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Porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus, Italy.

Porcine epidemic diarrhea virus (PEDV) and Transmissible gastroenteritis virus (TGEV) (family Coronaviridae, genus Alphacoronavirus) are enveloped viruses that contain a single-stranded, positive-sense RNA genome of [approximately equal to]28 kb. Infection with these viruses causes watery diarrhea, dehydration, and a high mortality rate among suckling pigs. Coronaviruses (CoVs) are prone to genetic evolution through accumulation of point mutations and homologous recombination among members of the same genus (1). Porcine respiratory coronavirus (PRCV), a mutant of TGEV, appeared in pigs in the 1980s (2). The spread of PRCV, which conserved most of the antigenic sites and causes cross-protection against TGEV (3), led to the gradual disappearance of TGEV. Newly emerging CoVs pose a potential threat to human and animal health because multiple human CoV infections have been derived from animal hosts. Emerging swine coronaviruses are of great concern to swine health because of the potential increase in viral pathogenesis.

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.

The Study

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).

Conclusions

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)

DOI: http://dx.doi.org/10.3201/eid2201.150544

References

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(3.) Wesley RD, Woods RD. Immunization of pregnant gilts with PRCV induces lactogenic immunity for protection of nursing piglets from challenge with TGEV. Vet Microbiol. 1993;38:31-40. http://dx.doi.org/10.1016/0378-1135(93)90073-G

(4.) Huang YW, Dickerman AW, Pyneyro P, Li L, Fang L, Kiehne R, et al. Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the United States. mBiol. 2013; 4:00737-13. http://dx.doi.org/10.1128/mBio.00737-13

(5.) Stevenson GW, Hoang H, Schwartz KJ, Burrough ER, Sun D, Madson D, et al. Emergence of porcine epidemic diarrhea virus in the United States: clinical signs, lesions, and viral genomic sequences. J Vet Diagn Invest. 2013;25:649-54. http://dx.doi.org/10.1177/1040638713501675

(6.) Vlasova AN, Marthaler D, Wang Q, Culhane MR, Rossow KD, Rovira A, et al. Distinct characteristics and complex evolution of PEDV strains, North America, May 2013-February 2014. Emerg Infect Dis. 2014;20:1620-8. http://dx.doi.org/10.3201/eid2010.140491

(7.) Sun M, Ma J, Wang Y, Wang M, Song W, Zhang W, et al. Genomic and epidemiological characteristics provide new insights into the phylogeographical and spatiotemporal spread of porcine epidemic diarrhea virus in Asia. J Clin Microbiol. 2015;53:148492. http://dx.doi.org/10.1128/JCM.02898-14

(8.) Martelli P, Lavazza A, Nigrelli AD, Merialdi G, Alborali LG, Pensaert MB. Epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in Italy. Vet Rec. 2008;162:307-10. http://dx.doi.org/10.1136/vr. 162.10.307

(9.) Hanke D, Jenckel M, Petrov A, Ritzmann M, Stadler J, Akimkin V, et al. Comparison of porcine epidemic diarrhea viruses from Germany and the United States, 2014. Emerg Infect Dis. 2015;21:493-6. http://dx.doi.org/10.3201/eid2103.141165

(10.) Grasland B, Bigault L, Bernard C, Quenault H, Toulouse O, Fablet C, et al. Complete genome sequence of a porcine epidemic diarrhea S gene indel strain isolated in France in December 2014. Genome Announc. 2015;3:e00535-15. http://dx.doi.org/10.1128/genomeA.00535-15

(11.) Theuns S, Conceijao-Neto N, Christiaens I, Zeller M, Desmarets LMB, Roukaerts IDM, et al. Complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in Belgium, January 2015. Genome Announc. 2015; 3:e00506-15. http://dx.doi.org/10.1128/genomeA.00506-15

(12.) Sozzi E, Luppi A, Lelli D, Moreno Martin A, Canelli E, Brocchi E, et al. Comparison of enzyme-linked immunosorbent assay and RTPCR or the detection of porcine epidemic diarrhoea virus. Res Vet Sci. 2010;88:166-8. http://dx.doi.org/10.1016/j.rvsc.2009.05.009

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(15.) Kim L, Chang KO, Sestak K, Parwani A, Saif LJ. Development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs. J Vet Diagn Invest. 2000;12:385-8. http://dx.doi.org/10.1177/104063870001200418

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: mariabeatrice.boniotti@izsler.it

M. Beatrice Boniotti, [1] Alice Papetti, [1] Antonio Lavazza, Giovanni Alborali, Enrica Sozzi, Chiara Chiapponi, Silvia Faccini, Paolo Bonilauri, Paolo Cordioli, [2] Douglas Marthaler

[1] These first authors contributed equally to this article.

[2] Deceased.

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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Title Annotation:DISPATCHES
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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