Properties of virion transactivator proteins encoded by primate cytomegaloviruses.

BACKGROUND: Human cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in situations where the immune system is immature or compromised. HCMV immediate early (IE) gene expression is stimulated by the virion phosphoprotein pp71, encoded by open reading frame (ORF) UL82, and this transactivation activity is important for the efficient initiation of viral replication. It is currently recognized that pp71 acts to overcome cellular intrinsic defences that otherwise block viral IE gene expression, and that interactions of pp71 with the cell proteins Daxx and ATRX are important for this function. A further property of pp71 is the ability to enable prolonged gene expression from quiescent herpes simplex virus type 1 (HSV-1) genomes. Non-human primate cytomegaloviruses encode homologs of pp71, but there is currently no published information that addresses their effects on gene expression and modes of action. RESULTS: The UL82 homolog encoded by simian cytomegalovirus (SCMV), strain Colburn, was identified and cloned. This ORF, named S82, was cloned into an HSV-1 vector, as were those from baboon, rhesus monkey and chimpanzee cytomegaloviruses. The use of an HSV-1 vector enabled expression of the UL82 homologs in a range of cell types, and permitted investigation of their abilities to direct prolonged gene expression from quiescent genomes. The results show that all UL82 homologs activate gene expression, and that neither host cell type nor promoter target sequence has major effects on these activities. Surprisingly, the UL82 proteins specified by non-human primate cytomegaloviruses, unlike pp71, did not direct long term expression from quiescent HSV-1 genomes. In addition, significant differences were observed in the intranuclear localization of the UL82 homologs, and in their effects on Daxx. Strikingly, S82 mediated the release of Daxx from nuclear domain 10 substructures much more rapidly than pp71 or the other proteins tested. All UL82 homologs stimulated the early release of ATRX from nuclear domain 10. CONCLUSION: All of the UL82 homolog proteins analysed activated gene expression, but surprising differences in other aspects of their properties were revealed. The results provide new information on early events in infection with cytomegaloviruses.

Evaluation of an interactive, case-based review session in teaching medical microbiology.

BACKGROUND: Oklahoma State University-Center for Health Sciences (OSU-CHS) has replaced its microbiology wet laboratory with a variety of tutorials including a case-based interactive session called Microbial Jeopardy!. The question remains whether the time spent by students and faculty in the interactive case-based tutorial is worthwhile? This study was designed to address this question by analyzing both student performance data and assessing students' perceptions regarding the tutorial. METHODS: Both quantitative and qualitative data were used in the current study. Part One of the study involved assessing student performance using archival records of seven case-based exam questions used in the 2004, 2005, 2006, and 2007 OSU-CHS Medical Microbiology course. Two sample t-tests for proportions were used to test for significant differences related to tutorial usage. Part Two used both quantitative and qualitative means to assess student's perceptions of the Microbial Jeopardy! session. First, a retrospective survey was administered to students who were enrolled in Medical Microbiology in 2006 or 2007. Second, responses to open-ended items from the 2008 course evaluations were reviewed for comments regarding the Microbial Jeopardy! session. RESULTS: Both student performance and student perception data support continued use of the tutorials. Quantitative and qualitative data converge to suggest that students like and learn from the interactive, case-based session. CONCLUSION: The case-based tutorial appears to improve student performance on case-based exam questions. Additionally, students perceived the tutorial as helpful in preparing for exam questions and reviewing the course material. The time commitment for use of the case-based tutorial appears to be justified.

Transient Compound Treatment Induces a Multigenerational Reduction of Oxysterol-Binding Protein (OSBP) Levels and Prophylactic Antiviral Activity.

Oxysterol-binding protein (OSBP) is a lipid transport and regulatory protein required for the replication of Enterovirus genus viruses, which includes many significant human pathogens. Short-term exposure (i.e., 1-6 h) to a low dose (i.e., 1 nM) of the natural product compound OSW-1 induces a reduction of cellular OSBP levels by ∼90% in multiple different cell lines with no measurable cytotoxicity, defect in cellular proliferation, or global proteome reduction. Interestingly, the reduction of OSBP levels persists multiple days after the low-dose, transient OSW-1 compound treatment is ended and the intracellular OSW-1 compound levels drop to undetectable levels. The reduction in OSBP levels is inherited in multiple generations of cells that are propagated after the OSW-1 compound treatment is stopped. The enduring multiday, multigenerational reduction of OSBP levels triggered by the OSW-1 compound is not due to proteasome degradation of OSBP or due to a reduction in OSBP mRNA levels. OSW-1 compound treatment induces transient autophagy in cells, but blocking autophagy does not rescue OSBP levels. Although the specific cellular mechanism of long-term OSBP repression is not yet identified, these results clearly show the existence of an OSBP specific cellular regulation process that is triggered upon treatment with an OSBP-binding compound. The stable reduction of OSBP levels upon short-term, transient OSW-1 compound treatment will be a powerful tool to understand OSBP regulation and cellular function. Additionally, the persistent reduction in OSBP levels triggered by the transient OSW-1 compound treatment substantially reduces viral replication in treated cells. Therefore, the long-term, compound-induced reduction of OSBP in cells presents a new route to broad spectrum anti- Enterovirus activity, including as a novel route to antiviral prophylactic treatment through small molecule targeting a human host protein.

Differing activities of oxysterol-binding protein (OSBP) targeting anti-viral compounds.

Oxysterol-binding Protein (OSBP) is a human lipid-transport protein required for the cellular replication of many types of viruses, including several human pathogens. The structurally-diverse small molecule compounds OSW-1, itraconazole (ITZ), T-00127-HEV2 (THEV) and TTP-8307 (TTP) inhibit viral replication through interaction with the OSBP protein. The OSW-1 compound reduces intracellular OSBP, and the reduction of OSBP protein levels persists multiple days after the OSW-1-compound treatment is stopped. The OSW-1-induced reduction of OSBP levels inhibited Enterovirus replication prophylactically in cells. In this report, the OSBP-interacting compounds ITZ, THEV, and TTP are shown not to reduce OSBP levels in cells, unlike the OSW-1-compound, and the OSW-1 compound is determined to be the only compound capable of providing prophylactic antiviral activity in cells. Furthermore, OSW-1 and THEV inhibit the binding of 25-hydroxycholesterol (25-OHC) to OSBP indicating that these compounds bind at the conserved sterol ligand binding site. The ITZ and TTP compounds do not inhibit 25-hydroxycholesterol binding to OSBP, and therefore ITZ and TTP interact with OSBP through other, unidentified binding sites. Co-administration of the THEV compound partially blocks the cellular activity of OSW-1, including the reduction of cellular OSBP protein levels; co-administration of the ITZ and TTP compounds have minimal effect on OSW-1 cellular activity further supporting different modes of interaction with these compounds to OSBP. OSW-1, ITZ, THEV, and TTP treatment alter OSBP cellular localization and levels, but in four distinct ways. Co-administration of OSW-1 and ITZ induced OSBP cellular localization patterns with features similar to the effects of ITZ and OSW-1 treatment alone. Based on these results, OSBP is capable of interacting with multiple structural classes of antiviral small molecule compounds at different binding sites, and the different compounds have distinct effects on OSBP cellular activity.

Evaluation of the sanitization effectiveness of a denture-cleaning product on dentures contaminated with known microbial flora. An in vitro study.

OBJECTIVES: To see if dentures contaminated with Staphylococcus aureus, Pseudomas aeruginosa, Bacillus cereus, Candida albicans, and herpes simplex virus 1 could be effectively decontaminated by using Medical Tabs for Dentures. METHOD AND MATERIALS: Ten methylmethacrylate dentures with processed soft liners (soft-liner dentures) and 10 methylmethacrylate dentures without processed soft liners (hard dentures) were aseptically fragmented and individually incubated with a target microorganism. Test denture fragments were immersed in Medical for 5 minutes, vortexed for 5 minutes, and serially diluted onto media. The control denture fragments were similarly treated in sterile water. For virus contamination, denture fragments were contaminated with 1.2 x 10(9) tissue culture infective dose (TCID)50/mL. They were treated with either Medical for 5 minutes (test fragments) or water (controls) for 5 minutes. Serial dilutions were performed and viral (TCID)50/mL titers were calculated using the Reed-Muench method. RESULTS: Medical treatments effectively eliminated C. albicans, S. aureus, and P. aeruginosa from soft-liner dentures. Treatment of hard dentures eradicated C. albicans and reduced the numbers of S. aureus and P. aeruginosa to < 10. B. cereus showed a reduction of 10 microorganisms in hard dentures while the soft-liner dentures did not show an appreciable reduction. Viral analyses found that both types of dentures retained large amounts of virus when washed with water, but no virus was recovered from any of the 40 samples treated with Medical. CONCLUSION: A single use of Medical Tabs for Dentures is effective in eliminating certain species of microorganisms, including selected viruses, in vitro.

A naturally occurring fatal case of Herpesvirus papio 2 pneumonia in an infant baboon (Papio hamadryas anubis).

Here we describe the unusual finding of herpesvirus pneumonia in a 7-d-old infant baboon (Papio hamadryas anubis). This animal had been separated from its dam the morning of its birth and was being hand-reared for inclusion in a specific pathogen-free colony. The baboon was presented for anorexia and depression of 2 d duration. Physical examination revealed a slightly decreased body temperature, lethargy, and dyspnea. The baboon was placed on a warm-water blanket and was given amoxicillin-clavulanate orally and fluids subcutaneously. The animal's clinical condition continued to deteriorate despite tube feeding, subcutaneous fluid administration, and antibiotic therapy, and it died 2 d later. Gross necropsy revealed a thin carcass and severe bilateral diffuse pulmonary consolidation. Histopathology of the lung revealed severe diffuse necrotizing pneumonia. Numerous epithelial and endothelial cells contained prominent intranuclear herpetic inclusion bodies. Virus isolated from lung tissue in cell culture was suspected to be Herpesvirus papio 2 (HVP2) in light of the viral cytopathic effect. Real-time polymerase chain reaction (PCR) analysis and DNA sequencing of PCR products both confirmed that the virus was HVP2. This case is interesting because the age at onset suggests perinatal transmission at or immediately after birth, and the disease course suggests inoculation of the virus into the respiratory tract.