Swine Macrophages Research Articles

A Single Amino Acid Substitution in the Matrix Protein (M51R) of Vesicular Stomatitis New Jersey Virus Impairs Replication in Cultured Porcine Macrophages and Results in Significant Attenuation in Pigs.

In this study, we explore the virulence of vesicular stomatitis New Jersey virus (VSNJV) in pigs and its potential relationship with the virus’s ability to modulate innate responses. For this purpose, we developed a mutant of the highly virulent strain NJ0612NME6, containing a single amino acid substitution in the matrix protein (M51R). The M51R mutant of NJ0612NME6 was unable to suppress the transcription of genes associated with the innate immune response both in primary fetal porcine kidney cells and porcine primary macrophage cultures. Impaired viral growth was observed only in porcine macrophage cultures, indicating that the M51 residue is required for efficient replication of VSNJV in these cells. Furthermore, when inoculated in pigs by intradermal scarification of the snout, M51R infection was characterized by decreased clinical signs including reduced fever and development of less and smaller secondary vesicular lesions. Pigs infected with M51R had decreased levels of viral shedding and absence of RNAemia compared to the parental virus. The ability of the mutant virus to infect pigs by direct contact remained intact, indicating that the M51R mutation resulted in a partially attenuated phenotype capable of causing primary lesions and transmitting to sentinel pigs. Collectively, our results show a positive correlation between the ability of VSNJV to counteract the innate immune response in swine macrophage cultures and the level of virulence in pigs, a natural host of this virus. More studies are encouraged to evaluate the interaction of VSNJV with macrophages and other components of the immune response in pigs.

Capsid proteins of foot-and-mouth disease virus interact with TLR2 and CD14 to induce cytokine production

The mechanism of recognition of the foot-and-mouth disease virus (FMDV) by host innate immune cells is not well-understood. In this study, we first found that binary ethylenimine inactivated-FMDV (BEI-FMDV) with structurally intact capsid activated TLR2, but not other TLRs, and this specific activation was blocked by anti-TLR2 Abs or knockout of TLR2. BEI-FMDV activated NF-κB to induce cytokines, notably interferon-β and IL-6, in a TLR2 and MyD88-dependent manner. Coexpression of TLR6 and CD14 showed additive effects on BEI-FMDV/TLR2-mediated activation of NF-κB. Further studies demonstrated that recombinant capsid proteins rVP1 and rVP3 of FMDV but not rVP0 bound directly with CD14 and TLR2. The rVP1- and rVP3-mediated activation of TLR2 and NF-κB were enhanced by the coexpression of TLR1 or TLR6. Immunoprecipitation of either rVP1 or rVP3 with mouse macrophage cell extracts revealed that rVP1 or rVP3 associated with TLR2, CD14 and TLR6 suggesting that rVP1 and rVP3 interact with CD14, TLR2/TLR1, and TLR2/TLR6 heterodimer. Additional study confirmed that rVP1 and rVP3 interacted with the swine TLR2 signaling pathway to induce IL-6 in swine macrophages. Our results identify VP1 and VP3 of FMDV as novel TLR agonists whose recognition by CD14, TLR2/TLR1, and TLR2/TLR6 of host innate immune cells is critical for the induction of cytokine production.

SERTA Domain Containing Protein 1 (SERTAD1) Interacts with Classical Swine Fever Virus Structural Glycoprotein E2, Which Is Involved in Virus Virulence in Swine.

E2 is the major structural glycoprotein of the classical swine fever virus (CSFV). E2 has been shown to be involved in important virus functions such as replication and virulence in swine. Using the yeast two-hybrid system, we previously identified several host proteins specifically interacting with CSFV E2. Here, we analyze the protein interaction of E2 with SERTA domain containing protein 1 (SERTAD1), a factor involved in the stimulation of the transcriptional activities of different host genes. We have confirmed that the interaction between these two proteins occurs in CSFV-infected swine cells by using a proximity ligation assay and confocal microscopy. Amino acid residues in the CSFV E2 protein that are responsible for mediating the interaction with SERTAD1 were mapped by a yeast two-hybrid approach using a randomly mutated E2 library. Using that information, a recombinant CSFV mutant (E2ΔSERTAD1v) that harbors substitutions in those residues mediating the protein-interaction with SERTAD1 was developed and used to study the role of the E2-SERTAD1 interaction in viral replication and virulence in swine. CSFV E2ΔSERTAD1v, when compared to the parental BICv, showed a clearly decreased ability to replicate in the SK6 swine cell line and a more severe replication defect in primary swine macrophage cultures. Importantly, 80% of animals infected with E2ΔSERTAD1v survived infection, remaining clinically normal during the 21-day observational period. This result would indicate that the ability of CSFV E2 to bind host SERTAD1 protein during infection plays a critical role in virus virulence.

Swine Host Protein Coiled-Coil Domain-Containing 115 (CCDC115) Interacts with Classical Swine Fever Virus Structural Glycoprotein E2 during Virus Replication.

Interactions between the major structural glycoprotein E2 of classical swine fever virus (CSFV) with host proteins have been identified as important factors affecting virus replication and virulence. Previously, using the yeast two-hybrid system, we identified swine host proteins specifically interacting with CSFV E2. In this report, we use a proximity ligation assay to demonstrate that swine host protein CCDC115 interacts with E2 in CSFV-infected swine cells. Using a randomly mutated E2 library in the context of a yeast two-hybrid methodology, specific amino acid mutations in the CSFV E2 protein responsible for disrupting the interaction with CCDC115 were identified. A recombinant CSFV mutant (E2ΔCCDC115v) harboring amino acid changes disrupting the E2 protein interaction with CCDC115 was produced and used as a tool to assess the role of the E2–CCDC115 interaction in viral replication and virulence in swine. CSFV E2ΔCCDC115v showed a slightly decreased ability to replicate in the SK6 swine cell line and a greater replication defect in primary swine macrophage cultures. A decreased E2–CCDC115 interaction detected by PLA is observed in cells infected with E2ΔCCDC115v. Importantly, animals intranasally infected with 105 TCID50 of E2ΔCCDC115v experienced a significantly longer survival period when compared with those infected with the parental Brescia strain. This result would indicate that the ability of CSFV E2 to bind host CCDC115 protein during infection plays an important role in virus replication in swine macrophages and in virus virulence during the infection in domestic swine.

A Novel Therapeutic Reagent, KA-1002 for Alleviating Lysophosphatidic Acid-Mediated Inflammation Related Gene Expression in Swine Macrophages.

Simple SummaryInflammatory diseases are a key factor reducing the productivity of animals in a livestock industrial environment. We have identified a novel lysophosphatidic acid signaling antagonist, KA-1002, which alleviates lysophosphatidic acid-mediated a broad range of inflammation related gene expression in swine macrophages. Specifically, we found that KA-1002 significantly alleviated LPA-induced genes related with inflammation such as a role of macrophages, fibroblasts and endothelial cells in rheumatoid arthritis and STAT3 signal pathway. Taken together, KA-1002 could be considered a novel therapeutic reagent candidate for swine inflammatory diseases.Stresses and various infectious reagents caused multiple inflammatory diseases in swine in a livestock industrial environment. Therefore, there is a need for an effective therapeutic or preventive agent that could alleviate chronic and acute inflammation. We found that lysophosphatidic acid (LPA), a stress-induced potent endogenous inflammatory molecule, causes a broad range-regulation of inflammation related genes inflammation in swine macrophages. We further investigated the genome scaled transcriptional regulatory effect of a novel LPA-signaling antagonist, KA-1002 on swine macrophages, inducing the alleviated LPA-mediated inflammation related gene expression. Therefore, KA-1002 could potentially serve as a novel therapeutic or preventive agent to maintain physiologically healthy and balanced conditions of pigs.

The MGF360-16R ORF of African Swine Fever Virus Strain Georgia Encodes for a Nonessential Gene That Interacts with Host Proteins SERTAD3 and SDCBP.

African swine fever virus (ASFV) causes a contagious and frequently lethal disease of pigs with significant economic consequences to the swine industry. The ASFV genome encodes for more than 160 genes, but only a few of them have been studied in detail. Here we report the characterization of open reading frame (ORF) MGF360-16R. Kinetic studies of virus RNA transcription demonstrated that the MGF360-16R gene is transcribed as a late virus protein. Analysis of host–protein interactions for the MGF360-16R gene using a yeast two-hybrid screen identified SERTA domain containing 3 (SERTAD3) and syndecan-binding protein (SDCBP) as host protein binding partners. SERTAD3 and SDCBP are both involved in nuclear transcription and SDCBP has been shown to be involved in virus traffic inside the host cell. Interaction between MGF360-16R and SERTAD3 and SDCBP host proteins was confirmed in eukaryotic cells transfected with plasmids expressing MGF360-16R and SERTAD3 or SDCBP fused to fluorescent tags. A recombinant ASFV lacking the MGF360-16R gene (ASFV-G-ΔMGF360-16R) was developed from the highly virulent field isolate Georgia2007 (ASFV-G) and was used to show that MGF360-16R is a nonessential gene. ASFV-G-ΔMGF360-16R had a similar replication ability in primary swine macrophage cell cultures when compared to its parental virus ASFV-G. Experimental infection of domestic pigs showed that ASFV-G-ΔMGF360-16R is as virulent as the parental virus ASFV-G.

Mechanisms of African swine fever virus pathogenesis and immune evasion inferred from gene expression changes in infected swine macrophages.

African swine fever (ASF) is a swine disease caused by a large, structurally complex, double-stranded DNA virus, African swine fever virus (ASFV). In domestic pigs, acute infection by highly virulent ASF viruses causes hemorrhagic fever and death. Previous work has suggested that ASFV pathogenesis is primarily mediated by host cytokines produced by infected monocytes and macrophages. To better understand molecular mechanisms mediating virus pathogenesis and immune evasion, we used transcriptome analysis to identify gene expression changes after ASFV infection in ex vivo swine macrophages. Our results suggest that the cytokines of TNF family including FASLG, LTA, LTB, TNF, TNFSF4, TNFSF10, TNFSF13B and TNFSF18 are the major causative cytokine factors in ASF pathogenesis via inducing apoptosis. Other up-regulated proinflammatory cytokines (IL17F and interferons) and down-regulated anti-inflammatory cytokine (IL10) may also significantly contribute to ASF pathogenesis and cause excessive tissue inflammatory responses. The differential expression of genes also indicates that ASFV could evade both the innate and adaptive immune responses by (i) inhibiting MHC Class II antigen processing and presentation, (ii) avoiding CD8+ T effector cells and neutrophil extracellular traps via decreasing expression of neutrophil/CD8+ T effector cell-recruiting chemokines, (iii) suppressing M1 activation of macrophages, (iv) inducing immune suppressive cytokines, and (v) inhibiting the processes of macrophage autophagy and apoptosis. These results provide novel information to further investigate and better understand the mechanism of pathogenesis and immune evasion of this devastating swine disease.

Differential Effect of the Deletion of African Swine Fever Virus Virulence-Associated Genes in the Induction of Attenuation of the Highly Virulent Georgia Strain.

African swine fever virus (ASFV) is the etiological agent of an often lethal disease of domestic pigs, African swine fever (ASF). The ASFV Georgia 2007 isolate (ASFV-G) is responsible for the current epidemic situation in Europe and Asia. Genetically modified ASFVs containing deletions of virulence-associated genes have produced attenuated phenotypes and induced protective immunity in swine. Here we describe the differential behavior of two viral genes, NL (DP71L) and UK (DP96R), both originally described as being involved in virus virulence. Deletion of either of these genes efficiently attenuated ASFV strain E70. We demonstrated that deletion of the UK gene from the ASFV-G genome did not decrease virulence when compared to the parental virus. Conversely, deletion of the NL gene produced a heterogeneous response, with early death in one of the animals and transient fever in the other animals. With this knowledge, we attempted to increase the safety profile of the previously reported experimental vaccine ASFV-GΔ9GL/ΔUK by deleting the NL gene. A triple gene-deletion virus was produced, ASFV-GΔ9GL/ΔNL/ΔUK. Although ASFV-GΔ9GL/ΔNL/ΔUK replicated in primary cell cultures of swine macrophages, it demonstrated a severe replication deficiency in pigs, failing to induce protection against challenge with parental ASFV-G.

Development and characterization of monoclonal antibodies against p30 protein of African swine fever virus

Among the structural proteins that compose the virion of African swine fever virus (ASFV), p30 is one of the most immunogenic proteins and is produced during early stage of ASFV infection. These two characteristics make p30 a good target for diagnostic assays to detect ASFV infection. In this study, we describe a panel of newly generated p30-specific monoclonal antibodies (mAbs). The reactivity of these mAbs was confirmed by immunoprecipitation and Western blot analysis in Vero cells infected with alphavirus replicon particles that express p30 (RP-p30). Furthermore, this panel of mAbs recognized ASFV strains BA71 V (Genotype I) and Georgia/2007 (Genotype II) in immunofluorescence assays on virus-infected Vero cells and swine macrophages, respectively. These mAbs also detected p30 expression by immunohistochemistry in tissue samples from ASFV-infected pigs. Epitope mapping revealed that a selected mAb from the panel recognized a linear epitope within the 32-amino acid region, 61–93. In contrast, two of the mAbs recognize the C-terminal region of the protein, which is highly hydrophilic, enriched in glutamic acid residues, and predicted to contain an intrinsically disordered protein region (IDPR). This panel of mAbs and mAb-based diagnostic assays potentially represent valuable tools for ASFV detection, surveillance and disease control.

Phenotypic characterization of swine peripheral blood monocyte-derived macrophages and ex vivo infection with Salmonella enterica serovar Typhimurium.

Macrophages are critical mediators of the inflammatory process, playing a relevant role in the pathogenesis of Salmonella Typhimurium. The protocols for isolation, culture, and differentiation of monocytes into macrophages and their interaction with Salmonella are well established in humans and murine models, but little information is available in swine. The aims of this study were to establish an efficient protocol for macrophage culture and to evaluate the interaction of the invA mutant strain and the wild type (WT) Salmonella Typhimurium with porcine macrophages. Peripheral blood monocyte-derived macrophages from pigs were obtained, separated by density-gradient centrifugation, and cultured in Teflon vials for 10days. After the differentiation period, cultures consisted of 92.4% CD14+ cells. In addition, these cells showed phagocytic ability, demonstrated by the presence of the same amount of WT and invA mutant Salmonella Typhimurium 1h after interaction with macrophages. The early cytotoxic effect was Salmonella pathogenicity island (SPI)-[1]dependent, in which log-phase WT strains were more efficient (p < 0.01) than the invA mutant strain at inducing the death of macrophages.

The L83L ORF of African swine fever virus strain Georgia encodes for a non-essential gene that interacts with the host protein IL-1β

African swine fever virus (ASFV) causes a contagious and frequently lethal disease of pigs causing significant economic consequences to the swine industry. The ASFV genome encodes for more than 150 genes, but only a few of them have been studied in detail. Here we report the characterization of open reading frame L83L which encodes a highly conserved protein across all ASFV isolates. A recombinant ASFV harboring a HA tagged L83L protein was developed (ASFV-G-L83L-HA) and used to demonstrate that L83L is a transiently expressed early virus protein. A recombinant ASFV lacking the L83L gene (ASFV-G-ΔL83L) was developed from the highly virulent field isolate Georgia2007 (ASFV-G) and was used to show that L83L is a non-essential gene. ASFV-G-ΔL83L had similar replication in primary swine macrophage cells when compared to its parental virus ASFV-G. Analysis of host-protein interactions for L83L identified IL-1β as its host ligand. Experimental infection of domestic pigs showed that ASFV-G-ΔL83L is as virulent as the parental virus ASFV-G.

CRISPR-Cas9, a tool to efficiently increase the development of recombinant African swine fever viruses

African swine fever virus (ASFV) causes a highly contagious disease called African swine fever. This disease is often lethal for domestic pigs, causing extensive losses for the swine industry. ASFV is a large and complex double stranded DNA virus. Currently there is no commercially available treatment or vaccine to prevent this devastating disease. Development of recombinant ASFV for producing live-attenuated vaccines or studying the involvement of specific genes in virus virulence has relied on the relatively rare event of homologous recombination in primary swine macrophages, causing difficulty to purify the recombinant virus from the wild-type parental ASFV. Here we present the use of the CRISPR-Cas9 gene editing system as a more robust and efficient system to produce recombinant ASFVs. Using CRISPR-Cas9 a recombinant virus was efficiently developed by deleting the non-essential gene 8-DR from the genome of the highly virulent field strain Georgia07 using swine macrophages as cell substrate.

CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus.

Gene editing of large DNA viruses, such as African swine fever virus (ASFV), has traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of CRISPR/Cas9 to edit ASFV. The use of CRISPR/Cas9 to modify the African swine fever virus genome resulted in a fast and relatively easy way to introduce genetic changes. To accomplish this goal we first infect primary swine macrophages with a field isolate, ASFV-G, and transfect with the CRISPR/Cas9 donor plasmid along with a plasmid that will express a specific gRNA that targets our gene to be deleted. By inserting a reporter cassette, we are then able to purify our recombinant virus from the parental by limiting dilution and plaque purification. We previously reported comparing the traditional homologous recombination methodology with CRISPR/Cas9, which resulted in over a 4 log increase in recombination.

Sophora subprosrate polysaccharide inhibited cytokine/chemokine secretion via suppression of histone acetylation modification and NF-κb activation in PCV2 infected swine alveolar macrophage

In the present study, effect of Sophora subprosrate polysaccharide on PCV2 infection-induced inflammation and histone acetylation modification in swine alveolar macrophage 3D4/2 cells was described for the first time. The relationship between histone acetylation modifications and inflammation response was investigated. The results showed that PCV2 infection induced inflammation by promoting the secretion of TNF-α, IL-1β, IL-6 and IL-10 in 3D4/2 cells. The production of TNF-α, IL-1β and IL-6 and their mRNA expression levels markedly decreased while the level and mRNA expression of IL-10 were elevated when the cells were treated with Sophora subprosrate polysaccharide. The SSP also decreased the activity of HATs, histone H3 acetylation (Ac-H3) and histone H4 acetylation (Ac-H4), p65 phosphorylation (P-p65) in the cells infected with PCV2 while HDACs activity was down-regulated, which involved in the inhibitory effect of SSP on histone acetylation and NF-κB signaling pathways activation. Down-regulation of HAT1 mRNA expression and up-regulation of HDAC1 mRNA expression further support the inhibitory effect of SSP on histone acetylation. In conclusion, Sophora subprosrate polysaccharide antagonized inflammatory responses induced by PCV2, via mechanisms involved in histone acetylation and NF-κB signaling pathways.

Development of a fluorescent ASFV strain that retains the ability to cause disease in swine

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.

Influence of Plasmid Type on the Replication of Rhodococcus equi in Host Macrophages.

The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a multihost, facultative intracellular pathogen of macrophages. When inhaled by susceptible foals, it causes severe bronchopneumonia. It is also a pathogen of pigs, which may develop submaxillary lymphadenitis upon exposure. R.equi isolates obtained from foals and pigs possess conjugative plasmids housing a pathogenicity island (PAI) containing a novel family of genes of unknown function called the virulence-associated protein or vap family. The PAI regions of the equine and swine plasmids differ in vap gene composition, with equine isolates possessing six vap genes, including the major virulence determinant vapA, while the PAIs of swine isolates house vapB and five other unique vap genes. Possession of the pVAPA-type virulence plasmid by equine isolates bestows the capacity for intramacrophage replication essential for disease development in vivo. Swine isolates of R.equi are largely unstudied. Here, we show that R.equi isolates from pigs, carrying pVAPB-type plasmids, are able to replicate in a plasmid-dependent manner in macrophages obtained from a variety of species (murine, swine, and equine) and anatomical locations. Similarly, equine isolates carrying pVAPA-type plasmids are capable of replication in swine macrophages. Plasmid swapping between equine and swine strains through conjugation did not alter the intracellular replication capacity of the parental strain, indicating that coevolution of the plasmid and chromosome is not crucial for this attribute. These results demonstrate that while distinct plasmid types exist among R.equi isolates obtained from equine and swine sources, this tropism is not determined by host species-specific intramacrophage replication capabilities. IMPORTANCE This work greatly advances our understanding of the opportunistic pathogen Rhodococcus equi, a disease agent of animals and immunocompromised people. Clinical isolates from diseased foals carry a conjugative virulence plasmid, pVAPA1037, that expresses Vap proteins, including VapA, essential for intramacrophage replication and virulence in vivo. The understudied R.equi isolates from pigs carry a related but different plasmid, pVAPB, expressing distinct Vap proteins, including VapB. In this work, we document for the first time that R.equi isolates carrying pVAPB-type plasmids are capable of intramacrophage replication. Moreover, we show that R.equi isolates carrying either plasmid type can replicate in both equine and swine macrophages, indicating that host species tropism is not due to species-specific intramacrophage replication capabilities defined by plasmid type. Furthermore, plasmid swapping between equine and swine strains did not alter intracellular replication capacity, indicating that coevolution of the plasmid and chromosome is not essential for intracellular growth.

Alteration of a Second Putative Fusion Peptide of Structural Glycoprotein E2 of Classical Swine Fever Virus Alters Virus Replication and Virulence in Swine.

This report describes the identification and characterization of a putative fusion peptide (FP) in the major structural protein E2 of classical swine fever virus (CSFV). The FP identification was performed by functional structural analysis of E2. We characterized the functional significance of this FP by using artificial membranes. Replacement of critical amino acid residues within the FP radically alters how it interacts with the artificial membranes. When we introduced the same mutations into the viral sequence, there was a reduction in replication in cell cultures, and when we infected domestic swine, the natural host of CSFV host, we observed that the virus was now completely attenuated in swine. In addition, the virus mutant that was attenuated in vivo efficiently protected pigs against wild-type virus. These results provide the proof of principle to support as a strategy for vaccine development the discovery and manipulation of FPs.

African swine fever virus Georgia isolate harboring deletions of 9GL and MGF360/505 genes is highly attenuated in swine but does not confer protection against parental virus challenge

African swine fever virus (ASFV) produces a contagious disease of domestic pigs that results in severe economic consequences to the swine industry. Control of the disease has been hampered by the unavailability of vaccines. We recently reported the development of two experimental vaccine strains (ASFV-G-Δ9GL and ASFV-G-ΔMGF) based on the attenuation of the highly virulent and epidemiologically relevant Georgia2007 isolate. Deletion of the 9GL gene or six genes of the MGF360/505 group produced two attenuated ASFV strains which were able to confer protection to animals when challenged with the virulent parental virus. Both viruses, although efficient in inducing protection, present concerns regarding their safety. In an attempt to solve this problem we developed a novel virus strain, ASFV-G-Δ9GL/ΔMGF, based on the deletion of all genes deleted in ASFV-G-Δ9GL and ASFV-G-ΔMGF. ASFV-G-Δ9GL/ΔMGF is the first derivative of a highly virulent ASFV field strain subjected to a double round of recombination events seeking to sequentially delete specific genes. ASFV-G-Δ9GL/ΔMGF showed a decreased ability to replicate in primary swine macrophage cultures relative to that of ASFV-G and ASFV-G-ΔMGF but similar to that of ASFV-G-Δ9GL. ASFV-G-Δ9GL/ΔMGF was attenuated when intramuscularly inoculated into swine, even at doses as high as 106 HAD50. Animals infected with doses ranging from 102 to 106 HAD50 did not present detectable levels of virus in blood at any time post-infection and they did not develop detectable levels of anti-ASFV antibodies. Importantly, ASFV-G-Δ9GL/ΔMGF does not induce protection against challenge with the virulent parental ASFV-G isolate. Results presented here suggest caution towards approaches involving genomic manipulations when developing rationally designed ASFV vaccine strains.

Lack of strong anti-viral immune gene stimulation in Torque Teno Sus Virus1 infected macrophage cells

While recent findings suggest that swine TTVs (TTSuVs) can act as primary or co-infecting pathogens, very little is known about viral immunity. To determine whether TTSuVs downregulate key host immune responses to facilitate their own survival, a swine macrophage cell line, 3D4/31, was used to over-express recombinant TTSuV1 viral particles or the ORF3 protein. Immune gene expression profiles were assessed by a quantitative PCR panel consisting of 22 immune genes, in cell samples collected at 6, 12, 24 and 48h post-transfection. Despite the upregulation of IFN-β and TLR9, interferon stimulated innate genes and pro-inflammatory genes were not upregulated in virally infected cells. The adaptive immune genes, IL-4 and IL-13, were significantly downregulated at 6h post-transfection. The ORF3 protein did not appear do not have a major immuno-suppressive effect, nor did it stimulate anti-viral immunity. Data from this study warrants further investigation into the mechanisms of TTV related immuno-pathogenesis.

Immune gene expression in swine macrophages expressing the Torque Teno Sus Virus1 (TTSuV1) ORF-1 and 2 proteins

Torque Teno viruses (TTVs) are small DNA viruses which are ubiquitous in nature. Recent reports indicate that swine torque teno viruses (TTSuVs) can act as primary pathogens or play a role in exacerbating co-infections. However, very little is known about the TTSuV host-viral interaction or how they so successfully establish chronic infections in the host. To determine whether the major viral proteins can modulate host immunity, recombinant TTSuV1 ORF1 and 2 proteins were expressed in a swine macrophage cell line (3D4/31). The differential expression of a panel of innate, adaptive, regulatory and inflammatory immune genes was studied by quantitative PCR; using cDNA samples collected at 6, 12, 24 and 48h post-transfection. The ORF1 protein induced an early anti-viral response. However, at 6h post-transfection it also upregulated IL-10, PD-1 and SOCS-1, the suppressors of T cell mediated immunity. An ensuing diminishment of the early protective response was noted. The TTSuV1 ORF2 protein suppressed IFN-β and IL-13 responses but did not significantly influence anti-viral immunity otherwise. These findings indicate that the TTSuV1 ORF1 protein plays a significant but dual role in viral immunity.