No abstract

The family Asfarviridae includes the single species African swine fever virus, isolates of which have linear dsDNA genomes of 170-194 kbp. Virions have an internal core, an internal lipid membrane, an icosahedral capsid and an outer lipid envelope.

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The disease is devastating the swine industry in Central Europe and East Asia, with current outbreaks caused by circulating strains of ASFV derived from the 2007 Georgia isolate (ASFV-G), a genotype II ASFV. In the absence of any available vaccines, African swine fever (ASF) outbreak containment relies on the control and culling of infected animals. Limited cross-protection studies suggest that in order to ensure a vaccine is effective, it must be derived from the current outbreak strain or at the very least from an isolate with the same genotype. Here, we report the discovery that the deletion of a previously uncharacterized gene, I177L, from the highly virulent ASFV-G produces complete virus attenuation in swine. Animals inoculated intramuscularly with the virus lacking the I177L gene, ASFV-G-ΔI177L, at a dose range of 102 to 106 50% hemadsorbing doses (HAD50), remained clinically normal during the 28-day observational period. All ASFV-G-ΔI177L-infected animals had low viremia titers, showed no virus shedding, and developed a strong virus-specific antibody response; importantly, they were protected when challenged with the virulent parental strain ASFV-G. ASFV-G-ΔI177L is one of the few experimental vaccine candidate virus strains reported to be able to induce protection against the ASFV Georgia isolate, and it is the first vaccine capable of inducing sterile immunity against the current ASFV strain responsible for recent outbreaks. IMPORTANCE Currently, there is no commercially available vaccine against African swine fever. Outbreaks of this disease are devastating the swine industry from Central Europe to East Asia, and they are being caused by circulating strains of African swine fever virus derived from the Georgia 2007 isolate. Here, we report the discovery of a previously uncharacterized virus gene, which when deleted completely attenuates the Georgia isolate. Importantly, animals infected with this genetically modified virus were protected from developing ASF after challenge with the virulent parental virus. Interestingly, ASFV-G-ΔI177L confers protection even at low doses (102 HAD50) and remains completely attenuated when inoculated at high doses (106 HAD50), demonstrating its potential as a safe vaccine candidate. At medium or higher doses (104 HAD50), sterile immunity is achieved. Therefore, ASFV-G-ΔI177L is a novel efficacious experimental ASF vaccine protecting pigs from the epidemiologically relevant ASFV Georgia isolate.

African swine fever virus (ASFV) causes a contagious and often lethal disease of feral and domestic swine. Experimental vaccines derived from naturally occurring, genetically modified, or cell culture-adapted ASFV have been evaluated, but no commercial vaccine is available to control African swine fever (ASF). We report here the genotypic and phenotypic analysis of viruses obtained at different passages during the process of adaptation of a virulent ASFV field isolate from the Republic of Georgia (ASFV-G) to grow in cultured cell lines. ASFV-G was successively passaged 110 times in Vero cells. Viruses obtained at passages 30, 60, 80, and 110 were evaluated in vitro for the ability to replicate in Vero cells and primary swine macrophages cultures and in vivo for assessing virulence in swine. Replication of ASFV-G in Vero cells increased with successive passages, corresponding to a decreased replication in primary swine macrophages cultures. In vivo, progressive loss of virus virulence was observed with increased passages in Vero cells, and complete attenuation of ASFV-G was observed at passage 110. Infection of swine with the fully attenuated virus did not confer protection against challenge with virulent parental ASFV-G. Full-length sequence analysis of each of these viruses revealed significant deletions that gradually accumulated in specific areas at the right and left variable ends of the genome. Mutations that result in amino acid substitutions and frameshift mutations were also observed, though in a rather limited number of genes. The potential importance of these genetic changes in virus adaptation/attenuation is discussed. IMPORTANCEThe main problem in controlling ASF is the lack of vaccines. Attempts to produce vaccines by adaptation of ASFV to cultured cell lines have been made. These attempts led to the production of attenuated viruses that conferred only homologous protection. Specifics regarding adaptation of these isolates to cell cultures have been insufficiently described. Details like the numbers of passages required to obtain attenuated viruses, genetic modifications introduced into the virus genomes along passages, and the extent of attenuation and induced protective efficacy are not readily available. In this study, we assessed the changes that lead to decreased growth in swine macrophages and to attenuation in swine. Loss of virulence, probably associated with limited replication in vivo, may lead to the lack of protective immunity in swine observed after challenge. This report provides valuable information that can be used to further the understanding of ASFV gene function, virus attenuation, and protection against infection.A frican swine fever (ASF) is a contagious disease of swine caused by ASF virus (ASFV), an enveloped virus containing a large (190-kbp) double-stranded DNA (dsDNA) genome. ASFV infection of domestic pigs can induce a spectrum of disease severity, from highly lethal to subclinical, depending on host characteristics and the particular circulating virus strain (1).A...

An African swine fever virus (ASFV) gene with similarity to the T-lymphocyte surface antigen CD2 has been found in the pathogenic African isolate Malawi Lil-20/1 (open reading frame [ORF] 8-DR) and a cell culture-adapted European virus, BA71V (ORF EP402R) and has been shown to be responsible for the hemadsorption phenomenon observed for ASFV-infected cells. The structural and functional similarities of the ASFV gene product to CD2, a cellular protein involved in cell-cell adhesion and T-cell-mediated immune responses, suggested a possible role for this gene in tissue tropism and/or immune evasion in the swine host. In this study, we constructed an ASFV 8-DR gene deletion mutant (Δ8-DR) and its revertant (8-DR.R) from the Malawi Lil-20/1 isolate to examine gene function in vivo. In vitro, Δ8-DR, 8-DR.R, and the parental virus exhibited indistinguishable growth characteristics on primary porcine macrophage cell cultures. In vivo,8-DR had no obvious effect on viral virulence in domestic pigs; disease onset, disease course, and mortality were similar for the mutant Δ8-DR, its revertant 8-DR.R, and the parental virus. Altered viral infection was, however, observed for pigs infected with Δ8-DR. A delay in spread to and/or replication of Δ8-DR in the draining lymph node, a delay in generalization of infection, and a 100- to 1,000-fold reduction in virus titers in lymphoid tissue and bone marrow were observed. Onset of viremia for Δ8-DR-infected animals was significantly delayed (by 2 to 5 days), and mean viremia titers were reduced approximately 10,000-fold at 5 days postinfection and 30- to 100-fold at later times; moreover, unlike in 8-DR.R-infected animals, the viremia was no longer predominantly erythrocyte associated but rather was equally distributed among erythrocyte, leukocyte, and plasma fractions. Mitogen-dependent lymphocyte proliferation of swine peripheral blood mononuclear cells in vitro was reduced by 90 to 95% following infection with 8-DR.R but remained unaltered following infection with Δ8-DR, suggesting that 8-DR has immunosuppressive activity in vitro. Together, these results suggest an immunosuppressive role for 8-DR in the swine host which facilitates early events in viral infection. This may be of most significance for ASFV infection of its highly adapted natural host, the warthog.