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 The C962R ORF of African Swine Fever Strain Georgia Is Non-Essential and Not Required for Virulence in Swine

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 676 ◽  
Author(s):  
Elizabeth. Ramirez-Medina ◽  
Elizabeth. A. Vuono ◽  
Ayushi. Rai ◽  
Sarah. Pruitt ◽  
Ediane. Silva ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Field Isolate ◽  
Protein A ◽  
African Swine Fever ◽  
Virus Genome ◽  
Economic Losses ◽  
Virus Protein ◽  
Swine Industry ◽  
Reading Frame

African swine fever virus (ASFV) is the causative agent of the African swine fever (ASF) epizootic currently affecting pigs throughout Eurasia, causing significant economic losses in the swine industry. The virus genome encodes for more than 160 genes, of which only a few have been studied in detail. Here we describe the previously uncharacterized ASFV open reading frame (ORF) C962R, a gene encoding for a putative NTPase. RNA transcription studies using infected swine macrophages demonstrate that the C962R gene is translated as a late virus protein. A recombinant ASFV lacking the C962R gene (ASFV-G-ΔC962R) demonstrates in vivo that the C962R gene is non-essential, since ASFV-G-ΔC962R has 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-ΔC962R produced a clinical disease similar to that caused by the parental ASFV-G, confirming that deletion of the C962R gene from the ASFV genome does not impact virulence.

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 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 Evaluation in Swine of a Recombinant Georgia 2010 African Swine Fever Virus Lacking the I8L Gene

Viruses ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 39
Author(s):  
Elizabeth Vuono ◽  
Elizabeth Ramirez-Medina ◽  
Sarah Pruitt ◽  
Ayushi Rai ◽  
Ediane Silva ◽  
...  
Keyword(s):  
Virus Replication ◽  
African Swine Fever Virus ◽  
Disease Outbreaks ◽  
African Swine Fever ◽  
Fever Virus ◽  
Transcriptional Analysis ◽  
Economic Losses ◽  
Conserved Gene

African swine fever virus (ASFV) is the causative agent of African swine fever, a disease currently causing significant economic losses in Europe and Asia. Specifically, the highly virulent ASFV strain Georgia 2010 (ASFV-G) is producing disease outbreaks in this large geographical region. The ASFV genome encodes for over 150 genes, most of which are still not experimentally characterized. I8L is a highly conserved gene that has not been studied beyond its initial description as a virus ORF. Transcriptional analysis of swine macrophages infected with ASFV-G demonstrated that the I8L gene is transcribed early during the virus replication cycle. To assess the importance of I8L during ASFV-G replication in vitro and in vivo, as well as its role in virus virulence in domestic swine, we developed a recombinant virus lacking the I8L gene (ASFV-G-ΔI8L). Replication of ASFV-G-ΔI8L was similar to parental ASFV-G replication in primary swine macrophage cultures, suggesting that the I8L gene is not essential for ASFV-G replication in vitro. Similarly, replication of ASFV-G-ΔI8L in swine intramuscularly inoculated with 102 HAD50 displayed replication kinetics similar to ASFV-G. In addition, animals inoculated with ASFV-G-ΔI8L presented with a clinical disease indistinguishable from that induced by the same dose of the virulent parental ASFV-G isolate. We conclude that deletion of the I8L gene from ASFV-G does not affect virus replication in vitro or in vivo, nor changes the disease outcome in swine.

scholarly journals Deletion of the L7L-L11L Genes Attenuates ASFV and Induces Protection against Homologous Challenge

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 255
Author(s):  
Jingyuan Zhang ◽  
Yanyan Zhang ◽  
Teng Chen ◽  
Jinjin Yang ◽  
Huixian Yue ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Clinical Signs ◽  
African Swine Fever ◽  
Biological Properties ◽  
High Dose ◽  
Swine Industry ◽  
Epidemic Disease ◽  
Intramuscular Inoculation ◽  
Homologous Challenge

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a major epidemic disease endangering the swine industry. Although a number of vaccine candidates have been reported, none are commercially available yet. To explore the effect of unknown genes on the biological characteristics of ASFV and the possibility of a gene-deleted isolate as a vaccine candidate, the strain SY18ΔL7-11, with deletions of L7L–L11L genes from ASFV SY18, was constructed, and its biological properties were analyzed. The results show that deletion of genes L7L-L11L did not affect replication of the virus in vitro. Virulence of SY18△L7-11 was significantly reduced, as 11 of the 12 pigs survived for 28 days after intramuscular inoculation with a low dose (103 TCID50) or a high dose (106 TCID50) of SY18ΔL7-11. All 11 surviving pigs were completely protected against challenge with the parental ASFV SY18 on 28 days postinoculation (dpi). Transient fever and/or irregularly low levels of genomic DNA in the blood were monitored in some pigs after inoculation. No ASF clinical signs or viremia were monitored after challenge. Antibodies to ASFV were induced in all pigs from 14 to 21 days postinoculation. IFN-γ was detected in most of the inoculated pigs, which is usually inhibited in ASFV-infected pigs. Overall, the results demonstrate that SY18ΔL7-11 is a candidate for further constructing safer vaccine(s), with better joint deletions of other gene(s) related to virulence.

scholarly journals Novel Swine Virulence Determinant in the Left Variable Region of the African Swine Fever Virus Genome

2002 ◽  
Vol 76 (7) ◽  
pp. 3095-3104 ◽  
Author(s):  
J. G. Neilan ◽  
L. Zsak ◽  
Z. Lu ◽  
G. F. Kutish ◽  
C. L. Afonso ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Gene Deletion ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Variable Region ◽  
Fever Virus ◽  
Marker Rescue ◽  
Virulence Determinant ◽  
Gene Deletion Mutant

ABSTRACT Previously we have shown that the African swine fever virus (ASFV) NL gene deletion mutant E70ΔNL is attenuated in pigs. Our recent observations that NL gene deletion mutants of two additional pathogenic ASFV isolates, Malawi Lil-20/1 and Pr4, remained highly virulent in swine (100% mortality) suggested that these isolates encoded an additional virulence determinant(s) that was absent from E70. To map this putative virulence determinant, in vivo marker rescue experiments were performed by inoculating swine with infection-transfection lysates containing E70 NL deletion mutant virus (E70ΔNL) and cosmid DNA clones from the Malawi NL gene deletion mutant (MalΔNL). A cosmid clone representing the left-hand 38-kb region (map units 0.05 to 0.26) of the MalΔNL genome was capable of restoring full virulence to E70ΔNL. Southern blot analysis of recovered virulent viruses confirmed that they were recombinant E70ΔNL genomes containing a 23- to 28-kb DNA fragment of the Malawi genome. These recombinants exhibited an unaltered MalΔNL disease and virulence phenotype when inoculated into swine. Additional in vivo marker rescue experiments identified a 20-kb fragment, encoding members of multigene families (MGF) 360 and 530, as being capable of fully restoring virulence to E70ΔNL. Comparative nucleotide sequence analysis of the left variable region of the E70ΔNL and Malawi Lil-20/1 genomes identified an 8-kb deletion in the E70ΔNL isolate which resulted in the deletion and/or truncation of three MGF 360 genes and four MGF 530 genes. A recombinant MalΔNL deletion mutant lacking three members of each MGF gene family was constructed and evaluated for virulence in swine. The mutant virus replicated normally in macrophage cell culture but was avirulent in swine. Together, these results indicate that a region within the left variable region of the ASFV genome containing the MGF 360 and 530 genes represents a previously unrecognized virulence determinant for domestic swine.

scholarly journals BA71ΔCD2: a New Recombinant Live Attenuated African Swine Fever Virus with Cross-Protective Capabilities

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Paula L. Monteagudo ◽  
Anna Lacasta ◽  
Elisabeth López ◽  
Laia Bosch ◽  
Javier Collado ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Sub Saharan Africa ◽  
Swine Industry ◽  
Lethal Challenge ◽  
Major Threat ◽  
Attenuated Viruses ◽  
Genotype Ii

ABSTRACT African swine fever is a highly contagious viral disease of mandatory declaration to the World Organization for Animal Health (OIE). The lack of available vaccines makes its control difficult; thus, African swine fever virus (ASFV) represents a major threat to the swine industry. Inactivated vaccines do not confer solid protection against ASFV. Conversely, live attenuated viruses (LAV), either naturally isolated or obtained by genetic manipulation, have demonstrated reliable protection against homologous ASFV strains, although little or no protection has been demonstrated against heterologous viruses. Safety concerns are a major issue for the use of ASFV attenuated vaccine candidates and have hampered their implementation in the field so far. While trying to develop safer and efficient ASFV vaccines, we found that the deletion of the viral CD2v (EP402R) gene highly attenuated the virulent BA71 strain in vivo. Inoculation of pigs with the deletion mutant virus BA71ΔCD2 conferred protection not only against lethal challenge with the parental BA71 but also against the heterologous E75 (both genotype I strains). The protection induced was dose dependent, and the cross-protection observed in vivo correlated with the ability of BA71ΔCD2 to induce specific CD8+ T cells capable of recognizing both BA71 and E75 viruses in vitro. Interestingly, 100% of the pigs immunized with BA71ΔCD2 also survived lethal challenge with Georgia 2007/1, the genotype II strain of ASFV currently circulating in continental Europe. These results open new avenues to design ASFV cross-protective vaccines, essential to fight ASFV in areas where the virus is endemic and where multiple viruses are circulating. IMPORTANCE African swine fever virus (ASFV) remains enzootic in most countries of Sub-Saharan Africa, today representing a major threat for the development of their swine industry. The uncontrolled presence of ASFV has favored its periodic exportation to other countries, the last event being in Georgia in 2007. Since then, ASFV has spread toward neighboring countries, reaching the European Union's east border in 2014. The lack of available vaccines against ASFV makes its control difficult; so far, only live attenuated viruses have demonstrated solid protection against homologous experimental challenges, but they have failed at inducing solid cross-protective immunity against heterologous viruses. Here we describe a new LAV candidate with unique cross-protective abilities: BA71ΔCD2. Inoculation of BA71ΔCD2 protected pigs not only against experimental challenge with BA71, the virulent parental strain, but also against heterologous viruses, including Georgia 2007/1, the genotype II strain of ASFV currently circulating in Eastern Europe.

scholarly journals Risk of African swine fever virus introduction into the United States through smuggling of pork in air passenger luggage

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Cristina Jurado ◽  
Lina Mur ◽  
María Sol Pérez Aguirreburualde ◽  
Estefanía Cadenas-Fernández ◽  
Beatriz Martínez-López ◽  
...  
Keyword(s):  
Western Europe ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
The United States ◽  
Economic Losses ◽  
Swine Industry ◽  
Virus Surveillance ◽  
Global Epidemiology ◽  
The Us ◽  
Mean Risk

Abstract African swine fever causes substantial economic losses in the swine industry in affected countries. Traditionally confined to Africa with only occasional incursions into other regions, ASF began spreading into Caucasian countries and Eastern Europe in 2007, followed by Western Europe and Asia in 2018. Such a dramatic change in the global epidemiology of ASF has resulted in concerns that the disease may continue to spread into disease-free regions such as the US. In this study, we estimated the risk of introduction of ASF virus into the US through smuggling of pork in air passenger luggage. Results suggest that the mean risk of ASFV introduction into the US via this route has increased by 183.33% from the risk estimated before the disease had spread into Western Europe or Asia. Most of the risk (67.68%) was associated with flights originating from China and Hong Kong, followed by the Russian Federation (26.92%). Five US airports accounted for >90% of the risk. Results here will help to inform decisions related to the design of ASF virus surveillance strategies in the US.

scholarly journals Genetic characterisation of African swine fever virus from 2017 outbreaks in Zambia: Identification of p72 genotype II variants in domestic pigs

2018 ◽  
Vol 85 (1) ◽  
Author(s):  
Edgar Simulundu ◽  
Yona Sinkala ◽  
Herman M. Chambaro ◽  
Andrew Chinyemba ◽  
Frank Banda ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Variable Region ◽  
Economic Losses ◽  
African Region ◽  
Swine Industry ◽  
African Countries ◽  
Central Variable Region ◽  
Close Relationship ◽  
Haemorrhagic Disease

African swine fever (ASF) is a contagious haemorrhagic disease associated with causing heavy economic losses to the swine industry in many African countries. In 2017, Zambia experienced ASF outbreaks in Mbala District (Northern province) and for the first time in Isoka and Chinsali districts (Muchinga province). Meanwhile, another outbreak was observed in Chipata District (Eastern province). Genetic analysis of part of the B646L gene, E183L gene, CP204L gene and the central variable region of the B602L gene of ASF virus (ASFV) associated with the outbreaks in Mbala and Chipata districts was conducted. The results revealed that the ASFV detected in Mbala District was highly similar to that of the Georgia 2007/1 isolate across all the genome regions analysed. In contrast, while showing close relationship with the Georgia 2007/1 virus in the B646L gene, the ASFV detected in Chipata District showed remarkable genetic variation in the rest of the genes analysed. These results suggest that the Georgia 2007/1-like virus could be more diverse than what was previously thought, underscoring the need of continued surveillance and monitoring of ASFVs within the south-eastern African region to better understand their epidemiology and the relationships between outbreaks and their possible origin.

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