Juan E. Ludert

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.

Endocytic pathway followed by dengue virus to infect the mosquito cell line C6/36 HT.

The endocytic pathway followed by dengue virus to infect the mosquito cells C6/36 HT was analyzed. Using DIL-labeled virions and real-time imaging it was determined that viral entry into C6/36 HT takes approximately 5 to 7 min. Pretreatment of C6/36 HT cells with sucrose and bafilomycin A, but not filipin, inhibited dengue virus infection up to 80%. Furthermore, the overexpression of dominant-negative mutants of Eps15, a molecule required for the formation of clathrin-coated vesicles, reduced dengue infection up to 50%, indicating that dengue virus entry is through clathrin-mediated endocytosis and is pH-dependent. By double-immunofluorescence assays, DIL-labeled particles were colocalized with early endosomes at 5 min and with lysosomes mainly at 30 min post-infection. Finally, disruption of the microtubule and microfilaments by nocodazole and by cytochalasin D reduced viral infection by more than 80%. Taken together these results indicate that dengue virions enter into C6/36 HT cells by clathrin-mediated endocytosis, using the endosomal pathway from early endosomes to acidic lysosomes before viral RNA is released into the cytoplasm.

A novel type of VP4 carried by a porcine rotavirus strain.

The gene encoding the VP8* trypsin-cleavage product of the VP4 protein of porcine rotavirus strain A34 was sequenced, and the predicted amino acid (aa) sequence was compared to the homologous region of all known P genotypes. The aa sequence of the VP8* of strain A34 shared low identity, ranging from 39% (bovine strain B223, P8[11]) to 76% (human strain 69M, P4[10]), with the homologous sequences of representative strains of the remaining 21 P genotypes. Phylogenetic relationships showed that the VP8* of strain A34 shares a common evolutionary lineage with those of human 69M (P4[10]) and equine H-2 (P4[12]) strains. Hyperimmune sera raised to strain A34 and to a genetic reassortant strain containing the VP4 gene from strain A34, both with high homologous neutralization titer via VP4, failed to neutralize strains representative of 15 different P genotypes. These results indicate that strain A34 should be considered as prototype of a new P genotype and serotype (P14[23]) and provide further evidence for the vast genetic and antigenic diversity of group A rotaviruses.

Initial Interaction of Rotavirus Strains with N-Acetylneuraminic (Sialic) Acid Residues on the Cell Surface Correlates with VP4 Genotype, Not Species of Origin

ABSTRACT We examined 41 human and animal rotavirus strains representative of all known P genotypes for their dependency on cellular N-acetylneuraminic (sialic) acid (SA) residues for infectivity. Our results showed that all rotaviruses studied, whether of animal or human origin, belonging to P genotypes [1], [2], [3], and [7] depended on SA residues on the cell surface for efficient infectivity but that all human and animal rotavirus strains representative of the remaining known P genotypes were SA independent. The SA residue requirement for efficient infectivity did not change for reassortant rotavirus strains with altered VP4-VP7 combinations. The initial interaction of rotavirus strains with SA residues on the cell surface correlated with VP4 genotype specifity, not with species of origin or VP7 G serotype specificity (P = 0.001; r2 = 1.00, Pearsons correlation coefficient). In addition to being a requirement for infectivity, the presence of SA residues on the cell surface is a requirement for efficient growth in cell culture; recognition of the association of specific P genotypes with the binding of rotavirus to SA residues will facilitate our understanding of the molecular basis of the early events of rotavirus-cell interactions in cell culture models and of pathogenicity in vivo.

Effect of rotavirus infection on intracellular calcium homeostasis in cultured cells

The effect of rotavirus infection on intracellular [Ca2+] was studied in a model system (MA-104 cells). In cells infected at high multiplicity with the OSU strain of rotavirus, production of infectious viruses was maximal at 6 hr postinfection. Cell death, as measured by incorporation of ethidium bromide, started at 6 hr and was complete at 15 hr postinfection. At 4 hr postinfection, intracellular [Ca2+], measured by quin2 fluorescence, was not modified, but Ca2+ permeability was increased. With progression of the infection, intracellular [Ca2+] and Ca2+ pools increased due to the failure of regulatory mechanisms to compensate increased Ca2+ entry. These effects were blocked by cycloheximide added up to 5 hr postinfection, but not by actinomycin D. Reduced extracellular [Ca2+] afforded protection of cell death induced by infection, under conditions at which production of infectious viruses was not affected. The cytopathic effect of rotavirus on host cells appears to be mediated by an increase in intracellular [Ca2+] induced by the synthesis of a viral product. The failure of ionic homeostasis of the enterocyte might be involved in the development of diarrhea.

Prevalence of enteric viruses in human immunodeficiency virus seropositive patients in Venezuela

The prevalence of enteric viruses associated with gastroenteritis was determined in 125 stool samples from patients infected with the human immunodeficiency virus (HIV), with or without diarrhea. Diagnostic assays included enzyme immunoassays for the identification of rotavirus, adenovirus, and Norwalk virus; polyacrylamide gel electrophoresis for atypical rotaviruses and picobirnaviruses and polymerase chain reaction for astrovirus. Enteric viruses were detected in 6.4% (8 of 125) of the stools collected: five (4.0%) samples positive for adenoviruses, and three (2.3%) samples positive for picobirnaviruses were detected. No rotavirus, astrovirus, or Norwalk virus were observed. Only one of the viruses identified (adenovirus) was found in a sample from a patient with diarrhea. Viruses were detected in 10% of the patients with AIDS, 14% of the symptomatic patients, and none of the asymptomatic persons. These results do not support a major role for enteric viruses in the diarrhea suffered by HIV‐infected patients. J. Med. Virol. 55:288–292, 1998.

Identification in porcine faeces of a novel virus with a bisegmented double stranded RNA genome

SummaryPolyacrylamide gel electrophoresis of nucleic acids extracted from porcine faecal samples revealed in several samples the presence of two discrete bands. The bands were resistant to digestion with DNase I and RNase T1, but not with RNase A in low salt conditions, indicating that they consisted of double stranded (ds) RNA. The two bands from different samples varied in sizes, in a range between 2.4–2.6 kbp and 1.7–1.9 kbp for the slow and fast moving band respectively. The bands cosedimented in CsCl gradients at an average density of 1.415 g/ml with icosahedral virus particles of a diameter of 34nm and a triangulation number equal to 3. Aggregates of virus, which appeared to be immunocomplexes, were seen in one sample. From 244 faecal samples collected in one farm, 27 (11.1%) were found to contain the characteristic dsRNA pattern, with a higher prevalence in samples from animals 15 to 35 days old. The agent was equally distributed among samples from diarrhoeic or non-diarrhoeic animals. These results confirm the circulation among pigs of a novel virus, possibly of vertebrates, with a bisegmented double stranded RNA genome, similar to viruses previously described in humans, wild rats, guinea pigs, pigs, and chickens, for which the name “picobirnavirus” has been proposed.

JNK phosphorylation, induced during dengue virus infection, is important for viral infection and requires the presence of cholesterol.

Infection with a broad diversity of viruses often activates host cell signaling pathways including the mitogen-activated protein kinase pathway. The present study established that dengue virus infection of human macrophages activates Jun NH(2)-terminal kinase (JNK) and the p38 MAPKs pathways. The activation was observed at early times after infection and occurs when either infectious or UV-inactivated dengue virus was used. The role of these activated kinases in dengue virus infection was evaluated using specific inhibitors. Inhibition of JNK and p38 kinases did result in a significant reduction in viral protein synthesis and in viral yield. Additionally, lipid rafts disruption induced a strong inhibition of JNK activation. These results suggest that, at early stages after dengue virus infection, MAPKs are activated and that activation of JNK and p38 is required for dengue virus infection.

The increase in cholesterol levels at early stages after dengue virus infection correlates with an augment in LDL particle uptake and HMG-CoA reductase activity

Several cellular molecules and components, specifically, cholesterol and lipid rafts have been described as necessary elements for dengue virus entry and signaling in several human cells. Thus, changes in lipid rafts formation and cholesterol levels were evaluated. Here we report that the amount of total cholesterol and lipid rafts formation increase early after infection of Huh-7 cells. This augment correlates with an increase in the amount of low density lipoprotein receptor (LDLr) on the surface of infected cells and also with a lower phosphorylation level of the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). None of the changes were observed in Huh 7 cells infected with VSV used as a control. These results suggest that dengue virus infection increases intracellular cholesterol levels at early times post infection by triggering the modulation of LDL particles uptake and the increase in the enzymatic activity of HMG-CoA reductase.

Dissecting Rotavirus Particle-Raft Interaction with Small Interfering RNAs: Insights into Rotavirus Transit through the Secretory Pathway

ABSTRACT Studies of rotavirus morphogenesis, transport, and release have shown that although these viruses are released from the apical surface of polarized intestinal cells before cellular lysis, they do not follow the classic exocytic pathway. Furthermore, increasing evidence suggests that lipid rafts actively participate in the exit of rotavirus from the infected cell. In this study, we silenced the expression of VP4, VP7, and NSP4 by using small interfering RNAs (siRNAs) and evaluated the effect of shutting down the expression of these proteins on rotavirus-raft interactions. Silencing of VP4 and NSP4 reduced the association of rotavirus particles with rafts; in contrast, inhibition of VP7 synthesis slightly affected the migration of virions into rafts. We found that inhibition of rotavirus migration into lipid rafts, by either siRNAs or tunicamycin, also specifically blocked the targeting of VP4 to rafts, suggesting that the association of VP4 with rafts is mostly mediated by the formation of viral particles in the endoplasmic reticulum (ER). We showed that two populations of VP4 exist, one small population that is independently targeted to rafts and a second large pool of VP4 whose association with rafts is mediated by particle formation in the ER. We also present evidence to support the hypothesis that assembly of VP4 into mature virions takes place in the late stages of transit through the ER. Finally, we analyzed the progression of rotavirus proteins in the exocytic pathway and found that VP4 and virion-assembled VP7 colocalized with ERGIC-53, suggesting that rotavirus particles transit through the intermediate compartment between the ER and the Golgi complex.