RESUMEN
The bovine viral diarrhea virus (BVDV-1) is a pathogen with the capacity to modulate the interferon type I system. To further investigate the effects of BVDV-1 on the production of the immune response, the Madin-Darby bovine kidney cell line was infected with the cytopathic CH001 field isolate of BVDV-1, and the IFNbeta expression profiles were analyzed. The results showed that cpBVDV-1 was able to induce the production of IFNbeta in a way similar to polyinosinic-polycytidylic acid, but with less intensity. Interestingly, all cpBVDV-1 activities were blocked by pharmacological inhibitors of the IRF-1, IRF-7, and NF-κB signaling pathway, and the level of IFNbeta decreased at the level of transcript and protein. These results, together with in silico analyses showing the presence of several regulatory consensus target motifs, suggest that cpBVDV-1 regulates IFNbeta expression in bovines through the activation of several key transcription factors. Collectively, the results suggest that during cpBVDV-1 infection, cross talk is evident between various signaling pathways involved in transcriptional activation of IFNbeta in cattle.
Asunto(s)
Diarrea Mucosa Bovina Viral/genética , Virus de la Diarrea Viral Bovina Tipo 1/inmunología , Regulación de la Expresión Génica/genética , Expresión Génica/genética , Factor 1 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/genética , FN-kappa B/genética , Animales , Diarrea Mucosa Bovina Viral/virología , Bovinos , Línea Celular , Células Epiteliales/inmunología , Células Epiteliales/virología , Expresión Génica/inmunología , Regulación de la Expresión Génica/inmunología , Factor 1 Regulador del Interferón/inmunología , Factor 7 Regulador del Interferón/inmunología , FN-kappa B/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
After initiation of an infective cycle, spread of virus infection can occur in two fundamentally different ways: (i) viral particles can be released into the external environment and diffuse through the extracellular space until they interact with a new host cell, and (ii) virions can remain associated with infected cells, promoting the direct passage between infected and uninfected cells that is referred to as direct cell-to-cell transmission. Although evidence of cell-associated transmission has accumulated for many different viruses, the ability of members of the genus Pestivirus to use this mode of transmission has not been reported. In the present study, we used a novel recombinant virus expressing the envelope glycoprotein E2 fused to mCherry fluorescent protein to monitor the spreading of bovine viral diarrhea virus (BVDV) (the type member of the pestiviruses) infection. To demonstrate direct cell-to-cell transmission of BVDV, we developed a cell coculture system that allowed us to prove direct transmission from infected to uninfected cells in the presence of neutralizing antibodies. This mode of transmission requires cell-cell contacts and clathrin-mediated receptor-dependent endocytosis. Notably, it overcomes antibody blocking of the BVDV receptor CD46, indicating that cell-to-cell transmission of the virus involves the engagement of coreceptors on the target cell.IMPORTANCE BVDV causes one of the most economically important viral infections for the cattle industry. The virus is able to cross the placenta and infect the fetus, leading to the birth of persistently infected animals, which are reservoirs for the spread of BVDV. The occurrence of persistent infection has hampered the efficacy of vaccination because it requires eliciting levels of protection close to sterilizing immunity to prevent fetal infections. While vaccination prevents disease, BVDV can be detected if animals with neutralizing antibodies are challenged with the virus. Virus cell-to-cell transmission allows the virus to overcome barriers to free virus dissemination, such as antibodies or epithelial barriers. Here we show that BVDV exploits cell-cell contacts to propagate infection in a process that is resistant to antibody neutralization. Our results provide new insights into the mechanisms underlying the pathogenesis of BVDV infection and can aid in the design of effective control strategies.
Asunto(s)
Diarrea Mucosa Bovina Viral/virología , Comunicación Celular , Virus de la Diarrea Viral Bovina Tipo 1/patogenicidad , Interacciones Huésped-Patógeno , Proteínas del Envoltorio Viral/metabolismo , Replicación Viral , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Diarrea Mucosa Bovina Viral/genética , Diarrea Mucosa Bovina Viral/metabolismo , Bovinos , Células Cultivadas , Proteínas del Envoltorio Viral/genética , Internalización del VirusRESUMEN
Bovine viral diarrhea virus 1, reclassified as Pestivirus A, causes an economically important cattle disease that is distributed worldwide. Pestivirus A may cause persistent infection in that calves excrete the virus throughout their lives, spreading the infection in the herd. Many persistently infected (PI) calves die in the first 2 years of life from mucosal disease (MD) or secondary infections, probably as a consequence of virus-induced immune depression. Here, high-throughput sequencing (HTS) was applied for evaluation of the total virome in sera of (i) PI calves displaying clinically apparent MD (n = 8); (ii) PI calves with no signs of MD (n = 8); and (iii) control, Pestivirus A-free calves (n = 8). All the groups were collected at the same time and from the same herd. Serum samples from calves in each of the groups were pooled, submitted to viral RNA/DNA enrichment, and sequenced by HTS. Viral genomes of Pestivirus A, Ungulate erythroparvovirus 1, bosavirus (BosV), and hypothetical circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses were identified. Specific real-time PCR assays were developed to determine the frequency of occurrence of such viruses in each of the groups. The absolute number of distinct viral genomes detected in both PI calf groups was higher than in the control group, as revealed by higher number of reads, contigs, and genomes, representing a wider range of taxons. Genomes representing members of the family Parvoviridae, such as U. erythroparvovirus 1 and BosV, were most frequently detected in all the three groups of calves. Only in MD-affected PI calves, we found two previously unreported Hypothetical single-stranded DNA genomes clustered along with CRESS-DNA viruses. These findings reveal that parvoviruses were the most frequently detected viral genomes in cattle serum; its frequency of detection bears no statistical correlation with the status of calves in relation to Pestivirus A infection, since clinically normal or MD-affected/non-affected PI calves were infected with similar U. erythroparvovirus 1 genome loads. Moreover, MD-affected PI calves were shown to support viremia of CRESS-DNA viral genomes; however, the meaning of such correlation remains to be established.
Asunto(s)
Diarrea Mucosa Bovina Viral/genética , Virus de la Diarrea Viral Bovina Tipo 1/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Pestivirus/genética , Animales , Anticuerpos Antivirales/sangre , Diarrea Mucosa Bovina Viral/sangre , Diarrea Mucosa Bovina Viral/fisiopatología , Diarrea Mucosa Bovina Viral/virología , Bovinos , ADN Viral/genética , Virus de la Diarrea Viral Bovina Tipo 1/aislamiento & purificación , Virus de la Diarrea Viral Bovina Tipo 1/patogenicidad , Genoma Viral/genética , Pestivirus/clasificación , Pestivirus/aislamiento & purificación , Pestivirus/patogenicidad , ARN Viral/genéticaRESUMEN
The bovine viral diarrhea virus (BVDV) is responsible for significant economic losses in the dairy and cattle industry; however, little is known about the protective and pathological responses of hosts to infection. The present study determined the principal molecular markers implicated in viral infection through meta-transcriptomic analysis using MDBK cells infected for two hours with a field isolate of BVDV-1. While several immune regulator genes were induced, genes involved in cell signaling, metabolic processes, development, and integrity were down-regulated, suggesting an isolation of infected cells from cell-to-cell interactions and responses to external signals. Analysis through RT-qPCR confirmed the expression of more than one hundred markers. Interestingly, there was a significant up-regulation of two negative NF-κB regulators, IER3 and TNFAIP3, indicating a possible blocking of this signaling pathway mediated by BVDV-1 infection. Additionally, several genes involved in the metabolism of reactive oxygen species were down-regulated, suggesting increased oxidative stress. Notably, a number of genes involved in cellular growth and development were also regulated during infection, including MTHFD1L, TGIF1, and Brachyury. Moreover, there was an increased expression of the genes ß-catenin, caprin-2, GSK3ß, and MMP-7, all of which are crucial to the Wnt signaling pathway that is implicated in the embryonic development of a variety of organisms. This meta-transcriptomic analysis provides the first data towards understanding the infection mechanisms of cytopathic BVDV-1 and the putative molecular relationship between viral and host components.
Asunto(s)
Diarrea Mucosa Bovina Viral/genética , Diarrea Mucosa Bovina Viral/inmunología , Transcriptoma , Animales , Bovinos , Línea Celular , Virus de la Diarrea Viral Bovina Tipo 1 , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cytokine production for immunological process is tightly regulated at the transcriptional and posttranscriptional levels. The NF-κB signaling pathway maintains immune homeostasis in the cell through the participation of molecules such as A20 (TNFAIP3), which is a key regulatory factor in the immune response, hematopoietic differentiation, and immunomodulation. Although A20 has been identified in mammals, and despite recent efforts to identify A20 members in other higher vertebrates, relatively little is known about the composition of this regulator in other classes of vertebrates, particularly for bovines. In this study, the genetic context of bovine A20 was explored and compared against homologous genes in the human, mouse, chicken, dog, and zebrafish chromosomes. Through in silico analysis, several regions of interest were found conserved between even phylogenetically distant species. Additionally, a protein-deduced sequence of bovine A20 evidenced many conserved domains in humans and mice. Furthermore, all potential amino acid residues implicated in the active site of A20 were conserved. Finally, bovine A20 mRNA expression as mediated by the bovine viral diarrhea virus and poly (I:C) was evaluated. These analyses evidenced a strong fold increase in A20 expression following virus exposure, a phenomenon blocked by a pharmacological NF-κB inhibitor (BAY 117085). Interestingly, A20 mRNA had a half-life of only 32min, likely due to adenylate- and uridylate-rich elements in the 3'-untranslated region. Collectively, these data identify bovine A20 as a regulator of immune marker expression. Finally, this is the first report to find the bovine viral diarrhea virus modulating bovine A20 activation through the NF-κB pathway.
Asunto(s)
Diarrea Mucosa Bovina Viral/genética , Diarrea Mucosa Bovina Viral/inmunología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , FN-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animales , Diarrea Mucosa Bovina Viral/virología , Dominio Catalítico , Bovinos , Línea Celular , Proteínas de Unión al ADN/química , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Ratones , Nitrilos/farmacología , Proteínas Nucleares/química , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacos , Sulfonas/farmacologíaRESUMEN
Infection with Bovine Viral Diarrhea Viruses (BVDV) in cattle results in a wide range of clinical manifestations, ranging from mild respiratory disease to fetal death and mucosal disease, depending on the virulence of the virus and the immune and reproductive status of the host. In this study 30 Argentinean BVDV isolates were characterized by phylogenetic analysis. The isolates were genotyped based on comparison of the 5' untranslated region (5' UTR) and the E2 gene. In both phylogenetic trees, 76% of the viruses were assigned to BVDV 1b, whereas BVDV 1a, 2a and 2b were also found. Eight of the BVDV 1b isolates were further characterized by cross-neutralization tests using guinea pig antisera and sera from bovines vaccinated with two different commercial vaccines. The results demonstrated the presence of a marked antigenic diversity among Argentinean BVDV isolates and suggest the need to incorporate BVDV 1b isolates in diagnostic strategies.
Asunto(s)
Variación Antigénica/inmunología , Diarrea Mucosa Bovina Viral/inmunología , Virus de la Diarrea Viral Bovina/inmunología , Filogenia , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Animales , Variación Antigénica/genética , Argentina , Secuencia de Bases , Diarrea Mucosa Bovina Viral/genética , Bovinos , Virus de la Diarrea Viral Bovina/genética , Cobayas , Datos de Secuencia Molecular , Pruebas de Neutralización/veterinaria , ARN Viral/química , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Alineación de Secuencia , Análisis de Secuencia de ADN , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
Due to the wide dissemination of bovine leukemia virus (BLV) infection among dairy cattle, control and eradication programs based on serological detection of infected cattle and subsequent culling face a major economic task. In Argentina, genetic selection of cattle carrying alleles of the bovine leukocyte antigen (BoLA) DRB3.2 gene associated with BLV-infection resistance, like *0902, emerges as the best additional tool toward controlling virus spread. A potential risk in expanding or segregating BoLA selected populations of cattle is that it might increase susceptibility to other common viruses. Special concern raises the strong association found between low proviral load and low antibody titer against major BLV structural proteins. This phenomenon might depend on host genetic factors influencing other viruses requiring, unlike BLV, strong and long-lasting humoral immune response to prevent infection. In this study, we demonstrate that there is no association among neutralizing antibody titers against foot and mouth disease virus, bovine viral diarrhea virus, or bovine herpesvirus type 1 and polymorphism of the BoLA DRB3.2 gene. Conversely, there is strong association between BoLA DRB3.2*0902 and low antibody titers against 2 BLV structural proteins--env gp51 and gag p24--to date, the best BLV resistance marker. There is also significant association between low antibody titers against gp51 and p24 and BoLA DRB3.2*1701 and low antibody titers against p24 and BoLA DRB3.2*1101 or 02. Our data suggest that increasing BoLA-selected BLV-resistant cattle or segregating BoLA-associated alleles to BLV susceptibility would not affect the resistance or the predisposition to bovine viral diarrhea virus, bovine herpesvirus type 1, or foot and mouth disease virus infection.
Asunto(s)
Anticuerpos Antivirales/inmunología , Formación de Anticuerpos/inmunología , Antígenos Virales/inmunología , Leucosis Bovina Enzoótica , Antígenos HLA , Inmunidad Innata/genética , Virus de la Leucemia Bovina/inmunología , Animales , Anticuerpos Antivirales/sangre , Diarrea Mucosa Bovina Viral/genética , Bovinos , Virus de la Diarrea Viral Bovina Tipo 1/inmunología , Leucosis Bovina Enzoótica/genética , Leucosis Bovina Enzoótica/inmunología , Femenino , Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/inmunología , Genotipo , Antígenos HLA/genética , Antígenos HLA/inmunología , Infecciones por Herpesviridae/genética , Herpesvirus Bovino 1/inmunología , Polimorfismo GenéticoRESUMEN
Single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products is a genetic screening technique for rapid detection of nucleotide substitutions in PCR-amplified genomic DNA or cDNA. It is based on the observation that partially formamide-denatured double-stranded DNA migrates as 2 single-stranded DNA molecules when electrophoresed in nondenaturing polyacrylamide gels. The mobility depends on the 3-dimensional conformation of the strand under the conditions used. It is possible to discriminate between DNA strands differing in only 1 nucleotide. The method was applied to the analysis of Bovine Viral Diarrhea Virus (BVDV) isolates. Reference and Argentinian strains were assessed for variations in their 5' untranslated region (5'-UTR). The PCR products of the 5'-UTR ends were formamide denatured and compared by SSCP analysis in nondenaturing 15% polyacrylamide and 15% polyacrilamide-5% glycerol gels. The reference strains SD-1, Singer, and Oregon C24V had differences in electrophoretic patterns. Despite the high conservation among the 5'-UTR of pestiviruses, the method allowed discrimination among all 9 Argentinian isolates. The 5'-UTR of a fetal kidney-derived isolate (1R93) was PCR amplified and cloned in a plasmid vector; the SSCP analysis of 30 PCR products obtained by direct amplification over randomly selected clones produced 5 different banding patterns, indicating the existence of viral quasispecies. The results show that SSCP may be used to identify and differentiate among BVDV isolates.
Asunto(s)
Diarrea Mucosa Bovina Viral/diagnóstico , Virus de la Diarrea Viral Bovina/genética , Polimorfismo Conformacional Retorcido-Simple , Regiones no Traducidas 5'/genética , Animales , Secuencia de Bases , Diarrea Mucosa Bovina Viral/genética , Bovinos , Riñón/patología , Riñón/virología , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa/veterinaria , Sensibilidad y EspecificidadRESUMEN
To ascertain the virulence of bovine viral diarrhea virus (BVDV) genotype II, isolate NY-93 was inoculated intranasally into 3 calves, 2 of which were treated with a synthetic glucocorticoid prior to and after virus inoculation. Anorexia, fever (up to 42 C), dyspnea, and hemorrhagic diarrhea developed 6 days after intranasal inoculation with BVDV NY-93. The condition of all calves deteriorated further until the end of the study on day 14 postinoculation. The most significant postmortem macroscopic changes in all calves were limited to the gastrointestinal tract and consisted of moderate to severe congestion of the mucosa with multifocal hemorrhages. Microscopic lesions found in the gastrointestinal tract were similar to those observed in mucosal disease, including degeneration and necrosis of crypt epithelium and necrosis of lymphoid tissue throughout the ileum, colon, and rectum. The basal stratum of the epithelium of tongue, esophagus, and rumen had scattered individual necrotic cells. Spleen and lymph nodes had lymphocytolysis and severe lymphoid depletion. Severe acute fibrinous bronchopneumonia was present in dexamethasone-treated calves. Abundant viral antigen was detected by immunohistochemistry in the squamous epithelium of tongue, esophagus, and forestomachs. BVDV antigen was prominent in cells of the media of small arteries and endothelial cells. The presence of infectious virus in tissues correlated with an absence of circulating neutralizing antibodies. These findings highlight the potential of BVDV genotype II to cause severe disease in normal and stressed cattle.