Sara E. Miller

Norwalk virus: How infectious is it?†

Noroviruses are major agents of viral gastroenteritis worldwide. The infectivity of Norwalk virus, the prototype norovirus, has been studied in susceptible human volunteers. A new variant of the hit theory model of microbial infection was developed to estimate the variation in Norwalk virus infectivity, as well as the degree of virus aggregation, consistent with independent (electron microscopic) observations. Explicit modeling of viral aggregation allows us to express virus infectivity per single infectious unit (particle). Comparison of a primary and a secondary inoculum showed that passage through a human host does not change Norwalk virus infectivity. We estimate the average probability of infection for a single Norwalk virus particle to be close to 0.5, exceeding that reported for any other virus studied to date. Infected subjects had a dose‐dependent probability of becoming ill, ranging from 0.1 (at a dose of 103 NV genomes) to 0.7 (at 108 virus genomes). A norovirus dose response model is important for understanding its transmission and essential for development of a quantitative risk model. Norwalk virus is a valuable model system to study virulence because genetic factors are known for both complete and partial protection; the latter can be quantitatively described as heterogeneity in dose response models. J. Med. Virol. 80:1468–1476, 2008.

Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells

Enterotoxigenic Escherichia coli (ETEC) is a prevalent cause of travelers diarrhea and infant mortality in third‐world countries. Heat‐labile enterotoxin (LT) is secreted from ETEC via vesicles composed of outer membrane and periplasm. We investigated the role of ETEC vesicles in pathogenesis by analyzing vesicle association and entry into eukaryotic cells. Fluorescently labeled vesicles from LT‐producing and LT‐nonproducing strains were compared in their ability to bind adrenal and intestinal epithelial cells. ETEC‐derived vesicles, but not control nonpathogen‐derived vesicles, associated with cells in a time‐, temperature‐, and receptor‐dependent manner. Vesicles were visualized on the cell surface at 4°C and detected intracellularly at 37°C. ETEC vesicle endocytosis depended on cholesterol‐rich lipid rafts. Entering vesicles partially colocalized with caveolin, and the internalized vesicles accumulated in a nonacidified compartment. We conclude that ETEC vesicles serve as specifically targeted transport vehicles that mediate entry of active enterotoxin and other bacterial envelope components into host cells. These data demonstrate a role in virulence for ETEC vesicles.

Diagnosis And Management Of Bk Polyomavirus Interstitial Nephritis In Renal Transplant Recipients

BACKGROUND Interstitial nephritis caused by BK polyomavirus is a recognized complication of renal transplantation. A study of renal transplant recipients at Duke University Medical Center was undertaken to evaluate diagnostic modalities and assess clinical outcomes in transplant polyomavirus infections. METHODS Polyomavirus nephritis was identified in 6 of 240 patients who received renal transplants between January 1996 and June 1998 and an additional patient who underwent transplantation in 1995. The clinical records of these seven patients were reviewed, as were all renal biopsy and nephrectomy specimens. Electron microscopy (EM) was performed on negatively stained urine samples from 6 patients with polyomavirus infection and 23 patients with other diagnoses. RESULTS Patients with polyomavirus infection shared several clinical features, including ureteral obstruction (5/7 patients), lymphocele (3/7), bacterial urinary tract infection (3/7), hematuria (3/7), cytomegalovirus infection (3/7), and immunosuppression with mycophenolate mofetil (6/7). All patients experienced elevations in serum creatinine, which stabilized or decreased in four patients with altered or decreased immunosuppression. The diagnosis of polyomavirus infection was established by renal biopsy and EM of urine in five patients, by biopsy alone in one, and by EM alone in one. Sequential examinations of urine by EM were used to monitor the course of infection in six patients. CONCLUSIONS Interstitial nephritis due to BK polyomavirus occurred in 2.5% of patients receiving renal transplants at our center since 1996. Polyomavirus infection can cause transplant dysfunction and graft loss, but progression of the infection can frequently be abrogated with alterations in immunosuppressive therapy. Both renal biopsy and EM of urine samples are useful in the diagnosis and monitoring of polyomavirus infections.

TDP-43 accumulation in inclusion body myopathy muscle suggests a common pathogenic mechanism with frontotemporal dementia

TAR DNA binding protein-43 (TDP-43) is found in ubiquitinated inclusions (UBIs) in some frontotemporal dementias (FTD-U). One form of FTD-U, due to mutations in the valosin containing protein (VCP) gene, occurs with an inclusion body myopathy (IBMPFD). Since IBMPFD brain has TDP-43 in UBIs, we looked for TDP-43 inclusions in IBMPFD muscle. In normal muscle, TDP-43 is present in nuclei. In IBMPFD muscle, TDP-43 is additionally present as large inclusions within UBIs in muscle cytoplasm. TDP-43 inclusions were also found in 78% of sporadic inclusion body myositis (sIBM) muscles. In IBMPFD and sIBM muscle, TDP-43 migrated with an additional band on immunoblot similar to that reported in FTD-U brains. This study adds sIBM and hereditary inclusion body myopathies to the growing list of TDP-43 positive inclusion diseases.

THE G PROTEIN-COUPLED RECEPTOR KINASE 2 IS A MICROTUBULE-ASSOCIATED PROTEIN KINASE THAT PHOSPHORYLATES TUBULIN

The G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase that phosphorylates and desensitizes agonist-occupied G protein-coupled receptors (GPCRs). Here we demonstrate that GRK2 is a microtubule-associated protein and identify tubulin as a novel GRK2 substrate. GRK2 is associated with microtubules purified from bovine brain, forms a complex with tubulin in cell extracts, and colocalizes with tubulin in living cells. Furthermore, an endogenous tubulin kinase activity that copurifies with microtubules has properties similar to GRK2 and is inhibited by anti-GRK2 monoclonal antibodies. Indeed, GRK2 phosphorylates tubulinin vitro with kinetic parameters very similar to those for phosphorylation of the agonist-occupied β2-adrenergic receptor, suggesting a functionally relevant role for this phosphorylation event. In a cellular environment, agonist occupancy of GPCRs, which leads to recruitment of GRK2 to the plasma membrane and its subsequent activation, promotes GRK2-tubulin complex formation and tubulin phosphorylation. These findings suggest a novel role for GRK2 as a GPCR signal transducer mediating the effects of GPCR activation on the cytoskeleton.

Modern Uses of Electron Microscopy for Detection of Viruses

SUMMARY Electron microscopy, considered by some to be an old technique, is still on the forefront of both clinical viral diagnoses and viral ultrastructure and pathogenesis studies. In the diagnostic setting, it is particularly valuable in the surveillance of emerging diseases and potential bioterrorism viruses. In the research arena, modalities such as immunoelectron microscopy, cryo-electron microscopy, and electron tomography have demonstrated how viral structural components fit together, attach to cells, assimilate during replication, and associate with the cellular machinery during replication and egression. These studies provide information for treatment and vaccine strategies.

Impaired Protein Aggregate Handling and Clearance Underlie the Pathogenesis of p97/VCP-associated Disease

Mutations in p97/VCP cause the multisystem disease inclusion body myopathy, Paget disease of the bone and frontotemporal dementia (IBMPFD). p97/VCP is a member of the AAA+ (ATPase associated with a variety of activities) protein family and has been implicated in multiple cellular processes. One pathologic feature in IBMPFD is ubiquitinated inclusions, suggesting that mutations in p97/VCP may affect protein degradation. The present study shows that IBMPFD mutant expression increases ubiquitinated proteins and susceptibility to proteasome inhibition. Co-expression of an aggregate prone protein such as expanded polyglutamine in IBMPFD mutant cells results in an increase in aggregated protein that localizes to small inclusions instead of a single perinuclear aggresome. These small inclusions fail to co-localize with autophagic machinery. IBMPFD mutants avidly bind to these small inclusions and may not allow them to traffic to an aggresome. This is rescued by HDAC6, a p97/VCP-binding protein that facilitates the autophagic degradation of protein aggregates. Expression of HDAC6 improves aggresome formation and protects IBMPFD mutant cells from polyglutamine-induced cell death. Our study emphasizes the importance of protein aggregate trafficking to inclusion bodies in degenerative diseases and the therapeutic benefit of inclusion body formation.

Carrier Detection in Duchenne Muscular Dystrophy

We measured endogenous phosphorylation of peak II (apparent molecular weight of 220,000 daltons) of the erythrocyte membrane in 21 mothers of patients with Duchenne muscular dystrophy. The mean values of mothers with affected sons were significantly increased over those of matched controls (77.0 and 55.8 pmoles per milligram of 15-minute incubation; P less than 0.01). Detailed testing of mothers of affected sons revealed proximal muscle weakness. Seven mothers of isolated patients who had normal levels of creatine phosphokinase and no daughters with elevated levels were identified as carriers, because their mean value of peak II phosphorylation was increased (75.9 pmoles per milligram per 15 minutes) and equivalent to the level demonstrated in the 14 acknowledged carriers. Our results suggest that cases of Duchenne muscular dystrophy previously considered to be new mutations are much less common than estimated.

Cryptosporidium, Enterocytozoon, and Cyclospora Infections in Pediatric and Adult Patients with Diarrhea in Tanzania

Cryptosporidiosis, microsporidiosis, and cyclosporiasis were studied in four groups of Tanzanian inpatients: adults with AIDS-associated diarrhea, children with chronic diarrhea (of whom 23 of 59 were positive [+] for human immunodeficiency virus [HIV]), children with acute diarrhea (of whom 15 of 55 were HIV+), and HIV control children without diarrhea. Cryptosporidium was identified in specimens from 6/86 adults, 5/59 children with chronic diarrhea (3/5, HIV+), 7/55 children with acute diarrhea (0/7, HIV+), and 0/20 control children. Among children with acute diarrhea, 7/7 with cryptosporidiosis were malnourished, compared with 10/48 without cryptosporidiosis (P < .01). Enterocytozoon was identified in specimens from 3/86 adults, 2/59 children with chronic diarrhea (1 HIV+), 0/55 children with acute diarrhea, and 4/20 control children. All four controls were underweight (P < .01). Cyclospora was identified in specimens from one adult and one child with acute diarrhea (HIV-). Thus, Cryptosporidium was the most frequent and Cyclospora the least frequent pathogen identified. Cryptosporidium and Enterocytozoon were associated with malnutrition. Asymptomatic fecal shedding of Enterocytozoon in otherwise healthy, HIV children has not been described previously.

Induction of Plasma (TRAIL), TNFR-2, Fas Ligand, and Plasma Microparticles after Human Immunodeficiency Virus Type 1 (HIV-1) Transmission: Implications for HIV-1 Vaccine Design

ABSTRACT The death of CD4+ CCR5+ T cells is a hallmark of human immunodeficiency virus (HIV) infection. We studied the plasma levels of cell death mediators and products—tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas ligand, TNF receptor type 2 (TNFR-2), and plasma microparticles—during the earliest stages of infection following HIV type 1 (HIV-1) transmission in plasma samples from U.S. plasma donors. Significant plasma TRAIL level elevations occurred a mean of 7.2 days before the peak of plasma viral load (VL), while TNFR-2, Fas ligand, and microparticle level elevations occurred concurrently with maximum VL. Microparticles had been previously shown to mediate immunosuppressive effects on T cells and macrophages. We found that T-cell apoptotic microparticles also potently suppressed in vitro immunoglobulin G (IgG) and IgA antibody production by memory B cells. Thus, release of TRAIL during the onset of plasma viremia (i.e., the eclipse phase) in HIV-1 transmission may initiate or amplify early HIV-1-induced cell death. The window of opportunity for a HIV-1 vaccine is from the time of HIV-1 transmission until establishment of the latently infected CD4+ T cells. Release of products of cell death and subsequent immunosuppression following HIV-1 transmission could potentially narrow the window of opportunity during which a vaccine is able to extinguish HIV-1 infection and could place severe constraints on the amount of time available for the immune system to respond to the transmitted virus.