key: cord-354729-dpaz01np
authors: Huan, Changchao; Pan, Haochun; Fu, Siyao; Xu, Weiyin; Gao, Qingqing; Wang, Xiaobo; Gao, Song; Chen, Changhai; Liu, Xiufan
title: Characterization and evolution of the coronavirus porcine epidemic diarrhoea virus HLJBY isolated in China
date: 2019-08-22
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.13321
sha: 
doc_id: 354729
cord_uid: dpaz01np

A strain of porcine epidemic diarrhoea virus (PEDV), namely HLJBY, was isolated in Heilongjiang province, China. To provide insight into the understanding of the phylogenetic and the current epidemiological status of PEDV, PEDV HLJBY was compared with CV777 and other PEDV strains deposited in the GenBank. The homology between the entire genomic nucleotide sequences of PEDV HLJBY and CV777 was 97.7%. The homology of M gene was the highest (99.0%). However, the homology of ORF3 gene was 97.7%, and protein of ORF3 was 90.1%. In addition, HLJBY showed the highest nucleotide identity (99.9%) with PEDV‐SX/China/2017 strain and lowest similarity (91.2%) to PEDV/Belgorod/dom/2008 strain. We analysed the changes in S gene and its protein of PEDV HLJBY with 65 historic PEDV strains. The highest nucleotide identity was 99.9% compared with PEDV‐SX/China/2017 strain, and the lowest nucleotide identity was 60.0% compared with PEDV/Belgorod/dom/2008 strain. The length of deduced amino acid sequences of S proteins varied from 1,372 to 1,390 amino acids (aa). Compared with most aa sequences of S proteins, HLJBY exhibited 5 aa deletions (position 55, 59–61, 144). Analysis and comparison of open reading frame 3 (ORF3) proteins between HLJBY strain and other PEDV strains were also focused in this study. We revealed that the length of deduced amino acid sequences of ORF3 proteins was 80–224 aa among tested strains and the identity of HLJBY ORF3 amino acids with other PEDV strains was 71.4%–98.9%. ORF3 protein of both HLJBY strain and PEDV‐SX/China/2017 strain consists of 91 aa, with 133 aa deletions at their C' end in relation to the other tested PEDV strains. The phylogenetic tree based on different proteins or genes resulted in different phylogenetic groups. For pathogenicity evaluation of PEDV HLJBY strain, colostrum deprivation piglets were challenged with PEDV HLJBY, and PEDV reference strain CV777 as a control, the results showed that animals challenged with either of these PEDV strains developed diarrhoea, and histopathological examination of small intestines of challenged animals showed acute viral enteritis with villous atrophy in either PEDV HLJBY‐P10 or PEDV CV777‐P8 inoculated piglets.

cluding a positive-sense single-strand RNA genome, which can cause a devastating enteric disease characterized with dehydration and watery diarrhoea (Pensaert & de Bouck, 1978) . PEDV is the causative agent of porcine epidemic diarrhoea (PED), which has high mortality in suckling piglets (Debouck & Pensaert, 1980; Pijpers, Nieuwstadt, Terpstra, & Verheijden, 1993) . This disease was initially documented in the United Kingdom in 1971. Since 1990s, PED was not serious. However, outbreak of PED suddenly occurred in the United States, Canada and Mexico causing huge economic losses (Mole, 2013; Stevenson et al., 2013; Vlasova et al., 2014) . In addition, PED caused tremendous economic losses to the swine industry in Europe and Asia, including China, Korea and Japan (Kocherhans, Bridgen, Ackermann, & Tobler, 2001; Sun et al., 2012) .

PEDV is a nonsegmented and infectious RNA virus. The genome of PEDV is 27-33 kb in length containing a 5'cap and a 3'polyadenylated tail (Pensaert & de Bouck, 1978) . In addition, the genome includes seven open reading frames (ORFs) encoding three nonstructural proteins (replicase 1a,1b and ORF3) and four structural proteins (the spike (S), envelope (E), membrane (M) and nucleoprotein (N)). These proteins arrange in the order of 5'-replicase(1a/1b)-S-ORF3-E-M-N-3' (Kocherhans et al., 2001) .

To reveal the characteristic and the diversity between PEDV strains currently circulating in China and other PEDV strains outside, the complete genomic sequence of PEDV HLJBY strain was determined and analysed, and the pathogenicity of PEDV HLJBY strain in newborn piglets was also evaluated.

PEDV HLJBY strain was isolated from the intestinal contents of a piglet with diarrhoea from Heilongjiang province, China at 2011. Vero cells were grown in Dulbecco's modified Eagle's medium (DMEM, HyClone) supplemented with 8% foetal bovine serum (FBS, GIBCO) and were maintained in maintenance medium (DMEM supplemented with 2% FBS) at 37 °C in a 5% CO 2 incubator. Strain HLJBY was passaged ten times in Vero cells. PEDV N-specific antibody used in immunofluorescence assay (IFA) was gifted by Professor Xiang Mao.

PEDV reference strain CV777 was purchased from China Institute of Veterinary Drug Control.

When PEDV-infected Vero cells showed 70%-80% cytopathic effect (CPE), cell culture flasks were frozen and thawed three times, and cell debris was pelleted by centrifugation for 30 min at 12,000 rpm.

Culture supernatants were collected and used for preparation of viral RNA. Total RNA was extracted using Trizol reagent (Vazyme Biotech) according to the manufacturer's instructions. Total RNA was used for synthesis of cDNA with HiScript reverse transcriptase (HiScript II 1st Strand cDNA synthesis kit; Vazyme Biotech) according to the manufacturer's instructions.

Specific primers for PEDV were designed based on PEDV-CV777 genome (Table 1) . PCR was performed to amplify for the six overlapping DNA fragments using LAmp DNA polymerase (Vazyme Biotech). The expected bands in agarose electrophoresis of PCR products were excised, and a GenClean Column gel extraction kit (Generay Biotech) was used to purify the synthesized DNA, and the DNA products were cloned into pJET1.2 vector (Thermo). The positive clone was sequenced by Biotechnology Co, Ltd. The validated genome sequence of PEDV HLJBY strain was submitted to GenBank and acquired accession number: KP403802.1.

Sequence data was employed to assemble and analyse by DNAStar software package (DNAStar Inc. genes of PEDV strains were used for sequence alignments and phylogenetic analyses. Nucleotide sequences of full-genomes, ORF3 genes and S genes of PEDV strains were aligned using the ClustalX 2.0 program (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997 ).

Vero cells grown on coverslips were infected with 0. 

Fifteen newborn piglets without colostrum were purchased from one pig farm and were free of PEDV, transmissible gastroenteritis virus, porcine deltacoronavirus, and porcine rotavirus. The piglets were divided randomly into three groups: the sham-inoculated control group (n = 5), CV777-P8-inoculated group (n = 5), and HLJBY-P10-inoculated group (n = 5). The piglets were fed commercial milk replacer (8 times daily).

The piglets in the challenge groups received an oral 1 ml dose of 10 7.0 TCID 50 /ml of PEDV CV777 or HLJBY. The sham-inoculated pigs were given DMEM medium (1 ml) orally. All animals were monitored for mortality and signs of vomiting and/or diarrhoea (observed and recorded for every 3 hr during whole experiment). All piglets were euthanized at 7 d post-challenge and checked for macroscopic and microscopic lesions. 

To reveal the characteristics of PEDV HLJBY strain and determine more precisely the relationship among the PEDV strains currently circulating in China and those from other nations, the full-length genome sequence of strain HLJBY was deduced by combining the sequences of several overlapping cDNA fragments. The genome sequence of strain HLJBY was 27,953 nucleotides (nt) in length, excluding the 3' poly(A) tail. The genomic organization was typical of previously sequenced PEDV strains and was summarized as 5'UTR-ORF1a/1b-S-ORF3-E-M-N-3'UTR ( Figure 1 ).

Of note, compared with classical PEDV CV777, the genome of HLJBY contains four deleted nucleotides or regions including 72 nt, 89-93 nt, 3403-3426 nt, 21092-21094 nt, respectively. The four deleted nucleotides or regions were located at 5'UTR, ORF1a/1b, S, ORF3, respectively. To investigate the molecular characteristics of PEDV HLJBY strain, UTR (5' and 3') and the nucleotide and predicted amino acid sequences of the nonstructural and structural proteins (replicase ORF1a/1b, S, ORF3, E, M, N) of PEDV HLJBY strain were compared with CV777. As shown in Figure 2a and Table 3 , the nucleotide of 5'UTR of PEDV HLJBY had 5 nt deletions and 1 nt insertion and the identity is 96.0% compared with CV777. 24 nt deletions were found in ORF1a/1b of PEDV HLJBY (Figure 2b ), and the nucleotide sequence identity was 98.0%, and the amino acid sequence identity was 98.0% (Table 3) . 3 nt deletions showed in S of PEDV HLJBY ( Figure 2c ), and the nucleotide sequence identity was 97.0%, and the amino acid sequence identity was 96.0% (Table 3) . 399 nt deletions exhibited in ORF3 of PEDV HLJBY (Figure 2d ), and nucleotide sequence identity was 91.7%, and the amino acid sequence identity was 90.1% (Table 3 ). In protein E, the nucleotide sequence identity was 97.0%, and the amino acid sequence identity was 97.0% (Table 3) , resulting in amino acid changes in E (11Val to Ala and 76 Val to Ile) ( Figure 2e ). In protein M, the nucleotide sequence identity was 99.0% and the amino acid sequence identity was 98.0% (Table 3) , resulting in 4 amino acid changes in M ( Figure 2f ). In protein N, the nucleotide sequence identity was 98.0% and the amino acid sequence identity was 98.0% (Table 3) , resulting in 9 amino acid changes in N ( Figure 2g ). The nucleotide sequence identity of 3'UTR was 97.0% (Table 3) .

The complete genome sequences of PEDV strains from different locations and years were compared, and the results revealed that HLJBY had a nucleotide identity of 99.9%-91.2% with other entire PEDV genomes available in GenBank ( 

The ORF3 protein (an accessory protein) was located between S and 

To investigate the evolution of PEDV, phylogenetic analysis based on the entire genomic nucleotide sequences of PEDV HLJBY strain The group Ⅲ was further divided into subgroup Ⅲa and Ⅲb. The The group Ⅱ included the other PEDV strains (Figure 7) . 

To 

In recent years, PEDV has re-emerged as one of the deadliest and most contagious pathogens in swine, causing large economic (Li et al., 2012) . Therefore, the relationship of (Wang et al., 2012) . ORF3 may contribute to the virulence of PEDV. The ORF3

was a key gene for PEDV culture in vitro. ORF3 gene was usually used to differentiate between field and vaccine-derived isolates and altered the virulence of PEDV (Park et al., 2008) . In addition, the PEDV S protein is an important viral gene for studying genetic relatedness among isolates and epidemiology of PEDV (Chen et al., 2014; Gerber et al., 2014; Lee, 2015; Lee et al., 2010; Oh et al., 2014) . Nucleotide sequencing of S gene analysis revealed that 3 nt deletions were found in S gene of PEDV HLJBY compared with CV777 ( Figure 2c ) and the nucleotide sequence identity is 97.0%. The amino acid sequence identity of S protein was 96.0% between PEDV HLJBY and CV777. 3 nt deletions of S gene were located in the amino terminus of S gene, which was suitable to analyse the significant differences in epidemiology of PEDV (Li et al., 2012; Temeeyasen et al., 2014; Vlasova et al., 2014) .

S gene functions as a receptor-binding domain (Belouzard, Millet, Licitra, & Whittaker, 2012) . and epidemiology of PEDV. Our study will provide more information about diversity, evolution, and in particular, the epidemic charac- In the phylogenetic tree of the whole genome, S aa and ORF3 aa, the phylogenetic tree based on both the whole genome and S aa was similar, but the phylogenetic tree of ORF3 aa was different from the phylogenetic trees for the S protein and genomic nucleotide sequences. All PEDV strains were divided into three groups based on the phylogenetic tree of the whole genome and S protein. In the phylogenetic tree of the whole genome and S protein, PEDV/Belgorod/ dom/2008 strain located at group Ⅰ, and PEDV HLJBY belonged to the group Ⅱ. In the phylogenetic tree of ORF3 protein, the group Ⅰ only harboured PEDV/Belgorod/dom/2008 and HLJBY. The phylogenetic tree based on different proteins implied the different evolution events. This will provide further information for virus evolution. The phylogenetic trees suggested that PEDV HLJBY might originate from a genetic recombination event between pandemic strains and classical strains. We need to explore the mechanism and/or cause of the different phylogenetic trees based on different proteins or genes. This will lay a foundation for the recombination and evolution of PEDV.

Biological characteristics of PEDV HLJBY including the cytopathic effect, and pathogenicity, indicated that PEDV HLJBY induced an apparent and classical cytopathic effect (CPE) in Vero cells, and caused acute viral enteritis with villous atrophy in small intestine of challenged piglets. But all HLJBY-P10 or CV777-P8 inoculated piglets had no deaths during the challenge experiment. These results suggested that PEDV HLJBY might have similar pathogenicity in experimentally infected piglets compared with that of PEDV reference strain CV777.

In summary, we found that the evolutionary trees are different based on different proteins or genes of PEDV strains. This phenomenon needs further study. Pathogenicity evaluation of PEDV HLJBY indicated that PEDV HLJBY potentially possessed equal pathogenicity compared with that of PEDV reference strain CV777. This study might provide a reference for the evolutionary and variation of PEDV.

We thank James Allen, PhD, for editing the English text of a draft of this manuscript. This work was supported by the Natural 

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All experimental protocols were approved by the Animal Care and 

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