key: cord-0960010-nrg8puqe
authors: Saeng‐chuto, Kepalee; Jermsutjarit, Patumporn; Stott, Christopher J.; Vui, Dam Thi; Tantituvanont, Angkana; Nilubol, Dachrit
title: Retrospective study, full‐length genome characterization and evaluation of viral infectivity and pathogenicity of chimeric porcine deltacoronavirus detected in Vietnam
date: 2019-09-13
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.13339
sha: 593d2c760dc9b6081415084fef822aa1197567c7
doc_id: 960010
cord_uid: nrg8puqe

Increased evidence of porcine deltacoronavirus (PDCoV) causing diarrhoea in pigs has been reported in several countries worldwide. The virus has currently evolved into three separated groups including US, China and Southeast Asia (SEA) groups. In Vietnam, PDCoV was first reported in 2015. Based on phylogenetic analyses of spike, membrane and nucleocapsid genes, it is suggested that Vietnam PDCoV is chimeric virus. In the present study, we retrospectively investigated the presence of PDCoV in Vietnam and the full‐length genomes of six PDCoV isolates identified in 2014–2016 were further characterized. The results demonstrated that Vietnam PDCoV was first detected as early as 2014. All six Vietnam PDCoV are in the SEA group and further divided into two separated subgroups including SEA‐1 and SEA‐2. Vietnam PDCoV in SEA‐2 was closely related to Thai and Lao PDCoV. Recombination analysis demonstrated that three isolates in SEA‐1 were a chimeric virus of which P12_14_VN_0814, the first Vietnam isolate, and US PDCoV isolates were major and minor parents, respectively. The recombination was further evaluated by phylogenetic construction based on 3 recombinant fragments. The first and third fragments, closely related to P12_14_VN_0814, were associated with ORF1a/1b and N genes, respectively. The second fragment, associated with S, E, and M genes, was closely related to US PDCoV isolates. High antigenic and hydrophobic variations were detected in S1 protein. Three‐day‐old pigs challenged with the chimeric virus displayed clinical diseases and villus atrophy. In conclusion, Vietnam PDCoV is genetically diverse influenced by an external introduction from neighbouring countries. The chimeric Vietnam PDCoV can induce a disease similar to Thai PDCoV.

. Clinical signs caused by PDCoV resemble those with two viruses in the genus Alphacoronavirus, family Coronaviridae, including porcine epidemic diarrhoea virus (PEDV) and transmissible gastroenteritis virus (TGEV) , but severity of clinical disease is lesser and piglet mortality is lower. PEDV and TGEV infect pigs of all ages and cause 100% morbidity, with 80%-100% mortality rates in piglets. The higher severity of PEDV and TGEV infection in pigs indicates that the two viruses are more suitable for infecting pigs. On the other hand, PDCoV may not be perfectly adapted to pigs, and the virus may be evolving towards better ability to infect a specific host. Recently, an experimental study in calves suggested that calves are susceptible to infection by PDCoV but not PEDV (Jung, Hu, & Saif, 2017) .

The full-length genome of PDCoV is approximately 25 kb in length and contains seven essential genes, including open reading frame 1a/1b (ORF1a/1b), spike (S), envelop (E), membrane (M), and nucleocapsid (N), and specific accessory genes including non-structural protein 6 (Nsp6) and Nsp7, flanked by a 5′-and 3′-untranslated region (UTR) (Woo et al., 2012) . ORF1a and ORF1b, occupying twothirds of the genome, encode 2 overlapping viral replicase/transcriptase polyproteins, 1a and 1b, which are cleaved into 15 Nsps, namely Nsp2-16 (Zhang & Yoo, 2016) . The S gene encodes S protein which consists of two domains called S1 and S2 domain. The S1 domain plays an important role in binding to specific host cell receptors and contains a neutralizing epitope. The S2 domain functions in membrane fusion (Shang et al., 2018) . The E and M genes are transmembrane proteins associated with viral envelope formation and virus release (Woo et al., 2010) . On the other hand, the functions of the N gene are associated with viral RNA replication and pathogenesis (Lee & Lee, 2015) .

PDCoV was first detected in Hong Kong (HKU15-44 and HKU15-155 isolates) in 2012 (Woo et al., 2012) . Although it was first detected, there was no association with clinical disease. The first evidence of PDCoV causing a disease was first evident in Ohio, USA in 2014. Soon after the first emergence in Ohio, USA, the virus was then reported in 18 states (Jung et al., 2016) . The rapid widespread of PDCoV was evident when the virus was subsequently reported in China, South Korea, Thailand, Laos, and Vietnam in 2015 (Le et al., 2018; Lee & Lee, 2014; Madapong et al., 2016; Marthaler, Jiang, Collins, & Rossow, 2014; Saeng-Chuto, Lorsirigool, et al., 2017; Song et al., 2015; Wang, Byrum, & Zhang, 2014a , 2014b . Although the first detection of PDCoV in those countries was in 2014-2015, the retrospective investigation of intestinal samples demonstrated that the presence of PDCoV in China, USA and Thailand was as early as 2004 , 2013 and 2013 , respectively (Saeng-Chuto, Stott, et al., 2017 Sinha, Gauger, Zhang, Yoon, & Harmon, 2015) .

Presently, PDCoV has evolved into three separated groups, including US, China and Southeast Asia (SEA). PDCoV isolates from Thailand and Laos were clustered in a novel PDCoV group, SEA. The SEA group is genetically distinct from US and China PDCoV groups (Madapong et al., 2016; Saeng-Chuto, Lorsirigool, et al., 2017; . In Vietnam, PDCoV was first detected in 2015 (Saeng-Chuto, Lorsirigool, et al., 2017) . Genetic analyses based on S, M, and N genes demonstrated that Vietnam PDCoV isolates were genetically different from the SEA group (Saeng-Chuto, Lorsirigool, et al., 2017 PDCoV were further characterized on the basis of heterogenicity, and recombination analyses together with virus infectivity in cell culture and pathogenicity in piglets were performed.

One hundred and eight intestinal samples collected from 49 swineherds with clinical diarrhoea outbreaks in different regions in Vietnam (Table 1) Inc.). Virus was isolated, and plaque purification was performed as described in Data S1.

Viral RNA was extracted from the supernatant using Nucleospin Viral RNA Extraction Kit (Macherey-Nagel Inc.) and converted to cDNA using M-MuLV Reverse Transcriptase (New England Biolabs Inc.). All samples were tested with the specific primer for S gene of PEDV and specific primer for N gene of TGEV as in previous studies (Lee & Lee, 2014; Park et al., 2007; Song et al., 2015; Temeeyasen et al., 2014; Wang, Byrum, & Zhang, 2014a; Woo et al., 2010) . PDCoV was detected by PCR using specific primer for M and N genes as previously described (Wang, Byrum, & Zhang, 2014b 

To determine the genetic relationship between the six Vietnam PDCoV isolates and other PDCoV isolates, the nucleotide (nt) and amino acid (aa) sequences were aligned using the CLUSTALW program (Thompson, Higgins, & Gibson, 1994) , together with 84 other PDCoV isolates available in GenBank (Table S2 ). The nt and aa identities were determined using the sequence identity matrix function implemented in BioEdit software (Hall, 1999) . The phylogenetic tree based on the full-length genome, open reading frame (ORF) 1a/1b, SEMN and S, M and N genes was separately built using the maximum likelihood (ML) method with 1,000 bootstrap replicates, and substitution models were selected by the best-fit substitution model function implemented in MEGA software version 6.0 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013) . TN93 + G+I model was used in phylogenetic trees based on full-length genome and SEMN gene construction. TN93 + G model was used in phylogenetic trees based on ORF1a/1b and S genes construction. K2 + G and K2 + G+I models were used in the construction of phylogenetic trees based on M and N genes, respectively.

Recombination of six Vietnam PDCoV isolates was further analysed together with the 84 reference PDCoV isolates using automated RDP, GENECONV, BOOTSCAN, MaxChi, CHIMAERA and SISCAN methods implemented in RDP4 software (Martin, Murrell, Golden, Khoosal, & Muhire, 2015) . Major and minor parents and breakpoints also were detected, and recombination fragments were generated with the RDP4 software (Martin et al., 2015) .

Phylogenetic trees based on recombinant fragments were separately constructed using the ML method with 1,000 bootstrap replicates, and substitution models were designated by the best-fit substitution model function implemented in MEGA software version 6.0 (Tamura et al., 2013) . TN93 + G+I model was used in phylogenetic tree based on all recombination fragments construction.

The sliding window, hydrophobicity and antigenicity were evaluated among the chimeric Vietnam PDCoV isolates in this study, and suspected major and minor parents of the chimeric Vietnam PDCoV, respectively ( Figure 2 ).

The nt sequence variation sites were predicted using dnaSP version 6 with 100 bp and a step size of 25 bp (Rozas et al., 2017) .

Antigenicity and hydrophobicity were also analysed using IEDB 

Eighteen 3-day-old healthy piglets were randomly divided into three groups: (i) mock control group (n = 6), (ii) chimeric Vietnam PDCoV (P29_15_VN_1215) isolate-infected group (n = 6), and (iii) NT1_1215

isolate-infected group (n = 6). Each piglet was inoculated with 5 ml of each virus at a titre of 10 3 TCID 50 /ml. Clinical signs were observed daily. Three piglets per group were euthanized at 3 and 5 days postinoculation (dpi.), and five segments of the small intestine, including duodenum, proximal jejunum, middle jejunum, distal jejunum and ileum, were collected and fixed in 10% formalin. Haematoxylin and eosin (H&E) staining was performed, and villus/crypt ratio was calculated. Comparison between each group was performed with oneway analysis of variance, followed by Tukey's multiple comparison test implemented in GraphPad Prism 7 (GraphPad Software Inc.).

p < .05 was considered significant.

One hundred and eight intestinal samples collected from pigs with diarrhoea were assayed for the presence of three viral pathogens including PEDV, TGEV and PDCoV using PCR assays (Table 1) . Of 108 intestinal samples tested, none were positive for TGEV. Eleven VN_0416 isolates were able to characterize their full-length genome sequences and further genetic analyses.

The full-length genome sequence of the six Vietnam PDCoV iso- 

Three recombination fragments were generated. The first and 

The full-length genome sequences of the five isolates including P29_15_VN_1215, P30_15_VN_1215, P1_16_VN_0116, 

Viral growth curves of the chimeric Vietnam PDCoV (P29_15_ VN_1215) and Thai (NT1_1215) isolates in ST cells were compared.

The two isolates had a similar pattern of viral growth curves, but virus titres were different ( Figure 6 ). Cytopathic effect, characterized by cell rounding and clumping, with both PDCoV isolates were first observed at 12 hpi with a virus titre of 10 1 TCID 50 /ml (mean log 10 TCID 50 /ml ± SD; 1 ± 0). At 24 hpi, the titre of P29_15_VN_1215 remained at 10 1 TCID 50 /ml (mean log 10 TCID 50 /ml ± SD; 1 ± 0), while the titre of NT1_1215 increased to 10 2 TCID 50 /ml (mean log 10 TCID 50 / ml ± SD; 2 ± 0). The titres of both PDCoV isolates slowly rose from 24 to 48 hpi. At 36 and 48 hpi, the titres of P29_15_VN_1215 were 10 2 (mean log 10 TCID 50 /ml ± SD; 2 ± 0.25) and 10 4 TCID 50 /ml (mean log 10 TCID 50 /ml ± SD; 4 ± 0.25), respectively, which were lower than those of NT1_1215 at 10 3 (mean log 10 TCID 50 /ml ± SD; 3 ± 0.25) and

10 5 TCID 50 /ml (mean log 10 TCID 50 /ml ± SD; 5 ± 0.25), respectively.

The titres of both PDCoV isolates rapidly declined to 10 1 TCID 50 / ml at 60 hpi (mean log 10 TCID 50 /ml ± SD; 1 ± 0.25). However, at 72 hpi., the titre of NT1_1215 increased to 10 2 TCID 50 /ml (mean log 10 TCID 50 /ml ± SD; 2 ± 0), but the titre of P29_15_VN_1215 remained at 10 1 TCID 50 /ml (mean log 10 TCID 50 /ml ± SD; 1 ± 0).

Pigs in the mock control group of which orally inoculated with culture media displayed no clinical disease throughout the study. In contrast, pigs orally inoculated with either Vietnam PDCoV isolates (P29_15_ VN_1215) or Thai PDCoV isolate (NT1_1215 isolate) displayed similar level of clinical diseases associated with PDCoV including severe diarrhoea, vomiting, dehydration, weakness and lethargy. Clinical diseases were observed in both Vietnam and Thai PDCoV infected groups at 1 dpi. However, no piglets died before euthanasia.

At 3 and 5 dpi, 3 piglets in each group were necropsied and five parts of small intestine including duodenum, proximal F I G U R E 3 (Continued) jejunum, middle jejunum, distal jejunum and ileum were collected for further histopathological examination. H&E staining was performed. All H&E-stained small intestine segments of piglets infected with P29_15_VN_1215 and NT1_1215 showed villous atrophy compared to that of the mock control group (G1) (Figure 7) . F I G U R E 4 Differences in full-length genome sequence between five isolates, including P29_15_VN_1215, P30_15_VN_1215, P1_16_ VN_0116, P12_VN_14_0814 (major parent) and USA/Minnesota159/2014 (minor parent), were analysed by sliding window method. The red arrows show high variation regions [Colour figure can be viewed at wileyonlinelibrary.com] F I G U R E 5 Hydrophobicity and antigenicity differences were found in S protein between the five isolates studied: P29_15_VN_1215, P30_15_VN_1215, P1_16_VN_0116, P12_VN_14_0814 (major parent) and USA/Minnesota159/2014 (minor parent) [Colour figure can be viewed at wileyonlinelibrary.com] Villus/crypt (V/C) ratios of piglets in each group were determined ( Figure 8) . At 3 dpi, the V/C ratio in the three jejunum segments of piglets infected with both PDCoV isolates was significantly lower compared to that of the mock control group (p < .05) (Figure 8b d). At 5 dpi, the V/C ratio in middle and distal jejunum segments of piglets infected with both PDCoV isolates was significantly lower than that in piglets in the mock control group (p < .05) (Figure 8c,d) .

Although no significant difference was observed, the V/C ratio in all parts part in both infected groups trend to reduced when compared between 3 and 5 dpi within the groups (Figure 8a ,c).

In Vietnam, the first detection of PDCoV was reported in 2015 in the southern region of Vietnam (Saeng-Chuto, Lorsirigool, et al., 2017) .

Genetic analyses based on S, M and N genes discovered contradictory results suggesting that Vietnam PDCoV could be a recombinant virus. However, the recombination of the virus has not been confirmed and the full-length genome was not attempt in the previous study. We therefore conducted a retrospective study investigating the presence of PDCoV in Vietnam, genetic diversity and characterize the full-length genome of detected PDCoV. The isolated chimeric virus was further evaluated for its pathogenicity.

In the present study, PDCoV was first detected in southern re- F I G U R E 7 Haematoxylin and eosin (H&E) staining of five segments of small intestine, including duodenum (a-c), proximal jejunum (d-f), middle jejunum (g-i), distal jejunum (j-l) and ileum (m-o). Villous atrophy was observed in all small intestine segments of piglets infected with the chimeric Vietnam PDCoV (P29_15_VN_1215) (b, e, h, k and n) or the Thai PDCoV (NT1_1215) (c, f, i, l and o) isolates, compared to mock control group (a, d, g, j and m). Statistical significance between groups annotated by asterisk. Differences in hydrophobicity and antigenicity were detected in six parts in the S1 domain (aa 1-552) among the five isolates. The S1

domain plays an important role in host receptor binding and contains a neutralizing epitope (Shang et al., 2018; Woo et al., 2010) . These results might support the proposition that the chimeric Vietnam shown to cause disease. The infected gnotobiotic pigs showed severe diarrhoea, vomiting and severe atrophic enteritis . Moreover, in 2018, piglets were challenged with PDCoV KNU16-07 isolate and showed similar clinical signs, namely diarrhoea, vomiting and viral enteritis (Jang et al., 2018) . The findings in our study confirmed that the chimeric Vietnam PDCoV infection could induce small intestinal enteritis.

In conclusion, Vietnam PDCoV is genetically diverse influencing from the external introduction. Vietnam PDCoV is further evolved into 2 separated subgroups including SEA-1 and SEA-2. Vietnam PDCoV in SEA-2 was closely related to Thai and Lao PDCoV.

Recombination analysis demonstrated that isolates in SEA-1 were a F I G U R E 8 Villus to crypt (V/C) ratio of five segments of small intestine, including duodenum (a), proximal jejunum (b), middle jejunum (c), distal jejunum (d) and ileum (e) chimeric virus. P12_14_VN_0814, the first Vietnam isolate, and US PDCoV isolates were major and minor parents, respectively. The chimeric Vietnam PDCoV could induce clinical diseases. Moreover, the partial funding was supported by Special Task Force for Activating Research (STAR), swine viral evolution and vaccine research (SVEVR), Chulalongkorn University.

The authors of this work have none connection with other institutions that unsuitable or bias in this work.

All utilizable international, national, and/or institutional guidelines for concern and usage of animals were followed. The animal challenge study was approved by Institutional Animal Care and Use

Committee of Chulalongkorn University (CU-IACUC) (1931018) .

Dachrit Nilubol https://orcid.org/0000-0002-7691-6790

The coronavirus spike protein is a class I virus fusion protein: Structural and functional characterization of the fusion core complex

BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT

Antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: Implication for vaccine design

Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States

Isolation and characterization of a Korean porcine deltacoronavirus strain KNU16-07

Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs

Porcine deltacoronavirus infection: Etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis

Calves are susceptible to infection with the newly emerged porcine deltacoronavirus, but not with the swine enteric alphacoronavirus, porcine epidemic diarrhea virus

A novel strain of porcine deltacoronavirus in Vietnam

Complete genome characterization of Korean Porcine Deltacoronavirus Strain KOR/KNU14-04

Functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus

Complete genome sequence of porcine deltacoronavirus isolated in Thailand

Complete genome sequence of strain SDCV/USA/Illinois121/2014, a porcine deltacoronavirus from the United States

Recombination in eukaryotic single stranded DNA viruses

RDP4: Detection and analysis of recombination patterns in virus genomes

Sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in Korea

DnaSP 6: DNA sequence polymorphism analysis of large data sets

Different lineage of porcine deltacoronavirus in Thailand, Vietnam and Lao PDR in 2015

Retrospective investigation and evolutionary analysis of a novel porcine deltacoronavirus strain detected in Thailand from

Cryo-electron microscopy structure of porcine deltacoronavirus spike protein in the Prefusion State

Why do RNA viruses recombine?

PCRbased retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in US swine

Newly emerged porcine deltacoronavirus associated with diarrhoea in swine in China: Identification, prevalence and full-length genome sequence analysis

MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution

Design strategies to address the effect of hydrophobic epitope on stability and in vitro assembly of modular virus-like particle

Genetic diversity of ORF3 and spike genes of porcine epidemic diarrhea virus in Thailand. Infection

CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice

Detection and genetic characterization of deltacoronavirus in pigs

Porcine coronavirus HKU15 detected in 9 US states

Improving influenza vaccine virus selection: Report of a WHO informal consultation held at WHO headquarters

Coronavirus genomics and bioinformatics analysis. Viruses

Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus

Immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling

Retrospective study, full-length genome characterization and evaluation of viral infectivity and pathogenicity of chimeric porcine deltacoronavirus detected in Vietnam