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Structure and Encapsidation of Transmissible Gastroenteritis Coronavirus (TGEV) Defective Interfering Genomes

  • Ana Méndez
  • Cristian Smerdou
  • Fátima Gebauer
  • Ander Izeta
  • Luis Enjuanes
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 380)

Abstract

Serial undiluted passages were performed with the PUR46 strain of TGEV in swine testis (ST) cells. Total cellular RNA was analyzed at different passages after orthophosphate metabolic labeling. Three new defective RNA species of 24, 10.5, and 9.5 kb (DI-A, DI-B, and DI-C respectively) were detected at passage 30, which were highly stable and significantly interfered with helper mRNA synthesis in subsequent passages. By Northern hybridization DIs A, B, and C were detected in purified virions at amounts similar to those of helper RNA. Standard and defective TGEV virions could be sorted in sucrose gradients, indicating that defective and full-length genomes are independently packaged. cDNA synthesis of DI-B and DI-C RNAs was performed by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments in each case. Cloning and sequencing of the DI-C PCR products showed that the smallest DI particle comprises 9.5 kb and has 4 discontinuous regions of the genome. It contains 2.1 kb from the 5′-end of the genome, about 7 kb from gene lb, the first 24 nucleotides of the S gene, 12 nucleotides of ORF 7, and the 0.4 kb of theUTR at the 3′-end.

Keywords

Infectious Bronchitis Virus Mouse Hepatitis Virus Sucrose Cushion Roller Bottle Discontinuous Region 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • Ana Méndez
    • 1
  • Cristian Smerdou
    • 1
  • Fátima Gebauer
    • 1
  • Ander Izeta
    • 1
  • Luis Enjuanes
    • 1
  1. 1.Department of Molecular and Cellular Biology, Centro Nacional de BiotecnologíaCampus Universidad AutónomaCanto Blanco, MadridSpain

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