Text S1 Endothelial cell specific markers included CD31 and CD54. CD80 and CD86 were included as non-endothelial cell specific markers, though a previous report has identified low level CD80 expression on primary murine lung endothelial cells [71] . Fluorescence was determined relative to unstained cells (grey). Cell morphology and surface expression was similar to previously characterized primary endothelial cells [71] . Found at: doi:10.1371/journal.ppat.1000152.s006 (0.89 MB TIF) Figure S5 Isolation and characterization of murine lung endothelial cells infected in vivo. (A) Primary murine lung endothelial cells were isolated for analysis of infection in vivo as described in Materials and Methods. Lung cells were stained for the endothelial cell marker CD31, as well as with a cocktail of PE labeleled antibodies specific to the following cell types (which includes potential contaminating infected cells): macrophages, granulocytes, CD8 + T lymphocytes, CD4 + T lymphocytes, and B lymphocytes. CD31 positive cells were enriched from total lung cells and then finally depleted of cells stained with the PE cocktail. (B) Flow cytometric analysis of lung cell separation following in vivo infection was performed on total lung cells and PE depleted/ CD31+ enriched cells. Left and middle panels show representative data (1610 6 cells per stain) from an infected mouse (Fig. 8D ). Gates were set on unstained cells, and data are representative of the four mice analyzed in Fig. 8D . Splenocytes (right panel) were included as a staining control. Found at: doi:10.1371/journal.ppat.1000152.s007 (0.20 MB TIF) Analysis of synonymous codon usage and evolution of begomoviruses Begomoviruses are single-stranded DNA viruses and cause severe diseases in major crop plants worldwide. Based on current genome sequence analyses, we found that synonymous codon usage variations in the protein-coding genes of begomoviruses are mainly influenced by mutation bias. Base composition analysis suggested that the codon usage bias of AV1 and BV1 genes is significant and their expressions are high. Fourteen codons were determined as translational optimal ones according to the comparison of codon usage patterns between highly and lowly expressed genes. Interestingly the codon usages between begomoviruses from the Old and the New Worlds are apparently different, which supports the idea that the bipartite begomoviruses of the New World might originate from bipartite ones of the Old World, whereas the latter evolve from the Old World monopartite begomoviruses. Synonymous codon usage bias has been investigated in many organisms, as the genetic code is degenerate. The synonymous codons are also non-randomly used in viruses infecting living organisms. Several factors such as mutational bias (Jenkins and Holmes, 2003; Gu et al., 2004; Zhou et al., 2005) , translational selection (Sau et al., 2005a; 2005b; 2005c) , gene function (Wang et al., 2002; Gu et al., 2004; Zhou et al., 2005) , gene length (Sau et al., 2005a) , and CpG island (Shackelton et al., 2006) were found to influence codon usage in animal viruses and phages, and mutational bias was found as the major determinant factor. Adams and Antoniw (2004) also suggested that mutational bias rather than translational selection was the major determinant of codon usage variation amongst plant viruses.