Generation and functional analysis of single chain variable fragments (scFvs) targeting the nucleocapsid protein of Porcine epidemic diarrhea virus

Abstract

Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, which can cause death in suckling piglets. Vaccines confer only partial protection against new mutant strains, whereas antibodies targeting virus-encoded proteins may be effective prophylactics. In this study, we constructed a recombinant single chain variable fragment (scFv) library from the spleens of two pigs immunized with a recombinant PEDV nucleocapsid (N) protein. Among the positive clones directed against PEDV N protein isolated from the library, four scFvs that showed higher affinity for N were functionally analyzed. These scFvs specifically bound to the PEDV N protein, but not to the transmissible gastroenteritis virus (TGEV) N protein. Their framework regions were highly conserved, whereas their complementarity-determining regions displayed clear diversity. An immunofluorescence assay showed the co-localization of the four scFvs with PEDV N protein in cells. They significantly suppressed PEDV replication, detected with reverse transcription (RT)-quantitative PCR (qPCR; P < 0.01). Two of them significantly reduced the viral titer at 48 hpi and 72 hpi ( P < 0.05). In addition, they observably suppressed the production of viral protein at 72 hpi. The expression of interferons, interferon regulatory factor 3 (IRF3), and IRF7 was assessed with RT-qPCR, which indicated that PEDV dramatically suppressed the transcription of interferon-λ1 and IRF7 and that the scFvs significantly upregulated their expression ( P < 0.05). These findings facilitated the investigation of the mechanism by which PEDV evaded the host immune response and suggested that these porcine scFvs were potential candidate agents for the prevention and treatment of porcine diarrhea caused by PEDV. Key points • Four scFvs targeting PEDV N protein were generated from porcine spleens • These scFvs co-localized with PEDV N protein and suppressed PEDV replication • These scFvs significantly upregulated IFN-λ1 expression Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00253-021-11722-z.

Adenovirus vector-mediated single chain variable fragments target the nucleocapsid protein of porcine epidemic diarrhea virus and protect against viral infection in piglets

“…Several studies reported that PEDV replication was suppressed when the N protein was degraded (49,58,59). Our scFvs targeting the PEDV N protein significantly suppressed PEDV replication in PEDV-infected cells, as described in our previous study (36). Previous studies indicated that PEDV infection in piglets caused fecal viral RNA shedding (39,60).…”

“…ScFvs targeting the PEDV N protein were previously generated and described ( 36 ). Briefly, a porcine recombinant scFv library was constructed and four scFvs targeting the PEDV N protein were generated.…”

“…Stool RNA Kit (Omega Bio-tek, Norcross, GA, USA) and served as a template for complementary DNA (cDNA) synthesis using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio, Shiga, Japan). PEDV M gene expression was analyzed by reverse transcription (RT)-quantitative PCR (qPCR) to quantify virus production in feces ( 36 ).…”

“…In our previous study, we reported that the four scFvs colocalized with the wild-type N protein in PEDV-infected cells (36). To determine scFv and wild-type N protein expression and distribution in vivo, jejunums from piglets orally administered rAdV-ZW and challenged with the prevalent PEDV strain (Table 2 and Figure 5A) were collected, fixed, and paraffinized.…”

“…We previously reported that four scFvs, ZW1-16, ZW3-21, ZW1-41, and ZW4-16, specifically targeted the PEDV N protein, significantly suppressing PEDV replication, and upregulating IFN-λ1 expression in host cells ( 36 ). Herein, to investigate scFv effects on PEDV-induced diarrhea in piglets, four recombinant adenoviruses expressing scFvs against the PEDV N protein were generated.…”