Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. Acute PEDV outbreaks have continually emerged in most swine-producing Asian countries and, recently, in the ...United States, causing significant economic losses in the pig industry. The spike (S) protein of PEDV is a type 1 transmembrane envelope glycoprotein and consists of the S1 and S2 domains, which are responsible for virus binding and fusion, respectively. Since the S1 domain is involved in a specific high-affinity interaction with the cellular receptor and induction of neutralizing antibody in the natural host, it is a primary target for the development of effective vaccines against PEDV. In this study, a codon-optimized PEDV S1 gene containing amino acid residues 25–738 was synthesized based on a multiple alignment of the S amino acid sequences of PEDV field isolates and used to establish a stable porcine cell line constitutively expressing the PEDV S1 protein. The purified recombinant S1 protein was found to mediate highly potent antibody responses in immunized rabbits. The antibodies strongly recognized the recombinant S1 protein from cell lysates and supernatants of S1-expressing cells, whereas they bound weakly to the authentic S protein of PEDV vaccine strain SM98-1. Furthermore, a serum neutralization test revealed that the rabbit antisera completely inhibit infection of the PEDV vaccine strain at a serum dilution of 1:16. We then tested the ability of vaccination with the recombinant S1 protein to protect piglets against PEDV. Late-term pregnant sows were inoculated intramuscularly with the purified S1 protein, and the outcome was investigated in passively immunized suckling piglets after a virulent PEDV challenge. The results showed that vaccination with S1 protein efficiently protected neonatal piglets against PEDV. Our data suggest that the recombinant S1 protein shows potential as an effective and safe subunit vaccine for PED prevention.
Since the 2013–2014 incursion of the virulent G2b porcine epidemic diarrhoea virus (PEDV) pandemic strains in South Korea, frequent moderate‐scale regional outbreaks have recurred. In particular, ...areas of Jeju Island with extensive swine production have faced repeated epidemics since the re‐emergence in 2014. The current study reports the complete genome sequences and molecular characterization of the representative PEDV strains responsible for the 2018 endemic outbreaks on Jeju Island. All isolates were determined to belong genetically to the highly pathogenic pandemic G2b group. Full‐length genome sizes of four isolates differed from that of the G2b epidemic field strain due to insertion or deletion (DEL) mutations in the non‐structural protein (nsp)‐ or spike (S) protein‐coding regions. The 2018 Jeju isolates shared 96.7%–98.7% and 98.5%–99.4% identity at the S gene and whole‐genome levels, respectively, compared to global G2b PEDV strains. Genetic and phylogenetic analyses indicated that the 2018 isolates were closest to the 2014 G2b re‐emergent Jeju strains, but appeared to have undergone substantial rapid independent evolution. Among the isolates, a notable nsp3 DEL variant strain, KOR/KNU‐1807/2018, was isolated and propagated by continuous passages in Vero cells, and displayed typical PEDV‐induced syncytia formation. Genomic sequencing identified a unique 8‐nt DEL in the extreme C‐terminal region of the S gene at the 4th passage (KNU‐1807‐P4) compared to its original sample. This DEL resulted in the premature termination of S by nine amino acid residues (EVFEKVHVQ), which contained a KxHxx motif that is a potential endoplasmic reticulum retrieval signal. In vivo animal studies showed that variant strain KNU‐1807 had decreased virulence in suckling piglets. These results advance our knowledge regarding the genetic variation and pathogenicity of the G2b PEDV endemic strains prevalent in Jeju swine herds in South Korea.
•Attenuated G2b PEDV strain S DEL5/ORF3 was pioneered to develop an MLV vaccine.•S DEL5/ORF3 did not revert to virulence after successive back passages in piglets.•Prime-boost maternal vaccinations ...conferred complete protection against PEDV.•Robust humoral immunity was observed in the sera and colostrum of vaccinated dams.•Sufficient lactogenic immunity provides passive protection in suckling neonates.
We have previously reported the generation of the attenuated KNU-141112-S DEL5/ORF3 virus by continuous propagation of highly virulent G2b porcine epidemic diarrhea virus (PEDV) in Vero cells. The present study aimed to assess the safety of S DEL5/ORF3 and to evaluate its effectiveness as a live vaccine for prime-booster vaccinations. Reversion to virulence experiments revealed that the S DEL5/ORF3 strain retains its attenuated phenotype and genetic stability after five successive passages in susceptible piglets. Pregnant sows were primed orally with an S DEL5/ORF3 live vaccine and boosted intramuscularly twice with a commercial killed vaccine at 2-week intervals prior to parturition. This sow vaccination regimen completely protected nursing piglets against virulent G2b challenge, as evidenced by the increase in survival rate from 0% to 100% and the significant reduction in diarrhea intensity, including the amount and duration of PEDV fecal shedding. In addition, despite a 2–3 day period of weight loss in piglets from vaccinated sows after challenge, their daily weight gain was recovered at 7 days post-challenge and became similar to that of unchallenged pigs from unvaccinated sows over the course of the experiment. Furthermore, strong antibody responses to PEDV were verified in the sera and colostrum of immunized sows with the prime-boost treatment and their offspring. Altogether, our data demonstrated that the attenuated S DEL5/ORF3 strain guarantees the safety to host animals with no reversion to virulence and is suitable as an effective primary live vaccine providing durable maternal lactogenic immunity for passive piglet protection.
Despite its deleterious effects on living cells, oxidative stress plays essential roles in normal physiological processes and provides signaling molecules for cell growth, differentiation, and ...inflammation. Macrophages are equipped with antioxidant mechanisms to cope with intracellular ROS produced during immune response, and Nrf2 (NF-E2-related factor 2)/HO-1 (heme oxygenase-1) pathway is an attractive target due to its protective effect against ROS-induced cell damage in inflamed macrophages. We investigated the effects of ethanol extract of A. villosum (AVEE) on lipopolysaccharide- (LPS-) stimulated inflammatory responses generated via the Nrf2/HO-1 signaling pathway in murine peritoneal macrophages and RAW 264.7 cells. AVEE was found to suppress the NF-κB signaling pathway, thus, to reduce proinflammatory cytokine, nitric oxide, and prostaglandin levels in peritoneal macrophages and Raw 264.7 cells treated with LPS, and to enhance HO-1 expression by activating Nrf2 signaling. Furthermore, these anti-inflammatory effects of AVEE were diminished when cells were pretreated with SnPP (a HO-1 inhibitor). HPLC analysis revealed AVEE contained quercetin, a possible activator of the Nrf2/HO-1 pathway. These results show A. villosum ethanol extract exerts anti-inflammatory effects by activating the Nrf2/HO-1 pathway in LPS-stimulated macrophages.
•A virulent G2b PEDV strain KNU-141112 was subjected to Vero cell culture passage.•Deletions (DEL) in S and ORF3 of KNU-141112 at high passage levels were identified.•Cell-adapted KNU-141112 DEL ...strains were highly attenuated and immunogenic in pigs.•Large deletions in ORF3 appear to be associated with PEDV attenuation in pigs.•High-cell-passage KNU-141112 DEL strains can be live, attenuated vaccine candidates.
Porcine epidemic diarrhea virus (PEDV) has emerged or re-emerged worldwide, posing a significant financial threat to major pig-producing countries. In the present study, a virulent Korean pandemic PEDV strain, KNU-141112, was serially propagated in Vero cells for up to 100 passages. Through cell culture adaptation, we obtained four distinct deletion (DEL) mutants by plaque purification followed by nucleotide sequencing of the spike (S)/ORF3 gene-coding region, which were designated KNU-141112-S DEL2, −S DEL5, −S DEL2/ORF3, and −S DEL5/ORF3. Further whole genome sequencing identified 12 or 14 amino acid changes in the cell-adapted DEL strains. Animal inoculation studies revealed that the virulence of both S DEL2/ORF3 and S DEL5/ORF3 viruses with a large 46-nt deletion in the intergenic portion of S and ORF3 was remarkably diminished, indicating viral attenuation in the natural host. Furthermore, these cell-adapted strains elicited potent neutralizing antibody responses in immunized pigs. Taken together, our data indicate that the cell-attenuated S DEL2/ORF3 and S DEL5/ORF3 strains are promising candidates for the development of a safe and effective live PEDV vaccine.
•A virulent PEDV G2b epidemic isolate was employed to develop an inactivated vaccine.•Vaccination of sows with the inactivated vaccine protects piglets against challenge.•Sow vaccination reduces ...diarrhea severity including fecal shedding in piglets.•Neutralizing antibody response to PEDV is present in vaccinated dams and offspring.•Lactogenic immunity in sows provides passive immune protection to their litters.
Massive outbreaks of porcine epidemic diarrhea virus (PEDV) recurred in South Korea in 2013–2014 and affected approximately 40% of the swine breeding herds across the country, incurring a tremendous financial impact on producers and consumers. Despite the nationwide use of commercially available attenuated and inactivated vaccines in South Korea, PEDV has continued to plague the domestic pork industry, raising concerns regarding their protective efficacies and the need for new vaccine development. In a previous study, we isolated and serially cultivated a Korean PEDV epidemic strain, KOR/KNU-141112/2014, in Vero cells. With the availability of a cell culture-propagated PEDV strain, we are able to explore vaccination and challenge studies on pigs. Therefore, the aim of the present study was to produce an inactivated PEDV vaccine using the KNU-141112 strain and evaluate its effectiveness in neonatal piglets. Pregnant sows were immunized intramuscularly with the inactivated adjuvanted monovalent vaccine at six and three weeks prior to farrowing. Six-day-old piglets born to vaccinated or unvaccinated sows were challenged with the homogeneous KNU-141112 virus. The administration of the inactivated vaccine to sows greatly increased the survival rate of piglets challenged with the virulent strain, from 0% to approximately 92% (22/24), and significantly reduced diarrhea severity including viral shedding in feces. In addition, litters from unvaccinated sows continued to lose body weight throughout the experiment, whereas litters from vaccinated sows started recovering their daily weight gain at 7 days after the challenge. Furthermore, strong neutralizing antibody responses to PEDV were verified in immunized sows and their offspring, but were absent in the unvaccinated controls. Altogether, our data demonstrated that durable lactogenic immunity was present in dams administrated with the inactivated vaccine and subsequently conferred critical passive immune protection to their own litters against virulent PEDV infection.
The recent emergence and re-emergence of porcine epidemic diarrhea virus (PEDV) underscore the urgent need for the development of novel, safe, and effective vaccines against the prevailing strain. In ...this study, we generated a cold-adapted live attenuated vaccine candidate (Aram-P29-CA) by short-term passage of a virulent PEDV isolate at successively lower temperatures in Vero cells. Whole genome sequencing identified 12 amino acid changes in the cold-adapted strain with no insertions and deletions throughout the genome. Animal inoculation experiments confirmed the attenuated phenotype of Aram-P29-CA virus in the natural host. Pregnant sows were orally administered P29-CA live vaccines two doses at 2-week intervals prior to parturition, and the newborn piglets were challenged with the parental virus. The oral homologous prime-boost vaccination of P29-CA significantly improved the survival rate of the piglets and notably mitigated the severity of diarrhea and PEDV fecal shedding after the challenge. Furthermore, strong antibody responses to PEDV were detected in the sera and colostrum of immunized sows and in the sera of their offspring. These results demonstrated that the cold-adapted attenuated virus can be used as a live vaccine in maternal vaccination strategies to provide durable lactogenic immunity and confer passive protection to litters against PEDV.
Amomum tsao-ko Crevost et Lemaire, used as a spice in Asia, is an important source of Chinese cuisine and traditional Chinese medicines. A. tsao-ko is reported to exert a variety of biological and ...pharmacological activities, including anti-proliferative, anti-oxidative and neuroprotective effects. In this study, NNMBS227, consisting of the ethanol extract of A. tsao-ko, exhibited potent anti-inflammatory activities in RAW264.7 macrophages. We investigated the effect of NNMBS227 in the suppression of pro-inflammatory mediators, including pro-inflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2) and cytokines (tumor necrosis factor-α and interleukin-1β) in LPS stimulated macrophages. NNMBS227 also inhibited the phosphorylation and degradation of IκB-α, as well as the nuclear translocation of nuclear factor kappa B (NF-κB) p65 caused by stimulation with LPS. In addition, NNMBS227 induced heme oxygenase (HO)-1 expression through the nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) in macrophages. Using tin protoporphyrin (SnPP), an HO activity inhibitor, we confirmed an association between the anti-inflammatory effects of NNMBS227 and the up-regulation of HO-1. These findings suggest that Nrf2-dependent increases in the expression of HO-1 induced by NNMBS227 conferred anti-inflammatory activities in LPS stimulated RAW264.7 macrophages.
Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis. Test-and-cull protocols and gross pathological examinations of abattoir animals as well as milk pasteurization have been ...implemented to prevent the spread of tuberculosis from animals to humans worldwide. Despite the importance of precise and rapid diagnostic tests, conventional methods including intradermal skin tests and γ-interferon assays are limited by the high rate of false-negative results for cattle in the late infectious stage and due to laborious and time-consuming procedures. Therefore, antibody detection methods such as enzyme-linked immunosorbent assay (ELISA) are urgently needed to supplement the established approaches and expand the diagnostic window. This study was conducted to develop a bTB ELISA by evaluating recombinant and native proteins and various assay parameters. We produced recombinant MPB70 and SahH (M70S) and a native 20-kDa protein (20K) and optimized the ELISA protocol. The 20K ELISA showed 94.4% sensitivity and 98.2% specificity with an optimal sample-to-positive ratio cut-off of 0.531. The sensitivity and specificity of M70S ELISA were 94.4% and 97.3%, respectively, with an optimal sample-to-negative ratio cut-off of 1.696. Both assays showed acceptable diagnostic efficiency and could be used for bTB diagnosis in combination with established methods for herd screening and to expand the diagnostic window.
•A virulent PRRSV nsp2 DEL CA-2 isolate was subjected to up to 100 serial passages.•Cell-adapted CA-2-P100 strain possesses altered phenotypic traits in vitro.•CA-2-P100 infection down-regulates ...expression of TNF-α and IL-8 genes in PAM cells.•CA-2-P100 virus is attenuated virologically and clinically in vivo.•CA-2-P100 contains no extra deletions and has 27 aa mutations throughout the genome.
Porcine reproductive and respiratory syndrome virus (PRRSV) is the most common and world-widespread viral pathogen of swine. We previously reported genomic sequences and pathogenicity of type 2 Korean PRRSV strains belonging to the virulent lineage 1 family, which contain remarkable amino acid deletions in nonstructural protein 2 (nsp2 DEL) compared to VR-2332. Here, a virulent type 2 Korean PRRSV nsp2 DEL strain, CA-2, was serially propagated in MARC-145 cells for up to 100 passages (CA-2-P100). As the passage number increased, the phenotypic characteristics of cell-adapted CA-2 strains were altered, in terms of higher viral titers and larger plaque sizes compared to the parental virus. Pro-inflammatory cytokine genes, including TNF-α, IL-8, MCP-1, and MCP-2, were found to be significantly down-regulated in PAM cells with the CA-2-P100 strain compared to its parental nsp2 DEL virus. Animal inoculation studies demonstrated that the virulence of CA-2-P100 was reduced significantly, with showing normal weight gain, body temperatures, and lung lesions comparable to the control group. Furthermore, high-passage CA-2-P100 showed declined and transient viremia kinetics, as well as delayed and low PRRSV-specific antibody responses in infected pigs. In addition, we determined whole genome sequences of low to high-passage derivatives of CA-2. The nsp2 DEL pattern was conserved for 100 passages, whereas no other deletions or insertions arose during the cell adaptation process. However, CA-2-P100 possessed 54 random nucleotide substitutions that resulted in 27 amino acid changes distributed throughout the genome, suggesting that these genetic drifts provide a possible molecular basis correlated with the cell-adapted features in vitro and the attenuated phenotype in vivo. Taken together, our data indicate that the cell-attenuated CA-2-P100 strain is a promising candidate for developing a safe and effective live PRRSV vaccine.
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