








key: cord-253850-e3l5xtc2
authors: Wang, M.; Wang, Y.; Baloch, A. R.; Pan, Y.; Tian, L.; Xu, F.; Shivaramu, S.; Chen, S.; Zeng, Q.
title: Detection and genetic characterization of porcine deltacoronavirus in Tibetan pigs surrounding the Qinghai–Tibet Plateau of China
date: 2018-01-23
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.12819
sha: 
doc_id: 253850
cord_uid: e3l5xtc2

Porcine deltacoronavirus (PDCoV) is a recently discovered RNA virus that belongs to the family Coronaviridae and genus Deltacoronavirus. This virus causes enteric disease in piglets that is characterized by enteritis and diarrhoea. In our present investigation, 189 diarrhoeic samples were collected between July 2016 and May 2017 from Tibetan pigs inhabiting in three different provinces surrounding the Qinghai–Tibet Plateau of China. We then applied the molecular‐based method of reverse transcription polymerase chain reactions (RT‐PCRs) to detect the presence of PDCoV in collected samples, and RT‐PCR indicated that the prevalence of PDCoV was 3.70% (7/189) in Tibetan pigs. Four of 7 PDCoV‐positive pigs were monoinfections of PDCoV, three samples were co‐infections of PDCoV with porcine epidemic diarrhoea virus (PEDV), and 52 (27.51%) samples were positive for PEDV. Four strains with different full‐length genomes were identified (CHN/GS/2016/1, CHN/GS/2016/2, CHN/GS‐/2017/1 and CHN/QH/2017/1), and their genomes were used to analyse the characteristics of PDCoV currently prevalent in Tibetan pigs. We found a 3‐nt insertion in the spike gene in four strains in Tibetan pigs. Phylogenetic analysis of the complete genome and spike and nucleocapsid gene sequences revealed that these strains shared ancestors with the strain CHN‐AH‐2004, which was found in pigs from the Anhui province of China mainland. However, PDCoV strains from Tibetan pigs formed different branches within the same cluster, implying continuous evolution in the field. Our present findings highlight the importance of epidemiologic surveillance to limit the spread of PDCoV in livestock at high altitudes in China.

The subfamily comprises four genera that is Alphacoronavirus, Betacoronavirus, Gamacoronavirus and Deltacoronavirus. Porcine deltacoronavirus (PDCoV) was first reported in Hong Kong in 2012 as an emerging genus prevalent in certain animal species, including swine (Woo et al., 2012) . Infection by PDCoV can be symptomatically compared with other porcine enteric coronavirus diseases caused by transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhoea virus (PEDV); however, PDCoV exhibits milder symptoms and lower mortality rates in affected neonatal piglets (Wang, Byrum, & Zhang, 2014) .

After the first report of PDCoV infection (Woo et al., 2012) , its prevalence was detected in pigs in several states of the United States, South Korea, mainland China, Southern China and Thailand with majority of piglets having clinical diarrhoeal disease (Dong et al., 2015; Lee et al., 2016; Mai et al., 2017; Saeng-Chuto et al., 2017; Wang et al., 2014) . The prevalence of PDCoV in certain regions of the world is intriguing with regard to its epidemiology, evolution and pathogenicity. Here, we are the first to report PDCoV infection in Tibetan pigs from the Qinghai-Tibet Plateau of China.

Tibetan pigs mainly live in Gansu, Qinghai, Sichuan and Tibet provinces, which surround the Qinghai-Tibet Plateau of China (altitude > 3,000 m, average annual temperature <0°C). Due to the cold and harsh environment, few viral infections have been reported in animals in these areas until recent years. Tibetan pigs had no history of travel to areas where a prevalence of CoVs had been reported earlier. Nevertheless, livestock such as pigs and yaks (Bos grunniens), which have been associated with clinical diarrhoeal disease have been reported in these provinces (Gong et al., 2014; Wang, Lan, & Yang, 2016 (Table 1) . Soon after sampling, 10% (wt/vol) faecal suspensions were prepared using sterile phosphate-buffered saline (PBS). Supernatants were separated after samples were centrifuged, and samples were then stored at À80°C for RNA extraction.

Total RNAs were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then used as templates to generate full-length cDNA by reverse transcription PCR (RT-PCR; SuperScript III Synthesis Kit, Invitrogen) according to the manufacturer's instructions. For higher specificity, two pairs of specific primers were used to detect PDCoV as described previously (Wang et al., 2014) but with some modifications (Table 2) . RT-PCR was performed in a 20-ll volume containing 1 ll of template and 0.1 lmol/l each primer; the reactions were subjected to 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 58°C for 25 s and 72°C for 30 s, with a final extension step of 10 min at 72°C. RT-PCR-amplified DNA fragments of expected sizes were submitted to a commercial company and sequenced in both directions by Sanger sequencing (Sangon Biotech, Shanghai, China). Before the presence of PDCoV was determined, the presence of PEDV and TGEV was examined with primers specific for the spike gene of PEDV and the N gene of TGEV as described previously (Kim, Choi, Kim, & Chae, 2000; Sinha, Gauger, Zhang, Yoon, & Harmon, 2015; Temeeyasen et al., 2014) . T A B L E 2 Primers used for detection and full-length genome amplification of porcine deltacoronavirus (PDCoV) in Tibetan pigs, as described previously (5), with some modifications

PDCoV-M-67F 5 0 -ATCCTCCARGGAGGCTATGC-3 0 23104-20597 494 PDCoV detection (4,5)

PDCoV-25420-R 5 0 -TGCTCCATCCCCCCTATAAG-3 0 of all examined samples were positive for PEDV. In seven PDCoVpositive samples, three were positive for PEDV; of these, one sample was from Gansu province, and two were from Qinghai province. The prevalence of PDCoV+PEDV was only 1.59% in Tibetan pigs that were associated with clinical diarrhoeal disease. We also found that all Tibetan pigs infected with PDCoV were under 1 month of age.

Nevertheless, both Tibetan pigs under 1 month of age and older than 1 month could be infected with PEDV.

Tibetan pigs were also located in the same separate subcluster (Figure 2b and c).

In our present study, we found that the prevalences of PDCoV, et al., 2015) . Additionally, all Tibetan pigs infected with PDCoV were under one month of age, therefore, indicating that the prevalence of PDCoV was related to the ages of Tibetan pigs (Table 1) , consistent with previous studies (Mai et al., 2017; Song et al., 2015) . Nevertheless, PDCoV-positive Tibetan pigs showed mild clinical diarrhoeal disease, and no mortality was recorded among the infected pigs with clinical diarrhoeal disease, which is inconsistent with previous reports (Janetanakit et al., 2016; Jang et al., 2017) . These results suggest that subclinical infection of PDCoV occurs in Tibetan pigs, which are emerging but imperfect hosts for the PDCoV.

Tibetan pigs have evolved for thousands of years as a unique and indigenous breed in China. Living in cold and harsh environments on the plateau for a long period of time, the Tibetan pigs have undergone a specific selection to enrich disease resistance-related genes in their genome (Li et al., 2013; Megens et al., 2008) . The lower prevalence of PDCoV and PEDV in Tibetan pigs that is demonstrated in our study is consistent with the prevalence of other pathogens infecting the Tibetan pigs (Fan et al., 2016; Liu et al., 2014 ing the continuous evolution and adaption of PDCoV to its hosts in special field conditions. Our results also complement the geographical lineage theory of global PDCoV distribution. Furthermore, investigating variations in other genes will also provide additional data to determine the diversity of the PDCoV genome, and we strongly suggest that effective vaccination against PDCoV is not ignored in Tibetan pigs in the studied areas.

We thank the China Animal Health and Epidemiology Center for their valuable assistance in sample collection.

The authors declare no conflict of interests. 

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