Abstract
Environmental surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) represents a key issue in control of the disease. CD151 has recently been recognized as one of several receptors for PRRSV. We describe here a novel method for concentration of PRRSV from the environmental samples by CD151-binding capture. After fusion to self-aggregating peptide ELK16, the large extracellular loop (LEL) of porcine CD151 and its two segments (namely N63 and C63) were expressed in E. coli as protein aggregates. The three fusion proteins were purified to high purities by regular centrifugation and washing with Triton X-100. Viral binding assay showed that the C63-ELK16 protein, but not ELK16-N63 protein, had the specific binding affinity for PRRSV. The C63-ELK16 protein could bind to, and eluted from, PRRSV in pH-, temperature-, and time-dependent manners with a final virus recovery of 44.7%. By using PRRSV-spiked and experimentally infected pig fecal slurry samples, the C63-ELK16 binding capture-combined quantitative RT-PCR was shown to have higher detection sensitivity than the conventional RT-PCR. Although the viral RNA could be detected in the experimentally infected pig samples with or without C63-ELK16 binding capture, infectious PRRSV was not isolated without C63-ELK16 binding capture. Therefore, the CD151-binding capture method established offers sufficient recovery and quickness and will facilitate environmental PRRSV surveillance.
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Acknowledgements
The authors wish to thank Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Agri-animal Husbandry Vocational College for providing the animal facility and technical assistance.
Funding
This work was supported by Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and Special Prospective Research Project of Jiangsu Province, China (BE2004380).
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The animal experiment was performed by following the animal ethics guidelines and approved protocols by Jiangsu Agri-Animal Husbandry Vocational College.
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Zong, Y., Zong, X., Xia, W. et al. A novel method for concentration of porcine reproductive and respiratory syndrome virus from the environmental samples using self-aggregating peptide-tagged CD151-binding capture. Appl Microbiol Biotechnol 101, 7987–7996 (2017). https://doi.org/10.1007/s00253-017-8477-0
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DOI: https://doi.org/10.1007/s00253-017-8477-0