Balance of RNA sequence requirement and NS3/NS3a expression of segment 10 of orbiviruses

Orbiviruses are insect-transmitted, non-enveloped viruses with a ten-segmented dsRNA genome of which the bluetongue virus (BTV) is the prototype. Viral non-structural protein NS3/NS3a is encoded by genome segment 10 (Seg-10), and is involved in different virus release mechanisms. This protein induces specific release via membrane disruptions and budding in both insect and mammalian cells, but also the cytopathogenic release that is only seen in mammalian cells. NS3/NS3a is not essential for virus replication in vitro with BTV Seg-10 containing RNA elements essential for virus replication, even if protein is not expressed. Recently, new BTV serotypes with distinct NS3/NS3a sequence and cell tropism have been identified. Multiple studies have hinted at the importance of Seg-10 in orbivirus replication, but the exact prerequisites are still unknown. Here, more insight is obtained with regard to the needs for orbivirus Seg-10 and the balance between protein expression and RNA elements. Multiple silent mutations in the BTV NS3a ORF destabilized Seg-10, resulting in deletions and sequences originating from other viral segments being inserted, indicating strong selection at the level of RNA during replication in mammalian cells in vitro. The NS3a ORFs of other orbiviruses were successfully exchanged in BTV1 Seg-10, resulting in viable chimeric viruses. NS3/NS3a proteins in these chimeric viruses were generally functional in mammalian cells, but not in insect cells. NS3/NS3a of the novel BTV serotypes 25 and 26 affected virus release from Culicoides cells, which might be one of the reasons for their distinct cell tropism

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

Bluetongue Disabled Infectious Single Animal (DISA) vaccine: Studies on the optimal route and dose in sheep

Bluetongue (BT) is a disease of ruminants caused by bluetongue virus (BTV) transmitted by biting midges of the Culicoides genus. Outbreaks have been controlled successfully by vaccination, however, currently available BT vaccines have several shortcomings. Recently, we have developed BT Disabled Infectious Single Animal (DISA) vaccines based on live-attenuated BTV without expression of dispensable non-structural NS3/NS3a protein. DISA vaccines are non-pathogenic replicating vaccines, do not cause viremia, enable DIVA and are highly protective. NS3/NS3a protein is involved in virus release, cytopathogenic effect and suppression of Interferon-I induction, suggesting that the vaccination route can be of importance. A standardized dose of DISA vaccine for serotype 8 has successfully been tested by subcutaneous vaccination. We show that 10 and 100 times dilutions of this previously tested dose did not reduce the VP7 humoral response. Further, the vaccination route of DISA vaccine strongly determined the induction of VP7 directed antibodies (Abs). Intravenous vaccination induced high and prolonged humoral response but is not practical in field situations. VP7 seroconversion was stronger by intramuscular vaccination than by subcutaneous vaccination. For both vaccination routes and for two different DISA vaccine backbones, IgM Abs were rapidly induced but declined after 14 days post vaccination (dpv), whereas the IgG response was slower. Interestingly, intramuscular vaccination resulted in an initial peak followed by a decline up to 21 dpv and then increased again. This second increase is a steady and continuous increase of IgG Abs. These results indicate that intramuscular vaccination is the optimal route. The protective dose of DISA vaccine has not been determined yet, but it is expected to be significantly lower than of currently used BT vaccines. Therefore, in addition to the advantages of improved safety and DIVA compatibility, the novel DISA vaccines will be cost–competitive to commercially available live attenuated and inactivated vaccines for Bluetongue

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

Stability of Seg-10 mutant viruses.

<p>(A) Stability of all Seg-10 deletion mutants was examined during three successive passages. Complete Seg-10 was amplified by RT-PCR, and Seg-10 stability was examined by gel electrophoresis. wtBTV1 was used as control. (B) Stability of Seg-10 of mutant virus ΔD(S2del) was confirmed for more than ten passages, by complete Seg-10 amplification using RT-PCR, gel electrophoresis and sequencing. (C) Stability of variants of Seg-10 mutant viruses with Seg-2 insertion during three successive passages. Seg-10 of ΔD(S2) and ΔD(S2reposition) were stable during three passages, whereas Seg-10 of ΔD(S2inverted) was not. Amplicons of the original Seg-10 mutant and Seg-10 mutant with additional inserted viral sequences are indicated by a dot and asterisks, respectively.</p

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