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Classical swine fever virus (CSFV) Core protein is involved in virus RNA protection, transcription regulation and virus virulence. To discover additional Core protein functions a yeast two-hybrid system was used to identify host proteins that interact with Core. Among the identified host proteins, the osteosarcoma amplified 9 protein (OS9) was further studied. Using alanine scanning mutagenesis, the OS9 binding site in the CSFV Core protein was identified, between Core residues 90IAIM93, near a putative cleavage site. Truncated versions of Core were used to show that OS9 binds a polypeptide representing the 12 C-terminal Core residues. Cells transfected with a double-fluorescent labeled Core construct demonstrated that co-localization of OS9 and Core occurred only on unprocessed forms of Core protein. A recombinant CSFV containing Core protein where residues 90IAIM93 were substituted by alanines showed no altered virulence in swine, but a significant decreased ability to replicate in cell cultures. [ABSTRACT FROM AUTHOR]

Foot and Mouth Disease is a highly contagious and economically important disease of livestock. While vaccination is often effective at controlling viral spread, failures can occur due to strain mismatch or viral mutation. Foot and Mouth Disease Virus (FMDV) possesses a hypervariable region within the G-H Loop of VP1, a capsid protein commonly associated with virus neutralization. Here, we investigate the effect of replacement of the G-H loop hypervariable epitope with a xenoepitope from PRRS virus on the immunogenicity and efficacy of an adenovirus vectored FMDV vaccine (Ad5-FMD). Pigs were vaccinated with Ad5-FMD, the modified Ad5-FMD xeno , or PBS, followed by intradermal challenge with FDMV strain O 1 Manisa at 21 days post-vaccination. While overall serum antibody titers were significantly higher in Ad5-FMD xeno vaccinated animals, neutralizing antibody titers were decreased in pigs that received Ad5-FMD xeno , when compared to those vaccinated with Ad5-FMD, prior to viral challenge, indicative of immune redirection away from VP1 towards non-neutralizing epitopes. As expected, animals vaccinated with unmodified Ad5-FMD were protected from lesions, fever, and viremia. In contrast, animals vaccinated with Ad5-FMD xeno developed clinical signs and viremia, but at lower levels than that observed in PBS-treated controls. No significant difference was found in nasal shedding of virions between the two Ad5-FMD vaccinated groups. This data suggests that the hypervariable epitope of the VP1 G-H loop contributes to protective immunity conferred by Ad5 vector-delivered FMD vaccines in swine, and cannot be substituted without a loss of immunogenicity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A human adenovirus (Ad5) vectored foot-and-mouth disease virus (FMDV) O1-Manisa subunit vaccine (Ad5-O1Man) was engineered to deliver FMDV O1-Manisa capsid and capsid-processing proteins. Swine inoculated with Ad5-O1Man developed an FMDV-specific humoral response as compared to animals inoculated with an empty Ad5-vector. Vaccinated animals were completely protected against homologous challenge at 7 or 21 days post-vaccination. Potency studies exhibited a PD50 of about 10 7 pfu/animal while a dose of 4×10 7 pfu/animal fully protected swine against FMDV intradermal challenge. In-vitro cross-neutralization analysis distinctly predicted that swine vaccinated with Ad5-O1Man would be protected against challenge with homologous FMDV O1Man Middle East-South Asia (ME-SA) topotype and also against recent outbreak strains of Mya-98 South East Asia (SEA) lineage including O1-UK-2001 and O1-South Korea-2010. These results indicate that recombinant Ad5-O1Man is an effective, safe and cross-reacting vaccine that could potentially be used preventively and in outbreak situations, to control FMDV O Mya-98 lineage in swine., (Published by Elsevier Inc.)

Unlabelled: 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., Importance: The 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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The role of non-structural protein 3A of foot-and-mouth disease virus (FMDV) on the virulence in cattle has received significant attention. Particularly, a characteristic 10-20 amino acid deletion has been implicated as responsible for virus attenuation in cattle: a 10 amino acid deletion in the naturally occurring, porcinophilic FMDV O1 Taiwanese strain, and an approximately 20 amino acid deletion found in egg-adapted derivatives of FMDV serotypes O1 and C3. Previous reports using chimeric viruses linked the presence of these deletions to an attenuated phenotype in cattle although results were not conclusive. We report here the construction of a FMDV O1Campos variant differing exclusively from the highly virulent parental virus in a 20 amino acid deletion between 3A residues 87-106, and its characterization in vitro and in vivo. We describe a direct link between a deletion in the FMDV 3A protein and disease attenuation in cattle., (Published by Elsevier Inc.)

The nonstructural protein p7 of classical swine fever virus (CSFV) is a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa. The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form transmembrane helices and a cytosolic loop, respectively. Using reverse genetics, partial in-frame deletions of p7 were deleterious for virus growth, demonstrating that CSFV p7 function is critical for virus production in cell cultures. A panel of recombinant mutant CSFVs was created using alanine scanning mutagenesis of the p7 gene harboring sequential three- to six-amino-acid residue substitutions spanning the entire protein. These recombinant viruses allowed the identification of the regions within p7 that are critical for virus production in vitro. In vivo, some of these viruses were partially or completely attenuated in swine relative to the highly virulent parental CSFV Brescia strain, indicating a significant role of p7 in CSFV virulence. Structure-function analyses in model membranes emulating the endoplasmic reticulum lipid composition confirmed that CSFV p7 is a pore-forming protein, and that pore-forming activity resides in the C-terminal transmembrane helix. Therefore, p7 is a viroporin which is clearly involved in the process of CSFV virulence in swine.

Here we show that IQGAP1, a cellular protein that plays a pivotal role as a regulator of the cytoskeleton interacts with Classical Swine Fever Virus (CSFV) Core protein. Sequence analyses identified residues within CSFV Core protein (designated as areas I, II, III and IV) that maintain homology to regions within the matrix protein of Moloney Murine Leukemia Virus (MMLV) that mediate binding to IQGAP1 [EMBO J, 2006 25:2155]. Alanine-substitution within Core regions I, II, III and IV identified residues that specifically mediate the Core-IQGAP1 interaction. Recombinant CSFV viruses harboring alanine substitutions at residues (207)ATI(209) (I), (210)VVE(212) (II), (213)GVK(215) (III), or (232)GLYHN(236) (IV) have defective growth in primary swine macrophage cultures. In vivo, substitutions of residues in areas I and III yielded viruses that were completely attenuated in swine. These data shows that the interaction of Core with an integral component of cytoskeletal regulation plays a role in the CSFV cycle., (Published by Elsevier Inc.)

Here we have identified host cell proteins involved with the cellular SUMOylation pathway, SUMO-1 (small ubiquitin-like modifier) and UBC9, a SUMO-1 conjugating enzyme that interact with classical swine fever virus (CSFV) Core protein. Five highly conserved lysine residues (K179, K180, K220, K221, and K246) within the CSFV Core were identified as putative SUMOylation sites. Analysis of these interactions showed that K179A, K180A, and K221A substitutions disrupt Core-SUMO-1 binding, while K220A substitution precludes Core-UBC9 binding. In vivo, Core mutant viruses (K179A, K180A, K220A, K221A) and (K220A, K221A) harboring those substitutions were attenuated in swine. These data shows a clear correlation between the disruption of Core protein binding to SUMO-1 and UBC9 and CSFV attenuation. Overall, these data suggest that the interaction of Core with the cellular SUMOylation pathway plays a significant role in the CSFV growth cycle in vivo., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Infection of domestic swine with highly virulent, classical swine fever virus (CSFV) strain Brescia, causes lethal disease in all infected animals. However, the molecular mechanisms involved in modulating the host cellular processes and evasion of the immune response have not been clearly established. To gain insight into, the early host response to CSFV, we analyzed the pattern of gene expression in infected swine macrophages, using custom designed swine microarrays. Macrophages, the target cell for CSFV infection, were isolated from primary cultures of peripheral blood mononuclear cells, allowing us to utilize identical uninfected macrophages at the same time points as CSFV-infected macrophages, allowing only genes induced by CSFV to be identified. First, microarray probes were optimized by screening 244,000 probes for hybridization with RNA from infected and uninfected macrophages. Probes that hybridized and passed quality control standards were used to design a 44,000 probe microarray for this study. Changes in expression levels of 79 genes (48 up- and 31 down-regulated) during the first 48h post-infection were observed. As expected many of the genes with an altered pattern of expression are involved in the development of an innate immune response. Several of these genes had differential expression in an attenuated strain NS4B.VGIv, suggesting that some of these differences are responsible for virulence. The observed gene expression profile might help to explain the immunological and pathological changes associated with infection of pigs with CSFV Brescia.