scholarly journals African Swine Fever Virus CD2v Protein Induces β-Interferon Expression and Apoptosis in Swine Peripheral Blood Mononuclear Cells

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1480
Author(s):  
Sabal Chaulagain ◽  
Gustavo A. Delhon ◽  
Sushil Khatiwada ◽  
Daniel L. Rock
Keyword(s):  
Mononuclear Cells ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Lymphoid Tissues ◽  
Hemorrhagic Disease ◽  
Peripheral Blood Mononuclear ◽  
Viral Virulence ◽  
Induced Apoptosis ◽  
In Cells

African swine fever (ASF) is a hemorrhagic disease of swine characterized by massive lymphocyte depletion in lymphoid tissues due to the apoptosis of B and T cells, a process likely triggered by factors released or secreted by infected macrophages. ASFV CD2v (EP402R) has been implicated in viral virulence and immunomodulation in vitro; however, its actual function(s) remains unknown. We found that CD2v expression in swine PK15 cells induces NF-κB-dependent IFN-β and ISGs transcription and an antiviral state. Similar results were observed for CD2v protein treated swine PBMCs and macrophages, the major ASFV target cell. Notably, treatment of swine PBMCs and macrophages with CD2v protein induced apoptosis. Immunoprecipitation and colocalization studies revealed that CD2v interacts with CD58, the natural host CD2 ligand. Additionally, CD58 knockdown in cells or treatment of cells with an NF-κB inhibitor significantly reduced CD2v-mediated NF-κB activation and IFN-β induction. Further, antibodies directed against CD2v inhibited CD2v-induced NF-κB activation and IFN-β transcription in cells. Overall, results indicate that ASFV CD2v activates NF-κB, which induces IFN signaling and apoptosis in swine lymphocytes/macrophages. We propose that CD2v released from infected macrophages may be a significant factor in lymphocyte apoptosis observed in lymphoid tissue during ASFV infection in pigs.

scholarly journals African Swine Fever Virus CD2v protein promotes β-Interferon expression and apoptosis in swine cells

2020 ◽  
Author(s):  
Sabal Chaulagain ◽  
Gustavo Delhon ◽  
Sushil Khatiwada ◽  
Daniel L. Rock
Keyword(s):  
Nuclear Translocation ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Lymphoid Tissues ◽  
Hemorrhagic Disease ◽  
Swine Industry ◽  
Lymphocyte Depletion ◽  
Viral Virulence ◽  
Induced Apoptosis

ABSTRACTAfrican swine fever (ASF) is a disease of swine characterized by massive lymphocyte depletion in lymphatic tissues due to apoptosis of B and T cells, most likely triggered by proteins or factors secreted by infected adjacent macrophages. Here we describe a role for the ASF virus (ASFV) protein CD2v in apoptosis induction in lymphocytes. CD2v is a viral homolog of host CD2 that has been implicated in viral virulence and immunomodulation in vitro; however, its actual function remains unknown. We show that CD2v is secreted into culture medium of CD2v-expressing swine cells; and expression of-or treatment with CD2v led to significant induction of IFN-β/ISGs transcription and antiviral state. CD2v expression led to enhanced NF-κB-p65 nuclear translocation in these cultures and incubation with a NF-κB inhibitor reduced CD2v-induced NF-κB-p65 nuclear translocation and IFN-β transcription. We show that CD2v binds CD58, the natural CD2 ligand, and that CD58 siRNA knock-down results in significant reduction in NF-κB-p65 nuclear translocation and IFN-β transcription. Treatment of swine PBMC with purified CD2v led to enhanced NF-κB-p65 nuclear translocation and induction of IFN-β transcription. Further, induction of caspase-3 and PARP1 cleavage was observed in these swine PBMC at later times, providing a mechanism for CD2v-induced apoptosis of lymphocytes. Finally, IFN-β induction and NF-κB activation was inhibited in swine PBMC treated with purified CD2v pre-incubated with antibodies against CD2v. Overall, our results indicate that ASFV CD2v is an immunomodulatory protein that, by promoting lymphocyte apoptosis, may contribute to bystander lymphocyte depletion observed during ASFV infection in pigs.IMPORTANCEASF, a severe hemorrhagic disease of domestic swine, represents a significant economic threat to swine industry worldwide. One critical pathological event observed in pigs infected with virulent isolates is an extensive destruction of lymphoid tissue and massive lymphocyte depletion. However, viral factor/s involved in this event are yet to be identified. Here we show that, by inducing NF-κB-dependent IFN signaling, ASFV CD2v is able to promote apoptosis in swine PBMC. We propose that CD2v released by ASFV-infected macrophages contributes to the massive depletion of lymphocytes observed in lymphoid tissues of infected pigs. Results here improve our understanding of ASFV pathogenesis and will encourage novel intervention approaches.

scholarly journals African Swine Fever Virus pE199L Induces Mitochondrial-Dependent Apoptosis

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2240
Author(s):  
Tingting Li ◽  
Gaihong Zhao ◽  
Taoqing Zhang ◽  
Zhaoxia Zhang ◽  
Xin Chen ◽  
...  
Keyword(s):  
Cell Death ◽  
Nuclear Dna ◽  
African Swine Fever Virus ◽  
Ectopic Expression ◽  
African Swine Fever ◽  
Lymphoid Tissues ◽  
Hemorrhagic Disease ◽  
Apoptotic Pathway ◽  
Induced Apoptosis

African swine fever (ASF) is a severe hemorrhagic disease in swine characterized by massive lymphocyte depletion and cell death, with apoptosis and necrosis in infected lymphoid tissues. However, the molecular mechanism regarding ASFV-induced cell death remains largely unknown. In this study, 94 ASFV-encoded proteins were screened to determine the viral proteins involved in cell death in vitro, and pE199L showed the most significant effect. Ectopic expression of pE199L in porcine cells (CRL-2843) and human cells (HEK293T and HeLa cells) induced cell death remarkably, showing obvious shrinking, blistering, apoptotic bodies, and nuclear DNA fragments. Meanwhile, cell death was markedly alleviated when the expression of pE199L was knocked down during ASFV infection. Additionally, the expression of pE199L caused a loss of mitochondrial membrane potential, release of cytochrome C, and caspase-9 and -3/7 activation, indicating that the mitochondrial apoptotic pathway was involved in pE199L-induced apoptosis. Further investigations showed that pE199L interacted with several anti-apoptotic BCL-2 subfamily members (such as BCL-XL, MCL-1, BCL-W, and BCL-2A1) and competed with BAK for BCL-XL, which promoted BAK and BAX activation. Taken together, ASFV pE199L induces the mitochondrial-dependent apoptosis, which may provide clues for a comprehensive understanding of ASFV pathogenesis.

scholarly journals Identification of Promiscuous African Swine Fever Virus T-Cell Determinants Using a Multiple Technical Approach

Vaccines ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 29
Author(s):  
Laia Bosch-Camós ◽  
Elisabet López ◽  
María Jesús Navas ◽  
Sonia Pina-Pedrero ◽  
Francesc Accensi ◽  
...  
Keyword(s):  
T Cell ◽  
Mononuclear Cells ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Cd8 T Cell ◽  
Protective Role ◽  
Full Length ◽  
T Cell Epitopes ◽  
Peripheral Blood Mononuclear

The development of subunit vaccines against African swine fever (ASF) is mainly hindered by the lack of knowledge regarding the specific ASF virus (ASFV) antigens involved in protection. As a good example, the identity of ASFV-specific CD8+ T-cell determinants remains largely unknown, despite their protective role being established a long time ago. Aiming to identify them, we implemented the IFNγ ELISpot as readout assay, using as effector cells peripheral blood mononuclear cells (PBMCs) from pigs surviving experimental challenge with Georgia2007/1. As stimuli for the ELISpot, ASFV-specific peptides or full-length proteins identified by three complementary strategies were used. In silico prediction of specific CD8+ T-cell epitopes allowed identifying a 19-mer peptide from MGF100-1L, as frequently recognized by surviving pigs. Complementarily, the repertoire of SLA I-bound peptides identified in ASFV-infected porcine alveolar macrophages (PAMs), allowed the characterization of five additional SLA I-restricted ASFV-specific epitopes. Finally, in vitro stimulation studies using fibroblasts transfected with plasmids encoding full-length ASFV proteins, led to the identification of MGF505-7R, A238L and MGF100-1L as promiscuously recognized antigens. Interestingly, each one of these proteins contain individual peptides recognized by surviving pigs. Identification of the same ASFV determinants by means of such different approaches reinforce the results presented here.

scholarly journals Deletion of a CD2-Like Gene, 8-DR, from African Swine Fever Virus Affects Viral Infection in Domestic Swine

1998 ◽  
Vol 72 (4) ◽  
pp. 2881-2889 ◽  
Author(s):  
M. V. Borca ◽  
C. Carrillo ◽  
L. Zsak ◽  
W. W. Laegreid ◽  
G. F. Kutish ◽  
...  
Keyword(s):  
Viral Infection ◽  
Mononuclear Cells ◽  
African Swine Fever Virus ◽  
Disease Onset ◽  
African Swine Fever ◽  
Fever Virus ◽  
Parental Virus ◽  
Viral Virulence

ABSTRACT 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.

scholarly journals Perifosine, an oral bioactive novel alkylphospholipid, inhibits Akt and induces in vitro and in vivo cytotoxicity in human multiple myeloma cells

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 4053-4062 ◽  
Author(s):  
Teru Hideshima ◽  
Laurence Catley ◽  
Hiroshi Yasui ◽  
Kenji Ishitsuka ◽  
Noopur Raje ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mononuclear Cells ◽  
Antitumor Agents ◽  
Mek Inhibitor ◽  
Peripheral Blood Mononuclear ◽  
Akt Phosphorylation ◽  
Human Multiple Myeloma ◽  
Induced Apoptosis

Perifosine is a synthetic novel alkylphospholipid, a new class of antitumor agents which targets cell membranes and inhibits Akt activation. Here we show that baseline phosphorylation of Akt in multiple myeloma (MM) cells is completely inhibited by perifosine [octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate] in a time- and dose-dependent fashion, without inhibiting phosphoinositide-dependent protein kinase 1 phosphorylation. Perifosine induces significant cytotoxicity in both MM cell lines and patient MM cells resistant to conventional therapeutic agents. Perifosine does not induce cytotoxicity in peripheral blood mononuclear cells. Neither exogenous interleukin-6 (IL-6) nor insulinlike growth factor 1 (IGF-1) overcomes Perifosine-induced cytotoxicity. Importantly, Perifosine induces apoptosis even of MM cells adherent to bone marrow stromal cells. Perifosine triggers c-Jun N-terminal kinase (JNK) activation, followed by caspase-8/9 and poly (ADP)-ribose polymerase cleavage. Inhibition of JNK abrogates perifosine-induced cytotoxicity, suggesting that JNK plays an essential role in perifosine-induced apoptosis. Interestingly, phosphorylation of extracellular signal–related kinase (ERK) is increased by perifosine; conversely, MEK inhibitor synergistically enhances Perifosine-induced cytotoxicity in MM cells. Furthermore, perifosine augments dexamethasone, doxorubicin, melphalan, and bortezomib-induced MM cell cytotoxicity. Finally, perifosine demonstrates significant antitumor activity in a human plasmacytoma mouse model, associated with down-regulation of Akt phosphorylation in tumor cells. Taken together, our data provide the rationale for clinical trials of perifosine to improve patient outcome in MM.

scholarly journals Identification of African Swine Fever Virus Transcription within Peripheral Blood Mononuclear Cells of Acutely Infected Pigs

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2333
Author(s):  
Ann Sofie Olesen ◽  
Miyako Kodama ◽  
Louise Lohse ◽  
Francesc Accensi ◽  
Thomas Bruun Rasmussen ◽  
...  
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Peripheral Blood Mononuclear ◽  
Virus Transcription ◽  
Blood Mononuclear Cells ◽  
Low Levels

African swine fever virus (ASFV) has become widespread in Europe, Asia and elsewhere, thereby causing extensive economic losses. The viral genome includes nearly 200 genes, but their expression within infected pigs has not been well characterized previously. In this study, four pigs were infected with a genotype II strain (ASFV POL/2015/Podlaskie); blood samples were collected before inoculation and at both 3 and 6 days later. During this period, a range of clinical signs of infection became apparent in the pigs. From the blood, peripheral blood mononuclear cells (PBMCs) were isolated. The transcription of the ASFV genes was determined using RNAseq on poly(A)+ mRNAs isolated from these cells. Only very low levels of virus transcription were detected in the PBMCs at 3 days post-inoculation (dpi) but, at 6 dpi, extensive transcription was apparent. This was co-incident with a large increase in the level of ASFV DNA within these cells. The pattern of the virus gene expression was very reproducible between the individual pigs. Many highly expressed genes have undefined roles. Surprisingly, some genes with key roles in virus replication were expressed at only low levels. As the functions of individual genes are identified, information about their expression becomes important for understanding their contribution to virus biology.

scholarly journals Development and In Vivo Evaluation of a MGF110-1L Deletion Mutant in African Swine Fever Strain Georgia

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 286
Author(s):  
Elizabeth Ramirez-Medina ◽  
Elizabeth Vuono ◽  
Sarah Pruitt ◽  
Ayushi Rai ◽  
Ediane Silva ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
African Swine Fever Virus ◽  
Field Isolate ◽  
African Swine Fever ◽  
Economic Losses ◽  
Swine Industry ◽  
In Vivo Evaluation ◽  
Viral Virulence

African swine fever (ASF) is currently causing an epizootic, affecting pigs throughout Eurasia, and causing significant economic losses in the swine industry. ASF is caused by African swine fever virus (ASFV) that consists of a large dsDNA genome that encodes for more than 160 genes; few of these genes have been studied in detail. ASFV contains four multi-gene family (MGF) groups of genes that have been implicated in regulating the immune response and host specificity; however, the individual roles of most of these genes have not been well studied. Here, we describe the evaluation of the previously uncharacterized ASFV MGF110-1L open reading frame (ORF) using a deletion mutant of the ASFV currently circulating throughout Eurasia. The recombinant ASFV lacking the MGF110-1L gene (ASFV-G-ΔMGF110-1L) demonstrated in vitro that the MGF110-1L gene is non-essential, since ASFV-G-ΔMGF110-1L had similar replication kinetics in primary swine macrophage cell cultures when compared to parental highly virulent field isolate Georgia2007 (ASFV-G). Experimental infection of domestic pigs with ASFV-G-ΔMGF110-1L produced a clinical disease similar to that caused by the parental ASFV-G, confirming that deletion of the MGF110-1L gene from the ASFV genome does not affect viral virulence.

scholarly journals An immortalized porcine macrophage cell line competent for the isolation of African swine fever virus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kentaro Masujin ◽  
Tomoya Kitamura ◽  
Ken -ichiro Kameyama ◽  
Kota Okadera ◽  
Tatsuya Nishi ◽  
...  
Keyword(s):  
Viral Replication ◽  
Cell Line ◽  
Genetic Manipulation ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Host Cells ◽  
Hemorrhagic Disease ◽  
Cytopathic Effects

AbstractAfrican swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a fatal hemorrhagic disease of domestic pigs and wild boar. The virus primarily infects macrophage and monocyte host cells, these do not grow in vitro. Many attempts have been made to establish sustainable ASFV-sensitive cell lines, but which supported only low viral replication levels of limited, mostly artificially attenuated strains of ASFV. Here, we examined the competence of a novel cell line of immortalized porcine kidney macrophages (IPKM) for ASFV infection. We demonstrated that IPKM cells can facilitate high levels (> 107.0 TCID50/mL) of viral replication of ASFV, and hemadsorption reactions and cytopathic effects were observed as with porcine alveolar macrophages when inoculated with virulent field isolates: Armenia07, Kenya05/Tk-1, and Espana75. These results suggested that IPKM may be a valuable tool for the isolation, replication, and genetic manipulation of ASFV in both basic and applied ASF research.

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