RESUMEN
Porcine hemagglutinating encephalomyelitis virus (PHEV) replicates in the upper respiratory tract and tonsils of pigs. Using an air-liquid interface porcine respiratory epithelial cells (ALI-PRECs) culture system, we demonstrated that PHEV disrupts respiratory epithelia homeostasis by impairing ciliary function and inducing antiviral, pro-inflammatory cytokine, and chemokine responses. This study explores the mechanisms driving early innate immune responses during PHEV infection through host transcriptome analysis. Total RNA was collected from ALI-PRECs at 24, 36, and 48 h post inoculation (hpi). RNA-seq analysis was performed using an Illumina Hiseq 600 to generate 100 bp paired-end reads. Differential gene expression was analyzed using DeSeq2. PHEV replicated actively in ALI-PRECs, causing cytopathic changes and progressive mucociliary disruption. Transcriptome analysis revealed downregulation of cilia-associated genes such as CILK1, DNAH11, LRRC-23, -49, and -51, and acidic sialomucin CD164L2. PHEV also activated antiviral signaling pathways, significantly increasing the expression of interferon-stimulated genes (RSAD2, MX1, IFIT, and ISG15) and chemokine genes (CCL5 and CXCL10), highlighting inflammatory regulation. This study contributes to elucidating the molecular mechanisms of the innate immune response to PHEV infection of the airway epithelium, emphasizing the critical roles of the mucociliary, interferon, and chemokine responses.
Asunto(s)
Betacoronavirus 1 , Células Epiteliales , Perfilación de la Expresión Génica , Interferones , Animales , Porcinos , Células Epiteliales/virología , Células Epiteliales/inmunología , Interferones/genética , Interferones/metabolismo , Interferones/inmunología , Betacoronavirus 1/inmunología , Betacoronavirus 1/genética , Inmunidad Innata , Replicación Viral , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/veterinaria , Citocinas/metabolismo , Citocinas/genética , Citocinas/inmunología , Transcriptoma , Mucosa Respiratoria/virología , Mucosa Respiratoria/inmunología , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/genética , Células Cultivadas , DeltacoronavirusRESUMEN
The upper respiratory tract is the primary site of infection by porcine hemagglutinating encephalomyelitis virus (PHEV). In this study, primary porcine respiratory epithelial cells (PRECs) were cultured in an air-liquid interface (ALI) to differentiate into a pseudostratified columnar epithelium, proliferative basal cells, M cells, ciliated cells, and mucus-secreting goblet cells. ALI-PRECs recreates a cell culture environment morphologically and functionally more representative of the epithelial lining of the swine trachea than traditional culture systems. PHEV replicated actively in this environment, inducing cytopathic changes and progressive disruption of the mucociliary apparatus. The innate immunity against PHEV was comparatively evaluated in ALI-PREC cultures and tracheal tissue sections derived from the same cesarean-derived, colostrum-deprived (CDCD) neonatal donor pigs. Increased expression levels of TLR3 and/or TLR7, RIG1, and MyD88 genes were detected in response to infection, resulting in the transcriptional upregulation of IFN-λ1 in both ALI-PREC cultures and tracheal epithelia. IFN-λ1 triggered the upregulation of the transcription factor STAT1, which in turn induced the expression of the antiviral IFN-stimulated genes OAS1 and Mx1. No significant modulation of the major proinflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α) was detected in response to PHEV infection. However, a significant upregulation of different chemokines was observed in ALI-PREC cultures (CCL2, CCL5, CXCL8, and CXCL10) and tracheal epithelium (CXCL8 and CXCL10). This study shed light on the molecular mechanisms driving the innate immune response to PHEV at the airway epithelium, underscoring the important role of respiratory epithelial cells in the maintenance of respiratory homeostasis and on the initiation, resolution, and outcome of the infectious process. IMPORTANCE The neurotropic betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) primarily infects and replicates in the swine upper respiratory tract, causing vomiting and wasting disease and/or encephalomyelitis in suckling pigs. This study investigated the modulation of key early innate immune genes at the respiratory epithelia in vivo, on tracheal tissue sections from experimentally infected pigs, and in vitro, on air-liquid interface porcine respiratory cell cultures. The results from the study underscore the important role of respiratory epithelial cells in maintaining respiratory homeostasis and on the initiation, resolution, and outcome of the PHEV infectious process.
Asunto(s)
Betacoronavirus 1/fisiología , Interferones/genética , Interleucina-8/inmunología , Receptores de Reconocimiento de Patrones/genética , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/virología , Replicación Viral , Animales , Animales Recién Nacidos , Betacoronavirus 1/inmunología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Interferones/inmunología , Interleucina-8/genética , Mucosa Respiratoria/patología , Porcinos , Regulación hacia Arriba , Replicación Viral/inmunologíaRESUMEN
Porcine hemagglutinating encephalomyelitis virus (PHEV) is a betacoronavirus that causes vomiting and wasting disease and/or encephalomyelitis in suckling pigs. This study characterized PHEV infection, pathogenesis, and immune response in cesarean-derived, colostrum-deprived (CDCD) neonatal pigs. Infected animals developed mild respiratory, enteric, and neurological clinical signs between 2 to 13 days postoronasal inoculation (dpi). PHEV did not produce viremia, but virus shedding was detected in nasal secretions (1 to 10 dpi) and feces (2 to 7 dpi) by reverse transcriptase quantitative PCR (RT-qPCR). Viral RNA was detected in all tissues except liver, but the detection rate and RT-qPCR threshold cycle (CT ) values decreased over time. The highest concentration of virus was detected in inoculated piglets necropsied at 5 dpi in turbinate and trachea, followed by tonsils, lungs, tracheobronchial lymph nodes, and stomach. The most representative microscopic lesions were gastritis lymphoplasmacytic, moderate, multifocal, with perivasculitis, and neuritis with ganglia degeneration. A moderate inflammatory response, characterized by increased levels of interferon alpha (IFN-α) in plasma (5 dpi) and infiltration of T lymphocytes and macrophages were also observed. Increased plasma levels of interleukin-8 (IL-8) were detected at 10 and 15 dpi, coinciding with the progressive resolution of the infection. Moreover, a robust antibody response was detected by 10 dpi. An ex vivo air-liquid CDCD-derived porcine respiratory cells culture (ALI-PRECs) system showed virus replication in ALI-PRECs and cytopathic changes and disruption of ciliated columnar epithelia, thereby confirming the tracheal epithelia as a primary site of infection for PHEV.IMPORTANCE Among the â¼46 virus species in the family Coronaviridae, many of which are important pathogens of humans and 6 of which are commonly found in pigs, porcine hemagglutinating encephalomyelitis remains one of the least researched. The present study provided a comprehensive characterization of the PHEV infection process and immune responses using CDCD neonatal pigs. Moreover, we used an ex vivo ALI-PRECs system resembling the epithelial lining of the tracheobronchial region of the porcine respiratory tract to demonstrate that the upper respiratory tract is a primary site of PHEV infection. This study provides a platform for further multidisciplinary studies of coronavirus infections.
Asunto(s)
Betacoronavirus 1/inmunología , Infecciones por Coronavirus/inmunología , Interferón-alfa/inmunología , Interleucina-8/inmunología , Enfermedades de los Porcinos/inmunología , Linfocitos T/inmunología , Animales , Línea Celular , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/veterinaria , Especificidad de Órganos/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Enfermedades de los Porcinos/patología , Linfocitos T/patología , Linfocitos T/virologíaRESUMEN
Porcine haemagglutinating encephalomyelitis virus (PHEV) is a member of coronavirus that causes acute infectious disease and high mortality in piglets. The transcription factor IRF3 is a central regulator of type I interferon (IFN) innate immune signalling. Here, we report that PHEV infection of RAW264.7 cells results in strong suppression of IFN-ß production in the early stage. A comparative analysis of the upstream effector of IFN-ß transcription demonstrated that deactivation of IRF3, but not p65 or ATF-2 proteins, is uniquely attributed to failure of early IFN-ß induction. Moreover, the RIG-I/MDA5/MAVS/TBK1-dependent protective response that regulates the IRF3 pathway is not disrupted by PHEV and works well underlying the deactivated IRF3-mediated IFN-ß inhibition. After challenge with poly(I:C), a synthetic analogue of dsRNA used to stimulate IFN-ß secretion in the TLR-controlled pathway, we show that PHEV and poly(I:C) regulate IFN-ß-induction via two different pathways. Collectively, our findings reveal that deactivation of IRF3 is a specific mechanism that contributes to termination of type I IFN signalling during early infection with PHEV independent of the conserved RIG-I/MAVS/MDA5/TBK1-mediated innate immune response.
Asunto(s)
Betacoronavirus 1/inmunología , Infecciones por Coronavirus/veterinaria , Factor 3 Regulador del Interferón/genética , Interferón beta/inmunología , Animales , Betacoronavirus 1/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Inmunidad Innata , Factor 3 Regulador del Interferón/inmunología , Ratones , Poli I-C/farmacología , Células RAW 264.7 , Transducción de Señal/inmunologíaRESUMEN
Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic coronavirus that invades the host central nervous system (CNS) and causes neurological dysfunction. Microglia are key immune cells in the CNS, however, whether and how they response to PHEV infection remains unclear. Herein, microglial activation and proliferation were detected in the CNS of PHEV-infected mice, as along with the proinflammatory response. Moreover, the production of proinflammatory cytokines induced by moderately activated microglia limited viral replication in the early stage of infection. Microglial depletion assays showed that during late infection, excess activation of microglia aggravated neurological symptoms, BBB destruction, and peripheral monocyte/macrophage infiltration into the CNS. Using an in vitro brain slice model, PHEV was identified to specifically and moderately induce microglial activation in the absence of peripheral immune cells infiltration. Consistently, macrophage clearance from circulating blood indicated that peripheral monocytes/macrophages crossing the BBB of mice were responsible for excess activation of microglia and CNS damage in late PHEV infection. Overall, our findings provide evidence supporting a dual role for microglia in the host CNS in response to coronavirus PHEV invasion.
Asunto(s)
Betacoronavirus 1/inmunología , Barrera Hematoencefálica/inmunología , Infecciones por Coronaviridae/inmunología , Macrófagos/inmunología , Microglía/inmunología , Monocitos/inmunología , Animales , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/virología , Macrófagos/patología , Macrófagos/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Microglía/patología , Microglía/virología , Monocitos/patología , Monocitos/virologíaRESUMEN
Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the genus Betacoronavirus and is the etiologic agent of encephalomyelitis or vomiting and wasting disease in neonatal pigs. Although there are only a few epidemiological studies that document the seroprevalence of PHEV infection, there are reports of sporadic outbreaks, including recent documentation of an influenza-like respiratory disease associated with PHEV in the United States. To address this issue, we have developed a new indirect enzyme linked immunosorbent assay (ELISA) for use in sero-epidemiological research of PHEV infection. One hundred and fifty porcine serum samples that were determined as antibody-positive or antibody-negative in virus neutralization (VN) tests were used in conjunction with PHEV-specific antigen extracted from virus-infected FS-L3 cells using RBS buffer containing 0.2 % NP-40 to develop this assay. The ELISA showed a high sensitivity (95.35 %) and specificity (96.88 %) by receiver operating characteristic (ROC) analysis, with an area under the curve (AUC) of 0.996 attesting to its accuracy. Our results revealed a strong correlation between the results of the indirect ELISA and VN test (R = 0.850, P < 0.05), with near-perfect agreement (kappa value = 0.932). These results indicate that this new indirect ELISA might be useful for diagnosis and sero-epidemiological tracking of PHEV infection.
Asunto(s)
Anticuerpos Antivirales/sangre , Betacoronavirus 1/inmunología , Infecciones por Coronavirus/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Enfermedades de los Porcinos/diagnóstico , Animales , Línea Celular , Infecciones por Coronavirus/diagnóstico , Sensibilidad y Especificidad , Estudios Seroepidemiológicos , Porcinos , Enfermedades de los Porcinos/virologíaRESUMEN
Members of family Coronaviridae cause a variety of diseases in birds and mammals. Porcine hemagglutinating encephalomyelitis virus (PHEV), a lesser-researched coronavirus, can infect naive pigs of any age, but clinical disease is observed in pigs ≤4 weeks of age. No commercial PHEV vaccines are available, and neonatal protection from PHEV-associated disease is presumably dependent on lactogenic immunity. Although subclinical PHEV infections are thought to be common, PHEV ecology in commercial swine herds is unknown. To begin to address this gap in knowledge, a serum IgG antibody enzyme-linked immunosorbent assay (ELISA) based on the S1 protein was developed and evaluated on known-status samples and then used to estimate PHEV seroprevalence in U.S. sow herds. Assessment of the diagnostic performance of the PHEV S1 ELISA using serum samples (n = 924) collected from 7-week-old pigs (n = 84; 12 pigs per group) inoculated with PHEV, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, or porcine deltacoronavirus showed that a sample-to-positive cutoff value of ≥0.6 was both sensitive and specific, i.e., all PHEV-inoculated pigs were seropositive from days postinoculation 10 to 42, and no cross-reactivity was observed in samples from other groups. The PHEV S1 ELISA was then used to estimate PHEV seroprevalence in U.S. sow herds (19 states) using 2,756 serum samples from breeding females (>28 weeks old) on commercial farms (n = 104) with no history of PHEV-associated disease. The overall seroprevalence was 53.35% (confidence interval [CI], ±1.86%) and herd seroprevalence was 96.15% (CI, ±3.70%).IMPORTANCE There is a paucity of information concerning the ecology of porcine hemagglutinating encephalomyelitis virus (PHEV) in commercial swine herds. This study provided evidence that PHEV infection is endemic and highly prevalent in U.S. swine herds. These results raised questions for future studies regarding the impact of endemic PHEV on swine health and the mechanisms by which this virus circulates in endemically infected populations. Regardless, the availability of the validated PHEV S1 enzyme-linked immunosorbent assay (ELISA) provides the means for swine producers to detect and monitor PHEV infections, confirm prior exposure to the virus, and to evaluate the immune status of breeding herds.
Asunto(s)
Anticuerpos Antivirales/sangre , Betacoronavirus 1/aislamiento & purificación , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/veterinaria , Enfermedades de los Porcinos/epidemiología , Animales , Anticuerpos Antivirales/inmunología , Betacoronavirus 1/inmunología , Infecciones por Coronavirus/diagnóstico , Reacciones Cruzadas/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Coronavirus Respiratorio Porcino/inmunología , Virus de la Diarrea Epidémica Porcina/inmunología , Estudios Seroepidemiológicos , Porcinos , Enfermedades de los Porcinos/diagnóstico , Virus de la Gastroenteritis Transmisible/inmunología , Estados Unidos/epidemiologíaRESUMEN
Lay bioethics is an applied ethic that allows us to face the problems generated by the tension between science and the human being.The current pandemic caused by an unknown infectious agent has highlighted the need for the application of this applied ethic, not located on the laurels of the ethereal or purely intellectual, but rather based in day-to-day reality. Decision-making from all areas of health care requires this fundamental frame of reference; the application of correct precepts in epidemiological, sanitary decisions and political measures cannot and should not be done without this frame of reference; or the result will be catastrophic.
La Bioética laica es en principio una ética aplicada que permite afrontar los problemas generados por la tensión existente entre la ciencia y el ser humano. La actual pandemia causada por un agente infeccioso no conocido ha puesto de relieve la necesidad de la aplicación de esta ética aplicada, no ubicada en los laureles de lo etéreo o puramente intelectual sino más bien asentada en la realidad del día a día. La toma de decisiones desde todos los ámbitos del quehacer sanitario requiere este marco referencial fundamental; aplicación de correctos preceptos en las decisiones epidemiológicas, sanitarias y medidas políticas no puede ni debe hacerse sin este marco referencial; otrora el resultado será catastrófico.(AU)
Asunto(s)
Humanos , Salud Global/estadística & datos numéricos , Discusiones Bioéticas/normas , Betacoronavirus 1/inmunología , Bioética , Reglamento Sanitario Internacional/estadística & datos numéricosRESUMEN
Equine coronavirus (ECoV) is considered to be involved in enteric diseases in foals. Recently, several outbreaks of ECoV infection have also been reported in adult horses from the USA, France and Japan. Epidemiological studies of ECoV infection are still limited, and the seroprevalence of ECoV infection in Europe is unknown. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) method utilizing ECoV spike S1 protein was developed in two formats, and further validated by analyzing 27 paired serum samples (acute and convalescent sera) from horses involved in an ECoV outbreak and 1084 sera of horses with unknown ECoV exposure. Both formats showed high diagnostic accuracy compared to virus neutralization (VN) assay. Receiver-operating characteristic (ROC) analyses were performed to determine the best cut-off values for both ELISA formats, assuming a test specificity of 99%. Employing the developed ELISA method, we detected seroconversion in 70.4% of horses from an ECoV outbreak. Among the 1084 horse sera, seropositivity varied from 25.9% (young horses) to 82.8% (adult horses) in Dutch horse populations. Further, sera of Icelandic horses were included in this study and a significant number of sera (62%) were found to be positive. Overall, the results demonstrated that the ECoV S1-based ELISA has reliable diagnostic performance compared to the VN assay and is a useful assay to support seroconversion in horses involved with ECoV outbreaks and to estimate ECoV seroprevalence in populations of horses.
Asunto(s)
Anticuerpos Antivirales/sangre , Betacoronavirus 1/aislamiento & purificación , Infecciones por Coronavirus/veterinaria , Enfermedades de los Caballos/diagnóstico , Pruebas Serológicas/veterinaria , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Betacoronavirus 1/inmunología , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/epidemiología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Enfermedades de los Caballos/epidemiología , Caballos , Islandia/epidemiología , Países Bajos/epidemiología , Curva ROC , Estudios Seroepidemiológicos , Pruebas Serológicas/métodosRESUMEN
A vaccine for equine coronavirus (ECoV) is so far unavailable. Bovine coronavirus (BCoV) is antigenically related to ECoV; it is therefore possible that BCoV vaccine will induce antibodies against ECoV in horses. This study investigated antibody response to ECoV in horses inoculated with BCoV vaccine. Virus neutralization tests showed that antibody titers against ECoV increased in all six horses tested at 14 days post inoculation, although the antibody titers were lower against ECoV than against BCoV. This study showed that BCoV vaccine provides horses with antibodies against ECoV to some extent. It is unclear whether antibodies provided by BCoV vaccine are effective against ECoV, and therefore ECoV challenge studies are needed to evaluate efficacy of the vaccine in the future.