Determinants of bluetongue virus serotype 26 that drive vector competence

Guimerà Busquets, Marc (2017) Determinants of bluetongue virus serotype 26 that drive vector competence. PhD thesis, University of Glasgow.

Due to Embargo and/or Third Party Copyright restrictions, this thesis is not available in this service.
Printed Thesis Information:


As an arbovirus, bluetongue virus (BTV) needs to replicate in its vector, biting midges of the genus Culicoides, in order to be transmitted between susceptible vertebrate hosts, which include wild and domestic ruminants. However, the latest identified serotypes of BTV, namely BTV-25, BTV 26 and BTV-27, have shown an inability to effectively amplify in BTV-susceptible Culicoides sonorensis derived KC cells, or in adult C. sonorensis midges. They also show a ‘non-conventional’ phenotype, specificity for small ruminants, where particularly in goats, longer viraemia, direct contact transmission and asymptomatic infection is observed. This, together with the failure of most earlier serological and RT PCR based assays to detect these ‘novel’, genetically distinct BTV serotypes, may help to explain why these viruses went unnoticed until recently. However, with the improvement of molecular diagnostic assays, increased awareness and surveillance, it appears that the current circulation of these non conventional viruses is more common than initially thought, and their number is expected to increase with reports of additional novel virus strains from Mongolia.
The work presented in this thesis is designed to further characterise virus isolate KUW2010/02, a BTV strain belonging to the non-conventional BTV serotype 26 (BTV 26), with particular interest in the viral determinants that restrict vector competence. A reverse genetics system for BTV, established in the Arbovirus Molecular Research Group at the Pirbright Institute, was used to generate specific reassortant viruses in mammalian BSR cells, containing genome segments derived from the BTV-1 reference strain and from BTV-26. The replication of these ‘engineered’ viruses was studied in KC cells, as a model for BTV vector interactions. Four genome segments from BTV-26 were identified that are associated with a restriction of BTV replication in both Culicoides cells (in vitro), and in adult Culicoides (in vivo). These include: genome segment 1 (Seg-1) encoding the viral RNA-dependent-RNA-polymerase VP1; Seg-2, encoding outer capsid protein VP2; Seg-3, encoding the sub-core shell protein VP3; and Seg 7, encoding core surface protein VP7.
Further investigations in vitro, revealed an inability of BTV-26, or the reassortant virus containing outer-capsid protein VP2 from BTV-26, to bind to the outer surface of KC cells even though they did bind efficiently to BSR cells. This indicates the absence of a suitable binding receptor for VP2 of BTV-26 on the surface of C. sonorensis cells.
The polymerase (VP1) and sub-core (VP3) proteins of BTV-26 were shown to be functional, at least during the initial stages of infection, in a Culicoides cell environment at 27 °C, with detection of low levels of transcription and non structural protein synthesis in these cells. However, a block at an unknown post transcriptional stage(s), restricts the successful completion/amplification of replication and dissemination to other cells, by the BTV viruses containing these genes (Seg 1/VP1, Seg-3/VP3).
The core surface protein (VP7) of BTV-26 caused only a partial restriction, detected as a delay in BTV replication in KC cells, with virus titres eventually reaching the same levels as for BTV-1. Mutagenesis studies of VP7 revealed that the lower domain of VP7 of BTV 26 was responsible for this delayed growth phenotype in KC cells.
Reassortment (exchange) of segments can occur between BTV-26 and a conventional isolate (BTV-1), confirming the classification of BTV-26 [KUW2010/02] as a member of the species Bluetongue virus. This also indicates the importance of providing a better understanding of these non-conventional viruses (such as BTV-26) since reassortment with co-circulating conventional strains could occur in the field, potentially generating progeny virus strains with novel and unpredictable phenotypes (e.g. with both higher pathogenesis and efficient direct contact transmission).
This work excludes adult C. sonorensis midges as a potential vector for BTV 26 strongly suggesting that it is a non vector borne virus. In general, the results obtained shed some light on BTV-Culicoides interactions, a field of research that has not been extensively studied, and demonstrate that these non-conventional viruses are powerful models for the further study of BTV replication in Culicoides systems.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Bluetongue virus, BTV-26, reverse genetics, vector competence.
Subjects: Q Science > QR Microbiology > QR355 Virology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
Supervisor's Name: Mertens, Prof. Peter and Palmarini, Prof. Massimo
Date of Award: 2017
Embargo Date: 21 December 2021
Depositing User: Marc Guimera Busquets
Unique ID: glathesis:2017-8638
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 04 Jan 2018 12:52
Last Modified: 04 Jan 2021 07:34
Related URLs:

Actions (login required)

View Item View Item


Downloads per month over past year