Cotton leafroll dwarf virus (CLRDV) was first reported in the United States (US) in 2017 from cotton plants in Alabama (AL) and has become widespread in cotton-growing states of the southern US. To investigate the genomic variability among CLRDV isolates in the US, complete genomes of the virus were obtained from infected cotton plants displaying mild to severe symptoms from AL, Florida, and Texas. Eight CLRDV genomes were determined, ranging in size from 5865 to 5867 bp, and shared highest nucleotide identity with other CLRDV isolates in the US, at 95.9–98.7%. Open reading frame (ORF) 0, encoding the P0 silencing suppressor, was the most variable gene, sharing 88.5–99.6% and 81.2–89.3% amino acid similarity with CLRDV isolates reported in cotton growing states in the US and in Argentina and Brazil in South America, respectively. Based on Bayesian analysis, the complete CLRDV genomes from cotton in the US formed a monophyletic group comprising three relatively divergent sister clades, whereas CLRDV genotypes from South America clustered as closely related sister-groups, separate from US isolates, patterns reminiscent of phylogeographical structuring. The CLRDV isolates exhibited a complex pattern of recombination, with most breakpoints evident in ORFs 2 and 3, and ORF5. Despite extensive nucleotide diversity among all available CLRDV genomes, purifying selection (dN/dS < 1) was implicated as the primary selective force acting on viral protein evolution.

Plantain and banana (Musa spp.) are among the most important staple crops for food and income generation for the rural and urban populations in the humid forest agroecological zone of West Africa. Until recently, Cucumber mosaic virus (genus Cucumovirus) and Banana streak virus (genus Badnavirus) were the only viruses reported to occur in Musa spp. in West Africa. In 2011, an outbreak of banana bunchy top disease (BBTD) caused by Banana bunchy top virus (BBTV; genus Babuvirus, family Nanoviridae) was reported in Ouémé Département (6°30′N and 2°36′E) in the Republic of Benin (2). BBTV is one of the most economically important pathogens of Musa spp. It is well established in Central Africa and also in Angola, Malawi, and Zambia in Southern Africa (2). Plants infected at early growth stages are severely dwarfed and do not bear fruit. BBTV is transmitted by the banana aphid Pentalonia nigronervosa, which is widespread in Africa (1). The regions in the Republic of Benin affected by BBTV border Ogun State (7°00′N and 3°35′E) of Nigeria. Epidemiological investigations were conducted during May 2012 at 31 locations in Ogun State to determine the potential risk of BBTV spreading into Nigeria. Plants with typical symptoms of BBTD (stunting, narrow and shortened leaves, chlorotic streaks on petioles and pseudostem) were observed in four locations: Ilashe, Odan-Itoro, Ido-Ologun, and Igbogila. Total DNA was extracted from 90 leaf samples randomly collected from symptomatic and asymptomatic banana and plantain plants in these areas. Samples were tested for BBTV by polymerase chain reaction (PCR) using primer pairs, mREP-F and mREP-R, which amplifies a 241-bp of BBTV DNA-mRep segment (1), and Scp-F and Scp-R specific for approximately 1075-bp BBTV DNA-S that encodes coat protein gene (1). The amplicons of expected size were obtained from 17 of 90 samples analyzed (18.8%). BBTV in the symptomatic plants was further confirmed by nucleic acid spot hybridization (NASH) assay using DIG-labeled 1,075-bp probe corresponding to coat protein gene and chromogenic detection as per the previously described protocol (3). The DIG-probe specifically reacted with nucleic acid from the symptomatic plants, but not with negative controls, providing conclusive evidence for the BBTV. The PCR products of DNA-mRep segment amplified from three banana plants infected with BBTV collected in Ilashe (Ipokia Local Government Area) were purified and sequenced in both directions. The sequences of these isolates were 100% identical with each other (GenBank Accession Nos. JX290301, JX290302, and JX290303). A BLASTn search revealed 100% nucleotide sequence identity with a BBTV isolate from Benin (JQ437548) and 99 to 100% identity with DNA-mRep sequences of several other BBTV isolates from Africa, Australia, India, and the South Pacific. Further analysis of the 241-bp mRep gene sequences with Neighbor-Joining phylogenetic analysis grouped the BBTV isolate with the South Pacific isolates. To our knowledge, this is the first report of BBTV in Nigeria. This underscores need for surveys to assess the extent of BBTV spread in Nigeria and strict implementation of phytosanitary measures, including restrictions on the movement of planting material from disease-affected regions, to prevent further spread of this important disease. References: (1) P. L. Kumar et al. Virus Res. 159:171, 2011. (2) B. Lokossou et al. New Dis. Rep. 25:13, 2012. (3) W. S. Xie and J. S. Hu. Phytopathol. 85:339, 1995.

No abstract

No abstract

Tospoviruses (genus Tospovirus, family Bunyaviridae) are responsible for major losses in an extensive range of crops worldwide. New species of these single-stranded, ambisense RNA viruses regularly emerge and have been shown to maintain heterogeneous populations with individual isolates having quite variable biological and virulence characteristics. Most tospovirus phylogenetic studies have focused on analysis of a single gene, most often the nucleocapsid protein gene. Complete genomic RNA segment amplification as a single fragment would facilitate more detailed analyses of genome-wide sequence variability, but obtaining such sequences for a large number of tospovirus isolates using traditional methods of amplification and cloning of small overlapping fragments is tedious, time consuming and expensive. In this study, protocols were optimized to amplify, clone and sequence full-length M- and S-RNA genome segments of Tomato spotted wilt virus and Impatiens necrotic spot virus. The strategy presented here is straightforward, scalable and offers several advantages over the previously commonplace and overlapping amplicon-based approach. Use of whole genome segments, instead of individual gene sequences or defined portions of genome segments, will facilitate a better understanding of the underlying molecular diversity of tospoviruses in mixed infections.

Grapevine red globe virus (GRGV; genus Maculavirus, family Tymoviridae) has been reported in grapevines (Vitis spp.) from Italy, Greece, France, China, Spain and Germany and in California, U.S.A. (Sabanadzovic et al. 2000; Cretazzo et al. 2017; Fan et al. 2016; Ruiz-Garcia et al., 2018). During surveys of grapevine nurseries, a total of 241 composite samples, each consisting of four petioles from mature leaves/vine from five asymptomatic grapevines, from 33 grapevine (Vitis vinifera) cultivars were collected. Total RNA isolated from these samples using Spectrum Total RNA isolation kit (Sigma-Aldrich, St. Louis, MO) was subjected to high-throughput sequencing (HTS) on an Illumina HiSeq2500 or Novaseq 6000 platforms in paired-end mode (Genomics Core Facility, Huntsman Cancer Institute, Utah University, Salt Lake City, UT). After trimming raw reads based on quality and ambiguity, the paired-end quality reads of approximately 120 (HiSeq) or 145 (Novaseq) base pair (bp) length were assembled de novo into a pool of contigs (CLC Genomics workbench 12). These contigs were subjected to BLASTn analysis against the nonredundant virus database from GenBank (http://www.ncbi.nlm.nih.gov/blast). A total of 49 contig sequences, ranging from 200 to 1645 bp in length with an average coverage ranging up to 418.7, aligning with GRGV genome were detected in cvs. Aglianico, Cabernet franc, Pinot gris and Riesling. BLASTn analysis of contigs greater than 500 bp length showed sequence identity between 88.5% and 95% with corresponding GRGV sequences reported from other countries. These results indicated the presence of genetically distinct isolates of GRGV. HTS data also revealed coinfection of GRGV in all samples with one or more of the following virus and/or viroids: grapevine rupestris stem pitting associated virus, grapevine rupestris vein feathering virus, hop stunt viroid or grapevine yellow speckle viroid-1. To further confirm infection by GRGV, total RNA was extracted from two asymptomatic Pinot gris vines previously tested positive in HTS using Spectrum Total RNA isolation kit and subjected to reverse transcription-PCR using primers specific to the replicase polyprotein gene of the virus (RG4847F: 5’-TGGTCTGTTGTTCGCATCTT-3’ and RG6076R: 5’ CGGAAGGGGAAGCATTGATCT-3’, Cretazzo et al., 2017). Sequence analysis of the approximately1,250 bp amplicons (accession number MT749359) showed 91.2 % nt sequence identity with corresponding sequence of GRGV isolate from Brazil (KX828704.1). To our knowledge, this is the first report of GRGV in Washington State. Together with the report of the occurrence of GRGV in California (Sabanadzovic et al. 2000), these/span> results indicate wide geographical distribution of the virus. Although GRGV can cause asymptomatic infections in grapevines (Martelli et al. 2002), the economic importance of GRGV as single or coinfections with other viruses needs to be examined to assess the potential significance of the virus to grape production and grapevine certification programs.

Tomato chlorotic spot virus (TCSV) is emerging as a significant constraint to vegetable and legume crops in the Americas. The complete genome sequence of a TCSV isolate naturally infecting peanut (Arachis hypogea) in Haiti was determined in the effort to build epidemiological knowledge of the virus.

Cacao Theobroma cacao L. (Malvaceae) is an economically important crop cultivated in tropical climates for the bean from which chocolate and other products are made (Zarrillo et al., 2018). Virus-like symptoms consisting of discoloration, leaf distortion with downward rolling of leaves, and yellow speckling or mottling (Fig. S1), were observed in imported cacao germplasm at the USDA-ARS-SHRS cacao quarantine facilities in the fall of 2020. Total RNA was isolated from leaves collected from four symptomatic plants using silica RNA extraction method (Rott and Jelkmann, 2001). Ribosomal RNA (rRNA)-depleted RNA samples were used for cDNA library construction, followed by high throughput sequencing on Illumina® NovaSeq 6000 platform (Novogene Corp., Sacramento, CA). Quality-filtered, 150-bp paired-ended reads (2,601,293-3,104,474) were assembled de novo using SPAdes v.3.14.1 (Nurk et al., 2013). The contigs (200,799 to 276,851) were queried against the NCBI virus reference sequence (RefSeq) database using the discontiguous megablast algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi?). The resultant contigs (n=1,344) were 150-nt to 1463-nt in length (k-mer coverage from 6.3x to 26,721.7x) and shared their highest nucleotide (nt) identity with potato leafroll virus (PLRV; NC_001747; genus Polerovirus; family Solemoviridae), at 69.1%-72.8%. The contigs pooled from the four samples were assembled into 15 scaffolds. BLASTn analyses of the 15 scaffolds against the RefSeq database indicated the best matches were to thirteen other polerovirus species, with top hits to cereal yellow dwarf virus-RPV (D10206) and pepper vein yellows virus (LC528383), having similarity scores of 66.2% and 100% respectively. The 15 scaffolds matched to the 5’ terminal end, ORF1-2, ORF3, ORF4 and ORF3-5 of the different polerovirus genomes. For confirmatory sequencing, total RNA was subjected to reverse transcription using SuperScript IV (Invitrogen, Carlsbad, CA), followed by RT-PCR amplification with general polerovirus primers PoconF/PoconcpR (Xiang et al., 2008) expected to yield an amplicon of ~1,400-bp located at the 3’ end of the RNA-dependent, RNA polymerase (RdRp), including the complete coat protein (CP) and movement protein (MP) genes. Amplicons were ligated to pGEM-T Easy vector (Promega, Madison, WI) and sequenced bi-directionally by Sanger sequencing (Eton Bio, Research Triangle Park, NC). BLASTn analysis of the polerovirus-like nt sequences (GenBank accession nos. (ON745771-ON745774) indicated the closest relatives were potato leafroll virus (OK058524) and cucumber aphid-borne yellows virus (FJ460218), at 71% and 73%, respectively. The CP amino acid (aa) sequence shared the greatest similarity to cereal yellow dwarf virus RPV (NP_840023), at 53%, and the MP aa sequence shared the greatest aa similarity to wheat yellow leaf dwarf virus-GPV (YP_003029842), at 38%. These results provide robust support for the association of a previously undescribed polerovirus with symptomatic cacao trees, herein named, cacao leafroll virus (Solemoviridae; Polerovirus). Although Koch’s postulates have not been completed to confirm causality, the presence of this virus in cacao germplasm undermine efforts to distribute pathogen-free germplasm and may pose a risk to cacao production in trees established from virus-infected plant material. To our knowledge, this is the first report of a polerovirus infecting cacao trees. All trees of these accessions at the quarantined facility in Miami, FL have been destroyed.