Epitope mapping of African swine fever virus (ASFV) structural protein p30

Since African swine fever (ASF) was first reported in 1921, it has brought huge economic losses to the world pig industry. No vaccine or therapy is available. Rapid and effective diagnostics are key steps in managing ASF. We generated three monoclonal antibodies (mAbs) against the African swine fever virus (ASFV) phosphoprotein p30 and designated these as 7D2, 8C8 and 2F6. Epitope mapping revealed that mAb 7D2 recognized VFHAG SLYNW of p30, and mAb 8C8 and 2F6 recognized MDFIL NISMK MEVIF KTDLR of p30. Furthermore, epitope MDFIL NISMK MEVIF KTDLR and VFHAG SLYNW could be well recognized by ASFV-positive sera from natural infected pigs, suggesting that they were natural linear B-cell epitope. Conservation analysis indicated that epitope MDFIL NISMK MEVIF KTDLR and VFHAG SLYNW were highly conserved among the different strains of ASFV. This is the first research to characterize specific mAbs against p30 protein. These findings may facilitate further understanding the function of p30 protein and development of diagnostic tools.

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Epitope mapping of African swine fever virus (ASFV) structural protein, p54

Virus Research ◽

10.1016/j.virusres.2020.197871 ◽

2020 ◽

Vol 279 ◽

pp. 197871 ◽

Cited By ~ 2

Author(s):

Vlad Petrovan ◽

Maria V. Murgia ◽

Ping Wu ◽

Andre D. Lowe ◽

Wei Jia ◽

...

Keyword(s):

Epitope Mapping ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Structural Protein

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Development and characterization of monoclonal antibodies against the N-terminal domain of African swine fever virus structural protein, p54

International Journal of Biological Macromolecules ◽

10.1016/j.ijbiomac.2021.03.059 ◽

2021 ◽

Author(s):

Aiping Wang ◽

Min Jiang ◽

Hongliang Liu ◽

Yankai Liu ◽

Jingming Zhou ◽

...

Keyword(s):

Monoclonal Antibodies ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Structural Protein ◽

Terminal Domain

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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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Development of a Directly Visualized Recombinase Polymerase Amplification–SYBR Green I Method for the Rapid Detection of African Swine Fever Virus

Frontiers in Microbiology ◽

10.3389/fmicb.2020.602709 ◽

2020 ◽

Vol 11 ◽

Author(s):

Shuai Zhang ◽

Aijun Sun ◽

Bo Wan ◽

Yongkun Du ◽

Yanan Wu ◽

...

Keyword(s):

African Swine Fever Virus ◽

African Swine Fever ◽

Sybr Green ◽

Fever Virus ◽

Diagnostic Methods ◽

Sybr Green I ◽

Recombinase Polymerase Amplification ◽

Economic Losses ◽

Field Samples ◽

Standard Plasmid

African swine fever (ASF) is a lethal disease in swine caused by etiologic African swine fever virus (ASFV). The global spread of ASFV has resulted in huge economic losses globally. In the absence of effective vaccines or drugs, pathogen surveillance has been the most important first-line intervention to prevent ASF outbreaks. Among numerous diagnostic methods, recombinase polymerase amplification (RPA)-based detection is capable of producing sensitive and specific results without relying on the use of expensive instruments. However, currently used gene-specific, probe-based RPA for ASFV detection is expensive and time-consuming. To improve the efficiency of ASFV surveillance, a novel directly visualized SYBR Green I-staining RPA (RPAS) method was developed to detect the ASFV genome. SYBR Green I was added to the amplified RPA products for direct visualization by the naked eye. The sensitivity and specificity of this method were confirmed using standard plasmid and inactivated field samples. This method was shown to be highly specific with a detection limit of 103 copies/μl of ASFV in 15 min at 35°C without any cross-reactions with other important porcine viruses selected. In summary, this method enables direct sample visualization with reproducible results for ASFV detection and hence has the potential to be used as a robust tool for ASF prevention and control.

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African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase

Journal of Virology ◽

10.1128/jvi.80.7.3157-3166.2006 ◽

2006 ◽

Vol 80 (7) ◽

pp. 3157-3166 ◽

Cited By ~ 36

Author(s):

Irene Rodríguez ◽

Modesto Redrejo-Rodríguez ◽

Javier M. Rodríguez ◽

Alí Alejo ◽

José Salas ◽

...

Keyword(s):

Disulfide Bonds ◽

Flavin Adenine Dinucleotide ◽

Virus Assembly ◽

Nonstructural Protein ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Structural Protein ◽

Sulfhydryl Oxidase ◽

Infected Cells

ABSTRACT Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway.

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African Swine Fever Virus Structural Protein pE120R Is Essential for Virus Transport from Assembly Sites to Plasma Membrane but Not for Infectivity

Journal of Virology ◽

10.1128/jvi.75.15.6758-6768.2001 ◽

2001 ◽

Vol 75 (15) ◽

pp. 6758-6768 ◽

Cited By ~ 40

Author(s):

Germán Andrés ◽

Ramón Garcı́a-Escudero ◽

Eladio Viñuela ◽

Marı́a L. Salas ◽

Javier M. Rodrı́guez

Keyword(s):

Plasma Membrane ◽

African Swine Fever Virus ◽

Growth Curves ◽

African Swine Fever ◽

Fever Virus ◽

Virus Yield ◽

Virus Infectivity ◽

Structural Protein ◽

Virus Growth ◽

Extracellular Virus

ABSTRACT This report examines the role of African swine fever virus (ASFV) structural protein pE120R in virus replication. Immunoelectron microscopy revealed that protein pE120R localizes at the surface of the intracellular virions. Consistent with this, coimmunoprecipitation assays showed that protein pE120R binds to the major capsid protein p72. Moreover, it was found that, in cells infected with an ASFV recombinant that inducibly expresses protein p72, the incorporation of pE120R into the virus particle is dependent on p72 expression. Protein pE120R was also studied using an ASFV recombinant in which E120R gene expression is regulated by the Escherichia coli lacrepressor-operator system. In the absence of inducer, pE120R expression was reduced about 100-fold compared to that obtained with the parental virus or the recombinant virus grown under permissive conditions. One-step virus growth curves showed that, under conditions that repress pE120R expression, the titer of intracellular progeny was similar to the total virus yield obtained under permissive conditions, whereas the extracellular virus yield was about 100-fold lower than in control infections. Immunofluorescence and electron microscopy demonstrated that, under restrictive conditions, intracellular mature virions are properly assembled but remain confined to the replication areas. Altogether, these results indicate that pE120R is necessary for virus dissemination but not for virus infectivity. The data also suggest that protein pE120R might be involved in the microtubule-mediated transport of ASFV particles from the viral factories to the plasma membrane.

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Characterization of the African Swine Fever Virus Structural Protein p14.5: A DNA Binding Protein

Virology ◽

10.1006/viro.1996.8434 ◽

1997 ◽

Vol 229 (1) ◽

pp. 201-211 ◽

Cited By ~ 15

Author(s):

Luisa Martinez-Pomares ◽

Carmen Simon-Mateo ◽

Carlos Lopez-Otin ◽

Eladio Viñuela

Keyword(s):

Dna Binding ◽

African Swine Fever Virus ◽

Binding Protein ◽

African Swine Fever ◽

Dna Binding Protein ◽

Fever Virus ◽

Structural Protein

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Production and application of mouse monoclonal antibodies targeting linear epitopes in pB602L of African swine fever virus

Archives of Virology ◽

10.1007/s00705-021-05335-0 ◽

2022 ◽

Author(s):

Pengfei Wang ◽

Chunguo Liu ◽

Shida Wang ◽

Lili Wen ◽

Zhibin Shi ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Immunohistochemical Staining ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Diagnostic Tools ◽

Hemorrhagic Disease ◽

Structural Protein ◽

Linear Epitopes ◽

Group 2 ◽

Basic And Applied Research

AbstractAfrican swine fever (ASF) is an acute hemorrhagic disease of domestic pigs. The causative agent of ASF, ASF virus (ASFV), is a double-stranded DNA virus, the sole member in the family Asfarviridae. The non-structural protein pB602L of ASFV is a molecular chaperone of the major capsid protein p72 and plays a key role in icosahedral capsid assembly. This protein is antigenic and is a target for developing diagnostic tools for ASF. To generate monoclonal antibodies (mAbs) against pB602L, a prokaryotically expressed recombinant pB602L protein was produced, purified, and used as an antigen to immunize mice. A total of eight mouse mAbs were obtained, and their binding epitopes were screened by Western blot using an overlapping set of polypeptides from pB602L. Three linear epitopes were identified and designated epitope 1 (366ANRERYNY373), epitope 2 (415GPDAPGLSI423), and epitope 3 (498EMLNVPDD505). Based on the epitope recognized, the eight mAbs were placed into three groups: group 1 (B2A1, B2F1, and B2D10), group 2 (B2H10, B2B2, B2D8, and B2A3), and group 3 (B2E12). The mAbs B2A1, B2H10, and B2E12, each representing one of the groups, were used to detect pB602L in ASFV-infected porcine alveolar macrophages (PAMs) and pig tissues, using an indirect fluorescence assay (IFA) and immunohistochemical staining, respectively. The results showed that pB602L was detectable with all three mAbs in immunohistochemical staining, but only B2H10 was suitable for detecting the proteins in ASFV-infected PAMs by IFA. In summary, we developed eight anti-pB602L mouse mAbs recognizing three linear epitopes in the protein, which can be used as reagents for basic and applied research on ASFV.

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An Isothermal Molecular Point of Care Testing for African Swine Fever Virus Using Recombinase-Aided Amplification and Lateral Flow Assay Without the Need to Extract Nucleic Acids in Blood

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.633763 ◽

2021 ◽

Vol 11 ◽

Author(s):

Yuhang Zhang ◽

Qingmei Li ◽

Junqing Guo ◽

Dongliang Li ◽

Li Wang ◽

...

Keyword(s):

African Swine Fever Virus ◽

Point Of Care ◽

African Swine Fever ◽

Lateral Flow ◽

Fever Virus ◽

Lateral Flow Assay ◽

Economic Losses ◽

Point Of Care Testing ◽

Viral Dna ◽

Blood Samples

African swine fever (ASF) is a highly contagious and usually deadly porcine infectious disease listed as a notifiable disease by the World Organization for Animal Health (OIE). It has brought huge economic losses worldwide, especially since 2018, the first outbreak in China. As there are still no effective vaccines available to date, diagnosis of ASF is essential for its surveillance and control, especially in areas far from city with limited resources and poor settings. In this study, a sensitive, specific, rapid, and simple molecular point of care testing for African swine fever virus (ASFV) B646L gene in blood samples was established, including treatment of blood samples with simple dilution and boiling for 5 min, isothermal amplification with recombinase-aided amplification (RAA) at 37°C in a water bath for 10 min, and visual readout with lateral flow assay (LFA) at room temperature for 10–15 min. Without the need to extract viral DNA in blood samples, the intact workflow from sampling to final diagnostic decision can be completed with minimal equipment requirement in 30 min. The detection limit of RAA-LFA for synthesized B646L gene-containing plasmid was 10 copies/μl, which was 10-fold more sensitive than OIE-recommended PCR and quantitative PCR. In addition, no positive readout of RAA-LFA was observed in testing classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, pseudorabies virus and porcine circovirus 2, exhibiting good specificity. Evaluation of clinical blood samples of RAA-LFA showed 100% coincident rate with OIE-recommended PCR, in testing both extracted DNAs and treated bloods. We also found that some components in blood samples greatly inhibited PCR performance, but had little effect on RAA. Inhibitory effect can be eliminated when blood was diluted at least 32–64-fold for direct PCR, while only a 2–4 fold dilution of blood was suitable for direct RAA, indicating RAA is a better choice than PCR when blood is used as detecting sample. Taken together, we established an sensitive, specific, rapid, and simple RAA-LFA for ASFV molecular detection without the need to extract viral DNA, providing a good choice for point of care testing of ASF diagnosis in the future.

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