in may 2014 , a pig fattening farm in southern germany ( federal state of baden - wuerttemberg ) that continuously houses 1,400 fattening pigs reported watery diarrhea in pigs in all age groups ( feeders to slaughter animals ) .
the first cases occurred after new feeder pigs from a large piglet producer were brought onto the farm .
fecal samples from diseased pigs were submitted to the regional laboratory for diagnosis , and coronaviruses were detected by electron microscopy ( figure 1 ) . additionally , 3 pigs with catarrhal enteritis were euthanized ; postmortem examination at the regional laboratory confirmed coronavirus infection in all 3 animals .  
porcine epidemic diarrhea virus particles seen by negative - stain electron microscopy of fecal samples .
subsequently , ped was diagnosed in a private laboratory ( ivd gmbh , hannover , germany ) by use of a published multiplex reverse transcription quantitative pcr ( 7 ) .
selected positive samples were submitted to the friedrich - loeffler - institut , isle of riems , germany , for confirmation and further virus characterization . at this institution , 2 fecal samples with high genome load , as determined by 2 independent , published ( 8,9 ) reverse transcription quantitative pcrs selective for pedv nucleocapsid ( n ) and spike ( s ) genes , were chosen for routine virologic testing and next - generation sequencing .
the phylogenetic tree , based on full - length genomes , was constructed by using phyml ( 11 ) in the geneious software suite ( http://www.geneious.com/ ) with a generalized time reversible substitution model , and the tree was supported by 1,000 bootstrapping replicates .
virus isolates were obtained after inoculation of cells of different permanent cell lines ( pig kidney [ pk]-15 and vero ) with clinical material from the pigs .
sequencing of nucleic acids isolated directly from diagnostic samples pedv / ger / l00719/2014 and pedv / ger / l00721/2014 resulted in 2 viral genomes ( table ) encompassing all typical pedv coding sequences .
each sequence encodes a large replicase polyprotein , a spike ( s ) protein , an alphacoronavirus - specific accessory membrane protein , an envelope protein , a membrane protein , and a nucleocapsid ( n ) protein . *
kj778615 , kf272920 , kf468753 , kj584361 , kj408801 , kf650374 , kf468752 , kf267450 , kc210147 , jn825712 , kc210145 ) .  #
aa 632f detected in reference strain kf267450 only . comparative analyses of the full genomes showed that the strains share a very high ( 99.5% ) identity with the new variant oh851 ( genbank accession no .
a more comprehensive comparison of 21 full - length pedv genomes from different years and locations revealed lower similarities ( 98.7% ) with currently circulating highly virulent strains from the united states and from china ( technical appendix figure , panel a ) .
in contrast , the new isolates from germany , pedv / ger / l00719/2014 and pedv / ger / l00721/2014 , are less similar ( 97.1% ) to the isolate from europe , cv777 , which dates back to the 1970s ( 12 ) . the nucleotide alignment of the obtained pedv genomes and the available references from the database revealed a region with high variability of the first 1,200 nt in the 5 portion of the s protein 
the n terminal s1 domain of the coronavirus s protein is necessary for virus attachment by interaction with host cell receptors ( 13 ) and might therefore be highly mutable .
although in - depth analysis of the deep - sequencing data for pedv / ger / l00721/2014 revealed a genetically homogenous population , this analysis for pedv / ger / l00719/2014 uncovered a mixed viral population with a total of 8 single - nucleotide variants .
one nonsynonymous single - nucleotide variant ( variant position g19042u , amino acid substitution s6348i ) was detected in the polyprotein coding sequences .
of note , 7 single - nucleotide variants are located in the aforementioned variable region in the s protein coding sequences , 5 of which are nonsynonymous ( table ) , thereby confirming the high variability in the n terminal part of the s protein . because quite extensive differences ( 50 aa ) were found between the recent n terminal s protein region of the isolates from germany and the highly virulent pedv strains from the united states and china , the isolates from germany described in this article seem to not be directly linked to the highly virulent pedv strains circulating in the united states ( figure 2 ) .
in contrast , the recent isolates from germany and strain oh851 share not only high identity over the entire genome , including the highly variable 5 region of the s protein coding sequences , but also their clinical phenotype observed under field conditions .
phylogenetic analysis based on 21 full - length porcine epidemic diarrhea virus ( pedv ) genomes .
the new strains from germany ( pedv / ger / l00719/2014 and pedv / ger / l00721/2014 , in boldface and italics ) and the new 2014 pedv variant from the united states ( oh851 , in boldface ) were included and compared with current circulating strains from the united states and china .
numbers above branches indicate proportions calculated from 1,000 bootstrap replicates : the scale bar represents nucleotide substitutions per site .
comparative analyses of full - length sequences revealed that the isolates from these pigs in germany show very high nucleotide similarity with strain oh851 found in the united states in 2014 .
however , differences exist that distinguish the strains from germany from the highly virulent pedv strains that caused the major losses in the united states .
given the fact that pedv surveillance has been lacking in germany , we can not exclude the possibility that the strains described here have already been circulating in europe for a longer time or were indeed recently introduced from the united states or asia to europe .
therefore , our report provides useful information about recent pedv strains in europe , but a comprehensive evaluation is still difficult because of a lack of data about additional strains .
future studies should therefore concentrate on analysis of additional pedv from different years and locations .
pairwise similarity matrix based on full - length genomes and schematic representation of the nucleotide sequence alignment of the complete spike protein coding sequences .
 since 2013 , highly virulent porcine epidemic diarrhea virus has caused considerable economic losses in the united states . to determine the relation of us strains to those recently causing disease in germany 
 , we compared genomes and found that the strain from germany is closely related to variants in the united states . 