1 SDS-polyacrylamide gel electrophoresis analysis of N protein purified through Ni-NTA columns. in viral pathogenesis and replication (10). The open reading frame coding for the N protein is located at the 3 end of the RNA genome (7). Monoclonal antibodies against the N protein protect mice from lethal infection and inhibit IDH-C227 viral transcription in vitro (12). The monoclonal antibodies against the N IDH-C227 protein of coronaviruses are generally nonneutralizing (3, 6). This is the first study in which monoclonal antibodies against the N protein of ECV have been produced and characterized (there are no previous reports on the detection and pathogenesis of ECV). We have found these monoclonal antibodies to be very effective for use with immunohistochemistry (IHC) for the detection of BCV and ECV in formalin-fixed tissues. The lesions caused by ruminant coronaviruses are subtle and are similar to those caused by other ruminant viruses, such as bovine viral diarrhea virus, a pestivirus. It is difficult to make a confirmed diagnosis on the basis of histopathology alone. Thus, IHC could provide a useful adjunct tool for the confirmation of coronavirus infections. MATERIALS AND METHODS Virus and cells. ECV WY-29 was IDH-C227 propagated in human rectal tumor-18 cells with trypsin and pancreatin in the culture medium (8, 9) and was plaque purified as described previously (11). Cloning of the nucleoprotein gene of ECV in prokaryotic expression vector. Reverse transcription and PCR were performed with a forward primer (5-TCTGGCATGGACACCGCATT-3) and a reverse primer (5-CCAGGTGCCGACATAAGGTT-3). The PCR product was ligated into pBluescript-SK (+) and was then subcloned into a prokaryotic expression vector (pQE-30; Qiagen Inc., Chatsworth, Calif.). The nucleoprotein inserts were sequenced by using the Sequitherm EXCEL Cycle Sequencing kit (Epicentre Technologies, Madison, Wis.) to IDH-C227 confirm the exactness of the N protein sequence and proper IDH-C227 in-frame ligation. The complete sequence of ECV N protein cDNA has been published previously (11). Expression and purification of recombinant ECV N protein. Single colonies of transformants were grown in Luria-Bertani medium (Difco, Detroit, Mich.) with ampicillin (100 g/ml) and kanamycin (25 g/ml). Protein expression was induced with 2 mM isopropyl–d-thiogalactopyranoside (IPTG) according to the instructions provided by the manufacturer (Qiagen Inc.). After 4 h of induction, the cells were harvested by centrifugation at 4,000 for 15 min and lysed by sonification in buffer B (8 M urea, 0.1 M NaH2PO4, 0.01 M and Tris-HCl [pH 8.0]). The recombinant N proteins were analyzed on a sodium dodecyl sulfate (SDS)C10% linear polyacrylamide gel. Recombinant ECV N proteins were purified with Ni-NTA columns (the polyhistidine tag at the amino terminus of the recombinant N protein binds to Ni-NTA resin). The recombinant N fusion protein was detected by Western blot analysis with mouse antipolyhistidine as the primary antibody and horse anti-mouse horseradish peroxidase (HRPO) labeled as the secondary antibody. 4-Chloro-1-naphthol (4-CN) (Pierce, Rockford, Ill.) chromogen was used to detect the bands. Hybridoma production. Six-week-old BALB/c mice (Cowan I cells, and the cells were incubated on ice for 2 h. The bacterial cells were pelleted by centrifugation at 4,000 for 10 min and washed once with TSA (1% Triton X-100 and 1% sodium RFC4 deoxycholic acid) and once with 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA. The cells were centrifuged, and the bacterial pellet was resuspended in 20 l of 1% SDS sample loading buffer and then electrophoresed on an SDSC10% polyacrylamide gel. The complexes were transferred to nitrocellulose membranes by electroblotting. The membranes were incubated with bovine anti-BCV serum as the primary antibody, followed by goat anti-bovine HRPO as the secondary antibody. The color was developed with 4-CN. IHC. Spiral colon sections taken from calves experimentally infected with BCV were used for IHC. Tissues were formalin fixed and paraffin embedded. Tissues were sectioned at 4 m and heat fixed at 55C for 30 min. Then, the slides were prepared by previously described procedures (15). Anti-nucleoprotein monoclonal antibodies were used as primary antibodies, and anti-mouse HRPO was used as the secondary antibody. The slides were washed with distilled water and were counterstained with hematoxylin for 30 s. The sections were dehydrated and then mounted with Permount (Fisher, St. Louis, Mo.) and examined by light microscopy. RESULTS The ECV nucleoprotein gene was subcloned in the pQE-30 expression vector, and the recombinant N protein with a polyhistidine tag at the amino terminus was induced by the addition.
