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High-Throughput Sequencing Reveals Further Diversity of Little Cherry Virus 1 with Implications for Diagnostics

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Abstract: Little cherry virus 1 (LChV1, Velarivirus, Closteroviridae) is a widespread pathogen of sweet or sour cherry and other Prunus species, which exhibits high genetic diversity and lacks a putative efficient transmission vector. Thus far, four distinct phylogenetic clusters of LChV1 have been described, including isolates from different Prunus species. The recent application of high throughput sequencing (HTS) technologies in fruit tree virology has facilitated the acquisition of new viral genomes and the study of virus diversity. In the present work, several new LChV1 isolates from different countries were fully sequenced using different HTS approaches.

Our results reveal the presence of further genetic diversity within the LChV1 species. Interestingly, mixed infections of the same sweet cherry tree with different LChV1 variants were identified for the first time. Taken together, the high intra-host and intra-species diversities of LChV1 might affect its pathogenicity and have clear implications for its accurate diagnostics.

Keywords: LChV1; Closteroviridae; intra-host variability; high-throughput sequencing; diagnostics
1. Introduction
Little cherry virus 1 (LChV1), a member of the genus Velarivirus (family Closteroviridae), is a graft-transmissible pathogen and its host range includes mainly sweet or sour cherry and other Prunus species [1]. Besides the typical reddening frequently observed in cherry leaves, some LChV1 isolates have been associated with various plant disorders [2,3]. LChV1 has a long, positive-sense, single-stranded RNA genome, which encodes eight open reading frames (ORFs) [4]. ORF 1a and ORF 1b, with ORF 1b expressed by a frameshift of ORF 1a, encode a large protein with papain-like proteinase (P-PRO), methyltransferase (MET) and Helicase (HEL) domains and a protein containing an RNA-dependent RNA polymerase (RdRp) conserved domain, respectively. ORF 2 encodes a small hydrophobic protein (4 kDa) which partially overlaps with the ORF 3 that encodes a 70 kDa heat-shock protein 70 homolog (HSP70h). ORF 4 encodes a polypeptide of 61 kDa and partially overlaps with ORF 3, ORF 5 and ORF 6 encode the coat protein (CP) and the CP minor (CPm), respectively, whereas ORF 7 and ORF 8 encode polypeptides of respectively 21 and 27 kDa, with currently unknown functions.
LChV1 exhibits high genetic diversity as revealed from partial or full genome sequencing of various isolates. So far, the complete genome of several LChV1 isolates has been determined [2,3,4,5,6,7]. The UW2 and ITMAR isolates are highly similar, whereas isolate V2356 differs significantly from them and represents the first fully sequenced one of a second phylogenetic group which also includes isolates from the US [2,3]. A third group is formed with the isolates Jerte, Ponferrada, Taian and YD [5,6,7]. Phylogenetic analyses using partial sequences of the RdRp, the HSP70h and the CP have classified LChV1 isolates into 4 clusters, including the three above mentioned ones [8]. Even though the intragroup diversity appears to be relatively low (3.3% to 7.4% in nts), the genetic distances between the different phylogenetic groups are higher (15% to 39% in nts) [3,5,6,7,8].
High throughput sequencing (HTS) has been used in plant virology since 2009 [9,10]. HTS provides highly efficient, rapid and cost-effective sequencing of the genomes of plant viruses and viroids [11]. Different approaches, such as sequencing of total RNA, double-stranded RNA (dsRNA) or small RNAs (sRNAs) have been developed. HTS was used in a number of studies in plant virology including, but not limited to, discovery of novel viruses and viroids as well as analysis of genome diversity and evolution, and study of pathogen epidemiology and population structure [12,13,14]. Another area where HTS has proven very valuable is in the detection of divergent variants of known viruses that escape existing detection procedures, particularly PCR or RT-PCR assays [15]. Because of its unbiased nature, the data obtained by HTS may provide a better knowledge of the polyvalence or specificity of existing assays and, if needed, facilitate the design of new detection primers of broader specificity, thus contributing to the improvement of classical detection methods.
In the present study, seven novel LChV1 isolates were fully sequenced using HTS technologies. One of the isolates (G15 3) was recently partially characterized and shown to exhibit the highest genetic divergence from all so far known isolates [8]. The sequences of the other LChV1 isolates described herein were acquired during HTS analysis of several sweet or flowering cherry samples from three different countries. Our results highlight the presence of further genetic diversity in the populations of LChV1, with clear implications for the diagnosis of this viral agent of quarantine status or included in cherry certification in a number of countries.


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Jul 24, 2018
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