Construction of a reverse genetic system for porcine astrovirus
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In order to construct a full-length infectious cDNA clone of porcine astrovirus, three fragments covering the complete genome of PAstV1-GX1 strain were amplified by RT-PCR. All three PCR-amplified fragments were cloned into T-Vector pMD19 (Simple), and subsequently assembled into a full-length cDNA clone by subcloning. A silent nucleotide change creating a PstI site was engineered into the full-length cDNA clone to distinguish the rescued virus from the parental virus. Upon transfection of BHK-21 cells with the in vitro transcripts of both the original and constructed cDNAs, typical cytopathic effects were observed on PK-15 cells after serial passaging of the cell supernatant. The construction and recovery of the infectious cDNA clone of porcine astrovirus will provide a valuable experimental system to study the genome function and pathogenesis of astroviruses.
We thank Yizhi Zhong for suggestions on the infectious clone construction. We are grateful to many colleagues for technical assistance and to Dr. Dev Sooranna, Imperial College London, for editing the manuscript. This work was supported by the National Natural Science Foundation of China (No. 31460671 and 31760735) and Innovation Driven Development Special Foundation of Guangxi (AA17204057-1).
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Conflict of interest
The authors declare they have no conflict of interest.
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