CA-CAS-01-A: A Permissive Cell Line for Isolation and Live Attenuated Vaccine Development Against African Swine Fever Virus.

African swine fever virus (ASFV) is the causative agent of the highly lethal African swine fever disease that affects domestic pigs and wild boars. In spite of the rapid spread of the virus worldwide, there is no licensed vaccine available. The lack of a suitable cell line for ASFV propagation hinders the development of a safe and effective vaccine. For ASFV propagation, primary swine macrophages and monocytes have been widely studied. However, obtaining these cells can be time-consuming and expensive, making them unsuitable for mass vaccine production. The goal of this study was to validate the suitability of novel CA-CAS-01-A (CAS-01) cells, which was identified as a highly permissive cell clone for ASFV replication in the MA-104 parental cell line for live attenuated vaccine development. Through a screening experiment, maximum ASFV replication was observed in the CAS-01 cell compared to other sub-clones of MA-104 with 14.89 and log10 7.5 ± 0.15 Ct value and TCID50/ml value respectively. When CAS-01 cells are inoculated with ASFV, replication of ASFV was confirmed by Ct value for ASFV DNA, HAD50/ml assay, TCID50/ml assay, and cytopathic effects and hemadsoption were observed similar to those in primary porcine alveolar macrophages after 5th passage. Additionally, we demonstrated stable replication and adaptation of ASFV over the serial passage. These results suggest that CAS-01 cells will be a valuable and promising cell line for ASFV isolation, replication, and development of live attenuated vaccines.

Coinfection with porcine epidemic diarrhea virus and Clostridium perfringens type A enhances disease severity in weaned pigs.

Clostridium perfringens is a constituent of the normal gut microbiome in pigs; however, it can potentially cause pre- and post-weaning diarrhea. Nevertheless, the importance of this bacterium as a primary pathogen of diarrhea in piglets needs to be better understood, and the epidemiology of C. perfringens in Korean pig populations is unknown. To study the prevalence and typing of C. perfringens, 203 fecal samples were collected from diarrheal piglets on 61 swine farms during 2021-2022 and examined for the presence of C. perfringens and enteric viruses, including porcine epidemic diarrhea virus (PEDV). We determined that the most frequently identified type of C. perfringens was C. perfringens type A (CPA; 64/203, 31.5%). Among the CPA infections, single infections with CPA (30/64, 46.9%) and coinfections with CPA and PEDV (29/64, 45.3%) were the most common in diarrheal samples. Furthermore, we conducted animal experiments to investigate the clinical outcome of single infections and coinfections with highly pathogenic (HP)-PEDV and CPA in weaned piglets. The pigs infected with HP-PEDV or CPA alone showed mild or no diarrhea, and none of them died. However, animals that were co-inoculated with HP-PEDV and CPA showed more-severe diarrheal signs than those of the singly infected pigs. Additionally, CPA promoted PEDV replication in coinfected piglets, with high viral titers in the feces. A histopathological examination revealed more-severe villous atrophy in the small intestine of coinfected pigs than in singly infected pigs. This indicates a synergistic effect of PEDV and CPA coinfection on clinical disease in weaned piglets.

Deletion of pentad residues in the N-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets.

Our previous study revealed that tissue culture-adapted porcine epidemic diarrhea virus (PEDV) strains, namely KNU-141112-S DEL2/ORF3 and -S DEL5/ORF3, were attenuated to different extents in vivo, suggesting that their independent deletion (DEL) signatures, including 2-amino acid (aa; residues 56-57) or 5-aa (residues 56-60) DEL in the N-terminal domain (NTD) of the spike (S) protein, may contribute to the reduced virulence of each strain. To investigate whether each DEL in the NTD of the S1 subunit is a determinant for the virulence of PEDV, we generated two mutant viruses, named icS DEL2 and icS DEL5, by introducing the identical double or quintuple aa DEL into S1 using reverse genetics with an infectious cDNA clone of KNU-141112 (icKNU-141112). We then orally inoculated conventional suckling piglets with icKNU-141112, icS DEL2, or icS DEL5 to compare their pathogenicities. The virulence of both DEL mutant viruses was significantly diminished compared to that of icKNU-141112, which causes severe clinical signs and 100 % mortality. Interestingly, the degree of attenuation differed between the two mutant viruses: icS DEL5 caused neither diarrhea nor mortality, whereas icS DEL2 caused mild to moderate diarrhea, higher viral titers in feces and intestinal tissues, and 25 % mortality. Furthermore, the icS DEL5-infected piglets displayed no remarkable macroscopic and microscopic intestinal lesions, while the icS DEL2-infected piglets showed histopathological changes in small intestine tissues, including moderate-to-severe villous atrophy. Our data indicate that the loss of the pentad (56GENQG60) residues in S alone can be sufficient to give rise to an attenuated phenotype of PEDV.

Efficacy of a Novel Multiepitope Vaccine Candidate against Foot-and-Mouth Disease Virus Serotype O and A.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease in cloven-hoofed animals. To prevent the spread of FMD virus (FMDV), traditional inactivated vaccines are used to immunize susceptible animals in disease-endemic countries. However, the inactivated FMD vaccine has several limitations, including safety concerns. To overcome these limitations, subunit proteins have been studied as alternative vaccine candidates. In this study, we designed two multiepitope recombinant proteins (OVM and AVM) containing antigenic sites (residue of VP1 132-162 and residue of VP1 192-212) of three topotypes of FMDV serotype O or three topotypes of FMDV serotype A. Each recombinant protein was efficiently expressed in Escherichia coli with high solubility, and the immunogenicity and protective efficacy of the proteins as FMD vaccine candidates were evaluated. The results showed that OVM and AVM emulsified with ISA201 adjuvant induced effective antigen-specific humoral and cell-mediated immune responses and successfully protected mice from O/Jincheon/SKR/2014, O/VET/2013, and A/Malaysia/97 viruses. In addition, intramuscular immunization of pigs with the OVM and AVM emulsified with ISA201 elicited effective levels of neutralizing antibodies to the viruses with homologous epitopes. Importantly, OVM-AVM emulsified with CAvant®SOE-X adjuvant conferred 100% protection against the O/Jincheon/SKR/2014 virus with homologous residues and 75% protection against A/SKR/GP/2018 with heterologous residues. The results presented in this study suggest that the combination of OVM and AVM protein with an effective adjuvant could yield an effective and safe vaccine candidate for the prevention and control of foot-and-mouth disease. In addition, our results provide a vaccine platform that can safely, cost-efficiently, and rapidly generate protective vaccine candidates against diverse FMDVs.

Pathological Evaluation of Porcine Circovirus 2d (PCV2d) Strain and Comparative Evaluation of PCV2d and PCV2b Inactivated Vaccines against PCV2d Infection in a Specific Pathogen-Free (SPF) Yucatan Miniature Pig Model.

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen that causes porcine circovirus-associated diseases (PCVADs). The objective of this study was to evaluate the use of specific pathogen-free Yucatan miniature pigs (YMPs) as an experimental model for PCV2d challenge and vaccine assessment because PCV2-negative pigs are extremely rare in conventional swine herds in Korea. In the first experiment, every three pigs were subjected to PCV2d field isolate or mock challenge. During three weeks of experiments, the PCV2d infection group exhibited clinical outcomes of PCVAD with high viral loads, lymphoid depletion, and detection of PCV2d antigens in lymphoid organs by immunohistochemistry. In the second experiment, three groups of pigs were challenged with PCV2d after immunization for three weeks: a nonvaccinated group (three pigs), a PCV2b-Vac group vaccinated with a commercial PCV2b-based inactivated vaccine SuiShot® Circo-ONE (five pigs), and a PCV2d-Vac group vaccinated with an experimental PCV2d-based inactivated vaccine (five pigs). During the three weeks of the challenge period, nonvaccinated pigs showed similar clinical outcomes to those observed in the PCV2d infection group from the first experiment. In contrast, both the PCV2b and PCV2d vaccinations produced good levels of protection against PCV2d challenge, as evidenced by reduced viral loads, improved growth performance, high virus-neutralizing antibody titers, and less development of PCV2-associated pathological lesions. Taken together, these data suggest that YMPs could be an alternative model for PCV2 challenge experiments, and these animals displayed typical clinical and pathological features and characteristics of protective immunity induced by the vaccines that were consistent with those resulting from PCV2 infections in conventional pigs.

Efficacy of bivalent vaccines of porcine circovirus type 2 and Mycoplasma hyopneumoniae in specific pathogen-free pigs challenged with porcine circovirus type 2d.

BACKGROUND: Porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (MHP) are economically significant pathogens in the pig industry. The use of combined vaccines against PCV2 and MHP is one of the most effective ways of protecting pigs from both diseases, and it has become a part of general management. OBJECTIVES: This study evaluated the efficacy of two new bivalent vaccines of PCV2 and MHP (Myco-X and Myco-XD) in SPF pigs. Myco-X and Myco-XD are a combined vaccine of MHP with PCV2b and PCV2d, respectively. METHODS: Sixteen pigs were divided into four groups: Myco-X-vaccinated challenged, Myco-XD-vaccinated challenged, unvaccinated challenged, and unvaccinated unchallenged. Two milliliters of Myco-X were administered intramuscularly, and 0.5 mL of Myco-XD was injected intradermally at 3 wk of age. The pigs were challenged with virulent PCV2d via the intramuscular and intranasal route 4 wk post-vaccination. RESULTS: All vaccinated pigs showed effective reduction of the clinical signs, the PCV2d load in the blood and nasal swab samples, as well as lung and lymphoid tissue lesions in the challenge test. Compared to unvaccinated challenged animals, the vaccinated challenged animals showed significantly higher (p < 0.05) levels of anti-PCV2 IgG, PCV2d-specific interferon-γ (IFN-γ), and anti-MHP IgG. CONCLUSIONS: Based on clinical, microbiological, serological, and pathological assessments, this study confirmed that both combined vaccines could protect pigs against PCV2 infection caused by PCV2d. On the other hand, further research on the efficacy evaluation of these new vaccines against the MHP challenge and PCV2d/MHP co-challenge is needed.

Comparison of the pathogenicity of porcine reproductive and respiratory syndrome virus (PRRSV)-1 and PRRSV-2 in pregnant sows.

To date, few studies related to the evaluation of the pathogenicity of different PRRSV isolates using a reproductive model have been undertaken, and the main focus has remained on respiratory models using young pigs. This study aimed to evaluate the pathogenicity of two PRRSV-1 isolates (D40 and CBNU0495) and two PRRSV-2 isolates (K07-2273 and K08-1054) in a reproductive model. Pregnant sows were experimentally infected with PRRSV at gestational day 93 or used as an uninfected negative control. Sera were collected at 0, 3, 7, 14, and 19 days post-challenge (dpc) for virological and serological assays. At 19 dpc, all sows were euthanized, and their fetuses were recovered by performing cesarean section and immediately euthanized for sample collection. Here, compared to the other isolates, the CBNU0495 isolate replicated most efficiently in the pregnant sows, and K07-2273 produced the highest rate of reproductive failure even though it did not replicate as efficiently as the other isolates in sows and fetuses, indicating that vertical transmission and reproductive failure due to PRRSV infection do not have any significant correlation with the viral loads in samples from sows and fetuses. Similarly, the viral loads and the histopathological lesions did not show any correlation with each other, as the PRRSV-2-infected groups displayed more prominent and frequent histopathological lesions with lower viral loads than the PRRSV-1-infected groups. However, viral loads in the myometrium/endometrium might be related to the spreading of PRRSV in the fetuses, which affected the birth weight of live fetuses. This study contributes to a better understanding of the pathogenicity of the most prevalent Korean PRRSVs in a reproductive model.

CAvant® WO-60 as an Effective Immunological Adjuvant for Avian Influenza and Newcastle Disease Vaccine.

Despite the immunogenicity of vaccines currently used in poultry, several pathogens, including avian influenza virus (AIV) and Newcastle disease virus (NDV), cause enormous economic losses to the global poultry industry. The efficacy of vaccines can be improved by the introduction of effective adjuvants. This study evaluated a novel water-in-oil emulsion adjuvant, CAvant® WO-60, which effectively enhanced both the immunogenicity of conserved influenza antigen sM2HA2 and inactivated whole H9N2 antigen (iH9N2). CAvant® WO-60 induced both humoral and cell-mediated immunity in mice and provided 100% protection from challenge with 10 LD50 of A/Aquatic bird/Korea/W81/2005 (H5N2) and A/Chicken/Korea/116/2004 (H9N2) AIV. Importantly, immunization of chickens with iH9N2 plus inactivated NDV LaSota (iNDV) bivalent inactivated vaccine emulsified in CAvant® WO-60 induced seroprotective levels of antigen-specific antibody responses. Taken together, these results suggested that CAvant® WO-60 is a promising adjuvant for poultry vaccines.

Evaluation of the Cross-Protective Efficacy of a Chimeric PRRSV Vaccine against Two Genetically Diverse PRRSV2 Field Strains in a Reproductive Model.

Despite the routine use of porcine reproductive and respiratory syndrome (PRRS)-modified live vaccines, serious concerns are currently being raised due to their quick reversion to virulence and limited cross-protection against divergent PRRS virus (PRRSV) strains circulating in the field. Therefore, a PRRS chimeric vaccine (JB1) was produced using a DNA-launched infectious clone by replacing open reading frames (ORFs) 3-6 with those from a mixture of two genetically different PRRSV2 strains (K07-2273 and K08-1054) and ORF1a with that from a mutation-resistant PRRSV strain (RVRp22) exhibiting an attenuated phenotype. To evaluate the safety and cross-protective efficacy of JB1 in a reproductive model, eight PRRS-negative pregnant sows were purchased and divided into four groups. Four sows in two of the groups were vaccinated with JB1, and the other 4 sows were untreated at gestational day 60. At gestational day 93, one vaccinated group and one nonvaccinated group each were challenged with either K07-2273 or K08-1054. All of the sows aborted or delivered until gestation day 115 (24 days post challenge), and the newborn piglets were observed up to the 28th day after birth, which was the end of the experiment. Overall, pregnant sows of the JB1-vaccinated groups showed no meaningful viremia after vaccination and significant reductions in viremia with K07-2273 and K08-1054, exhibiting significantly higher levels of serum virus-neutralizing antibodies than non-vaccinated sows. Moreover, the JB1-vaccinated groups did not exhibit any abortion due to vaccination and showed improved piglet viability and birth weight. The piglets from JB1-vaccinated sows displayed lower viral concentrations in serum and fewer lung lesions compared with those of the piglets from the nonvaccinated sows. Therefore, JB1 is a safe and effective vaccine candidate that confers simultaneous protection against two genetically different PRRSV strains.

The Potential Adjuvanticity of CAvant®SOE for Foot-and-Mouth Disease Vaccine.

Foot-and-mouth disease (FMD) is a notifiable contagious disease of cloven-hoofed mammals. A high potency vaccine that stimulates the host immune response is the foremost strategy used to prevent disease persistence in endemic regions. FMD vaccines comprise inactivated virus antigens whose immunogenicity is potentiated by immunogenic adjuvants. Oil-based adjuvants have clear advantages over traditional adjuvant vaccines; however, there is potential to develop novel adjuvants to increase the potency of FMD vaccines. Thus, we aimed to evaluate the efficacy of a novel water-in-oil emulsion, called CAvant®SOE, as a novel vaccine adjuvant for use with inactivated FMD vaccines. In this study, we found that inactivated A22 Iraq virus plus CAvant®SOE (iA22 Iraq-CAvant®SOE) induced effective antigen-specific humoral (IgG, IgG1, and IgG2a) and cell-mediated immune responses (IFN-γ and IL-4) in mice. Immunization of pigs with a single dose of iA22 Iraq-CAvant®SOE also elicited effective protection, with no detectable clinical symptoms against challenge with heterologous A/SKR/GP/2018 FMDV. Levels of protection are strongly in line with vaccine-induced neutralizing antibody titers. Collectively, these results indicate that CAvant®SOE-adjuvanted vaccine is a promising candidate for control of FMD in pigs.

Efficacy of Porcine Epidemic Diarrhea Vaccines: A Systematic Review and Meta-Analysis.

Porcine epidemic diarrhea (PED) is a devastating disease that causes considerable economic damage to the global pig industry. Although the causative agent, the porcine epidemic diarrhea virus (PEDV), was identified about a half century ago, there is still much debate on the preventive measures against the disease, especially regarding the PED vaccine. Recent reports on PEDV variants make the vaccination for PEDV more confusing. Therefore, we systematically reviewed published articles on PED and vaccines against the disease and performed a meta-analysis of vaccine efficacy based on the clinical signs, fecal score and survival rates. A total of 299 articles on the efficacy of PED vaccines were found online, and 21 articles were selected that fulfilled all the criteria. A meta-analysis was performed on the 21 articles based on the fecal scores and survival rates. This analysis showed the efficacy of PED vaccines, and no significant differences in the efficacy depending on vaccine type (killed vs. live) or administration route (intramuscular vs. oral) were found. The results from our study suggest that any vaccination against PED is a useful strategy to control the disease regardless of the type of vaccine and administration route.

Assessment of the safety and efficacy of an attenuated live vaccine based on highly virulent genotype 2b porcine epidemic diarrhea virus in nursing piglets.

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.

Development of Actinobacillus pleuropneumoniae ApxI, ApxII, and ApxIII-specific ELISA methods for evaluation of vaccine efficiency.

Among various vaccines against Actinobacillus pleuropneumoniae, subunit vaccines using recombinant proteins of ApxI, ApxII, and ApxIII as vaccine antigens have shown good efficacy in terms of safety and protection. Therefore, subunit vaccines are being applied worldwide and the development of new subunit vaccines is actively being conducted. To evaluate the efficacy of the subunit vaccines, it is important to measure immune responses to each Apx toxin separately. However, the cross-reactivity of antibodies makes it difficult to measure specific immune reactivity to each toxin. In the present study, specific antigen regions among the toxins were identified and cloned to solve this problem. The antigenicity of each recombinant protein was demonstrated by Western blot. Using the recombinant proteins, we developed enzyme-linked immunosorbent assay (ELISA) methods that can detect specific immune responses to each Apx toxin in laboratory guinea pigs. We suggest that the ELISA method developed in this study can be an important tool in the evaluation of vaccine efficiency and vaccine development.

Identification of novel immunogenic proteins against Streptococcus parauberis in a zebrafish model by reverse vaccinology.

Streptococcus parauberis is the major infectious agent of streptococcosis in the olive flounder (Paralichthys olivaceus), causing serious economic damage. In this study, we identified potential vaccine candidates against S. parauberis by reverse vaccinology. In total, the 2 out of 21 proteins were identified as vaccine candidates from two available S. parauberis genomes. The membrane-anchored protein SEC10/PgrA and the metal ABC transporter substrate-binding lipoprotein mtsA were potent antigenic proteins based on western blotting with mouse-derived antiserum against whole bacteria of S. parauberis serotypes I and II. In particular, metal ABC transporter substrate-binding lipoprotein (mtsA) showed similar protective immunity to that of whole-cell bacterins against S. parauberis in a zebrafish model. These results suggest that mtsA may be considered as a novel candidate in the development of vaccines against S. parauberis.

Microcrystalline Cellulose for Delivery of Recombinant Protein-Based Antigen against Erysipelas in Mice.

The study describes the development of a vaccine using microcrystalline cellulose (Avicel PH-101) as a delivery carrier of recombinant protein-based antigen against erysipelas. Recombinant SpaA, surface protective protein, from a gram-positive pathogen Erysipelothrix rhusiopathiae was fused to a cellulose-binding domain (CBD) from Trichoderma harzianum endoglucanase II through a S3N10 peptide. The fusion protein (CBD-SpaA) was expressed in Escherichia coli and was subsequently bound to Avicel PH-101. The antigenicity of CBD-SpaA bound to the Avicel was evaluated by enzyme-linked immunosorbent (ELISA) and confocal laser scanning microscope (CLSM) assays. For the examination of its immunogenicity, groups of mice were immunized with different constructs (soluble CBD-SpaA, Avicel coated with CBD-SpaA, whole bacterin of E. rhusiopathiae (positive control), and PBS (negative control)). In two weeks after immunization, mice were challenged with 1x107 CFU of E. rhusiopathiae and Avicel coated with CBD-SpaA induced protective immunity in mice. In conclusion, this study demonstrates the feasibility of microcrystalline cellulose as the delivery system of recombinant protein subunit vaccine against E. rhusiopathiae infection in mice.

Phenotypic and genotypic analyses of an attenuated porcine reproductive and respiratory syndrome virus strain after serial passages in cultured porcine alveolar macrophages.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a globally ubiquitous swine viral pathogen that causes major economic losses worldwide. We previously reported an over-attenuated phenotype of cell-adapted PRRSV strain CA-2-P100 in vivo. In the present study, CA-2-P100 was serially propagated in cultured porcine alveolar macrophage (PAM) cells for up to 20 passages to obtain the derivative strain CA-2-MP120. Animal inoculation studies revealed that both CA-2-P100 and CA-2-MP120 had decreased virulence, eliciting weight gains, body temperatures, and histopathologic lesions similar to those in the negative control group. However, compared to CA-2-P100 infection, CA-2-MP120 yielded consistently higher viremia kinetics and enhanced antibody responses in pigs. All pigs inoculated with CA-2-MP120 developed viremia and seroconverted to PRRSV. During 20 passages in PAM cells, CA-2-MP120 acquired 15 amino acid changes that were mostly distributed in nsp2 and minor structural protein-coding regions. Among these changes, 6 mutations represented reversions to the sequences of the reference CA-2 and parental CA-2-P20 strains. These genetic drifts may be hypothetical molecular markers associated with PRRSV macrophage tropism and virulence. Our results indicate that the PAM-passaged CA-2-MP120 strain is a potential candidate for developing a live, attenuated PRRSV vaccine.

Genetic characteristics, pathogenicity, and immunogenicity associated with cell adaptation of a virulent genotype 2b porcine epidemic diarrhea virus.

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.

Efficacy of an inactivated genotype 2b porcine epidemic diarrhea virus vaccine in neonatal piglets.

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.

Pathogenicity and genetic characteristics associated with cell adaptation of a virulent porcine reproductive and respiratory syndrome virus nsp2 DEL strain CA-2.

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.

Protective efficacy of Streptococcus iniae derived enolase against Streptococcal infection in a zebrafish model.

Enolase (ENO) is one of the surface-exposed proteins of Streptococcus iniae, which previously had been identified as a plasminogen-binding protein. In this study, ENO was evaluated to induce cross-protective immunity against S. iniae and Streptococcus parauberis which are major pathogens causing streptococcosis in fish. Immunoblot analysis shows that S. iniae recombinant ENO (rENO) produced in Escherichia coli was cross-reactive with antisera against S. iniae, and S. parauberis serotype I and II. In the challenge experiment of streptococcal infection after vaccination in zebrafish, rENO elicited a similar protection with a whole cell bacterin against S. iniae and S. parauberis, which suggests its feasibility as an efficient vaccine against streptococcosis.