measuring-dengue-virus-rna-culture-supernatant-infected-cells-real

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

Osaka Medical and Pharmaceutical University

Osaka Medical College

Electrolyzed products of sodium chloride solution were examined for their disinfection potential against hepatitis B virus (HBV) and human immunodeficiency virus (HIV) in vitro. Electrolysis of 0.05% NaCl in tap water was carried out for 45 min at room temperature using a 3 A electric current in separate wells installed with positive and negative electrodes. The electrolyzed products were obtained from the positive well. The oxidation reduction potential (ORP), pH and free chlorine content of the product were 1053 mV, pH 2.34 and 4.20 ppm, respectively. The products modified the antigenicity of the surface protein of HBV as well as the infectivity of HIV in time- and concentration-dependent manner. Although the inactivating potential was decreased by the addition of contaminating protein, recycling of the product or continuous addition of fresh product may restore the complete disinfection against bloodborne pathogens.

Disinfection potential of electrolyzed solutions containing sodium chloride at low concentrations.

Improvement of the sensitivity of detection systems for human immunodeficiency virus-1 (HIV-1) has been carried out. One approach to improve the sensitivity is purification and/or concentration of the virus from a specimen. In this study, a method for concentrating HIV-1 using polyethylene glycol (PEG) has been re-evaluated and the optimal protocol for concentrating the virus from low-titer specimens was determined. That is, to obtain a virus pellet, a mixture of equal volumes of a specimen and 20% PEG 20,000 solution in saline is incubated at 4 degrees C for 16 h and then centrifuged at 17860 x g in a microcentrifuge for 20 min. HIV-1 in the pellet could be detectable by HIV-1 p24 antigen capture assay for viral protein, reverse transcriptase (RT) assay for viral enzyme, reverse transcriptase polymerase chain reaction (RT-PCR) assay for viral RNA and a virus infectivity assay.

A new improved method for the concentration of HIV-1 infective particles.

Helicobacter pylori is an aetiological agent of gastric disease. Although the role of urease in gastric colonization of H. pylori has been shown, it remains unclear as to where urease is located in this bacterial cell. The purpose of this study was to define the urease-associated apparatus in the H. pylori cytoplasm. H. pylori was incubated at both a neutral and an acidic pH in the presence or absence of urea and examined by double indirect immunoelectron microscopy. The density of gold particles for UreA was greatest in the inner portion of the wild-type H. pylori cytoplasm at neutral pH but was greatest in the outer portion at acidic pH. This difference was independent of the presence of urea and was not observed in the ureI-deletion mutant. Also, the eccentric shift of urease in acidic pH was not observed in UreI. After a 2 day incubation period at acidic pH, it was observed that the urease gold particles in H. pylori assembled and were associated with UreI gold particles. Urease immunoreactivity shifted from the inner to the outer portion of H. pylori as a result of an extracellular decrease in pH. This shift was urea-independent and UreI-dependent, suggesting an additional role of UreI in urease-dependent acid resistance. This is the first report of the intracellular transport of molecules in bacteria in response to changes in the extracellular environment.

Medium pH-dependent redistribution of the urease of Helicobacter pylori.

Helicobacter pylori CagA modifies the signalling of host cells and causes gastric diseases. Although CagA is injected into gastric epithelial cells through the type IV secretion machinery, it remains unclear how CagA is transported towards the machinery in the bacterial cytoplasm. In this study, it was determined that the proton-dependent intracytoplasmic transport system correlates with the priming of CagA secretion from H. pylori. The cytotoxicity of neutral-pH- and acidic-pH-treated H. pylori was examined in the AGS cell line. The amount of phosphorylated CagA in AGS cells incubated with acidic-pH- and neutral-pH-treated H. pylori was determined by enzyme immunoassay and Western blot. The production of CagA and adherence of the treated bacteria were examined by enzyme immunoassay and light microscopy, respectively. To clarify how CagA is transported towards the inner membrane of the treated bacteria, the localization of CagA was analysed by immunoelectron microscopy. The proportion of hummingbird cells in the AGS cell line rapidly increased following the inoculation of acidic-pH-treated H. pylori but increased more slowly with neutral-pH-treated H. pylori, and the phenomenon correlated with the amount of phosphorylated CagA in AGS cells. CagA was densely localized near the inner membrane in the acidic-pH-treated bacterial cytoplasm, but this localization was not observed in the neutral-pH-treated bacterial cytoplasm, suggesting that CagA shifts from the centre to the peripheral portion of the cytoplasm as a result of an extracellular decrease in pH. This phenomenon depended on the presence of UreI, a proton-dependent urea channel, but not on the presence of urea. The pH treatments did not enhance CagA production or the adherence of the bacterium to AGS cells. The authors propose that H. pylori possesses a proton-dependent intracytoplasmic transport system that probably accelerates priming for CagA injection.

Intrabacterial proton-dependent CagA transport system in Helicobacter pylori.

Vibrio cholerae (V. cholerae) cholera toxin (CT), which causes a severe watery diarrheal illness, is secreted via the type II secretion machinery; it remains unclear, however, how this toxin is transported toward the machinery. In this study, we determined that the pH-dependent intrabacterial transport system correlates with the priming of CT secretion by V. cholerae. The secretion and production of V. cholerae treated at different pHs were examined by enzyme immunoassay. The localization of the CT was analyzed by immunoelectron microscopy. The CT secretion level rapidly increases in the alkaline-pH-treated V. cholerae but does so more slowly in neutral- and acidic-pH-treated V. cholerae. The CT was found to be densely localized near the membrane in the alkaline-pH-treated bacterial cytoplasm, suggesting that the CT shifts from the center to the peripheral portion of the cytoplasm following an extracellular rise in pH. The shift was observed in V. cholerae treated at alkaline pH for more than 10 min. The pH treatment did not enhance CT production at the same stage at which secretion and intrabacterial transport of the CT were enhanced. We propose that V. cholerae possesses a pH-dependent intrabacterial nanotransportation system that probably accelerates priming for CT secretion.

Nanotransportation system for cholera toxin in Vibrio cholerae 01.

A recent report has indicated that proteins and genes of simian virus 5 (SV5) are detected in a human gastric adenocarcinoma (AGS) cell line, which is widely provided for oncology, immunology, and microbiology research. However, the production of infective virions has not been determined in this cell line. In this study, the morphology and infectivity of the virus particles of the AGS cell line were studied by light and electron microscopy and virus transmission assay. The virus particles were approximately 176.0 ± 41.1 nm in diameter. The particles possessed projections 8-12 nm long on the surface and contained a nucleocapsid determined to be 13-18 nm in width and less than 1,000 nm in length. The virus was transmissible to the Vero cell line, induced multinuclear giant cell formation, and reproduced the same shape of antigenic virions. In this study, the persistently infected virus in the AGS cell line was determined to be infective and form reproducible virions, and a new morphological feature of SV5 was determined.

Morphology and infectivity of virus that persistently caused infection in an AGS cell line.

A new method was established for fine visualization of bacterial subcelluar filamentous structures by freezing the bacterial cells to displace cytoplasmic matrix granules to the periphery. This method was successfully applied in immunoelectron microscopy and electron microscopic tomography, and should be applicable for further studies of bacterial architecture and nanotransportation.

Fine visualization of filamentous structures in the bacterial cytoplasm.

Electrolyzed acid water (EAW) has been studied for its disinfective potential against pathogenic microbes; however, the bactericidal process against Mycobacteria has not been clearly presented. In this study, to clarify the disinfective process against Mycobacteria, EAW-treated bacteria were examined against laboratory strains of Mycobacterium bovis (M. bovis), Mycobacterium smegmatis (M. smegmatis), and Mycobacterium terrae (M. terrae) by recovery culture and observation of morphology, enzymatic assay, and the detection of DNA. All experiments were performed with the use of EAW containing 30 ppm free chlorine that kills Mycobacteria, including three pathogenic clinical isolates of Mycobacterium tuberculosis (M. tuberculosis) and six isolates of other Mycobacteria, within 5 min. In morphology, the bacterial surface became rough, and a longitudinal concavity-like structure appeared. The intrabacterial enzyme of EAW-contacted bacteria was inactivated, but chromosomal DNA was not totally denatured. These results suggest that the bactericidal effect of EAW against Mycobacteria occurs by degradation of the cell wall, followed by denaturation of cytoplasmic proteins, but degeneration of the nucleic acid is not always necessary.

Disinfective process of strongly acidic electrolyzed product of sodium chloride solution against Mycobacteria.

The recent development of antiviral drugs has led to concern that the release of the chemicals in surface water due to expanded medical use could induce drug-resistant mutant viruses in zoonosis. Many researchers have noted that the appearance of an oseltamivir (Tamiflu(®))-resistant avian influenza mutant virus, which may spread to humans, could be induced by oseltamivir contamination of surface water. Although past studies have reported electrolysis as a possible method for degradation of antineoplastics and antibacterials in water, the validity of the method for treatment of antiviral drugs is unknown. In this study, electrolysis was used to degrade an antiviral prodrug, oseltamivir, and a stable active form, oseltamivir carboxylate, and the degradation process was monitored with HPLC-UV and the neuraminidase inhibitory assay. HPLC-UV-detectable oseltamivir and oseltamivir carboxylate were decomposed by electrolysis within 60 min, and inhibitory activity of neuraminidase decreased below the detection limit of the assay used. Cytotoxic and genotoxic activity were not detected in electrolyzed fluid. These results indicate that electrolysis is a possible treatment for inactivation of the antiviral drug oseltamivir.

Application of electrolysis for inactivation of an antiviral drug that is one of possible selection pressure to drug-resistant influenza viruses.

Helicobacter pylori possesses intrabacterial nanotransportation systems (ibNoTSs) for CagA and urease. Both systems are UreI-dependent and urea-independent, and activated by extrabacterial acid. The activation occurs/appears within 15 min after exposure to extrabacterial acid stimulation. Although it has been clarified that VacA is secreted via the type-V secretion machinery, it remains unclear how this toxin is transported toward the machinery. To clarify the intrabacterial nanotransportation system for H. pylori VacA, immunoelectron microscopic analysis was performed in this study. VacA shifted to the periphery of the bacterial cytoplasm at 30 min after the extracellular pH change, whereas CagA and urease did so within 15 min. Studies using an ureI-deletion mutant revealed that unlike CagA and urease transport, VacA transport was not UreI-dependent. VacA secretion was accelerated without an increase in the production of VacA 30 min after the exposure to extrabacterial acid. These findings indicated that H. pylori possesses a novel type of ibNoTS for VacA, which is different from that for CagA or urease, in response time and dependency of UreI.

A new type of intrabacterial nanotransportation system for VacA in Helicobacter pylori.

Helicobacter pylori (H. pylori) possesses an intrabacterial nanotransportation system (ibNoTS) for transporting CagA and urease within the bacterial cytoplasm; this system is controlled by the extrabacterial environment. The transportation routes of the system have not yet been studied in detail. In this study, we demonstrated by immunoelectron microscopy that CagA localizes closely with the MreB filament in the bacterium, and MreB polymerization inhibitor A22 obstructs ibNoTS for CagA. These findings indicate that the route of ibNoTS for CagA is closely associated with the MreB filament. Because these phenomena were not observed in ibNoTS for urease, the route of ibNoTS for CagA is different from that of ibNoTS for urease as previously suggested. We propose that the route of ibNoTS for CagA is associated with the MreB filament in H. pylori.

Route of intrabacterial nanotransportation system for CagA in Helicobacter pylori.

We attempted to clarify in detail the conditions of disinfection using electrolyzed strongly acidic water (ESW) against Mycobacteria, and the recovery of the disinfection potential of inactivated ESW by re-electrolysis. We mixed ESW containing 10, 20, and 30 ppm free chlorine with M. bovis cells (10(5)-10(8) CFU/mL) for 0-7 min. The disinfection potential of ESW positively correlated with free chlorine concentration, and negatively correlated with the initial density of bacterial cells. To clarify the recovery of the disinfection potential of inactivated ESW by re-electrolysis, we mixed ESW containing 10 ppm free chlorine with M. bovis cells (10(7) CFU/mL) for 1 min. The number of viable cells decreased to 1/10(3), but the cells were still detected. After re-electrolysis for 7 min, viable cells were not detected. Moreover, we confirmed by reusing the re-electrolyzed water against M. bovis cells that it regained its disinfection potential. These findings indicate that ESW once inactivated during disinfection can be re-activated by re-electrolysis. In conclusion, we were able to clarify in detail the conditions of ESW against Mycobacteria, and found the recovery of the disinfection potential of inactivated ESW by re-electrolysis.

Disinfection potential of electrolyzed strongly acidic water against Mycobacteria: conditions of disinfection and recovery of disinfection potential by reelectrolysis.

Vibrio cholerae colonizes the lumen of the proximal small intestine, which has an alkaline environment, and secretes cholera toxin (CT) through a type II secretion machinery. V. cholerae possesses the intrabacterial nanotransportation system (ibNoTS) for transporting CT from the inner portion toward the peripheral portion of the cytoplasm, and this system is controlled by extrabacterial pH. Association of ATP with ibNoTS has not yet been examined in detail. In this study, we demonstrated by immunoelectron microscopy that ibNoTS of V. cholerae under the extrabacterial alkaline condition was inhibited by ATP inhibitors, 2,4-dinitrophenol (DNP), a protonophore, or 8-amino-adenosine which produces inactive form of ATP. The inhibition of CT transport can be reversed by neutralization of DNP. Those inhibitions were associated with decrease of CT secretion by which ibNoTS followed. We propose that ATP closely associates with V. cholerae ibNoTS for transporting CT.

ATP-association to intrabacterial nanotransportation system in Vibrio cholerae.

Erratum to: ATP-association to intrabacterial nanotransportation system in Vibrio cholerae.

Colonic epithelial cells are covered by thick inner and outer mucus layers. The inner mucus layer is free of commensal microbiota, which contributes to the maintenance of gut homeostasis. In the small intestine, molecules critical for prevention of bacterial invasion into epithelia such as Paneth-cell-derived anti-microbial peptides and regenerating islet-derived 3 (RegIII) family proteins have been identified. Although there are mucus layers providing physical barriers against the large number of microbiota present in the large intestine, the mechanisms that separate bacteria and colonic epithelia are not fully elucidated. Here we show that Ly6/PLAUR domain containing 8 (Lypd8) protein prevents flagellated microbiota invading the colonic epithelia in mice. Lypd8, selectively expressed in epithelial cells at the uppermost layer of the large intestinal gland, was secreted into the lumen and bound flagellated bacteria including Proteus mirabilis. In the absence of Lypd8, bacteria were present in the inner mucus layer and many flagellated bacteria invaded epithelia. Lypd8(-/-) mice were highly sensitive to intestinal inflammation induced by dextran sulfate sodium (DSS). Antibiotic elimination of Gram-negative flagellated bacteria restored the bacterial-free state of the inner mucus layer and ameliorated DSS-induced intestinal inflammation in Lypd8(-/-) mice. Lypd8 bound to flagella and suppressed motility of flagellated bacteria. Thus, Lypd8 mediates segregation of intestinal bacteria and epithelial cells in the colon to preserve intestinal homeostasis.

Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia.

It remains unclear why gastric disease does not develop in all cases of Helicobacter pylori infection. In this study, we analyzed whether simian virus 5 (SV5) enhanced adherence of H. pylori to adenocarcinoma epithelial cells (AGS). H. pylori in AGS (harboring SV5) and SV5-infected Vero cells, and an agglutination of H. pylori mixed with SV5 were observed by light microscopy, scanning and transmission electron microscopies. The adherent rate of H. pylori to SV5-infected Vero cells and treated with an anti-SV5 antibody was determined. H. pylori adhered to the surface of AGS cells near SV5 particles, as shown by scanning and transmission electron microscopies. The adherence of H. pylori to SV5-infected Vero cells was significantly enhanced compared with that to Vero cells. In contrast, the adherence of H. pylori to Vero cells was decreased by treatment with the anti-SV5 antibody. Agglutination of H. pylori mixed with SV5 was observed by scanning and transmission electron microscopies. Agglutination did not occur when SV5 was treated with the anti-SV5 antibody before mixing. These findings demonstrated that SV5 enhanced the adherence of H. pylori to host cells, suggesting that a persistently infected virus may be a factor enhancing the pathogenicity of H. pylori in humans.

Article	Year
Disinfection potential of electrolyzed solutions containing sodium chloride at low concentrations. Journal of virological methods\| PubMed ID: 10716349	2000
A new improved method for the concentration of HIV-1 infective particles. Journal of virological methods\| PubMed ID: 12393147	2002
Medium pH-dependent redistribution of the urease of Helicobacter pylori. Journal of medical microbiology\| PubMed ID: 12621085	2003
Intrabacterial proton-dependent CagA transport system in Helicobacter pylori. Journal of medical microbiology\| PubMed ID: 16278423	2005
Nanotransportation system for cholera toxin in Vibrio cholerae 01. Medical molecular morphology\| PubMed ID: 19294491	2009
Morphology and infectivity of virus that persistently caused infection in an AGS cell line. Medical molecular morphology\| PubMed ID: 22179184	2011
Fine visualization of filamentous structures in the bacterial cytoplasm. Journal of microbiological methods\| PubMed ID: 22504230	2012
Disinfective process of strongly acidic electrolyzed product of sodium chloride solution against Mycobacteria. Medical molecular morphology\| PubMed ID: 23224598	2012
Application of electrolysis for inactivation of an antiviral drug that is one of possible selection pressure to drug-resistant influenza viruses. Journal of virological methods\| PubMed ID: 23994466	2013
A new type of intrabacterial nanotransportation system for VacA in Helicobacter pylori. Medical molecular morphology\| PubMed ID: 24420644	2014
Route of intrabacterial nanotransportation system for CagA in Helicobacter pylori. Medical molecular morphology\| PubMed ID: 25707504	2015
Disinfection potential of electrolyzed strongly acidic water against Mycobacteria: conditions of disinfection and recovery of disinfection potential by reelectrolysis. Biomedical research (Tokyo, Japan)\| PubMed ID: 25876661	2015
ATP-association to intrabacterial nanotransportation system in Vibrio cholerae. Medical molecular morphology\| PubMed ID: 25986680	2015
Erratum to: ATP-association to intrabacterial nanotransportation system in Vibrio cholerae. Medical molecular morphology\| PubMed ID: 26134077	2015
Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia. Nature\| PubMed ID: 27027293	2016
Enhancement of adherence of Helicobacter pylori to host cells by virus: possible mechanism of development of symptoms of gastric disease. Medical molecular morphology\| PubMed ID: 28283804	2017

Hong Wu

Biography

JoVE Journal Publications

Other Publications