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

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 phosphate-buffered 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.

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 fragments ~ 500 bp size were selected. Libraries were loaded onto a paired-end flow cell and sequenced as 101 bp paired-end, 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.

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].

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.

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.

The four flocks tested comprised 2-week-, 3-week-, 4-week-, 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).

Five samples (05D72, 07D11, 13D62, 13Q45 and control) were sequenced, producing a total of 14,893,900–19,711,846 reads with an average read length of 162–193 bp, representing a total of 18,777–60,556 sequences. Homology-based (BLAST) classification showed a total of 48,979-1,766,642 viral sequence reads and identified ten different viruses with sequence identity to avian viruses and bacteriophages. Bacteriophage sequences comprised 3.3 to 100% of the viral sequences and were assigned to known families, including Leviviridae, Siphoviridae, Myoviridae and Podoviridae. Most of the samples had abundant Leviviridae sequences, but 13D62 and 13Q45 showed plentiful Siphoviridae sequences. All samples excluding 05D72 contained sequences from unassigned phages. The identified viral sequences were assigned to known viral families, including Picornaviridae, Astroviridae, Parvoviridae, Reoviridae, Caliciviridae, Picobirnaviridae, Adenoviridae, Coronaviridae and Anelloviridae. In all four samples from chickens diagnosed with RSS, Astroviridae and Picornaviridae were detected. Parvoviridae sequences were detected in three (07D11, 13D62, 13Q45) of the four samples, Reoviridae sequences were identified in two samples (07D11, 13D62), and Caliciviridae and Picobirnaviridae sequences were detected in two samples (13D62, 13Q45). Coronaviridae and Anelloviridae sequences were identified in sample 05D72, and Adenoviridae sequences were identified in sample 13D62. Sample 05D72 from chickens diagnosed with infectious bronchitis as well as RSS contained Coronaviridae (0.2%) with Picornaviridae (94.6%), Astroviridae (0.1%) and Anelloviridae (1.9%). Reoviridae (66.1%) and Picornaviridae (24.3%) families were abundant in sample 07D11, which also contained Parvoviridae (5.9%) and Astroviridae (2.1%) sequences. Sample 13D62, which was taken from chickens with a final diagnosis of inclusion body hepatitis and RSS, contained Astroviridae (75.2%) and Picornaviridae (11.8%) sequences, followed by sequences from Reoviridae (3.8%), Caliciviridae (1.1%), Picobirnaviridae (0.9%), Parvoviridae (0.8%) and Adenoviridae (0.2%) families at equal abundance. Sample 13Q45 showed a viral profile similar to that of sample 07D11 containing Picornaviridae (37.9%), Astroviridae (8.6%), Parvoviridae (5.3%), Caliciviridae (16.0%) and 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).

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.

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%).