However, the number of foci in the virus control was also reduced drastically (<15?per well) (data not shown), suggesting that additional parts in the serum may already affect the infectivity of the disease. Open in a separate window Fig. only (Pritchard et MIV-150 al., 1999). PEDV was first found out in 1971 and rapidly spread across Europe and parts of Asia. Even though incidence of PED outbreaks in MIV-150 Europe offers diminished significantly by 1990, it remains as one of the most important viral pathogens in swine among Asian countries (Pensaert, 1999). In Korea, seasonal outbreaks resulting in high mortality MIV-150 of neonatal piglets as well as weight loss in fattening pigs cause serious economic deficits in the swine market (Chae et al., 2000). Due to the economic importance of PEDV, several cell-adapted strains of the disease have been developed as candidate live attenuated vaccines (Kweon et al., 1999, Music et al., 2003, Park et al., 2007). Development of additional vaccine candidates possess focused on the CO-26K fragment equal (COE), a website found on the spike protein that is reported to contain the neutralizing epitope against PEDV (Chang et al., 2002, Kang et al., 2006). Recently, another B-cell epitope was reported to induce neutralizing antibodies against PEDV (Cruz et al., 2006). This epitope, characterized by the GPRLQPY motif, was mapped to the carboxy-terminal region of the spike protein using phageCpeptide library. The location of this antigenic motif within the cytoplasmic tail of the spike protein is definitely of great interest since it offers important implications within the possible involvement of this domain in disease attachment and access. In this study, the antigenicity and neutralizing activity of antibodies against the GPRLQPY motif was investigated. Antibodies were generated by immunizing BALB/c mice having a peptide possessing a linear sequence identical to the 24?a.a. carboxy-terminal region of the PEDV spike protein then characterized by ELISA and focus reduction neutralization test. The properties of these antibodies were compared with those of polyclonal antisera and 2C10 monoclonal antibodies to PEDV. Cell-adapted strain of PEDV (KPEDV-9) was propagated in African green monkey MIV-150 kidney cells (Vero, CCL-81) following a procedure explained by Hofmann and Wyler (1988). Briefly, 175?cm2 TC flasks (Nunc) comprising a confluent monolayer of Vero cells were inoculated with KPEDV-9 and cultured in minimal essential medium (MEM, Gibco Life Technology) comprising 10?g?ml?1 trypsin at 37?C for 24?h. Infected cells were harvested and placed in 100?mM NaCl, 10?mM TrisCHCl, 1?mM EDTA buffer (STE buffer, pH 7.4). Progeny virions caught in intracellular vesicles were released by repeated freezing/thawing and harvested in the supernatant by centrifugation at 10,000?? for 10?m. Harvested disease was titrated by focus formation assay as explained previously (Cruz and Shin, 2007). Briefly, two-fold dilutions of the disease stock were inoculated to Vero Mouse monoclonal to Influenza A virus Nucleoprotein cells cultivated to confluence in 96-well TC plate (Nunc). After adsorption at 37?C for 2?h, the inoculum was removed and the monolayer was overlaid with MEM containing 0.5% methyl cellulose and 10?g?ml?1 trypsin then kept at 37?C, 5% CO2 for 12?h. After fixing with 5% formaldehyde and permeating with 1% Nonidet P-40, the cell monolayer was probed with mouse anti-PEDV polyclonal antisera followed by biotin-conjugated anti-mouse IgG (Vector Laboratories). Foci of virus-infected cells were visualized by addition of avidin-biotinylated horseradish peroxidase (HRP, Vector Laboratories) and 3,3-diaminobenzidine tetrahydrochloride dihydrate (DAB, Vector Laboratories) in the presence of NiCl and H2O2. The number of foci, indicated by clusters of dark gray-stained cells were observed and counted under an inverted light microscope (Zeiss). Synthetic peptides S-CT24 and S-CT17 were designed and purchased from Anygen, Korea. These peptides were based on the putative neutralizing epitope recognized previously using a seven-mer phageCpeptide library (Cruz et al., 2006). The S-CT24 peptide (ACFSGCCRGPRLQPYEAFQKVHVQ) and S-CT17 peptide (ACFSGCCREAFQKVHVQ) were reconstituted in 10?mM phosphate-buffered saline (PBS, pH 7.4) and kept at ?20?C until use. Groups of six 6-weeks-old BALB/c mice (Samtako, Korea) were immunized intraperitoneally with 100?g S-CT24 peptide, S-CT17 peptide or 1??105 ?ffu KPEDV-9 whole disease. The antigens were mixed with Freund’s total adjuvant (SigmaCAldrich) for the initial immunization, then with Freund’s incomplete adjuvant (SigmaCAldrich) on succeeding immunizations given every 2 weeks for 6 weeks. Blood samples were collected from your retro-orbital sinus prior to immunization and 1 week after final immunization b. Mice sera were extracted by centrifugation at 1500?? g, 10?m and kept at ?20?C prior to use. Euthanasia was performed by cervical dislocation. Antibody reactions.
