








key: cord-289584-rbp7p8s9
authors: Zhou, Ling; Sun, Yuan; Lan, Tian; Wu, Ruiting; Chen, Junwei; Wu, Zixian; Xie, Qingmei; Zhang, Xiangbin; Ma, Jingyun
title: Retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern China
date: 2019-01-09
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.13008
sha: 
doc_id: 289584
cord_uid: rbp7p8s9

Swine acute diarrhoea syndrome coronavirus (SADS‐CoV), a novel coronavirus, was first discovered in southern China in January 2017 and caused a large scale outbreak of fatal diarrheal disease in piglets. Here, we conducted a retrospective investigation of 236 samples from 45 swine farms with a clinical history of diarrheal disease to evaluate the emergence and the distribution of SADS‐CoV in pigs in China. Our results suggest that SADS‐CoV has emerged in China at least since August 2016. Meanwhile, we detected a prevalence of SADS‐CoV (43.53%), porcine deltacoronavirus (8.83%), porcine epidemic diarrhoea virus (PEDV) (78.25%), rotavirus (21.77%), and transmissible gastroenteritis virus (0%), and we also found the co‐infection of SADS‐CoV and PEDV occurred most frequently with the rate of 17.65%. We screened and obtained two new complete genomes, five N and five S genes of SADS‐CoV. Phylogenetic analysis based on these sequences revealed that all SADS‐CoV sequences in this study clustered with previously reported SADS‐CoV strains to form a well defined branch that grouped with the bat coronavirus HKU2 strains. This study is the first retrospective investigation for SADS‐CoV and provides the epidemiological information of this new virus in China, which highlights the urgency to develop effective measures to control SADS‐CoV.

Swine acute diarrhoea syndrome coronavirus (SADS-CoV) is a newly discovered coronavirus which is an enveloped, positive and singlestranded sense RNA virus with a genome size of approximately 27 kb (Gong et al., 2017; Pan et al., 2017; Zhou et al., 2018) .

SADS-CoV belongs to the family Coronaviridae which contains four genera, Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (Woo, Huang, Lau, & Yuen, 2010; Woo et al., 2012) . So far, six coronaviruses have been identified from pigs, which include porcine epidemic diarrhoea virus (PEDV), porcine respiratory coronavirus (PRCV), SADS-CoV and transmissible gastroenteritis virus (TGEV) that all belong to the Alphacoronavirus genus, as well as one betacoronavirus, porcine hemagglutinating encephalomyelitis virus (PHEV) and one deltacoronavirus, porcine deltacoronavirus (PDCoV) (Lin, Saif, Marthaler, & Wang, 2016; Wesley, Woods, & Cheung, 1991; Woo et al., 2010) . Among these viruses, SADS-CoV is the most newly discovered coronavirus, which has been first reported in 2017 in China and is considered to be an HKU2-related coronavirus with a bat-origin (Gong et al., 2017; Zhou et al., 2018) . In January 2017, SADS-CoV was detected in a swine farm and subsequently spread rapidly to three other farms in *These authors contributed equally to this work. Guangdong Province and caused the fatal swine acute diarrhoea syndrome (SADS) characterized by the clinical signs with severe, acute diarrhoea and rapid weight loss of piglets. The symptoms of SADS-CoV are similar to those that caused by other swine enteric coronaviruses such as PDCoV and PEDV, but SADS-CoV is more harmful than these viruses because it has led to the death of almost 25,000 piglets in a short time and resulted in more significant economic losses (Dong et al., 2015; Sun, Wang, Wei, Chen, & Feng, 2016; Zhou et al., 2018) . So, it is urgent to investigate the molecular epidemiology and transmission patterns of SADS-CoV for establishing effective controls for this new coronavirus.

In the present study, we performed the retrospective PCR testing on diarrheal samples from 45 swine farms in Guangdong Province to evaluate the emergence and the distribution of SADS-CoV in pigs in China. The prevalence and co-infection information of SADS-CoV from eleven SADS-CoV-positive farms was provided. The sequences of SADS-CoV, including two complete genomes, five nucleocapsid protein (N) genes and five spike protein (S) genes, were also identified and characterized to investigate the phylogenetic relationships of SADS-CoV. 

Samples were homogenized in phosphate-buffered saline (PBS) (20% w/v), frozen and thawed three times, then centrifuged for 10 min at 10,000 g. Viral nucleic acid was extracted following the manufacturer's recommendations of AxyPrep TM Body Fluid Viral DNA/RNA Miniprep Kit (Axygen Scientific, Inc). The virus nucleic acid was stored at −80°C until PCR was performed. A pair of primers (forward primer 5′-GGTCCCTGTGACCGAAGTTTTAG-3′, reverse primer 5′-GCGTTCTGCGATAAAGCTTAAAACTATTA-3′) was designed to detected SADS-CoV based on the conserved N gene of this virus.

One step RT-PCR using PrimeScript ™ One Step RT-PCR Kit Ver.2 with Dye Plus (Takara, Biotechnology, Dalian, China) was carried out to amplify the target fragments by the following thermal profile of 50°C for 30 min, 94°C for 3 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, an extension at 72°C for 30 s, and a final step of 72°C for 5 min. Four other diarrheal pathogens including PEDV, PDCoV, rotavirus (RV), and TGEV from SADS-CoVpositive farms were also tested by RT-PCR according to the previously described methods (Liu, Zhu, Liao, Xu, & Zhou, 2015; Mai et al., 2017; Stevenson et al., 2013) .

Specific primer pairs based on reported SADS-CoV strains (GenBank accession numbers: MF094681-MF094684) were designed for S genes, N genes and complete genome amplifications, respectively (Table S1 ). PCR assays were performed with the following thermal profile: 95°C for 5 min, 35 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min 15 s, followed by a final 10 min extension at 72°C. The products were purified following the manufacturer's 

The nucleotide sequences were assembled and aligned using the DNASTAR program (DNAStar V7.1, Madison, WI, USA). Phylogenetic trees were constructed using the neighbour-joining method in MEGA 7.0 software with bootstrap analysis of 1,000 replicates. Percentages of replicate trees in which the associated taxa clustered are shown as nearby branches (Chenna et al., 2003; Kumar, Stecher, & Tamura, 2016; Tamura, Nei, & Kumar, 2004 ). 

The two complete genomes (accession number MG605090 and F I G U R E 4 Phylogenetic analysis of the S genes of SADS-CoV and reference coronavirus species. The tree was constructed as per Figure 2 above. Five new sequences of S genes studied in this work were indicated with "black solid circles" diarrhoea samples. Our results showed that the first SADS-CoV positive sample was collected in August 2016 from the farm LS with a history of diarrhoea, as well as from other two farms TP and ZW, which indicates that SADS-CoV has emerged in pigs in China at least since August 2016. And this time point is 5 months earlier than the first discovered time reported by our previous study (Zhou et al., 2018) . As the same time, clinical signs of SADS-CoV during the retrospective investigation included sever and acute vomiting and diarrhoea, leading to death in piglets that were less than 5 days of age with a mortality rate of around 50%. These clinical presentations were similar to those signs in the large scale outbreak of SADS-CoV reported by Zhou et al. (2018) , except the mortality rate in piglets later increased to 90%.

Based on the rates of infection documented in our work, it revealed that PEDV (78.25%) was still the primary cause of the porcine diarrhoea, which is consistent with previous studies that PEDV has been considered to be the major pathogen responsible for the porcine diarrhoea epidemic in China since 2010 (Ge et al., 2013; Sun et al., 2012; Zhao et al., 2016) . The phylogenetic relationships of SADS-CoV sequences were also identified in this study. The results showed that all SADS-CoV sequences clustered together to form an independent branch and separated from other viral sequences in the genus Alphacoronavirus.

Our results also indicated that both the complete genomes, N genes and S genes of all SADS-CoV strains shared the highest nucleotides identifies with those corresponding sequences of four bat coronavirus HKU2 strains. In this work, The phylogenetic trees of full length genomes and S genes of SADS-CoV sequences showed that the SADS-CoV branch clustered with these four HKU2 strains, which is same to previous results (Gong et al., 2017; Pan et al., 2017; Zhou et al., 2018) . Besides the genomes and S genes, the tree of N genes in our study revealed the identical result too. So far, a total of eight full-length genomes of SADS-CoV have been reported in Guangdong

Province of China (Gong et al., 2017; Pan et al., 2017; Zhou et al., 2018 ; this study). The two new genomes of SADS-CoV sequences in this work shared 100% nucleotides identities with the sequence MF167434 published by Gong et al. (2017) and our four previously reported sequences (Zhou et al., 2018) , and shared 99.8% nucleotides identities with the sequence MF370205 studied by Pan et al. (2017) . The results suggest that these eight SADS-CoV sequences may come from the same origin. Only the phylogenetic tree of S genes in our work showed that sequences of the AlphaCoV were divided into two sublineages, AlphaCoV1 which contained all SADS-CoV sequences and AlphaCoV2, clustering together with sequences of the BeltaCoV and the DelatCov, respectively. And this result was consistent with the study of Pan et al. (2017) . As a newly discovered coronavirus, the availability of SADS-CoV sequences data is limited which prevents better understandings of the molecular epidemiology of this virus. Meanwhile, being a RNA virus, SADS-CoV may mutate rapidly and exhibit high genetic differences (Drummond, Pybus, Rambaut, Forsberg, & Rodrigo, 2003; Kühnert, Wu, & Drummond, 2011 

The authors declare no conflict of interests with any organization.

Ma https://orcid.org/0000-0001-6285-312X

Infectious Diseases, 23, 1607-1609. https://doi.org/10.3201/eid2309.

Porcine enteric alphacoronavirus GDS04 | MF167434

Rhinolophus bat coronavirus HKU2 CH/GD-01/2017/P2 | MF370205

Bat coronavirus HKU2 | NC009988

Bat coronavirus HKU2 strain HKU2/HK/46

Bat coronavirus HKU2 strain HKU2/HK/33

Human coronavirus 229E | NC002645

Camel alphacoronavirus isolate Riyadh/Ry141/2015 | NC028752

229E-related bat coronavirus strain BtKY229E-1 | KY073747

Porcine enteric alphacoronavirus GDS04 | MF167434

Rhinolophus bat coronavirus HKU2 CH/GD-01/2017/P2 | MF370205

Bat coronavirus HKU2 strain HKU2/HK/33

Bat coronavirus HKU2 strain HKU2/HK/46

Bat coronavirus 1A | NC010437

Bat coronavirus HKU8 strain AFCD77 | EU420139

Rousettus bat coronavirus HKU10 | NC01887

Porcine epidemic diarrhoea virus strain GDS01 | KM089829

Porcine epidemic diarrhoea virus strain CV777 | KT323979

Porcine epidemic diarrhoea virus| NC003436

Human Coronavirus NL63 | NC005831.2 NL63-related bat coronavirus strain BtKYNL63-9a | NC032107

229E-related bat coronavirus strain BtKY229E-1 | KY073747

Camel alphacoronavirus isolate Riyadh/Ry141/2015 | NC028752

Human coronavirus 229E | NC002645

Porcine hemagglutinating encephalomyelitis virus | NC007732

Human coronavirus OC43 strain SC2481 | KY983583

Mouse hepatitis virus strain MHV-A59 C12 mutant | NC001846

Murine hepatitis virus strain JHM complete genome | AC000192

Middle East respiratory syndrome coronavirus | NC019843

Bat SARS coronavirus HKU3-1 | DQ022305

Bat SARS-like coronavirus RsSHC014 | KC881005

Bat SARS-like coronavirus WIV1 | KF367457

European turkey coronavirus 080385d | KR822424

Bulbul coronavirus HKU11-796 | FJ376620

Porcine deltacoronavirus isolate PDCoV/CHJXNI2/2015 | KR131621.1

Porcine coronavirus HKU15 strain HKU15-44 | NC016990

Rhinolophus bat coronavirus HKU2 CH/GD-01/2017/P2 | MF370205

Bat coronavirus HKU2 strain HKU2/HK/46

Bat coronavirus HKU2 strain HKU2/HK/33

Human coronavirus OC43 strain SC2481 | KY983583

Porcine hemagglutinating encephalomyelitis virus | NC007732

Mouse hepatitis virus strain MHV-A59 C12 mutant | NC001846

Murine hepatitis virus strain JHM complete genome | AC000192

Middle East respiratory syndrome coronavirus | NC019843

Bat SARS coronavirus HKU3-1 | DQ022305.2 SARS coronavirus | NC004718

Bat SARS-like coronavirus RsSHC014 | KC881005

Bat SARS-like coronavirus WIV1 | KF367457

European turkey coronavirus 080385d | KR822424

Porcine deltacoronavirus isolate PDCoV/CHJXNI2/2015 | KR131621.1

Porcine coronavirus HKU15 strain HKU15-44 | NC016990

Bulbul coronavirus HKU11-796 | FJ376620

Bat coronavirus HKU8 strain AFCD77 | EU420139

Bat coronavirus 1A | NC010437

Camel alphacoronavirus isolate Riyadh/Ry141/2015 | NC028752

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