Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain

PLoS One. 2016 Jun 1;11(6):e0156579. doi: 10.1371/journal.pone.0156579. eCollection 2016.

Abstract

The infectious disease caused by the duck Tembusu virus (DTMUV) has resulted in massive economic losses to the Chinese duck industry in China since 2010. Research on the molecular basis of DTMUV pathogenicity has been hampered by the lack of a reliable reverse genetics system for this virus. Here we developed a PCR-based reverse genetics system with high fidelity for the attenuated DTMUV strain FX2010-180P. The rescued virus was characterized by using both indirect immunofluorescence assays (IFA) and whole genome sequencing. The rescued virus (rFX2010-180P) grew to similar titers as compared with the wild-type virus in DF-1 cells, and had similar replication and immunogenicity properties in ducks. To determine whether exogenous proteins could be expressed from DTMUV, both an internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (eGFP) gene were introduced between the NS5 gene and the 3' non-coding sequence of FX2010-180P. A recombinant DTMUV expressing eGFP was rescued, but eGFP expression was unstable after 4 passages in DF-1 cells due to a deletion of 1,294 nucleotides. The establishment of a reliable reverse genetics system for FX2010-180P provides a foundation for future studies of DTMUV.

MeSH terms

  • 3' Untranslated Regions
  • Animals
  • Cell Line
  • Ducks / virology*
  • Flavivirus / genetics*
  • Fluorescent Antibody Technique, Indirect
  • Genome, Viral
  • Green Fluorescent Proteins / genetics
  • Plasmids
  • Polymerase Chain Reaction / methods*
  • Reverse Genetics*

Substances

  • 3' Untranslated Regions
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins

Grants and funding

This study was supported by the National Natural Science Foundation of China (no. 31172332, 31472205 and 31402150), the Shanghai Science and Technology Program for Agriculture (no. 2012-2-6), the "Innovation Action Plan" Key Basic Research Project of Shanghai Science and Technology Commission (no. 12JC1410600), the "Innovation Action Plan" Key Scientific and Technological Projects in the Field of Modern Agriculture of Shanghai Science and Technology Commission (no. 13391901601), and the Special Fund for Central Nonprofit Research Institutes Fundamental Research (no. 2011JB02). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.