Direct functional screening of a cDNA expression library derived from primary porcine alveolar macrophages (PAM) revealed that CD163 is capable of conferring a porcine reproductive and respiratory syndrome virus (PRRSV)-permissive phenotype when introduced into nonpermissive cells. Transient-transfection experiments showed that full-length CD163 cDNAs from PAM, human U937 cells (histiocytic lymphoma), African green monkey kidney cells (MARC-145 and Vero), primary mouse peritoneal macrophages, and canine DH82 (histocytosis) cells encode functional virus receptors. In contrast, CD163 splice variants without the C-terminal transmembrane anchor domain do not provide PRRSV receptor function. We established several stable cell lines expressing CD163 cDNAs from pig, human, and monkey, using porcine kidney (PK 032495), feline kidney (NLFK), or baby hamster kidney (BHK-21) as the parental cell lines. These stable cell lines were susceptible to PRRSV infection and yielded high titers of progeny virus. Cell lines were phenotypically stable over 80 cell passages, and PRRSV could be serially passed at least 60 times, yielding in excess of 10 5 50% tissue culture infective doses/ml.

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Twelve hybridomas secreting monoclonal antibodies (MAbs) against Miller virulent strain of transmissible gastroenteritis virus (TGEV) were generated and characterized. In a cell culture immunofluorescence (CCIF) assay, three MAbs directed against peplomer protein (E2) had perinuclear fluorescence and four unclassified MAbs showed cell membrane fluorescence. Six of these seven MAbs neutralized both attenuated and virulent TGEV, and the seventh (an unclassified MAb) neutralized only the latter virus. Two MAbs able to bind the cell membrane of infected cells had low neutralizing antibody titers (8 to 72) but were able to distinguish between virulent and attenuated TGEV (9- to 72-fold differences in neutralizing titers). Two E2-specific MAbs had higher neutralizing antibody titers (782 to 34,117) and showed 4- to 13-fold differences in titers against the attenuated and virulent TGEV strains. Five MAbs which were specific for nucleocapsid (N) protein had cytoplasmic, particulate fluorescence in CCIF, and did not neutralize TGEV. Comparison of CCIF antibody titers of MAbs to the virulent and attenuated strains of TGEV indicated that differences existed in titers of most E2 and all N-specific MAbs, with titers consistently higher against virulent TGEV (homologous strain). Hyperimmune antisera prepared in gnotobiotic pigs against the attenuated, virulent and a recent isolate of TGEV immunoprecipitated the 3 major structural proteins of both the attenuated and virulent TGEV strains. Relative mol. wt. differences in the E1 and E2 proteins between the two virus strains were revealed using either the hyperimmune pig sera or MAbs. In addition to the 48 K N protein, a 44 K protein was coimmunoprecipitated by the hyperimmune sera and MAbs, but mainly from lysates of attenuated TGEV.

polyacrylamide gels was found to react with viroplasms in SAil-infected monkey kidney cells (30). However, technical limitations (lack of a high-titered monospecific antiserum and lack of sufficient protein for direct characterization) prevented definitive proof of whether minor amounts of the gene 11 product were present in virus particles. We have pursued this question, because the presence of a third minor outer capsid protein in rotavirus particles could be important for understanding the biology of these viruses and for ongoing vaccine development programs. This report describes studies that used the baculovirus expression system to synthesize the gene 11 protein and antiserum produced to this expressed protein to characterize and clarify the properties of the SAil gene 11 product. MATERIALS AND METHODS Viruses and cells. The rotaviruses used in this study were simian rotavirus SAil, clone 3 (serotype 3), human Wa (serotype 1), human S2 (serotype 2), porcine Gottfried (serotype 4), porcine OSU (serotype 5), bovine Nebraska calf diarrhea virus (serotype 6), and human 69M (serotype 8). These rotaviruses were pretreated with 10 ,ug of trypsin per ml and propagated in fetal rhesus monkey kidney (MA104) cells as described previously (9). Wild-type baculovirus Aiutographa californica nuclear polyhedrosis virus (AcNPV) and recombinant virus pVL941/SA11-11 were propagated and assayed in confluent monolayers of Spodoptera frligiperda cells (IPLB-SF21-AE) at 27°C in Grace medium as previously described (8). Preparation and sequencing of gene 11 cDNA. A pBR322 plasmid containing a full-length cDNA of SAil gene 11 was synthesized from genomic RNA by addition of poly(A) tails as previously described (10). The cDNA was excised from EcoRI and HindIII sites and subcloned into transcription 3974

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