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BVDV- Determination of molecular and cellular mechanisms of virulence in field and emerging viruses - SE0777

Description
In the recent aftermath of several major epidemics of livestock diseases, there can be little doubting the importance of a national research capability to provide informed opinion to DEFRA and other policy makers. Virulence, persistence and surveillance of emerging pathogens were all critical issues for understanding the pathobiology of these infections and, thereafter, strategies for control. This proposal examines all these issues, using Bovine Viral Diarrhoea Virus (BVDV) as a viral exemplar of an infectious and damaging virus disease of livestock.

The proposed research, examining the pathogenesis of this important viral infection of UK livestock, supports and extends the national capability into field and potential emerging viruses, capable of causing severe, if not fatal, disease. This programme provides a multi-disciplinary collaborative platform, between the RVC, IAH, VLA & SAC, on which we can integrate existing and future research projects. It is distinguished by its active surveillance involvement in farm outbreaks, strength in molecular and cellular pathology and the ability to assess the immunological consequences of viral genomic alterations.

We shall determine molecular and cellular mechanisms of virulence in acutely haemorrhagic and damaging strains of BVDV. Such strains will be resourced from isolates within the VLA virus-bank. However, to study further the genomic site for mutation associated with virulence, we intend to make chimeric viruses from the infectious cDNA of BVDV Ho916 by constructing recombinants with exchange of a) the entire ORF, b) the structural proteins, c) the non-structural proteins for the attenuated Genogroup 1 strain, Ky1203 strain of BVDV and later from the virulent Genotype 2 CD87. Thereafter, the pathobiology of these viruses will be verified by in vitro and in vivo challenge. The clinical outcomes following in vivo challenge of calves with either field strains of different virulence or with the infectious cDNA rHo916 and its parental virus will allow us to study the mechanisms of virulence and immune perturbance. Samples will also be taken for assessment of cytokine levels, particularly of IL-2, IL-4 and IFN-y. This will provide crucial data for analysing the underlying process of immunosuppression that exacerbates other microbial infections (calfhood diseases and more recently bovine tuberculosis).

The virulence phenotypes of the three recombinant viruses (a, b and c above) will be used to assess the regions of the genome, which in isolation or in combination contribute to the severe disease?inducing characteristics of the Ho916 strain. This information will be used to guide single substitutions of individual proteins. Future work will investigate the structure-function relationships of the same proteins from strains, which induce different pathology and potentially allow the rational attenuation of vaccine strains of BVDV.Cellular immune responses to virulent field virus and their relevant molecular constructs will be characterised, with particular reference to the mechanisms of immunosuppression and thrombocytopenia. A number of new technologies will be used to perform these studies including: in-situ hybridisation for viral RNA, chemokines and cytokines known to play a role in acute infection; furthermore, immunofluorescence analysis using confocal microscopy will be undertaken in order to understand the cellular location and pathology of viruses of differing virulence. The dramatic consequences of virulent virus infection on leucopenia is documented but its influence on abrogating Antigen Presenting Cell (APC) function(s) as mediators of antigen presentation, T cell stimulation, balance between Th1 and Th2 mediated response, and possibly T cell tolerance is poorly understood. Gene expression profiling using microarrays will be employed to study the changes induced in bovine APC infected with different genotypes of BVDV, and the initial data will be subsequently corroborated using real-time PCR and studies of the intracellular signalling cascades. Over the period of the proposal, we will establish an active surveillance system to monitor BVD outbreaks in the field. This will establish animal health and economic outcomes. This will be associated with the existing Defra funded programmes on surveillance and biosecurity. The findings of this project will be incorporated into the recommendations on development of a BVDV control programme for the UK.

There is a final and underlying theme that this proposal addresses; it is to provide essential support of research expertise in livestock diseases. Defra must have access to the highest quality disease research at all times; this needs to be multidisciplinary and, with advantage, some needs to be independent and multi-centred. There is additional advantage for this support to be within a veterinary school in order to provide ‘cutting edge’ pathobiology thereby offering excellent hypotheses for post-graduate research study. It is the depth of molecular and cellular pathology at the RVC that can provide Defra with a sustainable and flexible resource to approach emerging and re-emerging diseases and, through our collaboration with IAH, a strength in livestock immune responses and through SAC interactive and applied involvement with the farming industry.

This proposal, with Defra support, will provide both a critical mass to support future research training positions and a focus for research on livestock disease.

1. BVD – Determination of molecular and cellular mechanisms of virulence in acutely haemorrhagic and damaging strains of BVDV (Margaret Collins, RVC)1.1 Construct an infectious cDNA of BVDV Ho916 and establish the clinical outcomes following in vivo challenge of calves with recovered virus rHo916 and with the non?recombinant parental virus. (24 months, start month 1)1.2 Construct recombinants with exchange of a) the entire ORF, b) the structural proteins, c) the non?structural proteins for the attenuated genogroup 1 strain, Ky1203 strain of BVDV (3 new recombinant viruses). Establish the clinical profiles following challenge of calves with each of the 3 new Ky1203?derived recombinant viruses. (36 months, start month 12)1.3 Establish the stable expression of structural proteins for the attenuated genogroup 1 strain, Ky1203 strain of BVDV in a suitable cell line eg MDBK cells and assess it's ability to induce protective immunity in BVDV naïve calves. STAFF:- RAIA (post-doctoral scientist) [RVC], RAIB (research assistance) [RVC], PhD (PhD 1) [RVC] and PhD (PhD 3) [CVL] will be appointed to the molecular biology project (1.1, 1.2, 1.3)2. BVD – Characterisation of the protective cellular immune responses to virulent field virus and their relevant molecular constructs (Dirk Werling, RVC & Bryan Charleston, IAH)2.1 Identify the cellular mechanisms of immunosuppression and thrombocytopenia (60 months, start month1)2.2 Investigate the immune response and cytokine profile of cattle to infection to field and chimeric viruses of different virulence (see 1.1, 1.2, 1.3 above) (36 months, start month 24)2.3 Assess the different antigen presenting cell subsets generated in cattle to different BVDV genotypes (36 months, start month 24) 2.4 Determine the duration of protective immunity following natural infection (36 months, start month 24) STAFF:- PhD (PhD 2) [RVC] and RAIB (25% FT) [RVC] will be appointed to the immunology project (2.1, 2.2, 2.3)3 BVD - Undertake pilot on-farm programmes of BVD control with assessment of animal health and economic outcomes, information critical to the development of regional and national strategies for control of BVDV (George Gunn, SAC & Michael McGowan, RVC)3.1 Develop a range of protocols for control of BVDV in beef and dairy herds and criteria for selection of herds (4 months, start month 1)3.2 Identify study herds, establish detailed baseline farm business information for three groups of cattle farms, define herd BVDV status and estimate pre-intervention economic losses associated with herd BVD infection (12 months, start month 5)3.3 Establish implement and maintain customised biosecurity recommendations for control of BVDV (36 months, start month 6)3.4 Assessment of the animal health and economic outcomes of the pilot BVDV control programmes with report to the industry (36 months, start month 24)STAFF:- RAIA (post-doctoral scientist) [I year at SAC]; PhD (PhD 4) [SAC] and RAIB (25% FT) [RVC] will be appointed to the pilot farm BVDV control projects (3.1, 3.2, 3.3, 3.4)
Objective
1) BVD – Determination of molecular and cellular mechanisms of virulence in acutely haemorrhagic and damaging strains of BVDV.
2) BVD – Characterisation of the protective cellular immune responses to virulent field virus and their relevant molecular constructs.
3) BVD - Undertake pilot on-farm programmes of BVD control with assessment of animal health and economic outcomes, information critical to the development of regional and national strategies for control of BVDV.
Project Documents
• FRP - Final Report : Defra final Report BVDV 050712   (831k)
Time-Scale and Cost
From: 2004

To: 2011

Cost: £1,534,492
Contractor / Funded Organisations
Royal Veterinary College
Keywords
Animal Diseases              
Animal Health              
Biosecurity              
Biotechnology              
BVDV              
Cattle              
GM Non-Food              
Pathogenosis              
Plants and Animals              
Fields of Study
Animal Health