scholarly journals Serodiagnostic potential of synthetic peptides derived from African swine fever virus putative protein pCP312R

2019 ◽  
Author(s):  
Sylvester Ochwo ◽  
David Kalenzi Atuhaire ◽  
Mathias Afayoa ◽  
Majid Kisseka ◽  
Phillip Kimuda Magambo ◽  
...  
Keyword(s):  
Synthetic Peptides ◽  
Indirect Elisa ◽  
African Swine Fever Virus ◽  
Prediction Method ◽  
African Swine Fever ◽  
Diagnostic Sensitivity ◽  
Hemorrhagic Disease ◽  
Putative Protein ◽  
First Time ◽  
Domestic Swine

AbstractAfrican swine fever (ASF) is a hemorrhagic disease of domestic swine, with often high mortality rates registered. To date there is still no vaccine produced against ASF, and disease management in countries including Uganda, where the disease is endemic is dependent on accurate and timely diagnosis programs and quarantine. This study aimed at contributing more knowledge towards ASF diagnosis by investigating the serodiagnostic potential of synthetic peptides of an ASF putative protein pCP312R. Antigenic regions of the pCP312R putative protein were identified using Kolaskar and Tongaonkar antigenicity prediction method and twelve (12) peptides were predicted, out of which four (4) peptides were selected and synthesised. An additional peptide derived from the carboxyl end of the ASFV p54 protein was also synthesised and used as a control. Polyclonal rabbit antibodies raised against each of the five peptides was used in immunohistochemistry, and each demonstrated ability to localize viral antigen in pig tissue albeit with slightly varying intensities, at a dilution of 1:200, with antibodies against peptides cpr1, cpr2, cpr3 and cpr4 all accurately staining infected macrophages. However all the peptides evaluated in this study performed moderately when used in indirect ELISA tests giving the following results; CP1; diagnostic sensitivity of 55% (95% CI, 0.3421-0.7418) and specificity of 96% (95% CI, 0.8046-0.9929), CP2; diagnostic sensitivity of 100% (95% CI, 0.8389-1) and specificity of 52% (95% CI, 0.335-0.6997), CP3; diagnostic sensitivity of 95% (95% CI, 76.39-99.11) and specificity of 88% (95% CI, 70.04-95.83), CP4; diagnostic sensitivity of 90% (95% CI: 0.699-0.9721) and specificity of 76% (95% CI: 0.5657-0.885) and p54; diagnostic sensitivity of 100% (95% CI, 0.8389-1) and specificity of 56% (95% CI, 0.3707-0.7333). This study presents the first time synthetic peptides have been successfully predicted, designed and evaluated for Serodiagnosis of African swine fever in domestic pigs. This study in addition showed that there is potential for use of polyclonal anti-peptide antibodies in the diagnosis of ASF using immunohistochemistry.

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 African Swine Fever Laboratory Diagnosis—Lessons Learned from Recent Animal Trials

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 177
Author(s):  
Jutta Pikalo ◽  
Paul Deutschmann ◽  
Melina Fischer ◽  
Hanna Roszyk ◽  
Martin Beer ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Clinical Signs ◽  
African Swine Fever ◽  
Laboratory Diagnosis ◽  
Lessons Learned ◽  
Hemorrhagic Disease ◽  
Passive Surveillance ◽  
Animal Trials ◽  
Fit For Purpose ◽  
The Impact

African swine fever virus (ASFV) causes a hemorrhagic disease in pigs with high socio-economic consequences. To lower the impact of disease incursions, early detection is crucial. In the context of experimental animal trials, we evaluated diagnostic workflows for a high sample throughput in active surveillance, alternative sample matrices for passive surveillance, and lateral flow devices (LFD) for rapid testing. We could demonstrate that EDTA blood is significantly better suited for early ASFV detection than serum. Tissues recommended by the respective diagnostic manuals were in general comparable in their performance, with spleen samples giving best results. Superficial lymph nodes, ear punches, and different blood swabs were also evaluated as potential alternatives. In summary, all matrices yielded positive results at the peak of clinical signs and could be fit for purpose in passive surveillance. However, weaknesses were discovered for some matrices when it comes to the early phase of infection or recovery. The antigen LFD showed variable results with best performance in the clinical phase. The antibody LFD was quite comparable with ELISA systems. Concluding, alternative approaches are feasible but have to be embedded in control strategies selecting test methods and sample materials following a “fit-for-purpose” approach.

DNA segments of African Swine Fever Virus detected for the first time in hard ticks from sheep and bovines

2019 ◽  
Vol 24 (1) ◽  
pp. 180 ◽  
Author(s):  
Ze Chen ◽  
Xiaofeng Xu ◽  
Yufeng Wang ◽  
Jinlong Bei ◽  
Xiufeng Jin ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Control Measures ◽  
Single Amino Acid ◽  
Small Rna Sequencing ◽  
Hard Ticks ◽  
Prevention And Control Measures ◽  
And Control ◽  
First Time

In this study, we detected African Swine Fever Virus (ASFV) in Dermacentor (Ixodidae) from sheep and bovines using small RNA sequencing. To validate this result, a 235-bp DNA segment was detected in a number of DNA samples from D. silvarum and sheep blood. This 235-bp segment had an identity of 99% to a 235-bp DNA segment of ASFV and contained three single nucleotide mutations (C38T, C76T and A108C). C38T, resulting in a single amino acid mutation G66D, suggests the existence of a new ASFV strain, which is different from all reported ASFV strains in the NCBI GenBank database and the ASFV strain (GenBank: MH713612.1) reported in China in 2018. To further confirm the existence of ASFV in Dermacentor ticks, three DNA segments of ASFV were detected in D. niveus females from bovines and their first generation ticks reared in our lab. These results also proved that transovarian transmission of ASFV occurs in hard ticks. This study revealed for the first time that ASFV has a wider range of hosts (e.g. sheep and bovines) and vectors (e.g. hard ticks), beyond the well-known Suidae family and Argasidae (soft ticks). Our findings pave the way toward further studies on ASFV transmission and the development of prevention and control measures.

scholarly journals 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

1999 ◽  
Vol 80 (10) ◽  
pp. 2693-2697 ◽  
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.

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.

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

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.

scholarly journals Establishment of a Blocking ELISA Detection Method for Against African Swine Fever Virus p30 Antibody

2021 ◽  
Vol 8 ◽  
Author(s):  
Xuexiang Yu ◽  
Xianjing Zhu ◽  
Xiaoyu Chen ◽  
Dongfan Li ◽  
Qian Xu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Confidence Interval ◽  
Roc Analysis ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Diagnostic Sensitivity ◽  
Cutoff Value ◽  
Blocking Elisa

African swine fever (ASF) is a highly lethal hemorrhagic viral disease of domestic pigs caused by African swine fever virus (ASFV). A sensitive and reliable serological diagnostic assay is required, so laboratories can effectively and quickly detect ASFV infection. The p30 protein is abundantly expressed early in cells and has excellent antigenicity. Therefore, this study aimed to produce and characterize p30 monoclonal antibodies with an ultimate goal of developing a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for ASFV antibody detection. Three monoclonal antibodies against p30 protein that were expressed in E. coli were generated, and their characterizations were investigated. Furthermore, a blocking ELISA based on a monoclonal antibody was developed. To evaluate the performance of the assay, 186 sera samples (88 negative and 98 positive samples) were analyzed and a receiver-operating characteristic (ROC) analysis was applied to determine the cutoff value. Based on the ROC analysis, the area under the curve (AUC) was 0.997 (95% confidence interval: 99.2 to 100%). Besides, a diagnostic sensitivity of 97.96% (95% confidence interval: 92.82 to 99.75%) and a specificity of 98.96% (95% confidence interval: 93.83 to 99.97%) were achieved when the cutoff value was set to 38.38%. Moreover, the coefficients of inter- and intra-batches were <10%, indicating the good repeatability of the method. The maximum dilution of positive standard serum detected by this ELISA method was 1:512. The blocking ELISA was able to detect seroconversion in two out of five pigs at 10 Dpi and the p30 response increasing trend through the time course of the study (0–20 Dpi). In conclusion, the p30 mAb-based blocking ELISA developed in this study demonstrated a high repeatability with maximized diagnostic sensitivity and specificity. The assay could be a useful tool for field surveillance and epidemiological studies in swine herd.

scholarly journals Development of an Indirect ELISA to Detect African Swine Fever Virus pp62 Protein-Specific Antibodies

2022 ◽  
Vol 8 ◽  
Author(s):  
Kexin Zhong ◽  
Mengmeng Zhu ◽  
Qichao Yuan ◽  
Zhibang Deng ◽  
Simeng Feng ◽  
...  
Keyword(s):  
Indirect Elisa ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Viral Disease ◽  
Fever Virus ◽  
Serum Samples ◽  
Swine Industry ◽  
Detection Techniques ◽  
Positive Serum ◽  
Great Reactivity

African swine fever (ASF) is a highly detrimental viral disease caused by African swine fever virus (ASFV). The occurrence and prevalence of this disease have become a serious threat to the global swine industry and national economies. At present, the detection volume of African swine fever is huge, more sensitive and accurate detection techniques are needed for the market. pp62 protein, as a protein in the late stage of infection, has strong antigenicity and a high corresponding antibody titer in infected pigs. In this study, the CP530R gene was cloned into expression vector pET-28a to construct a prokaryotic expression plasmid, which was induced by IPTG to express soluble pp62 protein. Western blot analysis showed that it had great reactivity. Using the purified recombinant protein as an antigen, an indirect ELISA method for detecting ASFV antibody was established. The method was specific only to ASFV-positive serum, 1:1600 diluted positive serum could still be detected, and the coefficients of variation (CV) of the intra assay and inter assay were both <10%. It turns out that the assays had excellent specificity, sensitivity, and repeatability. This provides an accurate, rapid, and economical method for the detection of ASFV antibody in clinical pig serum samples.

scholarly journals Optimization in the expression of ASFV proteins for the development of subunit vaccines using poxviruses as delivery vectors

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jaime Lopera-Madrid ◽  
Lex G. Medina-Magües ◽  
Douglas P. Gladue ◽  
Manuel V. Borca ◽  
Jorge E. Osorio
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Expression Vectors ◽  
Complex Nature ◽  
Hemorrhagic Disease ◽  
Mrna Levels ◽  
Subunit Vaccines ◽  
Protective Antigens ◽  
Delivery Vector ◽  
Efficacious Treatment

AbstractAfrican swine fever virus (ASFV) causes a highly contagious hemorrhagic disease that affects domestic pig and Eurasian wild boar populations. To date, no safe and efficacious treatment or vaccine against ASF is available. Nevertheless, there are several reports of protection elicited by experimental vaccines based on live attenuated ASFV and some levels of protection and reduced viremia in other approaches such as DNA, adenovirus, baculovirus, and vaccinia-based vaccines. Current ASF subunit vaccine research focuses mainly on delivering protective antigens and antigen discovery within the ASFV genome. However, due to the complex nature of ASFV, expression vectors need to be optimized to improve their immunogenicity. Therefore, in the present study, we constructed several recombinant MVA vectors to evaluate the efficiency of different promoters and secretory signal sequences in the expression and immunogenicity of the p30 protein from ASFV. Overall, the natural poxvirus PrMVA13.5L promoter induced high levels of both p30 mRNA and specific anti-p30 antibodies in mice. In contrast, the synthetic PrS5E promoter and the S E/L promoter linked to a secretory signal showed lower mRNA levels and antibodies. These findings indicate that promoter selection may be as crucial as the antigen used to develop ASFV subunit vaccines using MVA as the delivery vector.

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