Application of a monoclonal antibody recognizing protein p30 to detect African swine fever virus-infected cells in peripheral blood

1995 ◽  
Vol 55 (3) ◽  
pp. 339-345 ◽  
Author(s):  
F. Ramiro-Ibáñez ◽  
J.M. Escribano ◽  
C. Alonso
Keyword(s):  
Monoclonal Antibody ◽  
Peripheral Blood ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Infected Cells

A comparison of two indirect immune electron microscopy procedures using monoclonal antibody to localize specific African swine fever virus (ASFV) proteins in infected cells and virus

1983 ◽  
Vol 41 ◽  
pp. 800-801
Author(s):  
G. J. Letchworth III ◽  
T. C. Whyard ◽  
S. H. Wool
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Grid Method ◽  
Viral Proteins ◽  
Fever Virus ◽  
Immune Electron Microscopy ◽  
Infected Cells

The primary purpose of this study was to devise a procedure in which a large number of monoclonal antibodies could be tested against several African swine fever virus (ASFV) preparations (virus, infected cells). The procedure had to be rapid, use a small sample and provide for some TEM.Monoclonal antibodies were produced by standard methods using myeloma cells and lymphocytes from mice immunized against ASF viral proteins. Antibodies were characterized by immunoprecipitation of radio-labelled viral proteins (Fig 1).The viral preparations used in this study were either infected macrophages, culture fluid supernatant from ASFV-infected macrophages or red blood cells from infected pigs. Each of the three viral preparations was tested by two indirect immune electron microscopy (IEM) methods; a grid method and a test tube method. In the grid method, formvar coated grids were floated on a viral preparation then for one hour each on the mouse monoclonal antibody and finally on ferritin tagged sheep anti-mouse IgG with washes between each step.

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

2006 ◽  
Vol 80 (7) ◽  
pp. 3157-3166 ◽  
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.

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.

Detection of African swine fever virus in cell culture and wild boar tissues using a commercially available monoclonal antibody

2020 ◽  
Vol 282 ◽  
pp. 113886
Author(s):  
Levente Szeredi ◽  
Erika Bakcsa ◽  
Zoltán Zádori ◽  
István Mészáros ◽  
Ferenc Olasz ◽  
...  
Keyword(s):  
Cell Culture ◽  
Monoclonal Antibody ◽  
Wild Boar ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus

scholarly journals African swine fever virus A238L inhibitor of NF-κB and of calcineurin phosphatase is imported actively into the nucleus and exported by a CRM1-mediated pathway

2007 ◽  
Vol 88 (2) ◽  
pp. 411-419 ◽  
Author(s):  
Rhiannon N. Silk ◽  
Gavin C. Bowick ◽  
Charles C. Abrams ◽  
Linda K. Dixon
Keyword(s):  
Molecular Mass ◽  
Nuclear Import ◽  
Nuclear Export ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Infected Cells ◽  
Calcineurin Phosphatase

This study examined nuclear and cytoplasmic shuttling of the African swine fever virus (ASFV) A238L protein, which is an inhibitor of NF-κB and of calcineurin phosphatase. The results showed that the protein was present in both the nucleus and the cytoplasm in ASFV-infected cells and that the higher molecular mass 32 kDa form of the A238L protein was the predominant nuclear form, which accumulated later in infection. In contrast, both the 28 and 32 kDa forms of the A238L protein were present in the cytoplasm. The A238L protein was actively imported into the nucleus and exported by a CRM1-mediated pathway, although a pool of the protein remained in the cytoplasm and did not enter the nucleus. By using a recombinant ASFV from which the A238L gene had been deleted, it was shown that expression of A238L did not inhibit nuclear import of the NF-κB p50 or p65 subunit and did not inhibit nuclear export of p65 by a CRM1-mediated pathway. The results were consistent with a model in which A238L functions within both the nucleus and the cytoplasm.

scholarly journals Identification of African Swine Fever Virus Transcription within Peripheral Blood Mononuclear Cells of Acutely Infected Pigs

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2333
Author(s):  
Ann Sofie Olesen ◽  
Miyako Kodama ◽  
Louise Lohse ◽  
Francesc Accensi ◽  
Thomas Bruun Rasmussen ◽  
...  
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Peripheral Blood Mononuclear ◽  
Virus Transcription ◽  
Blood Mononuclear Cells ◽  
Low Levels

African swine fever virus (ASFV) has become widespread in Europe, Asia and elsewhere, thereby causing extensive economic losses. The viral genome includes nearly 200 genes, but their expression within infected pigs has not been well characterized previously. In this study, four pigs were infected with a genotype II strain (ASFV POL/2015/Podlaskie); blood samples were collected before inoculation and at both 3 and 6 days later. During this period, a range of clinical signs of infection became apparent in the pigs. From the blood, peripheral blood mononuclear cells (PBMCs) were isolated. The transcription of the ASFV genes was determined using RNAseq on poly(A)+ mRNAs isolated from these cells. Only very low levels of virus transcription were detected in the PBMCs at 3 days post-inoculation (dpi) but, at 6 dpi, extensive transcription was apparent. This was co-incident with a large increase in the level of ASFV DNA within these cells. The pattern of the virus gene expression was very reproducible between the individual pigs. Many highly expressed genes have undefined roles. Surprisingly, some genes with key roles in virus replication were expressed at only low levels. As the functions of individual genes are identified, information about their expression becomes important for understanding their contribution to virus biology.

scholarly journals Modulation of p53 Cellular Function and Cell Death by African Swine Fever Virus

2004 ◽  
Vol 78 (13) ◽  
pp. 7165-7174 ◽  
Author(s):  
Aitor G. Granja ◽  
María L. Nogal ◽  
Carolina Hurtado ◽  
José Salas ◽  
María L. Salas ◽  
...  
Keyword(s):  
Caspase 3 ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Tumor Suppressor P53 ◽  
Infected Cells ◽  
Cellular Dna ◽  
P53 Inactivation ◽  
Infectious Cycle ◽  
Active Caspase

ABSTRACT Modulation of the activity of tumor suppressor p53 is a key event in the replication of many viruses. We have studied the function of p53 in African swine fever virus (ASFV) infection by determining the expression and activity of this transcription factor in infected cells. p53 levels are increased at early times of infection and are maintained throughout the infectious cycle. The protein is transcriptionally active, stabilized by phosphorylation, and localized in the nucleus. p53 induces the expression of p21 and Mdm2. Strikingly, these two proteins are located at the cytoplasmic virus factories. The retention of Mdm2 at the factory may represent a viral mechanism to prevent p53 inactivation by the protein. The expression of apoptotic proteins, such as Bax or active caspase-3, is also increased following ASFV infection, although the increase in caspase-3 does not appear to be, at least exclusively, p53 dependent. Bax probably plays a role in the induction of apoptosis in the infected cells, as suggested by the release of cytochrome c from the mitochondria. The significance of p21 induction and localization is discussed in relation to the shutoff of cellular DNA synthesis that is observed in ASFV-infected cells.

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