








key: cord-352211-3ps5o8ji
authors: Mai, K.; Feng, J.; Chen, G.; Li, D.; Zhou, L.; Bai, Y.; Wu, Q.; Ma, J.
title: The detection and phylogenetic analysis of porcine deltacoronavirus from Guangdong Province in Southern China
date: 2017-03-27
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.12644
sha: 
doc_id: 352211
cord_uid: 3ps5o8ji

Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus that causes diarrhoea, vomiting and dehydration in sucking and nursing piglets. It was first reported in Hong Kong in 2012 and has since been discovered in the United States, Canada, South Korea, mainland China, Thailand and Laos. PDCoV has been experimentally proved to lead to diarrhoea in swine and it was detected positive in pigs in Guangdong, southern China. In our study, 252 faecal and intestinal samples from sucking piglets and sows with diarrhoea were surveyed for common enteric viruses. We found a prevalence of PDCoV (21.8%), porcine epidemic diarrhoea virus (65.5%), transmissible gastroenteritis virus (0%), rotavirus group A (25.0%) and porcine kobuvirus (68.7%). We isolated 13 PDCoV strains and discovered that PDCoV infections were often co‐infections with kobuvirus rather than the commonly linked porcine epidemic diarrhoea virus. Phylogenetic analysis of S gene and N gene revealed that 11 of 13 PDCoV strains belonged to Chinese lineage. As for the left two strains, one single strain (CHN‐GD16‐05) belonged to American and Korean lineages while another strain (CHN‐GD16‐03) was similar to a Thai strain, but only in the S gene. This suggested a possible recombination event between the Thai and the newly described Chinese strain.

industry (Chen, Gauger et al., 2015; Chen, Zhu et al., 2015; Homwong et al., 2016; Song et al., 2015; Wang et al., 2014) .

RT-PCR detection methods have provided data for the prevalence of PDCoV in China (Dong et al., 2015; Song et al., 2015; Zhai et al., 2016) . A previous study (Zhai et al., 2016) reported that the current prevalence in Southern China was 1.54% (5/390), and three of the five detected strains were from Guangdong Province, revealing that the information regarding the molecular epidemiology of PDCoVs in Guangdong was still limited. The current study further investigates the prevalence, epidemiology and genomic properties of PDCoV in Guangdong.

In order to monitor the prevalence and sequence properties of (Song et al., 2015) . The thermal cycling (worked on Bio-Rad T100, Forster City, CA) was operated with the following thermal profile: 94°C for 5 min, 30 cycles of 94°C for 45 s, 55°C for 30 s, 72°C for 1 min and a final step of 72°C for 10 min. Amplicons were analysed on 1% agarose gels. Moreover, on the basis of the four porcine enteric pathogens below, additional RT-PCR detections from the 13 tissue cultured purified PDCoV-positive samples targeting porcine bocavirus (PBV), porcine sapelovirus (PSV) and porcine astrovirus (PAstV) were added. Primers specific for PEDV, TGEV, Rotavirus A, PKV, PBV, PSV and PAstV have been described at Table S1. 2.2 | Amplification, cloning and sequencing the Spike (S) protein and Nucleocapsid protein (N) genes The complete S and N genes were amplified using three pairs of primers: PDCoV-S1, 5 0 -ATGCAGAGAGCTCTATTG-3 0 and 5 0 -TATTT CAACTTCGCCATC-3 0 ; PDCoV-S2, 5 0 -CGACCATCCATAGTTTCA-3 0 and PDCoV-S2, 5 0 -CTACCATTCCTTAAACTT-3 0 ; and PDCoV-N, 5 0 -ATGGCCGCACCAGTAGTC-3 0 and 5 0 -CTACGCTGCTGATTCCTG-3 0 (based on CHJXNI2/2015/China). PCR amplification was carried out using the LA Taq polymerase kit (Takara, Biotechnology, Dalian, China) using directions supplied by the manufacturer. PCR products were purified using the Gel Band Purification Kit (Omega Bio-Tek, USA) and then cloned into the PMD-19T vector (Takara) using an In-fusion PCR Cloning Kit (Takara). The recombinant plasmids were sequenced by the Beijing Genomics Institute (Shenzhen, Guangdong).

Nucleotide sequences were submitted to GenBank with accession numbers (KY078891-KY078903 for nucleocapsid gene and KY078904-KY078916 for spike gene of CHN-GD16-01 to CHN-GD16-13, respectively). Reference sequences included 33 strains for the S gene and 33 strains of N gene from different farms of global isolates obtained from GenBank (Table S2 and 

The secondary structure and surface properties of S1 protein of (Table. 1 ). In addition, co-infections with PEDV and PKV were the most common (44.4%, 79/178) ( Table 2) . PDCoV occurrence was examined further by an additional RT-PCR analysis using 13 PDCoV isolates we successfully purified in cell culture (Table 3) 

We identified 11 potential and distinct B-cell antigenic epitopes in the S1 protein ( Figure S1 ). Peptides less than 4 amino acid homology

were not computed. These predicted epitopes all contained mostly b structure and high hydrophilicity, strong accessibility, good flexibility and a high, predicted antigenicity. We also identified 17 potential N- 

Until now, numerous reports had revealed that PEDV was the most common porcine enteric pathogen in pigs due to its high prevalence in diarrhoeal samples. PDCoV-PEDV co-infection occurred at a rate greater than PDCoV-Rota A and PDCoV-TGEV. But in our study, co-infection of PDCoV and PKV was greatly higher than PDCoV and PEDV co-occurrence (45.4%, 25/55 vs. 3.6%, 2/55, Table. therefore, evidence is lacking to prove that PKV co-infects more frequently than PEDV with PDCoV. This warrants further investigation.

HKU15-44 and HKU15-155 were the first two PDCoV strains that were sequenced (Woo et al., 2012) . The whole S gene deletion (Chen, Gauger et al., 2015; Chen, Zhu et al., 2015; Dong et al., 2015; Song et al., 2015; Wang et al., 2015; Zhai et al., 2016) .

In strain CHN/AH/2004 (Dong et al., 2015) , a 3-nt (TAA) deletion existed in its 3 0 -UTR. The N gene, however, lacked any nucleotide deletions or insertions.

In our work, pairwise alignment analysis revealed that two S genes from our strains 03 and 05 lacked any deletions or insertions.

T A B L E 3 Detection of porcine enteric pathogens from 13 tissue cultured purified PDCoV-positive samples from 11 swine farms in Guangdong Two pairs of strains 07 and 08 from farm G and 10 and 11 from farm I were isolated from the same commercial swine farms, but at different times. Unlike 07 and 08 that were identical in nucleotide sequence, 10 and 11 showed nucleotide identities of 99.0% and 98.3%, respectively, resulting in 10 amino acid changes. Interestingly, these differences were in the S protein and concentrated within the S1 region. According to the two phylogenetic trees established for the S and N genes, 11 of 13 strains clustered within the Chinese category and had the highest nucleotide identities with five pub- Table S2 ) (Zhai et al., 2016) , which were collected from Guangdong and nearby Guangxi provinces. However, the N gene of this strain revealed a completely reverse conclusion;

that it belongs to the Chinese group with a low nt identity (96.7%) with the Thai strains S5011 and 5015L (Janetanakit et al., 2016) ( Fig. 2 and Table S3 ). Therefore, we inferred that a recombination event might occur in PDCoV strain 03.

Recombination events are often reported in PEDV studies and the majority are intrarecombinants with different strain lineages of the same kinds of enteric coronaviruses (Jarvis et al., 2015; Tian et al., 2014) . A recent study (Boniotti et al., 2016; Valerij et al., 2016) identified a swine enteric coronavirus (SeCoV) from Italy as more closely related to PEDV in the S gene, while the remainder of the genome shared highest identity with TGEV. Recombination events have not been reported in PDCoV strains. Since we were lack of the full-length genome of this PDCoV strain, the mechanism of the construction of this chimera as well as with the recombination in strain 03 is unknown, something suppose to be further work and explain with it.

The spike protein is an important surface glycoprotein of coronavirus and plays a significant role in receptor binding and fusion of the viral and cellular membranes (Chakraborti, Prabakaran, Xiao, & Dimitrov, 2005; Schwegmann-Weßels et al., 2009) . And also, it mediates interspecies transmission (Bosch, Van, de Haan, & Rottier, 2003) . All coronavirus spike proteins share the same two functional components: an N-terminal subunit and a membrane-anchored subunit (C-terminal) that are covalently bound. The S protein of PDCoV can be similarly subdivided into S1 (1-573 aa) and S2 (574-1161 aa)

regions (Thachil, Gerber, Xiao, Huang, & Opriessnig, 2015) . S1 is a dominant viral antigen and an ELISA is available for its detection (Thachil et al., 2015) . Three different groups of S1 proteins from coviral infections shared less than 12% amino acid sequence identities with each other (Wang, Deng et al., 2016) . The B-cell response is directed against the spike protein of coronaviruses (Cao et al., 2015) and it plays an important role in pathogenesis of virus infection.

In our study, 11 potential B-cell antigenic epitopes of PDCoV ( Figure S1 ) and 16 or 17 N-glycosylation sites were analysed. Interestingly, in amino acids 549-561 in strain 05, a T559I mutation greatly reduced the predicted level of its antigenicity, hydrophilicity, surface probability and flexibility ( Figure S2 & Table S4 ). Whether this mutation has altered its antigenicity will be interesting to test in the laboratory. Overall, further studies are needed to confirm whether these alterations of B-cell antigenic epitopes and N-glycosylation sites affect the pathogenicity and antigenicity of each PDCoV strain.

In conclusion, diarrhoeal samples collected from pigs in Guangdong Province were screened to detect the prevalence of PDCoV.

Phylogenetic analysis suggested that nearly all of the strains in mainland China were clustered into the Chinese lineage except one newly discovered PDCoV strain that had a close relationship with US and Korea strains. This complements the geographical lineage theory of global PDCoV distribution. The presence of another suspected recombinant strain will provide additional data to examine the diversity of the PDCoV genome.

The study was supported by The National Key Research and Development Program (2016YFD0501300) of China.

The authors declare no conflicts of interest.

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