Rapid Generation of Attenuated Infectious Bursal Disease Virus from Dual-Promoter Plasmids by Reduction of Viral Ribonucleoprotein Activity

Abstract

Infectious bursal disease virus (IBDV) of the Birnaviridae family leads to immunosuppression of young chickens by destroying B cells in the bursa of Fabricius (BFs). Given the increasing number of variant IBDV strains, we urgently require a method to produce attenuated virus for vaccine development. To accomplish this goal, the dual-promoter plasmids in which the RNA polymerase II and RNA polymerase I (Pol I) promoters were placed upstream of the IBDV genomic sequence, which was followed by mouse Pol I terminator and a synthetic polyadenylation signal, were developed for rapid generation of IBDV. This approach did not require trans-supplementation of plasmids for the expression of VP1 and VP3, the main components of IBDV ribonucleoprotein (RNP). Based on the finding in this study that the IBDV RNP activity was partially retained by VP1-FLAG, we successfully rescued the replication-competent IBDV/1FLAG expressing VP1-FLAG. Compared with its parental counterpart, IBDV/1FLAG formed smaller size plaques in cultured cells and induced the same 100% immune protection in vivo. However, neither retarded development nor severe BFs lesion was observed in the IBDV/1FLAG-inoculated chickens. Collectively, this is the first report that viral RNP activity was affected by the addition of an epitope tag on the componential viral proteins. Furthermore, this work demonstrates the rapid generation of attenuated IBDV from dual-promoter plasmids via reducing viral RNP activity by a fused FLAG tag on the C terminus of VP1. This would be a convenient strategy to attenuate epidemic variant IBDV strains for rapid and efficient vaccine development. IMPORTANCE Immunosuppression in chickens as a result of infectious bursal disease virus (IBDV) infection leads to significant economic losses in the poultry industry worldwide every year. Currently, vaccination is still the best way to prevent the prevalence of IBDV. However, with the occurrence of increasing numbers of variant IBDV strains, it is challenging to develop antigen-matched live attenuated vaccine. Here, we first developed a dual-promoter reverse-genetic system for the rapid generation of IBDV. Using this system, the attenuated IBDV/1FLAG expressing VP1-FLAG, which displays the decreased viral RNP activity, was rescued. Moreover, IBDV/1FLAG inoculation induced a similar level of neutralizing antibodies to that of its parental counterpart, protecting chickens against lethal challenge. Our study, for the first time, describes a dual-promoter reverse-genetic approach for the rapid generation of attenuated IBDV while maintaining entire parental antigenicity, suggesting a potential new method to attenuate epidemic variant IBDV strains for vaccine development.

Development of a Viral-Like Particle Candidate Vaccine Against Novel Variant Infectious Bursal Disease Virus

“…Humoral immunity is essential for the immune protection of IBDV [ 15 ]. The serum neutralizing antibody titer is an important indicator to evaluate the strength of the humoral immune response.…”

“…VP2, the main IBDV host-protective antigen [ 7 ] that possesses a series of neutralizing epitopes [ 8 , 9 ], is an important determinant for virulence, antigenic variation, and cell tropism of IBDV [ 10 , 11 ]. Segment B encodes VP1, an RNA-dependent RNA polymerase, which is responsible for the genome transcription and translation of IBDV [ 12 , 13 , 14 , 15 ].…”

The Over-40-Years-Epidemic of Infectious Bursal Disease Virus in China

“…Segment B has only one ORF encoding the protein VP1, which has RNA-dependent RNA polymerase activity and is an essential protein for viral transcription and replication. Segment B and its VP1 also play an important role in the genetic evolution of IBDV and have a significant impact on its virulence [ 20 , 21 , 22 , 23 ].…”

Ex vivo rescue of recombinant very virulent IBDV using a RNA polymerase II driven system and primary chicken bursal cells

“…Recently, a new approach for the recovery of IBDV was described, in which the trans-supplementation of the viral VP1 and VP3 proteins was required and sufficient to rescue infectious virus from RNA polymerase I (pol I) transcribed IBDV genomic RNA 23 . This strategy was further improved through the design of a dual promoter plasmid system, which includes, upstream of the pol I promoter ensuring genomic RNA synthesis , a pol II promoter that results in the expression of viral proteins 24 .…”