Biomedical subjects

M K Estes

Publications and source records attributed to M K Estes.

At least 91 records · Page 5Linked to original sources

PubMed1997

Study of Norwalk virus and Mexico virus infections at Ga-Rankuwa Hospital, Ga-Rankuwa, South Africa.

Small round-structured viruses (SRSVs) or Norwalk-like viruses have been implicated as an important causative agent of gastroenteritis outbreaks. We used the relatively newly developed recombinant enzyme immunoassays (EIAs) to determine the seroprevalence of Norwalk virus (NV) and Mexico virus (MxV) in a family-based cohort and an antenatal clinic cohort at Ga-Rankuwa Hospital, Ga-Rankuwa, South Africa. High prevalences (96 to 99%) of anti-NV and anti-MxV antibodies were detected in both cohorts. We also investigated the pattern of antibody acquisition in a cohort of infants and young children without gastroenteritis and found that by 48 months of age all children had acquired adult antibody levels to both these viruses. Lastly, we tested 276 stool specimens collected from infants and young children with gastroenteritis for the presence of NV or MxV antigen by recombinant EIAs to each virus, by electron microscopy (EM), and by reverse transcription (RT)-PCR. NV and MxV antigens were present in 1.8 and 4.3% of the stool specimens, respectively, by the recombinant EIAs; 9.2% were positive for SRSVs by EM, and 25% of these SRSVs gave a positive result by RT-PCR for primer pair 35-36 directed to a region of the RNA-dependent RNA polymerase gene. The seroprevalence studies indicate a high level of exposure to these viruses in both children and adults. Although the viral antigens are not highly prevalent in diarrheal stools, it was determined by the two assays for NV and MxV that children are, nevertheless, infected early in life.

Adolescent↗

PubMed1997

Three-dimensional structural analysis of recombinant rotavirus-like particles with intact and amino-terminal-deleted VP2: implications for the architecture of the VP2 capsid layer.

Rotaviruses are the leading cause of severe infantile gastroenteritis worldwide. These viruses are large, complex icosahedral particles consisting of three concentric capsid layers enclosing a genome of eleven segments of double-stranded RNA (dsRNA). The amino terminus of the innermost capsid protein VP2 possesses a nonspecific single-stranded RNA and dsRNA binding activity, and the amino terminus is also essential for the incorporation of the polymerase enzyme VP1 and guanylyltransferase VP3 into the core of the virion. Biochemical and structural studies have suggested that VP2, and especially the amino terminus, appears to act as a scaffold for proper assembly of the components of the viral core. To locate the amino terminus of VP2 within the core, we have used electron cryomicroscopy and image reconstruction to determine the three-dimensional structures of recombinant virus-like particles that contain either full-length or amino-terminal-deleted forms of VP2 coexpressed with the intermediate capsid protein VP6. A comparison of these structures indicates two significant changes along the inner surface of VP2 in the structure lacking the amino terminus: a loss of mass adjacent to the fivefold axes and a redistribution of mass along the fivefold axes. Examination of the VP2 layer suggests that the proteins are arranged as dimers of 120 quasi-equivalent molecules, with each dimer extending between neighboring fivefold axes. Our results indicate that the amino termini of both quasi-equivalent VP2 molecules are located near the icosahedral vertices.

Animals↗

PubMed1997

Biochemical characterization of a smaller form of recombinant Norwalk virus capsids assembled in insect cells.

The expression of the single capsid protein of Norwalk virus (NV) in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (VLPs) of two sizes, the predominant 38-nm, or virion-size VLPs, and smaller, 23-nm VLPs. Here we describe the purification and biochemical characterization of the 23-nm VLPs. The 23-nm VLPs were purified to 95% homogeneity from the medium of Sf9 cultures by isopycnic CsCl gradient centrifugation followed by rate-zonal centrifugation in sucrose gradients. The compositions of the purified 23- and 38-nm VLPs were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblots. VLPs of both sizes showed a doublet at 58 kDa, the size of the full-length capsid protein. Upon alkaline treatment, the 23-nm VLPs underwent dissociation into soluble intermediates that were able to reassemble into 23- and 38-nm VLPs upon dialysis, suggesting that the assembly of both types of structures has a common pathway. Antigenic and biochemical properties of the 38- and 23-nm VLPs were examined and found to be conserved. Immunoprecipitation assays using polyclonal and monoclonal antibodies indicated that immunodominant epitopes on the capsid protein as well as conformational epitopes are conserved in the two types of particles. The trypsin cleavage site at residue 227 was protected in the assembled particles of both sizes but exposed after alkaline dissociation. These results, and the conservation of the binding activity of both forms of recombinant NV VLPs to cultured cells (L. J. White, J. M. Ball, M. E. Hardy, T. N. Tanaka, N. Kitamoto, and M. K. Estes, J. Virol. 70:6589-6597, 1996), suggest that the tertiary folding of the capsid protein responsible for these properties is conserved in the two structures. We hypothesize that the 23-nm VLPs are formed when 60 units of the NV capsid protein assembles into a structure with T=1 symmetry.

Animals↗

PubMed1997

Rotavirus virus-like particles administered mucosally induce protective immunity.

We have evaluated the immunogenicity and protective efficacy of rotavirus subunit vaccines administered by mucosal routes. Virus-like particles (VLPs) produced by self-assembly of individual rotavirus structural proteins coexpressed by baculovirus recombinants in insect cells were the subunit vaccine tested. We first compared the immunogenicities and protective efficacies of VLPs containing VP2 and VP6 (2/6-VLPs) and G3 2/6/7-VLPs mixed with cholera toxin and administered by oral and intranasal routes in the adult mouse model of rotavirus infection. VLPs administered orally induced serum antibody and intestinal immunoglobulin A (IgA) and IgG. The highest oral dose (100 microg) of VLPs induced protection from rotavirus challenge (> or = 50% reduction in virus shedding) in 50% of the mice. VLPs administered intranasally induced higher serum and intestinal antibody responses than VLPs administered orally. All mice receiving VLPs intranasally were protected from challenge; no virus was shed after challenge. Since there was no difference in immunogenicity or protective efficacy between 2/6- and 2/6/7-VLPs, protection was achieved without inclusion of the neutralization antigens VP7 and VP4. We also tested the immunogenicities and protective efficacies of 2/6-VLPs administered intranasally without the addition of cholera toxin. 2/6-VLPs administered intranasally without cholera toxin induced lower serum and intestinal antibody titers than 2/6-VLPs administered with cholera toxin. The highest dose (100 microg) of 2/6-VLPs administered intranasally without cholera toxin resulted in a mean reduction in shedding of 38%. When cholera toxin was added, higher levels of protection were achieved with 10-fold less immunogen. VLPs administered mucosally offer a promising, safe, nonreplicating vaccine for rotavirus.

Adjuvants, Immunologic↗

PubMed1997

Prevalence of antibodies to human caliciviruses (HuCVs) in Kuwait established by ELISA using baculovirus-expressed capsid antigens representing two genogroups of HuCVs.

Baculovirus recombinant-expressed antigens of Norwalk viruses (rNV) and a Mexico strain (rMX) of the Snow Mountain serogroup of human caliciviruses (HuCVs) were used in enzyme immunoassays to study the antibody prevalence among the Kuwaiti population and foreign workers employed in Kuwait. The antibody titers in 16 different age groups which ranged from neonates to centenarians were investigated by testing eight different dilutions of each serum (1:200-1:25,600). The results indicate that NV infection is widespread in Kuwait and affects all age groups Ninety-eight percent of the 433 serum samples tested had antibodies to rNV. In the 50-79-year, old age group, the antibody levels to rNV were higher and significantly different from those in children 0-7 years old. In infants, the rNV antibodies did not diminish by 4 months of age and their titer steadily increased with age. When 414 of these sera samples were tested for antibodies to rMX, 96% positive serological responses were observed. Antibody titers to rMX were reduced in infants from 4 to 11 months; however, 95% of the samples were positive. These data indicate that children born in Kuwait are infected with Norwalk-like viruses at a very early age. Finally, antibodies to rNV and rMX were found in 98% of 151 and in 95% of 148 foreign workers, respectively.

Adolescent↗

PubMed1996

Rotavirus infection in infants as protection against subsequent infections.

BACKGROUND: Rotavirus is the leading cause of severe diarrhea in infants. To provide a base line for assessing the efficacy of rotavirus vaccines, we evaluated the protection that is conferred by natural rotavirus infection. METHODS: We monitored 200 Mexican infants from birth to two years of age by weekly home visits and stool collections. A physician assessed the severity of any episodes of diarrhea and collected additional stool specimens for testing by enzyme immunoassay and typing of strains. Serum collected during the first week of life and every four months thereafter was tested for antirotavirus IgA and IgG. RESULTS: A total of 316 rotavirus infections were detected on the basis of the fecal excretion of virus (56 percent) or a serologic response (77 percent), of which 52 percent were first and 48 percent repeated infections. Children with one, two, or three previous infections had progressively lower risks of both subsequent rotavirus infection (adjusted relative risk, 0.62, 0.40, and 0.34, respectively) and diarrhea (adjusted relative risk, 0.23, 0.17, and 0.08) than children who had no previous infections. No child had moderate-to-severe diarrhea after two infections, whether symptomatic or asymptomatic. Subsequent infections were significantly less severe than first infections (P=0.024), and second infections were more likely to be caused by another G type (P=0.054). CONCLUSION: In infants, natural rotavirus infection confers protection against subsequent infection. This protection increases with each new infection and reduces the severity of the diarrhea.

Antibodies, Bacterial↗

PubMed1996

Visualization of ordered genomic RNA and localization of transcriptional complexes in rotavirus.

In double-stranded-RNA (dsRNA) viruses found in animals, bacteria and yeast, the genome is transcribed within the structurally intact core of the virion with extraordinary efficiency. The structural organization of the genome and the enzymes involved in the transcription inside any of these viruses, critical for understanding this process, is not known. Here we report what we believe is the first three-dimensional characterization of the viral genome and the transcription complex in a prototypical dsRNA virus. Rotavirus is a large (diameter 1,000 A) icosahedral virus composed of three capsid protein layers and 11 dsRNA segments. It is the most important cause of gastroenteritis in children, accounting for over a million deaths annually. We show that viral dsRNA forms a dodecahedral structure in which the RNA double helices, interacting closely with the inner capsid layer, are packed around the enzyme complex located at the icosahedral 5-fold axes. The ordered RNA accounts for about 4,500 out of a total 18,525 base pairs in the genome, the largest amount of icosahedrally ordered RNA observed in any virus structure to date. We propose that the observed organization of the dsRNA is conducive for an orchestrated movement of the RNA relative to the enzyme complex during transcription.

Cell Line↗

PubMed1996

Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.

Alternatives to cell culture systems for production of recombinant proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the capsid protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The capsid protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The capsid protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed.

Administration, Oral↗

PubMed1996

Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein.

The rotavirus nonstructural glycoprotein NSP4 is an intracellular receptor that mediates the acquisition of a transient membrane envelope as subviral particles bud into the endoplasmic reticulum. NSP4 also causes an increase in intracellular calcium in insect cells. Purified NSP4 or a peptide corresponding to NSP4 residues 114 to 135 induced diarrhea in young (6 to 10 days old) CD1 mice. This disease response was age-dependent, dose-dependent, and specific. Electrophysiologic data from intestinal mucosa showed that the NSP4 114-135 peptide potentiates chloride secretion by a calcium-dependent signaling pathway. Diarrhea is induced when NSP4, acting as a viral enterotoxin, triggers a signal transduction pathway.

Aging↗

PubMed1996

Antigenic mapping of the recombinant Norwalk virus capsid protein using monoclonal antibodies.

Norwalk virus (NV) is the prototype strain of a group of noncultivatable caliciviruses that infect humans and cause outbreaks of epidemic acute nonbacterial gastroenteritis. The NV virion is composed of 180 copies of a single structural protein that, when expressed in insect cells infected with a recombinant baculovirus, assembles into empty recombinant Norwalk virus-like particles (rNV VLPs) which are morphologically and antigenically similar to native NV. We have begun to dissect the antigenic structure of the rNV particles using monoclonal antibodies made to the rNV VLPs. Ten MAbs made to rNV particles were characterized for their reactivity as detector antibodies by ELISA, as capture antibodies in an ELISA to detect NV in stools, by Western blot, and by immunoprecipitation. Seven of the MAbs recognize discontinuous epitopes, requiring the rNV capsid protein to remain at least partially folded, while the other three recognize continuous epitopes. Eight of the MAbs map to the C-terminal half of the capsid protein as they react by Western blot and by immunoprecipitation with a 32K trypsin cleavage product of the full-length 58K capsid protein, suggesting that the C-terminal half of the capsid protein may contain the immunodominant epitopes. The three MAbs that recognize continuous epitopes map to the extreme C terminus of the capsid protein, between amino acids 457 and 530, in a region that is relatively conserved among different human calicivirus capsid proteins. These MAbs which were assigned into three antigenic groups will be useful as tools to further dissect the structural and antigenic topography of the NV virion, and as unlimited reagents to detect NV in diagnostic assays.

Animals↗

PubMed1996

Rotavirus subunit vaccines.

We evaluated rotavirus subunit vaccines for use in humans and animals. Insect cells were co-infected with combinations of individual baculovirus recombinants expressing human, bovine or simian rotavirus VP2, VP4, VP6 or VP7 to produce virus-like particles (VLPs). To determine whether immunization with VLPs could induce active protective immunity, VLPs were administered parenterally to rabbits, and the immune response and protection from rabbit ALA rotavirus challenge were evaluated. Complete or partial protection was attained, showing that parenteral immunization with VLPs induces active protective immunity. We also examined whether heterotypic immune responses could be induced with a limited number of broadly reactive VP7 proteins or with chimeric particles (multiple VP7 types on individual particles). The feasibility of this approach was determined by immunizing mice with VLPs containing a G3 VP7 or G1 VP7 and chimeric G1/G3 VLPs. Broadly reactive neutralizing antibody was induced by the G1 VLPs. VLPs also have been successfully used to boost lactogenic (colostral and milk) immunity in dairy cows. Taken together, these results show that VLPs can be effective immunogens in rabbits, mice and dairy cattle when administered parenterally, a limited number of VLPs may be sufficient to produce a broadly protective vaccine, and G3 VLPs may serve as an effective subunit vaccine for use in bovines.

Animals↗

PubMed1996

Structure of Norwalk virus.

Norwalk virus is the major cause of epidemic viral gastroenteritis of humans. Attempts to grow this virus in laboratory cell lines have been unsuccessful. However, the Norwalk virus capsid protein, when expressed in insect cells infected with a recombinant baculovirus, spontaneously assembles into virus-like particles. We have determined the 3-dimensional structure of baculovirus-expressed Norwalk virus using electron cryomicroscopy and computer image reconstruction to a resolution of approximately 22 A. These particles, having a diameter of 380 A exhibit T = 3 icosahedral symmetry. The 3-dimensional structure is composed of 90 dimers of the 58000 molecular weight (58 K) capsid protein, each of which forms an arch-like capsomere. The structure of the protein subunit is modular with three distinct domains. The distal globular domain that appears bilobed is connected to the lower shell domain by a central stem-like domain. We also have been able to grow crystals of the baculovirus-expressed Norwalk virus particles suitable for high resolution X-ray crystallography.

Capsid↗

PubMed1996

Recombinant Norwalk virus-like particles as an oral vaccine.

Viruses that infect cells in the gastrointestinal tract are well suited for examining the immune response to oral delivery of antigen and for exploring the advantages and pitfalls of oral vaccines. Norwalk virus (NV) (family Caliciviridae, genus Calicivirus) causes acute gastroenteritis in all age groups. The NV capsid is composed of 180 copies of a single 58000 molecular weight protein which spontaneously forms virus-like particles (VLPs) that can be purified in extremely high yields (22 mg per 300 ml culture) when produced using the baculovirus expression system. We are testing the potential of these recombinant NV (rNV) particles for use as an oral vaccine by administering them to mice and volunteers. Mice were orally inoculated four times with rNV particles in concentrations ranging from 5 to 500 micrograms in the absence of adjuvant or from 5 to 200 micrograms with 10 micrograms of cholera toxin. Serum IgG and fecal IgA immune responses were monitored. rNV particles were found to be immunogenic when orally given to mice with or without adjuvant. These particles also were safe and immunogenic when orally given to volunteers. These studies show that rNV particles are an excellent model to test the oral delivery of mucosal immunogens in general, and that rNV particles are ideal candidates for vaccine development in particular.

Administration, Oral↗

PubMed1996

Genetic and antigenic diversity of human caliciviruses (HuCVs) using RT-PCR and new EIAs.

RT-PCR using primers from conserved regions of calicivirus genomes, followed by sequencing, permits characterization of genetic variation within the family. EIAs based on baculovirus-expressed viral capsid proteins and hyperimmune antisera against the capsid proteins were developed to detect HuCV antigens and antibodies. Serologic surveys using recombinant Norwalk virus (rNV) and recombinant Mexico virus (rMX, a SMA-like virus) EIAs showed that infections by HuCVs are common and that children acquire antibodies to HuCVs at an early age in both developed and developing countries. Three HuCV genogroups have been described that are represented by Norwalk virus (NV), Snow Mountain agent (SMA), and Sapporo virus, although recently accumulated sequences of HuCV strains indicate these genogroups can be further divided. These genogroups also correspond to distinct antigenic groups based on the results of the recombinant EIAs. The three genogroups co-circulate and have a worldwide distribution, although the SMA genogroup seems to be predominant currently. Application of these new assays for further characterization of the genetic and antigenic properties of HuCVs remains an important task for HuCV research.

Antibodies, Viral↗

PubMed1996

The epidemiology of human calicivirus/Sapporo/82/Japan.

Based on genome analysis of the RNA-dependent RNA polymerase region, it has been proposed that human caliciviruses (HuCV) can be classified into at least three genogroups: genogroup I is represented by Norwalk virus (NV), genogroup II by Snow Mountain agent (SMA) and genogroup III by HuCV/Sapporo/82/Japan (HuCV/Sa/82/J) virus. HuCV/Sa/82/J strain is genetically unique and more closely related to animal caliciviruses than are other known HuCVs, such as NV and SMA. HuCV/Sa/82/J strain was detected in four outbreaks of HuCV gastroenteritis occurring between 1977 and 1982 in an infant home in Sapporo. The HuCVs detected from these four outbreaks all showed a typical "Star of David" configuration by electron microscopy (EM), and they were identical antigenically and genetically. This strain has also been detected in other prefectures in Japan, as well as in the USA, UK, Saudi Arabia and Kenya. Seroepidemiological studies have shown a worldwide distribution of this virus, including Japan, USA, UK, Southeast Asia, Canada, China and Kenya. This virus has been circulating in Sapporo for at least 19 years (1977-1995). HuCV/Sa/82/J strain is thought to be one of the common causes of viral gastroenteritis worldwide. The HuCV/Sa/82/J strain has been detected mainly in infants. Age-related prevalence of antibody to this strain also shows that infections commonly occur in children less than 5 years old, although viruses in the NV and SMA genogroups commonly infect adults. The pattern of acquisition of antibodies to strain HuCV/Sa/82/J is similar to that of other common viral infections. HuCV/Sa/82/J strain is unique virologically and clinically among caliciviruses.

Age Factors↗

PubMed1996

Rotavirus structure: interactions between the structural proteins.

Structural studies on rotavirus using electron cryomicroscopy and computer image analysis have permitted visualization of each shell in the triple-layered rotavirus structure. Biochemical results have aided our interpretation of the structural organization of these layers and protein interactions seen in the three-dimensional structure, and have provided a better understanding of the structure-function relationships of the rotavirus structural proteins.

Capsid↗

PubMed1996

Identification of the minimal replicase and the minimal promoter of (-)-strand synthesis, functional in rotavirus RNA replication in vitro.

An in vitro replication system supporting the initiation and synthesis of complete rotavirus (-)-strands on (+)-strand template RNA (Chen et al., J Virol 68: 7030, 1994) was used to examine several parameters related to rotavirus RNA replication. Coexpression of VP1/2/3 in all possible combinations from baculovirus vectors revealed: [i] Virus-like particles (VLPs) were formed only if VP2 was present, and [ii] VP1/2 and VP1/2/3 VLPs had replicase activity in the in vitro system whereas VP2/3 and VP2 VLPs did not. Thus, the minimal replicase is composed of VP1 and VP2 and replicase activity is associated with VP1. In vitro replication reactions, using T7 transcripts of porcine rotavirus OSU genome segment 9 as reporter template, were performed to map cis-acting elements that regulate replication. Internal deletions and terminal truncations of the reporter RNA localized a replication signal, conferring full template activity, to the 5'-terminal 27 nucleotides (nt 1-27) and the 3'-terminal 26 nucleotides (nt 1037-1062). Further analysis showed that a minimal promoter of (-)-strand synthesis was contained in the 3'-terminal 7 nucleotides (nt 1056-1062); the sequence conserved at the 3'-terminus of all rotavirus genes. Hybrid constructs with this promoter had minimal, but detectable, template activity. This result indicated that upstream sequences between nucleotides 1037-1055 positively regulate the activity of the minimal promoter.

Animals↗

PubMed1996

Rotavirus protein expression is important for virus assembly and pathogenesis.

Rotaviruses have a unique morphogenesis in which particles obtain a transient membrane-envelope as newly made subviral particles bud into the endoplasmic reticulum (ER). This process is mediated by a viral nonstructural glycoprotein, NSP4. We have found that NSP4 has pleiotropic properties that became evident following expression of this protein in eukaryotic cells. NSP4 expressed in insect cells bound double-layered rotavirus particles in a manner similar to receptor-ligand interactions and this interaction is thought to trigger the particle budding process. Expression of NSP4 in insect cells also increases intracellular calcium ([Ca2+]i) levels and this effect may explain the toxicity of this protein in eukaryotic cells. Increases in [Ca2+]i levels in insect cells also are observed following exogenous addition to cells of purified NSP4 or of a synthetic peptide of NSP4. Experiments to determine the mechanism by which NSP4 causes an increase in [Ca2+]i showed that Ca2+ is released from a subset of the thapsigargin-sensitive store [endoplasmic reticulum (ER)]. However, exogenously added and endogenously expressed NSP4 use different mechanisms to alter the Ca2+ permeability of the ER membrane. We hypothesize that NSP4-mediated changes in ER membrane permeability trigger viral budding into the lumen of the ER, and eventually induce cell death and release of virus particles from infected cells. We also propose that release of NSP4 following cell lysis and the concomitant stimulation of a Ca2+ signal transduction pathway in neighboring cells contributes to altered ion transport in intestinal epithelium resulting in diarrheal disease.

Animals↗