Tatsuhiko Shinozaki

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.

Infantile convulsions with mild gastroenteritis

The development of sensitive new molecular genetic techniques has led to the detection of rotavirus in cerebrospinal fluid, stools and throat swabs from patients with gastroenteritis with accompanying clinical symptoms similar to infantile benign convulsions. Small round structured virus (SRSV) has also been found in stools of patients with similar clinical symptoms by a new procedure. However, the mechanism by which these viral infections induce benign convulsions remains to be elucidated. The present paper reviews recent virological and clinical studies of seizures probably caused by gastroenteritis viruses including rotavirus, SRSV and other viruses.

Epidemiology of enteric adenoviruses 40 and 41 in acute gastroenteritis in infants and young children in the tokyo area

82/2,223 stool specimens, collected 1982-1988, from children with enteritis (3.7%) were found to contain adenoviruses; 17 adenovirus-positive samples were provided from other institutes. 89 adenoviruses were isolated in Graham 293 cells from these 99 specimens and were typed by DNA restriction enzyme analysis with Sma I. 37 strains were typed as adenovirus 40 (AD40), and 37 strains as adenovirus 41 (Ad41). Although most strains had the same DNA profiles, a few strains had 3 kinds of different electropherotypes generated by Sma I. Five strains were identified as adenovirus 31. The remaining 10 strains were adenovirus 1 (2 strains), adenovirus 2 (3 strains), adenovirus 3 (1 strain), adenovirus 5 (1 strain), and a non-classified adenovirus (3 strains). Ad40 and Ad41 infections were found throughout the year, but peaked between September and November. 80% of the children with adenovirus infections were less than or equal to 2 years of age. The highest incidence of diarrhea caused by Ad40 or Ad41 was in 6-11 months old children. 1982-1984, the rate of Ad40 infection was 91.7%, while the rate of Ad41 infection was only 8.3%. The prevalence of Ad40 infection gradually diminished from 1985. During 1987 and 1988 the reverse ratios, 20.6% and 79.4%, respectively, of Ad40 and Ad41 infections were observed. Thereafter, Ad41 infection became predominant.

Neutralizing antibody to bovine rotavirus in various animal species

Abstract A total of 288 serum samples were collected from 12 species of animals in various localities of Japan from 1975 to 1977. Neutralizing antibody to bovine rotavirus was found in serum samples from all the species, viz., horses, cattle, sheep, goats, pigs, dogs, rabbits, guinea pigs, rats, mice, chickens and human beings. The incidence of neutralizing antibody in titers of 1 : 2 or higher ranged from 31 to 100%, and high incidences exceeding 70% were obtained in horses, cattle, sheep, pigs, dogs, rabbits and human beings. High titers were most common in horses, cattle, sheep and pigs. These serological results suggest that rotaviruses occur commonly in these species of animals.

Epidemiology of rotavirus infection in Tokyo during two winter seasons, as revealed by analyses of recovered viral RNA

Sir, - Rotaviruses are important etiologic agents of acute gastroenteritis in young children all over the world [9]. Gel electrophoresis can be used to detect and genotype rotavirus strains [2, 3, 4, 8]. We have investigated the relationships between rotavirus RNA electropherotypes and the epidemiology of acute gastroenteritis in Tokyo. Three hundred and seventy-four diarrheal stool specimens were collected from in- and out-patients (less than 4 years old, mainly between 6 months and 2 years old) at Teikyo University Hospital in Tokyo during the winter periods of 1981-1982 and 1982-1983. Outbreaks of rotavirus infection developed in two nursery rooms during this time. Twentyeight and 11 stool specimens were obtained from children in each nursery room, respectively. The extraction of virus from feces and

Coproantibody response to rotavirus in an outbreak in a day-care nursery

13 months and 10years 8 months. Patient no. 1 was first observed at a very advanced stage of the disease and died 19 months after the diagnosis of lung metastases. Patient no. 2 is still alive 10 years and 8 months after the diagnosis. Patients 3 and 4 are still alive after 6 years 11 months and patient no. 5 is well 6 years after the diagnosis. All the patients treated in the second period are still alive. Patients 6 and 8 are still in complete remission 35 and 13 months, respectively, after diagnosis. In patient no. 7 (alveolar RMS) a local relapse was noted 2 months after the completion of initial therapy. The subject was again treated with 5000 rads to the orbit, plus chemotherapy consisting of cisplatinum and tenixposide. After 7 months of treatment there were side-effects of renal toxicity with hyperuricaemia, increase in BUN and hyaline casts in the urine. Treatment was stopped and the patient is still in complete remission. Our results do not demonstrate any significant difference in life expectancy between patients treated with radical surgery and those treated conservatively, but the advantage of the latter treatment is so impressive cosmetically that this approach must be seriously taken into consideration. In our patients, the follow-up period is obviously too short to allow evaluation of long-term evolution and to draw definitive conclusions about the most suitable chemotherapy protocol. However, taking the quality of life, vision and psychological situation into consideration, we believe that chemotherapy and radiotherapy can offer a very valuable alternative in the therapy of RMS. Further trials involving this approach must be carefully considered.

A study of adenovirus gastroenteritis in the Tokyo area

Probable serotypes of faecal adenoviruses 40, 41, 31, 2 and 1 were found in the Tokyo area by rapid DNA restriction endonuclease analyses directly of stool specimens or after primary culture in Graham 293 cells. Some variations were found in both adenoviruses 40 and 41 by this method.

Comparison of five methods for detecting human rotavirus in stool specimens

Sir, Various methods have been developed for the detection of rotaviruses in stool specimens. For the rapid diagnosis of rotavirus infections, a simple latex agglutination (LA) test has recently become commercially available (Rotalex, Orion). Its sensitivity is comparable with that of polyacrylamide gel electrophoresis (PAGE) [3], and is four times greater than that of EM [11. We identified rotaviruses by electron microscopy (EM) after negative staining, by solid-phase immune microscopy (SPIEM) with protein-A [4], PAGE [5], LA, and reversed passive haemagglutination (RPHA) [2]. For the LA, antihuman rotavirus (strain Wa) rabbit serum was coated on latex beads. Stool samples were diluted with a solution of 0.2M Tris-HC1, 0.9% NaC1, and 0.1% bovine serum albumin. A commercially available RPHA kit was used (ROTA RPHA, Denka Seiken, Japan) with antihuman rotavirus (Wa strain) rabbit serum as antibody. Of 51 samples, 28 were positive and 18 were negative by all five techniques (Table 1). One sample was positive by four methods and negative by one, while one sample was negative by four methods and positive by one. The sensitivities of the tests were as follows: 30/51 (59%) for EM and SPIEM, 32/51 (63%) for PAGE, 31/51 (61%) for LA, and 29/51 (57%) for RPHA. EM and SPIEM were less sensitive than PAGE and LA. Although PAGE is the most sensitive technique, it takes 2-3 days before RNA genomes are visualized. All LA-positive specimens were positive in EM, SPIEM and/or PAGE. Though RPHA is the least sensitive test it is valuable for screening a large number of samples. LA is suitable for rapid bedside detection of rotavirus in stool specimens. This work was supported in part by the Ministry of Education Grant-in-Aid for Scientific Research 59440048, Japan, and by a grant from the Morinaga Hoshikai of Japan.

Neutralizing Antibody to Calf Diarrhea Coronavirus in Various Animal Species in Japan

Coronavirus infections associated with respiratory, intestinal or other diseases have been reported in a number of animal species, including man, cattle, pig, dog, cat, mouse, rat, turkey, and chicken (1-3,5,6,8, 10, 12, 13). Recently coronaviruses have been divided by immunofluorescence into two distinct groups on the basis of antigenic cross-reactivity (9), although coronaviruses, as a group, display complex serologic variability (5). In this paper we report on the presence of neutralizing antibodies against calf diarrhea coronavirus (6, 12) in serum specimens from various animal species. Calf diarrhea coronavirus passaged in bovine embryonic kidney cells (6) was kindly supplied by Dr. C.A. Mebus, University of Nebraska, U.S.A. In our laboratory the virus was shown to replicate readily and induce a cytopathic effect in cultures of a continuous cell line, BEK-l, derived from bovine embryonic kidney (4). In the present study the virus was used at the 7th passage level in BEK-l cells. The neutralization test was carried out by the serum dilution method in II X 100-mm tube cultures of BEK-l cells. The growth medium was Eagles minimum essential medium (MEM) containing 10% tryptose phosphate broth (TPB) and 10% inactivated calf serum, and the maintenance medium was MEM containing 10% TPB, 0.05% yeast extract, 0.5% sodium glutamate and 0.1 % glucose. Serial fourfold dilutions of the serum inactivated at 56 C for 30 min were made in maintenance medium and mixed with equal volumes of maintenance medium containing 200 TCIDso of virus per 0.1 ml. The serum-virus mixtures were incubated at 37 C for 1 hr before inoculation ofO.l-ml volumes into two tube cultures per serum dilution. The tests were read after incubation in a roller drum at 37 C for 4 days. The antibody titer was expressed as the reciprocal of the highest serum dilution which showed complete neutralization in at least one of the two tubes. Titers of 2 or higher were taken as positive. A total of 306 serum samples were collected from 12 animal species in various parts of Japan during the period from 1975 to 1977 (Table 1). These samples were stored at -20 C and inactivated at 56 C for 30 min before being tested. The results of neutralization tests on these serum samples are summarized 10

Evaluation of four tests for detecting human rotavirus in feces

Sir, Infantile diarrhea during the winter is mostly due to viruses and the majority of pathogenic viruses prove to be rotavirus. Various methods for diagnosing infections by rotavirus have been described. The migration patterns of RNA segments ascertained by polyacrylamide gel electrophoresis (PAGE) are sensitive for the detection of rotavirus and effective for the analysis of the molecular epidemiology of rotavirus infections [9]. A simple and specific technique for rapidly detecting rotaviruses in feces is still needed. Enzyme-linked immunosorbent assay (ELISA) kits for detecting rotaviruses (Abbott Laboratories, North Chicago, Ill., USA) and latex agglutination test kits (Orion Diagnostica, Helsinki, Finland) are commercially available and have recently been evaluated [1-5, 7, 10]. In the present study, four different techniques to detect rotaviruses in feces are compared. Sixty-two fecal specimens were obtained from patients with infantile diarrhea. Specimens were kept at -20~ Samples were suspended in phosphatebuffered solution (PBS), and centrifuged at 3000 rpm for 15rain at room temperature. Supernatant was used for all four techniques. Migration patterns of rotavirus RNA segments by PAGE were determined as described previously [9]. Virus isolation: MA104 cells and primary cynomolgus monkey kidney (PMK) cells were used. The isolation methods have been described previously [6, 8]. Latex agglutination (LA): The Rotalex kit was used according to the manufacturers instructions. The results were read under the inverted microscope (x 40, Chiyoda Kogaku). ELISA was carried out according to the manual of the commercially available Rotazyme kit. Results were read by measuring absorbance at 492nm (Quantum II, Abbott). Of 62 samples, 31 were positive and 10 were negative by all four techniques (Table 1). Thirteen samples were positive by three techniques and negative by one, while five samples were negative by three techniques and positive by one. The results for the 62 samples tested are as follows: 79% (49/62) for PAGE, 65% (40/62) for virus isolation, 73% (45/62) for LA, and 61% (37/62) for ELISA. Several different migration patterns of RNA segments can be ascertained by PAGE [8]. However, there were no distinct correlations between migration patterns and the results obtained in this study. The commercially available