Archives of Virology

, Volume 158, Issue 3, pp 711–715

Phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in China

Authors

  • Yueyi Gao
    • Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of AgrobiotechnologyChina Agricultural University
  • Qiuwen Kou
    • Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of AgrobiotechnologyChina Agricultural University
  • Xinna Ge
    • Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of AgrobiotechnologyChina Agricultural University
  • Lei Zhou
    • Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of AgrobiotechnologyChina Agricultural University
  • Xin Guo
    • Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of AgrobiotechnologyChina Agricultural University
    • College of Veterinary MedicineChina Agricultural University
Annotated Sequence Record

DOI: 10.1007/s00705-012-1541-2

Cite this article as:
Gao, Y., Kou, Q., Ge, X. et al. Arch Virol (2013) 158: 711. doi:10.1007/s00705-012-1541-2
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Abstract

Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), which is characterized by severe diarrhea, dehydration and high mortality in the affected pigs. Recently, clinical outbreaks of diarrhea in suckling piglets emerged in pig-producing areas of China. In this study, molecular detection of PEDV was conducted using RT-PCR (targeting the M gene) on samples collected from piglets with watery diarrhea from 15 pig farms, and phylogenetic analysis of PEDV field strains was carried out based on their M and S genes. In addition, the complete genome sequence of a PEDV field strain was determined. PEDV was detected in 92.7 % of the samples (267/288). The 15 M genes that were amplified shared 99.6-100 % nucleotide identity and 99.1-100 % amino acid similarity with each other. The 15 S genes exhibited 98.6-99.9 % homology, both at the nucleotide level and at the deduced amino acid level. Phylogenetic analysis showed that all of the amplified M genes grouped in cluster 3, together with some Chinese, Korean and Thai strains, while all of the amplified S genes were in cluster 3 and were closely related to Korean strains. Compared with previous PEDV strains, all of the S genes have common characteristics, namely, a 4-aa (GENQ) insertion between positions 55 and 56, a 1-aa (N) insertion between positions 135 and 136, and a 2-aa (DG) deletion between positions 155 and 156, similar or identical to Korean KNU-serial strains reported in recent years. The genome of the sequenced PEDV field strain is 28,038 nucleotides in length, excluding the poly (A) tail. Our findings suggest that a novel PEDV with a characteristic variant S gene is responsible for recent outbreaks of clinical diarrhea in piglets in China.

Porcine epidemic diarrhea (PED) is an acute, highly contagious viral enteric disease that is economically important for pig production. Clinically, it is characterized by severe diarrhea, dehydration and high mortality in the affected swine, especially in piglets. The disease has been documented mainly in either European or Asian countries since it was first recognized in an acute outbreak of diarrhea in pigs [17]. Porcine epidemic diarrhea virus (PEDV) was first recognized as the causative agent of PED in Belgium and the United Kingdom in 1978 [16]. The virus is an enveloped, single-stranded RNA virus belonging to the genus Alphacoronavirus, subfamily Coronavirinae, family Coronaviridae. The genome of the prototype PEDV strain CV777 was the first to be completely sequenced [8]. To date, limited complete genomic information about PEDV is available in the GenBank database, and most investigations have focused on molecular epidemiological analysis targeting single gene of PEDV. PEDV contains a polymerase gene that encodes a replicase polyprotein (P1), occupying 70 % of the genome, and four main structural protein genes that encode the spike (S) protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein [5, 6, 12, 22, 23].

PED is a sporadic viral disease that has affected Chinese pig production since transmissible gastroenteritis (TGE)-like outbreaks of acute diarrhea caused by PEDV were first observed in Shanghai in 1973. Bivalent killed or attenuated vaccines have been used to prevent TGE and PED in China. Surprisingly, clinical outbreaks of severe diarrhea have emerged in pig-producing areas of China in the past two years, resulting in great losses of suckling piglets. In the present study, we used RT-PCR (targeting the M gene) on clinical fecal and intestinal tissue samples collected from pig farms with diarrhea outbreaks to detect PEDV, and we performed phylogenetic analysis of PEDV field strains based on the amplified M and S genes and determined the complete genome sequence of a PEDV field strain.

A total of 288 fecal and intestine tissue samples from suckling piglets with severe watery diarrhea were collected from fifteen farms in the Beijing, Hebei and Zhejiang areas of China during 2011-2012. Fecal samples were suspended in DMEM, and intestinal samples were prepared as 1:10 (W/V) homogenate suspensions with DMEM. The suspensions were then vortexed and centrifuged for 20 min at 12,000 rpm. RT-PCR was performed to directly amplify a partial M gene sequence of PEDV from the clarified supernatants, using the primers listed in Appendix A (Supplementary materials) according to a method described previously [10]. Thirty amplified products with a size of 370 bp were randomly selected (two amplicons from each pig farm) for purification and were subjected to sequencing (Tsingke, Beijing, China). Nucleotide sequence identities were analyzed with DNAStar software. In addition, the complete M and S genes of PEDV were amplified from positive samples (one from each pig farm) by RT-PCR using the primers listed in Appendix A, and the amplicons were subjected to sequencing. The complete genome sequence of a PEDV field strain (designated BJ-2011-1) from a pig farm in Beijing was determined by RT-PCR amplification of fecal sample using the primers shown in Appendix A and sequencing the amplicons. Briefly, viral RNA was extracted from the clarified supernatants with TRIzol (Transgen, Beijing, China) according to the manufacturer’s instructions. Reverse transcription (RT) was performed using a Quantscript RT Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. Then, PCR was conducted to amplify each fragment using PrimeSTARTM HS DNA polymerase (TaKaRa, Japan) under the following conditions: denaturation at 98 °C for 30 s, 35 cycles of denaturation at 98 for 10 s, annealing at 55 °C for 30 s, and extension at 72 °C for 2 min. After gel purification, the amplified fragments were cloned into pEASY-Blunt Cloning Vector (Transgen) and sequenced in both directions using commercial vector-specific M13 primers. The nucleotide and deduced amino acid sequence of each gene for this strain were aligned and analyzed with Clustal X version 1.83 [21] and DNASTAR software. Phylogenetic trees were generated by the neighbour-joining method in MEGA 4.1 [9, 20], and the robustness of phylogenetic constructions was evaluated by bootstrapping with 1,000 replicates. A representative sequence of each complete gene for PEDV was downloaded from the GenBank database (Appendix B of Supplementary materials) and was used for homology comparison and phylogenetic analysis as a reference sequence.

Clinical observations were recorded at pig farms where outbreaks of watery diarrhea had occurred. On these farms, acute diarrhea occurred extensively in 3- to 10-day-old suckling piglets. The affected piglets exhibited watery diarrhea, severe dehydration, and 100 % morbidity. The mortality of the diseased piglets could reach 80-100 % within one week. In pigs of other ages, rare clinical signs were observed. Occasionally, sporadic cases with diarrhea could be seen in sows. In the majority of affected pig farms, the outbreak lasted more than four weeks.

The results obtained by RT-PCR detection showed that the prevalence of the M gene of PEDV in the collected samples was 92.7 % (267/288) (Table 1). Sequencing of the selected amplicons showed that the partial M genes shared 99.5-100 % nucleotide sequence identity and 100 % amino acid similarity with each other, indicating that the amplified partial PEDV M genes from the clinical samples were relatively conserved.
Table 1

Detection results of clinical samples from fifteen pig farms with watery diarrhea outbreaks in suckling piglets by RT-PCR

Pig farm no.

Geographic origin

Mortality in suckling piglets with watery diarrhea within one week (%)

Date of sample collection

Sample origin

Positive rate by RT-PCR

1

Beijing

87.9

2011 Jan

Feces

100 % (10/10)

2

Beijing

88.2

2011 Mar

Feces & intestine tissue

97 % (32/33)

3

Beijing

100

2011Dec

Feces

60 % (6/10)

4

Beijing

94.2

2012 Jan

Feces

93.3 % (14/15)

5

Beijing

96.1

2012 Feb

Feces

88 % (22/25)

6

Hebei

83.5

2011 Feb

Feces

100 % (20/20)

7

Hebei

85

2011 Feb

Feces

100 % (22/22)

8

Hebei

88.5

2011 Mar

Feces & intestine tissue

70 % (7/10)

9

Hebei

96.7

2011 Dec

Feces

92 % (23/25)

10

Hebei

94.9

2012 Jan

Feces

90 % (18/20)

11

Hebei

97.5

2012 Feb

Feces & intestine tissue

100 % (15/15)

12

Hebei

97.2

2012 Mar

Feces

94.4 % (17/18)

13

Hebei

94.1

2012 Mar

Feces

92 % (23/25)

14

Zhejiang

98

2011 Apr

Feces

86.7 % (13/15)

15

Zhejiang

98.1

2011 Apr

Feces

100 % (25/25)

     

Total, 92.7 % (267/288)

Detection of partial M gene using the primers listed in Table 1 by RT-PCR

As a structural membrane glycoprotein, the M protein of PEDV plays an important role in the assembly process [4, 13] and is able to induce antibodies that neutralize the virus in the presence of complement [19]. The M gene has been proposed to be used as a target gene for phylogenetic analysis of PEDV in the field [3, 14, 18]. The 15 complete M genes that were amplified were 681 nt in length (GenBank accession nos. JX435306-JX435320) and shared 99.6-100 % nucleotide sequence identity and 99.1-100 % amino acid similarity with each other. Phylogenetic analysis indicated that the representative strains of PEDV downloaded from GenBank grouped into three clusters (1-3) (Fig. 1A). All of the M genes sequenced in this study were grouped in cluster 3, together with some Chinese, Korean and Thai strains, while the majority of Korean strains and some previous Chinese strains were in cluster 2, most Chinese strains and some Korean strains were grouped in cluster 1, together with the PEDV prototype strain CV777. These analyses indicated that the M genes obtained in this study were relatively conserved and exhibited minor variations although the M gene of PEDV was genetically diverse.
https://static-content.springer.com/image/art%3A10.1007%2Fs00705-012-1541-2/MediaObjects/705_2012_1541_Fig1_HTML.gif
Fig. 1

Phylogenetic analysis of PEDV based on nucleotide sequences of M (A) and S (B) genes sequenced in this study (in boldface) and other PEDV reference strains. The phylogenetic tree was constructed using the MEGA 4.1 software. The scale bar represents nucleotide substitutions per site. Δ, Korean strains; ∇, Thai strains; ▲, Japanese strains; •, Chinese strains; ⋄, European strains. GenBank accession numbers of all the reference strains are listed in Appendix B of Supplementary materials

The S gene of PEDV encodes the spike glycoprotein, which is known to play a pivotal role in interacting with the cellular receptor to mediate viral entry and inducing neutralizing antibodies in pigs as an immunogenic protein [1, 2, 7]. The S gene can serve as a target gene for analyzing genetic variation of PEDV strains in the field [15, 18]. The 15 S genes that were amplified from the clinical samples (GenBank accession nos. JN825706-JN825712, JX435298-JX435305) had a size of 4161 nt. Phylogenetic analysis indicated that the representative complete S genes downloaded from GenBank could be divided into three clusters (Fig. 1B). All of the S genes sequenced in this study were grouped in cluster 3, together with Korean strains only. Cluster 2 comprised Korean and Japanese strains, and cluster 1 consisted of previous Chinese, Korean, Japanese and Thai strains, as well as European strains (CV777 and Br1/87). The 15 S genes in this study were highly homologous, with nucleotide identity and deduced amino acid similarity of 98.6-99.9 % to each other, suggesting that the same strain of PEDV is epidemic in these pig farms in China. These S genes were closely related to Korean strains, showing 96.9-97.2 % nucleotide sequence identity and 96.-96.9 % amino acid similarity to Korean strains in cluster 3, and 94.3-96 % nucleic acid and 92.8-95.2 % amino acid sequence identity to Korean and Japanese strains in cluster 2. The deduced amino acids of these S genes were further aligned with the representative S genes downloaded from GenBank (Appendix C of Supplementary materials). Interestingly, all S genes had 4-aa (GENQ) and 1-aa (N) insertions between positions 56 and 59, and 135 and 136, and a 2-aa (DG) deletion between positions 155 and 156 in their coding regions when compared with CV777, previous Chinese strains (LZC, CH/S, JS-2004-2, LJB-03 and DX), Korean strains (Chinju99) and Japanese strains (MK, 83P-5). Of these variations, the amino acid insertions between positions 155 and 156 were identical to those found in Korean KNU-serial strains (the representative KNU-0802) reported in recent years [11] and were similar to Korean strain Spk1, with the insertion GETQ, and Japanese strains NK and KH, with the insertion GEQQD/N, while the amino acid deletion between positions 155 and 156 was identical to that found in Korean KNU-serial strains [11]. In addition, the variations of individual amino acids in the 15 S genes were identical or similar to those observed in Korean and Japanese strains.

The genome of PEDV BJ-2011-1 sequenced in this study was 28,038 nucleotides in length (GenBank accession no. JN825712), excluding the poly (A) tail. Comparative analysis of the whole genome sequence indicated that this strain shared 97 % nucleotide sequence identity with strain CV777, 96.7 % with both LZC and SM98, 97.1 % with CH/S, and 97.9 % with DR13. In addition to the M and S genes, which were discussed above, the Pol 1 gene, with a size of 20,345 nt, had 97.4 %, 97.5 %, 97.6 %, 98.1 %, and 98.4 % nucleotide sequence identity to SM98, LZC, CV777, CH/S and DR13, respectively. The E gene, with 231 nt, had 97 % nucleotide sequence identity to CV777, 96.1-97.8 % to four Chinese strains (LZC, LJB/03, CH/S and DX), and 95.7-96.1 % to three Korean strains (SM98, DR13 and Chinju99). The ORF3 gene, with a size of 675 nt, exhibited 96-99.3 %, 97-99.1 %, and 97.3 % nucleotide sequence identity to 11 Chinese strains, 20 Korean strains, and CV777, respectively. The N gene, with a size of 1326 nt, shared 95.4-99.5 % nucleotide sequence identity with 19 Chinese strains, 94.8-97.0 % with two Korean strains, 96.1 % with one Japanese strain, and 95.6 % to CV777.

It is clear that the PEDV prevailing in China is a novel strain with a variant S gene that is distinct from previous Chinese strains. Currently, the PEDV used for preparation of vaccines in China is derived from strain CV777. Thus, the characteristic variations of the S gene of PEDV field strains might result in less-effective immunological protection induced by current vaccines because the S glycoprotein functions to induce neutralizing antibodies against PEDV. Further investigation of the efficacy of current vaccines against recently emerging PEDV strain is needed.

Based on molecular detection of a partial M gene of PEDV in clinical samples, our results showed that PEDV is the causative pathogen contributing to the outbreaks of clinical diarrhea in these pig farms, and the prevailing PEDV field strains have common molecular characteristics in their S genes, suggesting that a novel strain of PEDV is circulating in Chinese pig farms. The pathogenesis and immunogenicity of PEDV field strains need to be explored in order to better prevent and control the epidemic of PED caused by the virus.

Acknowledgments

This work was supported by China Agriculture Research System of China (CARS-36).

Supplementary material

705_2012_1541_MOESM1_ESM.doc (213 kb)
Supplementary material 1 (DOC 213 kb)

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© Springer-Verlag Wien 2012