Construction and evaluation of infectious cDNA clones and analysis of packaging determinants of Soybean dwarf virus

Soybean dwarf virus (SbDV) is a phloem-limited, aphid-transmitted luteovirus that causes dwarfing of soybean plants. In the present study, infectious clones of two dwarfing isolates of SbDV were constructed, and methods for Agrobacterium-mediated inoculation of plants were standardized and used to introduce them into fava bean, Nicotiana benthamiana, ???Puget??? pea and red clover plants. SbDV virions purified from N. benthamiana leaves infiltrated with either of these cDNA clones encapsidated genomic RNA (gRNA), but small subgenomic RNA (SsgRNA) was encapsidated by only one isolate and hence its packaging was isolate specific. Site-specific, deletion and domain-exchange mutagenesis indicated that, open reading frames (ORFs) 1, 2, 4, and 5 and interacting stem-loop structures in the 5??? and 3??? untranslated regions (UTRs) were required for replication of SbDV gRNA and that sequences in the 3??? UTR were involved in isolate-specific encapsidation of SbDV SsgRNA. Preferential trans-encapsidation of non-replicating mutant gRNAs and large subgenomic RNA (LsgRNA) by coat protein provided by replicating wild-type virus suggested that encapsidation of SbDV gRNA is replication independent, and that two interacting signals located in ORFs 1 and 5 differentially regulate encapsidation of SbDV gRNA and LsgRNA. The present study also showed that soybean aphids (Aphis glycines) can vector SbDV, that the readthrough protein (RTP), a minor structural protein involved in specificity of aphid transmission, is much more diverse than the major coat protein among Midwestern SbDV isolates, and that no mutations occurred in the RTP as a result of aphid transmission of SbDV from red clover into soybean plants

Soybean Thrips (Thysanoptera: Thripidae) Harbor Highly Diverse Populations of Arthropod, Fungal and Plant Viruses

Soybean thrips (Neohydatothrips variabilis) are one of the most efficient vectors of soybean vein necrosis virus, which can cause severe necrotic symptoms in sensitive soybean plants. To determine which other viruses are associated with soybean thrips, the metatranscriptome of soybean thrips, collected by the Midwest Suction Trap Network during 2018, was analyzed. Contigs assembled from the data revealed a remarkable diversity of virus-like sequences. Of the 181 virus-like sequences identified, 155 were novel and associated primarily with taxa of arthropod-infecting viruses, but sequences similar to plant and fungus-infecting viruses were also identified. The novel viruses were predicted to have positive-sense RNA, negative-stranded RNA, double-stranded RNA, and single-stranded DNA genomes. The assembled sequences included 100 contigs that represented at least 95% coverage of a virus genome or genome segment. Sequences represented 12 previously described arthropod viruses including eight viruses reported from Hubei Province in China, and 12 plant virus sequences of which six have been previously described. The presence of diverse populations of plant viruses within soybean thrips suggests they feed on and acquire viruses from multiple host plant species that could be transmitted to soybean. Assessment of the virome of soybean thrips provides, for the first time, information on the diversity of viruses present in thrips

A Virus in American Blackcurrant (Ribes americanum) with Distinct Genome Features Reshapes Classification in the Tymovirales

A novel virus with distinct genome features was discovered by high throughput sequencing in a symptomatic blackcurrant plant. The virus, tentatively named Ribes americanum virus A (RAVA), has distinct genome organization and molecular features bridging genera in the order Tymovirales. The genome consists of 7106 nucleotides excluding the poly(A) tail. Five open reading frames were identified, with the first encoding a putative viral replicase with methyl transferase (MTR), AlkB, helicase, and RNA dependent RNA polymerase (RdRp) domains. The genome organization downstream of the replicase resembles that of members of the order Tymovirales with an unconventional triple gene block (TGB) movement protein arrangement with none of the other four putative proteins exhibiting significant homology to viral proteins. Phylogenetic analysis using replicase conserved motifs loosely placed RAVA within the Betaflexiviridae. Data strongly suggest that RAVA is a novel virus that should be classified as a species in a new genus in the Betaflexiviridae or a new family within the order Tymovirales

Discovery of a Novel Member of the <em>Carlavirus</em> Genus from Soybean (<em>Glycine max</em> L. Merr.)

A novel member of the Carlavirus genus, provisionally named soybean carlavirus 1 (SCV1), was discovered by RNA-seq analysis of randomly collected soybean leaves in Illinois, USA. The SCV1 genome contains six open reading frames that encode a viral replicase, triple gene block proteins, a coat protein (CP) and a nucleic acid binding protein. The proteins showed highest amino acid sequence identities with the corresponding proteins of red clover carlavirus A (RCCVA). The predicted amino acid sequence of the SCV1 replicase was only 60.6% identical with the replicase of RCCVA, which is below the demarcation criteria for a new species in the family Betaflexiviridae. The predicted replicase and CP amino acid sequences of four SCV1 isolates grouped phylogenetically with those of members of the Carlavirus genus in the family Betaflexiviridae. The features of the encoded proteins, low nucleotide and amino acid sequence identities of the replicase with the closest member, and the phylogenetic grouping suggest SCV1 is a new member of the Carlavirus genus

Molecular Characterization of Divergent Closterovirus Isolates Infecting Ribes Species

Five isolates of a new member of the family Closteroviridae, tentatively named blackcurrant leafroll-associated virus 1 (BcLRaV-1), were identified in the currant. The 17-kb-long genome codes for 10 putative proteins. The replication-associated polyprotein has several functional domains, including papain-like proteases, methyltransferase, Zemlya, helicase, and RNA-dependent RNA polymerase. Additional open reading frames code for a small protein predicted to integrate into the host cell wall, a heat-shock protein 70 homolog, a heat-shock protein 90 homolog, two coat proteins, and three proteins of unknown functions. Phylogenetic analysis showed that BcLRaV-1 is related to members of the genus Closterovirus, whereas recombination analysis provided evidence of intraspecies recombination