Biomedical subjects

M K Estes

Publications and source records attributed to M K Estes.

At least 109 records · Page 6Linked to original sources

PubMed1996

Completion of the Norwalk virus genome sequence.

Norwalk virus (NV) is the prototype human calicivirus, and causes epidemic outbreaks of acute gastroenteritis. The sequence and predicted genome organization of NV and a NV-like virus [Southampton virus (SHV)] suggested they are similar viruses at the nucleotide and amino acid level, although SHV was reported to be antigenically distinct from NV. A recent review described the discovery of an additional 12 nucleotides at the 5' end of SHV and prompted us to investigate the possibility of additional nucleotides at the 5' end of the NV genome. The results obtained by homopolymeric tailing of NV cDNA with dCTP and dATP showed 12 additional nucleotides also are present on the NV genomic RNA. These data are important with respect to the biology of the virus, and make the genome sequence of NV complete.

Base Sequence↗

PubMed1996

Dot blot hybridization with a cDNA probe derived from the human calicivirus Sapporo 1982 strain.

A dot blot hybridization assay was developed for detection of human calicivirus/Sapporo/82/J (HuCV/Sa/82) or strains closely related to HuCV/Sa/82 in stool specimens. The cDNA derived from the RNA-dependent RNA polymerase (RDRP) region of HuCV/Sa/82 was used as a positive probe and the pBR322 DNA as a negative control probe. Both probes were labeled with digoxigenin and the products of hybridization reaction were detected with an anti-digoxigenin antibody-alkaline phosphatase conjugate. This assay was specific for HuCV/Sa/82 and for HuCV antigenically related to HuCV/Sa/82. The lower limit of sensitivity of this assay was estimated to be about 10(5) physical particles or 10 pg of cDNA, similar to that of the previously developed ELISA for HuCV. In 1273 stool specimens obtained from children with acute gastroenteritis in Sapporo, Japan, 110 (8.6%) contained small round structured viruses by EM and 23 (1.8%) were positive for HuCV antigenically related to HuCV/Sa/82 by either the hybridization assay or ELISA. A higher positive rate was obtained with the dot blot assay (21%) than by ELISA (10%), suggesting that the dot blot assay either detects HuCV more broadly than the ELISA or detects HuCV covered with fecal antibodies which interrupt antigen-antibody reactions in the ELISA. Negative results for detection of Norwalk virus (NV) cDNA and feline calicivirus (FCV) RNA by both this assay and the ELISA indicated that the HuCV/Sa/82 strain is distinct antigenically and genetically from NV and FCV.

Acute Disease↗

PubMed1996

Evaluation of a degenerate primer for the PCR detection of human caliciviruses.

Numerous outbreaks of gastroenteritis have been associated with Norwalk virus and Small Round Structured Viruses (SRSVs). These single-stranded RNA viruses, recently classified in the Caliciviridae, have been divided into three genogroups. Antigenic relationships also have been established among the different strains. As both an in vitro culture system and an animal model are lacking for these viruses, virus detection depends primarily on electron microscopy, immunological assays or molecular detection. In this study we first analyzed the genetic homology of the RNA polymerase region for 40 SRSV strains. From a consensus sequence for these strains, we designed a degenerate oligonucleotide to prime cDNA synthesis from viral RNA. We evaluated the degenerate primer in combination with three previously described primers in PCR reactions. A panel of 15 stools containing SRSVs, typed when possible by solid phase immune electron microscopy (SPIEM), were selected to represent all three genogroups and four different SPIEM antigenic types. Serial dilutions of the purified viral nucleic acids were amplified using the three different primer sets. Virus-specific probes were used to characterize the amplicons obtained. Virus-specific amplicons were obtained with at least one primer pair for each strain, but apparent viral RNA titers differed as much as 1000-fold between primer sets. Amplicons from all but one of the 15 strains were confirmed as virus-specific using a panel of 10 different probes. Correlations between the most sensitive primer pair and SPIEM type were seen. This study showed that a single degenerate primer could be used in cDNA synthesis for a variety of SRSVs but that the sensitivity of the RT-PCR assay depended upon the second primer and virus-specific probes used.

DNA Primers↗

PubMed1996

Isotype-specific antibody responses to rotavirus and virus proteins in cows inoculated with subunit vaccines composed of recombinant SA11 rotavirus core-like particles (CLP) or virus-like particles (VLP).

The isotype antibody responses to bovine IND P5, G6 and simian SA11 P2, G3 rotavirus and SA11 rotavirus proteins (VP4, VP6 and VP7) in serum, colostrum and milk were analysed by ELISA in three groups of vaccinated cows and nonvaccinated controls. Pregnant cows were vaccinated intramuscularly and intramammarily with recombinant baculovirus-expressed SA11 rotavirus VLP (triple-layered virus-like particles containing rotavirus VP2, VP4, VP6 and VP7); CLP (double-layered core-like particles containing rotavirus VP2 and VP6); or inactivated SA11 rotavirus, respectively. Rotavirus antigen titers were highest (30-200-fold) in ELISA in the VLP vaccine compared to the inactivated SA11 vaccine. The IgG1, IgG2 and IgM geometric mean antibody titers (GMT) to rotavirus (titers to bovine rotavirus vs SA11 rotavirus did not differ significantly for any isotype or group) and the IgG2 GMT to VP6 in serum at calving in the vaccinated groups were significantly (P < 0.05) higher than in the control group. In colostrum, IgG1 and IgA rotavirus antibody titers were significantly elevated for VLP (IgG1 GMT 832225; IgA GMT 16384), CLP (IgG1 GMT 660561; IgA GMT 10321) and SA11 (IgG1 GMT 131072; IgA GMT 1448) vaccinated cows compared to control cows (IgG1 GMT 11585; IgA GMT 45). The IgG1 and IgA GMT to rotavirus were significantly elevated (6-100-fold) in milk of VLP and CLP vaccinated cows compared to SA11 vaccinated or control cows. The isotype antibody responses to VP6 in serum, colostrum and milk paralleled the responses to rotavirus, but titers were approximately 2-10-fold lower. Only cows vaccinated with VLP had significantly enhanced serum, colostral and milk antibody titers to rotavirus VP4 and VP7. These results demonstrate that rotavirus antibody titers in serum, colostrum and milk are significantly enhanced by use of non-infectious VLP, CLP and inactivated SA11 rotavirus vaccines, but the VLP or CLP vaccines induced the highest antibody responses, corresponding to their higher rotavirus antigen titers measured by ELISA.

Animals↗

PubMed1996

Advances in molecular biology: impact on rotavirus vaccine development.

The first candidate rotavirus vaccine was a live attenuated oral vaccine made by the classical empirical method of serial passage of virus in tissue culture cells. Current tetravalent vaccine candidates that are in the final stages of efficacy testing in the United States were made by genetic reassortment. This article briefly highlights how advances in the basic understanding of the molecular biology of rotaviruses have facilitated vaccine development. New approaches for second-generation vaccines and improvements in vaccine efficacy based on further exploitation of the tools and knowledge of rotavirus molecular biology and pathogenesis are discussed.

Animals↗

PubMed1996

Virus-like particles as a rotavirus subunit vaccine.

Rotavirus subunit vaccines are being evaluated for use in humans. The virus-like particles (VLPs) for these vaccines are produced in insect cells coinfected with combinations of baculovirus recombinants expressing bovine RIF VP2 and simian SA11, VP4, VP6, or VP7 rotavirus proteins. VLPs were administered parenterally to mice and rabbits, and the immunogenicity and protective efficacy of the vaccines were evaluated. Rabbits vaccinated with VP2/4/6/7 or VP2/6/7 VLP combinations developed high levels of rotavirus-specific serum antibody and fecal IgG but not fecal IgA. The induction of fecal IgG was associated with total or partial protection from oral challenge with ALA rotavirus. Heterotypic serum and fecal neutralizing antibody was induced in mice vaccinated parenterally with G1 VP2/6/7 or VP2/4/6n VLPs. VLPs were highly immunogenic when administered in QS21 adjuvant, inducing serum neutralizing antibody titers comparable to those induced by SA11 virus. VLPs are effective immunogens when administered parenterally and may be an effective subunit vaccine.

Animals↗

PubMed1996

Collaborative evaluation of a method for the detection of Norwalk virus in shellfish tissues by PCR.

A multicenter, collaborative trial was performed to evaluate the reliability and reproducibility of a previously described method for the detection of Norwalk virus in shellfish tissues with the PCR (R.L. Atmar, F. H. Neill, J. L. Romalde, F. Le Guyader, C. M. Woodley, T. G. Metcalf, and M. K. Estes, Appl. Environ. Microbiol. 61:3014-3018, 1995). Virus was added to the stomachs and hepatopancreatic tissues of oysters or hard-shell clams in the control laboratory, the samples were shipped to the participating laboratories, and viral nucleic acids were extracted and then detected by reverse transcription-PCR. The sensitivity and specificity of the assay were 85 and 91%, respectively, when results were determined by visual inspection of ethidium bromide-stained agarose gels; the test sensitivity and specificity improved to 87 and 100%, respectively, after confirmation by hybridization with a digoxigenin-labeled, virus-specific probe. We have demonstrated that this method can be implemented successfully by several laboratories to detect Norwalk virus in shellfish tissues.

Animals↗

PubMed1996

Detection and analysis of a small round-structured virus strain in oysters implicated in an outbreak of acute gastroenteritis.

Outbreaks of shellfish-transmitted viral disease occur periodically, but frequently the causative agent is not identified. In November 1993, during investigation of a multistate outbreak of acute gastroenteritis, incriminated lots of oysters were collected. Oyster tissues (stomachs and digestive diverticula) were processed for virus extraction and nucleic acid purification. Human calicivirus sequences were sought by reverse transcriptase PCR using different primer sets. Amplicons were obtained from 9 of 10 shellfish samples from four different lots when primers specific for the outbreak virus strain were used. The specificity of the amplification was confirmed by hybridization. The amplicons from the nine positive oysters were cloned and sequenced. The sequence of each of the clones was identical to the others but showed some variation (7 of 81 bp) from the sequences obtained from the stools of three persons made III by the outbreak.

Animals↗

PubMed1996

Isolation of group B porcine rotavirus in cell culture.

While group A and C rotaviruses have been grown in cell culture, group B rotavirus has never been cultured. In this study we successfully isolated porcine group B rotavirus in swine kidney cells. Pancreatin treatment is essential for the propagation of group B rotavirus.

Animals↗

PubMed1996

Attachment and entry of recombinant Norwalk virus capsids to cultured human and animal cell lines.

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant Norwalk virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. We have used these rNV VLPs to examine virus-cell interactions. Binding and internalization of VLPs to cultured human and animal cell lines were studied in an attempt to identify potentially susceptible cell lines for virus propagation in vitro and to determine if early events in the replication cycle were responsible for the narrow host range and restriction of virus growth in cell culture. Radiolabeled VLPs specifically bound to a saturable number of binding molecules on the cell surface of 13 cell lines from different origins, including human intestine (differentiated and undifferentiated Caco-2) and insect (Spodoptera frugiperda 9) ovary. Differentiated Caco-2 cells bound significantly more rNV VLPs than the other cell lines. Variations in the amount of bound VLPs among the different cell lines did not correlate with the tissue or species of origin. VLP binding was specific, as determined by competition experiments with unlabeled rNV VLPs; however, only 1.4 to 6.8% of the specifically prebound radiolabeled VLPs became internalized into cells. Blocking experiments using polygonal and monoclonal anti-rNV sera and specific antipeptide sera were performed to map the domains on rNV VLPs involved in binding to cells. One monoclonal antibody (NV8812) blocked binding of rNV VLPs to human and animal cell lines. The binding site of monoclonal antibody NV8812 was localized to the C-terminal 300 to 384 residues of the capsid protein by immunoprecipitation with truncated and cleaved forms of the capsid protein. These data suggest that the C-terminal region of the capsid protein is involved in specific binding of rNV VLPs to cells.

Animals↗

PubMed1996

The rotavirus nonstructural glycoprotein NSP4 possesses membrane destabilization activity.

During a unique morphogenetic process, rotaviruses obtain a transient membrane envelope when newly synthesized subviral particles bud into the endoplasmic reticulum (ER). As rotavirus particles mature, they lose their transient membrane and a layer of the glycoprotein VP7 forms the virion outer capsid shell. The nonstructural glycoprotein NSP4 functions as an intracellular receptor in the ER membrane (K. S. Au, W. K. Chan, J. W. Burns, and M. K. Estes, J. Virol. 63:4553-4562, 1989), and it has been hypothesized that NSP4 is involved in the removal of the envelope during viral morphogenesis (M. K. Estes and J. Cohen, Microbiol. Rev. 53:410-449, 1989; B. L. Petrie, M. K. Estes, and D. Y. Graham, J. Virol. 46:270-274, 1983). The purpose of the present study was to determine if NSP4 has a direct membrane destabilization activity (MDA) by using liposome leakage assays and electron microscopic visualization of liposome, microsome, and viral envelope disruption. The fluorescent marker (calcein) incorporated into liposomes was released when the liposomes were incubated with purified NSP4. A region corresponding to amino acid residues 114 to 135 of NSP4 also released calcein from liposomes. NSP4(114-135) peptide-specific antibody completely blocked the MDA of the purified NSP4 protein. These results suggest that this region contains at least part of the functional domain of NSP4. Liposomes composed of phosphatidylcholine and microsomes (to simulate ER membranes) were broken when observed by electron microscopy after incubation with NSP4 or the NSP4(114-135) peptide. In contrast, the envelope of Sendai virus, which is derived from cytoplasmic membranes, and erythrocytes were not disrupted by NSP4 and the NSP4(114-135) peptide. These results provide direct evidence that NSP4 possesses MDA and suggest that it can cause ER membrane damage. Therefore, NSP4 might play an important role in the removal of the transient envelope from budding particles during viral morphogenesis. A model for the MDA of NSP4 in viral morphogenesis is proposed.

Amino Acid Sequence↗

PubMed1996

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants.

Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negative-stain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VPI/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VPl, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 single-layered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.

Animals↗

PubMed1995

Epidemiology of Norwalk virus during an outbreak of acute gastroenteritis aboard a US aircraft carrier.

A large outbreak of acute gastroenteritis occurred over a 5-week period aboard an aircraft carrier. The estimated cumulative attack rate was 13% among the 4,500-man crew. Eight percent of the crew sought medical attention, nearly all of whom missed 1 day or more of work. The risk of developing illness was 2 to 3 times greater for individuals living in more crowded sleeping quarters (> 50 persons per compartment). Occurrence of gastroenteritis was associated with a fourfold or more rise in Norwalk virus antibody levels, as measured by an enzyme-linked immunoassay utilizing a baculovirus expressed recombinant antigen. In addition, 27 nm Norwalk virus-like particles were visualized in two of six stools examined by immune electron microscopy. The presence of a low (< 1:50) or a high (> or = 1:6,400) pre-illness antibody level was associated with a lower incidence of illness. This investigation indicates that Norwalk virus can adversely impact operations of a military vessel and that crowding is a major risk factor in transmission.

Acute Disease↗

PubMed1995

Molecular characterization of a human calicivirus with sequence relationships closer to animal caliciviruses than other known human caliciviruses.

cDNA clones were produced from a morphologically typical human calicivirus (HuCV) in stool specimens collected in 1982 during an outbreak of gastroenteritis in Sapporo, Japan. The cDNA clones were generated separately in two laboratories by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers 35 and 36 derived from Norwalk virus. The RT-PCR product from six specimens was of the predicted size, had a continuous protein encoding frame on the positive strand, and contained GLPS and YGDD amino acid motifs at the predicted distance from the primers. RT-PCR amplification with primer 35 and a HuCV/Sapporo-specific primer 36 of four HuCV/Sapporo-positive stool specimens from a 1986 Houston day care center outbreak yielded products with 93% nucleotide and 99% predicted amino acid sequence identity with the HuCV/Sapporo strain from the 1982 outbreak. The HuCV/Sapporo strains are genetically distinct from previously characterized HuCVs and more closely related to known animal CVs than other known HuCVs.

Animals↗

PubMed1995

Antibody prevalence and immunoglobulin IgG subclass pattern to Norwalk virus in Sweden.

The antibody prevalence in Sweden to Norwalk virus (NV) was investigated using a baculovirus expressed capsid antigen. One hundred thirty-two serum samples were examined for IgA, total IgG and IgG subclass antibodies to Norwalk virus. In young children, NV IgG antibody prevalence was higher than the IgA prevalence, whereas no difference was found in individuals older than 21 years. The IgG antibody prevalence was 50% in children below 5 years of age and increased to > 80% in individuals older than 10 years of age. To examine the IgG antibody response in more detail, IgG subclass patterns were characterized. IgG 1 predominated in all age groups. IgG 4, usually detected after repeated exposure to antigen, was the second most prevalent subclass, but was only found in individuals older than 21 years of age. IgG 3 subclass antibodies were found in 13% and IgG 2 in 3% of the sera examined. IgG 3 subclass antibodies have been recognized as a marker for recent or ongoing viral infections.

Adolescent↗

PubMed1995

Characterization of SRSVs using RT-PCR and a new antigen ELISA.

Stool samples from 451 patients involved in volunteer studies, 26 outbreaks and approximately 175 sporadic cases of acute gastroenteritis from different geographical locations in the world were tested for Norwalk virus (NV) using a newly developed antigen ELISA and RT-PCR. NV was detected in most outbreaks previously characterized as being of NV origin. Overall, a low number of positives for NV was obtained using either RT-PCR with primers that amplified a unique region of the genome, or an ELISA with hyperimmune antisera made to the baculovirus-expressed recombinant NV capsid. However, a significant number of positives was obtained when these samples were tested by RT-PCR using primers that amplified the more highly conserved regions of the genome. Sequence analysis of the amplified viral cDNAs indicated that small round structured viruses (SRSVs) with a wide range of variable genomic sequences (44-87% nucleotide and 31-99% amino acid similarity to the 8Flla NV genome sequence) were responsible for these outbreaks. Several recent outbreaks from the US, Japan and the UK were related to the Snow Mountain Agent (SMA) by sequence analyses. Continued accumulation of sequence information will facilitate the design of new primers for virus detection and increase our understanding of the relationships and epidemiology of these viruses from different sources.

Amino Acid Sequence↗

PubMed1995

Group A rotavirus G type prevalence in two regions of Hungary.

Rotaviruses are a major cause of gastroenteritis in children world-wide. Rotaviruses are antigenically complex, with multiple serotypes (G types). The first longitudinal study of group A rotavirus serotype (G type) distribution in Hungary is reported. Neutralizing monoclonal antibodies specific for G1, G2, G3, and G4 were used in an enzyme immunoassay to determine the antigenic variation of group A rotaviruses in two collections of stool specimens assembled from 1984-1992 in Baranya County, southwest Hungary, and from 1988-1992 at the Central Hospital for Infectious Diseases in Budapest. Ninety-two percent of the 1215 virus-positive samples were typed as follows: G1 (81%), G2 (4%), G3 (1%), G4 (5%), or mixed type (1%). G1 was the predominant type during the entire study period with the exception of the 1988/1989 rotavirus season in Baranya County when G4 predominated. Among G1 strains, different electropherotypes were detected with a shift of the predominant G1 electropherotype(s) each 2 to 3 years. G typing from two longitudinal collections established regional differences within Hungary in the prevalence of rotavirus antigenic types among children with rotavirus-associated diarrhea. These are the first longitudinal rotavirus typing results for Hungary and Central Europe.

Antibodies, Monoclonal↗

PubMed1995

Viral shedding and fecal IgA response after Norwalk virus infection.

Protection is not conferred by preexposure to Norwalk virus (NV). By use of an ELISA with baculovirus-expressed recombinant NV (rNV) capsid protein, the pattern of NV fecal shedding and the protective effect of rNV-specific fecal IgA (flgA) were investigated in volunteers who were repeatedly challenged with NV. After the first challenge, ill volunteers were significantly more likely than well volunteers to have NV antigen in their stool (P < .05). After challenge, antigen shedding was detected on days 1-13; ill volunteers shed the antigen longer (P = .02). A higher prechallenge rNV-specific flgA geometric mean titer was found in ill compared with well volunteers (P < .05) and in infected versus noninfected volunteers (P < .05). NV shedding was common after infection and was present up to 2 weeks after challenge. Preexisting rNV-specific flgA, like serum IgG, is not protective and may be a marker for symptomatic disease.

Antibodies, Viral↗