In vitro inhibition of African swine fever virus-topoisomerase II disrupts viral replication

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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GS-441524 inhibits African swine fever virus infection in vitro

Antiviral Research ◽

10.1016/j.antiviral.2021.105081 ◽

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

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

10.1101/2021.04.16.439957 ◽

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.

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

Journal of Virology ◽

10.1128/jvi.72.4.2881-2889.1998 ◽

1998 ◽

Vol 72 (4) ◽

pp. 2881-2889 ◽

Cited By ~ 74

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.

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Differential vector competence of Ornithodoros soft ticks for African swine fever virus: What if it involves more than just crossing organic barriers in ticks?

Parasites & Vectors ◽

10.1186/s13071-020-04497-1 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Rémi Pereira De Oliveira ◽

Evelyne Hutet ◽

Renaud Lancelot ◽

Frédéric Paboeuf ◽

Maxime Duhayon ◽

...

Keyword(s):

Viral Replication ◽

Vertical Transmission ◽

African Swine Fever Virus ◽

Vector Competence ◽

African Swine Fever ◽

Realistic Model ◽

Molecular Data ◽

Fever Virus ◽

Genotype I ◽

Genotype Ii

Abstract Background Several species of soft ticks in genus Ornithodoros are known vectors and reservoirs of African swine fever virus (ASFV). However, the underlying mechanisms of vector competence for ASFV across Ornithodoros species remain to be fully understood. To that end, this study compared ASFV replication and dissemination as well as virus vertical transmission to descendants between Ornithodorosmoubata, O. erraticus, and O. verrucosus in relation to what is known about the ability of these soft tick species to transmit ASFV to pigs. To mimic the natural situation, a more realistic model was used where soft ticks were exposed to ASFV by allowing them to engorge on viremic pigs. Methods Ornithodoros moubata ticks were infected with the ASFV strains Liv13/33 (genotype I) or Georgia2007/1 (genotype II), O. erraticus with OurT88/1 (genotype I) or Georgia2007/1 (genotype II), and O. verrucosus with Ukr12/Zapo (genotype II), resulting in five different tick–virus pairs. Quantitative PCR (qPCR) assays targeting the VP72 ASFV gene was carried out over several months on crushed ticks to study viral replication kinetics. Viral titration assays were also carried out on crushed ticks 2 months post infection to confirm virus survival in soft ticks. Ticks were dissected. and DNA was individually extracted from the following organs to study ASFV dissemination: intestine, salivary glands, and reproductive organs. DNA extracts from each organ were tested by qPCR. Lastly, larval or first nymph-stage progeny emerging from hatching eggs were tested by qPCR to assess ASFV vertical transmission. Results Comparative analyses revealed higher rates of ASFV replication and dissemination in O. moubata infected with Liv13/33, while the opposite was observed for O. erraticus infected with Georgia2007/1 and for O. verrucosus with Ukr12/Zapo. Intermediate profiles were found for O. moubata infected with Georgia2007/1 and for O. erraticus with OurT88/1. Vertical transmission occurred efficiently in O. moubata infected with Liv13/33, and at very low rates in O. erraticus infected with OurT88/1. Conclusions This study provides molecular data indicating that viral replication and dissemination in Ornithodoros ticks are major mechanisms underlying ASFV horizontal and vertical transmission. However, our results indicate that other determinants beyond viral replication also influence ASFV vector competence. Further research is required to fully understand this process in soft ticks.

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

Journal of Virology ◽

10.1128/jvi.75.21.10372-10382.2001 ◽

2001 ◽

Vol 75 (21) ◽

pp. 10372-10382 ◽

Cited By ~ 31

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.

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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

Journal of General Virology ◽

10.1099/0022-1317-80-10-2693 ◽

1999 ◽

Vol 80 (10) ◽

pp. 2693-2697 ◽

Cited By ~ 28

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.

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