scholarly journals Deletion of CD2-Like (CD2v) and C-Type Lectin-Like (EP153R) Genes from African Swine Fever Virus Georgia-∆9GL Abrogates Its Effectiveness as an Experimental Vaccine

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1185
Author(s):  
Douglas P. Gladue ◽  
Vivian O’Donnell ◽  
Elizabeth Ramirez-Medina ◽  
Ayushi Rai ◽  
Sarah Pruitt ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Viral Gene ◽  
Gene Deletions ◽  
Reading Frame ◽  
Recombinant Viruses ◽  
Undetectable Viremia

African swine fever virus (ASFV) is currently the most dreaded infectious disease affecting the global swine production industry. There is no commercial vaccine available, making the culling of infected animals the current solution to control outbreaks. Effective experimental vaccines have been developed by deleting virus genes associated with virulence. Deletion of the ASFV 9GL gene (∆9GL) has resulted in the attenuation of different ASFV strains, although the degree of attenuation varies across isolates. Here, we investigated the possibility of the increased safety of the experimental vaccine strain ASFV-G-Δ9GL by deleting two additional virus genes involved in pathogenesis, CD2v, a CD2 like viral encoded gene from the EP402R open reading frame (ORF), and C-type lectin-like viral gene, encoded from the EP153R ORF. Two new recombinant viruses were developed, ASFV-G-Δ9GL/ΔCD2v and ASFV-G-Δ9GL/ΔCD2v/ΔEP153R, harboring two and three gene deletions, respectively. ASFV-G-Δ9GL/ΔCD2v/ΔEP153R, but not ASFV-G-Δ9GL/ΔCD2v, had a decreased ability to replicate in vitro in swine macrophage cultures when compared with parental ASFV-G-Δ9GL. Importantly, ASFV-G-Δ9GL/ΔCD2v and ASFV-G-Δ9GL/ΔCD2v/ΔEP153R induced almost undetectable viremia levels when inoculated into domestic pigs and failed to protect them against challenge with parental virulent ASFV-Georgia, while ASFV-G-Δ9GL offered robust protection during challenge. Therefore, the deletion of CD2-like and C-type lectin-like genes significantly decreased the protective potential of ASFV-G-Δ9GL as a vaccine candidate. This study constitutes an example of the unpredictability of genetic manipulation involving the simultaneous deletion of multiple genes from the ASFV genome.

Genetic manipulation of African swine fever virus: Construction of recombinant viruses expressing the β-galactosidase gene

Virology ◽  
1992 ◽  
Vol 188 (1) ◽  
pp. 67-76 ◽  
Author(s):  
Javier M. Rodríguez ◽  
Fernando Almazán ◽  
Eladio Viñuela ◽  
Jose F. Rodriguez
Keyword(s):  
Genetic Manipulation ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Recombinant Viruses

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.

GS-441524 inhibits African swine fever virus infection in vitro

2021 ◽  
pp. 105081
Author(s):  
Zhao Huang ◽  
Lang Gong ◽  
Zezhong Zheng ◽  
Qi Gao ◽  
Xiongnan Chen ◽  
...  
Keyword(s):  
Virus Infection ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus

scholarly journals Role of African swine fever virus (ASFV) proteins EP153R and EP402R in reducing viral persistence and virulence from attenuated BeninΔDP148R

2021 ◽  
Author(s):  
Vlad Petrovan ◽  
Anusyah Rathakrishnan ◽  
Muneeb Islam ◽  
Lynnette Goatley ◽  
Katy Moffat ◽  
...  
Keyword(s):  
Virus Replication ◽  
Vaccine Development ◽  
African Swine Fever Virus ◽  
Clinical Signs ◽  
African Swine Fever ◽  
Viral Persistence ◽  
Fever Virus ◽  
Post Immunization

The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress towards vaccine development. In this study we investigated the effect of deleting combinations of different genes from a previously attenuated virus, BeninΔDP148R on: virus replication in macrophages, virus persistence and clinical signs post immunization, and induction of protection against challenge. Deletion of either EP402R or EP153R genes individually or in combination from BeninΔDP148R did not reduce virus replication in vitro. However, deletion of EP402R dramatically reduced viral persistence in vivo, whilst maintaining high levels of protection against challenge. The additional deletion of EP153R (BeninΔDP148RΔEP153RΔEP402R) further attenuated the virus and no viremia or clinical signs were observed post immunization. This was associated with decreased protection and detection of moderate levels of challenge virus in blood. Interestingly, the deletion of EP153R alone from BeninΔDP148R did not result in further virus attenuation and a slight increase in virus genome copies in blood was observed at different times post immunization when compared with BeninΔDP148R. These results show that EP402R and EP153R have a synergistic role in promoting viremia, however EP153R alone does not seem to have a major impact on virus levels in blood.

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 Infection of Porcine Aortic Endothelial Cells Leads to Inhibition of Inflammatory Responses, Activation of the Thrombotic State, and Apoptosis

2001 ◽  
Vol 75 (21) ◽  
pp. 10372-10382 ◽  
Author(s):  
Isabelle Vallée ◽  
Stephen W. G. Tait ◽  
Penelope P. Powell
Keyword(s):  
Endothelial Cells ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Surface Expression ◽  
Fever Virus ◽  
Parp Cleavage ◽  
Aortic Endothelial Cells ◽  
Porcine Aortic Endothelial Cells ◽  
Aortic Endothelial

ABSTRACT African swine fever (ASF) is an asymptomatic infection of warthogs and bushpigs, which has become an emergent disease of domestic pigs, characterized by hemorrhage, lymphopenia, and disseminated intravascular coagulation. It is caused by a large icosohedral double-stranded DNA virus, African swine fever virus (ASFV), with infection of macrophages well characterized in vitro and in vivo. This study shows that virulent isolates of ASFV also infect primary cultures of porcine aortic endothelial cells and bushpig endothelial cells (BPECs) in vitro. Kinetics of early and late gene expression, viral factory formation, replication, and secretion were similar in endothelial cells and macrophages. However, ASFV-infected endothelial cells died by apoptosis, detected morphologically by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and nuclear condensation and biochemically by poly(ADP-ribose) polymerase (PARP) cleavage at 4 h postinfection (hpi). Immediate-early proinflammatory responses were inhibited, characterized by a lack of E-selectin surface expression and interleukin 6 (IL-6) and IL-8 mRNA synthesis. Moreover, ASFV actively downregulated interferon-induced major histocompatibility complex class I surface expression, a strategy by which viruses evade the immune system. Significantly, Western blot analysis showed that the 65-kDa subunit of the transcription factor NF-κB, a central regulator of the early response to viral infection, decreased by 8 hpi and disappeared by 18 hpi. Both disappearance of NF-κB p65 and cleavage of PARP were reversed by the caspase inhibitor z-VAD-fmk. Interestingly, surface expression and mRNA transcription of tissue factor, an important initiator of the coagulation cascade, increased 4 h after ASFV infection. These data suggest a central role for vascular endothelial cells in the hemorrhagic pathogenesis of the disease. Since BPECs infected with ASFV also undergo apoptosis, resistance of the natural host must involve complex pathological factors other than viral tropism.

Characterization of the African swine fever virus protein p49: a new late structural polypeptide

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Inmaculada Galindo ◽  
Eladio Viñuela ◽  
Angel L. Carrascosa
Keyword(s):  
Cell Attachment ◽  
African Swine Fever Virus ◽  
Rabbit Antiserum ◽  
African Swine Fever ◽  
Virus Genome ◽  
Fever Virus ◽  
Virus Protein ◽  
Significant Similarity ◽  
Reading Frame ◽  
Cell Extracts

The open reading frame B438L, located within the EcoRI B fragment of the African swine fever virus genome, is predicted to encode a protein of 438 amino acids with a molecular mass of 49·3 kDa. It presents a cell attachment RGD (Arg–Gly–Asp) motif but no other significant similarity to protein sequences in databases. Northern blot and primer extension analysis showed that B438L is transcribed only at late times during virus infection. The B438L gene product has been expressed in Escherichia coli, purified and used as an antigen for antibody production. The rabbit antiserum specific for pB438L recognized a protein of about 49 kDa in virus-infected cell extracts. This protein was synthesized late in infection by all the virus strains tested, was located in cytoplasmic virus factories and appeared as a structural component of purified virus particles.

scholarly journals An African swine fever virus ORF with similarity to C-type lectins is non-essential for growth in swine macrophages in vitro and for virus virulence in domestic swine

1999 ◽  
Vol 80 (10) ◽  
pp. 2693-2697 ◽  
Author(s):  
J. G. Neilan ◽  
M. V. Borca ◽  
Z. Lu ◽  
G. F. Kutish ◽  
S. B. Kleiboeker ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Virus Infectivity ◽  
Mrna Transcription ◽  
Host Cell Interactions ◽  
Lectin Family ◽  
Virus Replication Cycle ◽  
Domestic Swine

An African swine fever virus (ASFV) ORF, 8CR, with similarity to the C-type lectin family of adhesion proteins has been described in the pathogenic isolate Malawi Lil-20/1. The similarity of 8CR to cellular and poxvirus genes associated with cell adhesion, cell recognition and virus infectivity suggested that 8CR may be of significance to ASFV–host cell interactions. Sequence analysis of the 8CR ORF from additional pathogenic ASFV isolates demonstrated conservation among isolates from both pig and tick sources. Northern blot analysis demonstrated 8CR mRNA transcription late in the virus replication cycle. A Malawi Lil-20/1 8CR deletion mutant (Δ8CR) was constructed to analyse 8CR function further. The growth characteristics in vitro of Δ8CR in porcine macrophage cell cultures were identical to those observed for parental virus. In domestic swine, Δ8CR exhibited an unaltered parental Malawi Lil- 20/1 disease and virulence phenotype. Thus, although well conserved among pathogenic ASFV field isolates, 8CR is non-essential for growth in porcine macrophages in vitro and for virus virulence in domestic swine.

scholarly journals Salt inactivation of classical swine fever virus and African swine fever virus in porcine intestines confirms the existing in vitro casings model

2019 ◽  
Vol 238 ◽  
pp. 108424 ◽  
Author(s):  
Tinka Jelsma ◽  
Joris J. Wijnker ◽  
Bregtje Smid ◽  
Eline Verheij ◽  
Wim H.M. van der Poel ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Classical Swine Fever ◽  
Fever Virus

Vectors for the genetic manipulation of African swine fever virus

1995 ◽  
Vol 40 (2) ◽  
pp. 121-131 ◽  
Author(s):  
Ramón Garcia ◽  
Fernando Almazán ◽  
Javier María Rodríguez ◽  
Marta Alonso ◽  
Eladio Viñuela ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus
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