Arch Virol DOI 10.1007/s00705-016-2830-y ORIGINAL ARTICLE Deep sequencing of banana bract mosaic virus from flowering ginger (Alpinia purpurata) and development of an immunocapture RT-LAMP detection assay 1,2 1 2 1 Jingxin Zhang • Wayne B. Borth • Birun Lin • Kishore K. Dey • 1 2 2 2 Michael J. Melzer • Huifang Shen • Xiaoming Pu • Dayuan Sun • John S. Hu1 Received: 9 October 2015 / Accepted: 14 March 2016 Ó Springer-Verlag Wien 2016 Abstract Banana bract mosaic virus (BBrMV) has never hypothesis that the A. purpurata isolate arrived in Hawaii been reported in banana plants in Hawaii. In 2010, how- from Southeast Asia. ever, it was detected in a new host, flowering ginger (Alpinia purpurata). In this study, we characterize the A. purpurata isolate and study its spread in flowering ginger Introduction in Hawaii. A laboratory study demonstrated that BBrMV could be transmitted from flowering ginger to its natural Banana bract mosaic virus (BBrMV) was first isolated from host, banana, therefore raising a serious concern about the bananas (Musa spp. L., Musaceae, Zingiberales) in the potential risk to the rapidly growing banana industry of Philippines in 1979 [1]. It has subsequently been detected Hawaii. To quickly monitor this virus in the field, we in other countries including India, Samoa, Sri Lanka, developed a robust immunocapture reverse transcription Thailand, and Vietnam [2]. Recently, the virus was dis- loop-mediated isothermal amplification (IC-RT-LAMP) covered in Colombia and Ecuador [3], indicating that this assay. Deep sequencing of the BBrMV isolate from A. pathogen can spread quickly and cause significant eco- purpurata revealed a single-stranded RNA virus with a nomic losses worldwide. In June 2009, BBrMV was also genome of 9,713 nt potentially encoding a polyprotein of detected in red- and pink-flowering ginger (Alpinia pur- 3,124 aa, and another predicted protein, PIPO, in the ?2 purata (Vieill.) K. Schum.) on the island of Oahu, Hawaii, reading-frame shift. Most of the functional motifs in the USA. The virus induced symptoms of mosaic, streaking, Hawaiian isolate were conserved among the genomes of and a severe cupping of leaves, with a browning of flowers isolates from one found in the Philippines and India. and reduction in their size and shelf life [4]. However, the A. purpurata isolate had an amino acid BBrMV belongs to the genus Potyvirus, family Po- deletion in the Pl protein that was most similar to the tyviridae. It has a poly-(A) tract at the 3’ terminus of the Philippine isolate. Phylogenetic analysis of an eastern monopartite, linear, single-stranded RNA (ssRNA) positive Pacific subpopulation that included A. purpurata was sense (?) genome [5, 6]. The virions lack an envelope and closest in genetic distance to a Southeast Asian subpopu- are flexuous, filamentous rods 720 to 850 nm long and lation, suggesting frequent gene flow and supporting the 12-15 nm in diameter (Viralzone, http://viralzone.expasy. org/viralzone/all_by_species/50.html). Only two com- pletely sequenced BBrMV genomes, one from the Philip- & John S. Hu pines [7] and one from India [8], have been published. In [email protected] both instances, the virus was isolated from banana. The 1 genome isolated in the Philippines (BBrMV-PHI) was Department of Plant and Environmental Protection Sciences, obtained using degenerate primers for members of the College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu, HI, USA genus Potyvirus [7], and the second, from India (BBrMV- TRY), was obtained using primers based on the nucleotide 2 Key Laboratory of New Technique for Plant Protection in Guangdong, Institute of Plant Protection, Guangdong sequence of the BBrMV-PHI isolate [8]. In our previous Academy of Agricultural Sciences, Guangzhou, China study [4] cloning of partial sequences of the BBrMV 123 J. Zhang et al. isolate from A. purpurata revealed 95-98 % nucleotide and elected to develop a loop-mediated isothermal ampli- sequence identity to the available sequences of BBrMV in fication (LAMP) assay for BBrMV. LAMP is a newly NCBI, indicating that the isolate might differ from isolates emerging molecular tool that uses Bst DNA polymerase found in other plant hosts or geographical locations. More with DNA strand-displacement activity to generate ampli- importantly, BBrMV has not been reported infecting fication products within 60 minutes [25]. It is simple, rapid, bananas in Hawaii, nor has A. purpurata been previously specific and cost-effective when compared to PCR [26]. reported as a host of this virus. We have fully characterized RT-LAMP (reverse transcription LAMP) synthesizes the genome of this A. purpurata isolate from Hawaii to cDNA from a viral RNA template in a one-step reaction determine its relatedness to other known strains. We used a [27]. It has a high-throughput diagnostic capacity that different strategy from previous BBrMV studies, applying meets the needs of the agricultural industry and quarantine deep sequencing and beginning with the isolation of dou- inspection protocols. ble-stranded RNA (dsRNA) from infected A. purpurata. Deep sequencing has been used widely to identify and characterize novel plant viruses. Published techniques have Materials and methods used DNA [9, 10], RNA [11, 12], dsRNA [13, 14], or short- interfering RNA [15, 16] as templates. We have previously Isolation of double-stranded RNA used dsRNA from infected plants to deep-sequence the genomes of four closely related closteroviruses infecting a Leaves of A. purpurata with symptoms of BBrMV were single common green ti plant, Cordyline fruticosa L. [17]. collected from the University of Hawaii at Manoa campus These previous studies indicated the great potential for in Honolulu. We isolated dsRNA from symptomatic leaves diagnostics and discovery of viruses by deep sequencing using the procedures described by Morris and Dodds [28] [18]. We used this procedure not only to characterize the as modified by Hu et al. [29]. Five microliters of dsRNA genome of BBrMV from A. purpurata but also to help was analyzed using 1 % (w/v) agarose gel electrophoresis, understand the differences between this virus and similar and the remainder was stored at -80 °C. viruses infecting banana. A further goal was to detect any undiscovered RNA viruses that also might be associated Library generation and deep sequencing with the disease symptoms described above. Another important question was the prevalence of this One microliter of dsRNA was heat-denatured at 95 °C for disease on A. purpurata. A reverse transcription poly- 10 minutes with 10 pmol of primer universal-dN6 [30] and merase chain reaction (RT-PCR) assay revealed that then immediately chilled on ice. First-strand cDNA syn- BBrMV is widespread on A. purpurata in Hawaii [4]. Red- thesis was performed using AMV reverse transcriptase and pink-flowering ginger (A. purpurata) is an ornamental (RNaseH?) (Promega, USA) according to the manufac- plant in the diverse family Zingiberaceae [19]. It is com- turer’s instructions. cDNA was then concentrated on a YM- monly grown for cut flowers in the home and also is in 50 column (Millipore, USA) together with 200 llof commercial production in Hawaii, where sales were valued nanopure H2O. The eluate was used as a primer/template in at $1.61 million in 2005-2006 [20]. BBrMV is transmitted a 20-ll hot-start overlap-extension PCR reaction using the by several species of aphids in a non-persistent manner [1, Easy-AÒ high-fidelity PCR cloning enzyme (Stratagene, 21]. It is also spread by vegetative propagation of the USA), with the following program: 95 °C for 7 minutes; rhizome and its offshoots and thus is easily spread by 10 cycles at 95 °C for 60 seconds, 55 °C for 60 seconds, humans. and 72 °C for 60 seconds; followed by 72 °C for 7 min- Bananas are an essential source of food in many tropical utes. Using the Easy-AÒ high-fidelity PCR cloning and subtropical countries [22]. The Hawaiian Islands are by enzyme, a 200-ll PCR reaction with 20 pmol of universal far the largest banana producer in the United States, fol- rPCR primer as the single primer [30] and 10 llofthe lowed by Florida. Banana production in Hawaii reached overlap-extension PCR reaction as the template was run 8,090 million metric tonnes in 2010 [23]. In this study, we according to the following cycling program: 95 °C for also tested whether BBrMV could be transmitted from A. 7 minutes; 35 cycles at 95 °C for 60 seconds, 58 °C for 60 purpurata to bananas by aphids, to determine if this virus seconds, and 72 °C for 60 seconds; followed by 72 °C for isolate could be a serious threat to banana production in 7 minutes. PCR products were fractionated by size (400- Hawaii. 700 bp) by agarose gel electrophoresis, ligated into To monitor spread of the disease, a robust detection pGEMÒ-T Easy Vector (Promega), and sequenced to assay for this virus is necessary. We considered the existing determine if BBrMV sequences were present. Following range of diagnostic assays for use in our study, including treatment with ExoSAP-ITÒ (USB/Affymetrix), the puri- ELISA [6], RT-PCR [7] and immunocapture RT-PCR [24] fied PCR products were sequenced directly on an Ion PGM 123 Banana bract mosaic virus from flowering ginger System (Ion 314 chips) at the University of Hawaii’s using PhusionÒ High-Fidelity DNA Polymerase (NEB) in a Advanced Studies of Genomics, Proteomics, and Bioin- 20-ll reaction containing 2 ll of dA-tailed cDNA, 10 ll formatics Laboratory, Short-length (\65 nt) and low- PhusionÒ mix, 10 pmol oligo-dT primer [31], with incu- quality reads and primer sequences were trimmed, as were bation at 98 °C for 3 minutes, 45 °C for 10 minutes, and low-quality basecalls at the ends of reads.