Molecular Characterization and Population Structure of Blueberry Mosaic Associated Virus

Blueberry mosaic disease was first described in the 1950s but the causal agent has not been characterized to date. Next generation sequencing was employed in the identification of the causal agent and an undescribed ophiovirus, tentatively named Blueberry mosaic associated virus (BlMaV), was detected in diseased plants. The segmented genome of the virus is comprised of three negative-sense RNAs (RNAs 1-3) encoding four proteins. The population structure of the virus was studied in detail using 59 isolates collected from various blueberry growing regions of North America and Slovenia. The open reading frames of RNAs 2 and 3 were analyzed; revealing stringent purification selection for both proteins. The information acquired will provide valuable data for the development of reliable assays able to detect the widest arrays of BlMaV isolates. Index words: Blueberry mosaic, ophioviruses, negative-strand RNA virus, population structure INTRODUCTION Blueberry mosaic was first reported in Michigan, United States (Varney, 1957). The disease is now present in several regions of North America including Arkansas, British Columbia, Indiana, Kentucky, New Jersey, New York, Oregon and Washington as well as several other parts of the globe including South America, Europe, New Zealand and South Africa (Varney, 1957; Martin et al., 2009). Symptoms include mild to brilliant mottle and mosaic patterns on foliage that normally appear on few leaves but could expand to the majority of the canopy (Fig. 1). Initially it was thought to be genetic variegation, but a virus etiology was later suggested. However, subsequent research failed to identify the disease agent (Varney, 1957; Raniere, 1960; Ramsdell and Stretch, 1987). Next generation sequencing (NGS) was employed to identify a new ophiovirus associated with the disease. This communication reports the molecular characterization and population structure of the virus detected in 100% of mosaic samples and tentatively named Blueberry mosaic associated virus (BlMaV). MATERIALS AND METHODS Identification and characterization Total nucleic acids (NAs) were extracted from a ‘Duke’ symptomatic plant as described (Poudel et al., 2013) and used for degenerate oligonucleotide-primed reverse transcription (DOP-RT)PCR (Telenius et al., 1992). Initial virus sequences were obtained using the Illumina (San Diego, CA, USA) platform and the sequences of three genomic RNAs were completed by RT-PCR as previously described (Laney et al., 2011; Tzanetakis et al., 2007). Genome termini were obtained by RACE RT-PCRs (Tzanetakis et al., 2007). For the phylogenetic analysis, the RNA-dependent RNA polymerase (RdRp) proteins of BlMaV and other negative-strand viruses belonging to the genera Tenuivirus, Tospovirus, Rhabdovirus, and Varicosavirus were used in the Neighborjoining method with 1,000 bootstrap replicates on MEGA v.5 (Tamura et al, 2011). Some ophioviruses have four RNAs and for this reason we explored the potential of a BlMaV RNA 4. For this purpose, DOP-RT-PCR was conducted as described in Ho et al. (2014) using either total RNAs depleted of plant ribosomal RNAs as well as dsRNA-enriched material. The products were subjected to a total of four separate 454 Junior (Roche, Branford, CT, USA) sequencing runs. Primers were designed from 12 sequences that showed similarity to the Mirafiori lettuce big-vein virus (MiLBVV) and Lettuce ring necrosis virus (LRNV) RNA 4 proteins and RT-PCRs were conducted on 11 mosaic samples to determine whether those sequences were closely associated with the blueberry virus and disease. In addition, a conserved region of 13 amino acids (aa) was identified in the MiLBVV and LRNV 37 kDa protein orthologs coded by RNA 4. Degenerate forward primers were designed to amplify this region and three separate PCRs were conducted on cDNA synthesized from A-tailed viral RNAs. Association of virus and disease To confirm BlMaV association with the disease, virus specific primers were designed from genomic RNAs and RT-PCRs were conducted on more than 100 symptomatic samples collected from North America and Slovenia. Amplified products were purified and sequenced to verify BlMaV presence. Population structure For the diversity analysis 59 mosaic samples were used. A 1200 nucleotide (nt) region from each of the ORFs of RNA 2 and RNA 3 were amplified as described (Table 2; Thekke-Veetil et al., 2013). The amplicons were cloned, sequenced, and variation in nt and predicted amino acid (aa) sequences were determined as described earlier (Thekke-Veetil et al., 2013). The phylogenetic trees were constructed using the Maximum likelihood method with 1000 bootstrap replicates using MEGA v.5 (Tamura et al., 2011). Selection pressures on the proteins were estimated by calculating the dNs /dS ratio using the Synonymous Non-synonymous Analysis Program (SNAP; Korber, 2000). RESULTS AND DISCUSSION BlMaV has a segmented genome with three negative-sense RNAs (Fig. 2) and 11,467 nt (GenBank accession numbers are KJ704366-8). Multiple NGS reactions and PCRs reactions failed to detect a BlMaV RNA 4. RNA 1 is 7,963 nt long, coding for two ORFs of 272 kDa, the putative RNA-dependent-RNA polymerase (RdRp) and a small 23 kDa protein of unknown function. The coding regions are separated by a 107 nt intergenic region whereas the 5’ and 3’ untranslated regions (UTR) are 238 and 19 nt, respectively. The presence of two ORFs with the same polarity in the largest RNA is a distinct genomic feature of Ophioviridae when compared to all other segmented negative-strand viruses (Naum-Ongania et al., 2003). The RdRp contains all the signature motifs of the Mononegavirales orthologs and is most closely related to the Citrus psorosis virus (CPsV) ortholog (Table 1). The 23 kDa protein (nt 7,944-7,360) did not present any significant similarity to studied viral proteins. RNA 2 is 1,934 nt long and codes for the putative movement protein (MP; nt 1,865-318) of 58 kDa. The 5’ and 3’ UTRs of this RNA are 317 nt and 69 nt long, respectively. The MP shared 37 % identity with its CPsV counterpart (Table 1). RNA 3 encodes a 50 kDa nucleocapsid protein (NP; nt 1493-129) which shared 38% identity to the CPsV ortholog (Table 1). The 1,570 nt RNA 3 has 5’ and 3’ UTRs of 128 nt and 77 nt respectively. The 5’ terminal sequences of BlMaV genomic RNAs were not conserved but the 3’ termini had the last six nucleotides identical (5’-AAUAUC-3’). The extreme 3’ ends of RNAs 2 and 3 were more conserved compared to their RNA 1 counterpart having 25 of the last 29 nt identical. No complementarity was found between the 5’ and 3’ ends of each genomic RNA. Phylogenetic analysis performed on the RdRps of BlMaV and other viruses from the Mononegavirales placed the virus within the Ophioviridae, in the same clade with CPsV (Fig. 3). The family Ophioviridae is currently composed of six viruses; MiLBVV, LRNV, CPsV, Tulip mild mottle mosaic virus (TMMMV), Ranunculus white mottle virus (RWMV), and Freesia sneak virus (FreSV) (Morikawa et al., 1995; Vaira et al., 1997; Roggero, 2000; Sanchez de la Torre et al., 2002; Torok and Vetten, 2002; van der Wilk et al., 2002; Vaira et al., 2006). Similar to CPsV the genome of BlMaV includes three RNAs and genome termini do not show complementarity. Also, the protein sequences of BlMaV were more similar to their CPsV orthologs. BlMV is present in all five states assayed (AR, MI, NJ, KY and OR), British Columbia in Canada, and Slovenia. Sequence analysis of RNAs 2 and 3 (over 20% of the total genome) revealed significant sequence conservation among the isolates. The NP exhibited more variation than MP, reaching up to 13% and 5% diversity respectively in the nt and aa sequences. Two isolates from NJ were unique with a Gly95 insertion compared all other isolates. For the MP, identity among the isolates was 90-100% in the nt and 96-100% in the predicted aa sequences. Both MP and NP were under stringent purifying selection which was shown by a dNs /dS ratio of 0.016 for the NP and 0.041 for the MP. The presence of BlMaV was confirmed in all of the more than 100 mosaic samples assayed, revealing the close association of virus and disease. The identification and characterization of BlMaV provide crucial information on the biology of the mosaic agent that can be used for adopting better disease management practices. The information on the genetic variation will allow for the development of assays able to detect a wide range of isolates. These assays could be used effectively in the quarantine and certification programs worldwide to prevent the disease spread. ACKNOWLEDGEMENTSThe authors thank USDA-NCPN 12-8100-1572-CA and the Southern Region Small FruitConsortium for the funding provided for this study. REFERENCESHo, T., Sharma, A., Barabote R., Tzanetakis, I.E., Developing plant virus detection and discoverypipeline using next generation sequencing (submitted).Korber, B., 2000. HIV signature and sequence variation analysis. In: Rodrigo, A.G., Learn, G.H.(Eds.), Computational analysis of HIV molecular sequences. Kluwer AcademicPublishers, Dordrecht, Netherlands, pp. 55-72. 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Abbreviations: CPsVCitruspsorosis virus, FreSVFreesia sneak virus, LRNVLettuce ring necrosis virus, MiLBVV-Mirafiori lettuce big-vein virus, RWMVRanunculus white mottle virus, TMMMVTulip mildmottle mosaic virus. RdRpRNA dependent RNA polymerase; NPnucleocapsid protein; NA-Not available; *Only partial sequences of RdRp is available. Proteins RdRp NP 50-58 kDa 22-25 kDaMiLBVV 38 29 1812LRNV37 29 2116CPsV42 35 378TMMMV NA* 27 NA NARWMV NA* 33 NA NAFreSV NA* 27 NA NA Table 2: Primers and PCR conditions used for the amplification of BlMaV RNA 2 and RNA 3 for diversity analysis. PrimerpairsPrimer sequencesPCR conditions RNA2-2FRNA2-2R5'-TTCGATCCCAGCCCTCTCCC-3'5'-AGGCAAAGGGAAAGAAATTCAGGTGTC-3'Initial denaturation at 98° C for 30 s, followed by 35 cycles ofdenaturation at 98° C for 5 s and annealing and extension at 72°C for 30 s, and a final extension at 72° C for 1 min. with PhireHot Start II DNA Polymerase (New England Biolabs, MA).RNA2-1FRNA2-1RRNA2-3FRNA2-3RRNA2-3FRNA2-4RRNA2-1FRNA2-4R5'-GAAAGATAATATGTTCGATCCCA-3'5'-AGGTGTCTAAGCGTTTAGATG-3'5'-CATCTTTCAGCCACATCCTTC-3'5'-ATGAGCATCAGGTCAGGAAG-3'5'-CATCTTTCAGCCACATCCTTC-3'5'-GAAATTCAGGTGTCTAAGCGTT-3'5'-GAAAGATAATATGTTCGATCCCA-3'5'-GAAATTCAGGTGTCTAAGCGTT-3'Initial denaturation at 94°C for 3 min followed by 40 cycles of94°C-30 s, 56°C-30 s and 72°C-1 min. 30 s and a finalextension of 10 min. with Taq polymerase (GenScript USAInc., NJ) RNA3-1FRNA3-1R5'-GGTTGATGGATGCTTACGAA-3'5'-CTTCACTTACCACATTATACATCTC-3'Initial denaturation at 94°C for 3 min followed by 40 cycles of94°C-20 s, 50°C-20 s and 72°C-1 min 30 s with TaqpolymeraseRNA3-2FRNA3-2R5'-TTTGCATTCTGTGACCTTGTG-3'5'-CATACTAAGAGGCCCGACCA-3'Initial denaturation at 95°C for 1 min followed by 35 cycles of95°C-20 s, 50°C-15 s and 72°C-30 s with MyFi DNApolymerase. (Bioline USA Inc., MA) Fig. 1. Blueberry mosaic symptoms. Affected bush shows yellow, yellowish green or pink mottleor mosaic pattern on leaves. Fig. 2. Genome organization of Blueberry mosaic associated virus; ( -): negative-sense RNA(viral RNA); (+): positive sense RNA (viral complementary RNA); RdRp: RNA dependent RNApolymerase; MP: movement protein; NP: nucleocapsid protein. Fig. 3. Phylogenetic analysis using the RNA-dependent RNA polymerase protein sequences ofBlueberry mosaic associated virus (BlMaV) and other negative-strand RNA viruses. Branchlengths are proportional to genetic distances between sequences. The tree was generated by theneighbor-joining method and bootstrap values (indicated for each branch node) were estimatedusing 1,000 replicates. Branch lengths are proportional to genetic distances between sequencesand the scale bar represents substitutions per amino acid site. LRNVLettuce ring necrosis virus(Acc. No. YP_053236), RWMVRanunculus white mottle virus (Acc. No. AF335429_1),MiLBVVMirafiori lettuce big-vein virus (Acc. No. NP_848527), CPsVCitrus psorosis virus(Acc. No. YP_089661), TSWVTomato spotted wilt virus (Acc. No. AEB33901.1), SVNaV-Soybean vein necrosis associated virus (Acc. No. ADX01591), RGSVRice grassy stunt virus(Acc. No. NP_058528), RSVRice stripe virus (Acc. No. AFM93820), LBVaVLettuce big-veinassociated virus (Acc. No. AFA36170), SYNVSonchus yellow net virus (Acc. No. P31332),MMVMaize mosaic virus (Acc. No. Q6E0W6), RYSVRice yellow stunt virus (Acc. No.O10378).