Porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus, Italy.
In 1978, PEDV was first identified in Europe; subsequent reports occurred in many countries in Asia, including China, Japan, Korea, and Thailand. In 2010-2012, genetically different PEDV strains emerged, causing severe outbreaks in China (4). PEDV spread to the United States, Canada, and Mexico in 2013-2014, and genetically related strains were detected in South Korea and Taiwan (5-7). The PEDV outbreak caused large global economic losses to the swine industry. In Europe, a severe PEDV epidemic occurred in Italy during 2005-2006 (8), and in 2014-2015, PEDV was detected in Germany, France, and Belgium. These strains have a high nucleotide identity to PEDV strains that contain distinct insertions and deletions (INDELs) in the S gene (S-INDELs) from the United States (9-11). We report the detection and genetic characterization of swine enteric CoVs circulating in Italy during 2007-2014. We also report a recombinant TGEV and PEDV strain (identified as the species Swine enteric coronavirus [SeCoV]) circulating from June 2009 through 2012. Finally, we describe the phylogenetic relationship of the 2014 PEDV S-INDELs to the recent PEDV strains circulating in Europe.
During 2007-2014, we collected 27 fecal and 24 intestinal samples from pigs with suspected PEDV or TGEV infections; the pigs came from swine farms in northern Italy (Po Valley), which contains the regions of Piemonte, Lombardia, Emilia Romagna, and Veneto (online Technical Appendix Figure 1, http://wwwnc.cdc.gov/EID/ article/22/1/15-0544-Techapp1.pdf). The Po Valley contains 70% of Italy's swine. Clinical signs included watery diarrhea in sows and a death rate in piglets of 5%-10%, lower than is typical with PEDV or TGEV infections. Samples were submitted for testing by electron microscopy, PEDV ELISA, viral isolation, pan-CoV reverse transcription PCR (RT-PCR), and RT-PCR for PRCV and TGEV; selected positive pan-CoV samples were sequenced (1214) (online Technical Appendix).
Results of electron microscopy showed that 25 (49%) of the 51 samples contained CoV-like particles, but all samples were negative for viral isolation. Although only 38 samples (74%) were positive by pan-CoV RT-PCR, 47 (92%) were positive by the PEDV ELISA (Table 1) (12,13). Of the 38 pan-CoV-positive samples, 18 were selected for partial RNA-dependent RNA polymerase (RdRp), spike (S1) (14), and membrane (M) sequencing (Table 1). All samples were negative for PRCV and TGEV by RT-PCR, ruling out co-infection with PEDV and TGEV or PRCV (15).
On the basis of the partial sequences from RdRp and the S1 and M genes, the strains from Italy clustered into 3 temporally divided groups, suggesting 3 independent virus entries. Cluster I represents strains circulating from 2007 through mid-2009; cluster II represents strains circulating from mid-2009 through 2012; and cluster III represents strains circulating since 2014 (online Technical Appendix Figure 2, panels A-C). Cluster I was identified in Emilia Romagna (n = 1), Lombardia (n = 5), and Veneto (n = 1). Cluster II was identified in Emilia Romagna (n = 1) and Lombardia (n = 8). Cluster III was identified in Emilia Romagna at 2 swine farms. To help explain the temporal clustering, a single S1 gene segment was sequenced from clusters I and II (PEDV/Italy/7239/2009 and SeCoV/Italy/213306/2009, respectively). Because of the recent outbreak of PEDV in Europe, the 2 positive samples from cluster III (PEDV/ Italy/178509/2014 and PEDV/Italy/200885/2014) were sequenced (Figure 1, panel A).
One strain from each cluster was selected for whole genome sequencing (online Technical Appendix). Unfortunately, the whole genome was obtained from only clusters I and II (PEDV/Italy/7239/2009 and SeCoV/Italy/213306/2009, respectively; Figure 1, panel B). Recombination analysis was conducted on the 2 whole genomes and was not detected in PEDV/Italy/7239/2009. Recombination was detected in SeCoV/Italy/213306/2009 at position 20636 and 24867 of PEDV CV777 and at position 20366 and 24996 of TGEV H16 (Figure 2), suggesting the occurrence of a recombination event between a PEDV and a TGEV. The complete S gene of SeCoV/Italy/213306/2009 shared 92% and 90% nt identity with the prototype European strain PEDV CV777 and the original highly virulent North American strain Colorado 2013, respectively, and the remaining genome shared a 97% nt identity with the virulent strains TGEV H16 and TGEV Miller M6. Whole-genome analysis of PEDV/Italy/7239/2009 showed that it grouped with the global PEDV strains (6) and shared [approximately equal to] 97% nt identity with PEDV strains CV777, DR13 virulent, and North American S-INDEL strain OH851 (Table 2).
During 2007-2014, most (92%) samples collected from the Po Valley in Italy were positive for PEDV by ELISA; only 72% were positive by pan-CoV PCR. However, because we were investigating the presence of PEDV or TGEV in samples with clinical signs of diarrhea, the high occurrence of PEDV may not reflect the actual prevalence of PEDV in Italy. The increased percentage of PEDV found in samples tested by ELISA, compared with the proportion found by PCR, may be explained by the number of ambiguous bases in the pan-CoV primers; the ambiguous bases severely reduce the efficiency of the reaction. The swine enteric CoV strains from Italy in our study, including the recombinant strain, were reported in pigs with mild clinical signs, indicating that PEDV and SeCoV have been circulating in Italy and likely throughout Europe for multiple years but were underestimated as a mild form of diarrhea.
To understand the evolution of PEDV in Italy, the partial RdRp, S, and M genes were sequenced from 18 samples and grouped in 3 different temporal clusters. Cluster I (2007-mid 2009) resembles the oldest PEDV strains; cluster II resembles a new TGEV and PEDV recombinant variant; and cluster III, identified from 2 pig farms in northern Italy in 2014, resembles the PEDV S-INDEL strains identified in Germany, France, Belgium, and the United States. The >99.3% nt identity of the S1 gene within cluster III and in previously identified strains could suggest the spread of the S-INDEL strain into Europe. However, directionality of spread cannot be determined because of a lack of global and temporal PEDV sequences.
Although our findings could indicate 3 introductions of PEDV in Italy, the results more likely suggest the high ability of natural recombination among CoVs and the continued emergence of novel CoVs with distinct pathogenic properties. Further investigation is needed to determine the ancestor of the SeCoV strain or to verify whether the recombinant virus was introduced in Italy. Recombinant SeCoV was probably generated in a country in which both PEDV and TGEV are endemic, but because the presence of these viruses in Europe is unclear and SeCoV has not been previously described, it is difficult to determine the parental strains and geographic spread of SeCoV. Future studies are required to describe the pathogenesis of SeCoV and its prevalence in other countries.
This work was funded by the Italian Ministry of Health (project PRC2010010- E87G11000130001).
Dr. Boniotti is a scientist at the Istituto Zooprofilattico Sperimentale della Lombardia e Emilia Romagna; her primary research interests include molecular diagnosis and epidemiology of viral and bacterial infectious diseases. Dr. Papetti is a research scientist at the Istituto Zooprofilattico Sperimentale della Lombardia e Emilia Romagna; her major research interests are molecular diagnostic development and phylogenetic analysis of infectious diseases.
Author affiliations: Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Brescia, Italy (M.B. Boniotti, A. Papetti, A. Lavazza, G. Alborali, E. Sozzi, C. Chiapponi, S. Faccini, P. Bonilauri, P. Cordioli); University of Minnesota Veterinary Diagnostic Laboratory, St. Paul, Minnesota, USA (D. Marthaler)
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Address for correspondence: M. Beatrice Boniotti, Research and Development Laboratory, Istituto Zooprofilattico Sperimentale della Lombardia e Emilia Romagna, via Bianchi 9, 25124 Brescia (BS), Italy; email: email@example.com
M. Beatrice Boniotti,  Alice Papetti,  Antonio Lavazza, Giovanni Alborali, Enrica Sozzi, Chiara Chiapponi, Silvia Faccini, Paolo Bonilauri, Paolo Cordioli,  Douglas Marthaler
 These first authors contributed equally to this article.
Table 1. Distribution of test results of samples from pig farms in study of swine enteric coronaviruses in northern Italy, 2007-2014 * Sample Farm no. no. Year Region 222654 1 2007 Emilia Romagna 1448 2 2007 Emilia Romagna 19908 3 2007 Emilia Romagna 70323 4 2007 Lombardia 114372 5 2007 Lombardia 200079 6 2007 Lombardia 320855/5 7 2007 Lombardia 320855/6 7 2007 Lombardia 3936/1 8 2008 Lombardia 3936/2 8 2008 Lombardia 29177 9 2008 Veneto 43853 10 2008 Lombardia 7239 ([double 11 2009 Lombardia dagger]) 20001 12 2009 Lombardia 20416 13 2009 Lombardia 22603 14 2009 Lombardia 26199/2 15 2009 Lombardia 87565 16 2009 Emilia Romagna 111357/7 17 2009 Lombardia 137442 18 2009 Lombardia 205396 19 2009 Lombardia 208995 20 2009 Lombardia 213306 ([double 21 2009 Lombardia dagger]) 244945 22 2009 Emilia Romagna 245242 22 2009 Emilia Romagna 274771 23 2009 Veneto 307121 24 2009 Emilia Romagna 315994 25 2009 Lombardia 320695 26 2009 Lombardia 320825 26 2009 Lombardia 324345 27 2009 Lombardia 324374 27 2009 Lombardia 324397 27 2009 Lombardia 324507/1 28 2010 Lombardia 324507/2 28 2010 Lombardia 324507/3 28 2010 Lombardia 324507/4 28 2010 Lombardia 5448/2 29 2011 Emilia Romagna 28607 30 2012 Lombardia 29742 30 2012 Lombardia 30917 31 2012 Lombardia 35621/1 32 2012 Lombardia 35621/2 32 2012 Lombardia 41906 33 2012 Lombardia 44833 34 2012 Lombardia 67322 8 2012 Lombardia 273992 35 2012 Lombardia 32961 36 2013 Piemonte 32963 36 2013 Piemonte 178509 37 2014 Emilia Romagna 200885 38 2014 Emilia Romagna Sample PEDV Pan-CoV TGEV/ no. EM ELISA RT-PCR PRCV S1 222654 - + + NA 1448 - + - NA 19908 - + + -- 70323 + + + NA 114372 + + + NA 200079 - + + NA 320855/5 + + + -- 320855/6 + + + NA 3936/1 - + + -- 3936/2 - + + NA 29177 + + + -- 43853 + + - NA 7239 ([double - + + -- dagger]) 20001 - + + -- 20416 - + + NA 22603 - + + -- 26199/2 - + - NA 87565 - + + NA 111357/7 NA + - NA 137442 + + + -- 205396 + + - NA 208995 + - + NA 213306 ([double + + + -- dagger]) 244945 + + - NA 245242 + + + NA 274771 + - - NA 307121 + + + -- 315994 + - - NA 320695 + + + NA 320825 + + + -- 324345 + + + NA 324374 + + + NA 324397 + + + -- 324507/1 + + + -- 324507/2 + + + NA 324507/3 + + + NA 324507/4 + + + NA 5448/2 NA + - NA 28607 - + + NA 29742 + + + -- 30917 + + - NA 35621/1 + + - NA 35621/2 - + + NA 41906 - + + NA 44833 NA + + -- 67322 - + + NA 273992 - + + -- 32961 - + - NA 32963 + + - NA 178509 NA NA + -- 200885 + + + -- Sequences Sample no. RdRp S1 M 222654 NA NA NA 1448 NA NA NA 19908 Cluster I Cluster I Cluster I 70323 NA ([dagger]) NA 114372 NA NA NA 200079 NA ([dagger]) NA 320855/5 Cluster I Cluster I Cluster I 320855/6 ([dagger]) ([dagger]) NA 3936/1 Cluster I Cluster I Cluster I 3936/2 ([dagger]) NA NA 29177 Cluster I Cluster I Cluster I 43853 NA NA NA 7239 ([double Cluster I Cluster I Cluster I dagger]) 20001 Cluster I Cluster I Cluster I 20416 ([dagger]) ([dagger]) NA 22603 Cluster I Cluster I Cluster I 26199/2 NA NA NA 87565 NA ([dagger]) NA 111357/7 NA NA NA 137442 Cluster II Cluster II Cluster II 205396 NA NA NA 208995 ([dagger]) NA NA 213306 ([double Cluster II Cluster II Cluster II dagger]) 244945 NA NA NA 245242 ([dagger]) NA NA 274771 NA NA NA 307121 Cluster II Cluster II Cluster II 315994 NA NA NA 320695 NA ([dagger]) ([dagger]) 320825 Cluster II Cluster II Cluster II 324345 ([dagger]) ([dagger]) ([dagger]) 324374 ([dagger]) ([dagger]) ([dagger]) 324397 Cluster II Cluster II Cluster II 324507/1 Cluster II Cluster II Cluster II 324507/2 NA ([dagger]) ([dagger]) 324507/3 NA NA ([dagger]) 324507/4 NA ([dagger]) ([dagger]) 5448/2 NA NA NA 28607 NA ([dagger]) ([dagger]) 29742 Cluster II Cluster II Cluster II 30917 NA NA NA 35621/1 NA NA NA 35621/2 NA NA NA 41906 NA NA NA 44833 Cluster II Cluster II Cluster II 67322 NA NA ([dagger]) 273992 Cluster II Cluster II Cluster II 32961 NA NA NA 32963 NA NA NA 178509 Cluster III Cluster III Cluster III 200885 Cluster III Cluster III Cluster III * Cluster I represents strains circulating from 2007 through mid-2009; cluster II represents strains circulating from mid-2009 through 2012; and cluster III represents strains circulating since 2014. EM, electron microscopy; M, membrane; pan-CoV RT-PCR, pan-coronavirus reverse transcription PCR; PEDV, porcine epidemic diarrhea virus; PRCV, porcine respiratory coronavirus; RdRp, RNA-dependent RNA polymerase; S1, spike; TGEV, transmissible gastroenteritis virus; +, positive; -, negative; NA, not tested or sequenced. ([dagger]) Sequence available but not included in this study. ([double dagger]) Samples selected for whole genome sequencing. Table 2. Nucleotide identities of strains PEDV/Italy/7239/2009 and SeCoV/Italy/213306/2009, representative of clusters I and II, respectively, in study of swine enteric coronaviruses in Italy * ORF1 Spike Strain identification I II I II PEDV/Belgium/CV777/1977 97.3 57.8 96.3 92.7 PEDV/South Korea/DR13 vir/2009 98.1 58.0 97.3 93.1 PEDV/U SA/Colorado/2013 98.0 57.9 94.6 90.7 PEDV/USA/OH851/2014 98.1 57.9 96.9 91.9 PEDV/L00721/GER/2014 98.0 57.9 97.0 92.0 TGEV/USA/Miller M6/2006 57.9 96.8 52.0 52.5 PRCV/USA/ISU-1/2006 58.0 96.5 47.7 48.2 ORF3 Envelope Strain identification I II I II PEDV/Belgium/CV777/1977 98.1 43.6 97.6 43.0 PEDV/South Korea/DR13 vir/2009 99.3 43.7 99.6 43.0 PEDV/U SA/Colorado/2013 98.6 44.0 99.2 43.0 PEDV/USA/OH851/2014 98.7 43.8 99.2 43.0 PEDV/L00721/GER/2014 98.6 43.7 99.2 43.0 TGEV/USA/Miller M6/2006 40.0 89.1 42.6 96.4 PRCV/USA/ISU-1/2006 53.0 76.6 43.8 96.0 Membrane Nucleocapsid Strain identification I II I II PEDV/Belgium/CV777/1977 97.6 55.9 96.6 42.5 PEDV/South Korea/DR13 vir/2009 97.7 55.6 97.6 43.3 PEDV/U SA/Colorado/2013 97.8 55.3 96.8 43.4 PEDV/USA/OH851/2014 97.7 55.5 96.7 43.4 PEDV/L00721/GER/2014 97.8 55.3 96.8 43.4 TGEV/USA/Miller M6/2006 56.1 97.1 43.1 96.3 PRCV/USA/ISU-1/2006 56.2 96.3 42.8 95.7 * Cluster I represents strains circulating from 2007 through mid-2009; cluster II represents strains circulating from mid-2009 through 2012. Ger, Germany; ORF, open reading frame; PRCV, porcine respiratory coronavirus; PEDV, porcine epidemic diarrhea virus; SeCoV, swine enteric coronavirus; TGEV, transmissible gastroenteritis virus.
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|Author:||Boniotti, M. Beatrice; Papetti, Alice; Lavazza, Antonio; Alborali, Giovanni; Sozzi, Enrica; Chiappon|
|Publication:||Emerging Infectious Diseases|
|Date:||Jan 1, 2016|
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