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1.  The rotavirus nonstructural glycoprotein NSP4 possesses membrane destabilization activity. 
Journal of Virology  1996;70(10):6973-6981.
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.
PMCID: PMC190747  PMID: 8794341
2.  Porcine rotaviruses antigenically related to human rotavirus serotypes 1 and 2. 
Journal of Clinical Microbiology  1990;28(3):633-636.
Fecal samples from rotavirus-infected piglets were characterized by a serotyping enzyme-linked immunosorbent assay (ELISA) by using monoclonal antibodies (MAbs) specific to human serotypes 1, 2, 3, and 4 (D. O. Matson, M. K. Estes, J. W. Burns, H. B. Greenberg, K. Taniguchi, and S. Urasawa, submitted for publication). Rotavirus in 19 of 25 specimens tested from two herds of pigs from Buenos Aires province, Argentina, were classified antigenically as follows: one serotype 1, four serotype 2, two serotype 3, and no serotype 4. Six specimens reacted with both serotype 1 and 2 MAbs, and viruses in six specimens probably belonged to other serotypes because they reacted only with a VP7 common epitope MAb. Two porcine rotavirus fecal samples found to contain both serotype 1 and 2 viruses by the MAb-based test and one found to contain a serotype 2 virus were grown in tissue culture. When plaque-purified preparations of these tissue culture-adapted viruses were analyzed in the serotyping ELISA, the C60 and C86 preparations reacted only as serotype 1 viruses, indicating that the original fecal samples, which showed multiple VP7 reactivities, were heterogeneous and apparently contained two types of viruses. Testing of plaque-purified C134 virus confirmed its serotype 2 reactivity. The MAb-based serotype designations of these viruses also were confirmed by using a neutralization immunoperoxidase focus reduction assay. This is the first report of the occurrence of serotype 1 and 2 rotaviruses in animals. The MAbs originally developed to serotype human rotaviruses can be utilized to type animal rotaviruses.
PMCID: PMC269683  PMID: 2157739
3.  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. 
Journal of Clinical Microbiology  1994;32(12):3059-3063.
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.
PMCID: PMC264229  PMID: 7883902
4.  Biochemical characterization of a smaller form of recombinant Norwalk virus capsids assembled in insect cells. 
Journal of Virology  1997;71(10):8066-8072.
The expression of the single capsid protein of Norwalk virus (NV) in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (VLPs) of two sizes, the predominant 38-nm, or virion-size VLPs, and smaller, 23-nm VLPs. Here we describe the purification and biochemical characterization of the 23-nm VLPs. The 23-nm VLPs were purified to 95% homogeneity from the medium of Sf9 cultures by isopycnic CsCl gradient centrifugation followed by rate-zonal centrifugation in sucrose gradients. The compositions of the purified 23- and 38-nm VLPs were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblots. VLPs of both sizes showed a doublet at 58 kDa, the size of the full-length capsid protein. Upon alkaline treatment, the 23-nm VLPs underwent dissociation into soluble intermediates that were able to reassemble into 23- and 38-nm VLPs upon dialysis, suggesting that the assembly of both types of structures has a common pathway. Antigenic and biochemical properties of the 38- and 23-nm VLPs were examined and found to be conserved. Immunoprecipitation assays using polyclonal and monoclonal antibodies indicated that immunodominant epitopes on the capsid protein as well as conformational epitopes are conserved in the two types of particles. The trypsin cleavage site at residue 227 was protected in the assembled particles of both sizes but exposed after alkaline dissociation. These results, and the conservation of the binding activity of both forms of recombinant NV VLPs to cultured cells (L. J. White, J. M. Ball, M. E. Hardy, T. N. Tanaka, N. Kitamoto, and M. K. Estes, J. Virol. 70:6589-6597, 1996), suggest that the tertiary folding of the capsid protein responsible for these properties is conserved in the two structures. We hypothesize that the 23-nm VLPs are formed when 60 units of the NV capsid protein assembles into a structure with T=1 symmetry.
PMCID: PMC192173  PMID: 9311906
5.  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.
PMCID: PMC167792  PMID: 8572702
6.  The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum. 
Journal of Virology  1995;69(9):5763-5772.
We previously reported that expression of rotavirus nonstructural glycoprotein NSP4 is responsible for an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in Spodoptera frugiperda (Sf9) insect cells (P. Tian, Y. Hu, W. P. Schilling, D. A. Lindsay, J. Eiden, and M. K. Estes, J. Virol. 68:251-257, 1994). The purpose of the present study was to determine the mechanism by which NSP4 causes an increase in [Ca2+]i by measuring the permeability of the cytoplasmic and endoplasmic reticulum (ER) membranes in recombinant-baculovirus-infected Sf9 cells. No obvious change in plasmalemma permeability to divalent cations was observed in cells expressing NSP4 compared with that in cells expressing another rotaviral glycoprotein (VP7) when the influx of Ba2+, a Ca2+ surrogate, was monitored. The basal Ca2+ permeability of the internal Ca2+ store was evaluated by measuring the release of Ca2+ induced by ionomycin, a Ca2+ ionophore, or thapsigargin, an inhibitor of the ER Ca(2+)-ATPase pump, following suspension of the cells in Ca(2+)-free extracellular buffer. Releasable Ca2+ decreased with time to a greater extent in cells expressing NSP4 compared with that in cells expressing VP7, suggesting that NSP4 increases the basal Ca2+ permeability of the ER membrane. To determine the possible mechanism by which NSP4 increases ER permeability, purified NSP4 protein or a 22-amino-acid synthetic peptide consisting of residues 114 to 135 (NSP4(114-135) was added exogenously to noninfected Sf9 cells during measurement of [Ca2+]i. Both NSP4 and the NSP4(114-135 peptide produced a time-dependent increase in [Ca2+]i that was attenuated by prior inhibition of phospholipase C with U-73122. Pretreatment of the cells with thapsigargin completely blocked the increase in [Ca2+]i produced by NSP4(114-135, but the peptide only partially reduced the change in [Ca2+]i produced by thapsigargin. No changes in [Ca2+]i were seen in cells treated with control peptides. These results suggest that (i) exogenous NSP4 increases [Ca2+]i through the activation of phospholipase C, (ii) Ca2+ release by exogenous NSP4 is from a store that is a subset of the thapsigargin-sensitive compartment, and (iii) amino acid residues 114 to 135 of NSP4 are sufficient for this activity. In contrast to exogenous NSP4, the mechanism by which endogenously expressed NSP4 increases [Ca2+]1 appears to be unrelated to phospholipase C, since no effect of U-73122 was seen on the elevated [Ca2+]1 in cells expressing NSP4 and exogenously applied NSP4(114-135) caused a further increase in [Ca2+]1 in cells expressing NSP4 protein.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC189437  PMID: 7637021
7.  Detection of Norwalk virus and hepatitis A virus in shellfish tissues with the PCR. 
A method for the detection of Norwalk virus and hepatitis A virus from shellfish tissues by PCR was developed. Virus was added to the stomach and hepatopancreatic tissues of oysters or hard-shell clams, and viral nucleic acids were purified by a modification of a previously described method (R.L. Atmar, T.G. Metcalf, F.H. Neill, and M.K. Estes, Appl. Environ. Microbiol. 59:631-635, 1993). The new method had the following advantages compared with the previously described method: (i) more rapid sample processing; (ii) increased test sensitivity; (iii) decreased sample-associated interference with reverse transcription-PCR; and (iv) use of chloroform-butanol in place of the chlorofluorocarbon trichlorotrifluoroethane. In addition, internal standards for both Norwalk virus and hepatitis A virus were made which demonstrated when inhibitors to reverse transcription-PCR were present and allowed quantitation of the viral nucleic acids present in samples. This assay can be used to investigate shellfish-associated gastroenteritis outbreaks and to study factors involved in virus persistence in shellfish.
PMCID: PMC167576  PMID: 7487032
8.  Specific proteolytic cleavage of recombinant Norwalk virus capsid protein. 
Journal of Virology  1995;69(3):1693-1698.
Norwalk virus (NV) causes epidemic outbreaks of acute nonbacterial gastroenteritis in humans. The NV capsid is made up of a single protein, and expression of the capsid protein in baculovirus recombinants results in spontaneous assembly of the protein into virus-like particles (X. Jiang, M. Wang, D. Y. Graham, and M. K. Estes, J. Virol. 66:6527-6532, 1992). We have investigated whether the NV capsid protein undergoes a specific proteolytic cleavage. Recombinant NV (rNV) particles were digested with trypsin to determine if a specific cleavage occurred. A predominant band with a molecular weight of approximately 32,000 (32K protein) was observed when trypsin-treated rNV was electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Determination of the N-terminal sequence of this band showed that a trypsin-specific cleavage occurred at amino acid residue 227. Early studies identified two proteins with molecular weights of 59,000 and 30,000 (59K and 30K proteins) in the stool of NV-infected volunteers that were reactive with postinfection antiserum. (H. B. Greenberg, J. R. Valdesuso, A. R. Kalica, R. G. Wyatt, V. J. McAuliffe, A. Z. Kapikian, and R. M. Chanock, J. Virol. 37:994-999, 1981). We hypothesized that the 32K rNV cleavage product might be analogous to the 30K soluble protein detected in stools of NV-infected volunteers. Immunoprecipitation of soluble protein from these stool extracts with a rabbit polyclonal antiserum made against rNV, and Western blot detection with a mouse polyclonal antiserum made against rNV, revealed a single band with an apparent molecular weight of 30,000 that migrated similarly to the trypsin cleavage product observed in vitro. The N terminus of this band was identical to that of the 32K cleavage product of rNV capsid protein. These data show that the 30K protein in stool is produced by specific cleavage of the NV capsid protein in vivo. Trypsin cleavage of isolated soluble rNV 58K capsid protein and of assembled particles showed that only soluble 58K capsid protein is susceptible to cleavage. The presence of a large amount of soluble capsid protein may influence the immune response to or pathogenicity of NV infections.
PMCID: PMC188770  PMID: 7853506
9.  Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a cross-reactive, neutralizing VP4-specific epitope. 
Journal of Virology  1992;66(1):432-439.
We previously reported that the expression of rotavirus phenotypes by reassortants was affected by recipient genetic background and proposed specific interactions between the outer capsid proteins VP4 and VP7 as the basis for the phenotypic effects (D. Chen, J. W. Burns, M. K. Estes, and R. F. Ramig, Proc. Natl. Acad. Sci. USA 86:3743-3747, 1989). A neutralizing, cross-reactive VP4-specific monoclonal antibody (MAb), 2G4, was used to probe the protein-protein interactions. The VP4 specificity of 2G4 was confirmed by immunoblot analysis. MAb 2G4 reacted with both standard (SA11-C13) and variant rotavirus SA11 (SA11-4F) but did not react with bovine rotavirus B223 as determined by plaque reduction neutralization (PRN) and enzyme-linked immunosorbent assay (ELISA). When a panel of SA11-4F/B223 and SA11-Cl3/B223 reassortants in purified or crude lysate form that had been grown in the presence or absence of trypsin was analyzed with MAb 2G4 by PRN and ELISA, the results with some reassortants were unexpected. That is, MAb 2G4 reacted with VP4 of SA11 parental origin (4F or C13) when it was assembled into capsids with the homologous SA11 VP7 but failed to react with VP4 of SA11 assembled into capsids with heterologous B223 VP7. Conversely, MAb 2G4 failed to react with VP4 of B223 parental origin when it was assembled into capsids with homologous B223 VP7 but did react with B223 VP4 assembled into capsids with the heterologous SA11 VP7. Similar reactivity was observed when 2G4 was used to immunoprecipitate purified double-shelled virions. When soluble unassembled viral proteins were analyzed by ELISA, the 2G4 reactive pattern was as predicted from the parental origin of VP4. That is, 2G4 reacted with the soluble VP4 of reassortants having VP4 from SA11-Cl3 or SA11-4F and failed to react with VP4 of B223 origin, regardless of the origin of VP7. PRN and ELISA results obtained with nonglycosylated viruses revealed that the unexpected reactivity of 2G4 with virus particles was not the result of differential glycosylation of VP7 and epitope masking. These results indicate that the 2G4 epitope existed in the soluble form of VP4 encoded by SA11-Cl3 or SA11-4F but not in soluble B223 VP4. On the other hand, in assembled virions, the presentation of the 2G4 epitope on VP4 was unexpected in some reassortants and was affected by the specific interactions between VP4 and VP7 of heterologous parental origin.
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PMCID: PMC238303  PMID: 1370090
10.  Prevalence of antibodies to Norwalk virus in England: detection by enzyme-linked immunosorbent assay using baculovirus-expressed Norwalk virus capsid antigen. 
Journal of Clinical Microbiology  1993;31(4):1022-1025.
A total of 3,250 serum specimens collected in England in 1991 and 1992 were tested by an indirect enzyme-linked immunosorbent assay for antibody to Norwalk virus using baculovirus-expressed capsid antigen, and 2,382 (73.3%) were positive. The prevalence of Norwalk virus antibody differed regionally. It was lowest (24.6%) in 6- to 11-month-old infants and increased to 89.7% in persons over 60 years old.
PMCID: PMC263611  PMID: 8385148
11.  Norwalk virus-associated gastroenteritis traced to ice consumption aboard a cruise ship in Hawaii: comparison and application of molecular method-based assays. 
Journal of Clinical Microbiology  1994;32(2):318-322.
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.
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PMCID: PMC263031  PMID: 8150941
12.  Characterization of DNA-protein complexes from simian adenovirus SA7. 
Journal of Virology  1978;25(3):917-922.
DNA-protein complexes prepared from purified simian adenovirus SA7 virions and from lytically infected monkey kidney cells exhibited similar properties when compared with respect to size by sucrose gradient centrifugation, to configuration by electron microscopy, and to susceptibility to a variety of treatments by electron microscopy and electrophoresis in agarose gels.
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PMCID: PMC525987  PMID: 205679
13.  Electron microscopy procedure influences detection of rotaviruses. 
Journal of Clinical Microbiology  1987;25(10):1902-1906.
Technical parameters of electron microscope staining procedures (type of stain, pH of stain, and time of staining) influence particle integrity for three groups of rotaviruses. Simian rotavirus SA11 (group A), Chinese adult diarrhea rotavirus and porcine rotavirus-like agent (group B), and porcine pararotavirus (group C) were tested. All rotavirus strains were quite stable in uranyl acetate and phosphotungstic acid at pH 4.5 and relatively stable in ammonium molybdate. However, staining with phosphotungstic acid at higher pH values with increased staining time yielded a reduction in the number of particles and particles that were broken or degraded to single-shelled particles or core particles. The different staining procedures were also tested in immunoelectron microscopy experiments. Antibody molecules bound to rotavirus particles were observed clearly only with phosphotungstic acid staining and not with uranyl acetate. We therefore recommend that uranyl acetate and phosphotungstic acid at pH 4.5 be used for negative staining of rotaviruses; phosphotungstic acid at pH 4.5 is optimal for immunoelectron microscopy. These technical points may be critical for rotavirus detection and are important for studies pertaining to the epidemiology and clinical importance of the non-group A rotaviruses.
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PMCID: PMC269364  PMID: 2444622
14.  Comparison of methods for immunocytochemical detection of rotavirus infections. 
Infection and Immunity  1979;26(2):686-689.
Rotavirus infections in intestinal tissues of animals or in tissue culture cells were detected by the immunocytochemical unlabeled soluble enzyme peroxidase antiperoxidase method. Comparison of the immunofluorescence and peroxidase antiperoxidase immunological staining techniques revealed that the two methods are equally sensitive for detection of rotavirus-infected cells. The peroxidase antiperoxidase technique offers the advantages of negligible nonspecific staining reactions, the use of a standard light microscope, the production of permanent slides, and the conservation of immunological reagents. The ability to detect antigens in paraffin-embedded tissues enhances the usefulness of the peroxidase antiperoxidase test for both prospective and retrospective studies.
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PMCID: PMC414670  PMID: 232694
15.  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.
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PMCID: PMC201581  PMID: 8031087
17.  Subclass-specific serum antibody responses to recombinant Norwalk virus capsid antigen (rNV) in adults infected with Norwalk, Snow Mountain, or Hawaii virus. 
Journal of Clinical Microbiology  1993;31(6):1630-1634.
Subclass-specific antibody responses to the Norwalk virus capsid protein in adults challenged with Norwalk, Snow Mountain, or Hawaii virus were evaluated by solid-phase enzyme immunoassay using recombinant Norwalk virus capsid antigen (rNV). Fourfold or greater serum immunoglobulin G (IgG) antibody responses to rNV were detected in 15 of 20 volunteers challenged with Norwalk virus, and serum IgA and IgM antibody responses to rNV were seen in almost all subjects who had rNV IgG responses. Serum rNV IgG antibody responses also were detected in 6 of 15 volunteers challenged with Snow Mountain virus and 2 of 12 volunteers challenged with the Hawaii virus. However, the magnitude of antibody response and the geometric mean postchallenge rNV IgG antibody titers were lower in subjects challenged with Snow Mountain or Hawaii virus, and serum IgA and IgM responses generally did not occur.
PMCID: PMC265593  PMID: 8391025
18.  Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms 
Journal of Virology  2006;80(12):6061-6071.
Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.
doi:10.1128/JVI.02167-05
PMCID: PMC1472611  PMID: 16731945
19.  Seroepidemiology of adult diarrhea rotavirus in China, 1977 to 1987. 
Journal of Clinical Microbiology  1989;27(10):2180-2183.
In 1982, large outbreaks of diarrhea that were caused by group B adult diarrhea rotavirus (ADRV) occurred throughout the People's Republic of China. Until 1982, group B rotavirus had never been associated with disease in humans. To determine whether ADRV was a new virus introduced in 1982 or had been present before that time, we examined antibody titers of ADRV in gamma globulin (pooled immunoglobulin) pools that were prepared during 1977 to 1987 in four cities in the People's Republic of China (Shanghai, Lanzhou, Wuhan, and Chandu). ADRV antibodies were assayed by using a blocking enzyme-linked immunosorbent assay. Antibodies were present in most Chinese gamma globulins tested, including those collected in Shanghai before the 1982 epidemic, and absent from American reference pools. The highest titers of antibody to ADRV (3,200) were found in gamma globulins collected in 1983 in Shanghai just after the epidemic, and these were fourfold higher than titers present in the preceding years. The quality of the gamma globulins stored for up to 12 years was tested by measuring levels of immunoglobulin G to group A rotavirus; these were equally high in gamma globulin pools prepared in the United States and in all samples from the People's Republic of China. Serum samples from patients from an outbreak of ADRV had elevated titers to ADRV 3 and 16 months after the onset of symptoms. These findings, as well as other epidemiologic findings on ADRV, suggest that the organism is an important and continuing cause of diarrhea in the People's Republic of China, was present before the first epidemic in 1982, and represents a risk to surrounding populations in Asia.
PMCID: PMC266989  PMID: 2479654
21.  Infant morbidity in an Indian slum birth cohort 
Archives of disease in childhood  2007;93(6):479-484.
Objective
To establish incidence rates, clinic referrals, hospitalisations, mortality rates and baseline determinants of morbidity among infants in an Indian slum.
Design
A community-based birth cohort with twice-weekly surveillance.
Setting
Vellore, South India.
Subjects
452 newborns recruited over 18 months, followed through infancy.
Main outcome measures
Incidence rates of gastrointestinal illness, respiratory illness, undifferentiated fever, other infections and non-infectious morbidity; rates of community-based diagnoses, clinic visits and hospitalisation; and rate ratios of baseline factors for morbidity.
Results
Infants experienced 12 episodes (95% confidence interval (CI) 11 to 13) of illness, spending about one fifth of their infancy with an illness. Respiratory and gastrointestinal symptoms were most common with incidence rates (95% CI) of 7.4 (6.9 to 7.9) and 3.6 (3.3 to 3.9) episodes per child-year. Factors independently associated with a higher incidence of respiratory and gastrointestinal illness were age (3-5 months), male sex, cold/wet season and household involved in beedi work. The rate (95% CI) of hospitalisation, mainly for respiratory and gastrointestinal illness, was 0.28 (0.22 to 0.35) per child-year.
Conclusions
The morbidity burden due to respiratory and gastrointestinal illness is high in a South Indian urban slum, with children ill for approximately one fifth of infancy, mainly with respiratory and gastrointestinal illnesses. The risk factors identified were younger age, male sex, cold/wet season and household involvement in beedi work.
doi:10.1136/adc.2006.114546
PMCID: PMC2682775  PMID: 17916587
22.  Simian rotavirus SA11 replication in cell cultures. 
Journal of Virology  1979;31(3):810-815.
Understanding the basic virology of rotavirus infections has been hampered by the fastidiousness of most isolates and by the lack of a rapid quantitative assay method. The growth characteristics of the simian rotavirus SA11 were studied because it grows to high titers in tissue culture and infectivity can be quantitated by plaque assay. SA11 replication was analyzed in a variety of primary cell cultures or continuous cell lines derived from both homologous and heterologous hosts. Viral replication was observed in each of the cell cultured examined. The individual cell cultures demonstrated marked variability in their susceptibility to rotavirus infection. The highest titers were obtained with MA104, BSC-1, CV-1, and BGM cells. Observable cytopathic effect was found to correlate with the percentage of infected cells in the culture. This study presents growth curves of the simian rotavirus in a variety of cell cultures.
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PMCID: PMC353508  PMID: 229253
23.  Serological characterization of bovine rotaviruses isolated from dairy and beef herds in Argentina. 
Journal of Clinical Microbiology  1989;27(11):2619-2623.
Bovine rotaviruses isolated from beef and dairy herds in Argentina were serotyped by the immunoperoxidase focus reduction assay as previously described (G. Gerna, M. Battaglia, G. Milenesi, N. Passarani, E. Percivalle, and E. Cattaneo, Infect. Immun. 43:722-729, 1984). Three strains from beef herds were related to the UK and NCDV bovine rotavirus strains defined as serotype 6 (Y. Hoshino, R. G. Wyatt, H. B. Greenberg, J. Flores, and A. Z. Kapikian, J. Infect. Dis. 149:694-702, 1984). Two other strains from dairy herds were classified as bovine viruses related to the bovine B223 strain reported by Woode and co-workers (G. N. Woode, N. E. Kelso, T. F. Simpson, S. K. Gaul, L. E. Evans, and L. Babiuk, J. Clin. Microbiol. 18:358-364, 1983) in the United States. A serotyping antibody-capture enzyme-linked immunoassay to detect serotype 6 rotavirus using a serotype 6-specific monoclonal antibody was developed and evaluated for strain characterization. Characterization of 72 group A rotavirus-positive fecal samples from beef herds and 43 fecal samples from dairy herds showed a predominance of serotype 6 rotavirus in beef herds but both serotype 6 and non-serotype 6 rotaviruses in dairy herds. Analysis of genomic double-stranded RNA by polyacrylamide gel electrophoresis showed that when outbreaks were caused by one serotype only a single electropherotype was present in all samples.
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PMCID: PMC267089  PMID: 2553769
24.  In situ hybridization for quantitative assay of infectious hepatitis A virus. 
Journal of Clinical Microbiology  1989;27(5):874-879.
A method of in situ hybridization using single-stranded RNA probes of opposite polarity for quantitative enumeration of hepatitis A virus (HAV) in infected cells has been developed. Kinetic experiments showed that foci of infected cells appeared as early as day 2 postinfection. The absence of foci in cells examined immediately after virus adsorption indicated that foci detected subsequently were related to viral replication. Foci were detected by hybridization with RNA probes complementary to HAV genomic RNA but not with RNA probes identical to HAV genomic RNA. The number of foci observed was linearly related to the HAV dose inoculated. Focus formation was reduced when a virus inoculum was pretreated with guinea pig anti-HAV hyperimmune serum but not when it was pretreated with preimmune serum. The high resolution of hybridization signals and relative rapidity of the test indicated that this technique will be useful for measuring serum neutralizing antibodies and for quantitative assay of infectious HAV.
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PMCID: PMC267446  PMID: 2545742
25.  Characterization of monoclonal antibodies to human group B rotavirus and their use in an antigen detection enzyme-linked immunosorbent assay. 
Journal of Clinical Microbiology  1989;27(2):245-250.
Three monoclonal antibodies (MAbs)--B5C9, B5E4, and B10G10--to human group B rotavirus, an agent implicated in epidemic outbreaks of diarrhea in the People's Republic of China, primarily in adults, were prepared. MAb reactivity was decreased when virus preparations were treated with EDTA, suggesting reactivity with the outer-capsid protein(s). Competition experiments suggested that these MAbs recognize overlapping epitopes within a single antigenic site. A simple antigen detection enzyme-linked immunosorbent assay (ELISA) specific for the human group B rotavirus was established by using these MAbs as capture antibodies. Fifteen clinical samples obtained from three epidemic areas in the People's Republic of China and previously shown by Chinese scientists to contain group B virus were all positive in the MAb capture antigen detection ELISA, whereas none of the 57 samples lacking the group B virus reacted in the test. The results suggest that this MAb capture antigen detection ELISA will be useful to identify outbreaks caused by the human group B rotavirus and to monitor possible spread of the virus.
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PMCID: PMC267285  PMID: 2536755

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