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Biomedical subjects

M K Estes

Publications and source records attributed to M K Estes.

At least 217 records · Page 12Linked to original sources
PubMed1988

Detection of human calicivirus antigen and antibody by enzyme-linked immunosorbent assays.

Enzyme-linked immunosorbent assays (ELISAs) were developed to detect human calicivirus (HCV) antigen and antibody to HCV. The ELISAs were specific for HCV and as sensitive as a previously developed radioimmunoassay. These ELISAs were used to search for evidence of HCV infection in the United States, where HCV gastroenteritis has rarely been reported. One hundred sixty-three stool samples collected from children hospitalized with diarrhea were examined; one sample was positive in the ELISA. Typical calicivirus particles were found in this stool sample, and these particles reacted with a hyperimmune guinea pig anti-HCV serum by immune electron microscopy. The age-related acquisition of antibody to HCV in hospitalized infants and children (from birth to 19 years old) without gastroenteritis and in healthy adults was also evaluated. The pattern of acquisition of antibody to HCV was similar to that for group A rotaviruses, namely, beginning in infancy and becoming 100% by the age of 4 years. These data suggest that HCV is associated with infantile gastroenteritis in the United States, that infections with HCV are common, and that many infections with HCV (Sapporo strain) may not require hospitalization.

Adolescent↗
PubMed1988

Rabbit model of rotavirus infection.

A new small animal model was developed to study parameters of rotavirus infections, including the active immune response. Seronegative New Zealand White rabbits (neonatal to 4 months old) were inoculated orally with cultivatable rabbit rotavirus strains Ala, C11, and R2 and with the heterologous simian strain SA11. The course of infection was evaluated by clinical findings, virus isolation (plaque assay and enzyme-linked immunosorbent assay), and serologic response. All four strains of virus were capable of infecting rabbits as determined by isolation of infectious virus from intestinal contents or fecal samples, by seroconversion, or by a combination of these methods. The responses differed depending on the virus strain used for inoculation. Rabbits remained susceptible to primary infection to at least 16 weeks of age (upper limit examined). Virus excretion in intestinal contents was detected from 6 h to 7 days postinoculation. RNA electropherotypes of inocula and viruses isolated from rabbits were the same in all samples tested. Transmission of Ala virus and R2 virus but not SA11 virus from inoculated animals to uninoculated controls also occurred. In a challenge experiment with Ala virus, 74- and 90-day-old rabbits were rechallenged with Ala 5 weeks after a primary infection with Ala. Virus was excreted in feces from 2 to 8 days after the primary infection but was not excreted after challenge. These results indicate that the rabbit provides an ideal model to investigate both the primary and secondary active immune responses to rotavirus infections and to evaluate candidate vaccines.

Age Factors↗
PubMed1988

Functional and topographical analyses of epitopes on the hemagglutinin (VP4) of the simian rotavirus SA11.

An immunochemical analysis of the hemagglutinin (VP4) of the simian rotavirus SA11 was performed to better understand the structure and function of this molecule. Following immunization of mice with double-shelled virus particles and VP4-enriched fractions from CsCl gradients, a battery of anti-SA11 hybridomas was generated. A total of 13 clones secreting high levels of anti-VP4 monoclonal antibody (MAb) was characterized and compared with two cross-reactive anti-VP4 MAbs generated against heterologous rhesus (RRV) and porcine (OSU) rotavirus strains. These cross-reactive MAbs effectively neutralized SA11 infectivity in vitro. The epitopes recognized by these 15 MAbs were grouped into six antigenic sites on the SA11 hemagglutinin. These sites were identified following analysis of the MAbs by using a simple competitive binding enzyme-linked immunosorbent assay (ELISA) and biological assays. Three of the antigenic sites were involved in neutralization of virus infectivity in vitro. All the MAbs with neutralization activity and two nonneutralizing MAbs were able to inhibit viral hemagglutination of human erythrocytes. Competitive binding ELISA data showed a positive cooperative binding effect with some pairs of the anti-VP4 MAbs, apparently due to a conformational change induced by the binding of the first MAb. Some of the MAbs also bound better to trypsin-treated virus than to non-trypsin-treated virus. A topographic map for VP4 is proposed on the basis of the observed properties of each antigenic site.

Antibodies, Monoclonal↗
PubMed1987

Minimal infective dose of rotavirus.

We studied the minimal infective dose of the gastroenteritis virus, rotavirus. Increasingly lower doses [10(4), 10(3), 10(1), 1, 10(-2) plaque forming units (PFU)] of the OSU strain of porcine rotavirus were administered to highly susceptible (colostrum deprived, cesarean derived) newborn miniature swine piglets. In vitro studies showed that virus infectivity was inactivated in piglet gastric juice, both by low pH and by pH- and concentration-dependent factor(s). These factors remain unidentified, but to prevent intragastric viral inactivation, sodium bicarbonate was administered prior to oral virus inoculation of piglets with virulent (non-tissue culture passaged) virus. The lowest dose of virus to induce clinical illness or to demonstrate viral replication by recovery of significantly more infectious virus than was administered, or both, was 1 PFU. These results should help establish standards for virus contamination of water and recommendations for evaluating disinfection procedures for rotaviruses.

Animals↗
PubMed1987

Detection of hepatitis A virus by hybridization with single-stranded RNA probes.

An improved method of dot-blot hybridization to detect hepatitis A virus (HAV) was developed with single-stranded RNA (ssRNA) probes. Radioactive and nonradioactive ssRNA probes were generated by in vitro transcription of HAV templates inserted into the plasmid pGEM-1. 32P-labeled ssRNA probes were at least eightfold more sensitive than the 32P-labeled double-stranded cDNA counterparts, whereas biotin-labeled ssRNA probes showed a sensitivity comparable with that of the 32P-labeled double-stranded cDNA counterparts. Hybridization of HAV with the ssRNA probes at high stringency revealed specific reactions with a high signal-to-noise ratio. The differential hybridization reactions seen with probes of positive and negative sense (compared with HAV genomic RNA) were used to detect HAV in clinical and field samples. A positive/negative ratio was introduced as an indicator that permitted a semiquantitative expression of a positive HAV reaction. Good agreement of this indicator was observed with normal stool samples and with HAV-seeded samples. By using this system, HAV was detected in estuarine and freshwater samples collected from a sewage-polluted bayou in Houston and a saltwater tributary of Galveston Bay.

Child↗
PubMed1987

Investigation of association of mycobacteria with inflammatory bowel disease by nucleic acid hybridization.

We used DNA-DNA hybridization to characterize a mycobacterial isolate, strain Linda, that was obtained from a patient with Crohn's disease and that has been reported to cause ileitis in experimental animals. We also investigated the association of this mycobacterium with Crohn's disease. Our results identified Mycobacterium strain Linda as a strain of Mycobacterium paratuberculosis, the etiologic agent of Johne's disease, a disease of ruminants that has some superficial resemblance to Crohn's disease. Sequences that hybridized with strain Linda DNA probes were detected in DNA extracted from human intestinal tissues from patients with Crohn's disease, ulcerative colitis, and noninflammatory bowel disease. These hybridizing DNA sequences were more prevalent in the muscle layers than in the intestinal mucosa, making it unlikely that they represented DNA from bacterial contaminants in the intestinal lumen. Measurement of the melting temperatures of the DNA-DNA hybrids formed between strain Linda probes and tissue DNAs indicated that the related sequences detected were of mycobacterial origin but were not identical to each other or to strain Linda DNA. These results do not support the proposed specific relationship between Mycobacterium strain Linda and Crohn's disease. The possible etiologic role of mycobacteria in Crohn's disease is discussed.

Colitis, Ulcerative↗
PubMed1987

Electron microscopy procedure influences detection of rotaviruses.

Technical parameters of electron microscope staining procedures (type of stain, pH of stain, and time of staining) influence particle integrity for three groups of rotaviruses. Simian rotavirus SA11 (group A), Chinese adult diarrhea rotavirus and porcine rotavirus-like agent (group B), and porcine pararotavirus (group C) were tested. All rotavirus strains were quite stable in uranyl acetate and phosphotungstic acid at pH 4.5 and relatively stable in ammonium molybdate. However, staining with phosphotungstic acid at higher pH values with increased staining time yielded a reduction in the number of particles and particles that were broken or degraded to single-shelled particles or core particles. The different staining procedures were also tested in immunoelectron microscopy experiments. Antibody molecules bound to rotavirus particles were observed clearly only with phosphotungstic acid staining and not with uranyl acetate. We therefore recommend that uranyl acetate and phosphotungstic acid at pH 4.5 be used for negative staining of rotaviruses; phosphotungstic acid at pH 4.5 is optimal for immunoelectron microscopy. These technical points may be critical for rotavirus detection and are important for studies pertaining to the epidemiology and clinical importance of the non-group A rotaviruses.

Animals↗
PubMed1987

Detection of antibody to group B adult diarrhea rotaviruses in humans.

Group B rotaviruses have been responsible for annual epidemics of severe diarrhea affecting both adults and children in China. We developed a specific and sensitive enzyme-linked immunosorbent blocking assay to detect antibody to group B rotaviruses that will be useful to assess the role of group B rotavirus infections as a cause of human gastroenteritis. We tested 219 human sera and 18 immunoglobulin pools collected from eight countries for antibodies to both group A and group B rotaviruses. Overall, a low proportion (10 of 237 or 4.2%) of sera contained antibody to group B rotaviruses. Antibody to group B rotavirus was detected in only 1 of 155 serum samples from healthy or hospitalized individuals in the United States, including patients with the chronic inflammatory bowel diseases Crohn's disease and ulcerative colitis. No antibody was detected in 15 serum samples from Australia and from an outbreak of gastroenteritis on a cruise ship or in nine immunoglobulin pools from Japan and the United Kingdom. Antibody to group B rotaviruses was detected in 8 convalescent-(but not acute-)phase serum samples from Chinese patients with group B gastroenteritis, in five immunoglobulin pools from China, in 1 of 6 serum samples from Chinese students in the United States, and in 1 each of 10 serum samples from Kenya, 20 from Thailand, and 15 from Canada. In contrast, most of these samples (226 of 237 or 95.4%) had antibody to group A rotaviruses. These results indicate that human infection with group B rotavirus has not been widespread in areas outside China. Seroconversion observed between the acute-and convalescent-phase serum samples from China also suggests that infections with this virus are primary infections. Continued surveillance for this new group of rotaviruses should determine whether the many susceptible people become infected of whether other factors influence the severe pathogenicity of human infections with these viruses in China.

Adolescent↗
PubMed1987

The cellular secretory pathway is not utilized for biosynthesis, modification, or intracellular transport of the simian virus 40 large tumor antigen.

Unlike most proteins, which are localized within a single subcellular compartment in the eucaryotic cell, the simian virus 40 (SV40) large tumor antigen (T-ag) is associated with both the nucleus and the plasma membrane. Current knowledge of protein processing would predict a role for the secretory pathway in the biosynthesis and transport of at least a subpopulation of T-ag to account for certain of its chemical modifications and for its ability to reach the cell surface. We have examined this prediction by using in vitro translation and translocation experiments. Preliminary experiments established that translation of T-ag was detectable with as little as 0.1 microgram of the total cytoplasmic RNA from SV40-infected cells. Therefore, by using a 100-fold excess of this RNA, the sensitivity of the assays was above the limits necessary to detect the theoretical fraction of RNA equivalent to the subpopulation of plasma-membrane-associated T-ag (2 to 5% of total T-ag). In contrast to a control rotavirus glycoprotein, the electrophoretic mobility of T-ag was not changed by the addition of microsomal vesicles to the in vitro translation mixture. Furthermore, T-ag did not undergo translocation in the presence of microsomal vesicles, as evidenced by its sensitivity to trypsin treatment and its absence in the purified vesicles. Identical results were obtained with either cytoplasmic RNA from SV40-infected cells or SV40 early RNA transcribed in vitro from a recombinant plasmid containing the SP6 promoter. SV40 early mRNA in infected cells was detected in association with free, but not with membrane-bound, polyribosomes. Finally, monensin, an inhibitor of Golgi function, failed to specifically prevent either glycosylation or cell surface expression of T-ag, although it did depress overall protein synthesis in TC-7 cells. We conclude from these observations that the constituent organelles of the secretory pathway are not involved in the biosynthesis, modification, or intracellular transport of T-ag. The initial step in the pathway of T-ag biosynthesis appears to be translation on free cytoplasmic polyribosomes. With the exclusion of the secretory pathway, we suggest that T-ag glycosylation, palmitylation, and transport to the plasma membrane are accomplished by previously unrecognized cellular mechanisms.

Animals↗
PubMed1987

Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system.

Rotaviruses are the major pathogens that cause life-threatening diarrhea in young children and animals. We inserted a simian rotavirus SA11 gene 6 cDNA into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. The major capsid antigen (VP6) was expressed in high yields (20 to 150 micrograms/10(6) cells) when Spodoptera frugiperda cells were infected with baculovirus recombinants containing SA11 gene 6 inserts. Reactivity with monospecific polyclonal and monoclonal antibodies suggested that VP6, expressed intracellularly or found in the media, maintained native antigenic determinants. VP6 purified from the media from infected cells also possessed a native oligomeric structure, was immunogenic in guinea pigs, and was able to spontaneously assemble into morphologic subunits. Antisera from immunized guinea pigs failed to neutralize virus in plaque reduction assays, but detected homologous and heterologous rotavirus strains when tested by immunofluorescence, immunoprecipitation, and enzyme-linked immunosorbent assays.

Antibodies, Viral↗
PubMed1986

Two glycoproteins are produced from the rotavirus neutralization gene.

The major neutralization antigen of rotaviruses is an outer capsid glycoprotein, VP7, with an apparent molecular weight of 38,000 (38K). The simian rotavirus SA11 genome segment 9, which codes for VP7, contains two in-phase initiation codons, each of which is followed by a sequence that codes for a region of hydrophobic amino acids. We have determined that this gene is functionally bicistronic by analyzing the synthesis of VP7 in SA11-infected cells and in cell-free translation systems programmed with hybrid-selected, segment 9 specific mRNA and dog pancreatic microsomes. The translation of hybrid-selected gene 9 mRNA in wheat germ extracts yielded two distinct polypeptides of molecular weights 37K and 35.3K. In vitro translation in the presence of microsomes yielded one diffuse band of 38K that was converted into the 37K and 35.3K precursor bands by digestion with endoglycosidase H. Studies with a variant of SA11 that lacks the glycosylation site in VP7 confirmed these precursor-product relationships and extended them by indicating that the glycoprotein produced by translation from the first AUG contained a cleaved signal sequence whereas the glycoprotein produced by translation from the second AUG contained an uncleaved signal sequence. Immunoprecipitation with monospecific anti-VP7 serum and improved gel electrophoresis conditions allowed us to show that both VP7s were expressed at similar times in infected cells and both were found in purified virus particles of several different rotavirus strains. Whether these two VP7 glycoproteins are functionally distinct remains to be determined.

Antigens, Viral↗
PubMed1986

Antigenic characterization and ELISA detection of adult diarrhea rotaviruses.

Recently, severe epidemics of diarrhea among both adults and children in China have been associated with an agent called adult diarrhea rotavirus (ADRV). We have studied ADRV from two areas of China (Jinzhou and Guangxi) and compared them with prototype group A, B, and C rotaviruses. The viral genomes were compared by electrophoresis of the RNA genome segments in polyacrylamide gels, and the antigenic relatedness of these viruses was examined by immune electron microscopic studies using virus preparations of either double- or single-capsid particles. Hyperimmune antisera (guinea pig and rabbit) to one strain of ADRV reacted with high titers with other strains of ADRV and with single-shelled capsids of a porcine group B rotavirus. The antisera to ADRV did not react with either group A or group C rotaviruses. Antisera to either bovine or porcine group B rotaviruses reacted with ADRV but not with the group A or group C viruses. Antisera to group A or group C virus reacted only with their respective homologous virus. These results and those of immunofluorescence studies place the human ADRV rotaviruses from China among the group B rotaviruses. We also report the development of an ELISA to detect ADRV; this ELISA should be useful for defining the epidemiology of these recently described rotaviruses.

Adult↗
PubMed1986

Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes.

The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10(4) physical particles of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hybridization stringency, 32P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments.

Animals↗
PubMed1985

Rotavirus antigens.

The rotaviruses are important medical and veterinary pathogens that cause acute diarrheal disease in children and in the young of most mammalian and avian species. The successful development of a rotavirus vaccine should significantly reduce worldwide infant morbidity and mortality and economic losses and increase food-production. Recent successes in the cultivation and characterization of rotavirus strains from humans and animals have led to a better understanding of the major antigens of these viruses and the genes encoding these antigens. The products of three independent genes (1) the neutralization antigen (the outer capsid glycoprotein); (2) the subgroup antigen (the major inner capsid protein); and (3) the hemagglutinin (the outer capsid protease-sensitive protein) are the major viral antigens. The properties and structure of these antigens are discussed. Further knowledge should facilitate the synthesis and development of novel antigen or subunit vaccines.

Animals↗
PubMed1985

Assignment of simian rotavirus SA11 temperature-sensitive mutant groups B and E to genome segments.

Recombinant (reassortant) viruses were selected from crosses between temperature-sensitive (ts) mutants of simian rotavirus SA11 and wild-type human rotavirus Wa. The double-stranded genome RNAs of the reassortants were examined by electrophoresis in Tris-glycine-buffered polyacrylamide gels and by dot hybridization with a cloned DNA probe for genome segment 2. Analysis of replacements of genome segments in the reassortants allowed construction of a map correlating genome segments providing functions interchangeable between SA11 and Wa. The reassortants revealed a functional correspondence in order of increasing electrophoretic mobility of genome segments. Analysis of the parental origin of genome segments in ts+ SA11/Wa reassortants derived from the crosses SA11 tsB(339) X Wa and SA11 tsE(1400) X Wa revealed that the group B lesion of tsB(339) was located on genome segment 3 and the group E lesion of tsE(1400) was on segment 8.

Animals↗