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Biomedical subjects

M K Estes

Publications and source records attributed to M K Estes.

At least 163 records · Page 9Linked to original sources
PubMed1993

Fecal antibody responses to symptomatic and asymptomatic rotavirus infections.

The role of anti-rotavirus fecal IgA (RVflgA) in protecting children against natural rotavirus infections is unclear. Rotavirus outbreaks occurred in each of four day care centers attended by 129 children; 42% of the infections were asymptomatic. RVflgA titers were measured by EIA before infection and 4 weeks later in 50 children who excreted rotavirus (excretors) and in two samples 4 weeks apart from 50 children without detected virus excretion (nonexcretors). Forty-three (86%) excretors and 18 (36%) nonexcretors had a fourfold or greater RVflgA titer rise. Preexposure RVflgA titers were higher in not infected than symptomatic (P = .002), asymptomatic than symptomatic (P = .036), and not infected than asymptomatic children (P = .07). RVflgA titers after asymptomatic infections were slightly than after symptomatic infections (P = .087). In summary, higher RVflgA titers were associated with protection against infection and illness and increased fourfold or more in both asymptomatic and symptomatic children.

Age Factors↗
PubMed1993

Detection of enteric viruses in oysters by using the polymerase chain reaction.

A procedure for the detection of enteric viral nucleic acid in oysters by the polymerase chain reaction was developed. Known quantities of poliovirus type 1 were seeded into oysters. Virus was extracted and concentrated by using organic flocculation and polyethylene glycol precipitation. Inhibitors of reverse transcription-polymerase chain reaction were present in the oyster extracts, preventing amplification of target viral nucleic acid. The use of cetyltrimethylammonium bromide precipitation sufficiently removed inhibitors to allow detection of as few as 10 PFU of poliovirus. Norwalk virus also could be detected after being seeded into oysters. This methodology may be useful for the detection of these and other shellfish-borne viral pathogens.

Animals↗
PubMed1993

Detection of antibody to recombinant Norwalk virus antigen in specimens from outbreaks of gastroenteritis.

Norwalk virus and other small round-structured viruses are commonly associated with outbreaks of gastroenteritis. We used a recently described recombinant-expressed Norwalk virus (rNV) capsid protein in enzyme immunoassays to quantitatively measure immunoglobulin G (IgG) and IgA to Norwalk virus in serum pairs from patients involved in outbreaks of gastroenteritis. The outbreaks previously were classified, on the basis of the results of a blocking antibody assay, as Norwalk virus negative, serologically intermediate, or Norwalk virus positive. The rNV IgG assay was more sensitive than the blocking assay for detecting IgG to Norwalk virus in serum from patients in all outbreak classes. There was 79% concordance between seroconversions detected by the blocking antibody assay and those detected by the rNV IgG assay. The rNV IgA assay detected seroconversions to Norwalk virus primarily in patients involved in outbreaks previously classified as Norwalk virus positive.

Antibodies, Viral↗
PubMed1993

Prevalence of antibodies to Norwalk virus in England: detection by enzyme-linked immunosorbent assay using baculovirus-expressed Norwalk virus capsid antigen.

A total of 3,250 serum specimens collected in England in 1991 and 1992 were tested by an indirect enzyme-linked immunosorbent assay for antibody to Norwalk virus using baculovirus-expressed capsid antigen, and 2,382 (73.3%) were positive. The prevalence of Norwalk virus antibody differed regionally. It was lowest (24.6%) in 6- to 11-month-old infants and increased to 89.7% in persons over 60 years old.

Adolescent↗
PubMed1993

Subclass-specific serum antibody responses to recombinant Norwalk virus capsid antigen (rNV) in adults infected with Norwalk, Snow Mountain, or Hawaii virus.

Subclass-specific antibody responses to the Norwalk virus capsid protein in adults challenged with Norwalk, Snow Mountain, or Hawaii virus were evaluated by solid-phase enzyme immunoassay using recombinant Norwalk virus capsid antigen (rNV). Fourfold or greater serum immunoglobulin G (IgG) antibody responses to rNV were detected in 15 of 20 volunteers challenged with Norwalk virus, and serum IgA and IgM antibody responses to rNV were seen in almost all subjects who had rNV IgG responses. Serum rNV IgG antibody responses also were detected in 6 of 15 volunteers challenged with Snow Mountain virus and 2 of 12 volunteers challenged with the Hawaii virus. However, the magnitude of antibody response and the geometric mean postchallenge rNV IgG antibody titers were lower in subjects challenged with Snow Mountain or Hawaii virus, and serum IgA and IgM responses generally did not occur.

Adult↗
PubMed1993

Comparison of the reactivities of baculovirus-expressed recombinant Norwalk virus capsid antigen with those of the native Norwalk virus antigen in serologic assays and some epidemiologic observations.

Since the discovery of the Norwalk virus (NV) by immune electron microscopy (IEM) in 1972, serologic studies with this virus have relied on particle-positive fecal material from infected volunteers as the source of antigen because it has not been possible to propagate this virus in cell culture. However, the recent cloning of the NV (strain 8FIIa) genome and expression of the capsid protein in a baculovirus system to form "virus-like particles" has provided a consistent source of antigen (designated rNV). The purpose of the present study was to compare the antigenicities of these rNV particles with those of native NV antigen derived from human fecal material by using well-characterized sera obtained from earlier studies. In IEM studies, the rNV antigen reacted with NV-specific antibodies in a manner similar to that observed previously when particle-positive fecal material was used as antigen. In addition, a direct enzyme-linked immunosorbent assay, in which the rNV antigen was used as antigen, proved efficient and specific for the detection of serologic responses to NV compared with the previously established techniques of IEM and blocking antibody immunoassays in which particle-positive fecal material was used as the antigen. The availability of an unlimited source of antigen will enable serologic studies that will greatly increase our understanding of the epidemiology of NV and its role in human enteric illness.

Adult↗
PubMed1993

Rotavirus vaccine administered parenterally induces protective immunity.

We performed experiments to determine whether parenteral immunization with SA11 rotavirus can induce active protective immunity in a rabbit model of rotavirus infection. After one or two intramuscular injections of 1 ml of live or formalin-inactivated SA11 virus, we evaluated the mucosal and serologic immune response and protection from challenge with a high dose of live, virulent rabbit (Ala) rotavirus. Inactivated SA11 virus preparations, evaluated by enzyme-linked immunosorbent assay (ELISA) with a panel of VP4- and VP7-specific neutralizing and nonneutralizing monoclonal antibodies, did not show a loss of epitopes from the inactivation procedure compared with live virus. Administration of two doses of vaccine, one at zero days postvaccination (DPV) and a booster shot at 49 DPV, followed by challenge at 71 DPV with 3.5 x 10(5) PFU of Ala virus resulted in protection from challenge. None of the two-dose virus-vaccinated rabbits shed virus after challenge, while virus shedding was detected in all control rabbits (P = 0.001, Fisher's exact two-tailed test). Differences in total serum immunoglobulin (Ig) antirotavirus ELISA titers (P < 0.05, Wilcoxon's rank sum test) were observed between groups vaccinated with virus in aluminum phosphate or Freund's adjuvant but not between groups vaccinated with live or inactivated virus in either adjuvant. All rabbits given two doses of vaccine had detectable antirotavirus intestinal antibody of the IgG, but not IgA, isotype. After challenge, fourfold or greater increases in intestinal IgG antibody responses were observed in three rabbits, whereas all controls and all but one virus-vaccinated rabbit had an intestinal IgA antibody response. In contrast, vaccination of rabbits with one dose of SA11 followed by challenge at 21 DPV did not protect from challenge; no difference in the mean number of days of virus shedding between any of the vaccinated groups and controls was observed. A serologic, but not a mucosal, antibody response was observed after the one-dose vaccination regimen. Differences in serologic antibody titers were not observed between any of the one-dose virus-vaccinated groups. These data indicate that parenteral vaccination with two, but not one, doses of rotavirus in either Freund's adjuvant or aluminum phosphate can induce active protection from challenge.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

A truncated recombinant alpha subunit of Gi3 with a reduced affinity for beta gamma dimers and altered guanosine 5'-3-O-(thio)triphosphate binding.

The baculovirus-based expression system was adapted to express alpha subunits of the complete (alpha i3) and an amino-terminally truncated (alpha i3') form of Gi3 and of two complete forms of Gs (alpha s-L and alpha s-S). Subunits encoded in full length cDNAs were obtained with yields of 40-60 mg of recombinant protein/liter of cells, of which alpha i3 was between 30 and 50% soluble, but alpha s subunits were only 5-10% soluble. Only the complete alpha i3 was myristoylated. alpha i3 was purified in four steps. The purified protein bound 0.8-0.9 mol of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) per mol of protein and had one predominant contaminant which was identified as a truncated form that begins with methionine 18 instead of methionine 1. Both the full length alpha i3 and the truncated alpha i3' formed trimers with human erythrocyte beta gamma as seen by their migration in sucrose density gradients and by an increased rate of ADP ribosylation by pertussis toxin, but compared to alpha i3, alpha i3' interacted with beta gamma with a reduced affinity and dissociated upon warming. At 32 degrees C, only full length alpha i3 was ADP-ribosylated; at 4 degrees C, alpha i3 and alpha i3' were both ADP-ribosylated, with the truncated form requiring approximately 200-fold higher concentrations of beta gamma. A genetically engineered alpha i3' (alpha i3[18-354]) was also expressed in Sf9 cells. Yields, assessed as saturable GTP gamma S binding sites, were 3-5 mg per liter. Scatchard analysis showed that truncation of the amino terminus interferes with the ability of Mg2+ to promote high affinity binding of GTP gamma S. We conclude that the G protein alpha subunit amino terminus is not essential for interaction with beta gamma dimers, but rather is important in determining the affinity of the alpha subunit for both the beta gamma dimers and guanine nucleotide.

Amino Acid Sequence↗
PubMed1992

Studies on the interaction of alpha subunits of GTP-binding proteins with beta gamma dimers.

The interaction of several preparations of purified beta gamma dimers with two types of guanosine-nucleotide-binding-regulatory-(G)-protein alpha subunits, a recombinant bv alpha i3, made in Sf9 Spodoptera frugiperda cells by the baculovirus (bv) expression system, and alpha s, either purified from human erythrocyte Gs-type GTP-binding protein, and activated by NaF/AlCl3, or unpurified as found in a natural membrane, were studied. The beta gamma dimers used were from bovine rod outer segments (ROS), bovine brain, human erythrocytes (hRBC) and human placenta and contained distinct ratios of beta subunits that, upon electrophoresis, migrated as two bands with approximate M(r) of 35,000 and 36,000, as well as distinct complements of at least two gamma subunits each. When tested for their ability to recombine at submaximal concentrations with bv alpha i3, ROS, brain, hRBC and placental beta gamma dimers exhibited apparent affinities that were the same within a factor of two. When bovine brain, placental and ROS beta gamma dimers were tested for their ability to promote deactivation of Gs, brain and placental beta gamma dimers were equipotent and at least 10-fold more potent than that of ROS beta gamma dimers; likewise, brain beta gamma and placental dimers were equipotent in inhibiting GTP-activated and GTP-plus-isoproterenol-activated adenylyl cyclase, while ROS beta gamma dimers were less potent when assayed at the same concentration. The possibility that different alpha subunits may distinguish subsets of beta gamma dimers from a single cell was investigated by analyzing the beta gamma composition of three G proteins, Gs, Gi2 and Gi3, purified to near homogeneity from a single cell type, the human erythrocyte. No evidence for an alpha-subunit-specific difference in beta gamma composition was found. These findings suggests that, in most cells, alpha subunits interact indistinctly with a common pool of beta gamma dimers. However, since at least one beta gamma preparation (ROS) showed unique behavior, it is clear that there may be mechanisms by which some combinations of beta gamma dimers may exhibit selectivity for the alpha subunits they interact with.

Adenosine Diphosphate Ribose↗
PubMed1992

A bovine rotavirus serotype 1: serologic characterization of the virus and nucleotide sequence determination of the structural glycoprotein VP7 gene.

Bovine rotavirus T449 was isolated from feces of a calf with diarrhea. Serological characterization by serotype-specific monoclonal antibodies showed that the T449 virus belonged to serotype 1. This is the first report of a bovine rotavirus that does not belong to serotype 6, 8, or 10. The serotype 1 designation was confirmed by using an immunoperoxidase focus neutralization assay. The gene encoding the major neutralization antigen (VP7) was cloned and its nucleotide sequence was determined. The sequence obtained was 1062 bp in length and contained an open reading frame corresponding to 326 amino acid residues. Comparative analysis of the deduced amino acid sequence with the corresponding sequence of the human serotype 1 rotavirus strain, Wa, revealed a 90% identity. When compared to the predicted amino acid sequence of VP7 protein of the other serotypes an overall divergence of 20 to 25% was detected. These data show that the serological typing agrees with the result of the genetic analysis.

Amino Acid Sequence↗
PubMed1992

Rotavirus VP3 expressed in insect cells possesses guanylyltransferase activity.

We have examined the possible function(s) of the protein VP3 encoded by the rotavirus SA11 genomic segment 3. Viral-associated VP3 in double-shelled and single-shelled particles was shown to bind GTP covalently and reversibly. These properties are similar to the unique characteristics of eukaryotic and viral guanylyltransferases, suggesting that VP3 is associated with a capping enzyme activity. Previous studies have shown that intact viral particles are required for transcription, making it difficult to unequivocally identify the functions of individual proteins within such particles. Characterization of VP3 produced in the baculovirus expression system showed that the expressed VP3 covalently bound GTP. These studies suggest that VP3 alone is the guanylyltransferase. GTP binding also was seen in core virus-like particles and single-shelled virus-like particles that lacked viral nucleic acid and were assembled in insect cells.

Animals↗
PubMed1992

Characterization of an oligomerization domain and RNA-binding properties on rotavirus nonstructural protein NS34.

Intermolecular interactions between polypeptide chains often play essential roles in such biological phenomena as replication, transcription, translation, transport, ligand binding, and assembly. We have initiated studies of the functions of the rotavirus SA114F gene 7 product by sequence analysis and expression in insect cells. This nonstructural protein, NS34, is a slightly acidic protein, and its secondary structure is predicted to be 78% alpha-helix, with several heptad repeats of hydrophobic amino acids being present in its carboxy half. NS34 was found in oligomers when analyzed in insect cells, in SA11-infected MA104 cells, and in cell-free translation reactions. Investigation of the multiple electrophoretically distinct forms of NS34 showed they were all composed of homooligomers. Deletion mutants constructed and tested for oligomerization showed that the carboxy terminus of the protein, containing the predicted heptad repeats, was responsible for oligomerization. A basic region present in NS34 of group A rotaviruses, found to be 40% conserved in NS34 of group C rotavirus, is a candidate for a functional domain of this protein. NS34, which was found to be associated with the cytoskeleton fraction of cells, also interacts with viral RNA. These results make it likely that NS34 plays a central role in the replication and assembly of genomic RNA structures.

Amino Acid Sequence↗
PubMed1992

Extraintestinal rotavirus infections in children with immunodeficiency.

Some rotavirus strains, including vaccine candidates, have been demonstrated to cause hepatitis in immunodeficient and malnourished mice and to grow in human liver cells. To determine whether rotavirus spreads outside the intestine in naturally infected children, we examined tissues from four immunodeficient children affected with severe combined immunodeficiency disease, acquired immunodeficiency disease syndrome, or DiGeorge syndrome. Chronic rotavirus-related diarrhea, which persisted until death, had also developed in each child. Using indirect immunoperoxidase techniques, we identified rotavirus antigen in the liver and kidney with a hyperimmune guinea pig antiserum prepared to double-shelled rotavirus particles. Similar immunostaining with an antiserum to a rotavirus nonstructural protein (NS26) provided evidence of active virus replication. The observed reactivity was eliminated specifically when serial sections were immunostained with the same antiserum that had been absorbed with either double-shelled rotavirus particles or NS26. Immunostaining was not observed in the liver of children with other diseases (alpha 1-antitrypsin deficiency, inspissated bile syndrome, and acute rejection of a transplanted liver). These findings demonstrate that rotavirus infections in children can extend beyond the intestinal tract. Further studies are warranted to determine whether extraintestinal rotavirus replication occurs in children without severe immunodeficiency, such as malnourished children.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Description of an adenovirus type 8 outbreak in hospitalized neonates born prematurely.

An adenovirus (Ad) type 8 outbreak was prospectively studied in a neonatal intensive care unit. Nasopharyngeal secretions were cultured weekly for viruses and clinical information was obtained daily in each infant. Eleven of 112 neonates were infected with Ad; 8 of 9 available isolates represented one variant of Ad 8. The median age at infection was 54 days and the median duration of virus shedding was 2 days. Seven of 11 infants had onset of new symptoms and/or required acute respiratory support; only 2 infants had eye disease. Maternal characteristics, race, gender, age at entry into study and respiratory distress syndrome at birth were similar for both groups. Ad-infected infants tended to have earlier gestations and lower birth weights. Ad-infected neonates stayed longer in the neonatal intensive care unit, required more days of respiratory support and were more likely to develop bronchopulmonary dysplasia. Thus an association was established between lung disease in premature neonates and Ad infection.

Adenovirus Infections, Human↗
PubMed1992

Detection of Norwalk virus in stool by polymerase chain reaction.

A method of reverse transcription (RT) and polymerase chain reaction (PCR) for the detection of Norwalk virus in human stools was developed. A cationic detergent, cetyltrimethylammonium bromide, was found to effectively remove from stool extracts factors that inhibit the RT-PCR assay. The specificities of the tests were shown by hybridization of the amplified DNA with Norwalk virus-specific cDNA probes and a consistent correlation between virus detection in stools and infection of volunteers. RT-PCR detected virus in stool samples diluted 10(-4) and was about 100 times more sensitive than dot blot hybridization. In serial stool samples collected before and at different times after inoculation of 10 volunteers with Norwalk virus, 37 of 55 were positive by RT-PCR, but only 27 were positive by dot blot hybridization (chi 2 = 22.96; P less than 0.001). Further application of this method should allow detection of Norwalk virus in food or environmental samples such as shellfish and shellfish waters.

Adult↗
PubMed1992

Comparisons of rotavirus VP7-typing monoclonal antibodies by competition binding assay.

Three sets of neutralizing monoclonal antibodies (MAbs) used to type the outer capsid protein VP7 of four group A rotavirus serotypes (1 through 4) were compared in competition immunoassays. Reciprocal competition was observed for each of the VP7 type 2-, 3-, and 4-specific MAbs. The VP7 type 1 MAbs exhibited variable competition patterns with other VP7 type 1 MAbs. MAb RV4:3, which has been used to recognize antigenic variants within VP7 type 1 strains, showed reciprocal competition with the four VP7 type 3 MAbs (RV3:1, YO-1E2, 4F8, and 159) using a VP7 type 3 virus (SA11) as antigen. MAb 2C9, also prepared against VP7 type 1, reacted with VP7 type 3 strains and competed with a VP7 type 3 MAb, 159, using RRV as antigen. Use of the different sets of VP7 type-specific MAbs in the enzyme-linked immunosorbent assay permitted the recognition of six antigenic variants within VP7 types 1, 2, and 3 among specimens whose VP7 type could not be determined previously with only one set of typing MAbs. These results demonstrate differences of typing ability among these VP7-specific MAbs and emphasize the need to improve the sensitivity of typing systems by incorporating panels of MAbs reacting with several neutralizing epitopes.

Antibodies, Monoclonal↗
PubMed1992

Characterization of serum antibody responses to natural rotavirus infections in children by VP7-specific epitope-blocking assays.

Knowledge of the immune response to rotavirus is crucial for vaccine development. We compared an epitope-blocking assay (EBA) that uses VP7-specific monoclonal antibodies with neutralization assays (NAs) with polyclonal antisera for detecting serum antibody responses after natural rotavirus infection in children. Twenty-six serum pairs from children living in an orphanage with and without symptoms during two rotavirus outbreaks were evaluated for VP7 type 1-, 2-, 3-, and 4-specific antibody responses. In the first outbreak, which was caused by a VP7 type 3 strain, homotypic antibody responses were detected in 11 of 11 symptomatic children by NA and in 10 of 11 symptomatic children by EBA. Heterotypic antibody responses were detected more frequently (12 of 15 children) by NA than by EBA, and the heterotypic epitope-blocking antibody responses occurred in children older than 14 months of age. Antibody responses in asymptomatic children were more commonly detected by EBA than by NA. EBA results from the sera of children in the second outbreak indicated that it was caused by VP7 type 4, whereas NA results suggested it was caused by VP7 type 3. Our results confirm that EBA is a sensitive and specific method for determining VP7 type-specific immune responses after natural rotavirus infections.

Antibodies, Viral↗