Porcine epidemic diarrhea virus (PEDV), a member of Coronaviridae, is the etiological agent of enteropathogenic diarrhea in swine [1±3] . Although the clinical symptoms of PEDV infection are similar to transmissible gastroenteritis virus (TGEV) infection, PEDV has a wider variety of clinical signs in pigs [4] . Propagation of PEDV in vitro was rather limited until Vero cells were found to support the growth of virus in the presence of trypsin [5] . In this study, we described the derivation of an attenuated strain of PEDV, as a potential vaccine candidate, through cell adaptation.
Vero cells obtained from ATCC (Vero C1008) were regularly maintained in alpha-MEM supplemented with 5% fetal bovine serum, penicillin (100 unit/ml), streptomycin (100 unit/ml) and amphotericin (0.25 g/ ml). The isolate of PEDV was originated from a neonatal pig and plaque puri®ed in Vero cells. The isolate, designated KPEDV-9, was passaged in 80±90% monolayers of Vero cells in alpha-MEM with 0.02% yeast extract, 0.3% tryptose phosphate broth (TPB) and 1±2 mg of trypsin as described [5, 6] . Sequential passages of the virus were normally conducted in roller culture. Each passage level of virus was stored at À708C or freeze-dried with equal volume of stabilizer (0.217 M Vaccine 17 (1999) 2546±2553 0264-410X/99/$ -see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 -4 1 0 X ( 9 9 ) 0 0 0 5 9 -6 Lactose, 0.0038 M KHPO 4 , 0.0072 M K 2 HPO 4 , 0.0049 M monosodium glutamate, 1% Gelatin). The ®nal stock of the PEDV was characterized in two ways. Firstly, the culture supernatant was subjected to direct centrifugation at 60,000 Â g for 2 h and the pellet was resuspended to 1/200 of initial volume for morphological identi®cation by transmission electron microscopy. Secondly, the virus infected cells were subjected to reverse transcription polymerase chain reaction (RT-PCR) to detect speci®c PEDV sequences. Three primers for RT-PCR were selected from the sequences information of membrane protein (M) gene of Duarte et al. [7] . P1 (27mer); 5 '±CCCCAGTACTGTTAT-TGACGTATAAAC±3 ' (position 974±1000), P2 (24mer); 5 '±GTTTAGACTAAATGAAGCACTTTC± 3 ' (position 1665±1688) for PCR and P3 (25mer); 5 '± GCCATAAAGTTTCTGTTTAGACTAA±3' (1702± 1678) as the primer for synthesis of complementary DNA, respectively. The extraction of RNA and RT-PCR were conducted according to the instructions of commercially available kit (Stratagene). PCR reactions were performed in the conditions as described previously [8] . After ampli®cation, the PCR products were cloned into pUC19 vector for sequencing using Sequenase version 2.0 (USB, USA). In addition, the presence of adventitious virus such as porcine parvovirus (PPV), Japanese encephalitis virus (JEV), Hog cholera virus (HCV) and other cythopathogenic viruses were examined as described previously [9±11].
For the detection of attenuation, viral stocks at 90 passages were tested in 4 days old piglets. From 10 6.0 TCID 50 /ml to 10 8.0 TCID 50 /ml of cell adapted PEDV was inoculated into suckling piglets intramuscularly or through the oral route. In order to compare the pathogenicity, PEDV isolate before cell adaptation was prepared from the small intestines of neonatal piglet. The intestine was ground in phosphate buered saline (PBS, pH 7.4). The 10% suspension was then ®ltered using a 0.2-mm membrane ®lter (Acrodisk, Gelman) and further diluted to 5-, 10-and 20-fold in PBS. Four groups of ®ve piglets were orally fed with the suspensions of diluted stock of intestine. The animals were observed for clinical symptoms of diarrhea and mortality in the inoculated animals was observed for 10 days.
Three pregnant sows 4±5 weeks prior to farrowing were tested for the detection of immune responses. Pigs were inoculated intramuscularly with the attenuated viruses at the titer of at 10 7.0 TCID 50 /ml. Two pregnant sows remained as uninoculated control. After two weeks, second inoculations of same titer were followed. Each paired serum before and after inoculation was collected at two-week interval. The collected sera and colostrum at delivery were tested for the presence of antibodies by ELISA. After delivery, suckling piglets of 2 days old were orally challenged with 10 or 5 LD 50 of wild PEDV. The clinical signs of diarrhea and mortality of challenged piglets were observed for two weeks.
For the preparation of antigen, the cell adapted PEDV was concentrated with polyethylene glycol (PEG, MW 6000) as described [12] . The PEG treated viral solution was then precipitated and resuspended at 1/10th of original volume with TEN buer (0.01 M Tris, 0.001 M EDTA, 0.1 M NaCl, pH 7.4). The puri-®ed virus was then used for the ELISA. The procedures for ELISA were basically the same with a previous study [13] . Brie¯y, the dilution of antigen and second antibodies were adjusted to the optical density (OD) around 0.2 (A 490) using the negative porcine sera. Usually each well in 96-well microplate (Costar) was coated with 1±2 mg of protein in 50 mM carbonate buer (pH 9.6) at 58C overnight, followed by the blocking with 1% skim milk at 378C. The 1/400 diluted porcine sera in PBS with 0.01% Bovine serum albumin (BSA) and 0.05% Tween 20 (PBST) were reacted at 378C for 30 min and then washed extensively with PBST three times at 5-min intervals. The reacted plate was washed again at the same condition and incubated with 2000-fold diluted horseradish peroxidase (HRP) labelled anti-porcine IgG (KPL) for 1 h at 378C. The plate was developed in O-phenylenediamine (OPD) at room temperature for 20 min. The reaction was stopped with 2 M H 2 SO 4 before measuring OD at 490 nm.
A total of 63 pregnant sows were inoculated intramuscularly with 1 ml of the virus containing 10 7.0 TCID 50 /ml. Twenty-three pregnant sows received one injection 3±4 weeks prior to farrowing. In another farm, 40 pregnant sows received two injections at 2±3week intervals before farrowing. The average number of the litters were compared with the data of uninoculated pregnant sows at the corresponding farm during same period of time.
The sequence accession number for M protein of attenuated KPEDV-9 is GeneBank accession number AFO15888.
The PEDV was continuously passaged in Vero cells. Sequential passage of virus regularly conducted every 4±5 days postinfection in cells. The supernatant was harvested and used for next inoculation in Vero cells up to 93 passages. However, cytopathogenic eect (CPE) in Vero cells was not so clear that the culture supernatant of virus infected cell was subjected to morphological and genetic characterization. When the culture supernatant of virus infected cell was examined by the transmission electron microscopy, characteristic shape of coronavirus with diameter of 100±150 nm was possible to identify (Fig. 1 ). In addition, the com-parision of M gene of cell passaged virus showed the 98.97% in nucleotide and 98.24% in amino acid identity with previously reported PEDV strain (Fig. 2) . No adventitious viral contaminations were detected in the ®nal stock of PEDV.
The pathogenicity of attenuated virus was tested in the 1-day-old piglets before taking colostrum. Six separate litters of 53 piglets and 15 litters of 111 piglets were inoculated intramuscularly with virus of 10 7.0 and 10 6.0 TCID 50 /ml, respectively. In this experiment, all the inoculated piglets failed to show signs of diarrhea and symptoms related to PEDV infection.
In order to avoid any possible eects from the maternal immunity through the colostrum and detect the potential pathogenicity of the attenuated virus, eight piglets of 4 days old were infected orally with 10 ml of virus stock, which contained virus of 10 8.0 TCID 50 /ml, and were arti®cially fed with dairy milk. Although three piglets showed signs of anorexia and mild signs of diarrhea in two or three days after inoculation, nevertheless, the signs seemed to be transient.
In fact, all piglets recovered in the next 2±3 days. However, in the groups of piglets fed with wild virus before cell passages, all the piglets developed symp-toms of watery diarrhea in 2±3 days, and the mortality reached up to 10±100%, depending on the dilution of virus within one week as shown in Table 1 .
When the collected sera were tested for the presence of the antibodies by ELISA, all inoculated sows showed the rising ELISA titers (Fig. 3) . On the other hands, the antibody titers of control pigs decreased at the time of delivery. In addition, colostrum at delivery showed higher or similar level of antibodies of corresponding sows. After challenge exposures mortality of piglets were compared with uninoculated control. Although the mortality of piglets after challenge with 10 LD 50 of wild PEDV was reduced to 20% compared to 100% in control litter, all piglets survived in the litters after challenge experiment with 5 LD 50 of virus compared to 60% in control (Table 2) . However, mild signs of diarrhea were also detected in one litter of piglets 2 days after challenge, but they recovered the next day.
The safety test of the attenuated virus in pregnant sow was conducted in two separate farms. One farm has not had any history of epidemic diarrhea in last few years and another farm had the experience of PEDV outbreak in the previous year. A total of 63 pregnant sows were inoculated once or twice before farrowing. As shown in Table 3 , all the inoculated sows farrowed the same average numbers of litters of uninoculated control group without any clinical problems.
In this study, we investigated the attenuation of PEDV through serial passages in Vero cell cultures and its prophylactic eect in pregnant sows. After serial passages in Vero cells, the growth of virus was rather trypsin-independent and the detection of cytopathic eect (CPE) was rather variable, depending on clones of Vero cell lines (data not presented here). Since the SPF or gnotobiotic piglets were not used in this experiment, it might not reasonable to ®gure out the exact dierences in pathogenicity between the wild and the attenuated virus. Nevertheless, when compared with the wild PEDV, the animals inoculated with the high passage level of virus did not show any severe signs of diarrhea or death in piglets, supporting attenuation.
It is known that PEDV replicates mainly in the villi of small intestines [14] . Like attenuated TGEV, the replication of attenuated PEDV may be limited to the small portion of intestine with short duration of secretion compared to virulent virus [15] . In fact, the detection of signs of diarrhea from piglets inoculated orally with attenuated virus delayed at least by two days and lasted one day compared with the signs from piglets inoculated with wild virus.
When we tested the immunoprophylactic eect in pregnant sows, it was demonstrated that the vaccinated swine resulted in reduced piglet mortality after challenge experiment (20±100% in vaccinates compared with 0±40% in controls), indicating that the attenuated PEDV could induce the status of immunity in pregnant pigs, providing protection in piglets like other enteric disease in swine. Previously, we found that PEDV infections were related to more than 20% of diarrheal cases in the neonatal pigs, thus causing a considerable losses in pig industry [13] . In fact, ®eld application of attenuated virus as vaccine resulted in the overall reduction of mortality of neonatal pigs (2± Fig. 3 . Immune responses of pregnant sows inoculated with cell attenuated KPEDV-9 strain. Animals were inoculated twice at 2-week intervals and serum samples were tested by ELISA. Table 2 Survival of piglets from attenuated KPEDV-9 vaccinated sows (V) and unvaccinated control (C) after challenge exposure 52% compared with before vaccination) in the farms having PEDV outbreak [16] . Nevertheless, it is worthy to note that the ecacy of protection was rather complicated and con¯icting according to the challenge dose like other enteric diseases. Although the pregnant pigs inoculated with attenuated virus showed the increased immune status by ELISA as compared to uninoculated control, it is dicult to explain the exact relation to mucosal immunity for protection. Since it was well con®rmed that the mechanisms of the passive immunity are extensively related to the presence of IgA and IgM antibodies [17] , further experiments, including detection of antibody secreting cells of IgA and IgM, may give the practical information for immunoprophylaxis against PEDV.