Establishment of different plasmid only-based reverse genetics systems for the recovery of African horse sickness virus

Conradie, Andelé M.; Stassen, Liesel; Huismans, H.; Potgieter, Christiaan; Theron, Jacques

doi:10.1016/j.virol.2016.07.010

Cited by 15 publications

(10 citation statements)

References 63 publications

(80 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To determine the roles of RNA capping during reovirus infection, we took advantage of the existing cap-independent reverse genetics system for producing reovirus (30)(31)(32)(33)(34)(35)(36) and characterized the impact of adding a cytosolic capping enzyme. The reverse genetics system consists of plasmids that carry all 10 reovirus genes under the control of a T7 promoter (T7p).…”

mentioning

confidence: 99%

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

Eaton

Kobayashi

Dermody

et al. 2017

J Virol

View full text Add to dashboard Cite

Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5' nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation. Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure.

show abstract

mentioning

confidence: 99%

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

Eaton

Kobayashi

Dermody

et al. 2017

J Virol

View full text Add to dashboard Cite

show abstract

“…As plaque size has previously been correlated to virulence [42,43], it is conceivable that the genetic differences involved in plaque phenotype could be linked to the attenuation of the virus. Once identified, the effect of the genetic variations could be verified and incorporated into synthetic vaccine viruses using a reverse genetics system [57][58][59].…”

Section: Discussionmentioning

confidence: 99%

A correlation between capsid protein VP2 and the plaque morphology of African horse sickness virus in cell culture

2018

View full text Add to dashboard Cite

The attenuated live virus vaccine that is used in South Africa to protect against African horse sickness infection was developed more than 50 years ago. With the selection of the vaccine strains by cell culture passage, a correlation between the size of plaques formed in monolayer Vero cultures and attenuation of virus virulence in horses was found. The large plaque phenotype was used as an indication of cell culture adaptation and strongly correlated with attenuation of virulence in horses. There was never any investigation into the genetic causes of either the variation in plaque size, or the loss of virulence. An understanding of the underlying mechanisms of attenuation would benefit the production of a safer AHSV vaccine. To this end, the genomes of different strains of two African horse sickness isolates, producing varying plaque sizes, were compared and the differences between them identified. This comparison suggested that proteins VP2, VP3, VP5 and NS3 were most likely involved in the determination of the plaque phenotype. Comparison between genome sequences (obtained from GenBank) of low and high passage strains from two additional serotypes indicated that VP2 was the only protein with amino acid substitutions in all four serotypes. The amino acid substitutions all occurred within the same hydrophilic area, resulting in increased hydrophilicity of VP2 in the large plaque strains.

show abstract

“…Over the last decade, reverse genetics systems have been used to generate novel live virus-based BTV and AHSV vaccine candidates, engineered according to a rational design rather than by random serial passage attenuation [127,128,129,130,131,132,133,134,135,136,137,138]. These live vaccine strains depend on the availability of cloned cDNA copies of the viral genes and are produced in mammalian cell lines via a double transfection strategy.…”

Section: Prevention and Controlmentioning

confidence: 99%

African Horse Sickness: A Review of Current Understanding and Vaccine Development

Dennis

Meyers

Hitzeroth

et al. 2019

Viruses

View full text Add to dashboard Cite

African horse sickness is a devastating disease that causes great suffering and many fatalities amongst horses in sub-Saharan Africa. It is caused by nine different serotypes of the orbivirus African horse sickness virus (AHSV) and it is spread by Culicoid midges. The disease has significant economic consequences for the equine industry both in southern Africa and increasingly further afield as the geographic distribution of the midge vector broadens with global warming and climate change. Live attenuated vaccines (LAV) have been used with relative success for many decades but carry the risk of reversion to virulence and/or genetic re-assortment between outbreak and vaccine strains. Furthermore, the vaccines lack DIVA capacity, the ability to distinguish between vaccine-induced immunity and that induced by natural infection. These concerns have motivated interest in the development of new, more favourable recombinant vaccines that utilize viral vectors or are based on reverse genetics or virus-like particle technologies. This review summarizes the current understanding of AHSV structure and the viral replication cycle and also evaluates existing and potential vaccine strategies that may be applied to prevent or control the disease.

show abstract

Establishment of different plasmid only-based reverse genetics systems for the recovery of African horse sickness virus

Cited by 15 publications

References 63 publications

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

A correlation between capsid protein VP2 and the plaque morphology of African horse sickness virus in cell culture

African Horse Sickness: A Review of Current Understanding and Vaccine Development

Contact Info

Product

Resources

About