The Study On Cross-reactive Epitopes Of Porcine Epidemic Diarrhea Virus And Porcine Transmissible

Both porcine epidemic diarrhea virus(PEDV)and porcine transmissible gastroenteritis virus(TGEV),which cause high mortality in piglets and produce similar clinical symptoms and histopathological morphology,belong to the Alphacoronavirus genus.Both are transmitted primarily via the fecal-oral route and show similar clinical symptoms of diarrhea,vomiting and dehydration.Serological diagnosis plays an important role in distinguishing between two pathogens.Coronaviruses mainly encode four structural proteins: the spike(S),envelope(E),membrane(M)and nucleocapsid(N)proteins.The N protein is the most abundant coronavirus antigen produced during viral infection.The N protein lacks glycosylation sites and is easy to purify,which is used as the common antigen in the serological diagnosis of PEDV and TGEV.However,different cross-reactions between porcine coronaviruses induced by the N protein have attracted attention in recent years.This nonspecific binding affects the effective detection of these two pathogens.To establish more specific diagnostic methods,we performed IFA and Western blotting to investigate the antigenic relationship between PEDV and TGEV.By construction of truncated plasmids,the cross-reactive regions mapped on the PEDV N protein were further explored.Based on the predicted structure and sequence alignment of the PEDV N protein,the cross-reactive epitopes on the PEDV N protein were identified.All the findings are beneficial to develop the specific serological assays.The mainly results of the research can be divided into the following three aspects:1.The relationship between PEDV and TGEV was studied.To investigate cross-reactivity of PEDV and TGEV,cells infected with TGEV and PEDV were detected by IFA and Western blot using the hyperimmune swine antisera and anti-N polyclonal antibodies(PAbs)of PEDV or TGEV.The results showed that the anti-TGEV N PAb cross-reacted with PEDV and vice versa,indicating the two-way cross-reactivity between PEDV and TGEV.To further determine whether this cross-reaction was induced by the N protein,plasmids encoding either the PEDV or the TGEV N protein were transfected into HEK-293 T cells.Intracellular proteins were extracted and subjected to Western blotting.Moderate cross-reaction with the heterologous N protein was observed.2.Identification of the cross-reactive regions on the PEDV N protein.Based on the predicted secondary structure of the PEDV N protein,plasmids encoding different lengths of PEDV N protein were constructed.Using the anti-PEDV or the anti-TGEV N protein PAb as the primary antibody,truncation of amino acids(aa)1-170 or aa 124-301 was detected by the anti-TGEV N protein PAb.The results showed that the N-terminal domain and the central domain were essential for the cross-reaction.3.Identification of the cross-reactive epitopes on the PEDV N protein.Based on sequence alignment and the predicted tertiary structure,conserved residues on the surface of the N-terminal or the central domain were selected.Each residue in these candidate epitopes was replaced with alanine residues in the full-length PEDV N protein.Using the anti-PEDV or the anti-TGEV N protein PAb as the primary antibody,cross-reactivity of different mutants was detected by Western blot.Compared with the wild-type PEDV N protein,the mutant of the 58-RWRMRRGERIE-68 and the 78-LGTGPHAD-85 showed a significantly lower cross-reactivity to the anti-TGEV N PAb.Furthermore,mutation of this motif did not affect the strong binding ability to the anti-PEDV N protein PAb.The 78-LGTGPHAD-85,58-RWRM-61 and 58-RWRMRRGERIE-68 motifs on the N-terminal domain of the PEDV N protein were demonstrated to be cross-reactive epitopes.In conclusion,we analysed the antigenic relationship between PEDV and TGEV.Two cross-reactive regions and two dominant epitopes of PEDV N protein were further identified,which provided a new insight for the specific diagnosis methods of porcine coronavirus.