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Gray, A. G.

Publications and source records attributed to Gray, A. G..

2 recordsLinked to original sources
bioRxiv2019

The stronger downregulation of in vitro and in vivo innate antiviral responses by a very virulent strain of infectious bursal disease virus (IBDV), compared to a classical strain, is mediated, in part, by the VP4 protein.

IBDV is economically important to the poultry industry. Very virulent (vv) strains cause higher mortality rates than other strains for reasons that remain poorly understood. In order to provide more information on IBDV disease outcome, groups of chickens (n=18) were inoculated with the vv strain, UK661, or the classical strain, F52/70. Birds infected with UK661 had a lower survival rate (50%) compared to F52/70 (80%). There was no difference in peak viral replication in the bursa of Fabricius (BF), but the expression of chicken IFN{beta}, MX1 and IL-8 was significantly lower in the BF of birds infected with UK661 compared to F52/70 (p<0.05) as quantified by RTqPCR, and this trend was also observed in DT40 cells infected with UK661 or F52/70 (p<0.05). The induction of expression of type I IFN in DF-1 cells stimulated with polyI:C (measured by an IFN-{beta} luciferase reporter assay) was significantly reduced in cells expressing ectopic VP4 from UK661 (p<0.05), but was higher in cells expressing ectopic VP4 from F52/70. Cells infected with a chimeric recombinant IBDV carrying the UK661-VP4 gene in the background of PBG98, an attenuated vaccine strain that induces high levels of innate responses (PBG98-VP4UK661) also showed a reduced level of IFN and IL-8 compared to cells infected with a chimeric virus carrying the F52/70-VP4 gene (PBG98-VP4F52/70), and birds infected with PBG98-VP4UK661 also had a reduced expression of IFN in the BF compared to birds infected with PBG98-VP4F52/70. Taken together, these data demonstrate that UK661 induced the expression of lower levels of anti-viral type I IFN and proinflammatory genes than the classical strain in vitro and in vivo and this was, in part, due to strain-dependent differences in the VP4 protein.

microbiology↗
bioRxiv2019

Discrete virus factories form in the cytoplasm of cells co-infected with two strains of the segmented dsRNA virus, infectious bursal disease virus (IBDV), that subsequently coalesce

The Birnaviridae family, responsible for major economic losses to poultry and aquaculture, are non-enveloped viruses with a segmented double-stranded (ds)RNA genome that replicate in discrete cytoplasmic virus factories (VFs). Reassortment is common, however, the underlying mechanism remains unknown given that VFs may act as a barrier to genome mixing. In order to provide new information on VF trafficking during dsRNA virus co-infection, we rescued two recombinant infectious bursal disease viruses (IBDVs) of strain PBG98 containing either a split GFP11- or Tetracysteine (TC)- tag fused to the VP1 polymerase (PBG98-VP1-GFP11 and PBG98-VP1-TC). DF-1 cells transfected with GFP1-10 prior to PBG98-VP1-GFP11 infection, or stained with ReAsH following PBG98-VP1-TC infection, had green or red foci in the cytoplasm respectively that co-localised with VP3 and dsRNA, consistent with VFs. The average number of VFs decreased from a mean of 60 to 5 per cell between 10 and 24 hours post infection (hpi) (p<0.001), while the average area increased from 1.24 m2 to 45.01m2 (p<0.001), and live cell imaging revealed that the VFs were highly dynamic structures that coalesced in the cytoplasm. Small VFs moved faster than large (average 0.57m/s at 16 hpi compared to 0.22 m/s at 22 hpi), and VF coalescence was dependent on an intact microtubule network and actin cytoskeleton. During co-infection with PBG98-VP1-GFP11 and PBG98-VP1-TC viruses, discrete VFs initially formed from each input virus that subsequently coalesced 10-16 hours post-infection. We speculate that dsRNA virus reassortment requires VF coalescence, and the potential for reassortment increases at later time points in infection. ImportanceReassortment is common in viruses with segmented double stranded (ds)RNA genomes. However, these viruses typically replicate within discrete cytoplasmic virus factories (VFs) that may represent a barrier to genome mixing. We generated the first replication competent tagged reporter birnaviruses, infectious bursal disease viruses (IBDVs) containing a split GFP11 or tetracysteine (TC) tag and used the viruses to track the location and movement of IBDV VFs, in order to better understand the intracellular dynamics of VFs from two different strains of dsRNA virus during a co-infection. Discrete VFs initially formed from each virus that subsequently coalesced from 10 hours pi. We hypothesise that VF coalescence is required for the reassortment of dsRNA viruses and the potential for reassortment increases later in the replication cycle. This study provides new information that adds to our understanding of dsRNA virus VF trafficking.

microbiology↗