Generation of ΔNS4 Bluetongue viruses by reverse genetics.

<p>(A) BTV segment 9 open reading frames. VP6 amino acid residues are written in black, NS4 amino acid residues are written in grey. The nucleotides at positions 183 (T), 252 (G) and 381 (T) were mutated to C, A and A, respectively (bold). Note that whilst these mutations do not change any amino acid residues of VP6, they remove the initiation codon of NS4 (position182) and introduce two stop codons into the NS4 coding sequence at amino acid positions 24 and 67. (B) Transfected BSR cells with BTV transcripts generated <i>in vitro</i> (0.5×10¹¹ molecules per segment for BTV-1 and 1×10¹¹ molecules per segment for BTV-8). Cell monolayers were stained using crystal violet at 72 h post-transfection. As negative controls, ΔVP6 assays correspond to using a segment 9 containing a stop codon at position 79 in the VP6 gene. (C) Agarose gel (1.5%) of purified BTV genomic dsRNA. BSR cells infected at a MOI of 0.01 were collected at 72 h post infection and BTV dsRNA was purified as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002477#s4" target="_blank">Materials and Methods</a>. 2 µg of dsRNA was loaded in each lane. (D) Western blotting of cellular extracts (lysate) of BSR cells infected at a MOI of 0.01. Cells were analyzed 36 h post-infection and blots were incubated with antisera against VP7, NS4 and γ-tubulin as indicated. Note that the double NS4 band in the BTV-1 sample is not a feature observed consistently. (E) Electron microscopy of BSR cells infected by BTV1-ΔNS4. Note cells display all the major features of BTV-infected cells including NS1 tubules (T), viral inclusion bodies (VIB) and viral particles (arrows). Scale bar = 1 µm.