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Biomedical subjects

H F Clark

Publications and source records attributed to H F Clark.

At least 55 records · Page 3Linked to original sources
PubMed1986

Molecular basis of rotavirus virulence: role of gene segment 4.

Bovine rotavirus NCDV and simian rotavirus SA-11 exhibited markedly different patterns of gastrointestinal tract disease when inoculated orally into newborn mice. A genetic approach was used to define the molecular basis of these differences. The SA-11 strain of rotavirus was more virulent than the NCDV strain when inoculated orally into newborn mice; the dose of SA-11 required to cause diarrhea in 50% of infant mice was 50-fold less than that required for NCDV. Nineteen reassortant viruses were derived by coinfection of MA-104 cells in vitro with the SA-11 and NCDV strains. The parental origin of reassortant virus double-stranded RNA segments was determined by gene segment migration differences in polyacrylamide gels and hybridization with radioactively labeled parental viral transcripts. The neutralization antigen phenotype of reassortant viruses was determined by plaque reduction neutralization. We found that the dose of SA-11 and NCDV rotavirus required to induce gastroenteritis in newborn mice was determined by gene segment 4. The results suggest that rotavirus virulence may be manipulated by modification or reassortment of gene segment 4.

Antigens, Viral↗
PubMed1986

Reassortant rotaviruses containing structural proteins vp3 and vp7 from different parents induce antibodies protective against each parental serotype.

Genetic studies of reassortant rotaviruses have demonstrated that gene segments 4 and 9 each segregate with the serotype-specific neutralization phenotype in vitro. Reassortant rotaviruses derived by coinfection of MA-104 cells with the simian strain SA11 and the antigenically distinct bovine strain NCDV were used to determine which viral genes coded for proteins which induced a protective immune response in vivo. In addition, reassortant rotaviruses containing only the gene segment 4 or 9 protein products (vp3 and vp7, respectively) from SA11 or NCDV were used to determine the serotypic specificities of both vp3 and vp7 in several mammalian rotavirus strains. vp3 and vp7 from the murine strain Eb were shown to be indistinguishable from the corresponding proteins from strain SA11. Adult mice orally inoculated with strain Eb developed neutralizing antibodies to both vp3 and vp7. The two naturally occurring bovine rotavirus strains NCDV and UK were shown to contain antigenically similar vp7 but distinct vp3 proteins. Mouse dams orally immunized with a reassortant virus containing only gene 9 from NCDV passively protected their progeny against UK challenge, whereas mouse dams orally immunized with a reassortant virus containing only gene 4 from NCDV did not. Finally, we constructed reassortant viruses that immunized against rotaviruses of two distinct serotypes. SA11 X NCDV reassortants that contained vp3 and vp7 from different parents induced a protective immune response against both parental serotypes. vp3 and vp7 were independently capable of inducing a protective immune response after oral immunization. An understanding of the serotypic specificities of both vp3 and vp7 of human rotavirus isolates will be necessary for the development of successful strategies to protect infants against severe rotavirus infections.

Animals↗
PubMed1985

Maternal antibody-mediated protection against gastroenteritis due to rotavirus in newborn mice is dependent on both serotype and titer of antibody.

In order to evaluate the role of passively acquired, rotavirus-specific antibodies in protection against diarrhea, we inoculated mouse dams with rotaviruses of various serotypes, and their newborns were orally challenged with a primate rotavirus (simian SA-11). Dams were immunized by using a regimen that included repeated inoculations administered either orally or intraperitoneally with adjuvant. The serum antibody response detected in dams by radioimmunoassay and plaque-reduction neutralization after parenteral immunization was approximately 15-fold and 80-fold greater, respectively, than that found after oral "hyperimmunization." Parenteral immunization with rotavirus serotypes either homotypic or heterotypic to the challenge virus protected suckling mice against diarrhea; protection was closely correlated with the in vitro neutralizing activity of maternal serum against the challenge virus. Oral immunization with only rotavirus strains homotypic to the challenge virus afforded protection; the lower immune response after oral immunization with rotaviruses heterotypic to the challenge virus resulted in a titer of neutralizing antibody to the challenge virus below the protective threshold. From our current studies it appears that antibody-mediated passive protection against rotavirus challenge is dependent on both serotype and titer of antibody.

Animals↗
PubMed1985

Rapid inactivation of rotaviruses by exposure to acid buffer or acidic gastric juice.

Inactivation rates of three bovine and several primate-origin rotaviruses were determined during exposure to acid buffers at pH 2.0, pH 3.0 or pH 4.0. Each rotavirus was inactivated at pH 2.0 (the acidity most resembling the normal fasting stomach) very rapidly, with half-lives for infectivity determined to be 1 min or less. Each rotavirus was inactivated at a much slower rate at pH 3.0; inactivation at pH 4.0 was minimal. No remarkable differences in acid resistance between different rotavirus strains were detected. Although these determinations were performed at room temperature (23 degrees C), experiments at diverse temperatures indicated an even more rapid rate of viral inactivation by acid at normal body temperature (37 degrees C). Studies of rotavirus exposed to natural human gastric juice at pH 1.8 or pH 2.1 revealed a rate of virus inactivation similar to that observed with glycine buffer of identical pH.

Acids↗
PubMed1985

Epidemiology of rotavirus electropherotypes determined by a simplified diagnostic technique with RNA analysis.

The incidence and RNA electropherotypes of rotavirus in stools or rectal swabs of children with diarrhea were studied for three rotavirus seasons (1981 through 1984) in Philadelphia, Pa. We used a simplified RNA analysis method involving polyacrylamide gel electrophoresis followed by silver staining. Phosphate-buffered saline suspensions of the stools and swab eluates were examined directly by polyacrylamide gel electrophoresis-silver staining analysis and enzyme-linked immunoadsorbent assay (Rotazyme; Abbott Laboratories); electron microscopy was performed on solid stool specimens. The RNA analysis results were compared with electron microscopy and enzyme-linked immunosorbent assay results and exhibited a sensitivity and specificity greater than or equal to that of electron microscopy or the enzyme-linked immunosorbent assay. Ten different electropherotypes were detected among the 68 rotavirus RNA-positive specimens examined over the 3-year study. The predominant electropherotype was different in each season. Our results indicate that the polyacrylamide gel electrophoresis-silver nitrate strain RNA analysis of simple unextracted stool suspensions is a uniquely useful diagnostic technique; it rapidly provides both a definitive positive result and immediate determination of the RNA electropherotype, which is of value for epidemiological study.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Protection against rotavirus-induced gastroenteritis in a murine model by passively acquired gastrointestinal but not circulating antibodies.

Newborn mice suckled on dams immunized either orally or parenterally with primate rotavirus SA-11 were protected against diarrhea induced by SA-11 virus challenge. Experimental oral administration of milk from orally immunized dams protected suckling mice against challenge; protective activity was detected both in the anti-rotavirus immunoglobulin A (IgA) and IgG fractions, but IgA was more potent in vivo than IgG. Oral administration of milk from parentally immunized dams also protected suckling mice against challenge; in this case, protective activity was detected in the anti-rotavirus IgG fraction. In newborn mice foster-nursed by seronegative dams, circulating rotavirus-specific antibodies in high titer did not protect mice against oral SA-11 virus challenge. It appears that the most effective rotavirus vaccine will be that which induces an efficient production of antibodies active at the intestinal cell surface.

Animals↗
PubMed1984

Rotavirus-specific antibodies in fetal bovine serum and commercial preparations of serum albumin.

Rotavirus-specific antibodies were detected in fetal bovine serum, bovine serum albumin, and human serum albumin by radioimmunoprecipitation with the NCDV strain of bovine rotavirus as the detecting antigen. Fetal bovine sera neutralized bovine rotavirus in a plaque reduction neutralization test to titers of 1:20 or greater. Immunoglobulins purified from fetal bovine serum by protein A-agarose affinity chromatography precipitated rotavirus antigens but did not neutralize bovine rotavirus. Rotavirus antibodies in fetal bovine serum and in purified serum albumin preparations may interfere with diagnostic assays for the detection of rotavirus antigens or antibodies.

Animals↗
PubMed1984

A murine model for oral infection with a primate rotavirus (simian SA11).

Simian rotavirus SA11 was shown to replicate in the gastrointestinal tracts of infant mice after oral inoculation. Clinical symptoms, histopathological changes in the small intestinal mucosa, and the type-specific humoral immune response were all characteristic of rotavirus-induced gastroenteritis. The availability of this small animal model for the study of a primate rotavirus infection should expedite analysis of the immune response necessary for protection against challenge.

Animals↗
PubMed1983

The cultivation of human rotavirus, strain 'Wa', to high titer in cell culture and characterization of the viral structural polypeptides.

The structural proteins of the 'Wa' (serotype 2) strain of human rotavirus have not been described previously. Single-cycle virus growth in MA-104 cells using 5 micrograms/ml of trypsin in the growth medium was rapid with maximal viral yields (approximately 10(6) PFU/ml) obtained 10-12 h post-infection. There was a continuous progression of cytopathic effect (CPE) from 6- to 5-h post-infection. Under conditions of multiple-cycle growth, a greater concentration of trypsin (40 micrograms/ml) in the growth medium was required to obtain rapid progression of CPE and production of a high titer (approximately 10(7) PFU/ml) of infectious (double-shelled) virus. Single- and double-shelled virions were separated by isopycnic centrifugation in CsCl and analyzed by SDS-PAGE. Five proteins with molecular weights of 116,000, 92,000, 88,000, 84,000 and 41,000 were identified as components of the inner shell and four proteins with molecular weights of 60,000, 38,000, 32,000 and 27,000 were located in the outer shell.

Animals↗
PubMed1983

Response of mice to rotaviruses of bovine or primate origin assessed by radioimmunoassay, radioimmunoprecipitation, and plaque reduction neutralization.

Sera from (i) gnotobiotic BALB/c, CD-1, and CFW mice and (ii) conventional BALB/c mice were evaluated by radioimmunoassay, radioimmunoprecipitation, and plaque reduction neutralization, using the Wa, SA-11, and WC-3 (bovine) strains of rotavirus as the detecting antigens. The gnotobiotic mice had no antirotavirus antibody detectable by radioimmunoprecipitation and no neutralizing antibody at a dilution of 1:50 by plaque reduction neutralization. All sera from the conventional mice had rotavirus-specific antibodies detected by radioimmunoassay and by radioimmunoprecipitation at serum dilutions of 1:50 and 1:10,000, respectively. The antibodies were directed against viral proteins p116, p94, p88, and p84 of all three viruses, but had no neutralizing activity against heterologous rotaviruses at a dilution of 1:50. Conventional seropositive mice were parenterally immunized with the Wa, SA-11, or WC-3 strain of rotavirus. An approximate 100-fold increase in rotavirus-specific antibodies was detected by radioimmunoassay, and greater than 20-fold selective neutralization of the immunizing strain of virus was observed. Sera from the mice immunized with Wa virus had antibodies directed against inner and outer capsid proteins of all three rotaviruses. The mouse can be a useful model for studying the immune response to heterologous rotavirus infection; preexisting antibodies presumably directed towards murine rotavirus do not prevent the development of a type-specific immune response to a nonmurine rotavirus.

Animals↗
PubMed1982

Similarity in the bound carbohydrate groups of glycoproteins from cells of several vertebrate classes.

The carbohydrate groups of the glycoproteins of human, hamster, chick, reptile and fish cells growing in culture have been fractionated in succession according to size (Sephadex G-50), affinity for concanavalin A, charge (DEAE-Sephadex) and by thin-layer chromatography. It was found that despite the complexity of the array of separable glycopeptides in each type of cell, most of these structures seemed to be common to all of the cells. This suggests that they have existed in a relatively stable state for several hundreds of millions of years throughout the evolution of the vertebrates.

Animals↗
PubMed1981

Defective interfering particles of fixed rabies viruses: lack of correlation with attenuation or auto-interference in mice.

Six different fixed strains of rabies virus were analysed for their capacity to produce defective particles following acute infection of BHK-21 cells. Five of the six strains produced one or more defective particle populations with strain-specific sedimentation properties, particle length and abbreviated RNA genome size. These defective particles varied in their capacity to interfere with replication of standard rabies virus in cell culture. Each virus strain characteristically either killed adult mice according to a normal dose-response pattern or to an auto-interference type of pattern, or failed to kill mice. Different strains also varied in their capacity to induce a cytopathic effect in cell culture. However, there was no apparent correlation between the presence of defective particles and the pathogenic potential of rabies virus in mice or in cell culture.

Animals↗
PubMed1981

Integration pattern of hepatitis B virus DNA sequences in human hepatoma cell lines.

Four human hepatoma cell lines established from primary hepatocellular carcinomas were examined for the presence of hepatitis B virus DNA sequences. Reassociation kinetic analysis indicated that the cell lines HEp-3B 217, HEp-3B 14, HEp-3B F1, and PLC/PRF/5 contained two, one, one, and four genome equivalents per cell, respectively. Southern blot hybridization analysis demonstrated that hepatitis B virus DNA was integrated into the cellular DNAs of these cell lines. Further liquid hybridization studies with 32P-labeled HincII restriction fragments of hepatitis B virus DNA established that DNA sequences from all regions of the HBV genome were represented in the integrated viral sequences. Although the three HEp-3B cell lines were derived from the same tumor, they differed significantly in their patterns of integration of hepatitis B virus DNA, the number of copies of viral DNA per cell, and their ability to produce the virus-coded surface antigen.

Carcinoma, Hepatocellular↗
PubMed1980

Regeneration of DI particles of virulent and attenuated rabies virus: genome characterization and lack of correlation with virulence phenotype.

Two strains of fixed rabies virus were examined for their ability to regenerate defective interfering (DI) particles and for possible correlation of DI particle production with the expression of virulence. A plaque-purified stock of the attenuated ERA strain (ERApp), which characteristically caused an auto-interfering death response in adult mice inoculated i.c., was serially passed at a high m.o.i. inBHK-21 cells. By the sixth passage, DI particles were regenerated that corresponded in sedimentation velocity and DI/RNA size to the smallest of three sizes of DI particles produced by the parental stock virus. The regeneration of ERA DI particles in vivo was not detected during 15 serial high or low m.o.i. passages of infected newborn mouse brain, though the passaged virus consistently elicited an auto-interfering-type death response when assayed in adult mice. The attenuated Flury HEPpp strain regenerated up to three unique size classes of DI particles during serial passage in BHK-21 or murine neuroblastoma C1300 clone NA cells compared with the one band of DI particles produced by the parental Flury HEP stock virus. The BHK-21 cell-adapted Flury HEPpp virus failed to kill adult mice when inoculated at high concentrations after two serial passages in NA cells. However, the virus became fully virulent and a single band of regenerated DI particles was visible. Additional bands of defective particles were visible following the third serial passage in NA cells. Single-stranded RNA with a mol. wt. of 0.62 x 10(6) was extracted from the first DI particle population to be regenerated. This corresponded in mol. wt. to the DI/ssRNA characteristic of the parental attenuated Flury HEP virus. However, in the parental type DI/RNA, partially dsRNA could be isolated in addition to ssRNA. Double-stranded RNA could not be detected in the regenerated DI particles derived from the virulent NA cell-propagated Flura HEPpp virus. These results suggest that the virulence phenotype of fixed rabies viruses does not depend on the presence or absence of DI particles.

Animals↗
PubMed1980

Rabies serogroup viruses in neuroblastoma cells: propagation, "autointerference," and apparently random back-mutation of attenuated viruses to the virulent state.

Each of several strains of fixed rabies virus was found to replicate to high titers in C1300 mouse neuroblastoma (clone NA) cells, without adaptation. Rabies serogroup Lagos bat, Mokola, and Duvenhage viruses also replicated efficiently in NA cells. Kotonkan and Obodhiang viruses replicated efficiently after adaptation, to titers not previously obtained in vitro. Infection in NA cells was frequently more cytopathic than in BHK-21 cells, allowing titration of Kotonkan and Obodhiang viruses by plaque assay. Duvenhage virus caused syncytium formation. Serial propagation of rabies viruses at a high multiplicity of infection in NA cells led to a rapid decline in virus yields; similar "autointerference" has not previously been demonstrated with rabies virus in other cell systems. Rabies virus infection in NA cells exhibited extreme sensitivity to interference by experimentally added defective interfering virions. Although several strains of attenuated rabies virus consistently reverted rapidly to virulence after propagation in NA cells, other strains of attenuated rabies and rabies serogroup viruses acquired increased virulence at a more gradual rate or not at all, suggesting that diverse characters may control virulence. When attenuated Flury HEP rabies virus was serially propagated at a low multiplicity of infection in either NA cells or suckling mouse brain, virulence appeared at a very variable rate, indicating that these systems may selectively enhance replication of randomly occurring virulent virus mutants.

Animals↗
PubMed1979

Evolutionary relatedness of viper and primate endogenous retroviruses.

A retrovirus previously isolated from a tumored Russell's viper is shown by molecular hybridization to be an endogenous virus of this reptilian species. Radio-immunologic techniques revealed that the viper retrovirus is immunologically and, hence, evolutionarily related to endogenous type D retorviruses of Old World primates. These findings extend the number of vertebrate classes possessing endogenous retroviruses and suggest that type D retroviruses may even be more widely distributed in nature than type C retroviruses.

Animals↗