Tien-Jye Chang

Development of a loop-mediated isothermal amplification for rapid detection of orf virus.

A loop-mediated isothermal amplification (LAMP) assay using six primers targeting a highly conserved region of the B2L gene has been developed to diagnose orf virus. The assay produces a ladder-like pattern of products on an agarose gel that can be specifically digested with BsrGI enzyme. The sensitivity of the LAMP assay, which was determined to be a single copy of the standard plasmid, was 100 fold and 10 fold higher than PCR and nested PCR, respectively; furthermore, no cross-reactivity was founded with the other tested viruses. By staining the products directly in the tube with PicoGreen or ethidium bromide, the products can be visualized with a similar sensitivity as by gel electrophoresis. Clinical samples were tested using PCR, nested PCR and LAMP assay and the positive rates were 60%, 70% and 70%, respectively. The LAMP assay allows easy, rapid, accurate and sensitive detection of infection with orf virus and is especially applicable in a resource-limited situation.

DNA vaccination induces a long-term antibody response and protective immunity against pseudorabies virus in mice

SummaryIn order to investigate the mechanism of long-term immunity and the effect of protective immunity induced by DNA vaccination, we constructed the expression plasmid containing a pseudorabies virus (PRV) gD gene encoding an envelope glycoprotein. Intramuscular vaccination of mice with the plasmid DNA induced a strong antibody response which lasted for one year after final vaccination. An IgM to IgG class switch occurred, indicating helper T-lymphocyte activity. We further analyzed the persistence and expression of gD gene by polymerase chain reaction and reverse transcriptase polymerase chain reaction. The results showed that gD gene was present and expressed in the muscle cell up to one year after final booster injection. Furthermore, mice vaccinated with the plasmid DNA were protected against a subsequent lethal challenge with PRV. Therefore, the DNA vaccination does induce a protective immunity and long-term antibody response against PRV, which could be maintained by persistent expression of gD gene in muscle cells.

Luteolin inhibits viral-induced inflammatory response in RAW264.7 cells via suppression of STAT1/3 dependent NF-κB and activation of HO-1.

Luteolin is a common dietary flavonoid present in Chinese herbal medicines that has been reported to have important anti-inflammatory properties. Previous studies have shown that luteolin is an anti-inflammatory and anti-oxidative agent. In this study, the anti-virus inflammatory capacity of luteolin and its molecular mechanisms of action were analyzed. The cytotoxic effects of luteolin were assessed in the presence or absence of pseudorabies virus (PRV) via LDH and MTT assays. The results showed that luteolin (<10μM) had no toxic effects and there were tendencies toward higher cell survival. In PRV-infected RAW264.7 cells, luteolin potently inhibited the production of NO, iNOS, COX-2 and inflammatory cytokine production. Luteolin did not inhibit the phosphorylation of ERK 1/2, p38, and JNK 1/2 either. We found that PRV-induced NF-κB activation is regulated through inhibition of STAT1and STAT3 phosphorylation in response to luteolin. Additionally, luteolin caused the induction of HO-1 via upregulation of Nrf2, both of which are involved in the secretion of proinflammatory mediators. The blockade of HO-1 expression with SnPP, a HO-1 inhibitor, attenuated HO-1 induction by luteolin and thus mitigated its anti-inflammatory effects during PRV-infected RAW264.7 cells. Taken together, our data indicate that luteolin diminishes the proinflammatory mediators NO, inflammatory cytokines and the expression of their regulatory genes, iNOS and COX-2, in PRV-infected RAW264.7 cells by inhibiting STAT1/3 dependent NF-κB activation and inducing Nrf2mediated HO-1 expression.

Regulation of virus-induced inflammatory response by β-carotene in RAW264.7 cells

Carotenoids are effective antioxidants, which can quench singlet oxygen, suppress lipid peroxidation, and prevent oxidative damage. Both Pseudorabies virus (PRV) and human Herpes simplex virus (HSV) are DNA viruses, and their pathogenesis and immunobiology are similar. However, PRV does not infect humans. Therefore, PRV was used to infect murine macrophages (RAW264.7 cells), to mimic HSV-induced inflammation. Meanwhile, the influence of β-carotene on PRV-induced inflammation was also investigated. Results indicated that β-carotene inhibited (p<0.05) NO, IL-1β, IL-6, and MCP-1 production in PRV-infected RAW264.7 cells. β-Carotene also suppressed (p<0.05) NF-κB (p50 and p65), phosphorylation of extracellular-signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) expression. It could be concluded that the anti-inflammatory effect of β-carotene is mainly through a suppression of cytokine expression in PRV-induced inflammation, which results from NF-κB inactivation. β-Carotene can be considered a potential anti-inflammatory agent for DNA-virus infection.

Growth hormone gene polymorphisms and growth performance traits in duroc, Landrace and Tao-Yuan Pigs

This study investigated the restriction fragment length polymorphism (RFLP) of the porcine growth hormone (pGH) gene in Duroc, Landrace, and Tao-Yuan pigs and its effects on growth performance and levels of plasma growth hormone in peripheral circulation. Genomic DNA extracted from 81 Tao-Yuan, 60 Landrace and 48 Duroc pigs were subjected to Southern blot hybridization with a pGH cDNA probe. Polymorphism was detected with the restriction enzymes TaqI and DraI. A comparison of these three breeds showed significant differences in allelic frequencies. Blood samples for radioimmunoassay (RIA) of GH were collected biweekly during the experimental period from pigs 12 to 40 weeks of age. Tao-Yuan pigs showed a mean plasma GH level (2.51 +/- 1.23 ng/mL) that was much lower than that of the Landrace (3.80 +/- 1.52 ng/mL) and Duroc (4.20 +/- 1.03 ng/mL) pigs (P < 0.05). Moreover, the Tao-Yuan pigs also showed poorer growth performance than the Landrace and the Duroc pigs both in the daily weight gain (0.37 +/- 0.06 vs. 0.67 +/- 0.05 and 0.70 +/- 0.05 kg/day, P < 0.01) and feed efficiency (3.12 +/- 0.28 vs. 2.60 +/- 0.14 and 2.52 +/- 0.12, P < 0.05). These results suggest that the growth performance trait in these pigs is highly correlated with their growth hormone genotype.

Single nucleotide variation in exon 11 of canine BRCA2 in healthy and cancerous mammary tissue.

Germline mutations in the BRCA2 tumour suppressor gene are significant risk indicators of breast cancer in women, especially for hereditary breast cancer. The BRCA2 protein interacts via the BRC (breast cancer) domain with RAD51, an essential component of the cellular machinery for the maintenance of genome stability and double strand-breaks repair. Exon 11 is the largest exon of the BRCA2 gene and contains the region encoding eight repeats of the BRC domain. Little is known about the roles of BRCA2 exon 11 in canine mammary tumours. In present study, the entire BRCA2 exon 11 was sequenced in canine mammary tumours. Fifteen mammary gland samples were obtained from four normal mammary glands and 11 mammary tumours (10 malignant and one benign tumours). Comparing sequences of normal mammary glands with those in GenBank (AB043895 and Z75664), a single nucleotide polymorphism (SNP) at codon 2414 G>A (resulting in a lysine to an arginine substitution) was identified. When compared with the normal mammary gland, 19 sporadically distributed point mutations were found in mammary tumours, including 68% of missense and 32% of silent mutations. A high frequency of genetic variations in codon 511 A>C or 2414 A>G were identified in 6/11 cases, and two missense mutations (2414 A>G, 2383 A>C) were located at the fourth repeat of the BRC domains.

Growth enhancement of fowls by dietary administration of recombinant yeast cultures containing enriched growth hormone

In present study the methylotrophic yeast, Pichia pastoris, was used to express a recombinant growth hormone (rGH) gene of swine. A synthetic secretion cassette was constructed using the promoter of the alcohol oxidase1 gene (AOX1), and a alpha-factor signal peptide. After electroporatic transformation and zeocin selection, several clones exhibited high levels of rGH protein expression constituting more than 20% of total yeast protein. Over 95% of rGH was shown to be export into the culture supernatant. Yeast transformant containing the highest recombinant growth hormone level (rGH yeast) and native GS115 Pichia pastoris (non-rGH yeast, as a control) were separately cultured, harvested and adsorbed by wheat bran. Yeast cultures of four dosages (0.05, 0.1, 0.2, and 0.4%) were mixed respectively with chick basal diet and fed to simulated country chickens for 9 weeks. The results showed that, when compared to control chicks, the percentage of body weight gain was improved significantly (P<0.05) in chicks fed with diets containing 0.1 or 0.2% rGH-rich yeast culture at brooding stage, and in chicks fed with 0.4% rGH-rich yeast culture at growing stage. The average weight gain in rGH yeast treated groups for the full-term (0 to 63d) and short term (43 to 63d) of growth were 10.6 and 9.4%, respectively, better than the non-rGH yeast control group. These experimental data suggest that the use of rGH-containing yeast as a supplement in fed provided an alternative approach for growth improvement in simulated country chickens.

Analysis of upregulated cellular genes in pseudorabies virus infection: use of mRNA differential display

Virus infection usually alters the host cell and shuts off the synthesis of cellular macromolecules. In order to screen the upregulated cellular transcripts during pseudorabies virus (PRV) infection, we employed the mRNA differential display technique. The screen is based on positive selection at the mRNA level for genes expressed in normal cells but increased in corresponding PRV-infected cells. Over 14000 species of mRNA, isolated from mock-infected and PRV-infected Madin-Darby bovine kidney cell at 1 h post infection, were screened, and 40 candidate clones were recovered. Southern blot analysis revealed that 17 out of 40 candidate clones, were enhanced in PRV-infected cells. Partial DNA sequences demonstrated that 17 clones were distinct cellular genes, including those encoding the modulators of signal transduction (saposin, 14-3-3, adenylate kinase, adenylyl cyclase, protein kinase C-alpha), those encoding the components of translation (fau, ribosomal proteins S11, L31, L36), other cellular genes (peptidase, cyclin E, rch1, oligo-C-rich single-stranded nucleic acid binding protein, rap, arginyl-tRNA synthetase), and two unknown genes. Thus, this study identifies successfully the transcriptionally regulated cellular genes which are associated with PRV infection. Furthermore, this study provides support for the use of mRNA differential display as a method to rapidly isolate differentially expressed genes in virus infection.

The recombinant nucleocapsid protein of classical swine fever virus can act as a transcriptional regulator

The cDNA of the nucleocapsid (core) protein of classical swine fever virus (CSFV) was generated by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into a eukaryotic expression vector. The effect of the recombinant core protein on the transcriptional regulation of cellular as well as viral promoters was studied. Using transient transfection assay, our results demonstrated that the core protein can activate the promoter of human heat shock protein 70 gene, and suppressed the SV40 early promoter. These findings indicate that the core protein appears to function not only as a viral structural protein but also as a regulator of gene expression. The implications of core proteins on the viral maturation are discussed.

Programmed cell death induced by Japanese encephalitis virus YL vaccine strain or its recombinant envelope protein in varied cultured cells

Objective: The Japanese encephalitis virus YL vaccine strain (JEV-YL) was investigated as regards its organ tropism and the role of recombinant envelope glycoprotein in the induction of apoptosis was explored. Methods: Threevaried cell lines (HepG2, Vero and C6) were infected with JEV-YL or transfected with eukaryotic expression plasmids (pcE, pcF1R2, pcF1R1 and pcF2R2) which contain different parts of the envelope gene and phenotypic properties were examined by flow cytometry and DNA fragmentation analysis. Results: After JEV-YL infection, smaller plaque was produced on HepG2 cells than on Vero cells, whereas no cytopathic effect was observed on C6 cells; moreover, by apoptosis and DNA fragmentation assays, the hallmark cytopathic effects were detected in HepG2 and Vero cells but not in C6 cells. Furthermore, cells used in our study transfected with recombinant core plasmid, pcE, which include full-length E gene but the deleted forms (pcF1R2, pcF1R1 and pcF2R2) did not have similar results as JEV-YLs. Conclusions: The JEV-YL vaccine strain had changed cell tropism to liver cells different from other virulent strains which have neural tropism, and in this study we proved that the transient-expressed entire E protein of JEV-YL could induce apoptosis and the mutations of E protein may change the organ tropism of JEV-YL.