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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.
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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
Porcine deltacoronavirus (PDCoV) is an important enteric coronavirus that has caused major worldwide economic losses in the pig industry. Previous studies have shown that cyclophilin A (CypA), a key player in aetiological agent infection, is involved in regulating viral infection. However, the role of CypA during PDCoV replication remains unknown. Therefore, in this study, the role of CypA in PDCoV replication was determined. The results demonstrated that PDCoV infection increased CypA expression in LLC-PK1 cells. CypA overexpression substantially promoted PDCoV replication. Proteomic analysis was subsequently used to assess changes in total protein expression levels after CypA overexpression. Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to further determine the mechanisms by which CypA affects viral replication. Proteomic analysis revealed that CypA protein overexpression significantly upregulated 75 differentially expressed proteins and significantly downregulated 172 differentially expressed proteins. The differentially expressed proteins were involved mainly in autophagy and activation of the host innate immune pathway. Subsequent experimental results revealed that the CypA protein promoted viral replication by reducing the levels of natural immune cytokines and mitigated the inhibitory effect of chloroquine (CQ) on viral replication. Further investigation revealed that CypA could activate the Ras/AKT/NF-κB pathway, mediate autophagy signalling and promote PDCoV replication. In summary, the findings of this study may help elucidate the role of CypA in PDCoV replication.
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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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The coding-complete genome sequence of bovine viral diarrhea virus (BVDV) isolate NX2023 that originated from a calf in China was determined. Phylogenetic analysis showed that the NX2023 strain belongs to the BVDV-1d subgenotype.
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BACKGROUND & AIMS: The spread of foot-and-mouth disease virus (FMDV) through aerosol droplets among cloven-hoofed ungulates in close contact is a major obstacle for successful animal husbandry. Therefore, the development of suitable mucosal vaccines, especially nasal vaccines, to block the virus at the initial site of infection is crucial. PATIENTS AND METHODS: Here, we constructed eukaryotic expression plasmids containing the T and B-cell epitopes (pTB) of FMDV in tandem with the molecular mucosal adjuvant Fms-like tyrosine kinase receptor 3 ligand (Flt3 ligand, FL) (pTB-FL). Then, the constructed plasmid was electrostatically attached to mannose-modified chitosan-coated poly(lactic-co-glycolic) acid (PLGA) nanospheres (MCS-PLGA-NPs) to obtain an active nasal vaccine targeting the mannose-receptor on the surface of antigen-presenting cells (APCs). RESULTS: The MCS-PLGA-NPs loaded with pTB-FL not only induced a local mucosal immune response, but also induced a systemic immune response in mice. More importantly, the nasal vaccine afforded an 80% protection rate against a highly virulent FMDV strain (AF72) when it was subcutaneously injected into the soles of the feet of guinea pigs. CONCLUSIONS: The nasal vaccine prepared in this study can effectively induce a cross-protective immune response against the challenge with FMDV of same serotype in animals and is promising as a potential FMDV vaccine.
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Administración Intranasal , Quitosano , Virus de la Fiebre Aftosa , Fiebre Aftosa , Nanosferas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Vacunas Virales , Animales , Quitosano/química , Quitosano/administración & dosificación , Virus de la Fiebre Aftosa/inmunología , Virus de la Fiebre Aftosa/genética , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Fiebre Aftosa/prevención & control , Fiebre Aftosa/inmunología , Ratones , Nanosferas/química , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Ratones Endogámicos BALB C , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Femenino , Ácidos Nucleicos/administración & dosificación , Inmunidad Mucosa , Sistemas de Liberación de MedicamentosRESUMEN
Inactivated vaccines lack the capability to serologically differentiate between infected and vaccinated animals, thereby impeding the effective eradication of pathogen. Conversely, vaccines based on virus-like particles (VLPs) emulate natural viruses in both size and antigenic structure, presenting a promising alternative to overcome these limitations. As the complexity of swine infectious diseases increases, the increase of vaccine types and doses may intensify the stress response. This exacerbation can lead to diminished productivity, failure of immunization, and elevated costs. Given the critical dynamics of co-infection and the clinically indistinguishable symptoms associated with foot-and-mouth disease virus (FMDV) and senecavirus A (SVA), there is a dire need for an efficacious intervention. To address these challenges, we developed a combined vaccine composed of three distinct VLPs, specifically designed to target SVA and FMDV serotypes O and A. Our research demonstrates that this trivalent VLP vaccine induces antigen-specific and robust serum antibody responses, comparable to those produced by the respective monovalent vaccines. Moreover, the immune sera from the combined VLP vaccine strongly neutralized FMDV type A and O, and SVA, with neutralization titers comparable to those of the individual vaccines, indicating a high level of immunogenic compatibility among the three VLP components. Importantly, the combined VLPs vaccines-immunized sera conferred efficient protection against single or mixed infections with FMDV type A and O, and SVA viruses in pigs. In contrast, individual vaccines could only protect pigs against homologous virus infections and not against heterologous challenges. This study presents a novel combined vaccines candidate against FMD and SVA, and provides new insights for the development of combination vaccines for other viral swine diseases.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Virus de la Fiebre Aftosa , Fiebre Aftosa , Picornaviridae , Enfermedades de los Porcinos , Vacunas de Partículas Similares a Virus , Vacunas Virales , Animales , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Fiebre Aftosa/prevención & control , Fiebre Aftosa/inmunología , Virus de la Fiebre Aftosa/inmunología , Porcinos , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Ratones , Picornaviridae/inmunología , Infecciones por Picornaviridae/prevención & control , Infecciones por Picornaviridae/inmunología , Infecciones por Picornaviridae/veterinaria , Femenino , Vacunas Combinadas/inmunología , Vacunas Combinadas/administración & dosificación , Coinfección/prevención & control , Coinfección/inmunologíaRESUMEN
In order to develop a safe and effective broad-spectrum vaccine for foot-and-mouth disease (FMDV), here, we developed a recombinant FMD multiple-epitope trivalent vaccine based on three distinct topotypes of FMDV. Potency of the vaccine was evaluated by immune efficacy in pigs. The results showed that the vaccine with no less than 25 µg of antigen elicited FMDV serotype O specific antibodies and neutralization antibodies by primary-booster regime, and offered immune protection to pigs. More importantly, the vaccine elicited not only the same level of neutralization antibodies against the three distinct topotypes of FMDV, but also provided complete protection in pigs from the three corresponding virus challenge. None of the fully protected pigs were able to generate anti-3ABC antibodies throughout the experiment, which implied the vaccine can offer sterilizing immunity. The vaccine elicited lasting-long high-level antibodies and effectively protected pigs from virulent challenge within six months of immunization. Therefore, we consider that this vaccine may be used in the future for the prevention and control of FMD.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Epítopos , Virus de la Fiebre Aftosa , Fiebre Aftosa , Serogrupo , Enfermedades de los Porcinos , Vacunas Sintéticas , Vacunas Virales , Animales , Virus de la Fiebre Aftosa/inmunología , Virus de la Fiebre Aftosa/genética , Fiebre Aftosa/prevención & control , Fiebre Aftosa/inmunología , Fiebre Aftosa/virología , Porcinos , Vacunas Virales/inmunología , Vacunas Virales/genética , Vacunas Virales/administración & dosificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Epítopos/inmunología , Epítopos/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Eficacia de las VacunasRESUMEN
Porcine epidemic diarrhea virus (PEDV) is considered the cause for porcine epidemic diarrhea (PED) outbreaks and hefty losses in pig farming. However, no effective commercial vaccines against PEDV mutant strains are available nowadays. Here, we constructed three native-like trimeric candidate nanovaccines, i.e., spike 1 trimer (S1-Trimer), collagenase equivalent domain trimer (COE-Trimer), and receptor-binding domain trimer (RBD-Trimer) for PEDV based on Trimer-Tag technology. And evaluated its physical properties and immune efficacy. The result showed that the candidate nanovaccines were safe for mice and pregnant sows, and no animal death or miscarriage occurred in our study. S1-Trimer showed stable physical properties, high cell uptake rate and receptor affinity. In the mouse, sow and piglet models, immunization of S1-Trimer induced high-level of humoral immunity containing PEDV-specific IgG and IgA. S1-Trimer-driven mucosal IgA responses and systemic IgG responses exhibited high titers of virus neutralizing antibodies (NAbs) in vitro. S1-Trimer induced Th1-biased cellular immune responses in mice. Moreover, the piglets from the S1-Trimer and inactivated vaccine groups displayed significantly fewer microscopic lesions in the intestinal tissue, with only one and two piglets showing mild diarrhea. The viral load in feces and intestines from the S1-Trimer and inactivated vaccine groups were significantly lower than those of the PBS group. For the first time, our data demonstrated the protective efficacy of Trimer-Tag-based nanovaccines used for PEDV. The S1-Trimer developed in this study was a competitive vaccine candidate, and Trimer-Tag may be an important platform for the rapid production of safe and effective subunit vaccines in the future.
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The internal ribosome entry site (IRES) element constitutes a cis-acting RNA regulatory sequence that recruits the ribosomal initiation complex in a cap-independent manner, assisted by various RNA-binding proteins and IRES trans-acting factors. Foot-and-mouth disease virus (FMDV) contains a functional IRES element and takes advantage of this element to subvert host translation machinery. Our study identified a novel mechanism wherein RALY, a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family belonging to RNA-binding proteins, binds to the domain 3 of FMDV IRES via its RNA recognition motif residue. This interaction results in the downregulation of FMDV replication by inhibiting IRES-driven translation. Furthermore, our findings reveal that the inhibitory effect exerted by RALY on FMDV replication is not attributed to the FMDV IRES-mediated assembly of translation initiation complexes but rather to the impediment of 80S ribosome complex formation after binding with 40S ribosomes. Conversely, 3Cpro of FMDV counteracts RALY-mediated inhibition by the ubiquitin-proteasome pathway. Therefore, these results indicate that RALY, as a novel critical IRES-binding protein, inhibits FMDV replication by blocking the formation of 80S ribosome, providing a deeper understanding of how viruses recruit and manipulate host factors. IMPORTANCE: The translation of FMDV genomic RNA driven by IRES element is a crucial step for virus infections. Many host proteins are hijacked to regulate FMDV IRES-dependent translation, but the regulatory mechanism remains unknown. Here, we report for the first time that cellular RALY specifically interacts with the IRES of FMDV and negatively regulates viral replication by blocking 80S ribosome assembly on FMDV IRES. Conversely, RALY-mediated inhibition is antagonized by the viral 3C protease by the ubiquitin-proteasome pathway. These results would facilitate further understanding of virus-host interactions and translational control during viral infection.
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Virus de la Fiebre Aftosa , Animales , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Unión al ARN/genética , Ribosomas/genética , Endopeptidasas/metabolismo , Sitios Internos de Entrada al Ribosoma , Proteasas Virales 3C , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
Foot-and-mouth disease virus (FMDV) remains a challenge for cloven-hooved animals. The currently licensed FMDV vaccines induce neutralizing antibody (NAb)-mediated protection but show defects in the early protection. Dendritic cell (DC) vaccines have shown great potency in inducing rapid T-cell immunity in humans and mice. Whether DC vaccination could enhance early protection against FMDV has not been elaborately explored in domestic pigs. In this study, we employed DC vaccination as an experimental approach to study the roles of cellular immunity in the early protection against FMDV in pigs. Autologous DCs were differentiated from the periphery blood mononuclear cells of each pig, pulsed with inactivated FMDV (iFMDV-DC) and treated with LPS, and then injected into the original pigs. The cellular immune responses and protective efficacy elicited by the iFMDV-DC were examined by multicolor flow cytometry and tested by FMDV challenge. The results showed that autologous iFMDV-DC immunization induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells (CTLs), high NAb titers, compared to the inactivated FMDV vaccine, and accelerated the development of memory CD4 and CD8 T cells, which was concomitantly associated with early protection against FMDV virulent strain in pigs. Such early protection was associated with the rapid proliferation of secondary T-cell response after challenge and significantly contributed by secondary CD8 effector memory T cells. These results demonstrated that rapid induction of cellular immunity through DC immunization is important for improving early protection against FMDV. Enhancing cytotoxic CD8+ T cells may facilitate the development of more effective FMDV vaccines.IMPORTANCEAlthough the currently licensed FMDV vaccines provide NAb-mediated protection, they have defects in early immune protection, especially in pigs. In this study, we demonstrated that autologous swine DC immunization augmented the cellular immune response and induced an early protective response against FMDV in pigs. This approach induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells, high NAb titers, and rapid development of memory CD4 and CD8 T cells. Importantly, the early protection conferred by this DC immunization is more associated with secondary CD8+ T response rather than NAbs. Our findings highlighted the importance of enhancing cytotoxic CD8+ T cells in early protection to FMDV in addition to Th1 response and identifying a strategy or adjuvant comparable to the DC vaccine might be a future direction for improving the current FMDV vaccines.
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Virus de la Fiebre Aftosa , Fiebre Aftosa , Vacunas Virales , Animales , Humanos , Ratones , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Linfocitos T CD8-positivos , Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Virus de la Fiebre Aftosa/fisiología , Porcinos , VacunaciónRESUMEN
Classical swine fever virus (CSFV) is a highly contagious pathogen causing significant economic losses in the swine industry. Conventional inactivated or attenuated live vaccines for classical swine fever (CSF) are effective but face biosafety concerns and cannot distinguish vaccinated animals from those infected with the field virus, complicating CSF eradication efforts. It is noteworthy that nanoparticle (NP)-based vaccines resemble natural viruses in size and antigen structure, and offer an alternative tool to circumvent these limitations. In this study, we developed an innovative vaccine delivery scaffold utilizing self-assembled mi3 NPs, which form stable structures carrying the CSFV E2 glycoprotein. The expressed yeast E2-fused protein (E2-mi3 NPs) exhibited robust thermostability (25 to 70 °C) and long-term storage stability at room temperature (25 °C). Interestingly, E2-mi3 NPs made with this technology elicited enhanced antigen uptake by RAW264.7 cells. In a rabbit model, the E2-mi3 NP vaccine against CSFV markedly increased CSFV-specific neutralizing antibody titers. Importantly, it conferred complete protection in rabbits challenged with the C-strain of CSFV. Furthermore, we also found that the E2-mi3 NP vaccines triggered stronger cellular (T-lymphocyte proliferation, CD8+ T-lymphocytes, IFN-γ, IL-2, and IL-12p70) and humoral (CSFV-specific neutralizing antibodies, CD4+ T-lymphocytes, and IL-4) immune responses in pigs than the E2 vaccines. To sum up, these structure-based, self-assembled mi3 NPs provide valuable insights for novel antiviral strategies against the constantly infectious agents.
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Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Lagomorpha , Nanopartículas , Animales , Conejos , Porcinos , Nanovacunas , Peste Porcina Clásica/prevención & control , Vacunas Atenuadas , Proteínas FúngicasRESUMEN
Porcine epidemic diarrhea virus (PEDV) is an acute and highly contagious porcine enteric coronavirus. It has caused serious economic losses of pig industry in China. Here we insolated a current PEDV field strain named GS2022, analyzed the characters of genetic variation and pathogenicity. The results demonstrated that the GS2022 strain was belong to a newly defined subgroup G2 d, forming an independent branch which mainly contains strains isolated in China from 2017 to 2023. Notably, there are multiple mutations and extensive N-glycosylation compared to CV777 strain and PT-P5 strain, therefore the structure of GS2022 strain is different from 6U7K and 7W6M. Animal pathogenicity test showed that GS2022 strain could cause severe clinical signs and the high level of virus shedding in 7-day-old piglets. But recovery of diarrhea after 5 days, and no pathological damage to important organs. Further study on 3-day-old piglets also indicated GS2022 strain have pathogenicity. In this study no piglets died, which make it possible for that GS2022 strain become a candidate vaccine. These results are helpful to understand the epidemiology, molecular characteristics, evolution, and antigenicity of PEDV circulating in China. It also provides reference for designing effective vaccines against PEDV.
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Infecciones por Coronavirus , Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Animales , Porcinos , Virulencia , Filogenia , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/veterinaria , China/epidemiología , Recombinación Genética , Diarrea/veterinariaRESUMEN
Porcine deltacoronavirus (PDCoV), a new porcine enteric coronavirus, has seriously endangered the pig breeding industry and caused great economic losses. However, a PDCoV vaccine is not commercially available. Therefore, new and efficient PDCoV vaccines must be developed without delay. In this study, we used the ExpiCHO eukaryotic expression system to express and purify the following 3 structural proteins of PDCoV: S, N and M. Subsequently, the level of humoral and cellular immunity induced by the S protein (immunization with the S protein alone) and a protein mixture (immunization with a mixture of S, N and M proteins) were evaluated in mice and piglets, respectively, and the performances of the 2 immunizations in a challenge protection test were assessed in piglets. The results showed that both the S protein and the protein mixture induced the production of high levels of specific IgG antibodies and neutralizing antibodies and effectively neutralized PDCoV-infected LLC-PK cells in vitro. Furthermore, compared with the S protein, the N and M proteins in the protein mixture promoted the expression of CD8+ T cells and IFN-γ, induced a stronger cellular immune response, and effectively protected 4/5 of the piglets from PDCoV infection. In conclusion, the results of this study showed that the N and M proteins play important roles in inducing an immunoprotective response. Using N and M antigens as effective antigenic components in the development of PDCoV vaccines in the future will effectively increase the immune efficacy of the vaccines.
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Infecciones por Coronavirus , Coronavirus , Enfermedades de los Porcinos , Animales , Porcinos , Ratones , Linfocitos T CD8-positivos , Coronavirus/genética , Coronavirus/metabolismo , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/veterinaria , Vacunas de SubunidadRESUMEN
To further enhance the immune effect of the foot-and-mouth disease (FMD) virus-like particles (VLPs) vaccine, this study prepared FMDV VLPs-zeolitic imidazolate (framework-8, ZIF-8) complexes with different particle sizes. We used a biomimetic mineralization method with Zn2+ and 2-methylimidazole in different concentration ratios to investigate the effect of size on the immunization effect. The results showed that FMDV VLPs-ZIF-8 with three different sizes were successfully prepared, with an approximate size of 70 nm, 100 nm, and 1 000 nm, respectively. Cytotoxicity and animal toxicity tests showed that all three complexes exhibited excellent biological safety. Immunization tests in mice showed that all three complexes enhanced the titers of neutralizing and specific antibodies, and their immune effects improved as the size of the complexes decreased. This study showed that ZIF-8 encapsulation of FMDV VLPs significantly enhanced their immunogenic effect in a size-dependent manner.
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Virus de la Fiebre Aftosa , Fiebre Aftosa , Vacunas de Partículas Similares a Virus , Vacunas Virales , Animales , Ratones , Fiebre Aftosa/prevención & control , Anticuerpos Neutralizantes , Inmunidad Humoral , Inmunización , Anticuerpos AntiviralesRESUMEN
Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.
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Virus de la Fiebre Aftosa , Fiebre Aftosa , Vacunas Virales , Animales , Virus de la Fiebre Aftosa/genética , Proteínas de la Cápside , Proteínas Virales/metabolismo , Fiebre Aftosa/prevención & control , Tetraciclinas/metabolismo , Anticuerpos Antivirales , Mamíferos/metabolismoRESUMEN
Nanovaccines based on self-assembling nanoparticles (NPs) can show conformational epitopes of antigens and they have high immunogenicity. In addition, flagellin, as a biological immune enhancer, can be fused with an antigen to considerably enhance the immune effect of antigens. In improving the immunogenicity and stability of a foot-and-mouth disease virus (FMDV) antigen, novel FMDV NP antigens were prepared by covalently coupling the VP1 protein and truncated flagellin containing only N-terminus D0 and D1 (N-terminal aa 1-99, nFLiC) with self-assembling NPs (i301). The results showed that the fusion proteins VP1-i301 and VP1-i301-nFLiC can assemble into NPs with high thermal tolerance and stability, obtain high cell uptake efficiency, and upregulate marker molecules and immune-stimulating cytokines in vitro. In addition, compared with monomeric VP1 antigen, high-level cytokines were stimulated with VP1-i301 and VP1-i301-nFLiC nanovaccines in guinea pigs, to provide clinical protection against viral infection comparable to an inactivated vaccine. This study provides new insight for the development of a novel FMD vaccine.
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Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes severe watery diarrhea, vomiting, dehydration and high mortality in piglets, resulting in significant economic losses by the global pig industry. Recently, PDCoV has also shown the potential for cross-species transmission. However, there are currently few vaccine studies and no commercially available vaccines for PDCoV. Hence, here, two novel human adenovirus 5 (Ad5)-vectored vaccines expressing codon-optimized forms of the PDCoV spike (S) glycoprotein (Ad-PD-tPA-Sopt) and S1 glycoprotein (Ad-PD-oriSIP-S1opt) were constructed, and their effects were evaluated via intramuscular (IM) injection in BALB/c mice with different doses and times. Both vaccines elicited robust humoral and cellular immune responses; moreover, Ad-PD-tPA-Sopt-vaccinated mice after two IM injections with 108 infectious units (IFU)/mouse had significantly higher anti-PDCoV-specific neutralizing antibody titers. In contrast, the mice immunized with Ad-PD-tPA-Sopt via oral gavage (OG) did not generate robust systemic and mucosal immunity. Thus, IM Ad-PD-tPA-Sopt administration is a promising strategy against PDCoV and provides useful information for future animal vaccine development.
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Vacunas contra el Adenovirus , Infecciones por Coronavirus , Enfermedades de los Porcinos , Vacunas , Humanos , Animales , Porcinos , Ratones , Glicoproteínas , Inmunidad Celular , Adenoviridae/genética , Enfermedades de los Porcinos/prevención & controlRESUMEN
Widely utilized in the chemical industry and agriculture, hydrazine is easily absorbed by living things and can cause physical harm when in touch for an extended period of time. As a result, a novel cinnamaldehyde chalcone C5 was produced by Friedel Crafts process and aldol condensation reaction. Triphenylamine was used as the raw material for hydrazine determination in both reactions. Chalcone C5 exhibits significant AIE behavior in a mixed mixture of ethanol and water in addition to having great selectivity and a low detection limit (0.119 nm) for hydrazine. The solvent effect test revealed a linear relationship between the Stokes shift of C5 in the solvent and the rise in solvent orientation polarization. It is important to note that C5 is not harmful to MCF-7 cells, mouse kidney cells, or pig kidney cells. Furthermore, research on cell imaging has demonstrated that probe C5 may be utilized to image the fluorescence of hydrazine in active MCF-7 cells.
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Vacuolar protein sorting 28 (Vps28), a component of the ESCRT-I (endosomal sorting complex required for transport I), plays an important role in the pathogen life cycle. Here, we investigated the reciprocal regulation between Vps28 and the foot-and-mouth disease virus (FMDV). Overexpression of Vps28 decreased FMDV replication. On the contrary, the knockdown of Vps28 increased viral replication. Subsequently, the mechanistic study showed that Vps28 destabilized the replication complex (RC) by associating with 3A rather than 2C protein. In addition, Vps28 targeted FMDV VP0, VP1, and VP3 for degradation to inhibit viral replication. To counteract this, FMDV utilized tactics to restrict Vps28 to promote viral replication. FMDV degraded Vps28 mainly through the ubiquitin-proteasome pathway. Additional data demonstrated that 2B and 3A proteins recruited E3 ubiquitin ligase tripartite motif-containing protein 21 to degrade Vps28 at Lys58 and Lys25, respectively, and FMDV 3Cpro degraded Vps28 through autophagy and its protease activity. Meantime, the 3Cpro-mediated Vps28 degradation principally alleviated the ability to inhibit viral propagation. Intriguingly, we also demonstrated that the N-terminal and C-terminal domains of Vps28 were responsible for the suppression of FMDV replication, which suggested the elaborated counteraction between FMDV and Vps28. Collectively, our results first investigate the role of ESCRTs in host defense against picornavirus and unveil underlying strategies utilized by FMDV to evade degradation machinery for triumphant propagation. IMPORTANCE ESCRT machinery plays positive roles in virus entry, replication, and budding. However, little has been reported on its negative regulation effects during viral infection. Here, we uncovered the novel roles of ESCRT-I subunit Vps28 on FMDV replication. The data indicated that Vps28 destabilized the RC and impaired viral structural proteins VP0, VP1, and VP3 to inhibit viral replication. To counteract this, FMDV hijacked intracellular protein degradation pathways to downregulate Vps28 expression and thus promoted viral replication. Our findings provide insights into how ESCRT regulates pathogen life cycles and elucidate additional information regarding FMDV counteraction of host antiviral activity.
Asunto(s)
Virus de la Fiebre Aftosa , Fiebre Aftosa , Animales , Virus de la Fiebre Aftosa/metabolismo , Proteínas Virales/metabolismo , Transducción de Señal , Transporte de Proteínas , Replicación Viral/fisiologíaRESUMEN
Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV), members of the genus Coronavirus, mainly cause acute diarrhea, vomiting and dehydration in piglets, and thus lead to serious economic losses. In this study, we investigated the effects of nicotinamide (NAM) on PEDV and PDCoV replication and found that NAM treatment significantly inhibited PEDV and PDCoV reproduction. Moreover, NAM plays an important role in replication processes. NAM primarily inhibited PEDV and PDCoV RNA and protein synthesis rather than other processes. Furthermore, we discovered that NAM treatment likely inhibits the replication of PEDV and PDCoV by downregulating the expression of transcription factors through activation of the ERK1/2/MAPK pathway. Overall, this study is the first to suggest that NAM might be not only an important antiviral factor for swine intestinal coronavirus, but also a potential candidate to be evaluated in the context of other human and animal coronaviruses.