Complete Genome Sequence of Mulberry Vein Banding Associated Virus, a New Tospovirus Infecting Mulberry

<div><p>Mulberry vein banding associated virus (MVBaV) that infects mulberry plants with typical vein banding symptoms had been identified as a tentative species of the genus <i>Tospovirus</i> based on the homology of N gene sequence to those of <i>tospoviruses</i>. In this study, the complete sequence of the tripartite RNA genome of MVBaV was determined and analyzed. The L RNA has 8905 nucleotides (nt) and encodes the putative RNA-dependent RNA polymerase (RdRp) of 2877 aa amino acids (aa) in the viral complementary (vc) strand. The RdRp of MVBaV shares the highest aa sequence identity (85.9%) with that of <i>Watermelon silver mottle virus</i> (WSMoV), and contains conserved motifs shared with those of the species of the genus <i>Tospovirus</i>. The M RNA contains 4731 nt and codes in ambisense arrangement for the NSm protein of 309 aa in the sense strand and the Gn/Gc glycoprotein precursor (GP) of 1,124 aa in the vc strand. The NSm and GP of MVBaV share the highest aa sequence identities with those of Capsicum chlorosis virus (CaCV) and <i>Groundnut bud necrosis virus</i> (GBNV) (83.2% and 84.3%, respectively). The S RNA is 3294 nt in length and contains two open reading frames (ORFs) in an ambisense coding strategy, encoding a 439-aa non-structural protein (NSs) and the 277-aa nucleocapsid protein (N), respectively. The NSs and N also share the highest aa sequence identity (71.1% and 74.4%, respectively) with those of CaCV. Phylogenetic analysis of the RdRp, NSm, GP, NSs, and N proteins showed that MVBaV is most closely related to CaCV and GBNV and that these proteins cluster with those of the WSMoV serogroup, and that MVBaV seems to be a species bridging the two subgroups within the WSMoV serogroup of tospoviruses in evolutionary aspect, suggesting that MVBaV represents a distinct tospovirus. Analysis of S RNA sequence uncovered the highly conserved 5’-/3’-ends and the coding regions, and the variable region of IGR with divergent patterns among MVBaV isolates.</p></div

Primers used for amplification of the MVBaV genome.

<p>Primers used for amplification of the MVBaV genome.</p

List of accession number and abbreviation of Tospoviruses used in this study.

<p>List of accession number and abbreviation of Tospoviruses used in this study.</p

Electron micrograph of a MVBaV-infected mulberry plant leaves showing typical tospovirus-like particle morphology.

<p>Typical spherical, enveloped virions are shown accumulating in the endoplasmic reticulum (A) or dispersing in the cytoplasm as single particles in leaf cells (B). The scale bar represents 200 nm.</p

Cloning strategies for L, M, and S RNAs.

<p>Arrows indicate the annealing positions of each primer. To determine the 5′-terminal sequences, total RNAs were denatured by heating at 65°C for 5 min and then mixed with the first primer, LRNA 5–1 for L RNA, MRNA 5–1 for M RNA, and SRNA 5–1 for S RNA, respectively. After removal of template RNAs by RNaseH digestion, PCR amplification of the 5′-cDNAs was performed with Ex Taq DNA polymerase (Takara Bio, Dalian, China) using UPM primer (provided with the kits) and a nested primer, LRNA 5–2 for L RNA, MRNA 5–2 for M RNA, and SRNA 5–2 for S RNA, respectively. To determine the 3′-terminal sequence, first strand cDNA was synthesized by using the primer Tos-UPA. PCR amplification of the 3′-cDNAs was performed with Ex Taq DNA polymerase using the primers Tos-UPA and the LRNA 3 primer for L RNA, MRNA 3 for M RNA, and SRNA 3 for S RNA, respectively. The primers used for RACE and amplifying the MVBaV Genome are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136196#pone.0136196.t001" target="_blank">Table 1</a>.</p

Phylogenetic trees of tospoviruses.

<p>Amino acid sequences of the RNA-dependent RNA Polymerase (RdRp), GP protein (GP),NSm protein (NSm),NSs protein (NSs), and N protein (N) were used to constructed the trees. The dendrograms were produced using the Neighbour—Joining algorithm with 1000 bootstrap replicates. The tospovirus species belonging to WSMoV serogroup are circled. The scale bar represents a relative genetic distance. Numbers above critical branches are significant bootstrap values (>70%). Abbreviations and accession numbers of the analyzed sequences of tospoviruses are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136196#pone.0136196.t002" target="_blank">Table 2</a>.</p

The NSm conserved motifs in viruses of the genus <i>Tospovirus</i>.

<p>The positions of the conserved motifs in NSm are indicated. Abbreviations and accession numbers of the analyzed sequences of tospoviruses are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136196#pone.0136196.t002" target="_blank">Table 2</a>.</p

The symptoms of MVBaV-infected mulberry leaves.

<p>(A) vein banding, (B) mosaic, (C) chlorotic spots, (D) leaf deformation and (E) vein necrosis. The photos were taken over a period of eight months from April to November in 2013.</p

The NSs conserved motifs in viruses of the genus <i>Tospovirus</i>.

<p>The positions of the conserved motifs in NSs are indicated. Abbreviations and accession numbers of the analyzed sequences of tospoviruses are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136196#pone.0136196.t002" target="_blank">Table 2</a>.</p

Colony morphology and virulence assay of mutant strains.

<p>The strains were cultured on PDA at 25°C and photographs were taken on the 14th day. Virulence assays were performed on dormant stems of Chinese chestnut (<i>Castanea mollissima</i>). The inoculated stems were kept at room temperature in a plastic bag to maintain moisture, and the cankers were measured at 4 weeks post-inoculation and photographed. The assays were performed with six replicates per fungal strain.</p