17 research outputs found
Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis
Validation of Arxula Yeast Estrogen Screen assay for detection of estrogenic activity in water samples: Results of an international interlaboratory study
Requirements and comparative analysis of reverse genetics for bluetongue virus (BTV) and African horse sickness virus (AHSV)
<p>Background: Bluetongue virus (BTV) and African horse sickness virus (AHSV) are distinct arthropod borne virus species in the genus Orbivirus (Reoviridae family), causing the notifiable diseases Bluetongue and African horse sickness of ruminants and equids, respectively. Reverse genetics systems for these orbiviruses with their ten-segmented genome of double stranded RNA have been developed. Initially, two subsequent transfections of in vitro synthesized capped run-off RNA transcripts resulted in the recovery of BTV. Reverse genetics has been improved by transfection of expression plasmids followed by transfection of ten RNA transcripts. Recovery of AHSV was further improved by use of expression plasmids containing optimized open reading frames. Results: Plasmids containing full length cDNA of the 10 genome segments for T7 promoter-driven production of full length run-off RNA transcripts and expression plasmids with optimized open reading frames (ORFs) were used. BTV and AHSV were rescued using reverse genetics. The requirement of each expression plasmid and capping of RNA transcripts for reverse genetics were studied and compared for BTV and AHSV. BTV was recovered by transfection of VP1 and NS2 expression plasmids followed by transfection of a set of ten capped RNAs. VP3 expression plasmid was also required if uncapped RNAs were transfected. Recovery of AHSV required transfection of VP1, VP3 and NS2 expression plasmids followed by transfection of capped RNA transcripts. Plasmid-driven expression of VP4, 6 and 7 was also needed when uncapped RNA transcripts were used. Irrespective of capping of RNA transcripts, NS1 expression plasmid was not needed for recovery, although NS1 protein is essential for virus propagation. Improvement of reverse genetics for AHSV was clearly demonstrated by rescue of several mutants and reassortants that were not rescued with previous methods. Conclusions: A limited number of expression plasmids is required for rescue of BTV or AHSV using reverse genetics, making the system much more versatile and generally applicable. Optimization of reverse genetics enlarge the possibilities to rescue virus mutants and reassortants, and will greatly benefit the control of these important diseases of livestock and companion animals.</p
RNA elements in open reading frames of the bluetongue virus genome are essential for virus replication.
Members of the Reoviridae family are non-enveloped multi-layered viruses with a double stranded RNA genome consisting of 9 to 12 genome segments. Bluetongue virus is the prototype orbivirus (family Reoviridae, genus Orbivirus), causing disease in ruminants, and is spread by Culicoides biting midges. Obviously, several steps in the Reoviridae family replication cycle require virus specific as well as segment specific recognition by viral proteins, but detailed processes in these interactions are still barely understood. Recently, we have shown that expression of NS3 and NS3a proteins encoded by genome segment 10 of bluetongue virus is not essential for virus replication. This gave us the unique opportunity to investigate the role of RNA sequences in the segment 10 open reading frame in virus replication, independent of its protein products. Reverse genetics was used to generate virus mutants with deletions in the open reading frame of segment 10. Although virus with a deletion between both start codons was not viable, deletions throughout the rest of the open reading frame led to the rescue of replicating virus. However, all bluetongue virus deletion mutants without functional protein expression of segment 10 contained inserts of RNA sequences originating from several viral genome segments. Subsequent studies showed that these RNA inserts act as RNA elements, needed for rescue and replication of virus. Functionality of the inserts is orientation-dependent but is independent from the position in segment 10. This study clearly shows that RNA in the open reading frame of Reoviridae members does not only encode proteins, but is also essential for virus replication
Overview of Seg-10 deletion mutants with insertions.
<p>Stability of Seg-10 deletion mutants during virus growth is indicated. For unstable mutants, changes in Seg-10 are indicated and specified for segment number of origin and nucleotide numbering (between brackets) of the respective segment. The location of the insertion is indicated by the nucleotide number of full length Seg-10.</p><p>* BTV mutant with the ΔA deletion in Seg-10 was not viable.</p
Phenotype and growth of wild type, AUG1+2 and ΔD(S2)del virus on BSR cells.
<p>(A) BSR cells, 1dpi, infected with MOI 0.1. CPE is clearly visible in BSR cells infected with BTV1. Upper row: Typical BTV1 CPE is indicated (arrows). Cells infected with the double ATG mutant (AUG1+2) also show CPE, but delayed. The ΔD(S2del) virus shows no CPE and infected cells look comparable to uninfected cells. Lower row: Infected monolayers were immunostained with αVP7 MAb. For BTV1 all cells are positive, AUG1+2 shows less positive cells and ΔD(S2del) only shows immunostaining of single cells or small groups of cells. (B) Virus titers of infected cells were examined in medium and cell fractions at time points up to 54 hpi. Virus titers in cell fractions are not significantly different for both viruses, except for 22 hpi. However, virus release in medium is significantly delayed and reduced for ΔD(S2del) virus compared to BTV1. Error bars represent SEM and asterisks indicate a significant difference in virus titer between ΔD(S2del) virus compared to BTV1 with p<0.05.</p
Stability of ΔD(S2delGFP) mutant virus.
<p>(A) ΔD(S2del) virus with the GFP sequence inserted (ΔD(S2delGFP)) was generated. GFP expression was obvious during several successive virus passages in BSR cells, as shown for passage 6 and 7 (p6, p7). GFP expression was less obvious after subsequent passages, as shown for passages 8 and 9 (p8, p9). (B) Genetic stability of Seg-10 of ΔD(S2delGPF) during ten passages was studied by RT-PCR amplification of Seg-10. The original Seg-10 of ΔD(S2delGFP) mutant virus was identified (.), but in subsequent passages additional smaller amplicons became more prominent (*). The middle small band has a deletion in the GFP sequence, the smallest amplicon has a larger deletion in the GFP sequence, and in the largest of the small amplicons, the Seg-2 insertion is also deleted, but a Seg-6 sequence is inserted instead.</p
dsRNA of Seg-10 deletion mutant viruses.
<p>dsRNA was isolated from cells infected with passage 4 of all Seg-10 deletion mutant viruses. Black dots indicate the segments 1–10 of BTV1, with Seg-5 and Seg-6 almost at the same position in the gel. A black dot also indicates the band with the expected size of Seg-10 based on the deletion, for the different mutant viruses. White dots indicate Seg-10 bands of mutant viruses, different from deletion Seg-10 of the expected size. All mutant viruses contain a band with the size of the original deletion Seg-10. All Seg-10 deletion mutant viruses contain Seg-10 variants, except for ΔB and ΔH. Note that the ladder used is made of dsDNA, so the height in the gel of the dsRNA cannot be used to determine the exact size of the band.</p
Representative result of rescue of mutant BTV with a deletion in Seg-10.
<p>BSR cells transfected with all segments of BTV1, BTV1 with Seg-10 ΔE or ΔG and untransfected control 2dpt stained with αVP7 MAb. Almost all cells transfected with the BTV1 segments were infected as was shown by immunostaining in purple. Smaller plaques of positive cells were visible in transfections with mutant Seg-10.</p
Deletion mutant Seg-10 used in reverse genetics for virus rescue.
<p>Deletions were made throughout the ORF of Seg-10. Mutant ΔA was not viable as indicated by a cross. Protein domains encoded by Seg-10 are indicated using different colours. BD = binding domain, LD = late domain, IC = intracellular, TM = trans membrane, EC = extracellular. Nucleotide positions are indicated with numbers. Segment length is indicated next to the illustrations. (A) Mutant segments with consecutive deletions throughout the original Seg-10. (B) Mutants based on segment ΔD, but with inserted viral sequences. Insertions of Seg-1 and Seg-2 are shown in purple and pink, respectively. The orientation of insertions are indicated by arrows. (C) ΔD(S2) segments, but with the insertion in a different location or orientation or with an additional deletion or with the GFP sequence(bright green) inserted.</p