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Objective: To develop a one-step RT-PCR method for identification of wild virus strain and gene-deleted attenuated vaccine virus strain TJM-F92 of porcine reproductive and respiratory syndrome virus (PRRSV). Methods: A pair of primers were designed according to the nsp2 gene sequences of highly pathogenic PRRSV TJ strain and gene-deleted attenuated vaccine virus strain TJM-F92, based on which a one-step RT-PCR method for identification of various wild and gene-deleted attenuated strains of PRRSV was developed and verified for specificity, sensitiveness, sensitivity and reproducibility. Results: The gene fragment at a length of 1 100 bp was amplified from TJ strain, while that at a length of 740 bp from TJM-F92 strain, indicating that the two strains were identified correctly. The developed one-step RT-PCR method showed high sensitiveness, by which PRRSV-TJ vaccine strain at 101 TCID 50/ml was detected. Both the coincidence rates of determination results of known positive and negative serum samples of piglets were 100%, indicating high sensitivity of the method. The method also showed high reproducibility. Conclusion: The developed one-step RT-PCR method might be used for clinical identification of wild PRRSV infection and PPRSV vaccination in pigs inoculated by vaccine prepared with TJM-F92 strain, which was helpful to evaluation of immune effect of vaccine and early diagnosis of wild PRRSV infection. Source


Zhang S.,Chinese Academy of Agricultural Sciences | Tan B.,Chinese Academy of Agricultural Sciences | Ding Y.,Inner Mongolia Agricultural University | Wang F.,Chinese Academy of Agricultural Sciences | And 5 more authors.
Virology Journal | Year: 2014

Background: Bovine viral diarrhea virus (BVDV) is a pathogen found worldwide in calves. It can cause significant economic losses in agriculture. Many BVDV strains have been isolated in China. However, the pathogenesis and complete gene characteristics of BVDV isolate have yet not been reported in China. Here, a BVDV isolate was isolated and its pathogenesis and complete genome were studied. Results: A new isolate of bovine viral diarrhea virus, named JL-1, was isolated from the spleen of a sick cow with diarrhea using MDBK cell culture. The complete genome of JL-1 is 12,276 nucleotides and contains a 5′-UTR of 382 nucleotides, a 3′-UTR of 188 nucleotides, and a large ORF encoding a polyprotein consisting of 3,901 amino acids. Genomic comparison and phylogenetic analyses of complete genomic sequence clearly showed that JL-1 fell into the BVDV-1b subtype. The result of pathogenesis of JL-1 strain showed that all infected calves developed clinical signs of elevated rectal temperatures, decreased leucopenia, and viral discharge. Viral antigen was detected in infected animal tissues using immunohistochemistry. Animals in the mock were normal. These results demonstrated that BVDV JL-1 was a virulent strain. Conclusions: This is the first study to report the pathogenesis and complete gene characterization of the BVDV strain in China. This report may set a good foundation for further study of BVDV in China. © 2014 Zhang et al.; licensee BioMed Central Ltd. Source


Liu Y.,Chinese Academy of Agricultural Sciences | Wang F.-X.,Chinese Academy of Agricultural Sciences | Wen Y.-J.,Chinese Academy of Agricultural Sciences | Li Z.-G.,Chinese Academy of Agricultural Sciences | And 7 more authors.
Intervirology | Year: 2015

Background: Highly pathogenic (HP) porcine reproductive and respiratory syndrome virus (PRRSV) causes prolonged high fever, red discoloration of the body, blue ears and a high mortality. Previously, we found that the PRRSV vaccine strain TJM contained a deletion of 120 amino acids (aa 628-747) in nonstructural protein 2 (Nsp2). We aimed to explore the replication features of PRRSV after adding the transiently expressed product of these 120 aa in vitro. Methods: We constructed seven eukaryotic expression plasmids containing different parts of the 120-aa sequence, transfected them into Marc-145 cells and then inoculated the cells with 103 TCID50 TJM per well. We detected virus replication at mRNA and protein level by real-time RT-PCR and Western blotting, respectively, and determined the virus titer. Results: The transiently expressed 120 aa and one of its truncated polypeptides inhibited PRRSV TJM propagation on Marc-145 cells. The complete 120-aa sequence induced a remarkable decrease in PRRSV replication, causing a reduction in structural protein levels between 36 and 48 h after infection. Additionally, aa 628-727 partly reduced the replication of PRRSV on Marc-145 cells. Conclusions: The 120 aa from Nsp2, especially aa 628-727, play a negative role in PRRSV TJM proliferation. © 2015 S. Karger AG. Source


Patent
Sinovet Beijing Biotechnology Co. | Date: 2012-05-25

The present disclosure provides vaccine compositions comprising a PRRSV vaccine and a second porcine vaccine, which are substantially free from immuno-inhibition against each other. The second porcine virus vaccine can be CSFV and/or PRV. The preparation methods for the vaccines and the formulations are also provided. The vaccine compositions provided herein confer protective immunity to pigs against porcine reproductive and respiratory syndrome, classical swine fever, and/or pseudorabies.


Zhang H.,Chinese Academy of Agricultural Sciences | Zhang H.,Sinovet Beijing Biotechnology Co. | Zhang Z.,Sinovet Beijing Biotechnology Co. | Zhang Z.,Jinan University | And 6 more authors.
Archives of Virology | Year: 2015

In this study, a porcine rotavirus was isolated from a fecal sample from a diarrheic piglet in Jiangsu Province, China. Rotavirus-specific cytopathic effects were observed after 12 blind passages on MA-104 cells, yielding a virus titer of 106.125 TCID50/ml. By applying an 80 % nucleotide cutoff value and the RotaC2.0 automated genotyping tool, the Vp4 genotype of the new isolate was identified as P[7]. The Vp7 genotype was identified as G[9], lineage VI, and sublineage c. Experimentally infected piglets showed severe diarrhea symptoms 16–24 h post-inoculation, indicating that this new porcine rotavirus isolate is a pathogenic strain. © 2015, Springer-Verlag Wien. Source

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