Monoclonal antibodies (mAbs) were prepared against one UK isolate of turkey rhinotracheitis virus (TRTV). Those which were virus-neutralizing were selected and used, together with polyclonal antisera raised to each isolate in turkeys, in cross-neutralization tests against TRTV strains isolated in the UK and elsewhere. Whilst the polyclonal antisera showed that there was some diversity between them, all strains examined belonged to one serotype. The TRTV strains isolated in the UK could clearly be differentiated from those isolated elsewhere by some of the mAbs. Isolates of TRTV made in South Africa in 1978 and UK in 1985 were more closely related than were isolates made in UK and France within a few months. TRTV strains isolated from turkeys and chickens could not be differentiated. Some mAbs were found to be group-specific in that they neutralized all TRTV strains examined. All mAbs were of either the IgGl or IgG2a isotype and recognized the surface G glycoprotein.

SUMMARYSix interspecific hybridomas (heterohybridomas) secreting bovine monoclonal antibodies (MAbs) against respiratory syncytial (RS) virus were produced. Four of the heterohybridomas were formed using the mouse myeloma cell line NS1 as the fusion partner, one using NS0, and the remaining heterohybridoma was formed using a bovine × murine hybridoma as the fusion partner. Five heterohybridomas secreted bovine IgG1 and one secreted IgG2. All six MAbs recognized human subtype A and B viruses as well as bovine RS virus. They were specific for the fusion glycoprotein and reacted with a 140K dimer and a 70K monomer in a Western blot of native antigen; three also bound to the 46K F~ component and its 22K cleavage product in a blot of reduced antigen. Two of these MAbs neutralized RS virus infectivity, inhibited virusinduced fusion, lysed RS virus-infected cells in the presence of complement and protected mice against RS virus challenge.

Genes encoding fowlpox virus (FWPVAvipoxviruses, which replicate only in avian cells, have been developed as safe recombinant vectors for human vaccination (23,30). In mammalian cells, avipoxvirus replication is blocked but early gene expression occurs (45,46). Late gene expression occurs in some mammalian cell types infected by fowlpox virus (FWPV), the prototypic avipoxvirus, without productive virus replication, as morphogenesis appears to be blocked (42). FWPV has been much less intensively studied than vaccinia virus (VACV). It appears to form extracellular enveloped virus (EEV) by budding rather then wrapping (6,16 (27). The complete sequence of a pathogenic strain of FWPV (1) includes genes encoding homologs of at least 21 known VACV structural proteins, but FWPV appears to lack homologs of intracellular mature virus (IMV) proteins encoded by A27L and D8L as well as an intracellular enveloped virus protein encoded by A36R and the EEV proteins encoded by B5R, A33R, and A56R (1).Although genes encoding FWPV structural proteins have been identified by sequence homology, very little is known about the proteins they encode. We produced monoclonal antibodies (MAbs) and polyclonal antibodies against FWPV proteins as markers to study FWPV morphogenesis and identified genes encoding three FWPV immunodominant structural proteins. MATERIALS AND METHODSVirus and cells. The origins, propagation, and purification of FWPV strains (FP9, HP1, and Poxine) and canarypox virus (CNPV) have been described previously (5).MAbs. BALB/c mice were immunized twice intraperitonally 3 weeks apart with a crude preparation of Poxine in Quil A, a saponin-derived adjuvant (Superfos, Vedbaek, Denmark). For the second fusion, mice were injected twice 4 weeks apart with 100 g of sucrose gradient-purified FP9 virus (5). Hybridoma supernatants were screened by enzyme-linked immunosorbent assay or by indirect immunofluorescence assay, with Poxine-infected or noninfected chicken embryo fibroblasts (CEFs) as the antigen. The 3D9 MAb directed against the 39-kDa protein was kindly provided by C. P. Czerny.Polyclonal sera. Chicken hyperimmune sera against FP9, Poxine, or CNPV were produced as described previously (5).Radioimmunoprecipitation. Infected (multiplicity of infection, 10) or uninfected CEFs were incubated in methionine-free medium containing 2% dialyzed fetal calf serum. Two hours later, the medium was replaced with fresh medium containing 1.85 MBq of [ 35 S]methionine/ml and incubated for 2 h. The medium was removed, and the cells were washed once with phosphate-buffered saline (PBS) and lysed in 2 ml of radioimmunoprecipitation assay (RIPA) buffer (2.5 mM Tris [pH 7.2], 0.15 M NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 1% sodium deoxycholate). Lysates were centrifuged for 15 min (Eppendorf centrifuge), and supernatants were stored at Ϫ70°C.Ascitic fluid (10 l) or hybridoma supernatant (500 l) was incubated for 1 h at 4°C with 50 l of 10% protein A-Sepharose CL-4B (Pharmacia) in 0.1 M sodium phosphate buffer, pH 8.1. After...

Abstract. Gut associated lymphoid tissue of the small and large intestine of calves and cows has been compared morphologically and quantitatively using monoclonal antibodies to bovine lymphocytes. B cells were significantly decreased in the ileum of the cow compared to the calf. Significantly increased numbers of T cells were present in cell suspensions of all lymphoid areas of the cow compared to the calf. T lymphocyte subsets were quantified into cryostat sections of lymphoid tissues expressing BoT4, and BoT8 antigens demonstrated increased numbers in follicular and dome areas of the discrete Peyer's patches of the small and large intestine of the cow. BoT4+, BoT8+, and the non-BoT4+/BoT8+ T cell subsets were increased in the mucosa of the jejunum, ileum, and cecum, while only BoT8+ lymphocytes were increased in the colonic mucosa of the cow as compared to the calf. Similarities in structure and lymphocyte composition of the discrete Peyer's patches of the small intestine, cecum and colon and isolated single follicles in the large intestine suggest similar functional properties.Gut associated lymphoid tissue (GALT) consists of the lymphoid cells in the epithelium, the lymphoid and reticuloendothelial cells in the lamina propria, and the organized lymphoid aggregates in the mucosae, which include Peyer's patches and are the primary site of antigen uptake in the intestine.25At 40 weeks gestation, the bovine fetus contains up to 76 discrete Peyer's patches (DPP) in the duodenum and jejunum. A single continuous Peyer's patch (CPP) that extends into the cecum is present in the terminal jejunum and ileum. Another single discrete Peyer's patch is present in the proximal colon.1z There are 18 to 40 DPP in adult cattle." The CPP, however, involutes at puberty in cattleg and and the ruminant CPP may be a primary lymphatic organ, with a function similar to that of the bursa of Fabricius in birds.z7Monoclonal antibodies (mAbs) that recognize leukocyte differentiation antigens on bovine T lymphocytes have been described recently, and it has been possible to identify three major subsets of lymphocytes in peripheral blood.z In cattle, two of the major subpopulations of T lymphocytes are the bovine homologues BoT2+, Bo5+, BoT4+ T helperhducer cells and the BoT2+, Bo5+, BoT8+ T suppressor/cytotoxic cells.A third lymphocyte subset, which is BoT2-, BoT4-, BoT8-and expresses the Bo5 antigen but at a lower intensity than the BoT2+ cells, is also present. These BoT4-, BoT8-cells may comprise up to 50% of the peripheral blood mononuclear cells of calves. The function of this subpopulation has not been defined, although it is stimulated in the autologous mixed lymphocyte r e a c t i~n~J~J~~~~ and is the bovine homologue of the SBU-T19+ cells in sheep.22 In this study, the GALT of neonatal calves and adult cattle is compared. Using the mAbs to label cells in cryostat sections and cell suspensions from GALT, the distribution of the major subpopulations of lymphocytes within the gut of calves and adult cattle has been ascertained.

A murine IgG1 monoclonal antibody (mAb), CC76, has been produced that, based on findings of the relative molecular mass of polypeptides that it recognized, staining of leukocytes in blood and tissues, and the biological properties of the T lymphocyte subpopulations with which it reacts, is considered to identify an isoform of the leukocyte common antigen (LCA) family of molecules in cattle. The mAb is more similar to human CD45R which detect products requiring the presence of the B exon within the LCA gene and to the anti-rat mAb MRC-OX22, than to CD45RA or CD45R0. mAb CC76 reacts with an antigen expressed by subpopulations of cells in bovine blood that express BoCD2 and either the BoCD4 or BoCD8 antigens. T cells that express the gamma/delta T cell receptor identified with mAb to BoWC1 antigen did not react with CC76. The molecule detected is expressed on B cells but not on monocytes or granulocytes. Only 2% of cells in thymic suspensions stained with mAb CC76. Immature cortical thymocytes that were BoCD1+ did not react with CC76 and 90% of the cells in thymic suspensions that were CC76+ had the phenotype of mature thymocytes. These cells were primarily in the medulla. The LCA isoform detected thus appears to be acquired by mature cells shortly before emigration from the thymic medulla into the periphery. Expression of the molecule detected by mAb CC76 on cells from lymph nodes was similar to that in blood, but expression on cells from the gut mucosa was quite different. Almost all, 95% and 93% respectively, of the BoCD4+ cells in the gut mucosa or discrete Peyer's patches were CC76-. A greater proportion of BoCD8+ cells from these sites, 35% and 26%, expressed the antigen. Lymphocytes from animals that had been immunized with Trypanosoma brucei were sorted into BoCD4+, CC76+ and BoCD4+, CC76- populations and cultured in vitro with the variable surface glycoprotein antigen from the parasite. Lymphocyte transformation responses were entirely within the CC76- population indicating that the mAb distinguished naive from memory BoCD4+ T cells in cattle. Major histocompatibility complex (MHC) class I-restricted cytotoxic precursor cells that expressed the BoCD8 antigen sorted from cattle that were immune to Theileria parva were both CC76+ and CC76- indicating that different isoforms of the LCA may be expressed on MHC class I- and class II-restricted memory cells and that BoCD8 memory cells are heterogeneous with respect to the LCA isoform that they express.