key: cord-0975334-0c8znvcf
authors: Jang, G.; Lee, K.‐K.; Kim, S.‐H.; Lee, C.
title: Prevalence, complete genome sequencing and phylogenetic analysis of porcine deltacoronavirus in South Korea, 2014–2016
date: 2017-07-30
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.12690
sha: 6bcdb389936a3e1937aa0d707ec657b4b4f6c4b3
doc_id: 975334
cord_uid: 0c8znvcf

Porcine deltacoronavirus (PDCoV) is a newly emerged enterotropic swine coronavirus that causes enteritis and diarrhoea in piglets. Here, a nested reverse transcription (RT)‐PCR approach for the detection of PDCoV was developed to identify and characterize aetiologic agent(s) associated with diarrhoeal diseases in piglets in South Korea. A PCR‐based method was applied to investigate the presence of PDCoV in 683 diarrhoeic samples collected from 449 commercial pig farms in South Korea from January 2014 to December 2016. The molecular‐based survey indicated a relatively high prevalence of PDCoV (19.03%) in South Korea. Among those, the monoinfection of PDCoV (9.66%) and co‐infection of PDCoV (6.30%) with porcine epidemic diarrhoea (PEDV) were predominant in diarrhoeal samples. The full‐length genomes or the complete spike genes of the most recent strains identified in 2016 (KNU16‐07, KNU16‐08 and KNU16‐11) were sequenced and analysed to characterize PDCoV currently prevalent in South Korea. We found a single insertion‐deletion signature and dozens of genetic changes in the spike (S) genes of the KNU16 isolates. Phylogenetic analysis based on the entire genome and spike protein sequences of these strains indicated that they are most closely related to other Korean isolates grouped with the US strains. However, Korean PDCoV strains formed different branches within the same cluster, implying continuous evolution in the field. Our data will advance the understanding of the molecular epidemiology and evolutionary characteristics of PDCoV circulating in South Korea.

Porcine deltacoronavirus (PDCoV) is a newly emerging enterotropic swine coronavirus that causes acute enteritis in nursing piglets Woo et al., 2012) . PDCoV infection is characterized by marked villous atrophy in the small intestine, which results in watery diarrhoea and vomiting, leading to dehydration and death in newborn piglets Ma et al., 2015) . This disease is symptomatically comparable to, but reportedly milder than, other porcine enteric coronavirus diseases caused by transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhoea virus (PEDV), with lower mortality rates in affected neonatal piglets . PDCoV belongs to the genus Deltacoronavirus within the family Coronaviridae of the order Nidovirales. PDCoV is a large, enveloped virus possessing a single-stranded, positive-sense RNA genome of approximately 25.4-kb long with a 5 0 cap and a 3 0 polyadenylated tail, which is the smallest genome size among porcine coronaviruses (de Groot et al., 2011; Woo et al., 2012) . The PDCoV genome consists of only six canonical coronaviral genes, except for open reading frame (ORF) 3, in the following conserved order: 5 0 untranslated region (UTR)-ORF1a-ORF1b-S-E-M-N-3 0 UTR. ORF1a and 1b occupy the 5 0 -proximal two-thirds of the genome encoding two overlapping viral replicase polyproteins, 1a and 1ab, which are proteolytically processed into mature non-structural proteins (nsps).

The production of polyproteins requires a À1 ribosomal frame shift, which C-terminally extends polyprotein 1a into polyprotein 1ab during translation of the genomic RNA. The 3 0 -proximal last third of the genome codes for the four structural proteins, spike (S), envelope (E), membrane (M) and nucleocapsid (N), as well as two accessory genes, non-structural gene 6 (NS6) and NS7, located between M and N, and within N, respectively (Lai, Perlman, & Anderson, 2007; Lee & Lee, 2014; Li et al., 2014; Woo et al., 2012) .

PDCoV was first discovered in Hong Kong, China in a territorial surveillance study of coronaviruses in mammals and birds in 2012 (Woo et al., 2012) . A PCR-based survey of diarrhoea samples from pigs in mainland China revealed the prevalence of PDCoV across the country since 2012. This study reported that the monoinfection of PDCoV and coninfection of PDCoV with PEDV are most common in pig herds in China (Song et al., 2015) . In February 2014, the presence of PDCoV was first announced in Ohio, United States, in conjunction with diarrhoea outbreaks without other aetiologic agents.

Molecular surveillance indicated that this novel coronavirus was present in 20 US states and nearly 80% of the tested samples corresponded to cases of co-infection of PDCoV with other enteric viral pathogens such as a rotavirus or PEDV (Li et al., 2014; Ma et al., 2015; Marthaler, Raymond, et al., 2014; Wang, Byrum, & Zhang, 2014) . Although the origin of PDCoV in the US remains unclear, sequence analyses suggest the possible introduction of a Chinese PDCoV strain into US swine (Li et al., 2014; Wang et al., 2014) . However, recent retrospective evaluation revealed that PDCoV antibodies could be detected in archival serum samples collected in 2010, suggesting that the virus may have already existed as early as 2010 (Thachil, Gerber, Xiao, Huang, & Opriessnig, 2015) . Shortly after its emergence in the US, PDCoV was also detected in South Korea from two diarrhoea samples independently collected in April and June 2014, which were positive for porcine rotavirus (PRV) and negative for other enteric viruses, respectively (Lee & Lee, 2014) .

Genetic and phylogenetic analyses showed that the Korean strains are more closely related to the US strains than to the Hong Kong HKU15 strains, suggesting that Korean PDCoV originated from the US (Lee & Lee, 2014) . In this study, we aimed to further investigate the prevalence and full-length genome sequence analysis of PDCoV from clinical cases associated with diarrhoea from Korean swine farms.

Small intestine or stool specimens (n = 683) were collected from piglets showing acute enteritis and watery diarrhoea from January 2014 through December 2016 (Table S1 ). A list of the sampling provinces is present in Table S1 . Intestinal homogenates were prepared as 10% (wt/vol) suspensions in phosphate-buffered saline (PBS) using a MagNA Lyser (Roche Diagnostics, Mannheim, Germany) by three repetitions of 15 s at a speed of 8,000 g. Faecal samples were also diluted with PBS to be 10% (wt/vol) suspensions. The suspensions were then vortexed and centrifuged for 10 min at 4,500 9 g (Hanil Centrifuge FLETA5, Incheon, South Korea). The clarified supernatants were stored at À80°C for RNA extraction until use. ing at 55°C for 30 s and extension at 72°C for 40 s, followed by a final extension step at 72°C for 7 min. For nested PCR, the second round of amplification was conducted using 1 ll of initial PCR product, internal primers and TaKaRa Ex Taq DNA polymerase (TaKaRa, Otsu, Japan) under the following conditions: denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 30 s, followed by a final extension step at 72°C for 10 min.

The complete genomes of representative PDCoV field strains, desig- (Table S2 ). Nine overlapping cDNA fragments spanning the entire viral genome were RT-PCR amplified using gene-specific primer sets as described above. The 5 0 and 3 0 ends of the PDCoV genome were determined by RACE as described previously (Lee & Lee, 2013) . The individual PCR amplicons were gel-purified, cloned into the pGEM-T easy vector (Promega, Madison, WI, USA), and sequenced in both directions using primers for the T7 and SP6 promoters, as well as PDCoVspecific primers. General procedures for DNA manipulation and cloning were performed according to standard procedures (Sambrook & Russell, 2001) . The complete genomic sequences of the KNU16-07 and KNU16-11 viruses and the S gene sequence of KNU16-08 were deposited in the GenBank database under accession numbers KY364365 and KY926512, and KY926511, respectively.

The sequences of the 38 fully sequenced S genes and 33 complete genomes of PDCoV isolates were independently used in sequence alignments and phylogenetic analyses. The multiple sequence alignments were generated with the ClustalX 2.0 program (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997) , and the percentages of the nucleotide sequence divergences were further assessed using the same software program. Phylogenetic trees were constructed from the aligned nucleotide or amino acid sequences using the neighbour-joining method and subsequently subjected to bootstrap analysis with 1,000 replicates in order to determine the percentage reliability values of each internal node of the tree (Saitou & Nei, 1987) . All figures involving phylogenetic trees were generated using the Mega 4.0 software (Tamura, Dudley, Nei, & Kumar, 2007) . The per cent full-length genome identity (nucleotides) was shown in the upper right, and the per cent of S protein identity (amino acid) was presented in the lower left. ND, Not determined. (Table 2; Table S3 ).

In contrast, the full-length genomes of the PDCoV KNU16 isolates had the lowest nucleotide identity to Chinese strains including two Hong Kong HKU15 strains, ranging from 98.4% to 99.2% (Table 2; Table S3 ). Comparing the complete genomes of the Korean KNU16 series to the Hong Kong HKU15 strains, all contained additional seven nucleotides (ACATGGG) at position 1, corresponding to the 5 0 UTR (Fig. S1 ). This insertion was reported in other global strains, including Chinese strains (Lee & Lee, 2014) . The

Korean KNU16 (Fig. S3) . These genetic drifts led to a relatively low aa identity to other Korean strains, ranging from 97.8% to 98.7% (Table 2 ).

For studies to establish the genetic relationships involved, phylogenetic analyses were carried out using the nucleotide sequences of the full-length genome and the S gene of the KNU16 isolates, which were determined in this study and those available from GenBank, together with selected coronavirus sequences from other genera (Figure 1 ).

The phylogeny based on the complete genome sequence indicated that the KNU16 strains were clustered into the deltacoronavirus group, forming a clade with nine other swine-origin PDCoV strains, which was distinct from deltacoronaviruses of avian-origin (Figure 1a ).

In agreement with previous studies (Marthaler, Raymond, et al., 2014; Wang, Hayes, Sarver, Byrum, & Zhang, 2016) and birds (Cavanagh, 1997; Mayo, 2002; Siddell & Snijder, 2008; Spaan et al., 2005) . Coronaviruses are divided into four genera:

Alpha-, Beta-, Gamma-and Deltacoronavirus, based on the phylogenetic distances of highly conserved domains (Woo et al., 2012) .

Although birds are the primary reservoirs for gamma-and deltacoronaviruses, these avian-origin coronaviruses may jump and adapt to some mammalian species including pigs. Indeed, a study to investigate the presence of deltacoronavirus identified a novel PDCoV genome from the faecal samples of pigs in Hong Kong, China in 2012 (Woo et al., 2012) . In early 2014, PDCoV was detected and reported in the US and South Korea (Lee & Lee, 2014; Wang et al., 2014) .

Soon thereafter, the pathogenicity and pathogenesis of this novel virus were elucidated in gnotobiotic and conventional piglets under different experimental conditions (Chen et al., 2015; Jung et al., 2015; Ma et al., 2015) . In South Korea, pigs have been prone to various diarrhoeic diseases for years, predominantly by PEDV (Lee, 2015; Lee & Lee, 2014 PDCoV/PRV co-infection, which was more prevalent in US pig herds (Marthaler, Raymond, et al., 2014) . Further study is needed to elucidate the interactions between PDCoV and PEDV or PDCoV and PRV. In the US, PDCoV infection was reported to be associated with significant mortality rates in the field (Ma et al., 2015; Song et al., 2015; Wang et al., 2014) . Similarly, a recent survey study revealed that a high number of piglet deaths occurring during an outbreak in China involved PDCoV infections and symptoms of severe diarrhoea (Song et al., 2015) . In contrast to the US and China, PDCoV infection appears to be less prevalent in South Korea, and PDCoV strains circulating in South Korea may not typically cause the severity and mortality of PDCoV-associated disease. Because PED is the most common enteric disease in South

Korea and PDCoV causes clinical signs indistinguishable from those of PEDV, it is possible that PDCoV-induced disease might be neglected and draw less attention for investigation. Some coronaviruses can retain some potential to infect different animal species, and subsequently can be adapted and maintained in the host by exploiting or sharing various cell surface components or other undefined factors (Su et al., 2016) . As PDCoV has a non-swine ancestor and is not swine-borne, a potential interspecies transmission of deltacoronavirus may have occurred between wild birds or small mammals and domestic pigs (Ma et al., 2015; Woo et al., 2012) .

Although pigs may have initially served as a susceptible host, PDCoV

may not yet be fully adapted to pigs and appears to continue to undergo genetic drift to become more completely adapted to pigs (Jung, Hu, & Saif, 2017 

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