Alfalfa virus S, a new species in the family Alphaflexiviridae.

A new species of the family Alphaflexiviridae provisionally named alfalfa virus S (AVS) was discovered in alfalfa samples originating from Sudan. A complete nucleotide sequence of the viral genome consisting of 8,349 nucleotides excluding the 3' poly(A) tail was determined by high throughput sequencing (HTS) on an Illumina platform. NCBI BLAST searches revealed that the virus shares the greatest degree of sequence identity with members of the family Alphaflexiviridae, genus Allexivirus. The AVS genome contains six computationally-predicted open reading frames (ORF) encoding viral replication protein, triple gene block protein 1 (TGB1), TGB2, TGB3-like protein, unknown 38.4 kDa protein resembling serine-rich 40 kDa protein characteristic for allexiviruses, and coat protein (CP). AVS lacks a clear 3' proximal ORF that encodes a nucleic acid-binding protein typical for allexiviruses. The identity of the virus was confirmed by RT-PCR with primers derived from the HTS-generated sequence, dot blot hybridization with DIG-labeled virus-specific RNA probes, and Western blot analysis with antibodies produced against a peptide derived from the CP sequence. Transmission electron microscopic observations of the infected tissues showed the presence of filamentous particles similar to allexiviruses in their length and appearance. To the best of our knowledge, this is the first report on the identification of a putative allexivirus in alfalfa (Medicago sativa). The genome sequence of AVS has been deposited in NCBI GenBank on 03/02/2016 as accession № KY696659

Western blot analysis of the alfalfa samples.

<p>Membranes were probed with antibody produced against the peptide CP1. M, ColorBurst electrophoresis marker (Sigma). Lane 1 to 9: alfalfa samples 98.3A (hybridization-positive), 89.2AS, 89.3H, 89.1H, 89.1AS, 98.1A (hybridization-positive), 98.3A(0) (hybridization-positive), 98.2RR and 97.2A, respectively. Arrow indicates bands specific to the AVS CP.</p

Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

Dot blot hybridization assay with DIG-labeled RNA probes.

<p>RNA probes derived from the AVS CP. (A) and p38.4 (B) ORFs. Total RNA was extracted from the following alfalfa samples: 1, 98.3A; 2, 89.2AS; 3, 89.3H; 4, 89.1H; 5, 89.1AS; 6, 98.1A; 7, 98.3A (0); 8, 98.2RR and 9, 97.2A.</p

Electron micrographs of viral particles observed in the AVS-infected alfalfa samples 98.3A, 98.1A and 98.3A(0).

<p>Scale bars represent 100 nm and 200 nm (left panel and right panel, respectively).</p

Phylogenetic relationship between alfalfa virus S (highlighted), classified allexiviruses and unassigned members of the family <i>Alphaflexiviridae</i>.

<p>The tree was built based on the available complete nucleotide sequences using MEGA 7 software (version 7.0.21) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178222#pone.0178222.ref025" target="_blank">25</a>] and the Neighbor-Joining method. The optimal tree with the sum of branch length = 3.10976014 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches.</p

Adjusted open reading frame finder output (ORFfinder, NCBI, https://www.ncbi.nlm.nih.gov/orffinder/) showing genome organization of alfalfa virus S.

<p>ORF1, encoding RNA-dependent RNA polymerase (RdRp); ORF 2,3, and 4, encoding triple gene block proteins (TGB) 1, 2 and TGB3-like protein, respectively; ORF5, encoding p38.4, an unknown protein; ORF6, encoding AVS coat protein (CP).</p

Effects of tissue type and season on the detection of regulated sugarcane viruses by high throughput sequencing

Abstract High throughput sequencing (HTS) can supplement and may replace diagnostic tests for plant pathogens. However, the methodology and processing of HTS data must first be optimized and standardized to ensure the sensitivity and repeatability of the results. Importation of sugarcane into the United States is highly regulated, and sugarcane plants are subjected to strict quarantine measures and diagnostic testing, especially for the presence of certain viruses of regulatory concern. Here, we tested whether HTS could reliably detect four RNA and three DNA sugarcane viruses over three seasons (fall, winter, and spring) and in three tissue types (root, stem, and leaves). Using HTS on ribosomal depleted total RNA samples, we reliably detected RNA viruses in all tissue types and across all seasons, but we failed to confidently detect DNA viruses in some samples. We recommend that future optimization be employed to ensure the robust and reliable detection of all regulated sugarcane viruses by HTS

Identification and characterization of Miscanthus yellow fleck virus, a new polerovirus infecting Miscanthus sinensis.

Miscanthus sinensis is a grass used for sugarcane breeding and bioenergy production. Using high throughput sequencing technologies, we identified a new viral genome in infected M. sinensis leaf tissue displaying yellow fleck symptoms. This virus is most related to members of the genus Polerovirus in the family Luteoviridae. The canonical ORFs were computationally identified, the P3 coat protein was expressed, and virus-like particles were purified and found to conform to icosahedral shapes, characteristic of the family Luteoviridae. We propose the name Miscanthus yellow fleck virus for this new virus