RESUMEN
Classical swine fever (CSF) re-emerged in Japan in 2018 for the first time in 26 years. The disease has been known to be caused by a moderately pathogenic virus, rather than the highly pathogenic virus that had occurred in the past. However, the underlying pathophysiology remains unknown. This study conducted an experimental challenge on specific pathogen-free (SPF) pigs in a naïve state for 2, 4, and 6 weeks and confirmed the disease state during each period by clinical observation, virus detection, and pathological necropsy. We revealed the pathological changes and distribution of pathogens and virus-specific antibodies at each period after virus challenge. These results were comprehensively analyzed and approximately 70% of the pigs recovered, especially at 4- and 6-week post-virus challenge. This study provides useful information for future countermeasures against CSF by clarifying the pathogenicity outcomes in unvaccinated pigs with moderately pathogenic genotype 2.1 virus.
Asunto(s)
Anticuerpos Antivirales , Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Genotipo , Animales , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/patogenicidad , Virus de la Fiebre Porcina Clásica/aislamiento & purificación , Peste Porcina Clásica/virología , Porcinos , Japón , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Organismos Libres de Patógenos Específicos , VirulenciaRESUMEN
Classical Swine Fever (CSF), a highly contagious viral disease affecting pigs and wild boar, results in significant economic losses in the swine industry. In endemic regions, prophylactic vaccination and stamping-out strategies are used to control CSF outbreaks. However, sporadic outbreaks and persistent infections continue to be reported. Although the conventional attenuated CSF vaccines protect pigs against the disease, they do not allow for the differentiation of infected from vaccinated animals (DIVA), limiting their use as an eradication tool. In this study, three targeted attenuation strategies were employed to generate vaccine candidates based on the current prevalent CSFV group 2 strains GD18 and QZ07: a single deletion of H79 in Erns (QZ07-sdErnsH-KARD), double deletion of H79 and C171 in Erns (GD18-ddErnsHC-KARD and QZ07-ddErnsHC-KARD), and deletion of H79 in Erns combined with a 5-168 amino acids deletion of Npro (GD18-ddNpro-ErnsH-KARD). Additionally, a negative serological marker with four substitutions in a highly conserved epitope in E2 recognized by the monoclonal antibody 6B8 was introduced in each candidate for DIVA purposes. The safety of these four resulting vaccine candidates was evaluated in pregnant sows. Two candidates, GD18-ddErnsHC-KARD and QZ07-sdErnsH-KARD were found to be safe for pregnant sows and unlikely to cause vertical transmission. Both candidates also demonstrated potential to be used as DIVA vaccines, as was shown using a proprietary blocking ELISA based on the 6B8 monoclonal antibody. These results, together with our previous work, constitute a proof-of-concept for the rational design of CSF antigenically marked modified live virus vaccine candidates.
Asunto(s)
Anticuerpos Antivirales , Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Vacunas Atenuadas , Vacunas Virales , Animales , Peste Porcina Clásica/prevención & control , Peste Porcina Clásica/virología , Peste Porcina Clásica/inmunología , Porcinos , Femenino , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/efectos adversos , Virus de la Fiebre Porcina Clásica/inmunología , Virus de la Fiebre Porcina Clásica/genética , Vacunas Virales/inmunología , Vacunas Virales/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/efectos adversos , Embarazo , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Vacunas Marcadoras/inmunología , Vacunas Marcadoras/administración & dosificación , Vacunas Marcadoras/genética , Vacunación/veterinaria , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunologíaRESUMEN
Chimeric marker vaccine candidates, vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns, have been generated and their efficacy and capability to differentiate infected from vaccinated animals were confirmed in previous studies. The safety profile of the two chimeric marker vaccine candidates, particularly in the potential reversion to virulence, was evaluated. Each virus was administered to pigs with a dose equivalent to the vaccination dose, and pooled tonsil homogenates were subsequently inoculated into further pigs. Chimeric virus vGPE-/PAPeV Erns displayed the most substantial attenuation, achieving this within only two passages, whereas vGPE-/PhoPeV Erns was detectable until the third passage and disappeared entirely by the fourth passage. The vGPE- strain, assessed alongside, consistently exhibited stable virus recovery across each passage without any signs of increased virulence in pigs. In vitro assays revealed that the type I interferon-inducing capacity of vGPE-/PAPeV Erns was significantly higher than that of vGPE-/PhoPeV Erns and vGPE-. In conclusion, the safety profile of the two chimeric marker vaccine candidates was affirmed. Further research is essential to ensure the stability of their attenuation and safety in diverse pig populations.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Vacunas Atenuadas , Vacunas Virales , Animales , Porcinos , Virulencia , Peste Porcina Clásica/prevención & control , Peste Porcina Clásica/virología , Peste Porcina Clásica/inmunología , Vacunas Virales/inmunología , Vacunas Virales/efectos adversos , Vacunas Virales/administración & dosificación , Virus de la Fiebre Porcina Clásica/inmunología , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/patogenicidad , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/administración & dosificación , Vacunas Marcadoras/inmunología , Vacunas Marcadoras/genética , Vacunas Marcadoras/administración & dosificación , VacunaciónRESUMEN
Background: Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. Methods: A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Results: Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. Conclusion: The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Genotipo , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , ARN Viral , Sensibilidad y Especificidad , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/aislamiento & purificación , Virus de la Fiebre Porcina Clásica/clasificación , Animales , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/economía , Peste Porcina Clásica/diagnóstico , Peste Porcina Clásica/virología , Porcinos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/economía , ARN Viral/genética , ARN Viral/aislamiento & purificación , Cartilla de ADN/genética , Colorimetría/métodos , TemperaturaRESUMEN
We devised a method to detect the classical swine fever virus (CSFV) in tail-wiped swabs from wild boars. The CSFV gene in swabs was detected with high sensitivity using nested real-time polymerase chain reaction (PCR), which is a combination of reverse transcription-PCR (RT-PCR) and real-time PCR. We compared CSFV gene detection from boar tissue using the conventional and our tail-wiped swab method. The tail-wiped swab method showed sensitivity and specificity of 100% (26/26) and 98.8% (172/174), respectively compared to the conventional method. Thus, the swab-based CSFV detection method was considered to have detection sensitivity comparable to that of conventional methods. Additionally, we conducted surveillance for CSFV in wild boars on Awaji Island. CSFV was detected in 10.7% (45/420) of samples.
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Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Sus scrofa , Animales , Virus de la Fiebre Porcina Clásica/aislamiento & purificación , Virus de la Fiebre Porcina Clásica/genética , Porcinos , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sus scrofa/virología , Peste Porcina Clásica/diagnóstico , Peste Porcina Clásica/virología , Cola (estructura animal)/virología , Japón , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
The FTA card has emerged as a promising alternative for nucleic acid extraction. The FTA card is a filter paper impregnated with chemicals that preserve and stabilize the genetic material present in the sample, allowing for its storage and transport at room temperature. The aim of this study was to test the card for the detection of RNA and DNA nucleic acids. Two RNA viruses (Senecavirus A and classical swine fever virus) and two DNA viruses (African swine fever virus and suid alphaherpesvirus 1) were tested, and in all cases, there was a decrease in sensitivity. The methods exhibited good repeatability and demonstrated a rapid and practical use for sample transport and nucleic acid extraction.
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Virus de la Fiebre Porcina Africana , Animales , Porcinos , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/aislamiento & purificación , Herpesvirus Suido 1/aislamiento & purificación , Herpesvirus Suido 1/genética , ARN Viral/genética , ARN Viral/aislamiento & purificación , Medicina Veterinaria/métodos , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/diagnóstico , Virus ADN/genética , Virus ADN/aislamiento & purificación , Picornaviridae/genética , Picornaviridae/aislamiento & purificación , Picornaviridae/clasificación , Sensibilidad y Especificidad , ADN Viral/genética , Virus ARN/genética , Virus ARN/aislamiento & purificación , Virus ARN/clasificación , Infecciones por Virus ADN/veterinaria , Infecciones por Virus ADN/diagnóstico , Infecciones por Virus ADN/virología , Manejo de Especímenes/métodos , Manejo de Especímenes/instrumentaciónRESUMEN
Several cellular factors have been reported to be required for replication of classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae. However, many steps of its replication cycle are still poorly understood. The low-density lipoprotein receptor (LDLR) is involved in cell entry and post-entry processes of different viruses including other members of the Flaviviridae. In this study, the relevance of LDLR in replication of CSFV and another porcine pestivirus, Bungowannah pestivirus (BuPV), was investigated by antibody-mediated blocking of LDLR and genetically engineered porcine cell lines providing altered LDLR expression levels. An LDLR-specific antibody largely blocked infection with CSFV, but had only a minor impact on BuPV. Infections of the genetically modified cells confirmed an LDLR-dependent replication of CSFV. Compared to wild type cells, lower and higher expression of LDLR resulted in a 3.5-fold decrease or increase in viral titers already 20â h post infection. Viral titers were 25-fold increased in LDLR-overexpressing cells compared to cells with reduced LDLR expression at 72â h post infection. The varying LDLR expression levels had no clear effect on permissivity to BuPV. A decoy receptor assay using recombinant soluble LDLR provided no evidence that LDLR may function as a receptor for CSFV or BuPV. Differences in their dependency on LDLR suggest that CSFV and BuPV likely use different mechanisms to interact with their host cells. Moreover, this study reveals similarities in the replication cycles of CSFV and other members of the family Flaviviridae that are dependent on LDLR.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Pestivirus , Porcinos , Animales , Virus de la Fiebre Porcina Clásica/genética , Pestivirus/fisiología , Línea Celular , Lipoproteínas LDL/metabolismo , Replicación ViralRESUMEN
Exosomes, which are small membrane-enclosed vesicles, are actively released into the extracellular space by a variety of cells. Growing evidence indicates that exosomes derived from virus-infected cells can selectively encapsulate viral proteins, genetic materials, or even entire virions. This enables them to mediate cell-to-cell communication and facilitate virus transmission. Classical swine fever (CSF) is a disease listed by the World Organisation for Animal Health (WOAH) Terrestrial Animal Health Code and must be reported to the organisation. It is caused by classical swine fever virus (CSFV) belonging to the Flaviviridae family. Recent studies have demonstrated that extracellular vesicles originating from autophagy can facilitate the antibody-resistant spread of classical swine fever virus. However, due to the extreme difficulty in achieving a complete separation from virions, the role of exosomes during CSFV infection and proliferation remains elusive. In this study, we ingeniously chose to perform immunoprecipitation (IP) targeting the CSFV E2 protein, thereby achieving the complete removal of infectious virions. Subsequently, we discovered that the purified exosomes are shown to contain viral genomic RNA and partial viral proteins. Furthermore, exosomes secreted by CSFV-infected cells can evade CSFV-specific neutralizing antibodies, establish subsequent infection, and stimulate innate immune system after uptake by recipient cells. In summary, exosomes play a critical role in CSFV transmission. This is of great significance for in-depth exploration of the characteristics of CSFV and its complex interactions with the host.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Exosomas , Enfermedades de los Porcinos , Porcinos , Animales , Virus de la Fiebre Porcina Clásica/genética , Anticuerpos Neutralizantes , Proteínas Virales , Inmunidad InnataRESUMEN
Classical Swine Fever (CSF), caused by the Classical Swine Fever Virus (CSFV), inflicts significant economic losses on the global pig industry. A key factor in the challenge of eradicating this virus is its ability to evade the host's innate immune response, leading to persistent infections. In our study, we elucidate the molecular mechanism through which CSFV exploits m6A modifications to circumvent host immune surveillance, thus facilitating its proliferation. We initially discovered that m6A modifications were elevated both in vivo and in vitro upon CSFV infection, particularly noting an increase in the expression of the methyltransferase METTL14. CSFV non-structural protein 5B was found to hijack HRD1, the E3 ubiquitin ligase for METTL14, preventing METTL14 degradation. MeRIP-seq analysis further revealed that METTL14 specifically targeted and methylated TLRs, notably TLR4. METTL14-mediated regulation of TLR4 degradation, facilitated by YTHDF2, led to the accelerated mRNA decay of TLR4. Consequently, TLR4-mediated NF-κB signaling, a crucial component of the innate immune response, is suppressed by CSFV. Collectively, these data effectively highlight the viral evasion tactics, shedding light on potential antiviral strategies targeting METTL14 to curb CSFV infection.
Asunto(s)
Adenina , Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Animales , Virus de la Fiebre Porcina Clásica/genética , Inmunidad Innata , Porcinos , Receptor Toll-Like 4RESUMEN
The early detection of classical swine fever (CSF) remains a key challenge, especially when outbreaks are caused by moderate and low-virulent CSF virus (CSFV) strains. Oral fluid is a reliable and cost-effective sample type that is regularly surveilled for endemic diseases in commercial pig herds in North America. Here, we explored the possibility of utilizing oral fluids for the early detection of CSFV incursions in commercial-size pig pens using two independent experiments. In the first experiment, a seeder pig infected with the moderately-virulent CSFV Pinillos strain was used, and in the second experiment, a seeder pig infected with the highly-virulent CSFV Koslov strain was used. Pen-based oral fluid samples were collected daily and individual samples (whole blood, swabs) every other day. All samples were tested by a CSFV-specific real-time RT-PCR assay. CSFV genomic material was detected in oral fluids on the seventh and fourth day post-introduction of the seeder pig into the pen, in the first and second experiments, respectively. In both experiments, oral fluids tested positive before the contact pigs developed viremia, and with no apparent sick pigs in the pen. These results indicate that pen-based oral fluids are a reliable and convenient sample type for the early detection of CSF, and therefore, can be used to supplement the ongoing CSF surveillance activities in North America.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Porcinos , Animales , Virus de la Fiebre Porcina Clásica/genética , Viremia/diagnóstico , Viremia/veterinaria , Viremia/epidemiología , Brotes de Enfermedades/veterinaria , Vacunación/veterinariaRESUMEN
Pestiviruses are classified into two biotypes based on their cytopathogenicity. As the majority of pestivirus field isolates are noncytopathogenic, their titration requires alternative methods rather than direct observation of cytopathogenic effects, such as immunostaining using specific antibodies or interference with cytopathogenic strains. However, these methods require microscopic observation to assess virus growth, which is time- and labor-intensive, especially when handling several samples. In this study, we developed a novel luciferase-based pestivirus titration method using the superinfection exclusion phenomenon with recombinant reporter pestiviruses that possessed an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). In this method, swine kidney cells were inoculated with classical swine fever virus (CSFV) and superinfected with the reporter CSFV vGPE-/HiBiT 5 days postinoculation. Virus titer was determined based on virus growth measured in luminescence using the culture fluid 3 days after superinfection; the resultant virus titer was comparable to that obtained by immunoperoxidase staining. Furthermore, this method has proven to be applicable for the titration of border disease virus (BDV) by superinfection with both the homologous reporter BDV and heterologous reporter CSFV, suggesting that this novel virus titration method is a simple technique for automated virus detection based on the luciferase system.
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Virus de la Fiebre Porcina Clásica , Pestivirus , Sobreinfección , Enfermedades de los Porcinos , Animales , Porcinos , Pestivirus/genética , Sobreinfección/veterinaria , Virus de la Fiebre Porcina Clásica/genética , Luciferasas/genéticaRESUMEN
Classical swine fever virus (CSFV) can dampen the host innate immunity by destabilizing IRF3 upon its binding with viral Npro. High mobility group box 1 (HMGB1), a non-histone nuclear protein, has diverse functions, including inflammation, innate immunity, etc., which are closely related to its cellular localization. We investigated potential mutual interactions between CSFV and HMGB1 and their effects on virus replication. We found that HMGB1 at the protein level, but not at mRNA level, was markedly reduced in CSFV-infected or Npro-expressing IPEC-J2 cells. HMGB1 in the nuclear compartment is anti-CSFV by promoting IFN-mediated innate immune response, as evidenced by overexpression of nuclear or cytoplasmic dominant HMGB1 mutant in IPEC-J2 cells stimulated with poly(I:C). However, CSFV Npro upregulates HMGB1 acetylation, a modification that promotes HMGB1 translocation into the cytoplasmic compartment where it is degraded by lysosomes. Ethyl pyruvate could downregulate HMGB1 acetylation and prevent Npro-mediated HMGB1 reduction. Inhibition of deacetylase HDAC1 with MS275 or by RNA silencing could promote Npro-mediated HMGB1 degradation. Taken together, our study elucidates the mechanism with which HMGB1 in the nuclei initiates antiviral innate immune response to suppress CSFV replication and elaborates the pathway by which CSFV uses its Npro to evade from HMGB1-mediated antiviral immunity through upregulating HMGB1 acetylation with subsequent translocation into cytoplasm for lysosomal degradation.
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Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Proteína HMGB1 , Porcinos , Animales , Virus de la Fiebre Porcina Clásica/genética , Acetilación , Línea Celular , Lisosomas , Replicación Viral/fisiologíaRESUMEN
The classical swine fever virus (CSFV) is a species member of the family Flaviviridae. CSFV is widely distributed in the world causing a severe impact on pig industry. This pathogen is considered restricted to domestic and wild suids. However, some reports from 2014 to 2018 showed the presence of the CFSV antigen in the bovine species. The virus was found in commercialized batches of fetal bovine serum (FBS) of Chinese origin and in bovine herds in in the provinces of Henan and Jiangsu, China, and in Tamil Nadu and Meghalaya, southern and northeastern states of India, respectively. Detection was done using antigen capture ELISA and RTPCR tests. In certain cases, animals with natural infection showed clinical signs and reproduction was also affected. Genetic characterization was performed considering the 5'UTR sequences of the bovine strains. In addition, the entire CSFV E2 genomic region could be amplified from two positive animals. The bovine strains were genetically related to the Chinese CSFV live attenuated hog cholera lapinized vaccine (HCLV) strain used in pigs, sharing sequence characteristics. The vaccine strain HCLV was widely used in China to protect bovines and yaks from bovine viral diarrhea, and, as a possible consequence, inducing an adaptation in cattle and a further natural diffusion. Furthermore, a contaminant strain from China was genetically distant from all other previously described genotypes of the CSFV. This suggests also the occurrence of micro evolutive step in the species related to geographical segregation. These observations deserve attention and further investigations, especially relevant in countries where CSFV control and eradication strategies are applied.
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Enfermedades de los Bovinos , Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Enfermedades de los Porcinos , Vacunas Virales , Bovinos , Animales , Porcinos , Virus de la Fiebre Porcina Clásica/genética , India/epidemiología , Peste Porcina Clásica/epidemiología , Peste Porcina Clásica/prevención & control , China/epidemiologíaRESUMEN
BACKGROUND: Classical swine fever (CSF) is a fatal contagious disease affecting pigs caused by classical swine fever virus (CSFV). The disease can be transmitted by pigs and wild boars, and it is difficult to prevent and control. To obtain necessary information to establish the CSFV resistant animals in a future study, we designed lentiviral vector-delivered short hairpin RNAs (shRNAs) targeting the conserved domain III of the internal ribosomal entry site (IRES) of the CSFV genomic RNA. RESULTS: First, we confirmed the effects of siRNAs on CSFV-IRES activity. We observed significant inhibition of CSFV-IRES activity by si42 (domain IIIa), si107 (domain IIIc), and si198 (domain IIIf) in SK-L cells and si56 (domain IIIb), si142 (domain IIId1) and si198 in HEK293 cells without affecting the amount of luciferase RNA. Next, we constructed lentiviral vectors expressing shRNA based on siRNA sequences. Treatment with shRNA-expressing lentivirus was examined at 7 and 14 days post infection in SK-L cells and HEK293 cells, and CSFV-IRES was significantly suppressed at 14 days (sh42) post infection in HEK293 cells without significant cytotoxicity. Next, we examined the silencing effect of siRNA on CSFV replicon RNA and observed a significant effect by si198 after 2 days of treatment and by shRNA-expressing lentivirus (sh56, sh142, and sh198) infection after 14 days of treatment. Treatment of sh198-expressing lentivirus significantly suppressed CSFV infection at 3 days after infection. CONCLUSION: The IRES targeting sh198 expressing lentivirus vector can be a candidate tool for CSFV infection control.
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Virus de la Fiebre Porcina Clásica , Humanos , Animales , Porcinos , ARN Interferente Pequeño/genética , Virus de la Fiebre Porcina Clásica/genética , Células HEK293 , Genómica , Lentivirus/genéticaRESUMEN
A previous study proved that vGPE- mainly maintains the properties of classical swine fever (CSF) virus, which is comparable to the GPE- vaccine seed and is a potentially valuable backbone for developing a CSF marker vaccine. Chimeric viruses were constructed based on an infectious cDNA clone derived from the live attenuated GPE- vaccine strain as novel CSF vaccine candidates that potentially meet the concept of differentiating infected from vaccinated animals (DIVA) by substituting the glycoprotein Erns of the GPE- vaccine strain with the corresponding region of non-CSF pestiviruses, either pronghorn antelope pestivirus (PAPeV) or Phocoena pestivirus (PhoPeV). High viral growth and genetic stability after serial passages of the chimeric viruses, namely vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns, were confirmed in vitro. In vivo investigation revealed that two chimeric viruses had comparable immunogenicity and safety profiles to the vGPE- vaccine strain. Vaccination at a dose of 104.0 TCID50 with either vGPE-/PAPeV Erns or vGPE-/PhoPeV Erns conferred complete protection for pigs against the CSF virus challenge in the early stage of immunization. In conclusion, the characteristics of vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns affirmed their properties, as the vGPE- vaccine strain, positioning them as ideal candidates for future development of a CSF marker vaccine.
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Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Pestivirus , Vacunas Virales , Porcinos , Animales , Vacunas Marcadoras , Anticuerpos Antivirales , Vacunas Atenuadas , Virus de la Fiebre Porcina Clásica/genética , Pestivirus/genéticaRESUMEN
C-strain, also known as the HCLV strain, is a well-known live attenuated vaccine against classical swine fever (CSF), a devastating disease caused by classical swine fever virus (CSFV). Vaccination with C-strain induces a rapid onset of protection, which is associated with virus-specific gamma interferon (IFN-γ)-secreting CD8+ T cell responses. The E2 protein of CSFV is a major protective antigen. However, the T cell epitopes on the E2 protein remain largely unknown. In this study, eight overlapping nonapeptides of the E2 protein were predicted and synthesized to screen for potential T cell epitopes on the CSFV C-strain E2 protein. Molecular docking was performed on the candidate epitopes with the swine leukocyte antigen-1*0401. The analysis obtained two highly conserved T cell epitopes, 90STEEMGDDF98 and 331ATDRHSDYF339, which were further identified by enzyme-linked immunospot assay. Interestingly, the mutants deleting or substituting the epitopes are nonviable. Further analysis demonstrated that 90STEEMGDDF98 is crucial for the E2 homodimerization, while CSFV infection is significantly inhibited by the 331ATDRHSDYF339 peptide treatment. The two novel T cell epitopes can be used to design new vaccines that are able to provide rapid-onset protection.
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Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Enfermedades de los Porcinos , Vacunas Virales , Porcinos , Animales , Virus de la Fiebre Porcina Clásica/genética , Epítopos de Linfocito T , Simulación del Acoplamiento Molecular , Peste Porcina Clásica/prevención & control , Proteínas del Envoltorio Viral/genética , Linfocitos T CD8-positivos , Interferón gamma , Anticuerpos AntiviralesRESUMEN
Classical swine fever virus (CSFV) is a highly pathogenic RNA virus belonging to the Flaviviridae family that can cause deadly classical swine fever (CSF) in pigs. However, the molecular details of virus replication in the host are still unclear. Our previous studies have reported that several Rab proteins mediate CSFV entry into host cells, but it is unknown whether CSFV hijacks other Rab proteins for effective viral infection. Here, we systematically studied the role of Rab14 protein in regulating lipid metabolism for promoting viral assembly. First, Rab14 knockdown and overexpression significantly affected CSFV replication, indicating the essential role of Rab14 in CSFV infection. Interestingly, Rab14 could significantly affect virus replication in the late stage of infection. Mechanistically, CSFV NS5A recruited Rab14 to the ER, followed by ceramide transportation to the Golgi apparatus, where sphingomyelin was synthesized. The experimental data of small molecule inhibitors, RNA interference, and replenishment assay showed that the phosphatidylinositol-3-kinase (PI3K)/AKT/AS160 signaling pathway regulated the function of Rab14 to affect the transport of ceramide. More importantly, sphingomyelin on the Golgi apparatus contributed to the assembly of viral particles. Blockage of the Rab14 regulatory pathway induced the reduction of the content of sphingomyelin on the Golgi apparatus, impairing the assembly of virus particles. Our study clarifies that Rab14 regulates lipid metabolism and promotes CSFV replication, which provides insight into a novel function of Rab14 in regulating vesicles to transport lipids to the viral assembly factory. IMPORTANCE The Rab protein family members participate in the viral replication of multiple viruses and play important roles in the virus infection cycle. Our previous research focused on Rab5/7/11, which regulated the trafficking of vesicles in the early stage of CSFV infection, especially in viral endocytosis. However, the role of other Rab proteins in CSFV replication is unclear and needs further clarification. Strikingly, we screened some Rabs and found the important role of Rab14 in CSFV infection. Virus infection mobilized Rab14 to regulate the vesicle to transport ceramide from the ER to the Golgi apparatus, further promoting the synthesis of sphingomyelin and facilitating virus assembly. The treatment of inhibitors showed that the lipid transport mediated by Rab14 was regulated by the PI3K/AKT/AS160 signaling pathway. Knockdown of Rab14 or the treatment with PI3K/AKT/AS160 inhibitors reduced the ceramide content in infected cells and hindered virus assembly. Our study is the first to explain that vesicular lipid transport regulated by Rab promotes CSFV assembly, which is conducive to the development of antiviral drugs.
Asunto(s)
Ceramidas , Virus de la Fiebre Porcina Clásica , Proteínas de Unión al GTP Monoméricas , Ensamble de Virus , Animales , Ceramidas/metabolismo , Peste Porcina Clásica , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/fisiología , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esfingomielinas/metabolismo , Porcinos , Replicación ViralRESUMEN
Neutralizing antibodies (nAbs) can be used before or after infection to prevent or treat viral diseases. However, there are few efficacious nAbs against classical swine fever virus (CSFV) that have been produced, especially the porcine-originated nAbs. In this study, we generated three porcine monoclonal antibodies (mAbs) with in vitro neutralizing activity against CSFV, aiming to facilitate the development of passive antibody vaccines or antiviral drugs against CSFV that offer the advantages of stability and low immunogenicity. Pigs were immunized with the C-strain E2 (CE2) subunit vaccine, KNB-E2. At 42 days post vaccination (DPV), CE2-specific single B cells were isolated via fluorescent-activated cell sorting (FACS) baited by Alexa Fluor™ 647-labeled CE2 (positive), goat anti-porcine IgG (H + L)-FITC antibody (positive), PE mouse anti-pig CD3ε (negative) and PE mouse anti-pig CD8a (negative). The full coding region of IgG heavy (H) chains and light (L) chains was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Overall, we obtained 3 IgG H chains, 9 kappa L chains and 36 lambda L chains, which include three paired chains (two H + κ and one H + λ). CE2-specific mAbs were successfully expressed in 293T cells with the three paired chains. The mAbs exhibit potent neutralizing activity against CSFVs. They can protect ST cells from infections in vitro with potent IC50 values from 14.43 µg/mL to 25.98 µg/mL for the CSFV C-strain, and 27.66 µg/mL to 42.61 µg/mL for the CSFV Alfort strain. This study is the first report to describe the amplification of whole-porcine IgG genes from single B cells of KNB-E2-vaccinated pig. The method is versatile, sensitive, and reliable. The generated natural porcine nAbs can be used to develop long-acting and low-immunogenicity passive antibody vaccine or anti-CSFV agents for CSF control and prevention.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Vacunas Virales , Porcinos , Animales , Ratones , Virus de la Fiebre Porcina Clásica/genética , Anticuerpos Monoclonales , Anticuerpos Antivirales , Anticuerpos Neutralizantes , Inmunoglobulina G , Proteínas del Envoltorio Viral/genéticaRESUMEN
Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases in pigs. Most pig farms in China are vaccinated against these two diseases. Gene-deleted pseudorabies virus (PRV) can be used to develop promising and economical multivalent live attenuated viral vector vaccines. It has been reported that recombinant PRV can express a truncated E2 protein (1-338 aa), but it has not been reported that recombinant PRV can express a full-length E2 protein. We constructed nine groups of E2 proteins with different expression forms and found that the E2 protein could be expressed in vitro only when the transmembrane region of E2 was removed and the signal peptide was added. Analysis of the transmembrane region of E2 revealed that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. By mutating an amino acid to reduce the hydrophobicity of the transmembrane region, it was found that the full-length mutant of E2 (E2FL-muta3 or E2FL-muta4) could be expressed. The expressed full-length mutant E2 could also localize to the cell membrane. Mice immunized with a PRV vector vaccine expressing E2FL-muta3 or E2FL-muta4 developed specific cellular immunity to the E2 protein and stimulated higher levels of E2 antibody than mice immunized with a PRV vector expressing truncated E2. After immunizing the rabbits, the lethal challenge by PRV-ZJ2013 and the febrile response elicited by CSFV were simultaneously prevented. These results suggest that rPRV-dTK/gE-E2FL-muta4 is a promising bivalent vaccine against CSFV and PRV infections.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Herpesvirus Suido 1 , Seudorrabia , Enfermedades de los Porcinos , Vacunas Virales , Animales , Porcinos , Ratones , Conejos , Herpesvirus Suido 1/genética , Virus de la Fiebre Porcina Clásica/genética , Aminoácidos/genética , Vacunas Virales/genética , Anticuerpos Antivirales , Inmunización , Seudorrabia/prevención & control , Mutación , Proteínas del Envoltorio Viral/genéticaRESUMEN
To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5'- and 3'-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV Erns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV Erns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.