Novel Porcine Epidemic Diarrhea Virus (PEDV) Variants with Large Deletions in the Spike (S) Gene Coexist with PEDV Strains Possessing an Intact S Gene in Domestic Pigs in Japan: A New Disease Situation

<div><p>Since late 2013, after an absence of seven years, outbreaks of porcine epidemic diarrhea virus (PEDV) infection have reemerged and swept rapidly across Japan, resulting in significant economic losses. In this study, we report the emergence, mixed infection, and genetic characterization of 15 novel field PEDV variants with large genomic deletions. The sizes of deletion varied between 582 nt (194 aa) and 648 nt (216 aa) at positions 28–714 (10–238) on the S gene (protein). Among 17 PEDV samples isolated from individual pigs, all of them contained at least two distinct genotypes with large genomic deletions, and 94.1% of them were found to consist of strains with an intact S gene. These variants were found in eight primary and nine recurrent outbreaks, and they might be associated with persistent PEDV infection in the farms. Full-length S and ORF3 genes of eight variants derived from 2 samples were characterized. This is the first report of mixed infections caused by various genotypes of PEDV and would be important for the studies of viral isolation, pathogenesis, and molecular epidemiology of the disease.</p></div

PCR product patterns amplified by the primer pair CS1-F/CS1-R targeting the partial S gene.

<p>Samples from left to right: (1) JAi-23, (2) JMi-69, (3) JMi-124, (4) JKa-230, (5) JA0-56, (6) JMi-231, (M) DNA marker. The dashed arrows indicate products of PEDV variants with large genomic deletions (approximately 0.2–0.3 kbp) and the solid arrow indicates the predicted product (872 bp).</p

The primers utilized in this study.

<p>The primers utilized in this study.</p

Sixty-seven PEDV field strains collected in Japan during outbreaks from December 2013 to June 2015.

<p>Sixty-seven PEDV field strains collected in Japan during outbreaks from December 2013 to June 2015.</p

Fifteen genotypes with large deletions in the S gene of the Japanese PEDV strains in this study.

<p>Fifteen genotypes with large deletions in the S gene of the Japanese PEDV strains in this study.</p

IFN-related responses in the parental and V protein-expressing H358 cells.

<p>(A) The parental H358, H358-V-Tyr₂₆₇-11, and H358-V-Cys₂₆₇-6 cells were infected with 007Lm-VDS, and CDV antigens (green) and IRF-3 (red) were detected by immunofluorescence confocal microscopy. The nuclei (blue) were detected by counterstaining with DAPI. (B) The parental H358, H358-V-Tyr₂₆₇-11, and H358-V-Cys₂₆₇-6 cells were untreated (0 h) or treated with 100 U/ml of IFN-β for 2, 4, and 8 h. Total RNAs were purified and the relative amounts of mRNAs for three ISGs (ISG15, OAS1, and ISG56) were measured by RT-qPCR.</p

Replication kinetics in H358 cells. H358 cells were infected with 007Lm-VDS or 007Lm-H358p8 at a MOI of 0.01. At 1, 3, 5, and 7 days p.i., the virus titers were determined by plaque assays.

<p>Data represent the means ± standard deviations of the results from triplicate samples.</p

Acquired and selected nucleotides during passages in NCI-H358 cells.

<p>^a Acquired and selected nucleotides are underlined.</p

Virus growth in the parental and V protein-expressing H358 cells.

<p>(A) Immunoblotting. The parental H358, H358-V-Tyr₂₆₇-11, H358-V-Cys₂₆₇-5, and H358-V-Cys₂₆₇-6 cells were lysed in RIPA buffer, and subjected to SDS-PAGE followed by immunoblotting for detection of the V protein. Tubulin was detected as an internal control. (B, C) The parental H358, H358-V-Tyr₂₆₇-11, and H358-V-Cys₂₆₇-6 cells were infected with 007Lm-VDS (B) or MVΔV (C) at a MOI of 0.01. At 5 days p.i., the virus titers (PFUs and FFUs, respectively) were determined. (D) Observation of EGFP-expressing MVΔV-infected cells (syncytia) using a fluorescence microscope.</p

Additional file 1: of Molecular characterization of US-like and Asian non-S INDEL strains of porcine epidemic diarrhea virus (PEDV) that circulated in Japan during 2013–2016 and PEDVs collected from recurrent outbreaks

Table S1. Highly-specific N-glycosylation sites in the spike protein of the vaccine and field strains. (DOCX 13 kb