Veterinary Microbiology 211 (2017) 15–21 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic Discovery of a novel swine enteric alphacoronavirus (SeACoV) in southern MARK China Yongfei Pana,b, Xiaoyan Tianb, Pan Qina, Bin Wanga, Pengwei Zhaoa, Yong-Le Yanga, ⁎⁎ ⁎ Lianxiang Wangb, Dongdong Wangb, Yanhua Songb, Xiangbin Zhangb,c, , Yao-Wei Huanga, a Institute of Preventive Veterinary Medicine and Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou 310058, China b Hog Production Division, Guangdong Wen’s Foodstuffs Group Co, Ltd, Xinxing, 527439, China c College of Animal Sciences, South China Agricultural University, Guangzhou 510642, China ARTICLE INFO ABSTRACT Keywords: Outbreaks of diarrhea in newborn piglets without detection of transmissible gastroenteritis virus (TGEV), por- Swine enteric alphacoronavirus (SeACoV) cine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV), have been recorded in a pig farm in Bat southern China since February 2017. Isolation and propagation of the pathogen in cell culture resulted in dis- Spike glycoprotein covery of a novel swine enteric alphacoronavirus (tentatively named SeACoV) related to the bat coronavirus Cross-species transmission HKU2 identified in the same region a decade ago. Specific fluorescence signal was detected in Vero cells infected with SeACoV by using a positive sow serum collected in the same farm, but not by using TGEV-, PEDV- or PDCoV-specific antibody. Electron microscopy observation demonstrated that the virus particle with surface projections was 100–120 nm in diameter. Complete genomic sequencing and analyses of SeACoV indicated that the extreme amino-terminal domain of the SeACoV spike (S) glycoprotein structurally similar to the domain 0 of the alphacoronavirus NL63, whereas the rest part of S structurally resembles domains B to D of the betacor- onavirus. The SeACoV-S domain 0 associated with enteric tropism had an extremely high variability, harboring 75-amino-acid (aa) substitutions and a 2-aa insertion, compared to that of HKU2, which is likely responsible for the extended host range or cross-species transmission. The isolated virus was infectious in pigs when inoculated orally into 3-day-old newborn piglets, leading to clinical signs of diarrhea and fecal virus shedding. These results confirmed that it is a novel swine enteric coronavirus representing the fifth porcine coronavirus. 1. Introduction fecal samples in Europe (Akimkin et al., 2016; Belsham et al., 2016; Boniotti et al., 2016), implying that novel SeCoV pathogens could Coronavirus (CoV) is an enveloped, single-stranded, positive-sense emerge by inter-CoV recombination under co-infection. The S gene RNA virus of the order Nidovirales, family Coronaviridae, subfamily encodes a glycoprotein, forming trimer projections on the viral surface, Coronavirinae, which comprises four genera, Alpha-, Beta-, Gamma-, and which is a major structural protein critical for CoV attachment and Delta-CoV. CoVs infect humans, other mammals, and birds, causing entry into the host cell (Hulswit et al., 2016). subclinical or respiratory and gastrointestinal diseases (de Groot et al., In addition to recombination events between two distinct CoVs, 2011; Woo et al., 2012). As of date, three types of swine enteric CoVs amino acid (aa) mutations in the S protein may alter the tropism of the (SeCoVs): transmissible gastroenteritis virus (TGEV), porcine epidemic virus. For example, 21-aa substitutions and a 7-aa insertion in the diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV), have amino-terminal domain (NTD) of the S glycoprotein of a murine he- been identified to induce clinical diarrhea in young pigs (Jung et al., patitis CoV (MHV) variant confer the ability to bind and in some cases 2016; Pensaert and de Bouck, 1978). In particular, emergences of infect cells of nonmurine species including swine cells (Schickli et al., variant PEDV fatal to newborn piglets in China in late 2010 (Pan et al., 2004). In this study, we report the isolation and genetic characteriza- 2012), and later in the United States in 2013 (Huang et al., 2013; Tian tion of a novel swine enteric alphacoronavirus (tentatively named et al., 2014), have posed a serious threat to the pork industry. Most SeACoV), related to a bat enteric coronavirus, from a pig farm that recently, several chimeric SeCoV strains with a TGEV genomic back- reported newborn-piglet diarrhea in southern China in 2017. This is yet bone replaced by a PEDV spike (S) gene were identified from swine another example to corroborate that the extended host range of CoV, ⁎ Corresponding author. Institute of Preventive Veterinary Medicine and Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou 310058, China. ⁎⁎ Corresponding author at: College of Animal Sciences, South China Agricultural University, Guangzhou 510642, China. E-mail addresses: [email protected] (X. Zhang), [email protected] (Y.-W. Huang). http://dx.doi.org/10.1016/j.vetmic.2017.09.020 Received 5 August 2017; Received in revised form 27 September 2017; Accepted 27 September 2017 0378-1135/ © 2017 Elsevier B.V. All rights reserved. Y. Pan et al. Veterinary Microbiology 211 (2017) 15–21 here from bat to pig, is likely associated with aa substitutions at the visualized under a fluorescence microscope. NTD of the S glycoprotein. Furthermore, we conducted a pilot experi- For antibody cross-reactivity test, Vero cells infected with SeACoV mental infection study with this novel SeACoV, confirming its in- or PEDV (ZJU/G2/2013 strain; GenBank accession no. KU558701), fectivity and ability to induced clinical signs of diarrhea in piglets. Vero-pAPN cells infected with TGEV (Purdue strain; a gift from Dr. Rong Ye at Shanghai Medical College of Fudan University), and LLC- 2. Materials and methods PK1 cells infected with PDCoV (Hunan strain; GenBank accession no. KY513724) were stained with the anti-PEDV-N, anti-TGEV-N and anti- 2.1. Cell lines and cell cultures PDCoV-N monoclonal antibody (purchased from Medgene Labs, Brookings, SD, USA), respectively. The FITC-conjugated goat anti-mice Baby hamster kidney fibroblast cell line BHK-21 (ATCC CCL-10), IgG (Thermo Fisher Scientific, USA) was used as the secondary antibody swine testis cell line ST (ATCC CRL-1746), porcine kidney epithelial cell followed by DAPI staining. line LLC-PK1 (ATCC CL-101), and African green monkey kidney epi- thelial Vero cell (ATCC CCL-81) were individually grown in Dulbecco’s 2.6. Genomic cloning and bioinformatics analyses modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin, streptomycin, w/v). A Vero Total RNA was extracted from the isolated virus with TRIzol re- cell line stably expressing the TGEV receptor porcine aminopeptidase N agent, and cDNAs were subsequently amplified by SuperScript II with (Vero-pAPN) was cultured in DMEM supplemented with 10 μg/ml specific primers according to the manufacturer’s instructions (Thermo puromycin and antibiotics (unpublished data). All cells were grown at Fisher Scientific). A total of 16 primer pairs based upon the bat CoV 37 °C with 5% CO2. HKU2 strain GD430-2006 (GenBank accession no. EF203064; Supplemental Table S1) were designed to amplify the complete genome 2.2. RT-PCR detections of SeACoV. PCR products were purified and cloned into a pCR-Blunt vector (Thermo Fisher Scientific). For each amplicon, three to five in- A pan-CoV RT-PCR assay was used to detect the unknown pathogen dividual clones were sequenced to determine the consensus sequence. with a pair of primers: Cor-FW (5′-ACWCARHTVAAYYTNAARTAYGC- The sequences were assembled and analyzed using the DNASTAR pro- 3′) and Cor-RV (5′-TCRCAYTTDGGRTARTCCCA-3′) as described (Moes gram. Multiple alignments of the full-length genomes, non-structural et al., 2005). After the pathogen (SeACoV) was identified, specific protein genes and S genes with representative CoV sequences and primers targeting the SeACoV-nucleocapsid (N) gene (the forward phylogenetic analyses were performed using the neighbor-joining primer SEAF: 5′-ATGGATAAACCTGAATGGAAGCG-3′, and the reverse method in MEGA5.2, respectively. Structure homology-modeling of primer SEAR: 5′-CACCATCTCAACCTCTTCCTCAG-3′) were used for SeACoV S glycoprotein was performed by the SWISS-MODEL server virus detection during isolation and subsequent passages. (https://www.swissmodel.expasy.org/). 2.3. Virus isolation 2.7. SeACoV infectivity study in neonatal piglets Fecal specimens collected from diarrheic piglets and positive for A pilot animal experiment was approved by the Experimental SeACoV RNA were homogenized in DMEM containing antibiotics fol- Animal Ethics Committee of Zhejiang University (approval no. lowed by centrifugation at 4000 × g for 15 min. The supernatants was ZJU20170026). Briefly, ten 3-day-old conventional piglets, free of inoculated onto confluent monolayers of BHK-21, ST, LLC-PK1 or Vero SeACoV, PEDV, TGEV, and PDCoV RNA in the feces, were assigned into cells cultured with the maintenance medium plus trypsin (MMT) at two groups with 5 in each. Piglets in each group were housed with their 37 °C and 5% CO2. The MMT consisted of DMEM supplemented with mothers (SeACoV RNA and serum antibody negative as determined by 10% FBS, 1% antibiotics and 5 μg/ml trypsin (Sigma). Cells were ob- IFA) without any artificially supplemental colostrum or milk. Piglets in served daily to record the development of cytopathic effect