RESUMEN
Porcine deltacoronavirus (PDCoV), an enteropathogenic coronavirus, causes severe watery diarrhoea, dehydration and high mortality in piglets, which has the potential for cross-species transmission in recent years. Growth factor receptor-bound protein 2 (Grb2) is a bridging protein that can couple cell surface receptors with intracellular signal transduction events. Here, we investigated the reciprocal regulation between Grb2 and PDCoV. It is found that Grb2 regulates PDCoV infection and promotes IFN-ß production through activating Raf/MEK/ERK/STAT3 pathway signalling in PDCoV-infected swine testis cells to suppress viral replication. PDCoV N is capable of interacting with Grb2. The proline-rich motifs in the N- or C-terminal region of PDCoV N were critical for the interaction between PDCoV-N and Grb2. Except for Deltacoronavirus PDCoV N, the Alphacoronavirus PEDV N protein could interact with Grb2 and affect the regulation of PEDV replication, while the N protein of Betacoronavirus PHEV and Gammacoronavirus AIBV could not interact with Grb2. PDCoV N promotes Grb2 degradation by K48- and K63-linked ubiquitin-proteasome pathways. Overexpression of PDCoV N impaired the Grb2-mediated activated effect on the Raf/MEK/ERK/STAT3 signal pathway. Thus, our study reveals a novel mechanism of how host protein Grb2 protein regulates viral replication and how PDCoV N escaped natural immunity by interacting with Grb2.
Asunto(s)
Proteína Adaptadora GRB2 , Proteínas de la Nucleocápside , Replicación Viral , Animales , Porcinos , Proteína Adaptadora GRB2/metabolismo , Proteína Adaptadora GRB2/genética , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/genética , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/metabolismo , Deltacoronavirus/metabolismo , Deltacoronavirus/genética , Sistema de Señalización de MAP Quinasas , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/metabolismo , Humanos , Transducción de Señal , Línea Celular , Quinasas raf/metabolismo , Quinasas raf/genética , Células HEK293RESUMEN
This study aims to develop an effective bivalent subunit vaccine that is promising to prevent both porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV). The receptor-binding domains (RBDs) of PDCoV and PEDV were fused and cloned into the eukaryotic expression vector pCDNA3.1(+). The fusion protein PDCoV-RBD-PEDV-RBD (pdRBD-peRBD) was expressed by the ExpiCHOTM expression system and purified. Mice were immunized with the fusion protein at three different doses (10, 20, and 30 µg). The humoral immune response and cellular immune response induced by the fusion protein were evaluated by ELISA and flow cytometry. The neutralization titers of the serum of immunized mice against PDCoV and PEDV were determined by the microneutralization test. The results showed that high levels of IgG antibodies were induced in the three different dose groups after booster immunization, and there was no significant difference in the antibody level between different dose groups, indicating that the immunization dose of 10 µg could achieve the fine immune effect. The results of flow cytometry showed that the immunization groups demonstrated increased proportion of CD3+CD4+ T cells and decreased proportion of CD3+CD8+ T cells, which was consistent with the expectation about the humoral immune response induced by the subunit vaccine. At the same time, the levels of interleukin (IL)-2, IL-4, and interferon (IFN)-γ in the serum were determined. The results showed that the fusion protein induced both humoral immune effect and cellular immune response. The results of the neutralization test showed that the antibody induced by 10 µg fusion protein neutralized both PDCoV and PEDV in vitro, with the titers of 1:179.25 and 1:141.21, respectively. The above results suggested that the pdRBD-peRBD could induce a high level of humoral immune response at a dose of 10 µg, and the induced antibody could neutralize both PDCoV and PEDV. Therefore, the fusion protein pdRBD-peRBD is expected to be an effective subunit vaccine that can simultaneously prevent PDCoV and PEDV.
Asunto(s)
Anticuerpos Antivirales , Infecciones por Coronavirus , Virus de la Diarrea Epidémica Porcina , Proteínas Recombinantes de Fusión , Vacunas Virales , Animales , Virus de la Diarrea Epidémica Porcina/inmunología , Virus de la Diarrea Epidémica Porcina/genética , Ratones , Porcinos , Vacunas Virales/inmunología , Vacunas Virales/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Deltacoronavirus/inmunología , Deltacoronavirus/genética , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/inmunología , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/genética , Ratones Endogámicos BALB C , Femenino , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Dominios Proteicos , Inmunogenicidad Vacunal , Inmunidad HumoralRESUMEN
This study evaluated the use of endemic enteric coronaviruses polymerase chain reaction (PCR)-negative testing results as an alternative approach to detect the emergence of animal health threats with similar clinical diseases presentation. This retrospective study, conducted in the United States, used PCR-negative testing results from porcine samples tested at six veterinary diagnostic laboratories. As a proof of concept, the database was first searched for transmissible gastroenteritis virus (TGEV) negative submissions between January 1st, 2010, through April 29th, 2013, when the first porcine epidemic diarrhea virus (PEDV) case was diagnosed. Secondly, TGEV- and PEDV-negative submissions were used to detect the porcine delta coronavirus (PDCoV) emergence in 2014. Lastly, encountered best detection algorithms were implemented to prospectively monitor the 2023 enteric coronavirus-negative submissions. Time series (weekly TGEV-negative counts) and Seasonal Autoregressive-Integrated Moving-Average (SARIMA) were used to control for outliers, trends, and seasonality. The SARIMA's fitted and residuals were then subjected to anomaly detection algorithms (EARS, EWMA, CUSUM, Farrington) to identify alarms, defined as weeks of higher TGEV-negativity than what was predicted by models preceding the PEDV emergence. The best-performing detection algorithms had the lowest false alarms (number of alarms detected during the baseline) and highest time to detect (number of weeks between the first alarm and PEDV emergence). The best-performing detection algorithms were CUSUM, EWMA, and Farrington flexible using SARIMA fitted values, having a lower false alarm rate and identified alarms 4 to 17 weeks before PEDV and PDCoV emergences. No alarms were identified in the 2023 enteric negative testing results. The negative-based monitoring system functioned in the case of PEDV propagating epidemic and in the presence of a concurrent propagating epidemic with the PDCoV emergence. It demonstrated its applicability as an additional tool for diagnostic data monitoring of emergent pathogens having similar clinical disease as the monitored endemic pathogens.
Asunto(s)
Infecciones por Coronavirus , Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Virus de la Gastroenteritis Transmisible , Animales , Porcinos , Virus de la Gastroenteritis Transmisible/genética , Virus de la Gastroenteritis Transmisible/aislamiento & purificación , Virus de la Diarrea Epidémica Porcina/aislamiento & purificación , Virus de la Diarrea Epidémica Porcina/genética , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/epidemiología , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/diagnóstico , Estudios Retrospectivos , Gastroenteritis Porcina Transmisible/diagnóstico , Gastroenteritis Porcina Transmisible/virología , Gastroenteritis Porcina Transmisible/epidemiología , Reacción en Cadena de la Polimerasa/métodos , Deltacoronavirus/genética , Deltacoronavirus/aislamiento & purificación , Estados Unidos/epidemiologíaRESUMEN
Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, is a serious threat to piglets and has zoonotic potential. Here, we aimed to further explore the role of aminopeptidase N (APN) as a receptor for PDCoV and test the inhibitory effect of a chimeric APN protein strategy on PDCoV infection. PK-15 cells and LLC-PK1 cells expressing chimeric APN were selected and infected with PDCoV. Viral replication was significantly decreased in these chimeric APN cells compared with that in control group cells. To further characterize the effect of the chimeric APN strategy on PDCoV infection in vitro, primary intestinal epithelial cells isolated from chimeric APN pigs were inoculated with PDCoV. Viral challenge of these cells led to decreased PDCoV infection. More importantly, virally challenged chimeric APN neonatal piglets displayed reduced viral load, significantly fewer microscopic lesions in the intestinal tissue, and no diarrhea. Taken together, these findings deepen our understanding of the mechanism of PDCoV infection and provide a valuable model for the production of disease-resistant animals. IMPORTANCE: Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhea in piglets and possesses the potential to infect humans. However, there are currently no effective measures for the prevention or control of PDCoV infection. Here, we have developed PK-15 cells, LLC-PK1 cells, and primary intestinal epithelial cells expressing chimeric APN, and viral challenge of these cells led to decreased PDCoV infection. Furthermore, virally challenged chimeric APN neonatal piglets displayed reduced viral load, significantly fewer microscopic lesions in the intestinal tissue, and no diarrhea. These data show that chimeric APN is a promising strategy to combat PDCoV infection.
Asunto(s)
Animales Recién Nacidos , Antígenos CD13 , Infecciones por Coronavirus , Deltacoronavirus , Enfermedades de los Porcinos , Replicación Viral , Animales , Porcinos , Antígenos CD13/genética , Antígenos CD13/metabolismo , Enfermedades de los Porcinos/virología , Deltacoronavirus/genética , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/prevención & control , Carga Viral , Edición Génica/métodos , Línea Celular , Células Epiteliales/virología , Diarrea/virologíaRESUMEN
Porcine deltacoronavirus (PDCoV) is an important enteric coronavirus that has caused enormous economic losses in the pig industry worldwide. However, no commercial vaccine is currently available. Therefore, developing a safe and efficacious live-attenuated vaccine candidate is urgently needed. In this study, the PDCoV strain CH/XJYN/2016 was continuously passaged in LLC-PK cells until passage 240, and the virus growth kinetics in cell culture, pathogenicity in neonatal piglets, transcriptome differences after LLC-PK infection, changes in the functional characteristics of the spike (S) protein in the high- and low-passage strains, genetic variation of the virus genome, resistance to pepsin and acid, and protective effects of this strain when used as a live-attenuated vaccine were examined. The results of animal experiments demonstrated that the virulent PDCoV strain CH/XJYN/2016 was completely attenuated and not pathogenic in piglets following serial cell passage. Genome sequence analysis showed that amino acid mutations in nonstructural proteins were mainly concentrated in Nsp3, structural protein mutations were mainly concentrated in the S protein, and the N, M, and E genes were conserved. Transcriptome comparison revealed that compared with negative control cells, P10-infected LLC-PK cells had the most differentially expressed genes (DEGs), while P0 and P240 had the least number of DEGs. Analysis of trypsin dependence and related structural differences revealed that the P10 S protein interacted more strongly with trypsin and that the P120 S protein interacted more strongly with the APN receptor. Moreover, the infectivity of P240 was not affected by pepsin but was significantly decreased after exposure to low pH. Furthermore, the P240-based live-attenuated vaccine provided complete protection to piglets against the challenge of virulent PDCoV. In conclusion, we showed that a PDCoV strain was completely attenuated through serial passaging in vitro. These results provide insights into the potential molecular mechanisms of PDCoV attenuation and the development of a promising live-attenuated PDCoV vaccine.IMPORTANCEPorcine deltacoronavirus (PDCoV) is one of the most important enteropathogenic pathogens that cause diarrhea in pigs of various ages, especially in suckling piglets, and causes enormous economic losses in the global commercial pork industry. There are currently no effective measures to prevent and control PDCoV. As reported in previous porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus studies, inactivated vaccines usually elicit less robust protective immune responses than live-attenuated vaccines in native sows. Therefore, identifying potential attenuation mechanisms, gene evolution, pathogenicity differences during PDCoV passaging, and immunogenicity as live-attenuated vaccines is important for elucidating the mechanism of attenuation and developing safe and effective vaccines for virulent PDCoV strains. In this study, we demonstrated that the virulence of the PDCoV strain CH/XJYN/2016 was completely attenuated following serial cell passaging in vitro, and changes in the biological characteristics and protection efficacy of the strain were evaluated. Our results help elucidate the mechanism of PDCoV attenuation and support the development of appropriate designs for the study of live PDCoV vaccines.
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Infecciones por Coronavirus , Deltacoronavirus , Genoma Viral , Pase Seriado , Enfermedades de los Porcinos , Vacunas Atenuadas , Animales , Porcinos , Deltacoronavirus/genética , Deltacoronavirus/patogenicidad , Vacunas Atenuadas/inmunología , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/veterinaria , Enfermedades de los Porcinos/virología , Virulencia , Vacunas Virales/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Línea Celular , MutaciónRESUMEN
Porcine deltacoronavirus (PDCoV) is an emergent enteric coronavirus, primarily inducing diarrhea in swine, particularly in nursing piglets, with the additional potential for zoonotic transmission to humans. Despite the significant impact of PDCoV on swine populations, its pathogenic mechanisms remain incompletely understood. Complement component 3 (C3) plays a pivotal role in the prevention of viral infections, however, there are no reports concerning the influence of C3 on the proliferation of PDCoV. In this study, we initially demonstrated that PDCoV is capable of activating the C3 and eliciting inflammatory responses. The overexpression of C3 significantly suppressed PDCoV replication, while inhibition of C3 expression facilitated PDCoV replication. We discovered that nonstructural proteins Nsp7, Nsp14, and M, considerably stimulated C3 expression, particularly Nsp14, through activation of the p38-MAPK-C/EBP-ß pathway. The N7-MTase constitutes a significant functional domain of the non-structural protein Nsp14, which is more obvious to upregulate C3. Furthermore, functional mutants of the N7-MTase domain suggested that the D44 and T135 of N7-Mtase constituted a pivotal amino acid site to promote C3 expression. This provides fresh insights into comprehending how the virus manipulates the host immune response and suggests potential antiviral strategies against PDCoV.
Asunto(s)
Complemento C3 , Deltacoronavirus , Proteínas no Estructurales Virales , Replicación Viral , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , Complemento C3/genética , Complemento C3/metabolismo , Complemento C3/inmunología , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Porcinos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Deltacoronavirus/genética , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/genética , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/inmunología , Sistema de Señalización de MAP Quinasas , Humanos , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genéticaRESUMEN
Introduction: Porcine deltacoronavirus (PDCoV) is a zoonotic pathogen with a global distribution, capable of infecting both pigs and humans. To mitigate the risk of cross-species transmission and potential outbreaks, it is crucial to characterize novel antiviral genes, particularly those from human hosts. Methods: This research used HIEC-6 to investigate PDCoV infection. HIEC-6 cells were infected with PDCoV. Samples were collected 48 h postinfection for proteomic analysis. Results: We discovered differential expression of MRPS6 gene at 48 h postinfection with PDCoV in HIEC-6 cells. The gene expression initially increased but then decreased. To further explore the role of MRPS6 in PDCoV infection, we conducted experiments involving the overexpression and knockdown of this gene in HIEC-6 and Caco2 cells, respectively. Our findings revealed that overexpression of MRPS6 significantly inhibited PDCoV infection in HIEC-6 cells, while knockdown of MRPS6 in Caco2 cells led to a significant increase of virus titer. Furthermore, we investigated the correlation between PDCoV infection and the expression of MRPS6. Subsequent investigations demonstrated that MRPS6 exerted an augmentative effect on the production of IFN-ß through interferon pathway activation, consequently impeding the progression of PDCoV infection in cellular systems. In conclusion, this study utilized proteomic analysis to investigate the differential protein expression in PDCoV-infected HIEC-6 cells, providing evidence for the first time that the MRPS6 gene plays a restrictive role in PDCoV virus infection. Discussion: Our findings initially provide the validation of MRPS6 as an upstream component of IFN-ß pathway, in the promotion of IRF3, IRF7, STAT1, STAT2 and IFN-ß production of HIEC-6 via dual-activation from interferon pathway.
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Deltacoronavirus , Humanos , Animales , Porcinos , Deltacoronavirus/fisiología , Deltacoronavirus/genética , Células CACO-2 , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/inmunología , Línea Celular , Interacciones Huésped-Patógeno/inmunología , Proteómica/métodos , Transducción de Señal , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunologíaRESUMEN
Porcine deltacoronavirus (PDCoV) is an enteropathogenic coronavirus that has been reported to use various strategies to counter the host antiviral innate immune response. The cGAS-STING signalling pathway plays an important role in antiviral innate immunity. However, it remains unclear whether PDCoV achieves immune evasion by regulating the cGAS-STING pathway. Here, we demonstrated that the nonstructural protein 2 (nsp2) encoded by PDCoV inhibits cGAS-STING-mediated type I and III interferon (IFN) responses via the regulation of porcine STING (pSTING) stability. Mechanistically, ectopically expressed PDCoV nsp2 was found to interact with the N-terminal region of pSTING. Consequently, pSTING was degraded through K48-linked ubiquitination and the proteasomal pathway, leading to the disruption of cGAS-STING signalling. Furthermore, K150 and K236 of pSTING were identified as crucial residues for nsp2-mediated ubiquitination and degradation. In summary, our findings provide a basis for elucidating the immune evasion mechanism of PDCoV and will contribute to the development of targets for anti-coronavirus drugs.
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Deltacoronavirus , Proteínas no Estructurales Virales , Animales , Porcinos , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Deltacoronavirus/genética , Deltacoronavirus/fisiología , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/metabolismo , Interferón Tipo I/metabolismo , Interferón Tipo I/genética , Inmunidad Innata , Células HEK293 , Evasión Inmune , UbiquitinaciónRESUMEN
Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/µL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2 % (11/5483), 8.82 % (485/5483), 1.22 % (67/5483), and 4.94 % (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39 %, 0.11 %, 0.01 %, and 0.03 %, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi.
Asunto(s)
Virus de la Diarrea Epidémica Porcina , Reacción en Cadena en Tiempo Real de la Polimerasa , Rotavirus , Sensibilidad y Especificidad , Enfermedades de los Porcinos , Virus de la Gastroenteritis Transmisible , Animales , Porcinos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Virus de la Gastroenteritis Transmisible/genética , Virus de la Gastroenteritis Transmisible/aislamiento & purificación , Virus de la Diarrea Epidémica Porcina/genética , Virus de la Diarrea Epidémica Porcina/aislamiento & purificación , Virus de la Diarrea Epidémica Porcina/clasificación , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/diagnóstico , Rotavirus/genética , Rotavirus/aislamiento & purificación , Rotavirus/clasificación , Gastroenteritis Porcina Transmisible/diagnóstico , Gastroenteritis Porcina Transmisible/virología , Deltacoronavirus/genética , Deltacoronavirus/aislamiento & purificación , Diarrea/virología , Diarrea/veterinaria , Diarrea/diagnóstico , Coronavirus/genética , Coronavirus/aislamiento & purificación , Coronavirus/clasificación , Heces/virología , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virologíaRESUMEN
Porcine Deltacoronavirus (PDCoV) is a newly identified coronavirus that causes severe intestinal lesions in piglets. However, the understanding of how PDCoV interacts with human hosts is limited. In this study, we aimed to investigate the interactions between PDCoV and human intestinal cells (HIEC-6) by analyzing the transcriptome at different time points post-infection (12 h, 24 h, 48 h). Differential gene analysis revealed a total of 3560, 5193, and 4147 differentially expressed genes (DEGs) at 12 h, 24 h, and 48 h, respectively. The common genes among the DEGs at all three time points were enriched in biological processes related to cytokine production, extracellular matrix, and cytokine activity. KEGG pathway analysis showed enrichment of genes involved in the p53 signaling pathway, PI3K-Akt signaling pathway, and TNF signaling pathway. Further analysis of highly expressed genes among the DEGs identified significant changes in the expression levels of BUB1, DDIT4, ATF3, GBP2, and IRF1. Comparison of transcriptome data at 24 h with other time points revealed 298 DEGs out of a total of 6276 genes. KEGG analysis of these DEGs showed significant enrichment of pathways related to viral infection, specifically the PI3K-Akt and P38 MAPK pathways. Furthermore, the genes EFNA1 and KITLG, which are associated with viral infection, were found in both enriched pathways, suggesting their potential as therapeutic or preventive targets for PDCoV infection. The enhancement of PDCoV infection in HIEC-6 was observed upon inhibition of the PI3K-Akt and P38 MAPK signaling pathways using sophoridine. Overall, these findings contribute to our understanding of the molecular mechanisms underlying PDCoV infection in HIEC-6 cells and provide insights for developing preventive and therapeutic strategies against PDCoV infection.
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Perfilación de la Expresión Génica , Transducción de Señal , Transcriptoma , Animales , Humanos , Línea Celular , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/genética , Deltacoronavirus/genética , Interacciones Huésped-Patógeno/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Porcinos , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/genéticaRESUMEN
Coronaviruses have been identified as pathogens of gastrointestinal and respiratory diseases in humans and various animal species. In recent years, the global spread of new coronaviruses has had profound influences for global public health and economies worldwide. As highly pathogenic zoonotic viruses, coronaviruses have become the focus of current research. Porcine Deltacoronavirus (PDCoV), an enterovirus belonging to the family of coronaviruses, has emerged on a global scale in the past decade and significantly influenced the swine industry. Moreover, PDCoV infects not only pigs but also other species, including humans, chickens and cattles, exhibiting a broad host tropism. This emphasizes the need for in-depth studies on coronaviruses to mitigate their potential threats. In this review, we provided a comprehensive summary of the current studies on PDCoV. We first reviewed the epidemiological investigations on the global prevalence and distribution of PDCoV. Then, we delved into the studies on the pathogenesis of PDCoV to understand the mechanisms how the virus impacts its hosts. Furthermore, we also presented some exploration studies on the immune evasion mechanisms of the virus to enhance the understanding of host-virus interactions. Despite current limitations in vaccine development for PDCoV, we highlighted the inhibitory effects observed with certain substances, which offers a potential direction for future research endeavors. In conclusion, this review summarized the scientific findings in epidemiology, pathogenesis, immune evasion mechanisms and vaccine development of PDCoV. The ongoing exploration of potential vaccine candidates and the insights gained from inhibitory substances have provided a solid foundation for future vaccine development to prevent and control diseases associated with PDCoV.
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Infecciones por Coronavirus , Deltacoronavirus , Evasión Inmune , Enfermedades de los Porcinos , Vacunas Virales , Animales , Porcinos , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/epidemiología , Deltacoronavirus/patogenicidad , Deltacoronavirus/inmunología , Deltacoronavirus/genética , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/epidemiología , Vacunas Virales/inmunología , Desarrollo de Vacunas , HumanosRESUMEN
Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that caused diarrhea and/or vomiting in neonatal piglets worldwide. Coronaviruses nucleocapsid (N) protein is the most conserved structural protein for viral replication and possesses good antigenicity. In this study, three monoclonal antibodies (mAbs), 3B4, 4D3, and 4E3 identified as subclass IgG2aκ were prepared using the lymphocytic hybridoma technology against PDCoV N protein. Furthermore, the B-cell epitope recognized by mAb 4D3 was mapped by dozens of overlapping truncated recombinant proteins based on the western blotting. The polypeptide 28QFRGNGVPLNSAIKPVE44 (EP-4D3) in the N-terminal of PDCoV N protein was identified as the minimal linear epitope for binding mAb 4D3. And the EP-4D3 epitope's amino acid sequence homology study revealed that PDCoV strains are substantially conserved, with the exception of the Alanine43 substitution Valine43 in the China lineage, the Early China lineage, and the Thailand, Vietnam, and Laos lineage. The epitope sequences shared high similarity (94.1%) with porcine coronavirus HKU15-155 (PorCoV HKU15), Asian leopard cats coronavirus (ALCCoV), sparrow coronavirus HKU17 (SpCoV HKU17), and sparrow deltacoronavirus. In contrast, the epitope sequences shared a very low homology (11.8 to 29.4%) with other porcine CoVs (PEDV, TGEV, PRCV, SADS-CoV, PHEV). Overall, the study will enrich the biological function of PDCoV N protein and provide foundational data for further development of diagnostic applications. KEY POINTS: ⢠Three monoclonal antibodies against PDCoV N protein were prepared. ⢠Discovery of a novel B-cell liner epitope (28QFRGNGVPLNSAIKPVE44) of PDCoV N protein. ⢠The epitope EP-4D3 was conserved among PDCoV strains.
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Infecciones por Coronavirus , Coronavirus , Enfermedades de los Porcinos , Porcinos , Animales , Deltacoronavirus/genética , Epítopos de Linfocito B/genética , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Coronavirus/genética , Infecciones por Coronavirus/veterinaria , Anticuerpos MonoclonalesRESUMEN
Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are the two most prevalent swine enteric coronaviruses worldwide. They commonly cause natural coinfections, which worsen as the disease progresses and cause increased mortality in piglets. To better understand the transcriptomic changes after PEDV and PDCoV coinfection, we compared LLC porcine kidney (LLC-PK) cells infected with PEDV and/or PDCoV and evaluated the differential expression of genes by transcriptomic analysis and real-time qPCR. The antiviral efficacy of interferon-stimulated gene 20 (ISG20) against PDCoV and PEDV infections was also assessed. Differentially expressed genes (DEGs) were detected in PEDV-, PDCoV-, and PEDV + PDCoV-infected cells at 6, 12, and 24 h post-infection (hpi), and at 24 hpi, the number of DEGs was the highest. Furthermore, changes in the expression of interferons, which are mainly related to apoptosis and activation of the host innate immune pathway, were found in the PEDV and PDCoV infection and coinfection groups. Additionally, 43 ISGs, including GBP2, IRF1, ISG20, and IFIT2, were upregulated during PEDV or PDCoV infection. Furthermore, we found that ISG20 significantly inhibited PEDV and PDCoV infection in LLC-PK cells. The transcriptomic profiles of cells coinfected with PEDV and PDCoV were reported, providing reference data for understanding the host response to PEDV and PDCoV coinfection.
Asunto(s)
Coinfección , Virus de la Diarrea Epidémica Porcina , Animales , Porcinos , Virus de la Diarrea Epidémica Porcina/genética , Coinfección/veterinaria , Deltacoronavirus/genética , Perfilación de la Expresión Génica , Interferones/genéticaRESUMEN
Porcine deltacoronavirus (PDCoV) is an enteric virus that was first identified in 2012. Although PDCoV has been detected worldwide, there is little information about its circulation in western China. In this study, fecal samples were collected from piglets with watery diarrhea in western China between 2015 and 2018 for the detection of PDCoV. The positive rate was 29.9%. A PDCoV strain (CHN/CQ/BN23/2016, BN23) was isolated and selected for further investigation. Phylogenetic analysis showed that this strain formed an individual cluster between the early Chinese lineage and the Chinese lineage. RDP4 and SimPlot analysis demonstrated that strain BN23 is a recombinant of Thailand/S5015L/2015 and CHN-AH-2004. The pathogenicity of BN23 was evaluated in 3-day-old piglets. Challenged piglets developed serious clinical signs and died at 3 days post-inoculation. Our data show that PDCoV is prevalent in western China and that strain BN23 is highly pathogenic to newborn piglets. Therefore, more attention should be paid to emerging PDCoV strains in western China.
Asunto(s)
Deltacoronavirus , Animales , China , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Deltacoronavirus/genética , Deltacoronavirus/aislamiento & purificación , Deltacoronavirus/patogenicidad , Diarrea/veterinaria , Genómica , Filogenia , Porcinos , Enfermedades de los Porcinos/virología , VirulenciaRESUMEN
Avian species often serve as transmission vectors and sources of recombination for viral infections due to their ability to travel vast distances and their gregarious behaviors. Recently a novel deltacoronavirus (DCoV) was identified in sparrows. Sparrow deltacoronavirus (SpDCoV), coupled with close contact between sparrows and swine carrying porcine deltacoronavirus (PDCoV) may facilitate recombination of DCoVs resulting in novel CoV variants. We hypothesized that the spike (S) protein or receptor-binding domain (RBD) from sparrow coronaviruses (SpCoVs) may enhance infection in poultry. We used recombinant chimeric viruses, which express S protein or the RBD of SpCoV (icPDCoV-SHKU17, and icPDCoV-RBDISU) on the genomic backbone of an infectious clone of PDCoV (icPDCoV). Chimeric viruses were utilized to infect chicken derived DF-1 cells, turkey poults, and embryonated chicken eggs (ECEs) to examine permissiveness, viral replication kinetics, pathogenesis and pathology. We demonstrated that DF-1 cells in addition to the positive control LLC-PK1 cells are susceptible to SpCoV spike- and RBD- recombinant chimeric virus infections. However, the replication of chimeric viruses in DF-1 cells, but not LLC-PK1 cells, was inefficient. Inoculated 8-day-old turkey poults appeared resistant to icPDCoV-, icPDCoV-SHKU17- and icPDCoV-RBDISU virus infections. In 5-day-old ECEs, significant mortality was observed in PDCoV inoculated eggs with less in the spike chimeras, while in 11-day-old ECEs there was no evidence of viral replication, suggesting that PDCoV is better adapted to cross species infection and differentiated ECE cells are not susceptible to PDCoV infection. Collectively, we demonstrate that the SpCoV chimeric viruses are not more infectious in turkeys, nor ECEs than wild type PDCoV. Therefore, understanding the cell and host factors that contribute to resistance to PDCoV and avian-swine chimeric virus infections may aid in the design of novel antiviral therapies against DCoVs.
Asunto(s)
Infecciones por Coronavirus , Gorriones , Enfermedades de los Porcinos , Animales , Pollos , Deltacoronavirus/genética , Aves de Corral , Glicoproteína de la Espiga del Coronavirus/genética , Porcinos , PavosRESUMEN
Porcine deltacoronavirus (PDCoV) can cause diarrhea and dehydration in newborn piglets. Here, we developed a double antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-ELISA) for detection of PDCoV by using a specific monoclonal antibody against the PDCoV N protein and an anti-PDCoV rabbit polyclonal antibody. Using DAS-ELISA, the detection limit of recombinant PDCoV N protein and virus titer were approximately 0.5 ng/mL and 103.0 TCID50/mL, respectively. A total of 59 intestinal and 205 fecal samples were screened for the presence of PDCoV by using DAS-ELISA and reverse transcriptase real-time PCR (RT-qPCR). The coincidence rate of the DAS-ELISA and RT-qPCR was 89.8%. DAS-ELISA had a sensitivity of 80.8% and specificity of 95.6%. More importantly, the DAS-ELISA could detect the antigen of PDCoV inactivated virus, and the viral antigen concentrations remained unchanged in the inactivated virus. These results suggest that DAS-ELISA could be used for antigen detection of clinical samples and inactivated vaccines. It is a novel method for detecting PDCoV infections and evaluating the PDCoV vaccine.
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Anticuerpos Antivirales/sangre , Antígenos Virales/sangre , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/veterinaria , Deltacoronavirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Enfermedades de los Porcinos/diagnóstico , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Deltacoronavirus/genética , Deltacoronavirus/aislamiento & purificación , Conejos , Porcinos , Enfermedades de los Porcinos/sangre , Enfermedades de los Porcinos/virologíaRESUMEN
Coronaviruses have caused three major epidemics since 2003, including the ongoing SARS-CoV-2 pandemic. In each case, the emergence of coronavirus in our species has been associated with zoonotic transmissions from animal reservoirs1,2, underscoring how prone such pathogens are to spill over and adapt to new species. Among the four recognized genera of the family Coronaviridae, human infections reported so far have been limited to alphacoronaviruses and betacoronaviruses3-5. Here we identify porcine deltacoronavirus strains in plasma samples of three Haitian children with acute undifferentiated febrile illness. Genomic and evolutionary analyses reveal that human infections were the result of at least two independent zoonoses of distinct viral lineages that acquired the same mutational signature in the genes encoding Nsp15 and the spike glycoprotein. In particular, structural analysis predicts that one of the changes in the spike S1 subunit, which contains the receptor-binding domain, may affect the flexibility of the protein and its binding to the host cell receptor. Our findings highlight the potential for evolutionary change and adaptation leading to human infections by coronaviruses outside of the previously recognized human-associated coronavirus groups, particularly in settings where there may be close human-animal contact.
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Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Deltacoronavirus/aislamiento & purificación , Porcinos/virología , Zoonosis Virales/epidemiología , Zoonosis Virales/virología , Secuencia de Aminoácidos , Animales , Teorema de Bayes , Niño , Chlorocebus aethiops , Secuencia Conservada , Infecciones por Coronavirus/sangre , Deltacoronavirus/clasificación , Deltacoronavirus/genética , Deltacoronavirus/patogenicidad , Femenino , Haití/epidemiología , Humanos , Masculino , Modelos Moleculares , Mutación , Filogenia , Células Vero , Zoonosis Virales/sangreRESUMEN
Circular RNAs (circRNAs) are a newly discovered class of noncoding RNAs (ncRNAs) present in various tissues and cells. However, the functions of most circRNAs have not been verified experimentally. Here, using deltacoronavirus as a model, differentially expressed circRNAs in cells with or without deltacoronavirus infection were analyzed by RNA sequencing to characterize the cellular responses to RNA virus infection. More than 57,000 circRNA candidates were detected, and seven significantly dysregulated circRNAs were quantitated by real-time reverse transcription-PCR. We discovered a previously unidentified circRNA derived from the TNFAIP3 gene, named circTNFAIP3, which is distributed and expressed widely in various tissues. RNA viruses, including deltacoronaviruses, rather than DNA viruses tend to activate the expression of endogenous circTNFAIP3. Overexpression of circTNFAIP3 promoted deltacoronavirus replication by reducing the apoptosis, while silencing of circTNFAIP3 inhibited deltacoronavirus replication by enhancing the apoptosis. In summary, our work provides useful circRNA-related information to facilitate investigation of the underlying mechanism of deltacoronavirus infection and identifies a novel circTNFAIP3 that promotes deltacoronavirus replication via regulating apoptosis. IMPORTANCE CircRNAs, a new class of ncRNAs, play important roles in cell growth, neural development, carcinogenesis, and anticarcinogenesis. Porcine deltacoronavirus is an emerging enteropathogenic coronavirus that causes diarrhea, but the role of host circRNAs in regulating its infection is unknown. Here, we performed expression profiling of circRNAs in mock- and deltacoronavirus- infected cells and identified the novel differentially expressed circular RNA circTNFAIP3. We demonstrate that circTNFAIP3 promotes deltacoronavirus replication by inhibiting apoptosis. Our findings first illustrate that circRNA can act as an apoptosis negative regulator during RNA virus infection and help to explore the underlying mechanism of deltacoronavirus infection.
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Deltacoronavirus/genética , Interacciones Microbiota-Huesped/genética , ARN Circular/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Replicación Viral/genética , Apoptosis , Línea Celular , Deltacoronavirus/fisiología , Perfilación de la Expresión Génica , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , ARN Circular/inmunología , Análisis de Secuencia de ARN , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Coronaviruses (CoVs) are widespread and highly diversified in wildlife and domestic mammals and can emerge as zoonotic or epizootic pathogens and consequently host shift from these reservoirs, highlighting the importance of veterinary surveillance. All genera can be found in mammals, with α and ß showing the highest frequency and diversification. The aims of this study were to review the literature for features of CoV surveillance in animals, to test widely used molecular protocols, and to identify the most effective one in terms of spectrum and sensitivity. We combined a literature review with analyses in silico and in vitro using viral strains and archive field samples. We found that most protocols defined as pan-coronavirus are strongly biased towards α- and ß-CoVs and show medium-low sensitivity. The best results were observed using our new protocol, showing LoD 100 PFU/mL for SARS-CoV-2, 50 TCID50/mL for CaCoV, 0.39 TCID50/mL for BoCoV, and 9 ± 1 log2 ×10-5 HA for IBV. The protocol successfully confirmed the positivity for a broad range of CoVs in 30/30 field samples. Our study points out that pan-CoV surveillance in mammals could be strongly improved in sensitivity and spectrum and propose the application of a new RT-PCR assay, which is able to detect CoVs from all four genera, with an optimal sensitivity for α-, ß-, and γ-.
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Alphacoronavirus/genética , Infecciones por Coronavirus/veterinaria , Deltacoronavirus/genética , Gammacoronavirus/genética , SARS-CoV-2/genética , Animales , Animales Salvajes/virología , Betacoronavirus/genética , COVID-19/veterinaria , Quirópteros/virología , Genoma Viral/genética , Humanos , Ganado/virología , Roedores/virologíaRESUMEN
Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.