RESUMEN

BACKGROUND: Senecavirus A (SVA) is the only member of the genus Senecavirus in the family Picornaviridae, and is one of the pathogens of porcine blistering disease. SVA has been reported in the United States, Canada, China, Thailand, and Colombia. METHODS: In this study, positive SVA infection was detected by RT-PCR in sick materials collected from pig farms of different sizes in Anhui Province. RESULTS: In this study, a virulent strain of SVA was successfully obtained by viral isolation on BHK21 cells and named SVA-CH-AHAU-1. Meanwhile, a simple, rapid and accurate nano-PCR method for the detection of SVA infection was established in this study, using the recombinant plasmid pClone-SVA-3D as a template. CONCLUSIONS: The complete genome of SVA-CH-AHAU-1 is 7286 bp, including a 5' non-coding region (UTR), an open reading frame (ORF) of 6546 nucleotides, encoding 2182 amino acids (aa), and a 3' UTR with Poly(A) features, and phylogenetic analysis showed that this isolate had the highest nucleotide homology (97.9 %) with the US isolate US-15-41901SD. In this study, the virulent strain SVA-CH-AHAU-1 was found to recombine in the ORF region with isolates SVA-CH-SDGT-2017 and SVA/Canada/ON/FMA-2015-0024 T2/2015. The complete genome has been submitted to GeneBank with the accession number OM654411. In addition, our results suggest that the established nano-PCR assay can be used as an economical, reliable and sensitive method for the field diagnosis of SVA method, especially in resource-limited areas.

RESUMEN

Viruses deploy sophisticated strategies to hijack the host's translation machinery to favor viral protein synthesis and counteract innate cellular defenses. However, little is known about the mechanisms by which Senecavirus A (SVA) controls the host's translation. Using a series of sophisticated molecular cell manipulation techniques, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as an essential host factor involved in translation control in SVA-infected cells. It was also determined that the SVA structural protein, VP3, binds to and relocalizes hnRNPA2B1, which interferes with the host's protein synthesis machinery to establish a cellular environment that facilitates viral propagation via a two-pronged strategy: first, hnRNPA2B1 serves as a potent internal ribosome entry site (IRES) trans-acting factor, which is selectively co-opted to promote viral IRES-driven translation by supporting the assembly of translation initiation complexes. Second, a strong repression of host cell translation occurs in the context of the VP3-hnRNPA2B1 interaction, resulting in attenuation of the interferons response. This is the first study to demonstrate the interaction between SVA VP3 and hnRNPA2B1, and to characterize their key roles in manipulating translation. This novel dual mechanism, which regulates selective mRNA translation and immune evasion of virus-infected cells, highlights the VP3-hnRNPA2B1 complex as a potential target for the development of modified antiviral or oncolytic reagents. IMPORTANCE: Viral reproduction is contingent on viral protein synthesis, which relies entirely on the host's translation machinery. As such, viruses often need to control the cellular translational apparatus to favor viral protein production and avoid host innate defenses. Senecavirus A (SVA) is an important virus, both as an emerging pathogen in the pork industry and as a potential oncolytic virus for neuroendocrine cancers. Here, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as a critical regulator of the translational landscape during SVA infection. This study supports a model whereby the VP3 protein of SVA efficiently subverts the host's protein synthesis machinery through its ability to bind to and relocalize hnRNPA2B1, not only selectively promoting viral internal ribosome entry site-driven translation but also resulting in global translation shutdown and immune evasion. Together, these data provide new insights into how the complex interactions between translation machinery, SVA, and innate immunity contribute to the pathogenicity of the SVA.

Asunto(s)

Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Inmunidad Innata , Sitios Internos de Entrada al Ribosoma , Picornaviridae , Biosíntesis de Proteínas , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Humanos , Picornaviridae/inmunología , Interacciones Huésped-Patógeno/inmunología , Células HEK293 , Replicación Viral , Evasión Inmune , Infecciones por Picornaviridae/inmunología , Infecciones por Picornaviridae/virología , Infecciones por Picornaviridae/metabolismo , Línea Celular

RESUMEN

Senecavirus A (SVA) is an emerging virus that poses a threat to swine herds worldwide. To date, the role of tripartite motif 5 (TRIM5) in the replication of viruses has not been evaluated. Here, TRIM5 was reported to inhibit SVA replication by promoting the type I interferon (IFN) antiviral response mediated by retinoic acid-inducible gene I (RIG-I). TRIM5 expression was significantly upregulated in SVA-infected cells, and TRIM5 overexpression inhibited viral replication and promoted IFN-α, IFN-ß, interleukin-1beta (IL-1ß), IL-6, and IL-18 expression. Conversely, interfering with the expression of TRIM5 had the opposite effect. Viral adsorption and entry assays showed that TRIM5 did not affect the adsorption of SVA but inhibited its entry. In addition, TRIM5 promoted the expression of RIG-I and RIG-I-mediated IFNs and proinflammatory cytokines, and this effect was also proven by inhibiting the expression of TRIM5. These findings expand the scope of knowledge on host factors inhibiting the replication of SVA and indicate that targeting TRIM5 may aid in the development of new agents against SVA.

Asunto(s)

Interferón Tipo I , Picornaviridae , Replicación Viral , Animales , Interferón Tipo I/metabolismo , Porcinos , Picornaviridae/fisiología , Picornaviridae/inmunología , Proteínas de Motivos Tripartitos/metabolismo , Proteínas de Motivos Tripartitos/genética , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología

RESUMEN

Senecavirus A (SVA) is a picornavirus that is endemic in swine, causing a vesicular disease clinically indistinguishable from other vesicular diseases, like foot-and-mouth disease. The widespread viral circulation, constant evolution, and economic losses caused to the swine industry emphasize the need for measures to control the agent. In this study, we evaluated the immunogenicity of a whole-virus-inactivated vaccine using a representative contemporary Brazilian SVA strain in Balb/ByJ mice. The animals were vaccinated with two doses by an intramuscular route. The humoral response induced by the vaccination was evaluated by an in-house ELISA assay for IgG detection. The cellular response was assessed by flow cytometry after in vitro SVA stimulation in splenocyte cultures from vaccinated and non-vaccinated groups. Protection against SVA was assessed in the experimental groups following an oral challenge with the homologous virus. The vaccination induced high levels of IgG antibodies and the proliferation of CD45R/B220+sIgM+, CD3e+CD69+, and CD3e+CD4+CD44+CD62L- cells. These results indicate the immunogenicity and safety of the vaccine formulation in a murine model and the induction of humoral and cellular response against SVA.

RESUMEN

Background and Aims: Hepatocellular carcinoma (HCC) is a highly aggressive tumor with limited treatment options and high mortality. Senecavirus A (SVA) has shown potential in selectively targeting tumors while sparing healthy tissues. This study aimed to investigate the effects of SVA on HCC cells in vitro and in vivo and to elucidate its mechanisms of action. Methods: The cell counting kit-8 assay and colony formation assay were conducted to examine cell proliferation. Flow cytometry and nuclear staining were employed to analyze cell cycle distribution and apoptosis occurrence. A subcutaneous tumor xenograft HCC mouse model was created in vivo using HepG2 cells, and Ki67 expression in the tumor tissues was assessed. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay and hematoxylin and eosin staining were employed to evaluate HCC apoptosis and the toxicity of SVA on mouse organs. Results: In vitro, SVA effectively suppressed the growth of tumor cells by inducing apoptosis and cell cycle arrest. However, it did not have a notable effect on normal hepatocytes (MIHA cells). In an in vivo setting, SVA effectively suppressed the growth of HCC in a mouse model. SVA treatment resulted in a significant decrease in Ki67 expression and an increase in apoptosis of tumor cells. No notable histopathological alterations were observed in the organs of mice during SVA administration. Conclusions: SVA inhibits the growth of HCC cells by inducing cell cycle arrest and apoptosis. It does not cause any noticeable toxicity to vital organs.

RESUMEN

Senecavirus A (SVA) is an emerging virus that causes the vesicular disease in pigs, clinically indistinguishable from other high consequence vesicular diseases. This virus belongs to the genus Senecavirus in the family Picornaviridae. Its genome is a positive-sense, single-stranded RNA, approximately 7,300 nt in length, with a 3' poly(A) tail but without 5'-end capped structure. SVA can efficiently propagate in different cells, including some non-pig-derived cell lines. A wild-type SVA was previously rescued from its cDNA clone using reverse genetics in our laboratory. In the present study, the BSR-T7/5 cell line was inoculated with the passage-5 SVA. At 12 h post-inoculation, SVA-infected and non-infected cells were independently collected for the analysis on comparative transcriptomics. The results totally showed 628 differentially expressed genes, including 565 upregulated and 63 downregulated ones, suggesting that SVA infection significantly stimulated the transcription initiation in cells. GO and KEGG enrichment analyses demonstrated that SVA exerted multiple effects on immunity-related pathways in cells. Furthermore, the RNA sequencing data were subjected to other in-depth analyses, such as the single-nucleotide polymorphism, transcription factors, and protein-protein interactions. The present study, along with our previous proteomics and metabolomics researches, provides a multi-omics insight into the interaction between SVA and its hosts.

RESUMEN

Senecavirus A (SVA) is a causative agent that can cause vesicular disease in swine, which causes a great threat to the swine husbandry in the world. Therefore, it is necessary to develop a vaccine that can effectively prevent the spread of SVA. In this study, we developed a 24-polymeric nano-scaffold using ß-annulus peptide from tomato bushy effect virus (TBSV) by coupling this antigen to SVA B cell epitope VP121-26 and VP2 proteins via linkers, respectively. The SVA-based nanoparticle protein of the VP1(B)-ß-VP2 was expressed and purified by low-cost prokaryotic system to prepare a SVA nanoparticle vaccine. The immunological protective effect of SVA nanoparticle vaccine was evaluated in mouse and swine models, respectively. The results suggested that both mice and swine could induce high levels SVA neutralizing antibodies and IgG antibodies after two doses immunization. In addition, the swine challenge protection experiment showed that the protection rate of immune SVA nanoparticle vaccine and SVA inactivated vaccine both were 80 %, while the negative control had no protection effect. It demonstrated that SVA nanoparticle vaccine effectively prevented SVA infection in swine. In summary, the preparation of SVA vaccine by using ß-annulus peptide is a promising candidate vaccine for prevent SVA transmission, and provides a new idea for the development of novel SVA vaccines.

Asunto(s)

Anticuerpos Neutralizantes , Anticuerpos Antivirales , Nanovacunas , Infecciones por Picornaviridae , Picornaviridae , Enfermedades de los Porcinos , Vacunas Virales , Animales , Femenino , Ratones , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Proteínas de la Cápside/inmunología , Ratones Endogámicos BALB C , Nanovacunas/administración & dosificación , Nanovacunas/inmunología , Picornaviridae/inmunología , Infecciones por Picornaviridae/veterinaria , Infecciones por Picornaviridae/prevención & control , Infecciones por Picornaviridae/inmunología , Infecciones por Picornaviridae/virología , Porcinos , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Proteínas Estructurales Virales/inmunología , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación

RESUMEN

Senecavirus A (SVA), a picornavirus, causes vesicular diseases and epidemic transient neonatal losses in swine, resulting in a multifaceted economic impact on the swine industry. SVA counteracts host antiviral response through multiple strategies facilitatng viral infection and transmission. However, the mechanism of how SVA modulates interferon (IFN) response remains elusive. Here, we demonstrate that SVA 3C protease (3Cpro) blocks the transduction of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway to antagonize type I IFN response. Mechanistically, 3Cpro selectively cleaves and degrades STAT1 and STAT2 while does not target JAK1, JAK2, and IRF9, through its protease activity. Notably, SVA 3Cpro cleaves human and porcine STAT1 on a Leucine (L)-Aspartic acid (D) motif, specifically L693/D694. In the case of STAT2, two cleavage sites were identified: glutamine (Q) 707 was identified in both human and porcine, while the second cleavage pattern differed, with residues 754-757 (Valine-Leucine-Glutamine-Serine motifs) in human STAT2 and Q758 in porcine STAT2. These cleavage patterns by SVA 3Cpro partially differ from previously reported classical motifs recognized by other picornaviral 3Cpro, highlighting the distinct characteristics of SVA 3Cpro. Together, these results reveal a mechanism by which SVA 3Cpro antagonizes IFN-induced antiviral response but also expands our knowledge about the substrate recognition patterns for picornaviral 3Cpro.IMPORTANCESenecavirus A (SVA), the only member in the Senecavirus genus within the Picornaviridae family, causes vesicular diseases in pigs that are clinically indistinguishable from foot-and-mouth disease (FMD), a highly contagious viral disease listed by the World Organization for Animal Health (WOAH). Interferon (IFN)-mediated antiviral response plays a pivotal role in restricting and controlling viral infection. Picornaviruses evolved numerous strategies to antagonize host antiviral response. However, how SVA modulates the JAK-STAT signaling pathway, influencing the type I IFN response, remains elusive. Here, we identify that 3Cpro, a protease of SVA, functions as an antagonist for the IFN response. 3Cpro utilizes its protease activity to cleave STAT1 and STAT2, thereby diminishing the host IFN response to promote SVA infection. Our findings underscore the significance of 3Cpro as a key virulence factor in the antagonism of the type I signaling pathway during SVA infection.

Asunto(s)

Cisteína Endopeptidasas , Infecciones por Picornaviridae , Picornaviridae , Factor de Transcripción STAT1 , Factor de Transcripción STAT2 , Transducción de Señal , Proteínas Virales , Animales , Porcinos , Factor de Transcripción STAT2/metabolismo , Humanos , Infecciones por Picornaviridae/virología , Infecciones por Picornaviridae/inmunología , Infecciones por Picornaviridae/metabolismo , Factor de Transcripción STAT1/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas Virales/metabolismo , Proteasas Virales 3C , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Células HEK293 , Interferón Tipo I/antagonistas & inhibidores , Interferón Tipo I/metabolismo , Línea Celular , Quinasas Janus/metabolismo , Quinasas Janus/antagonistas & inhibidores

RESUMEN

BACKGROUND: Senecavirus A (SV-A) is an RNA virus that belongs to the genus Senecavirus within the family Picornaviridae. This study aimed to analyze factors that can influence the molecular diagnosis of Senecavirus A, such as oligonucleotides, RNA extraction methods, and RT-qPCR kits. METHODS: Samples from suspected cases of vesicular disease in Brazilian pigs were analyzed for foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. All tested negative for these diseases but positive for SV-A. RT-qPCR tests were used, comparing different reagent kits and RNA extraction methods. Sensitivity and repeatability were evaluated, demonstrating efficacy in detecting SV-A in clinical samples. RESULTS: In RNA extraction, significant reduction in Cq values was observed with initial dilutions, particularly with larger supernatant volumes. Trizol and Maxwell showed greater sensitivity in automated equipment protocols, though results varied in tissue tests. RT-qPCR kit comparison revealed differences in amplification using viral RNA but minimal differences with plasmid DNA. Sensitivity among methods was comparable, with slight variations in non-amplified samples. Repeatability tests showed consistent results among RT-qPCRs, demonstrating similarity between methods despite minor discrepancies in Cq values. CONCLUSIONS: Trizol, silica columns, and semi-automated extraction were compared, as well as different RT-qPCR kits. The study found significant variations that could impact the final diagnosis.

Asunto(s)

Infecciones por Picornaviridae , Picornaviridae , ARN Viral , Enfermedades de los Porcinos , Animales , Picornaviridae/genética , Picornaviridae/aislamiento & purificación , Porcinos , Infecciones por Picornaviridae/diagnóstico , Infecciones por Picornaviridae/veterinaria , Infecciones por Picornaviridae/virología , ARN Viral/genética , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/diagnóstico , Sensibilidad y Especificidad , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enfermedad Vesicular Porcina/diagnóstico , Enfermedad Vesicular Porcina/virología , Fiebre Aftosa/diagnóstico , Fiebre Aftosa/virología , Brasil , Reproducibilidad de los Resultados

RESUMEN

BACKGROUND: Senecavirus A (SVA), identified in 2002, is known to cause porcine idiopathic vesicular disease (PIVD), which presents with symptoms resembling other vesicular diseases. This similarity complicates field diagnosis. Conventional molecular diagnostic techniques are limited by their cost, sensitivity, and requirement for complicated instrumentation. Therefore, developing an effective and accurate diagnostic method is crucial for timely identification and isolation of affected pigs, thereby preventing further disease spread. METHODS: In this study, we developed a highly-specific and ultra-sensitive SVA detection method powered by CRISPR/Cas12a. To enhance the availability in laboratories with varied equipment conditions, microplate reader and ultraviolet light transilluminator were introduced. Moreover, PCR amplification has also been incorporated into this method to improve sensitivity. The specificity and sensitivity of this method were determined following the preparation of the recombinant Cas12a protein and optimization of the CRISPR/Cas12a-based trans-cleavage system. RESULTS: The method demonstrated no cross-reactivity with ten kinds of viruses of swine. The minimum template concentration required to activate substantial trans-cleavage activity was determined to be 106 copies/µL of SVA templates. However, when PCR amplification was incorporated, the method achieved a detection limit of one copy of SVA templates per reaction. It also exhibited 100% accuracy in simulated sample testing. The complete testing process does not exceed three hours. CONCLUSIONS: Importantly, this method utilizes standard laboratory equipment, making it accessible for use in resource-limited settings and facilitating widespread and ultra-sensitive screening during epidemics. Overall, the development of this method not only broadens the array of tools available for detecting SVA but also holds significant promise for controlling the spread of PIVD.

Asunto(s)

Sistemas CRISPR-Cas , Picornaviridae , Sensibilidad y Especificidad , Enfermedades de los Porcinos , Animales , Porcinos , Picornaviridae/aislamiento & purificación , Picornaviridae/genética , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/diagnóstico , Infecciones por Picornaviridae/veterinaria , Infecciones por Picornaviridae/diagnóstico , Infecciones por Picornaviridae/virología , Reacción en Cadena de la Polimerasa/veterinaria , Reacción en Cadena de la Polimerasa/métodos , Proteínas Asociadas a CRISPR/genética

RESUMEN

Senecavirus A (SVA) is an important emerging swine pathogen that causes vesicular lesions in swine and acute death in newborn piglets. VP2 plays a significant role in the production of antibodies, which can be used in development of diagnostic tools and vaccines. Herein, the aim of the current study was to identify B-cell epitopes (BCEs) of SVA for generation of epitope-based SVA marker vaccine. Three monoclonal antibodies (mAbs), named 2E4, 1B8, and 2C7, against the SVA VP2 protein were obtained, and two novel linear BCEs, 177SLGTYYR183 and 266SPYFNGL272, were identified by peptide scanning. The epitope 177SLGTYYR183 was recognized by the mAb 1B8 and was fully exposed on the VP2 surface, and alanine scanning analysis revealed that it contained a high continuity of key amino acids. Importantly, we confirmed that 177SLGTYYR183 locates on "the puff" region within the VP2 EF loop, and contains three key amino acid residues involved in receptor binding. Moreover, a single mutation, Y182A, blocked the interaction of the mutant virus with the mAb 1B8, indicating that this mutation is the pivotal point for antibody recognition. In summary, the BCEs that identified in this study could be used to develop diagnostic tools and an epitope-based SVA marker vaccine.

RESUMEN

Porcine idiopathic vesicular disease (PIVD), one of several clinically indistinguishable vesicular diseases of pigs, is caused by the emerging pathogen Senecavirus A (SVA). Despite the widespread prevalence of porcine SVA infection, no effective commercial vaccines for PIVD prevention and control are available, due to high costs associated with vaccine testing in pigs, considerable SVA diversity, and SVA rapid evolution. In this study, SVA CH/JL/2022 (OP562896), a novel mutant SVA strain derived from an isolate obtained from a pig farm in Jilin Province, China, was inactivated then combined with four adjuvants, MONTANIDETM GEL02 PR (GEL 02), MONTANIDETM ISA 201 VG (ISA 201), MONTANIDETM IMG 1313 VG N (IMS1313), or Rehydragel LV (LV). The resulting inactivated SVA CH/JL/2022 vaccines were assessed for efficacy in mice and found to induce robust in vivo lymphocyte proliferation responses and strong IgG1, IgG2a, and neutralizing antibody responses with IgG2a/IgG1 ratios of <1. Furthermore, all vaccinated groups exhibited significantly higher levels of serum cytokines IL-2, IL-4, IL-6, and IFN as compared to unvaccinated mice. These results indicate that all vaccines elicited both Th1 and Th2 responses, with Th2 responses predominating. Moreover, vaccinated mice exhibited enhanced resistance to SVA infection, as evidenced by reduced viral RNA levels and SVA infection-induced histopathological changes. Collectively, our results demonstrate that the SVA-GEL vaccine induced more robust immunological responses in mice than did the other three vaccines, thus highlighting the potential of SVA-GEL to serve an effective tool for preventing and controlling SVA infection.

RESUMEN

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. This virus possesses a positive-sense, single-stranded RNA genome, approximately 7200 nt in length, composed of a single 5' untranslated region, encoding region and 3' untranslated region. In this study, a recombinant SVA tagged with enhanced green fluorescent protein (eGFP) sequence, rSVA-eGFP, was rescued from its cDNA clone using reverse genetics. The passage-5 (P5) rSVA-eGFP was totally subjected to 55 rounds of consecutive fluorescent plaque-to-fluorescent plaque (FP-FP) transfers, and one extra common passaging in vitro. The P61 viral stock was analyzed by next-generation sequencing. The result showed ten single-nucleotide mutations (SNMs) in the rSVA-eGFP genome, including nine transitions and only one transversion. The P61 progeny still showed a complete eGFP sequence, indicating no occurrence of copy-choice recombination within the eGFP region during serial FP-FP transfers. In other words, this progeny was genetically deficient in the recombination of eGFP sequence (RES), namely, an RES-deficient strain. Out of ten SNMs, three were missense mutations, leading to single-amino acid mutations (SAAMs): F15V in L protein, A74T in VP2, and E53R in 3D protein. The E53R was predicted to be spatially adjacent to the RNA channel of 3D protein, perhaps involved in the emergence of RES-deficient strain. In conclusion, this study uncovered a global landscape of rSVA-eGFP genome after serial FP-FP transfers, and moreover shed light on a putative SAAM possibly related to the RES-deficient mechanism.

Asunto(s)

Genoma Viral , Proteínas Fluorescentes Verdes , Picornaviridae , Proteínas Fluorescentes Verdes/genética , Genoma Viral/genética , Picornaviridae/genética , Genética Inversa/métodos , ARN Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Recombinación Genética , Ensayo de Placa Viral

RESUMEN

Senecavirus A (SVA) is a newly identified picornavirus associated with swine vesicular disease and neonatal mortality. The development of an SVA incorporating an exogenous reporter gene provides a powerful tool for viral research. In this study, we successfully constructed a recombinant SVA expressing Gaussia Luciferase (Gluc), termed rSVA-Gluc. The growth kinetics of rSVA-Gluc in BHK-21 cells were found to be comparable to those of the parental virus, and Gluc activity paralleled the virus growth curve. Genetic analysis revealed stable inheritance of the inserted reporter protein genes for at least six generations. We evaluated the utility of rSVA-Gluc in antiviral drug screening, and the results highlighted its potential as an effective tool for such purposes against SVA. DATA AVAILABILITY STATEMENT: The data that support the findings of this study are available on request from the corresponding author.

Asunto(s)

Antivirales , Genes Reporteros , Luciferasas , Picornaviridae , Picornaviridae/genética , Picornaviridae/efectos de los fármacos , Animales , Antivirales/farmacología , Línea Celular , Luciferasas/genética , Luciferasas/metabolismo , Cricetinae , Evaluación Preclínica de Medicamentos/métodos

RESUMEN

Senecavirus A (SVA) causes outbreaks of vesicular disease in pigs, which imposes a considerable economic burden on the pork industry. As current SVA prevention measures are ineffective, new strategies for controlling SVA are urgently needed. Circular (circ)RNA is a newly characterized class of widely expressed, endogenous regulatory RNAs, which have been implicated in viral infection; however, whether circRNAs regulate SVA infection remains unknown. To investigate the influence of circRNAs on SVA infection in porcine kidney 15 (PK-15) cells, RNA sequencing technology was used to analyze the circRNA expression profiles of SVA-infected and uninfected PK-15 cells, the interactions between circRNAs, miRNAs, and mRNAs potentially implicated in SVA infection were predicted using bioinformatics tools. The prediction accuracy was verified using quantitative real-time (qRT)-PCR, Western blotting, as well as dual-luciferase reporter and RNA pull-down assays. The results showed that 67 circRNAs were differentially expressed as a result of SVA infection. We found that circ_8521 was significantly upregulated in SVA-infected PK-15 cells and promoted SVA infection. circ_8521 interacted with miR-324. miR-324 bound to LC3A mRNA which inhibited the expression of LC3A. Knockdown of LC3A inhibited SVA infection. However, circ_8521 promoted the expression of LC3A by binding to miR-324, thereby promoting SVA infection. We demonstrated that circ_8521 functioned as an endogenous miR-324 sponge to sequester miR-324, which promoted LC3A expression and ultimately SVA infection.

Asunto(s)

MicroARNs , Picornaviridae , Humanos , Animales , Porcinos , MicroARNs/genética , MicroARNs/metabolismo , ARN Circular/genética , Picornaviridae/genética , ARN Mensajero/metabolismo

RESUMEN

Senecavirus A (SVA) is a non-enveloped, positive sense, single-stranded RNA virus that causes vesicular diseases in pigs. Interferon-induced transmembrane 3 (IFITM3) is an interferon-stimulated gene (ISG) that exhibits broad antiviral activity. We investigated the role of IFITM3 in SVA replication. Both viral protein expression and supernatant virus titer were significantly increased when endogenous IFITM3 was knocked down by approximately 80% in human non-smallcell lung carcinoma cell line (NCI-H1299) compared to silencing RNA control. Interestingly, overexpression of exogenous IFITM3 in NCI-H1299 cells also significantly enhanced viral protein expression and virus titer compared to vector control, which was positively correlated with induction of autophagy mediated by IFITM3 overexpression. Overall, our results indicate an antiviral role of endogenous IFITM3 against SVA. The exact molecular mechanisms by which endogenous IFITM3 limits SVA replication remain to be determined in future studies.

RESUMEN

The role of host factors in the replication of emerging senecavirus A (SVA) which induced porcine idiopathic vesicular disease (PIVD) distributed worldwide remains obscure. Here, interferon-induced transmembrane (IFITM) protein 1 and 2 inhibit SVA replication by positive feedback with RIG-I signaling pathway was reported. The expression levels of IFITM1 and IFITM2 increased significantly in SVA infected 3D4/21 cells. Infection experiments of cells with over and interference expression of IFITM1 and IFITM2 showed that these two proteins inhibit SVA replication by regulating the expression of interferon beta (IFN-ß), IFN-stimulated gene 15 (ISG-15), interleukin 6 (IL-6), IL-8, tumor necrosis factor alpha (TNF-α), IFN regulatory factor-3 (IRF3), and IRF7. Further results showed that antiviral responses of IFITM1 and IFITM2 were achieved by activating retinoic acid-inducible gene I (RIG-I) signaling pathway which in turn enhanced the expression of IFITM1 and IFITM2. It is noteworthy that conserved domains of these two proteins also paly the similar role. These findings provide new data on the role of host factors in infection and replication of SVA and help to develop new agents against the virus.

Asunto(s)

Antígenos de Diferenciación , Interferón beta , Proteínas de la Membrana , Picornaviridae , Transducción de Señal , Animales , Retroalimentación , Interferón beta/genética , Porcinos , Replicación Viral/genética , Antígenos de Diferenciación/metabolismo , Proteínas de la Membrana/metabolismo

RESUMEN

BACKGROUND: Senecavirus A (SVA) causes an emerging vesicular disease (VD) with clinical symptoms indistinguishable from other vesicular diseases, including vesicular stomatitis (VS), foot-and-mouth disease (FMD), and swine vesicular disease (SVD). Currently, SVA outbreaks have been reported in Canada, the U.S.A, Brazil, Thailand, Vietnam, Colombia, and China. Based on the experience of prevention and control of FMDV, vaccines are the best means to prevent SVA transmission. RESULTS: After preparing an SVA inactivated vaccine (CH-GX-01-2019), we evaluated the immunogenicity of the SVA inactivated vaccine mixed with Imject® Alum (SVA + AL) or Montanide ISA 201 (SVA + 201) adjuvant in mice, as well as the immunogenicity of the SVA inactivated vaccine combined with Montanide ISA 201 adjuvant in post-weaned pigs. The results of the mouse experiment showed that the immune effects in the SVA + 201 group were superior to that in the SVA + AL group. Results from pigs immunized with SVA inactivated vaccine combined with Montanide ISA 201 showed that the immune effects were largely consistent between the SVA-H group (200 µg) and SVA-L group (50 µg); the viral load in tissues and blood was significantly reduced and no clinical symptoms occurred in the vaccinated pigs. CONCLUSIONS: Montanide ISA 201 is a better adjuvant choice than the Imject® Alum adjuvant in the SVA inactivated vaccine preparation, and the CH-GX-01-2019 SVA inactivated vaccine can provide effective protection for pigs.

Asunto(s)

Adyuvantes Inmunológicos , Compuestos de Alumbre , Manitol/análogos & derivados , Aceite Mineral , Ácidos Oléicos , Picornaviridae , Animales , Ratones , Porcinos , Vacunas de Productos Inactivados

RESUMEN

Senecavirus A (SVA) is constantly associated with vesicular disease in pigs, and the clinical symptoms of pig infection with SVA are indistinguishable from other porcine vesicular diseases. Vaccine is one of the best methods to eliminate and control the spread of SVA. Virus-like particles (VLPs) can play important roles in prevention for infectious diseases. Here, the SVA VLPs was assembled by the baculovirus expression vector system, and the immunogenicity of the SVA VLPs mixed with different adjuvants were evaluated in mice and pigs. Two recombinant baculoviruses (rPFBD-VP1-VP3 and rPFBD-VP2-VP4) were constructed, which co-infected with Sf9 suspension cells to assemble SVA VLPs successfully. SVA VLPs mixed with ISA201 adjuvant and ISA201 +Poly(I:C) adjuvant produced higher levels of neutralizing antibody, specific antibody (total IgG, IgG1, IgG2a and IgG2b) and cytokines in the T cells. And there was no significant difference between SVA VLPs+ 201 group and SVA VLPs+Poly(I:C)+ 201 group. Pigs immunized with high dose of SVA VLPs mixed with ISA201 adjuvant could produce higher titers of neutralizing antibody and SVA-specific antibody. Furthermore, the protection rates of SVA VLPs-H and SVA VLPs-L were 100% and 80%, and the viral load of SVA VLPs-H group is the lowest in all SVA VLPs groups. It is the first time to develop the SVA VLPs using the baculovirus expression vector system, which may lay the foundation for the research and development of SVA vaccine.

Asunto(s)

Picornaviridae , Vacunas de Partículas Similares a Virus , Ratones , Animales , Porcinos , Anticuerpos Antivirales , Adyuvantes Inmunológicos , Anticuerpos Neutralizantes