The stronger downregulation ofin vitroandin vivoinnate 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

Abstract

word count: 279 14 15 Text word Count: 5,698 16 17 Abstract 18IBDV is economically important to the poultry industry. Very virulent (vv) strains cause higher 19 mortality rates than other strains for reasons that remain poorly understood. In order to provide 20 more information on IBDV disease outcome, groups of chickens (n=18) were inoculated with the vv 21 strain, UK661, or the classical strain, F52/70. Birds infected with UK661 had a lower survival rate 22 (50%) compared to F52/70 (80%). There was no difference in peak viral replication in the bursa of 23 Fabricius (BF), but the expression of chicken IFNβ, MX1 and IL-8 was significantly lower in the BF of 24 birds infected with UK661 compared to F52/70 (p<0.05) as quantified by RTqPCR, and this trend was 25 also observed in DT40 cells infected with UK661 or F52/70 (p<0.05). The induction of expression of 26 in vivo and this was, in part, due to strain-dependent differences in the VP4 protein. 36 37 39 (c) strains have circulated worldwide, however, in the 1980s, so-called "very virulent" (vv) strains 53 emerged, complicating IBDV control efforts [6, 7]. The vvIBDV strains cause a far higher mortality 54 rate than classical strains, reaching up to 60-70% in some flocks [8]. However, the molecular basis for 55 the difference in disease outcome remains poorly understood, although it has been demonstrated 56 that both segments A and B contribute to virulence [9]. Segment A encodes the non-structural 57 protein, VP5, and a polyprotein (VP2-VP4-VP3) which is co-translationally cleaved by the protease, 58 VP4 [10]. VP2 is the capsid protein and VP3 is a multifunctional scaffolding protein that binds the 59 genome. The single ORF on Segment B encodes VP1, the RNA-dependent RNA polymerase. 60 61The innate immune response to IBDV infection is characterised by the production of type I IFN 62responses, including the upregulation of IFNα and IFNβ, that lead to the induction of interferon 63 stimulated genes (ISGs), including MX1, which is one of the top ISGs identified in chicken cells ranked 64 by fold change [11]. The IFN-response aims to provide an antiviral state in infected and bystander 65 cells. In addition, pro-inflammatory cytokines, for example IL-6, IL-8 and IL-1β are produced 66 following IBDV infection that recruit immune cells into the infected bursa of Fabricius (BF) [12][13][14][15]. 67 68We sought to identify IBDV virulence determinants in order to better understand the molecular basis 69 of phenotypic differences between vv-and c-IBDV strains. Here we report that vvIBDV UK661 down-70 regulated the expression of antiviral type I IFN responses and pro-inflammatory cytokines compared 71 to cIBDV F52/70 in vitro and in vivo. Moreover, we demonstrate that the differences in IFN 72 antagonism were, in part, due to strain-dependent differences in the VP4 proteins. 73 74 Materials and methods 75 76 Cells and viruses. DF-1 cells (chicken embryonic fibroblast cells, ATCC number CRL-12203) were 77 sustained in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich...