The S Gene Is Necessary but Not Sufficient for the Virulence of Porcine Epidemic Diarrhea Virus Novel Variant Strain BJ2011C

Abstract

The recently emerged highly virulent variants of porcine epidemic diarrhea virus (PEDV) have caused colossal economic losses to the worldwide swine industry. In this study, we investigated the viral virulence determinants by constructing a series of chimeric mutants between the highly virulent strain BJ2011C and the avirulent strain CHM2013. When tested in the 2-day-old piglet model, wild-type (WT) BJ2011C caused severe diarrhea and death of the piglets within 72 h. In contrast, its chimeric derivative carrying the S gene from CHM2013 (BJ2011C-S_CHM) was avirulent to the piglets. Moreover, reciprocal substitution of the BJ2011C S gene (CHM2013-S_BJ) did not enable CHM2013 to gain any virulence. However, when the whole structural protein-coding region of BJ2011C (CHM2013-SP_BJ) was swapped, CHM2013 started to gain the ability to efficiently colonize the intestinal tract and caused diarrhea in piglets. A further gain of virulence required additional acquisition of the 3' untranslated region (UTR) of BJ2011C, and the resultant virus (CHM2013-SP + 3UTR_BJ) caused more severe diarrhea and death of piglets. Together, our findings suggest that the virulence of PEDV epidemic strains is a multigenic event and that the S gene is only one of the necessary determinants.IMPORTANCE The recently emerged highly virulent PEDV variants are the major cause of the global porcine epidemic diarrhea (PED) pandemic. The S gene of the variants undergoes remarkable variations and has been thought to be the virulence determinant for the enhanced pathogenesis. Our studies here showed that the S gene is only part of the story and that full virulence requires cooperation from other genes. Our findings provide insight into the pathogenic mechanism of the highly virulent PEDV variants and have implications for future vaccine development.

Keywords: 3′ UTR; PEDV; S gene; structural protein; virulence determinants.

FIG 1

Construction of chimeric viruses of PEDV. (A) DNA-launched reverse genetics BAC system for PEDV strains BJ2011C and CHM2013. The critical restriction enzymes used for molecular cloning and their relative positions in the genome are indicated. CMV, cytomegalovirus; HDV, hepatitis D virus; BGH, bovine growth hormone polyadenylation signal. (B) Diagram of chimeric infectious cDNA clones with S genes exchanged. (C) Diagram of chimeric infected cDNA clones with exchanged structural protein (SP)-coding regions. (D) Diagram of chimeric clones with SP regions and 3′ UTRs swapped. The boxes represent the genomic fragments of parental backbone viruses CHM2013 (blue) and BJ2011C (green).

FIG 2

In vitro characterization of PEDV chimeric viruses. (A) Virus-induced CPE and immunofluorescence staining. Vero cells were infected with the indicated viruses at an MOI of 0.01. The virus-induced CPE was monitored and recorded with an inverted microscope. At 24 h postinfection, the cells were fixed and stained with mouse monoclonal antibody 4E3 to PEDV N protein. Nuclei were stained with DAPI (blue). (B) Multistep growth curves of PEDV wild-type and chimeric viruses on Vero cells at an MOI of 0.01. Virus titers at different time points, as indicated, were determined by endpoint dilution assay. The error bars indicate standard deviations.

FIG 3

Pathogenicity analysis of PEDV chimeras. (A) Average weight gain of piglets on the day of death or euthanasia at the final time points. (B) Clinical mental state scores of piglets of different groups. Criteria for evaluation: 0, normal; 1, mild lethargy (slow to move; head down); 2, moderate lethargy (stands but tends to lie down); 3, heavier lethargy (lies down; occasionally stands); 4, severe lethargy (recumbent; moribund). (C) Fecal scores of piglets of different groups. Evaluation standards: 0, normal; 1, soft (cowpie); 2, very soft and tends to be liquid; 3, liquid with some solid content; 4, watery diarrhea with no solid content. (D) Survival rates of piglets of each group. The survival curves of piglets infected with rescued viruses in each group are shown. Asterisks indicate significant differences between BJ2011C and CHM2013 (***, P < 0.001). Pound signs indicate significant differences between chimeric viruses CHM2013-SP_BJ and CHM2013 (#, P < 0.05; ##, P < 0.01; ###, P < 0.001). Thetas indicate significant differences between chimeric viruses CHM2013-SP + 3UTR_BJ and CHM2013 (θ, P < 0.05; θθ, P < 0.01; θθθ, P < 0.001). The error bars indicate standard deviations.

FIG 4

Gross lesions of piglets inoculated with the recombinant viruses. The intestinal lesions were examined on the day of death or euthanasia at the final time points. The necropsy images show transparent intestines observed in piglets inoculated with BJ2011C, CHM2013-SP_BJ, and CHM2013-SP + 3UTR_BJ but not in the CHM2013-, CHM2013-S_BJ-, BJ2011C-S_CHM-, BJ2011C-SP_CHM-, and BJ2011C-SP + 3UTR_CHM-infected or mock-inoculated piglets.

FIG 5

Histopathological examination of intestines of recombinant PEDV-infected piglets. Different segments, including duodenum, jejunum, cecum, colon, and rectum, of intestines from each group were taken and then processed for H&E staining, and representative images are shown.

FIG 6

Virus shedding in feces. Daily virus shedding in feces of different groups was measured by reverse transcription (RT-PCR) detection of virus genomes in fecal swabs. The length of the target fragment is 618 bp.

FIG 7

Quantification of viral loads in different intestine segments from PEDV-infected piglets. The segments were collected and used for total RNA extraction. Virus abundance was quantified by TaqMan real-time RT-PCR targeting the PEDV N gene. Asterisks indicate significant differences between BJ2011C and CHM2013 (***, P < 0.001). Pound signs indicate significant differences between chimeric viruses CHM2013-SP_BJ and CHM2013 (###, P < 0.001). Thetas indicate significant differences between chimeric viruses CHM2013-SP + 3UTR_BJ and CHM2013 (θθθ, P < 0.001). Sigmas indicate significant differences between chimeric viruses BJ2011C-S_CHM and CHM2013 (σσσ, P < 0.001). The error bars indicate standard deviations.