Minoru Matumoto

Isolation of human rotavirus in cell cultures

SummaryThree cytopathic rotavirus strains were isolated in MA104 cells from faecal specimens of pediatric patients with acute gastroenteritis. Pre-treatment of virus with trypsin and incorporation of a small amount of trypsin in maintenance medium were important for establishment of the strains in these cells. The isolates were antigenically closely related with strain Wa of human rotavirus and had some antigenic relationship with strain Lincoln of bovine rotavirus.

Serologic evidence for etiologic role of Akabane virus in epizootic abortion-arthrogryposis-hydranencephaly in cattle in Japan, 1972–1974

SummaryIn the outbreak of abortions, premature births, stillbirths and congenital arthrogryposis-hydranencephaly (AH) syndrome in Japan during the summer through winter of 1972–73 and 1973–74, precolostral sera from calves with congenital AH syndrome and normal calves were tested for neutralizing antibodies against some arboviruses,i. e. Akabane, Aino, Getah and Japanese encephalitis (JE) viruses. The incidence of antibody for Akabane virus was very high in calves with AH syndrome (49/59 or 83 per cent) as compared with normal calves (3/11 or 27 per cent), indicating an intimate correlation between the AH syndrome and precolostral anti-Akabane antibody. Three stillborn fetuses also had anti-Akabane antibody. On the other hand, no precolostral serum antibody for the other viruses was detected in any of the calves tested. The mothers of these calves, normal and with AH syndrome, had anti-Akabane antibody in high percentages (44/52 or 85 per cent and 7/8 or 88 per cent), whereas a few of the mothers had antibodies for the other viruses.Serological surveys indicate a wide dissemination of Akabane virus in epizootic areas during the summer months of 1972 and 1973. Thus, 8 groups of cattle in epizootic areas showed high rates of seroconversion for Akabane virus during the 1972 or 1973 summer. Very high incidences of Akabane antibody were shown among cattle in epizootic areas but extremely low incidences in near-by non-epizootic areas. The geographic distribution of anti-Akabane antibody among cattle throughout the country in the 1973 spring generally agrees with the pattern of case distribution in the 1972–73 outbreak.All these findings strongly suggest that Akabane virus is the etiological agent of the outbreaks. Further studies are needed, particularly isolation of the virus, demonstration of infection with the virus in lesions by immunofluorescence and production of intrauterine infection by experimental infection of pregnant cows.

Epizootic congenital arthrogryposis-hydranencephaly syndrome in cattle: Isolation of Akabane virus from affected fetuses

SummaryPrevious serological studies strongly suggested Akabane virus to be the etiologic agent of epizootic abortion and congenital arthrogryposis-hydranencephaly in cattle, and this view was further corroborated in this study by the isolation of the virus from an aborted fetus in an epizootic of the disease and from a fetus extracted from a cow which was suggested by serologic tests to have a recent infection with the virus. The latter fetus had histological changes of encephalomyelitis and polymyositis, and specific antigens of Akabane virus was shown by the immunofluorescent technique in brain tissues as well as skeletal muscular tissues. The virus was recovered from various fetal tissues and fluids, and in relatively large amounts from brain, spinal cord, cerebral fluid, skeletal muscles and fetal placenta. The intracranial inoculation of suckling mice, 1–2 days of age, was the most sensitive system for Akabane virus isolation and Hm Lu-1, a continuous cell line from hamster lung, seemed almost as sensitive as suckling mice.

Comparison of mouse hepatitis virus strains for pathogenicity in weanling mice infected by various routes

SummaryThe pathogenicity for mice of nine strains of mouse hepatitis virus was studied in mice free from the virus by the intracerebral, intraperitoneal, intravenous and intranasal routes of inoculation.

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

SummaryAntigenic 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.

Varicella virus isolation from spinal ganglion

There is strong support for the view that herpes zoster of skin represents a reactivation of varicella virus which has remained latent in one or more of the spinal or cranial ganglia following an episode of varicella infection (5, 6). However, information regarding the presence of virus in nervous tissues is extremely meager despite the regular demonstration of virus in the skin lesions. The present paper describes the isolation of varicella virus from a zoster patient not only from the skin lesion but also from the corresponding spinal ganglion which had typical histological lesions and varicella antigens demonstrable by immunofluorescence. A 71-year-old Japanese female developed vesicles of herpes zoster on the right chest 3 days before her death due to bacterial pneumonia. Autopsy revealed pneumonia in the left lung and the right lower lobe. Multiple fresh vesicles covered an area of the right lateral chest corresponding roughly to the 7th and 8th thoracic dermatomes. The 7th, 8th and 9th thoracic ganglia on both sides, the corresponding segments of the spinal cord and the affected skin region were removed at autopsy. The skin specimen was then subjected to virus isolation. The other specimens were cut into 2 portions, one for virus isolation and the other for histological and immunofluorescent examinations, and stored at 8 0 ~ in Eagles minimum essential medium (MEM) containing 10 per cent fetal calf serum (FCS) and 7 per cent dimethylsulfoxide until use. The conventional histological examination revealed tha t the right 7th thoracic ganglion was severely affected, while the other ganglia and the spinal cord showed no changes. The central portion of the affected ganglion showed complete hemorrhagic necrosis, which was surrounded by a less damaged zone showing degeneration of ganglion cells, satellite cells, Schwann cells and nerve fibers, accompanied by infiltration of lymphocytes and polymorphonuelear leukoeytes and congestion of blood vessels. Ghosts of ganglion cells were identified on account

A new in vitro method (END) for detection and measurement of hog cholera virus and its antibody by means of effect of HC virus on newcastle disease virus in swine tissue culture

A newin vitro method of neutralization test for hog cholera virus by means of the END method was established. The test is based on the finding that immune serum against hog cholera virus specifically inhibits the exalting effect of hog cholera virus on Newcastle disease virus in swine testicular cell culture. The serum-virus mixtures are incubated at 37° C for 60 minutes or at 4° C overnight and tested for infectivity by the END method described in the previous papers of this series. The test is not only simple enough for the routine use, but also highly reproducible and specific. The test may achieve wide application in studies of hog cholera virus.

Replication of bovine coronavirus in cell line BEK-1 culture.

SummaryBovine coronavirus readily multiplied and induced marked cytopathic effect in BEK-1 cultures, thus providing a sensitive, practical assay system for viral infectivity and neutralizing antibody and a satisfactory source of the virus.

Serologic survey of equine rhinopneumonitis virus infection among horses in various countries

Horse serum samples from 388 horses in 17 countries exhibited high incidence of antibodies for equine rhinopneumonitis virus which had been isolated and studied in USA and subsequently in Japan. Complement fixing antibody was shown in 75.1% of 386 horses for viral antigen of the virus and in 36.5% for soluble antigen. In neutralization tests two serologically distinct types of the virus were used; 85.2% of 384 horses were positive with the Kentucky D strain (American) and 81.7% of 312 horses with the H-45 strain (Japanese). The high incidences of antibodies for each of the countries studied, i. e. USSR, Norway, Sweden, Holland, Germany, Switzerland, Italy, England, South Africa, Egypt, India, Malaya, Taiwan, Japan, New Zealand, Argentine, and Canada, strongly suggest world-wide dissemination of equine rhino-pneumonitis virus or closely related viruses. The present serologic data, together with the clinical and pathologic findings reported by many authors, appear to indicate that the virus abortion, associated with acute respiratory infection, recorded in European horses is caused by equine rhinopneumonitis virus. The correlation diagrams of anti-Kentucky D and anti H-45 titers in individual horses gave evidence for circulation of at least these two serotypes of the virus in various countries.

Hemagglutination with Nebraska Calf Diarrhea Virus

Nebraska calf diarrhea (NCD) virus or reovirus-like agent of calf diarrhea has been shown to be a primary etiological agent of neonatal calf diarrhea by Mebus and his associates (1, 4, 5). Recently Spence et al (6) prepared hemagglutinin from African green-monkey kidney cells infected with NCD virus. This antigen agglutinated human group O cells. We also examined hemagglutination (HA) with NCD virus grown in calf kidney (BK) cells and found that the virus agglutinated erythrocytes of human and some other animals. This paper describes our observations on the HA with this virus and its inhibition (HI) by specific antiserum. Lincoln strain of NCD virus (4) maintained by serial passage in BK cells, was supplied by Dr. C.A. Mebus, University of Nebraska, Lincoln, Nebraska, and used in the present study after several passages in BK cells in our laboratory. BK cell cultures were prepared as described previously (2). The growth medium used was LE medium (Earles solution containing 0.5% lactalbumin hydrolysate) supplemented with 10% calf serum. Primary BK cell monolayers prepared in 500-ml bottles were inoculated with 0.1 TCID50 per cell of virus and incubated at 37 C for 2 days with LE medium containing 0.1 % yeast extract. As described later, in some preliminary experiments, culture fluid harvested from infected cells was used as HA antigen after centrifugation to remove coarse debris. In subsequent experiments, however, HA antigens concentrated from infected culture fluid were used. HA and HI tests were carried out by the microtiter method. The diluent used was VBS (veronal-buffered saline, pH 7.0, containing 0.1 % bovine serum albumin and 0.001% gelatin), unless otherwise stated. Blood was obtained in Alsevers solution and stored at 4 C. Erythrocytes from horse, cattle, sheep and goat were used as 0.3% suspensions, and those from other species as 0.5% suspensions. In HA tests, serial 2-fold dilutions of HA antigen were prepared in 0.05-ml amounts, and mixed with 0.025 ml of an erythrocyte suspension. The mixtures were incubated at 37 C for 1 hr before the results were read, unless otherwise stated. The HA titer was expressed as the reciprocal of the highest antigen dilution showing complete HA. For HI tests, 0.2 ml of the serum was inactivated by heating at 56 C for 30 min, diluted 5-fold, mixed with 0.2 ml of packed erythrocytes and incubated at 37 C for 1 hr. After removal of the cells by centrifugation, the supernatant fluid was mixed with 0.5 ml of 25% kaolin, incubated at room temperature for 1 hr, and centrifuged to remove kaolin. The resulting supernatant fluid was taken as the 10-fold dilution