key: cord-265631-b0kg6qpo
authors: Saeng-chuto, Kepalee; Stott, Christopher J.; Wegner, Matthew; Senasuthum, Raweewan; Tantituvanont, Angkana; Nilubol, Dachrit
title: Retrospective investigation and evolutionary analysis of a novel porcine deltacoronavirus strain detected in Thailand from 2008 to 2015
date: 2017-03-23
journal: Arch Virol
DOI: 10.1007/s00705-017-3331-3
sha: 
doc_id: 265631
cord_uid: b0kg6qpo

Porcine deltacoronavirus (PDCoV) in Thailand was first detected in 2015. We performed a retrospective investigation of the presence of PDCoV in intestinal samples collected from piglets with diarrhea in Thailand from 2008 to 2015 using RT-PCR. PDCoV was found to be present as early as February 2013. Phylogenetic analysis demonstrated that all PDCoV variants from Thailand differ from those from other countries and belong to a novel group of PDCoV that is separate from the US and Chinese PDCoV variants. Evolutionary analysis suggested that the Thai PDCoV isolates probably diverged from a different ancestor from that of the Chinese and US PDCoV isolates and that this separation occurred after 1994. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00705-017-3331-3) contains supplementary material, which is available to authorized users.

Porcine deltacoronavirus (PDCoV) causes an enteric disease in pigs that is characterized by diarrhea and is clinically similar to porcine epidemic diarrhea (PED) and transmissible gastroenteritis (TGE) [4] . PDCoV, a recently emerged RNA virus of the family Coronaviridae, genus

Deltacoronavirus, was first reported in Hong Kong in 2012 during an investigation to identify novel coronaviruses [14] . In February 2014, PDCoV was first detected in pigs with clinical diarrheal disease on farms in Ohio, USA. A subsequent retrospective investigation reported revealed that PDCoV had been present in the US as early as 2013 [11] . PDCoV has since been identified in Canada, China, South Korea and Laos [1, 6, 9] . In Thailand, PDCoV was first reported in 2015 [10] . However, it is unknown whether PDCoV was present in Thailand prior to 2015. We therefore conducted a retrospective study to detect the presence of PDCoV intestinal samples from Thailand that had been submitted for the identification of enteric diseases. Positive samples were subjected to full-length genome characterization followed by genetic analyses to compare Thai strains of PDCoV to those from other countries and to investigate the genetic similarity and the evolutionary relationships among them.

A total of 241 intestinal samples were assayed for the presence of PDCoV using RT-PCR. Intestinal samples were from 3-to 4-day-old piglets with clinical diarrheal disease collected from January 2008 to December 2015 and stored at -80°C. The presence of other viruses causing enteric disease, including porcine epidemic diarrhea virus, and transmissible gastroenteritis virus was detected using RT-PCR. In brief, viral RNA was extracted from the samples using NucleoSpin RNAVirus (Macherey-Nagel Inc., PA, USA) in accordance with the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from the extracted RNA using M-MuLV reverse transcriptase (New England BioLabs Inc., Ipswich, MA, USA). PCR amplification was performed on cDNA using specific primers for the N gene of PDCoV, the spike gene of PEDV, and the N gene of TGEV as described previously [5, 10, 12] .

Samples positive for PDCoV were then subjected to fulllength genome sequence characterization. Twenty-six pairs of primers were used to amplify the different regions of PDCoV [8] . PCR amplification was performed using Platinum Ò Taq DNA Polymerase High Fidelity (Invitrogen, CA, USA) in accordance with the manufacturer's protocol. The PCR products were purified using a NucleoSpin Plasmid kit (Macherey-Nagel Inc., Bethlehem, PA, USA). The 5 0 -and 3 0 -terminal regions were determined by using a kit for rapid amplification of 5 0 and 3 0 cDNA ends (5 0 and 3 0 -RACE, Clontech, Japan). Sequencing was performed at First BASE Laboratory SdnBhd (Selangor, Malaysia) using an ABI Prism 3730XL DNA sequencer.

The full-length PEDV genome sequence was assembled, and nucleotide and deduced amino acid sequences were aligned and analyzed using the CLUSTALW program [13] . The percentages of homology between the sequences at the nucleotide and amino acid levels were calculated. To investigate its evolutionary relationships to foreign PDCoV strains and estimate the divergence time of PDCoV in Thailand, phylogenetic analysis of the full-length nucleotide sequence of the PDCoV isolate collected in this study, together with 21 other PDCoV isolate sequences (Supplementary Table 1 ), was performed using the Bayesian Markov chain Monte Carlo (BMCMC) method implemented in the program BEAST v1.8.3 [2, 3] . A BEAST run was performed based on the GTR?G?I model with a coalescent Bayesian skyline tree prior and a clock model for each analysis using 200 million generations with sampling of every 10,000 generations and the first 10% discarded as burn-in. Tracer v1.6 was used to confirm that post-burn-in trees yielded an effective sample size (ESS) of [200 for all parameters. The resulting tree was viewed and generated in FigTree v1.4.2.

The results of testing for PDCoV, PEDV, and TGEV in intestinal samples are shown in Table 1 . Of 241 intestinal Lopburi and Chonburi. These four provinces contain three major swine-producing areas of Thailand. These results suggest that PDCoV is widespread throughout Thailand. It is noteworthy that 144 samples (59.75%) were positive for PEDV and that samples that were positive for PDCoV were also positive for PEDV. The results suggest the presence of mixed infections with PEDV and PDCoV in pigs. The production records of the herd in which PDCoV was first detected showed that there had been three diarrhea outbreaks during 2014 and 2015, and both PEDV and PDCoV were detected. In three diarrhea outbreaks, the pre-weaning mortality rate was 56.9, 52.1 and 65.3%, respectively. However, we were unable to define the relationship between PEDV and PDCoV regarding to the primary or secondary causes of the diarrhea outbreaks. Whether coinfection with these viruses would necessitate more-complex control methods or would result in more-frequent, repetitive outbreaks requires further investigation. The pairwise nucleotide and amino acid sequence identity values of the full-length genome sequences of the four Thai PDCoV isolates are shown in Table 3 . These sequences were 98. .1% identical at the nucleotide and 97.4-97.9% identical at the amino acid level and were more closely related to each other and to variants from China and the United States than to Thai strains isolated in 2015, suggesting that the Thai PDCoV population is continuously changing.

Phylogenetic analysis based on the full-length genome sequence demonstrated that PDCoV isolates are divided into two groups, the Chinese-and US-like-groups, as was reported previously [1] . All four Thai PDCoV isolates were grouped in a novel cluster separate from variants from the United States and China but were more closely related to the Chinese isolates than to the US PDCoV isolates (Fig. 1) . The currently circulating isolates of PDCoV share a common ancestor that was present around 1994, with 95% HPD values covering 30 years from 1975 to 2004 (Fig. 1 (Fig. 1) . The analysis of amino acid changes also suggests that they are from a different lineage.

In conclusion, we retrospectively investigated the presence of PDCoV in Thailand and found that PDCoV has been present in Thailand since at least February 2013 and that it is present in mixed infections with PEDV. Interestingly, the PDCoV variants in Thailand are different from variants in other countries and belong to a novel group of PDCoV strains. Evolutionary analysis suggested that the Thai PDCoV isolates likely diverged from a different ancestor from that of the Chinese and US PDCoV isolates, and the separation occurred after 1994. This study provides interesting results associated with the emergence and the evolution of PDCoV. Further investigation, including more retrospective studies of available samples, will provide a better understanding of this pathogen.

Porcine deltacoronavirus in Mainland China

BEAST: Bayesian evolutionary analysis by sampling trees

Bayesian phylogenetics with BEAUti and the BEAST 1.7

Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs

Detection and differentiation of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus in clinical samples by multiplex RT-PCR

Complete genome characterization of Korean porcine deltacoronavirus strain KOR/KNU14-04

Full-length genome sequence of porcine deltacoronavirus strain USA/IA/2014/8734

The genetic diversity and complete genome analysis of two novel porcine deltacoronavirus isolates in Thai

The first detection and full-length genome sequence of porcine deltacoronavirus isolated in Lao PDR

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

PCRbased retrospective evaluation of diagnostic samples for Porcine deltacoronavirus in Thailand 2107 emergence of porcine deltacoronavirus in US swine

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

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

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

Acknowledgements This work was supported by the National Research Council of Thailand (Grant Number 5080001) and the Agricultural Research Development Agency (public organization). We gratefully acknowledge the graduate scholarship program of Chulalongkorn University and the funding provided by the Special Task Force for Activating Research (STAR), Swine Viral Evolution and Vaccine Research (SVEVR), Chulalongkorn University.

The study was funded by the Thailand Research Fund (Grant Number MRG5080323) and the National Research Council of Thailand (Grant Number 5080001).

Conflict of interest The authors declare that they have no conflicts of interest related to this work.Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.