Takehiro Kohno

Disinfection potential of electrolyzed solutions containing sodium chloride at low concentrations

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.

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

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 highly sensitive chemiluminescent reverse transcriptase assay for human immunodeficiency virus

A simple and highly sensitive reverse transcriptase (RT) assay was developed by combining a previously reported non-radioisotopic RT assay with the use of a template-primer-immobilized microplate, an enzyme capture protocol, product digestion and a chemiluminescent substrate. The assay was able to detect directly the RT activity in serum samples, plasma and cell culture medium without the need for concentration and extraction of the enzyme. The assay was able to detect RT activity equivalent to 100 virions/ml of HIV-1. These results suggest that this highly sensitive chemiluminescent RT assay can be used not only for virological investigation but also for routine screening of biopharmaceuticals.

Contrast-enhanced immunoelectron microscopy for Helicobacter pylori

Since a method of contrast enhancement for immunoelectron microscopy has not been available in bacteriology, the morphological localization of proteins of Helicobacter pylori is not well known. In this report, we established a method of contrast enhancement in immunoelectron microscopy in this organism. Immunostained ultrathin sections are stained with a mixture of alcian blue and osmium tetroxide prior to staining with uranyl acetate. This method of staining provided good contrast enhancement of the bacterial cell wall and membrane without any loss of immunolabeled gold particles on the ultrathin section.

Contrast-enhancement for the image of human immunodeficiency virus from ultrathin section by immuno electron microscopy

A simple contrast-enhancement method is described for electron microscopic imaging of the human immunodeficiency virus (HIV) from a sample embedded in Lowicryl K4M resin, by immuneelectron microscopy. Ultrathin sections were treated with a mixture of ruthenium red dye (RR) and osmium tetroxide (OSO4). This treatment provided good contrast enhancement of the entire ultrastructural image of virus particles without the loss of immunolabelling. RR/OsO4 solution is simple to prepare and provides a better contrast than that which is achieved during conventional post-embedding immunoelectron microscopy. Treatment of ultrathin sections from low temperature-embedded samples with RR/OsO4 solution is recommended.

Nanotransportation system for cholera toxin in Vibrio cholerae 01.

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.

Targets of a protease inhibitor, KNI-272, in HIV-1-infected cells

The targets of a protease inhibitor, KNI‐272, in the HIV‐1 life cycle were investigated in this study. Neither expression of HIV‐1 Gag proteins nor production of virus particles was detected in cells infected acutely with HIV‐1 cultured in the presence of KNI‐272. Although HIV‐1 proviral DNA was detected in the cells by PCR, the inhibitor depressed the amount of the proviral DNA in a concentration dependent manner. These results indicate that one of the targets of KNI‐272 occurs in the stage before the expression of viral structural proteins. No direct inhibition of reverse transcription was found with the inhibitor. To confirm the inhibition of viral protease, persistently HIV‐1‐infected cells were cultured in the presence of the inhibitor and examined by electron microscopy for the morphology of HIV‐1 particles. Doughnut‐shaped immature particles were observed in the extracellular space of the cells, and disrupted semicircular shaped particles were also seen at the higher concentration of KNI‐272. A bioassay for infectivity showed that the virus particles were not infectious, and immunofluorescent assay using anti‐p17 antibody, that does not react with the precursor of Gag protein, revealed that Gag precursor p55 protein in the cells was not processed. Thus, KNI‐272 blocked the maturation of viral particles. Consequently, KNI‐272 has at least two inhibition targets in the stages of the HIV‐1 life cycle. J. Med. Virol. 63:203–209, 2001.

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

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.