Construction of a reverse genetic system for porcine astrovirus

Arch Virol. 2018 Jun;163(6):1511-1518. doi: 10.1007/s00705-018-3771-4. Epub 2018 Feb 15.

Abstract

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.

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line
  • Cloning, Molecular
  • Cricetulus
  • DNA, Complementary / genetics
  • DNA, Complementary / metabolism
  • Epithelial Cells / pathology
  • Epithelial Cells / virology*
  • Genome, Viral*
  • Kidney / pathology
  • Kidney / virology
  • Mamastrovirus / genetics*
  • Mamastrovirus / growth & development
  • Mamastrovirus / metabolism
  • Mamastrovirus / pathogenicity
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Point Mutation
  • RNA, Viral / genetics*
  • RNA, Viral / metabolism
  • Reverse Genetics / methods*
  • Swine

Substances

  • DNA, Complementary
  • RNA, Viral

Supplementary concepts

  • Mamastrovirus 3