PRRSV Vaccine Strain-Induced Secretion of Extracellular ISG15 Stimulates Porcine Alveolar Macrophage Antiviral Response against PRRSV

Porcine reproductive and respiratory syndrome virus (PRRSV) has disrupted the global swine industry since the 1980s. PRRSV-host interactions are largely still unknown but may involve host ISG15 protein. In this study, we developed a monoclonal antibody (Mab-3D5E6) specific for swine ISG15 (sISG15) by immunizing mice with recombinant sISG15. A sandwich enzyme-linked immunosorbent assay (ELISA) incorporating this sISG15-specific Mab was developed to detect sISG15 and provided a lower limit of sISG15 detection of 200 pg/mL. ELISA results demonstrated that infection of porcine alveolar macrophages (PAMs) with low-virulence or attenuated PRRSV vaccine strains induced intracellular ISG15 expression that was independent of type I IFN production, while PAMs infection with a PRRSV vaccine strain promoted extracellular ISG15 secretion from infected PAMs. Conversely, the addition of recombinant sISG15 to PAMs mimicked natural extracellular ISG15 effects whereby sISG15 functioned as a cytokine by activating PAMs. Once activated, PAMs could inhibit PRRSV replication and resist infection with PRRSV vaccine strain TJM. In summary, a sandwich ELISA incorporating homemade anti-ISG15 Mab detected ISG15 secretion induced by PAMs infection with a PRRSV vaccine strain. Recombinant ISG15 added to cells exhibited cytokine-like activity that stimulated PAMs to assume an anti-viral state that enabled them to inhibit PRRSV replication and resist viral infection.

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The Function of the PRRSV–Host Interactions and Their Effects on Viral Replication and Propagation in Antiviral Strategies

Vaccines ◽

10.3390/vaccines9040364 ◽

2021 ◽

Vol 9 (4) ◽

pp. 364

Author(s):

Jun Ma ◽

Lulu Ma ◽

Meiting Yang ◽

Wei Wu ◽

Wenhai Feng ◽

...

Keyword(s):

Immune Response ◽

Molecular Mechanisms ◽

Immune Escape ◽

Rna Virus ◽

Economic Losses ◽

Respiratory Syndrome Virus ◽

Swine Industry ◽

Live Virus ◽

Virus Challenge ◽

Host Interactions

Porcine reproductive and respiratory syndrome virus (PRRSV) affects the global swine industry and causes disastrous economic losses each year. The genome of PRRSV is an enveloped single-stranded positive-sense RNA of approximately 15 kb. The PRRSV replicates primarily in alveolar macrophages of pig lungs and lymphatic organs and causes reproductive problems in sows and respiratory symptoms in piglets. To date, studies on how PRRSV survives in the host, the host immune response against viral infections, and pathogenesis, have been reported. PRRSV vaccines have been developed, including inactive virus, modified live virus, attenuated live vaccine, DNA vaccine, and immune adjuvant vaccines. However, there are certain problems with the durability and effectiveness of the licensed vaccines. Moreover, the high variability and fast-evolving populations of this RNA virus challenge the design of PRRSV vaccines, and thus effective vaccines against PRRSV have not been developed successfully. As is well known, viruses interact with the host to escape the host’s immune response and then replicate and propagate in the host, which is the key to virus survival. Here, we review the complex network and the mechanism of PRRSV–host interactions in the processes of virus infection. It is critical to develop novel antiviral strategies against PRRSV by studying these host–virus interactions and structures to better understand the molecular mechanisms of PRRSV immune escape.

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Porcine reproductive and respiratory syndrome virus infection upregulates negative immune regulators and T-cell exhaustion markers

Journal of Virology ◽

10.1128/jvi.01052-21 ◽

2021 ◽

Author(s):

Jayeshbhai Chaudhari ◽

Chia-Sin Liew ◽

Jean-Jack M. Riethoven ◽

Sarah Sillman ◽

Hiep L. X. Vu

Keyword(s):

T Cell ◽

Fluorescent Protein ◽

Ex Vivo ◽

Acute Stage ◽

Respiratory Syndrome Virus ◽

Porcine Alveolar Macrophage ◽

Host Immune System ◽

T Cell Exhaustion ◽

Swine Industry ◽

Cell Exhaustion

Porcine alveolar macrophage (PAM) is one of the primary cellular targets for PRRSV, but less than 2% of PAMs are infected with the virus during the acute stage of infection. To comparatively analyze the host transcriptional response between PRRSV-infected PAMs and bystanders PAMs that remained uninfected but were exposed to the inflammatory milieu of an infected lung, pigs were infected with a PRRSV strain expressing green fluorescent protein (PRRSV-GFP) and GFP + (PRRSV infected) and GFP – (bystander) cells were sorted for RNA-sequencing (RNA-seq). Approximately 4.2% of RNA reads from GFP + and 0.06% reads from GFP – PAMs mapped to the PRRSV genome, indicating that PRRSV-infected PAMs were effectively separated from bystander PAMs. Further analysis revealed that inflammatory cytokines, interferon-stimulated genes, and antiviral genes were highly upregulated in GFP + as compared to GFP – PAMs. Importantly, negative immune regulators including NF-κB inhibitors (NFKBIA, NFKBID, NFKBIZ, and TNFAIP3), and T-cell exhaustion markers (PD-L1, PD-L2, IL10, IDO1, and TGFB2) were highly upregulated in GFP + cells as compared to GFP – cells. By using in situ hybridization assay, RNA transcripts of TNF and NF-κB inhibitors were detected in PRRSV-infected PAMs cultured ex vivo and lung sections of PRRSV-infected pigs during the acute stage of infection. Collectively, the results suggest that PRRSV infection upregulates expression of negative immune regulators and T-cell exhaustion markers in PAMs to modulate the host immune response. Our findings provide further insight into PRRSV immunopathogenesis. Importance PRRSV is widespread in many swine producing countries, causing substantial economic loses to the swine industry. PAM is considered the primary target for PRRSV replication in pigs. However, less than 2% of PAM from an acutely infected pigs are infected with the virus. In the present study, we utilized a PRRSV-GFP strain to infect pigs and sorted infected- and bystander- PAMs from the pigs during the acute stage of infection for transcriptome analysis. PRRSV infected PAMs showed a distinctive gene expression profile and contained many uniquely activated pathways compared to bystander PAMs. Interestingly, upregulated expression of and NF-κB signaling inhibitors and T-cell exhaustion molecules were observed in PRRSV-infected PAMs. Our findings provide additional knowledge on the mechanisms that PRRSV employs to modulate the host immune system.

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Porcine 2′,5′-oligoadenylate synthetase-like protein inhibits replication of porcine reproductive and respiratory syndrome virus

10.21203/rs.2.18823/v1 ◽

2019 ◽

Author(s):

huawei li ◽

ruining wang ◽

wenjia wang ◽

yinfeng kang ◽

mengmeng zhao

Keyword(s):

Alveolar Macrophages ◽

Respiratory Syndrome Virus ◽

Type I ◽

Oligoadenylate Synthetase ◽

Swine Industry ◽

Exogenous Expression ◽

Polymerase Chain ◽

Interfering Rna ◽

Inducible Gene

Abstract Background : Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious pathogen that causes $664 million losses per year to the swine industry. There are few useful vaccines that can provide protection against PRRSV infection. 2′, 5′-oligoadenylate synthetase-like protein (OASL) has antiviral activity, this has not been shown for PRRSV and the mechanism is unknown. Methods : Expression of OASL in porcine alveolar macrophages induced by interferon (IFN)-b stimulation and PRRSV infection was examined by real-time polymerase chain reaction. Exogenous expression and knockdown of OASL were used to determine the role of OASL in the PRRSV replication cycle. The type I IFN signaling pathway was evaluated after OASL overexpression. Results : In this study, we found that the expression of OASL in porcine alveolar macrophages was significantly increased by IFN-b stimulation and PRRSV infection. Porcine-OASL-specific small interfering RNA (siRNA) promoted PRRSV replication, whereas exogenous expression of porcine OASL inhibited replication of the virus. The anti-PRRSV activity of porcine OASL was lost after knockdown of retinoic acid-inducible gene I ( DDX58 , also known as RIG-I ). Conclusions : Porcine OASL suppresses PRRSV replication.

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Monoclonal antibodies specific for prothrombin fragment 1.2 and their use in a quantitative enzyme-linked immunosorbent assay

Clinical Chemistry ◽

10.1093/clinchem/39.4.583 ◽

1993 ◽

Vol 39 (4) ◽

pp. 583-591 ◽

Cited By ~ 13

Author(s):

M J Hursting ◽

B T Butman ◽

J P Steiner ◽

B M Moore ◽

M C Plank ◽

...

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Human Plasma ◽

Sandwich Elisa ◽

Peptide Sequence ◽

Carboxyl Terminus ◽

Enzyme Linked Immunosorbent Assay ◽

Thrombotic Risk ◽

Prothrombin Fragment ◽

Amino Terminal

Abstract Prothrombin fragment 1.2 (F1.2) is an activation peptide generated during a critical event of blood coagulation, the conversion of prothrombin to thrombin. As a marker of thrombin generation, F1.2 has clinical potential in assessing thrombotic risk and monitoring anticoagulant therapy. In developing a highly specific, monoclonal antibody-based immunoassay of human plasma F1.2, we generated six murine anti-F1.2 monoclonal antibodies, using as immunogen a synthetic peptide (sequence: CGSD-RAIEGR) similar to the unique carboxyl terminus of F1.2. Each antibody bound F1.2 but not prothrombin. Epitope mapping studies with one antibody (5-3B) showed that optimum binding required six to eight amino acids plus a terminal arginine to emulate the F1.2 carboxyl terminus. A quantitative sandwich ELISA for human plasma F1.2 was configured with monoclonal antibody 5-3B as the capture antibody and peroxidase-labeled polyclonal antibodies to the F1.2 amino-terminal region as detector antibodies. Calibrators were prepared by adding purified F1.2, 0-10 nmol/L, to F1.2-depleted plasma. Assay characteristics included the following: mean (+/- SD) analytical recovery of 98% +/- 13%; no interference from lipemia, hemolysis, icterus, or thrombolytic agents; 0.08 nmol/L sensitivity; and mean intra- and interassay imprecision (three lots) < 12% at both low and high concentrations of F1.2.

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Sandwich enzyme immunoassay of osteocalcin in serum with use of an antibody against human osteocalcin

Clinical Chemistry ◽

10.1093/clinchem/39.6.942 ◽

1993 ◽

Vol 39 (6) ◽

pp. 942-947 ◽

Cited By ~ 19

Author(s):

D A Monaghan ◽

M J Power ◽

P F Fottrell

Keyword(s):

Monoclonal Antibody ◽

Enzyme Immunoassay ◽

Solid Phase ◽

Sandwich Elisa ◽

Normal Subjects ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Standard Curve ◽

Microtiter Plates ◽

Human Osteocalcin

Abstract We have developed and thoroughly validated a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) on microtiter plates for osteocalcin in human serum with use of an antibody raised against human osteocalcin. We used a monoclonal antibody against bovine osteocalcin as the capture antibody; the second antibody was a polyclonal antibody against human osteocalcin. The amount of bound second antibody was determined with use of swine anti-rabbit antibody labeled with horseradish peroxidase. We demonstrated independence of volume and determined the recovery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 micrograms/L and the midpoint of the standard curve ranged from 14 to 17 micrograms/L. The intraassay CV was < or = 8% in the range 2.7-52 micrograms/L; the interassay CV was usually < or = 15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently > 90%. Values for osteocalcin measured in serum from 44 normal subjects were similar to those obtained with a competitive enzyme immunoassay (EIA) that used a monoclonal antibody against bovine osteocalcin. There was a good correlation between the two assays [r2 = 0.877, slope and intercept (+/- SE) = 0.88(+/- 0.051) and 0.316(+/- 0.523), respectively]. The range and mean (+/- SD) for the sandwich ELISA and the competitive EIA were 1.7-18.1 micrograms/L [8.7(+/- 4.4) micrograms/L] and 1.9-22.8 micrograms/L [9.1(+/- 4.4) micrograms/L], respectively.

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Development of a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay for detecting canine distemper virus

Applied Microbiology and Biotechnology ◽

10.1007/s00253-020-10997-y ◽

2020 ◽

Vol 104 (24) ◽

pp. 10725-10735

Author(s):

Yuan Zhang ◽

Gang Xu ◽

Lu Zhang ◽

Jiakai Zhao ◽

Pinpin Ji ◽

...

Keyword(s):

Monoclonal Antibody ◽

Immunosorbent Assay ◽

Colloidal Gold ◽

Canine Distemper Virus ◽

Sandwich Elisa ◽

Enzyme Linked Immunosorbent Assay ◽

Test Results ◽

Canine Distemper ◽

Serum Samples ◽

Ideal Method

Abstract Canine distemper virus (CDV) infection causes mass mortality in diverse carnivore species. For effective virus surveillance, rapid and sensitive assays are needed to detect CDV in field samples. In this study, after BABL/c mice were immunized with recombinant CDV-fusion (F) protein, monoclonal antibodies (mAbs) against recombinant CDV-F protein (designated 1A5, 1A6, and 7D5) were produced using traditional hybridoma cell technology. Next, capture antibody (1A6, 800 ng/well) and horseradish peroxidase (HRP)–conjugated detection antibody (HRP-7D5, 1:100, 500 ng/well) were used in a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay (ELISA) for CDV detection after optimization of both mAb amounts per well using a checkerboard titration test. Based on sandwich ELISA test results for 120 known CDV-negative samples, the cutoff value for a positive result was set to an OD450 nm value ≥ 0.196. As compared with test results obtained from commercial immune colloidal gold test strips, the low limits of detection for the two assays were revealed to be 100 TCID50 per 100 μL. In addition, the sandwich ELISA agreed 100% and 96.4% with commercial immune colloidal gold test strips when testing serum and stool samples. The sandwich ELISA assay provided statistically similar CDV detection. Thus, the sandwich ELISA developed here to detect CDV in fecal and serum samples provided good sensitivity, high specificity, and good reproducibility and should serve as an ideal method for large-scale surveillance of CDV infections in carnivores. Key points • Three CDV mAbs that recognized different epitopes and bound to virion were generated. • The sandwich ELISA based mAbs to detect CDV in fecal and serum samples was developed. • The sandwich ELISA is an ideal method for detecting CDV infections in the field.

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Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

Clinical and Vaccine Immunology ◽

10.1128/cvi.00234-12 ◽

2012 ◽

Vol 19 (9) ◽

pp. 1480-1486 ◽

Cited By ~ 8

Author(s):

Meng Ge ◽

Wei Luo ◽

Daliang Jiang ◽

Runcheng Li ◽

Wenwei Zhao ◽

...

Keyword(s):

Capsid Protein ◽

Immunosorbent Assay ◽

Sandwich Elisa ◽

Test Procedure ◽

Porcine Circovirus ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Swine Industry ◽

Elisa Kit ◽

Porcine Circovirus 2

ABSTRACTA double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 91.62% compared with IIF and 94.38, 95.28, and 94.67% compared with the commercial ELISA kit. Assay of 12 PCV-free pigs over a 5-week period produced only PCV2-negative titers by all 3 methods. These results and the seroprofiles of 4 pig farms obtained by both the commercial ELISA kit and the double-antigen sandwich ELISA indicate that the sandwich ELISA is a reliable method for detection of antibodies to PCV2. Additionally, the method described here permits the use of undiluted test serum samples simultaneously loaded with horseradish peroxidase (HRP)-conjugated antigen into the test well, and the complete test procedure can be performed in less than 90 min. This double-antigen sandwich ELISA should be a useful tool to aid swine industry professionals in deciding the intervention strategies for the control of PCV2-associated diseases.

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Evasion of Antiviral Innate Immunity by Porcine Reproductive and Respiratory Syndrome Virus

Frontiers in Microbiology ◽

10.3389/fmicb.2021.693799 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tong-Yun Wang ◽

Ming-Xia Sun ◽

Hong-Liang Zhang ◽

Gang Wang ◽

Guoqing Zhan ◽

...

Keyword(s):

Innate Immunity ◽

Immune Responses ◽

Viral Infections ◽

Respiratory Syndrome Virus ◽

Type I ◽

Swine Industry ◽

Interferon Stimulated Genes ◽

Β Receptor ◽

Underlying Mechanisms ◽

Antiviral Innate Immunity

Innate immunity is the front line for antiviral immune responses and bridges adaptive immunity against viral infections. However, various viruses have evolved many strategies to evade host innate immunity. A typical virus is the porcine reproductive and respiratory syndrome virus (PRRSV), one of the most globally devastating viruses threatening the swine industry worldwide. PRRSV engages several strategies to evade the porcine innate immune responses. This review focus on the underlying mechanisms employed by PRRSV to evade pattern recognition receptors signaling pathways, type I interferon (IFN-α/β) receptor (IFNAR)-JAK-STAT signaling pathway, and interferon-stimulated genes. Deciphering the antiviral immune evasion mechanisms by PRRSV will enhance our understanding of PRRSV’s pathogenesis and help us to develop more effective methods to control and eliminate PRRSV.

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Immunodiagnosis of paramphistomosis using monoclonal antibody-based sandwich ELISA for detection ofParamphistomum gracilecirculating 16 kDa antigen

Parasitology ◽

10.1017/s003118201600264x ◽

2017 ◽

Vol 144 (7) ◽

pp. 899-903 ◽

Cited By ~ 4

Author(s):

PANAT ANURACPREEDA ◽

KULLANID TEPSUPORNKUL ◽

RUNGLAWAN CHAWENGKIRTTIKUL

Keyword(s):

Monoclonal Antibody ◽

Detection Method ◽

Sandwich Elisa ◽

Enzyme Linked Immunosorbent Assay ◽

Microtitre Plate ◽

Serum Samples ◽

Fecal Samples ◽

Lower Detection Limit ◽

Lower Detection ◽

Sensitivity Specificity

SUMMARYIn this study, we have produced a monoclonal antibody (MoAb) against 16 kDa antigen ofParamphistomum gracile(16 kDaAgPg), and developed an accurate sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for the detection of circulating 16 kDaAg in the serum and fecal samples from cattle naturally infected withP. gracile. MoAb 1D10 was immobilized on a microtitre plate, and the antigen in the samples was captured and detected with biotinylated rabbit anti-16 kDaAgPg antibody. The lower detection limit of sandwich ELISA was 3·5 pg mL−1, and no cross-reaction with other parasite antigens was evaluated. The reliability of the assay was examined using the serum and fecal samples from cattle naturally infected withP. gracile, Fasciola gigantica, Moniezia benedeni, Trichurissp.,Strongyloidessp., strongylids and non-infected animals. The sandwich ELISA showed the sensitivity, specificity and accuracy at 98·33, 100 and 99·55% (serum samples), and 96·67, 100 and 99·09% (fecal samples). Therefore, this detection method is a rapid and excellent potential assay for the accurate diagnosis of paramphistomosis.

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Immunodiagnostic monoclonal antibody-based sandwich ELISA of fasciolosis by detection ofFasciola giganticacirculating fatty acid binding protein

Parasitology ◽

10.1017/s0031182016001104 ◽

2016 ◽

Vol 143 (11) ◽

pp. 1369-1381 ◽

Cited By ~ 6

Author(s):

PANAT ANURACPREEDA ◽

RUNGLAWAN CHAWENGKIRTTIKUL ◽

PRASERT SOBHON

Keyword(s):

Monoclonal Antibody ◽

Fatty Acid ◽

Fatty Acid Binding Protein ◽

Binding Protein ◽

Sandwich Elisa ◽

Parasite Burden ◽

Enzyme Linked Immunosorbent Assay ◽

Fatty Acid Binding ◽

Acid Binding Protein ◽

Low Sensitivity

SUMMARYUp to now, parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Hence, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In the present study, a monoclonal antibody (MoAb) against recombinantFasciola giganticafatty acid binding protein (rFgFABP) has been produced. As well, a reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) has been developed for the detection of circulating FABP in the sera of mice experimentally and cattle naturally infected withF. gigantica. MoAb 3A3 and biotinylated rabbit anti-recombinant FABP antibody were selected due to their high reactivities and specificities. The lower detection limit of sandwich ELISA was 5 pg mL−1, and no cross-reaction with other parasite antigens was observed. This assay could detectF. giganticainfection from day 1 post infection. In experimental mice, the sensitivity, specificity and accuracy of this assay were 93·3, 100 and 98·2%, while in natural cattle they were 96·7, 100 and 99·1%. Hence, this sandwich ELISA method showed high efficiencies and precisions for diagnosis of fasciolosis byF. gigantica.

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