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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 (link)], 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.
Table 1

Primers and PCR instrumental conditions for detecting enteric viruses

Virus	Target gene	Primer sequences	PCR condition	Product size (bp)
parvovirus	NS	PV-CLC-F1 5′-ACGAAGGTAAGAATGAGG-3′ PV-CLC-R1 5′-GGAGATTGATTGGGGATG-3’	PCR 95 °C, 5 min - 30 cycles(94 °C, 30s, 55 °C, 1 min, 72 °C, 1 min) - 72 °C, 5 min	364
picornavirus	NS	Picorna-F3 5’-TACCCGAGAAAACGACCC-3′ Picorna-R3 5′-ACACCTCAGCTACAAGAA-3’	Multiplex RT-PCR 50 °C, 30 min - 94 °C, 2 min - 40 cycles(94 °C, 30s, 50 °C, 30s, 72 °C, 1 min) - 72 °C, 5 min	504
astrovirus	ORF1a	Astro-F1 5’-ATTCCAGTTCAGCTCTTC-3′ Astro-R1 5′-TCCTGTTGGTAAGAGAGT-3’		138
calicivirus	VP1	Calici-CLC-F3 5’-AGAAGCGTGACTACATGGA-3′ Calici-CLC-R1 5′-TTGTGTTGTTTGTGGGGT-3’		1247
rotavirus A	VP6	RotaA-CLC-F2 5’-CTCCTCAATCTAATGCACT-3′ RotaA-CLC-R2 5′-CCGAACCATTATTTAGCCA-3’	Multiplex RT-PCR 50 °C, 30 min - 94 °C, 2 min - 40 cycles(94 °C, 30s, 60 °C, 30s, 72 °C, 1 min) - 72 °C, 5 min	230
rotavirus Da	VP6	RotaD-F 5’-GGAGGCGCTGTCTTCAATTGCG-3′ RotaD-R 5′-TGGCCAATAGTGTGTGGCAGCT-3’		742
rotavirus F	VP6	RotaF-CLC-F2 5’-GTGGAGTCGGAAATATGG-3′ RotaF-CLC-R2 5′-CAGGTACAACATAAGGATTC-3’		330

^aprimers for detecting rotavirus D were used according to the reference [14 (link)]

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.

For conventional RT-PCR, total RNA was extracted from cells using the RNeasy Mini Kit (QIAGEN, Tokyo, Japan), followed by RT-PCR using PrimeScript One Step RT-PCR Kit Ver.2 (TaKaRa) with primer nos. 7 and 8 (for detecting full-length AS1-S) or 9 and 10 (for GAPDH). The PCR conditions were as follows: 50°C for 30 min, 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s.
RNAs encoding U6 and TYR were used as controls for nuclear and cytoplasmic RNA, respectively, and were quantified by real-time RT-PCR using the One Step TB Green PrimeScript RT-PCR Kit II (Perfect Real Time) (TaKaRa) and QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) with primers no. 11 and 12 or 13 and 14. AS1-S RNA in the established MDBK CAG AS1-S and MDBK 3´LTR AS1-S cells were quantified in the same manner using primers no. 15 and 16. For quantification of AS1-S RNA in BL3.1 cells, strand-specific real-time RT-PCR was performed as previously reported (47 (link)) to determine the sense and anti-sense transcript levels. Briefly, RNA samples were reverse-transcribed using the Prime Script RT Reagent Kit (TaKaRa) in combination with an AS1-specific tagged primer (no. 17). The resultant reaction mixture was then 10fold diluted and subjected to quantitative PCR using the TB Green Premix Ex Taq II Kit (TaKaRa) in combination with a tag primer (no. 18) and an AS1-specific reverse primer (no. 19). All PCR conditions were in accordance with the manufacturers’ instructions.
For validation of the high-throughput sequencing analyses, such as the transcriptome and RIP-seq analyses, target genes were quantified by real-time RT-PCR using the One Step TB Green PrimeScript RT-PCR Kit II (Perfect Real Time) (TaKaRa) and QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) with primers no. 20–41. All PCR conditions were in accordance with the manufacturers’ instructions.
The BLV proviral load was measured as previously described (45 (link)). Briefly, a commercial real-time PCR kit for BLV detection (RC202A; TaKaRa) was used to determine the copy numbers of the BLV-pol gene and the bovine RPPH1 gene in DNA samples by multiplex real-time PCR according to the manufacturer’s instructions. The PCR conditions were as follows: 25°C for 10 min and 95°C for 30 s, followed by a two-step procedure for 45 cycles at 95°C for 5 s and 60°C for 30 s. The proviral load data were normalized to the number of BLV-pol gene copies per 100 cells, which was calculated based on the copy number of the bovine RPPH1 gene (two copies per cell).

RNA transcripts of RSV-A, RSV-B, and human ACTB were used as standards to determine the assay sensitivity. Primers for RSV-A, RSV-B, and ACTB containing the T7 promoter sequence were used to amplify the target DNAs with the PrimeScript One Step RT–PCR Kit ver. 2 (TaKaRa, Dalian, China): RSV-A forward primer: 5′-TAATACGACTCACTATAACTGCAATCAYACAAGATGCAACRA-3′; RSV-A reverse primer: 5′-CAGATTGRAGAAGCTGATTCCA-3′; RSV-B forward primer: 5′-TAATACGACTCACTATAACTTACCTTACTCAAGTCTCACCAGAAA-3′; RSV-B reverse primer: 5′-TTGTRGCTGARTTTGTGTGGAT-3′; human ACTB forward primer: 5′-TAATACGACTCACTATAGGCATCCACGAAACTACCTT-3′; and human ACTB reverse primer: 5′-GCCGGACTCGTCATACTCCT-3′. The target DNAs were purified with a TIANprep DNA gel extraction kit (Tiangen, Beijing, China), and the RNAs were transcribed in vitro with a TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific, Lithuania). The purity and concentrations of the RNA transcripts were established with a NanoDrop spectrophotometer (Thermo Scientific, NanoDrop Technologies, USA). The sensitivity and standard curves for the multiplex real-time PCR were established for RSV-A, RSV-B, and human ACTB by testing the transcripts of known concentrations serially diluted in diethyl-pyrocarbonate-treated water.

First, we evaluated the sensitivity and specificity of conventional RT-PCR using an LN34 primer set for viral RNA detection with the DFAT as the reference test. The LN34 assay was used for the amplification of short-fragment nucleoprotein (N) gene products (165 bp) (21 (link)). We used the PrimeScript one-step RT-PCR kit version 2 with a modified protocol (TaKaRa Bio, Inc., Shiga, Japan). The reaction mixture (22.5 μL) contained 7 μL of double-distilled water (ddH₂O), 12.5 μL of one-step buffer, 1.25 μL of LN34 primers, 0.5 μL of PrimeScript one-step enzyme mix, and 2.5 μL of the RNA template. Reactions were performed using the Veriti thermal cycler (Applied Biosystems, USA) under the following conditions: 50°C for 30 min; 94°C for 2 min; 40 cycles of 94°C for 15 s, 50°C for 30 s, and 68°C for 2 min; and a final extension step at 68°C for 5 min. PCR products were visualized on a 1.5% agarose gel using Biotium gel green nucleic acid stain. A positive PCR result was expected to be observed as an amplicon of 165 bp (see Table S1 in the supplemental material).

Pooled mosquitoes were homogenized in Minimum Essential Medium containing 2% fetal bovine serum using the BioMasher (Nippi, Japan). RNA was extracted from 100 µL of supernatants of mosquito homogenates using the Direct-Zol kit (Zymo research, USA) according to the manufacturer’s instructions. The remaining mosquito homogenates were filtered and inoculated onto cells for virus isolation. To detect multiple phleboviruses, we designed degenerate primer sets targeting the conserved RdRP gene region; L-2779F 5'-CARCATGGWGGTYTDAGRGARATCTA-3' and L-3287R 5'-TGCARKATKCCYTGCATCATHCCWG-3′^{9 (link)}. RNA samples were amplified using a PrimeScript One-step RT-PCR kit Ver.2 (Takara, Japan) and 1 µM of primer sets. The cycling protocol was comprised of 30 min of incubation at 50 °C for cDNA synthesis, followed by 2 min of incubation at 94 °C, 43 cycles each of 94 °C for 30 s, 52 °C for 30 s and 72 °C for 30 s, and 72 °C for 5 min. The PCR products were sequenced using a BigDye Terminator v3.0 Cycle Sequencing kit on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, USA).

Second, we used the P1-304 primer set to amplify the long fragment (1,506 bp) of the entire N gene (22 (link), 23 (link)). We used the TaKaRa PrimeScript one-step RT-PCR kit, and the volume for the master mix and the RNA template was the same as that used in the above-described procedures for the LN34 RT-PCR assay; a 1.5% agarose gel was used to visualize the positive result with an expected size of 1,506 bp. Before subjecting the samples to sequencing, specific bands were extracted and purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). All RT-PCR products with successful amplification were sent to Macrogen, Inc., Philippines, for Sanger sequencing in both the forward and reverse directions. In addition to the above-mentioned primer sets, a cocktail of JW6 DPL (Duvenhage virus, rabies virus strain Pasteur and Lagos bat virus), JW6 M (Mokola virus), and JW6 E (European bat lyssavirus-1 and -2) primers was used for sequencing (see Table S1 in the supplemental material) (24 (link)).

The modified primers were validated using serially diluted fetal bovine serum (FBS) spiked with viral particles of DENV1, DENV2, and YFV17D to a final concentration ranging from 10 to 10⁵ PFU/mL. Viral RNA was extracted from samples using QIAamp viral RNA mini kit (QIAGEN), according to the manufacturer’s instruction. One-step RT-PCR was carried out using PrimeScript One Step RT-PCR Kit Ver.2 (Takara) according to the manufacturer’s instruction, in a total reaction of 15 µL containing 2 µL template RNA, 250 nM of each sense primers, and 500 nM anti-sense primer. Temperature conditions for the RT-PCR was as follows: 50 °C for 30 min (cDNA synthesis), 94 °C for 30 s, followed by 43 cycles of 94 °C, 53 °C, 72 °C, 30 s each, and lastly 72 °C for 5 min. The amplicons (1 µL) were then visualized on 1.5% agarose gels alongside Gene Ladder100, a 100 bp DNA marker (Nippongene).

To determine the transmission status of medically important mosquito-transmitted arboviruses in Ghana, Aedes and Culex mosquito pools were screened for DENV, CHIKV, or WNV by RT-PCR. Screening was carried out using the PrimeScript One Step RT-PCR Kit ver. 2 (Takara Bio) and the species-specific primers DENV (D1: 5’-TCAATATGCTGAAACGCGCGAGAAACCG-3’ and D2: 5’-TTGCACCAACAGTCAATGTCTTCAGGTTC-3’); CHIKV (Chik10294s, 5’-ACG CAA TTG AGC GAA GCA CAT-3’ and Chik10573c, 5’-AAA TTG TCC TGG TCT TCC TG-3’ for CHIKV); and WNV (WNNY514, 5’-CGG CGC CTT CAT ACA CW-3’ and WNNY904, 5’-GCC TTT GAA CAG ACG CCA TA-3’ for WNV) [22 (link),23 (link),24 (link)]. The cycle conditions used were 50 °C for 30 min, 94 °C for 2 min, and 35 cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 30 s. The resulting products were analyzed by agarose gel electrophoresis.

Viral RNA was extracted using a QIAamp viral RNA Mini Kit (Qiagen, Valencia, CA, the USA), following the protocol provided by the manufacturer. Then, we amplified the whole E25 genome by reverse transcription-polymerase chain reaction (RT-PCR) using a PrimeScript One-Step RT-PCR Kit Ver.2 (TaKaRa, Shiga, China) and 18 E25 strains using primers of our design (Supplementary Table S2). PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and then amplicons were sequenced in both directions using the ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, the USA). Primers design for amplifying the full length of E25 were designed using NCBI, and primers for 5' UTR and 3' UTR amplification were obtained from other sources^{22 (link)}. Fragment sequences were edited and spliced using Sequence 5.4.5 software (Gen Codes, Ann Arbor, MI, the USA). Sequences are stored in the China National Microbiology Data Center (NMDC) with accession numbers NMDCN0001DRD–NMDCN0001DRU (https://nmdc.cn/resource/genomics/sequence).

For molecular serotyping, viral RNA was extracted from the viral isolate by using a QIAamp Viral RNA Mini Kit (Qiagen. Germany) and was stored at −80 °C until use. Reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the VP1 coding region using PrimeScript One Step RT-PCR Kit Ver.2 (TaKaRa, Dalian, China) with primer pairs E012 as forward and E011 as reverse36 (link). The PCR products were purified using the QIAquick PCR purification kit (Qiagen, Germany) and subjected to nucleotide sequencing. Sequencing was performed in both directions using an ABI 3130 Genetic Analyzer (Applied Biosystems, USA), and every nucleotide position was sequenced at least once from each strand. The sequences were analysed with the Basic Local Alignment Search Tool server at the National Center for Biotechnology Information and EV serotype was determined according to a previously described molecular serotyping method20 (link).

Viral RNA was extracted from samples using TRIzol Reagent (Invitrogen, CA, USA), resuspended in nuclease-free water, and kept at −70 °C until further use. Reverse transcription was performed at 50 °C for 30 min in a reaction mixture consisting of 2.5 μl RNA (0.2 μg), 1 μl primer (10 pmol), 1 μl Prime Script One Step Enzyme mix, 8 μl RNase-free H₂O, and 12.5 μl 2 × One Step Buffer. The cycling conditions for the PCR were 94 °C for 2 min, followed by 32 cycles of denaturation (94 °C for 10 s), annealing (58 °C for 30 s), and extension (72 °C for 1 min), followed by a final extension at 72 °C for 7 min. Both the reverse transcription and the polymerase chain reaction were conducted using a PrimeScript One Step RT-PCR Kit (TaKaRa, Japan).

All collected ticks were thoroughly surface-sterilized with 70% ethanol, followed by distilled sterile water, and then each individual tick and the aliquot of each organ of wild small mammals were homogenized by using small steel balls as an abrasive. Total viral nucleic acids were extracted by using All Prep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. A one-step reverse transcription-polymerase chain reaction (RT-PCR) system based on two primer sets (HRTV and TBPV) that were universal for the detection of TBPV was applied to test the extracted RNAs according to a previous report (Table 1; Matsuno et al., 2015 (link)). PCR amplification was performed using the PrimeScript one-step RT-PCR kit version 2 (TaKaRa) following the manufacturer’s instructions, under the following program: 50°C for 30 min; 94°C for 2 min; 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and 72°C for 5 min. The amplified products were detected by agarose gel electrophoresis to confirm their sizes and then subjected to Sanger sequencing.
Primers (5′-CCATCAATCTGTACACCAGG-3′ and 5′- ACACAAAGTCCGCCCATTAC-3′) targeting the 895 bp fragment of the large (L) gene were used to confirm the positive results (Table 2). PCR amplification was performed using the PrimeScript one-step RT-PCR kit version 2 (TaKaRa) following the manufacturer’s instructions, under the following program: initial denaturation: 50°C for 30 min; 94°C for 2 min; 13 cycles, decreasing the annealing temperature 0.5°C each cycle: 94°C, 30 s; 57°C, 30 s (0.5°C/cycle); 72°C, 1 min; 37 cycles: 94°C for 30 s, 51°C for 30 s, and 72°C for 1 min; and 72°C for 5 min. The amplified products were detected by agarose gel electrophoresis and then subjected to Sanger sequencing. All positive and negative results were reconfirmed by real-time RT-PCR with the MKWV-specific primers (L-6314-6443F, L-6314-6443R) (Table 1). All PCR tests were conducted in parallel with positive control (RNA from positive sample) and negative control (RNase-free water).

We amplified 3120-bp HCV sequences between the NS3 and NS5A genes by using the PrimeScript One Step RT-PCR kit (Takara Bio, Shiga, Japan) and PrimeSTAR HS kit (Takara) according to the manufacturer’s protocol (Fig. 1, Supplementary Materials). Also, we prepared two samples of HCV-containing plasmids for control experiments and amplified the NS3-to-NS5A region with the above method. The primers for RT-PCR are shown in Supplementary Table S8. The PacBio DNA library was constructed from purified DNA product (5 µg) using a DNA Template Prep Kit 3.0 (Pacific Biosciences, Menlo Park, CA, USA) according to the PacBio standard template prep protocol (Pacific Biosciences)²⁹ . The DNA library was sequenced using PacBio RS II following the protocol from Pacific Biosciences. We used P6C4 polymerase for the sequencing reaction and 6-h movie windows for signal detection.

RT-PCR was performed employing a PrimeScript One Step RT-PCR Kit (TaKaRa, Japan). Primers S-F (5′-GCTTTTTGAGTCCTCAGCGTG-3′) and S-R (5′-GATGAATAGGCGAGTCCCGC-3′) were used to amplify the corresponding S3 segment encoding sigma B gene. For RT-PCR, 2.5 µL of extracted viral RNA was mixed with a reaction mixture containing 1 µL primer (10 pmol), 1 µL Prime Script One Step Enzyme mix, 8 µL RNase-free H2O and 12.5 µL 2 × One Step Buffer. Reverse transcription was performed at 50 °C for 30 min. The cycling conditions for the PCR were 94 °C for 2 min, followed by 32 cycles of denaturation (94 °C for 10 s), annealing (58 °C for 30 s) and extension (72 °C for 1 min), followed by a final extension at 72 °C for 7 min.

We utilized a previously reported one-step RT-PCR system that detects a wide-range of TBPVs from RNA samples from collected ticks (14 (link)). Briefly, one-step RT-PCR was performed with the PrimeScript One Step RT-PCR kit, version 2 (Dye Plus) (TaKaRa), with 1 µl of total tick RNA and 4 pmol of primers, HRT-GOUL2759F (5′-CAGCATGGIGGIYTIAGRGAAATYTATGT-3′) and HRT-GOUL3276R (5′-GAWGTRWARTGCAGGATICCYTGCATCAT-3′) in 10 µl of reaction solution with the following incubation program: 50°C for 30 min; 94°C for 2 min; 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and 72°C for 5 min. Amplified RT-PCR products were purified and sequenced with a 3130 Genetic Analyzer (ABI, Thermo Fisher Scientific).

Viral RNA was directly extracted from the clinical samples using a commercial nucleic acid extraction kit according to the manufacturer’s instructions (Tianlong Biotechnology Co., Ltd., China, CAT: ZTLJB-Y64). The coding sequence of the HN gene (1,716 nucleotides [nt]) from the HPIV2-positive samples was amplified by reverse transcription PCR (RT-PCR) using two in-house paired primers (Table S1) with the PrimeScript One-Step RT-PCR kit (TaKaRa, Dalian, China). The PCR products were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany) and sequenced using an ABI 3130 genetic analyzer (Applied Biosystems, Foster City, CA, USA). The sequencing results were assembled to obtain complete HN sequences using Sequencher v5.4.5 software (GeneCode, Ann Arbor, MI, USA).

Viral RNAs were extracted using the high pure viral RNA kit (Roche Diagnostics) from SIVs pre-ART and post-ART, the culture supernatants of CD8^- cells derived from PBMCs at weeks 10 and 34 post-infection in individual animals. Viral RNAs were also extracted from plasma samples at weeks 12 and 34. Viral gag and vif cDNAs were amplified from these RNAs by reverse transcription and nested PCR using the PrimeScript one-step RT-PCR kit version 2 (Takara) and KOD-Plus version 2 (Toyobo), and subjected to direct sequencing by using dye terminator chemistry and an automated DNA sequencer (Applied Biosystems) as described before^{37 (link)}.

Viral RNA was extracted directly from AIV-positive allantoic fluid with MagaBio plus Virus RNA Purification Kit (BIOER, China). The whole-genome of AIV isolates were sequenced using Next generation sequencing (NGS)^{13 (link)}. Briefly, RT-PCR and DNA synthesis were performed using the PrimeScript One Step RT-PCR kit (Takara). Next, the sequencing libraries were prepared. The libraries were sequenced on the BGI500 and Illumina HiSeq 4000. Sequencers by 200 bp or 250 bp paired-end sequencing, and sequencing depth for AIV isolates was about 0.2G per sample. The accuracy of the NGS method was confirmed by the published qRT-PCR method^{60 (link)} and qRT-PCR kits (Mabsky Biotech Co., Ltd.) with reference samples.

The assembled library was used to identify husaviruses by real-time (RT)-PCR assays using previously described husavirus-specific probes and primers [11 (link)]. After confirming husavirus in a sample, RT-PCR was performed to amplify the partial coding region using the PrimeScript One Step RT-PCR Kit Ver.2 (TaKaRa, Dalian, China) with specific primers (Table S1, Supplementary Materials). The PCR products were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany). The ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) was used for sequencing in both directions. The acquired partial genomic sequences were analyzed using BLAST against the GenBank database. A total of nine husavirus strains were confirmed based on their sequence information.

To determine the full genome of AToV, a set of specific primers were designed (Supplementary Table S1) based on contigs obtained by RNA sequencing. The total RNA of the AToV-positive fecal sample was used as the template. All RT-PCR amplifications were performed using PrimeScript One Step RT-PCR kit and ExTaq DNA polymerase (TaKaRa, Japan). The terminal ends of the genome were confirmed using a 5'/3'RACE Kit (Roche, Switzerland). The amplicons were sequenced after ligation into the pGEM-T vector (Promega, United States). Sequences were assembled with SeqMan (version 7.1.0) to produce the full-length virus genome sequences.