Invasion and spread of equine herpesvirus 9 in the olfactory pathway of pigs after intranasal inoculation.

The neuropathogenesis of equine herpesvirus 9 (EHV-9) in pigs was investigated by intranasal inoculation of the virus together with intramuscular administration of dexamethasone (DM). All infected pigs developed characteristic meningo-encephalitis, accompanied by basophilic intranuclear inclusion bodies in the neuronal cells. One non-DM-treated and two DM-treated pigs had prominent malacic lesions in the rhinencephalon. Associated with the encephalitic lesions, there was invariably an increase in the number of nucleated cells in the cerebrospinal fluid (CSF). EHV-9 antigen was first detected in the nasal and olfactory epithelial cells in the nasal cavity, and in the neuroglial cells in the olfactory bulb. Subsequently it was demonstrated in the amygdaloid and caudate nuclei, and putamen. The virus was not isolated from the CSF. These results suggest that, after intranasal inoculation, EHV-9 replicates in the olfactory epithelial cells, spreading to the central nervous system via the olfactory pathway.

Brain lesions and transmission of experimental equine herpesvirus type 9 in pigs.

We demonstrated that pigs are susceptible to acute infection by equine herpesvirus type 9 (EHV-9). Six 8-week-old SPF pigs were inoculated intranasally and four were inoculated orally with different doses of EHV-9, and observed for 6 days. Although neurological signs did not develop in any of the infected pigs, the six intranasally infected pigs and one of the orally infected pigs developed lesions of encephalitis consisting of neuronal necrosis, neuronophagia, and intranuclear inclusion bodies, distributed mainly in the rhinencephalon. EHV-9 antigen was localized in the necrotic neuronal cells and was closely associated with the presence of inclusion bodies. These findings clearly demonstrate that pigs are fully susceptible to EHV-9 infection following intranasal inoculation (but less so following oral inoculation), and that EHV-9 in pigs has a highly neurotropic nature.

Structure-activity relationship and conformational analysis of monoglycosylceramides on the syngeneic mixed leukocyte reaction.

We examined effects of alpha-, beta-galactosylceramides (CalCers) and alpha-, beta-glucosylceramides (GlcCers) on the syngeneic mixed leukocyte reaction (MLR) using spleen cells (responder cells) and dendritic cells (DC, stimulator cells). The DC pretreated with these alpha-monoglycosylceramides markedly stimulated the proliferation of spleen cells, in contrast to the little stimulatory effects produced by the DC pretreated with the corresponding beta-anomers. In addition, when we compared the effects of alpha-GalCer derivatives on the syngeneic MLR, it appeared that the 2"- and 3-hydroxyl groups in alpha-GalCers play a critical role in their stimulation of the MLR response. Based on these results, we performed a computer-aided molecular modeling study, and found that the orientations of the 2"-, 4"- and 3-hydroxyl groups common to alpha-GalCer and alpha-GlcCer are not accessible to those of inactive monoglycosyleeramides such as beta-GalCer. These results suggest that there might be a receptor-like site for alpha-monoglycosylceramides on the cells which are involved in the MLR response.

Immunohistochemical demonstration of spread of Aujeszky's disease virus to the porcine central nervous system after intestinal inoculation.

Aujeszky's disease virus (ADV) was injected into the duodenal lumen of eight specific pathogen-free pigs aged 5 weeks. The infected pigs did not show any diarrhoea or nervous symptoms, but they developed characteristic necrotizing enteritis and myenteric plexitis, accompanied by follicular necrosis in the Peyer's patches. ADV antigen was detected in the submucosa of the dome area of Peyer's patches, lymphatic follicles, Meissner's and Auerbach's plexuses, solar ganglia and thoracic spinal ganglia. These findings suggest that ADV spreads from the intestinal mucosa to the central nervous system via the autonomic nerves.

Effects of alpha- and beta-galactosylated C2-ceramides on the immune system.

In contrast to the immunosuppressive effects of C2-ceramide (C2-Cer), alpha-galactosylceramides with ceramides having more than 10 carbons in fatty acid chains have immunostimulatory activities. We therefore synthesized alpha- and beta-galactosylated C2-Cers in order to examine their effects on the immune system. beta-Galactosylated C2-Cer and C2-Cer suppressed the allogeneic mixed leukocyte reaction (MLR) responses, but alpha-galactosylated C2-Cer stimulated the MLR response.

Immunostimulatory activities of mono- or diglycosylated alpha-galactosylceramides.

We examined the effects of 2"- or 3"-monoglycosylated alpha-galactosylceramides (alpha-GalCers) and 2",3"-diglycosylated alpha-GalCers on allogeneic MLR and the proliferation of murine spleen cells. It was found that their ceramide portions greatly affect their immunostimulatory activities, and that the 3"-hydroxyl group plays a more important role in the immunostimulatory effects of alpha-GalCer derivatives than the 2"-hydroxyl group.

Immunohistopathological characterization of pig pneumonia caused by a combined Aujeszky's disease virus and Actinobacillus pleuropneumoniae infection.

Nine pigs were inoculated endobronchially with Actinobacillus pleuropneumoniae serotype 1 (App-1) 6 days after infection with Aujeszky's disease virus (ADV); four died within 3 days and the remainder were killed after 1-6 days. Immunohistopathologically, there were two types of pneumonic lesion: pleuropneumonia, characterized by coagulative necrosis, oedema and fibrinous thrombosis; and necrotizing interstitial pneumonia, characterized by bronchitis, bronchiolitis and alveolitis. The former type of lesion was associated with App-1 antigen, and the latter with ADV antigen. These results indicated that a combined ADV and App-1 infection produced severe haemorrhagic pleuropneumonia; and that ADV and App-1 each produced a characteristic pneumonic lesion.

Immunostimulatory activities of monoglycosylated alpha-D-pyranosylceramides.

We compared the immunostimulatory effects of chemically synthesized alpha-galactosylceramides (alpha-GalCers), alpha-glucosylceramides (alpha-GluCers), 6"-monoglycosylated alpha-GalCer and 6"- or 4"-monoglycosylated alpha-GluCer and made the following observations: (1) the length of the fatty acid side chain in the ceramide portions greatly affects the immunostimulatory effects of alpha-GalCers and alpha-GluCers; (2) the configuration of the 4"-hydroxyl group of the inner pyranose moiety plays an important role in the immunostimulatory effects of monoglycosylated alpha-D-pyranosylceramides; (3) the free 4"-hydroxyl group of the inner pyranose of monoglycosylated alpha-D-pyranosylceramides plays a more important role in their immunostimulatory effects than the free 6"-hydroxyl group.

cDNA cloning of porcine interleukin-2 receptor-alpha gene.

Porcine interleukin-2 receptor-alpha subunit (IL-2R alpha) cDNA was cloned from the cDNA library of Con A-stimulated PBMC. The coding sequence of porcine IL-2R alpha, including the signal peptide sequence, is 813 b.p. in length. The identities of the sequence when it was compared with ovine, murine, feline and human sequences were 72.2, 62.4, 69.8 and 68.9% at nucleotide level and 58.9, 44.6, 54.6 and 55.6% at amino acid level, respectively. Then, the coding sequence of porcine IL-2R alpha was subcloned into the COS expression vector, pcDNA3.1/Zeo(+), and transfected into COS-7 cells. The expressed protein was specifically reactive to the mAb, 231-3B2, which seemed to be specific for porcine IL-2R alpha. This result reciprocally confirmed that the mAb, 231-3B2, recognizes porcine IL-2R alpha on a molecular basis.

Pneumonia in pigs infected with pseudorabies virus and Haemophilus parasuis serovar 4.

Six HPCD (hysterectomy-produced, colostrum-deprived) pigs were inoculated endobronchially with pseudorabies virus (PRV) in the right caudal lobe by means of a bronchoscope. Two pigs, killed on days 5 and 7, had severe purulent pneumonia in the right caudal lobe, associated with an accidental Haemophilus parasuis serovar 4 infection. The three surviving animals were treated with antibiotics. The pigs infected with PRV had necrotizing bronchiolitis and alveolitis. PRV antigen was closely associated with necrotic foci, and was sometimes surrounded by profuse H. parasuis antigen. PRV antigen and IgG- and IgA-containing cells were also detected in bronchioalveolar lavage fluid. These results suggested that the PRV infection destroyed respiratory epithelial cells and allowed H. parasuis to proliferate in the lungs.

Pneumonia in pigs inoculated with pseudorabies virus by means of a bronchoscope.

Pigs inoculated endobronchially (EB) with 2 ml of virus suspension containing 10(4) TCID50 per ml of the YS-81 strain of pseudorabies virus (PRV), by means of a bronchoscope, all developed viral pneumonia. No pneumonic lesions were observed in intranasally inoculated pigs. Macroscopical and microscopical lesions were localized to the middle to caudal parts of the right caudal lobe and were closely associated with the site at which the inoculum was deposited. PRV became attached to all types of cells and caused destruction of epithelial cells, and viral antigen persisted in the alveolar macrophages. After PRV infection, the total cell number in broncho-alveolar lavage (BAL) fluid was slightly increased and a high titre of PRV was found in the cells of BAL fluid in EB infected pigs. The findings suggest that PRV infection leads to dysfunction of alveolar macrophages before cell death is produced by virus replication.

Reverse interference method for measurement of hog cholera virus (HCV) and anti-HCV antibody.

A new procedure was developed for the assay of the hog cholera virus (HCV) and anti-HCV antibody. Initially, the suppression effect of HCV on interferon (IFN) by HCV production was confirmed. Swine kidney cell cultures preinfected with HCV produced no IFN, even following the addition of IFN inducers. However the sensitivity of the cell to IFN was not influenced by the infection with this virus. Based on these results, a new method, named reverse interference method, was established. In this method, infective titer of HCV was determined by the appearance of cell pathogenic effects (CPE) induced by vesicular stomatitis virus (VSV), which is caused by the suppression effect on the heterologous interference of GPE- strain of HCV against VSV infection in swine kidney cell cultures. This method showed nearly the same sensitivity as the END method. There was no difference in the infective titer of HCV and antibody titer against HCV as estimated by this method and the END method. The reverse interference method had advantages in rapidity and objectivity compared with the END method.

Synthesis of chemically modified sialic acid-containing sialyl-LeX ganglioside analogues recognized by the selectin family.

Sialyl Lewis X ganglioside analogues containing 5-acetamido-3,5-dideoxy-L-arabino-2-heptulopyranosylonic acid (C7-Neu5Ac), 5-acetamido-3,5-dideoxy-D-galacto-2-octulopyranosylonic acid (C8-Neu5Ac), and 5-acetamido-3,5-dideoxy-L-glycero-D-galacto-1-2-nonulopyranosylonic++ + acid (8-epi-Neu5Ac) in place of N-acetylneuraminic acid (Neu5Ac) have been synthesized. Glycosylation of 2-(trimethylsilyl)ethyl 6-O-benzoyl-beta-D-galactopyranoside with the phenyl or methyl 2-thioglycoside derivatives of the respective sialic acids, using N-iodosuccinimide (NIS)-trifluoromethanesulfonic acid as a promoter in acetonitrile, gave the three required 2-(trimethylsilyl)ethyl (2S)-sialyl-(2-->3)-beta-galactopyranosides. These were converted via O-benzoylation, selective transformation of the 2-(trimethylsilyl)ethyl group to acetyl, and introduction of the methylthio group with methylthiotrimethylsilane into the corresponding glycosyl donors. Glycosylation of 2-(trimethylsilyl)ethyl O-(2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl)-(1-->3)-O-(2-acetamido-6-O- benzyl- 2-deoxy-beta-D-glucopyranosyl)-(1-->3)-2,4,6-tri-O-benzyl-beta-D- galactopyranoside with these donors in the presence of dimethyl(methylthio)sulfonium triflate (DMTST) afforded the expected beta-glycosides, which were converted into the corresponding alpha-trichloroacetimidates, and these, on coupling with (2S, 3R, 4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol, gave the required beta-glycosides. Finally, these were transformed via selective reduction of the azide group, condensation with octadecanoic acid, O-deacylation, and de-esterification into the target compounds in good yields.