A Nonessential African Swine Fever Virus Gene UK Is a Significant Virulence Determinant in Domestic Swine

African swine fever (ASF) is a highly lethal and economically significant disease of domestic pigs for which there is no vaccine or disease control strategy other than animal quarantine and slaughter. The causative agent of ASF, a large enveloped double-stranded DNA virus (ASFV), is the sole member of an unnamed family of animal viruses (7, 10, 16). Although the icosahedral morphology of the ASFV virion resembles those of iridoviruses, both the ASFV genomic organization, which includes terminal cross-links and inverted terminal repeats, and the cytoplasmic replication strategy indicate a close relationship to the Poxviridae (20, 35, 41). ASFV is the only known DNA arbovirus (7, 10, 16). In nature, the perpetuation and transmission of this virus involve the cycling of virus between two highly adapted hosts, Ornithodoros ticks and wild pig populations (warthogs and bushpigs) in sub-Saharan Africa (37, 38, 50, 54). In the warthog host, ASFV infection is subclinical, characterized by low viremia titers (39, 49). In domestic pigs the severity of ASF ranges from a highly lethal hemorrhagic disease to subclinical infection, depending on contributing viral and host factors (9, 31, 39). ASFV infects cells of the mononuclear-phagocytic system, including highly differentiated fixed-tissue macrophages and specific lineages of reticular cells; affected tissues show extensive damage after infection with highly virulent viral strains (9, 26, 27, 31, 32). This ability to replicate and induce marked cytopathology in these cell types in vivo appears to be critical for ASFV virulence. The natures of viral and host factors responsible for the differing outcomes of infection with strains of high virulence and of lesser virulence are largely unknown. High degrees of variation in genomic size and restriction pattern are observed among different ASFV isolates. Like poxviruses, variations within the ASFV genome are primarily localized to the terminal regions (4, 5, 53). Poxvirus genes located in the terminal variable regions are often nonessential for viral replication in cell culture, performing instead functions related to viral host range (30). ASFV terminal variable regions comprise the left 35-kb and the right 15-kb ends of the genome and contain at least five multigene families (MGF): MGF100, MGF110, MGF300, MGF360, and MGF530 (2, 12, 21, 51, 56). Variation within these regions, including gene deletion events, is observed during ASFV adaptation to monkey cell lines (4, 44) and appears to be associated with reduction of viral virulence (44). Given the similarities with poxviruses, it is likely that ASFV variable region genes are associated with important host range functions in either the pig or tick host. Previously, we described an ASFV right variable region gene, NL-S, with similarity to the neurovirulence-associated gene (ICP34.5) of herpes simplex virus and demonstrated, using a viral gene deletion mutant, that NL-S, while being nonessential for replication in swine macrophages in vitro, is a significant viral virulence factor. Deletion of this gene from the European pathogenic isolate E70 resulted in almost complete attenuation of the virus in the domestic swine host (57). Consistent with a host range function, NL was found to be highly conserved among diverse pathogenic ASFV isolates, existing in either a long (184-amino-acid) or a short (70- to 72-amino acid) form (57). Here, we describe a second ASFV right variable region gene, UK, associated with pig virulence. Our data indicate that (i) the UK gene is highly conserved among African and European ASFV isolates; (ii) UK is a novel gene, showing no similarity to other known genes in the current sequence databases; (iii) UK encodes a 15-kDa protein that is expressed in virus-infected macrophages at early times postinfection; and (iv) although it is nonessential for growth in porcine macrophage cell cultures, UK is a significant viral virulence determinant in domestic swine. Thus, the right variable region of the ASFV genome contains at least two genes, NL-S and UK, with functions involving pig virulence and swine host range.