PEDV CHYJ130330 strain

The highly virulent CHYJ130330 strain had shown cytopathic effect on Vero cell line after 24 h having characteristic features like fusion of cells and syncytia formation (Fig. 1). The 2–7 day old piglets orally infected with 10TCID50 (50% tissue culture infective dose) of this strain had induced severe diarrhea within 48 h [9].

Fig. 1

Identification of PEDV CHYJ130330 strain in Vero cells. The panels show cells infected with CHYJ130330 strain (A) Control (uninfected) cells (B) Cytopathic effects (CPE) at 16 h after inoculation (×10) (C) CPE at 24 h after inoculation

PEDV (CHYJ130330 strain) particles in Vero cell culture, when observed under electron microscope showed typical Coronavirus particles in cell culture media and in the surface sections of infected vero cells. Circular shaped virions 80–120 nm in diameter with characteristic surface projections were observed (Fig. 2).

Fig. 2

Electron micrograph of purified PEDV virus. Left image is the virus at ×20,000 and right image is magnification at ×60,000 PEDV virions

Clinical signs in experimental animals

The CHYJ130330 strain has strong virulence affecting 3 day old piglets after oral administration of 1 ml of 100 TCID50 virus solution (morbidity 100%) (Tables 3 and 4). There was vomiting and watery diarrhea in affected piglets that occurs within 36 h (Fig. 3). All 104 TCID50 group piglets died of dehydration within 5 days. There was thinning of small intestine with presence of yellowish fluid in affected animals (Fig. 4).

Table 3 PEDV shedding in rectal swabs of pigletsTable 4 Virulence of CHYJ130330 strain 3 day old piglets after oral administration of 1 ml of TCID50 virus solutionsFig. 3

Clinical signs in experimentally infected piglets (A, B) Hind Quarter of piglets showing signs of watery diarrhea (C) healthy animal

Fig. 4

Necropsy examinations of the intestine of piglets. A, B Inoculated with PEDV-CHYJ130330 strain. The small intestine was thin-walled and contained soft to watery yellowish contents. C Inoculated with control medium. No intestinal lesions were found in the uninfected piglets

Histopathological findings

On histopathology, the intestine showed stunting and fusion of villi (Fig. 5) with necrosis and sloughing of the area (Fig. 5A, B). Mucosal epithelial cells were found necrotic and lysed; having nuclear changes suggesting cell death i.e., pyknosis, karyorrhexis, and karyolysis. The number of intestinal villi was reduced, the length was shortened and the length of the intestine villi and crypt was about 2:1.

Fig. 5

Photomicrographs of intestines of PEDV piglets. A, B Atrophy of villi with extensive intestinal epithelial degeneration as well as sloughing and necrosis of the villi. C No lesions in intestines of uninfected piglets. HE Stain ×100

Whole genome sequence comparison and phylogenetic analysis

The CHYJ130330 strain possessed a 28,038 nucleotides long complete genome sequence excluding 3′ poly (A) tail, having unique arrangement as 5′ UTR (1 to 292 nt), a replicase gene (1a/1b) (293 to 20,637 nt), a spike gene (20,634 to 24,794 nt), ORF3 (24,794 to 25,468 nt), an envelope gene (25,449 to 25,679 nt), a membrane gene (25,687 to 26,367 nt), a nucleoprotein gene (26,379 to 27,704 nt), and a 3′ UTR (27,705 to 28,038 nt) [9].

CHYJ130330 strain had 96.5–97.2% nt identity with the pre-2013 strains and 97–99% nt identity with the post-2013 strains reported from China. The exceptions included the CH/ZMDZY/2011(KC196276) strain reported in 2011 having 98.75% identity with the whole genome nt sequence reported from our lab. The whole genome sequences reported from other regions included CV777, vir.DR13/S.Korea, SM98/S.Korea and atten.DR13/S.Korea sharing 96.8%, 97.6%, 96.6 and 97.1% identity with CHYJ130330 respectively while all other selected reference strains shared 98–99% identity with our sequence.

The phylogenetic analysis of 43 reference strains including highly virulent CHYJ130330 had clearly distinguished them into two groups namely G1 and G2. G1 had three while G2 was further split into two subgroups respectively. In addition to 1a and 1b subgroup; G1 also had a tentative cluster comprising virulent DR13 (South Korea/1999) and CH/S (China/1986) strains placed in R group (Fig. 6A). The prototype strain of CV777, Korean (attenuated DR13, virulent DR13 and SM98) and earlier Chinese strains (CH/Atten. Vaccine, CH/S, CH/85-7mutant, JS2008, LZC and SD-M/2012) comprised the G1 group. More importantly all the strains reported after 2013 from China till date were fallen in group G2 except CH/JS-2/2015(KX534206).

Fig. 6

A–F Phylogenetic trees based on A complete genome of 43 PEDVs (B–F) Amino acid sequence of S glycoprotein gene, envelope, membrane, nucleocapsid and ORF3 genes respectively. The trees were constructed by the distance-based neighbor-joining algorithm using MEGA software. Bootstrap was set in 1000 replicates with a value > 70% to assess the significance of the tree topology. A bar of 0.002/0.005/0.05 indicated nucleotide or amino acid substitutions per site. “white circle” indicated strains from USA, “white diamond” indicated strains from South Korea, “white down-pointing triangle” indicated strains from China after 2013, “black down-pointing triangle” indicated strain from this study, “black diamond” indicated strains from Belgium, “black up-pointing triangle” indicated strains from China before 2013, “No symbol” indicated strains from other parts of world.

Comparative analysis of structural genesSpike (S) protein gene

The full-length of S gene of CHYJ130330 was 4,160 nt in size, which was 8-nt longer than that of the prototype of PEDV CV777 strain. CHYJ130330 strain has 92.7–93.3% aa identity with the pre-2013 strains except CH/ZMDZY/2011(KC196276) reported from central China having 98.1% sequence similarity. The post-2013 PEDV strains have 95.2–98.4% aa identity except CH/85-7mutant (KX839247) and CH/JS-2/2015(KX534206) sharing 92% to 93% similarity reported from China. CHYJ130330 strain shared 93.7% aa identity with CV777, 92.9–94.6% identity with three S. Korean strains which also included the cell adapted PEDV vaccine strain, 95.6–98.7% identity with the other 19 strains of PEDV selected from various regions of the world which also included four strains of PEDV reported from USA.

The S protein targeted phylogenetic tree using 43 strains of PEDV showed two major groups, having two subgroups each. The strain CHYJ130330 belonged to subgroup 2b, which included all other strains reported from China post-2013 except one strain JS-2/2015 which was a part of group (G1). All other strains reported from China pre-2013 belong to group G1; including the attenuated Vaccine strain, KC189944 reported from Hubei, China. The S protein of PEDV strains reported from USA also belongs to Group G2 (Fig. 6B).

Envelope (E) protein gene

The full-length of E gene of CHYJ130330 was 230 nt, encoding 76-aa protein. CHYJ130330 strain has 94.7–97.4% aa identity with the pre-2013 strains and 98.7–100% aa identity with the post-2013 strains reported from China except 85-7mutant(KX839247), ZL29/2015(KU847996), HNQX-3/14(KR095279) and JS-2/2015(KX534206) strains which have 95.6–97.4% similarity with our sequence. CHYJ130330 has 98.6–100% similarity with all other sequences including CV777. The exceptions include SM98/S.Korea (96%), atten.DR13/S.Korea (95%), NB1/S.Korea (97.4%).

The aa deduced phylogenetic tree depicted two groups, that is, Group 1 and Group 2 (Fig. 6C). The strain CHYJ130330 belonged to group G2, which included all PEDV strains selected in this study reported from China post-2013 along with other globally reported E protein sequences. The G1 group included the CV777, attenuated Vaccine strain (KC189944), 85-7mutant(KX839247) and JS-2/2015(KX534206) and other strains reported from China pre-2013.

Membrane (M) protein gene

The full-length of M gene of CHYJ130330 was 680 nt, encoding 226 amino acids. M protein of CHYJ130330 strain has 96.9–99.6% aa identity with the pre-2013 and more than 97.3% aa identity with the post-2013 strains reported from China, with many having 100% identity indicating that the sequence of M protein is highly conserved. CHYJ130330 strain shared 98.7% aa identity with CV777, 97.8–100% identity with other reference PEDV strains.

The phylogenetic analysis of M protein confirmed two groups, i.e., Group 1 and Group 2 in the chosen strains (Fig. 6D). Group 2 contained 12 Chinese strains, including CHYJ130330, and US strains. The G1 group includes the CV777, attenuated Vaccine strain (KC189944), JS-2/2015(KX534206) and other strains reported from China pre-2013.

Nucleocapsid (N) protein gene

The full-length of N gene of CHYJ130330 was 1325 nt. N protein of CHYJ130330 strain has 95.5–96.6% aa identity with the pre-2013 Chinese strains except 98.6% aa identity with ZMDZY(KC196276) strain. The post-2013 strains reported from China have 96.4–99.5% aa identity. In addition to CV777, few S. Korean and Thailand strains and almost all other strains shared 98–99.3% similarity with CHYJ130330. The CV777 has 96.1% while others (S. Korean and Thailand strains) have 95.9–96.8% identity with amino acid sequence of N protein reported in this study (Fig. 6E).

The phylogenetic analysis showed that CHYJ130330 strain is present in the group 1b along with other major nucleoprotein sequences reported from China. CV777, attenuated vaccine strain and predominant sequences reported from China pre-2013 belong to Group (G1).

ORF3 protein gene

The full-length of ORF3 gene of CHYJ130330 was 674 nt, encoding 224 amino acids. CHYJ130330 strain has 90.1–94.1% aa identity with the pre-2013 strains except CH/S/1986(JN547228) and ZMDZY/2011(KC196276) which have 99.5% and 99.1% homology respectively with our strain. The post-2013 strains have 96.4–100% aa identity with CHYJ130330 except CH/JS-2/2015(KX534206) from Shanghai sharing 86.8% identity with ORF3 protein.

CHYJ130330 strain shared 96% aa identity with CV777, more than 98% identity with other reference strains from various parts of the world except VN/KCHY/2013(KJ960180), atten.DR13/S.Korea(JQ023162) and Thailand/CBR2(KR610994) sharing 96%, 91.2% and 96.4% homology respectively (Fig. 6F).