Effect of enzymes on the growth of human and animal rotaviruses.

The growth of group A human, bovine, equine and porcine rotaviruses were enhanced by pretreatment of virus with pancreatin, trypsin, protease, alkaline phosphatase or pepsin and incorporation of these enzymes in maintenance medium. In contrast, alpha-amylase or lipase inhibited the growth of equine and porcine rotaviruses. The other enzymes, adenosine deaminase, lactase, lysozyme, ribonuclease or triose-phosphate isomerase gave little or no change in the growth of all four rotaviruses.

Growth activity of bovid herpesvirus 1 in bovine follicular oocytes with cumulus cells.

Bovine follicular oocytes collected from bovine ovaries were exposed to bovid herpesvirus 1 (BHV-1). After washings, these oocytes were cultured to mature. As a result BHV-1 could not be removed from the oocytes and could replicate in the oocytes with cumulus cells, but not in the oocytes without the cells. Moreover, the specific fluorescence for BHV-1 was detected in the cumulus cells by a indirect immunofluorescent technique. Therefore these findings suggested BHV-1 could be absorbed in the oocytes but the replication of BHV-1 was done in the cumulus cells.

Detection of antibody to pseudorabies virus in swine sera by indirect immunoperoxidase plaque staining.

A new method for the detection of pseudorabies antibody in swine sera was developed by use of the indirect immunoperoxidase plaque staining test. The indirect immunoperoxidase plaque staining test demonstrated serologic responses in pigs that were subcutaneously infected with pseudorabies virus. The test had a sensitivity and a specificity comparable to those of the complement-dependent neutralization test and appeared to be somewhat more sensitive than the neutralization test and the enzyme-linked immunosorbent assay. The test was simple to perform, and the results could be read with the unaided eye under ordinary light, making the test particularly suitable for the routine testing of large numbers of serum samples.

Appearance of slow-reacting and complement-requiring neutralizing antibody in cattle infected with Akabane virus.

Slow-reacting complement-requiring neutralizing (NT) antibody was detected in sera from cattle 2 weeks after infection with Akabane virus. Bovine sera obtained 3 or 4 weeks after infection contained slow-reacting noncomplement-requiring NT antibody. The slow-reacting complement-requiring NT antibody was sensitive to 2-mercaptoethanol (2-ME), whereas the slow-reacting noncomplement-requiring NT antibody was resistant to 2-ME. The initial phase may represent the IgM response and the later phase a change to IgG. A NT test was developed in which virus-serum mixtures were incubated at 4 degrees C for 48 h and then with complement at 37 degrees C for 60 min; this gave an improved sensitivity over the previous incubation at 37 degrees C for 60 min.

Detection of bovine coronavirus in feces by reversed passive hemagglutination.

A reversed passive hemagglutination (RPHA) method was developed for the detection of bovine coronavirus in fecal specimens. Sheep erythrocytes fixed with glutaraldehyde, and then treated with tannic acid were coated with anti-bovine coronavirus rabbit antibodies purified by affinity chromatography using bovine coronavirus linked to Sepharose 4B. The RPHA test was carried out by a microtiter method. Erythrocytes coated with purified specific antibodies were agglutinated by bovine coronavirus, but not by bovine rotavirus or enterovirus. The reaction was inhibited by antiserum to bovine coronavirus, confirming the specificity of the reaction. The RPHA test detected bovine coronavirus in 13 of 22 fecal specimens (59 per cent), from natural cases of diarrhea, while the positive rates were only 14 per cent (3/22) and 22 per cent (5/22) for immunofluorescent staining of primary cultures of calf kidney cells infected with the specimens, and immune electron microscopy respectively. The advantages of the RPHA method are its simplicity, high sensitivity and rapidity.

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

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 noncytotoxic 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 dissemination of haemagglutinating encephalomyelitis virus or antigenically-related viruses in Japanese pigs.

Seroepizootiological survey on bluetongue virus infection in cattle in Japan.

Bovine sera collected in various parts of Japan were subjected to seroepizootiological tests with bluetongue virus type 1 (BTV1), type 12 (BTV12), and type 20 (BTV20). All these viruses have been widely disseminated among cattle in the southern part of Japan in 1974. Relatively high incidences of neutralizing (NT) antibody against the three viruses were shown among cattle in the Kyushu district, including Okinawa Prefecture, or the southern part of Japan, but extremely low or incidences in Hokkaido, or the northern part of Japan. The incidence of reactors was higher in old animals. Cattle in Okinawa Prefecture showed a high rate of seroconversion for all the viruses during the summer of 1979. None of the animals seroconverted, however, manifested any sign of disease. Seroepizootiological investigation made it clear that BTV1, BTV12 and BTV20 had existed in Japan and that the epizootic of bluetongue virus infection started during a period from summer through early autumn.

Isolation of lapine rotavirus in cell cultures. Brief report.

Two cytopathic rotavirus strains were isolated in MA 104 cells from diarrheal rabbits. The isolates showed marked cross reactions with strain Lincoln of bovine rotavirus in immunofluorescence, but no cross reaction in neutralization.

Antigenic relationships between rotaviruses from different species as studied by neutralization and immunofluorescence.

Antigenic relationships between the Wa, Hochi, Ito, Hosokawa and Nemoto strains of human rotavirus, the SA-11 strain of simian rotavirus, the Lincoln strain of bovine rotavirus, the OSU strain of porcine rotavirus and the R-2 strain of lapine rotavirus, which were all established in cell culture, were studied by neutralization (NT) and immunofluorescence (IF) using guinea pig antisera against these strains. The strains could not be distinguished by indirect IF staining of infected cells because of marked cross reactions. On the other hand, the NT test clearly distinguished these strains, although there was a one-way antigenic relationship between some of them. The five strains of human rotavirus were clearly distinguished from each other by NT test. These human strains exhibited much higher homologous titers than heterologous titers, the former being at least eight-fold higher than the latter. The presence of at least five serotypes of human rotavirus was indicated.

Hemagglutination of epizootic hemorrhagic disease virus.

Hemagglutination of epizootic hemorrhagic disease virus (EHDV) with a variety of erythrocyte species at 4 degrees C, room temperature and 37 degrees C was dependent on the NaCl molarity and the pH of the diluent. The hemagglutination inhibition test was used to identify EHDV serotypes.

Propagation of the Kakegawa strain of bovine coronavirus in suckling mice, rats and hamsters.

The Kakegawa strain of bovine coronavirus was easily propagated in suckling mice. Infected animals died with nervous symptoms, and serial passage was readily accomplished by intracerebral inoculation with brain emulsions. The 3rd passage viral material from infected mice evoked the same disease in suckling mice, rats and hamsters inoculated by the intracerebral or by the subcutaneous route. Viruses recovered from mice, rats and hamsters could be clearly differentiated from mouse hepatitis virus strain 2 by the neutralization test.