Molecular determinants of IBDV pathogenesis and modulation of the host innate response

Viruses are known to interact with the innate immune pathways and, in some cases, strains that differ in virulence are known to interact with these pathways in different ways. This thesis aimed to directly compare IBDV strains of differing virulence to determine key interactions with the innate immune response that may contribute to disease outcome. Infection of chicken B cells with the very virulent s UK661 strain, suppressed type I IFN responses compared to both infection with the cell-adapted IBDV strain, D78 in primary bursal cells, and infection with the classical strain, F52/70 in DT40 cells. Birds infected with UK661 also had down-regulated type I IFN and pro-inflammatory responses in the bursa of Fabricius (BF), compared to infection with F52/70. No difference in the peak virus titres was detected in the BF or spleen, although UK661 reached higher titres in the caecal tonsils than F52/70. Increased type I IFN production following F52/70 infection coincided with a reduced mortality in these birds, indicating a protective role of this immune response. The UK661 VP4 protein was found to suppress IFNβ production in vitro compared to the F52/70 VP4, which instead suppressed Mx1 production, indicating that the IBDV VP4 from different strains impairs either IFN production or signalling pathways. Upon knocking out the protease function of UK661 VP4, IFNβ production remained suppressed, and multiple amino acids are likely responsible for the different phenotype between strains. This work demonstrates that UK661 and F52/70 have strain-specific differences in their interactions with the innate immune response, mediated by the VP4 protein, therefore differences in this protein between strains may contribute to virulence. This information could be useful in the development of recombinant rationally designed live attenuated IBDV vaccines, by generating a vvIBDV backbone containing a VP4 from a classical or cell-adapted strain, as a vaccine candidate.Open Acces

Genetic characterisation and phylogenetic analysis of bovine astroviruses and kobuviruses

Calf diarrhoea remains the most important cause of economic loss to both dairy and beef cattle industries worldwide, with approximately 50% of deaths among weaning calves resulting from diarrhoeal disease. Complex etiopathogenesis involving the infection of one or multiple pathogens, as well as other non-infectious factors such as the environment and nutrition, contributes to its devastating effects. Astroviruses (AstVs) and Kobuviruses (KoVs) are two single-stranded, positive-sense RNA viruses previously detected in healthy and diarrhoeic calves. AstV was identified in healthy and diarrhoeic calves in similar proportions, while KoV was predominantly associated with diarrhoeic individuals. In order to investigate the KoV strains found in diarrhoeic calves, the full genome of KoV from a diarrhoeic calf was sequenced. This KoV was then compared with the bovine U-1, porcine KoV and Aichi virus strains. Specifically-designed PCRs were used to target the full KoV genome of positive samples, and amplicons were cloned to allow the internal sequencing of one single KoV detected among possible mixed infections. Upon assembly of the genome sequences, some animals were found to be co-infected with multiple KoVs. The main region of diversity, the VP1 (capsid) region, was amplified from multiple samples to determine the diversity of KoV in Scotland. The genome sequenced in this study will be used to produce an infectious clone for future challenge studies to establish the potential role of bovine KoV in calf diarrhoea. Another aspect of the study was to explore the diversity and epidemiology of AstV in diarrhoeic and healthy calves by capsid gene analysis. As the AstV capsid protein is the major target of host antibody production, a serological test for AstV infection could then be developed. Following amplification of AstV capsid genes by PCR, phylogenetic analysis identified 4 lineages from which capsids from 2 of these lineages were successfully cloned ready for use in the baculovirus expression system. This information and the expression plasmids containing representative AstV capsid genes can then be used to develop serological tests for AstV, enabling estimation of the prevalence of AstV in the British cattle population

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, IFNβ, 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-β 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

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 &lt; 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.

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

Pandemic norovirus in coastal blue mussels during summer in UK 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