RESUMO
Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.
Assuntos
Vírus da Doença Hemorrágica Epizoótica/genética , Genética Reversa/métodos , Virologia/métodos , Animais , Linhagem Celular , DNA Complementar/genética , Vírus da Doença Hemorrágica Epizoótica/crescimento & desenvolvimento , Mesocricetus , Plasmídeos , RNA Viral/genética , Recombinação Genética , Infecções por Reoviridae/virologiaRESUMO
BACKGROUND: Bluetongue virus (BTV), an emerging insect vector mediated pathogen affecting both wild ruminants and livestock, has a genome consisting of 10 linear double-stranded RNA genome segments. BTV has a severe economic impact on agriculture in many parts of the world. Current reverse genetics (RG) strategy to rescue BTV mainly rely on in vitro synthesis of RNA transcripts from cloned complimentary DNA (cDNA) corresponding to viral genome segments with the aid of helper plasmids. RNA synthesis is a laborious job which is further complicated with a need for expensive reagents and a meticulous operational procedure. Additionally, the target genes must be cloned into a specific vector to prepare templates for RNA transcription. RESULT: In this study, we have developed a PCR based BTV RG system with easy two-step transfection. Viable viruses were recovered following a first transfection with the seven helper plasmids and a second transfection with the 10 PCR products on the BSR cells. Further, recovered viruses were characterized with indirect immunofluorescence assays (IFA) and gene sequencing. And the proliferation properties of these viruses were also compared with wild type BTV. Interestingly, we have identified that viruses containing the segment 2 of the genome from reassortant BTV, grew slightly slower than the others. CONCLUSION: In this study, a convenient PCR based RG platform for BTV is established, and this strategy could be an effective alternative to the original available BTV rescue methods. Furthermore, this RG strategy is likely applicable for other Orbiviruses.
Assuntos
Vírus Bluetongue/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Genética Reversa/métodos , Virologia/métodos , Animais , Vírus Bluetongue/genética , Linhagem Celular , Cricetinae , Viabilidade Microbiana , Plasmídeos , TransfecçãoRESUMO
Bluetongue (BT) is an arbovirus-borne disease of ruminants caused by bluetongue virus (BTV) that has the potential to have a serious economic impact. Currently available commercial vaccines include attenuated vaccines and inactivated vaccines, both of which have achieved great success in the prevention and control of BTV. However, these vaccines cannot distinguish between infected animals and immunized animals. To control outbreaks of BTV, the development of labeled vaccines is urgently needed. In this study, we used the plasmid-based reverse genetics system (RGS) of BTV to rescue four recombinant viruses in which HA (influenza hemagglutinin) tags were inserted at different sites of VP2. In vitro, the recombinant tagged viruses exhibited morphologies, plaque, and growth kinetics similar to the parental BTV-16, and expressed both VP2 and HA tag. Subsequently, the selected recombinant tagged viruses were prepared as inactivated vaccines to immunize IFNAR(-/-) mice and sheep, and serological detection results of anti-HA antibody provided discriminative detection. In summary, we used plasmid-based RGS to rescue BTV recombinant viruses with HA tags inserted into VP2, and detected several sites on VP2 that can accommodate HA tags. Some of the recombinant tagged viruses have potential to be developed into distinctive inactivated vaccines.