Anthony R. Kalica

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.

Identification of the rotaviral gene that codes for hemagglutination and protease-enhanced plaque formation

Temperature-sensitive mutants of bovine rotavirus, UK Compton strain, and rhesus monkey rotavirus, MMU18006 strain, were used to derive 16 reassortants by coinfection of MA104 cells. The parental viruses differed phenotypically in their neutralization specificity, their ability to hemagglutinate, and their requirement for exogenous trypsin for infectivity. When the reassortants were assayed for neutralization specificity and hemagglutination, four phenotypes were observed, indicating that these two rotaviral functions segregated independently. Protease-enhanced infectivity phenotype segregated with the HA phenotype indicating that these two functions were manifestations of the same gene product. In order to determine the gene responsible for these rotaviral functions, the reassortants were genotyped by hybridizing 32P-labeled parental transcripts and denatured reassortant genomic RNAs and analyzing the resulting hybrids by gel electrophoresis. The fourth RNA segment was clearly shown to code for HA and protease enhanced plaque formation in MA104 cells. The neutralization antigen was linked to the eighth and ninth RNA segments that comigrated during gel electrophoresis and thus could not be differentiated.

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.

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.

Differentiation of human and calf revoiruslike agents associated with diarrhea using polyacrylamide gel electrophoresis of RNA

Abstract By polyacrylamide gel electrophoresis of RNA it was possible to distinguish between the human and calf reoviruslike agents associated with diarrhea. These two viruses were seen to differ in as many as five of their RNA genome segments on the basis of electrophoretic mobility on 9% gels. In addition, comparison of the RNA segments of two other related viruses (the “O” agent and SA-11 virus) indicated that these viruses differed from the human and calf viruses as well as from each other. Evidence that all four viruses contain 11 double-stranded RNA segments was also suggested from electrophoretic patterns. Based on contour length measurement, the RNA segments of the human and calf viruses were divided into four size classes in contrast to the three size classes seen for reovirus type 2.

Induction of diarrhea in colostrum-deprived newborn rhesus monkeys with the human reovirus-like agent of infantile gastroenteritis.

SummaryDiarrhea developed in five newborn rhesus monkeys(Macaca mulatta) inoculated orally on the first day of life with the human reovirus-like agent of infantile gastroenteritis. Incubation period ranged from 2–5 days; virus particles were detected in stools in association with illness, and virus shedding lasted between 1 and 3 days. Virus derived from monkeys that developed illness following inoculation was infectious for other monkeys but did not induce diarrhea which could be associated temporally with virus shedding. Viral antigens were also detected in tissues of the grossly abnormal small intestine of an acutely-ill monkey. Serum antibody responses were demonstrated in two of the ill animals by complement-fixation and/or immunofluorescence.

Isolation of a recombinant between simian and bovine rotaviruses.

A recombinant between simian rotavirus, simian agent 11 (SA-11) and bovine rotavirus, neonatal calf diarrhoea virus (NCDV), was obtained by mixed infection of MA-104 cells with NCDV and u.v.-irradiated SA-11 virus, and isolation of a plaque formed in the presence of anti-NCDV serum. The genome of the recombinant contained dsRNA segments 4, 5 and 10 derived from SA-11 virus and segments 1, 2, 3, 6 and 11 derived from NCDV, and segments 7, 8 and 9 of undetermined origin. Polypeptides VP4, VP5, VP7a, VP7b, NCVP1 and MCVP3 were derived from SA-11 virus and polypeptides VP1, VP2, VP3, VP6, VP8, NCVP2a and NCVP2b from NCDV. Haemagglutination of the recombinant was inhibited and its infectivity neutralized by the antiserum against SA-11 virus but not by anti-NCDV serum.

Use of transcription probes for genotyping rotavirus reassortants

Abstract Radioactively labeled transcripts (probes) synthesized in vitro from rotavirus particles were employed to genotype rotavirus reassortants obtained from cells coinfected with a cultivatable bovine rotavirus (UK strain) and a noncultivatable human rotavirus (“Wa” or DS-1 strain). The technique consists of denaturing the genomic double-stranded RNA (ds RNA) from the reassortant and hybridizing it to labeled single-stranded RNA (ss [ 32P ]RNA) prepared from either parent by in vitro transcription. Electrophoresis and autoradiography of the hybrids permit recognition of the parental origin of each of the genes of the reassortant. By using this approach, we have confirmed that gene 9 of the “Wa” virus codes for the specific antigen that reacts with neutralizing antibody. In addition, an analysis of a series of reassortants suggests that in the two human viruses studied, gene 4 may be responsible for restriction of growth in tissue culture.

Inhibition of Respiratory syncytial, parainfluenza 3 and measles viruses by 2-deoxy-D-glucose.

Abstract 2-Deoxy- d -glucose was found to inhibit the replication and cytopathic effects of respiratory syncytial, parainfluenza 3 and measles viruses. The effects of the drug were completely reversible and appeared directed against viral functions occurring in the second half of the viral growth cycle. At least some viral functions appeared to be unaffected by the agent. Electron microscopic studies with a temperature-sensitive mutant of respiratory syncytial virus indicated that 2-deoxy- d -glucose produced a morphological change. Surface projections were greatly reduced in virions grown in the presence of the drug.