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Viral Replication: Lytic Cycle01:20

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Bacteriophages, or phages, are viruses that specifically infect bacteria. Among them, T-even bacteriophages, such as T4, exhibit a well-characterized lytic replication cycle in Escherichia coli (E. coli). This process ensures the rapid proliferation of the virus while ultimately leading to the destruction of the bacterial host.Attachment and DNA InjectionThe infection process begins with the recognition and binding of the T4 phage to the E. coli cell surface. Tail fibers of the phage...
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An in vitro workflow to create and modify infectious clones using replication cycle reaction.

Jeffrey M Marano1, Chelsea Cereghino2, Carla V Finkielstein3

  • 1Translational Biology, Medicine, and Health Graduate Program, Virginia Tech, Roanoke, VA, USA; Department of Biomedical Sciences and Pathobiology, Virginia Tech, VA-MD Regional College of Veterinary Medicine, Blacksburg, VA, United States; Center for Emerging, Zoonotic, and Arthropod-borne Pathogens, Virginia Tech, Blacksburg, VA, United States.

Virology
|June 18, 2023
PubMed
Summary

This study introduces a new in vitro method for creating infectious clones of difficult viruses, overcoming bacterial toxicity issues. This novel workflow enables easier manipulation and distribution of viral genetic material for research.

Keywords:
Dengue virusInfectious clonesMutagenesisReverse geneticsSARS-CoV-2

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Area of Science:

  • Virology
  • Molecular Biology
  • Infectious Diseases

Background:

  • Reverse genetics systems are crucial for understanding viral disease mechanisms.
  • Traditional bacterial cloning methods face challenges with toxic viral sequences, leading to mutations.

Purpose of the Study:

  • To develop a novel in vitro workflow for generating infectious clones of viruses that are difficult to handle using traditional methods.
  • To demonstrate the utility of this workflow for creating and manipulating viral infectious clones.

Main Methods:

  • Leveraging gene synthesis and replication cycle reactions for in vitro workflow.
  • Producing supercoiled infectious clone plasmids for easy distribution and manipulation.
  • Developing infectious clones for Dengue virus serotype 2 and SARS-CoV-2 (USA-WA1/2020 strain).

Main Results:

  • Successfully generated infectious clones for Dengue virus and SARS-CoV-2.
  • The generated clones replicated similarly to their parental viruses.
  • A medically relevant SARS-CoV-2 mutant (Spike D614G) was created using this workflow.

Conclusions:

  • The novel in vitro workflow is a viable alternative for generating and manipulating infectious clones of viruses problematic for bacterial cloning.
  • This method facilitates the study of viral genetics and the development of countermeasures against emerging viruses.