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Rapid Detection Tests for FMD and Rift Valley Fever

After an outbreak of Foot and Mouth Disease (FMD) has been confirmed, the emergency response program to eradicate the disease involves sometimes massive culling of infected or exposed herds. The FAZD Center is developing rapid, accurate, inexpensive field tests that will distinguish between infected and uninfected animals at chute site within minutes. This will eliminate unnecessary loss of uninfected animals, saving hundreds of thousands of animals in large outbreaks.

Tilahun YilmaPrincipal Investigator: Dr. Tilahun Yilma, University of California, Davis. Director, International Laborator of molecular Biology for Tropical Disease Agents and Professor of Virology.  Research interests in Rift Valley fever and foot and mouth disease. Published research

 

 

 

 

 

 

 

 

Rapid diagnostic test for FMD

Foot-and-Mouth Disease is one of the most economically devastating diseases of livestock. Rapid identification of the cause of an outbreak of a vesicular disease in cattle, swine and sheep is an essential tool for controlling outbreaks of this disease. We are currently constructing a rapid diagnostic kit that will detect antigen to the virus that causes foot- and- mouth disease. This kit is based on technology similar to that used in commercially available pregnancy testing kits and will be about the size of a matchbox. The kit will be very simple to use and no special equipment or trained laboratory technicians are required to perform the test. A drop of blood or vesicular fluid from an animal in a suspected outbreak is all that is needed to detect foot-and-mouth disease viral antigen in just a few minutes using this technology. Current tests require sending samples to diagnostic laboratories and results are typically available in days or weeks. Thus, this kit will greatly increase the speed of detection of this virus in the event of an outbreak or for routine epidemiological surveys.

To construct the kit we will use a battery of monoclonal antibodies against a highly conserved viral protein (the viral RNA polymerase or P3D protein) bound to a chromatographic strip. Since this protein is nearly identical in all strains of the foot-and-mouth disease virus it will enable the detection of all serotypes. Thus, only one kit needs to be constructed. To screen for monoclonal antibodies to this protein, it was expressed at high levels in an insect virus (baculovirus), a technique that allows large quantities of a viral protein to be obtained and purified inexpensively and safely. The baculovirus-expressed P3D protein was purified and used to coat ELISA plates as a rapid screening test for monoclonal antibodies recognizing this protein.

Monoclonal antibodies to FMDV-P3D will be developed as capture antigen for the rapid test kit. The antigen-capture monoclonal antibodies will be bound to a chromatographic matrix strip and the reagents for the kit will be optimized. Once the kit is optimized, it will be produced in quantity and validated by using it to test large numbers of blood samples from animals positive or negative to foot-and-mouth disease virus.

We have extensive experience using the baculovirus system for the expression of proteins for use in diagnostic kits; we have developed diagnostic kits for three other diseases of livestock using this technique.

 

 

Rapid diagnostic kit for Rift Valley fever

Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, infects most mammalian species, especially small ruminants, with high morbidity and mortality, and is also highly infectious to humans with potentially fatal consequences. This virus is transmitted and maintained by numerous mosquito genera including several native to North America (Aedes, Culex, and Anopheles). Thus, if RVFV is accidentally or deliberately introduced into this country, the disease is highly likely to become endemic in North America. Consequently, this virus has enormous potential as a bioterrorist agent to cause devastating economic losses in the livestock industry as well as illness and death in humans.

In the event of an outbreak of RVF in the US, rapid diagnostic techniques would be required to control disease.
We propose using molecular biology techniques to produce an inexpensive and safe source of reagents for an indirect ELISA kit. This test can be run in two to four hours. We have developed three diagnostic kits similar to the one proposed for RVFV that are currently being use to detect antibody to other viral causes of diseases of ruminants: rinderpest virus, peste des petites ruminants virus, and vesicular stomatitis virus. We used the baculovirus expression system to safely produce large quantities of inexpensive reagents for these diagnostic tests that are currently in use in the United States and Africa.

We are using the same techniques to express large amounts of a highly immunogenic protein (nucleocapsid) of the RVFV to develop a similar test. We have cloned the gene for this protein into a baculovirus plasmid transfer vector, co-transfected with a baculovirus expression system in Sf9 cells and recombination confirmed. We are in the process of producing anti-RVFV serum in mice for further characterization of the N protein antigen as well as for the standardization of the RVFV-ELISA.