1.Diagonsis establishment of fluorescen quantitative PCR assay for pseudorabies wild-type virus and vaccine virus.
Li ZHAO ; Baoan CUI ; Hongying CHEN ; Zhanyong WEI ; Lanlan ZHENG ; Xiaoli LÜ ; Yanyan JIA ; Xuyong ZHAO
Chinese Journal of Biotechnology 2008;24(7):1149-1154
We designed two pairs of primers and their corresponding TaqMan probes according to gH, gE gene of PRV. By optimizing the probe's concentration, Mg2+ concentration, primers concentration and sample DNA extraction, real-time fluorescent quantitative PCR (FQ-PCR) which can quickly identity field virus and vaccine virus of PRV was established. According to our results, the dynamic range of the FQ-PCR assay is between 10 x 10(1) copies/microL and 10 x l0(8) copies/microL, and the detection limit of FQ-PCR is 1.0 x 10(1) copies/microL, which is 100 fold higher than that of conventional PCR. We detected 60 doubtful tissue samples using the FQ-PCR assay, serum neutralization and conventional PCR. In conclusion, the FQ-PCR method is rapid, sensitive, specific and accurate, and can be used to detect field strains of PRV rapidly. The closed-tube format of the assay minimized the risk of contamination of subsequent reaction and the assay can be performed in 2 h or less. Development of real-time quantitative PCR provides the basis for the early and rapid detection and analyzing quantitatively the infectious degree of PRV.
Animals
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Fluorescent Dyes
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Herpesvirus 1, Suid
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genetics
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isolation & purification
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Polymerase Chain Reaction
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methods
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Pseudorabies
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diagnosis
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prevention & control
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virology
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Pseudorabies Vaccines
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immunology
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isolation & purification
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Swine
2.Polarization of protective immunity induced by replication-incompetent adenovirus expressing glycoproteins of pseudorabies virus.
Young Woo HAN ; Abi G ALEYAS ; Junu A GEORGE ; Seon Ju KIM ; Hye Kyung KIM ; Hyun A YOON ; Dong Jin YOO ; Seong Ho KANG ; Koanhoi KIM ; Seong Kug EO
Experimental & Molecular Medicine 2008;40(6):583-595
Replication-incompetent adenoviruses expressing three major glycoproteins (gB, gC, and gD) of pseudorabies virus (PrV) were constructed and used to examine the ability of these glycoproteins to induce protective immunity against a lethal challenge. Among three constructs, recombinant adenovirus expressing gB (rAd-gB) was found to induce the most potent immunity biased to Th1-type, as determined by the IgG isotype ratio and the profile of the Th1/Th2 cytokine production. Conversely, the gC-expressing adenovirus (rAd-gC) revealed Th2-type immunity and the gD-expressing adenovirus (rAd-gD) induced lower levels of IFN-gamma and IL-4 production than other constructs, except IL-2 production. Mucosal delivery of rAd-gB induced mucosal IgA and serum IgG responses and biased toward Th2-type immune responses. However, these effects were not observed in response to systemic delivery of rAd-gB. In addition, rAd-gB appeared to induce effective protective immunity against a virulent viral infection, regardless of whether it was administered via the muscular or systemic route. These results suggest that administration of replication-incompetent adenoviruses can induce different types of immunity depending on the expressed antigen and that recombinant adenoviruses expressing gB induced the most potent Th1-biased humoral and cellular immunity and provided effective protection against PrV infection.
Adenoviridae/genetics/*immunology/metabolism
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Animals
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Antibody Formation
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Cell Line
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Cytokines/immunology
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Female
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Glycoproteins/biosynthesis/genetics/*immunology
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Herpesvirus 1, Suid/genetics/*immunology/physiology
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Immunity, Cellular
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Immunoglobulin G/immunology
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Mice
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Mice, Inbred C57BL
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Pseudorabies/*immunology/prevention & control
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Pseudorabies Vaccines/administration & dosage/*immunology
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Swine
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Th1 Cells/immunology
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Th2 Cells/immunology
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*Virus Replication