key: cord-0743431-t3t40nty
authors: Diep, N. V.; Sueyoshi, M.; Izzati, U.; Fuke, N.; Teh, A. P. P.; Lan, N. T.; Yamaguchi, R.
title: Appearance of US‐like porcine epidemic diarrhoea virus (PEDV) strains before US outbreaks and genetic heterogeneity of PEDVs collected in Northern Vietnam during 2012–2015
date: 2017-07-30
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.12681
sha: be320ef72da0a9379816b5b6684d70528828bf0c
doc_id: 743431
cord_uid: t3t40nty

Porcine epidemic diarrhoea virus (PEDV) is the aetiologic agent of porcine epidemic diarrhoea (PED), a highly contagious enteric disease that is threatening the swine industry globally. Since PED was first reported in Southern Vietnam in 2009, the disease has spread throughout the country and caused substantial economic losses. To identify PEDVs responsible for the recent outbreaks, the full‐length spike (S) gene of 25 field PEDV strains collected from seven northern provinces of Vietnam was sequenced and analysed. The sequence analysis revealed that the S genes of Vietnamese PEDVs were heterogeneous and classified into four genotypes, namely North America and Asian non‐S INDEL, Asian non‐S INDEL, new S INDEL and classical S INDEL. This study reported the pre‐existence of US‐like PEDV strains in Vietnam. Thirteen Vietnamese variants had a truncated S protein that was 261 amino acids shorter than the normal protein. We also detected one novel variant with an 8‐amino acid insertion located in the receptor‐binding region for porcine aminopeptidase N. Compared to the commercial vaccine strains, the emerging Vietnamese strains were genetically distant and had various amino acid differences in epitope regions and N‐glycosylation sites in the S protein. The development of novel vaccines based on the emerging Vietnamese strains may be contributive to the control of the current PED outbreaks.

Porcine epidemic diarrhoea virus (PEDV) is the aetiologic agent of porcine epidemic diarrhoea (PED), a highly contagious enteric disease that is threatening the swine industry globally. Since PED was first reported in Southern Vietnam in 2009, the disease has spread throughout the country and caused substantial economic losses.

To identify PEDVs responsible for the recent outbreaks, the full-length spike (S) gene of 25 field PEDV strains collected from seven northern provinces of Vietnam was sequenced and analysed. The sequence analysis revealed that the S genes of ized by acute enteritis and severe watery diarrhoea followed by dehydration leading to death, and the virus is associated with high mortality rates in piglets (Song & Park, 2012) . PEDV belongs to the family Coronaviridae in the order Nidovirales. This virus has a single-stranded positive-sense RNA genome of approximately 28 kb with a 5 0 cap and a 3 0 polyadenylated tail (Kocherhans, Bridgen, Ackermann, & Tobler, 2001; Pensaert & de Bouck, 1978) . The PEDV genome is composed of the 5 0 untranslated region and at least seven open reading frames (ORFs) that encode four structural proteins, namely spike (S), envelope (E), membrane (M) and nucleocapsid (N), and three major non-structural proteins (1a, 1ab and ORF3) (Song & Park, 2012) . Of the structural proteins, the S glycoprotein is the The S protein has been utilized as an effective component for studying genetic relatedness among PEDV strains, examining their epidemiological status and facilitating vaccine development (Chiou et al., 2015; Lee, Park, Kim, & Lee, 2010; Puranaveja et al., 2009; Temeeyasen et al., 2014) .

Porcine epidemic diarrhoea was first recognized in England in 1971 and later reported in several European countries (Pensaert & de Bouck, 1978; Wood, 1977) . In Asia, PED has been reported since the 1980s, and it has become an economic concern for the swine industry in Japan (Takahashi, Okada, & Ohshima, 1983) , China (Sun, Wang, Wei, Chen, & Feng, 2016) , South Korea (Kweon et al., 1993) , Thailand (Puranaveja et al., 2009) and Taiwan (Lin et al., 2014) . Before 2010, the prevalence of PEDV infection was comparatively low with mostly isolated outbreaks (Song & Park, 2012) . Since late 2010, new PEDV strains with increased pathogenicity compared to the classical strains emerged in China, and they are regarded as the first pandemic strains . In April 2013, highly pathogenic PEDV (US prototype, North American type) was first detected in the US, and it rapidly spread to 31 states as well as Mexico and Canada (Oka et al., 2014) .

Later, the second type of PEDV in the US with lower virulence in the field, designated S INDEL PEDV, was detected in samples collected beginning in June 2013 (Vlasova et al., 2014) . Subsequently, US-like strains have been reported in South Korea, Japan, Taiwan and European countries such as Ukraine, Belgium, Germany, France, Slovenia and Netherlands (Lin, Saif, Marthaler, & Wang, 2016) . In Vietnam, PED outbreaks were firstly confirmed by reverse transcription polymerase chain reaction (RT-PCR) in the southern provinces in early 2009 (Duy, Toan, Puranaveja, & Thanawongnuwech, 2011) . For Northern Vietnam, PEDV has been detected in severe outbreaks since the beginning of 2010 using commercial immunochromatographic assay kits (Diep, Lan, Hoa, & Yamaguchi, 2014) . A previous study of PED in Northern Vietnam conducted in 2012 and early 2013 revealed that the disease primarily occurred in the cold season lasting from November to April. Pigs of all ages were affected, manifesting diarrhoea at a rate of 92.0%-100% in herds, and the mortality rate is as high as 93.8% (68.6% on average) in suckling pigs . A commercial PED live vaccine (strain has been used in pig farms in Northern Vietnam since 2011. Another more common immunoprophylaxis applied in the field is the artificial infection of sows (i.e., the feedback method) during pregnancy using pooled faeces and intestine collected from infected piglets (Song, Moon, & Kang, 2015) . However, the effectiveness of these preventive measurements in the field is questionable because PED still occurred and frequently recurred in pig farms in which vaccination or the feedback method was applied. The persistence and reoccurrence of PED have been becoming more common in infected farms. To date, PEDV continues to spread widely and cause severe economic losses for the national swine industry. To identify the PEDV strains responsible for the recent outbreaks occurred from 2012 to 2015 in Northern Vietnam, we sequenced and analysed the full-length S gene of PEDVs obtained from 25 affected pig farms. This result will provide useful insights into the molecular epidemiology in Vietnam and serve as the basis for the development of effective vaccines for controlling the disease.

2 | MATERIALS AND METHODS 2.1 | Sample collection, RNA extraction and PEDV detection Twenty-five PEDV-positive samples as confirmed by RT-PCR collected from 25 herds located in seven different northern provinces of Vietnam ( Fig. S1 ) between December 2012 and July 2015 were used in this study. The number of samples from each province was as follows: Hung Yen (n = 9), Ha Noi (n = 6), Hoa Binh (n = 3), Thai Binh (n = 3), Vinh Phuc (n = 2), Quang Ninh (n = 1) and Son La (n = 1).

Small intestine and stool specimens were taken from sucking piglets, post-weaning pigs and sows exhibiting acute watery diarrhoea at 25 pig farms. Intestine samples were collected from dead piglets, and the faecal samples were non-invasively collected immediately after excretion. Therefore, no aggressive operation was conducted in pigs for sampling purposes. Samples were prepared in 20% suspensions through homogenization with Dulbecco's Modified Eagle's Medium with a low concentration of glucose. The suspensions were then vortexed and centrifuged at 2300 g for 10 min at 4°C. The clarified supernatants were stored at À80°C until use. Viral RNA was extracted from 300 ll of the samples and eluted in 30 ll of RNasefree water using ReliaPrep TM RNA Cell Miniprep kits (Promega Corporation, Madison, WI, USA) in accordance with the manufacturer's instructions. PEDV was detected in the collected samples by RT-PCR using a newly designed primer pair (pM-F/pM-R) for amplification of a partial M gene (Table S1 ). The expected size of amplified product was 463 bp. One-tube RT-PCR was performed using AccessQuick TM RT-PCR System kits (Promega). Exactly 4 ll of RNA template was mixed with the reaction mixture, which contained 12.5 ll of Access-Quick TM Master Mix (29), 0.5 ll of each specific primer (10 lM) and 0.5 ll of AMV reverse transcriptase (5 u/ll). Then, 7 ll of nucleasefree water was added to reach the total reaction volume of 25 ll.

RT-PCR was performed using a PCR Thermal Cycler (Takara, Japan).

Following a reverse transcription step of 45°C for 45 min and an incubation step of 94°C for 2 min, 35 cycles were performed as follows: 94°C for 30 s, 53°C for 30 s and 72°C for 30 s. The cycles were followed by a final 10-min extension step at 72°C. The RT-PCR products were analysed by 1.2% agarose gel electrophoresis and visualized by ultraviolet illumination after ethidium bromide staining.

From extracted RNA, RT was first performed using random hexamer primers and oligo primers from Reverse Transcription System Kits (Promega). The full-length S gene of PEDV was amplified using the e84 | primer pair FS-F/FS-R (Table S1 ) and KOD FX neo Kit (Toyobo Co., Japan). The PCR conditions were as follows: denaturation at 94°C for 2 min; 35 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at 68°C for 2.5 min; and a final extension step at 68°C for 10 min. Then, the PCR products were used as templates for nested PCR to amplify five DNA fragments spanning the entire S gene using five published primer pairs (CS1-CS5) (Diep, Norimine, Sueyoshi, Lan, & Yamaguchi, 2017) . The amplified PCR products were purified using a FastGene Gel/PCR Extraction Kit (NIPPON Genetics Co., Ltd, Japan) according to the manufacturer's protocol. All sequencing reactions were performed in duplicate, and sequences were determined in both directions using BigDye â Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems, CA, USA). The products were analysed using ABI PRISM 3130xl Genetic Analyzers (Applied Biosystems).

Nucleotide and deduced amino acid sequences were assembled and aligned using Geneious version 9.1.6 software (http://www.gene ious.com). The percentage sequence divergences at the nucleotide and amino acid levels were further calculated using the same software application. The obtained nucleotide sequences were deposited in GenBank under the accession numbers KX982553-KX982577 as shown in Table 1 . Unrooted phylogenetic trees were constructed using molecular evolutionary genetics analysis (MEGA) software version 6.06 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013) with maximum likelihood method and bootstrap tests of 1,000 replicates.

The best-fit nucleotide substitution models for analysis were assessed. Phylogenetic trees based on the nucleotide sequences of the full-length S gene were generated using the Tamura-Nei substitution model with a discrete gamma distribution (TN93+G). Prediction of N-glycosylation sites was performed using services available at http://www.cbs.dtu.dk/services/NetNGlyc. To identify high-specificity N-glycosylation sites, any potential crossing 0.5 and Jury agreement (9/9) or potential greater 0.75 for asparagines that occurred within the Asn-Xaa-Ser/Thr triplet was used. The potential phosphorylation sites were determined using the NetPhos 3.1 server ( | e85 3 | RESULTS

To investigate the diversity and relatedness of PEDV strains circulating in Northern Vietnam, the full-length S gene of Vietnamese PEDVs derived from 25 field samples collected in seven Northern provinces of Vietnam were sequenced and analysed. The phylogenetic tree of the identified S genes was constructed together with other classical and recent strains isolated in Europe, Asia and North

America. Phylogenetic analysis based on the nucleotide sequences of the S gene revealed two major clusters that were designated genogroup 1 (G1) and genogroup 2 (G2). G2 was further divided into G2-1 and G2-2, and G1 was divided into G1-1, G1-2 and G1-3 as shown in Figure 

The sequence data revealed that the S genes of the Vietnamese field strains are 4,143-4,182 nucleotides in length (Table 1) At least four epitope regions exhibiting neutralizing activities against PEDV have been identified in the S glycoprotein (Chang et al., 2002; Cruz, Kim, & Shin, 2008; Sun et al., 2008) . 

The numbers identifying N-glycosylation sites correspond to aa position within the consensus S protein sequence of the Vietnamese and Vaccine strains. N: N-glycosylation is predicted to be formed at that site; h: No N-glycosylation is predicted to be formed at the site; *: Prediction of N-glycosylation was not applied at that site of the truncated S protein. Although NA non-S INDEL strains were proposed to have evolved from different emerging Chinese strains via recombination (Tian et al., 2014; Vlasova et al., 2014) , no NA non-S INDEL strains were isolated in China (Lin et al., 2016) . The timing and mechanism of the appearance and global spread of the NA PEDV type remain unclear.

In this study, we identified NA PEDV strains from PED outbreaks in Deng et al., 2016) . It is revealed that the classical PEDV exhibited weaker sugar-binding activity than the recently uncovered G2 isolates (Deng et al., 2016) . Amino acid substitutions were also reported in the receptor-binding region . Whether Diep et al., 2015) . Therefore, the possibility that multiple parental PEDV strains introduced into Japan originated from Vietnam is quite notable. Considering this international dissemination route, we may recheck the quarantine process against PEDV and other epizootic agents.

In a previous study (Kim, Lim et al., 2015) , five PEDV strains iso- (Keating & Striker, 2012) . Phosphorylation is considered to introduce a change in the charge of the protein which results in a conformational change and alters the activation status of the protein (Keck, Ataey, Amaya, Bailey, & Narayanan, 2015) . Therefore, the result suggested that these mutations in the epitope regions may lead to changes in the antigenicity of prevailing Vietnamese PEDVs and consequently influence the efficacy of the vaccines.

Along with phosphorylation, other post-translational modifications such as N-glycosylation often strongly affect protein function.

The changes in N-glycosylation sites might influence the survival and transmission of the virus. They also play a major role in interactions with receptors and result in interference with virus recognition by the immune system of the host, therefore, influencing viral replication and infectivity (Meunier et al., 1999; Vigerust & Shepherd, 2007) . A study of a lactate dehydrogenase-elevating virus belonging to Coronaviridae reported that the acquisition or loss of N-glycosylation sites in an envelope glycoprotein (VP-3P) could result in changes in virulence and cellular tropism (Li et al., 2000) . N-glycosylation sites in the S gene of severe acute respiratory syndrome coronavirus were demonstrated to be crucial to virus infection (Han, Lohani, & Cho, 2007) . In our study, variations in high-specificity Nglycosylation sites were found among the prevailing Vietnamese PEDVs as well as between the Vietnamese strains and vaccine strains. In particular, the high-specificity sites 514 ( In conclusion, there are at least four genotypes of the PEDVs circulating in northern provinces of Vietnam. This study reported the prevalence of US-like strains in South-East Asia, and in particular, the NA type had already existed in Northern Vietnam before its first appearance in the US. Compare to the vaccine strains, Vietnamese DIEP ET AL.

PEDVs were genetically distant and had a variation in the epitope regions as well as N-glycosylation sites in the S proteins. The development of novel vaccines based on the circulating Vietnamese strains may be necessary to protect pigs against the emerging PEDV strains. Our present study also provides useful insights into the molecular epidemiology of PED that could improve disease control in the future.

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The work was supported by grants from The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan for global human resource and prevention of livestock epidemics by the Center for Animal Disease Control, University of Miyazaki (2014)(2015).

The authors declare no conflict of interests.