Two strains of African swine fever virus (ASFV), the high virulence Lisboa60 (L60) and the low virulence NH/P68 (NHV), which have been used previously in effective immunization/protection studies, were sequenced. Both were isolated in Portugal in the 11-year period after the introduction of ASFV into the European continent in 1957. The predicted proteins coded by both strains were compared, and where differences were found, these were also compared to other strains of known virulence. This highlighted several genes with significant alterations in low virulence strains of ASFV that may constitute virulence factors, several of which still lacking knowledge regarding their function. Phylogenetic analysis grouped L60 and NHV closest with other P72 genotype I ASFV strains from Europe and West Africa, consistent with the assumed West-African origin of all European strains. Interestingly, a relatively lower genomic identity exists between L60 and NHV, both isolated in a similar geographical location 8-years apart, than with other European and West-African strains isolated later in time and in more distant locations. This may reflect the intensive passage in tissue culture during the early 60’s of a Portuguese isolate to obtain an attenuated vaccine, which may have led to NHV. This study contributes to a better understanding of the evolution of ASFV and defines additional potential virulence genes for future studies of pathogenesis towards the development of effective vaccines.

African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Triton-insoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (−78.3%), and reduced transcription of a late viral gene (−47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV. IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently available. Remarkably, ASFV is the only known mammalian virus that putatively codes for a histone-like protein (pA104R) that shares extensive sequence homology with bacterial histone-like proteins. In this study, we characterized the DNA-binding properties of pA104R, analyzed the functional importance of two conserved residues, and showed that pA104R and ASFV topoisomerase II cooperate and display DNA-supercoiling activity. Moreover, pA104R is expressed during the late phase of infection and accumulates in viral DNA replication sites, and its downregulation revealed that pA104R is required for viral DNA replication and transcription. These results suggest that pA104R participates in the modulation of viral DNA topology and genome packaging, indicating that A104R deletion mutants may be a good strategy for vaccine development against ASFV.

African swine fever (ASF) is, once more, spreading throughout the world. After its recent reintroduction in Georgia, it quickly reached many neighboring countries in Eastern Europe. It was also detected in Asia, infecting China, the world’s biggest pig producer, and spreading to many of the surrounding countries. Without any vaccine or effective treatment currently available, new strategies for the control of the disease are mandatory. Its etiological agent, the African swine fever virus (ASFV), has been shown to code for a type II DNA topoisomerase. These are enzymes capable of modulating the topology of DNA molecules, known to be essential in unicellular and multicellular organisms, and constitute targets in antibacterial and anti-cancer treatments. In this review, we summarize most of what is known about this viral enzyme, pP1192R, and discuss about its possible role(s) during infection. Given the essential role of type II topoisomerases in cells, the data so far suggest that pP1192R is likely to be equally essential for the virus and thus a promising target for the elaboration of a replication-defective virus, which could provide the basis for an effective vaccine. Furthermore, the use of inhibitors could be considered to control the spread of the infection during outbreaks and therefore limit the spreading of the disease.

Mitotic chromatin is largely assumed incompatible with transcription due to changes in the transcription machinery and chromosome architecture. However, the mechanisms of mitotic transcriptional inactivation and their interplay with chromosome assembly remain largely unknown. By monitoring ongoing transcription in Drosophila early embryos, we reveal that eviction of nascent mRNAs from mitotic chromatin occurs after substantial chromosome compaction and is not promoted by condensin I. Instead, we show that the timely removal of transcripts from mitotic chromatin is driven by the SNF2 helicase-like protein Lodestar (Lds), identified here as a modulator of sister chromatid cohesion defects. In addition to the eviction of nascent transcripts, we uncover that Lds cooperates with Topoisomerase 2 to ensure efficient sister chromatid resolution and mitotic fidelity. We conclude that the removal of nascent transcripts upon mitotic entry is not a passive consequence of cell cycle progression and/or chromosome compaction but occurs via dedicated mechanisms with functional parallelisms to sister chromatid resolution.