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Development of rapid detection and differentiation tests for West Nile Virus and other flaviviruses in different species - SE4106

West Nile Virus (WNV) is a positive-stranded arthropod-borne virus belonging to the family Flaviviridae. It is a notifiable exotic virus and is classified as a ACDP 3 / SAPO 3 agent. The virus naturally cycles between mosquitos and wild birds, but can also be transmitted to humans and equine species where it can cause a fatal encephalitis. WNV was first isolated in Uganda in 1937 but has since been found throughout North Africa and the Middle East. For reasons, which have yet to be determined, the virus appears to be spreading north and in recent years, outbreaks have been observed in Romania (Savage et al., 1999, Am. J. Trop. Med. 61, 600) and France (Murgue, et al., 2000, Emerg. Inf. Dis. 7, 792) It has also inadvertently been transported to North America where, from an initial case in New York in 1999 (Lanciotti, at. al., 1999, Science 286, 2333), it has spread throughout the continent causing numerous human cases and resulted in a large number of avian deaths. As a result, the United States maintains extensive surveillance for the presence of WNV. Preliminary surveys using serological and sensitive molecular techniques of British birds have suggested that WNV is present within native species in the UK (Buckley et al., 2003, J. Gen. Virol. 84, 2807) and could present a public health hazard to humans and domestic livestock, especially equines.

This proposal aims to develop rapid WNV detection methods capable of high throughput in order to ensure that the UK is capable of responding to a large outbreak of the virus. This work would involve the development of a network of links to scientist around the world with the purpose of sharing expertise and obtaining panels of viral isolates and serum to enable validation of selected tests. These would include virus detection tests, including Taqman PCR, and serological assays (virus neutralisation and IgM ELISA) for use with both avian and equine samples. Further aims would develop a WNV sequence database from existing sequences in the public domain and sequences generated by the VLA. This data would, through the use of molecular epidemiological techniques, enable the comparison of outbreak viruses to other epidemics of WNV from around the world and the tracing of an outbreak to distinguish between the spread of a single introduction or multiple introductions. Finally, this project would investigate the pathogenic mechanisms that allow WNV to cross the blood-brain-barrier and cause disease within the susceptible host. Of particular interest is the role of a family of protein receptors within the brain, known as toll-like receptors, in the early response to viral infection. It has been reported that these receptors actually enhance the entry of WNV entry into the brain by an unspecified mechanism (Wang et al., 2004, Nature Medicine, 10, 1366). This would allow the development of an appropriate disease model at the VLA and lead to a clearer understanding of the host immune respons and WNV pathogenesis. Such work could enable the development of future diagnostic tests.

This will directly support Defra policy through:

The development of validated tests (National Reference Methods) that will enhance quality standards for the surveillance of viruses with zoonotic potential within the UK.

The establishment of diagnostic assays capable of delivering rapid high-throughput testing required for the proposed contingency plan for Specified Type Equine Exotic Diseases (STEED).

Enhanced quality standards for tests supporting International Trade.

Improved consultancy for Defra through a network of expertise linked to the Veterinary Laboratories Agency.
The overall objective is to develop the ability of the VLA to respond to both single cases and large-scale outbreaks of West Nile Virus. In both cases, the need for rapid, accurate diagnosis is paramount. This will directly support Defra policy for the proposed Specified Type Equine Exotic Diseases (STEED) contingency plan. This work will also enhance the quality standards for UK surveillance of emerging viruses with zoonotic potential through the validation of National Reference Methods (NRM).

To achieve this development, this proposal has been divided into four general objectives:

1. Establish a network of scientific links to existing researchers in the field of West Nile Virus research and surveillance. The purpose of this is to bring expert knowledge to the VLA and to obtain reagents necessary for test development and validation.

2. Develop and validate rapid diagnostic tests for West Nile Virus. A Taqman PCR offers rapid detection of the viral genome and the ability to measure large volumes of samples. Additional tests include the validation of the plaque neutralisation assay and a commercially available IgM ELISA for the rapid detection of WNV positive antisera.

3. The establishment of a West Nile Virus genomic database using existing viral sequences and new sequences obtained for the viral envelope (E) gene. This information would be used in tracking outbreaks and enable the distinction between WNV and other viruses from the Japanese Encephalitis complex of viruses that are capable of causing encephalitis.

4. Develop experimental models for studying WNV pathogenesis, specifically those that allow investigation of the ability of the virus to cross the blood-brain-barrier. Specific emphasis will be given to the host response to viral infection of the brain. This will enhance our understanding of disease mechanism in both avian and mammalian hosts and enable the development of future diagnostic tests.
Project Documents
• Final Report : Final Report for SE4106   (592k)
Time-Scale and Cost
From: 2006

To: 2009

Cost: £296,229
Contractor / Funded Organisations
Veterinary Laboratories Agency
Animal Diseases              
Animal Health              
Plants and Animals              
Vector-Borne Viral Diseases              
Fields of Study
Animal Health