Comparison of α-glucosyl hesperidin of citrus fruits and epigallocatechin gallate of green tea on the Loss of Rotavirus Infectivity in Cell Culture.

A number of secondary plant metabolites (e.g., flavonoids) possess antiviral/antimicrobial activity. Most flavonoids, however, are difficult to study, as they are immiscible in water-based systems. The relatively new semisynthetic α-glucosyl hesperitin (GH), and the natural plant product epigallocatechin gallate (EGCG) are unique among most flavonoids, as these flavonoids are highly soluble. The antiviral activity of these plant metabolites were investigated using the rotavirus as a model enteric virus system. Direct loss of virus structural integrity in cell-free suspension and titration of amplified RTV in host cell cultures was measured by a quantitative enzyme-linked immunosorbent assay (qEIA). After 30 min. 100 × 10(3) µg/ml GH reduced RTV antigen levels by ca. 90%. The same compound reduced infectivity (replication in cell culture) by a similar order of magnitude 3 to 4 days post inoculation. After 3 days in culture, EGCG concentrations of 80, 160, and 320 µg/ml reduced RTV infectivity titer levels to ca. 50, 20, and 15% of the control, respectively. Loss of RTV infectivity titers occurred following viral treatment by parallel testing of both GH and EGCG, with the latter, markedly more effective. Cytotoxicity testing showed no adverse effects by the phenolic concentrations used in this study. The unique chemical structure of each flavonoid rather than each phenolic's inherent solubility may be ascribed to those marked differences between each molecule's antiviral (anti-RTV) effects. The solubility of EGCG and GH obviated our need to use potentially confounding or obfuscating carrier molecules (e.g., methanol, ethanol, DMSO) denoting our use of a pure system environ. Our work further denotes the need to address the unique chemical nature of secondary plant metabolites before any broad generalizations in flavonoid (antiviral) activity may be proposed.

Effect of pH on anti-rotavirus activity by comestible juices and proanthocyanidins in a cell-free assay system.

Cranberry (Vaccinium macrocarpon) and grape (Vitis labrusca) juices, and these species' secondary plant metabolites [i.e., proanthocyanidins (PACs)] possess antiviral activity. An understanding of the mechanism(s) responsible for these juices and their polyphenolic constituents' direct effect on enteric virus integrity, however, remains poorly defined. Using the rotavirus (RTV) as a model enteric virus system, the direct effect of manufacturer-supplied and commercially purchased juices [Ocean Spray Pure Cranberry 100 % Unsweetened Juice (CJ), Welch's 100 % Grape Juice (GJ), 100 % Concord (PG) and 100 % Niagara juices (NG)] and these species' cranberry (C-PACs) and grape PACs (G-PACs) was investigated. Loss of viral capsid integrity in cell-free suspension by juices and their PACs, and as a factor of pH, was identified by an antigen (RTV) capture enzyme-linked immunosorbent assay. At native and an artificially increased suspension at or near pH 7, loss of viral infectivity occurred after 5 min, in the order CJ > NG = GJ > PG, and PG > GJ = NG = CJ, respectively. Antiviral activity of CJ was inversely related to pH. Grape, but not cranberry PACs, displayed a comparatively greater anti-RTV activity at a suspension pH of 6.7. Anti-RTV activity of C-PACs was regained upon reduction of RTV-cranberry PAC suspensions to pH 4. An alteration or modification of Type A PAC (of V. macrocarpon) structural integrity at or near physiologic pH is suggested to have impacted on this molecule's antivirus activity. Type B PACs (of V. labrusca) were refractive to alternations of pH. Significantly, findings from pure system RTV-PAC testing paralleled and in turn, supported those RTV-juice antiviral studies. Electron microscopy showed an enshroudment by PACs of RTV particles, suggesting a blockage of viral antigenic binding determinants. The implications of our work are significant, especially in the interpretation of PAC (and PAC-containing food)-RTV interactions in the differing [pH] conditions of the gastrointestinal tract.

The recording of student performance in the microbiology laboratory as a training, tutorial, and motivational tool.

The laboratory component of a microbiology course consists of exercises which mandate a level of proficiency and manual dexterity equal to and often beyond that recognized among other biology courses. Bacterial growth, maintenance, identification (e.g., Gram stain, biochemical tests, genomics), as well as the continuous need to maintain laboratory safety and sterile technique, are only a few skills/responsibilities critical to the discipline of microbiology. Performance of the Gram stain remains one of the most basic and pivotal skills that must be mastered in the microbiology laboratory. However, a number of students continually have difficulty executing the Gram stain and preparative procedures associated with the test. In order to address this issue, we incorporated real-time digital recording as a supplemental teaching aid in the microbiology laboratory. Our use of the digital movie camera in the teaching setting served to enhance interest, motivate students, and in general, improve student performance.

Cranberry and Grape Juices Affect Tight Junction Function and Structural Integrity of Rotavirus-Infected Monkey Kidney Epithelial Cell Monolayers.

Cranberry juice (CJ) and grape juice (GJ) from Vaccinium macrocarpon and Vitis labrusca, respectively, and purified proanthocyanidins (PACs) from these species are recognized to possess antiviral activity. The effects of CJ and GJ on tight junction (TJ) structure and function among rotavirus-infected monkey kidney epithelial cells (MA-104) in monolayer cultures were evaluated. Antiviral activity by cranberry PACs of rotavirus in cell-free suspension was investigated by a rotavirus antigen [i.e., viral capsid protein 6 (VP6)] capture enzyme-linked immunosorbent assay (ELISA) and by transmission electron microscopy (TEM). MA-104 monolayers were treated with CJ, GJ, or cranberry juice cocktail (CJC) drink before inoculation with rotavirus. TJ function and structural integrity were measured by changes in transepithelial electrical resistance (TEER) and by reduction of signal intensity of the TJ α-claudin 1 by immunofluorescence. The inhibitory activity of CJ and GJ on viral RNA synthesis, as a function of viral concentration, was determined by reverse transcription polymerase chain reaction (rtPCR). After 4 days, virus-infected monolayers pretreated with GJ (Concord and Niagara GJs) had TEER readings similar to uninfected controls. CJ and CJC also had a significant protective effect (P < 0.05) on TJ function, but to a lesser extent than GJ. Disorganization of TJ integrity commenced at 24- to 36-h post-viral inoculation, but this effect was reduced by pretreatment with CJ or GP of monolayer cultures. TEM showed aggregation of rotavirus by cranberry PACs. The destruction of rotavirus capsid proteins VP6, in cell-free suspension was inversely related to the concentration of cranberry PACs (C-PAC). Loss of rotavirus RNA by CJ or GJ was inversely related to viral infectivity titers. CJ, GJ, or PAC-associated antiviral activity has been linked to modifications in cellular physiologic events and to physical factors (e.g., PAC-mediated viral aggregation) that probably compromise viral infectivity. Multiple cell physiological and physical events must be considered when determining the mechanisms associated with the antiviral (i.e., rotavirus) activity of CJ, GJ, and PACs.

Cranberry cocktail juice, cranberry concentrates, and proanthocyanidins reduce reovirus infectivity titers in African green monkey kidney epithelial cell cultures.

Studies were performed to investigate the effect of several cranberry and grape juice extracts on the inhibition of reovirus infectivity following cell culture inoculation. Infectivity testing was performed utilizing cranberry juice extracts NutriCran-100 and NutriCran-90. At 5% extract concentrations, titers were reduced by ca. 50%. Cranberry cocktail juice caused an infectivity loss of ca. 10%. We ascribe these data to higher concentrations of proanthocyanidins (PACs) in the cranberry extracts. Further testing was performed utilizing purified high and low molecular weight cranberry PAC fractions (CB HMW and CB LMW, respectively), a cranberry flavonol glycoside (CB EToAc), cranberry anthocyanins (CB CA), and a grape PAC extract. Reovirus titers were reduced to undetectable levels at PAC concentrations < or =0.2%. CB CA had no effect on the inhibition of infectivity titers. Loss of infectivity titers was in the order: GP PAC>CB HMW>CB LMW>CB EToAc. Probe homogenization of CB HMW enhanced the extract to efficacy levels equal to that of grape PAC. Reovirus dsRNA segments were undetectable 96-h postcranberry cocktail juice pretreatment of MA-104 cell cultures. This study indicates an inhibition of reovirus infectivity titers by cranberry or grape juices or their purified PAC extracts. Viral inhibition probably occurs at the host cell surface.

Viral studies on amniotic fluid from fetuses with and without abnormalities detected by prenatal sonography.

OBJECTIVE: To study the prevalence of viruses (cytomegalovirus [CMV] adenoviruses and enteroviruses) in amniotic fluid samples from fetuses with and without anomalies detected by prenatal sonography. STUDY DESIGN: Fluid samples obtained aseptically from 474 women undergoing genetic amniocentesis at our institutions from 1995 to 1996 were stored at -20 degrees C. Fetal anomalies (renal, central nervous system, gastrointestinal and cardiac) were detected by ultrasound in 162 of the fetuses. At a later date, the samples were retrieved, blinded, and tested by virus isolation techniques for CMV, adenoviruses and enteroviruses. Fisher's exact test was used for statistical analysis. RESULTS: The prevalence of viral isolation in amniotic fluid samples in fetuses with anomalies was 2.5% for CMV, 1.3% for adenovirus and 1.2% for enterovirus. Structurally normal fetuses had prevalences of 0.3%, 0% and 0%, respectively. CONCLUSION: The prevalence of viruses, especially CMV, appears to be higher in amniotic fluid from fetuses with sonographically detected anomalies.

Survival of spumavirus, a primate retrovirus, in laboratory media and water.

The persistence of a previously characterized spumavirus strain (strain SV-522) was investigated utilizing various laboratory media and waters, including Eagle's minimal essential medium (EMEM) plus 0% fetal bovine serum (EMEM-0%), EMEM-2%, EMEM-10%, Chlamydia transport medium (CTM), phosphate-buffered saline, distilled, estuarine, and marine water, human serum, and the germicides, ethyl alcohol (70%) and sodium hypochlorite (10%). Experiments were performed at 4 degrees C and/or 23 degrees C. Infectivity endpoints were determined in stock aliquots upon initiation of testing and then after 3, 5, 7, and 10 days. The virus was reisolated from all diluents after 5 days at 23 degrees C and in EMEM-10% after 7 days. The virus was detected in CTM, EMEM-2%, EMEM-10%, and estuarine and marine waters after 7 days at 4 degrees C. Differences in the persistence of the virus may be ascribed to temperature and organic load. Water ionic strengths (e.g., estuarine vs. marine water) had no effect on modifying persistence of viral particles. Infectivity of spumavirus was undetectable after 30 s in 70% ethanol or 10% sodium hypochlorite. After 30 min at 23 degrees C, spumavirus infectivity in normal but not heat-inactivated human serum increased by almost 100-fold. Persistence of infectivity of primate spumavirus after 7 days in media and waters, and the agent's infectious potential in the human host, emphasize a need for cautious recognition during the manipulation of primate cells/organs and in the handling of primates themselves.