Biomedical subjects

Mary K Estes

Publications and source records attributed to Mary K Estes.

At least 19 recordsLinked to original sources

PubMed2026

Culture of infectious human norovirus isolated from live contaminated oysters.

Human noroviruses are a major cause of foodborne outbreaks worldwide. Filter-feeding shellfish, such as oysters, can bioaccumulate these viruses in their digestive tissue when grown in sewage-impacted coastal areas and are often implicated in norovirus foodborne outbreaks. Despite the high sensitivity of current molecular assays, these methods for norovirus detection in shellfish fail to distinguish between infectious and non-infectious particles. Assessing norovirus infectivity in shellfish remains a challenge due to the lack of suitable isolation methods that maintain capsid integrity. In this study, a protocol for isolating infectious norovirus from oyster tissues, based on chloroform-butanol elution and polyethylene glycol concentration (CB-PEG), was optimized for the recovery of human norovirus GI and GII. While CB-PEG method recovered various norovirus GI and GII genotypes, it was less efficient at the genomic level than a protocol based on proteinase K elution (adapted from ISO 15216) and showed genotype-dependent viral recovery rates. By optimizing the flocculation step, we improved the method's compatibility with human intestinal enteroid (HIE) cultures. Using this approach, we successfully quantified infectious norovirus GII.3 titers recovered from artificially-contaminated live oysters. Interestingly, infectious virus was better isolated following a freezing step of the digestive tissues, with titers ranging from 13 to 40 TCID50/mL for positive samples. In conclusion, this study established an optimized methodological approach for the relative quantification of infectious norovirus GII.3 in shellfish, paving the way for future research on viral persistence and inactivation strategies in this foodstuff.

Norovirus↗

PubMed2007

Expression of rotavirus NSP4 alters the actin network organization through the actin remodeling protein cofilin.

Rotavirus is a major cause of infantile gastroenteritis with a multifactorial pathogenesis. As with many other pathogens, rotavirus infection and replication leads to rearrangement of the cytoskeleton with disorganization of cytoskeletal elements such as actin and cytokeratin through a calcium-dependent process that has not been fully characterized. The rotavirus enterotoxin NSP4, shown previously to elevate intracellular calcium levels when added exogenously as well as when expressed intracellularly, is a key player in intracellular calcium regulation during rotavirus infection. Here, we investigated the role NSP4 may play in actin rearrangement. Expression of NSP4 fused to enhanced green fluorescent protein (NSP4-EGFP), but not expression of EGFP alone, caused stabilization of long cellular projections in fully confluent HEK 293 cells. Cells expressing NSP4-EGFP for 24 h were also resistant to cell rounding induced by cytochalasin D. Quantification of filamentous actin (F-actin) content by using rhodamine-conjugated phalloidin and flow cytometry showed an elevated F-actin content in NSP4-EGFP-expressing and rotavirus-infected cells in comparison with that in nonexpressing and noninfected cells. Normalization of intracellular calcium levels prevented alterations of F-actin content. Observed changes in F-actin amounts correlated with the increased activation of the actin-remodeling protein cofilin. These calcium-dependent actin rearrangements induced by intracellular NSP4 expression may contribute to rotavirus pathogenesis by interfering with cellular processes dependent on subcortical actin remodeling, including ion transport and viral release.

Actin Depolymerizing Factors↗

PubMed2006

Geographic information systems and genotyping in identification of rotavirus G12 infections in residents of an urban slum with subsequent detection in hospitalized children: emergence of G12 genotype in South India.

Rotavirus infections by G12 strains in several countries have recently been described. In this study, we report the emergence of G12 strains in south India. Fourteen cases of G12 infection were identified between June and September 2005. G12 was seen in combination with P[6], P[8], or nontypeable P type. Nine cases, including five symptomatic infections and four asymptomatic infections, were identified as part of routine surveillance for rotavirus infections in a birth cohort in the community between June and July 2005. Significant temporal and time-space clustering of eight of these cases represents a possible recent introduction of a new rotavirus VP7 genotype. Previous rotavirus infections had been documented for six of the nine children in the community. In the following 2 months, five cases of G12 infection were identified among children presenting to a referral hospital with diarrhea. This is the first description of symptomatic and asymptomatic G12 infections in children in the community. The detection of G12 strains from different parts of the world in recent years suggests the possibility of its emergence as an important global genotype. Monitoring of cocirculating rotavirus strains and detection of emerging strains is important in the context of the availability of rotavirus vaccines.

Child, Preschool↗

PubMed2006

Antibody prevalence and titer to norovirus (genogroup II) correlate with secretor (FUT2) but not with ABO phenotype or Lewis (FUT3) genotype.

BACKGROUND: Histo-blood group antigens and secretor status have been associated with susceptibility to Norovirus infections, which suggests that antibody prevalence and titer might correlate with these phenotypes. METHODS: Plasma samples (n = 105) from Swedish blood donors that had been genotyped for secretor (FUT2) and Lewis (Le; FUT3) genotypes and phenotyped for ABO and Le blood groups were analyzed for immunoglobulin G antibody prevalence and titers to norovirus genogroup (GG) II.4. RESULTS: The results showed that nonsecretors (se4128se428) and Lea+b- individuals not only had significantly lower antibody titers than did secretors (P < .0001) and Lea-b+ individuals (P < .0002) but were also significantly more often antibody negative (P < .05). Antibody titers in secretors were not significantly different between individuals of different Le (FUT3) genotypes or different ABO phenotypes. CONCLUSIONS: Nonsecretors and Lea+b- individuals are significantly less prone to be infected with GGII noroviruses. This new information extends previous knowledge and supports the hypothesis that nonsecretors are relatively but not absolutely resistant to norovirus infections.

ABO Blood-Group System↗

PubMed2006

Cryoelectron microscopy structures of rotavirus NSP2-NSP5 and NSP2-RNA complexes: implications for genome replication.

The replication and packaging of the rotavirus genome, comprising 11 segments of double-stranded RNA, take place in specialized compartments called viroplasms, which are formed during infection and involve a coordinated interplay of multiple components. Two rotavirus nonstructural proteins, NSP2 (with nucleoside triphosphatase, single-stranded RNA [ssRNA] binding and helix-destabilizing activities) and NSP5, are essential in these events. Previous structural analysis of NSP2 showed that it is an octamer in crystals, obeying 4-2-2 crystal symmetry, with a large 35-A central hole along the fourfold axis and deep grooves at one of the twofold axes. To ascertain that the solution structure of NSP2 is the same as that in the crystals and investigate how NSP2 interacts with NSP5 and RNA, we carried out single-particle cryoelectron microscopy (cryo-EM) analysis of NSP2 alone and in complexes with NSP5 and ssRNA at subnanometer resolution. Because full-length NSP5 caused severe aggregation upon mixing with NSP2, the deletion construct NSP566-188 was used in these studies. Our studies show that the solution structure of NSP2 is same as the crystallographic octamer and that both NSP566-188 and ssRNA bind to the grooves in the octamer, which are lined by positively charged residues. The fitting of the NSP2 crystal structure to cryo-EM reconstructions of the complexes indicates that, in contrast to the binding of NSP566-188, the binding of RNA induces noticeable conformational changes in the NSP2 octamer. Consistent with the observation that both NSP5 and RNA share the same binding site on the NSP2 octamer, filter binding assays showed that NSP5 competes with ssRNA binding, indicating that one of the functions of NSP5 is to regulate NSP2-RNA interactions during genome replication.

Binding Sites↗

PubMed2006

X-ray structure of a native calicivirus: structural insights into antigenic diversity and host specificity.

Caliciviruses, grouped into four genera, are important human and veterinary pathogens with a potential for zoonosis. In these viruses, capsid-related functions such as assembly, antigenicity, and receptor interactions are predominantly encoded in a single protein that forms an icosahedral capsid. Understanding of the immunologic functions and pathogenesis of human caliciviruses in the Norovirus and Sapovirus genera is hampered by the lack of a cell culture system or animal models. Much of our understanding of these viruses, including the structure, has depended on recombinant capsids. Here we report the atomic structure of a native calicivirus from the Vesivirus genus that exhibits a broad host range possibly including humans and map immunological function onto a calicivirus structure. The vesivirus structure, despite a similar architectural design as seen in the recombinant norovirus capsid, exhibits novel features and indicates how the unique modular organization of the capsid protein with interdomain flexibility, similar to an antibody structure with a hinge and an elbow, integrates capsid-related functions and facilitates strain diversity in caliciviruses. The internally located N-terminal arm participates in a novel network of interactions through domain swapping to assist the assembly of the shell domain into an icosahedral scaffold, from which the protruding domain emanates. Neutralization epitopes localize to three hypervariable loops in the distal portion of the protruding domain surrounding a region that exhibits host-specific conservation. These observations suggest a mechanism for antigenic diversity and host specificity in caliciviruses and provide a structural framework for vaccine development.

Amino Acid Sequence↗

PubMed2006

The epidemiologic and clinical importance of norovirus infection.

Noroviruses are a major cause of sporadic cases and epidemic outbreaks of gastroenteritis. The development of molecular diagnostic assays has led to an increased recognition of the significance of these viruses as causes of gastroenteritis in all age groups. This article reviews the epidemiology, clinical manifestations and pathogenesis of norovirus infection, and it describes the diagnostic and therapeutic approaches to this disease.

Caliciviridae Infections↗

PubMed2006

Ricin toxin B subunit enhancement of rotavirus NSP4 immunogenicity in mice.

A 90-amino acid peptide from the simian rotavirus SA-11 nonstructural protein, NSP4 was linked to the N-terminus of the Ricinus communis A-B toxin B subunit protein (RTB) and synthesized in Escherichia coli. Recombinant RTB and the NSP4(90)::RTB fusion protein bound artificial receptor glycoprotein asialofetuin in an in vitro enzyme-linked immunosorbent assay (ELISA), demonstrating biological activity of the recombinant protein. Mice co-inoculated with purified recombinant RTB plus NSP4(90) peptide proteins or heat denatured NSP4(90)::RTB fusion protein generated higher titers of serum anti-NSP4(90) IgG antibodies than mice immunized with NSP4(90) peptide alone, indicating the presence of adjuvant functions for N-terminal linked RTB. Serum anti-NSP4(90) IgG titers were highest in mice immunized with native recombinant NSP4(90)::RTB fusion protein, confirming the immunostimulatory function of RTB. Results of experiments described here demonstrate the feasibility of using RTB mediated adjuvant functions for stimulation of the antigenicity of a rotavirus nonstructural protein. The ability of recombinant NSP4(90)::RTB fusion protein synthesized in E. coli to bind glycoprotein receptor molecules effectively indicates that protein linkage to the RTB N-terminus and synthesis of the recombinant NSP4(90)::RTB fusion protein in bacteria do not interfere with the immunostimulatory properties of the RTB subunit.

Animals↗

PubMed2006

Noroviruses everywhere: has something changed?

PURPOSE OF REVIEW: Noroviruses causing gastroenteritis in humans have increasingly been described in both the scientific literature and the lay press. This review summarizes new information about where and why these viruses are detected as well as new developments to prevent or treat these infections. RECENT FINDINGS: Noroviruses are highly infectious and quite stable. Noroviruses infect all age groups, with particularly severe disease occurring in young children, the elderly, and persons with chronic diseases. Noroviruses also cause persistent infections in immunosuppressed patients. Host susceptibility factors have been identified, including histo-blood group antigens that likely function as initial receptors for some noroviruses. The patterns of susceptibility are complex, however, and not completely understood for all virus strains. X-ray crystallographic information on the virus capsid and other viral-encoded proteins provides targets for structure-based drug development. SUMMARY: The availability of new methods of detecting noroviruses has resulted in increased detection of these pathogens. Advances in understanding of virus replication and structure should lead to development of strategies to prevent and treat these infections. Such efforts may be challenged by the emergence of new virus strains that appear through mechanisms that remain to be understood.

Antiviral Agents↗

PubMed2006

High efficiency cross-reactive monoclonal antibody production by oral immunization with recombinant norwalk virus-like particles.

Monoclonal antibodies (MAbs) are important for in-depth antigenic characterization and diagnosis of infections with human caliciviruses that cause almost all outbreaks of nonbacterial gastroenteritis. We compared different routes of immunization with nonreplicating virus-like particles (VLPs) from recombinant Norwalk virus (rNV) and recombinant Mexico virus (rMX) administered to BALB/c mice to determine the efficiency of hybridoma production. Oral immunization with VLPs without adjuvant resulted in high yields of MAb-secreting hybridomas (90%) to these VLPs of IgG (61%), IgM (29%) and IgA (10%) isotypes. Fusions with mesenteric lymph node lymphocytes yielded MAbs of various subclasses including IgG2a, IgG3, IgM and IgA. These results suggest that an immunization route that mimics the natural route of viral infection pathway may facilitate MAb technology by increasing the yields of antibody secreting hybridoma cells.

Administration, Oral↗

PubMed2006

Tomato is a highly effective vehicle for expression and oral immunization with Norwalk virus capsid protein.

Norwalk virus (NV) is an important agent of epidemic gastroenteritis, and an oral subunit vaccine shows potential for protection. Recombinant Norwalk virus (rNV) capsid protein expressed in plants assembles virus-like particles (VLPs) that are orally immunogenic in mice and humans. In this article we examine rNV expression in tomato and potato using a plant-optimized gene, and test the immunogenicity of dried tomato fruit and potato tuber fed to mice. The synthetic gene increased rNV expression fourfold in tomato and potato plants, which assembled VLP. Four doses of 0.4 g freeze-dried tomato fruit containing 64 microg rNV (40 microg VLPs) induced NV-specific serum IgG and mucosal IgA in > or = 80% of mice, while doses of 0.8 g elicited systemic and mucosal antibody responses in all mice. Feedings of 1 g freeze-dried potato tuber containing 120 microg rNV (90 microg VLPs) were required to produce 100% responsiveness. Oxidation of phenolic compounds upon rehydration of dried tuber caused significant VLP instability, thus decreasing immunogenicity. Air-dried tomato fruit stimulated stronger immune responses than freeze-dried fruit of the same mass, perhaps by limiting the destruction of plant cell matrix and membrane systems that occurs with freeze-drying. Thus, rNV in dried transgenic tomato fruit was a more potent immunogen than that in dried potato tubers, based on the total VLPs ingested. These findings support the use of stabilized, dried tomato fruit for oral delivery of subunit vaccines.

Administration, Oral↗

PubMed2006

Comparative study of the epidemiology of rotavirus in children from a community-based birth cohort and a hospital in South India.

Rotavirus gastroenteritis is the major cause of severe dehydrating diarrhea in children worldwide. This study compares rotavirus diarrhea in 351 children in a community-based cohort and 343 children admitted to a hospital during the same period. Clinical information and fecal specimens were obtained during diarrheal episodes. Fecal samples were screened for VP6 antigen, and the positive samples were G and P typed by reverse transcription-PCR. Rotavirus was detected in 82/1,152 (7.1%) episodes of diarrhea in the community and 94/343 (27.4%) cases in the hospital. The median age of affected children (7.5 versus 10.5 months) and the mean severity of symptoms (Vesikari score, 7.6+/-3.4 versus 11+/-2.5) were lower in the community. A larger proportion of children in the community were breast-fed than were children admitted to the hospital (73% versus 34.8%). In the community, the genotypes identified in symptomatic patients, in order of frequency, were G1 (36.5%), G10 (17.1%), G2 (15.9%), and G9 (7.3%) and mixed infections (7.3%). The most common G-P combinations were G1P[8], G2P[4], G1P[4], and G10P[11]. The distribution of G types from hospitalized children was G1 (46.8%), G9 (19.1%), G2 (8.5%), G10 (1.1%), and 4.3% mixed infections. The most common G-P combinations were G1P[8] and G9P[8]. This study documents significant genetic heterogeneity of rotaviruses in the community and the hospital. G10P[11] strains resembling a vaccine candidate strain caused disease in the community, indicating the need for careful epidemiological studies as well as safety studies for the vaccine candidates.

Age Factors↗

PubMed2006

Rotavirus viremia and extraintestinal viral infection in the neonatal rat model.

Rotaviruses infect mature, differentiated enterocytes of the small intestine and, by an unknown mechanism, escape the gastrointestinal tract and cause viremia. The neonatal rat model of rotavirus infection was used to determine the kinetics of viremia, spread, and pathology of rotavirus in extraintestinal organs. Five-day-old rat pups were inoculated intragastrically with an animal (RRV) or human (HAL1166) rotavirus or phosphate-buffered saline. Blood was collected from a subset of rat pups, and following perfusion to remove residual blood, organs were removed and homogenized to analyze rotavirus-specific antigen by enzyme-linked immunosorbent assay and infectious rotavirus by fluorescent focus assay or fixed in formalin for histology and immunohistochemistry. Viremia was detected following rotavirus infection with RRV and HAL1166. The RRV 50% antigenemia dose was 1.8 x 10(3) PFU, and the 50% diarrhea dose was 7.7 x 10(5) PFU, indicating that infection and viremia occurred in the absence of diarrhea and that detecting rotavirus antigen in the blood was a more sensitive measure of infection than diarrhea. Rotavirus antigens and infectious virus were detected in multiple organs (stomach, intestines, liver, lungs, spleen, kidneys, pancreas, thymus, and bladder). Histopathological changes due to rotavirus infection included acute inflammation of the portal tract and bile duct, microsteatosis, necrosis, and inflammatory cell infiltrates in the parenchymas of the liver and lungs. Colocalization of structural and nonstructural proteins with histopathology in the liver and lungs indicated that the histological changes observed were due to rotavirus infection and replication. Replicating rotavirus was also detected in macrophages in the lungs and blood vessels, indicating a possible mechanism of rotavirus dissemination. Extraintestinal infectious rotavirus, but not diarrhea, was observed in the presence of passively or actively acquired rotavirus-specific antibody. These findings alter the previously accepted concept of rotavirus pathogenesis to include not only gastroenteritis but also viremia, and they indicate that rotavirus could cause a broad array of systemic diseases in a number of different organs.

Animals↗

PubMed2006

X-ray crystallographic structure of the Norwalk virus protease at 1.5-A resolution.

Norwalk virus (NV), a member of the Caliciviridae family, is the major cause of acute, epidemic, viral gastroenteritis. The NV genome is a positive sense, single-stranded RNA that encodes three open reading frames (ORFs). The first ORF produces a polyprotein that is processed by the viral cysteine protease into six nonstructural proteins. We have determined the structure of the NV protease to 1.5 and 2.2 A from crystals grown in the absence or presence, respectively, of the protease inhibitor AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride]. The protease, which crystallizes as a stable dimer, exhibits a two-domain structure similar to those of other viral cysteine proteases with a catalytic triad composed of His 30, Glu 54, and Cys 139. The native structure of the protease reveals strong hydrogen bond interactions between His 30 and Glu 54, in the favorable syn configuration, indicating a role of Glu 54 during proteolysis. Mutation of this residue to Ala abolished the protease activity, in a fluorogenic peptide substrate assay, further substantiating the role of Glu 54 during proteolysis. These observations contrast with the suggestion, from a previous study of another norovirus protease, that this residue may not have a prominent role in proteolysis (K. Nakamura, Y. Someya, T. Kumasaka, G. Ueno, M. Yamamoto, T. Sato, N. Takeda, T. Miyamura, and N. Tanaka, J. Virol. 79:13685-13693, 2005). In the structure from crystals grown in the presence of AEBSF, Glu 54 undergoes a conformational change leading to disruption of the hydrogen bond interactions with His 30. Since AEBSF was not apparent in the electron density map, it is possible that these conformational changes are due to subtle changes in pH caused by its addition during crystallization.

Binding, Competitive↗

PubMed2006

Synthesis of a ricin toxin B subunit-rotavirus VP7 fusion protein in potato.

A gene encoding the outer capsid glycoprotein (VP7) of simian rotavirus SA11, was genetically linked to the amino terminus of the ricin toxin B subunit (RTB) isolated from castor-oil plant (Ricinus communis) seeds. To assess fusion protein expression in plant cells, the VP7::RTB fusion gene was transferred into potato (Solanum tuberosum) cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The fusion gene was detected in transformed potato genomic DNA by polymerase chain reaction DNA amplification methods. Immunoblot analysis with anti-SA11 antiserum as the primary antibody verified the presence of VP7::RTB fusion protein in transformed potato tuber tissues. The plant-synthesized fusion protein bound RTB membrane receptors as measured by asialofetuin-enzyme-linked immunosorbent assay (ELISA). The ELISA results indicated that the VP7::RTB fusion protein was biologically active and made up approx 0.03% of total soluble transformed tuber protein. The biosynthesis of receptor binding VP7::RTB fusion protein in potato tissues demonstrates the feasibility of producing monomeric ricin toxin B subunit adjuvant-virus antigen fusion proteins in crop plants for enhanced immunity.

Agrobacterium tumefaciens↗

PubMed2006

Norwalk virus-specific binding to oyster digestive tissues.

The primary pathogens related to shellfish-borne gastroenteritis outbreaks are noroviruses. These viruses show persistence in oysters, which suggests an active mechanism of virus concentration. We investigated whether Norwalk virus or viruslike particles bind specifically to oyster tissues after bioaccumulation or addition to tissue sections. Since noroviruses attach to carbohydrates of the histo-blood group family, tests using immunohistochemical analysis were performed to evaluate specific binding of virus or viruslike particles to oyster tissues through these ligands. Viral particles bind specifically to digestive ducts (midgut, main and secondary ducts, and tubules) by carbohydrate structures with a terminal N-acetylgalactosamine residue in an alpha linkage (same binding site used for recognition of human histo-blood group antigens). These data show that the oyster can selectively concentrate a human pathogen and that conventional depuration will not eliminate noroviruses from oyster tissue.

ABO Blood-Group System↗

PubMed2005

Oral immunization with a shiga toxin B subunit: rotavirus NSP4(90) fusion protein protects mice against gastroenteritis.

A fusion protein containing the shiga toxin-1 B subunit (STB) linked to a 90 amino acid peptide (aa residues 86--175) from simian rotavirus (SA--11) nonstructural protein NSP4 was synthesized in Escherichia coli. Mice orally inoculated with 60 microg of STB::NSP4(90) fusion protein per dose generated higher humoral and intestinal antibody titers than mice inoculated with 30 microg of NSP4 alone. Serum anti-NSP4 IgG2a isotype titers were substantially greater than IgG1 titers, suggesting a dominant Th1 immune response. ELISA measurement of cytokines secreted from splenocytes isolated from immunized mice confirmed the STB::NSP4(90) fusion protein stimulation of a strong Th1 cell mediated immune response. Diarrhea in SA-11 rotavirus challenged neonates suckling from STB::NSP4 immunized dams was significantly reduced in severity and duration in comparison with virus challenged neonates from unimmunized mice. Together, our experiments demonstrate for the first time that the shiga toxin B subunit provides ligand mediated delivery of virus antigens to the gut-associated lymphoid tissues for enhanced stimulation of humoral and cellular responses against rotavirus gastroenteritis.

Administration, Oral↗

PubMed2005

Mucosal immunization with a ricin toxin B subunit-rotavirus NSP4 fusion protein stimulates a Th1 lymphocyte response.

The castor-oil plant Ricinus communis A-B dimeric toxin B subunit (RTB) was genetically linked at its N-terminus with a 90 amino acid peptide from simian rotavirus SA-11 non-structural protein NSP4(90) and produced in Escherichia coli BL21 cells. Biologically active recombinant NSP4(90)-RTB fusion protein was shown to bind glycoprotein asialofetuin receptor molecules in an in vitro enzyme-linked immunosorbent assay (ELISA). Oral inoculation of the purified NSP4(90)-RTB ligand-antigen fusion protein delivered the chimeric protein to intestinal epidermal cells for mucosal immunization against rotavirus infection. Mice fed the NSP4(90)-RTB fusion protein generated higher humoral and intestinal antibody titers than mice inoculated with NSP4(90) alone. Titers of serum IgG2a antibodies were significantly higher than IgG1 titers suggesting a dominant Th1 lymphocyte immune response. ELISA measurement of cytokines secreted from splenocyte isolated from immunized mice confirmed NSP4(90)-RTB fusion protein stimulates a strong Th1 cell-mediated immune response. The experimental results demonstrate that the ricin toxin B subunit N-terminus can be used as a site for delivery of virus antigens to the gut associated lymphoid tissues for RTB-mediated immune stimulation of antiviral mucosal immune responses.

Administration, Oral↗