22 uM filters to remove remaining cell fragments and bacteria. The resulting eluates were subsequently subjected to nuclease therapy with one hundred U of DNase I at 37 C for one particular hour to clear away all nucleic acids that are not protected inside virions. The resulting virion enriched samples were used for viral RNA extrac tion employing the QIAamp Viral RNA Mini Kit in accordance for the manufacturers guidelines. Sequence independent single primer amplification was performed fundamentally as previously described with some modifications. Briefly, the extracted RNA was converted into single stranded cDNA using the Transcriptor Initially Strand cDNA Synth esis Kit and one particular uM ran dom primer FR26RV N. Ten ul extracted RNA was denatured at 95 C for five minutes during the presence of primer FR26RV N, straight away followed by cooling on ice.

The remaining reagents were extra. The 20 ul response mix contained one Transcriptor Reverse Transcriptase Response Buffer, dNTP mix, 20 U Protector RNase Inhibitor, 10 units Transcriptor Reverse Transcriptase and 1 ul PCR grade H2O. The reaction was incubated following website at 25 C for 10 minutes followed by 50 C for 60 minutes. Following a reverse transcriptase inactivation stage at 85 C for five minutes and chilling on ice, 2. five U of three 5 exo Klenow Fragment of DNA polymerase had been extra for second strand synthesis employing random primer FR26RV N for 1 hour at 37 C. An enzyme inactivation phase was performed at 75 C for ten minutes. Five microliters of the response mix was utilized as tem plate to get a subsequent PCR amplification. The 50 ul reaction combine consisted of one AmpliTaq Gold 360 DNA buffer, two.

five mM MgCl2, dNTP mix, 2. five U AmpliTaq kept Gold 360 DNA poly merase, 32. seven ul RNase absolutely free water and 1. six uM FR20RV primer. This PCR primer is comple mentary for the amplification tag of FR26RV. The reac tion was incubated at 95 C for 10 minutes, 40 cycles at 95 C for 1 minute, 48 C for one particular minute and 72 C for two minutes followed by a ultimate elongation for 7 minutes at 72 C. The random amplified DNA fragments had been visualised on a 1 % agarose gel. Fragments of 400 1000 base pairs were excised and purified through the gel with the Large Pure PCR Item Purification Kit. The purified PCR fragments have been quantified by spectro photometry. Sequencing Five micrograms of size chosen purified random amplified DNA was sequenced on a GS FLX through the Genomics Core from the University Hospital, University of Leuven, Belgium.

They applied multiplex identifier identification dur ing library planning and GS FLX Titanium series reagents according to their stan dard procedures, aiming for 5000 10000 reads per library. Briefly, adaptors such as common MID tag sequences had been ligated towards the dimension picked double stranded DNA library, followed by single stranded DNA library isolation and library top quality assessment and quantitation. The resulting libraries were then pooled with other MID recognized libraries and emulsion PCR clonal amplification was performed as described through the provider. The amplified libraries had been then loaded on the Pico Titer Plate for sequencing by the Genome Sequencer FLX. Data were offered towards the authors by secured ftp server. Data Analysis The obtained raw sequence information had been assembled applying SeqMan NGen edition three. 0. The reads have been trimmed to take out primer sequences as well as low top quality ends. Conventional assem bling and filtering parameters have been used. Initial we per formed a de novo assembly and entered the resulting contigs into a Blastn similarity search towards public sequence data bases for identification.