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*

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

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 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

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

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 culture, isolate and identify new bunyavirus in Vero cell line.

METHODSSamples of 164 new bunyavirus positive by real time RT-PCR detection and well preserved serum specimens were selected from cases of fever, thrombocytopenia and leukopenia syndrome (FTLS) in Xinyang, Henan province in 2009 - 2011. These sera were cultured in Vero cell line and new bunyavirus were detected by observing cytopathic effect (CPE), Real-time RT-PCR, indirect immunofluorescence assay (IFA) and thin-section electron microscopy observation. A total of 10 positive PCR products were selected randomly for sequencing and the results were compared with sequence in Genbank.

RESULTSAmong 164 FTLS serum specimens cultured in Vero cell line, no special CPE were observed and 67 strains (40.85%) were positive detected by Real-time RT-PCR. Nucleic acid similarity of 10 specimens were 97.8% - 100% and there's also a high similarity (> 99%) between specimens and new bunyavirus isolates (Accession No. HQ141600.1). Among 67 positive strains, 58 of them showed specific fluorescence particles by IFA. The viral particles were observed to be spheres with a diameter of 80 - 100 nm by electron microscopy.

CONCLUSIONVero cell line is suitable for culture, isolation and identification of new bunyavirus.

Animals ; Cercopithecus aethiops ; Humans ; Orthobunyavirus ; growth & development ; isolation & purification ; Serum ; virology ; Vero Cells ; virology ; Virus Cultivation ; methods

This study inquired about the influences on the gene delivery efficiency of polyethylenimine. pSVbeta plasmids were transferred into COS-7 and NIH3T3 cells with polyethylenimine. Influences of plasmids factor, albumin, serum, cell density, operation methods and polyethylenimine/DNA preserve factors on transfection efficiency were investigated. Inhibitors of biological activities in plasmids could be removed by ultrafiltration with cutoff molecular weight of 3000 or 10000. Linear plasmids lowered transfection efficiency. Serum and albumin in the culture medium decreased the transfection efficiency of polyethylenimine/DNA. Cell density was associated with PEI/DNA transfection efficiency. Incubation of PEI/DNA complexes with the cells for 8 h and then aspiration for removal of the complexes could obtain an optimal transfection effect. Freezing of PEI/DNA complexes significantly decreased transfection efficiency. In conclusion, polyethylenimine could obtain optimal and reduplicate transfection results by controlling related factors.
3T3 Cells ; Animals ; COS Cells ; Cercopithecus aethiops ; DNA ; genetics ; Genetic Vectors ; genetics ; Mice ; Plasmids ; genetics ; Polyethyleneimine ; chemistry ; Transfection ; Ultrafiltration

OBJECTIVETo evaluate the effectiveness of small interfering RNA (siRNA) on inhibiting severe acute respiratory syndrome (SARS)-associated coronavirus replication, and to lay bases for the future clinical application of siRNA for the treatment of viral infectious diseases.

METHODSVero-E6 cells was transfected with siRNA before SARS virus infection, and the effectiveness of siRNA interference was evaluated by observing the cytopathic effect (CPE) on Vero-E6 cells.

RESULTSFive pairs of siRNA showed ability to reduce CPE dose dependently, and two of them had the best effect.

CONCLUSIONsiRNA may be effective in inhibiting SARS-associated coronavirus replication.

Animals ; Cercopithecus aethiops ; RNA, Small Interfering ; pharmacology ; SARS Virus ; drug effects ; Transfection ; Vero Cells ; Virus Replication ; drug effects

OBJECTIVETo compare the substrate specificity of three murine GDP fucose: beta-galactoside alpha1,2-fucosyltransferases (alpha1,2-FT).

METHODSThree members of MFUT- I, -II and -III, coding for a alpha1,2-FT, a GDP-fucose, were cloned from a cDNA of murine small intestine by reverse transcription-polymerase chain reaction. The coding regions were ligated into mammalian expression vector pcDNA 3.1 (pcDNA3.1-MFUT-I, pcDNA3.1-MFUT- II , and pcDNA3.1-MFUT- III) and were transiently transfected into COS-7 cells using a cellphect transfection kit. Then the cells were analyzed for expression and function of alpha1,2-FT and the substrate specificity of three alpha1,2-FT was compared.

RESULTSMFUT- I, -II, and -III exhibited sequence homology with human H (77%), Se (79%), and Sec1 (75%) genes, respectively. COS-7 cells transfected with pcDNA3.1-MFUT- I and pcDNA3.1-MFUT- II showed alpha1,2-FT activity, but no activity was detected in COS-7 cells transfected with pcDNA3.1- MFUT-III. MFUT- II showed alpha1,2-FT activity with both asialo-monosialoteterahexosyl ganglioside (GA1) and monosialoteterahexosyl ganglioside (GM1) as substrates to produce fucosyl GA1(FGA1) and fucosyl GM1(FGM1), respectively, but MFUT- I only showed alpha1,2-FT activity with GA1. The relative activity of MFUT- II with GA1 was 80-90-folds higher compared with MFUT- I, and the relative activity of MFUT- II with GA1 was 10-20-folds higher than that of GM1. The fucosyltransferase encoded by the MFUT- II gene showed the enzyme activity not only responsible for the synthesis of type 4-H antigens FGA1 and FGM1, but also responsible for the synthesis of type 1-H and 2-H antigens with lactotetraosylceramide and neolactotetraosylceramide as substrates.

CONCLUSIONMFUT- II is the main alpha1,2-FT in mouse and MFUT- II can product type 4-H antigen FGA1 and FGM1, but MFUT- I only synthesizes FGA1. MFUT-III has no alpha1,2-FT activity.

Animals ; Antigens, Bacterial ; biosynthesis ; COS Cells ; Cercopithecus aethiops ; Cloning, Molecular ; Fucosyltransferases ; chemistry ; genetics ; metabolism ; Gangliosides ; metabolism ; Mice ; Substrate Specificity ; Transfection