African swine fever virus infects macrophages, the natural host cells, via clathrin- and cholesterol-dependent endocytosis

African swine fever (ASF) is highly contagious, causes high mortality in domestic and feral swine, and has a significant economic impact on the global swine industry due to the lack of a vaccine or an effective treatment. African swine fever virus (ASFV) encodes more than 150 polypeptides, which may have intricate and delicate interactions with the host for the benefit of the virus to evade the host’s defenses. However, currently, there is still a lack of information regarding the roles of the viral proteins in host cells. Here, our data demonstrated that the p17, encoded by D117L gene could suppress porcine cGAS-STING signaling pathway, exhibiting the inhibitions of TBK1 and IRF3 phosphorylations, downstream promoter activities, cellular mRNA transcriptions and ISG56 induction, and antiviral responses. Further, we found that p17 was located in endoplasmic reticulum (ER) and Golgi apparatus, and interacted with STING, perturbing it in the recruitment of TBK1 and IKKϵ Additionally, it appeared that the transmembrane domain (amino acids 39–59) of p17 could be required for interacting with STING and inhibiting cGAS-STING pathway. Taken together, p17 could inhibit the cGAS-STING pathway through its interaction with STING and interference with STING in the recruitment of TBK1 and IKKϵ

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Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens

Clinical and Vaccine Immunology ◽

10.1128/cvi.00395-16 ◽

2016 ◽

Vol 23 (11) ◽

pp. 888-900 ◽

Cited By ~ 17

Author(s):

Shehnaz Lokhandwala ◽

Suryakant D. Waghela ◽

Jocelyn Bray ◽

Cameron L. Martin ◽

Neha Sangewar ◽

...

Keyword(s):

Immune Responses ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Host Cells ◽

Hemorrhagic Disease ◽

Virulent Strains ◽

Challenge Study ◽

Cellular Immune

ABSTRACTThe African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (CTLs) could be the key to complete protection. Hence, generation of an efficacious subunit vaccine depends on identification of CTL targets along with a suitable delivery method that will elicit effector CTLs capable of eliminating ASFV-infected host cells and confer long-term protection. To this end, we evaluated the safety and immunogenicity of an adenovirus-vectored ASFV (Ad-ASFV) multiantigen cocktail formulated in two different adjuvants and at two immunizing doses in swine. Immunization with the cocktail rapidly induced unprecedented ASFV antigen-specific antibody and cellular immune responses against all of the antigens. The robust antibody responses underwent rapid isotype switching within 1 week postpriming, steadily increased over a 2-month period, and underwent rapid recall upon boost. Importantly, the primed antibodies strongly recognized the parental ASFV (Georgia 2007/1) by indirect fluorescence antibody (IFA) assay and Western blotting. Significant antigen-specific gamma interferon-positive (IFN-γ+) responses were detected postpriming and postboosting. Furthermore, this study is the first to demonstrate induction of ASFV antigen-specific CTL responses in commercial swine using Ad-ASFV multiantigens. The relevance of the induced immune responses in regard to protection needs to be evaluated in a challenge study.

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The determinants of African Swine Fever Virus Virulence – the Georgia 2007/1 strain and the host macrophage response

10.1101/2021.07.26.453801 ◽

2021 ◽

Author(s):

Gwenny Cackett ◽

Raquel Portugal ◽

Dorota Matelska ◽

Linda Dixon ◽

Finn Werner

Keyword(s):

Gene Expression ◽

African Swine Fever Virus ◽

Case Fatality ◽

African Swine Fever ◽

Fever Virus ◽

Host Cells ◽

Macrophage Response ◽

Transcription Start Sites ◽

Nucleotide Resolution ◽

Cap Analysis

African swine fever virus (ASFV) has a major global economic impact. With a case fatality in domestic pigs approaching 100%, it currently presents the largest threat to animal farming. Although genomic differences between attenuated and highly virulent ASFV strains have been identified the molecular determinants for virulence at the level of gene expression have remained opaque. Here we characterise the transcriptome of ASFV genotype II Georgia 2007/1 (GRG) during infection of the physiologically relevant host cells, porcine macrophages. In this study Cap Analysis Gene Expression sequencing (CAGE-seq) was used to map the 5’ ends of mRNAs at nucleotide resolution, transcription start sites (TSSs) and the global promoter landscape of GRG at early and late times, 5 and 16 hours, post-infection. We then compared transcriptomic maps between the GRG isolate against the lab-attenuated BA71V strain. GRG-specific transcripts identified potential determinants of virulence including members of early expressed multi-gene family members (MGFs), including two we newly characterised in MGF 100 (I7L and I8L). We have importantly shown the response of the host transcriptome to infection, which highlighted a pro-inflammatory immune response with the upregulation of NF-kB activated genes, innate immunity- as well as lysosome components such as S100 proteins.

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Spatiotemporally Orchestrated Interactions between Viral and Cellular Proteins Involved in the Entry of African Swine Fever Virus

Viruses ◽

10.3390/v13122495 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2495

Author(s):

Kehui Zhang ◽

Su Li ◽

Sheng Liu ◽

Shuhong Li ◽

Liang Qu ◽

...

Keyword(s):

Intracellular Transport ◽

Early Stage ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Host Cells ◽

Hemorrhagic Disease ◽

Multilayered Structures ◽

Viral Receptor ◽

Molecular Events

African swine fever (ASF) is a highly contagious hemorrhagic disease in domestic pigs and wild boars with a mortality of up to 100%. The causative agent, African swine fever virus (ASFV), is a member of the Asfarviridae family of the nucleocytoplasmic large DNA viruses. The genome size of ASFV ranges from 170 to 194 kb, encoding more than 50 structural and 100 nonstructural proteins. ASFV virions are 260–300 nm in diameter and composed of complex multilayered structures, leading to an intricate internalization pathway to enter host cells. Currently, no commercial vaccines or antivirals are available, due to the insufficient knowledge of the viral receptor(s), the molecular events of ASFV entry into host cells, and the functions of virulence-associated genes. During the early stage of ASFV infection, the fundamental aspects of virus-host interactions, including virus internalization, intracellular transport through the endolysosomal system, and membrane fusion with endosome, are precisely regulated and orchestrated via a series of molecular events. In this review, we summarize the currently available knowledge on the pathways of ASFV entry into host cells and the functions of viral proteins involved in virus entry. Furthermore, we conclude with future perspectives and highlight areas that require further investigation. This review is expected to provide unique insights for further understanding ASFV entry and facilitate the development of vaccines and antivirals.

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Dynamin- and Clathrin-Dependent Endocytosis in African Swine Fever Virus Entry

Journal of Virology ◽

10.1128/jvi.01557-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 2100-2109 ◽

Cited By ~ 74

Author(s):

Bruno Hernaez ◽

Covadonga Alonso

Keyword(s):

Actin Polymerization ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Cell Types ◽

Fever Virus ◽

Dominant Negative ◽

Endocytic Pathway ◽

Host Cells ◽

Productive Infection ◽

Cellular Compartments

ABSTRACT African swine fever virus (ASFV) is a large DNA virus that enters host cells after receptor-mediated endocytosis and depends on acidic cellular compartments for productive infection. The exact cellular mechanism, however, is largely unknown. In order to dissect ASFV entry, we have analyzed the major endocytic routes using specific inhibitors and dominant negative mutants and analyzed the consequences for ASFV entry into host cells. Our results indicate that ASFV entry into host cells takes place by clathrin-mediated endocytosis which requires dynamin GTPase activity. Also, the clathrin-coated pit component Eps15 was identified as a relevant cellular factor during infection. The presence of cholesterol in cellular membranes, but not lipid rafts or caveolae, was found to be essential for a productive ASFV infection. In contrast, inhibitors of the Na+/H+ ion channels and actin polymerization inhibition did not significantly modify ASFV infection, suggesting that macropinocytosis does not represent the main entry route for ASFV. These results suggest a dynamin-dependent and clathrin-mediated endocytic pathway of ASFV entry for the cell types and viral strains analyzed.

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

Scientific Reports ◽

10.1038/s41598-021-84237-2 ◽

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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