Tracking the intracellular localization of African swine fever virus genomic DNA using DNAscope in situ hybridization.

The genome of African swine fever virus (ASFV) is characterized by a single linear double-stranded DNA molecule. However, the question of whether ASFV genomic DNA enters the nucleus has been a subject of debate. In this study, DNAscope in situ hybridization (ISH) technology was employed to trace the genomic DNA of ASFV. Specific probes targeting the DNA of the ASFV HLJ/18 strain were designed and synthesized, facilitating the successful detection of ASFV DNA in ASFV-infected porcine alveolar macrophages (PAMs). Detection of ASFV genomic DNA commenced as early as 2 h post-infection (hpi), with a progressive increase in DNA signals over time. At 8 hpi, ASFV DNA predominantly localized around nuclear virus factories (VFs), and with continued viral replication increased, the fluorescence signals associated with the detected DNA intensified and extended to additional cytoplasmic areas beyond the VFs. After 20 hpi, the fluorescence signals reached a state of stabilization. Furthermore, confocal Z-stack imaging revealed that ASFV DNA signals were absent from the nucleus throughout all the stages of ASFV infection, providing substantial evidence regarding ASFV genome localization.