Nogales Aitor, Chiem Kevin, Lorenzo María M, Rangel-Moreno Javier, de la Luz Garcia-Hernandez Maria, Park Jun-Gyu, Blasco Rafael, Martinez-Sobrido Luis
Department of Microbiology and Immunology, University of Rochester, Rochester, NY, USA.
Animal Health Research Centre (CISA), Centro Nacional Instituto de Investigación y Tecnología Agraria y Alimentaria (INIA, CSIC), Madrid, Spain.
Methods Mol Biol. 2025;2860:157-174. doi: 10.1007/978-1-0716-4160-6_11.
The family Poxviridae comprises multiple viruses with large double-stranded (ds) DNA genomes that can infect numerous vertebrate and invertebrate hosts, including humans. The development of genetic engineering methods for Vaccinia virus (VACV), the prototypic member in the family, have allowed the manipulation of the genomes of poxviruses for the generation of recombinant (r)VACV expressing easily traceable luciferase and/or fluorescent reporter genes. These recombinant viruses have significantly contributed to progress in the field of poxvirus research and accelerated the development of novel prophylactic vaccines and therapeutic antiviral treatments. Recently, we described two reporter rVACV expressing luciferase (Nluc) and fluorescent (GFP or Scarlet) proteins to easily track viral infections in different systems, overcoming the limitations associated with the use of rVACV expressing a single luciferase or fluorescent reporter gene. Here, we describe the experimental procedures to carry out in vitro, in vivo and ex vivo studies using these novel bireporter-expressing rVACV, which also represent an excellent option to study the biology of VACV, including the use of these reporter viruses for testing new antivirals and vaccines, using cultured cells and/or well-characterized animal models of infection.
痘病毒科包含多种病毒，其基因组为大型双链（ds）DNA，可感染包括人类在内的众多脊椎动物和无脊椎动物宿主。痘病毒科的原型成员痘苗病毒（VACV）基因工程方法的发展，使得人们能够对痘病毒基因组进行操作，以产生表达易于追踪的荧光素酶和/或荧光报告基因的重组（r）VACV。这些重组病毒为痘病毒研究领域的进展做出了重大贡献，并加速了新型预防性疫苗和治疗性抗病毒疗法的开发。最近，我们描述了两种表达荧光素酶（Nluc）和荧光（GFP或Scarlet）蛋白的报告rVACV，以便在不同系统中轻松追踪病毒感染，克服了使用表达单一荧光素酶或荧光报告基因的rVACV的局限性。在这里，我们描述了使用这些新型双报告基因表达rVACV进行体外、体内和离体研究的实验程序，这也是研究VACV生物学的一个绝佳选择，包括使用这些报告病毒在培养细胞和/或特征明确的感染动物模型中测试新的抗病毒药物和疫苗。
