Elsevier

Virus Research

Volume 155, Issue 2, February 2011, Pages 397-405
Virus Research

Complete sequence and genetic characterization of Raspberry latent virus, a novel member of the family Reoviridae,☆☆

https://doi.org/10.1016/j.virusres.2010.11.008Get rights and content

Abstract

A new virus isolated from red raspberry plants and detected in the main production areas in northern Washington State, USA and British Columbia, Canada was fully sequenced and found to be a novel member of the family Reoviridae. The virus was designated as Raspberry latent virus (RpLV) based on the fact that it is symptomless when present in single infections in several Rubus virus indicators and commercial raspberry cultivars. RpLV genome is 26,128 nucleotides (nt) divided into 10 dsRNA segments. The length of the genomic segments (S) was similar to those of other reoviruses ranging from 3948 nt (S1) to 1141 nt (S10). All of the segments, except S8, have the conserved terminal sequences 5′-AGUU----GAAUAC-3′. A point mutation at each terminus of S8 resulted in the sequences 5′-AGUA----GAUUAC-3′. Inverted repeats adjacent to each conserved terminus as well as stem loops and extended pan handles were identified by analyses of secondary structures of the non-coding sequences. All segments, except S3 and S10, contained a single open reading frame (ORF) on the positive sense RNAs. Two out-of-frame overlapping ORFs were identified in segments S3 (ORF S3a and S3b) and S10 (ORF S10a and S10b). Amino acid (aa) alignments of the putative proteins encoded by the main ORF in each segment revealed a high identity to several proteins encoded by reoviruses from different genera including Oryzavirus, Cypovirus, and Dinovernavirus. Alignments of the polymerase, the most conserved protein among reoviruses, revealed a 36% aa identity between RpLV and Rice ragged stunt virus (RRSV), the type member of the genus Oryzavirus, indicating that these two viruses are closely related. Phylogenetic analyses showed that RpLV clusters with members of the genera Oryzavirus, Cypovirus, Dinovernavirus and Fijivirus. These genera belong to the subfamily Spinareovirinae which includes reoviruses with spiked core particles (‘turreted’ reoviruses). In addition, two nucleotide binding motifs, regarded as ‘signature’ sequences among turreted reoviruses, were also found in RpLV P8, suggesting that RpLV is a novel dicot-infecting reovirus in the subfamily Spinareovirinae.

Introduction

The family Reoviridae contains viruses with genomes composed of multiple (9–12) segments of linear double-stranded RNA (dsRNA). This family is the most diverse of the dsRNA virus families, as it includes species isolated from a wide range of hosts including mammals, birds, fish, insects, arachnids, marine protists, crustaceans, fungi and plants (Brussaard et al., 2004, Hillman et al., 2004, Mertens et al., 2005).

More than 75 virus species have been classified as members of 12 well-established genera: Orbivirus, Orthoreovirus, Rotavirus, Coltivirus, Aquareovirus, Cypovirus, Fijivirus, Phytoreovirus, Oryzavirus, Seadornavirus, Mycoreovirus and Idnoreovirus (Mertens et al., 2005). The genera Cardoreovirus, Dinovernavirus, and Mimoreovirus, have been established recently and include viruses isolated from crabs, mosquitoes and marine protists, respectively (Attoui et al., 2005, Attoui et al., 2006, Zhang et al., 2004).

Currently, there are three genera of plant reoviruses; Fijivirus, Oryzavirus and Phytoreovirus. The genus Fijivirus includes members with 10 segments and is divided into 5 groups based on host, vectors and serological properties (Isogai et al., 1998a, Mertens et al., 2000). Members include viruses that infect monocots and insects (Hunter et al., 2009, Nakashima et al., 1996, Noda and Nakashima, 1995). The genus Oryzavirus includes two members; Rice ragged stunt virus (RRSV) and Echinochloa ragged stunt virus (ERSV), also comprised of 10 segments (Chen et al., 1989, Holmes et al., 1995, Shikata et al., 1979, Uyeda and Milne, 1995, Yan et al., 1992). Phytoreovirus includes viruses with 12 segments that infect both monocots and dicots with Wound tumor virus (WTV), Rice dwarf virus (RDV) and Rice gall dwarf virus (RGDV) being the recognized members of the genus (Black, 1945, Mertens et al., 2000, Nuss and Dall, 1990). The recent characterization of Tobacco leaf enation virus (TLEV) (Picton et al., 2007, Rey et al., 1999), and Homalodisca vitripennis reovirus (HoVRV), isolated from the hemipteran Homalodisca vitripennis (Stenger et al., 2009), have expanded this genus now to include insect viruses.

In 1988, a novel reovirus was identified in red raspberry in Washington State, USA. Partial sequence was obtained and used to design detection primers (Jelkmann and Martin, 1989). The virus has been detected commonly in the main raspberry production areas in the Pacific Northwest (PNW), especially in areas where raspberry crumbly fruit disease is prominent (Murant et al., 1974, Martin, 1999).

The new reovirus does not cause symptoms when grafted onto raspberry indicator plants and thus, the name Raspberry latent virus (RpLV) is proposed. The characterization of RpLV has been necessary in order to elucidate possible interactions with other viruses and its implication in crumbly fruit and other raspberry diseases.

Section snippets

Type isolate source

Root cuttings from ‘Meeker’ raspberry were obtained from production fields in northern Washington State, USA. The cuttings were originated from 3-and 4-year-old plants, some of which had shown crumbly fruit symptoms in previous seasons. A total of 90 root cuttings were planted, grown in 1 gal pots and maintained in a greenhouse under standard conditions (12 h day light at 25 °C). It is important to point out that we did not know the status of each cutting at the time of planting, i.e. whether each

Electrophoretic analysis of double-stranded RNAs

Double-stranded RNA from RpLV singly-infected plants was separated by 1.5% agarose gel electrophoresis (AGE) and 10% PAGE. The AGE profile was similar to those of other reoviruses and revealed the presence of multiple segments arranged in three groups, based on their molecular masses (high, intermediate and low) (Fig. 1). The PAGE profile allowed for the separation of three distinct dsRNA segments that co-migrated as a single band in AGE. S1–S4 form the high molecular mass group, the

Discussion

RpLV was first isolated from raspberry in 1988 and has been found at high incidence in the main production areas in the northern Washington State, USA and British Columbia, Canada. The suggested name derives from the fact that RpLV is symptomless in single infections in standard Rubus virus indicators or in the red raspberry cultivars tested. However, RpLV is implicated in a new virus complex responsible for causing severe crumbly fruit in red raspberries (Quito-Avila et al., 2009). The high

Acknowledgements

This work was funded partially by the US Department of Agriculture under the Specialty Crops Research Initiative (SCRI), award number 2009-51181-06022. We are grateful to Dr. Sead Sabanadzovic from Mississippi State University for sharing unpublished data on a closely related reovirus, Simone Alt and Svenja Leible (Julius Kuhn Institute for plant protection in fruit crops and viticulture, Dossenheim, Germany) for initial sequencing work, and to growers in northern Washington State for providing

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