Yasuo Miura

Properties of a coronavirus isolated from a cow with epizootic diarrhea

Abstract A coronavirus (Kakegawa isolate) isolated from a cow with epizootic diarrhea was grown in BEK-1 cells and examined for biophysical and biochemical properties. The Kakegawa isolate was able to replicate in the presence or absence of 5-iodo-2′-deoxy-uridine, indicating that its viral nucleic acid was RNA. It was highly sensitive to ether and chloroform, and moderately sensitive to trypsin and heat. It was, however, readily stabilized by treatment with cation at 50°C for 1 h. Its infectivity was slightly reduced at pH 3.0. The virus passed through a membrane filter of 200 nm pore size, but not through one of 100 nm pore size. The buoyant density of the virus was determined in a sucrose density gradient. The peak of infectivity and hemagglutinin activity was found at a density of 1.182. Neutralization and hemagglutination inhibition tests showed a close serological relationship between the Kakegawa isolate and the American strain of calf diarrhea coronavirus.

Canine coronavirus induces apoptosis in cultured cells

Abstract Canine coronavirus (CCoV) is widespread in dogs in several countries and causes mild enteric illness evolving to severe enteritis in young pups. In in vitro cultures canine coronaviruses generally induce extensive cell death, however nature of the events leading to cell death remains largely unknown. We analysed the induction of cytopathic effect by CCoV in a canine fibrosarcoma cell line (A-72) in order to characterize the apoptotic effect in homologous cell system. Following CCoV infection A-72 cell line, which is permissive to CCoV, showed reduced growth rate, as detected by MTT assay, a standard colorimetric assay for measuring cellular proliferation, and underwent to apoptotic death. Starting from 24h after CCoV infection, cells morphology appeared dramatically changed, with cells rounding and detachment from culture surface. Morphologic and biochemical features of apoptosis, such as blebbing of the plasma membrane, translocation of phosphatidilserine to cell surface and annexin V positive staining, nuclear fragmentation, apoptotic bodies formation and DNA laddering, were detected in CCoV-infected cells. Propidium iodide staining of infected culture indicated the appearance of hypodiploid DNA peak corresponding to apoptotic cell population. Commonly to other animal coronavirus infection caspase-3 is likely to contribute to the execution phase of apoptosis induced by CCoV in A-72 cells since we found activation of enzymatic activity as well as procaspase-3 activating cleavage. Apoptotic death of infected cells is detrimental as it causes cell and tissue destruction as well as inflammatory responses. Therefore in the case of CCoV associated gastroenteritis, apoptosis of epithelial mucosa cells may be responsible for pathology induced by CCoV infection.

Serological survey of equine viral diseases in Mongolia.

Three hundred sera were collected from horses in various parts of Mongolia in 2007 and seroepidemiological surveys for several equine viruses performed on them. Equid herpesvirus 1 and equine rhinitis A virus were prevalent, and equine arteritis virus and equid herpesvirus 3 were detected over a wide area though their rates of antibody‐positivity were not high. Equine infectious anemia was distributed locally. The rates of horses antibody‐positive for Japanese encephalitis virus and equine influenza virus were low, but these were detected. Bovine coronavirus antibodies were detected at a high rate, but it was not clear whether they were due to horse coronavirus.

A Low-molecular-weight Fraction of Bovine Colostrum and Milk Enhances the Oxidative Burst Activity of Polymorphonuclear Leukocytes

Bovine colostrum and milk contain many immunomodulatory components. The low-molecular-weight fraction (<10 kDa) was separated from colostrum and milk by gel filtration chromatography, and its effect on the oxidative burst of bovine polymorphonuclear leukocytes (PMNL) was investigated in vitro. The oxidative burst activity induced by Staphylococcus aureus was considerably enhanced when PMNLs were incubated with this low-molecular-weight fraction. However, phorbol 12-myristate 13-acetate did not trigger a burst after priming with this fraction. The oxidative burst activity enhanced by this fraction was reduced after heating. These results confirmed that a low-molecular-weight substance(s) of less than 10 kDa, present in bovine milk and colostrum, enhances the oxidative burst activity of PMNL.

Hemagglutination-inhibition test applied to the study of akabane virus infection in domestic animals

Abstract Some factors affecting the hemagglutination-inhibition (HI) reaction with Akabane virus were investigated and an HI test developed. The test was proven to be useful in studies of antibody responses in cattle and other domestic animals infected with Akabane virus. HI antibody titers of individual animals were shown to be closely correlated with their neutralizing antibody titers and to remain undiminished for a relatively long time. In some early sera from domestic animals infected with Akabane virus, HI antibody sensitive to 2-mercaptoethanol was demonstrated.

Development of loop-mediated isothermal amplification method for diagnosis of bovine leukemia virus infection

A rapid, sensitive loop-mediated isothermal amplification (LAMP) assay was established for diagnosis of bovine leukemia virus (BLV) infection. The LAMP assay for targeting the BLV-LTR region can detect at least 2 copies of proviral DNA in a 2microl sample and its sensitivity is equivalent to or greater than the conventional single PCR. In addition, amplification is obtained in less than 1h by incubating a single tube in a water bath. The data obtained by the LAMP assay applied to field samples were compared with PCR and serological tests for BLV. The results showed a high level of agreement with these serological methods, but there was one animal positive only by the LAMP assay. When the blood was collected from the cow after 6 months, the BLV antibody was detected. This suggested that the LAMP assay could help the detection of the cattle in the early stage of BLV infection. The LAMP assay is a rapid, sensitive and simple method for the diagnosis of BLV infection as reported for other pathogens, and is available for use in local laboratories without any special equipment.

Inhibitors of bovine parvovirus, coronavirus and rotavirus in precolostral and fetal bovine sera

Abstract Eleven, 11 and 2 of 11 precolostral sera from normal calves and 13, 14 and 9 of 14 sera from normal fetuses, 7 to 10 months of gestation, neutralized bovine parvovirus, coronavirus and rotavirus, respectively. When assayed by single radial immunodiffusion, all the sera contained IgG at 360 to 1400 mg/dl, and some of them had much smaller amounts of IgM or IgA. Most of the neutralizing activities against bovine coronavirus and rotavirus were readily inactivated by treatment with acetone or 2-mercaptoethanol. Some sera fractionated by Sephadex G-200 gel filtration or starch block electrophoresis had neutralizing activities against bovine parvovirus or coronavirus in fractions containing no detectable amounts of immunoglobulins. These observations seem to indicate the presence of substance(s), other than immunoglobulins, capable of inhibiting replication of bovine parvovirus, coronavirus or rotavirus. The chemical nature and the mode of action of the inhibitors await elucidation.

Inducement of cytopathic changes and plaque formation by porcine haemagglutinating encephalomyelitis virus

Abstract ESK cells were shown to be a good medium for propagating the 67N strain of porcine haemagglutinating encephalomyelitis virus, although no cytopathic effect was observed. The virus induced a readily recognizable cytopathic effect in ESK cells, when a non-cytotoxic amount of diethylaminoethyl-dextran (DEAE-dextran) was incorporated in the culture medium. Based on this finding, a sensitive, practical assay method for the virus was developed. When DEAE-dextran was incorporated in the agar overlay medium, 67N virus formed plaques in ESK cell monolayers. The cytopathic effect as well as the plaque formation were specifically inhibited by antisera against the virus. Neutralization tests were developed on the basis of these findings. Neutralization and haemagglutination-inhibition tests on swine serum samples indicated a wide dissemation of haemagglutinating encephalomyelitis virus or antigenically-related viruses in Japanese pigs.

A mutant of pseudorabies virus with deletion of glycoprotein gIII gene prepared from a Japanese isolate: It fails to agglutinate mouse erythrocytes

A mutant with deletion of the glycoprotein gIII gene was produced from a Japanese isolate of pseudorabies virus (PrV) and characterized. Viral titers of the mutant propagated in PK-15 cells were always lower than those of the parental virus. The parental virus agglutinated BALB/c mouse erythrocytes, whereas the deletion mutant showed no hemagglutinating activity. Pigs inoculated with the parental virus produced not only neutralizing but also hemagglutination-inhibiting antibodies. On the other hand, the mutant induced high titers of neutralizing antibody comparable to the parental virus but no hemagglutination-inhibiting antibody in inoculated pigs, suggesting that glycoprotein gIII is an essential component for hemagglutination of PrV. Finally, no evidence that the deletion mutant lost virulence for mice was obtained.

Guinea pig herpesvirus detected as a Guinea pig kidney cell culture contaminant in Mexico.

A cytopathic virus was isolated from the primary kidney cell cultures of apparently healthy guinea pigs reproduced in Mexico between 1982 and 1983. This virus produced intranuclear type A inclusion bodies in the infected cell cultures. Viral replication was inhibited by IUdR, indicating that the viral nucleic acid was DNA. The virus was sensitive to ether, chloroform and acid (pH 3.0), and passed readily through a 200 nm filter, but not through a 100 nm filter. Electron microscopy showed a spherical particle with envelope. These properties were consistent with those of Herpesviridae. In serologic survey of guinea pigs in Mexico and Japan, and of mice, hamsters, rats, rabbits, pigs, goats, horses and calves in Japan, neutralizing antibody against the isolate GPM 83-11 was detected in 4 of 46 infant guinea pigs about 3-4 weeks of age and in 9 of 30 breeding guinea pigs reproduced in Mexico, but neither in any of guinea pigs nor other animals in Japan.