Enzyme-linked immunosorbent assay for identification of rotaviruses from different animal species.

Rotaviruses cause gastroenteritis in man and a wide variety of animal species. They cross-react in many immunologic tests and have a similar appearance by electron microscopy, making differentiation among them difficult. Rotaviruses derived from different host species were distinguished by postinfection serum blocking virus activity in an enzyme-linked immunosorbent assay (ELISA). Thirty-three rotavirus isolates from children living in three different parts of the world could not be differentiated by this technique, but they were distinct from four strains recovered from calves, and a series of strains isolated from piglets, foals, monkeys, and infant mice. The four bovine strains were similar, but they could be differentiated from the other animal strains, each of which exhibited a distinct pattern when tested by the ELISA blocking technique.

Rotaviral immunity in gnotobiotic calves: heterologous resistance to human virus induced by bovine virus.

The possibility of immunizing human infants against rotaviruses, which cause severe dehydrating diarrheal disease, may depend on the use of a related rotavirus, derived from another animal species, as a source of antigen. To test the feasibility of this approach, calves were infected in utero with a bovine rotavirus and challenged with bovine or human type 2 rotavirus shortly after birth. Infection in utero with bovine rotavirus induced resistance to diarrheal disease caused by the human virus as well as the homologous bovine virus. These data suggest that the bovine virus is sufficiently related antigenically to the human type 2 virus to warrant further evaluation of the former as a source of vaccine.

Human rotavirus type 2: cultivation in vitro.

A strain of type 2 human rotavirus (Wa) was grown to relatively high titer through 14 passages in primary cultures of African green monkey kidney (AGMK) cells. This passage series was initiated with virus that had been passaged 11 times serially in newborn gnotobiotic piglets. In contrast, virus present in the stool of patient Wa as well as virus from the first, second, or third passage in piglets could not be propagated successfully in African green monkey kidney cells. Prior to each passage in cell culture, the virus was treated with trypsin and the inoculated cultures were centrifuged at low speed. Cultivation of a type 2 human rotavirus should aid attempts to characterize this virus and to develop a means of immunoprophylaxis for a serious diarrheal disease of human infants.

Bronchoalveolar lavage. The report of an international conference.

The application of the method of bronchoalveolar lavage to an increasing array of pulmonary diseases was evident, and the use of sophisticated technology to study cells and measure minute amounts of protein and other components in bronchoalveolar lavage fluid indicated that more meaningful information may be gained about diseased airways and alveolar spaces than suspected. That new techniques are being developed to make assays more sensitive and specific was evident. The popularity of this research approach was underscored by the interest and participation of colleagues in the United States and especially in Europe (notably France, England, Italy, and West Germany) and in Japan. This meeting was an opportune time to reflect on what bronchoalveolar lavage analysis has contributed to date and to focus attention on new applications that will be forthcoming and will improve our understanding of immunopathogenic mechanisms in an expanding number of pulmonary diseases.

Approaches to the treatment of pulmonary fibrosis.

There was a general consensus at the workshop that pulmonary fibrosis is a highly lethal lung disorder and that current therapies for this disease have little effect on the natural history of the disease. There was also broad consensus that much has been learned about the pathogenesis of this disease in recent years and this information can be used to direct new therapies for this disease. As a complement to the findings from studies on pulmonary fibrosis itself, there has also been significant development of new therapies for other disorders that might be useful in treating patients with pulmonary fibrosis. The following is a list of some of the issues and recommendations that were generated from this workshop.

Polypeptides of simian rotavirus (SA-11) determined by a continuous polyacrylamide gel electrophoresis method.

Simian rotavirus (SA-11) isolated from infected African green monkey kidney cells was separated into two virus fractions in a CsCl density gradient and their proteins analysed on a continuous phosphte buffered polyacrylamide gel electrophoresis system. One peak (buoyant density 1.37 g/ml) contained double capsid virus particles which were radioimmunoassay (RIA)- and haemagglutinin (HA)- positive and yielded eight polypeptides whose mol. wt. ranged from 48,000 to 128,000. The second peak (buoyant density 1.39 g/ml) which contained 70% single capsid particles and was RIA-positive but HA-negative, yielded only five polypeptides. The three polypeptides missing in the second peak are associated presumably with the outer capsid of SA-11 virus particles and one or more of these is assumed to be the HA of SA-11 rotavirus.

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.

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.

Hemagglutination by simian rotavirus.

A simian rotavirus (SA-11) was shown to hemagglutinate human group "O" and guinea pig erythrocytes. The hemagglutinin appeared to be associated with the outer capsid of the SA-11 virus and was inhibited by specific hyperimmune anti-SA-11 guinea pig serum.

Ultrastructural lesions in Mycoplasma pneumoniae membranes produced by antibody and complement.

Lesions in Mycoplasma pneumoniae membranes were produced by antibody and either human or guinea pig complement and were observed by electron microscopy after negative staining. These defects measured approximately 9.0 to 10.0 nm in diameter-slightly more for human complement than for guinea pig complement. The development of these small lesions was accompanied by a decrease in the viability of the organism.

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.

Pattern of shedding of the Norwalk particle in stools during experimentally induced gastroenteritis in volunteers as determined by immune electron microscopy.

In 11 of 23 volunteers the Norwalk virus-like particle was visualized by immune electron microscopy in at least one stool specimen obtained during the acute phase of experimentally induced nonbacterial gastroenteritis. Examination of multiple stool specimens obtained during the course of illness in these 11 volunteers revealed maximal concentration of Norwalk virus-like particle at the onset of illness and shortly thereafter; in no case was the Norwalk particle visualized in stools obtained before the onset of illness. This finding further suggests that the Norwalk particle was the etiological agent of the Norwalk gastroenteritis outbreak. The limit of reliability of our immune electron microscopy assay system for particle counting was examined.

Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis.

A 27-nm particle was observed by immune electron microscopy in an infectious stool filtrate derived from an outbreak in Norwalk, Ohio, of acute infectious nonbacterial gastroenteritis. Both experimentally and naturally infected individuals developed serological evidence of infection; this along with other evidence suggested that the particle was the etiological agent of Norwalk gastroenteritis.

Detection by immune electron microscopy of 26- to 27-nm viruslike particles associated with two family outbreaks of gastroenteritis.

Viruslike particles 26-27 nm in size were detected by immune electron microscopy in stools of volunteers who were ill after administration of bacteria-free fecal filtrates derived from two separate family outbreaks of acute epidemic nonbacterial gastroenteritis. Fluorocarbon treatment and concentration of the filtrates were necessary to provide enough antigen to test sera by immune electron microscopy. Serum antibody responses were detected in both naturally occurring and experimentally induced cases of illness. The Montgomery County viruslike particle appeared to be related to the previously described Norwalk particle, whereas the Hawaii particle appeared to be unrelated to the Norwalk particle.

Isolation and characterization of an equine rotavirus.

A rotavirus, designated as the H-1 strain, was isolated from a diarrheic foal in primary African green monkey kidney cells and MA104 cells. This cell culture-adapted strain hemagglutinated erythrocytes of human group O, rhesus monkeys, guinea pigs, and sheep. It was found to be similar, if not identical, to porcine rotaviruses (strains OSU, EE, and A-580) by plaque reduction neutralization and hemagglutination inhibition tests, and, in addition, it was found to belong to subgroup 1. This equine rotavirus has an RNA electrophoretic migration pattern which was distinct from those of the three strains of porcine rotavirus. The serological relationship established by plaque reduction neutralization and hemagglutination inhibition tests between the equine (H-1) and porcine (OSU, EE, and A-580) rotaviruses is an example of a rotavirus of the same serotype being isolated from different species. The H-1 strain was distinct from four human rotavirus serotypes (Wa, DS-1, P, and St. Thomas 4) as well as from bovine rotavirus NCDV, simian rotavirus MMU18006, and canine rotavirus CU-1 by plaque reduction neutralization tests. This equine isolate (H-1) was found to be related antigenically to canine CU-1 and bovine NCDV rotaviruses in a one-way fashion by hemagglutination inhibition tests.

Isolation, propagation, and characterization of a second equine rotavirus serotype.

A rotavirus designated strain H-2 was isolated in primary African green monkey kidney cells from a foal with diarrhea. This cell culture-adapted strain was found to be similar, if not identical, to simian rotavirus (strains MMU18006 and SA-11) and canine rotavirus (strain CU-1) and, in addition, demonstrated a one-way antigenic relationship with five human rotavirus strains (P, B, no. 14, no. 15, and YO) of the third human rotavirus serotype by the plaque reduction neutralization test. This is the fifth example of an animal rotavirus which shares serotypic specificity with a human rotavirus. The H-2 strain is distinct from the H-1 strain (Y. Hoshino et al., J. Clin. Microbiol., in press) of equine rotavirus not only in serotypic specificity by neutralization but also in subgroup specificity, hemagglutinating activity, and RNA electrophoretic migration pattern, thus establishing the existence of a second equine rotavirus serotype. This H-2 isolate is also distinct by neutralization from three other human rotavirus serotypes, 1 (Wa), 2 (DS-1), and 4 (St. Thomas no. 4), as well as bovine (NCDV), and porcine (OSU) rotaviruses.

Rescue and serotypic characterization of noncultivable human rotavirus by gene reassortment.

Thirty-three of 50 noncultivable human rotavirus strains from a variety of locations were successfully rescued by gene reassortment. The serotype of each of the 33 strains was investigated by a qualitative cytopathic effect neutralization assay. Nineteen strains resembled the previously characterized human rotavirus serotype Wa, whereas three strains were serologically related to the DS-1 strain. Eleven strains appeared to be serotypically distinct from the Wa and DS-1 strains and thus apparently represent one or more new human rotavirus serotypes.

Serological comparison of canine rotavirus with various simian and human rotaviruses by plaque reduction neutralization and hemagglutination inhibition tests.

By the plaque reduction neutralization test, the CU-1 strain of canine rotavirus was similar, if not identical, to three strains (no. 14, no. 15, and P) of the tentatively designated third human rotavirus serotype. In addition, strain CU-1 demonstrated a one-way antigenic relationship with two other strains (M and B) of the third human rotavirus serotype. The CU-1 strain of canine rotavirus hemagglutinated human group O, rhesus monkey, dog, sheep, and guinea pig erythrocytes. A two-way antigenic relationship between canine (CU-1) and simian (MMU 18006 and SA11) rotaviruses demonstrated previously by the plaque reduction neutralization test was confirmed further with two additional isolates (A79-10 and LSU 79C-36) of canine rotavirus by the plaque reduction neutralization test and the hemagglutination inhibition test. The CU-1 strain of canine rotavirus, which is known to be distinct from two well-characterized human rotavirus serotypes (Wa and DS-1), was also found to be distinct from the St. Thomas no. 4 strain, which is a newly defined fourth human rotavirus serotype. Thus, this canine strain, which is related antigenically to one of four human rotavirus serotypes, is another example of an animal rotavirus which shares serotype specificity with a human rotavirus.