antigens were co-expressed. Moreover, vaccines candidates co-expressing VP2 or VP7 along with NS1 and NS2-Nt provided multiserotype protection. We assessed protective efficacy of both vaccine candidates in sheep against virulent challenge with BTV-4M. Discussion: Immunization with MVA-VP7-NS1-2A-NS2-Nt partially dumped viral replication and clinical disease whereas administration of MVA-VP2-NS1-2A-NS2-Nt promoted a complete protection, preventing viraemia and the pathology produced by BTV infection.
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Vírus Bluetongue , Bluetongue , Proteínas do Capsídeo , Vetores Genéticos , Receptor de Interferon alfa e beta , Vaccinia virus , Proteínas não Estruturais Virais , Vacinas Virais , Animais , Vírus Bluetongue/imunologia , Vírus Bluetongue/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/genética , Bluetongue/prevenção & controle , Bluetongue/imunologia , Bluetongue/virologia , Camundongos , Vacinas Virais/imunologia , Vacinas Virais/genética , Vaccinia virus/genética , Vaccinia virus/imunologia , Receptor de Interferon alfa e beta/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Camundongos Knockout , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , FemininoRESUMO
Bluetongue virus (BTV) is the causative agent of the important livestock disease bluetongue (BT), which is transmitted via Culicoides bites. BT causes severe economic losses associated with its considerable impact on health and trade of animals. By reverse genetics, we have designed and rescued reporter-expressing recombinant (r)BTV expressing NanoLuc luciferase (NLuc) or Venus fluorescent protein. To generate these viruses, we custom synthesized a modified viral segment 5 encoding NS1 protein with the reporter genes located downstream and linked by the Porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site. Therefore, fluorescent signal or luciferase activity is only detected after virus replication and expression of non-structural proteins. Fluorescence or luminescence signals were detected in cells infected with rBTV/Venus or rBTV/NLuc, respectively. Moreover, the marking of NS2 protein confirmed that reporter genes were only expressed in BTV-infected cells. Growth kinetics of rBTV/NLuc and rBTV/Venus in Vero cells showed replication rates similar to those of wild-type and rBTV. Infectivity studies of these recombinant viruses in IFNAR(-/-) mice showed a higher lethal dose for rBTV/NLuc and rBTV/Venus than for rBTV indicating that viruses expressing the reporter genes are attenuated in vivo. Interestingly, luciferase activity was detected in the plasma of viraemic mice infected with rBTV/NLuc. Furthermore, luciferase activity quantitatively correlated with RNAemia levels of infected mice throughout the infection. In addition, we have investigated the in vivo replication and dissemination of BTV in IFNAR (-/-) mice using BTV/NLuc and non-invasive in vivo imaging systems.IMPORTANCEThe use of replication-competent viruses that encode a traceable fluorescent or luciferase reporter protein has significantly contributed to the in vitro and in vivo study of viral infections and the development of novel therapeutic approaches. In this work, we have generated rBTV that express fluorescent or luminescence proteins to track BTV infection both in vitro and in vivo. Despite the availability of vaccines, BTV and other related orbivirus are still associated with a significant impact on animal health and have important economic consequences worldwide. Our studies may contribute to the advance in orbivirus research and pave the way for the rapid development of new treatments, including vaccines.
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Vírus Bluetongue , Vacinas , Chlorocebus aethiops , Animais , Camundongos , Vírus Bluetongue/genética , Genes Reporter , Células Vero , Proteínas Virais/genética , Luciferases/genéticaRESUMO
Although receiving adjuvant chemotherapy after radical surgery, a disappointing proportion of patients with colorectal cancer will develop tumor recurrence. Probability of relapse is currently predicted from pathological staging, there being a need for additional markers to further select high-risk patients. This study was aimed to identify a gene-expression signature to predict tumor recurrence in patients with Stages II and III colon cancer treated with 5'fluoruracil (5FU)-based adjuvant chemotherapy. Two-hundred and twenty-eight patients diagnosed with Stages II-III colon cancer and treated with surgical resection and 5FU-based adjuvant chemotherapy were included. RNA was extracted from formalin-fixed, paraffin-embedded tissue samples and expression of 27 selected candidate genes was analyzed by RT-qPCR. A tumor recurrence predicting model, including clinico-pathological variables and gene-expression profiling, was developed by Cox regression analysis and validated by bootstrapping. The regression analysis identified tumor stage and S100A2 and S100A10 gene expression as independently associated with tumor recurrence. The risk score derived from this model was able to discriminate two groups with a highly significant different probability of tumor recurrence (HR, 2.75; 95%CI, 1.71-4.39; p = 0.0001), which it was maintained when patients were stratified according to tumor stage. The algorithm was also able to distinguish two groups with different overall survival (HR, 2.68; 95%CI, 1.12-6.42; p = 0.03). Identification of a new gene-expression signature associated with a high probability of tumor recurrence in patients with Stages II and III colon cancer receiving adjuvant 5FU-based chemotherapy, and its combination in a robust, easy-to-use and reliable algorithm may contribute to tailor treatment and surveillance strategies.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Recidiva Local de Neoplasia/genética , Idoso , Quimioterapia Adjuvante , Neoplasias do Colo/patologia , Neoplasias do Colo/cirurgia , Feminino , Fluoruracila/administração & dosagem , Humanos , Masculino , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Estadiamento de Neoplasias , Valor Preditivo dos Testes , TranscriptomaRESUMO
Epizootic Hemorrhagic Disease (EHD) of ruminants is a viral pathology that has significant welfare, social, and economic implications. The causative agent, epizootic hemorrhagic disease virus (EHDV), belongs to the Orbivirus genus and leads to significant regional disease outbreaks among livestock and wildlife in North America, Asia, Africa, and Oceania, causing significant morbidity and mortality. During the past decade, this viral disease has become a real threat for countries of the Mediterranean basin, with the recent occurrence of several important outbreaks in livestock. Moreover, the European Union registered the first cases of EHDV ever detected within its territory. Competent vectors involved in viral transmission, Culicoides midges, are expanding its distribution, conceivably due to global climate change. Therefore, livestock and wild ruminants around the globe are at risk for this serious disease. This review provides an overview of current knowledge about EHDV, including changes of distribution and virulence, an examination of different animal models of disease, and a discussion about potential treatments to control the disease.
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The COVID-19 pandemic has underscored the importance of swift responses and the necessity of dependable technologies for vaccine development. Our team previously developed a fast cloning system for the modified vaccinia virus Ankara (MVA) vaccine platform. In this study, we reported on the construction and preclinical testing of a recombinant MVA vaccine obtained using this system. We obtained recombinant MVA expressing the unmodified full-length SARS-CoV-2 spike (S) protein containing the D614G amino-acid substitution (MVA-Sdg) and a version expressing a modified S protein containing amino-acid substitutions designed to stabilize the protein a in a pre-fusion conformation (MVA-Spf). S protein expressed by MVA-Sdg was found to be expressed and was correctly processed and transported to the cell surface, where it efficiently produced cell-cell fusion. Version Spf, however, was not proteolytically processed, and despite being transported to the plasma membrane, it failed to induce cell-cell fusion. We assessed both vaccine candidates in prime-boost regimens in the susceptible transgenic K18-human angiotensin-converting enzyme 2 (K18-hACE2) in mice and in golden Syrian hamsters. Robust immunity and protection from disease was induced with either vaccine in both animal models. Remarkably, the MVA-Spf vaccine candidate produced higher levels of antibodies, a stronger T cell response, and a higher degree of protection from challenge. In addition, the level of SARS-CoV-2 in the brain of MVA-Spf inoculated mice was decreased to undetectable levels. Those results add to our current experience and range of vaccine vectors and technologies for developing a safe and effective COVID-19 vaccine.
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Modified vaccinia virus Ankara (MVA) is employed widely as an experimental vaccine vector for its abortive replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a wide range of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination , and the capacity to deliver substantial amounts of heterologous antigens. rMVAs encoding proteins of Bluetongue virus (BTV), an orbivirus that infects domestic and wild ruminants through transmission by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter, we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of BTV . The included protocols cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of rMVAs, the titration of virus working stocks, and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification procedure.
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Vírus Bluetongue , Bluetongue , Vacinas Virais , Animais , Bluetongue/prevenção & controle , Vírus Bluetongue/genética , Proteínas do Capsídeo/genética , Mamíferos , Ovinos , Vaccinia virus/genéticaRESUMO
Bluetongue virus (BTV) and African horse sickness virus (AHSV) are widespread arboviruses that cause important economic losses in the livestock and equine industries, respectively. In addition to these, another arthropod-transmitted orbivirus known as epizootic hemorrhagic disease virus (EHDV) entails a major threat as there is a conducive landscape that nurtures its emergence in non-endemic countries. To date, only vaccinations with live attenuated or inactivated vaccines permit the control of these three viral diseases, although important drawbacks, e.g., low safety profile and effectiveness, and lack of DIVA (differentiation of infected from vaccinated animals) properties, constrain their usage as prophylactic measures. Moreover, a substantial number of serotypes of BTV, AHSV and EHDV have been described, with poor induction of cross-protective immune responses among serotypes. In the context of next-generation vaccine development, antigen delivery systems based on nano- or microparticles have gathered significant attention during the last few decades. A diversity of technologies, such as virus-like particles or self-assembled protein complexes, have been implemented for vaccine design against these viruses. In this work, we offer a comprehensive review of the nano- and microparticulated vaccine candidates against these three relevant orbiviruses. Additionally, we also review an innovative technology for antigen delivery based on the avian reovirus nonstructural protein muNS and we explore the prospective functionality of the nonstructural protein NS1 nanotubules as a BTV-based delivery platform.
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PURPOSE: Local excision of malignant rectal tumors remains controversial due to the lack of prospective studies. The principal aim of this paper is to analyze survival and recurrence of patients with rectal cancer who were operated by transanal endoscopic microsurgery with curative intention. METHODS: In 1997, we started a prospective protocol for patients who had T1/T2 rectal tumors: transanal local full-thickness excision was considered curative in T1 low risk (group A); patients with T1 high-risk and T2 low-risk tumors received postoperative radiotherapy (group B). From 1997 to 2006, 88 patients were enrolled. Sixty eight entered the study after the preoperative workup and 20 patients with an initial diagnosis of adenoma after postoperative definitive pathological assessment. RESULTS: After definitive histological findings, 54 patients were to group A, 28 to group B, and 6 had immediate radical surgery. One patient was lost for follow-up. At a mean follow-up of 71 months, 7 (4 from group A and 3 from group B) out of 81 patients recurred. Five-year overall survival was of 94% and cancer-specific survival of 96%. CONCLUSIONS: Our data support that transanal endoscopic microsurgery is an adequate treatment for T1 low-risk tumor, and no additional measures are required. For T2 low-risk lesions, our study showed a higher local recurrence rate than that reported after radical surgery but a similar survival outcome.
Assuntos
Canal Anal/cirurgia , Endoscopia , Microcirurgia/métodos , Neoplasias Retais/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Cuidados Pré-Operatórios , Neoplasias Retais/patologia , Análise de Sobrevida , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Influenza A viruses (IAV) can infect a broad range of mammalian and avian species. However, the host innate immune system provides defenses that restrict IAV replication and infection. Likewise, IAV have evolved to develop efficient mechanisms to counteract host antiviral responses to efficiently replicate in their hosts. The IAV PA-X and NS1 non-structural proteins are key virulence factors that modulate innate immune responses and virus pathogenicity during infection. To study the determinants of IAV pathogenicity and their functional co-evolution, we evaluated amino acid differences in the PA-X and NS1 proteins of early (1996-1997) and more recent (since 2016) H5N1 IAV. H5N1 IAV have zoonotic and pandemic potential and represent an important challenge both in poultry farming and human health. The results indicate that amino acid changes occurred over time, affecting the ability of these two non-structural H5N1 IAV proteins to inhibit gene expression and affecting virus pathogenicity. These results highlight the importance to monitor the evolution of these two virulence factors of IAV, which could result in enhanced viral replication and virulence.
Assuntos
Virus da Influenza A Subtipo H5N1/genética , Proteínas Repressoras/metabolismo , Seleção Genética , Proteínas não Estruturais Virais/metabolismo , Animais , Aves , Feminino , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Vírus da Influenza A/genética , Influenza Aviária , Influenza Humana/virologia , Interferons/metabolismo , Masculino , Camundongos , Infecções por Orthomyxoviridae/virologia , Proteínas Repressoras/genética , Proteínas não Estruturais Virais/genética , Virulência , Replicação Viral/genéticaRESUMO
The identification of transmission routes for bluetongue virus (BTV) is essential to improve the control of the disease. Although BTV is primarily transmitted by several species of Culicoides biting midges, there has been evidence of transplacental and oral transmission. We now report that IFNAR((-/-)) mice are susceptible to oral infection by BTV-8. Viraemia, clinical manifestations and tissue lesions are similar to those in intravenously infected mice. In addition, we show that the oral cavity and oesophagus are susceptible to BTV infection and replication, suggesting that these organs are possible entry routes during BTV oral infection.