Thanuja Thekke-Veetil

A new ophiovirus is associated with blueberry mosaic disease.

Blueberry mosaic disease (BMD) was first described more than 60 years ago and is caused by a yet unidentified graft transmissible agent. A combination of traditional methods and next generation sequencing disclosed the presence of a new ophiovirus in symptomatic plants. The virus was detected in all BMD samples collected from several production areas of North America and was thus named blueberry mosaic associated virus. Phylogenetic analysis, supported by high bootstrap values, places the virus within the family Ophioviridae. The genome organization resembles that of citrus psorosis virus, the type member of the genus Ophiovirus. The implications of this discovery in BMD control and blueberry virus certification schemes are also discussed.

Molecular characterization and population structure of blackberry vein banding associated virus, new ampelovirus associated with yellow vein disease.

Blackberry yellow vein disease is the most important viral disease of blackberry in the United States. Experiments were conducted to characterize a new virus identified in symptomatic plants. Molecular analysis revealed a genome organization resembling Grapevine leafroll-associated virus 3, the type species of the genus Ampelovirus in the family Closteroviridae. The genome of the virus, provisionally named blackberry vein banding associated virus (BVBaV), consists of 18,643 nucleotides and contains 10 open reading frames (ORFs). These ORFs encode closterovirid signature replication-associated and quintuple gene block proteins, as well as four additional proteins of unknown function. Phylogenetic analyses of taxonomically relevant products consistently placed BVBaV in the same cluster with GLRaV-3 and other members of the subgroup I of the genus Ampelovirus. The virus population structure in the U.S. was studied using the replication associated polyprotein 1a, heat shock 70 homolog and minor coat proteins of 25 isolates. This study revealed significant intra-species variation without any clustering among isolates based on their geographic origin. Further analyses indicated that these proteins are under stringent purifying selections. High genetic variability and incongruent clustering of isolates suggested the possible involvement of recombination in the evolution of BVBaV.

Population structure of blueberry mosaic associated virus: Evidence of reassortment in geographically distinct isolates.

The population structure of blueberry mosaic associated virus (BlMaV), a putative member of the family Ophioviridae, was examined using 61 isolates collected from North America and Slovenia. The studied isolates displayed low diversity in the movement and nucleocapsid proteins and low ratios of non-synonymous to synonymous nucleotide substitutions, indicative of strong purifying selection. Phylogenetic analyses revealed grouping primarily based on geography with some isolates deviating from this rule. Phylogenetic incongruence in the two regions, coupled with detection of reassortment events, indicated the possible role of genetic exchange in the evolution of BlMaV.

Transmission attributes and resistance to rose rosette virus

Rosette caused by rose rosette virus (RRV) is a devastating disease of rose in the United States. The virus was discovered in 2011 and Kochs postulates completed in 2015. Because of these recent discoveries, assumptions about the disease including movement, transmission and resistance are based on visual observations of material that may or may have not been infected by the virus. This study addresses several aspects of virus and disease dynamics. Twenty rose genotypes were screened for mite and/or virus resistance. Phyllocoptes fructiphilus (Keifer), the only known vector of RRV, was able to establish, lay eggs and develop nymphs and adults in all rose genotypes. ‘Stormy Weather’ shows resistance to the virus as assessed in both mite and cleft-grafting transmission experiments. Mites showed a long acquisition/latent period but a rapid inoculation time for RRV. Knowledge of resistance as well as transmission attributes will assist in better management of vector and disease. The identified resistant genotype would be used in areas with high disease pressure to minimize spread, for identification of the mechanisms behind resistance or as breeding parent to incorporate virus resistance to new cultivars. The short inoculation access period suggests that chemical control for this vector may be challenging to undertake. This article is protected by copyright. All rights reserved.

Development of reliable detection assays for blueberry mosaic- and blackberry vein banding- associated viruses based on their population structures

Blueberry mosaic associated virus (BlMaV), the presumed causal agent of the homonymous disease and blackberry vein banding associated virus (BVBaV), a component of the blackberry yellow vein disease complex, are recently characterized RNA viruses. There is a need for efficient and sensitive detection protocols for the two viruses, not only for screening during the nursery propagation process but also in commercial fields to better understand virus epidemiology and minimize disease spread. RNA viruses display significant nucleotide variation forming quasi-species. Therefore, sequence-based detection methodologies, even though sensitive, may lead to false negative results. For this reason, information on the genetic diversity of virus populations is essential to develop diagnostic assays that have the potential to detect all variants. Detection assays for BlMaV and BVBaV were developed based on existing genetic diversity data and were validated by screening samples from different geographical areas in the United States. These detection tests provide sensitivity and specificity and will serve as the protocols of choice for virus screening in Vaccinium and Rubus certification programs in the United States and elsewhere. Given the increasing global trade of both blueberry and blackberry these tests will be valuable in avoiding virus introductions to new areas.

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. ACKNOWLEDGEMENTS The authors thank USDA-NCPN 12-8100-1572-CA and the Southern Region Small Fruit Consortium for the funding provided for this study. REFERENCES Ho, T., Sharma, A., Barabote R., Tzanetakis, I.E., Developing plant virus detection and discovery pipeline 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 Academic Publishers, Dordrecht, Netherlands, pp. 55-72. 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