Development and application of a recombinant M protein-based indirect ELISA for the detection of porcine deltacoronavirus IgG antibodies

Abstract

Porcine deltacoronavirus (PDCoV) is a recently identified coronavirus in the genus Deltacoronavirus that can cause enteric disease including diarrhea, vomiting, dehydration and mortality in neonatal piglets. Serological assays to detect anti-PDCoV antibodies are presently limited to certain laboratories and geographic regions. In this study, a recombinant M protein-based indirect enzyme-linked immunosorbent assay (PDCoV-rM ELISA) was developed and utilized to determine the prevalence of anti-PDCoV IgG in Hebei province. The PDCoV-rM ELISA showed no cross-reaction with antisera against transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PRV), porcine circovirus 2 (PCV2), classical swine fever virus (CSFV) or porcine reproductive and respiratory syndrome virus (PRRSV). The diagnostic sensitivity was 90.6% and the diagnostic specificity was 93.3%. A total of 871 serum samples collected in Hebei from January 2015 to October 2016 were checked for presence of antibodies against PDCoV using the novel PDCoV-rM ELISA. Anti-PDCoV IgG antibodies were detected in 11% (96/871) of the samples and in 25% (10/40) of the investigated farms. The data suggest that PDCoV has a low seroprevalence in pig population in Hebei province, China.

Identification of the immunodominant neutralizing regions in the spike glycoprotein of porcine deltacoronavirus

“…The recombinant plasmids were separately transformed into Transetta (DE3) cells, protein expression was induced with 0.8 mM IPTG for 3-6 h. Protein expression was analyzed by SDS-PAGE. The recombinant proteins were purified as previous described (Luo et al, 2017). The concentration of the purified proteins was determined using an enhanced BCA protein assay kit (Beyotime, China).…”

“…Rabbits were inoculated with the purified recombinant proteins (NTD, CTD, or S2) for production of polyclonal antibodies, as previously described with some modifications (Luo et al, 2017). Briefly, rabbits were inoculated with 1 mg/rabbit of recombinant protein in Montanide™ Gel 01 PR adjuvant (SEPPIC, France), then boosted three times with the same immunogen and adjuvant at 2-week intervals.…”

Development of a multiplex RT‐PCR for the detection of major diarrhoeal viruses in pig herds in China

“…Although several standard detection methods, for example virus isolation, virus neutralization tests, and indirect immunofluorescence assay, are available for the detection of viruses, these techniques are time‐consuming and not suitable for detecting large‐scale samples. Currently, polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay (ELISA) methods for the detection of these viruses have been reported (Luo et al, ; Ma et al, ; Zhu, Wang, Cui, & Cui, ). Due to the high pathogenicity of these viruses to suckling piglets, which have immature immune systems and few antibodies, ELISA is inefficient for detecting these viruses compared to PCR.…”

Point‐of‐care diagnostic assay for rapid detection of porcine deltacoronavirus using the recombinase polymerase amplification method

“…At present, several diagnostic methods for rapid detection of PDCoV have been developed. Serological assays such as virus neutralization test, indirect fluorescent antibody assay and enzyme linked immunosorbent assay were reported to detect PDCoV‐specific antibodies (Hu et al., ; Luo et al., ; Zhang, ). However, the serological methods were not suitable for the detection of antibodies during the early stage of virus infection as antibody generation required a few days.…”