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The immunogenic potency of an experimental vaccine against rotavirus infection of cattle was studied under production conditions. Cows with calves were vaccinated in the second period of pregnancy once, twice and three times. The study showed the schedule of immunization of pregnant cows including three vaccinations at 50-40, 30-20, and 15-10 days before delivery to be technological, providing a high protective effect in newborn calves.

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An enzyme-linked immunosorbent assay (ELISA) based on peplomer glycoprotein E2 was developed for the detection of antibodies to transmissible gastroenteritis virus (TGEV). Purified preparations of E2 were isolated by solubilization of the viral membrane with nonion detergent Nonidet P-40, followed by sucrose density gradient sedimentation. ELISA optical density values with E2 antigen significantly exceeded the indices when other TGEV protein or intact virion antigen was used. It was shown that a virus protein concentration in the E2 preparation of 500 ng per well is sufficient to sensitize the solid phase of microplates. A comparison of the ELISA and the virus neutralization test for the detection of TGEV antibodies was conducted. A significant correlation between the ELISA and the virus neutralization test was shown (r = 0.97). This serological test may be successfully used for various immunologic investigations.

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Two different methods were used to prepare subunit components of transmissible gastroenteritis (TGE) virus. The ultrastructure of the peplomer glycoprotein E2 of TGE virus was studied. The presence of detergent NP-40 was shown to prevent the formation of this glycoprotein aggregates. In dialysed preparations, the peplomers are aggregated to form multimer structures, the so-called "rosettes". The orientation of the peplomers in the "rosettes" and in the whole virion was identical. The antigenic activity of viral subunits was studied in guinea pigs. Preparations of isolated peplomers were shown to induce enhanced production of virus-neutralizing antibodies whose mean titre exceeded that in the group of animals immunized with whole virion-based preparations.

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Immunobiological properties of swine rotaviruses were studied. A field isolate of this virus was markedly virulent for newborn colostrum-less piglets. An attenuated strain of swine rotavirus was obtained by serial passages in continuous cell cultures (MA-104 and SPEV). The virus of the 60th passage was nonvirulent for colostrum-less piglets although produced marked seroconversion. Naso-oral immunization of piglets with the attenuated strain prevented the manifestation of clinical disease upon challenge with the virulent swine rotavirus. Besides, the swine rotavirus of the 60th passage showed cross-protective activity against virulent human rotavirus in modelling human rotavirus infection in colostrum-less piglets.

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Two variants of enzyme immunoassay (EIA) using purified swine transmissible gastroenteritis virus antigen for sensitization of the solid phase and using infected cells on the solid phase have been developed for quantitation of specific antibodies to swine transmissible gastroenteritis (STG) virus. Both variants of the method were found to have approximately similar sensitivity and to be highly specific. The developed EIA proved to be approximately 100 times as sensitive as neutralization test. The optimal concentration of purified antigen for sensitization of microplates, as well as the multiplicity of infection of the heterologous cell culture grown in microplate wells have been determined. The above methods may be widely used in virological and epizootiological studies.

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Direct and indirect variants of enzyme immunoassay (EIA) were developed for the detection of specific antigen of swine transmissible gastroenteritis virus (TGEV). Preparation of IgG were obtained from highly specific hyperimmune sera of guinea pigs and gnotobiotic piglets and their optimal concentration for the tests were determined. For the direct EIA, a peroxidase conjugate was prepared from guinea pig IgG and its working dilution was determined. The developed EIA systems proved to be highly sensitive for the detection of TGEV antigen both in infected cell cultures and clinical specimens from sick piglets detecting 5 ng of the virus-specific protein.

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On the basis of porcine rotavirus a heterologous EIA test system was worked out and tested for diagnosis of human rotavirus infection. A high sensitivity and specificity of the test system was demonstrated, its results were compared with those of electron microscopy, diffuse precipitation test, and RNA electrophoresis. Out of 201 specimens (fecal filtrates) collected from children ranging in ages from 14 days to 10 years, rotavirus antigen was detected in 68 (33.8%) RNA electrophoresis demonstrated the rotaviruses circulating in Moscow to have basically the same electrophoretic type, although rotaviruses with other electrophoretic types, including the "short" electrophoretic type, were also detected.

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Pretreatment of the transmissible gastroenteritis virus of swine (TGE) (coronavirus) with trypsin increased the yield of the infectious virus approximately 100-fold when propagated in roller cultures of SPEV cells, the time required for the development of marked CPE being twice shorter than without treatment. The method of concentration and purification of TGE virus is described which yields a preparative amount of highly purified virions retaining their structure. Such preparations were used for immunization of guinea pigs. It is concluded that purified preparations of TGE virus possess marked antigenic potency and may be used for generation of highly active and specific hyperimmune sera.

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The genome structure and polypeptide composition of a rotavirus virion isolated from piglets is described. Proteins p90 and p38 were localized in the external capsid of the virion, and the group-specific antigen of rotaviruses, protein p42, in the internal capsid. Minor polypeptides p116, p98, and p92 were associated with the virus core. A scheme of the structure of porcine rotavirus virion is proposed on the basis of the virion ultrastructure and localization of viral proteins in the capsid. Attention is drawn to the fact that the "wild" strain of swine rotavirus which is virulent for piglets differed in electrophoretic mobility of at least 3 segments of virus genome from the same strain which had undergone 70 passages in cell culture. The virus with altered RNA-segments showed no virulence. The change in the molecular weight of the RNA-segment 4 correlated with the change in polypeptide p90 coded for by it. In the authors' opinion, variability of the major product of protein p90 proteolysis caused the loss of rotavirus virulence for the naturally susceptible host.

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The data on the structural and functional organization of human rotaviral virion and rotaviral virions of different species of animals obtained by the native and foreign researchers for the last five years of study have been summarized. The virion ultrastructure, the genomic structure and polypeptide composition of rotaviruses are discussed. The data are presented on the transcription and molecular mapping of rotaviral genome. Special attention is paid to localization of rotaviral proteins in the virion capsid, to their function and derivation. The data on the role of endogenous enzymes in rotaviral transcription and replication are presented separately.

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The use of the dot-hybridization assay for the diagnosis of human rotavirus infection was shown to be principally possible. 32P-labeled RNA segments of porcine rotavirus transcribed in an in vitro system by activation of endogenous RNA-dependent RNA-polymerase were used as hybridization probes. The transcripts were synthesized to all 11 segments of the parental virus genome. The method proved to be highly specific and very sensitive detecting 10 picograms of viral RNA in fecal filtrates of the patients.

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A swine rotavirus capable of inducing the cytopathic effect was isolated in a roller culture of Macaca rhesus kidney cells (line MA-104) after two preliminary passages in gnotobiotic piglets and colostrum-free piglets, and the isolate was designated strain K. For virus isolation, fecal specimens were treated with trypsin, and besides, trypsin was added into the maintenance medium. After 20 passages in MA-104 cell culture the swine rotavirus was adapted to pig embryo kidney cell cultures (SPEV line) in which the maximum virus accumulation, 8.0 log TCD50/ml, was achieved within 24 hours after inoculation. The virus accumulation was most marked in the presence of 10 micrograms/ml trypsin in the maintenance medium. In the roller culture, the virus multiplied to a much higher titre (approximately 100-fold) than in the stationary culture. In the course of passages the virus was shown to lose its pathogenic properties. A scheme of swine rotavirus virion structure is suggested on the basis of ultramicroscopic studies.

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