Suzanne M. Matsui

Etiology and outcome of diarrhea after marrow transplantation: A prospective study

BACKGROUND/AIMS Acute diarrhea after marrow transplant is usually ascribed to acute graft-vs.-host disease (GVHD) or infection, with a reported 40%-50% incidence of infection. The aim of this study was to determine the incidence of acute diarrhea after transplantation, its causes, and its outcome. METHODS Two hundred ninety-six patients were followed up; patients with diarrhea were studied using standard evaluation of stool plus immunoelectron microscopy; assays for astrovirus, picobirnavirus, and Norwalk virus; and gene-probe methods for toxin-producing Escherichia coli. In 38 patients with diarrhea, intestinal biopsy specimens and duodenal fluid were also analyzed. RESULTS One hundred fifty acute diarrheal episodes developed in 126 patients (an incidence of 43%). Intestinal infection was found in 20 of 150 episodes: viruses (astrovirus, adenovirus, cytomegalovirus, and rotavirus) in 12 patients, nosocomially acquired bacteria (Clostridium difficile and Aeromonas) in 7 patients, and mixed infection in 1 patient. Acute GVHD was responsible for 72 of 150 episodes (48%). Clinical signs and symptoms of infection and GVHD were similar. In 58 of 150 episodes (39%), no clear etiology could be found for self-limited diarrhea. CONCLUSIONS Intestinal infection accounted for 13% and acute GVHD for 48% of diarrheal episodes. The most common infecting organisms were astrovirus, C. difficile, and adenovirus. Most cases of diarrhea after marrow transplant are not caused by infection.

Population-Based Prevalence of Symptomatic and Asymptomatic Astrovirus Infection in Rural Mayan Infants

Symptomatic and asymptomatic astrovirus infection was prospectively determined in a 3-year birth cohort of Mayan infants. Stool samples from 271 infants and 268 older siblings were tested for astrovirus, adenovirus 40/41, rotavirus and Salmonella, Shigella and Campylobacter species. Concurrent diarrhea, vomiting, fever, or anorexia were noted. Astrovirus was detected in 164 infants (61%) and 20 siblings (7%). Rotavirus (4%) and adenovirus 40/41 (13%) were isolated less frequently. Of all diarrheal episodes reported at a visit, 26% (78/305) were associated with astrovirus; 17% (78/452) of astrovirus infections were associated with diarrhea and 9% with other symptoms. Only diarrhea was associated with astrovirus infection (odds ratio, 1.4; 95% confidence interval [CI], 1.07-1.92; P = .01). Of infants with astrovirus, 70% shed at multiple visits over a period of 2-17 weeks (median, 5). The point prevalence of astrovirus infection was significantly higher among infants than siblings (relative risk, 6.18; 95% CI, 3.93-9.72; P < .0001, chi2). Astrovirus was identified throughout the year, peaked in March and May, and decreased in September. In this population, astrovirus was the most common enteric pathogen isolated; symptomatic infection was prevalent among infants.

Molecular determinant of rotavirus neutralization and protection

Publisher Summary Human group A rotaviruses were first identified by Bishop et al. in 1973. In the ensuing 15 years, a great deal was learned about these important pathogens. Rotaviruses are members of the Reoviridae family. They are icosahedral viruses with two concentric capsid layers and a diameter of approximately 75 nm. The outer capsid of the virus contains a t least two primary gene products, VP4 and VP7, the products of genes 4 and 8 or 9, respectively. The chapter takes into account the recently revised nomenclature for rotavirus proteins. Genetic studies have demonstrated that VP4 is the viral hemagglutinin. Rotaviruses are the single most important cause of severe, dehydrating diarrhea in children under the age of 2. These viruses are a major cause of infantile mortality in less developed countries and the most frequent cause of morbidity due to pediatric gastroenteritis in developed countries. Rotaviruses can be isolated from the young of many, if not most, mammalian species, and in many cases these viruses are the cause of infantile diarrhea in the mammalian species from which they are isolated.

The isolation and characterization of a Norwalk virus-specific cDNA.

Norwalk virus, an important cause of epidemic, acute, nonbacterial gastroenteritis in adults and children, has eluded adaptation to tissue culture, the development of an animal model, and molecular cloning. In this study, a portion of the Norwalk viral genome encoding an immunoreactive region was cloned from very small quantities of infected stool using sequence-independent single primer amplification. Six overlapping complementary DNA (cDNA) clones were isolated by immunologic screening. The expressed recombinant protein from a representative clone reacted with six of seven high titer. Norwalk-specific, postinfection sera but not with corresponding preinfection sera. Nucleic acid sequence for all clones defined a single open reading frame contiguous with the lambda gt11-expressed beta-galactosidase protein. Only oligonucleotide probes specific for the positive strand (defined by the open reading frame) hybridized to an RNaseA-sensitive, DNaseI-resistant nucleic acid sequence extracted from Norwalk-infected stool. Furthermore, RNA extracted from serial postinfection, but not preinfection, stools from three of five volunteers hybridized to a Norwalk virus cDNA probe. Clone-specific oligonucleotide probes hybridized with cesium chloride gradient fractions containing purified Norwalk virion. In conclusion, an antigenic, protein-coding region of the Norwalk virus genome has been identified. This epitope has potential utility in future sero- and molecular epidemiologic studies of Norwalk viral gastroenteritis.

Characterization of homotypic and heterotypic VP7 neutralization sites of rhesus rotavirus

The gene 9 nucleotide sequence was determined for rhesus rotavirus and each of 14 viral variants selected for their resistance to neutralizing monoclonal antibodies. Each variant contains a single gene 9, VP7, mutation which permits viral growth in the presence of the antibody. Variant mutations were identified in two distinct neutralization regions. Region A was identified by monoclonal antibodies that are involved in both serotype-specific and serotype cross-reactive neutralization. Region C was identified by serotype-specific neutralizing monoclonal antibodies. Heterotypic neutralizing monoclonal antibody 57-8 selected variants with a mutation at amino acid 94 in the A region, the same amino acid location selected by serotype-specific monoclonal antibodies. Monoclonal antibody 3 selected a VP7 mutation at amino acid 99 resulting in additional N-linked glycosylation of the VP7 protein. Despite the added VP7 glycosylation, variant v3 was not broadly resistant to additional VP7-specific neutralizing monoclonal antibodies.

Processing of Nonstructural Protein 1a of Human Astrovirus

ABSTRACT Astrovirus contains three open reading frames (ORF) on its genomic RNA, ORF1a, ORF1b, and ORF2. ORF1a encodes a 920-amino-acid (aa) nonstructural protein, nsP1a, which displays a 3C-like serine protease motif. Little is known about the processing of nsP1a or whether the protease it contains is active and involved in autocatalytic processing. Here we address both of these matters. Intact and N-terminally deleted forms of ORF1a from human astrovirus serotype 1 were expressed in BHK cells, and nsP1a-derived processing products were immunoprecipitated with an nsP1a-specific antibody or an antibody specific for an N-terminally linked epitope tag. The mapping of the main processing products, p20 and p27, suggests cleavage sites near aa 170, 410, and 655 of nsP1a. Cleavages at around aa 410 and 655, but not aa 170, were abolished when a 9-aa substitution was introduced into the protease motif in nsP1a. The p27 processing product was also found in Caco-2 cells that had been infected with human astrovirus serotype 1, confirming the presence of the cleavage sites at approximately aa 410 and 655.

Studies on intracellular processing of the capsid protein of human astrovirus serotype 1 in infected cells.

Astroviruses are non-enveloped, positive-strand RNA viruses. Their structural (capsid) protein is processed extracellularly into several smaller fragments which are found on the mature viral particle. In addition, intracellular cleavage of the capsid protein has been proposed. However, analysis of capsid protein processing has been hampered by the lack of antibodies to regions near the N and C termini of the protein. Here we describe the construction of two infectious mutants of human astrovirus serotype 1 (HAstV-1), in which amino acids (aa) 11-30 or aa 783-787, respectively, of the 787 aa capsid protein were replaced by tag sequences. Processing of the tagged capsid proteins in infected Caco-2 cells was analysed by immunoprecipitation with specific reagents directed against the tags or against native internal regions of the capsid protein. No intracellular processing of the capsid protein in infected cells could be detected, while assembled viral particles were readily observed within cells.

Host and Viral Factors Affecting the Decreased Immunogenicity of Sabin Type 3 Vaccine after Administration of Trivalent Oral Polio Vaccine to Rural Mayan Children

Abstract Factors affecting immunogenicity of the first 2 doses of oral poliovirus vaccine (OPV) among unimmunized Mayan infants were prospectively evaluated. The relative impact of multiple variables, including mass or routine vaccination, concurrent enteric bacterial (salmonella, shigella, and campylobacter) and viral (adenovirus 40/41, astrovirus, nonpolio enteroviruses, and rotavirus) infections, interference among Sabin vaccine viruses, and preexisting poliovirus antibodies were studied. Sera were available from 181 infants after 2 OPV doses. Seroresponses were 86% to Sabin type 1, 97% to Sabin type 2, and 61% to Sabin type 3 vaccines. Mass versus routine vaccination and preexisting poliovirus antibodies did not affect immunogenicity. By multiple logistic regression analysis, fecal shedding of homologous Sabin strains was associated with increased seroresponses to all Sabin types, especially to Sabin type 3. Decreased OPV immunogenicity was primarily attributable to interference of Sabin type 3 by Sabin type 2. OPV formulations with higher doses of Sabin type 3 could improve immunogenicity among infants in developing countries.

Immature and mature human astrovirus: structure, conformational changes, and similarities to hepatitis E virus.

Abstract Human astroviruses (HAstVs) are a major cause of gastroenteritis. HAstV assembles from the structural protein VP90 and undergoes a cascade of proteolytic cleavages. Cleavage to VP70 is required for release of immature particles from cells, and subsequent cleavage by trypsin confers infectivity. We used electron cryomicroscopy and icosahedral image analysis to determine the first experimentally derived, three-dimensional structures of an immature VP70 virion and a fully proteolyzed, infectious virion. Both particles display T =3 icosahedral symmetry and nearly identical solid capsid shells with diameters of ~350Å. Globular spikes emanate from the capsid surface, yielding an overall diameter of ~440Å. While the immature particles display 90 dimeric spikes, the mature capsid only displays 30 spikes, located on the icosahedral 2-fold axes. Loss of the 60 peripentonal spikes likely plays an important role in viral infectivity. In addition, immature HAstV bears a striking resemblance to the structure of hepatitis E virus (HEV)-like particles, as previously predicted from structural similarity of the crystal structure of the astrovirus spike domain with the HEV P-domain [Dong, J., Dong, L., Méndez, E. & Tao, Y. (2011). Crystal structure of the human astrovirus capsid spike. Proc. Natl. Acad. Sci. USA 108, 12681–12686]. Similarities between their capsid shells and dimeric spikes and between the sequences of their capsid proteins suggest that these viral families are phylogenetically related and may share common assembly and activation mechanisms.

Studies of the Astrovirus Signal That Induces (−1) Ribosomal Frameshifting

Human astroviruses use a (-1) ribosomal frameshift mechanism to regulate expression of the viral RNA-dependent RNA polymerase gene. To evaluate the efficiency of the astrovirus frameshift signal in cell culture, different regions of the frameshift signal were cloned into the rhesus rotavirus VP4 gene and expressed in an infection-transfection transient expression cell-culture system. The various astrovirus-VP4 constructs were transfected into BHK-21 cells infected with a recombinant vaccinia virus that expresses T7 RNA polymerase (vTF7-3). All constructs contain a T7 promoter, a picornavirus internal ribosome entry site, and a T7 terminator. Frameshifted and non-frameshifted proteins were distinguished by immunoprecipitation with monoclonal antibodies specific for either the VP4 amino- or carboxy-terminus. Frameshifting efficiency was calculated as the ratio of radioactive counts in the frameshifted protein to the total counts in both the frameshifted and nonframeshifted proteins as determined by Phosphorimager analysis. We found the efficiency of astrovirus frameshifting in intact cells to be 25-28%, significantly greater than the 5-7% efficiency reported previously in a cell-free uncoupled translation system. Since the transfected plasmid is expressed in the cytoplasm in the infection-transfection system, the frameshifting efficiency determined by this assay may be a more accurate reflection of the level of frameshifting for this RNA virus in which transcription and translation are likely coupled in the cytoplasm of infected cells. This hypothesis is supported by the observation that the level of astrovirus frameshifting is increased three-fold when evaluated in a cell-free coupled transcription-translation system. These studies also confirm in intact cells what was previously determined in cell-free studies: the shifty heptamer is an absolute requirement for astrovirus ribosomal frameshifting, but deletion of sequences downstream of the stem-loop that are potentially involved in pseudoknot formation does not affect the efficiency of frameshifting.