Zhanyong Wei

Simultaneous detection of porcine parvovirus and porcine circovirus type 2 by duplex real-time PCR and amplicon melting curve analysis using SYBR Green

The development of a SYBR Green-based duplex real-time PCR is described for simultaneous detection of porcine parvovirus (PPV) and porcine circovirus type 2 (PCV-2) genomes. Viral genomes were identified in the same sample by their distinctive melting temperature (T(m)) which is 77.5°C for PPV VP2 313bp amplicon and 82.3°C for PCV-2 ORF2 171bp amplicon, respectively. The detection limit of the method was 0.01TCID(50)/mL for PPV and PCV-2, about 10 times more sensitive than conventional PCR. In addition, PPV and PCV-2 viral load were measured in 126 field samples, confirming the sensitivity and specificity, and the result showed that 70/126 samples were positive for PPV and 92/126 samples were positive for PCV2 by the duplex real-time PCR. This method may be a useful alternative rapid and reliable method for the detection of PPV/PCV-2 co-infection.

A TaqMan-based real-time polymerase chain reaction for the detection of porcine parvovirus.

A real-time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect porcine parvovirus (PPV). Real-time PCR was optimized to quantify PPV using a detection system (Rotor Gene 2000 detector) and a dual-labeled fluorogenic probe. The gene-specific labeled fluorogenic probe for the VP2 gene of PPV was used to detect PPV. Quantitation of PPV was accomplished by a standard curve plotting cycle threshold values (Ct) against each dilution of standard plasmids. When the specificity of the assay using specific PPV primers was evaluated by testing the PPV standard strain and other viruses, no cross-reactions were detected with non-PPV reference viruses. The detection limit of real-time PCR for PPV was 2.08log10 genome copy equivalent (gce). In this study, a real-time PCR assay was performed on 80 clinical samples and compared with a conventional PCR assay. In 48 of 80 samples, PPV DNA was detected by the conventional PCR assay. All samples positive for PPV DNA by the conventional PCR assay were also positive by the real-time PCR assay, and 12 of 32 samples that tested negative for PPV DNA by the conventional method tested positive by the real-time PCR assay. Using the real-time PCR assay, the number of samples in which PPV was detected increased by 15%. Therefore, it is considered to be a useful tool for the detection of PPV.

Enhancement of the immunogenicity of an infectious laryngotracheitis virus DNA vaccine by a bicistronic plasmid encoding glycoprotein B and interleukin-18

A DNA vaccine against infectious laryngotracheitis virus (ILTV) can induce specific humoral and cell-mediated immunity. However, compared to conventional vaccines, DNA vaccines usually induce poor antibody responses. To determine if co-expression of a cytokine can result in a more potent ILTV DNA vaccine, immunogenicity and protective efficacy of a monocistronic vector encoding the glycoprotein B (gB) of ILTV was compared to that of a bicistronic vector separately encoding the gB and chicken interleukin-18. Humoral and cellular responses induced by the DNA vaccines administered to the quadriceps muscle of chickens were evaluated. There were significant differences in antibody levels elicited by either monocistronic or bicistronic DNA vaccines as determined by ELISA. The percentages of CD3(+), CD3(+)CD8(+) and CD3(+)CD4(+) subgroups of peripheral blood T-lymphocytes in chickens immunized with the bicistronic DNA vaccine were higher than those in chickens immunized with monocistronic DNA vaccine. When chickens were challenged with a virulent CG strain of ILTV, the protective efficacy was enhanced significantly after immunization with the bicistronic DNA vaccine. These results demonstrated that co-expression of an adjuvant cytokine from a bicistronic DNA vaccine may be an effective approach to increasing ILTV DNA vaccine immunogenicity.

Inhibitory effects of indigowoad root polysaccharides on porcine reproductive and respiratory syndrome virus replication in vitro.

BACKGROUND Indigowoad root polysaccharide (IRPS) is a natural polysaccharide isolated from the traditional Chinese medicinal herb Radix Isatidis, and has many kinds of biological activities. However, the IRPS antiviral activity, especially the anti-porcine reproductive and respiratory syndrome virus (PRRSV) effect, has not been evaluated. METHODS PRRSV was propagated in the MARC-145 cell line, and viral titre was determined by cytopathic effect and expressed as the 50% tissue culture infection dose (TCID(50)) in the current study. The cell cytotoxic effect of IRPS toward MARC-145 was evaluated by MTT assay firstly, then the inhibitory effects of IRPS on PRRSV replication in vitro were investigated by determining the effect of IRPS upon a single replicative cycle of PRRSV in MARC-145 cells. The effects of IRPS on viral RNA and protein synthesis in PRRSV-infected cells were investigated using real-time PCR and double-antibody (sandwich) ELISA. RESULTS IRPS was able to effectively suppress the infectivity of the PRRSV in a dose-dependent manner, especially by adding IRPS during the PRRSV infection. IRPS could affect the attachment of PRRSV to MARC-145 cells, and also inhibit the viral RNA and protein synthesis. CONCLUSIONS IRPS has an antiviral effect on PRRSV replication in MARC-145 cells and might be useful in medical development for antiviral research. However, the precise mechanism of the host and viral targets of IRPS are unknown, so further studies should be conducted to investigate the precise mechanism of IRPS inhibitory effect on PRRSV infection.

Enhancing immune responses to inactivated porcine parvovirus oil emulsion vaccine by co-inoculating porcine transfer factor in mice.

Inactivated porcine parvovirus (PPV) vaccines are available commercially and widely used in the breeding herds. However, inactivated PPV vaccines have deficiencies in induction of specific cellular immune response. Transfer factor (TF) is a material that obtained from the leukocytes, and is a novel immune-stimulatory reagent that as a modulator of the immune system. In this study, the immunogenicity of PPV oil emulsion vaccine and the immuno-regulatory activities of TF were investigated. The inactivated PPV oil emulsion vaccines with or without TF were inoculated into BALB/c mice by subcutaneous injection. Then humoral and cellular immune responses were evaluated by indirect enzyme-linked immunosorbent assays (ELISA), fluorescence-activated cell sorter analyses (FACS). The results showed that the PPV specific immune responses could be evoked in mice by inoculating with PPV oil emulsion vaccine alone or by co-inoculation with TF. The cellular immune response levels in the co-inoculation groups were higher than those groups receiving the PPV oil emulsion vaccine alone, with the phenomena of higher level of IFN-γ, a little IL-6 and a trace of IL-4 in serum, and a vigorous T-cell response. However, there was no significant difference in antibody titers between TF synergy inactivated vaccine and the inactivated vaccine group (P>0.05). In conclusion, these results suggest that TF possess better cellular immune-enhancing capability and would be exploited into an effective immune-adjuvant for inactivated vaccines.

Cloning, in vitro expression, and bioactivity of interleukin-18 isolated from a domestic porcine breed found in Henan

To evaluate the effects of recombinant porcine interleukin-18 (rpIL-18) on the replication of viruses in host cells and proliferation of lymphocytes, porcine IL-18 (pIL-18) isolated from a domestic big-white porcine breed found in the Henan province (HN) was cloned using a reverse transcriptase-PCR. The cloned HN pIL-18 contained an ORF of 579 base pairs encoding a 192-amino-acid precursor protein. The amino acid sequence of HN pIL-18 was compared with all the other pIL-18 amino acid sequences and varied by at least one amino acid to the consensus of all the others available. HN pIL-18 mature protein gene was inserted into a prokaryotic vector pGEX-4T-1 and expressed in Escherichia coli BL21. The expression of glutathione-S-transferase-pIL18 fusion protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The rpIL-18 induced in vitro proliferation of concanavalin-A-stimulated porcine splenocytes, as revealed by the MTT assay. We studied the antiviral activities of the rpIL-18 on the replication of porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine parvovirus (PPV) cultured in two homologous cell lines. The results suggested that rpIL-18 can stimulate the proliferation of lymphocytes and inhibit viral pathogens infecting the porcine population.

Nitric oxide inhibits the replication cycle of porcine parvovirus in vitro

This study investigated the inhibitory effect and mechanism of nitric oxide (NO) on porcine parvovirus (PPV) replication in PK-15 cells. The results showed that two NO-generating compounds, S-nitroso-l-acetylpenicillamine (SNAP) and l-arginine (LA), at a noncytotoxic concentration could reduce PPV replication in a dose-dependent manner and that this anti-PPV effect could be reversed by the NO synthase (NOS) inhibitor N-nitro-l-arginine methyl ester (l-NAME). By assaying the steps of the PPV life cycle, we also show that NO inhibits viral DNA and protein synthesis. This experiment provides a frame of reference for the study of the anti-viral mechanism of NO.

Use of a Multiplex RT-PCR Assay for Simultaneous Detection of the North American Genotype Porcine Reproductive and Respiratory Syndrome Virus, Swine Influenza Virus and Japanese Encephalitis Virus

Abstract A multiplex reverse transcriptase-polymerase chain reaction (multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously, in swine. Specific primers for each of the 3 RNA viruses, North American genotype porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus, and swine influenza virus, were used in the testing procedure. The assay was shown to be highly sensitive because it could detect as little as 10−5 ng of each of the respective amplicons in a single sample containing a composite of all 3 viruses. The assay was also effective in detecting one or more of the same viruses in various combinations in specimens, including lymph nodes, lungs, spleens, and tonsils, collected from clinically ill pigs and in spleen specimens collected from aborted pig fetuses. The results from the multiplex RT-PCR were confirmed by virus isolation. The relative efficiency (compared to the efficiency of separate assays for each virus) and apparent sensitivity of the multiplex RT-PCR method show that this method has potential for application in routine molecular diagnostic procedures.

Construction and immunogenicity of a recombinant pseudorabies virus co-expressing porcine circovirus type 2 capsid protein and interleukin 18

A novel recombinant pseudorabies virus (PRV) expressing porcine circovirus type 2 (PCV2) capsid protein and IL-18 was constructed. The PCV2 open reading frame 2 (ORF2) and porcine IL-18 genes were amplified by PCR and then inserted into the PRV transfer vector (pG) to produce a recombinant plasmid (pGO18). Plasmid pGO18 was transfected into porcine kidney cell (PK15) pre-infected with PRV HB98 vaccine strain to generate a recombinant virus. The recombinant virus PRV-ORF2-IL18 was purified by green fluorescent plaque purification and the inserts were confirmed by PCR, enzyme digestion, sequencing, and Western blot. The humoral and cellular responses induced by the recombinant virus were assessed in mice. Mice (n=10) were immunized with PRV-ORF2-IL18, PRV-ORF2, PRV HB98, or inactivated PCV2. PRV-ORF2-IL18 elicited high titers of ELISA and serum neutralizing antibodies and strong cell-mediated immune responses in mice as indicated by anti-PCV2 ELISA, PRV-neutralizing assay, PCV2 specific lymphocyte proliferation assay, CD3(+), CD4(+) and CD8(+) T lymphocytes analysis, respectively. And PRV-ORF2-IL18 immunization protected mice against a lethal challenge of a virulent PRV Fa strain and significantly reduced the amount of PCV2 viremia. These results suggest an adjuvant effect of IL-18 on cellular immune responses. The recombinant virus might be an attractive candidate vaccine for preventing PCV2 and PRV infections in pigs.

A nano silicon adjuvant enhances inactivated transmissible gastroenteritis vaccine through activation the Toll-like receptors and promotes humoral and cellular immune responses

Inactivated transmissible gastroenteritis virus (TGEV) vaccines are widely used in swine herds in China. These are limited, however, by the need to elicit both humoral and cellular immunity, as well as the efficiency of adjuvants. In this study, a 70-nm nano silicon particle was applied with inactivated TGEV vaccine in mice, and its immune-enhancing effects and mechanism of action investigated. We found that nano silicon applied with inactivated TGEV vaccine induced high antibody titers, increase IL-6, TNF-α and IFN-γ expression, and stimulate CD3+ T cell proliferation with a high CD4+/CD8+ T lymphocyte ratio. Nano silicon could quickly activate innate and adaptive immunity by stimulating Toll-like receptor signaling pathways, indicating that the nano silicon adjuvant enhanced long-term humoral and early cellular immune responses when combined with inactivated TGEV vaccine. Nano silicon could be considered for use as an antigen- carrier and adjuvant for veterinary vaccines.