Differential gene expression in chicken primary B cells infected ex vivo with attenuated and very virulent strains of infectious bursal disease virus (IBDV).

Infectious bursal disease virus (IBDV) belongs to the family Birnaviridae and is economically important to the poultry industry worldwide. IBDV infects B cells in the bursa of Fabricius (BF), causing immunosuppression and morbidity in young chickens. In addition to strains that cause classical Gumboro disease, the so-called 'very virulent' (vv) strain, also in circulation, causes more severe disease and increased mortality. IBDV has traditionally been controlled through the use of live attenuated vaccines, with attenuation resulting from serial passage in non-lymphoid cells. However, the factors that contribute to the vv or attenuated phenotypes are poorly understood. In order to address this, we aimed to investigate host cell-IBDV interactions using a recently described chicken primary B-cell model, where chicken B cells are harvested from the BF and cultured ex vivo in the presence of chicken CD40L. We demonstrated that these cells could support the replication of IBDV when infected ex vivo in the laboratory. Furthermore, we evaluated the gene expression profiles of B cells infected with an attenuated strain (D78) and a very virulent strain (UK661) by microarray. We found that key genes involved in B-cell activation and signalling (TNFSF13B, CD72 and GRAP) were down-regulated following infection relative to mock, which we speculate could contribute to IBDV-mediated immunosuppression. Moreover, cells responded to infection by expressing antiviral type I IFNs and IFN-stimulated genes, but the induction was far less pronounced upon infection with UK661, which we speculate could contribute to its virulence.

Transcriptomic Analysis of Inbred Chicken Lines Reveals Infectious Bursal Disease Severity Is Associated with Greater Bursal Inflammation In Vivo and More Rapid Induction of Pro-Inflammatory Responses in Primary Bursal Cells Stimulated Ex Vivo.

In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p < 0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

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 IFNα, IFNß, 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-ß 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 IFNα and IL-8 compared to cells infected with a chimeric virus carrying the F52/70-VP4 gene (PBG98-VP4F52/70) (p < 0.01), and birds infected with PBG98-VP4UK661 also had a reduced expression of IFNα in the BF compared to birds infected with PBG98-VP4F52/70 (p < 0.05). 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.

Molecular screening of blue mussels indicated high mid-summer prevalence of human genogroup II Noroviruses, including the pandemic "GII.4 2012" variants in UK coastal waters during 2013.

This molecular study is the first report, to the best of our knowledge, on identification of norovirus, NoV GII.4 Sydney 2012 variants, from blue mussels collected from UK coastal waters. Blue mussels (three pooled samples from twelve mussels) collected during the 2013 summer months from UK coastal sites were screened by RT-PCR assays. PCR products of RdRP gene for noroviruses were purified, sequenced and subjected to phylogenetic analysis. All the samples tested positive for NoVs. Sequencing revealed that the NoV partial RdRP gene sequences from two pooled samples clustered with the pandemic "GII.4 Sydney variants" whilst the other pooled sample clustered with the NoV GII.2 variants. This molecular study indicated mussel contamination with pathogenic NoVs even during mid-summer in UK coastal waters which posed potential risk of NoV outbreaks irrespective of season. As the detection of Sydney 2012 NoV from our preliminary study of natural coastal mussels interestingly corroborated with NoV outbreaks in nearby areas during the same period, it emphasizes the importance of environmental surveillance work for forecast of high risk zones of NoV outbreaks.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

Molecular screening of blue mussels indicated high mid-summer prevalence of human genogroup II Noroviruses, including the pandemic "GII. 4 2012" variants in UK coastal waters during 2013

Abstract This molecular study is the first report, to the best of our knowledge, on identification of norovirus, NoV GII.4 Sydney 2012 variants, from blue mussels collected from UK coastal waters. Blue mussels (three pooled samples from twelve mussels) collected during the 2013 summer months from UK coastal sites were screened by RT-PCR assays. PCR products of RdRP gene for noroviruses were purified, sequenced and subjected to phylogenetic analysis. All the samples tested positive for NoVs. Sequencing revealed that the NoV partial RdRP gene sequences from two pooled samples clustered with the pandemic "GII.4 Sydney variants" whilst the other pooled sample clustered with the NoV GII.2 variants. This molecular study indicated mussel contamination with pathogenic NoVs even during mid-summer in UK coastal waters which posed potential risk of NoV outbreaks irrespective of season. As the detection of Sydney 2012 NoV from our preliminary study of natural coastal mussels interestingly corroborated with NoV outbreaks in nearby areas during the same period, it emphasizes the importance of environmental surveillance work for forecast of high risk zones of NoV outbreaks.