Chen et al. Virology Journal (2018) 15:95 https://doi.org/10.1186/s12985-018-1001-z RESEARCH Open Access Metagenomic analysis of the RNA fraction of the fecal virome indicates high diversity in pigs infected by porcine endemic diarrhea virus in the United States Qi Chen1†, Leyi Wang2†, Ying Zheng1, Jianqiang Zhang1, Baoqing Guo1, Kyoung-Jin Yoon1, Phillip C. Gauger1, Karen M. Harmon1, Rodger G. Main1 and Ganwu Li1* Abstract Background: Emergence and re-emergence of porcine epidemic diarrhea virus (PEDV) in North America, Asia and Europe has caused severe economic loss to the global swine industry. However, the virome of PEDV infected pigs and its effect on disease severity remains unknown. The advancements of sequencing technology have made it possible to characterize the entire microbiome of different body sites for any host. Methods: The objective of this study was to characterize the RNA virome in PEDV-positive pigs using the hypothesis-free metagenomics approach based on next-generation sequencing. Specifically, 217 PEDV-positive swine fecal swab samples collected from diarrheic piglets over 17 US states during 2015–2016 were analyzed. Results: A Kraken algorithm-based bioinformatics analysis revealed the presence of up to 9 different RNA genera besides PEDV (Alphacoronavirus genus), including Mamastrovirus (52%, 113/217), Enterovirus (39%, 85/217), Sapelovirus (31%, 67/217), Posavirus (30%, 66/217), Kobuvirus (23%, 49/217), Sapovirus (13%, 28/217), Teschovirus (10%, 22/217), Pasivirus (9%, 20/217), and Deltacoronavirus (3%, 6/217). There were 58 out of 217 piglets (27%) have PEDV infection alone whereas the remaining 159 (73%) shed 2 up to 9 different viruses. Conclusion: These findings demonstrated that PEDV infected diarrheic pigs had an extensive RNA viral flora consisting of four different families: Astroviridae, Picornaviridae, Caliciviridae, and Coronaviridae. Keywords: PEDV, RNA virome, Coinfection, Metagenomic analysis Background and porcine astrovirus as well as others [1]. Among Diarrhea in pigs, especially in piglets, is a major issue af- them, PEDV, TGEV, PDCoV and PRoV have been well fecting global swine industry. A diverse array of viral characterized for their role in clinical diarrhea and have pathogens are implicated in piglet diarrhea, including been considered as major swine enteric viral pathogens porcine epidemic diarrhea virus (PEDV), transmissible while the causative role of the other agents in diarrheic gastroenteritis virus (TGEV), porcine deltacoronavirus disease has been characterized to a significantly less ex- (PDCoV), porcine rotavirus (PRoV), porcine bocavirus, tent or remains to be further studied. Aichivirus C (formerly named as porcine kobuvirus 1 PEDV is an enveloped, single-stranded, positive-sense [PKV-1]), porcine sapovirus, porcine sapelovirus (PSV), RNA virus belonging to the Order Nidovirales, the fam- ily Coronaviridae. Initially identified in Europe in 1970’s, * Correspondence: [email protected] PEDV was later detected in many Asian countries where †Qi Chen and Leyi Wang contributed equally to this work. 1Department of Veterinary Diagnostic and Production Animal Medicine, it causes severe problems in pig populations. In North College of Veterinary Medicine, Iowa State University, 1907 ISU C-Drive, America, PEDV was first detected in the United States VMRI#1, Ames, IA 50011, USA (US) in 2013, which rapidly spread across the US in the Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chen et al. Virology Journal (2018) 15:95 Page 2 of 9 subsequent 2 years [2–4]. The diarrhea caused by PEDV XP Beads (Beckman Coulter Life Sciences, Indianapolis, can result in dehydration in suckling piglets and eventu- IN) following the manufacturer’s instruction. ally up to 100% mortality, which caused significant eco- nomic loss to the US swine industry [5, 6]. Library preparation and sequencing Using virus-specific PCRs, viruses such as PDCoV and Reverse transcription was performed on purified RNA PRoV have been detected along with PEDV in some diar- using NEXTflex™ Rapid RNA-Seq Kit (Bioo Scientific rheic cases submitted to the Iowa State University Veter- Corp, Austin, TX). Double-stranded cDNA was purified inary Diagnostic Laboratory (ISUVDL) and other with Agencourt® AMPure® XP Beads (Beckman Coulter diagnostic laboratories of different states [7, 8]. However, Life Sciences, Indianapolis, IN) per manufacturer’srec- there have been no reports thoroughly investigating vi- ommended procedure, and was re-suspended into 9 μl ruses concurrent co-infections with PEDV in diarrheic re-suspension buffer. The concentration of cDNA was pigs. The random primers-based, hypothesis-free determined by Qubit® 2.0 Fluorometer and Qubit® High-throughput sequencing (HTS) enables simultaneous dsDNA HS Assay Kit (Thermo Fisher Scientific, detection of multiple microorganisms, making it possible Waltham, MA) and adjusted to 0.2 ng/μl for sequencing to identify not only known pathogens, but also novel and/ library preparation. Sequencing library was prepared or uncharacterized potential pathogens. In this study, a using Nextera® XT library preparation kit (Illumina, San HTS-based metagenomics approach was used to Diego, CA) with dual-indexing and sequencing was per- characterize the fecal RNA virome in diarrheic pigs in- formed on an Illumina MiSeq® instrument (Illumina, San fected with PEDV. Some previously uncharacterized vi- Diego, CA) according to user manual of “MiSeq Sequen- ruses were detected and genetically characterized in the cing System Guide” for 2 × 150 base pair (bp) reads [9]. present study. Each library composed of 24 pooled samples or fewer without negative control samples for cross contamin- ation assessment. Methods Clinical specimens Sequence data analysis Two hundred and seventeen fecal samples, stored at − The output of sequence data was demultiplixed based 80 °C upon use, were selected from submissions to on default setting of MiSeq. The primary workflow used ISUVDL between February 2015 and February 2016. All to identify mixed infections is outlined in Fig. 1. The de- of the fecal samples were collected from pigs with acute fault parameters were used to perform analysis on all the diarrhea, tested positive for PEDV RNA by virus-specific softwares used in the study. Raw sequencing reads from real-time reverse transcription PCR (rRT-PCR) with Ct MiSeq were subjected to sequencing quality analysis values ranging 8–26, and requested for whole genome with FastQC pre-installed in the Illumina MiSeq® instru- sequencing of PEDV with HTS. Fecal samples negative ment with defaulting parameter setting. Reads with low with PEDV rRT-PCR was not included in this study as sequencing quality, sequencing adaptors and primers negative control. Of the 217 samples, 173 samples were were removed from further bio-informative analysis with submitted from swine operations in 17 US states while Trimmomatic v0.36 [10]. Cleaned reads were classified the remaining 44 samples were submitted without geo- with the standard database Kraken v0.10.5-beta includ- graphical information. ing pig host, refseq virus, bacteria, and archaea se- quences (Fig. 1)[11]. KronaTools-2.6 was then used to Nucleic acid extraction and DNA removal generate the interactive charts for the hierarchical classi- Fecal swabs collected with Synthetic-Tipped Applicators fication results and any classified organisms can be (Thermo Fisher Scientific, Waltham, MA) were viewed in these pie charts [12]. To confirm the classifi- re-suspended by being swirled in 1 ml PBS (Thermo cation and to control the quality of assembling results of Fisher Scientific, Waltham, MA), followed by total gen- Kraken method, Kaiju program version 1.6.2 was used as etic materials extraction using MagMAX™ viral RNA iso- a secondary validating method, which determines taxo- lation kit (Life Technologies, Carlsbad, CA) on a nomic classification on protein level [13]. Kingfisher magnetic particle processor (Thermo Fisher To further characterize each virus, all de-multiplexed Scientific, Waltham, MA) following the manufacturer’s reads of an interested taxon were filtered, extracted, and instructions with minor modification as described previ- de novo assembled with SPAdes Genome Assembler ver- ously [2]. Specifically, extracted DNA/RNA was eluted sion 3.11.1 for Linux with the assembly options for into 50 μl elution buffer. Extracted nucleic acids were paired end reads and Burrows Wheeler Aligner mis- then treated by RNase-free DNase I (Qiagen, Hilden, match correction [14]. To further validate the assembled Germany) for 15 min at ambient temperature to remove contigs, the contigs assembled from each viral genus DNA, and was then purified by Agencourt® RNAclean® were blasted against a customized database built Chen et al. Virology Journal (2018) 15:95 Page 3 of 9 Total reads Trimmomatic;FastQC Clean reads Kraken classification Identified viruses Visualized