Bluetongue virus infection induces aberrant mitosis in mammalian cells.

BACKGROUND: Bluetongue virus (BTV) is an arbovirus that is responsible for 'bluetongue', an economically important disease of livestock. Although BTV is well characterised at the protein level, less is known regarding its interaction with host cells. During studies of virus inclusion body formation we observed what appeared to be a large proportion of cells in mitosis. Although the modulation of the cell cycle is well established for many viruses, this was a novel observation for BTV. We therefore undertook a study to reveal in more depth the impact of BTV upon cell division. METHODS: We used a confocal microscopy approach to investigate the localisation of BTV proteins in a cellular context with their respective position relative to cellular proteins. In addition, to quantitatively assess the frequency of aberrant mitosis induction by the viral non-structural protein (NS) 2 we utilised live cell imaging to monitor HeLa-mCherry tubulin cells transfected with a plasmid expressing NS2. RESULTS: Our data showed that these 'aberrant mitoses' can be induced in multiple cell types and by different strains of BTV. Further study confirmed multiplication of the centrosomes, each resulting in a separate mitotic spindle during mitosis. Interestingly, the BTV NS1 protein was strongly localised to the centrosomal regions. In a separate, yet related observation, the BTV NS2 protein was co-localised with the condensed chromosomes to a region suggestive of the kinetochore. Live cell imaging revealed that expression of an EGFP-NS2 fusion protein in HeLa-mCherry tubulin cells also results in mitotic defects. CONCLUSIONS: We hypothesise that NS2 is a microtubule cargo protein that may inadvertently disrupt the interaction of microtubule tips with the kinetochores during mitosis. Furthermore, the BTV NS1 protein was distinctly localised to a region encompassing the centrosome and may therefore be, at least in part, responsible for the disruption of the centrosome as observed in BTV infected mammalian cells.

Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparison to other European strains.

During 2006 the first outbreak of bluetongue ever recorded in northern Europe started in Belgium and the Netherlands, spreading to Luxemburg, Germany and north-east France. The virus overwintered (2006-2007) reappearing during May-June 2007 with greatly increased severity in affected areas, spreading further into Germany and France, reaching Denmark, Switzerland, the Czech Republic and the UK. Infected animals were also imported into Poland, Italy, Spain and the UK. An initial isolate from the Netherlands (NET2006/04) was identified as BTV-8 by RT-PCR assays targeting genome segment 2. The full genome of NET2006/04 was sequenced and compared to selected European isolates, South African vaccine strains and other BTV-8 strains, indicating that it originated in sub-Saharan Africa. Although NET2006/04 showed high levels of nucleotide identity with other 'western' BTV strains, it represents a new introduction and was not derived from the BTV-8 vaccine, although its route of entry into Europe has not been established.

Conservation of L and 3C proteinase activities across distantly related aphthoviruses.

The foot-and-mouth disease virus (FMDV) leader (L) proteinase is an important virulence determinant in FMDV infections. It possesses two distinct catalytic activities: (i) C-terminal processing at the L/VP4 junction; and (ii) induction of the cleavage of translation initiation factor eIF4G, an event that inhibits cap-dependent translation in infected cells. The only other member of the Aphthovirus genus, equine rhinitis A virus (ERAV), also encodes an L protein, but this shares only 32% amino acid identity with its FMDV counterpart. Another more distantly related picornavirus, equine rhinitis B virus (ERBV), which is not classified as an aphthovirus, also encodes an L protein. Using in vitro transcription and translation analysis, we have shown that both ERAV and ERBV L proteins have C-terminal processing activity. Furthermore, expression of ERAV L, but not ERBV L, in BHK-21 cells resulted in the efficient inhibition of cap-dependent translation in these cells. We have shown that the ERAV and FMDV L proteinases induce cleavage of eIF4GI at very similar or identical positions. Interestingly, ERAV 3C also induces eIF4GI cleavage and again produces distinct products that co-migrate with those induced by FMDV 3C. The ERBV L proteinase does not induce eIF4GI cleavage, consistent with its inability to shut down cap-dependent translation. We have also shown that another unique feature of FMDV L, the stimulation of enterovirus internal ribosome entry site (IRES) activity, is also shared by the ERAV L proteinase but not by ERBV L. The functional conservation of the divergent ERAV and FMDV proteinases indicates the likelihood of a similar and important role for these enzymes in the pathogenesis of infections caused by these distantly related aphthoviruses.