An Insect Nidovirus Emerging from a Primary Tropical Rainforest

Zirkel, Florian; Kurth, Andreas; Quan, Phenix-Lan; Briese, Thomas; Ellerbrok, Heinz; Pauli, Georg; Leendertz, Fabian H.; Lipkin, W. Ian; Ziebuhr, John; Drosten, Christian; Junglen, Sandra

Genome characterization and phylogenetic analyses. The nucleotide sequence of the CAVV genome was analyzed for ORFs and translated. Nucleotide and amino acid sequences were compared with other sequences by BLASTn, BLASTx, tBLASTx, and psiBLAST with the GenBank database (http://www.ncbi.nlm.Nih.gov/Genbank), and protein motifs were identified by web-based comparison to the Pfam database (http: //www.pfam.janelia.org). Identification of cleavage sites for signal peptides was accomplished by using signalP-NN (http://www.cbs.dtu.dk /services/SignalP). Prediction of the hydropathy profile was performed by TMHMM v2.0 (http://www.cbs.dtu.dk/services/TMHMM/), and N-linked glycosylation sites were identified using the NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc). RNA folding was modeled by using the Mfold server (http://mfold.bioinfo.rpi.edu/cgi-bin/rna -form1.cgi) (65) . For phylogenetic analysis, CAVV amino acid sequences were aligned with representative sequences of other nidoviruses in MEGA v5.0 (66) . Alignments were optimized according to published crystal structure predictions. Phylogenetic analysis of amino acid sequences was conducted by the neighbor-joining (NJ) algorithm with the BLOSUM62 substitution matrix for distance correction with 1,000 bootstrap replicates in MEGA v5.0. Maximum-likelihood analyses were done with Fasttree selected genomic regions. Phylogenetic analyses were performed using the NJ algorithm, a BLOSUM62 substitution matrix, and no distance correction. Indels were fully deleted. Significance was tested by bootstrap analysis using 1,000 resampling steps as implemented in MEGA 5.0 (66) . Bootstrap values are shown above nodes. Analyses were also performed using the maximumlikelihood algorithm in FastML with the same settings. Results of this analysis are not shown because of congruent topologies. Bootstrap support values (1,000 replicates) from maximum-likelihood analysis are shown in grey below nodes. (67) , and tree files were displayed in MEGA versus 5.0. Evolutionary divergence over sequence pairs was estimated in MEGA versus 5.0.