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

Viruses ◽  
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.

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

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1078 ◽  
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.

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

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.

scholarly journals I267L Is Neither the Virulence- Nor the Replication-Related Gene of African Swine Fever Virus and Its Deletant Is an Ideal Fluorescent-Tagged Virulence Strain

Viruses ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 53
Author(s):  
Yanyan Zhang ◽  
Junnan Ke ◽  
Jingyuan Zhang ◽  
Huixian Yue ◽  
Teng Chen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
African Swine Fever Virus ◽  
Clinical Signs ◽  
Case Fatality ◽  
African Swine Fever ◽  
Fever Virus ◽  
Economic Losses ◽  
Effective Vaccine ◽  
Domestic Pigs

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF) which reaches up to 100% case fatality in domestic pigs and wild boar and causes significant economic losses in the swine industry. Lack of knowledge of the function of ASFV genes is a serious impediment to the development of the safe and effective vaccine. Herein, I267L was identified as a relative conserved gene and an early expressed gene. A recombinant virus (SY18ΔI267L) with I267L gene deletion was produced by replacing I267L of the virulent ASFV SY18 with enhanced green fluorescent protein (EGFP) cassette. The replication kinetics of SY18ΔI267L is similar to that of the parental isolate in vitro. Moreover, the doses of 102.0 TCID50 (n = 5) and 105.0 TCID50 (n = 5) SY18ΔI267L caused virulent phenotype, severe clinical signs, viremia, high viral load, and mortality in domestic pigs inoculated intramuscularly as the virulent parental virus strain. Therefore, the deletion of I267L does not affect the replication or the virulence of ASFV. Utilizing the fluorescent-tagged virulence deletant can be easy to gain a visual result in related research such as the inactivation effect of some drugs, disinfectants, extracts, etc. on ASFV.

scholarly journals Structure of African Swine Fever Virus and Associated Molecular Mechanisms Underlying Infection and Immunosuppression: A Review

2021 ◽  
Vol 12 ◽  
Author(s):  
Yue Wang ◽  
Weifang Kang ◽  
Wenping Yang ◽  
Jing Zhang ◽  
Dan Li ◽  
...  
Keyword(s):  
Immune Response ◽  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
African Swine Fever Virus ◽  
Animal Health ◽  
African Swine Fever ◽  
Fever Virus ◽  
Economic Losses ◽  
Cell Immune Response ◽  
Effective Vaccine

African swine fever (ASF) is an acute, highly contagious, and deadly infectious disease. The mortality rate of the most acute and acute ASF infection is almost 100%. The World Organization for Animal Health [Office International des épizooties (OIE)] lists it as a legally reported animal disease and China lists it as class I animal epidemic. Since the first diagnosed ASF case in China on August 3, 2018, it has caused huge economic losses to animal husbandry. ASF is caused by the African swine fever virus (ASFV), which is the only member of Asfarviridae family. ASFV is and the only insect-borne DNA virus belonging to the Nucleocytoplasmic Large DNA Viruses (NCLDV) family with an icosahedral structure and an envelope. Till date, there are still no effective vaccines or antiviral drugs for the prevention or treatment of ASF. The complex viral genome and its sophisticated ability to regulate the host immune response may be the reason for the difficulty in developing an effective vaccine. This review summarizes the recent findings on ASFV structure, the molecular mechanism of ASFV infection and immunosuppression, and ASFV-encoded proteins to provide comprehensive proteomic information for basic research on ASFV. In addition, it also analyzes the results of previous studies and speculations on the molecular mechanism of ASFV infection, which aids the study of the mechanism of clinical pathological phenomena, and provides a possible direction for an intensive study of ASFV infection mechanism. By summarizing the findings on molecular mechanism of ASFV- regulated host cell immune response, this review provides orientations and ideas for fundamental research on ASFV and provides a theoretical basis for the development of protective vaccines against ASFV.

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

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.

scholarly journals Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Xiaoying Wang ◽  
Sheng He ◽  
Na Zhao ◽  
Xiaohong Liu ◽  
Yongchang Cao ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Diagnostic Technique ◽  
African Swine Fever ◽  
Cross Reactivity ◽  
Fever Virus ◽  
Effective Vaccine ◽  
Highly Sensitive ◽  
Resource Poor ◽  
Good Consistency ◽  
Virus Genotypes

Abstract Background As no treatment or effective vaccine for African swine fever virus (ASFV) is currently available, a rapid, highly sensitive diagnostic is urgently needed to curb the spread of ASFV. Results Here we designed a novel CRISPR-Cas12a based assay for ASFV detection. To detect different ASFV genotypes, 19 crRNAs were designed to target the conserved p72 gene in ASFV, and several crRNAs with high activity were identified that could be used as alternatives in the event of new ASFV variants. The results showed that the sensitivity of the CRISPR-Cas12a based assay is about ten times higher than either the commercial quantitative PCR (qPCR) kit or the OIE-recommended qPCR. CRISPR-Cas12a based assay could also detect ASFV specifically without cross-reactivity with other important viruses in pigs and various virus genotypes. We also found that longer incubation times increased the detection limits, which could be applied to improve assay outcomes in the detection of weakly positive samples and new ASFV variants. In addition, both the CRISPR-Cas12a based assay and commercial qPCR showed very good consistency. Conclusions In summary, the CRISPR-Cas12a based assay offers a feasible approach and a new diagnostic technique for the diagnosis of ASFV, particularly in resource-poor settings.

scholarly journals Rapid Sequence-Based Characterization of African Swine Fever Virus by Use of the Oxford Nanopore MinION Sequence Sensing Device and a Companion Analysis Software Tool

2019 ◽  
Vol 58 (1) ◽  
Author(s):  
Vivian K. O’Donnell ◽  
Frederic R. Grau ◽  
Gregory A. Mayr ◽  
Tracy L. Sturgill Samayoa ◽  
Kimberly A. Dodd ◽  
...  
Keyword(s):  
Real Time ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Full Genome Sequence ◽  
Effective Vaccine ◽  
Fast Analysis ◽  
Rapid Acquisition ◽  
Oxford Nanopore

ABSTRACT African swine fever virus (ASFV) is the causative agent of a severe and highly contagious viral disease of pigs that poses serious economic consequences to the swine industry due to the high mortality rate and impact on international trade. There is no effective vaccine to control African swine fever (ASF), and therefore, efficient disease control is dependent on early detection and diagnosis of ASFV. The large size of the ASFV genome (∼180 kb) has historically hindered efforts to rapidly obtain a full-genome sequence. Rapid acquisition of data is critical for characterization of the isolate and to support epidemiological efforts. Here, we investigated the capacity of the Oxford Nanopore MinION sequence sensing device to act as a rapid sequencing tool. When coupled with our novel companion software script, the African swine fever fast analysis sequencing tool (ASF-FAST), the analysis of output data was performed in real time. Complete ASFV genome sequences were generated from cell culture isolates and blood samples obtained from experimentally infected pigs. Removal of the host-methylated DNA from the extracted nucleic acid facilitated rapid ASFV sequence identification, with reads specific to ASFV detected within 6 min after the initiation of sequencing. Regardless of the starting material, sufficient sequence was available for complete genome resolution (up to 100%) within 10 min. Overall, this paper highlights the use of Nanopore sequencing technology in combination with the ASF-FAST software for the purpose of rapid and real-time resolution of the full ASFV genome from a diagnostic sample.

scholarly journals Structure of African Swine Fever Virus Late Promoters: Requirement of a TATA Sequence at the Initiation Region

2000 ◽  
Vol 74 (17) ◽  
pp. 8176-8182 ◽  
Author(s):  
Ramón García-Escudero ◽  
Eladio Viñuela
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
African Swine Fever Virus ◽  
Point Mutations ◽  
African Swine Fever ◽  
Initiation Site ◽  
Fever Virus ◽  
Transcriptional Initiation ◽  
Viral Promoters ◽  
Tata Sequence

ABSTRACT A number of mutations, including deletions, linker scan substitutions, and point mutations, were performed in the promoter of the late African swine fever virus (ASFV) gene coding for the capsid protein p72. The consequences of the mutations in terms of promoter activity were analyzed by luciferase assays using plasmids transfected into infected cells. The results showed that the promoter function is contained between nucleotides −36 and +5 relative to the transcription initiation site. Moreover, two major essential regions for promoter activity, centered at positions −13 and +3, were located along the 41-bp sequence, the latter mapping in the transcription start site. Sequence alignment with other ASFV late promoters showed homology in the region of transcriptional initiation, where the presence of the sequence TATA was observed in most of the promoters. Substitution of these four residues in three other late viral promoters strongly reduced their respective activities. These results show thatcis-acting control elements of ASFV p72 gene transcription are restricted to a short sequence of about 40 bp and suggest that transcription of late genes is initiated around a TATA sequence that would function as an initiator element.

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

2004 ◽  
Vol 78 (18) ◽  
pp. 9731-9739 ◽  
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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