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. 2023 Sep 28;97(9):e0084723.
doi: 10.1128/jvi.00847-23. Epub 2023 Sep 8.

Deletion of a 7-amino-acid region in the porcine epidemic diarrhea virus envelope protein induces higher type I and III interferon responses and results in attenuation in vivo

Zhiwei Li #  1   2 , Zhiqian Ma #  2 , Weiguo Han  1 , Chuanzhe Chang  2 , Yang Li  2 , Xuyang Guo  1 , Zifang Zheng  2 , Yingtong Feng  1 , Lele Xu  1 , Haixue Zheng  2 , Xinglong Wang  1 , Shuqi Xiao  1   2
Affiliations

Deletion of a 7-amino-acid region in the porcine epidemic diarrhea virus envelope protein induces higher type I and III interferon responses and results in attenuation in vivo

Zhiwei Li et al. J Virol. .

Abstract

Porcine epidemic diarrhea virus (PEDV) leads to enormous economic losses for the pork industry. However, the commercial vaccines failed to fully protect against the epidemic strains. Previously, the rCH/SX/2016-SHNXP strain with the entire E protein and the rCH/SX/2015 strain with the deletion of 7-amino-acid (7-aa) at positions 23-29 in E protein were constructed and rescued. The pathogenicity assay indicated that rCH/SX/2015 is an attenuated strain, but rCH/SX/2016-SHNXP belongs to the virulent strains. Then, the recombination PEDV (rPEDV-EΔaa23-aa29)strain with a 7-aa deletion in the E protein was generated, using the highly virulent rCH/SX/2016-SHNXP strain (rPEDV-Ewt) as the backbone. Compared with the rPEDV-Ewt strain, the release and infectivity of the rPEDV-EΔaa23-aa29 strain were significantly reduced in vitro, but stronger interferon (IFN) responses were triggered both in vitro and in vivo. The pathogenicity assay showed that the parental strain resulted in severe diarrhea (100%) and death (100%) in all piglets. Compared with the parental strain group, rPEDV-EΔaa23-aa29 caused lower mortality (33%) and diminished fecal PEDV RNA shedding. At 21 days, all surviving pigs were challenged orally with rPEDV-Ewt. No pigs died in the two groups. Compared with the mock group, significantly delayed and milder diarrhea and reduced fecal PEDV RNA shedding were detected in the rPEDV-EΔaa23-aa29 group. In conclusion, the deletion of a 7-aa fragment in the E protein (EΔaa23-aa29) attenuated PEDV but retained its immunogenicity, which can offer new ideas for the design of live attenuated vaccines and provide new insights into the attenuated mechanism of PEDV. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets and remains a large challenge to the pork industry. Unfortunately, no safe and effective vaccines are available yet. The pathogenesis and molecular basis of the attenuation of PEDV remain unclear, which seriously hinders the development of PEDV vaccines. This study found that the rPEDV carrying EΔaa23-aa29 mutation in the E protein induced significantly higher IFN responses than the parental virus, partially attenuated, and remained immunogenic in piglets. For the first time, PEDV E was verified as an IFN antagonist in the infection context and identified as a virulence factor of PEDV. Our data also suggested that EΔaa23-aa29 mutation can be a good target for the development of live attenuated vaccines for PEDV and also provide new perspectives for the attenuated mechanism of PEDV.

Keywords: PEDV; coronavirus; envelope; interferon; reverse genetic analysis; the molecular basis of the attenuation of PEDV; virulence factor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Analysis of the pathogenicity of the rCH/SX/2015 and rCH/SX/2016-SHNXP strains. (A) Survival rates of piglets in each group. (B) Fecal scores of piglets in different groups. Fecal scores were scored as follows: 0, solid; 1, pasty; 2, semiliquid; and 3, liquid. Each line indicates the mean scores of a group. (C) Average weight gain of piglets on the day of death or euthanasia at the final time points. (D) Intestinal lesions of piglets inoculated with the different viruses. The intestinal lesions were analyzed on the day of death or euthanasia at the final time points.
Fig 2
Fig 2
Amino acid sequence alignments and transmembrane prediction of the E protein. (A) Alignment of E protein amino acid sequences from several different PEDV strains. (B) The Clustal W method in MegAlign software was used to analyze the sequence of the S protein. (C) Transmembrane prediction of the E protein and EΔaa23–aa29 mutation with TMHMM server v.2.0.
Fig 3
Fig 3
Construction and rescue of the recombinant strain with deletions of amino acids 23–29 in the E protein. (A) Schematic illustration of constructing the rPEDV-EΔaa23–aa29 strain. (B) MARC145 cells were infected with rPEDV-Ewt and rPEDV-EΔaa23–aa29. Infected cells were fixed at 36 hpi and immunolabeled with fluorescein (FITC)-AffiniPure goat anti-mouse IgG (H + L). Nuclei were labeled with 4–6-diamidino-2-phenylindole (blue). (C) MARC145 cells were infected with rPEDV-Ewt and rPEDV-EΔaa23–aa29. Infected cells were harvested at 36 hpi. The total RNA was extracted and reverse-transcribed. Then the rescue of the rPEDV-EΔaa23–aa29 strain was identified by RT-PCR. (D) MARC145 cells were infected with rPEDV-Ewt and rPEDV-EΔaa23–aa29. Infected cells were harvested at 36 hpi. The total RNA was extracted and reverse-transcribed. Then the rescue of the rPEDV-EΔaa23–aa29 strain was identified by Sanger sequencing.
Fig 4
Fig 4
In vitro characterization of the rPEDV-EΔaa23–aa29. MARC145 cells were infected with PEDV. (A) CPE was observed under a light microscope. (B) The plaque assay was performed. (C) MARC145 cells in 12-well plates were infected with rPEDV-Ewt and rPEDV-EΔaa23–aa29 at an MOI of 0.05. The supernatant was harvested at 12, 24, 36, 48, and 60 hpi and titrated on MARC145 cells. (D) LLC-PK1 cells in 12-well plates were infected with rPEDV-Ewt and rPEDV-EΔaa23–aa29 at an MOI of 0.1. The supernatant was harvested at 12, 24, 36, 48, and 60 hpi and titrated on MARC145 cells. (E) MARC145 cells in 12-well plates were infected with rPEDV-Ewt and rPEDV-EΔaa23–aa29 at an MOI of 1. Cells were harvested at 9 and 12 hpi. The relative expression of the genomic RNA was determined by RT-quantitative PCR (qPCR). (F) MARC145 cells in 12-well plates were infected with rPEDV-Ewt and rPEDV-EΔaa23–aa29 at an MOI of 0.05. The supernatant was harvested at 24 and 48 hpi. The viral genomic RNA copies were detected by RT-qPCR. (G) MARC145 cells in 12-well plates were infected with rPEDV-Ewt and rPEDV-EΔaa23–aa29 at an MOI of 0.05. Then, the supernatants were collected, and the cell samples with added DMEM were frozen, thawed for one cycle, and centrifuged. The tissue culture infectious dose 50 (TCID50)of the supernatants and cells was titrated to calculate the ratio of supernatant titer to intracellular titer. (H) MARC145 cells in 12-well plates were infected with rPEDV-Ewt and rPEDV-EΔaa23–aa29 at an MOI of 0.05. Then, we scratched cells from the plates and collected both cells and supernatant at 24 and 48 hpi. Then, the samples were frozen, thawed, and centrifuged, and the supernatants were used to detect and calculate the genomic RNA/TCID50 ratios. Error bars indicate standard deviations. The significance level was expressed as *P < 0.05, **P < 0.01, or ***P < 0.001.
Fig 5
Fig 5
Evaluation of the interferon responses to rPEDV infection in MARC145 cells. MARC145 cells in 12-well plates were infected with rPEDV-Ewt and rPEDV-EΔaa23–aa29 at an MOI of 0.05. (A–E) Cells were collected at 12 and 24 hpi, and RT-qPCR was used to detect the relative expression of the indicated mRNA. Error bars indicate standard deviations. The significance level was expressed as *P < 0.05, **P < 0.01, or ***P < 0.001.
Fig 6
Fig 6
Evaluation of the interferon responses to rPEDV infection in LLC-PK1 cells. LLC-PK1 cells in 12-well plates were infected with rPEDV-Ewt and rPEDV-EΔaa23–aa29 at an MOI of 0.05. (A–H) Cells were collected at 12 and 24 hpi, and RT-qPCR was used to detect the relative expression of the indicated mRNA. Error bars indicate standard deviations. The significance level was expressed as *P < 0.05, **P < 0.01, or ***P < 0.001.
Fig 7
Fig 7
Pathogenicity of the rPEDV-EΔaa23–aa29 strain in piglets. (A) Fecal scores of pigs. Fecal scores were scored as follows: 0, solid; 1, pasty; 2, semiliquid; and 3, liquid. Each line indicates the mean score of a group. (B) Survival curves of piglets. (C) Fecal shedding of PEDV RNA. Viral RNA was isolated from rectal swab samples daily and subjected to RT-qPCR to determine the PEDV N gene RNA copies. (D) IFN-β levels in the fecal swabs on day 1 and day 3 were detected using a commercial Porcine IFN-β Elisa Kit. (E) H&E staining of the jejunum and ileum from piglets euthanized at 2 dpi. (F) Immunohistochemistry staining of PEDV N proteins in the jejunum and ileum. Error bars indicate standard deviations. The significance level was expressed as *P < 0.05. Ns represents no significant difference.
Fig 8
Fig 8
Protection induced by the EΔaa23–aa29 mutant in pigs against rPEDV-Ewt challenge. (A) The levels of anti-PEDV N IgG in sera collected at 14 and 21 dpi. (B) Virus-neutralizing antibody titers in sera collected at 14 and 21 dpi. (C) Fecal scores of pigs post-challenge. Fecal scores were scored as follows: 0, solid; 1, pasty; 2, semiliquid; and 3, liquid. Each line indicates the mean score of a group. (D) Survival curves of piglets post-challenge. (E) Evaluation of fecal PEDV RNA shedding in pigs post-challenge. Viral RNA was isolated from rectal swab samples daily and subjected to RT-qPCR to determine the PEDV N gene RNA copies. Error bars indicate standard deviations. The significance level was expressed as ***P < 0.001.
Fig 9
Fig 9
Genetic stability of the EΔaa23–aa29 mutant. (A) rPEDV-EΔaa23–aa29 was serially passaged in MARC145 cells to P15, and cells were lysed to collect total RNA for cDNA synthesis. RT-PCR was used to evaluate the genetic stability of the EΔaa23–aa29 mutant in vitro. (B) Fecal swabs from the rPEDVEΔaa23–aa29 strain-infected pigs were collected at 2–8 dpi. RT-PCR was used to evaluate the genetic stability of the EΔaa23–aa29 mutant in vivo.
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