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Evidence of Intragenic Recombination in African Horse Sickness Virus.

ABSTRACT: Intragenic recombination has been described in various RNA viruses as a mechanism to increase genetic diversity, resulting in increased virulence, expanded host range, or adaptability to a changing environment. Orbiviruses are no exception to this, with intragenic recombination previously detected in the type species, bluetongue virus (BTV). African horse sickness virus (AHSV) is a double-stranded RNA virus belonging to the Oribivirus genus in the family Reoviridae. Genetic recombination through reassortment has been described in AHSV, but not through homologous intragenic recombination. The influence of the latter on the evolution of AHSV was investigated by analyzing the complete genomes of more than 100 viruses to identify evidence of recombination. Segment-1, segment-6, segment-7, and segment-10 showed evidence of intragenic recombination, yet only one (Segment-10) of these events was manifested in subsequent lineages. The other three hybrid segments were as a result of recombination between field isolates and the vaccine derived live attenuated viruses (ALVs).

SUBMITTER: Ngoveni HG

PROVIDER: S-EPMC6669442 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Evidence of Intragenic Recombination in African Horse Sickness Virus.

Ngoveni Harry G HG van Schalkwyk Antoinette A Koekemoer J J Otto JJO

Viruses 20190718 7

Intragenic recombination has been described in various RNA viruses as a mechanism to increase genetic diversity, resulting in increased virulence, expanded host range, or adaptability to a changing environment. Orbiviruses are no exception to this, with intragenic recombination previously detected in the type species, bluetongue virus (BTV). African horse sickness virus (AHSV) is a double-stranded RNA virus belonging to the <i>Oribivirus</i> genus in the family <i>Reoviridae</i>. Genetic recombi ...[more]

PMID: 31323749

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Project description:The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essential for virus replication in vitro In addition, in-frame replacement of RNA sequences in Seg-2 by that of green fluorescence protein (GFP) resulted in AHSV expressing GFP, which further confirmed that VP2 is not essential for virus replication. In contrast to virus replication without VP2 expression in mammalian cells, virus replication in insect cells was strongly reduced, and virus release from insect cells was completely abolished. Further, the other outer capsid protein, VP5, was not copurified with virions for virus mutants without VP2 expression. AHSV without VP5 expression, however, could not be recovered, indicating that outer capsid protein VP5 is essential for virus replication in vitro Our results demonstrate for the first time that a structural viral protein is not essential for orbivirus replication in vitro, which opens new possibilities for research on other members of the Reoviridae family. Members of the Reoviridae family cause major health problems worldwide, ranging from lethal diarrhea caused by rotavirus in humans to economic losses in livestock production caused by different orbiviruses. The Orbivirus genus contains many virus species, of which bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus (AHSV) cause notifiable diseases according to the World Organization of Animal Health. Recently, it has been shown that nonstructural proteins NS3/NS3a and NS4 are not essential for virus replication in vitro, whereas it is generally assumed that structural proteins VP1 to -7 of these nonenveloped, architecturally complex virus particles are essential. Here we demonstrate for the first time that structural protein VP2 of AHSV is not essential for virus replication in vitro Our findings are very important for virologists working in the field of nonenveloped viruses, in particular reoviruses.

Project description:BackgroundBluetongue 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.ResultsPlasmids 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.ConclusionsA 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.

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Evidence of Intragenic Recombination in African Horse Sickness Virus.

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Evidence of Intragenic Recombination in African Horse Sickness Virus.

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