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The full-length equine interferon-gamma (eIFN-gamma) cDNA, including the secretion signal peptide coding region, was recloned into baculovirus transfer vector pAcYM1. This vector was co-transfected with Autographa californica nuclear polyhedrosis virus DNA or hybrid nuclear polyhedrosis virus DNA into Spodoptera frugiperda cells. The recombinant viruses, named AcEIFN-gamma and HyEIFN-gamma, were then recovered. Recombinant eIFN-gamma (reIFN-gamma) was accumulated in the culture fluid of the AcEIFN-gamma or HyEIFN-gamma infected Tricoplusia ni -derived cell line, BTI TN 5B1-4, and hemolymph of HyEIFN-gamma infected silkworm larvae. These reIFN-gamma forms were shown to be 14, 16, 18 and 20kDa proteins, and glycosylated as confirmed by SDS-PAGE and tunicamycin treatment. Both reIFN-gamma proteins, showed high-level biological activities to vesicular stomatitis virus by cytopathic effect reduction assay, and MHC class II antigen induction on the equine fetal kidney-78 cell line.

We investigated the ability of a baculovirus-insect cell system to produce sialylated glycoproteins. Despite the presence of enzymes for synthesizing complex-type N-glycans, the most frequent structure of insect N-glycan is the paucimannosidic type, Man(3)GlcNAc(2)(+/-Fuc). The reason for the overwhelming assembly of paucimannosidic N-glycans is not yet well understood. We hypothesized that this predominance might be due to insect-specific, Golgi-associated beta-N-acetylglucosaminidase (GlcNAcase)-mediated removal of N-acetylglucosamine residues from the precursor N-glycan, thereby preventing its galactosylation and terminal sialylation. As we expected, the suppression of intrinsic GlcNAcase activity with a specific inhibitor, 2-acetamido-1,2-dideoxynojirimycin, allowed the accumulation of sialylated glycoproteins in the supernatants of insect cell cultures after baculoviral infection. Our observation indicates that GlcNAcase-dependent depletion of N-acetylglucosamine residues from intermediate N-glycans is critical for the assembly of paucimannosidic N-glycans in insect cells and, more importantly, that insect cells (under specific conditions) retain the ability to construct sialylated N-glycans like those in mammalian cells.

The immune response to a naked plasmid DNA encoding the nucleoprotein (N protein) of porcine transmissible gastroenteritis virus (TGEV) was investigated in this study. A complementary DNA of the entire N gene was amplified by RT-PCR, and inserted into a mammalian expression vector (pcDNA3.1) to construct a recombinant plasmid (pcDNA/N). To evaluate the immunogenicity of the construct, BALB/c mice were intramuscularly immunized with different doses (50, 100 and 200 μg/mouse) of pcDNA/N twice at a 5-week interval. An optimal antibody response was achieved with 100 μg of pcDNA/N. The response lasted at least 11 weeks after primary immunization. By western blotting analysis, the antibodies specifically recognized a 47 kDa protein corresponding to the viral N protein, but they did not reveal neutralizing activity against infectious TGEV in vitro. Immunoglobulin G2a was predominant among these antibodies, which was indicative of Th1 type cell activation in pcDNA/N immunized mice. Moreover, spleen cells from these mice showed stronger immune responses than those from live vaccine or parental vector immunized mice. These results suggest that the construct can elicit both humoral and cell-mediated immune (CMI) responses against TGEV N protein in mice.

Eleven aborted bovine fetuses and five calves suspected as having neosporosis were necropsied and tissues from these animals were inoculated into bovine cardiopulmonary aortic endothelial cells and monkey kidney cells and maintained at 37°C with 5 per cent CO2. Neospora tachyzoites were observed in one cell 49 days after inoculation. The isolated parasite ( JPA1 ) was morphologically identical to the previously reported bovine Neospora species ( BPA1 ) and confirmed by its strong antigenic reactivity with bovine control antisera to Neospora species and its lack of reactivity with Toxoplasma gondii and Sarcocystis cruzi antisera. This is the first bovine Neospora species isolate in Asia and further studies with this isolate are now expected.