Citation

  • Authors: Marano JM. et al.
  • Year: 2020
  • Journal: Virology 551 58-63
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell types:
    1. Name: BHK-21
      Description: Hamster Syrian Kidney Fibroblast
      Known as: BHK21, BHK 21
    2. Name: HEK-293T
      Description: Human embryonic kidney Fibroblast
      Known as: HEK293T, 293T
    3. Name: VERO
      Description: African green monkey kidney cells

Method

Small scale virus production (Mayaro virus strain TRVL 4675)

Abstract

Alphaviruses (genus Alphavirus; family Togaviridae) are a medically relevant family of viruses that include chikungunya virus and Mayaro virus. Infectious cDNA clones of these viruses are necessary molecular tools to understand viral biology. Traditionally, rescuing virus from an infectious cDNA clone requires propagating plasmids in bacteria, which can result in mutations in the viral genome due to bacterial toxicity or recombination and requires specialized equipment and knowledge to propagate the bacteria. Here, we present an alternative- rolling circle amplification (RCA), an in vitro technology. We demonstrate that the viral yield of transfected RCA product is comparable to midiprepped plasmid, albeit with a slight delay in kinetics. RCA, however, is cheaper and less time-consuming. Further, sequential RCA did not introduce mutations into the viral genome, subverting the need for glycerol stocks and retransformation. These results indicate that RCA is a viable alternative to traditional plasmid-based approaches to viral rescue.

Pubmed