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. 2019 Nov 19;16(1):139.
doi: 10.1186/s12985-019-1243-4.

Intra-species recombination among strains of the ampelovirus Grapevine leafroll-associated virus 4

Jati Adiputra  1   2 , Sridhar Jarugula  1 , Rayapati A Naidu  3
Affiliations

Intra-species recombination among strains of the ampelovirus Grapevine leafroll-associated virus 4

Jati Adiputra et al. Virol J. .

Abstract

Background: Grapevine leafroll disease is one of the most economically important viral diseases affecting grape production worldwide. Grapevine leafroll-associated virus 4 (GLRaV-4, genus Ampelovirus, family Closteroviridae) is one of the six GLRaV species documented in grapevines (Vitis spp.). GLRaV-4 is made up of several distinct strains that were previously considered as putative species. Currently known strains of GLRaV-4 stand apart from other GLRaV species in lacking the minor coat protein.

Methods: In this study, the complete genome sequence of three strains of GLRaV-4 from Washington State vineyards was determined using a combination of high-throughput sequencing, Sanger sequencing and RACE. The genome sequence of these three strains was compared with corresponding sequences of GLRaV-4 strains reported from other grapevine-growing regions. Phylogenetic analysis and SimPlot and Recombination Detection Program (RDP) were used to identify putative recombination events among GLRaV-4 strains.

Results: The genome size of GLRaV-4 strain 4 (isolate WAMR-4), strain 5 (isolate WASB-5) and strain 9 (isolate WALA-9) from Washington State vineyards was determined to be 13,824 nucleotides (nt), 13,820 nt, and 13,850 nt, respectively. Multiple sequence alignments showed that a 11-nt sequence (5'-GTAATCTTTTG-3') towards 5' terminus of the 5' non-translated region (NTR) and a 10-nt sequence (5'-ATCCAGGACC-3') towards 3' end of the 3' NTR are conserved among the currently known GLRaV-4 strains. LR-106 isolate of strain 4 and Estellat isolate of strain 6 were identified as recombinants due to putative recombination events involving divergent sequences in the ORF1a from strain 5 and strain Pr.

Conclusion: Genome-wide analyses showed for the first time that recombinantion can occur between distinct strains of GLRaV-4 resulting in the emergence of genetically stable and biologically successful chimeric viruses. Although the origin of recombinant strains of GLRaV-4 remains elusive, intra-species recombination could be playing an important role in shaping genetic diversity and evolution of the virus and modulating the biology and epidemiology of GLRaV-4 strains.

Keywords: Ampelovirus; Grapevine leafroll disease; Grapevine leafroll-associated virus 4.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Analysis of recombination events in the genome of GLRaV-4 strains. (a) Graphical representation (not drawn to scale) of the generalized genome map of GLRaV-4. Individual open reading frames (ORFs) are shown as boxes with associated protein designations used for closteroviruses [2] and numbered 1 to 6 above the diagram. Abbreviations of ORFs: L-Pro, papain-like leader protease; MET, methyltransferase domain; HEL, RNA helicase domain; AlkB, the AlkB domain; RdRp, RNA-dependent RNA polymerase; p5, 5 kDa protein; Hsp70h, heat shock protein 70 homolog; CP, coat protein; p23, 23 kDa protein. Lines at the genome extremities represent non-translated regions. b Putative recombinant events in isolates LR106 and Estellat. (B-1) and (B-2) represent, respectively, recombination event-1 (nt 4105–5240) and event-2 (nt 627–1551) in ORF1a of the LR106 isolate and (B-3) represents recombinant event (nt 1–6312) in the genome of the Estellat isolate identified by the RDP. The X-axis indicates the nucleotide position in the alignment and the Y-axis shows informative nucleotide pairwise identity between parental and recombinant isolates. The color key of the parental isolates is shown next to the plots
Fig. 2
Fig. 2
Multiple sequence alignment of the (a) 5′ and (b) 3′ nontranslated regions of GLRaV-4 strains. Asterisk (*) indicates conserved residues. The conserved nt at the 5' and 3' ends is highlighted. The alignment was adjusted manually and gaps (shown as '-') introduced for optimal alignment of sequences
Fig. 3
Fig. 3
Phylogenetic evidence for recombination among GLRaV-4 strains. Nucleotide sequence corresponding to (a) the CP, (b) ORF1a, recombinant regions identified for putative (c) event-1 (nt 4105–5240) and (d) event-2 (nt 627–1551) in ORF1a of the LR106 isolate, and (e) event-3 (nt 1–6312) in the Estellat isolate were used for constructing the Maximum-likelihood method-besed trees with 1000 replicates, using the MEGA 7 software. Recombinant isolates showing phylogenetic discordance are indicated in red color. Refer to Fig. 1b and Table 2 for details of putative recombinant event-1, event-2 and event3
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