Biomedical subjects

M K Estes

Publications and source records attributed to M K Estes.

At least 145 records · Page 8Linked to original sources

PubMed1994

Norwalk virus infection of volunteers: new insights based on improved assays.

Norwalk virus infection is a common cause of gastroenteritis in humans. The clinical features and virologic and immunologic responses following oral administration of Norwalk virus to 50 volunteers were monitored. New ELISAs using recombinant virus particles as the antigen source were used to assess the pattern of virus shedding and the specific immune responses. Forty-one subjects (82%) became infected; 68% were symptomatic and 32% were asymptomatic. The proportion of subjects infected was similar for those with and without preexisting antibody (82% vs. 60%; P > .2). The magnitude of seroconversion was highest in subjects who had vomiting. The peak of viral shedding was between 25 and 72 h, and virus first appeared in stool at 15 h. Specimens collected 7 days after inoculation remained positive. These results show a higher infection rate, more subclinical infections, and longer virus excretion following Norwalk virus inoculation than previously recognized.

Adult↗

PubMed1994

Acquisition of serum isotype-specific and G type-specific antirotavirus antibodies among children in day care centers.

The acquisition of serum antirotavirus antibodies among children in day care centers was monitored through two rotavirus seasons. Twenty-six children were monitored daily for diarrhea and weekly for stool rotavirus excretion through a rotavirus season of infections with serotype G1 and a successive season of infections with both G1 and G3. Sera were collected before and after each rotavirus season and tested for antirotavirus IgA and IgG and for G type-specific blocking antibody. The prevalence of protective serum IgA and IgG titers increased from 36% and 45% before Season 1 to 77% and 96% after Season 2, respectively (P < 0.02 and 0.001). G type-specific antibodies also increased (G1, P < 0.001; G2, P = 0.005; G3, P = 0.003; G4, P = 0.006), including for noncirculating types. Homotypic and heterotypic antibodies increased as the number of rotavirus infections experienced by a child increased. The group of children with two proven infections developed protective isotype-specific and G type-specific antibodies. These results indicate that in first exposures to rotavirus G types, children develop predominantly homotypic antibody. However, as the number of rotavirus infections increase, children develop heterotypic antibody to G types at levels that correlate with broad protection against rotavirus infection and illness, despite exposure to a restricted number of G types.

Antibodies, Viral↗

PubMed1994

In situ detection of hepatitis A virus in cell cultures and shellfish tissues.

An in situ transcription method was developed to detect hepatitis A virus RNA in both cell cultures and shellfish tissues. Radiolabeled cDNA copies were synthesized in situ by reverse transcriptase-directed transcription after annealing with a specific primer to the viral RNA. Both tritium (3H) and 35S were useful in the in situ transcription reaction, but the use of 3H resulted in a lower background and finer detail in the localization of viral particles. Application of the method to different organs of oysters which had bioaccumulated hepatitis A virus allowed the first in situ localization of the virus, specifically in stomach and hepatopancreatic tissues.

Animals↗

PubMed1994

Epidemiological study of Norwalk virus infections in Japan and Southeast Asia by enzyme-linked immunosorbent assays with Norwalk virus capsid protein produced by the baculovirus expression system.

In this study, we investigated Norwalk virus (NV) antigen and antibody to recombinant NV (rNV) in human populations in Japan and Southeast Asia by enzyme-linked immunosorbent assays (ELISAs). Baculovirus-expressed recombinant NV (rNV) capsid protein was used for preparing antisera to rNV or used as an antigen for detecting antibody to rNV. The ELISAs were specific for NV and had sensitivities equivalent to or higher than those of the previously developed radioimmunoassays. In 159 stool samples obtained from children, mainly younger than 10 years old, with acute gastroenteritis due to small round structured viruses in Japan, only 1 was positive for NV antigen. The pattern of acquisition of antibody to rNV was quite different from those of antibodies to group A rotavirus and human calicivirus Sapporo (HuCV-Sa) strain. The prevalence of antibody to rNV remained at a low level throughout childhood and then showed a steep rise during school age and early adulthood in Japan. A high prevalence of antibody was observed in samples collected from healthy adults in Japan and Southeast Asia. These results suggested that NV infection is common in adults in Japan and Southeast Asia but may be rare in infants in Japan. The HuCV-Sa strain was negative by the ELISA, and no serological relationship between NV and the HuCV-Sa strain was found. NV may be quite different from the HuCV-Sa strain, although both viruses are classified in the family Caliciviridae.

Adolescent↗

PubMed1994

Detection of immunoglobulin M (IgM), IgA, and IgG Norwalk virus-specific antibodies by indirect enzyme-linked immunosorbent assay with baculovirus-expressed Norwalk virus capsid antigen in adult volunteers challenged with Norwalk virus.

Pre- and postexposure sera collected from 17 adult volunteers challenged with Norwalk virus as described previously (D. Y. Graham, X. Jiang, T. Tanaka, A. Opekun, P. Madore, and M. K. Estes, J. Infect. Dis. 170:34-43, 1994) were examined for Norwalk virus-specific immunoglobulin M (IgM), IgA, and IgG by indirect enzyme-linked immunosorbent assays with recombinant Norwalk virus antigen bound to the solid phase. Sixteen of the 17 volunteers had evidence of past infection, all presenting with preexisting IgG antibody of high avidity; only one volunteer had no evidence of previous infection. Virus infection was detected in 14 of the 16 volunteers with evidence of past infection, and 9 of the infected volunteers had symptomatic illness. A significant rise in both virus-specific IgA and IgG titers was detected after challenge in all of the volunteers who became ill. Five of the asymptomatic volunteers who were infected had rising titers of virus-specific IgG, but only two of the five had a concomitant rise in their virus-specific IgA antibody titers. Antibody rises were detectable in eight of nine ill volunteers 8 to 11 days after challenge but in the asymptomatic volunteers only after more than 15 days had elapsed. Virus-specific IgM was detected after challenge in all 14 infected volunteers. Between symptomatic and asymptomatic volunteers there were no significant differences in titers of virus-specific IgG and IgA in serum before challenge; however, there were significantly higher titers in symptomatic volunteers between 8 and > 90 days after challenge for virus-specific IgG and 8 and 24 days after challenge for virus-specific IgA.

Adult↗

PubMed1994

Norwalk virus-associated gastroenteritis traced to ice consumption aboard a cruise ship in Hawaii: comparison and application of molecular method-based assays.

Investigation of an outbreak of acute nonbacterial gastroenteritis on a cruise ship provided an opportunity to assess new molecular method-based diagnostic methods for Norwalk virus (NV) and the antibody response to NV infection. The outbreak began within 36 h of embarkation and affected 30% of 672 passengers and crew. No single meal, seating, or food item was implicated in the transmission of NV, but a passenger's risk of illness was associated with the amount of ice (but not water) consumed (chi-square for trend, P = 0.009). Of 19 fecal specimens examined, 7 were found to contain 27-nm NV-like particles by electron microscopy and 16 were positive by PCR with very sensitive NV-specific primers, but only 5 were positive by a new highly specific antigen enzyme immunoassay for NV. Ten of 12 serum specimen pairs demonstrated a fourfold or greater rise in antibody titer to recombinant baculovirus-expressed NV antigen. The amplified PCR band shared only 81% nucleotide sequence homology with the reference NV strain, which may explain the lack of utility of the fecal specimen enzyme immunoassay. This report, the first to document the use of these molecular method-based assays for investigation of an outbreak, demonstrates the importance of highly sensitive viral diagnostics such as PCR and serodiagnosis for the epidemiologic investigation of NV gastroenteritis.

Antibodies, Viral↗

PubMed1994

Application of PCR to detect Norwalk virus in fecal specimens from outbreaks of gastroenteritis.

Norwalk virus (NV) and other small round-structured viruses (SRSVs) are frequent causes of gastroenteritis outbreaks. The recent cloning and sequencing of the NV genome has made it possible to detect NV and Norwalk-related viruses from fecal specimens by reverse transcription (RT)-PCR. We applied this technique to the examination of a total of 139 fecal specimens from 19 outbreaks characterized by NV serology, including 56 samples from 7 NV outbreaks, 36 from 6 Norwalk-related virus outbreaks, and 47 from 6 outbreaks with SRSVs visualized by electron microscopy that were serologically unrelated to NV. Three primer pairs were evaluated: two pairs in the polymerase region of NV and one pair near the 3' end of the genome. When one set of primers (primer pair 51-3) from the polymerase region was used, 40% of all samples were positive by RT-PCR and specimens from the NV outbreaks were more likely to be positive (64%) than those from outbreaks associated with Norwalk-related viruses (44%) or SRSVs (8%). To determine the relationship of the outbreak strains to NV, we compared the sequences of a 145-base portion of the polymerase gene from 10 specimens obtained from five different outbreaks characterized as NV by serology. No two outbreak strains had the same sequence in this 145-base portion of the polymerase gene, and the identities of the nucleotide and amino acid sequences of these products compared with the sequences of the corresponding region of NV ranged from 62 to 79% and 69 to 90%, respectively. Because of sequence diversity in the polymerase region, the successful application of RT-PCR to investigations of outbreaks of suspected NV-associated gastroenteritis will depend on the use of either multiple primer pairs or primers made against regions of the genome that are more conserved.

Amino Acid Sequence↗

PubMed1994

The nonstructural glycoprotein of rotavirus affects intracellular calcium levels.

Rotavirus infection of monkey kidney cells has been reported to result in a significant increase in the concentration of intracellular calcium. This increase in intracellular calcium was associated with viral protein synthesis and cytopathic effects in infected cells. We tested the effect of individual rotavirus proteins on intracellular calcium concentrations in insect Spodoptera frugiperda (Sf9) cells. Insect cells were infected with wild-type baculovirus or baculovirus recombinants that contained an individual rotavirus gene. The cells were harvested at different times postinfection, and the intracellular calcium concentration was measured by using fura-2 as a fluorescent calcium indicator. We found that the concentration of intracellular calcium was increased nearly fivefold in infected Sf9 cells that expressed the nonstructural glycoprotein (NSP4) of group A rotavirus, and this increase in intracellular calcium concentration coincided with NSP4 expression. A similar result was observed in insect cells expressing NSP4 from a group B rotavirus, suggesting the conservation of this function among rotavirus groups. Expression of the other 10 rotavirus proteins or of wild-type baculovirus proteins in Sf9 cells did not significantly increase intracellular calcium levels. These results suggest that the nonstructural glycoprotein NSP4 is responsible for the increase in cytosolic calcium observed in rotavirus-infected cells.

Animals↗

PubMed1994

Template-dependent, in vitro replication of rotavirus RNA.

A template-dependent, in vitro rotavirus RNA replication system was established. The system initiated and synthesized full-length double-stranded RNAs on rotavirus positive-sense template RNAs. Native rotavirus mRNAs or in vitro transcripts, with bona fide 3' and 5' termini, derived from rotavirus cDNAs functioned as templates. Replicase activity was associated with a subviral particle containing VP1, VP2, and VP3 and was derived from native virions or baculovirus coexpression of rotavirus genes. A cis-acting signal involved in replication was localized within the 26 3'-terminal nucleotides of a reporter template RNA. Various biochemical and biophysical parameters affecting the efficiency of replication were examined to optimize the replication system. A replication system capable of in vitro initiation has not been previously described for Reoviridae.

Animals↗

PubMed1994

Identification of minireovirus as a Norwalk-like virus in pediatric patients with gastroenteritis.

In 1977, 30- to 32-nm virus-like particles, named minireovirus because of their unique morphologic appearance, were detected by electron microscopy in the stools of infants and young children with gastroenteritis. Sequence analysis of approximately 2,800 consecutive bases derived from overlapping PCR clones of a recent minireovirus clinical isolate showed 52% nucleotide sequence identity with the Norwalk virus sequence and, in addition, demonstrated that the genomic organizations of these two viruses were similar. Our data show that minireovirus is a Norwalk-like virus and should now also be included in the Caliciviridae family.

Amino Acid Sequence↗

PubMed1994

Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.

Amino Acid Sequence↗

PubMed1994

Three-dimensional structure of baculovirus-expressed Norwalk virus capsids.

The three-dimensional structure of the baculovirus-expressed Norwalk virus capsid has been determined to a resolution of 2.2 nm using electron cryomicroscopy and computer image processing techniques. The empty capsid, 38.0 nm in diameter, exhibits T = 3 icosahedral symmetry and is composed of 90 dimers of the capsid protein. The striking features of the capsid structure are arch-like capsomeres, at the local and strict 2-fold axes, formed by dimers of the capsid protein and large hollows at the icosahedral 5- and 3-fold axes. Despite its distinctive architecture, the Norwalk virus capsid has several similarities with the structures of T = 3 single-stranded RNA (ssRNA) viruses. The structure of the protein subunit appears to be modular with three distinct domains: the distal globular domain (P2) that appears bilobed, a central stem domain (P1), and a lower shell domain (S). The distal domains of the 2-fold related subunits interact with each other to form the top of the arch. The lower domains of the adjacent subunits associate tightly to form a continuous shell between the radii of 11.0 and 15.0 nm. No significant mass density is observed below the radius of 11.0 mm. It is suspected that the hinge peptide in the adjoining region between the central domain and the shell domain may facilitate the subunits adapting to various quasi-equivalent environments. Architectural similarities between the Norwalk virus capsid and the other ssRNA viruses have suggested a possible domain organization along the primary sequence of the Norwalk virus capsid protein. It is suggested that the N-terminal 250 residues constitute the lower shell domain (S) with an eight-strand beta-barrel structure and that the C-terminal residues beyond 250 constitute the protruding (P1+P2) domains. A lack of an N-terminal basic region and the ability of the Norwalk virus capsid protein to form empty T = 3 shells suggest that the assembly pathway and the RNA packing mechanisms may be different from those proposed for tomato bushy stunt virus and southern bean mosaic virus but similar to that in tymoviruses and comoviruses.

Baculoviridae↗

PubMed1994

Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells.

Rotaviruses are triple-layered particles that contain four major capsid proteins, VP2, VP4, VP6, and VP7, and two minor proteins, VP1 and VP3. We have cloned each of the rotavirus genes coding for a major capsid protein into the baculovirus expression system and expressed each protein in insect cells. Coexpression of different combinations of the rotavirus major structural proteins resulted in the formation of stable virus-like particles (VLPs). The coexpression of VP2 and VP6 alone or with VP4 resulted in the production of VP2/6 or VP2/4/6 VLPs, which were similar to double-layered rotavirus particles. Coexpression of VP2, VP6, and VP7, with or without VP4, produced triple-layered VP2/6/7 or VP2/4/6/7 VLPs, which were similar to native infectious rotavirus particles. The VLPs maintained the structural and functional characteristics of native particles, as determined by electron microscopic examination of the particles, the presence of nonneutralizing and neutralizing epitopes on VP4 and VP7, and hemagglutination activity of the VP2/4/6/7 VLPs. The production of VP2/4/6 particles indicated that VP4 interacts with VP6. Cell binding assays performed with each of the VLPs indicated that VP4 is the viral attachment protein. Chimeric particles containing VP7 from two different G serotypes also were obtained. The ability to express individual proteins or to coexpress different subsets of proteins provides a system with which to examine the interactions of the rotavirus structural proteins, the role of individual proteins in virus morphogenesis, and the feasibility of a subunit vaccine.

Animals↗

PubMed1993

Two successive outbreaks of SRSV-associated gastroenteritis in South Africa.

Two successive outbreaks of gastroenteritis in South Africa were investigated to identify the aetiological agents. Some patients were involved in both outbreaks. Enteropathogenic bacteria or parasites were not evident in either outbreak. Small round structured viruses (SRSVs) were demonstrated in both outbreaks by direct electron microscopy. SRSV UK3/Hawaii virus was identified by immune electron microscopy as the causative agent in the first outbreak. Using new recombinant Norwalk virus (rNV) immunoassays for antibodies and antigen, Norwalk virus was implicated in the second outbreak. Preexisting antibodies to Norwalk virus were not protective and there was no cross protection between Hawaii and Norwalk viruses. There was no anamnestic response to Norwalk virus following the SRSV UK3/Hawaii outbreak although those affected had preexisting antibodies to Norwalk virus. To our knowledge, this is the first definitive diagnosis of SRSV-associated gastroenteritis in South Africa.

Adult↗

PubMed1993

A subviral particle binding domain on the rotavirus nonstructural glycoprotein NS28.

The single-shelled particle binding domain(s) on NS28 was examined by testing the ability of different truncated forms of NS28 to bind single-shelled particles (ssp). Deletion of amino acids (aa) 161 to 175 of NS28 abolished ssp binding activity. Deletion of the last three aa (173-175) of NS28 diminished, but did not abolish, the ligand binding activity in our assay conditions. An internal deletion of NS28 (aa 110 to 155) also significantly diminished ssp binding activity in standard binding assays. As an alternative approach to study the ssp binding domain on NS28, we mapped the epitope of binding of monoclonal antibody BA/55, which was found to block ssp binding to NS28. Immunoprecipitation experiments done with truncated mutants of NS28 located the epitope of BA/55 to aa 149-160 of NS28, immediately adjacent to or partially overlapping the putative ssp binding domain. Experiments using synthetic peptides mimicking the carboxy end of NS28, found these peptides were not able to compete for ssp binding. Together, these results suggest that the ssp binding site in NS28 (aa 161-172) is highly dependent on the conformational integrity of the cytoplasmic C-terminus of NS28. NS28 truncation mutants also were assayed for interactions with rotavirus VP4 expressed in baculovirus. Amino acids 112 to 148 of NS28 were found to be critical for NS28-VP4 binding. Unexpectedly, aa 149 to 175 not only were nonessential for interaction with VP4, but mutants lacking those aa showed improved binding activity. We hypothesize that the VP4 binding domain may be buried in the NS28 cytoplasmic domain, and that the binding of ssp and VP4 may be an interdependent process that functions in conjunction with triggering of the budding of the whole complex into the endoplasmic reticulum. These results demonstrate the pleiotropic properties of NS28 in the unique rotavirus morphogenetic process.

Animals↗

PubMed1993

Sequence and genomic organization of Norwalk virus.

A library of overlapping cDNAs obtained from Norwalk virus purified from stools of human volunteers (Jiang et al., 1990, Science 250, 1580-1583) was used to obtain the nucleotide sequence of the viral genome. The sequence has a total of 7642 nucleotides, excluding the 3' poly(A) tail, and has a base composition of 48% G + C. Three open reading frames (ORF) are predicted in the sequence. The longest ORF (ORF1, nucleotides (nt) 146 to 5359) is predicted to encode a polyprotein precursor to nonstructural proteins based on identification of sequences similar to the picornavirus 2C protein, 3C protease, and 3D RNA-dependent RNA polymerase. ORF2 (nt 5346 to 6935) is predicted to encode a polypeptide with a predicted molecular weight of 56,571 (56.6K, close to the expected size of the viral capsid protein), and it contains a short region of sequence similarity to the picornavirus structural protein VP3. A third potential ORF (nt 6938 to 7573) could encode a small polypeptide of 22.5K. The genomic organization found in Norwalk virus shares striking similarities with the genome of two caliciviruses, the feline calicivirus and the rabbit hemorrhagic disease virus. The morphology, size, polarity, and genomic organization of the Norwalk virus indicate it is a member of the Caliciviridae family.

Base Sequence↗

PubMed1993

Expression of two bovine rotavirus non-structural proteins (NSP2, NSP3) in the baculovirus system and production of monoclonal antibodies directed against the expressed proteins.

Studies on rotavirus non-structural proteins have been hampered in the past by difficulties in obtaining monospecific reagents. To make such reagents available, we have expressed in the baculovirus system NSP2 and NSP3 (formerly called NS35 and NS34, respectively) of the bovine rotavirus RF and produced hybridomas against these proteins. Full-length DNA copies of RNA segments 7 (coding for NSP3) and 8 (coding for NSP2) of the virus strain RF were cloned and sequenced. Each cDNA was inserted in the transfer vector pVL941 and used to transfect Spodoptera frugiperda cells (Sf9). Recombinant baculoviruses encoding these proteins were obtained. Infection of Sf9 cells with these recombinant viruses resulted in a high level of expression of NSP2 and NSP3 (range of 1 microgram per 10(6) cells). Monoclonal antibodies (MAbs) were elicited by immunization of BALB/c mice with adjuvented, unpurified recombinant proteins in the rear foot pads. Fusion was performed using lymphocytes from popliteal lymph nodes with SP2/O-Ag14 myeloma line. Screening was by differential indirect immunofluorescent staining on monolayers of Sf9 cells infected with each recombinant virus. Two MAbs proved to be reactive against NSP3 and a single one against NSP2. They showed high specificity by immunofluorescence, immunoprecipitation and Western blot. The isotype of these MAbs was IgG1. Oligomeric forms of NSP3 and NSP2 proteins were detected and the existence of intra-chain disulfide bridge in NSP2 protein was suggested. The levels of synthesis and cellular localization of NSP3 and NSP2 proteins were different as shown by immunoprecipitation and immunofluorescence.

Animals↗

PubMed1993

Alterations in the sequence of the gene 4 from a human rotavirus after multiple passages in HepG2 liver cells.

The human Wa strain of rotaviruses, initially unable to grow in liver cells, was adapted by multiple passages to grow in HepG2 cells. The genome segment 4 of both the parental and passaged strains was cloned and sequenced. Five amino acid differences (residues 38, 120, 421, 525, and 618) were found in the HepG2-passaged variant compared to the parental Wa strain. Our results support the hypothesis that viral variants that have improved capabilities for infecting liver cells can be generated during infection.

Amino Acid Sequence↗