key: cord-0972323-h5jfrj2p authors: Than, Van T.; Choe, Se‐Eun; Vu, Thi T. H.; Do, Tien D.; Nguyen, Thi L.; Bui, Thi T. N.; Mai, Thi N.; Cha, Ra M.; Song, Daesub; An, Dong‐Jun; Le, Van P. title: Genetic characterization of the spike gene of porcine epidemic diarrhea viruses (PEDVs) circulating in Vietnam from 2015 to 2016 date: 2020-03-11 journal: Vet Med Sci DOI: 10.1002/vms3.256 sha: 02d0ce9f28d04f69024ffbace2a931a05cb44555 doc_id: 972323 cord_uid: h5jfrj2p BACKGROUND: Porcine epidemic diarrhea (PED) is a highly contagious swine disease caused by the PED virus (PEDV), which is a member of the family Coronaviridae. Since the first outbreaks in Belgium and the United Kingdom were reported in 1971, PED has spread throughout many countries around the world and causing significant economic loss. This study was conducted to investigate the recent distribution of PEDV strains in Vietnam during the 2015–2016 seasons. METHODS: A total of 30 PED‐specific PCR‐positive intestinal and faecal samples were collected from unvaccinated piglets in Vietnam during the 2015–2016 seasons. The full length of the spike (S) gene of these PEDV strains were analysed to determine their phylogeny and genetic relationship with other available PEDV strains globally. RESULTS: Phylogenetic analysis of the complete S gene sequences revealed that the 28 Vietnamese PEDV strains collected in the northern and central regions clustered in the G2 group (both G2a and G2b sub‐groups), while the other 2 PEDV strains (HUA‐PED176 and HUA‐PED254) collected in the southern region were clustered in the G1/G1b group/sub‐group. The nucleotide (nt) and deduced amino acid (aa) analyses based on the complete S gene sequences showed that the Vietnamese PEDV strains were closely related to each other, sharing nt and aa homology of 93.2%–99.9% and 92.6%–99.9%, respectively. The N‐glycosylation patterns and mutations in the antigenic region were observed in Vietnamese PEDV strains. CONCLUSIONS: This study provides, for the first time, up‐to‐date information on viral circulation and genetic distribution, as well as evidence to assist in the development of effective PEDV vaccines in Vietnam. Porcine epidemic diarrhea (PED) is a highly contagious swine disease caused by PED virus (PEDV), a member of the family Coronaviridae. Pathologically, the disease is characterized by causing vomiting, enteritis, and watery diarrhoea with high mortality in pigs of all ages, although the most severe signs are reported in piglets less than 2 weeks old (Bridgen, Duarte, Tobler, Laude, & Ackermann, 1993) . PEDV is an enveloped coronavirus with a single-stranded RNA genome about 28 kb in length. The PEDV genome is composed of seven open reading frames (ORFs) encoding for four structural proteins (spike, S; envelope, E; membrane, M; nucleocapsid, N) and three nonstructural proteins (ORF1a, −1b and ORF3) (Song & Park, 2012) . Among viral proteins, the S protein is a glycoprotein peplomer on the viral surface which plays an important role in induction of neutralizing antibodies and interaction with cellular receptors in the host. It is cleaved by host-derived proteases into two subunits, namely S1 (binds to the receptor) and S2 (responsible for fusion activity) (Spaan, Cavanagh, & Horzinek, 1988) . Since the first outbreaks in Belgium and the United Kingdom were reported in 1971, PED has spread throughout many countries in Europe (Pensaert & de Bouck, 1978; Wood, 1977) and Asia (Kusanagi et al., 1992; Lin et al., 2014; Puranaveja et al., 2009 ). Recently, a new PEDV strain with high morbidity and mortality rates in infected suckling piglets emerged in China in 2010 (Chen et al., 2013) before rapidly spreading to the United States in 2014 Jarvis et al., 2016) , followed by other countries including Canada (Ojkic et al., 2015) , Mexico (Vlasova et al., 2014) , Japan , Korea (Lee, & Lee., 2014; Sun et al., 2015) and Taiwan (Lin et al., 2014) . In addition, new variant of PEDVs, the mild virulence PEDV strains, has insertions and/or deletions (INDEL) in the N-terminal region of the S genes have been identified in the United States (Wang, Byrum, & Zhang, 2014) , South Korea (Park, Kim, Song, & Park, 2014) and Japan (Masuda et al., 2015) . In Vietnam, since the first outbreak reported in 2009, PEDV has spread widely throughout country, causing significant economic loss (Duy, Toan, Puranaveja, & Thanawongnuwech, 2011) . Phylogenetic study indicated that PEDV circulating in northern and central Vietnam during 2013-2014 could be divided into two separate groups, G2 and G1 (Kim et al., 2015) . On the other hand, these emerging PEDV strains are genetically distant from PEDV vaccine strains at the neutralizing epitope regions, suggesting a novel vaccine is needed to control the current PED outbreaks in Vietnam (Diep et al., 2018) . In this study, for the first time, we investigated the molecular characterization of nearly the full spike glycoprotein gene (S gene) of 30 PEDV strains collected from endemic outbreaks during 2015-2016 in 11 different provinces, representing the three parts of Vietnam (the northern, central and southern regions). Furthermore, phylogenetic trees were constructed to determine the relationship between the Vietnamese PEDV strains and other PEDV strains circulating worldwide. The results of this study provide up-to-date information on recent circulating PEDVs in Vietnam, as well as genetic information for downstream vaccine development. In order to achieve representative sampling for the entire country, thirty PED-specific PCR-positive intestinal and faecal samples were opportunely collected from unvaccinated piglets from 11 different provinces in Vietnam during the 2015-2016 seasons (Table 1, Figure S1 ). The number of samples from each province was as follows: the northern: Hanoi (n = 2), Hai Phong (n = 4), Hung Yen (n = 4), Lao Cai (n = 2), Thai Nguyen (n = 4), Thai Binh (n = 1), Tuyen Quang (n = 1) and Vinh Phuc (n = 2); the central: Nghe An (n = 1); and the southern: Dong Nai (n = 5), Ho Chi Minh city (n = 4). The viral RNA was extracted from infected cell culture supernatants using the QIAamp viral RNA mini kit (Qiagen), according to the manufacturer's instructions. cDNA was synthesized by SuperScript ™ III First-strand Synthesis SuperMix (Invitrogen), according to the manufacturer's instructions. In order to assay the complete S-gene, a set of previous published primers were used to perform the PCR (Tian et al., 2013) . Briefly, the reaction was carried out at 42°C for 60 min (for reverse transcription), 95°C for 5 min (for denaturation), 35 cycles of 95°C for 30 s (for denaturation), 52°C for 30 s (for annealing) and 72°C for 90 s (for extension), followed by 72°C for 10 min (for final extension). The PCR products were separated on 1.2% SeaKem LE agarose gel and viewed on a BioRad Gel Doc XR image analysis system. The PCR products were cloned into the pGEM-T Vector System II (Cat. No. A3610; Promega) according to the manufacturer's instructions. The cloned genes were sequenced with T7 and SP6 sequencing primers on an ABI PRISM ® 3730xl DNA Sequencer at Macrogen Institute (Macrogen Co., Ltd.). The raw sequences were assembled by the SeqMan program (DNAstar package). The complete sequences were aligned using BioEdit v7.2.5 (Hall, 1999) . The obtained nucleotide sequences were deposited in GenBank database under the accession numbers as shown in Table 1 . Glycosylation sites were predicted using the web-based program N-Glycosite (http://www.hiv.lanl.gov/conte nt/seque nce/GLYCO SITE/glyco site. html) using aa alignment data (Zhang et al., 2004) . The complete nucleotide sequences of the S-genes from all 30 PEDV samples examined in this study were compared against a representative S-gene from the available PEDV sequences in the GenBank database. Recombination Detection Program version 4 (RDP v4) containing seven recombination detection methods (RDP, Bootscan, Geneconv, MaxChi, Chimaera, SISCAN, and 3Seq) was used to predict recombination events (Martin, Murrell, Golden, Khoosal, & Muhire, 2015) . Only the recombination events detected by more than five methods implemented in RDP v4 were considered to be reliable. SimPlot 3.5.1 software was further used to confirm putative recombination strains and points (Lole et al., 1999) . The analyses were performed with default settings (the window width and the step size to 200 bp and 20 bp, respectively). The phylogenetic tree was reconstructed by the MEGA 6.06 software package using the NEIGHBOR-JOINING method with the Kimura 2 parameter substitution model (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013) . The BOOTSTRAP method, with 1,000 replicates, was used to evaluate the reliability of the phylogenetic tree. In this study, 30 completed S-genes from PEDV collected in 11 different provinces throughout Vietnam were successfully sequenced. The Results from RDP v4 and SimPlot 3.5.1 analysis indicated that the PED strains in this study revealed no recombination event among the reference strains (data not shown). Phylogenetic analysis indicated that all PEDV isolates collected in Vietnam during the 2015-2016 outbreaks belonged to two distinct groups, G2 and G1 (Figure 1 ). In brief, two strains (HUA-PED176 and HUA-PED254) collected in Ho Chi Minh City in 2016 were clustered into the G1b sub-group, while the remaining isolates clustered into the G2 group (both G2a and G2b sub-groups) which included PEDV strains that caused recent outbreaks in China, United States and Thailand. Interestingly, while HUA-PED94, HUA-PED96, HUA-PED146 and HUA-PED120 were closed related to PEDV strains recently circulating in United States, the other strains were more genetically related to PEDV strains recently circulating in China (Figure 1 ). There are four major epitopes capable of inducing neutralizing antibodies located in the S-protein: the COE region, SS2, SS6 and 2C10. (Table S2 ). There were 14 significant differences in the N-glycosylation patterns (5 and 9 N-glycosylation patterns in the PEDV strains collected in the 2015 and 2016 outbreaks, respectively) which were mainly located in the N-terminal region of S1 among PEDV strains isolated in Vietnam (Table S2 ). These results suggest that the effects of changes in N-glycosylation sites on biological characteristics, pathogenicity and antigenicity of Vietnamese PEDVs need to be evaluated for further information regarding PEDV circulation and control strategies in Vietnam. Similar to other coronavirus S proteins, the PEDV S protein, a surface spike glycoprotein, is critical for regulating the interactions of the virus with specific host cell receptor glycoproteins, which mediate viral entry (Bosch, Zee, Haan, & Rottier, 2003) . Thus, the S protein is a primary target for vaccines against PEDV. The S protein is also the major envelope glycoprotein of the virion and it is important for understanding the genetic relationships among strains as well as their epidemiological status (Chen et al., 2012; Li, Zhu, et al., 2012; Song & Park, 2012) . In previous studies, phylogenetic analyses showed that PEDV strains could be divided into two main groups, G1 (G1a and G1b sub-groups) and G2 (G2a and G2b sub-groups) (Kim et al., 2015; Song, Moon, & Kang, 2015) . In this study, the two PEDV strains (HUA-PED176, HUA-PED254) collected from Ho Chi Minh city located in the south of Vietnam belonged to sub-group G1b, which includes mild classical US-PEDV strains, while the other PEDV strains collected from the northern and central regions of Vietnam belonged to group G2 (G2a and G2b sub-groups) which includes strains that recently emerged in the United States, China and other countries (Lin, Saif, Marthaler, & Wang, 2016) . Interestingly, three PEDV strains isolated from Hung Yen province (HUA-PED94, HUA-PED96, HUA-PED120) and one strain isolated from Hanoi (HUA-PED146) located in the north of Vietnam were distantly related to strains collected from other northern provinces. Similar results were reported in a pandemic that recently occurred in South Korea (Lee, 2015) . These results indicate that emergent, highly virulent PEDV strains could be divided into two different groups, one including US-like strains appearing currently worldwide and the other tending to restrictively circulate in China, Vietnam and Thailand (Carvajal et al., 2015; Chiou et al., 2017; Lin et al., 2016) . As a result, we conclude that PEDV strains belonging to the G2 group were the major agent of PED outbreaks in Vietnam as a whole, but G1b-PEDV strains still exist in the centre and south of Vietnam. Therefore, further investigation into the molecular characterization of Vietnamese PEDV strains is needed to monitor outbreaks. In the present study, aa insertions and deletions were found in the S protein of Vietnamese PEDV strains. The PEDV strains in the G2 group contained five aa insertions at residues 59-62 and 143 and two aa deletions at residues 167-168 when compared to the G1 group (G1b sub-group). Most of the sequence variations were in the N-terminal region of S1. This finding was similar to previous reports showing that F I G U R E 2 Amino acid alignment of the four major epitopes which are capable of inducing neutralizing antibodies located in S-protein (COE, SS2, SS6 and 2C10) of the Vietnamese PEDV strains, with topotype of classical PEDV strain CV777, topotype of emergent mild pathogenic strain and topotype of emergent severe pathogenic strains. PEDV, porcine epidemic diarrhea virus the N-terminal region of S1 was the most variable region of the S protein (Kim et al., 2015; Lee, Park, Kim, & Lee, 2010; Park et al., 2007) . Several regions of the S protein were shown to be related to neutralizing antigenic sites. Antigenic analysis showed that the aa sequences of the SS2 and 2C10 epitopes were conserved among Vietnamese PEDV strains, and they were identical to that of the prototype PEDV CV777 strain. However, the substitution mutations in the SS6 epitope and COE domain changed their antigenic characteristics. The present results suggest that these mutations might be related to virus evolution and immune evasion. This finding is also in agreement with the results of previous studies and suggests that the PED vaccine strains, DR13, e.g., may only induce partial neutralizing antibodies against emergent PED field virus strains (Chung et al., 2016; Hao, Xue, He, Wang, & Cao, 2014; Horie, Kabemura, Masatani, Matsuu, & Ozawa, 2016) . N-glycosylation of the virus plays an important role in host cell attachment and release, glycan shielding and infectivity (Vigerust & Shepherd, 2007 Genetic analysis showed that the S gene sequences of Vietnamese The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article. VPL and VTT conceived and designed the proposal . VPL and TTHV performed the experiments. VTT, SEC, TTHV, TDD, TLN, TTNB, TNM, RMC, DS, DJA and VPL participated in analysing the data. VTT and VPL wrote the paper. All authors have read and approved the final manuscript. The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate internal ethics review committee approvals has been received. Van T. 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