Amaryllis (Hippeastrum hybridum Hort.) is a perennial bulbous plant of Amaryllidaceae and has attracted worldwide attention due to its charming and colorful flowers. The Netherlands, South Africa, Japan, Brazil and the United States are the world’s major exporters . Amaryllis is a very popular garden flower in Taiwan. Virus-like symptoms can often be observed on amaryllis plants. The orthotospovirus capsicum chlorosis virus, the cucumovirus cucumber mosaic virus, the potyvirus hippeastrum mosaic virus (HiMV), and the carlavirus nerine latent virus (NeLV) infecting amaryllis have been reported in Taiwan, and the mixed infection of HiMV and NeLV is common in the field [2–4].
In April 2011, amaryllis plants showing chlorotic ringspots were found at a private garden in Changhua County, Taiwan (Fig. 1a). A virus isolate, denoted AV-N1, was isolated from a diseased amaryllis plant by three successive single lesion transfers on Chenopodium quinoa leaves. AV-N1 induced local chlorotic spots on C. quinoa leaves 7 days post-inoculation (dpi) (Fig. 1b) and caused systemic mosaic and curling symptoms on Nicotiana benthamiana plants 10 dpi (Fig. 1c). The virus was maintained in C. quinoa as a local-lesion host and N. benthamiana as a systemic host by mechanical transmission under temperature-controlled conditions (25-28°C) in the greenhouse for study.
To determine the genomic sequence of AV-N1, 100 mg of AV-N1-infected C. quinoa leaf tissue 7 dpi was used to purify total RNA using the Plant Total RNA Miniprep Purification kit (GMbiolab, Taichung, Taiwan) following the manufacturer’s instructions. The total RNA was used as a template to prepare the random primer-primed cDNA library, following the Illumina TruSeq® RNA Sample Preparation v2 Guide as previously described . Sequencing was conducted at Genetech Biotech Co., Ltd. (Taipei, Taiwan) using an Illumina MiSeq sequencing platform to synthesize paired-end reads. After the trimming of adaptors and low-quality sequences, the raw reads were randomly clipped into 23-mers using CLC Genomics Workbench 6.0.2 (CLC bio, Aarhus, Denmark) to facilitate de novo sequence assembly. The contigs were then subjected to the Basic Local Alignment Search Tool (BLAST) searches against the non-redundant protein database of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov). A contig with 9377 nt, containing a putative open reading frame (ORF) of the potyviral polyprotein, was annotated as Amazon lily mosaic virus (ALiMV), a Potyvirus species, by sharing 95.5% nt identity with a partial genomic sequence of ALiMV (acc. no. AB158523) available in the GenBank of NCBI. Based on sequence homology, AV-N1 was identified as an amaryllis isolate of ALiMV and renamed ALiMV-TW.
Furthermore, both 5´ and 3´ rapid amplification of cDNA ends (RACEs) were performed as the previously described methods  to exlucidate the complete genome of ALiMV-TW. Specific primers designed from the nt sequence of the ALiMV contig were used in RACEs. In 5´ RACE, the primer ALiMV-P1-1269R (5´-CCGCTATCCCCTTTCCGTAAAGTA-3´) was used to synthesize the first cDNA strand that was then tailed with PolyC(3c3t11c) (5´-CCCTTTCCCCCCCCCCC-3´) . Subsequently, the primers ALiMV-P1-1137R (5´-GTATGCGTGCTGCTTCTCGATTG-3´) and PolyG(11g3a3g) (5´-GGGGGGGGGGGAAAGGG-3´)  were used to amplify the 5´ end of the ALiMV genome. The primer ALiMV-NIb-8642F (5´-AACACTGGTACTTCCGGGACGCA-3´) paired with Oligo-dT (5´-TTTTTTTTTTTTTTTTTTTTTTTTB-3´) were used in 3´ RACE. Revere transcription (RT) was conducted at 50°C for 30 min, and then inactivation at 94°C for 2 min. Polymerase chain reaction (PCR) was run with the conditions of hot start at 94°C for 2 min, and then 30 cycles of 94°C for 1 min, 60°C for 2 min and 72°C for 3 min, and a final extension of 72°C for 7 min. All amplicons were cloned by the TOPO TA Cloning kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) using standard procedures, and then sequenced by Mission Biotech company (Taipei, Taiwan) using an ABI 3730 DNA sequencer. Three clones for each fragment were sequenced. The obtained nt sequences of the 5´- and 3´-ends were assembled with the contig sequence to obtain the complete genome sequence of ALiMV-TW, deposited in the GenBank database (acc. no. OL355031).
Analysis of the genome structure indicated that the ALiMV-TW genome is 9618 nt long and contains a single ORF, ranging from nt 126 to nt 9359, encoding a polyprotein of 3077 amino acids (aa) (350.8 kDa). According to the consensus sequences for P1, HC-Pro and NIa protease cleavage sites , the positions of P1 (nt 126-992), HC-Pro (nt 993-2360), P3 (nt 2361-3467), 6K1 (nt 3468-3623), CIP (nt 3624-5525), 6K2 (nt 5526-5681), VPg (nt 5682-6245), NIa (nt 6246-6983), NIb (nt 6984-8540) and CP (nt 8541-9356) were deduced (Fig. 2a).
The complete genome sequences of 136 potyviruses available in the GenBank database (Table S1) were used for sequence comparison with ALiMV-TW. Multiple sequence alignments were performed using the ClustalW method in the MEGA X software (http://www.megasoftware.net) . Sequence Demarcation Tool Version 1.2 (SDTv1.2) was used to perform pairwise alignments and generate color-coded pairwise matrices and nt and aa identity calculations . Phylogenetic analyses were conducted in the MEGA X software using the Neighbor-Joining method with 1000 bootstrap replicates, and the maximum composite likelihood method was used to compute the evolutionary distances. The results revealed that the full-length genome and polyprotein of ALiMV share 52.3%-65.1% nt identity and 30.1%-64.2% aa identity, respectively, with those of other potyviruses (Fig. 2b & c). And, ALiMV is close to the potato virus Y (PYV) clade (Fig. 2d & e).
Using the One-Step RT-PCR kit (GMbiolab), a one-step RT-PCR method with the primer pair ALiMV-P1-967F (5´-CAATAAGACACCTAGTGCCCGAAAGC-3´)/ALiMV-P1-1269R was developed to specifically detect ALiMV in plants. The amplification conditions were set as RT at 50°C for 30 min, inactivation at 94°C for 2 min, and then PCR at 35 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 1 min, and a final extension of 72°C for 7 min. An expected 303-bp DNA fragment was amplified and its nt sequence was directly sequenced by Mission Biotech company using an ABI 3730 DNA sequencer to verify the correctness.
ALiMV was first discovered in Amazon lily (Eucharis grandiflora) in Japan. Before this report, only part of the genome sequence was available in the GenBank database [10–11]. Here, we have identified the amaryllis virus as ALiMV and elucidated the whole genome sequence of the virus. Although an ALiMV isolate (acc. no. AY590143) was previously reported in Taiwan , that virus should be re-identified as HiMV due to the over 90% nt identity. Therefore, this is also the first report of ALiMV in Taiwan.