Biomedical subjects

M K Estes

Publications and source records attributed to M K Estes.

At least 37 records · Page 2Linked to original sources

PubMed2001

Identification and characterization of a transcription pause site in rotavirus.

In rotavirus, transcription of the 11 double-stranded RNA genome segments occurs within the structurally intact subviral particle, and nascent transcripts are released through channels penetrating the two capsid layers at the icosahedral vertices. To gain insight into the early molecular events in transcription, we used high-resolution polyacrylamide gel electrophoresis to investigate the length distribution of transcription products at various times following initiation. We observed that, in the subviral particle under normal conditions, transcript initiation and capping are followed by a momentary pause in elongation after the addition of 6 to 7 nucleotides. In the absence of the capping reaction cofactor S-adenosylmethionine, conditions under which the rate of nucleotide incorporation is reduced, we observe a significant decrease in the ratio of paused to full-length transcripts. We propose that this pause site may represent the point at which specific molecular events take place to facilitate processive elongation. Furthermore, our results indicate that the presence of specific ligands on the viral surface, such as VP7 in the mature virion, inhibits polymerase function. From the perspective of the viral replication cycle, this inhibition may serve to ensure that transcription occurs with greatest efficiency only after the virus has entered the cytoplasm and assumed the form of a double-layered particle.

Electrophoresis, Polyacrylamide Gel↗

PubMed2001

Microbes and microbial toxins: paradigms for microbial-mucosal interactions. VIII. Pathological consequences of rotavirus infection and its enterotoxin.

Rotaviral infection in neonatal animals and young children leads to acute self-limiting diarrhea, but infected adults are mainly asymptomatic. Recently, significant in-roads have been made into our understanding of this disease: both viral infection and virally manufactured nonstructural protein (NSP)4 evoke intracellular Ca(2+) ([Ca(2+)]i) mobilization in native and transformed gastrointestinal epithelial cells. In neonatal mouse pup mucosa models, [Ca(2+)]i elevation leads to age-dependent halide ion movement across the plasma membrane, transepithelial Cl(-) secretion, and, unlike many microbial enterotoxins, initial cyclic nucleotide independence to secretory diarrhea. Similarities between rotavirus infection and NSP4 function suggest that NSP4 is responsible for these enterotoxigenic effects. NSP4-mediated [Ca(2+)]i mobilization may further facilitate diarrhea by signaling through other Ca(2+)-sensitive cellular processes (cation channels, ion and solute transporters) to potentiate fluid secretion while curtailing fluid absorption. Apart from these direct actions in the mucosa at the onset of diarrhea, innate host-mediated defense mechanisms, triggered by either or both viral replication and NSP4-induced [Ca (2+)]i mobilization, sustain the diarrheal response. This secondary component appears to involve the enteric nervous system and may be cyclic nucleotide dependent. Both phases of diarrhea occur in the absence of significant inflammation. Thus age-dependent rotaviral disease represents an excellent experimental paradigm for understanding a noninflammatory diarrhea.

Animals↗

PubMed2000

Human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes.

A new approach for delivering vaccine antigens is the use of inexpensive, plentiful, plant-based oral vaccines. Norwalk virus capsid protein (NVCP), assembled into virus-like particles, was used as a test antigen, to determine whether immune responses could be generated in volunteers who ingested transgenic potatoes. Twenty-four healthy adult volunteers received 2 or 3 doses of transgenic potato (n=20) or 3 doses of wild-type potato (n=4). Each dose consisted of 150 g of raw, peeled, diced potato that contained 215-751 microgram of NVCP. Nineteen (95%) of 20 volunteers who ingested transgenic potatoes developed significant increases in the numbers of specific IgA antibody-secreting cells. Four (20%) of 20 volunteers developed specific serum IgG, and 6 (30%) of 20 volunteers developed specific stool IgA. Overall, 19 of 20 volunteers developed an immune response of some kind, although the level of serum antibody increases was modest.

Animals↗

PubMed2000

Seroprevalence and seroincidence of Norwalk-like virus infection among Brazilian infants and children.

To determine the importance of Norwalk-like viruses (NLVs) as pediatric pathogens in a developing country, the seroprevalence and seroincidence of this group of viruses in a cohort of children less than 4 years of age in an urban shantytown in northeastern Brazil was examined. Serum samples were collected approximately every 6 months from 135 children who were surveyed three times each week for diarrhea and vomiting. NLV IgG was measured by an enzyme immunosorbent assay (EIA) with recombinant Norwalk virus capsid protein. Overall NLV seroprevalence was 71%, and the overall NLV seroconversion rate was 0.7 seroconversions per child-year. The highest age-specific NLV seroconversion rate (0.8 seroconversions per child-year) was observed in the 13-24-month age group. For all study children, the incidence of diarrhea and vomiting was significantly greater (P < 0.01) during time periods spanned by serum pairs that indicated NLV seroconversion compared with time periods without NLV seroconversion. However, NLV seroconversion was not associated with gastrointestinal symptoms during the first year of life.

Brazil↗

PubMed2000

Species specificity and interspecies relatedness of NSP4 genetic groups by comparative NSP4 sequence analyses of animal rotaviruses.

Previous sequence analyses of the rotavirus nonstructural NSP4 from human and some animal rotavirus strains revealed the presence of three distinct NSP4 alleles or genetic groups. To examine the species of origin relatedness and diversity of NSP4, the nucleotide and deduced amino acid sequences of the gene encoding the NSP4 from 15 animal rotavirus strains of porcine, equine, bovine, lapine and canine origin were determined and compared to human and other animal strains sequenced previously. Lapine and equine strains were shown to belong to the NSP4 genotype A. Murine NSP4 sequences formed a previously unrecognized fourth distinct NSP4 genotype (genotype D) that was more divergent compared to NSP4 genotype A, B, and C than the latter three are among each other. Within NSP4 genotypes, strains isolated from rabbits, horses, cows (genotype A) and pigs (genotype B) clustered according to species of origin, suggesting a conserved pattern of evolution within species. NSP4 sequence comparison among one wildtype and two tissue culture-adapted lapine strains, known to cause disease in neonatal rabbits, failed to identify amino acid changes within the variable region spanning amino acids 130 to 141, suggesting that disease in rabbits is the result of the lapine virus infection and replication, including production of the NSP4 enterotoxin.

Amino Acid Sequence↗

PubMed2000

Mechanism of genome transcription in segmented dsRNA viruses.

Genome transcription is a critical stage in the life cycle of a virus, as this is the process by which the viral genetic information is presented to the host cell protein synthesis machinery for the production of the viral proteins needed for genome replication and progeny virion assembly. Viruses with dsRNA genomes face a particular challenge in that host cells do not produce proteins which can transcribe from a dsRNA template. Therefore, dsRNA viruses contain all of the necessary enzymatic machinery to synthesize complete mRNA transcripts within the core without the need for disassembly. Indeed one of the more striking observations about genome transcription in dsRNA viruses is that this process occurs efficiently only when the transcriptionally competent particle is fully intact. This observation suggests that all of the components of the TCP, including the viral genome, the transcription enzymes, and the viral capsid, function together to produce and release mRNA transcripts and that each component has a specific and critical role to play in promoting the efficiency of this process. This review has examined the process of genome transcription in dsRNA viruses from the perspective of rotavirus as a model system. However, despite numerous architectural and organizational differences among the families of dsRNA viruses, numerous studies suggest that the basic mechanism of mRNA production may be similar in most, if not all, viruses having dsRNA genomes. Important functional similarities include (1) the presence of a capsid-bound RNA-dependent RNA polymerase, which produces single-stranded mRNA transcripts from the dsRNA genome and regenerates the dsRNA genome from single-stranded RNA templates; (2) in viruses infecting eukaryotic hosts, the presence of all the enzymatic activities needed to generate the 5' cap required by the eukaryotic translation machinery; (3) the high degree of structural order present in the packaged genome, suggesting the requirement for organization in the viral core; (4) the role of the innermost capsid protein as a scaffold on which the core components of the transcription apparatus are assembled; and (5) the release of nascent mRNA transcripts through channels at the icosahedral vertices. The process of genome transcription in dsRNA viruses will become better understood as structural studies progress to higher resolution and as more viruses become amenable to study using site-directed mutagenesis coupled with viral reconstitution to generate recombinant particles having precise functional and structural changes. Future studies will dissect important intermolecular interactions required for efficient mRNA synthesis and will shed further light on the reasons for which the viral core must be structurally intact in order for transcription to occur efficiently. Structural studies of the capping enzymes at atomic resolution will reveal how multiple enzyme activities reside within a single polypeptide and how they act in concert to synthesize the 5' cap on the end of each mature transcript. Perhaps most interestingly, high resolution structural studies of actively transcribing virions will provide insight into the conformational changes that occur within the core during mRNA synthesis. Together, these studies will clarify the function of this complex macromolecular machine and will also shed additional light on the basic principles of virus architecture and assembly, as well as provide avenues for the design of antiviral therapies.

Animals↗

PubMed2000

Development of an immunomagnetic capture reverse transcription-PCR assay for the detection of Norwalk virus.

Norwalk virus (NV) is the prototype human virus of the family Caliciviridae. A rapid immunomagnetic capture/reverse transcription-(IMC/RT-)PCR assay was developed for the detection of NV. Immunomagnetic capture (IMC) utilizes paramagnetic beads coupled to a virus-specific antibody and allows separation of virus from contaminating materials and virus concentration in a single step. The detection limit of the developed assay was approximately 250-750 genomic equivalents/ml of 10% stool suspension. The detection limit of the assay was not altered by the presence of excess hepatitis A virus (HAV), although non-specific binding of HAV to the paramagnetic beads was observed. A panel of 100 stools from experimental human infections was screened for NV using a previously described heat release method, an antigen ELISA, or IMC/RT-PCR. NV was detected in 65/100 of these samples by IMC/RT-PCR compared to only 38/99 by heat release and 31/95 by antigen detection ELISA. All samples that were negative by IMC were also negative by both heat release and antigen ELISA. The number of samples in which RT-PCR was inhibited was greatly reduced by the use of IMC/RT-PCR compared to the heat release method (1/100 and 16/95 samples inhibited, respectively). The ability of IMC to concentrate virus (> or =2000-fold greater than heat release) and effectively remove inhibitory substances gives this assay distinct advantages over both the heat release and antigen ELISAs.

Caliciviridae Infections↗

PubMed2000

Structural studies of recombinant Norwalk capsids.

Norwalk virus is the major cause of epidemic viral gastroenteritis in humans. Attempts to grow this human 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. The x-ray crystallographic structure of these recombinant Norwalk particles has been determined to 3.4 A, using a 22-A electron cryomicroscopy structure as a phasing model. The recombinant capsids, 380 A in diameter, exhibit a T=3 icosahedral symmetry. The capsid is formed by 90 dimers of the capsid protein, each of which forms an arch-like capsomere. The capsid protein has two distinct domains-a shell (S) and a protruding (P) domain-that are connected by a flexible hinge. Although the S domain has a classical beta-sandwich fold, the structure of the P domain is unlike any other viral protein. One of the subdomains in the P domain formed by the most variable part of the sequence is located at the exterior of the capsid.

Capsid↗

PubMed2000

Diagnosis of human caliciviruses by use of enzyme immunoassays.

The application of molecular technologies, such as the expression of viral proteins in baculovirus, has provided a powerful approach to the diagnosis of human calicivirus (HuCV) infections. The baculovirus-expressed HuCV capsid protein self-assembles into virus-like particles, providing excellent reagents for immunologic assays, such as enzyme immunoassays (EIAs). Following the expression of the capsid protein of Norwalk virus, the capsid proteins of 8 other HuCV strains have been expressed in baculovirus. The unlimited supply of baculovirus-produced reagents for HuCVs allows these EIAs to be applied in large-scale clinical and epidemiological studies. Both the antigen and antibody-detection EIAs are highly sensitive. The antigen-detection EIAs are highly specific, but the antibody-detection EIAs are more broadly reactive. This article reviews baculovirus expression techniques used to produce HuCV capsid antigens, development of EIAs using these antigens, and application of these EIAs in studies of HuCV infection and illness.

Animals↗

PubMed2000

Norwalk virus vaccines: challenges and progress.

Human caliciviruses (HuCVs) are the major cause of outbreaks of acute nonbacterial gastroenteritis throughout the world. An increasing recognition of the clinical significance of these viruses as human pathogens causing foodborne and waterborne disease indicates that an effective vaccine would be useful. This article reviews the current challenges that exist for the development of a vaccine for the HuCVs as well as the status of development of a candidate vaccine. HuCVs are viruses that exhibit a restricted tropism for infection of the gastrointestinal tract of humans, and a volunteer model of infection and disease is available. As pathogens with a restricted host range, the HuCVs are excellent models for understanding the mechanisms that mediate and regulate viral infection of the gastrointestinal tract and mucosal immunity in humans.

Caliciviridae Infections↗

PubMed2000

Norwalk-like virus infection in military forces: epidemic potential, sporadic disease, and the future direction of prevention and control efforts.

The impact of Norwalk-like virus (NLV) infection on military forces is evaluated in this report. NLVs were a major cause of both outbreaks and sporadic disease among crowded US ground troops in the 1991 war with Iraq. NLVs also have been found to be a cause of acute gastroenteritis in other ground and shipboard deployments. Four large outbreaks of acute gastroenteritis were investigated aboard US Navy aircraft carriers between 1992 and 1997. In these outbreaks, NLVs were identified as the probable cause, and crowding was a major risk factor for transmission. An evaluation of a routine shipboard deployment also suggests that NLVs cause sporadic gastroenteritis. These data indicate that NLV infection is a major cause of acute morbidity in military forces. Because of the limitations of available prevention and control methods, development of a vaccine against these viruses may be the best solution in the military environment.

Caliciviridae Infections↗

PubMed2000

Taxonomy of the caliciviruses.

The International Committee on Taxonomy of Viruses (ICTV) has recently approved several proposals submitted by the present Caliciviridae Study Group. These proposals include the division of the family into 4 new genera designated Lagovirus, Vesivirus, "Norwalk-like viruses (NLVs), and "Sapporo-like viruses (SLVs); the latter 2 genera were assigned temporary names until acceptable names can be determined by the scientific community. The genera have been further divided into the following species: Feline calicivirus and Vesicular exanthema of swine virus (genus Vesivirus), Rabbit hemorrhagic disease virus and European brown hare syndrome virus (genus Lagovirus), Norwalk virus (genus NLV), and Sapporo virus (genus SLV). In addition, the ICTV approved a proposal to remove the hepatitis E virus from the Caliciviridae into an "unassigned classification status.

Animals↗

PubMed2000

Simian rhesus rotavirus is a unique heterologous (non-lapine) rotavirus strain capable of productive replication and horizontal transmission in rabbits.

Simian rhesus rotavirus (RRV) is the only identified heterologous (non-lapine) rotavirus strain capable of productive replication at a high inoculum dose of virus (>10(8) p.f.u.) in rabbits. To evaluate whether lower doses of RRV would productively infect rabbits and to obtain an estimate of the 50% infectious dose, rotavirus antibody-free rabbits were inoculated orally with RRV at inoculum doses of 10(3), 10(5) or 10(7) p.f.u. Based on faecal virus antigen or infectious virus shedding, RRV replication was observed with inoculum doses of 10(7) and 10(5) p.f.u., but not 10(3) p.f.u. Horizontal transmission of RRV to one of three mock-inoculated rabbits occurred 4-5 days after onset of virus antigen shedding in RRV-infected rabbits. Rabbits infected at 10(7) and 10(5), but not 10(3), p.f.u. of RRV developed rotavirus-specific immune responses and were completely (100%) protected from lapine ALA rotavirus challenge. These data confirm that RRV can replicate productively and spread horizontally in rabbits. In attempts to elucidate the genetic basis of the unusual replication efficacy of RRV in rabbits, the sequence of the gene encoding the lapine non-structural protein NSP1 was determined. Sequence analysis of the NSP1 of three lapine rotaviruses revealed a high degree of amino acid identity (85-88%) with RRV. Since RRV and lapine strains also share similar VP7s (96-97%) and VP4s (69-70%), RRV might replicate efficiently in rabbits because of the high relatedness of these three gene products, each implicated in host range restriction.

Amino Acid Sequence↗

PubMed2000

Development of methods to detect "Norwalk-like viruses" (NLVs) and hepatitis A virus in delicatessen foods: application to a food-borne NLV outbreak.

"Norwalk-like viruses" (NLVs) and hepatitis A virus (HAV) are the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for the detection of NLVs and HAV in foodstuffs. Although reverse transcription (RT)-PCR methods have been useful in detecting NLVs and HAV in bivalve mollusks implicated in outbreaks, to date such methods have not been available for other foods. To address this need, we developed a method to detect NLVs and HAV recovered from food samples. The method involves washing of food samples with a guanidinium-phenol-based reagent, extraction with chloroform, and precipitation in isopropanol. Recovered viral RNA is amplified with HAV- or NLV-specific primers in RT-PCRs, using a viral RNA internal standard control to identify potential sample inhibition. By this method, 10 to 100 PCR units (estimated to be equivalent to 10(2) to 10(3) viral genome copies) of HAV and Norwalk virus seeded onto ham, turkey, and roast beef were detected. The method was applied to food samples implicated in an NLV-associated outbreak at a university cafeteria. Sliced deli ham was positive for a genogroup II NLV as determined by using both polymerase- and capsid-specific primers and probes. Sequence analysis of the PCR-amplified capsid region of the genome indicated that the sequence was identical to the sequence from virus detected in the stools of ill students. The developed method is rapid, simple, and efficient.

Animals↗

PubMed2000

Identification of an epitope common to genogroup 1 "norwalk-like viruses".

A panel of 10 monoclonal antibodies (MAbs) to recombinant Norwalk virus (NV) capsid protein were tested in competition enzyme-linked immunosorbent assays. Patterns of competition indicated that these MAbs recognize six to eight epitopes covering five nonoverlapping regions of the capsid protein. A single epitope, recognized by NV MAbs NV3901, NV3912, and NV2461 was found to occur in the majority of genogroup 1 (G1) but not genogroup 2 (G2) "Norwalk-like viruses" (NLVs). This observation supports the subdivision of human NLVs into two genogroups and provides an assay for the rapid identification of G1 NLVs in fecal specimens.

Antibodies, Monoclonal↗

PubMed2000

Norwalk virus open reading frame 3 encodes a minor structural protein.

Norwalk virus (NV) is a causative agent of acute epidemic nonbacterial gastroenteritis in humans. The inability to cultivate NV has required the use of molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characterization of the NV ORF 3 protein expressed in a cell-free translation system and in insect cells and show its association with recombinant virus-like particles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbit reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3' end of the genome beginning with the ORF 2 coding sequences (ORFs 2+3), and the second recombinant contained ORF 3 alone. Expression from the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with ORF 3-specific peptide antisera. However, expression from a construct containing only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect-immunofluorescence studies using an ORF 3 peptide antiserum showed that the ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purified from the media of insect cells infected with a baculovirus recombinant containing the entire 3' end of the NV genome. Western blot analysis of NV purified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple higher-molecular-weight bands specifically recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.

Animals↗

PubMed2000

Intranasal administration of 2/6-rotavirus-like particles with mutant Escherichia coli heat-labile toxin (LT-R192G) induces antibody-secreting cell responses but not protective immunity in gnotobiotic pigs.

We investigated the immunogenicity of recombinant double-layered rotavirus-like particle (2/6-VLPs) vaccines derived from simian SA11 or human (VP6) Wa and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were administered to gnotobiotic pigs intranasally (i.n.) with a mutant Escherichia coli heat-labile toxin, LT-R192G (mLT), as mucosal adjuvant. Pigs were challenged with virulent Wa (P1A[8],G1) human rotavirus at postinoculation day (PID) 21 (two-dose VLP regimen) or 28 (three-dose VLP regimen). In vivo antigen-activated antibody-secreting cells (ASC) (effector B cells) and in vitro antigen-reactivated ASC (derived from memory B cells) from intestinal and systemic lymphoid tissues (duodenum, ileum, mesenteric lymph nodes [MLN], spleen, peripheral blood lymphocytes [PBL], and bone marrow lymphocytes) collected at selected times were quantitated by enzyme-linked immunospot assays. Rotavirus-specific immunoglobulin M (IgM), IgA, and IgG ASC and memory B-cell responses were detected by PID 21 or 28 in intestinal and systemic lymphoid tissues after i.n. inoculation with two or three doses of 2/6-VLPs with or without mLT. Greater mean numbers of virus-specific ASC and memory B cells in all tissues prechallenge were induced in pigs inoculated with two doses of SA11 2/6-VLPs plus mLT compared to SA11 2/6-VLPs without mLT. After challenge, anamnestic IgA and IgG ASC and memory B-cell responses were detected in intestinal lymphoid tissues of all VLP-inoculated groups, but serum virus-neutralizing antibody titers were not significantly enhanced compared to the challenged controls. Pigs inoculated with Wa-RF 2/6-VLPs (with or without mLT) developed higher anamnestic IgA and IgG ASC responses in ileum after challenge compared to pigs inoculated with SA11 2/6-VLPs (with or without mLT). Three doses of SA 11 2/6-VLP plus mLT induced the highest mean numbers of IgG memory B cells in MLN, spleen, and PBL among all groups postchallenge. However, no significant protection against diarrhea or virus shedding was evident in any of the 2/6-VLP (with or without mLT)-inoculated pigs after challenge with virulent Wa human rotavirus. These results indicate that 2/6-VLP vaccines are immunogenic in gnotobiotic pigs when inoculated i.n. and that the adjuvant mLT enhanced their immunogenicity. However, i.n. inoculation of gnotobiotic pigs with 2/6-VLPs did not confer protection against human rotavirus challenge.

Animals↗

PubMed2000

Direct inhibitory effect of rotavirus NSP4(114-135) peptide on the Na(+)-D-glucose symporter of rabbit intestinal brush border membrane.

The direct effect of a rotavirus nonstructural glycoprotein, NSP4, and certain related peptides on the sodium-coupled transport of D-glucose and of L-leucine was studied by using intestinal brush border membrane vesicles isolated from young rabbits. Kinetic analyses revealed that the NSP4(114-135) peptide, which causes diarrhea in young rodents, is a specific, fully noncompetitive inhibitor of the Na(+)-D-glucose symporter (SGLT1). This interaction involves three peptide-binding sites per carrier unit. In contrast, the Norwalk virus NV(464-483) and mNSP4(131K) peptides, neither of which causes diarrhea, both behave inertly. The NSP4(114-135) and NV(464-483) peptides inhibited Na(+)-L-leucine symport about equally and partially via a different transport mechanism, in that Na(+) behaves as a nonobligatory activator. The selective and strong inhibition caused by the NSP4(114-135) peptide on SGLT1 in vitro suggests that during rotavirus infection in vivo, NSP4 can be one effector directly causing SGLT1 inhibition. This effect, implying a concomitant inhibition of water reabsorption, is postulated to play a mechanistic role in the pathogenesis of rotavirus diarrhea.

Amino Acid Sequence↗