Chi-Young Wang

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.

Development of multiplex PCR for simultaneous detection of six swine DNA and RNA viruses.

Uniplex and multiplex reverse transcription-polymerase chain reaction (RT-PCR) and PCR protocols were developed and evaluated subsequently for its effectiveness in detecting simultaneously single and mixed infections in swine. Specific primers for three DNA viruses and three RNA viruses, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV) were used for testing procedure. A single nucleic acid extraction protocol was adopted for the simultaneous extraction of both RNA and DNA viruses. The multiplex PCR consisted with two-step procedure which included reverse transcription of RNA virus and multiplex PCR of viral cDNA and DNA. The multiplex PCR assay was shown to be sensitive detecting at least 450pg of viral genomic DNA or RNA from a mixture of six viruses in a reaction. The assay was also highly specific in detecting one or more of the same viruses in various combinations in specimens. Thirty clinical samples and aborted fetuses collected from 4- to 12-week-old piglets were detected among 39 samples tested by both uniplex and multiplex PCR, showing highly identification. Because of the sensitivity and specificity, the multiplex PCR is a useful approach for clinical diagnosis of mixed infections of DNA and RNA viruses in swine.

Detection of Duck Hepatitis B Virus DNA on Filter Paper by PCR and SYBR Green Dye-Based Quantitative PCR

ABSTRACT Duck hepatitis B virus (DHBV) belongs to the Hepadnaviridae family, which includes human Hepatitis B virus (HBV) and Woodchuck hepatitis virus. It is widely distributed in wild and domestic ducks due to congenital transmission. HBV is a worldwide health problem, with carriers at risk of developing cirrhosis and liver cancer. Medical staff and scientists working with HBV must be vaccinated because of its contagious nature. DHBV is a safe surrogate for HBV because of their similarities. Collection of serum and blood samples on filter paper has been used to screen for metabolic disorders, genetic diseases, and viral infection and for evolutionary studies of the genome. In this study, DHBV from serum and blood dried on filters was detected by PCR. A 0.1-μl sample was sufficient for detection. The immobilization potential of filter papers for DHBV was examined, and the highest yield of PCR products was observed with Whatman paper. Dried serum was stable under different storage temperatures for 4 weeks, but the yields of PCR products decreased when the temperature was ⩾4°C. The optimal condition for storage was −70°C. A newly developed quantitative PCR based on monitoring the amplification by measuring the increase in fluorescence caused by the binding of SYBR green dye to double-stranded products was applied herein. DHBV genomic DNA cloned in a plasmid was used for the generation of standard DHBV DNA for quantitative PCR. It validated results from PCR in terms of the copy number of DHBV particles. The specificity of PCR was demonstrated by melting curve analysis, and the differentiation of two DHBV isolates amplified from dried serum was demonstrated based on their melting temperatures determined by GC contents and sequence. It was easier and simpler than other PCR-based DNA techniques. The use of serum dried on filters allows samples from distant field for which cold storage and transportation are a problem to be mailed to the diagnostic laboratory. Samples can be archived for comparison and used as a source of DNA for cloning and sequencing.

Rapid and sensitive detection of infectious bursal disease virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick

Infectious bursal disease (IBD), an immunosuppressive disease that affects all ages of chickens, results in significant losses in the poultry industry. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with a chromatographic lateral flow dipstick (LFD) for the detection of infectious bursal disease virus (IBDV) was developed. The whole process of testing can be completed in less than 70 min using biotin-labeled primers, an FITC-labeled DNA probe, and the LFD. The detection limits for IBDV using RT-LAMP and RT-LAMP-LFD were the same at 10(-1)plaque forming units (PFU). When other unrelated viruses and cells were tested, no false positive results were observed. In addition, the amplification efficiency of RT-LAMP was enhanced when a loop primer was used. The RT-LAMP-LFD product started to be detected after 40 min. Clinical samples were used to compare assays using RT-PCR, nested RT-PCR, RT-LAMP, and RT-LAMP-LFD and the positive rates were 16%, 40%, 40%, and 40%, respectively. In conclusion, this assay is an easy, rapid, accurate, and sensitive method for the detection of IBDV and will improve the screening of field samples, especially when veterinarians have limited resources.

AGE-BSA down-regulates endothelial connexin43 gap junctions

BackgroundAdvanced glycation end products generated in the circulation of diabetic patients were reported to affect the function of vascular wall. We examined the effects of advanced glycation end products-bovine serum albumin (AGE-BSA) on endothelial connexin43 (Cx43) expression and gap-junction communication.ResultsIn human aortic endothelial cells (HAEC) treated with a series concentrations of AGE-BSA (0-500 μg/ml) for 24 and 48 hours, Cx43 transcript and Cx43 protein were reduced in a dose dependent manner. In addition, gap-junction communication was reduced. To clarify the mechanisms underlying the down-regulation, MAPKs pathways in HAEC were examined. Both a MEK1 inhibitor (PD98059) and a p38 MAPK inhibitor (SB203580) significantly reversed the reductions of Cx43 mRNA and protein induced by AGE-BSA. Consistently, phosphorylation of ERK and p38 MAPK was enhanced in response to exposure to AGE-BSA. However, all reversions of down-regulated Cx43 by inhibitors did not restore the functional gap-junction communication.ConclusionsAGE-BSA down-regulated Cx43 expression in HAEC, mainly through reduced Cx43 transcription, and the process involved activation of ERK and p38 MAPK.

Purification and functional motifs of the recombinant ATPase of orf virus

Our previous study showed that the recombinant ATPase encoded by the A32L gene of orf virus displayed ATP hydrolysis activity as predicted from its amino acids sequence. This viral ATPase contains four known functional motifs (motifs I-IV) and a novel AYDG motif; they are essential for ATP hydrolysis reaction by binding ATP and magnesium ions. The motifs I and II correspond with the Walker A and B motifs of the typical ATPase, respectively. To examine the biochemical roles of these five conserved motifs, recombinant ATPases of five deletion mutants derived from the Taiping strain were expressed and purified. Their ATPase functions were assayed and compared with those of two wild type strains, Taiping and Nantou isolated in Taiwan. Our results showed that deletions at motifs I-III or IV exhibited lower activity than that of the wild type. Interestingly, deletion of AYDG motif decreased the ATPase activity more significantly than those of motifs I-IV deletions. Divalent ions such as magnesium and calcium were essential for ATPase activity. Moreover, our recombinant proteins of orf virus also demonstrated GTPase activity, though weaker than the original ATPase activity.

Development of ELISA kits for antibodies against avian reovirus using the σC and σB proteins expressed in the methyltropic yeast Pichia pastoris

Both the sigmaC and sigmaB proteins of avian reovirus (ARV) can induce type- and group-specific neutralizing antibodies, respectively. In this study, the full-length of S1133 sigmaC, 1071-1 sigmaC, S1133 sigmaB, and S1133 sigmaC-sigmaB fusion genes of ARV were cloned into a secreted vector pPICZalphaA and then integrated into the chromosome of Pichia pastoris for induced expression. Western blot assay showed that ARV sigmaC, sigmaB, and sigmaC-sigmaB fusion proteins were expressed and secreted into the medium. Two types of ELISA kits using equal mixtures of 1071-1sigmaC and S1133 sigmaB and S1133 sigmaC-sigmaB fusion proteins as antigens were developed. After a checker board titration for optimal conditions, the cut-off values of positive results for the 1071-1sigmaC/S1133 sigmaB and S1133 sigmaC-sigmaB ELISA kits were 0.24 and 0.12, respectively. Forty-four serum neutralization test-positive and twenty-eight serum neutralization-negative samples from vaccinated and commercial farm chickens were tested by the new ELISA kits and by the conventional ELISA. The new ELISA kits have higher positive rates than the conventional ELISA. The results revealed that the correlation rates for the serum neutralization titer and the absorbance values with the new ELISA kits and the conventional ELISA were 100% and 95.8%, respectively.

Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) for detection of Newcastle disease virus

Due to appearance of new genotypes of Newcastle disease virus (NDV) with no cross-protection and with vaccine strains, some outbreaks have been reported in Taiwan that caused significant damage to the poultry industry. A reliable assay protocol, (RAPID)-bioactive amplification with probing (BAP), for detection of NDV that uses a nested PCR and magnetic bead-based probe to increase sensitivity and specificity, was developed. Primers and probes were designed based on the conserved region of the F protein-encoding gene sequences of all NDV Taiwan isolates. The optimal annealing temperature for nested reverse transcription-polymerase chain reaction (RT-PCR) to amplify the gene was 61 degrees C and optimal hybridization occurred when buffer 1x SSC and 0.5% SDS were used at 50 degrees C. The sensitivity of RAPID-BAP was 1 copy/microl for standard plasmids and 10 copy/mul for transcribed F protein-encoding gene of NDV with comparable linearity (R(2)=0.984 versus R(2)=0.99). This sensitivity was superior to that of other techniques currently used. The assay was also highly specific because the negative controls, including classical swine fever virus, avian influenza virus, avian reovirus, and infectious bursa disease virus could not be detected. Thirty-four field samples were tested using conventional RT-PCR, nested RT-PCR, real-time quantitative RT-PCR, and RAPID-BAP assay and the positive rates were 24%, 30%, 41%, and 53%, respectively. The developed assay allows for rapid, correct, and sensitive detection of NDV and fulfils all of the key requirements for clinical applicability. It could reliably rule out false negative results from antibody-based assays and also facilitate a rapid diagnosis in the early phase of the disease for emergency quarantine that may help prevent large-scale outbreaks.

Complete Genome Sequence of a New H9N2 Avian Influenza Virus Isolated in China.

ABSTRACT The complete genomic sequence of a new H9N2 avian influenza virus (AIV), isolated in northwestern China, was determined. Sequence and phylogenetic analyses based on the sequences of eight genomic segments revealed that the isolate is phylogenetically related to the Y280-like sublineage.

The genotyping of Infectious bronchitis virus in Taiwan by a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction

Infectious bronchitis virus (IBV; Avian coronavirus) causes acute respiratory and reproductive and urogenital diseases in chickens. Following sequence alignment of IBV strains, a combination of selective primer sets was designed to individually amplify the IBV wild-type and vaccine strains using a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) approach. This system was shown to discriminate the IBV wild-type and vaccine strains. Moreover, an ARMS real-time RT-PCR (ARMS qRT-PCR) was combined with a high-resolution analysis (HRMA) to establish a melt curve analysis program. The specificity of the ARMS RT-PCR and the ARMS qRT-PCR was verified using unrelated avian viruses. Different melting temperatures and distinct normalized and shifted melting curve patterns for the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were detected. The new assays were used on samples of lung and trachea as well as virus from allantoic fluid and cell culture. In addition to being able to detect the presence of IBV vaccine and wild-type strains by ARMS RT-PCR, the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were distinguished using ARMS qRT-PCR by their melting temperatures and by HRMA. These approaches have acceptable sensitivities and specificities and therefore should be able to serve as options when carrying out differential diagnosis of IBV in Taiwan and China.