Viral metagenomic analysis of chickens with runting-stunting syndrome in the Republic of Korea

Runting-stunting syndrome (RSS) in chickens, also known as malabsorption syndrome, which is characterized by mild to severe enteritis and diagnosed through typical histopathologic examination as well as clinical signs, results in considerable economic losses. Despite the many studies carried out over decades to determine the etiologic agents of RSS involved in the disease, several outbreaks remained without the elucidation of, potentially multiple, etiologies involved. We performed comparative analysis of viral metagenomes from four chicken flocks affected with RSS using next-generation sequencing. Primers for the detection of chicken enteric viruses were designed from the sequencing data obtained with metagenomics. Multiplex reverse transcription–polymerase chain reaction (PCR) and PCR were performed to detect a variety of etiological agents previously described in natural cases of RSS. The most abundant viral families identified in this study were Astroviridae, Picornaviridae, Parvoviridae, Caliciviridae, Reoviridae and Picobirnaviridae. Chicken astrovirus sequences were present in all four samples, suggesting an association between chicken astrovirus and RSS and chicken astrovirus as a candidate pathogen responsible for RSS. Picobirnavirus and the newly identified chapparvovirus were found in chickens in the Republic of Korea for the first time, and the genetic diversity of enteric viruses and viral communities was showed. Chicken astrovirus was consistently detected in broilers affected with RSS and the result of this study may contribute to knowledge of enteric diseases and viruses in chickens.


Introduction
Infectious intestinal diseases affecting young chickens and turkeys are characterized by mild to severe enteritis and result in considerable economic losses. Viral enteritis increases susceptibility to other diseases, decreases feed conversion efficiency, and prolongs the time to market [1,2]. Runting-stunting syndrome (RSS), also known as malabsorption syndrome, is diagnosed based on clinical signs (diarrhea, dehydration, growth depression, unevenness in size) and histopathological lesions (distension of the crypts of the small intestine, inflammatory cell infiltration in the lamina propria adjacent to affected crypts and necrotic cellular debris in crypts) [3,4].
Metagenomics provides the opportunity to simultaneously detect a large number of sequences from different microbes, including viruses [17]. Metagenomics has enabled a recent increase in understanding of viral diversity, and environmental and clinical metagenomics have contributed to the discovery of novel viruses [18]. In addition, studies are necessary to determine sequence differences by comparison with high-throughput data obtained from a variety of genomic regions, and samples through various protocols that differ in terms of sample preparation, cDNA synthesis, library preparation, sequencing and data analysis [19].
Here, Illumina sequencing was applied to explore viral communities in the small intestines and intestinal contents of four chicken flocks with RSS and one specific pathogen-free (SPF) chicken flock in the Republic of Korea.

Samples and purification
The small intestines and intestinal contents of broiler chickens from four flocks diagnosed with RSS (05D72, 07D11, 13D62 and 13Q45) were inspected in this study. The chickens were submitted to the Avian Disease Division (ADD) of the Animal and Plant Quarantine Agency (APQA) for disease diagnosis between 2005 and 2013 and diagnosed by an APQA diagnostic protocol on the basis of clinical manifestation and the presence of gross lesions. The small intestines (duodenum, jejunum and ileum) of broiler chickens in flocks 05D72 and 07D11 were collected after necropsy, processed promptly via blending into a 10% homogenate in sterile phosphatebuffered saline (PBS) containing 0.4 mg gentamicin per ml, and stored at − 80°C until analysis. The intestinal contents of broiler chickens in flocks 13D62 and 13Q45 and control (SPF) chickens were collected from the small intestine after necropsy, mixed with an equal volume of sterile PBS containing 0.4 mg gentamicin per ml, and stored at 2~5°C until analysis. RSS-positive and control samples were centrifuged at 3500 r.p.m. and 13, 000 r.p.m. for 10 min each. To remove large particles and bacteria, the supernatants of intestine homogenates were filtered with 0.8, 0.45, and 0.22 μm syringe filters, and feces filtrates were concentrated using an Amicon ultrafiltration apparatus (Amicon chamber 8400 with a 30 kDa MWCO UF membrane, Millipore, USA). Viral particles were pelleted by ultracentrifugation (30, 000 r.p.m., 5 h, 4°C), resuspended in 500 μL of 1 M Tris-Cl (pH 7.4) and treated with 2.5 units of DNase I (AMPD1, Sigma-Aldrich, USA) for 3 h at 37°C to eliminate free DNA. The samples were concentrated and washed twice using a Microcon 30 column (Millipore, USA). DNase activity was inhibited by adding 0.5 M EDTA to a concentration of 20 mM.

High-throughput sequencing and analysis
Total RNA was extracted from the purified samples using a Viral Gene-spin viral DNA/RNA extraction kit (iNtRON Biotechnology, Republic of Korea) according to the manufacturer's instructions. cDNA synthesis and PCR amplification of nucleic acid were carried out in a 50 μL mixture containing 5 μg of RNA, 0.5 μM random primer K (GAC CAT CTA GCG ACC TCC AC) and 0.5 μM primer KN (GAC CAT CTA GCG ACC TCC CAN NNN NNN N) as described previously [20] using the Access RT-PCR system (Promega, USA). The products were purified using an UltraClean PCR Clean-Up Kit (MO BIO, USA) and sequenced at Theragen Etex (Suwon, Republic of Korea). Sample libraries were prepared using an Illumina TruSeq DNA sample preparation kit (Illumina, USA), and DNA was fragmented using a Covaris adaptive focused acoustics device to generate double-stranded DNA fragments 300-400 bp in size. The ends were repaired, phosphorylated, and 3′end adenylated. Paired-end DNA adaptors (Illumina) were ligated, and the resulting construct fragments5 00 bp size were selected. Libraries were loaded onto a paired-end flow cell and sequenced as 101 bp pairedend, indexed reads on an Illumina HiSeq 2000 instrument. The raw read sequences were filtered using the following criteria: 1) the presence of ambiguous bases (letter N) in excess of 10%, 2) an average quality below 20, 3) more than 5% of nucleotides with quality inferior to 20, and 4) the presence of an adapter sequence (Supplementary data 1). Host genome (galgal4)-filtered reads were aligned with known viral, bacterial, and fungal genome database from NCBI using Burrows-Wheeler Aligner software. Data from each sample were assembled using MetaVelvet (k-mer size 51) and Bambus2. Homology-based (BLAST) classification based on the nucleotide sequence was performed using the 'nucleotide' database from NCBI to annotate the scaffolds.

Sequence analysis
Nucleotide and amino acid sequences were aligned using Vector NTI 10 software. The aligned fragments from Astroviridae, Picornaviridae, Parvoviridae, Caliciviridae and Picobirnaviridae were trimmed, based on lengths of 302, 1421, 3661, 7315, and 812 bp, respectively, using BioEdit software. Phylogenetic trees were generated by the neighbor-joining method using MEGA 4.0 software [21] with 1000 bootstrap replications. Assignment of genotypes to rotavirus A sequences was performed using the online tool RotaC [22].

Trial for whole-genome analysis of a parvovirus
The design of primers for bridging contigs identified by Illumina, and the resequencing of these regions, were carried out at Cosmo Genetech Co., Ltd. (Seoul, Republic of Korea) using an ABI 3730 DNA sequencer. Nested PCR and rapid amplification of cDNA ends (RACE) [23] were attempted to obtain sequences of the 5′ and 3′ ends of a parvo-like virus genome.

Detection of enteric viruses
Sequences revealed through high-throughput sequencing indicated a variety of etiologies related to RSS. Multiplex reverse transcription (RT)-polymerase chain reaction (PCR) and PCR were used to distinguish these agents from the original chicken intestine homogenate using primer sets. Most primers were designed using CLC Main Workbench 6 with the high-throughput sequences obtained in this study, and the primers for rotavirus D used were chosen according to a published report [24], as described in Table 1. DNA and RNA from individual enteric samples were extracted using a Viral Gene-spin Viral DNA/RNA Extraction kit (iNtRON Biotechnology, Republic of Korea) according to the manufacturer's instructions. Multiplex RT-PCR for the detection of picornavirus, astrovirus and calicivirus was carried out using the PrimeScript One Step RT-PCR kit ver.2 (TaKaRa, Japan) following the manufacturer's instructions, with each primer at 0.5 μM and 2 μL of RNA. Thermocycling conditions were as follows: 50°C for 30 min and 94°C for 2 min, followed by 40 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, and a final step at 72°C for 5 min. Multiplex RT-PCR for the detection of rotaviruses A, D and F was carried out using the PrimeScript One Step RT-PCR kit ver.2 (TaKaRa), with each primer at 0.5 μM and 2 μL of RNA. Thermocycling conditions were as follows: 50°C for 30 min and 94°C for 2 min, followed by 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min and a final step at 72°C for 5 min. To detect the parvovirus, PCR was performed using AccuPower PCR Premix (Bioneer, Republic of Korea) with the following conditions: 95°C for 5 min; 30 cycles of 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min; and final incubation at 72°C for 5 min. The sensitivity and specificity of the primer pairs for each agent were tested by uniplex PCR. All PCR products were purified from agarose gels using a QIAquick gel extraction kit (Qiagen, USA) and sequenced directly using an ABI 3730 DNA sequencer at Cosmo Genetech Co., Ltd. using the respective PCR primers.
These detection methods were used to identify the distribution of these enteric viruses in non-RSS-infected chickens. Intestinal samples were randomly collected from 86 non-RSS-infected broiler chickens less than 6  weeks old submitted to the ADD of the APQA for diagnosis. These specimens were diagnosed with a variety of diseases, viral diseases (infectious bronchitis, inclusion body hepatitis, infectious bursal disease, etc.), bacterial diseases (necrotic enteritis, bacterial arthritis, colibacillosis, etc.), or complicated disease. Multiplex RT-PCR and PCR were carried out as described above.

Diagnosis of RSS
The four flocks tested comprised 2-week-, 3-week-, 4week-, and 6-week-old broiler chickens with poor growth, retarded feathering, diarrhea and various clinical signs. Thin-welled intestines filled with undigested feed at postmortem examination and characteristic microscopic lesions (distension of the crypts in the duodenum and jejunum lined with flattened epithelium, exfoliated cells in the crypts and inflammatory cell infiltration in the adjacent lamina propria) were observed, but villous atrophy was not observed (Fig. 1a). One flock (07D11) was diagnosed with only RSS, whereas the other three flocks (05D72, 13D62 and 13Q45) had been infected with additional diseases, such as infectious bronchitis (IB), inclusion body hepatitis (IBH) and coccidiosis ( Table 2). IB was identified by virus isolation and RT-PCR, IBH was confirmed by PCR and histopathology (observation of intranuclear inclusions in the liver), and coccidiosis was confirmed by the presence of oocysts in the cecum using microscopic examination. Electron microscopy identified many small, round and nonenveloped viral particles of various sizes in all four samples (Fig. 1b).     Picobirnaviridae (11.1%) sequences, but no sequences were assigned to Reoviridae. Sequences from the control sample differed from those of all other samples and contained no viral reads assigned to known avian viruses and only bacteriophage sequences (Table 2 Supplementary data 2).

Enteric virus genome sequence analysis
Sixty-six contigs from six viral families 251 to 7315 nucleotides (nts) in length were obtained from the four RSS-positive chicken samples. These genome sequences were deposited into GenBank under accession numbers KM254161-KM254224.

Astroviridae
Four contigs with similarity to genomes of the Avastrovirus genus were identified; specifically, chicken astrovirus sequences were found in all four samples. The contigs identified in samples 05D72, 07D11 and 13D62 were partial non-structural (NS) polyprotein 302, 305, and 2266 nt in length, respectively. The 13Q45 contig consisted of full NS and partial capsid protein sequences 5084 nt in length. The four NS sequences from the chicken astrovirus genome detected showed 84.2~86.7% nt homology to the Chinese strain (GenBank No. HM029238) [25] and were clustered differently from other Avastrovirus genus (avastrovirus 1, 2, and 3) in the phylogenetic tree (Fig. 2a).

Parvoviridae
Two nearly complete Aveparvovirus genome sequences 4782 and 5034 nt in length were identified from samples 07D11 and 13D62, respectively. Both contigs showed 92.7 and 97.1% nt identity with the ABU-P1 strain (Gen-Bank No. GU214704) and contained three ORFs that encode nonstructural (NS1, NP1) and capsid (VP1, VP2) proteins. A parvo-like virus (3661 nt sequence length) was obtained from sample 13Q45 and found to contain the complete NS1 CDS (Fig. 2c). Sequence comparison with the ABU-P1 strain revealed a high level of divergence with 46.6% nt identify and 13.6% aa identify. The DNA coding sequences was confirmed by Sanger sequencing of amplicons obtained by primer walking. Nested PCR and RACE were conducted to obtain the sequences of the 5′ and 3′ ends of a parvo-like virus genome in sample 13Q45 but yielded no products. In the Parvoviridae phylogenetic tree, this identified virus was clustered with the recently classified genus Chapparvovirus [26].

Reoviridae
A total of 47 viral contigs belonging to three rotavirus species (rotaviruses A, D, and F) were detected in samples 07D11 and 13D62. All eleven genome segments of rotaviruses A, D and F were compared with those of the German strains 02V0002G3, 05 V0049 and 03 V0568. The sequences of contigs from rotavirus A obtained from sample 13D62 were similar to sequences of strain 02V0002G3 (GenBank No. FJ169853-169863), and each viral segment showed 91.2~96.3% nt identity with the corresponding segment in strain 02V0002G3, but the VP4 gene showed a low degree of identity (74.9%) and a different genotype (P31). The percent identities of rotavirus D segment-associated contigs with strain 05 V0049 (GenBank No.GU733443-733453) varied (79.5-95.5%). Rotavirus F segments from sample 13D62 showed between 72.6 and 91.7% nt identity to strain 03 V0568 (GenBank No. FJ169853-169863). The sequences of contigs from rotavirus species in sample 07D11 were similar to those of contigs in sample 13D62 (Table 3).

Caliciviridae
Two nearly complete chicken calicivirus genome sequences with lengths of 7865 and 7315 nt were found in samples 13D62 and 13Q45. Both contigs contained two coding open reading frames (polyprotein and the VP2 protein) and were closely related in a cluster of the genus Nacovirus (Fig. 2d).

Picobirnaviridae
Five picobirnavirus contigs were identified from samples 13D62 and 13Q45. Two or three distinct partial RNAdependent RNA polymerase gene sequences were found in each sample. Three sequences were clustered with genogroup I, and the other sequence clustered with genogroup II (Fig. 2e).

Detection of enteric viruses in the non-RSS-affected flock using multiplex RT-PCR and PCR
Intestinal samples from 86 non-RSS-affected broiler flocks were examined to detect enteric viruses using the developed multiplex RT-PCR method and PCR. Parvoviruses and chicken astroviruses were detected with very high positive detection rates of 75.6 and 62.8%, respectively (Table 4). Picornaviruses, caliciviruses and rotavirus species were also found to have low to moderate positive detection rates (7.0-39.5%).

Discussion
High-throughput sequencing data generated by Illumina sequencing indicated the presence of several different viruses in four chicken flocks affected by RSS. Chicken astrovirus, parvovirus, calicivirus and rotavirus have been identified as causes of gastrointestinal tract infections in poultry, such as RSS, which is also called malabsorption syndrome [27]. A few studies using viral metagenomics could not specify any particular pathogen from the viral community in the chicken flocks with RSS because the distributions of enteric viruses in diseased and healthy flocks were not significantly different [17,28,29]. Chicken astrovirus has been distributed worldwide for decades and is nearly ubiquitous constituent of the chicken gut [30]. We also found a high positive detection rate (62.8%) for this virus in the non-RSS-affected chickens. On the other hand, metagenomics analysis in this study showed that only chicken astroviruses were common to chickens that had been diagnosed with RSS via pathological lesions in the crypts of the small intestine. In addition, chicken astrovirus was not identified in the fecal virome of healthy chickens in Brazil [31]. Chicken astrovirus has been suggested as an etiological agent for RSS, but it could be detected in chickens without microscopic lesions. Recently, the pathogenesis of chicken astrovirus in broilers was revealed. Serial passages of the virus from chicken to chicken induced the increased virulence, as shown by decreased weight gain and the presence of histopathological lesions [32]. Kang et al. provided strong evidence of chicken astrovirus as an etiological agent of RSS, although the mechanism for its reproduction via bird-to-bird passage is not known. Our metagenomics results support the notion of chicken astrovirus as a major etiology of RSS. Chicken megriviruses of the Picornaviridae family detected in two samples tested in this study were shown to be similar to viruses identified from chickens with transmissible viral proventriculitis [33]. These cases were thought to be cases of RSS associated with proventriculitis [34], but the two cases did not exhibit histopathological lesions in the proventriculus. Viral sequences closely related to members of Gallivirus were identified, suggesting the diversity of chicken picornaviruses, but the role of these viruses in chicken disease is still unknown [35].
Parvovirus, calicivirus and rotavirus have been suggested as etiological agents of RSS in chickens [6,7,36] and were shown to be widely distributed in the non-RSS-affected chicken flocks in this study. As of yet, these viruses have not specified their pathogenesis and reproduced of the pathogenicity; therefore, further research is needed. Independently, we also identified the full NS gene sequence from chapparvovirus in a single chicken intestine sample. Chapparvovirus, which was recently classified, has been found in various vertebrate animals, such as birds, chickens, pigs, bats and dogs, throughout the world, and many studies have been performed to reveal the properties of this virus [37]. Although trials to identify the whole genome of this virus failed, we have  the opportunity for novel viral discovery through environmental metagenomics. In this study, chicken picobirnaviruses from the Republic of Korea were firstly identified using metagenomics, and viral sequences were grouped into genogroups I and II. Avian picobirnaviruses in genogroup I have primarily been noted to date, and those in genogroup II are rarely detected in chickens [17,29,38]. We could not design a primer set to detect picobirnaviruses due to the high genetic diversity between chicken picobirnaviruses. The high level of picobirnavirus sequence diversity in various hosts and environmental samples suggests the evolution of heterologous strains [39]. Coronavirus was found in sample 05D72, which was taken from chickens diagnosed with IB and RSS, and adenovirus was also found in sample 13D62, which was taken from chickens diagnosed with inclusion body hepatitis and RSS. Although the viral reads were not high in number, these findings indicate that viral metagenomics can help to determine the clinical etiological agents of chicken diseases.

Conclusions
Chickens are a major protein source for human consumption, and their diseases are closely connected with public health concerns and economic losses. RSS in chickens is disease that impairs productivity in the Republic of Korea. In the present study, we used an unbiased metagenomic approach for viral pathogen discovery, the detection of novel viruses and the development of a molecular diagnostic tool to detect pathogens from the obtained sequences. We suggest that RSS in chickens, which is also called malabsorption syndrome, is caused by a chicken astrovirus. In addition, the multifactorial etiologies as a cause of RSS, as well as astrovirus enteritis could be ruled out through further studies of other enteric viruses, such as parvovirus, rotavirus and calicivirus.
Additional file 1. Base quality and filter result of high-throughput data obtained from samples in this study.
Additional file 2. Taxonomy annotation of viral reads obtained from samples in this study.