The recombinant Erns and truncated E2-based indirect enzyme-linked immunosorbent assays to distinguishably test specific antibodies against classical swine fever virus and bovine viral diarrhea virus

Abstract

Background Classical swine fever (CSF) virus is the causative agent of an economically important, highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV). BVDV infection in pigs can mimic CSF clinical signs, which cause difficulty in differentiation. Serological test for detection of virus specific antibodies is a valuable tool for diagnosis and surveillance of CSFV and BVDV infections in animals. The aim of this study was to develop the CSFV Erns and BVDV tE2 -based ELISAs to distinguishably test specific antibodies against CSFV and BVDV. Methods The CSFV Erns and truncated E2 (tE2, residues 690–865) of BVDV were expressed in E. coli and purified by Ni–NTA affinity chromatography, respectively. Employing Erns or tE2 protein as diagnostic antigen, indirect ELISAs were developed to distinguishably test specific antibodies against CSFV and BVDV. The specificity and sensitivity of ELISAs were evaluated using a panel of virus specific sera of pigs, immunized rabbits and immunized mice. A total 150 clinical serum samples from farm pigs were measured by the developed ELISAs and compared with virus neutralizing test (VNT). Results Indirect ELISA was established based on recombinant CSFV Erns or BVDV tE2 protein, respectively. No serological cross-reaction between antibodies against CSFV and BVDV was observed in sera of immunized rabbits, immunized mice or farm pigs by detections of the Erns and tE2 -based ELISAs. Compared to VNT, the CSFV Erns -based ELISA displayed a high sensitivity (93.3%), specificity (92.0%) and agreement rate (92.7%), and the sensitivity, specificity and agreement rate of BVDV tE2 -based ELISA was 92.3%, 95.2% and 94.7%, respectively. Conclusion The newly developed ELISAs are highly specific and sensitive and would be valuable tools for serological diagnosis for CSFV and BVDV infections.

Development of a dual immunochromatographic test strip to detect E2 and Erns antibodies against classical swine fever

“…In protein manipulation, previous studies have indicated that plant-derived transient expression enhances high protein expression and yield, immunogenicity in animal experiments, and high binding affinity for antibody detection absent in certain conventional expression systems ( Sohn et al, 2018 ; Yamamoto et al, 2018 ; Xu et al, 2022 ). Compared to the transgenic approach for CSFV E2 protein expression using baculovirus ( Wei et al, 2021 ; Bai et al, 2022 ), mammalian cells ( Ji et al, 2018 ; Chen et al, 2023 ), or the E. coli system ( Li et al, 2012 ; Yi et al, 2022 ), the CSFV E2-expressing transgenic plant-derived system was preferable in terms of a large amount and stable expression of the target protein and inexpensiveness, as in recent studies ( Sohn et al, 2018 ; Laughlin et al, 2019 ; Park et al, 2019 , 2020 ; Xu et al, 2022 ). This approach facilitated the purification of recombinant E2 from N. benthamiana extracts.…”

Virus-like particles vaccines based on glycoprotein E0 and E2 of bovine viral diarrhea virus induce Humoral responses

“…SignalP 4.1 server and TMHMM Serverv.2.0 software analysis showed that BVDV-LC E0 had no signal peptide and transmembrane domain, and BVDV-LC E2 and XJ-04 E2 proteins had no signal peptide, but there were transmembrane regions at 340–362 and 343–365 amino acids, respectively. Some studies have also found that optimizing codon usage preference can enhance the expression and formation of VLPs ( Wang et al, 2020 ; Yi et al, 2022 ). Therefore, the E0 + E2 and E2 + E2 fusion genes were synthesized according to the codon usage preference of insect cells and linked to pMD19-T vector, and then linked to pFastBac1 vector after double enzyme digestion.…”

“…Therefore, we next evaluated the immunogenicity of VLPs in BALB/c mice and their neutralization potential against BVDV NADL strain. VNT is the gold standard for serological diagnosis of BVDV infection, and it is also used as a reference efficacy test for commercial vaccines ( Chimeno et al, 2007 ; Yi et al, 2022 ). The results of VNT showed that the neutralizing antibody levels of mice immunized with vaccines were significantly higher compared with control ( p < 0.0001; 3 and 5 weeks), and even the neutralizing antibody levels of mice immunized with VLPs E0 + E2 or E2 + E2 were significantly higher compared with commercial vaccine ( p < 0.01; 3 or 5 weeks; Figure 3B ).…”

“…Virus neutralization test (VNT) is the gold standard for serological diagnosis of BVDV and classical swine fever virus (CSFV) infection, and it is also used as a reference efficacy test for commercial vaccines ( Chimeno et al, 2007 ; Yi et al, 2022 ). VNT as previously reported ( Hoppe et al, 2019 ; Wang et al, 2021 ), the serum samples were heat-inactivated at 56°C for 30 min in a water bath and serially diluted twofold (2 −1 –2 −8 ) in DMEM medium, and then an equal volume of DMEM-diluted BVDV virus containing 100 TCID 50 was added and samples were incubated at 37°C for 2 h. Then the mixtures were removed and cultured with 1% FBS for 5–7 days (37°C, 5% CO 2 ), and the cytopathic effects were observed.…”

The Establishment and Application of Indirect 3AB-ELISA for the Detection of Antibodies against Senecavirus A

“…Test results were evaluated using an optical microscope. Titer was read as the highest dilution of serum, resulting in 50% CPE reduction [31]. Sera with neutralizing titers > 1/64 were considered positive.…”