A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus

Abstract

Recently, porcine deltacoronavirus (PDCoV) has been proven to be associated with enteric disease in piglets. Diagnostic tools for serological surveys of PDCoV remain in the developmental stage when compared with those for other porcine coronaviruses. In our study, an indirect enzyme-linked immunosorbent assay (ELISA) (rPDCoV-N-ELISA) was developed to detect antibodies against PDCoV using a histidine-tagged recombinant nucleocapsid (N) protein as an antigen. The rPDCoV-N-ELISA did not cross-react with antisera against porcine epidemic diarrhea virus, swine transmissible gastroenteritis virus, porcine group A rotavirus, classical swine fever virus, porcine circovirus-2, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus; the receiver operating characteristic (ROC) curve analysis revealed 100% sensitivity and 90.4% specificity of the rPDCoV-N-ELISA based on samples of known status (n=62). Analyses of field samples (n=319) using the rPDCoV-N-ELISA indicated that 11.59% of samples were positive for antibodies against PDCoV. These data demonstrated that the rPDCoV-N-ELISA can be used for epidemiological investigations of PDCoV and that PDCoV had a low serum prevalence in pig population in Heilongjiang province, northeast China.

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

“…The detection results showed that 11% of samples were positive for antibodies against PDCoV. The total positive rate of the PDCoV antibodies in samples is similar to that reported in Heilongjiang province (11.6%) from January 2014 to June 2015 (Su et al, 2015). When compared with samples from farms with occurrence of diarrhea, the positive rate in Hebei province (11%) was much lower than that in Heilongjiang province (27.5%) (Su et al, 2015).…”

“…No crossreactivity was detected by virus neutralization, indirect immunofluorescence, and immunostaining assays using pig hyperimmune antisera to PEDV or PDCoV in that study (Ma et al, 2016) and the PEDV whole virus-based ELISA and the S1 protein-based ELISA showed no cross-reaction with pig antisera against PDCoV, PRCV or TGEV Purdue strain (Chen et al, 2016). While the S protein gene has a high degree of variability in the members of coronaviruses (Su et al, 2015), which could lead to decreased sensitivity, N protein gene is conserved, which could lead to cross-reaction to porcine coronaviruses (Ma et al, 2016).…”

“…ELISA is suitable for testing a large number of samples (Zhang, 2016). A recombinant PDCoV S1 polypeptide-based ELISA and a recombinant PDCoV N protein based ELISA have been developed to detect PDCoV IgG antibodies (Thachil et al, 2015;Su et al, 2015). Neither ELISA methods crossreacted with antisera against PEDV, TGEV and other pig pathogens and these results were supported by other research studies using indirect immunofluorescence assays and immunohistochemical staining assays Ma et al, 2015).…”

“…Considering that there is possibility that antibodies produced by PDCoV infection may cross-react to other porcine coronaviruses antigen, and that it may affect the validation of the developed ELISA, serum samples used to establish the cutoff value were negative for antibodies against TGEV, PEDV and PRCV. The ROC curve assay showed that the relative sensitivity and specificity of the PDCoV-rM ELISA were 90.6% and 93.3%, respectively, indicating a potential diagnostic application compared to previously developed ELISAs (Thachil et al, 2015;Su et al, 2015).…”

Isolation, genomic characterization, and pathogenicity of a Chinese porcine deltacoronavirus strain CHN-HN-2014

“…With increased concern about this newly emerging enteric disease, reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, nested RT-PCR, and singleplex RT insulated isothermal PCR methods have been developed to detect PDCoV in clinical samples Wang et al, 2014a;Song et al, 2015;Zhang et al, 2015Zhang et al, , 2016. Enzyme-linked immunosorbent assays for PDCoV-specific serum antibodies, based on the PDCoV S1 or nucleocapsid protein, have also been used to determine the prevalence of anti-PDCoV immunoglobulin G (IgG) in pig serum (Su et al, 2015;Thachil et al, 2015).…”