Institut für molekulare Virologie und Zellbiologie, Friedrich-Loeffler-Institut, 17493, Greifswald-Insel Riems, Germany,
Arch Virol. 2014 Sep;159(9):2421-8. doi: 10.1007/s00705-014-2095-2. Epub 2014 May 3.
Manipulation of African swine fever virus (ASFV) genomes, in particular those from field strains, is still a challenge. We have shown recently that generation of a green-fluorescent-protein-expressing, thymidine-kinase-negative (TK-) mutant of the low-pathogenic African swine fever virus field strain NHV was supported by a TK- Vero cell line. Since NHV, like other ASFV field strains, does not replicate well in Vero cells, a bromodeoxyuridine (BrdU)- resistant cell line derived from wild boar lung (WSL) cells, named WSL-Bu, was selected. WSL cells were used because they are suitable for productive replication of NHV and other ASFV field strains. Here, we show that WSL-Bu cells enable positive selection of both TK- and TK+ ASFV recombinants, which allows for novel strategies for construction of ASFV mutants. We further demonstrate for a low-pathogenic ASFV strain that TK expression is required for infectious replication in macrophages infected at low multiplicity and that vaccinia TK fully complements ASFV TK in this respect.
操纵非洲猪瘟病毒（ASFV）基因组，特别是那些来自野外分离株的基因组，仍然是一个挑战。我们最近表明，通过一株胸苷激酶阴性（TK-）的非洲猪瘟弱毒 NHV 野毒株的 TK-Vero 细胞系，支持表达绿色荧光蛋白的 TK-突变体的产生。由于 NHV 与其他 ASFV 野毒株一样，在 Vero 细胞中不能很好地复制，因此选择了一株来自野猪肺（WSL）细胞的溴脱氧尿苷（BrdU）抗性细胞系 WSL-Bu。选择 WSL 细胞是因为它们适合 NHV 和其他 ASFV 野毒株的有效复制。在这里，我们表明 WSL-Bu 细胞能够对 TK-和 TK+的 ASFV 重组体进行正向选择，这为构建 ASFV 突变体提供了新的策略。我们进一步证明，对于一株低致病性 ASFV 株，在低感染复数感染的巨噬细胞中，TK 表达是感染性复制所必需的，而痘苗病毒 TK 在此方面完全可以替代 ASFV TK。
