Development of a dual immunochromatographic test strip to detect E2 and Erns antibodies against classical swine fever.

Background: It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting Erns antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/Erns dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed. Methods: Recombinant E2 or Erns protein was transiently expressed in the plant benthamiana using Agrobacterium tumefaciens. ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or Erns protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and Erns antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates. Results: E2 and Erns proteins were successfully expressed in N. benthamiana-produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and Erns antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for Erns antibody detection. ICS confirmed the absence of CSFV Erns-specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates. Conclusion: E2 and Erns proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/Erns dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and Erns antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates.

Landscape analysis of available European data sources amenable for machine learning and recommendations on usability for rare diseases screening.

BACKGROUND: Patient registries and databases are essential tools for advancing clinical research in the area of rare diseases, as well as for enhancing patient care and healthcare planning. The primary aim of this study is a landscape analysis of available European data sources amenable to machine learning (ML) and their usability for Rare Diseases screening, in terms of findable, accessible, interoperable, reusable(FAIR), legal, and business considerations. Second, recommendations will be proposed to provide a better understanding of the health data ecosystem. METHODS: In the period of March 2022 to December 2022, a cross-sectional study using a semi-structured questionnaire was conducted among potential respondents, identified as main contact person of a health-related databases. The design of the self-completed questionnaire survey instrument was based on information drawn from relevant scientific publications, quantitative and qualitative research, and scoping review on challenges in mapping European rare disease (RD) databases. To determine database characteristics associated with the adherence to the FAIR principles, legal and business aspects of database management Bayesian models were fitted. RESULTS: In total, 330 unique replies were processed and analyzed, reflecting the same number of distinct databases (no duplicates included). In terms of geographical scope, we observed 24.2% (n = 80) national, 10.0% (n = 33) regional, 8.8% (n = 29) European, and 5.5% (n = 18) international registries coordinated in Europe. Over 80.0% (n = 269) of the databases were still active, with approximately 60.0% (n = 191) established after the year 2000 and 71.0% last collected new data in 2022. Regarding their geographical scope, European registries were associated with the highest overall FAIR adherence, while registries with regional and "other" geographical scope were ranked at the bottom of the list with the lowest proportion. Responders' willingness to share data as a contribution to the goals of the Screen4Care project was evaluated at the end of the survey. This question was completed by 108 respondents; however, only 18 of them (16.7%) expressed a direct willingness to contribute to the project by sharing their databases. Among them, an equal split between pro-bono and paid services was observed. CONCLUSIONS: The most important results of our study demonstrate not enough sufficient FAIR principles adherence and low willingness of the EU health databases to share patient information, combined with some legislation incapacities, resulting in barriers to the secondary use of data.

Child-to-adult transition: a survey of current practices within the European Reference Network for Rare Neurological Diseases (ERN-RND).

BACKGROUND: Transition from child-centered to adult-centered healthcare is a gradual process that addresses the medical, psychological, and educational needs of young people in the management of their autonomy in making decisions about their health and their future clinical assistance. This transfer is challenging across all chronic diseases but can be particularly arduous in rare neurological conditions. AIM: To describe the current practice on the transition process for young patients in centers participating in the European Reference Network for Rare Neurological Diseases (ERN-RND). METHODS: Members of the ERN-RND working group developed a questionnaire considering child-to-adult transition issues and procedures in current clinical practice. The questionnaire included 20 questions and was sent to members of the health care providers (HCPs) participating in the network. RESULTS: Twenty ERN-RND members (75% adult neurologists; 25% pediatricians; 5% nurses or study coordinators) responded to the survey, representing 10 European countries. Transition usually occurs between 16 and 18 years of age, but 55% of pediatric HCPs continue to care for their patients until they reach 40 years of age or older. In 5/20 ERN-RND centers, a standardized procedure managing transition is currently adopted, whereas in the remaining centers, the transition from youth to adult service is usually assisted by pediatricians as part of their clinical practice. CONCLUSIONS: This survey demonstrated significant variations in clinical practice between different centers within the ERN-RND network. It provided valuable data on existing transition programs and highlighted key challenges in managing transitions for patients with rare neurological disorders.

Performance of a Differentiation of Infected from Vaccinated Animals (DIVA) Classical Swine Fever Virus (CSFV) Serum and Oral Fluid Erns Antibody AlphaLISA Assay.

Classical swine fever virus (CSFV) is an OIE-listed disease that requires effective surveillance tools for its detection and control. The aim of this study was to develop and evaluate the diagnostic performance of a novel CSFV Erns IgG AlphaLISA for both serum and oral fluid specimens that would likewise be compatible with the use of CSFV E2 DIVA vaccines. Test performance was evaluated using a panel of well-characterized serum (n = 760) and individual (n = 528) or pen-based (n = 30) oral fluid samples from four groups of animals: (1) negative controls (n = 60 pigs); (2) inoculated with ALD strain wild-type CSFV (n = 30 pigs); (3) vaccinated with LOM strain live CSFV vaccine (n = 30 pigs); and (4) vaccinated with live CSFV marker vaccine on commercial farms (n = 120 pigs). At a cutoff of S/P ≥ 0.7, the aggregate estimated diagnostic sensitivities and specificities of the assay were, respectively, 97.4% (95% CI 95.9%, 98.3%) and 100% for serum and 95.4% (95% CI 92.9%, 97.0%) and 100% for oral fluid. The Erns IgG antibody AlphaLISA combined DIVA capability with solid diagnostic performance, rapid turnaround, ease of use, and compatibility with both serum and oral fluid specimens.

[Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis].

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

A Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Protect Pigs against a Virulent CSFV Challenge.

Classical swine fever (CSF) remains one of the most economically significant viral diseases affecting domestic pigs and wild boars worldwide. To develop a safe and effective vaccine against CSF, we have constructed a triple gene-deleted pseudorabies virus (PRVtmv)-vectored bivalent subunit vaccine against porcine circovirus type 2b (PCV2b) and CSFV (PRVtmv+). In this study, we determined the protective efficacy of the PRVtmv+ against virulent CSFV challenge in pigs. The results revealed that the sham-vaccinated control group pigs developed severe CSFV-specific clinical signs characterized by pyrexia and diarrhea, and became moribund on or before the seventh day post challenge (dpc). However, the PRVtmv+-vaccinated pigs survived until the day of euthanasia at 21 dpc. A few vaccinated pigs showed transient diarrhea but recovered within a day or two. One pig had a low-grade fever for a day but recovered. The sham-vaccinated control group pigs had a high level of viremia, severe lymphocytopenia, and thrombocytopenia. In contrast, the vaccinated pigs had a low-moderate degree of lymphocytopenia and thrombocytopenia on four dpc, but recovered by seven dpc. Based on the gross pathology, none of the vaccinated pigs had any CSFV-specific lesions. Therefore, our results demonstrated that the PRVtmv+ vaccinated pigs are protected against virulent CSFV challenge.

Challenges in mapping European rare disease databases, relevant for ML-based screening technologies in terms of organizational, FAIR and legal principles: scoping review.

Background: Given the increased availability of data sources such as hospital information systems, electronic health records, and health-related registries, a novel approach is required to develop artificial intelligence-based decision support that can assist clinicians in their diagnostic decision-making and shorten rare disease patients' diagnostic odyssey. The aim is to identify key challenges in the process of mapping European rare disease databases, relevant to ML-based screening technologies in terms of organizational, FAIR and legal principles. Methods: A scoping review was conducted based on the PRISMA-ScR checklist. The primary article search was conducted in three electronic databases (MEDLINE/Pubmed, Scopus, and Web of Science) and a secondary search was performed in Google scholar and on the organizations' websites. Each step of this review was carried out independently by two researchers. A charting form for relevant study analysis was developed and used to categorize data and identify data items in three domains - organizational, FAIR and legal. Results: At the end of the screening process, 73 studies were eligible for review based on inclusion and exclusion criteria with more than 60% (n = 46) of the research published in the last 5 years and originated only from EU/EEA countries. Over the ten-year period (2013-2022), there is a clear cycling trend in the publications, with a peak of challenges reporting every four years. Within this trend, the following dynamic was identified: except for 2016, organizational challenges dominated the articles published up to 2018; legal challenges were the most frequently discussed topic from 2018 to 2022. The following distribution of the data items by domains was observed - (1) organizational (n = 36): data accessibility and sharing (20.2%); long-term sustainability (18.2%); governance, planning and design (17.2%); lack of harmonization and standardization (17.2%); quality of data collection (16.2%); and privacy risks and small sample size (11.1%); (2) FAIR (n = 15): findable (17.9%); accessible sustainability (25.0%); interoperable (39.3%); and reusable (17.9%); and (3) legal (n = 33): data protection by all means (34.4%); data management and ownership (22.9%); research under GDPR and member state law (20.8%); trust and transparency (13.5%); and digitalization of health (8.3%). We observed a specific pattern repeated in all domains during the process of data charting and data item identification - in addition to the outlined challenges, good practices, guidelines, and recommendations were also discussed. The proportion of publications addressing only good practices, guidelines, and recommendations for overcoming challenges when mapping RD databases in at least one domain was calculated to be 47.9% (n = 35). Conclusion: Despite the opportunities provided by innovation - automation, electronic health records, hospital-based information systems, biobanks, rare disease registries and European Reference Networks - the results of the current scoping review demonstrate a diversity of the challenges that must still be addressed, with immediate actions on ensuring better governance of rare disease registries, implementing FAIR principles, and enhancing the EU legal framework.

Together4RD position statement on collaboration between European reference networks and industry.

Notwithstanding two decades of policy and legislation in Europe, aimed to foster research and development in rare conditions, only 5-6% of rare diseases have dedicated treatments. Given with the huge number of conditions classed as rare (which is increasing all the time), this equates to major unmet need for patients (over 30 million in the EU alone). Worryingly, the pace of Research and Innovation in Europe is lagging behind other regions of the world, and a seismic shift in the way in which research is planned and delivered is required, in order to remain competitive and-most importantly-bring meaningful, disease-altering treatments to those who desperately need them. The European Reference Networks (ERNs), launched in 2017, hold major potential to alleviate many of these challenges, and more, but only if adequately supported (financially, technically, and via robust policies and infrastructure) to realise that potential: and even then, only if able to forge robust collaborations harnessing the expertise, resources, knowledge and data of all stakeholders involved in rare disease, including Industry. To-date, however, ERN-Industry interactions have been largely limited, for a range of reasons (concerning barriers both tangible and perceived). This Position Statement analyses these barriers, and explains how Together4RD is seeking to move the needle here, by learning from case studies, exploring frameworks for collaboration, and launching pilots to explore how best to plan and deliver multistakeholder interactions addressing real research needs.

Identification of key proteins of cytopathic biotype bovine viral diarrhoea virus involved in activating NF-κB pathway in BVDV-induced inflammatory response.

Bovine viral diarrhoea virus (BVDV) is the etiologic agent of bovine viral diarrhea-mucosal disease, one of the most important viral diseases in cattle, with inflammatory diarrhea, enteritis, and mucosa necrosis as the major clinical manifestations. NF-κB is an important transcription complex that regulates the expression of genes involved in inflammation and immune responses. NLRP3 inflammasome plays a key role in the development of inflammatory diseases. However, whether the activation of NF-κB is crucial for BVDV infection-induced inflammatory responses remains unclear. The results of our present study showed that BVDV infection significantly activated the NF-κB pathway and promoted the expression of NLRP3 inflammasome components (NLRP3, ASC, pro-caspase 1) as well inflammatory cytokine pro-IL-1ß in BVDV-infected bovine cells, resulting in the cleavage of pro-caspase 1 and pro-IL-1ß into active form caspase 1 and IL-1ß. However, the levels of the NLRP3 inflammasome components and inflammatory cytokines were obviously inhibited, as well the cleavage of pro-caspase 1 and pro-IL-1ß in the pre-treated bovine cells with NF-κB-specific inhibitors after BVDV infection. Further, cytopathic biotype BVDV (cpBVDV) Erns and NS5A proteins with their key functional domains contributed to BVDV-induced inflammatory responses via activating the NF-κB pathway were confirmed experimentally. Especially, the NS5A can promote cholesterol synthesis and accelerate its augmentation, further activating the NF-κB signalling pathway. Conclusively, our data elucidate that the activation of NF-κB signaling pathway plays a crucial role in cpBVDV infection-induced inflammatory responses.

Characterization of monoclonal antibodies that specifically differentiate field isolates from vaccine strains of classical swine fever virus.

Classical swine fever virus (CSFV) is a major animal pathogen threatening the global pork industry. To date, numerous anti-CSFV monoclonal antibodies (mAbs) and their recognizing epitopes have been reported. However, few mAbs were systematically characterized for the capacity to differentiate field CSFV isolates from CSF vaccine strains, and the molecular basis associated with antigenic differences between vaccines and field isolates is still largely unknown. In the present study, recombinant CSFV structural glycoproteins E2 of both virulent and vaccine strains and Erns of vaccine strain were expressed using eukaryotic cells and murine mAbs generated against E2 and Erns. After serial screening and cloning of the hybridomas, the viral spectra of mAbs were respectively determined by indirect fluorescent antibody assay (IFA) using 108 CSFVs, followed by Western blot analysis using expressed glycoproteins of all CSFV sub-genotypes including vaccine strains. The antigenic structures recognized by these mAbs were characterized by epitope mapping using truncated, chimeric, and site-directed mutated E2 and Erns proteins. We have identified two vaccine-specific, one field isolate-specific, and two universal CSFV-specific mAbs and five novel conformational epitopes with critical amino acid (aa) motifs that are associated with these five mAbs: 213EPD215, 271RXGP274, and 37LXLNDG42 on E2 and 38CKGVP42, W81, and D100/V107 on Erns. Particularly, E213 of E2 is field isolate-specific, while N40 of E2 and D100/V107 of Erns are vaccine strain-specific. Results from our study further indicate that N40D of E2 mutation in field strains was likely produced under positive selection associated with long-term mass vaccination, leading to CSFV evasion of host immune response. Taking together, this study provides new insights into the antigenic structure of CSFV E2 and Erns and the differentiating mAbs will contribute to the development of a diagnostic strategy to differentiate C-strain vaccination from natural infection (DIVA) of CSFV in terms of elimination of CSF in China.

A Novel Competitive ELISA for Specifically Measuring and Differentiating Immune Responses to Classical Swine Fever C-Strain Vaccine in Pigs.

Classical swine fever can be controlled effectively by vaccination with C-strain vaccine. In this study, we developed a novel competitive enzyme-linked immunosorbent assay (cELISA) based on a C-strain Erns specific monoclonal antibody (mAb 1504), aiming to serologically measure immune responses to C-strain vaccine in pigs, and finally to make the C-strain become a DIVA-compatible vaccine. The cELISA system was established based on the strategy that mAb 1504 will compete with the C-strain induced antibodies in the pig serum to bind the C-strain Erns protein. The cELISA was optimized and was further evaluated by testing different categories of pig sera. It can efficiently differentiate C-strain immunized from wild-type CSFV-infected pigs and lacks cross-reaction with other common swine viruses and viruses in genus Pestivirus such as Bovine viral diarrhea virus (BVDV). The C-strain antibody can be tested in pigs 7-14 days post vaccination with this cELISA. The sensitivity and specificity of the established cELISA were 100% (95% confidence interval: 95.60 to 100%) and 100% (95% confidence interval: 98.30 to 100%), respectively. This novel cELISA is a reliable tool for specifically measuring and differentiating immune responses to C-strain vaccine in pigs. By combining with the wild-type CSFV-specific infection tests, it can make the C-strain have DIVA capability.

The recombinant Erns and truncated E2-based indirect enzyme-linked immunosorbent assays to distinguishably test specific antibodies against classical swine fever virus and bovine viral diarrhea virus.

BACKGROUND: Classical swine fever (CSF) virus is the causative agent of an economically important, highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV). BVDV infection in pigs can mimic CSF clinical signs, which cause difficulty in differentiation. Serological test for detection of virus specific antibodies is a valuable tool for diagnosis and surveillance of CSFV and BVDV infections in animals. The aim of this study was to develop the CSFV Erns and BVDV tE2 -based ELISAs to distinguishably test specific antibodies against CSFV and BVDV. METHODS: The CSFV Erns and truncated E2 (tE2, residues 690-865) of BVDV were expressed in E. coli and purified by Ni-NTA affinity chromatography, respectively. Employing Erns or tE2 protein as diagnostic antigen, indirect ELISAs were developed to distinguishably test specific antibodies against CSFV and BVDV. The specificity and sensitivity of ELISAs were evaluated using a panel of virus specific sera of pigs, immunized rabbits and immunized mice. A total 150 clinical serum samples from farm pigs were measured by the developed ELISAs and compared with virus neutralizing test (VNT). RESULTS: Indirect ELISA was established based on recombinant CSFV Erns or BVDV tE2 protein, respectively. No serological cross-reaction between antibodies against CSFV and BVDV was observed in sera of immunized rabbits, immunized mice or farm pigs by detections of the Erns and tE2 -based ELISAs. Compared to VNT, the CSFV Erns -based ELISA displayed a high sensitivity (93.3%), specificity (92.0%) and agreement rate (92.7%), and the sensitivity, specificity and agreement rate of BVDV tE2 -based ELISA was 92.3%, 95.2% and 94.7%, respectively. CONCLUSION: The newly developed ELISAs are highly specific and sensitive and would be valuable tools for serological diagnosis for CSFV and BVDV infections.

Domain-Specific Common Data Elements for Rare Disease Registration: Conceptual Approach of a European Joint Initiative Toward Semantic Interoperability in Rare Disease Research.

BACKGROUND: With hundreds of registries across Europe, rare diseases (RDs) suffer from fragmented knowledge, expertise, and research. A joint initiative of the European Commission Joint Research Center and its European Platform on Rare Disease Registration (EU RD Platform), the European Reference Networks (ERNs), and the European Joint Programme on Rare Diseases (EJP RD) was launched in 2020. The purpose was to extend the set of common data elements (CDEs) for RD registration by defining domain-specific CDEs (DCDEs). OBJECTIVE: This study aims to introduce and assess the feasibility of the concept of a joint initiative that unites the efforts of the European Platform on Rare Disease Registration Platform, ERNs, and European Joint Programme on Rare Diseases toward extending RD CDEs, aiming to improve the semantic interoperability of RD registries and enhance the quality of RD research. METHODS: A joint conference was conducted in December 2020. All 24 ERNs were invited. Before the conference, a survey was communicated to all ERNs, proposing 18 medical domains and requesting them to identify highly relevant choices. After the conference, a 3-phase plan for defining and modeling DCDEs was drafted. Expected outcomes included harmonized lists of DCDEs. RESULTS: All ERNs attended the conference. The survey results indicated that genetic, congenital, pediatric, and cancer were the most overlapping domains. Accordingly, the proposed list was reorganized into 10 domain groups and recommunicated to all ERNs, aiming at a smaller number of domains. CONCLUSIONS: The approach described for defining DCDEs appears to be feasible. However, it remains dynamic and should be repeated regularly based on arising research needs.

A Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Protects Pigs against PCV2b Challenge and Induces Serum Neutralizing Antibody Response against CSFV.

Porcine circovirus type 2 (PCV2) is endemic worldwide. PCV2 causes immunosuppressive infection. Co-infection of pigs with other swine viruses, such as pseudorabies virus (PRV) and classical swine fever virus (CSFV), have fatal outcomes, causing the swine industry significant economic losses in many if not all pig-producing countries. Currently available inactivated/modified-live/vectored vaccines against PCV2/CSFV/PRV have safety and efficacy limitations. To address these shortcomings, we have constructed a triple gene (thymidine kinase, glycoprotein E [gE], and gG)-deleted (PRVtmv) vaccine vector expressing chimeric PCV2b-capsid, CSFV-E2, and chimeric Erns-fused with bovine granulocytic monocyte-colony stimulating factor (Erns-GM-CSF), designated as PRVtmv+, a trivalent vaccine. Here we compared this vaccine's immunogenicity and protective efficacy in pigs against wild-type PCV2b challenge with that of the inactivated Zoetis Fostera Gold PCV commercial vaccine. The live PRVtmv+ prototype trivalent subunit vaccine is safe and highly attenuated in pigs. Based on PCV2b-specific neutralizing antibody titers, viremia, viral load in lymphoid tissues, fecal-virus shedding, and leukocyte/lymphocyte count, the PRVtmv+ yielded better protection for vaccinated pigs than the commercial vaccine after the PCV2b challenge. Additionally, the PRVtmv+ vaccinated pigs generated low to moderate levels of CSFV-specific neutralizing antibodies.

Patient-centered approaches for patients with systemic autoimmune rheumatic diseases: development and evolution.

INTRODUCTION: European Reference Networks (ERNs) are dedicated to rare complex diseases. Systemic autoimmune rheumatic diseases (SARDs) comprise a group of disorders, some of which are rare, complex, and chronic, characterized by relapsing-remitting course and requiring targeted treatments for long periods; SARDs are also associated with various co-morbidities and therefore health-care infrastructures, at the highest level of expertise are required. AREAS COVERED: For the current work, literature on the basic characteristics of a center of excellence dedicated to SARDs, its advantages over the existing health infrastructures in order to improve health and social care, its contribution to the education of health-care workers, and the related research opportunities are presented. In addition, our experience, vision, and initiatives as a new member of the ERNs are reported. EXPERT OPINION: A restructure in healthcare policy and resource allocation, based on centers of expertise, is necessary to improve the medical care of patients with SARDs.

Bungowannah Pestivirus Chimeras as Novel Double Marker Vaccine Strategy against Bovine Viral Diarrhea Virus.

Marker or DIVA (differentiation of infected from vaccinated animals) vaccines are beneficial tools for the eradication of animal diseases in regions with a high prevalence of the designated disease. Bovine viral diarrhea virus (BVDV)-1 (syn. Pestivirus A) is a flavivirus that infects predominantly cattle resulting in major economic losses. An increasing number of countries have implemented BVDV eradication programs that focus on the detection and removal of persistently infected cattle. No efficient marker or DIVA vaccine is yet commercially available to drive the eradication success, to prevent fetal infection and to allow serological monitoring of the BVDV status in vaccinated farms. Bungowannah virus (BuPV, species Pestivirus F), a related member of the genus Pestivirus with a restricted prevalence to a single pig farm complex in Australia, was chosen as the genetic backbone for a marker vaccine candidate. The glycoproteins E1 and E2 of BuPV were substituted by the heterologous E1 and E2, which are major immunogens, of the BVDV-1 strain CP7. In addition, the candidate vaccine was further attenuated by the introduction of a deletion within the Npro protein coding sequence, a major type I interferon inhibitor. Immunization of cattle with the chimeric vaccine virus BuPV_ΔNpro_E1E2 CP7 (modified live or inactivated) followed by a subsequent experimental challenge infection confirmed the safety of the prototype strain and provided a high level of clinical protection against BVDV-1. The serological discrimination of vaccinated cattle could be enabled by the combined detection of BVDV-1 E2- in the absence of both BVDV NS3- and BVDV Erns-specific antibodies. The study demonstrates for the first time the generation and application of an efficient BVDV-1 modified double marker vaccine candidate that is based on the genetic background of BuPV accompanied by commercially available serological marker ELISA systems.

Genetic diversity and molecular characterization of classical swine fever virus envelope protein genes E2 and Erns circulating in Vietnam from 2017 to 2019.

Classical swine fever virus (CSFV) is an RNA virus that incurs severe economic costs to swine industries worldwide. This study was conducted to investigate the genetic diversity among CSFV strains circulating in Vietnam, with a focus on their genetic variants relative to four vaccine strains. Samples from clinical cases were collected from different provinces of Central and Southern Vietnam from 2017 to 2019. 21 CSFV-positive samples were selected for amplification and sequencing of the full-length Erns and E2 genes. Phylogenetic analyses of these two genes showed that most CSFV strains circulating in Central and Southern Vietnam from 2017 to 2019 belong to subgroup 2.1c, whereas the remaining strains cluster into subgroup 2.2. All CSFV field strains in this study were genetically distant from group 1 strains. Analysis of the E2 and Erns genes indicated that all CSFV field strains have low sequence identity with the vaccine strains (80-83.5% and 82.3-86% sequence identity for E2 and Erns, respectively). Likewise, amino acid-level sequence analysis showed 87.3-91.1% and 87.6-91.6% sequence identity for E2 and Erns, respectively. Together, our findings indicate that CSFV strains circulating in Vietnam belong to subtypes 2.2 and 2.1c, and we also provide novel insights into the epidemiology, molecular characteristics, genetic diversity, and evolution of these circulating CSFV strains.

Abrogation of the RNase activity of Erns in a low virulence classical swine fever virus enhances the humoral immune response and reduces virulence, transmissibility, and persistence in pigs.

The prevalence of low virulence classical swine fever virus (CSFV) strains makes viral eradication difficult in endemic countries. However, the determinants for natural CSFV attenuation and persistence in the field remain unidentified. The aim of the present study was to assess the role of the RNase activity of CSFV Erns in pathogenesis, immune response, persistent infection, and viral transmission in pigs. To this end, a functional cDNA clone pPdR-H30K-36U with an Erns lacking RNase activity was constructed based on the low virulence CSFV field isolate Pinar de Rio (PdR). Eighteen 5-day-old piglets were infected with vPdR-H30K-36U. Nine piglets were introduced as contacts. The vPdR-H30K-36U virus was attenuated in piglets compared to the parental vPdR-36U. Only RNA traces were detected in sera and body secretions and no virus was isolated from tonsils, showing that RNase inactivation may reduce CSFV persistence and transmissibility. The vPdR-H30K-36U mutant strongly activated the interferon-α (IFN-α) production in plasmacytoid dendritic cells, while in vivo, the IFN-α response was variable, from moderate to undetectable depending on the animal. This suggests a role of the CSFV Erns RNase activity in the regulation of innate immune responses. Infection with vPdR-H30K-36U resulted in higher antibody levels against the E2 and Erns glycoproteins and in enhanced neutralizing antibody responses when compared with vPdR-36U. These results pave the way toward a better understanding of viral attenuation mechanisms of CSFV in pigs. In addition, they provide novel insights relevant for the development of DIVA vaccines in combination with diagnostic assays for efficient CSF control.

Induction of Robust and Specific Humoral and Cellular Immune Responses by Bovine Viral Diarrhea Virus Virus-Like Particles (BVDV-VLPs) Engineered with Baculovirus Expression Vector System.

Bovine viral diarrhea virus (BVDV) is an important animal pathogen that affects cattle. Infections caused by the virus have resulted in substantial economic losses and outbreaks of BVDV are reported globally. Virus-like particles (VLPs) are promising vaccine technology largely due to their safety and strong ability to elicit robust immune responses. In this study, we developed a strategy to generate BVDV-VLPs using a baculovirus expression vector system (BEVS). We were able to assemble BVDV-VLPs composed of dimerized viral proteins E2 and Erns, and the VLPs were spherical particles with the diameters of about 50 nm. Mice immunized with 15 µg of VLPs adjuvanted with ISA201 elicited higher levels of E2-specific IgG, IgG1, and IgG2a antibodies as well as higher BVDV-neutralizing activity in comparison with controls. Re-stimulation of the splenocytes collected from mice immunized with VLPs led to significantly increased levels of CD3+CD4+T cells and CD3+CD8+T cells. In addition, the splenocytes showed dramatically enhanced proliferation and the secretion of Th1-associated IFN-γ and Th2-associated IL-4 compared to that of the unstimulated control group. Taken together, our data indicate that BVDV-VLPs efficiently induced BVDV-specific humoral and cellular immune responses in mice, showing a promising potential of developing BVDV-VLP-based vaccines for the prevention of BVDV infections.

Generation and immunogenicity analysis of recombinant classical swine fever virus glycoprotein E2 and Erns expressed in baculovirus expression system.

Classical swine fever (CSF) caused by the classical swine fever virus (CSFV) is a highly contagious swine disease resulting in large economical losses worldwide. The viral envelope glycoprotein E2 and Erns are major targets for eliciting antibodies against CSFV in infected animals. In this report, the glycoprotein E2 and Erns were expressed using the baculovirus system and their protective immunity in rabbits were tested. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with CSFV-E2, CSFV-Erns, or their combination (CSFV-E2 + Erns). Besides, a commercial CSFV vaccine (C-strain) and PBS were used as positive or negative controls, respectively. Four weeks after the second immunization, all the rabbits were challenged with 100 RID50 of CSFV C-strain. High levels of CSFV E2-specific antibody, neutralizing antibody and cellular immune responses to CSFV were elicited in the rabbits inoculated with C-strain, CSFV-E2, and CSFV-E2 + Erns. And the rabbits inoculated with the three vaccines received complete protection against CSFV C-strain. However, no neutralizing antibody was detected in the Erns vaccinated rabbits and the rabbits exhibited fever typical of CSFV, suggesting the Erns alone is not able to induce a protective immune response. Taken together, while the Erns could not confer protection against CSFV, E2 and E2 + Erns could not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits.