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Bovine viral diarrhea (BVD) is a single-stranded, positive-sense ribonucleic acid (RNA) virus belonging to the genus Pestivirus of the Flaviviridae family. BVD frequently causes economic losses to farmers. Among bovine viral diarrhea virus (BVDV) strains, BVDV-1b is predominant and widespread in Hanwoo calves. Reverse-transcription polymerase chain reaction (RT-PCR) is an essential method for diagnosing BVDV-1b and has become the gold standard for diagnosis in the Republic of Korea. However, this diagnostic method is time-consuming and requires expensive equipment. Therefore, Clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems have been used for point-of-care (POC) testing of viruses. Developing a sensitive and specific method for POC testing of BVDV-1b would be advantageous for controlling the spread of infection. Thus, this study aimed to develop a novel nucleic acid detection method using the CRISPR-Cas13 system for POC testing of BVDV-1b. The sequence of the BVD virus was extracted from National Center for Biotechnology Information (NC_001461.1), and the 5' untranslated region, commonly used for detection, was selected. CRISPR RNA (crRNA) was designed using the Cas13 design program and optimized for the expression and purification of the LwCas13a protein. Madin Darby bovine kidney (MDBK) cells were infected with BVDV-1b, incubated, and the viral RNA was extracted. To enable POC viral detection, the compatibility of the CRISPR-Cas13 system was verified with a paper-based strip through collateral cleavage activity. Finally, a colorimetric assay was used to evaluate the detection of BVDV-1b by combining the previously obtained crRNA and Cas13a protein on a paper strip. In conclusion, the CRISPR-Cas13 system is highly sensitive, specific, and capable of nucleic acid detection, making it an optimal system for the early point-of-care testing of BVDV-1b.
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Introduction: Bovine viral diarrhea virus (BVDV) brings great economic loss to the cattle industry worldwide. Developing a control/prevention strategy requires the prior assessment of certain epidemiological parameters. To determine the BVD incidence rate and associated risk factors, a dairy cattle herd in the eastern region of Saudi Arabia was monitored between 2020 and 2022. Methods: Nasal swabs (n = 190), rectal swabs (n = 190), and sera (n = 190) were collected from 79 cows in this herd. Collected sera and swabs were tested using the commercially available ELISAs for the BVDV antibodies and antigens, respectively. Collected sera were also tested for the presence of BVDV nucleic acids using commercial real-time RT-PCR kits. Results and discussion: Our data show BVDV seroprevalence (18.8%, 15%, and 8.2%) in the tested animals in 2020-2022, respectively. None of the collected nasal swabs, rectal swabs, or sera tested positive for the BVDV antigen, whereas 10.1%, 10%, and 18.1% of the tested sera were positive for BVDV nucleic acid in 2020-2022, respectively. The incidence rate was estimated at 0.02446 new cases/year despite the detection of BVDV in seronegative animals on single or two occasions at ≥6-month intervals. Young calves and bulls remained apparently unexposed to BVDV despite their presence with BVDV-infected females, with no significant physical separation. Both seropositivity and nucleic acid detectability showed significant positive and negative correlations, respectively, with reproductive performance. Collectively, the present study provides useful clues about the transmissibility of BVDV in the presence of possibly persistently infected animals. To the best of our knowledge, this is the first longitudinal study of BVDV in the Eastern Region of Saudi Arabia. Further detailed characterization of the circulating BVDVs is encouraged.
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Bovine viral diarrhea virus (BVDV) is a significant pathogen that causes great economic losses in the global livestock industry. During the long-term interactions between BVDV and its hosts, the virus has evolved multiple strategies to evade the host's innate immunity and adaptive immunity, thereby promoting viral survival and replication. This review focuses on the most recent research on immune evasion strategies employed by BVDV, including evading type I IFN signaling pathway, evading host adaptive immunity, mediating NF-κB signaling pathway, mediating cell apoptosis and inducing autophagy. Unraveling BVDV's immune evasion strategies will enhance our understanding of the pathogenesis of BVDV and contribute to the development of more effective therapies for the prevention, control and eradication of BVDV.
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Virus de la Diarrea Viral Bovina , Evasión Inmune , Humanos , Inmunidad Innata , Inmunidad Adaptativa , DiarreaRESUMEN
Introduction: Bovine viral diarrhea virus (BVDV) significantly impacts the bovine industries, both dairy and beef sectors. BVDV can infect various domestic and wild animals, most notably cattle. The dynamic variations among BVDV serotypes due to the continuous genetic diversity, especially in BVDV1 (BVDV1), reduce the effectiveness of the currently available vaccines and reduce the specificity/sensitivity of the diagnostic assays. The development of novel, safe, and effective vaccines against BVDV requires deep knowledge of the antigenicity and virulence of the virus. Previous studies on the antigenicity and the virulence of BVDV serotypes have been mainly focused on one or a few BVDV proteins. While however, little is known about the orchestration of all BVDV in the context of viral virulence and immunogenicity. The main aim of the current study was to do a comparative computational evaluation of the immunogenicity, and virulence for all the encoded proteins of both BVDV1 and BVDV2 and their sub-genotypes. Methods: To achieve this goal, 11,737 protein sequences were retrieved from Virus Pathogen Resource. The analysis involved a total of 4,583 sequences after the removal of short sequences and those with unknown collection time. We used the MP3 tool to map the pathogenic proteins across different BVDV strains. The potential protective and the epitope motifs were predicted using the VaxiJen and EMBOSS antigen tools, respectively. Results and discussion: The virulence prediction revealed that the NS4B proteins of both BVDV1 and BVDV2 likely have essential roles in BVDV virulence. Similarly, both the capsid (C) and the NS4-A proteins of BVDV1 and the Npro and P7 proteins of BVDV2 are likely important virulent factors. There was a clear trend of increasing predicted virulence with the progression of time in the case of BVDV1 proteins, but that was not the case for the BVDV2 proteins. Most of the proteins of the two BVDV serotypes possess antigens predicted immunogens except Npro, P7, and NS4B. However, the predicted antigenicity of the BVDV1 was significantly higher than that of BVDV2. Meanwhile, the predicted immunogenicity of the immunodominant-E2 protein has been decreasing over time. Based on our predicted antigenicity and pathogenicity studies of the two BVDV serotypes, the sub-genotypes (1a, 1f, 1k, 2a, and 2b) may represent ideal candidates for the development of future vaccines against BVDV infection in cattle. In summary, we identified some common differences between the two BVDV genotypes (BVDV1 and BVDV2) and their sub-genotypes regarding their protein antigenicity and pathogenicity. The data presented here will increase our understanding of the molecular pathogenesis of BVDV infection in cattle. It will also pave the way for developing some novel diagnostic assays and novel vaccines against BVDV in the near future.
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BACKGROUND: The European bison (Bison bonasus) is a near threatened species and requires health monitoring. The aim of the present study was to determine the prevalence of antibodies to pathogens known to cause respiratory and digestive illness in ruminants. RESULTS: In the studied 328 European bison, the highest seroprevalence was observed for Bovine herpesvirus-1 (BoHV-1) (50.27%), Bovine Coronavirus (BCoV) (26.36%), and Bluetongue Virus (BTV) (12.83%). For Mycoplasma bovis strains and Bovine Viral Diarrhea Virus (BVDV), positive results were rare. Interestingly, a higher prevalence of BTV antibodies was noted in the northeastern populations and older animals. CONCLUSIONS: Our findings indicate that the Polish European bison population appears to have considerable contact with BoHV-1; however, this does not appear to be of great significance, as clinical symptoms and post-mortem lesions are rarely noted in Polish European bison population. The high seroprevalence of BTV in the north-east of Poland is an ongoing trend, also noted in previous studies. It is possible that European bison may perpetuate the virus in this region. This is the first report of antibodies for BCoV in European bison.
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Bison , Herpesvirus Bovino 1 , Animales , Polonia/epidemiología , Estudios Seroepidemiológicos , Anticuerpos Antivirales , Sistema DigestivoRESUMEN
The development of potent non-nucleoside inhibitors (NNIs) could be an alternate strategy to combating infectious bovine viral diarrhea virus (BVDV), other than the traditional vaccination. RNA-dependent RNA polymerase (RdRp) is an essential enzyme for viral replication; therefore, it is one of the primary targets for countermeasures against infectious diseases. The reported NNIs, belonging to the classes of quinolines (2h: imidazo[4,5-g]quinolines and 5m: pyrido[2,3-g] quinoxalines), displayed activity in cell-based and enzyme-based assays. Nevertheless, the RdRp binding site and microscopic mechanistic action are still elusive, and can be explored at a molecular level. Here, we employed a varied computational arsenal, including conventional and accelerated methods, to identify quinoline compounds' most likely binding sites. Our study revealed A392 and I261 as the mutations that can render RdRp resistant against quinoline compounds. In particular, for ligand 2h, mutation of A392E is the most probable mutation. The loop L1 and linker of the fingertip is recognized as a pivotal structural determinant for the stability and escape of quinoline compounds. Overall, this work demonstrates that the quinoline inhibitors bind at the template entrance channel, which is governed by conformational dynamics of interactions with loops and linker residues, and reveals structural and mechanistic insights into inhibition phenomena, for the discovery of improved antivirals.
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Bovine viral diarrhea virus (BVDV) belongs to the family Flaviviridae genus pestivirus. The viral genome is a single-stranded, positive-sense RNA that encodes four structural proteins (i.e., C, Erns, E1, and E2) and eight non-structural proteins (NSPs) (i.e., Npro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Cattle infected with BVDV exhibit a number of different clinical signs including diarrhea, abortion, and other reproductive disorders which have a serious impact on the cattle industry worldwide. Research on BVDV mainly focuses on its structural protein, however, progress in understanding the functions of the NSPs of BVDV has also been made in recent decades. The knowledge gained on the BVDV non-structural proteins is helpful to more fully understand the viral replication process and the molecular mechanism of viral persistent infection. This review focuses on the functions of BVDV NSPs and provides references for the identification of BVDV, the diagnosis and prevention of Bovine viral diarrhea mucosal disease (BVD-MD), and the development of vaccines.
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Diarrea Mucosa Bovina Viral , Virus de la Diarrea Viral Bovina Tipo 1 , Virus de la Diarrea Viral Bovina , Animales , Bovinos , Proteínas no Estructurales Virales/metabolismo , ARN Viral/genética , Línea Celular , Virus de la Diarrea Viral Bovina/genética , Virus de la Diarrea Viral Bovina/metabolismo , Diarrea/veterinaria , Virus de la Diarrea Viral Bovina Tipo 1/genéticaRESUMEN
The genus Pestivirus, family Flaviviridae, includes four historically accepted species, i.e., bovine viral diarrhea virus (BVDV)-1 and -2, classical swine fever virus (CSFV), and border disease virus (BDV). A large number of new pestivirus species were identified in recent years. A common feature of most members is the presence of two unique proteins, Npro and Erns, that pestiviruses evolved to regulate the host's innate immune response. In addition to its function as a structural envelope glycoprotein, Erns is also released in the extracellular space, where it is endocytosed by neighboring cells. As an endoribonuclease, Erns is able to cleave viral ss- and dsRNAs, thus preventing the stimulation of the host's interferon (IFN) response. Here, we characterize the basic features of soluble Erns of a large variety of classified and unassigned pestiviruses that have not yet been described. Its ability to form homodimers, its RNase activity, and the ability to inhibit dsRNA-induced IFN synthesis were investigated. Overall, we found large differences between the various Erns proteins that cannot be predicted solely based on their primary amino acid sequences, and that might be the consequence of different virus-host co-evolution histories. This provides valuable information to delineate the structure-function relationship of pestiviral endoribonucleases.
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Endorribonucleasas/metabolismo , Evasión Inmune , Inmunidad Innata , Pestivirus/inmunología , Pestivirus/patogenicidad , Proteínas del Envoltorio Viral/metabolismo , Animales , Línea Celular , Endocitosis , Endorribonucleasas/química , Endorribonucleasas/genética , Interferones/antagonistas & inhibidores , Interferones/biosíntesis , Mutación , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , Pestivirus/metabolismo , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
The genus Pestivirus, family Flaviviridae, includes four economically important viruses of livestock, i.e., bovine viral diarrhea virus-1 (BVDV-1) and -2 (BVDV-2), border disease virus (BDV) and classical swine fever virus (CSFV). Erns and Npro, both expressed uniquely by pestiviruses, counteract the host's innate immune defense by interfering with the induction of interferon (IFN) synthesis. The structural envelope protein Erns also exists in a soluble form and, by its endoribonuclease activity, degrades immunostimulatory RNA prior to their activation of pattern recognition receptors. Here, we show that at least three out of four positively-charged residues in the C-terminal glycosaminoglycan (GAG)-binding site of BVDV-Erns are required for efficient cell entry, and that a positively charged region more upstream is not involved in cell entry but rather in RNA-binding. Moreover, the C-terminal domain on its own determines intracellular targeting, as GFP fused to the C-terminal amino acids of Erns was found at the same compartments as wt Erns. In summary, RNase activity and uptake into cells are both required for Erns to act as an IFN antagonist, and the C-terminal amphipathic helix containing the GAG-binding site determines the efficiency of cell entry and its intracellular localization.
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Aminoácidos/química , Endorribonucleasas/metabolismo , Evasión Inmune , Pestivirus/genética , Pestivirus/fisiología , Internalización del Virus , Aminoácidos/metabolismo , Animales , Bovinos , Células Cultivadas , Endorribonucleasas/farmacología , Interacciones Microbiota-Huesped , Pestivirus/enzimología , Pestivirus/inmunología , ARN Viral/genética , Cornetes Nasales/citología , Cornetes Nasales/efectos de los fármacos , Cornetes Nasales/virología , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Bovine viral diarrhea virus (BVDV) is affecting cattle populations all over the world causing acute disease, immunosuppressive effects, respiratory diseases, gastrointestinal, and reproductive failure in cattle. The virus is taken up via the oronasal route and infection of epithelial and immune cells contributes to the dissemination of the virus throughout the body. However, it is not known how the virus gets across the barrier of epithelial cells encountered in the airways. Here, we analyzed the infection of polarized primary bovine airway epithelial cells (BAEC). Infection of BAEC by a non-cytopathogenic BVDV was possible via both the apical and the basolateral plasma membrane, but the infection was most efficient when the virus was applied to the basolateral plasma membrane. Irrespective of the site of infection, BVDV was efficiently released to the apical site, while only minor amounts of virus were detected in the basal medium. This indicates that the respiratory epithelium can release large amounts of BVDV to the environment and susceptible animals via respiratory fluids and aerosols, but BVDV cannot cross the airway epithelial cells to infect subepithelial cells and establish systemic infection. Further experiments showed that the receptor, bovine CD46, for BVDV is expressed predominantly on the apical membrane domain of the polarized epithelial cells. In a CD46 blocking experiment, the addition of an antibody directed against CD46 almost completely inhibited apical infection, whereas basolateral infection was not affected. While CD46 serves as a receptor for apical infection of BAEC by BVDV, the receptor for basolateral infection remains to be elucidated.
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Polaridad Celular , Virus de la Diarrea Viral Bovina/patogenicidad , Células Epiteliales/virología , Sistema Respiratorio/citología , Animales , Bovinos , Línea Celular , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/fisiología , Sistema Respiratorio/virologíaRESUMEN
BACKGROUND: Bovine viral diarrhea virus (BVDV) is one of the main causes of infectious diseases in cattle and causes large financial losses to the cattle industry worldwide. In this study, Lactobacillus casei strain W56 (Lc W56) was used as antigen deliver carrier to construct a recombinant Lactobacillus vaccine pPG-E2-ctxB/Lc W56 constitutively expressing BVDV E2 protein fused with cholera toxin B subunit (ctxB) as an adjuvant, and its immunogenicity against BVDV infection in mice model by oral route was explored. RESULTS: Our results suggested that pPG-E2-ctxB/Lc W56 can effectively activate dendritic cells (DCs) in the Peyer's patches, up-regulate the expression of Bcl-6, and promote T-follicular helper (Tfh) cells differentiation, as well as enhance B lymphocyte proliferation and promote them differentiate into specific IgA-secreting plasma cells, secreting anti-E2 mucosal sIgA antibody with BVDV-neutralizing activity. Moreover, significant levels (p < 0.01) of BVDV-neutralizing antigen-specific serum antibodies were induced in the pPG-E2-ctxB/LC W56 group post-vaccination. The recombinant Lactobacillus vaccine can induce cellular immune responses, and significant levels (p < 0.01) of Th1-associated cytokines (IL-2, IL-12, and IFN-γ), Th2-associated cytokines (IL-4, IL-10) and Th17-associated cytokine (IL-17) were determined in the serum of vaccinated mice. Significantly, the recombinant Lactobacillus vaccine provides immune protection against BVDV infection, which can be cleared effectively by the vaccine post-challenge in orally vaccinated animals. CONCLUSIONS: The genetically engineered Lactobacillus vaccine constructed in this study is immunogenic in mice and can induce mucosal, humoral, and cellular immune responses, providing effective anti-BVDV immune protection. It thus represents a promising strategy for vaccine development against BVDV.
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Diarrea Mucosa Bovina Viral/prevención & control , Toxina del Cólera/inmunología , Virus de la Diarrea Viral Bovina/inmunología , Lacticaseibacillus casei/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Adyuvantes Inmunológicos , Administración Oral , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Formación de Anticuerpos/inmunología , Diarrea Mucosa Bovina Viral/patología , Bovinos , Citocinas/inmunología , Tracto Gastrointestinal/patología , Tracto Gastrointestinal/virología , Inmunidad Celular , Lacticaseibacillus casei/genética , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/inmunología , Organismos Libres de Patógenos Específicos , Vacunas Sintéticas/inmunología , Carga ViralRESUMEN
Bovine viral diarrhea virus (BVDV) entry into a host cell is mediated by the interaction of the viral glycoprotein E2 with the cellular transmembrane CD46 receptor. In this study, we generated a stable Madin-Darby Bovine Kidney (MDBK) CD46-knockout cell line to study the ability of different pestivirus A and B species (BVDV-1 and -2) to escape CD46-dependent cell entry. Four different BVDV-1/2 isolates showed a clearly reduced infection rate after inoculation of the knockout cells. However, after further passaging starting from the remaining virus foci on the knockout cell line, all tested virus isolates were able to escape CD46-dependency and grew despite the lack of the entry receptor. Whole-genome sequencing of the escape-isolates suggests that the genetic basis for the observed shift in infectivity is an amino acid substitution of an uncharged (glycine/asparagine) for a charged amino acid (arginine/lysine) at position 479 in the ERNS in three of the four isolates tested. In the fourth isolate, the exchange of a cysteine at position 441 in the ERNS resulted in a loss of ERNS dimerization that is likely to influence viral cell-to-cell spread. In general, the CD46-knockout cell line is a useful tool to analyze the role of CD46 for pestivirus replication and the virus-receptor interaction.
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Virus de la Diarrea Viral Bovina Tipo 1/fisiología , Virus de la Diarrea Viral Bovina Tipo 2/fisiología , Proteína Cofactora de Membrana/genética , Receptores Virales/genética , Proteínas del Envoltorio Viral/genética , Adaptación Biológica , Sustitución de Aminoácidos , Animales , Sistemas CRISPR-Cas , Bovinos , Virus de la Diarrea Viral Bovina Tipo 1/genética , Virus de la Diarrea Viral Bovina Tipo 2/genética , Perros , Técnicas de Inactivación de Genes , Interacciones Huésped-Patógeno , Células de Riñón Canino Madin Darby , Proteína Cofactora de Membrana/metabolismo , Multimerización de Proteína , Receptores Virales/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Replicación ViralRESUMEN
BACKGROUND: As a pestivirus of the Flaviviridae family, bovine viral diarrhea virus (BVDV), has imposed a large burden on animal husbandry worldwide, and such virus can be transmitted mainly through direct contact with other infected animals and probably via aerosols. In the present study, we aimed to develop a real-time RT-PCR method for detection of BVDV-1 in aerosol samples. METHODS: A pair of primers specific for highly conserved regions of the BVDV-1 5'-UTR was designed. The standard curve and sensitivity of the developed assay were assessed based on 10-fold serial dilutions of RNA molecular standard. The specificity of the assay was evaluated with other pestiviruses and infectious bovine viruses. The clinical performance was examined by testing 169 aerosol samples. RESULTS: The results showed that a good linear relationship existed between the standard curve and the concentration of template. The lowest detection limit was 5.2 RNA molecules per reaction. This assay was specific for detection of BVDV-1, and no amplification was found for other pestiviruses such as classical swine fever virus (CSFV), border disease virus (BDV), and common infectious bovine viruses, including BVDV-2, infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine ephemeral fever virus (BEFV) and bovine coronavirus (BcoV). The assay was highly reproducible with low variation coefficient values (CVs) for intra-assay and inter-assay. A total of 169 aerosol samples collected from six dairy herds were tested using this method. The results showed that the positive detection rate of BVDV-1 was 17.2% (29/169), which was significantly higher compared with the conventional RT-PCR. Additionally, the positive samples (n = 29) detected by real-time RT-PCR were verified by BVDV RPA-LFD, and a concordance rate of 100% was obtained between them. CONCLUSIONS: Taken together, we developed a real-time RT-PCR assay for quantitative analysis of BVDV-1 in aerosol samples, and our finding provided valuable insights into the risk on aerosol transmission of BVDV-1.
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Virus de la Diarrea Viral Bovina Tipo 1/aislamiento & purificación , Genotipo , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Regiones no Traducidas 5'/genética , Aerosoles , Microbiología del Aire , Animales , Virus de la Diarrea Viral Bovina Tipo 1/clasificación , Virus de la Diarrea Viral Bovina Tipo 1/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y EspecificidadRESUMEN
Acute infection of bovine viral diarrhea virus (BVDV) is associated with immune dysfunction and can cause peripheral blood lymphopenia and lymphocyte apoptosis. Our previous study has confirmed that programmed death-1 (PD-1) blockade inhibits peripheral blood lymphocyte (PBL) apoptosis and restores proliferation and anti-viral immune functions of lymphocytes after BVDV infection in vitro. However, the immunomodulatory effects of PD-1 pathway on major PBL subsets are unclear and their underlying molecular mechanisms need to be further studied. Therefore, in this study, we examined PD-1 expression in bovine PBL subsets after BVDV infection in vitro and analyzed the effects of PD-1 blockade on the apoptosis and proliferation of CD4+ and CD8+ T cells and expression of PD-1 downstream signaling molecules. The results showed that PD-1 expression was enhanced on CD4+ and CD8+ T cells, but not on CD21+ B cells after cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain KD) infection in vitro and PD-1 blockade significantly reduced the apoptosis of CD4+ and CD8+ T cells after these two strains infection. Remarkably, PD-1 blockade significantly increased the proliferation of CD4+ and CD8+ T cells after CP BVDV infection, but only significantly increased the proliferation of CD4+ T cells after NCP BVDV infection. In addition, we confirmed that PD-1-mediated PI3K/Akt/mTOR, caspase 9/caspase 3 and ERK pathways are involved in regulating the apoptosis and proliferation of CD4+ and CD8+ T cells during BVDV infection in vitro. Notably, ERK is involved in the regulation mechanism PD-1 mediated only when the cells are infected with CP BVDV. Our findings provide a scientific basis for exploring the molecular mechanism of immune dysfunction caused by acute BVDV infection.
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Diarrea Mucosa Bovina Viral/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Virus de la Diarrea Viral Bovina/fisiología , Receptor de Muerte Celular Programada 1/metabolismo , Animales , Apoptosis , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Bovinos , Proliferación Celular , Células Cultivadas , Sistema de Señalización de MAP Quinasas , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Bovine viral diarrhea caused by bovine viral diarrhea virus (BVDV) is an important disease in cattle, resulting in significant economic losses to the cattle industry worldwide. In order to develop an effective vaccine against BVDV infection, we constructed a dendritic cell (DC)-targeting oral probiotic vaccine (pPG-E2-DCpep/LC W56) using Lactobacillus casei as antigen delivery carrier to express BVDV glycoprotein E2 fused with DC-targeting peptide, and the immunogenicity of orally administered probiotic vaccine was evaluated in mice model. Our results showed that after immunization with the probiotic vaccine, significantly levels of antigen-specific sera IgG and mucosal sIgA antibodies (p < 0.05) with BVDV-neutralizing activity were induced in vivo. Challenge experiment showed that pPG-E2-DCpep/LC W56 can provide effective immune protection against BVDV, and BVDV could be effectively cleared from the intestine of immunized mice post-challenge. Moreover, the pPG-E2-DCpep/LC W56 could efficiently activate DCs in the intestinal Peyer's patches, and significantly levels of lymphoproliferative responses, Th1-associated IFN-γ, and Th2-associated IL-4 were observed in mice immunized with pPG-E2-DCpep/LC W56 (p < 0.01). Our results clearly demonstrate that the probiotic vaccine could efficiently induce anti-BVDV mucosal, humoral, and cellular immune responses via oral immunization, indicating a promising strategy for the development of oral vaccine against BVDV.
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Diarrea Mucosa Bovina Viral/prevención & control , Células Dendríticas/inmunología , Virus de la Diarrea Viral Bovina/inmunología , Portadores de Fármacos , Lacticaseibacillus casei/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Administración Oral , Animales , Anticuerpos Neutralizantes/análisis , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Bovinos , Células Dendríticas/metabolismo , Virus de la Diarrea Viral Bovina/genética , Modelos Animales de Enfermedad , Vectores Genéticos , Inmunoglobulina A Secretora/sangre , Inmunoglobulina G/sangre , Lacticaseibacillus casei/metabolismo , Ratones , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Resultado del Tratamiento , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Bovine viral diarrhea virus (BVDV) is an important virus that can cause extensive economic losses in both dairy and beef industry worldwide. Acute infection with BVDV results in peripheral blood lymphopenia, apoptosis and immunosuppression. Up-regulated programmed death-1 (PD-1) expression induces functional exhaustion of lymphocytes, inhibition of proliferation and apoptosis of lymphocytes during acute and chronic viral infections, such as HIV and HCV. However, there are no reports showing the role of PD-1 in peripheral blood lymphopenia, apoptosis and immunosuppression after acute BVDV infection. Accordingly, we measured the mRNA and protein expression of PD-1 and programmed death-ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) infected with BVDV, and analyzed the effects of PD-1 blockade on immune-associated function and activity in peripheral blood lymphocytes (PBLs). The results showed that both cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain KD) infection stimulated the mRNA and protein expression of PD-1 and PD-L1 significantly. The upregulation of PD-1/PD-L1 was accompanied by the decreased PBLs proliferation and increased apoptosis. Additionally, PD-1 blockade restored proliferation, inhibited apoptosis, increased IFN-γ production and decreased BVDV load. Remarkably, the PD-1/PD-L1 interaction has a more substantial effect on the immunoregulation of inhibiting proliferation induced by CP BVDV infection. Our findings confirm that PD-1 plays a vital role in peripheral blood lymphopenia and apoptosis caused by acute BVDV infection, and provide new insights into exploring the immunopathological mechanisms of BVDV or other members of the Flaviviridae family, and a potential therapeutic strategy to control BVDV infection.
Asunto(s)
Virus de la Diarrea Viral Bovina Tipo 1/inmunología , Leucocitos Mononucleares/virología , Linfocitos/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Animales , Anticuerpos Monoclonales/farmacología , Apoptosis , Diarrea Mucosa Bovina Viral/virología , Bovinos , Proliferación Celular , Técnicas In Vitro , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Linfocitos/inmunología , Linfocitos/fisiología , Linfocitos/virología , Receptor de Muerte Celular Programada 1/inmunologíaRESUMEN
Esta pesquisa avaliou a TIP e a dinâmica de anticorpos (ACs) específicos em bezerros naturalmente expostos aos agentes causadores da doença respiratória bovina (DRB). Foram selecionados 19 bezerros Holandeses alimentados com colostro proveniente de doadoras vacinadas para DRB. Amostras de soro foram obtidas antes e após a ingestão do colostro (48h) para a soroneutralização (SN). Os valores médios (log2) detectados após colostragem foram de 11,5±1,6 (BVDV), 8,8±1,3 (BoHV-1), 5,5±1,6 (BRSV) e 8,4±1,5 (BPIV-3). Cinco bezerros foram criados do nascimento aos 240 dias de vida, observando-se decréscimo nos títulos de ACs para BVDV, BoHV-1 e BPIV-3 ao longo do tempo (P≤0,001). As taxas de infecções detectadas entre o D14 e o D240 foram de 40% (2/5), 20% (1/5), 80% (4/5), e 60% (3/5), respectivamente, para BVDV, BoHV-1, BRSV e BPIV-3. A maioria dos bezerros manifestou broncopneumonia após as infecções virais. Os bezerros apresentaram ACs para todas as viroses às 48 horas de vida, porém os títulos adquiridos para o BRSV foram baixos. A susceptibilidade para as infecções variou de acordo com os níveis e a duração dos títulos de ACs maternos.(AU)
This research evaluated the PIT and the dynamics of specific antibody (Ab) for calves naturally exposed to the viral agents involved in Bovine Respiratory Disease (BRD). Nineteen Holstein calves fed colostrum from vaccinated donors for DRB. Serum samples were obtained before and after colostrum intake (48h) for serum neutralization (SN). Mean values (log2) detected after colostrum feeding were 11.5±1.6 (BVDV), 8.8 ±1.3 (BoHV-1) 5.5±1.6 (BRSV) and 8.4±1.5 (BPIV-3). Five calves were raised from birth to 240 days of life and presented a decrease in Ab titers for BVDV, BoHV-1 and BPIV-3 over time (P≤ 0.001). Infection rates from D14 to D240 were of 40% (2/5), 20% (1/5), 80% (4/5) and 60% (3/5), respectively for BVDV, BoHV-1, BRSV and BPIV-3. Most of the calves presented bronchopneumonia after seroconversion to the virus. Calves presented Ab for all viruses at 48 hours of life, however BRSV Ab titer were low. Levels and persistence of maternal antibody titers determined the susceptibility to viral infections.(AU)
Asunto(s)
Animales , Bovinos , Bovinos/inmunología , Inmunización Pasiva/veterinaria , Virosis/inmunología , Herpesvirus Bovino 1RESUMEN
Assuring viral safety of horse plasma-derived products is fundamental for ethical and regulatory reasons. We previously demonstrated the ability of pepsin digestion at low pH to inactivate West Nile and Sindbis viruses in horse plasma. The present study further examined the efficiency of pepsin digestion to inactivate four additional viruses: HSV-1 and BVDV (lipid-enveloped), BPV and Reo-3 (nonenveloped). These viruses were spiked into hyperimmunized horse plasma against botulinum toxin and subjected to low pH (3.2) alone or combined with pepsin digestion (1200 units/ml). Peptic digestion inactivated the lipid-enveloped viruses, whereas the nonenveloped viruses were unaffected. Interestingly, HSV-1 was rapidly inactivated by acidic pH alone (≥4.9 ± 0.6 log10), whereas a non-robust but meaningful BVDV inactivation (2.9 ± 0.7 log10) was achieved by combined low pH and pepsin. The current study demonstrated the ability of low pH alone and in combination with pepsin digestion to inactivate enveloped viral contaminants in anti-toxin horse plasma.
Asunto(s)
Antitoxina Botulínica/química , Virus de la Diarrea Viral Bovina , Contaminación de Medicamentos/prevención & control , Herpesvirus Humano 1 , Pepsina A/química , Plasma/química , Inactivación de Virus , Animales , Antitoxina Botulínica/inmunología , Caballos , Concentración de Iones de Hidrógeno , Plasma/inmunología , Plasma/virologíaRESUMEN
A series of N4-arylsubstituted thiosemicarbazones derived from 1-indanones and a set of compounds lacking such substitution in the N4 position of the thiosemicarbazone moiety were synthesized and evaluated for their anti-bovine viral diarrhea virus (BVDV) activity. Among these, derivatives 2 and 15 displayed high activity (EC50=2.7±0.4 and 0.7±0.1µM, respectively) as inhibitors of BVDV replication. Novel key structural features related to the anti-BVDV activity were identified by structure-activity relationship (SAR) analysis. In a previous study, the thiosemicarbazone of 5,6-dimethoxy-1-indanone (5,6-TSC) was characterized as a non-nucleoside inhibitor (NNI) of the BVDV RNA-dependent RNA polymerase. In the present work, cross-resistance assays were performed with the most active compounds. Such studies were carried out on 5,6-TSC resistant BVDV (BVDV-TSCr T1) carrying mutations in the viral polymerase. This BVDV mutant was also resistant to compound 15. Molecular docking studies and MM/PBSA calculations were performed to assess the most active derivatives at the 5,6-TSC viral polymerase binding site. The differences in the interaction pattern and the binding affinity of derivative 15 either to the wild type or BVDV-TSCr T1 polymerase were key factors to define the mode of action of this compound.