Requirements and comparative analysis of reverse genetics for bluetongue virus (BTV) and African horse sickness virus (AHSV)
Reverse genetics (RG) has been developed for viruses of many families including those with double stranded RNA (dsRNA) segmented genomes. The latter includes members of Birnaviridae [52, 53], and different genera within the Reoviridae family [25]. RG for BTV, the prototype member of the Orbivirus genus has also been developed. This RG method followed two subsequent transfections of in vitro synthesized positive single stranded capped run-off RNA transcripts [23]. Capped RNAs expressing VP1, 3, 4, 6, and NS1 and NS2 were used for primary protein expression followed by transfection of a set of ten uncapped RNA transcripts to recover BTV containing these genome segments [26]. However, a similar RG system for AHSV was not successful [12]. These results indicate that recovery of BTV is more efficient than for AHSV.
Primary protein expression by the first transfection of RNA transcripts can be replaced by plasmid-driven expression for BTV recovery [42]. A similar method was also successful for AHSV [12, 24]. Apparently, RG by plasmid-driven expression followed by RNA transfection is more efficient than by transfection of exclusively RNA transcripts. Further, increase of the primary protein expression changes the amount of plasmids critical for AHSV recovery. Use of optimized-ORF expression plasmids, excludes the need of VP7 by which the requirements for RG of AHSV becomes similar to that of BTV (Tables 2 and 3).
NS1 expression by the first transfection is not essential for recovery of BTV and AHSV (Tables 2 and 3) [42]. NS1 increases protein synthesis from BTV RNAs [9], which explains that NS1 expression is not required for virus recovery with plasmid-driven protein expression. Expectedly, Seg-5 and its expressed NS1 protein of AHSV are essential for AHSV replication, since AHSV without Seg-5 or with mutated Seg-5 abolishing NS1 expression were not rescued (Table 2). In further agreement to BTV, initial VP7 expression is not required for AHSV recovery (Tables 2 and 3). AHSV-VP7 is however essential for RG with authentic ORF expression plasmids (Table 2). Others have suggested that VP7 stabilizes the BTV replication complex [42]. Similarly, it can be suggested that AHSV-VP7 is beneficial for AHSV recovery by stabilizing the replication complex as observed with suboptimal conditions, like the lower AHSV protein expression from authentic ORFs compared to optimized ORFs. These results indicate that initial NS1 and VP7 expression are not essential, but beneficial for virus recovery.
The minimal requirements of plasmid-driven expression differs between BTV and AHSV (Tables 2 and 3). VP1 and NS2 expression plasmids are essential for recovery of BTV and AHSV, irrespective of capped RNAs in the second transfection. VP3 expression plasmid is also essential, although BTV was recovered without VP3 expression plasmid in combination with capped RNA transcripts. Apparently, expression of VP1, VP3 and NS2 is essential for recovery of both orbiviruses, although delayed and lower expression of VP3 is sufficient for recovery of BTV.
Similar to the enhancement in recovery by VP7, we suggest that VP3 enhances orbivirus recovery by increase of the initial replication activity by stabilization of premature replication complexes and the formation of subcore particles. Consequently, suboptimal expression of VP1 and NS2 requires expression of VP3 and VP7 to efficiently recovery virus. In full agreement with this, in vitro reconstitution of infectious BTV core particles showed that VP1, VP4 and VP6 form a transcription complex that does not need other proteins for its activity [54]. NS2 recruits mRNA from the cytoplasm to the replication complex and forms virus inclusion bodies in vivo [10], and is therefore not needed for in vitro reconstitution of core particles.
Small amounts of VP4, 6 and 7 expressed from capped RNAs after 18–22 h are sufficient to initiate recovery of AHSV. These capped RNA transcripts are not needed for BTV recovery (Tables 2 and 3). Others have shown that VP6 is essential for BTV replication and acts early in the replication cycle of BTV [55]. We suggest that very low expression levels of these proteins from either uncapped RNA or after in vivo capping of transfected uncapped RNA transcripts are sufficient to initiate BTV recovery in our system. We have noticed that not all attempts with these conditions were successful suggesting that the efficiency of virus recovery was much lower.
Summarizing, VP1 and NS2 expression plasmids are essential for an optimized RG system of BTV and AHSV. VP3 expression plasmid is also essential for BTV recovery if uncapped RNAs are used. In contrast, only NS1 expression plasmid could be omitted in combination with uncapped RNAs for AHSV recovery. Generally, we conclude that recovery of BTV is more efficient than of AHSV. In order to increase the success rate for rescue of mutants and reassortants, the most optimal system is the combination of all seven expression plasmids with optimized ORFs and capped RNA transcripts for the second transfection. BTV1 and BTV8 variants were generated using BTV6 based ORF-optimized expression plasmids [35], and virulent AHSV5 was similarly recovered with AHSV4LP based expression plasmids (published elsewhere). We propose that one set of expression plasmids of the respective orbivirus species in combination with a set of ten even uncapped run-off RNA transcripts will be sufficient to quickly recover new orbivirus variants. A reproducible and efficient RG system is a prerequisite to conclude the lethality of mutations or genome constellations. Still, the essentiality of viral proteins or lethality of mutations could be conclusively demonstrated by use of in trans complementation followed by virus passage on ordinary cells [55].
Requirements for RG of BTV and AHSV showed many similarities as well as differences, likely caused by the difference in efficiency of virus recovery, but fundamental differences between these orbivirus species cannot be excluded. This comparative analysis of reverse genetics for orbivirus prototypes BTV and AHSV will contribute to development of RG methods for other orbiviruses, such as the OIE listed, notifiable epizootic haemorrhagic disease virus, but also for other orbiviruses and reoviruses. This will increase our understanding of the complex replication cycle and assembly process of these viruses with a segmented double stranded RNA genome.