Infection, Genetics and Evolution 21 (2014) 205–213
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Genetic diversity of ORF3 and spike genes of porcine epidemic diarrhea virus in Thailand Gun Temeeyasen a, Anchalee Srijangwad a, Thitima Tripipat a, Pavita Tipsombatboon b, Jittima Piriyapongsa b, Waranyoo Phoolcharoen c, Taksina Chuanasa c, Angkana Tantituvanont d, Dachrit Nilubol a,⇑ a
Department of Veterinary Microbiology, Faculty of Veterinary Science, Bangkok 10330, Thailand Genome Institute, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand d Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand b c
a r t i c l e
i n f o
Article history: Received 21 June 2013 Received in revised form 2 November 2013 Accepted 5 November 2013 Available online 26 November 2013 Keywords: Porcine epidemic diarrhea virus Spike gene ORF3 gene Diversity Thailand
a b s t r a c t Porcine epidemic diarrhea virus (PEDV) has become endemic in the Thai swine industry, causing economic losses and repeated outbreaks since its first emergence in 2007. In the present study, 69 Thai PEDV isolates were obtained from 50 swine herds across Thailand during the period 2008–2012. Both partial and complete nucleotide sequences of the spike (S) glycoprotein and the nucleotide sequences of ORF3 genes were determined to investigate the genetic diversity and molecular epidemiology of Thai PEDV. Based on the analysis of the partial S glycoprotein genes, the Thai PEDV isolates were clustered into 2 groups related to Korean and Chinese field isolates. The results for the complete spike genes, however, demonstrated that both groups were grouped in the same cluster. Interestingly, both groups of Thai PEDV isolates had a 4-aa (GENQ) insertion between positions 55 and 56, a 1-aa insertion between positions 135 and 136, and a 2-aa deletion between positions 155 and 156, making them identical to the Korean KNU series and isolates responsible for outbreaks in China in recent years. In addition to the complete S sequences, the ORF3 gene analyses suggested that the isolates responsible for outbreaks in Thailand are not vaccine related. The results of this study suggest that the PEDV isolates responsible for outbreaks in Thailand since its emergence represent a variant of PEDV that was previously reported in China and Korea. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Porcine epidemic diarrhea virus (PEDV) is the etiologic agent of porcine epidemic diarrhea (PED), a devastating enteric disease that is characterized by vomiting, acute severe watery diarrhea and dehydration, which results in significant high mortality in piglets under 7 days of age (Pensaert and de Bouck, 1978). In addition to causing enteric problem in pigs, PEDV infection in sows can lead to a reduction of subsequent reproductive performance (Olanratmanee et al., 2010). PED was first recognized in Belgium and the United Kingdom in 1976–1978 (Pensaert and de Bouck, 1978; Wood, 1977). Post-emergence, disease outbreaks have been reported in several European countries (Chasey and Cartwright, 1978). At present, PED disease has continued to causing devastating enteric disease and severe economic losses in many Asian ⇑ Corresponding author. Address: Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University, Henry Dunant Road, Pathumwan, Bangkok 10330, Thailand. Tel.: +66 2 218 9583; fax: +66 2 251 1656. E-mail address: [email protected] (D. Nilubol). 1567-1348/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.11.001
countries, including China, Korea, Japan and Thailand (Gao et al., 2013; Li et al., 2012b; Park et al., 2007a, 2011; Sato et al., 2011). PEDV is an enveloped, single-stranded RNA virus belonging to the genus Alphacoronavirus, family Coronaviridae and order Nidovirales. The PEDV genome is approximately 28 kb in length and is composed of 7 open reading frames (ORF) (Kocherhans et al., 2001). ORF1a and ORF1b cover 70% of the entire genome and encode the non-structural replicase gene. The remaining 5 ORFs code for major structural proteins including the spike (S) glycoprotein, ORF3, envelope protein, membrane protein and nucleocapsid protein. Among the proteins encoded by these genes, the S glycoprotein is a glycosylated protein located on the envelope of the virus. It is involved with pathogenesis by binding to cell receptors on the host cell membranes and penetrating cells by membrane fusion. The S glycoprotein consists of 2 domains, including S1 and S2 domains. Whereas the S1 domain is involved in viral attachment, the S2 domain undergoes conformational changes following S1 attachment to facilitate fusion of the virus envelope with the host membrane, allowing entry of the viral genome into the host.
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Because of its important role in viral attachment and entry, the S1 domain was characterized and demonstrated to contain epitopes involved with neutralizing antibodies (Chang et al., 2002; Cruz et al., 2006, 2008; Sun et al., 2008). Because of its role in the induction of neutralizing antibodies, the S protein is the primary target for the development of a vaccine against PED (Kang et al., 2006; Oszvald et al., 2007). Several investigators have reported a high degree of genetic diversity observed in the S glycoprotein gene (Lee et al., 2010; Li et al., 2012b; Park et al., 2007a, 2011). Thus, this gene is important for understanding the genetic relatedness of PEDV field isolates, the epidemiological status of the virus and vaccine development. In addition to the S glycoprotein gene, the accessory ORF3 gene has received a large amount of attention in the context of PEDV virulence. The virulence of PEDV can be reduced by altering the ORF3 gene through cell culture adaptation (Park et al., 2007b, 2008). The ORF3 genes have been used to differentiate between field and vaccine-derived isolates. The vaccine-derived isolates have unique characteristics at amino acids 82–99, where a continuous deletion of 17 amino acids has been observed (Park et al., 2008). Therefore, this deletion can be used to differentiate between field and attenuated vaccine viruses. The differentiation of the ORF3 gene can be used for molecular epidemiology studies of PEDV. In Thailand, a PED outbreak was first reported in 1995 in a farm located in the southern region of the country (Srinuntapunt et al., 1995). The disease did not spread to other swine-producing areas at that time; however, in late 2007, the disease re-emerged in Nakorn Pathom, a province with the highest pig densities in Thailand (Puranaveja et al., 2009). The source of viral introduction has not been identified to date. There is only speculation concerning contaminated pork meat and bone meal, the illegal introduction of the vaccine or both. Since its re-emergence, PED has spread throughout the country, causing pandemic PED outbreaks in Thailand resulting in >90% of Thai swine farms being infected. Currently, the disease develops until reaching an endemic stage in which many herds experience recurrent outbreaks. To develop a more successful control and prevention program, further investigation related to the genetic diversity and molecular epidemiology of PEDV in Thailand is needed. The objectives of the present study were to investigate the genetic diversity of the S glycoprotein and ORF3 genes of PEDV in Thailand. The results of a study on the genetic differences of the S genes may provide information on the heterogeneity of the outbreak. Moreover, such a study may also provide a basis of knowledge that can be used for the development of an effective vaccine.
2. Materials and methods 2.1. Source of specimens and PEDV isolation A total of 120 porcine intestinal samples were collected from 50 pig farms that experienced PED outbreaks from 2008 to 2012. In each outbreak, intestinal samples were collected from 3- to 4-day-old piglets that displayed the clinical features associated with PED, including vomiting, watery diarrhea and dehydration. The intestinal samples were subjected to PEDV isolation using the continuous Vero cell line (ATCC, CCL-81) (Hofmann and Wyler, 1988). Briefly, the intestinal samples of piglets were minced into small pieces and suspended in 10 ml of phosphate buffer saline (PBS; 0.1 M, pH 7.2). The suspended samples were vortexed and clarified by centrifugation at 1350g for 10 min. The supernatant was filtered through 0.45-lm filters and stored at 80 °C until use. Five hundred microliters of supernatant samples was diluted with maintenance media (MM) consisted of Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10 lg/
ml trypsin (Gibco, USA) and inoculated into a 25-cm2 flask that had been previously plated with Vero cells for 2 days or until full confluence was reached. The inoculated 25-cm2 flask was incubated at 37 °C in 5% CO2 for 1 h. Then, the media was replaced with 10 ml of MM, and incubated again for a period of 2–3 days. Cytopathic effect (CPE) was observed with a light microscope. Then, the 25-cm2 flasks were frozen and thawed 2 times before being centrifuged at 1350g for 10 min to collect the supernatant, which was stored at 80 °C. The clarified supernatants were subjected to RT-PCR. Partial S, complete S and ORF3 genes were amplified and sequenced. The intestinal samples were considered negative for PEDV isolation following 3 blind passages. 2.2. Reverse transcription-polymerase chain reaction Total RNA was extracted from the supernatant from the previous procedure using the NucleospinÒ RNA Virus kit (Macherey-Nagel Inc., PA, USA) in accordance with the manufacturer’s instructions. cDNA was synthesized from the extracted RNA using M-MuLV Reverse Transcriptase (New England BioLabs Inc., MA, USA). PCR amplification was performed on the cDNA. To amplify the partial S genes, the following primers were utilized: PEDF (50 -TTC TGA GTC ACG AAC AGC CA-30 ) and PEDR (50 -CAT ATG CAG CCT GCT CTG AA-30 ) (Park et al., 2007a). PCR amplification was performed using PlatinumÒ Tag DNA polymerase High Fidelity (Invitrogen, CA, USA). After the initial incubation at 94 °C for 5 min, the reactions were subjected to 35 cycles of PCR as follows: 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. These cycles were followed by a terminal 7-min extension at 72 °C. To amplify the complete S genes, the sequence was separately constructed for S1 and S2,with the primers S-F-1 (50 -ACG TAA ACA AAT GAG GTC TTT-30 ) and S-R-1 (50 -ATA CAC CAA CAC AGG CTC TGT-30 ) used to amplify S1 and the primers S-F-2 (50 -GGT TTC TAC CAT TCT AAT GAC G-30 ) and S-R-2 (50 -GTA TTG AAA AAG TCC AAG AAA CA-30 ) used to amplify S2 (Lee et al., 2010). PCR amplification was performed using PlatinumÒ Tag DNA polymerase High Fidelity (Invitrogen, CA, USA). After an initial incubation at 94 °C for 5 min, the reactions were subjected to 35 cycles of PCR as follows: 94 °C for 30 s, 55 °C (58 °C for S2) for 30 s, and 72 °C for 2 min. These cycles were followed by a terminal 10-min extension at 72 °C. The PCR products were visualized by agarose gel electrophoresis. To amplify the ORF3 genes, primers ORF3F (50 -CCT AGA CTT CAA CCT TAC GA-30 ) and ORF3R (50 -CAG GAA AAA GAG TAC GAA AA-30 ) were used, and PCR amplification was performed using PlatinumÒ Tag DNA polymerase High Fidelity (Invitrogen, CA, USA). After an initial incubation at 94 °C for 5 min, the reactions were subjected to 30 cycles of PCR as follows: 94 °C for 60 s, 50 °C for 60 s, and 72 °C for 60 s. These cycles were followed by a terminal 10-min extension at 72 °C. The PCR products were visualized by agarose gel electrophoresis. 2.3. Cloning, plasmid purification and sequence determination The PCR products were cloned into plasmid vectors for the subsequent transformation of Escherichia coli cells by using a commercial kit (pGEM-TÒ Easy Vector System I (Promega, WI, USA), and the controls were included at all stages of cloning and transformation. Bacterial transformant colonies were randomly selected from each sample for plasmid purification using the NucleospinÒ Plasmid kit (Macherey-Nagel Inc., PA, USA), and the 10 elected colonies were grown in LB broth for 24 h and subjected to plasmid isolation and sequencing. Sequencing reactions were performed by Biobasic Inc. (Ontario, Canada) using an ABI Prism 3730XL DNA sequencer.
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2.4. Sequence analyses The nucleotide and deduced amino acid sequences were aligned using the CLUSTALW program (Thompson et al., 1994). The percentage of identity between every pair of isolate sequences at the nucleotide and amino acid levels was calculated using MEGA 5.2 (Tamura et al., 2011). The analyses included only sequences in which nucleotide sequence data are complete and accession numbers are available in GenBank (Supplementary Material 1). Phylogenetic analyses of the partial S, complete S, and ORF3 genes was separately performed using a Bayesian Markov chain Monte Carlo (BMCMC) method implemented in the program BEAST v1.7.4 (Drummond et al., 2012). The analysis of the partial S gene included the nucleotide sequences from the present study, previously reported partial S sequences of the Thai PED (Puranaveja et al., 2009) and isolates from Belgium, United Kingdom (UK), China and Korea (Park et al., 2007a). The analysis of the complete S and ORF3 genes included the nucleotide sequences from the present study and isolates from Belgium, UK, China and Korea (Gao et al., 2013; Lee et al., 2010; Li et al., 2012a). The strict molecular clock model with a coalescent constantsize tree prior and empirical base frequencies were used for all analyses. The best fitted substitution model and among-site rate heterogeneity model for each gene data set was determined using Model Generator v0.85 (Keane et al., 2006). For each analysis, three independent BEAST runs were performed; each consisting of 50 million generations with sampling of every 1000 generations and the first 10% discarded as burn-in. LogCombinerv1.7.4 (http:// beast.bio.ed.ac.uk/LogCombiner) was used to combine logand tree files from independent runs of BEAST. Tracer v1.5 (http://beast.bio.ed.ac.uk/Tracer) was used to confirm that post-burn-in trees yielded an effective sample size (ESS) of >200 for all parameters. A 50% majority-rule consensus tree was generated from the tree file using SumTrees v.3.3.1 (Sukumaran and Holder, 2010). The resulting tree was viewed in FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/).
3. Results 3.1. Phylogenic analysis of the partial spike gene Of the 120 porcine intestinal samples collected in the years 2008–2012, 69 PEDV isolates were collected and the origins of those Thai isolates are shown in Fig. 1. All isolates produced CPE characterized by cell fusion and syncytia formation in the first passage (Supplementary Material 2). To investigate the heterogeneity of these 69 Thai PEDV isolates, the partial spike genes of the 69 field isolates were amplified. The nucleotide sequences revealed that these partial S glycoprotein genes of the 69 field isolates consisted of 657 nucleotides encoding a protein of 219 amino acids located on the full length of the S glycoprotein gene at nucleotide positions 1462–2118 or amino acid positions 488–706. Identical partial S nucleotide sequences were identified and excluded, resulting in further genetic analysis of 30 genes. The phylogenetic tree of 30 partial S glycoprotein genes was constructed together with another 16 previously reported Thai PEDV isolates (Puranaveja et al., 2009) as well as some partial S glycoprotein genes that were previously isolated in other countries (Park et al., 2007a). Based on the previously reported phylogenetic analysis (Park et al., 2007a), the PEDV isolates were further divided into 3 groups, designated G1, G2 and G3; group G1 was further divided into 3 subgroups, which were designated G1-1, G1-2 and G1-3. When using the clustering system based on this phylogenetic analysis (Park et al., 2007a), the phylogenetic tree demonstrated that the Thai PEDV isolates were further divided into 2 groups
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(Fig. 2). Both groups of Thai PEDV isolates were in G1 but differed in subgroup. The most dominant isolates were clustered in subgroup G1-1, which comprised all 16 previously reported Thai PEDV isolates along with 27 of 30 current Thai isolates. Another subgroup designated G1-4 comprised 3 of 30 isolates, including AGPED0609_1, AGPED0609_2 and SPPED0111_1. This subgroup did not fit into the previously reported clustering system. It sat between subgroups 1-2 and 1-3. Therefore, we named it subgroup G1-4. After comparing the nucleotide and amino acid sequences of all Thai field PEDV isolates, it was demonstrated that the nucleotide and amino acid sequence identity of the all Thai field PEDV isolates ranged from 94.4–99.9% and 85.8–100.0%, respectively. The percentage of identity between nucleotide and amino acid sequences of the Thai G1-1 isolates ranged from 96.4–99.7% and (92.5–100.0%), respectively. The nucleotide and amino acid sequences of isolates in G1-1 were more divergent compared to those of G1-4, in which all 3 isolates had 100.0% identity at the nucleotide and amino acid level. Subgroups G1-1 and G1-4 had 94.4–95.5% and 85.8–88.5% identity, respectively, at the nucleotide and amino acid level. 3.2. Genetic analyses of complete spike gene To investigate the genetic diversity of 2 groups of Thai field PEDV isolates and their genetic relationship with PEDV isolates from other countries, 13 different Thai field PEDV isolates, including 10 isolates from subgroup G1-1 and 3 isolates from subgroup G1-4, were randomly selected, and the full-length nucleotide sequences of their S glycoprotein genes were investigated. The phylogenetic tree of their complete S genes was constructed together with those of other PEDV isolates from other countries (Supplementary Material 1). The phylogenetic analysis revealed that the complete S glycoprotein genes were further divided into 3 clusters, including clusters 1, 2 and 3 (Fig. 3). Cluster 1 comprised Chinese isolates (CH/FJND-3, CH1, CH8, CHGD-01, HB-2011-1, HB-2011-2, HB-2011-3, HB-2011-4, HB-2012-1, HB-2012-2, HB-2012-3, HB2012-4, BJ-2011-2, BJ-2011-3, BJ-2012-1, BJ-2012-2, ZJ-2011-1 and ZJ-2011-2) and Korean field isolates (KNU-0802,KNU-0902, CNU-091222-01 and CNU-091222-02). Based on the results of the partial S gene comparisons, all 13 PEDV Thai isolates in subgroups G1-1 and G1-4 were included in cluster 1. Cluster 2 comprised 7 isolates from Korea (KNU-0801, KNU-0901, KNU-0903, KNU-0904, KNU-0905, Spk1 and Chinju99). Cluster 3 comprised 16 reference strains, including isolates from Korea (DR13 and SM98), China (CH2, CH3, CH4, CH5, CH6, CH7, CH/FJND-1, CH/ FJND-2, JS-2004-2, LZC, LJB03 and DX) and Europe (CV777 and Br1/87). The sequence homology between the S glycoprotein genes was measured. The nucleotide and amino acid sequence identity of the cluster 1 isolates ranged from 94.4–99.9% and 93.1–99.9%, respectively (Supplementary Material 3). The isolates had 93.0–97.8% (90.7–96.5%) and 92.4–98.1% (90.1–97.7%) nucleotide (deduced amino acid) sequence identity with members of groups 2 and 3, respectively. The percentage of identity between nucleotide and amino acid sequences of all 13 Thai field PEDV isolates ranged from 94.9–99.8% and 94.0-99.7%, respectively. All 13 Thai PEDV isolates displayed 94.9–99.9% and 94.0–99.9% identity to Chinese isolates in this cluster at the nucleotide and amino acid levels, respectively. In addition, all 13 Thai PEDV isolates displayed 94.9–99.8% and 94.0–99.7% identity to Korean isolates (KNU0902, CNU-09122201 and CNU-091222-02) at the nucleotide and amino acid levels, respectively. It was demonstrated that the S glycoprotein genes of the Thai PEDV isolates are 4158 nucleotides in length and encode 1386 amino acid residues. The nucleotide and deduced amino acid sequences of the complete S genes were aligned separately to
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Fig. 1. Map of Thailand. Red dots represent the provinces where the porcine epidemic diarrhea virus were isolated and used for sequencing in this study. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
investigate the amino acid differences between the clusters, especially the hypervariable regions and the 4 major epitope regions. The largest number of amino acid differences was clustered in the N-terminal region of the S1 domain. The N-terminal region of the S1 gene demonstrated the lowest similarity with the PEDV reference strain. The isolates in cluster 1 and 2, except isolate Chinju99, had 2 insertions of 4 (56GENQ59) and 1 (140N) amino acids at amino acid positions 55–60 and 140, which are located in the hypervariable domain in the N-terminus of the S1 region (Supplementary Material 4). In addition, 1 deletion of 2 amino acids was observed at amino acid positions 160 and 161. All 13 Thai isolates in cluster 1 examined in this study have similar patterns of amino acid insertion and deletion. Several studies have identified 4 regions in the S glycoprotein that contains major epitopes (Cruz et al., 2008; Kang et al., 2006; Sun et al., 2008). Those 4 regions include amino acid positions 504–643 (Kang et al., 2006); 753YSNIGVCK760 and 769LQDGQVKI776 (Sun et al., 2008); and 1373GPRLQPY1379 (Cruz et al., 2008). Therefore, we analyzed these 3 regions. It was demonstrated that amino
acid position 753YSNIGVCK760 and 1373GPRLQPY1379 were conserved between the Thai PEDV isolates and isolates from other countries. However, the amino acid positions 504–643 and 769LQDGQVKI776 were demonstrated to be variable. The largest number of amino acid differences was observed in amino acid positions 504–643. In addition, amino acid differences were observed in the 769 LQDGQVKI776 region, with the substitutions of S ? L and S ? D/ Y at amino acid positions 769 and 771, respectively. 3.3. Genetic analyses of ORF3 genes To investigate the genetic relationship between the Thai field PED isolates and modified live vaccine, the nucleotide sequences of the ORF3 genes of 13 Thai PEDV isolates were investigated. Identical sequences were excluded, resulting in 7 isolates for further analysis. The phylogenetic tree of the complete ORF3 genes was constructed along with those of other reference isolates (Supplementary Material 1). The phylogenetic analysis revealed that, based on the ORF3 gene, the PEDV isolates were further divided
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Fig. 2. Phylogenetic analysis of the porcine epidemic diarrhea virus isolates based on the nucleotide sequences of the partial spike glycoprotein genes. The tree includes nucleotide sequences of Thai isolates from the present study (light blue font) and a previous study (red font) as well as sequences from China (pink font), Korea (dark blue font) and European countries (green font). The 50% majority-rule consensus tree was constructed using Bayesian MCMC method. Each internal node with the posterior probability of the corresponding clade >0.5 is labeled. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
into 3 clusters: clusters 1, 2 and 3 (Fig. 4). Cluster 1 was further divided into 2 sub-clusters: sub-clusters 1-1 and 1-2. All Thai field isolates were in cluster 1 and sub-cluster 1-1, as were the isolates from China. Sub-cluster 1-2 consisted of Chinju99 and DR13, isolates from Korea. Cluster 2 consisted of CV777 and isolates with a unique truncated portion, a 17-amino acid deletion at position 82–98, which represented attenuated virus vaccines, and no Thai PEDV isolate was included in this group. Cluster 3 consisted of MC, which was isolated in China. The sequence identity of the ORF3 genes in cluster 1 ranged from 96.0–100.0% and 93.7–100.0%, at the nucleotide and amino acid levels, respectively (Supplementary Material 5). They have
94.8–99.0.5% (92.4–99.5%) and 94.1–96.9% (93.7–97.3%) nucleotide (deduced amino acid) sequence identity with members of groups 2 and 3, respectively. The ORF3 genes of the Thai PEDV isolates were relatively conserved. Only point substitution was observed (Supplementary Material 6).
4. Discussion Since its emergence in late 2007, porcine epidemic diarrhea virus has continued to cause economic damage to the Thai swine industry. The disease has developed to an endemic stage. Several
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Fig. 3. Phylogenetic analysis of the porcine epidemic diarrhea virus isolates based on the nucleotide sequences of the complete spike glycoprotein genes. The tree includes nucleotide sequences from Thailand (light blue font), China (pink font), Korea (dark blue font) and European countries (green font). The 50% majority-rule consensus tree was constructed using Bayesian MCMC method. Each internal node with the posterior probability of the corresponding clade >0.5 is labeled. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
herds continue to experience repeated outbreaks, but the disease severity, as demonstrated by piglet mortality, has lessened. Although several control methods including vaccination and strict biosecurity have been implemented, these measures have had varying degrees of success. The recurrence of PED outbreaks has led to the necessity for further investigation into the genetic diversity of PEDV. A better understanding of the genetic diversity of PEDV may facilitate the development of a more successful control program and vaccines. To investigate the heterogeneity of PEDV in Thailand, the partial and complete S genes were characterized. The partial S gene was initially characterized because it can be used to characterize a very large number of samples, unlike the characterization of the com-
plete S gene, in which the characterization is more complicated due to the large size of the gene. In addition, previous studies have reported the use of the partial S gene, including the neutralization epitope regions of PEDV, to investigate the genetic diversity of PEDV and to divide it into clusters; these studies have also demonstrated that this region displays high similarity when using complete S glycoprotein genes (Park et al., 2007a). Therefore, we initially chose to study the partial S genes to understand the heterogeneity of PEDV in Thailand. Based on the characterization of the partial S glycoprotein gene, the results of the present study demonstrated that Thai PEDV isolates are further divided into 2 groups. However, those 2 groups were grouped in the same cluster, cluster 1, based on the characterization of the complete S genes. In cluster
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Fig. 4. Phylogenetic analysis of the porcine epidemic diarrhea virus isolates based on the nucleotide sequences of the ORF3 genes. The trees include nucleotide sequences from Thailand (light blue font), China (pink font), Korea (dark blue font) and European countries (green font). The 50% majority-rule consensus tree was constructed using Bayesian MCMC method. Each internal node with the posterior probability of the corresponding clade >0.5 is labeled. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
1, which appeared to be the most prevalent cluster of virus in Thailand, PEDV consisted of isolates genetically related to KNU-serial isolates, PEDV field isolates from Korea, and isolates that have caused outbreaks in China in recent years. A previous report suggested that Thai PEDV isolates were China-like isolates and similar to an isolate named JS2004 that was isolated in China. The results of the present study are in contrast with that previous report. The results of the current study, based on the comparison of the complete S glycoprotein genes of PEDV field isolates, which are the isolates that continue to cause economic damage to the Thai swine industry, indicate that the Thai isolates belong to cluster 1. The Thai PEDV isolates collected from 2008–2012 possess a unique and common genome characteristic defined by the insertion of 4 amino acids (GENQ) between positions 55 and 56, a 1-aa (N) insertion between positions 135 and 136, and the deletion of 2 amino acids between positions 155 and 156. These unique isolates were genetically related to or identical to previously reported Korean KNU-serial isolates (Lee et al., 2010) and isolates that were responsible for recent severe
outbreaks in China (Gao et al., 2013; Li et al., 2012a). In addition, the isolates with unique characteristics, which were reported to be a variant of PEDV, emerged in China and caused more severe outbreaks (Li et al., 2012a). The isolate JS2004, however, does not possess the 2 discontinuous insertions and the one deletion shared by the above mentioned isolates. The discrepancy of the results between the present study and previous studies could be explained by the current study’s investigation of both partial and complete S glycoprotein genes, whereas the previous study reported on only the partial S genes. These contradictions may have arisen because the phylogenetic analysis of the partial S gene was based on only 657 nucleotides that are involved in coding for amino acid positions 488–706 of the complete S genes. This coverage level was not sufficient to represent the whole gene of the virus. In addition, the 2 discontinuous deletions and the one insertion mentioned above are located in the N-terminus of S1 gene region. The deduced amino acid, therefore, highlights the disadvantage of phylogenetic analysis using only partial S genes to investigate the molecular epidemiology or heterogeneity of PEDV isolates.
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The method of PEDV introduction to Thailand is not known. It has been speculated that imported pork meat and bone materials from countries where PEDV is endemic is a possible route of introduction. The outbreak was first reported in Nakorn Pathom province, which is located in the central region of Thailand, an area very distant from the border. In addition, no genetic materials such as gilts or boar were imported to this farm. The only observed changes were that the raw material for manufacturing feed was changed from fish meal to pork meat and bone meal due to high fish meal prices. To investigate whether the isolates are closely related to field isolates in Korea or to vaccine isolates, the ORF3 genes of Thai field isolates were sequenced. The ORF3 genes have been used to differentiate between field and vaccine-derived isolates. The vaccinederived isolates have a unique characteristic at nucleotide position 245–293, a continuous deletion of 49 NT. The results of this study demonstrated that the Thai field isolates do not possess that unique 49-nucleotide deletion suggesting that they are not vaccine-related. PEDV has become endemic and thus continues to cause damage to the Thai swine industry. Although the severity of the disease has been reduced and the mortality following repeated outbreaks has been contained to piglets farrowed from primi-parous sows, the disease continues to cause economic losses. Repeated outbreaks in herds occur from every 3–6 months. Herds with repeated outbreaks have higher production costs compared with herds with no repeated outbreaks. To investigate whether the heterogeneity of PEDV plays an important role in disease recurrence, the S genes of 10 isolates from 3 herds with repeated outbreaks were characterized. The genetic analysis suggested that all 10 isolates were grouped in cluster 1 and had only few amino acid differences in the S genes. These results suggest that the heterogeneity of the S gene might not contribute to disease recurrence in herds. Other factors, including the immune status of replacement gilts prior to introduction, might play a more important role in disease recurrence. Several management strategies including vaccination have been implemented to control PEDV in swine herds. Most common vaccination protocol includes 2 dosages of modified live and killed vaccines in gilts prior to introduction and pre-farrow vaccination at 12 and 14 weeks of gestation with KV. Although PEDV vaccines are not commercially available in Thailand, we have noticed the use of these vaccines, illegally smuggled, from China and Korea. Their efficacy is questionable, as herds with heavy vaccination programs, including whole herd vaccination and pre-farrow vaccination, still experience repeated outbreaks. Because the neutralizing epitopes of Thai PEDV isolates are hypervariable compared with PEDV isolates in vaccines, these amino acid differences could be the cause of vaccination failure. The use of smuggled vaccines that are manufactured from old seed stock needs to be further investigated to determine the efficacy of such vaccine. The results of the present study demonstrated that Thai PEDV isolates possess a unique characteristic in the S gene defined by two insertions and one deletion that are genetically similar to those of isolates responsible for recent outbreaks in China and Korean field isolates. In addition, the Thai PEDV isolates displayed a high degree of genetic heterogeneity, especially in the neutralizing epitope region. This finding may explain why the current illegal use of the PEDV vaccine, which is based on old seed stock including strains from China and KPED9 from Korea, has not resulted in successful PED control. Confirmation of this explanation could lead to the development of a new vaccine that is more suitable for PEDV outbreaks in Thailand.
Acknowledgements The authors are grateful to the National Research Council of Thailand, the National Science and Technology Development Agency and the Thailand Research Fund for funding this research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2013. 11.001. References Chang, S.H., Bae, J.L., Kang, T.J., Kim, J., Chung, G.H., Lim, C.W., Laude, H., Yang, M.S., Jang, Y.S., 2002. Identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus. Mol. Cells 14, 295–299. Chasey, D., Cartwright, S.F., 1978. Virus-like particles associated with porcine epidemic diarrhoea. Res. Vet. Sci. 25, 255–256. Cruz, D.J., Kim, C.J., Shin, H.J., 2006. Phage-displayed peptides having antigenic similarities with porcine epidemic diarrhea virus (PEDV) neutralizing epitopes. Virology 354, 28–34. Cruz, D.J., Kim, C.J., Shin, H.J., 2008. The GPRLQPY motif located at the carboxyterminal of the spike protein induces antibodies that neutralize Porcine epidemic diarrhea virus. Virus Res. 132, 192–196. Drummond, A.J., Suchard, M.A., Xie, D., Rambaut, A., 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29, 1969–1973. Gao, Y., Kou, Q., Ge, X., Zhou, L., Guo, X., Yang, H., 2013. Phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in China. Arch. Virol. 158, 711–715. Hofmann, M., Wyler, R., 1988. Propagation of the virus of porcine epidemic diarrhea in cell culture. J. Clin. Microbiol. 26, 2235–2239. Keane, T.M., Creevey, C.J., Pentony, M.M., Naughton, T.J., McLnerney, J.O., 2006. Assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified. BMC Evol Biol 6, 29. Kang, T.J., Han, S.C., Yang, M.S., Jang, Y.S., 2006. Expression of synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat-labile enterotoxin in tobacco plants. Protein Expr. Purif. 46, 16–22. Kocherhans, R., Bridgen, A., Ackermann, M., Tobler, K., 2001. Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence. Virus Genes 23, 137–144. Lee, D.K., Park, C.K., Kim, S.H., Lee, C., 2010. Heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in Korea. Virus Res. 149, 175–182. Li, W., Li, H., Liu, Y., Pan, Y., Deng, F., Song, Y., Tang, X., He, Q., 2012a. New variants of porcine epidemic diarrhea virus, China, 2011. Emerg. Infect. Dis. 18, 1350–1353. Li, Z.L., Zhu, L., Ma, J.Y., Zhou, Q.F., Song, Y.H., Sun, B.L., Chen, R.A., Xie, Q.M., Bee, Y.Z., 2012b. Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in south China. Virus Genes 45, 181–185. Olanratmanee, E.O., Kunavongkrit, A., Tummaruk, P., 2010. Impact of porcine epidemic diarrhea virus infection at different periods of pregnancy on subsequent reproductive performance in gilts and sows. Anim. Reprod. Sci. 122, 42–51. Oszvald, M., Kang, T.J., Tomoskozi, S., Tamas, C., Tamas, L., Kim, T.G., Yang, M.S., 2007. Expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat labile enterotoxin in rice endosperm. Mol. Biotechnol. 35, 215–223. Park, S.J., Kim, H.K., Song, D.S., Moon, H.J., Park, B.K., 2011. Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field isolates in Korea. Arch. Virol. 156, 577–585. Park, S.J., Moon, H.J., Luo, Y., Kim, H.K., Kim, E.M., Yang, J.S., Song, D.S., Kang, B.K., Lee, C.S., Park, B.K., 2008. Cloning and further sequence analysis of the ORF3 gene of wild- and attenuated-type porcine epidemic diarrhea viruses. Virus Genes 36, 95–104. Park, S.J., Moon, H.J., Yang, J.S., Lee, C.S., Song, D.S., Kang, B.K., Park, B.K., 2007a. Sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in Korea. Virus Genes 35, 321–332. Park, S.J., Song, D.S., Ha, G.W., Park, B.K., 2007b. Cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus DR13. Virus Genes 35, 55–64. Pensaert, M.B., de Bouck, P., 1978. A new coronavirus-like particle associated with diarrhea in swine. Arch. Virol. 58, 243–247. Puranaveja, S., Poolperm, P., Lertwatcharasarakul, P., Kesdaengsakonwut, S., Boonsoongnern, A., Urairong, K., Kitikoon, P., Choojai, P., Kedkovid, R.,
G. Temeeyasen et al. / Infection, Genetics and Evolution 21 (2014) 205–213 Teankum, Thanawongnuwech, R., . Chinese-like strain of porcine epidemic diarrhea virus, Thailand. Emerg. Infect. Dis. 15, 1112–1115. Sato, T., Takeyama, N., Katsumata, A., Tuchiya, K., Kodama, T., Kusanagi, K., 2011. Mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo. Virus Genes 43, 72–78. Srinuntapunt, S., Trongwongsa, L., Antarasena, C., Sangsuwan, W., Prommuang, P., 1995. Porcine epidemic diarrhea in Trang province. J. Thai. Vet. Med. Assoc. 46, 11–19. Sukumaran, J., Holder, M.T., 2010. DendroPy: a Python library for phylogenetic computing. Bioinformatics 26, 1569–1571. Sun, D., Feng, L., Shi, H., Chen, J., Cui, X., Chen, H., Liu, S., Tong, Y., Wang, Y., Tong, G., 2008. Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein. Vet. Microbiol. 131, 73–81.
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Genetic diversity of ORF3 and spike genes of porcine epidemic diarrhea virus in Thailand Gun Temeeyasen a, Anchalee Srijangwad a, Thitima Tripipat a, Pavita Tipsombatboon b, Jittima Piriyapongsa b, Waranyoo Phoolcharoen c, Taksina Chuanasa c, Angkana Tantituvanont d, Dachrit Nilubol a,⇑ a
Department of Veterinary Microbiology, Faculty of Veterinary Science, Bangkok 10330, Thailand Genome Institute, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand d Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand b c
a r t i c l e
i n f o
Article history: Received 21 June 2013 Received in revised form 2 November 2013 Accepted 5 November 2013 Available online 26 November 2013 Keywords: Porcine epidemic diarrhea virus Spike gene ORF3 gene Diversity Thailand
a b s t r a c t Porcine epidemic diarrhea virus (PEDV) has become endemic in the Thai swine industry, causing economic losses and repeated outbreaks since its first emergence in 2007. In the present study, 69 Thai PEDV isolates were obtained from 50 swine herds across Thailand during the period 2008–2012. Both partial and complete nucleotide sequences of the spike (S) glycoprotein and the nucleotide sequences of ORF3 genes were determined to investigate the genetic diversity and molecular epidemiology of Thai PEDV. Based on the analysis of the partial S glycoprotein genes, the Thai PEDV isolates were clustered into 2 groups related to Korean and Chinese field isolates. The results for the complete spike genes, however, demonstrated that both groups were grouped in the same cluster. Interestingly, both groups of Thai PEDV isolates had a 4-aa (GENQ) insertion between positions 55 and 56, a 1-aa insertion between positions 135 and 136, and a 2-aa deletion between positions 155 and 156, making them identical to the Korean KNU series and isolates responsible for outbreaks in China in recent years. In addition to the complete S sequences, the ORF3 gene analyses suggested that the isolates responsible for outbreaks in Thailand are not vaccine related. The results of this study suggest that the PEDV isolates responsible for outbreaks in Thailand since its emergence represent a variant of PEDV that was previously reported in China and Korea. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Porcine epidemic diarrhea virus (PEDV) is the etiologic agent of porcine epidemic diarrhea (PED), a devastating enteric disease that is characterized by vomiting, acute severe watery diarrhea and dehydration, which results in significant high mortality in piglets under 7 days of age (Pensaert and de Bouck, 1978). In addition to causing enteric problem in pigs, PEDV infection in sows can lead to a reduction of subsequent reproductive performance (Olanratmanee et al., 2010). PED was first recognized in Belgium and the United Kingdom in 1976–1978 (Pensaert and de Bouck, 1978; Wood, 1977). Post-emergence, disease outbreaks have been reported in several European countries (Chasey and Cartwright, 1978). At present, PED disease has continued to causing devastating enteric disease and severe economic losses in many Asian ⇑ Corresponding author. Address: Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University, Henry Dunant Road, Pathumwan, Bangkok 10330, Thailand. Tel.: +66 2 218 9583; fax: +66 2 251 1656. E-mail address: [email protected] (D. Nilubol). 1567-1348/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.11.001
countries, including China, Korea, Japan and Thailand (Gao et al., 2013; Li et al., 2012b; Park et al., 2007a, 2011; Sato et al., 2011). PEDV is an enveloped, single-stranded RNA virus belonging to the genus Alphacoronavirus, family Coronaviridae and order Nidovirales. The PEDV genome is approximately 28 kb in length and is composed of 7 open reading frames (ORF) (Kocherhans et al., 2001). ORF1a and ORF1b cover 70% of the entire genome and encode the non-structural replicase gene. The remaining 5 ORFs code for major structural proteins including the spike (S) glycoprotein, ORF3, envelope protein, membrane protein and nucleocapsid protein. Among the proteins encoded by these genes, the S glycoprotein is a glycosylated protein located on the envelope of the virus. It is involved with pathogenesis by binding to cell receptors on the host cell membranes and penetrating cells by membrane fusion. The S glycoprotein consists of 2 domains, including S1 and S2 domains. Whereas the S1 domain is involved in viral attachment, the S2 domain undergoes conformational changes following S1 attachment to facilitate fusion of the virus envelope with the host membrane, allowing entry of the viral genome into the host.
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Because of its important role in viral attachment and entry, the S1 domain was characterized and demonstrated to contain epitopes involved with neutralizing antibodies (Chang et al., 2002; Cruz et al., 2006, 2008; Sun et al., 2008). Because of its role in the induction of neutralizing antibodies, the S protein is the primary target for the development of a vaccine against PED (Kang et al., 2006; Oszvald et al., 2007). Several investigators have reported a high degree of genetic diversity observed in the S glycoprotein gene (Lee et al., 2010; Li et al., 2012b; Park et al., 2007a, 2011). Thus, this gene is important for understanding the genetic relatedness of PEDV field isolates, the epidemiological status of the virus and vaccine development. In addition to the S glycoprotein gene, the accessory ORF3 gene has received a large amount of attention in the context of PEDV virulence. The virulence of PEDV can be reduced by altering the ORF3 gene through cell culture adaptation (Park et al., 2007b, 2008). The ORF3 genes have been used to differentiate between field and vaccine-derived isolates. The vaccine-derived isolates have unique characteristics at amino acids 82–99, where a continuous deletion of 17 amino acids has been observed (Park et al., 2008). Therefore, this deletion can be used to differentiate between field and attenuated vaccine viruses. The differentiation of the ORF3 gene can be used for molecular epidemiology studies of PEDV. In Thailand, a PED outbreak was first reported in 1995 in a farm located in the southern region of the country (Srinuntapunt et al., 1995). The disease did not spread to other swine-producing areas at that time; however, in late 2007, the disease re-emerged in Nakorn Pathom, a province with the highest pig densities in Thailand (Puranaveja et al., 2009). The source of viral introduction has not been identified to date. There is only speculation concerning contaminated pork meat and bone meal, the illegal introduction of the vaccine or both. Since its re-emergence, PED has spread throughout the country, causing pandemic PED outbreaks in Thailand resulting in >90% of Thai swine farms being infected. Currently, the disease develops until reaching an endemic stage in which many herds experience recurrent outbreaks. To develop a more successful control and prevention program, further investigation related to the genetic diversity and molecular epidemiology of PEDV in Thailand is needed. The objectives of the present study were to investigate the genetic diversity of the S glycoprotein and ORF3 genes of PEDV in Thailand. The results of a study on the genetic differences of the S genes may provide information on the heterogeneity of the outbreak. Moreover, such a study may also provide a basis of knowledge that can be used for the development of an effective vaccine.
2. Materials and methods 2.1. Source of specimens and PEDV isolation A total of 120 porcine intestinal samples were collected from 50 pig farms that experienced PED outbreaks from 2008 to 2012. In each outbreak, intestinal samples were collected from 3- to 4-day-old piglets that displayed the clinical features associated with PED, including vomiting, watery diarrhea and dehydration. The intestinal samples were subjected to PEDV isolation using the continuous Vero cell line (ATCC, CCL-81) (Hofmann and Wyler, 1988). Briefly, the intestinal samples of piglets were minced into small pieces and suspended in 10 ml of phosphate buffer saline (PBS; 0.1 M, pH 7.2). The suspended samples were vortexed and clarified by centrifugation at 1350g for 10 min. The supernatant was filtered through 0.45-lm filters and stored at 80 °C until use. Five hundred microliters of supernatant samples was diluted with maintenance media (MM) consisted of Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10 lg/
ml trypsin (Gibco, USA) and inoculated into a 25-cm2 flask that had been previously plated with Vero cells for 2 days or until full confluence was reached. The inoculated 25-cm2 flask was incubated at 37 °C in 5% CO2 for 1 h. Then, the media was replaced with 10 ml of MM, and incubated again for a period of 2–3 days. Cytopathic effect (CPE) was observed with a light microscope. Then, the 25-cm2 flasks were frozen and thawed 2 times before being centrifuged at 1350g for 10 min to collect the supernatant, which was stored at 80 °C. The clarified supernatants were subjected to RT-PCR. Partial S, complete S and ORF3 genes were amplified and sequenced. The intestinal samples were considered negative for PEDV isolation following 3 blind passages. 2.2. Reverse transcription-polymerase chain reaction Total RNA was extracted from the supernatant from the previous procedure using the NucleospinÒ RNA Virus kit (Macherey-Nagel Inc., PA, USA) in accordance with the manufacturer’s instructions. cDNA was synthesized from the extracted RNA using M-MuLV Reverse Transcriptase (New England BioLabs Inc., MA, USA). PCR amplification was performed on the cDNA. To amplify the partial S genes, the following primers were utilized: PEDF (50 -TTC TGA GTC ACG AAC AGC CA-30 ) and PEDR (50 -CAT ATG CAG CCT GCT CTG AA-30 ) (Park et al., 2007a). PCR amplification was performed using PlatinumÒ Tag DNA polymerase High Fidelity (Invitrogen, CA, USA). After the initial incubation at 94 °C for 5 min, the reactions were subjected to 35 cycles of PCR as follows: 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. These cycles were followed by a terminal 7-min extension at 72 °C. To amplify the complete S genes, the sequence was separately constructed for S1 and S2,with the primers S-F-1 (50 -ACG TAA ACA AAT GAG GTC TTT-30 ) and S-R-1 (50 -ATA CAC CAA CAC AGG CTC TGT-30 ) used to amplify S1 and the primers S-F-2 (50 -GGT TTC TAC CAT TCT AAT GAC G-30 ) and S-R-2 (50 -GTA TTG AAA AAG TCC AAG AAA CA-30 ) used to amplify S2 (Lee et al., 2010). PCR amplification was performed using PlatinumÒ Tag DNA polymerase High Fidelity (Invitrogen, CA, USA). After an initial incubation at 94 °C for 5 min, the reactions were subjected to 35 cycles of PCR as follows: 94 °C for 30 s, 55 °C (58 °C for S2) for 30 s, and 72 °C for 2 min. These cycles were followed by a terminal 10-min extension at 72 °C. The PCR products were visualized by agarose gel electrophoresis. To amplify the ORF3 genes, primers ORF3F (50 -CCT AGA CTT CAA CCT TAC GA-30 ) and ORF3R (50 -CAG GAA AAA GAG TAC GAA AA-30 ) were used, and PCR amplification was performed using PlatinumÒ Tag DNA polymerase High Fidelity (Invitrogen, CA, USA). After an initial incubation at 94 °C for 5 min, the reactions were subjected to 30 cycles of PCR as follows: 94 °C for 60 s, 50 °C for 60 s, and 72 °C for 60 s. These cycles were followed by a terminal 10-min extension at 72 °C. The PCR products were visualized by agarose gel electrophoresis. 2.3. Cloning, plasmid purification and sequence determination The PCR products were cloned into plasmid vectors for the subsequent transformation of Escherichia coli cells by using a commercial kit (pGEM-TÒ Easy Vector System I (Promega, WI, USA), and the controls were included at all stages of cloning and transformation. Bacterial transformant colonies were randomly selected from each sample for plasmid purification using the NucleospinÒ Plasmid kit (Macherey-Nagel Inc., PA, USA), and the 10 elected colonies were grown in LB broth for 24 h and subjected to plasmid isolation and sequencing. Sequencing reactions were performed by Biobasic Inc. (Ontario, Canada) using an ABI Prism 3730XL DNA sequencer.
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2.4. Sequence analyses The nucleotide and deduced amino acid sequences were aligned using the CLUSTALW program (Thompson et al., 1994). The percentage of identity between every pair of isolate sequences at the nucleotide and amino acid levels was calculated using MEGA 5.2 (Tamura et al., 2011). The analyses included only sequences in which nucleotide sequence data are complete and accession numbers are available in GenBank (Supplementary Material 1). Phylogenetic analyses of the partial S, complete S, and ORF3 genes was separately performed using a Bayesian Markov chain Monte Carlo (BMCMC) method implemented in the program BEAST v1.7.4 (Drummond et al., 2012). The analysis of the partial S gene included the nucleotide sequences from the present study, previously reported partial S sequences of the Thai PED (Puranaveja et al., 2009) and isolates from Belgium, United Kingdom (UK), China and Korea (Park et al., 2007a). The analysis of the complete S and ORF3 genes included the nucleotide sequences from the present study and isolates from Belgium, UK, China and Korea (Gao et al., 2013; Lee et al., 2010; Li et al., 2012a). The strict molecular clock model with a coalescent constantsize tree prior and empirical base frequencies were used for all analyses. The best fitted substitution model and among-site rate heterogeneity model for each gene data set was determined using Model Generator v0.85 (Keane et al., 2006). For each analysis, three independent BEAST runs were performed; each consisting of 50 million generations with sampling of every 1000 generations and the first 10% discarded as burn-in. LogCombinerv1.7.4 (http:// beast.bio.ed.ac.uk/LogCombiner) was used to combine logand tree files from independent runs of BEAST. Tracer v1.5 (http://beast.bio.ed.ac.uk/Tracer) was used to confirm that post-burn-in trees yielded an effective sample size (ESS) of >200 for all parameters. A 50% majority-rule consensus tree was generated from the tree file using SumTrees v.3.3.1 (Sukumaran and Holder, 2010). The resulting tree was viewed in FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/).
3. Results 3.1. Phylogenic analysis of the partial spike gene Of the 120 porcine intestinal samples collected in the years 2008–2012, 69 PEDV isolates were collected and the origins of those Thai isolates are shown in Fig. 1. All isolates produced CPE characterized by cell fusion and syncytia formation in the first passage (Supplementary Material 2). To investigate the heterogeneity of these 69 Thai PEDV isolates, the partial spike genes of the 69 field isolates were amplified. The nucleotide sequences revealed that these partial S glycoprotein genes of the 69 field isolates consisted of 657 nucleotides encoding a protein of 219 amino acids located on the full length of the S glycoprotein gene at nucleotide positions 1462–2118 or amino acid positions 488–706. Identical partial S nucleotide sequences were identified and excluded, resulting in further genetic analysis of 30 genes. The phylogenetic tree of 30 partial S glycoprotein genes was constructed together with another 16 previously reported Thai PEDV isolates (Puranaveja et al., 2009) as well as some partial S glycoprotein genes that were previously isolated in other countries (Park et al., 2007a). Based on the previously reported phylogenetic analysis (Park et al., 2007a), the PEDV isolates were further divided into 3 groups, designated G1, G2 and G3; group G1 was further divided into 3 subgroups, which were designated G1-1, G1-2 and G1-3. When using the clustering system based on this phylogenetic analysis (Park et al., 2007a), the phylogenetic tree demonstrated that the Thai PEDV isolates were further divided into 2 groups
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(Fig. 2). Both groups of Thai PEDV isolates were in G1 but differed in subgroup. The most dominant isolates were clustered in subgroup G1-1, which comprised all 16 previously reported Thai PEDV isolates along with 27 of 30 current Thai isolates. Another subgroup designated G1-4 comprised 3 of 30 isolates, including AGPED0609_1, AGPED0609_2 and SPPED0111_1. This subgroup did not fit into the previously reported clustering system. It sat between subgroups 1-2 and 1-3. Therefore, we named it subgroup G1-4. After comparing the nucleotide and amino acid sequences of all Thai field PEDV isolates, it was demonstrated that the nucleotide and amino acid sequence identity of the all Thai field PEDV isolates ranged from 94.4–99.9% and 85.8–100.0%, respectively. The percentage of identity between nucleotide and amino acid sequences of the Thai G1-1 isolates ranged from 96.4–99.7% and (92.5–100.0%), respectively. The nucleotide and amino acid sequences of isolates in G1-1 were more divergent compared to those of G1-4, in which all 3 isolates had 100.0% identity at the nucleotide and amino acid level. Subgroups G1-1 and G1-4 had 94.4–95.5% and 85.8–88.5% identity, respectively, at the nucleotide and amino acid level. 3.2. Genetic analyses of complete spike gene To investigate the genetic diversity of 2 groups of Thai field PEDV isolates and their genetic relationship with PEDV isolates from other countries, 13 different Thai field PEDV isolates, including 10 isolates from subgroup G1-1 and 3 isolates from subgroup G1-4, were randomly selected, and the full-length nucleotide sequences of their S glycoprotein genes were investigated. The phylogenetic tree of their complete S genes was constructed together with those of other PEDV isolates from other countries (Supplementary Material 1). The phylogenetic analysis revealed that the complete S glycoprotein genes were further divided into 3 clusters, including clusters 1, 2 and 3 (Fig. 3). Cluster 1 comprised Chinese isolates (CH/FJND-3, CH1, CH8, CHGD-01, HB-2011-1, HB-2011-2, HB-2011-3, HB-2011-4, HB-2012-1, HB-2012-2, HB-2012-3, HB2012-4, BJ-2011-2, BJ-2011-3, BJ-2012-1, BJ-2012-2, ZJ-2011-1 and ZJ-2011-2) and Korean field isolates (KNU-0802,KNU-0902, CNU-091222-01 and CNU-091222-02). Based on the results of the partial S gene comparisons, all 13 PEDV Thai isolates in subgroups G1-1 and G1-4 were included in cluster 1. Cluster 2 comprised 7 isolates from Korea (KNU-0801, KNU-0901, KNU-0903, KNU-0904, KNU-0905, Spk1 and Chinju99). Cluster 3 comprised 16 reference strains, including isolates from Korea (DR13 and SM98), China (CH2, CH3, CH4, CH5, CH6, CH7, CH/FJND-1, CH/ FJND-2, JS-2004-2, LZC, LJB03 and DX) and Europe (CV777 and Br1/87). The sequence homology between the S glycoprotein genes was measured. The nucleotide and amino acid sequence identity of the cluster 1 isolates ranged from 94.4–99.9% and 93.1–99.9%, respectively (Supplementary Material 3). The isolates had 93.0–97.8% (90.7–96.5%) and 92.4–98.1% (90.1–97.7%) nucleotide (deduced amino acid) sequence identity with members of groups 2 and 3, respectively. The percentage of identity between nucleotide and amino acid sequences of all 13 Thai field PEDV isolates ranged from 94.9–99.8% and 94.0-99.7%, respectively. All 13 Thai PEDV isolates displayed 94.9–99.9% and 94.0–99.9% identity to Chinese isolates in this cluster at the nucleotide and amino acid levels, respectively. In addition, all 13 Thai PEDV isolates displayed 94.9–99.8% and 94.0–99.7% identity to Korean isolates (KNU0902, CNU-09122201 and CNU-091222-02) at the nucleotide and amino acid levels, respectively. It was demonstrated that the S glycoprotein genes of the Thai PEDV isolates are 4158 nucleotides in length and encode 1386 amino acid residues. The nucleotide and deduced amino acid sequences of the complete S genes were aligned separately to
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Fig. 1. Map of Thailand. Red dots represent the provinces where the porcine epidemic diarrhea virus were isolated and used for sequencing in this study. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
investigate the amino acid differences between the clusters, especially the hypervariable regions and the 4 major epitope regions. The largest number of amino acid differences was clustered in the N-terminal region of the S1 domain. The N-terminal region of the S1 gene demonstrated the lowest similarity with the PEDV reference strain. The isolates in cluster 1 and 2, except isolate Chinju99, had 2 insertions of 4 (56GENQ59) and 1 (140N) amino acids at amino acid positions 55–60 and 140, which are located in the hypervariable domain in the N-terminus of the S1 region (Supplementary Material 4). In addition, 1 deletion of 2 amino acids was observed at amino acid positions 160 and 161. All 13 Thai isolates in cluster 1 examined in this study have similar patterns of amino acid insertion and deletion. Several studies have identified 4 regions in the S glycoprotein that contains major epitopes (Cruz et al., 2008; Kang et al., 2006; Sun et al., 2008). Those 4 regions include amino acid positions 504–643 (Kang et al., 2006); 753YSNIGVCK760 and 769LQDGQVKI776 (Sun et al., 2008); and 1373GPRLQPY1379 (Cruz et al., 2008). Therefore, we analyzed these 3 regions. It was demonstrated that amino
acid position 753YSNIGVCK760 and 1373GPRLQPY1379 were conserved between the Thai PEDV isolates and isolates from other countries. However, the amino acid positions 504–643 and 769LQDGQVKI776 were demonstrated to be variable. The largest number of amino acid differences was observed in amino acid positions 504–643. In addition, amino acid differences were observed in the 769 LQDGQVKI776 region, with the substitutions of S ? L and S ? D/ Y at amino acid positions 769 and 771, respectively. 3.3. Genetic analyses of ORF3 genes To investigate the genetic relationship between the Thai field PED isolates and modified live vaccine, the nucleotide sequences of the ORF3 genes of 13 Thai PEDV isolates were investigated. Identical sequences were excluded, resulting in 7 isolates for further analysis. The phylogenetic tree of the complete ORF3 genes was constructed along with those of other reference isolates (Supplementary Material 1). The phylogenetic analysis revealed that, based on the ORF3 gene, the PEDV isolates were further divided
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Fig. 2. Phylogenetic analysis of the porcine epidemic diarrhea virus isolates based on the nucleotide sequences of the partial spike glycoprotein genes. The tree includes nucleotide sequences of Thai isolates from the present study (light blue font) and a previous study (red font) as well as sequences from China (pink font), Korea (dark blue font) and European countries (green font). The 50% majority-rule consensus tree was constructed using Bayesian MCMC method. Each internal node with the posterior probability of the corresponding clade >0.5 is labeled. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
into 3 clusters: clusters 1, 2 and 3 (Fig. 4). Cluster 1 was further divided into 2 sub-clusters: sub-clusters 1-1 and 1-2. All Thai field isolates were in cluster 1 and sub-cluster 1-1, as were the isolates from China. Sub-cluster 1-2 consisted of Chinju99 and DR13, isolates from Korea. Cluster 2 consisted of CV777 and isolates with a unique truncated portion, a 17-amino acid deletion at position 82–98, which represented attenuated virus vaccines, and no Thai PEDV isolate was included in this group. Cluster 3 consisted of MC, which was isolated in China. The sequence identity of the ORF3 genes in cluster 1 ranged from 96.0–100.0% and 93.7–100.0%, at the nucleotide and amino acid levels, respectively (Supplementary Material 5). They have
94.8–99.0.5% (92.4–99.5%) and 94.1–96.9% (93.7–97.3%) nucleotide (deduced amino acid) sequence identity with members of groups 2 and 3, respectively. The ORF3 genes of the Thai PEDV isolates were relatively conserved. Only point substitution was observed (Supplementary Material 6).
4. Discussion Since its emergence in late 2007, porcine epidemic diarrhea virus has continued to cause economic damage to the Thai swine industry. The disease has developed to an endemic stage. Several
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Fig. 3. Phylogenetic analysis of the porcine epidemic diarrhea virus isolates based on the nucleotide sequences of the complete spike glycoprotein genes. The tree includes nucleotide sequences from Thailand (light blue font), China (pink font), Korea (dark blue font) and European countries (green font). The 50% majority-rule consensus tree was constructed using Bayesian MCMC method. Each internal node with the posterior probability of the corresponding clade >0.5 is labeled. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
herds continue to experience repeated outbreaks, but the disease severity, as demonstrated by piglet mortality, has lessened. Although several control methods including vaccination and strict biosecurity have been implemented, these measures have had varying degrees of success. The recurrence of PED outbreaks has led to the necessity for further investigation into the genetic diversity of PEDV. A better understanding of the genetic diversity of PEDV may facilitate the development of a more successful control program and vaccines. To investigate the heterogeneity of PEDV in Thailand, the partial and complete S genes were characterized. The partial S gene was initially characterized because it can be used to characterize a very large number of samples, unlike the characterization of the com-
plete S gene, in which the characterization is more complicated due to the large size of the gene. In addition, previous studies have reported the use of the partial S gene, including the neutralization epitope regions of PEDV, to investigate the genetic diversity of PEDV and to divide it into clusters; these studies have also demonstrated that this region displays high similarity when using complete S glycoprotein genes (Park et al., 2007a). Therefore, we initially chose to study the partial S genes to understand the heterogeneity of PEDV in Thailand. Based on the characterization of the partial S glycoprotein gene, the results of the present study demonstrated that Thai PEDV isolates are further divided into 2 groups. However, those 2 groups were grouped in the same cluster, cluster 1, based on the characterization of the complete S genes. In cluster
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Fig. 4. Phylogenetic analysis of the porcine epidemic diarrhea virus isolates based on the nucleotide sequences of the ORF3 genes. The trees include nucleotide sequences from Thailand (light blue font), China (pink font), Korea (dark blue font) and European countries (green font). The 50% majority-rule consensus tree was constructed using Bayesian MCMC method. Each internal node with the posterior probability of the corresponding clade >0.5 is labeled. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
1, which appeared to be the most prevalent cluster of virus in Thailand, PEDV consisted of isolates genetically related to KNU-serial isolates, PEDV field isolates from Korea, and isolates that have caused outbreaks in China in recent years. A previous report suggested that Thai PEDV isolates were China-like isolates and similar to an isolate named JS2004 that was isolated in China. The results of the present study are in contrast with that previous report. The results of the current study, based on the comparison of the complete S glycoprotein genes of PEDV field isolates, which are the isolates that continue to cause economic damage to the Thai swine industry, indicate that the Thai isolates belong to cluster 1. The Thai PEDV isolates collected from 2008–2012 possess a unique and common genome characteristic defined by the insertion of 4 amino acids (GENQ) between positions 55 and 56, a 1-aa (N) insertion between positions 135 and 136, and the deletion of 2 amino acids between positions 155 and 156. These unique isolates were genetically related to or identical to previously reported Korean KNU-serial isolates (Lee et al., 2010) and isolates that were responsible for recent severe
outbreaks in China (Gao et al., 2013; Li et al., 2012a). In addition, the isolates with unique characteristics, which were reported to be a variant of PEDV, emerged in China and caused more severe outbreaks (Li et al., 2012a). The isolate JS2004, however, does not possess the 2 discontinuous insertions and the one deletion shared by the above mentioned isolates. The discrepancy of the results between the present study and previous studies could be explained by the current study’s investigation of both partial and complete S glycoprotein genes, whereas the previous study reported on only the partial S genes. These contradictions may have arisen because the phylogenetic analysis of the partial S gene was based on only 657 nucleotides that are involved in coding for amino acid positions 488–706 of the complete S genes. This coverage level was not sufficient to represent the whole gene of the virus. In addition, the 2 discontinuous deletions and the one insertion mentioned above are located in the N-terminus of S1 gene region. The deduced amino acid, therefore, highlights the disadvantage of phylogenetic analysis using only partial S genes to investigate the molecular epidemiology or heterogeneity of PEDV isolates.
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The method of PEDV introduction to Thailand is not known. It has been speculated that imported pork meat and bone materials from countries where PEDV is endemic is a possible route of introduction. The outbreak was first reported in Nakorn Pathom province, which is located in the central region of Thailand, an area very distant from the border. In addition, no genetic materials such as gilts or boar were imported to this farm. The only observed changes were that the raw material for manufacturing feed was changed from fish meal to pork meat and bone meal due to high fish meal prices. To investigate whether the isolates are closely related to field isolates in Korea or to vaccine isolates, the ORF3 genes of Thai field isolates were sequenced. The ORF3 genes have been used to differentiate between field and vaccine-derived isolates. The vaccinederived isolates have a unique characteristic at nucleotide position 245–293, a continuous deletion of 49 NT. The results of this study demonstrated that the Thai field isolates do not possess that unique 49-nucleotide deletion suggesting that they are not vaccine-related. PEDV has become endemic and thus continues to cause damage to the Thai swine industry. Although the severity of the disease has been reduced and the mortality following repeated outbreaks has been contained to piglets farrowed from primi-parous sows, the disease continues to cause economic losses. Repeated outbreaks in herds occur from every 3–6 months. Herds with repeated outbreaks have higher production costs compared with herds with no repeated outbreaks. To investigate whether the heterogeneity of PEDV plays an important role in disease recurrence, the S genes of 10 isolates from 3 herds with repeated outbreaks were characterized. The genetic analysis suggested that all 10 isolates were grouped in cluster 1 and had only few amino acid differences in the S genes. These results suggest that the heterogeneity of the S gene might not contribute to disease recurrence in herds. Other factors, including the immune status of replacement gilts prior to introduction, might play a more important role in disease recurrence. Several management strategies including vaccination have been implemented to control PEDV in swine herds. Most common vaccination protocol includes 2 dosages of modified live and killed vaccines in gilts prior to introduction and pre-farrow vaccination at 12 and 14 weeks of gestation with KV. Although PEDV vaccines are not commercially available in Thailand, we have noticed the use of these vaccines, illegally smuggled, from China and Korea. Their efficacy is questionable, as herds with heavy vaccination programs, including whole herd vaccination and pre-farrow vaccination, still experience repeated outbreaks. Because the neutralizing epitopes of Thai PEDV isolates are hypervariable compared with PEDV isolates in vaccines, these amino acid differences could be the cause of vaccination failure. The use of smuggled vaccines that are manufactured from old seed stock needs to be further investigated to determine the efficacy of such vaccine. The results of the present study demonstrated that Thai PEDV isolates possess a unique characteristic in the S gene defined by two insertions and one deletion that are genetically similar to those of isolates responsible for recent outbreaks in China and Korean field isolates. In addition, the Thai PEDV isolates displayed a high degree of genetic heterogeneity, especially in the neutralizing epitope region. This finding may explain why the current illegal use of the PEDV vaccine, which is based on old seed stock including strains from China and KPED9 from Korea, has not resulted in successful PED control. Confirmation of this explanation could lead to the development of a new vaccine that is more suitable for PEDV outbreaks in Thailand.
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