Identification of a Functional Small Noncoding RNA of African Swine Fever Virus

ABSTRACT African swine fever virus (ASFV) causes a lethal hemorrhagic disease of domestic pigs, against which no vaccine is available. ASFV has a large, double-stranded DNA genome that encodes over 150 proteins. Replication takes place predominantly in the cytoplasm of the cell and involves complex interactions with host cellular components, including small noncoding RNAs (sncRNAs). A number of DNA viruses are known to manipulate sncRNA either by encoding their own or disrupting host sncRNA. To investigate the interplay between ASFV and sncRNAs, a study of host and viral small RNAs extracted from ASFV-infected primary porcine macrophages (PAMs) was undertaken. We discovered that ASFV infection had only a modest effect on host miRNAs, with only 6 miRNAs differentially expressed during infection. The data also revealed 3 potential novel small RNAs encoded by ASFV, ASFVsRNA1-3. Further investigation of ASFVsRNA2 detected it in lymphoid tissue from pigs with ASF. Overexpression of ASFVsRNA2 led to an up to 1-log reduction in ASFV growth, indicating that ASFV utilizes a virus-encoded small RNA to disrupt its own replication. IMPORTANCE African swine fever (ASF) poses a major threat to pig populations and food security worldwide. The disease is endemic to Africa and Eastern Europe and is rapidly emerging into Asia, where it has led to the deaths of millions of pigs in the last 12 months. The development of safe and effective vaccines to protect pigs against ASF has been hindered by lack of understanding of the complex interactions between ASFV and the host cell. We focused our work on characterizing the interactions between ASFV and sncRNAs. Although comparatively modest changes to host sncRNA abundances were observed upon ASFV infection, we discovered and characterized a novel functional ASFV-encoded sncRNA. The results from this study add important insights into ASFV host-pathogen interactions. This knowledge may be exploited to develop more effective ASFV vaccines that take advantage of the sncRNA system.

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Identification of a functional small non-coding RNA encoded by African swine fever virus

10.1101/865147 ◽

2019 ◽

Cited By ~ 2

Author(s):

Laura E. M. Dunn ◽

Alasdair Ivens ◽

Christopher L. Netherton ◽

David A. G. Chapman ◽

Philippa M. Beard

Keyword(s):

Small Rnas ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Dna Viruses ◽

Log Reduction ◽

Complex Interactions ◽

Non Coding Rna ◽

Haemorrhagic Disease ◽

Cellular Components

AbstractAfrican swine fever virus (ASFV) causes a lethal haemorrhagic disease of domestic pigs, to which there is no vaccine available. ASFV has a large, double-stranded DNA genome that encodes over 150 proteins. Replication takes place in the cytoplasm of the cell and involves complex interactions with host cellular components including small non-coding RNAs (sncRNAs). A number of DNA viruses are known to manipulate sncRNA either by encoding their own or disrupting host sncRNA. In order to investigate the interplay between ASFV and sncRNAs, study of host and viral small RNAs extracted from ASFV-infected primary porcine macrophages (PAMs) was undertaken. We discovered that ASFV infection had only a modest effect on host miRNAs, with only 6 miRNAs differentially expressed during infection. The data also revealed 3 potential novel small RNAs encoded by ASFV, ASFVsRNA1-3. Further investigation of ASFVsRNA2 detected it in lymphoid tissue from pigs with ASF. Overexpression of ASFVsRNA2 led to up to a 1 log reduction in ASFV growth indicating that ASFV utilises a virally-encoded small RNA to disrupt its own replication. This study describes the modest impact of ASFV on host sncRNAs and the identification of a functional ASFV-encoded sncRNA.ImportanceAfrican swine fever (ASF) poses a major threat to pig populations and food security worldwide. The disease is endemic in Africa and Eastern Europe and rapidly emerging into Asia where it has led to the deaths of millions of pigs in the past 12 months. The development of safe and effective vaccines to protect pigs against ASF has been hindered by lack of understanding of the complex interactions between ASFV and the host cell. We focused our work on characterising the interactions between ASFV and sncRNAs. We found only modest changes to host sncRNA abundance after ASFV infection, and discovered a functional ASFV-encoded sncRNA. The knowledge from this study can be exploited to develop more effective ASFV vaccines that take advantage of the sncRNA system.

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Computational Analysis of African Swine Fever Virus Protein Space for the Design of an Epitope-Based Vaccine Ensemble

Pathogens ◽

10.3390/pathogens9121078 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1078 ◽

Cited By ~ 1

Author(s):

Albert Ros-Lucas ◽

Florencia Correa-Fiz ◽

Laia Bosch-Camós ◽

Fernando Rodriguez ◽

Julio Alonso-Padilla

Keyword(s):

T Cell ◽

Vaccine Development ◽

De Novo ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Economic Losses ◽

Virus Protein ◽

Hemorrhagic Disease ◽

Starting Point

African swine fever virus is the etiological agent of African swine fever, a transmissible severe hemorrhagic disease that affects pigs, causing massive economic losses. There is neither a treatment nor a vaccine available, and the only method to control its spread is by extensive culling of pigs. So far, classical vaccine development approaches have not yielded sufficiently good results in terms of concomitant safety and efficacy. Nowadays, thanks to advances in genomic and proteomic techniques, a reverse vaccinology strategy can be explored to design alternative vaccine formulations. In this study, ASFV protein sequences were analyzed using an in-house pipeline based on publicly available immunoinformatic tools to identify epitopes of interest for a prospective vaccine ensemble. These included experimentally validated sequences from the Immune Epitope Database, as well as de novo predicted sequences. Experimentally validated and predicted epitopes were prioritized following a series of criteria that included evolutionary conservation, presence in the virulent and currently circulating variant Georgia 2007/1, and lack of identity to either the pig proteome or putative proteins from pig gut microbiota. Following this strategy, 29 B-cell, 14 CD4+ T-cell and 6 CD8+ T-cell epitopes were selected, which represent a starting point to investigating the protective capacity of ASFV epitope-based vaccines.

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Two African Swine Fever Virus Proteins Derived from a Common Precursor Exhibit Different Nucleocytoplasmic Transport Activities

Journal of Virology ◽

10.1128/jvi.78.18.9731-9739.2004 ◽

2004 ◽

Vol 78 (18) ◽

pp. 9731-9739 ◽

Cited By ~ 9

Author(s):

A. Eulálio ◽

I. Nunes-Correia ◽

A. L. Carvalho ◽

C. Faro ◽

V. Citovsky ◽

...

Keyword(s):

Nuclear Import ◽

Mammalian Cells ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Nucleocytoplasmic Transport ◽

Proteolytic Processing ◽

Fever Virus ◽

Hemorrhagic Disease ◽

Common Precursor ◽

Green Fluorescent

ABSTRACT African swine fever virus (ASFV), a large icosahedral deoxyvirus, is the causative agent of an economically relevant hemorrhagic disease that affects domestic pigs. The major purpose of the present study was to investigate the nuclear transport activities of the ASFV p37 and p14 proteins, which result from the proteolytic processing of a common precursor. Experiments were performed by using yeast-based nucleocytoplasmic transport assays and by analysis of the subcellular localization of different green fluorescent and Myc fusion proteins in mammalian cells. The results obtained both in yeast and mammalian cells clearly demonstrated that ASFV p14 protein is imported into the nucleus but not exported to the cytoplasm. The ability of p37 protein to be exported from the nucleus to the cytoplasm of both yeast and mammalian cells was also demonstrated, and the results clearly indicate that p37 nuclear export is dependent on the interaction of the protein with the CRM-1 receptor. In addition, p37 was shown to exhibit nuclear import activity in mammalian cells. The p37 protein nuclear import and export abilities described here constitute the first report of a nucleocytoplasmic shuttling protein encoded by the ASFV genome. Overall, the overlapping results obtained for green fluorescent protein fusions and Myc-tagged proteins undoubtedly demonstrate that ASFV p37 and p14 proteins exhibit nucleocytoplasmic transport activities. These findings are significant for understanding the role these proteins play in the replication cycle of ASFV.

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Pathogenesis of African swine fever virus inOrnithodorosticks

Animal Health Research Reviews ◽

10.1079/ahrr200133 ◽

2001 ◽

Vol 2 (2) ◽

pp. 121-128 ◽

Cited By ~ 39

Author(s):

Steven B. Kleiboeker ◽

Glen A. Scoles

Keyword(s):

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Sub Saharan Africa ◽

Hemorrhagic Disease ◽

Sylvatic Cycle ◽

Infectious Dose ◽

Domestic Pigs ◽

Ornithodoros Porcinus Porcinus ◽

Sub Saharan

AbstractAfrican swine fever virus (ASFV) is the only known DNA arbovirus and the sole member of the family Asfarviridae. It causes a lethal, hemorrhagic disease in domestic pigs. ASFV is enzootic in sub-Saharan Africa and is maintained in a sylvatic cycle by infecting both wild members of the Suidae (e.g. warthogs) and the argasid tickOrnithodoros porcinus porcinus. The pathogenesis of ASFV inO. porcinus porcinusticks is characterized by a low infectious dose, lifelong infection, efficient transmission to both pigs and ticks, and low mortality until after the first oviposition. ASFV pathogenesis in warthogs is characterized by an inapparent infection with transient, low viremic titers. ThusO. porcinus porcinusticks probably constitute the most important natural vector of ASFV, although both the mammalian and tick hosts are probably required for the maintenance of ASFV in the sylvatic cycle. The mechanism of ASFV transmission from the sylvatic cycle to domestic pigs is probably through infected ticks feeding on pigs. In addition toO. porcinus porcinus, a number of North American, Central American and Caribbean species ofOrnithodoroshave been shown to be potential vectors of ASFV.

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Swift and Reliable “Easy Lab” Methods for the Sensitive Molecular Detection of African Swine Fever Virus

International Journal of Molecular Sciences ◽

10.3390/ijms22052307 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2307

Author(s):

Ahmed Elnagar ◽

Jutta Pikalo ◽

Martin Beer ◽

Sandra Blome ◽

Bernd Hoffmann

Keyword(s):

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Molecular Diagnostic ◽

Economic Losses ◽

Hemorrhagic Disease ◽

Tissue Samples ◽

Wild Boars ◽

Field Samples ◽

Edta Blood

African swine fever (ASF) is a contagious viral hemorrhagic disease of domestic pigs and wild boars. The disease is notifiable to the World Organisation for Animal Health (OIE) and is responsible for high mortality and serious economic losses. PCR and real-time PCR (qPCR) are the OIE-recommended standard methods for the direct detection of African swine fever virus (ASFV) DNA. The aim of our work was the simplification and standardization of the molecular diagnostic workflow in the lab. For validation of this “easy lab” workflow, different sample materials from animal trials were collected and analyzed (EDTA blood, serum, oral swabs, chewing ropes, and tissue samples) to identify the optimal sample material for diagnostics in live animals. Based on our data, the EDTA blood samples or bloody tissue samples represent the best specimens for ASFV detection in the early and late phases of infection. The application of prefilled ready-to-use reagents for nucleic acid extraction or the use of a Tissue Lysis Reagent (TLR) delivers simple and reliable alternatives for the release of the ASFV nucleic acids. For the qPCR detection of ASFV, different published and commercial kits were compared. Here, a lyophilized commercial kit shows the best results mainly based on the increased template input. The good results of the “easy lab” strategy could be confirmed by the ASFV detection in field samples from wild boars collected from the 2020 ASFV outbreak in Germany. Appropriate internal control systems for extraction and PCR are key features of the “easy lab” concept and reduce the risk of false-negative and false-positive results. In addition, the use of easy-to-handle machines and software reduces training efforts and the misinterpretation of results. The PCR diagnostics based on the “easy lab” strategy can realize a high sensitivity and specificity comparable to the standard PCR methods and should be especially usable for labs with limited experiences and resources.

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African Swine Fever Virus A528R Inhibits TLR8 Mediated NF-κB Activity by Targeting p65 Activation and Nuclear Translocation

Viruses ◽

10.3390/v13102046 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2046

Author(s):

Xueliang Liu ◽

Da Ao ◽

Sen Jiang ◽

Nengwen Xia ◽

Yulin Xu ◽

...

Keyword(s):

Promoter Activity ◽

Nuclear Translocation ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Viral Proteins ◽

Fever Virus ◽

Economic Losses ◽

Effective Vaccine ◽

Hemorrhagic Disease ◽

Host Innate Immunity

African swine fever (ASF) is mainly an acute hemorrhagic disease which is highly contagious and lethal to domestic pigs and wild boars. The global pig industry has suffered significant economic losses due to the lack of an effective vaccine and treatment. The African swine fever virus (ASFV) has a large genome of 170–190 kb, encoding more than 150 proteins. During infection, ASFV evades host innate immunity via multiple viral proteins. A528R is a very important member of the polygene family of ASFV, which was shown to inhibit IFN-β production by targeting NF-κB, but its mechanism is not clear. This study has shown that A528R can suppress the TLR8-NF-κB signaling pathway, including the inhibition of downstream promoter activity, NF-κB p65 phosphorylation and nuclear translocation, and the antiviral and antibacterial activity. Further, we found the cellular co-localization and interaction between A528R and p65, and ANK repeat domains of A528R and RHD of p65 are involved in their interaction and the inhibition of p65 activity. Therefore, we conclude that A528R inhibits TLR8-NF-κB signaling by targeting p65 activation and nuclear translocation.

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Cytokine Storm in Domestic Pigs Induced by Infection of Virulent African Swine Fever Virus

Frontiers in Veterinary Science ◽

10.3389/fvets.2020.601641 ◽

2021 ◽

Vol 7 ◽

Author(s):

Shuchao Wang ◽

Jingyuan Zhang ◽

Yanyan Zhang ◽

Jinjin Yang ◽

Lidong Wang ◽

...

Keyword(s):

Inflammatory Cytokines ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Post Inoculation ◽

Hemorrhagic Disease ◽

Cytokine Storm ◽

Domestic Pigs ◽

Tnf Α ◽

Anti Inflammatory

African swine fever, caused by African swine fever virus (ASFV), is a highly contagious hemorrhagic disease of domestic pigs. The current continent-wide pandemic has persisted for over 10 years, and its economy-devastating effect was highlighted after spreading to China, which possesses half of the world pig industry. So far, development of an effective and safe vaccine has not been finished largely due to the knowledge gaps in pathogenesis and immunology, particularly the role of cytokines in the host's immune response. Therefore, we performed experiments in domestic pigs to analyze the kinetics of representative circulating interferons (IFNs), interleukins (ILs), growth factors, tumor necrosis factors (TNFs), and chemokines induced by infection of type II virulent ASFV SY18. Pigs infected with this Chinese prototypical isolate developed severe clinical manifestations mostly from 3 days post inoculation (dpi) and died from 7 to 8 dpi. Serum analysis revealed a trend of robust and sustained elevation of pro-inflammatory cytokines including TNF-α, IFN-α, IL-1β, IL-6, IL-8, IL-12, IL-18, RANTES (regulated upon activation, normal T cell expressed and secreted), and IFN-γ-induced protein 10 (IP-10) from 3 dpi, but not the anti-inflammatory cytokines IL-10 and transforming growth factor-β (TGF-β). Moreover, secondary drastic increase of the levels of TNF-α, IL-1β, IL-6, and IL-8, as well as elevated IL-10, was observed at the terminal phase of infection. This pattern of cytokine secretion clearly drew an image of a typical cytokine storm characterized by delayed and dysregulated initiation of the secretion of pro-inflammatory cytokine and imbalanced pro- and anti-inflammatory response, which paved a way for further understanding of the molecular basis of ASFV pathogenesis.

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African Swine Fever Virus Protein pE199L Mediates Virus Entry by Enabling Membrane Fusion and Core Penetration

mBio ◽

10.1128/mbio.00789-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Tania Matamoros ◽

Alí Alejo ◽

Javier María Rodríguez ◽

Bruno Hernáez ◽

Milagros Guerra ◽

...

Keyword(s):

Membrane Fusion ◽

Host Cell ◽

Virus Assembly ◽

African Swine Fever Virus ◽

Virus Entry ◽

African Swine Fever ◽

Viral Envelope ◽

Fever Virus ◽

Virus Protein ◽

Hemorrhagic Disease

ABSTRACT African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) causing a lethal hemorrhagic disease that currently threatens the global pig industry. Despite its relevance in the infectious cycle, very little is known about the internalization of ASFV in the host cell. Here, we report the characterization of ASFV protein pE199L, a cysteine-rich structural polypeptide with similarity to proteins A16, G9, and J5 of the entry fusion complex (EFC) of poxviruses. Using biochemical and immunomicroscopic approaches, we found that, like the corresponding poxviral proteins, pE199L localizes to the inner viral envelope and behaves as an integral transmembrane polypeptide with cytosolic intramolecular disulfide bonds. Using an ASFV recombinant that inducibly expresses the E199L gene, we found that protein pE199L is not required for virus assembly and egress or for virus-cell binding and endocytosis but is required for membrane fusion and core penetration. Interestingly, similar results have been previously reported for ASFV protein pE248R, an inner membrane virion component related to the poxviral L1 and F9 EFC proteins. Taken together, these findings indicate that ASFV entry relies on a form of fusion machinery comprising proteins pE248R and pE199L that displays some similarities to the unconventional fusion apparatus of poxviruses. Also, these results provide novel targets for the development of strategies that block the first stages of ASFV replication. IMPORTANCE African swine fever virus (ASFV) causes a highly lethal swine disease that is currently present in many countries of Eastern Europe, the Russian Federation, and Southeast Asia, severely affecting the pig industry. Despite extensive research, effective vaccines or antiviral strategies are still lacking and relevant gaps in knowledge of the fundamental biology of the viral infection cycle exist. In this study, we identified pE199L, a protein of the inner viral membrane that is required for virus entry. More specifically, pE199L is necessary for the fusion event that leads to the penetration of the genome-containing core in the host cell. Our results significantly increase our knowledge of the process of internalization of African swine fever virus, which may instruct future research on antiviral strategies.

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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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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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