A novel diagnostic approach to detecting porcine epidemic diarrhea virus: The lateral immunochromatography assay.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in sucking piglets and has a high mortality rate. An immunochromatography (IC) assay, known as a lateral flow test, is a simple device intended to detect the presence of target pathogens. Here, we developed an IC assay that detected PEDV antigens with 96.0% (218/227) sensitivity and 98.5% (262/266) specificity when compared with real-time reverse transcriptase (RT)-PCR using FAM-labeled probes based on sequences from nucleocapsid genes. The detection limits of the real-time RT-PCR and IC assays were 1×10(2) and 1×10(3) copies, respectively. The IC assay developed herein did not detect non-specific reactions with other viral or bacterial pathogens, and the assay could be stored at 4°C or room temperature for 15 months without affecting its efficacy. Thus, the IC assay may result in improved PED detection and control on farms, and is a viable alternative to current diagnostic tools for PEDV.

The CSFV DNAChip: a novel diagnostic assay for classical swine fever virus.

A novel assay, the CSFV DNAChip, was developed to clearly and rapidly discriminate three genotypes of classical swine fever virus (CSFV). Total RNA was extracted from clinical samples and then subjected to a one-step reverse-transcription polymerase chain reaction (RT-PCR) using Cy3-labeled primers from the 5' non-coding region (NCR) of CSFV. Amplicons were hybridized to the CSFV DNAChip and fluorescence scanning was performed for detection of CSFV. A cut-off fluorescence intensity value of 5000 was determined by two-graph receiver operating curve (TG-ROC) analysis. The limit of detection values for the developed DNA chip assay were 0.313ng/µL for amplicon concentration and 1TCID50/100µL for virus titer. Using the developed DNA chip, 157 field samples (91 CSFV-positive and 66 CSFV-negative) were investigated. The genotypes determined by the CSFV DNAChip agreed completely with those determined by nucleotide sequence analysis of the viral genome. The developed CSFV DNAChip will be helpful in implementing a CSFV eradication strategy, as it provides a rapid and accurate diagnostic assay that can discriminate easily among CSFV genotypes.

9G DNAChip: microarray based on the multiple interactions of 9 consecutive guanines.

We introduce the phenomenon of molecular recognition to immobilize oligonucleotides on AMCA slides for the production of 9G DNAChips. Facile and efficient method for the immobilization of the oligonucleotides appended with consecutive nine guanine bases is described. The 9G DNAChips shows more than 90% hybridization efficiency at 25 °C in 30 min.

One-step multiplex reverse-transcriptase PCR for detecting pandemic (H1N1) 2009 influenza virus.

Pandemic (H1N1) 2009 influenza has spread throughout the world since April 2009 and has caused many human deaths since its first report in humans. Pandemic (H1N1) 2009 influenza virus was first identified in a Canadian pig herd in April 2009 and has been reported in more than ten countries, including Korea. We developed a one-step multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay based on the matrix gene that discriminates pandemic (H1N1) 2009 influenza virus from endemic swine influenza viruses. The sensitivity of this assay was 100 copies of in vitro-transcribed target RNA and 0.01 tissue culture infective dose (TCID(50)/ml) of virus and was as high as those of conventional influenza A virus common matrix reverse transcriptase PCR assays and real-time reverse transcriptase PCR assays (1 to 200 copies) developed for detecting pandemic (H1N1) 2009 influenza viruses from human and pig samples. This one-step multiplex RT-PCR assay would be a good tool in monitoring pandemic (H1N1) 2009 influenza virus among pig herds on a regular basis.

Induction of apoptosis by KI0477959 through activation of caspase-3 in human leukemia cell line, HL-60 cells.

KI0477959 (Herbkines) has been used for the purpose of development of physical strength in wasting diseases, like cancer. In the present study, apoptosis-inducing activities of butanol fraction of KI0477959 were studied in human leukemia cell line, HL-60 cells. KI0477959 increased cytotoxicity but had less effect on human peripheral blood mononuclear cells. KI0477959-induced apoptosis was accompanied by activation of caspase-3 and specific proteolytic cleavage of poly-ADP-ribose polymerase. Increased apoptosis was reduced by treatment with p38 and extracellular signal-regulated protein kinase (ERK) inhibitors. These results suggest that KI0477959 induces apoptosis through activation of caspase-3, p38, and ERK in HL-60 cells.

Vascular endothelial growth factor is regulated by hypoxic stress via MAPK and HIF-1 alpha in the inner ear.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. The iron-chelator desferrioxamine (DFX) increased the expression of hypoxia-inducible factor (HIF)-1alpha in the hair cell line, HEI-OC1. The increased VEGF production by DFX was inhibited by iron. DFX also induced the activation of mitogen-activated protein kinase (MAPK) on HEI-OC1. The increased VEGF production by DFX was inhibited by a specific inhibitor of MAPK. In addition, DFX induced the VEGF production and HIF-1alpha stabilization in vivo. These results indicate that VEGF production is regulated via MAPK and HIF-1alpha under hypoxic condition in the inner ear.

Induction of apoptosis by Korean medicine Gagam-whanglyun-haedoktang through activation of caspase-3 in human leukemia cell line, HL-60 cells.

Gagam-whanglyun-haedoktang (GWH) is a newly designed herbal drug formula based on the traditional oriental pharmacological knowledge for the purpose of treating tumorous diseases. Apoptosis is an evolutionarily conserved suicide program residing in cells. In the present study, apoptosis inducing activities of the decocted water extract of GWH were studied. Results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that GWH had a strong cytotoxic effect on HL-60 cells. The number of live cells was less than 20% after exposure to 1 mg/ml GWH for 48 h. GWH increased cytotoxicity of HL-60 cells in a dose- and time-dependent manner. The percentage of apoptotic cells by flow cytometric analysis of the DNA-stained cells increased to 28%, 31%, and 37% at 24 h and to 37%, 44%, and 81% at 48 h after treatment with 0.01, 0.1, and 1 mg/ml GWH, respectively. DNA fragmentation also occurred in apoptosis and was characterized by a ladder pattern on agarose gel. In addition, GWH increased the secretion of tumor necrosis factor-alpha. GWH-induced apoptosis was accompanied by activation of caspase-3. These results suggest that GWH induces activation of caspase-3 and eventually leads to apoptosis.

Immune-enhancement effect of the herbal combination Allergina.

BACKGROUND: The herbal formulation, Allergina, has long been used for various diseases. It is known to have an anti-microbial and anti-virus activity. However, it is still unclear how Allergina has these effects in experimental models. We investigated the effect of Allergina on the proliferation of T cell and production of cytokines in human T-cell line, MOLT-4 cells, and mouse peritoneal macrophages. METHODS: The MOLT-4 cells were cultured for 24 h in the presence or absence of Allergina. Allergina significantly increased the cell viability by 26.9+/-5.4% (P<0.05) and interleukin (IL)-2, IL-4 and interferon (IFN)-gamma production compared with media control (about 4-fold for IL-2, 2.5-fold for IL-4 and 3.4-fold for IFN-gamma, P<0.05). Maximal effective concentration of Allergina was 1 mg/ml for IL-2 and, 0.01 mg/ml for IL-4 and IFN-gamma. Allergina alone or Allergina plus recombinant IFN-gamma (rIFN-gamma) increased the production of tumor necrosis factor (TNF)-alpha, but Allergina decreased the production of TNF-alpha on rIFN-gamma plus LPS-stimulated macrophages. In addition, Allergina increased the production of IL-12 on mouse peritoneal macrophages and peripheral blood mononuclear cells. CONCLUSION: Allergina may have an immune-enhancement effect through the cytokine production.