Computational protocol: A dsRNA virus with filamentous viral particles

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[…] The cDNA sequences of genomic dsRNAs were determined as previously described. The 5′- and 3′-terminal sequences of the dsRNAs were obtained from dsRNAs extracted and individually separated from purified virus particles (see below), as previously described. At least three independent clones of each fragment were sequenced in both directions. Sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd, China, and every nucleotide was determined in at least three independent overlapping clones in both orientations. The full-length of the shorter dsRNAs (dsRNAs 7 and 8) were also determined as previously described with minor modifications. Briefly, the 3′ terminus of each dsRNA strand was ligated with the closed adaptor primer RACE-OLIGO with a phosphorylated (p) 5′ end and an NH2 3′ end (5′-p-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2)-3′), reverse transcribed with M-MLV reverse transcriptase (Promega Corporation) using the oligo REV (5′-GGCAATTAACCCTCACTAAAG-3′, complementary to the adaptor at positions 46–26), amplified with PCR using primer O5RACE-2 (5′-TCACTAAAGAATTCGATCGATC-3′, complementary to the adaptor at positions 34–17), followed by nested PCR amplication using O5RACE-3 (5′-CGATCGATCATGATGCAATGC-3′, complementary to the adaptor at positions 17–1), and cloned as described above.Sequence similarity searches were performed using National Center for Biotechnology Information (NCBI) databases with the BLAST program. Multiple alignments of nucleic and amino-acid sequences were conducted using MAFFT version 6.85, as implemented at http://www.ebi.ac.uk/Tools/msa/mafft/ with default settings except for refinement with 10 iterations. Identity analyses were conducted using the MegAlign program (version 5.00) with the Clustal W method (DNASTAR Inc.). The phylogenetic tree for RdRp sequences was constructed as previously described. ORFs were deduced using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/). Functions of the deduced proteins were predicted with hidden Markov models implemented in the HHpred (Homology Detection & Structure Prediction by HMM-HMM comparison) package (http://toolkit.tuebingen.mpg.de/hhpred). [...] A mycelial plug was grown at 25 °C in the dark for 8 days in sterilized cellophane membranes placed on PDA. After harvest, 30 g mycelia was ground to a fine powder in liquid nitrogen, and the resulting powder was subjected to extraction. Briefly, the powder was mixed with 100 mM phosphate buffer (PB; 8.0 mM Na2HPO4, 2.0 mM NaH2PO4, pH 7.0) and centrifuged at 12,096 × g at 4 °C for 30 min to remove cellular debris. The supernatant was further ultracentrifuged (Optima LE-80K; Beckman Coulter, Inc.) at 110,000 × g at 4 °C for 1 h to collect the sediment, which was resuspended in 100 mM PB buffer. The crude extract was further subjected to virus purification following sucrose gradient centrifugation as previously described, or by centrifugation at 55,000 × g at 4 °C for 1.5 h through a cushion of CsCl (1.45 g/cm3), as previously described. The crude or purified virus particle preparations were negatively stained with 1% uranyl acetate on carbon-coated 400-mesh copper grids and examined by TEM (H-7000FA; Hitachi) before storage at −70 °C. The widths and lengths of the particles were measured using ImageJ 1.43, and the width of each particle was determined based on at least five measurements along the particles selected at random. [...] Descriptive statistics were determined, and χ 2-tests, one-way analysis of variance, and Tukey post hoc tests were performed using SPSS Statistics 17.0 (WinWrap Basic; http://www.winwrap.com). p < 0.05 was considered to indicate statistical significance. […]

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