LETTERS

Porcine diagnosis of diarrheal disease from transcription PCR (Figure). Of those farms in 10 US states. We report the 109 samples, 19 (17%) were also posi- HKU15 detection and phylogenetic analy- tive for porcine epidemic diarrhea vi- Detected in 9 US ses of PorCoV HKU15 strains from rus. This result suggests that PorCoV States, 2014 these samples. HKU15 is prevalent among pig popu- Between February 7, 2014, when lations in the major pig-producing To the Editor: Porcine corona- PorCoV HKU15-OH1987 was first US states. (PorCoV) HKU15 is a single- identified in Ohio (2), and April 9, To determine the genetic diver- stranded, positive-sense, enveloped 2014, the Animal Disease Diagnos- sity of PorCoV HKU15 strains from RNA virus belonging to the genus tic Laboratory of the Ohio Depart- the 9 states, we conducted whole- (family Coronaviri- ment of Agriculture received >2,000 genome sequencing for 1 strain dae). PorCoV HKU15 was first iden- swine samples from farms in 10 US from each state by using 16 pairs of tified in 2012 in a surveillance study states for diagnosis of diarrheal dis- previously described, overlapping from China (1). Until February 2014, ease. The states from which samples primers (2). Strain names were des- however, the role of this virus strain had been submitted were Minnesota, ignated by the state abbreviation and in clinical diseases of pigs had not South Dakota, Nebraska, Illinois, In- case number. The complete sequence been reported. diana, Michigan, Kentucky, Pennsyl- for HKU15-OH1987 was reported We recently reported the de- vania, Maryland, and Ohio. Among previously (2). Sequence analysis tection of PorCoV strain HKU15- those samples, 435 were selected to showed that strains from South Da- OH1987 in feces samples from sows be tested for the presence of PorCoV kota (SD3424), Nebraska (NE3579), and intestine samples from piglets in HKU15. A real-time reverse transcrip- Illinois (IL2768), Indiana (IN2847), Ohio, United States; the infected ani- tion PCR assay targeting the mem- Kentucky (KY4813), Michi- mals were from swine farms where brane protein gene was used to iden- gan (MI6148), and Pennsylvania outbreaks of diarrheal disease had oc- tify PorCoV HKU15. Samples with (PA3148) have the same genome size curred in late January and early Febru- a cycle threshold value of <35 were (25,422 nt) as OH1987, and whole- ary 2014 (2). Genetic analysis showed considered positive on the basis of genome pairwise comparison showed that HKU15-OH1987 is closely re- validation data using the cloned mem- that they share a high nucleotide lated to 2 deltacoronavirus strains that brane protein gene (data not shown). similarity (>99.8%). Furthermore, all were detected in Hong Kong, China, Of the 435 samples, 109 (25%) from 9 of the isolates share high nucleotide in 2012: HKU15-155 and HKU15-44 states (all states mentioned above, ex- similarity (98.9%–99.2%) with the 2 (2). We also demonstrated the pres- cluding Maryland) were positive for PorCoV HKU15 strains in GenBank, ence of histopathologic lesions in the PorCoV HKU15 by real-time reverse HKU15-155 and HKU15-44. small intestines of PorCoV HKU15– infected piglets with diarrhea (L. Wang, unpub. data). In April 2014, a novel swine enteric coronavirus dis- ease caused by PorCoV HKU15 or porcine epidemic diarrhea virus was reported to the World Animal Health Organization by the US Department of Agriculture (http://www.oie.int/ wahis_2/public/wahid.php/Review report/Review?page_refer=MapFullE ventReport&reportid=15133). PorCoV HKU15 is now recog- nized as a key pathogenic cause of di- arrheal diseases in pigs in the United States. However, the geographic dis- tribution and genotype diversity of PorCoV HKU15 in this country are Figure. US states with swine samples positive for porcine coronavirus (PorCoV) HKU15, between February and April 2014. A total of 435 samples from 10 states were selected to still not clear. To further our knowl- be tested for the presence of PorCoV. Of those samples, 109 (25%) from 9 states (dark edge of the virus, we analyzed swine gray) were positive for PorCoV HKU15. Another recent article reported the presence of samples that had been submitted for PorCoV HKU15 in Iowa (white) (3). Strain names are indicated below state abbreviations.

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A phylogenetic tree constructed Acknowledgments Schistosomiasis by using the entire sequence showed We acknowledge and appreciate the that all PorCoV HKU15 strains from excellent technical help provided by Jason Haematobium, the United States clustered together in Herr and Kerri Lawrence. Corsica, France 1 clade of the genus Deltacoronavi- rus with HKU15-155 and HKU15-44 Leyi Wang,1 Beverly Byrum, To the Editor: In Europe, urinary (online Technical Appendix Figure, and Yan Zhang1 schistosomiasis (1) has previously panel A, http://wwwnc.cdc.gov/EID/ Author affiliation: Ohio Department of Agri- been detected only in Portugal, where article/20/9/14-0756-Techapp1.pdf). culture, Reynoldsburg, Ohio, USA this focus disappeared during the This finding indicates that 1 genotype 1950s (2). However, freshwater snails of PorCoV HKU15 is currently cir- DOI: http://dx.doi.org/10.3209/eid2009.140756 of the species Bulinus contortus, B. culating in multiple US states. This truncatus, and Planorbarius metidjen- result was further supported by phylo- References sis, which are recognized intermediate genetic trees constructed by using the 1. Woo PC, Lau SK, Lam CS, Lau CC, hosts for Schistosoma haematobium full-length amino acids of spike and Tsang AK, Lau JH, et al. Discovery of sev- trematodes, have been found in Portu- nucleocapsid proteins (online Tech- en novel mammalian and avian coronavi- gal (3), Spain (4), and Corsica (5,6). nical Appendix Figure, panels B, C). ruses in the genus Deltacoronavirus sup- This finding suggested that autochtho- ports bat as the gene source Because of limitation of the samples of and Betacoronavi- nous schistosomiasis could re-emerge received, only a partial genome se- rus and avian coronaviruses as the gene in southern Europe if these mollusks quence was determined for strain source of and Delta- become infected. We report a probable MN3092 from Minnesota. However, coronavirus. J Virol. 2012;86:3995–4008. focus for transmission of schistosomi- http://dx.doi.org/10.1128/JVI.06540-11 on the basis of the spike and nucleo- 2. Wang L, Byrum B, Zhang Y. Detection asis haematobium in Corsica, France. capsid protein sequence analyses, it is and genetic characterization of a delta- In March 2014, a 4-year-old highly likely that the entire genome coronavirus in pigs in the United States. girl (index case-patient) from France of the Minnesota strain is genetically Emerg Infect Dis. 2014; was referred to the Toulouse Univer- 3. Li G, Chen Q, Harmon KM, Yoon KJ, identical to that of the other 8 strains Schwartz KJ, Hoogland MJ, et al. Full- sity Hospital (Toulouse, France), with (online Technical Appendix Figure, length genome sequence of porcine del- gross hematuria. Ultrasonography and panels B, C). tacoronavirus strain USA/IA/2014/8734. cystoscopic examination of the blad- In addition to the 9 states report- Genome Announc. 2014;2:e00278-14. der detected a polyp. Examination of http://dx.doi.org/10.1128/genomeA. ed in this study, Iowa has also had a 00278-14 the polyp for parasites identified bod- recent detection of PorCoV HKU15 4. Chu DK, Leung CY, Gilbert M, ies that were consistent with schisto- (3). Thus, PorCoV HKU15 has been Joyner PH, Ng EM, Tse TM, et al. some eggs. Parasitologic examination detected in 10 of the 50 US states, and Avian coronavirus in wild aquatic birds. of urine confirmed schistosomiasis by J Virol. 2011;85:12815–20. http://dx.doi. those 10 states mainly cluster in the org/10.1128/ JVI.05838-11 detecting viable S. haematobium eggs. midwestern United States (Figure). The parents of the girl (family As with data collected for porcine Address for correspondence: Yan Zhang, A) did not report any stay or travel epidemic diarrhea outbreaks by the Animal Disease Diagnostic Laboratory, Ohio in an area to which urinary schisto- US National Animal Health Labo- Department of Agriculture, 8995 East Main St, somiasis was endemic; they reported ratory Network (http://www.nahln. Bldg. 6, Reynoldsburg, OH 43068, USA; email: summer holidays only in Mallorca in org/default/), data collected on the [email protected] the Balearic Islands (Spain) and Cor- geographic location and numbers of sica. However, her father reported that 1These authors were co–principal PorCoV HKU15 cases is also required since 2012, he had experienced gross investigators. to be reported weekly. hematuria that had been evaluated by Earlier reports support avian standard urologic investigations but coronaviruses as the gene source for not by cystoscopy; no etiology was Deltacoronavirus spp. (1,4). To con- determined. Parasitologic urinalysis firm this, surveillance for PorCoV in our hospital department showed nu- should be carried out among birds. merous viable S. haematobium eggs in Moreover, effective control strate- the father’s urine. gies, including vaccine development, The parents of the index case-pa- should be in place for prevention tient also reported that an 8-year-old and control of infections caused by boy in a friend’s family (family B), PorCoV HKU15. who shared summer vacations with

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Article DOI: http://dx.doi.org/10.3201/eid2009.140756 Porcine Coronavirus HKU15 Detected in 9 US States, 2014

Technical Appendix

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Technical Appendix Figure. Phylogenetic tree constructed on the basis of the whole genome sequences (A), spike protein (B) and nucleocapsid protein (C) of 4 genera of coronaviruses (Alphacoronavirus, , Gammacoronavirus, and Deltacoronavirus), including the 9 US strains of porcine coronavirus HKU15 (indicated by red triangle). The dendrogram was constructed by using the neighbor- joining method in the MEGA software package, version 6.05 (http://www.megasoftware.net). Bootstrap resampling (1,000 replications) was performed, and bootstrap values are indicated for each node. Reference sequences obtained from GenBank are indicated by strain name and accession number. Scale bar represents 0.5 nt (A) or 0.1 aa (B, C) substitutions per site. PEDV, porcine epidemic diarrhea virus; PHEV, porcine hemagglutinating encephalomyelitis virus; PRCV, porcine respiratory coronavirus; TGEV, transmissible gastroenteritis coronavirus; SARS, severe acute respiratory syndrome.

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