Characterization of complete genome and small RNA profile of pagoda yellow mosaic associated virus, a novel badnavirus in China.

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ARTICLE IN PRESS

VIRUS 96275 1–6

Virus Research xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Virus Research journal homepage: www.elsevier.com/locate/virusres

Characterization of complete genome and small RNA profile of pagoda yellow mosaic associated virus, a novel badnavirus in China

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Yilun Wang a,1 , Xiaofei Cheng b,c,1 , Xiaoxia Wu d , Aiming Wang c , Xiaoyun Wu a,∗ a

College of Agricultural and Food Science, Zhejiang Agricultural and Forestry University, Lin’an 311300, Zhejiang, PR China College of Life and Environmental Science, Hangzhou Normal University, Hangzhou 310036, Zhejiang, PR China Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London N5V 4T3, Ontario, Canada d College of Agriculture, Northeast Agricultural University, Key Laboratory of Soybean Biology, Ministry of Education, Harbin 150030, Heilongjiang, PR China

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Article history: Received 13 January 2014 Received in revised form 6 April 2014 Accepted 9 April 2014 Available online xxx

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Keywords: Badnavirus Deep sequencing 19 Genome 20 Pagoda 21 Phylogeny 22 23 Q3 RNA silencing Small RNA 24 17 18

A new badnavirus was discovered from pagoda trees showing yellow mosaic symptoms on the leaves by high throughput sequencing of small RNAs. The complete genome of this virus was determined to comprise 7424 nucleotides, and the virus shared 40.4–45.1% identity with that of other badnaviruses. The genome encodes five open reading frames (ORFs) on the plus strand, which includes three conserved badnaviral ORFs. These results suggest that this virus is a new member of the genus Badnavirus in the family Caulimoviridae. The virus is tentatively named pagoda yellow mosaic associated virus (PYMAV). Phylogenetic analysis suggested that this virus together with gooseberry vein banding virus (GVBV) and grapevine vein-clearing virus (GVCV) forms a separate group that is distinct two other well characterized badnaviral groups. Additionally, the viral derived small RNA (vsRNA) profile of PYMAV was analyzed and compared with that of viruses within the same family. Results showed that the most abundant PYMAV vsRNAs were 21-nt, whereas other viruses in the same family have a predominance of 22- or 24-nt vsRNA. The percentage of sense PYMAV vsRNA was almost equal to that of antisense vsRNA, whereas vsRNAs of other viruses in the family display preferences toward the sense strand of their genome. Furthermore, PYMAV vsRNAs were symmetrically distributed along the genome with no obvious vsRNA generating hotspots. © 2014 Published by Elsevier B.V.

1. Introduction

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High throughput sequencing of small RNAs has been proved to be a useful method for discovering novel pathogens, especially viruses (Singh et al., 2012; Wu et al., 2010). This method is based on that viruses are targeted by host RNA silencing, a universal gene regulatory and anti-viral mechanism of eukaryotes (Axtell et al., 2007; Baulcombe, 2002; Ding and Voinnet, 2007). The key step of RNA silencing is the production of 21–24 nt small RNAs from sense or anti-sense viral genomes. These small RNAs are loaded into socalled RNA induced silencing complexes (RISCs) and in turn guide the RISCs to cleave the viral genome and finally attenuate viral infection. In the virus infected cells, viral derived small RNAs (vsRNAs) can cover the entire sense and antisense of the viral genome (Kreuze et al., 2009; Liu et al., 2011; Wu et al., 2010). Therefore, the

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∗ Corresponding author. Tel.: +86 13326891266. E-mail address: [email protected] (X. Wu). 1 These authors contributed equally.

viral genome can be assembled through sequencing and analysis of the small RNAs from plants infected by that virus. From January to May 2013, we carried out a survey of plant viruses on common crops, field weeds and ornamental plants in Zhejiang Province, PR China. During the survey, we noticed that many pagoda trees [Styphnolobium japonicum (L.) Schott] showed typical viral symptoms. The pagoda tree, native to eastern Asia, is a popular ornamental and street tree all over the world. It has also been used in traditional Chinese medicine for thousands of years as it contains medicinal flavonoids and galactomannans (Kite et al., 2007, 2009; Smirnova et al., 2004). Despite its important economic value, little attention has been given to its diseases. Therefore, we further investigated the causal agent of this disease and a novel badnavirus was discovered from the diseased pagoda trees. Badnaviruses are emerging as important pathogens of agriculture and horticulture, and have induced serious diseases and crop losses in recent years (Borah et al., 2013; Harper et al., 2005; Huang and Hartung, 2001). The genus Badnavirus consists of a group of plant viruses with a circular double-stranded DNA genome of about 7–8 kb in size (Bouhida et al., 1993). Typical badnaviral genome encodes three open reading frames (ORFs) which are

http://dx.doi.org/10.1016/j.virusres.2014.04.006 0168-1702/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Wang, Y., et al., Characterization of complete genome and small RNA profile of pagoda yellow mosaic associated virus, a novel badnavirus in China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.04.006

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ARTICLE IN PRESS Y. Wang et al. / Virus Research xxx (2014) xxx–xxx

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2.3. Sequencing assembling The adaptor sequences of raw reads from high-throughput sequencing were trimmed using SOAP (Li et al., 2008). This step also removed the low quality reads that contained no or partial adaptor sequence. The resulting clean reads were then assembled into larger contigs using Velvet 0.7.31 (Zerbino and Birney, 2008) with the k-mer value of 17 or 19. 2.4. Isolation of total plant DNA and determination of viral genome

Fig. 1. Symptoms of yellow mosaic on pagoda leaves. A closer view of the leaf is shown on the left bottom.

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tandemly arranged on the plus strand. ORF1 encodes a small protein of unknown function; ORF2 encodes a protein with nucleic acid-binding activities (Jacquot et al., 1996); and ORF3 encodes a large polypeptide which is post-translationally cleaved into several proteins, including the movement protein (MP), coat protein (CP), viral aspartic protease (AP), reverse transcriptase (RT), and ribonuclease H (RNase H) (Harper and Hull, 1998). In this study, the complete genome of this virus was determined and its phylogenetic relationship with other badnaviruses was analyzed. Besides, the viral derived small RNA profile of this virus was further analyzed and compared with that of viruses within the same family.

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2. Materials and methods

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2.1. Source of plant materials

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Pagoda samples with yellow mosaic symptom (Fig. 1) and raspberry leaves showing mottling and stunting symptoms were collected from Lin’an, Zhejiang Province, PR China. Leaf samples were used for RNA isolation immediately or stored at −80 ◦ C until use.

2.2. RNA isolation and deep sequencing Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. The small RNA library was constructed as described by Yang et al. (2011). In brief, the low molecular weight (LMW) RNAs were enriched using NaCl and PEG 8000 and then separated by 15% denaturing polyacrylamide gel electrophoresis (PAGE). The small RNAs of 19–28 nt were recovered and ligated to 3 and 5 adapters. After each ligation step, small RNAs were purified using 15% denaturing PAGE as described above. The final purified ligation products were reverse-transcribed into cDNA using Superscript III reverse transcriptase (Invitrogen). The first strand cDNA was PCR amplified using DNA Taq polymerase (Roche, Mannheim, Germany) and DNA fragments were purified for high-throughput sequencing using the Solexa platform (Illumina, San Diego, CA).

For total plant DNA isolation, the leaf tissue was ground in liquid nitrogen and total DNA was extracted using hexadecyltrimethylammonium bromide (CTAB) method (Springer, 2010). PCR was performed in a total volume of 50 ␮L with an ABI thermal cycler Veriti 9902 (Life Science, Foster City, CA) with Phusion® highfidelity DNA polymerase (New England BioLabs, Ipswich, MA). Primers (Supplementary Table 1) were designed based on the contig sequences using Primer3 (http://simgene.com/Primer3). The amplified fragment was recovered with the AxyPrepTM DNA Gel Extraction Kit (Axygen Biosciences, Hangzhou, China) and inserted into pGEM-T Easy Vector (Promega, Madison, WI). The positive plasmid was further confirmed by DNA sequencing. 2.5. Phylogenetic analysis Pair-wise comparison was performed using DNAman version 6 (Lynnon Corporation) with default parameters. Multiple sequence alignment was performed using Clustal X 1.83 software (Chenna et al., 2003) and colorized by ESPript (Gouet et al., 1999). Phylogenetic trees were constructed by the Neighbor-joining method using MEGA 5.0 software (Tamura et al., 2011). The substitution model was set to the Maximum Composite Likelihood (MCL) model for nucleotide sequences and Jones–Taylor–Thornton (JTT) model for amino acid sequences. 2.6. Small RNA profile Viral derived small RNAs were selected from the small RNA library and mapped to the genome of PYMAV with house-written BioPython scripts, which are available upon request. 2.7. Detection of cherry virus A and plum bark necrosis stem pitting-associated virus The total RNA isolated from pagoda or raspberry leaves were reverse-transcribed into cDNA using Oligo dT18 (Invitrogen) or Random Hexamers (Invitrogen). PCR reactions were performed with primers were designed based on the contig sequences of cherry virus A (CVA-2506f and CVA-3780r) or plum bark necrosis stem pitting-associated virus (Plum-13686f and Plum-14045r) (Supplementary Table 1). After PCR reaction, the mixture was observed by 1% agarose gel electrophoresis. The amplified fragment was ligated, inserted into pGEM-T Easy Vector (Promega, Madison, WI) and sequenced.

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3. Results

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3.1. Deep sequencing and small RNA characterization

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The small RNA library was prepared from an equal ratio leaf mixture of pagoda leaves with yellow mosaic symptom and raspberry leaves with mottling and stunting symptoms that were collected in the same survey. A total of 4,363,601 clean reads with length between 17 and 29 nt was obtained from the small RNA library.


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Fig. 2. Genome structure of pagoda yellow mosaic virus. The putative ORFs of pagoda yellow mosaic virus represented by rectangles, the five contigs obtained from small RNAs were shown by gray lines, and the four fragments amplified by PCR were indicated by dashed line. The relative positions of primers were also indicated by arrows. Note that the contif 1 and DNA fragment 4 were shown as broken lines.

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For reducing calculating time, the known miRNAs, rRNAs, snoRNAs, snRNAs, and tRNAs were not included in further analysis. The final pool contained 4,228,083 clean reads. These small RNAs were assembled by Velvet software with the parameters stated in Section 2. Contigs longer than 150 bp were searched by BlastN and BlastX (Altschul et al., 1997). Results showed that five contigs shared some similarities with the genome sequences of badnaviruses, such as gooseberry vein banding virus (GVBV), grapevine vein-clearing virus (GVCV) and dracaena mottle virus (DrMV), suggesting that one or more badnaviruses were present in the two samples.

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3.2. Complete genome characterization

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Since the small RNA library was prepared from a mixture of pagoda and raspberry leaves, we performed PCR reactions with

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both the total DNA isolated from the pagoda and raspberry leaves. Interestingly, the DNA fragments can only be amplified from the DNA isolated from pagoda leaves with yellow and mosaic symptoms, but not from the DNA isolated from raspberry leaves or healthy pagoda leaves (data not shown). These results indicate that this virus is from the pagoda tree with mosaic symptoms on leaves. The amplified DNA fragments were cloned and sequenced. After assembling, the complete genome of this virus was 7424 bp in length with the GC content of 48.4% (Genbank accession Number: KJ013302). BlastN search results showed that the virus was most similar to viruses of the genus Badnavirus (family Caulimoviridae). Pair-wised genome similarity comparison with 31 other badnaviruses having full genome sequences available in GenBank showed genomic similarities from 40.4% (sweet potato badnavirus B, SPBV-B) to 45.1% (sugarcane bacilliform virus, SCBV). Furthermore, a plant cytoplasmic initiator methionine tRNA sequence was also found at position 1–18 (5 -TGGTATCAGAGCTCAACA-3 ) with 12 of the 18 nucleotides complementary to the consensus sequence of plant tRNAMet (3 -ACCAUAGUCUCGGUCCAA-5 ). Together, these results suggest that this virus belongs to the genus Badnavirus.

3.3. Genome structure analysis All badnaviruses reported thus far have three conserved ORFs, namely ORF1, ORF2, and ORF3 (Bouhida et al., 1993). These three ORFs are tandemly arranged on the plus strand and also have overlapping start and stop codons (5 -ATGA-3 ) (Kalischuk et al., 2013). ORF scanning of the sense strand of this virus also revealed the three ORFs at similar positions to other badnaviruses (Fig. 2). ORF1 starts at 213 and ends at 674 nt and encodes a peptide of 153 amino acids (aa), ORF2 (671–1111 nt) encodes a peptide of 146 aa, whereas ORF3 (1108–6915 nt) encodes a polypeptide of 1935 aa. Furthermore, like other badnaviruses, the ORFs 1, 2 and 3 of this Q4 virus also have overlapping start and stop codons. Interestingly, two

Fig. 3. Comparison of the highly conserved amino acid motifs of the putative protein encoded by ORF2 of badnaviruses. Sequences were aligned by ClustalX 1.83 (Chenna et al., 2003) and colorized by ESPript (Gouet et al., 1999). Functional conserved amino acid residues were highlighted with yellow backgrounds and complete consistent residues were highlighted in white and with red backgrounds. The start and end positions of each motif of PYMAV were also indicated. Acronyms are as follows: BSV, banana streak virus (NC 008018); BSCAV, banana streak CA virus (NC 015506); BSGFV, banana streak GF virus (NC 007002); BSIMV, banana streak IM virus (NC 015507); BSMYV, banana streak Mysore virus (NC 006955); BSOLV, banana streak OL virus (NC 003381); BSUAV, banana streak UA virus (NC 015502); BSUIV, banana streak UI virus (NC 015503); BSULV, banana streak UL virus (NC 015504); BSUMV, banana streak UM virus (NC 015505); BSVNV, banana streak VN virus (NC 007003); BSCVBV, bougainvillea spectabilis chlorotic vein-banding virus (NC 011592); CiYMV, citrus yellow mosaic virus (NC 003382); ComYMV, commelina yellow mottle virus (NC 001343); CSSV, cacao swollen shoot virus (NC 001574); CyLNV, cycad leaf necrosis virus (NC 011097); DiBV, dioscorea bacilliform virus (NC 009010); DrMV, dracaena mottle virus (NC 008034); FiBV-1, fig badnavirus 1 (NC 017830); GVBV, gooseberry vein banding virus (NC 018105); GVCV, grapevine vein-clearing virus (NC 015784); KTSV, kalanchoe top-spotting virus (NC 004540); PVBV, pelargonium vein banding virus (NC 013262); PYMV, piper yellow mottle virus strainish-1 (NC 022365); PBCV, pineapple bacilliform comosus virus Q7 (NC 014648); SCBIMV, sugarcane bacilliform IM virus (NC 003031); SCBMOV, sugarcane bacilliform MO virus (NC 008017); SCBV, sugarcane bacilliform virus (NC 013455); TaBV, taro bacilliform virus (NC 004450). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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Fig. 4. Neighbor-joining phylogenetic trees of badnaviruses based on full genomic sequences (A) or deduced amino acid sequences of ORF3 (B). The phylogenetic tree was rooted to the genome sequence of the rice tungro bacilliform virus (RTBV, NC 001914; A) or polypeptide of RTBV (B). Branch numbers represent a percentage of the bootstrap values in 1000 sampling replicates. SPBV-A, sweet potato badnavirus A (NC 015655); SPBV-B, sweet potato badnavirus B (NC 012728); other viral acronyms are the same as in Fig. 3. The position of PYMAV was indicated by an arrow.

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additional ORFs (ORF4 and ORF5) which encode more than 100 aa were found on the plus strand (Fig. 2). ORF4 partially overlapped ORF3 (6534–6995nt) and encodes a peptide of 153 aa (Fig. 2). This ORF is in −1 translational frame relative to the 1809 aa (Glu) of ORF1. Some badnaviruses, such as citrus yellow mosaic virus (CYMV) and cacao swollen shoot virus (CSSV), also contain a similar ORF at this location (Borah et al., 2009; Hagen et al., 1993). The ORF5 is located within ORF3 (3557–3880 nt) and encodes a hypothetical peptide of 108 aa.

motif of MP, CP, AP, RT and RNase H (Xu et al., 2011; Yang et al., 2003) was also present in the polypeptide (Supplemental Fig. 3). The peptide encoded by ORF4 has no sequence similarity with that encoded by other badnaviruses, however blast search showed that it shares similarities with several nucleotide metabolism proteins, such as hypothetical ATP-dependent DNA helicase (YP 004692129) and type I site-specific deoxyribonuclease (YP 362924). The protein encoded by ORF5 does not have any similarities with any other proteins in GenBank.

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3.4. Amino acid analysis

3.5. Phylogenetic relationship

Although ORF1 was found at the similar location as that of other badnaviruses, the peptide encoded by ORF1 shares barely similarities (9.6–15.7%) with the counterparts of other badnaviruses. The protein encoded by ORF2 shows low similarities (15.5–29.0%) with the corresponding proteins encoded by other badnaviruses. However, multiple-sequence alignment revealed three conserved sequence motifs were present in these proteins (Fig. 3 and Supplementary Fig. 2), suggesting that this protein is more conserved among badnaviruses. Compared to the proteins encoded by ORF1 and ORF2, the polypeptide encoded by ORF3 is conserved in all badnaviruses, and shared 34.6–43.6% identities with other badnaviruses. Multiple sequence alignment showed that the conserved

In order to understand the phylogenetic relationship between this virus and other badnaviruses, two Neighbor-Joining phylogenetic trees, one based on the full genome sequence and the other based on the deduced amino acid sequence of ORF3, were constructed (Fig. 4A and B). In both phylogenetic trees, the 32 badnaviruses were clustered into four major groups, namely groups 1–4. This virus together with GVCV and GVBV formed a separated cluster within group 3 in both phylogenetic trees. Taken together, all the results above showed that the virus identified in this study is a new member of the genus Badnavirus within the family Caulimoviridae. As a result, the name pagoda yellow mosaic associated virus (PYMAV) is proposed.

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amplified from the trees showing yellow mosaic on leaves (Supplementary Fig. 3A). Furthermore, we also inspected the presence of circular DNA by PCR with two pairs of back-to-back primers (Bad256f/r and Bad-4424f/r; Supplementary Table 1). DNA fragments equal to the viral genome size can only be amplified from the total DNA isolated from pagoda leaves with yellow mosaic symptoms, but not the total DNA isolated from healthy pagoda plants (Supplementary Fig. 3B). Subsequently sequencing results confirmed that the amplified DNA fragments only have few nucleotide substitutions with the full genome sequence obtained (data not shown). Taken together, these results clearly showed that this virus is episomal and the yellow mosaic symptom on the pagoda tree is closely related to this virus.

4. Discussion

Fig. 5. Small RNA profile of PYMAV from infected pagoda leaves. (A) Bar graph shows the proportion of mapped PYMAV vsRNA size distribution. (B) Genome-wide mapping of PYMAV vsRNA. A linear genome map of PYMAV was shown in the middle.

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3.6. Viral derived small RNA profile The small RNA profile of PYMAV was also analyzed with the small RNAs obtained from the deep sequencing. A total of 33,311 reads can be mapped to the PYMAV genomic sequence, which account for 0.76% of the small RNAs. The size classes of the viral derived small RNAs were mostly 21–24 nt with 21-nt being the most abundant size class (Fig. 5A). The viral derived sRNAs (vsRNA) were then classified according to their orientations on the genome. Interestingly, the number of vsRNAs derived from sense genomic sequence was almost equal to that from antisense viral genome (17,974 vs 15,337 reads). When all these vsRNAs were mapped to the PYMAV genome, it was apparent that the vsRNAs covered almost all of both sense and antisense strands of the PYMAV genome (Fig. 5B). Interestingly, unlike the vsRNA profiles of most viruses that contain some vsRNA generating hotspots within their genomes (Donaire et al., 2009; Qi et al., 2009), the vsRNAs of PYMAV were equally distributed along the sense and antisense of the genome with no obvious vsRNA generating hotspots (Fig. 5B). 3.7. Relationship between pagoda yellow mosaic disease and PYMAV To determine the relationship between pagoda yellow mosaic disease and this virus, we further analyzed the assembled contigs of length less than 150 bp as no other viral sequences were obtained from 13 contigs longer than 150 bp. Two additional viruses were discovered from the contigs. The contigs of one virus showed high level of nucleotide identity (>97%) with plum bark necrosis stem pitting-associated virus (genus Ampelovirus, family Closteroviridae), whereas the contigs of the other virus showed 70–80% nucleotide identity with the genome of cherry virus A (genus Capillovirus, family Betaflexiviridae). Subsequent RT-PCR showed that both of these viruses were associated with raspberry, but not pagoda (data not shown). PCR analysis was also performed with the DNA of pagoda trees with yellow mosaic symptom or without symptom on leaves. The results showed that the specific DNA fragment can only be

Badnaviruses are receiving more and more attention since many serious crop diseases have been associated with this class of viruses in recent (Borah et al., 2013; Harper et al., 2005; Huang and Hartung, 2001; Yang et al., 2003). Integrated badnaviruses, also known as endogenous pararetroviruses (EPRVs), have been found in the genome of various plant species (Chabannes et al., 2013; Hany et al., 2014; Laney et al., 2012; Lyttle et al., 2011), suggesting the ubiquitous existence of this class of viruses. In the present study, a new badnavirus (PYMAV) was characterized and its genome sequence was determined. The genome of PYMAV is 7424 bp in length and shares 34.6–45.7% sequence identity with known badnaviruses. Genomic structure analysis showed that, similar as other badnaviruses, the three tandemly arranged ORFs are present on the plus strand. Phylogenetic relationship analyses showed that this virus is distinct from the two well characterized branches of badnaviruses, and together with GVBV and GVCV form a distinct sub-branch. PYMAV also showed many unique characteristics when compared to other badnaviruses. Firstly, the protein encoded by ORF1 shares no similarity with the corresponding proteins encoded by other badnaviruses. Secondly, besides the three conserved ORFs, two additional ORFs (ORF4 and ORF5) were found on the plus strand of the genome. An ORF at a similar position to PYMAV ORF4 was also found in some badnaviruses, however the peptide encoded by this ORF has no similarity with that of other badnaviruses. Considering ORF4 is −1 translational frame relative to the Glu-1809 of ORF3 and the encoded peptide shows obvious similarities with several nucleotide metabolism proteins in the GenBank database, it is reasonable to believe that this ORF may be functional. The peptide encoded by ORF5 has no similarity to any proteins in the GenBank database; therefore the authenticity of this ORF still needs to be verified experimentally. The small RNA profile of several viruses in the Caulimoviridae, including cauliflower mosaic virus (CaMV), PYNV, SPBV-A and SPBV-B, have been studied in detail (Blevins et al., 2011; Kalischuk et al., 2013; Kreuze et al., 2009; Moissiard and Voinnet, 2006). These studies showed that although the 21-nt class of vsRNA is predominant in plants infected by other RNA viruses (Donaire et al., 2009; Qi et al., 2009), within the family Caulimoviridae vsRNA were mainly either 24-nt in length (CaMV) or 22-nt vsRNAs (PYNV, SPBV-A and SPBV-B). Also, vsRNAs of CaMV, PYNV, SPBV-A and SPBV-B were more abundant in the sense strand of the genome. Furthermore, several vsRNA generating hot spots were found in the sense strand of the genomes of these viruses. In comparison with these viruses, the vsRNA profile of PYMAV showed several unique properties. Firstly, 21-nt vsRNA, but not 22-nt or 24-nt vsRNA were the predominant class of vsRNAs. Secondly, the ratio of sense and antisense vsRNAs was almost equal. Thirdly, both the sense and antisense vsRNAs were distributed approximately equally along the genome


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with no obvious vsRNA generating hot spots. It remains to be illustrated if these different characteristics of the small RNA profile of PYMAV are caused by the unique pagoda interfering RNA pathway 337 or by specific interaction between PYMAV and the pagoda. 338 Regardless of the fact that our results showed that the yel339 low mosaic symptom on the pagoda tree is closely related to 340 Q5 PYMAV (Fig. 6), full confirmation by inoculating this virus back 341 to the healthy pagoda plants is still needed. Unsuccessful of sap 342 inoculation suggests that this virus may be difficult or unable to 343 be transmitted by mechanical transmission. At present, we are 344 attempting to construct an infectious clone of this virus, which will 345 be used to inoculate its natural host through either agro-infiltration 346 or biolistic bombardment. 347 335 336

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Acknowledgement This study was supported by the National Natural Science Foundation of China (Grant Nos. 31101417, 31101415 and 31071438), the Natural Science Foundation of Heilongjiang Province (Grant No. ZD201117) and the Research Foundation of Education Bureau of Heilongjiang Province (Grant No. 12531Z001). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.04.006. References Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25 (17), 3389–3402. Axtell, M.J., Snyder, J.A., Bartel, D.P., 2007. Common functions for diverse small RNAs of land plants. Plant Cell 19 (6), 1750–1769. Baulcombe, D., 2002. RNA silencing. Curr. Biol. 12 (3), R82–R84. Blevins, T., Rajeswaran, R., Aregger, M., Borah, B.K., Schepetilnikov, M., Baerlocher, L., Farinelli, L., Meins Jr., F., Hohn, T., Pooggin, M.M., 2011. Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense. Nucleic Acids Res. 39 (12), 5003–5014. Borah, B.K., Johnson, A.M., Sai Gopal, D.V., Dasgupta, I., 2009. Sequencing and computational analysis of complete genome sequences of Citrus yellow mosaic badna virus from acid lime and pummelo. Virus Genes 39 (1), 137–140. Borah, B.K., Sharma, S., Kant, R., Johnson, A.M., Saigopal, D.V., Dasgupta, I., 2013. Bacilliform DNA-containing plant viruses in the tropics: commonalities within a genetically diverse group. Mol. Plant Pathol. 14 (8), 759–771. Bouhida, M., Lockhart, B.E., Olszewski, N.E., 1993. An analysis of the complete sequence of a sugarcane bacilliform virus genome infectious to banana and rice. J. Gen. Virol. 74 (Pt 1), 15–22. Chabannes, M., Baurens, F.C., Duroy, P.O., Bocs, S., Vernerey, M.S., Rodier-Goud, M., Barbe, V., Gayral, P., Iskra-Caruana, M.L., 2013. Three infectious viral species lying in wait in the banana genome. J. Virol. 87 (15), 8624–8637. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T.J., Higgins, D.G., Thompson, J.D., 2003. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 31 (13), 3497–3500. Ding, S.W., Voinnet, O., 2007. Antiviral immunity directed by small RNAs. Cell 130 (3), 413–426. Donaire, L., Wang, Y., Gonzalez-Ibeas, D., Mayer, K.F., Aranda, M.A., Llave, C., 2009. Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes. Virology 392 (2), 203–214.

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