In order to survey the occurrence of waterborne viruses in Korean surface water, a total of 192 water samples from July 2003 to January 2006 were collected and analyzed. The presence of waterborne viruses was investigated by total culturable virus assay (TCVA) using buffalo green monkey kidney (BGMK) cells. The results showed that 63 of 192 samples (32.8%) were positive for waterborne viruses with the average concentration of 3.1+/-18 most probable numbers (MPN)/100 L. The relationship between the occurrence of the viruses and the physicochemical environmental factors revealed that there was a significant correlation between the turbidity of water and the occurrence of the viruses. It was also noted that the water temperature might have some relationship with the occurrence of the viruses, as the frequency of the viruses was higher in low temperature or winter season. Therefore, the occurrence of waterborne viruses in Korean surface water might be affected by the physicochemical environmental factors such as turbidity and water temperature.
Buffaloes ; Cercopithecus aethiops ; Kidney ; Seasons ; Water*

The purpose of this research was to construct a lentiviral vector containing pir-b gene, and to detect the expression of pir-b gene in 293T cells. The open reading frame (ORF) of pir-b gene from the mRNA of mouse was cloned, then inserted into a sequencing vector. The pir-b gene was subcloned into the transfer plasmid of the lentivirus system, which was transfected together with the packaging plasmids into 293T cells by Lipofectin 2000, thereafter, the supernatant was collected and concentrated to transfect 293T cells. Western blot was used to detect the expression of the exogenous PIR-B after 293T cells were infected by the virus, while the lentivirus harboring egfp gene was packaged as the control group. The result indicated that the ORF of the pir-b gene was successfully cloned, the sequence of which was consistent to that was expected and the PIR-B protein could be expressed in 293T cells normally. It is concluded that the lentiviral vector containing pir-b gene was constructed successfully, which would contribute to illustrating the important role of pir-b gene in the immunological regulation.
Animals ; COS Cells ; Cercopithecus aethiops ; Genetic Vectors ; Lentivirus ; genetics ; Mice ; RNA, Messenger ; genetics ; Receptors, Immunologic ; genetics

OBJECTIVETo investigate the mitochondrial injury in enterovirus 71 (EV71)-infected Vero cells and explore the possible mechanism.

METHODSA clinical isolate of EV71 was inoculated to Vero cells and the EV71 antigen was detected by immunofluorescence assay. The morphological changes of Vero cells were observed using optical microscopy and transmission electron microscopy. The diameter and area density of the viral particles and the ratio and area density of vacuolated mitochondria in the cells were measured on the ultrastructural images.

RESULTSEV71-infected Vero cells underwent obvious changes and to a spherical morphology followed by cell death EV71 particles were detected in the cytoplasm by immunofluorescence. Ultrastructurally, the infected cells contained a large number of viral particles in the cytoplasm, with a clustered distribution and lattice-like arrangement. The diameter of the particles were 16.3 nm and the mean area density was 38.3%. Most of the mitochondria presented with swelling, vacuoles and degeneration. The ratio of the vacuolated mitochondria was 90.9% with a mean area density of 89.2%. Viral particles were also found in some mitochondria.

CONCLUSIONEV71 proliferates in the cytoplasm and invades the mitochondria of infected Vero cells leading to mitochondrial injury and cell death, suggesting that mitochondria are the targets for EV71 infection.

Animals ; Cercopithecus aethiops ; Cytoplasm ; virology ; Enterovirus ; Enterovirus Infections ; pathology ; Humans ; Mitochondria ; pathology ; virology ; Vero Cells ; virology

OBJECTIVETo explore the driving force of the oral astringency produced by the interaction of theaflavin (TF) and epigallocatechin-3-gallate (EGCG) to human salivary a-amylase(HSA).

METHODSThe constants of the rate, equilibrium of reaction (k(a), k(d), K(A), and K(D)) and Langmuir, Freundlich adsorption isotherm (K(L), K(f), and Mm) were determined by surface plasmon resonance (SPR) technique and adsorption kinetics.

RESULTSBoth of Langmuir and Freundlich models could be used for describing the binding processes of TF and EGCG onto HSA surfaces, and there were no significant differences of the correlation coefficient of determination between these two models (P > 0.05). The constants of adsorption isotherm, the rate and equilibrium constants of the association for TF were higher than those of EGCG (P < 0.05). The rate and equilibrium constants of the dissociation for TF were lower than those of EGCG (P < 0.05). The affinity of TF to HSA was higher than that of EGCG.

CONCLUSIONThe sorely oral astringency is much easily produced by TF from the black tea rather than EGCG from the green tea. The driving force of the oral astringency is attributed to the hydrogen bonds of hydroxyl groups and hydrophobic interaction of galloyl groups in polyphenolic structures.

Adsorption ; Animals ; Antioxidants ; Biflavonoids ; Catechin ; analogs & derivatives ; Cercopithecus aethiops ; Humans ; Salivary alpha-Amylases ; Tea ; alpha-Amylases

OBJECTIVEElectron microscopical study of infected cells to identify the pathogenic agent of SARS.

METHODSVero E6 cells infected with lung autopsy samples or nasopharyngeal swabs from SARS patients of Beijing and Guangzhou were inoculated. The supernatant and cultured cells exhibiting identifiable cytopathic effect (CPE) were prepared for electron microscopic study.

RESULTSExamination of CPE cells on thin-section revealed characteristic coronavirus particles within the cisternae of endoplasmic reticulum, Golgi apparatus, vesicles and extracellular space. They were mainly spherical or oval in shape, annular or dense, about 80 nm in diameter. Negative-stain electron microscopy identified coronavirus particles in culture supernatant, 80 - 120 nm in diameter, with club-shaped surface projections. Elongated, rod-, kidney- or other irregular shaped virons with the size of 100 - 200 nm by 60 - 90 nm were also found in the cultured cells infected with the lung samples from the Guangdong patients. Infectious virons entered cells by endocytosis or membrane fusion and released through a budding process.

CONCLUSIONThese data indicate a novel coronavirus as the causative agent of SARS. Most viral particles showed typical characteristics of coronavirus. The potential role of special shape viruses is expected to be further investigated.

Animals ; Cercopithecus aethiops ; Humans ; Microscopy, Electron ; SARS Virus ; ultrastructure ; Severe Acute Respiratory Syndrome ; virology ; Vero Cells

OBJECTIVETo investigate the expression and analysis of its activity of anti-bacterial peptide gloverin in COS-7 cells.

METHODSThe appearance frequency of all genetic codes in the cDNA sequence from the same species of protein Attacin A was analyzed, and its cDNA sequence was synthesized by PCR overlapping extension method in conjunction with the designation of the known protein sequence of gloverin. The genes were inserted into pCDSI, an eukaryotic vector, after being identified correctly. As a result, the vector pBZHG was constructed. Thereafter, the liposome FuGENE( trade mark ) 6 was employed as the vector, and the COS-7 cells were transfected with liposome pBZHG and blank vector pCDSI. The normal cells were taken as the control. The supernatant was collected for the detection of its bactericidal activity after 72 PBHs.

RESULTSThe gloverin cDNA sequence designed artificially was expressed in COS-7 cells. The supernatant of the cells transfected by pBZHG exhibited bactericidal activity to E. coli J5 when compared with that from normal cells and in cells transfected with blank vectors.

CONCLUSIONThe designed cDNA sequence of gloverin was proved to be genuine, and it provided the basis for future study of its antibiotic and anti-endotoxin activities.

Animals ; Anti-Bacterial Agents ; pharmacology ; COS Cells ; Cercopithecus aethiops ; Proteins ; genetics ; pharmacology ; Sequence Analysis, DNA ; Transfection

OBJECTIVETo investigate the differentiation of human testicular spermatogenic cells during in vitro culture.

METHODSTesticular cells of obstructive azoospermic patients' testis biopsies were dispersed employing mechanic methods. Then, (1) mixed testicular cells were applied to in vitro culture, and changes of the ratio of elongating spermatids and all round cells were analyzed during mixed cell culture; (2) round spermatids were picked up from the mixed cells employing micromanipulator, followed by differentiation of the isolated round spermatids during microdrop culture.

RESULTSThe ratio of the elongating spermatids increased significantly (P < 0.05) after 24 hours of mixed cell culture in HTF medium supplemented with FSH and testosterone. During single round spermatid culture, transformation of the round spermatid to elongating spermatid with newly formed flagellum was observed, and the transformation ratio within 48 hours of microdrop culture was 3.54%. The differentiation of human testicular spermatogenic cells cultured in Vero cell conditioned medium was similar to that cultured in HTF medium.

CONCLUSIONHuman testicular round spermatids can differentiate to elongating spermatids during in vitro culture. Vero cell conditioned medium does not promote the differentiation of human testicular round spermatids to elongating spermatids.

Animals ; Cell Differentiation ; Cells, Cultured ; Cercopithecus aethiops ; Humans ; Infertility, Male ; pathology ; Male ; Spermatids ; cytology ; Testis ; cytology ; Vero Cells

BACKGROUNDTo express and purify human alpha3-integrin to serve as the antigen to prepare its antibody and to separate the Vero cell clones without expression of alpha3-integrin.

METHODSThe human alpha3-integrin gene was amplified by using RT-PCR, then subcloned into a pQE30 expression vector and expressed in E. coli. The gene expression was confirmed by Western blot assay. Rabbit was inoculated with purified antigen to stimulate the antibody generation. The target Vero cells were separated by negative selection using antibody plus complement mediated cytolysis. The separated cell clones were confirmed by immunofluorescence and Western blot assay.

RESULTSThe alpha3- integrin gene was cloned and expressed effetively, Western blot assay revealed that the expressed protein held good immune reactivity. High titer antibody was generated. However the expression of alpha3-integrin was not detected on Vero, VeroE6, Hep-2, 2BS and 293 cells.

CONCLUSIONSThe results of the study suggested that hantavirus has other receptors on Vero cells beside alpha 3-integrin.

Animals ; Cercopithecus aethiops ; Cloning, Molecular ; Gene Expression ; Hantavirus ; Integrin beta3 ; biosynthesis ; genetics ; immunology ; Rabbits ; Receptors, Virus ; Vero Cells ; metabolism

OBJECTIVETo find a sensitive cytotoxic response to reflect the bio-toxicity of trace organic pollutants, the sensitivity and reliability of morphological change and proliferation inhibition of Vero cells exposed to 2, 4, 6-trichlorophenol (TCP) and the leachate from products related to drinking water (PRDW) were compared, and the mechanism of the morphological change in Vero cells exposed to chemical pollutants was studied.

METHODSVero cells were treated by different concentration of TCP and the leachate from PRDW. Methylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT) assay was carried out for proliferation inhibition. Bioluminescence method was carried out as another method to test the toxicity of TCP. Flow Cytometry assay was used to test cell Apoptosis and damage of cell-membrane.

RESULTS0.25 mg/L TCP had an effect on cell morphology, and the proportion of morphologically changed cells increased with increasing TCP concentration. At low TCP concentrations, inhibition of cell proliferation did not seem to correlate to TCP concentration, and was negative when TCP concentration was <1.0 mg/L. After exposure to leachate from PRDW extracted at different temperatures, the percentage of morphologically changed cells increased with extracting temperature, but the inhibition of cell proliferation failed to reflect the correlation between extracting temperature and proliferation inhibition of Vero cells. Although the Sensitivity of bioluminescence method seems to be similar to morphological change in Vero cells, the bacterial in this method is not homologous enough with human body cells to reflect the toxicity to human body. These imply cell morphological change is a more sensitive and reliable method to reflect bio-toxicity of organic pollutants than proliferation inhibition. Flow cytometry analysis and cell rejuvenation experiments indicated cell membrane damage, which results in cell morphological change, was an early and sensitive cytotoxic response comparing with necrosis.

CONCLUSIONThese results indicated that the cell membrane toxicity represented by morphological changes is a more sensitive and reliable method to indicate the composite bio-toxicity of trace chemicals than proliferation inhibition, inhibition on bioluminescence and necrosis. Nevertheless, the quantification of morphological change should be studied further.

Animals ; Cell Division ; drug effects ; Cell Survival ; drug effects ; Cercopithecus aethiops ; Vero Cells ; Water Pollutants, Chemical ; toxicity

OBJECTIVEThis study aimed at constructing secretory eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and investigating the transcription and expression of recombined plasmid VR1020/KGPcd in mammalian cells.

METHODSEukaryotic expression plasmid VR1020/KCPcd was constructed by using molecular cloning methods. Then, the VR1020/KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer's instruction. The transcription of VR1020/KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcd was analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISA method.

RESULTSIt proved that the VR1020/KGPcd could be transcribed and translated into transfected COS7 cells. The expressed targeted protein could be secreted into cultural supernatant and could be detected by ELISA.

CONCLUSIONThe eukaryotic expression plasmid of VR1020/KGPcd was constructed successfully and its product can be expressed in mammalian cells. The results indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidates in the development of anti-periodontitis and paved the way for further study.

Animals ; Bacterial Proteins ; genetics ; COS Cells ; Cercopithecus aethiops ; Cysteine Endopeptidases ; genetics ; Genetic Vectors ; Plasmids ; Porphyromonas gingivalis ; genetics ; Transfection