En-Min Zhou

Northwest A & F University, Yang-ling-chen, Shaanxi, China

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Publications (4)12.29 Total impact

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    ABSTRACT: The PiggyBac (PB) transposon system is a non-viral DNA-transfer system in which a transposase directs integration of a PB transposon into a TTAA site in the genome. Transgenic expression of porcine CD163 is necessary and sufficient to confer non-permissive cells susceptible to infection with porcine reproductive and respiratory syndrome virus (PRRSV). Such permissive cells can be used as a tool for PRRSV cellular receptor and other studies. One of the problems in studying PRRSV is the lack of porcine cell lines. In this study, efficient transfection and expression of porcine CD163 in PK-15 cells by PB transposition was demonstrated. The stable PK-15(CD163) cell line was used in PRRSV infection assays. The data indicated that the average PB transgene copy number per genome was approximately ten. In line with previous literature the integration of PB into the genome had a bias toward the TTAA chromosomal site. The PK-15(CD163) cell line was susceptible to infection by different PRRSV strains and the virus grew to similar titers compared to the Marc-145 cell line. This simplification of PK-15(CD163) cell line production will provide a valuable tool to facilitate PRRSV cellular receptor studies and to accelerate existing vectors for PK-15 cell-based gene transfer and expression.
    Journal of virological methods 07/2013; · 2.13 Impact Factor
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    ABSTRACT: The coronavirus nucleocapsid (N) protein plays a multifunctional role in the virus life cycle from regulation of replication and transcription, genome packaging and modulation of host cell processes. These functions are likely to be facilitated by interactions with host cell proteins. The potential interactome of the infectious bronchitis virus (IBV) N protein was mapped using stable isotope labeling with amino acids in cell culture (SILAC) coupled to a GFP-nanotrap and LC-MS/MS. The addition of the SILAC label allowed discrimination of proteins that were likely to specifically bind to the N protein over background binding. Overall, 142 cellular proteins were selected as potentially binding to the N protein, many as part of larger possible complexes. These included ribosomal proteins, nucleolar proteins, translation initiation factors, helicases and hnRNPs. The association of selected cellular proteins with IBV N protein was confirmed by immunoblotting, co-sedimentation and confocal microscopy. Further, the localization of selected proteins in IBV infected cells as well as their activity during virus infection was assessed by siRNA-mediated depletion, demonstrating the functional importance of cellular proteins in the biology of IBV. This interactome confirms previous observations made with other coronavirus and IBV N proteins both on over-expressed proteins and using infectious virus, but also provides novel data that can be exploited to understand the interaction between the virus and the host cell.
    Journal of Virology 05/2013; · 5.08 Impact Factor
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    ABSTRACT: An indirect enzyme-linked immunosorbent assay (iELISA) that could detect immunoglobulin G antibodies against avian hepatitis E virus (HEV) was developed. This assay employs a truncated C-terminal 268-amino acid recombinant ORF2 protein from an avian HEV genotype 3 strain isolated in China (CaHEV) as the coating antigen. The antigen concentration and serum dilution were optimized using a checkerboard titration. A cut-off value of 0.368 at OD450 nm was determined by testing 120 positive and 200 negative chicken sera for avian HEV antibodies using the two-graph receiver operating characteristic (TG-ROC) analysis. This iELISA has a sensitivity of 96.1% and a specificity of 95.8%. The overall agreement between the iELISA and a corresponding Western blot was 97%. The iELISA was used to evaluate the seroprevalence of avian HEV in poultry farms in the Shandong province. The avian HEV seropositive rate of 35.9% was determined by testing 1871 serum samples that were collected from 10 chicken flocks ranged from 10 to 60 weeks of age. The iELISA that was developed in this study can be used for detection of immunoglobulin G antibodies against avian HEV.
    Journal of Virological Methods. 01/2013; 187(1):32–36.
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    ABSTRACT: Following the 2006 outbreaks of the highly pathogenic porcine reproductive and respiratory syndrome, the causative agent was identified as the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). To investigate whether the HP-PRRSV variant continues circulating and accelerating evolution, we sequenced and analyzed the complete genome of the identified HP-PRRSV field strain SD16. The sequence data indicate that the HP-PRRSV variant continues to prevail and accelerate evolution, especially in the nonstructural protein.
    Journal of Virology 08/2012; 86(16):8906. · 5.08 Impact Factor