African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Successful experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically modified live attenuated ASFV. Recombinant viruses harboring engineered deletions of specific virulence-associated genes induce solid protection against challenge with parental viruses. Deletion of the 9GL (B119L) gene in the highly virulent ASFV isolates Malawi Lil-20/1 (Mal) and Pretoriuskop/96/4 (Δ9GL viruses) resulted in complete protection when challenged with parental isolates. When similar deletions were created within the ASFV Georgia 2007 (ASFV-G) genome, attenuation was achieved but the protective and lethal doses were too similar. To enhance attenuation of ASFV-G, we deleted another gene, UK (DP96R), which was previously shown to be involved in attenuation of the ASFV E70 isolate. Here, we report the construction of a double-gene-deletion recombinant virus, ASFV-G-Δ9GL/ΔUK. When administered intramuscularly (i.m.) to swine, there was no induction of disease, even at high doses (106 HAD50). Importantly, animals infected with 104 50% hemadsorbing doses (HAD50) of ASFV-G-Δ9GL/ΔUK were protected as early as 14 days postinoculation when challenged with ASFV-G. The presence of protection correlates with the appearance of serum anti-ASFV antibodies, but not with virus-specific circulating ASFV-specific gamma interferon (IFN-γ)-producing cells. ASFV-G-Δ9GL/ΔUK is the first rationally designed experimental ASFV vaccine that protects against the highly virulent ASFV Georgia 2007 isolate as early as 2 weeks postvaccination. IMPORTANCE Currently, there is no commercially available vaccine against African swine fever. Outbreaks of the disease are devastating to the swine industry and are caused by circulating strains of African swine fever virus. Here, we report a putative vaccine derived from a currently circulating strain but containing two deletions in two separate areas of the virus, allowing increased safety. Using this genetically modified virus, we were able to vaccinate swine and protect them from developing ASF. We were able to achieve protection from disease as early as 2 weeks after vaccination, even when the pigs were exposed to a higher than normal concentration of ASFV.

African swine fever is a contagious and often lethal disease for domestic pigs with a significant economic impact for the swine industry. The etiological agent, African swine fever virus (ASFV), is a highly structurally complex double stranded DNA virus. No effective vaccines or antiviral treatment are currently commercially available. We present here the development of a strain of ASFV that has been shown to retain its ability to cause disease in swine, efficiently replicate in swine macrophage and that is fluorescently tagged. The insertion of an EGFP cassette replacing the reading frames for two neighboring genes, MGF360-13L and MGF360-14L, in highly virulent field isolate Georgia/2007, did not affect virus replication in cell cultures and did not affect disease progression in swine, the natural host for ASFV. A virulent fluorescently tagged ASFV is a suitable tool to conduct pathogenesis studies in swine, study on virus-macrophage interaction and to run large scale screens that require a sensitive high throughput output. Utilizing an EGFP reporter system for observing ASFV replication and infectivity can circumvent the time and labor-intensive steps associated with viral antigen-based assays such as the observation of hemadsorption or cytopathic effect.

28Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the 29 host innate immune response. Previous studies in bovine cells have demonstrated that deletion 30 (LLV, leaderless) or point mutations in Lpro results in increased expression of interferon (IFN) 31 and IFN-stimulated genes (ISG) including among others, the ubiquitin-like protein modifier 32 ISG15 and the ubiquitin specific peptidase USP18. In addition to its conventional papain-like 33 protease activity, Lpro acts as a deUbiquitinase (DUB) and deISGylase. In this study, we 34 identified a conserved residue in Lpro that is involved in its interaction with ISG15. Mutation 35W105A rendered bacterial-expressed Lpro unable to cleave the synthetic substrate pro-ISG15, 36 while preserving cellular eIF4G cleavage. Interestingly, mutant FMDV W105A was viable. 37 Overexpression of ISG15 and the ISGylation machinery in porcine cells resulted in moderate 38 inhibition of FMDV replication along with a decrease of the overall state of ISGylation in WT 39 infected cells. In contrast, reduced deISGylation was observed upon infection with W105A and 40 leaderless virus. Reduction in the levels of deubiquitination were also observed in cells infected 41 with FMDV LproW105A mutant. Surprisingly, similarly to WT, infection with W105A 42 inhibited IFN/ISG expression despite displaying an attenuated phenotype in vivo in mice. 43Altogether, our studies indicate that abolishing/reducing the deISGylase/DUB activity of Lpro 44 causes viral attenuation independently of its ability to block expression of IFN and ISGs mRNA. 45Furthermore, our studies highlight the potential of ISG15 for development as a novel 46 biotherapeutic against FMD. 47 48 IMPORTANCE 49 on July 4, 2020 by guest http://jvi.asm.org/ Downloaded from 3 In this study, we have identified an aromatic hydrophobic residue in FMDV Lpro (W105) that is 50 involved in the interaction with ISG15. Mutation in Lpro W105 (A12-LproW105A) resulted in 51 reduced deISGylation in vitro and in porcine-infected cells when compared to WT. Impaired 52 deISGylase activity in Lpro correlated with viral attenuation in vitro and in vivo and did not 53 affect Lpro ability to block expression of type I IFN and other IFN-stimulated genes. Moreover, 54 overexpression of ISG15 resulted in reduction of FMDV viral titers. Thus, our study highlights 55 the potential of Lpro mutants with modified deISGylase activity for the development of live 56 attenuated vaccine candidates and novel biotherapeutics against FMD. 57 58 59 60 61 Foot-and-mouth disease virus (FMDV) is a member of the Aphthovirus genus within 62 the Picornaviridae family and it is the etiologic agent of FMD, a disease of cloven-hoofed 63 animals (1). The virus contains a single-stranded, positive-sense RNA genome of approximately 64 8500 nucleotides surrounded by a non-enveloped icosahedral capsid. FMDV is genetically 65 highly variable and as such, it displays seven distinct serotypes: A, Asia-1, C, O and Southern 66 African Territories 1...

The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence. IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.