Date Published: January 17, 2017
Publisher: Public Library of Science
Author(s): Nguyen Van Diep, Junzo Norimine, Masuo Sueyoshi, Nguyen Thi Lan, Ryoji Yamaguchi, Yongchang Cao.
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.
Porcine epidemic diarrhea (PED) is a highly contagious enteric disease characterized by vomiting, acute watery diarrhea, and in particular, a high mortality rate in suckling piglets, resulting in substantial economic losses . The etiologic agent of this disease is the PED virus (PEDV), which is an enveloped, positive-sense, single-stranded RNA virus that belongs to the order Nidovirales, family Coronaviridae, and genus Alphacoronavirus. The disease was initially reported in England in 1971  and Belgium in 1978 . Since then, it has been identified in many swine-producing countries such as those in Europe and Asia, notably Belgium, Czech Republic, Hungary, Italy, China, South Korea, Japan, Thailand, and Taiwan [1, 4]. Between 2008 and 2010, new PEDV strains characterized by multiple insertions/deletions or mutations on the spike (S) protein compared with previous endemic strains have been reported in South Korea  and China [6, 7]. In April 2013, PED was identified in the US for the first time, and it rapidly spread across the country, causing the deaths of more than eight million newborn piglets during a one year-epidemic period [8, 9]. US-like PEDV strains have been reported in Germany , France , Belgium , South Korea , Taiwan , and Japan . Currently, there are two PEDV types identified in the US: the original highly pathogenic (North American) PEDV  and the S INDEL PEDV, which has insertions and deletions in the N-terminal region of the S protein, with reportedly mild virulence in the field [9, 16]. Three novel PEDV strains with large deletions in the S gene have recently been identified in the US , South Korea , and Japan .
A total of 248 field samples (70.7%) collected from animals at 145 farms (82.4%) experiencing acute diarrhea in six prefectures were positive for PEDV. The result indicated that PEDV has a high prevalence rate in Japanese pig herds. Notably, PED outbreaks were commonly observed in PED-vaccinated farms, and there was a significant difference in the mortality rate (0–100%) for piglets and durations of diarrhea among PEDV-infected farms. This prompted critical questions regarding the molecular characteristics and virulence of circulating PEDV as well as efficacy of PEDV vaccines that have been used in Japan.
This is the first report describing PED outbreaks with mixed infections of various genotypes. Our study identified 15 field PEDV genotypes with large deletions in the S gene that have been circulating in Japan from 2013 to 2015. The full-length S gene and ORF3 gene of eight variants and three strains with an intact S gene were characterized. The variants coexisted with intact S strains in high frequency and they might be associated with persistent PEDV infection in pig farms in Japan. Such a reemerging and mixed infection of the variants in this study have put PED in a new situation indicating this disease has become more complex in terms of viral isolation, pathogenesis, and epidemiology. The mechanism by which the PEDV variants were generated and evolved remains unclear. Hence, further studies are required to elucidate biological properties such as changes in tissue tropism and the virulence patterns of the new variants.