Protective evaluation of the commercialized porcine reproductive and respiratory syndrome virus vaccines in piglets challenged by NADC34-like strain.

In China, the porcine reproductive and respiratory syndrome virus (PRRSV) has undergone several variations over the decades and contributed to the diversity of the clinical epidemic PRRSV strains. This has complicated the prevention and control of PRRS. In particular, the efficacy of the currently available commercial vaccines against the highly pathogenic NADC34-like strains is unclear. Therefore, the objective of this study was to evaluate the protection efficacy of three commercial PRRS modified-live virus (MLV) vaccines derived from classical PRRS VR2332 MLV and R98 MLV against challenge with a heterologous NADC34-like PRRSV strain, JS2021NADC34, which has high pathogenicity in pigs. PRRSV- and antibody-free piglets were immunized with the PRRS VR2332 MLV vaccine or either of two R98 MLV vaccines (from different manufacturers) and were challenged with the JS2021NADC34 strain 28 days after immunization. Rectal temperature, clinical symptoms, viremia and viral shedding from the nose, gross lesions in the thymus and lungs, microscopic lesions and viral distribution in the lungs, as well as the humoral immune response and mortality rates were recorded over a 14-day post-challenge period. The results showed that PRRS VR2332 MLV had better efficacy against the JS2021NADC34 challenge than PRRS R98 MLV, with vaccinated piglets in the former group showing transient and mild symptoms, mild pathological lesions in the lungs, mild thymic atrophy, and low viral levels in sera and nasal swabs, as well as better growth performance and a 100% survival rate. In contrast, two PRRS R98 MLVs exhibited limited efficacy against the JS2021NADC34 challenge, with the piglets in two R98 groups showing obvious clinical symptoms and pathological changes in the lungs and thymus; moreover, there were two deaths caused by PRRS in two R98 groups, respectively. Despite this, the mortality rate was lower than that of the unvaccinated piglets that were challenged with JS2021NADC34. The cumulative results demonstrate that PRRS VR2332 MLV was partly effective against the highly pathogenic PRRSV NADC34-like strain based on the observations over the 14-day post-challenge period. Thus, it might be a viable option among the commercially available vaccines for control of NADC34-like virus infections in swine herds.

Effect of cardiolipin on the lamellarity and elongation of liposomes hydrated in PBS.

Lamellarity and shape are important factors in the formation of vesicles and determine their role in biological systems and pharmaceutical applications. Cardiolipin (CL) is a major lipid in many biological membranes and exerts a great influence on their structural organization due to its particular structure and physico-chemical properties. Here, we used small-angle X-ray and neutron scattering to study the effects of CL with different acyl chain lengths and saturations (CL14:0, CL18:1, CL18:2) on vesicle morphology and lamellarity in membrane models containing mixtures of phosphatidylcholine and phosphatidylethanolamine with different acyl chain lengths and saturations (C14:0 and C 18:1). Measurements were performed in the presence of Phosphate Buffer Saline (PBS), at 37°C, to better reflect physiological conditions, which resulted in strong effects on vesicle morphology, depending on the type and amount of CL used. The presence of small quantities of CL (from 2.5%) reduced inter-membrane correlations and increased perturbation of the membrane, an effect which is enhanced in the presence of matched shorter saturated acyl chains, and mainly unilamellar vesicles (ULV) are formed. In extruded vesicles, employed for SANS experiments, flattened vesicles are observed partly due to the hypertonic effect of PBS, but also influenced by the type of CL added. Our experimental data from SAXS and SANS revealed a strong dependence on CL content in shaping the membrane microstructure, with an apparent optimum in the PC:CL mixture in terms of promoting reduced correlations, preferred curvature and elongation. However, the use of PBS caused distinct differences from previously published studies in water in terms of vesicle shape, and highlights the need to investigate vesicle formation under physiological conditions in order to be able to draw conclusions about membrane formation in biological systems.

Effect of vaccination route (intradermal vs. intramuscular) against porcine reproductive and respiratory syndrome using a modified live vaccine on systemic and mucosal immune response and virus transmission in pigs.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a viral disease with worldwide distribution and an enormous economic impact. To control PRRS virus (PRRSV) infection, modified live vaccines (MLVs) are widely used in the field, mainly administered via an intramuscular (IM) route. Currently, some MLVs are authorized for intradermal (ID) administration, which has many practical and welfare advantages. The objectives of the study were to compare the immune responses (systemic in blood and mucosal in lungs) and vaccine efficacy in preventing challenge strain transmission after IM or needle-free ID immunization of piglets with an MLV against PRRSV-1 (MLV1). METHODS: Groups of sixteen 5-week-old specific pathogen-free piglets were vaccinated with Porcilis PRRS® (MSD) either by an IM (V+ IM) or ID route (V+ ID) using an IDAL®3G device or kept unvaccinated (V-). Four weeks after vaccination, in each group, 8 out of the 16 piglets were challenged intranasally with a PRRSV-1 field strain, and one day later, the inoculated pigs were mingled by direct contact with the remaining 8 sentinel noninoculated pigs to evaluate PRRSV transmission. Thus, after the challenge, each group (V+ IM, V+ ID or V-) included 8 inoculated and 8 contact piglets. During the postvaccination and postchallenge phases, PRRSV replication (RT-PCR), PRRSV-specific antibodies (ELISA IgG and IgA, virus neutralization tests) and cell-mediated immunity (ELISPOT Interferon gamma) were monitored in blood and bronchoalveolar lavages (BALs). RESULTS: Postvaccination, vaccine viremia was lower in V+ ID pigs than in V+ IM pigs, whereas the cell-mediated immune response was detected earlier in the V+ ID group at 2 weeks postvaccination. In the BAL fluid, a very low mucosal immune response (humoral and cellular) was detected. Postchallenge, the vaccine efficacy was similar in inoculated animals with partial control of PRRSV viremia in V+ ID and V+ IM animals. In vaccinated sentinel pigs, vaccination drastically reduced PRRSV transmission with similar estimated transmission rates and latency durations for the V+ IM and V+ ID groups. CONCLUSIONS: Our results show that the tested MLV1 induced a faster cell-mediated immune response after ID immunization two weeks after vaccination but was equally efficacious after IM or ID immunization towards a challenge four weeks later. Considering the practical and welfare benefits of ID vaccination, these data further support the use of this route for PRRS MLVs.

Multilab Validation Report for the Verification and Subtyping of Listeria monocytogenes Using qPCR.

Listeria monocytogenes (Lm) is a Gram-positive bacterium that causes invasive listeriosis, an illness with high mortality and hospitalization rates. Due to the severity of illness associated with Lm, rapid identification and characterization of isolates from foods and the food-processing environment are critical to properly identify and track the pathogen and quickly remove adulterated foods from the market. Prior methods can rely on time-consuming biochemical or sera-agglutination assays to perform these tasks. Development of a high-throughput method that would rapidly perform these tasks is critical to improve response to contamination events. Previously, a single laboratory validation of a qPCR-based method was presented that could rapidly verify Lm isolates and characterize them into six molecular serogroups. In the current study, a multi-laboratory validation (MLV) was performed to evaluate the reliability of the qPCR method for identification and serogrouping of Lm isolates. Sixteen collaborating laboratories independently analyzed a panel of 43 blinded isolates plus three control strains using the qPCR method. This panel was comprised of representatives for non-Listeria (n = 7), Listeria sp. (n = 8), and Lm (n = 28) strains. The Lm isolates contained representatives of the six serogroups: 2A, 2B, 2C, 4B, NT, and 4bV/IVb-v1, with five strains for each serogroup except 4bV/IVb-v1 (n = 3). The results generated by 16 laboratories showed high sensitivity, specificity, and accuracy, generally ≥97%, for both the genus-species and serogrouping qPCRs. Results from one laboratory lowered the sensitivity of the non-Listeria group to 93%. These results indicated the method was highly reliable. However, only the previously evaluated serogroups were tested within the MLV panel, though there is the potential for other serogroup results. Sequence Read Archive (SRA) files for Lm isolates were evaluated to determine the frequency of other potential serogroup profiles. This effort identified a low percentage of isolates with atypical qPCR serogroups (0.30%) that are consistent with Lm and were generally associated with lineage II and the natural environment. In summary, the results indicate that the proposed qPCR method is reliable and has a high degree of sensitivity, accuracy, and specificity, while also decreasing hands-on analysis time and increasing throughput of the analysis.

Transmission Dynamics of Imported Vaccine-Origin PRRSV-2 within and between Commercial Swine Integrations in Hungary.

This study reports on the molecular epidemiology of Ingelvac-PRRS-MLV-associated cases in Hungary for the period 2020-2021. Field epidemiology investigations led the experts to conclude that imported pigs, which were shipped through transit stations in Denmark, introduced the vaccine virus. The movement of fatteners and the neglect of disease control measures contributed to the spread of the virus to PRRS-free pig holdings in the vicinity. Deep sequencing was performed to genetically characterize the genes coding for the virion antigens (i.e., ORF2 through ORF7). The study isolates exhibited a range of 0.1 to 1.8% nucleotide sequence divergence from the Ingelvac PRRS MLV and identified numerous polymorphic sites (up to 57 sites) along the amplified 3.2 kilo base pair genomic region. Our findings confirm that some PRRSV-2 vaccine strains can accumulate very high number of point mutations within a short period in immunologically naive pig herds.

Genomic similarity and antibody-dependent enhancement of immune serum potentially affect the protective efficacy of commercial MLV vaccines against NADC30-like PRRSV.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant diseases affecting the pig industry worldwide. The PRRSV mutation rate is the highest among the RNA viruses. To date, NADC30-like PRRSV and highly pathogenic PRRSV (HP-PRRSV) are the dominant epidemic strains in China; however, commercial vaccines do not always provide sufficient cross-protection, and the reasons for insufficient protection are unclear. This study isolated a wild-type NADC30-like PRRSV, SX-YL1806, from Shaanxi Province. Vaccination challenge experiments in piglets showed that commercial modified live virus (MLV) vaccines provided good protection against HP-PRRSV. However, it could not provide sufficient protection against the novel strain SX-YL1806. To explore the reasons for this phenomenon, we compared the genomic homology between the MLV strain and HP-PRRSV or NADC30-like PRRSV and found that the MLV strain had a lower genome similarity with NADC30-like PRRSV. Serum neutralization assay showed that MLV-immune serum slightly promoted the homologous HP-PRRSV replication and significantly promoted the heterologous NADC30-like PRRSV strain replication in vitro, suggesting that antibody-dependent enhancement (ADE) might also play a role in decreasing MLV protective efficacy. These findings expand our understanding of the potential factors affecting the protective effect of PRRSV MLV vaccines against the NADC30-like strains.

Histopathological Pulmonary Lesions in 1st-Day Newborn Piglets Derived from PRRSV-1 MLV Vaccinated Sows at the Last Stage of Gestation.

Modified live virus (MLV) vaccines for the control of porcine respiratory and reproductive syndrome virus (PRRSV) have been associated with the vertical and horizontal transmission of vaccine viruses. The present study aimed to describe pathological lung lesions in piglets born by gilts vaccinated with PRRSV-1 MLV. In total, 25 gilts were vaccinated at late gestation (100th day) and were divided into five groups according to the different vaccines (Vac) used: no vaccine-control group, Vac-1-strain DV, Vac-2-strain VP-046 BIS, Vac-3-strain 94881, Vac-4-strain 96V198. Within the first 0-9 h of the farrowing, blood samples were collected from all newborn piglets and lung samples were exanimated grossly, histopathologically and with scanning electron microscopy. PRRSV (RT-PCR-positive) and antibodies were detected in the serum of piglets from gilts vaccinated with Vac-2. In these piglets, moderate to severe interstitial pneumonia with thickened alveolar septa was noticed. Type II pneumocyte hyperplasia was also observed. The rest of the trial piglets showed unremarkable lung lesions. Phylogenetic analysis revealed the 98.7% similarity of the PRRSV field strain (GR 2019-1) to the PRRS MLV vaccine strain VP-046 BIS. In conclusion, the Vac-2 PRRSV vaccine strain can act as an infectious strain when vaccination is administrated at late gestation, causing lung lesions.

Unfolded protein response inhibits KAT2B/MLKL-mediated necroptosis of hepatocytes by promoting BMI1 level to ubiquitinate KAT2B.

Unfolded protein response (UPR) plays an important role in the pathogenesis of many liver diseases. BMI1 has a liver protection effect, but whether it participates in the regulation of hepatocyte death through UPR is not well defined. Herein, the endoplasmic reticulum stress model was established by inducing hepatocyte line (MIHA) with tunicamycin (TM, 5 µg/ml). Cell counting kit-8 assay and flow cytometry were used to evaluate the viability and apoptosis of hepatocytes. The expression levels of BMI1, KAT2B, and proteins related to UPR (p-eIF2α, eIF2α, ATF4, and ATF6), NF-κB (p65 and p-p65), apoptosis (cleaved caspase-3, bcl-2, and bax) and necroptosis (p-MLKL and MLKL) were determined by Western blot. The relationship between KAT2B and BMI1 was determined by co-immunoprecipitation and ubiquitination assay. The results showed that TM not only promoted UPR, apoptosis, and necroptosis in hepatocytes but also upregulated the expression levels of BMI1 and KAT2B and activated NF-κB pathway. BAY-117082 reversed the effects of TM on viability, apoptosis, NF-κB pathway, and BMI1 but strengthened the effects of TM on KAT2B/MLKL-mediated necroptosis. BMI1 promoted the ubiquitination of KAT2B, and BMI1 overexpression reversed the effects of TM on viability, apoptosis, and KAT2B/MLKL-mediated necroptosis. In summary, overexpression of BMI1 promotes the ubiquitination of KAT2B to block the MLKL-mediated necroptosis of hepatocytes.

Multi-laboratory validation study of a real-time PCR method for detection of Salmonella in baby spinach.

The FDA Bacteriological Analytical Manual (BAM) Salmonella culture method takes at least 3 days for a presumptive positive result. The FDA developed a quantitative PCR (qPCR) method to detect Salmonella from 24-h preenriched cultures, using ABI 7500 PCR system. The qPCR method has been evaluated as a rapid screening method for a broad range of foods by single laboratory validation (SLV) studies. The present multi-laboratory validation (MLV) study was aimed to measure the reproducibility of this qPCR method and compare its performance with the culture method. Sixteen laboratories participated in two rounds of MLV study to analyze twenty-four blind-coded baby spinach test portions each. The first round yielded ∼84% and ∼82% positive rates across laboratories for the qPCR and culture methods, respectively, which were both outside the fractional range (25%-75%) required for fractionally inoculated test portions by the FDA's Microbiological Method Validation Guidelines. The second round yielded ∼68% and ∼67% positive rates. The relative level of detection (RLOD) for the second-round study was 0.969, suggesting that qPCR and culture methods had similar sensitivity (p > 0.05). The study demonstrated that the qPCR yields reproducible results and is sufficiently sensitive and specific for the detection of Salmonella in food.

A recombinant porcine reproductive and respiratory syndrome virus type 2 field strain derived from two PRRSV-2-modified live virus vaccines.

A porcine reproductive and respiratory syndrome virus (PRRSV) type 2 (PRRSV-2) isolate was obtained from lung samples collected from a 4.5-month-old pig at a wean-to-finish site in Indiana, USA, although no gross or microscopic lesions suggestive of PRRSV infection were observed in the lung tissue. Phylogenetic and molecular evolutionary analyses based on the obtained virus sequences indicated that PRRSV USA/IN105404/2021 was a natural recombinant isolate from Ingelvac PRRS® MLV and Prevacent® PRRS, which are PRRSV-2-modified live virus vaccines commercially available in the United States. This study is the first to report the detection of a PRRSV-2 recombinant strain consisting entirely of two modified live virus vaccine strains under field conditions. Based on clinical data and the absence of lung lesions, this PRRSV-2 recombinant strain was not virulent in swine, although its pathogenicity needs to be confirmed by clinical trials.

Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors.

To date, the establishment of high-titer stable viral packaging cells (VPCs) at large scale for gene therapeutic applications is very time- and cost-intensive. Here we report the establishment of three human suspension 293-F-derived ecotropic MLV-based VPCs. The classic stable transfection of an EGFP-expressing transfer vector resulted in a polyclonal VPC pool that facilitated cultivation in shake flasks of 100 mL volumes and yielded high functional titers of more than 1 × 106 transducing units/mL (TU/mL). When the transfer vector was flanked by transposon terminal inverted repeats (TIRs) and upon co-transfection of a plasmid encoding for the transposase, productivities could be slightly elevated to more than 3 × 106 TU/mL. In contrast and using mRNA encoding for the transposase, as a proof of concept, productivities were drastically improved by more than ten-fold exceeding 5 × 107 TU/mL. In addition, these VPC pools were generated within only 3 weeks. The production volume was successfully scaled up to 500 mL employing a stirred-tank bioreactor (STR). We anticipate that the stable transposition of transfer vectors employing transposase transcripts will be of utility for the future establishment of high-yield VPCs producing pseudotype vector particles with a broader host tropism on a large scale.

Effects of concurrent administration of modified live viral vaccines with RB51 on immune responses to RB51.

Brucella abortus is a gram negative, zoonotic pathogen that can cause abortions and stillbirths in the cattle industry and has contributed to significant economic losses to cow-calf producers. Cell mediated immunity (CMI) is an important component of the immune response associated with protection against Brucella abortus and other intracellular pathogens. Brucellosis and viral modified live vaccines (vMLV) are licensed individually but may be used concurrently under field conditions. Peripheral blood mononuclear cells (PBMC) from non-vaccinated cattle and cattle vaccinated with either Brucella abortus strain RB51, a vMLV or both RB51 and a vMLV vaccine were isolated. The frequency of CD4+, CD8+ and γδ+ T cell populations within PBMC, and the frequency of interferon gamma (IFN-γ) production within these cell types was characterized via flow-cytometry. The goal of this study was to characterize immune responses to RB51 vaccination and determine the effect of concurrent vaccine administration. Although immune responses were greatest in PBMC from cattle vaccinated with only RB51, cattle vaccinated with both RB51 and vMLV demonstrated measurable T cell responses associated with protective immunity. Data suggests a lack of significant biological differences between the groups in protective immune responses. Collectively, our data demonstrated a lack of vaccine interference following concurrent administration of vMLV and RB51. Although concurrent administration of individually licensed vaccines may influence immune responses and contribute to vaccine interference, potential vaccine combinations should be evaluated for biological effects.

Pseudotyped Viruses for Mammarenavirus.

Mammarenaviruses are classified into New World arenaviruses (NW) and Old World arenaviruses (OW). The OW arenaviruses include the first discovered mammarenavirus-lymphocytic choriomeningitis virus (LCMV) and the highly lethal Lassa virus (LASV). Mammarenaviruses are transmitted to human by rodents, resulting in severe acute infections and hemorrhagic fever. Pseudotyped viruses have been widely used as a tool in the study of mammarenaviruses. HIV-1, SIV, FIV-based lentiviral vectors, VSV-based vectors, MLV-based vectors, and reverse genetic approaches have been applied in the construction of pseudotyped mammarenaviruses. Pseudotyped mammarenaviruses are commonly used in receptor research, neutralizing antibody detection, inhibitor screening, viral virulence studies, functional analysis of N-linked glycans, and studies of viral infection, endocytosis, and fusion mechanisms.

Lamellarity-Driven Differences in Surface Structural Features of DPPS Lipids: Spectroscopic, Calorimetric and Computational Study.

Although single-lipid bilayers are usually considered models of eukaryotic plasma membranes, their research drops drastically when it comes to exclusively anionic lipid membranes. Being a major anionic phospholipid in the inner leaflet of eukaryote membranes, phosphatidylserine-constituted lipid membranes were occasionally explored in the form of multilamellar liposomes (MLV), but their inherent instability caused a serious lack of efforts undertaken on large unilamellar liposomes (LUVs) as more realistic model membrane systems. In order to compensate the existing shortcomings, we performed a comprehensive calorimetric, spectroscopic and MD simulation study of time-varying structural features of LUV made from 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), whereas the corresponding MLV were examined as a reference. A substantial uncertainty of UV/Vis data of LUV from which only Tm was unambiguously determined (53.9 ± 0.8 °C), along with rather high uncertainty on the high-temperature range of DPPS melting profile obtained from DSC (≈50-59 °C), presumably reflect distinguished surface structural features in LUV. The FTIR signatures of glycerol moiety and those originated from carboxyl group serve as a strong support that in LUV, unlike in MLV, highly curved surfaces occur continuously, whereas the details on the attenuation of surface features in MLV were unraveled by molecular dynamics.

Genome stability assessment of PRRS vaccine strain with new ARTIC-style sequencing protocol.

A tiling amplicon sequencing protocol was developed to analyse the genome sequence stability of the modified live PRRSV vaccine strain, Porcilis MLV. The backbone of the ARTIC-style protocol was formed by 34 individual primer pairs, which were divided into two primer pools. Primer pairs were designed to amplify 532 to 588 bp fragments of the corresponding genomic region. The amplicons are suitable for sequencing on Illumina DNA sequencers with available 600-cycle sequencing kits. The concentration of primer pairs in the pools was optimized to obtain a balanced sequencing depth along the genome. Deep sequencing data of three vaccine batches were also analysed. All three vaccine batches were very similar to each other, although they also showed single nucleotide variations (SNVs) affecting less than 1 % of the genome. In the three vaccine strains, 113 to 122 SNV sites were identified; at these sites, the minority variants represented a frequency range of 1 to 48.7 percent. Additionally, the strains within the batches contained well-known length polymorphisms; the genomes of these minority deletion mutants were 135 to 222 bp shorter than the variant with the complete genome. Our results show the usefulness of ARTIC-style protocols in the evaluation of the genomic stability of PRRS MLV strains.

Field evaluation of the safety and immunogenicity of a classical swine fever virus E2 subunit vaccine in breeding and nursery animals on Jeju Island, South Korea.

Purpose: Classical swine fever (CSF) reemerged on CSF-free Jeju Island where vaccination is not practiced by the unintentional injection of a live attenuated vaccine (modified live attenuated vaccines-low-virulence Miyagi [MLV-LOM]) in 2014. Since the Jeju provincial authority is considering adopting a voluntary immunization policy using a CSF-E2 subunit vaccine to combat LOM-derived CSF endemic, this study aimed to evaluate in Jeju herds. Materials and Methods: Two vaccination trials using the Bayovac CSF-E2 vaccine licensed for use in South Korea assessed the safety and humoral immunity of the CSF-E2 vaccine in breeding (trial 1) and nursery animals (trial 2) under farm application conditions. Results: Neither local nor systemic (including reproductive) adverse effects were objectively observed in pregnant sows and young piglets following a respective vaccination regime at pregnancy or weaning, respectively. Trial 1 showed that sows immunized with the CSF-E2 vaccine possessed high and consistent E2-specific and neutralizing antibody levels. The CSF-E2 vaccine-immunized pregnant sows subsequently conferred appropriate and steady passive immunity to their offspring. In trial 2, a double immunization scheme of the CSF-E2 vaccine in piglets at 40 and 60 days of age could elicit a consistent and long-lasting adequate antibody response. Additionally, the two trials detected no Erns-specific antibody responses, indicating that CSF-E2 vaccine can differentiate infected from vaccinated animals (DIVA). Conclusion: Our trial data collectively provide invaluable information on applying the CSF-E2 subunit vaccine to circumvent the possible drawbacks associated with the MLV-LOM concerning the safety, efficacy, and DIVA, in the LOM-endemic field farms and contribute to advanced CSF eradication on Jeju Island.

Safety, efficacy, and DIVA feasibility on a novel live attenuated classical swine fever marker vaccine candidate.

Classical swine fever virus (CSFV) is the etiological agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. Systemic prophylactic immunization with the live attenuated vaccine, the C-strain vaccine, is one of the effective measures for CSF control. However, one of the limitations of the C-strain vaccine is that the field strains-infected animals cannot be differentiated from the C-strain vaccinated herds by serological tests (DIVA). This constraint hampers the practical usage of the C-strain vaccine to eradicate the CSF in China. In the current study, a novel CSF modified live marker vaccine candidate was constructed based on the attenuation of the prevalent 2.1 genotype strain by the deletion of two virulence associated functional residues in the CSFV Erns, H79, and C171. Meanwhile, four residues S14, G22, E24, and E25 were identified specifically for the 6B8 mAb binding to the CSFV E2 as the novel conformational epitope. Then four substitutions of S14K, G22A, E24R, and G25D were further incorporated in the double deletion construct as a negative serological marker. Finally, the double-deletion marker MLV candidate GD18-ddErnHC-KARD was rescued, and its safety and efficacy profiles were evaluated in piglets. The safety study results indicated that the candidate did not induce fever, clinical signs, or pathological lesions with a high dose of 105.0 TCID50, and in addition, no virus shedding was detected until 21 days post-inoculation. Meanwhile, the efficacy study results demonstrated that at a low dose of 103.0 TCID50, it conferred complete clinical protection and no virus shedding was detected after the challenge with a highly virulent Shimen strain. Importantly, the infected animals were differentiated using the accompanied DIVA ELISA. These results constitute a proof-of-concept for rationally designing a CSF antigenically marked modified live vaccine candidate.

Dysmyelination and glycolipid interference caused by phenylalanine in phenylketonuria.

Phenylketonuria (PKU) is a metabolic disorder connected to an excess of phenylalanine (Phe) in the blood and tissues, with neurological consequences. The disease's molecular bases seem to be related to the accumulation of Phe at the cell membrane surface. Radiological outcomes in the brain demonstrate decreased water diffusivity in white matter, involving axon dysmyelination of not yet understood origin. We used a biophysical approach and model membranes to extend our knowledge of Phe-membrane interaction by clarifying Phe's propensity to affect membrane structure and dynamics based on lipid composition, with emphasis on modulating cholesterol and glycolipid components to mimic raft domains and myelin sheath membranes. Phe showed affinity for the investigated membrane mimics, mainly affecting the Phe-facing membrane leaflet. The surfaces of our neuronal membrane raft mimics were strong anchoring sites for Phe, showing rigidifying effects. From a therapeutic perspective, we further investigated the role of doxycycline, known to disturb Phe packing, unveiling its action as a competitor in Phe interactions with the membrane, suggesting its potential for treatment in the early stages of PKU. Our results suggest how Phe accumulation in extracellular fluids can impede normal growth of myelin sheaths by interfering with membrane slipping and by remodulating free water and myelin-associated water contents.

Pathogenicity of a novel classical swine fever LOM vaccine-derived virus isolated on Jeju Island, South Korea.

BACKGROUND: Reemergent local outbreaks of classical swine fever (CSF) occurred simultaneously in multiple pig farms on CSF-free Jeju Island, South Korea, in 2014 because of inadvertent injection of a commercial CSF (LOM) vaccine into pregnant sows. The LOM virus has since spread across the island and has become endemic in Jeju herds, raising concern about possible reversion to the virulence of the LOM vaccine. We previously isolated LOM-derived field CSF virus (CSFV) strains with unique insertion-deletion (INDEL) mutations in the 3'-untranslated region (UTR), designated LOM-derived Jeju 3'-UTR INDEL variants, from CSF-recurrent swine farms on Jeju Island in 2019. METHODS: The present study conducted animal experiments to investigate whether a 2019 emergent LOM 3'-UTR INDEL variant, KNU-1905, has reverted to a pathogenic form in conventional pigs (n = 10). RESULTS: Experimental animal infection showed that pigs inoculated with the commercial LOM vaccine strain developed no adverse effects compared to the sham-infected pigs. However, KNU-1905 displayed pathogenic characteristics in pigs, including clinical symptoms (e.g., lethargy, conjunctivitis, nasal discharge, and diarrhoea), weight loss, and gross lesions. Moreover, viremia, virus shedding in faeces and nasal fluids, and viral loads in various tissues of all the KNU-1905-infected pigs were highly significant, in contrast to those of the LOM-infected group in which CSFV RNA was detected only in the serum, nasal, and tonsil samples of one identical pig. CONCLUSIONS: Overall, the LOM-derived field isolate with molecular variations induced clinical adverse events in pigs, which commonly shed considerable amounts of CSFV. This study provides evidence that the genetic evolution of the LOM-derived CSFV circulating on Jeju Island might have allowed the LOM vaccine to recover its primary prototype and that these variants might have induced chronic or persistent infection in pigs that can shed CSFV in field farms leading to a risk of transmission among pigs or farms in this former CSF-free region.

Development of a child-friendly oral drug formulation using liposomal multilamellar vesicle technology.

Many medicines are only available in solid dosage forms suitable for adults, and extemporaneous compounding is required to prepare formulations for children. However, this common practice often results in inaccurate dosing and unpleasant taste, reducing the medication adherence. Here, we report the development of a new method to prepare and compound child-friendly oral formulations based on a liposomal multilamellar vesicle (MLV) platform. MLVs composed of a phospholipid (DSPC) and cholesterol (55/45, molar ratio) were prepared using the standard thin film hydration method with 300 mM citric acid (pH 2), followed by an addition of aqueous sodium carbonate to adjust the exterior pH to 8-10 for creating a transmembrane pH gradient. Weak-base drugs, such as chloroquine (CQ) and hydroxychloroquine (HCQ), could be actively and completely loaded into the MLVs at a drug-to-lipid ratio of 15-20 wt%. This technique formulated weak-base drugs from the powder or tablet form into a liquid preparation, and the complete drug encapsulation would prevent contact between the drug molecules and the taste buds. The gradient MLV formulation could be preserved by lyophilization and stored at room temperature for at least 8 weeks. Upon reconstitution with water, the MLV formulation could completely encapsulate CQ at 20 wt%, which was comparable to the freshly prepared MLVs. The CQ-loaded MLV formulation could be stored at 4 °C for 2 weeks without drug leakage. In vitro release studies indicated that MLV could retain CQ in the simulated saliva, but released up to 50% and 30% of the drug in the simulated gastric and intestinal fluids, respectively. The orally delivered MLV-CQ formulation displayed higher CQ absorption in mice, with a 2-fold increase in the area under the curve (AUC) of the plasma profile compared to CQ solution. Our data suggest that the new MLV method could serve as a platform to prepare child-friendly oral formulation for weak-base drugs.