Development of an indirect ELISA for the immunoprotection evaluation of E2 antibodies against classical swine fever virus.

The Chinese government's reclassification of Classical Swine Fever (CSF) from a class Ⅰ to a class Ⅱ animal infectious disease, now also including CSF under the disease eradication program, reflects the significant progress made through extensive immunization with CSF vaccines. In light of this advancement, there is an imperative need for an expedient and accurate method to assess the levels of immunoprotection against classical swine fever virus (CSFV) in vaccinated pigs, a critical component in the campaign to eradicate the disease. This study develops an indirect enzyme-linked immunosorbent assay (iELISA) based on a highly glycosylated E2 protein stable expressed in CHO-K1 mammalian cells. Statistical analysis revealed strong positive correlations between the iELISA and VNT results (r = 0.9063, p < 0.0001) that were much greater than those between the IDEXX ELISA and VNT results (r = 0.8126, p < 0.0001). Taking the VNT data as the standard, the consistency of the iELISA (κ =0.880) was greater than that of the IDEXX ELISA (κ =0.699). In summary, the iELISA provides a more efficient and precise method for assessing CSFV immunity in pigs. Its reliable detection of immunoprotection levels against CSFV makes it an essential tool for optimizing CSF vaccination strategies. Consequently, its application can significantly support the ongoing efforts to eradicate CSF.

Safety and DIVA Capability of Novel Live Attenuated Classical Swine Fever Marker Vaccine Candidates in Pregnant Sows.

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.

Assessment of the Safety Profile of Chimeric Marker Vaccine against Classical Swine Fever: Reversion to Virulence Study.

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.

Recent decline in hepatitis E virus prevalence among wild boars in Japan: Probably due to countermeasures implemented in response to outbreaks of classical swine fever virus infection.

Previous studies have emphasized the necessity of surveillance and control measures for hepatitis E virus (HEV) infection in wild boars, an important reservoir of HEV. To assess the current situation of HEV infection in wild boars in Japan, this study investigated the prevalence and genetic diversity of HEV among wild boars captured in 16 prefectures of Japan during 2018-2023. Serum samples from 968 wild boars were examined for anti-HEV IgG antibodies and HEV RNA. The prevalence of anti-HEV IgG varied geographically from 0 % to 35.0 %. HEV RNA was detected in 3.6 % of boars, with prevalence varying by prefecture from 0 % to 22.2 %. Genotype 3 was the most prevalent genotype (91.9 %), followed by genotype 4 (5.4 %), with one strain closely related to genotype 6. The prevalence of HEV infection among wild boars decreased from 2018/2019 to 2022/2023 with significant declines in levels of anti-HEV IgG antibodies (14.5 % vs. 6.2 %, P < 0.0001) and HEV RNA (7.6 % vs. 1.5 %, P < 0.0001). Regional analysis showed varying trends, with no HEV RNA-positive boars found in several regions in recent years. A plausible factor contributing to the decline in HEV infection is the application of countermeasures, including installing fences to prevent intrusion into pig farms, implemented in response to the emergence of classical swine fever virus (CSFV) infection in wild boars and domestic pigs, with incidents reported annually since 2018. Further investigation is warranted to explore the association between countermeasures to CSFV infection and the decrease in HEV infection among wild boars.

Enhancement of systemic virus-specific T lymphocyte responses in pigs supplemented with algae-derived ß-glucan.

Algae-derived ß-glucan has been widely used as a feed additive in the swine industry. The supplementation of ß-glucan aims to improve growth performance and modulate the immunity of pigs. However, the potential effects of supplementing ß-glucan from algae on immune responses in pigs-specifically antigen-specific immunity-must be determined. In this study, the effects of algae-derived ß-glucan supplementation on growth performance, virus neutralising antibody and virus-specific T lymphocytes responses were investigated in pigs. Piglets (n=112 per treatment) were assigned to three treatments including non-supplemented group (control), ß-glucan 100 g/ton supplemented group (BG100), and ß-glucan 200 g/ton supplemented group (BG200). In this study, production performance of pigs was not found to be different between the experimental groups. Pigs supplemented with ß-glucan exhibited high levels of classical swine fever virus (CSFV)-specific producing T lymphocytes and neutralising antibody titer, compared to the control group. Interestingly, supplementation of ß-glucan significantly enhanced porcine reproductive and respiratory syndrome virus (PRRSV)-specific interferon-gamma (IFN-γ) producing T lymphocytes, including CD4+, CD8+, and CD4+CD8+ T lymphocyte subpopulations. Moreover, PRRS modified live vaccine (MLV) viremia was reduced in earlier for ß-glucan-supplemented pigs compared to the control group. The findings indicate that the algae-derived ß-glucan possesses biological potential as an immunomodulatory substance to enhance antiviral immunity, which may contribute to disease resistance in pigs.

Application of machine learning with large-scale data for an effective vaccination against classical swine fever for wild boar in Japan.

Classical swine fever has been spreading across the country since its re-emergence in Japan in 2018. Gifu Prefecture has been working diligently to control the disease through the oral vaccine dissemination targeting wild boars. Although vaccines were sprayed at 14,000 locations between 2019 and 2020, vaccine ingestion by wild boars was only confirmed at 30% of the locations. Here, we predicted the vaccine ingestion rate at each point by Random Forest modeling based on vaccine dissemination data and created prediction surfaces for the probability of vaccine ingestion by wild boar using spatial interpolation techniques. Consequently, the distance from the vaccination point to the water source was the most important variable, followed by elevation, season, road density, and slope. The area under the curve, model accuracy, sensitivity, and specificity for model evaluation were 0.760, 0.678, 0.661, and 0.685, respectively. Areas with high probability of wild boar vaccination were predicted in northern, eastern, and western part of Gifu. Leave-One-Out Cross Validation results showed that Kriging approach was more accurate than the Inverse distance weighting method. We emphasize that effective vaccination strategies based on epidemiological data are essential for disease control and that our proposed tool is also applicable for other wildlife diseases.

Self-Assembling E2-Based Nanoparticles Improve Vaccine Thermostability and Protective Immunity against CSFV.

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.

Elucidation of novel SNPs affecting immune response to classical swine fever vaccination in pigs using immunogenomics approach.

The host genetic makeup plays a significant role in causing the within-breed variation among individuals after vaccination. The present study was undertaken to elucidate the genetic basis of differential immune response between high and low responder Landlly (Landrace X Ghurrah) piglets vis-à-vis CSF vaccination. For the purpose, E2 antibody response against CSF vaccination was estimated in sampled animals on the day of vaccination and 21-day post-vaccination as a measure of humoral immune response. Double-digestion restriction associated DNA (ddRAD) sequencing was undertaken on 96 randomly chosen Landlly piglets using Illumina HiSeq platform. SNP markers were called using standard methodology. Genome-wide association study (GWAS) was undertaken in PLINK program to identify the informative SNP markers significantly associated with differential immune response. The results revealed significant SNPs associated with E2 antibody response against CSF vaccination. The genome-wide informative SNPs for the humoral immune response against CSF vaccination were located on SSC10, SSC17, SSC9, SSC2, SSC3 and SSC6. The overlapping and flanking genes (500Kb upstream and downstream) of significant SNPs were CYB5R1, PCMTD2, WT1, IL9R, CD101, TMEM64, TLR6, PIGG, ADIPOR1, PRSS37, EIF3M, and DNAJC24. Functional enrichment and annotation analysis were undertaken for these genes in order to gain maximum insights into the association of these genes with immune system functionality in pigs. The genetic makeup was associated with differential immune response against CSF vaccination in Landlly piglets while the identified informative SNPs may be used as suitable markers for determining variation in host immune response against CSF vaccination in pigs.

Infection of PRRSV inhibits CSFV C-strain replication by inducing macrophages polarization to M1.

It is a common sense that porcine reproductive and respiratory syndrome virus (PRRSV) infection could cause immune failure of classical swine fever (CSF) vaccine, and porcine alveolar macrophages (PAMs) are the target cells of both. To elucidate the role of macrophage polarization in PRRSV infection induced CSF vaccine failure, an immortal porcine alveolar macrophage line PAM39 cell line was used to investigate the effect of PRRSV or/and CSFV C-strain (CSFV-C) infection on macrophage polarization in vitro. Interestingly, PRRSV single infection or PRRSV co-infection with CSFV-C promoted PAM39 cells to M1, while CSFV-C single infection induced PAM39 cells to M2. After the construction of M1 and M2 PAM39 cells polarization models, M1 polarized PAM39 cells were found to inhibit the replication of CSFV-C, and Chinese medicine such as matrine, ginsenosides and astragalus polysaccharides could alleviate the polarization of PAM39 cells and the replication of CSFV-C. Furthermore, interferon (IFN)-γ and lipopolysaccharide (LPS) co-stimulation induced NF-κB activation while matrine treatment blocked M1 polarization-induced NF-κB pathway activation. These findings provided a theoretical basis for designing a new strategy to improve the immune effect of CSFV-C based on porcine alveolar macrophage polarization subtypes.

Efficacy of an orally administered classical swine fever live marker vaccine (Flc-LOM-BErns strain) in pigs.

In several countries, classical swine fever (CSF) has not been detected in domestic pigs, but has been detected in wild boars, making the disease difficult to control. To overcome this problem, we inoculated pigs with a CSF live marker vaccine (Flc-LOM-BErns strain), which has "distinguish infection from vaccinated animals (DIVA)" function, to determine whether it is suitable as an oral vaccine specifically for wild boars. Pigs inoculated intramuscularly or orally with the Flc-LOM-BErns vaccine were challenged 2 or 4 weeks later, respectively, with virulent CSFV. Pigs administered the oral Flc-LOM-BErns strain (105.0 and 6.0 TCID50/dose), and those vaccinated intramuscularly (103.0 TCID50/dose), had normal numbers of leukocytes and normal body temperature. Also, they generated protective neutralizing antibodies and anti-BVDV Erns antibodies. In addition, all pigs in these groups survived, with no CSFV RNA detected in feces, spleen, or other organs. Thus, the Flc-LOM-BErns vaccine shows excellent safety and efficacy, while having DIVA function and suitability for oral inoculation.

Circulation of classical swine fever virus (CSFV) strains of bovine origin in China and India.

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.

Analysis of effective spatial range of oral vaccination against classical swine fever for wild boar.

Classical swine fever (CSF) re-emerged in Gifu Prefecture, central Japan, in September 2018 and is currently widespread in wild boar populations. Due to its widespread in wild boars, an oral mass vaccination strategy was initiated in March 2019, employing a commercial bait vaccine that is a live attenuated vaccine. To enhance the effectiveness of oral vaccination, it is crucial to determine the vaccine's effective spatial range. This understanding is essential for devising a comprehensive vaccination strategy, which should also include a preliminary investigation of wild boar habitats before vaccination. This study aimed to estimate the effective range of oral vaccination for wild boars against CSF by analyzing the geographical relationship between immune wild boars and vaccination points within the vaccination areas in Gifu Prefecture. This study utilized oral vaccination data from April 2021 to March 2022. The prevalence of CSF infections in wild boars remained below 5% in this period, suggesting limited disease transmission and immune wild boars were considered to be induced by the effect of vaccination. Two vaccination campaigns were conducted during this period, with almost 2000 vaccination points each. To investigate the factors associated with the intensity (i.e., density) of immune wild boar, the nearest distances to a vaccination point and to a susceptible wild boar were evaluated as explanatory variables. The Rhohat procedure and point process model were utilized to analyze the relationship between the intensity of immune wild boars and the explanatory variables. The point process model revealed a significant decrease in the intensity of immune wild boars when the distance from the nearest vaccination point exceeded 500 m, indicating that the effective spatial range of bait vaccination is within 500 m of the vaccination point. Although the distance to the nearest susceptible animal did not show significance in the model, Rhohat plots indicated that the intensity of immune wild boars decreased at distances greater than 1200 m from the nearest susceptible wild boar. This finding highlights the importance of investigating susceptible wild boar populations within a range of at least 1200 m from a vaccination point before implementation. The present study revealed the effective range of oral vaccination for wild boars against CSF and indicated the importance of investigating susceptible wild boar habitats around vaccination points before the implementation of vaccination. These findings may help improve the effectiveness of oral vaccinations.

Development and application of classical swine fever virus monoclonal antibodies derived from single B cells.

Vaccination with E2 subunit vaccines is currently the main measure to control classical swine fever virus (CSFV), which is an endemic disease, and detection of antibodies against CSFV E2 is the most effective way to evaluate herd immunity. In the present study, the E2 protein was expressed by a baculovirus expression system, and two monoclonal antibodies (mAbs), namely, 3A9 and 4F7, were successfully produced using techniques for the isolation of single B cells from splenocytes from mice immunized with the E2 protein. Moreover, two linear B-cell epitopes, 25GLTTTWKEYSHDLQL39 and 259GNTTVKVHASDERGP273, reactive to 3A9 and 4F7, respectively, were identified using epitope mapping of the E2 protein. In addition, the diagnostic performance of the two mAbs was evaluated using blocking enzyme-linked immunosorbent assay (bELISA), and the results showed that the two mAbs had high diagnostic specificity (96.08%, 94.38%) and diagnostic sensitivity (97.49%, 95.97%). Together, these findings identify two ideal candidate peptides and matching mAbs for a new method of CSFV diagnosis, which will contribute to the control and eradication of classical swine fever.

Assessing mammal fence crossing and local fence management in relation to classical swine fever spread in Japan.

Infectious diseases at the wildlife-livestock-human interface have become a crucial issue, and evidence-based measures are growing increasingly important. One countermeasure against animal diseases in wildlife is using fencing to contain and reduce disease spread and transmission rates between wild populations; however, quantitative assessments on fencing are rare. Moreover, existing research on fencing has highlighted knowledge gaps on the social and ecological aspects relevant to the use and design of fences. To control the spread of classical swine fever, fences were installed from the east to west in Gifu Prefecture, Japan, by March 2019, with the aim of restricting wild boar movement. To clarify the process of installation and maintenance of the fences, we conducted semi-structured interviews with prefectural government officers in Gifu Prefecture. Additionally, we installed infrared-triggered cameras at fence locations with and without gaps to evaluate the fence permeability of mammals. We used a generalized linear mixed model to evaluate the relationship between the presence of gaps and the relative abundance and permeability of each mammal. Our findings showed that the occurrence of gaps was inevitable during the installation and management of wide-area perimeter fence in Japan, partly because of social factors. For example, fences could not be installed on roads that were frequently used by residents and were not adequately maintained owing to budgetary reasons in some cases. Analysis of footage from the infrared-triggered cameras revealed that several mammal species crossed the fence at gaps and even had the ability to cross the gapless parts of the fences. Wild boars crossed through the gaps regularly. It is possible that Sika deer, Japanese serows, raccoons, Japanese badgers, raccoon dogs, Japanese macaques, and feral cats crossed through fence gaps because their relative abundance was high at gap locations. In contrast, Japanese hares slipped through the fence mesh rather than crossing through the gaps. In conclusion, we suggest that coordination and collaboration among related parties, a sufficient supply of fence materials, and securing a budget for fence maintenance are important for fence installation and maintenance. Furthermore, as fence gaps are inevitable, technical development of countermeasures for these gaps could be effective.

Rice-produced classical swine fever virus glycoprotein E2 with herringbone-dimer design to enhance immune responses.

Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone-dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 µg provided effective protection in pigs without interference from pre-existing antibodies. Crystal structure and small-angle X-ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines.

A Genetically Engineered Bivalent Vaccine Coexpressing a Molecular Adjuvant against Classical Swine Fever and Porcine Epidemic Diarrhea.

Classical swine fever (CSF) and porcine epidemic diarrhea (PED) are highly contagious viral diseases that pose a significant threat to piglets and cause substantial economic losses in the global swine industry. Therefore, the development of a bivalent vaccine capable of targeting both CSF and PED simultaneously is crucial. In this study, we genetically engineered a recombinant classical swine fever virus (rCSFV) expressing the antigenic domains of the porcine epidemic diarrhea virus (PEDV) based on the modified infectious cDNA clone of the vaccine strain C-strain. The S1N and COE domains of PEDV were inserted into C-strain cDNA clone harboring the mutated 136th residue of Npro and substituted 3'UTR to generate the recombinant chimeric virus vC/SM3'UTRN-S1NCOE. To improve the efficacy of the vaccine, we introduced the tissue plasminogen activator signal (tPAs) and CARD domain of the signaling molecule VISA into vC/SM3'UTRN-S1NCOE to obtain vC/SM3'UTRN-tPAsS1NCOE and vC/SM3'UTRN-CARD/tPAsS1NCOE, respectively. We characterized three vaccine candidates in vitro and investigated their immune responses in rabbits and pigs. The NproD136N mutant exhibited normal autoprotease activity and mitigated the inhibition of IFN-ß induction. The introduction of tPAs and the CARD domain led to the secretory expression of the S1NCOE protein and upregulated IFN-ß induction in infected cells. Immunization with recombinant CSFVs expressing secretory S1NCOE resulted in a significantly increased in PEDV-specific antibody production, and coexpression of the CARD domain of VISA upregulated the PEDV-specific IFN-γ level in the serum of vaccinated animals. Notably, vaccination with vC/SM3'UTRN-CARD/tPAsS1NCOE conferred protection against virulent CSFV and PEDV challenge in pigs. Collectively, these findings demonstrate that the engineered vC/SM3'UTRN-CARD/tPAsS1NCOE is a promising bivalent vaccine candidate against both CSFV and PEDV infections.

Identification of two novel T cell epitopes on the E2 protein of classical swine fever virus C-strain.

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.

Enhanced passive surveillance for early detection of African and classical swine fevers.

African swine fever (ASF) and classical swine fever (CSF) are transboundary animal diseases (TADs) of pigs. Much effort and resources are regularly put into preventing these diseases' introduction in free areas. Passive surveillance activities bring the highest chances for the early detection of TAD incursions because they are routinely and widely conducted at farms, and because these activities focus on the time between introduction and when the first sample is sent for diagnostic testing. The authors proposed the implementation of an enhanced passive surveillance (EPS) protocol based on collecting data through participatory surveillance actions using an objective and adaptable scoring system to aid the early detection of ASF or CSF at the farm level. The protocol was applied in two commercial pig farms for ten weeks in the Dominican Republic, which is a CSF- and ASF-infected country. This study was a proof of concept, based on the EPS protocol to aid detection of substantial variations in the risk score triggering testing. One of the followed farms had score variation, which triggered testing of the animals, although the test results were negative. The study enables assessment of some of the weaknesses associated with passive surveillance and provides lessons applicable to the problem. Results demonstrate the potential for overcoming some issues preventing the broad application of EPS protocols and suggest that standardised approaches may contribute to the early detection of CSF and ASF introductions.

La peste porcine africaine (PPA) et la peste porcine classique (PPC) sont des maladies animales transfrontalières touchant les porcs. De nombreux efforts et ressources sont régulièrement alloués pour prévenir l'introduction de ces maladies dans des zones indemnes. Les activités de surveillance passive offrent les meilleures perspectives de détection précoce des incursions de maladies animales transfrontalières parce qu'elles sont menées de manière systématique et exhaustive dans les élevages, et parce qu'elles se concentrent sur la période entre l'introduction de la maladie et le moment où le premier échantillon est envoyé au laboratoire pour analyse. Les auteurs proposent la mise en oeuvre d'un protocole de surveillance passive renforcée fondé sur la collecte de données via des actions de surveillance participative utilisant un système de notation objectif et adaptable, en vue d'une détection précoce de la PPA et de la PPC dans les élevages. Ce protocole a été appliqué en République dominicaine, pays infecté par la PPA et la PPC, dans deux élevages porcins commerciaux pendant dix semaines. Cette étude était destinée à valider le principe de la méthode et se fondait sur le protocole de surveillance passive renforcée pour mieux détecter les variations substantielles de la note de risque qui conduisent à tester les animaux. L'un des élevages suivis a présenté une variation de cette note, ce qui a conduit à tester les animaux mais les tests se sont révélés négatifs. L'étude permet d'évaluer certaines des faiblesses associées à la surveillance passive et apporte des enseignements applicables à ce problème. Les résultats illustrent le potentiel de l'approche à surmonter certaines des problématiques empêchant l'application extensive des protocoles de surveillance passive renforcée. Ils suggèrent également que des approches normalisées pourraient contribuer à la détection précoce des cas d'introduction de la PPC et de la PPA.

La peste porcina africana (PPA) y la peste porcina clásica (PPC) son enfermedades animales transfronterizas que afectan al cerdo. Periódicamente se dedican grandes esfuerzos y cuantiosos recursos a evitar que estas enfermedades penetren en zonas que están exentas de ellas. Las actividades de vigilancia pasiva son las más eficaces para detectar con prontitud toda incursión de enfermedades animales transfronterizas, no solo por la regularidad y amplitud con que se llevan a cabo en las explotaciones, sino también porque inciden específicamente en el intervalo entre la penetración de una enfermedad y el momento en que se envía la primera muestra para que sea sometida a pruebas de diagnóstico. Los autores propusieron que se aplicara un protocolo de vigilancia pasiva reforzada que reposaba en la obtención de datos mediante actividades de vigilancia participativa, empleando para ello un sistema objetivo y adaptable de puntuación que ayudaba a detectar con prontitud la presencia en las explotaciones de PPA o PPC. Dicho protocolo fue aplicado a lo largo de diez semanas en dos explotaciones porcinas industriales de la República Dominicana, país en el que ambas infecciones están presentes. El estudio, que sirvió para poner a prueba la idea, pasaba por la aplicación del protocolo de vigilancia pasiva reforzada para ayudar a detectar variaciones sustanciales de la puntuación del nivel de riesgo que activa la realización de pruebas. En una de las explotaciones estudiadas se produjo una variación de la puntuación, cosa que activó la realización de pruebas en los animales, aunque estas arrojaron resultado negativo. El estudio aquí descrito permite evaluar algunos de los puntos débiles de la vigilancia pasiva y extraer enseñanzas aplicables al problema. Los resultados demuestran que es posible salvar algunas de las dificultades que impiden la aplicación generalizada de protocolos de vigilancia pasiva reforzada y dejan pensar que quizá el uso de planteamientos normalizados pueda ayudar a detectar con prontitud los casos de penetración de PPC o PPA.

Development of stable HEK293T cell pools expressing CSFV E2 protein: A potential antigen expression platform.

Large quantities of antigens are required since protective antigens, such as classical swine fever virus (CSFV) E2 protein, are widely used in diagnostic reagents and subunit vaccines. Compared to clonal cell lines and transient gene expression, stable cell pools provide a potential alternative platform to rapidly produce large amounts of antigens. In this work, firstly, Human embryonic kidney 293 T (HEK293T) cell pools expressing E2 protein were developed by transduction of lentiviral vectors. On the one hand, the SP7 was selected from 7 well-performing signal peptides to remarkably increase the production of E2 protein. On the other hand, it was found that high MOI could improve the expression of E2 protein by increasing gene copy numbers. Moreover, the HEK293T cell pools were evaluated for stability by passages and batch cultures, demonstrating that the cell pools were stable for at least 90 days. And then, the performance of the cell pools in batch, fed-batch, and semi-perfusion was studied. Among them, the titer of E2 protein was up to 2 g/L in semi-perfusion, which is currently the highest to the authors' knowledge. Finally, the aggregations and immunogenicity of the E2 protein were analyzed by SDS-PAGE and immunization of mice, respectively. There was no significant difference in aggregations and antibody titers of E2 protein in three culture methods. These results suggest that stable HEK293T cell pools are a promising and robust platform for rapid and efficient production of recombinant proteins.

Construction and efficacy of a new live chimeric C-strain vaccine with DIVA characteristics against classical swine fever.

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.