Biomedical subjects

M K Estes

Publications and source records attributed to M K Estes.

At least 127 records · Page 7Linked to original sources

PubMed1995

Effect of maternal rotavirus immunization on milk and serum antibody titers.

This prospective study evaluated human milk and serum antirotavirus antibody concentrations following maternal rotavirus immunization. Postpartum women (33) were randomized into 3 groups and received a single oral dose of rhesus rotavirus monovalent reassortant vaccine (10(4) pfu), tetravalent vaccine (10(4) pfu), or placebo. Milk (secretory [s] IgA) and serum (IgA and IgG) specimens were tested for antirotavirus isotype-specific antibody. Sera also were tested for G1- to G4-specific antibody. Prevaccine milk and serum isotype-specific antibody concentrations were not significantly different in the 3 groups. Postvaccine sIgA log titers were significantly greater in the 2 vaccine groups than the placebo group (P = .002). Mean log10 titers at 1 week were 2.1 (95% confidence interval [CI], 2.0-2.3) in the 2 vaccine groups and 1.7 (95% CI, 1.5-1.9) in the placebo group. Milk titers did not differ between vaccine groups. There was no difference in reactogenicity between groups. The significantly higher milk concentrations of antibody to rotavirus in postpartum women who received rotavirus immunization persisted for 4 months.

Adolescent↗

PubMed1995

Detection of Norwalk virus and hepatitis A virus in shellfish tissues with the PCR.

A method for the detection of Norwalk virus and hepatitis A virus from shellfish tissues by PCR was developed. Virus was added to the stomach and hepatopancreatic tissues of oysters or hard-shell clams, and viral nucleic acids were purified by a modification of a previously described method (R.L. Atmar, T.G. Metcalf, F.H. Neill, and M.K. Estes, Appl. Environ. Microbiol. 59:631-635, 1993). The new method had the following advantages compared with the previously described method: (i) more rapid sample processing; (ii) increased test sensitivity; (iii) decreased sample-associated interference with reverse transcription-PCR; and (iv) use of chloroform-butanol in place of the chlorofluorocarbon trichlorotrifluoroethane. In addition, internal standards for both Norwalk virus and hepatitis A virus were made which demonstrated when inhibitors to reverse transcription-PCR were present and allowed quantitation of the viral nucleic acids present in samples. This assay can be used to investigate shellfish-associated gastroenteritis outbreaks and to study factors involved in virus persistence in shellfish.

Animals↗

PubMed1995

Monoclonal antibodies for detection of Norwalk virus antigen in stools.

Monoclonal antibodies against the prototype 8FIIa strain of Norwalk virus were prepared and applied to an enzyme immunoassay (EIA) for detecting Norwalk virus in stool specimens. The monoclonal antibodies immunoprecipitated a 58-kDa protein which had been produced by in vitro transcription-translation of Norwalk virus cloned cDNA, and they reacted by EIA with recombinant Norwalk virus capsid protein at a sensitivity level of 1 ng/ml. The EIA detected virus in all tested samples from 15 different Norwalk virus-infected volunteers. No cross-reactions were seen in stools containing other caliciviruses or in stools containing rotaviruses, astroviruses, or enteric adenoviruses.

Antibodies, Monoclonal↗

PubMed1995

Specific proteolytic cleavage of recombinant Norwalk virus capsid protein.

Norwalk virus (NV) causes epidemic outbreaks of acute nonbacterial gastroenteritis in humans. The NV capsid is made up of a single protein, and expression of the capsid protein in baculovirus recombinants results in spontaneous assembly of the protein into virus-like particles (X. Jiang, M. Wang, D. Y. Graham, and M. K. Estes, J. Virol. 66:6527-6532, 1992). We have investigated whether the NV capsid protein undergoes a specific proteolytic cleavage. Recombinant NV (rNV) particles were digested with trypsin to determine if a specific cleavage occurred. A predominant band with a molecular weight of approximately 32,000 (32K protein) was observed when trypsin-treated rNV was electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Determination of the N-terminal sequence of this band showed that a trypsin-specific cleavage occurred at amino acid residue 227. Early studies identified two proteins with molecular weights of 59,000 and 30,000 (59K and 30K proteins) in the stool of NV-infected volunteers that were reactive with postinfection antiserum. (H. B. Greenberg, J. R. Valdesuso, A. R. Kalica, R. G. Wyatt, V. J. McAuliffe, A. Z. Kapikian, and R. M. Chanock, J. Virol. 37:994-999, 1981). We hypothesized that the 32K rNV cleavage product might be analogous to the 30K soluble protein detected in stools of NV-infected volunteers. Immunoprecipitation of soluble protein from these stool extracts with a rabbit polyclonal antiserum made against rNV, and Western blot detection with a mouse polyclonal antiserum made against rNV, revealed a single band with an apparent molecular weight of 30,000 that migrated similarly to the trypsin cleavage product observed in vitro. The N terminus of this band was identical to that of the 32K cleavage product of rNV capsid protein. These data show that the 30K protein in stool is produced by specific cleavage of the NV capsid protein in vivo. Trypsin cleavage of isolated soluble rNV 58K capsid protein and of assembled particles showed that only soluble 58K capsid protein is susceptible to cleavage. The presence of a large amount of soluble capsid protein may influence the immune response to or pathogenicity of NV infections.

Amino Acid Sequence↗

PubMed1995

The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum.

We previously reported that expression of rotavirus nonstructural glycoprotein NSP4 is responsible for an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in Spodoptera frugiperda (Sf9) insect cells (P. Tian, Y. Hu, W. P. Schilling, D. A. Lindsay, J. Eiden, and M. K. Estes, J. Virol. 68:251-257, 1994). The purpose of the present study was to determine the mechanism by which NSP4 causes an increase in [Ca2+]i by measuring the permeability of the cytoplasmic and endoplasmic reticulum (ER) membranes in recombinant-baculovirus-infected Sf9 cells. No obvious change in plasmalemma permeability to divalent cations was observed in cells expressing NSP4 compared with that in cells expressing another rotaviral glycoprotein (VP7) when the influx of Ba2+, a Ca2+ surrogate, was monitored. The basal Ca2+ permeability of the internal Ca2+ store was evaluated by measuring the release of Ca2+ induced by ionomycin, a Ca2+ ionophore, or thapsigargin, an inhibitor of the ER Ca(2+)-ATPase pump, following suspension of the cells in Ca(2+)-free extracellular buffer. Releasable Ca2+ decreased with time to a greater extent in cells expressing NSP4 compared with that in cells expressing VP7, suggesting that NSP4 increases the basal Ca2+ permeability of the ER membrane. To determine the possible mechanism by which NSP4 increases ER permeability, purified NSP4 protein or a 22-amino-acid synthetic peptide consisting of residues 114 to 135 (NSP4(114-135) was added exogenously to noninfected Sf9 cells during measurement of [Ca2+]i. Both NSP4 and the NSP4(114-135 peptide produced a time-dependent increase in [Ca2+]i that was attenuated by prior inhibition of phospholipase C with U-73122. Pretreatment of the cells with thapsigargin completely blocked the increase in [Ca2+]i produced by NSP4(114-135, but the peptide only partially reduced the change in [Ca2+]i produced by thapsigargin. No changes in [Ca2+]i were seen in cells treated with control peptides. These results suggest that (i) exogenous NSP4 increases [Ca2+]i through the activation of phospholipase C, (ii) Ca2+ release by exogenous NSP4 is from a store that is a subset of the thapsigargin-sensitive compartment, and (iii) amino acid residues 114 to 135 of NSP4 are sufficient for this activity. In contrast to exogenous NSP4, the mechanism by which endogenously expressed NSP4 increases [Ca2+]1 appears to be unrelated to phospholipase C, since no effect of U-73122 was seen on the elevated [Ca2+]1 in cells expressing NSP4 and exogenously applied NSP4(114-135) caused a further increase in [Ca2+]1 in cells expressing NSP4 protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1995

Environmental virology: from detection of virus in sewage and water by isolation to identification by molecular biology--a trip of over 50 years.

Environmental virology began with efforts to detect poliovirus in sewage and water more than 50 years ago. Since that time, cell-culture methods useful for detection of enteroviruses have been replaced by molecular biology techniques for detection of pathogens (hepatitis A and E viruses, caliciviruses, rotaviruses, and astroviruses) that do not grow in cell culture or grow with great difficulty. Amplification of viral nucleic acid using the polymerase chain reaction (PCR) is the current preferred method. PCR or RT-PCR (to detect RNA viral genomes) is rapid, sensitive, specific, and quantitative. Method shortcomings include potential inhibition by substances in some environmental samples and an inability of test results to distinguish between infectious and noninfectious virus. Current questions involving use of PCR/RT-PCR tests for public health purposes include: What is the public health significance of a positive test, and should direct tests for viruses replace current public health-monitoring programs?

DNA, Viral↗

PubMed1994

Characterization of rotavirus VP2 particles.

Rotavirus particles consist of three concentric proteinaceous capsid layers. The innermost capsid (core) is made of VP2. The genomic RNA and the two minor proteins VP1 and VP3 are encapsidated within this layer. Empty rVP2 particles are produced when insect cells are infected with a recombinant baculovirus which contains the bovine Rf rotavirus gene 2 (Labbé et al., 1991, J. Virol. 65, 2946-2952). Analysis of expressed rVP2 particles by SDS-PAGE showed these particles were composed of three major VP2-related proteins, called bands A, B, and C, with apparent molecular weights of 94K, 85K, and 77K, respectively. N-Terminal amino acid sequence analysis of each band showed that band A and band B were blocked, and band C lacked 92 amino acids from the N terminus. Bands B and C were predicted to also lack an approximately 10K peptide fragment from the C terminus. Electron microscopy (EM) showed negatively stained rVP2 particles to be spherical with icosahedral symmetry, 520 +/- 20 A in diameter. Highly concentrated rVP2 particles were converted to unusual forms, including elongated bristly structures, helix-like structures, and sheet-like helix structures. These unusual forms apparently resulted from a structural conversion of individual rVP2 particles. This conversion was reversible both in solution or on a collodion-carbon-coated grid support. The reconstituted rVP2 particles possessed normal morphology and reacted with purified VP6 to form rVP2/6 empty double-layered (previously called single-shelled) virus-like particles with an association constant Ka approximately 10(11) M-1. Native viral core particles lacking RNA were obtained by dialysis of full cores prepared from purified SA11-4F rotavirus double-layered particles against a hypotonic buffer in the presence of EDTA. EM showed both the full and empty native viral cores to be spherical with icosahedral symmetry. Highly concentrated SA11-4F full and empty cores also were converted into elongated and bead-like structures. However, in contrast to rVP2 particles, the conversion of SA11-4F cores was not reversible. These results provide some helpful clues to understanding VP2 functions, the assembly of VP2 particles, the assembly of VP2/6 double-layered particles, and the transport of metabolites inside and outside of the core particle.

Amino Acid Sequence↗

PubMed1994

Prevalence of antibodies to Norwalk virus among Amerindians in isolated Amazonian communities.

The seroepidemiology of Norwalk virus infections was examined among Amerindians belonging to eight relatively isolated communities in the Amazon region by means of a new enzyme immunoassay using recombinant Norwalk virus antigen. The seroprevalence of antibodies to Norwalk virus ranged from 39% in the Maiogong to 100% in the Kubenkrankrein. The distribution of antibody levels varied greatly among groups; five of the eight communities had an antibody prevalence greater than 90% with many high values (> 100 units), while three had both a low seroprevalence and a preponderance of low values (< 100 units). While few children less than 5 years of age were sampled, no significant differences in antibody prevalence were noted among age groups, and the prevalence of antibody among children 5-10 years of age approached that of the older age groups. The low prevalence of titers of antibodies to Norwalk virus in several tribes living in these isolated Indian communities suggests that Norwalk virus may have been only recently introduced.

Adolescent↗

PubMed1994

Seroprevalence studies using a recombinant Norwalk virus protein enzyme immunoassay.

A recombinant Norwalk virus (NV) protein enzyme immunoassay was used to study the age of acquisition of NV IgG in various populations. In London, England, there was little evidence of infection during the first 2 years of life. However, the prevalence of NV IgG rose steadily throughout the period that children attend school, reaching a peak of 70% in the group aged 11-16 years. High levels of maternal antibody were detected in infants aged < 3 months. Comparison of the acquisition of antibodies to three strains of human calicivirus in Japanese children in northern Japan indicated that although the majority had experienced infection with strains Japan and UK1 by the age of 12 years, only 22% possessed antibodies to NV. In Australian aborigines NV infection occurs early in life; by the age of 6 years over 90% of children were seropositive.

Adolescent↗

PubMed1994

Sequence similarity of human caliciviruses and small round structured viruses.

The application of reverse transcription-polymerase chain reaction (RT-PCR) using primers directed to the RNA dependent RNA polymerase region within ORF1 of Norwalk virus (NV) showed that 31 percent of morphologically typical human caliciviruses (HuCV) and 57% of small round structured viruses (SRSVs) produced a product of 470 bp similar to the NV control, NV 8FIIa/68/US. Alignment of the amino acid sequences of morphologically typical HuCVs with previously published sequences for SRSVs, NV, and Snow Mountain agent (SMA) showed a high degree of homology (90-92%) with SMA and a lesser extent of homology with NV (60-61%). The amino acid sequence of two strains of HuCV, HuCV/3C/92/UK, and HuCV/5C/92/UK differed by only one or two amino acids respectively in the RNA dependent RNA polymerase region from that of two strains of SRSV obtained from children in the United Kingdom, SRSV/4S/90/UK and Japan, SRSV/OTH-25/89/J which were found to have identical amino acid sequences. The use of an EIA for detection of NV antigen employing antisera raised to recombinant NV protein indicated that HuCVs and SRSVs obtained from children and adults in the United Kingdom were antigenically distinct from the prototype Norwalk virus, NV/8fIIa/68/US.

Adult↗

PubMed1994

Genomic diversity of small round structured viruses in the United Kingdom.

Fifty-two faecal specimens collected in the United Kingdom between 1986 and 1992, which contained small round structured virus (SRSV) particles, were tested by reverse transcriptase polymerase chain reaction assays using two primer pairs derived from sequences of Snow Mountain Agent and Norwalk virus. There was poor correlation between results obtained with each primer pair. Twenty specimens (38%) gave positive bands with SM51/31 primers and 18 (34%) were positive with SM52/32 primers, with a total of 30 specimens (57.7%) giving amplification products of the expected size with one or both primer pairs. Genomic variation was investigated by sequencing a 266 bp region of the RNA polymerase gene from nine strains which had been antigenically typed by solid phase immune electron microscopy (SPIEM). RNA sequence identities ranged from 53 to 99%. Three genomic groups were suggested by phylogenic analysis, the first of which contained Norwalk virus, Southampton virus, and strains typed by SPIEM as SRSV UK2. The second contained Snow Mountain agent and strains typed as either SRSV UK3 or UK4. The third contained strains typed as SRSV UK1 and strains untypeable by SPIEM. Some correlation was demonstrated when antigen typing by SPIEM and phylogenic grouping based on sequence data were compared.

Adult↗

PubMed1994

Molecular characterization and expression of the capsid protein of a Norwalk-like virus recovered from a Desert Shield troop with gastroenteritis.

Norwalk virus (NV) infection was recently found to be associated with gastroenteritis in U.S. military troops stationed in Saudi Arabia during the 1990 Desert Shield Operation. We identified a Norwalk-like virus in the stools of two military personnel with gastroenteritis by ELISA and IEM. By RT-PCR and sequence analysis, the nucleotide sequence of part of the polymerase region of each of these two "Desert Shield" strains (DSV275 and DSV395) was found to be 73% identical to the corresponding region of NV. In addition, one of the strains (DSV395), which underwent sequence analysis of approximately 2900 consecutive bases, had a genomic organization characteristic of the Caliciviridae. Comparison of the DSV395 amino acid sequence of the capsid region with that of three other viruses in the Norwalk group (Norwalk, Southampton, and Toronto viruses) showed amino acid identity of 47-68%. Consensus sequence analysis of these capsid proteins identified two regions of conserved amino acids that flanked an area of variable amino acids. In addition, the proteins corresponding to the capsid regions of DSV395 and NV were expressed in an in vitro translation system. Immunoprecipitation studies using the expressed capsid proteins and paired DSV395 or NV infection sera indicated the presence of shared antigenic sites between the capsid proteins of DSV395 and NV. However, hyperimmune sera specific for the self-assembled recombinant NV capsid protein did not react with DSV stool antigen in an ELISA, suggesting that there may also be unique antigenic sites not shared between DSV395 and NV.

Amino Acid Sequence↗

PubMed1994

Rotavirus vaccines and vaccination potential.

The development of a successful rotavirus vaccine is a complex problem. Our review of rotavirus vaccine development shows that many challenges remain, and priorities for future studies need to be established. For example, the evaluation of administration of a vaccine with OPV or breast milk might receive less emphasis until a vaccine is made that shows clear efficacy against all virus serotypes. Samples remaining from previous trials should be analyzed to determine epitope-specific serum and coproantibody responses to clarify why only some trials were successful. Detailed evaluation of the antigenic properties of the viruses circulating and causing illness in vaccinated children also should be performed for comparisons with the vaccine strains. In future trials, sample collection should include monitoring for asymptomatic infections and cellular immune responses should be analyzed. The diversity of rotavirus serotype distribution must be monitored before, during, and after a trial in the study population and placebo recipients must be matched carefully to vaccine recipients. Epidemiologic and molecular studies should be expanded to document, or disprove, the possibility of animal to human rotavirus transmission, because, if this occurs, vaccine protection may be more difficult in those areas of the world where cohabitation with animals occurs. We also need to have an accurate assessment of the rate of protection that follows natural infections. Is it realistic to try to achieve 90% protective efficacy with a vaccine if natural infections with these enteric pathogens only provide 60% or 70% protection? Subunit vaccines should be considered to be part of vaccine strategies, especially if maternal antibody interferes with the take of live vaccines. The constraints on development of new vaccines are not likely to come from molecular biology. The challenge remains whether the biology and immunology of rotavirus infections can be understood and exploited to permit effective vaccination. Recent advances in developing small animal models for evaluation of vaccine efficacy should facilitate future vaccine development and understanding of the protective immune response(s) (Ward et al. 1990b; Conner et al. 1993).

Animals↗

PubMed1994

Comparison of the polymerase region of small round structured virus strains previously classified in three antigenic types by solid-phase immune electron microscopy.

We have used a reverse transcription-polymerase chain reaction with nested sets of primers to determine the nucleotide sequences of a 166 base pair segment of the RNA polymerase region of seven strains of small round structured viruses (SRSVs) from the United Kingdom. These SRSV strains were previously classified by solid-phase immune electron microscopy into three antigenic types--UK2, UK3 and UK4, which are comparable to the prototype strains Norwalk virus, Hawaii agent, and Snow Mountain agents, respectively. Based on their sequences, the seven strains from the United Kingdom could be divided into two groups. The first group included two strains of the UK2 type along with Norwalk virus and Southampton virus and the second group included three strains of UK3 and two strains of UK4 types. Viruses in the first group showed 75.3%-77.1% nucleotide and 89.1%-94.6% amino acid identity with Norwalk virus while those of the second group showed 60.8%-63.3% nucleotide and 67.3%-69.1% amino acid identity. Nucleotide and amino acid identity within the second group ranged between 91.6%-99.4% and 96.4%-100%, respectively. These results suggest that the SRSVs antigenically related with Norwalk virus, Hawaii agent, and Snow Mountain agent, can be classified into two genotypes on the basis of their sequences in the RNA polymerase region.

Adult↗

PubMed1994

Identification of Norwalk virus in artificially seeded shellfish and selected foods.

A rotavirus dsRNA purification protocol was adapted to extract Norwalk ssRNA from artificially contaminated shellfish, and a sensitive reverse transcription-polymerase chain reaction assay for Norwalk virus was devised to identify an estimated 20-200 genomic copies. The technique includes deproteinization with guanidinium isothiocyanate, adsorption of RNA to hydroxyapatite, and sequential precipitation with cetyltrimethylammonium bromide and ethanol. The protocol allows high recovery of viral RNA free of enzymatic inhibitors from oysters, clams, and a variety of food matrices. Norwalk virus sequences were copied and amplified by using primers selected from the polymerase gene. Digestion of the amplified products with restriction enzymes ensured the specificity of the test. This rapid and sensitive assay may significantly improve the prospect for the routine screening of the uncultivatable Norwalk virus in food stuffs.

Animals↗

PubMed1994

Assessment of epitope-blocking assays for measuring antibody to rotavirus.

Criteria for determining the presence of antibody and of a response to infection in the epitope-blocking assay for anti-rotavirus antibody were evaluated using 222 sera from children younger than 30 months of age. The children were monitored for rotavirus diarrhea by means of daily symptom records and weekly stool specimen collection, whether or not symptoms occurred. Sera were collected at 6-month intervals. Forty-three serum pairs were collected before and after documented rotavirus infections. The remaining 136 sera were collected from children with no identified infections in the monitoring interval. Use of a 50% cutoff-point, as in prior reports, was too stringent a criterion for determining the presence of blocking antibody. The absolute percent blocking at the 1:10 serum dilution was a better measure of antibody content than end-point titration using the 50% cutoff-point.

Antibodies, Viral↗

PubMed1994

Anti-rotavirus G type-specific and isotype-specific antibodies in children with natural rotavirus infections.

Serum VP7 (G) type- and isotype-specific anti-rotavirus antibodies were assessed among children monitored longitudinally over one or two rotavirus seasons in day care centers. Seventy-five pairs of blood specimens from 63 children were tested for anti-rotavirus antibodies. Stool specimens were collected weekly and tested for rotavirus antigen. G typing of detected rotaviruses showed that seven outbreaks of G1 and one of G3 occurred during the two seasons. G type-specific responses to the outbreak strain occurred among 79% of infected children and 9% of children with infection not detected (P < .001). Of children infected with G1, 54% had a heterotypic response; they were older (P = .048) and had higher preexisting G1 antibody levels than children who had only homotypic responses (P = .012). Higher IgA, IgG, and homotypic antibody levels to the antigenic site C of the G1 and G3 VP7s correlated with protection against infection and illness, homotypic antibody independently of IgA or IgG titers.

Antibodies, Viral↗

PubMed1994

Detection of Norwalk virus or Norwalk-like virus infections in Finnish infants and young children.

Norwalk virus (NV) and Norwalk-like viruses are important causes of epidemic nonbacterial gastroenteritis in older children and adults. Serologic responses to NV of 154 Finnish infants and young children participating in a rotavirus vaccine study were examined by ELISA with a recently available baculovirus-expressed recombinant NV capsid protein. In 4 serially collected sera (at the median ages of 3, 4, 14, and 23 months), 49% of children had at least one NV infection over the approximately 2-year study period. Children with low NV-specific IgG titers (< 1:50) at the median age of 4 or 14 months were significantly more likely to acquire an NV infection by the median age of 14 or 23 months, respectively, than children who had higher NV IgG titers (> 1:50) (P < .05). Thus, NV or Norwalk-like virus infections are more common in infants and young children than previously believed, and antibody to NV may be protective against such infections.

Caliciviridae Infections↗