Specific recombinant proteins of porcine epidemic diarrhea virus are immunogenic, revealing their potential use as diagnostic markers

Abstract

A B S T R A C TGiven the highly contagious and acute nature of porcine epidemic diarrhea (PED), especially in piglets, there is an urgent need for the development of rapid and sensitive diagnostic assays. The diagnostic potentials of specific porcine epidemic diarrhea virus (PEDV) accessory and nonstructural proteins, if any, have not yet been investigated. In order to determine and compare which of the viral proteins may be useful as diagnostic antigens, whole virus (WV) particles and a panel of structural and nonstructural PEDV proteins [spike subunit 1 (S1), the C-terminal part of ORF3 (ORF3C), envelope (E), nonstructural protein 1 (Nsp1), Nsp2, Ac (acidic domain of Nsp3), and ADRP (ADP-ribose-1-monophosphatase domain of Nsp3), expressed individually in bacterial and/or mammalian cells] were tested for reactivity with sera from PEDV-infected pigs by ELISA and/or western blot analysis. According to western blots, serum antibody interactions with the S1 protein were relatively more sensitive and specific than ORF3C, E and Ac. Furthermore, a total of 851 serum samples from diarrheal pigs of different ages were analyzed by ELISA, with most showing immune-reactivity towards the WV, S1, ORF3C, and E proteins. The earliest IgG antibody response was observed in the one-week-old piglets, with similar antibody ontogeny and patterns of seroconversion for S1, ORF3C, E, and WV antigens. In addition, the pattern of neutralizing antibody was more similar to that of IgA in weaning piglets after PEDV infection. Collectively, these data provide more reliable information on the host immune response to different viral proteins, which will be useful for development of novel serological assays and for design of vaccines that better stimulate protective immunity.

A novel spike subunit 1‐based enzyme‐linked immunosorbent assay reveals widespread porcine torovirus infection in eastern China

“…Our results, with lower seropositive rates than those from European farms (Alonso‐Padilla et al., 2012), are consistent with previous findings from the molecular and serological investigation. In these same serum samples, we also detected IgG against two other viruses commonly associated with diarrhoea, PEDV and MRV3 (Lei et al., 2019; Qin et al., 2017). The high prevalence of antibodies against other viruses in these samples suggests that PToV may thrive during co‐infections with PEDV, MRV3, or other enteric pathogens.…”

“…Indirect ELISA assays based on the recombinant purified PToV S1 protein as antigens were developed and optimized by checkerboard titrations using protocols developed previously (Huang et al., 2012; Lei et al., 2019; Qin et al., 2017). Briefly, purified recombinant proteins were diluted in carbonate coating buffer (pH 9.6) and used to coat 96‐well ELISA plates (100 μl/well).…”

“…Positive anti‐sera from pigs infected with PEDV (Lei et al., 2019), PDCoV (Pan et al., 2017) or MRV (Qin et al., 2017) were used to test the cross‐reactivity of the S1‐based ELISA. A total of 96 samples from diarrheic multiparous sows were tested by S1‐ and N‐based ELISA.…”

“…However, these ELISA methods may have some disadvantages such as poor specificity due to the potential cross‐reactivity, making them inappropriate for widespread use. According to experiences with porcine CoVs (Lei et al., 2019; Ma et al., 2016), ELISA diagnostic methods based upon the PToV S1 protein (a major viral envelope protein supposedly binding to the cellular receptor) with good reactivity and high sensitivity/specificity would likely be more accurate. In this study, we have established the first‐ever indirect ELISA method based on the spike S1, and performed the first large‐scale survey of PToV seropositivity in Chinese swine herds.…”

Porcine epidemic diarrhea virus: Molecular mechanisms of attenuation and vaccines

“…However, whether the PEDV E protein affects the viral virulence and whether the E protein is absolutely necessary for PEDV remain to be further studied. The E protein is also a diagnostic maker, which will be helpful to the development of novel serological assays as well as the design of vaccines [ 131 ].…”