Shiow-Suey Lai

Phylogenetic analysis of classical swine fever virus in Taiwan

Summary.Two envelope glycoprotein (Erns and E2) regions of the classical swine fever virus (CSFV) were amplified by RT-PCR and sequenced directly from 158 specimens collected between 1989 and 2003 in Taiwan. Phylogenetic analysis of the two regions revealed a similar tree topology and the Erns region provided better discrimination than the E2 region. One hundred and fifteen isolates out of the 158 isolates were clustered within subgroup 2.1 (further classified as 2.1a and 2.1b) and 2.2, which were considered to be likely of the introduced strains, whereas the remaining 43 isolates were clustered within subgroup 3.4 and were considered to be of the endemic strains. The subgroup 2.1a viruses were first detected in 1994 and predominated from 1995 onwards. However, subgroup 3.4 viruses were prevalent in the early years, not being isolated after 1996. We have observed a dramatic switch in genotype from subgroup 3.4 to 2.1a. The subgroup 2.1a isolates are closely related to the Paderborn and Lao isolates, whereas 2.1b isolates have a close relationship to the Chinese Guangxi isolates. The phylogenetic tree of 27 CSFV sequences based on the complete envelope glycoprotein gene (Erns–E2) displayed better resolution than that based on the complete open reading frame.

Prevalence and genetic variation of porcine circovirus type 2 in Taiwan from 2001 to 2011

Porcine circovirus type 2 (PCV2) is the major causative agent of postweaning multisystemic wasting syndrome (PMWS) in Taiwanese pig farms. We analyzed the complete genomes of 571 Taiwanese PCV2 isolates in Taiwan from 2001 to 2011 and divided the isolates into 2 distinct genotypes (PCV2a and PCV2b) with 6 clusters (1A, 1B, 1C, 2B, 2D, and 2E). Of the 571 Taiwanese PCV2 isolates, 22.9% (131/571) belonged to PCV2a and 77.1% (440/571) to PCV2b. In this study, PCV2a isolates were the most common in 2001, and then PCV2b isolates became predominate thereafter and widely distributed in pig farms since 2003. Sequence comparisons among the 571 isolates indicated that 89.6-100% had nucleotide identity for complete genome and 87.3-100% for open reading frames 2 (ORF2). The results suggest that a higher genetic variation and shift occurred among PCV2 isolates collected from 2001 to 2011 in Taiwan.

Highly permissive subclone of the porcine kidney cell line for porcine circovirus type 2 production.

This study established a highly permissive and decontaminated cell line for growing porcine circovirus type 2 (PCV2). A porcine kidney-15 cell line (PK-15) contaminated with porcine circovirus type 1 (PCV1) was decontaminated by neutralizing with rabbit anti-PCV1 hyperimmune serum. Subsequently, by limiting dilution and cell subcloning, four PCV1-free monoclonal cells were grown to monolayers. Each cell clone and PK-15 cell were infected with PCV2. The PKKC cell clone yielded up to 10(6.8)TCID(50)/ml at 6 days post-infection. In addition, PKKC was free of extraneous viral contamination and exhibited a cytopathic effect (CPE) to PCV2 at 6 days post-infection. The advantages of the PKKC cell are that it can grow a high PCV2 titer and exhibit CPE; therefore, it can be used for PCV2 cultivation, vaccine production, and diagnostic purposes.

The nuclear localization signal sequence of porcine circovirus type 2 ORF2 enhances intracellular delivery of plasmid DNA.

The phospholipid bilayer of the cell membrane is a natural barrier that prevents large molecules from entering the cell. Cationic liposomes are commonly used for transfection of plasmid DNA but they have high cost and toxicity. Many reports have shown that cell-penetrating peptides (CPP) are able to translocate across the cell membrane efficiently. The VP22 peptide of herpes simplex virus (HSV) was synthesized as a CPP. Two fusion protein candidates, containing binding/condensing protein (VP22-TmHU) and porcine circovirus type 2 nuclear localization signal (VP22-TmHU-PCV2.NLS), were constructed and expressed in E. coli in an attempt to improve delivery of plasmid DNA (pDNA). Firstly, as shown by the electrophoretic mobility shift assay (EMSA), VP22-TmHU (VT) and VP22-TmHU-PCV2.NLS (VTN) were able to bind to pDNA (pEGFP-N1) effectively. Secondly, intracellular transport of pEGFP-N1 was observed by fluorescence microscopy and quantified by flow cytometry after transfection. VTN was successful in delivering pEGFP-N1 intracellularly but VT was not. Thirdly, two protein candidates were combined with Lipofectamine, and both VT and VTN enhanced the transfection rate to 65%, compared to 25% with Lipofectamine alone. Lastly, mice were injected intramuscularly with PBS, pcDNA3-ORF2, pcDNA3-ORF2 plus Lipofectamine, pcDNA3-ORF2 plus VT, pcDNA3-ORF2 plus VT plus Lipofectamine, pcDNA3-ORF2 plus VTN, and pcDNA3-ORF2 plus VTN plus Lipofectamine. The highest level of antibodies raised against PCV2 ORF2 Cap protein was detected with pcDNA3-ORF2 plus VTN. Contrary to the in vitro results, VTN delivered pDNA effectively in vivo without Lipofectamine. In summary, the nuclear localization signal sequence of porcine circovirus type 2 ORF2 can enhance intracellular delivery of pDNA.

The presence of RNA splicing signals in the cDNA construct of the E2 gene of classical swine fever virus affected its expression

E2 is the major neutralizing antigen for classical swine fever virus (CSFV) infection. Previously, we have cloned and sequenced the E2 cDNA of Taiwan strain p97 by the reverse transcription-polymerase chain reaction (RT-PCR) method from CSFV-infected tissue. The presence of RNA splicing donor and acceptor sites were found in the cDNA sequence. In this study, transfection of E2 cDNA into mammalian cells resulted in the production of a spliced RNA. Site-directed mutagenesis of the donor and acceptor sites prevented the RNA splicing event and generated a full length transcript in COS7 cells. Although the spliced E2 transcript has not been reported in natural infection of CSFV, this study suggested that the potential splicing sites affected the E2 gene expression when the plasmid-based E2 gene was introduced into mammalian cells.

Development and evaluation of a loop-mediated isothermal amplification method for rapid detection and differentiation of two genotypes of porcine circovirus type 2.

BACKGROUND Porcine circovirus type 2 (PCV2) is one of the major swine viral diseases and has caused significant economic loss to pig producers. PCV2 has been divided into two major genotypes: PCV2a, PCV2b. A loop-mediated isothermal amplification (LAMP) method was developed for the detection and differentiation of PCV2a and PCV2b in clinical samples. METHODS LAMP-specific primer sets were designed based on six PCV2a and six PCV2b reference isolates. To determine the analytical specificity of LAMP, DNA samples extracted from 36 porcine virus isolates were tested by LAMP, including eight PCV2a, 11 PCV2b, four PCV type 1, two porcine parvovirus, three pseudorabies virus, and eight porcine reproductive and respiratory virus. To evaluate the analytical sensitivity of the assay, 10-fold serial dilutions of PCV2a and PCV2b recombinant plasmids were performed to prepare the dilutions at concentration from 10(6) to 1 copy(ies)/μL, and each dilution was tested by both LAMP and nested polymerase chain reaction (nested PCR). A total of 168 clinical samples were analyzed by both LAMP and nested PCR, and the relative sensitivity and specificity of LAMP compared to nested PCR were calculated. RESULTS Using different primer sets of LAMP, LAMP could be completed within 50 minutes. This method was found to be highly analytically specific for PCV2a and PCV2b; only the target gene was detected without cross-reaction. The analytical sensitivity of LAMP for PCV2a and PCV2b were 10 copies/μL, demonstrating analytical sensitivity comparable to that obtained using nested PCR. In addition, the sensitivity and specificity of LAMP relative to those of nested PCR were 97.7% and 100.0%, respectively. The percentage of observed agreement was 98.2%, and the κ statistic was 0.949. CONCLUSION LAMP is a rapid, specific, and sensitive diagnostic method for the detection and differentiation of PCV2a and PCV2b in clinical samples.

Detoxified Pseudomonas Exotoxin A Enhanced the Immune Response of a Porcine Circovirus type 2 (PCV2) ORF2Recombinant Protein

In this study, five different fragments of porcine circovirus type 2 (PCV2) ORF2 antigenic regions were cloned, over-expressed in E. coli. Rats were immunized with the fragments to evaluate the immunogenicity of the PCV2 ORF2 recombinant proteins. Results showed that the ORF2 F2 fragment (residues 78-156) was the most immunogenic in terms of the antibody response. Detoxified Pseudomonas exotoxin A (PE) and KDEL signal peptide were fused with F2 fragment at the N and C terminuses, respectively. This study evaluated the application of using the binding and translocation domains of PE as a vehicle for PCV2 F2 fragment, resulting in the construction of a PE-F2- KDEL recombinant protein. F2 and PE-F2-KDEL recombinant proteins were intraperitoneally injected to mice. Results showed that PE-F2-KDEL induced significantly higher levels of anti-PCV2 serum IgG antibodies than F2. Additionally, PE-F2-KDEL recombinant protein also stimulated significantly higher levels of PCV2-specific IgG compared to inactivated PCV2 whole virus antigen. These results showed that detoxified Pseudomonas exotoxin A could enhance the immune response of a PCV2 ORF2 recombinant protein.