Richard G. Wyatt

Human Reovirus-like Agent as the Major Pathogen Associated with Winter Gastroenteritis in Hospitalized Infants and Young Children

We found a human reovirus-like agent in the stools of 42 per cent of 143 infants and young children hospitalized with acute gastroenteritis between January, 1974, and June, 1975. Half the patients studied by electron microscopy and serologic technics had evidence of infection with the agent. The infection had a seasonal pattern: 59 per cent of those admitted during the cooler months (November to April) shed the agent, with a peak of 78 per cent in December, 1974, and January, 1975, combined. None of the patients admitted during the warmer months (May to October) shed the agent. None of 275 Escherichia coli isolates from 32 patients with diarrhea produced heat-labile enterotoxin, whereas 17 of the 32 had evidence of infection with the reovirus-like agent. In addition, 14 of 40 parents of 37 patients with diarrhea associated with the reovirus-like agent were also infected, but most infectious were inapparent. This agent appears to be the major cause of diarrheal illness in the young during the cooler months.

Biological properties of Norwalk agent of acute infectious nonbacterial gastroenteritis.

Summary Acute infectious nonbacterial gastroenteritis was induced in adult volunteers through three serial passages by oral administration of bacteria-free stool filtrates. This suggested that a replicating agent of subbacterial size was responsible for the observed disease. The biophysical properties of the agent, as assayed in volunteers, were consistent with those of a small virus, most closely related to the parvovirus group among known animal viruses. The agent appeared to have a diameter less than 66 nm and likely less than 36 nm. It appeared to lack a lipid containing envelope and was acid stable. It was stable to heating at 60° for 30 min. The agent appeared to be relatively host specific for man and conferred at least short-term immunity. Because of the high frequency of disease induced in unselected volunteers, widespread natural immunity to this agent may be absent or perhaps incomplete. Preliminary evidence suggested that the Norwalk agent replicated in an in vitro system, human fetal intestinal organ culture. We gratefully acknowledge the cooperation of the volunteers and staff at the Maryland State House of Correction. We also thank Drs. David Fedson and Sheldon Wolff of the Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, for their help with the study, Dr. Samuel Formal of the Walter Reed Army Institute of Medical Research for performing enterotoxin assays and monkey inoculations, and Dr. Robert Purcell of the Laboratory of Infectious Diseases for performing Australia antigen determinations.

Reoviruslike Agent in Stools: Association with Infantile Diarrhea and Development of Serologic Tests

Reoviruslike particles were visualized by electron microscopy in stool filtrates prepared from stools of infants and young children with severe acute gastroenteritis. Patients who had such particles in their stools and whose paired acute and convalescent serums were tested developed an antibody response to the reoviruslike agent, which was measured by immune electron microscopy and by complement fixation. The reoviruslike agent was antigenically related to the epizootic diarrhea of infant mice virus and the Nebraska calf diarrhea virus.

Genes of human (strain Wa) and bovine (strain UK) rotaviruses that code for neutralization and subgroup antigens.

Abstract Sixteen rotaviral reassortants recovered from mixed infection with a ts mutant of bovine rotavirus (UK strain) and noncultivatable or cultivatable human rotavirus (Wa strain) were genotyped by polyacrylamide gel electrophoresis. Analysis of the reassortants showed that the ninth RNA gene segment regularly segregated with the neutralization specificity and the sixth RNA gene segment regularly segregated with the subgroup antigenic specificity detected by immune adherence hemagglutination assay. This indicates that the ninth RNA segment codes for the protein that induces and reacts with neutralizing antibodies while the sixth RNA segment codes for the subgroup antigen. In addition, it appears that the fourth and/or fifth RNA segments may be responsible for restriction of growth of the noncultivatable human rotavirus in cell culture.

Gene coding assignments for growth restriction, neutralization and subgroup specificities of the W and DS-1 strains of human rotavirus.

Gene coding assignments for growth restriction, neutralization and subgroup specificities were determined for two human rotavirus strains, DS-1 and W, which represent two distinct serotypes. The 4th gene segment of both viruses was associated with restriction of growth in cell culture. The 9th gene segment of W virus and 8th segment of DS-1 were associated with serotype specificity, while the 6th gene segment of W virus was associated with subgroup specificity.

Comparison of human and animal rotavirus strains by gel electrophoresis of viral RNA.

Abstract Many rotavirus strains have been detected but few have been grown in vitro and this has hampered the development of serologic tests and antigenic comparison of strains obtained from the same or different host species. Because of this limitation of growth in vitro a different approach for distinguishing rotaviruses was undertaken. The rotavirus genome could be separated into 11 discrete RNA segments by polyacrylamide gel electrophoresis. Differences in RNA migration pattern were observed among human strains as well as between human and animal strains; the number of interspecies differences was greater than the number of intraspecies differences. Three distinct patterns were observed among the eight human rotaviruses obtained from each of four successive annual rotavirus epidemics in the Washington, D.C. area. Each of four animal rotaviruses also had distinct patterns which differed from the human patterns in the mobility of from four to seven RNA segments.

Viral gastroenteritis induced by the Hawaii agent: Jejunal histopathology and serologic response

Peroral jejunal biopsies were performed in seven normal volunteer subjects prior to, 48 hours after and two weeks after the administration of the Hawaii agent of viral gastroenteritis. Light and electron microscopic examination revealed an intact mucosa with blunted villi, shortened and distorted microvilli, swollen mitochondria and intercellular edema. These histologic changes were seen only in acutely ill volunteer subjects and were absent two weeks after illness in three of four who were previously ill. This reversible lesion was similar to, but not identical with, that previously described in viral gastroenteritis induced by the Norwalk agent. Serum antibody increases in response to the Hawaii agent as measured by immune electron microscopy were present in three of four ill volunteer subjects and in none of three who remained well.

Antigenic relationships among five reovirus-like (RVL) agents by complement fixation (CF) and development of new substitute CF antigens for the human RVL agent of infantile gastroenteritis.

Summary The human reovirus-like (HRVL) agent, Nebraska calf diarrhea virus (NCDV), epizootic diarrhea of infant mice (EDIM) virus, simian agent (SA)-11, and the “O” (offal) agent were found to be similar, if not identical, in reciprocal complement fixation (CF) tests employing hyperimmune animal sera. In addition, in CF tests with paired sera from 35 diarrhea patients who shed the HRVL agent, 74% developed serologic evidence of infection with the HRVL antigen, 43% with NCDV, 51% with EDIM virus, 57% with SA-11, and 71% with the “O” agent. Thus, in addition to the NCDV, which had previously been described as a suitable substitute CF antigen for the HRVL agent, the SA-11, “O,” and EDIM viruses may also be utilized as substitute antigens for the HRVL agent. However, the “O” agent appears to be the most efficient of the four substitute CF antigens and thus should be used preferentially when the HRVL agent is not available. The “O” agent was about as efficient as the HRVL agent and significantly more efficient than the NCDV for detecting seroresponses. The greatest efficiency for detecting infection with the HRVL agent resulted when sera were tested with both the HRVL and “O” agents as 31 (89%) of the patients developed serologic evidence of infection with one or both antigens. The finding of additional substitute CF antigens for the HRVL agent may have implications in the immuno-prophylaxis against human disease. We thank Dr. D. W. Alling for assistance in statistical analyses and Mr. C. E. Banks, Mr. E. Williams, Mr. B. Andrews, Mr. J. P. Jackson, Miss D. M. Bertran, Mrs. S. J. Kelly, Mr. Robert P. Chames, Mrs. L. P. Kendrick, Mr. C. S. Osborne, Mrs. B. V. Armiger, and Mrs. C. L. Armiger for assistance in these studies.

Secretory antibody directed against rotavirus in human milk—measurement by means of enzyme-linked immunosorbent assay

Human milk contains antibodies to a variety of enteropathic agents. We utilized the method of enzyme-linked immunosorbent assay to investigate anti-rotavirus secretory IgA in 113 human milk and colostral specimens from a rural area in Guatemala, 32 colostral specimens from an urban area of Costa Rica, and 12 from an urban area of the United States. Anti-rotavirus SCIgA was found in all colostral samples and in 94% of the milk specimans. Both the absolute concentration of anti-rotavirus SCIgA and concentration relative to total SCIgA were highest in colostrum, falling to lower but detectable levels from one week to two years after birth. No significant differences were noted in the results from the specimens from the three different geographic areas. The possible role of this antibody in immunity to rotavirus infections is discussed.

Isolation and characterization of a canine rotavirus

SummaryCanine rotavirus particles were visualized by direct electron microscopy in the feces from a clinically normal dog. The virus was subsequently propagated in cell cultures; it was chracterized and compared with rotaviruses from other species. Replication of the virus in cell culture was found to be less dependent upon trypsin than that of human, bovine and porcine rotaviruses. Reproducible, sharp-edged plaques of various sizes were produced by the canine rotavirus in an established cell line of fetal rhesus monkey kidney, MA104, under overlays of carboxymethyl cellulose or agarose. Intracytoplasmic inclusion bodies of different sizes and shapes were produced in infected MA104 cells. By plaque reduction neutralization assay, a two-way antigenic relationship was found between the canine (CU-1) and simian (rhesus MMU 18006 and SA-11) rotaviruses. The canine rotavirus had a one-way antigenic relationship with feline (Taka), bovine (NCDV), and porcine (OSU) rotaviruses.