[A rapid and accurate method for herpesviral gnome editing]

Area of Science:

• Virology
• Molecular Biology
• Genomics

Background:

• Marek's disease virus (MDV) poses a significant threat to poultry health.
• Efficient genome manipulation tools are crucial for studying MDV pathogenesis and developing control strategies.

Purpose of the Study:

• To construct an infectious bacterial artificial chromosome (BAC) clone of the very virulent MDV strain Md5.
• To establish a platform for rapid and accurate genome manipulation of MDV.
• To investigate the role of the gene lorf10 in MDV replication.

Main Methods:

• Homologous recombination was used to insert the entire MDV Md5 genome into a BAC.
• Recombinant DNA was introduced into DH10B competent cells and verified using PCR and RFLP.
• Transfection into chicken embryo fibroblast (CEF) cells generated infectious virus.
• Red-mediated homologous recombination was employed to create a lorf10 deletion mutant and a revertant strain.

Main Results:

• An infectious BAC clone of MDV (Md5BAC) was successfully constructed and rescued.
• A lorf10 deletion mutant and its revertant were generated.
• Indirect immunofluorescence assay confirmed successful packaging of recombinant viruses.
• Growth kinetics and plaque size assays indicated that lorf10 is dispensable for in vitro MDV propagation.

Conclusions:

• The study successfully developed an infectious BAC clone of MDV, enabling efficient genome manipulation.
• The gene lorf10 is not essential for MDV replication in vitro.
• This BAC system provides a valuable tool for future research on MDV and potentially other herpesviruses.