key: cord-0809359-47czdddb
authors: Liu, B.‐J.; Zuo, Y.‐Z.; Gu, W.‐Y.; Luo, S.‐X.; Shi, Q.‐K.; Hou, L.‐S.; Zhong, F.; Fan, J.‐H.
title: Isolation and phylogenetic analysis of porcine deltacoronavirus from pigs with diarrhoea in Hebei province, China
date: 2018-01-24
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.12821
sha: a2c6efff9a3cd78c88a6acea51d07f8dc7c27207
doc_id: 809359
cord_uid: 47czdddb

Porcine deltacoronavirus (PDCoV) is a recently identified coronavirus in the genus Deltacoronavirus that can cause enteric disease with clinical signs including diarrhoea, vomiting, dehydration and mortality in neonatal piglets. Although evidence of the prevalence of PDCoV in China is accumulating, little published information about Chinese PDCoV isolates is available. In this study, we investigated the presence of PDCoV in 49 faecal/intestinal samples from piglets with diarrhoea on different farms in Hebei province. Five samples (10.2%) were positive for PDCoV, but no coinfection of PDCoV with other enteropathogens was observed. A PDCoV strain named HB‐BD was successfully isolated from the intestinal contents of a diarrhoeic piglet and serially propagated in swine testicular (ST) cells for >40 passages. The complete genome of the HB‐BD strain was sequenced and analysed. Genomic analysis showed that the HB‐BD strain had a closer relationship with Chinese strains than those from other countries and was grouped within the Chinese PDCoV cluster. The results of this study will be valuable for further research of PDCoV genetic evolution and development of effective diagnostic reagents, assays and potential vaccines against newly emerged PDCoV strains.

effective treatments or vaccines available to control PDCoV (Hu, Jung, et al., 2015) .

The PDCoV is an enveloped, positive-sense, single-stranded RNA virus. The full genome of PDCoV is approximately 25.4 kb in length (Woo et al., 2012) , encodes two polymerase proteins (ORF1a/b) gene, four major structural proteins for the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins, as well as non-structural protein 6 (NS6) and non-structural protein 7 (NS7) (Lee & Lee, 2014; Li et al., 2014) . The functions of PDCoV individual proteins have not been elucidated. However, among the structural proteins of the other coronaviruses, the S protein is known to play pivotal roles in interacting with the cellular receptor to mediate viral entry and is an antigenic target for neutralizing antibodies (Park, Song, Ha, & Park, 2007; Sato et al., 2011) . The M protein is a transmembrane protein that is responsible for the transport of nutrients across the membrane. In addition, the M protein plays an important role in viral assembly, as well as the induction of virus neutralization antibodies (Fan, Zuo, Li, & Pei, 2012) . The coronavirus N protein binds viral RNA and highly conserved. It is involved in several of the biological activities of the virus (Chang et al., 2006; Molenkamp & Spaan, 1997) . The S, M and N protein genes have been targeted for the development of virological and serological diagnostic assays for PDCoV (Chen, Gauger, et al., 2015; Ma et al., 2015; Marthaler et al., 2014; Song et al., 2015; Su et al., 2016; Wang et al., 2014) . Therefore, it is important to understand the molecular characteristics of the prevalent strains and its relevance to the development of diagnostic reagents, assays and potential vaccines against emergent PDCoV strains. Although some publications have reported the detection of PDCoV RNA in domestic pigs in mainland China (Chen, Zhu, et al., 2015; Song et al., 2015; Wang, Yue, Fang, & Huang, 2015) , a few have reported on the isolation of PDCoV or the characteristics of those isolates.

In this study, five of 49 samples collected from different swine farms reporting cases of diarrhoea in Hebei province were PDCoV positive. A PDCoV strain, HB-BD, was successfully isolated and serially propagated in swine testicular (ST) cells. The complete genome of the HB-BD strain was sequenced and analysed. RT-PCR primers (F: 5 0 -TAACTCCGCCATCAAAC-3 0 and R: 5 0 -CCACTTCCACGCTCCT-3 0 ) targeting the N gene were used with the following reaction conditions: 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 50°C for 30 s and 72°C for 30 s, with a final extension at 72°C for 10 min. In addition, molecular detection of the other three diarrhoea-related enteric viruses, PEDV, TGEV and porcine rotavirus (PRoV), was performed using previously reported methods for further evaluation of possible coinfection status with PDCoV (Hu, Li, et al., 2015; Jeong et al., 2009; Sun, Leng, Zhai, Chen, & Song, 2014 ).

Isolation of PDCoV was attempted on samples that were positive for PDCoV alone. The isolated strain was used for sequence analysis and phylogenetic analysis, the details of which are presented in For the first inoculation, cells were cultured in a T25 flask. When the ST cell reached 80% confluence in the flask, cells were washed twice with maintenance medium and were used for virus inoculation.

A volume of 900 ll of maintenance medium together with 100 ll of the filtered sample was added to the flask. After incubation of the virus for 60 min at 37°C in 5% CO 2 incubator, the cells were washed twice, and 10 ml maintenance medium with 1% pancreatin was added to each flask. The cells were then cultured continuously at 37°C in 5% CO 2 incubator and were observed daily for the cytopathic effect (CPE). When an obvious CPE was observed in around 90% of the cell monolayers 2-3 days after inoculation, the flasks were frozen at À80°C and thawed three times. The supernatants and cells were harvested together and stored at À80°C. These samples were used as seed stocks for subsequent passage and for the detection of PDCoV and other enteric viruses. The isolated PDCoV strain, HB-BD, was plaque purified as previously described (Hu, Jung, et al., 2015) .

For PDCoV serial propagation, 80% confluent ST cells in a T25 flask were washed twice with maintenance medium, and then, 1 ml of the virus inoculum containing 1% pancreatin was added to the flask. The cells were cultured at 37°C in 5% CO 2 incubator . After 2-3 days, 80% of the virus-infected cells showed CPE, and the cultures were collected after three frozen-thawed cycles. The supernatants were stored at À80°C and used for PDCoV propagation.

Sequence(5 0 -3 0 ) Nucleotide position a Product size (bp) Nucleotide position is numbered based on the PDCoV/NH strain (KU981059).

T A B L E 1 Oligonucleotide primers used for amplification of the complete genome of PDCoV strain HB-BD by RT-PCR Viral titre was measured using 50% tissue culture infectious dose (TCID 50 ) assays on ST cells in a 96-well plate, as previously described (Hu, Jung, et al., 2015) .

PDCoV-infected ST cells were fixed with 4% paraformaldehyde at 4°C for 30 min then washed three times with PBS and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. After blocked with 5% bovine serum albumin (BSA) at 37°C for 1 hr, the cells were incubated with PDCoV polyclonal antibodies which were produced from PDCoV HB-BD-immunized BALB/c mice. After 1 hr incubation at 37°C, the cells were washed three times with PBS and incubated with FITC-labelled goat anti-mouse secondary antibody for 1 hr. The stained cells were examined using a fluorescence microscope.

The 5th passage of PDCoV HB-BD cell culture sample was centrifuged at 4,000 g for 5 min. Sequence alignment analysis was processed using the Clustal W program in the DNAStar software, and the phylogenetic tree was constructed by the neighbour-joining method with 1000 bootstrap replications using the MEGA 7.0.14 software. The PDCoV/HB-BD strain and the reference strains used for the sequence analysis are presented in Table 2 .

The nucleotide sequences of the complete M, N, S gene and complete genome of the PDCoV strain, HB-BD, were deposited in the 

During serial passages, the PDCoV infectious titres were determined using a TCID 50 assay on ST cells. Four passages of the isolate (P 5 , P 9 , P 13 and P 19 ) were detected. The results showed that the virus titres for these four passages were 10 5.3 TCID 50 /ml, 10 6.2 TCID 50 / ml, 10 6.9 TCID 50 /ml and 10 6.5 TCID 50 /ml, respectively. Korea and Thailand (Chen, Zhu, et al., 2015) . and C459T) that were similar to those of the strains from Thailand.

In addition, the HB-BD strain had a unique nucleotide mutation (C63T). However, these nucleotide mutations did not lead to changes in the predicted amino acid sequences of the PDCoV isolates. strains. In addition, HB-BD had one further aa mutation (A 308 V).

To analyse the phylogenetic relationships between the HB-BD isolate and the reference strains, a phylogenetic tree was constructed. The representative tree is shown in Figure 3 . In the context of the complete genome phylogenetic tree, all 29 PDCoV strains were separated into the genus Deltacoronavirus, a cluster that was distinct from the PEDV and TGEV strains.

Further analysis demonstrated that the PDCoV isolate, HB-BD, was more closely related to other Chinese PDCoV isolates than to those isolated from the United States, South Korea and Thailand ( Figure 3 ).

PDCoV is novel swine enteropathogenic coronaviruses that have emerged in several pig breeding countries in recent years (Homwong In China, as PDCoV HKU15-44 and HKU15-155 strains were first identified in Hong Kong in 2012 (Woo et al., 2012) , PDCoV has been reported in some local provinces and the prevalence of PDCoV has been investigated (Chen, Zhu, et al., 2015; Dong et al., 2015; Song et al., 2015; Wang et al., 2015) . In one study, 64 faecal and intestinal samples collected from six pig farms in the Shanxi, Guangdong and Hubei provinces in China, were examined for enteropathogens using RT-PCR (Chen, Zhu, et al., 2015) . Other general enteropathogens, such as PEDV, TGEV and PRoV, F I G U R E 3 Phylogenetic tree of the nucleotide sequences of PDCoV isolates based on the complete genome. All the reference sequences used in this study were obtained from the GenBank database. The names of the strains, places of isolation and GenBank accession numbers are shown in Table 1 . The Tree was constructed by the neighbour-joining method with 1,000 bootstrap replications using MEGA 7.0.14 software. The isolate identified in this study is indicated by black triangle, and the Chinese reference strains are indicated by black dots were also examined. (Hu, Jung, et al., 2015; Dong et al., 2016) . In the present study, we attempted to isolate the virus from two intestinal content samples and three faecal samples that were positive for PDCoV. However, in the ST cells, only the PDCoV HB-BD strain was successfully isolated from intestinal contents. The success rate of virus isolation is relatively low. These results are consistent with those of previous reports (Dong et al., 2016; Hu, Jung, et al., 2015) .

To understand the genetic characteristics of the HB-BD isolate, the complete genome of the isolate was sequenced, and phylogenetic analysis was performed with known sequences selected from the GenBank database. The results showed that all PDCoV strains were distinct from PEDV and TGEV, and clustered within the genus

Deltacoronavirus. The HB-BD strain had a closer relationship with other Chinese strains than those from other countries and was grouped within the Chinese PDCoV cluster in the phylogenetic trees.

When compared with the Chinese strains, HB-BD strain showed most closely related to the NH strain (emerged in Heilongjiang province) and the HKU15-155 strain (isolated in Hong Kong). Based on the S gene, HB-BD shared the highest aa identity (99.2%) with the NH strain. In addition, both strains have two aa mutations (L45H and Y123H). However, based on the N gene, the HB-BD strain had the highest aa identity (99.4%) with HKU15-155 and shared a similar aa substitution (V43A). Further analysis showed that HB-BD had four further aa mutations (H149Y, R888T, A894T and G910C) in the S gene, one further aa mutation (A308V) in the N gene and a unique nucleotide mutation (C63T) in the M gene. Studies have shown that the S protein of coronaviruses is associated with receptor binding and host adaptation (Graham & Baric, 2010; Sato et al., 2011) and the N protein participates in viral transcription and replication (Brian & Baric, 2005) . Further research in HB-BD should be conducted to understand whether these mutations can lead to changes in virulence, pathogenicity and antigenicity of PDCoV.

In conclusion, the presence of PDCoV in Hebei province was confirmed, and the PDCoV strain, HB-BD, was successfully isolated and serially passaged in ST cells. The complete genome of HB-BD was sequenced and characterized. These results will be valuable for further research of PDCoV genetic evolution. The immunogenicity and pathogenicity of the HB-BD strain require further study for the development of effective diagnostic reagents, assays and potential vaccines against newly emerged PDCoV strains. 

The authors declare no conflict of interest.

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