A viral pseudotype based approach to measuring morbillivirus neutralising antibodies

The goal of this project is to develop a diagnostic test that is rapid, sensitive and specific. This test will be used to diagnose a viral infection of sheep and goats called PPR, or peste des petits ruminants. PPR is a threat to the livestock industry worldwide and has been declared a priority target for global control and eradication by vaccination. The success of a global control and vaccination programme against peste des petits ruminants virus (PPRV) will require the availability of a highly accurate and rapid diagnostic test that can be performed without the need for expensive specialized laboratory containment facilities since it will have to be carried out in laboratories across the world. However, the current prescribed test for PPRV is the live virus neutralisation test, a test that is slow, requires a laboratory operating under a high level of biocontainment, and is limited in both sensitivity and flexibility.

In order to develop an improved PPRV diagnostic test, we have used a technique in which PPRV surface glycoproteins are expressed on a harmless carrier virus derived from vesicular stomatitis virus (VSV) to generate VSV(PPRV) "pseudotypes". A viral pseudotype consists of the structural proteins and genetic material of one virus (in this case VSV), carrying the surface proteins of another, different virus (in this assay we use the PPRV H and F proteins). The VSV(PPRV) pseudotypes can be used to measure PPRV specific antibodies more rapidly than conventional live virus tests (three days compared with 1 to 2 weeks). The assay displays greater sensitivity (at least 10-times more sensitive) than live virus tests and can measure antibody responses against diverse strains of virus representative of distinct viral lineages in circulation. Finally, the assay circumvents the requirement for high level biocontainment and may be performed safely in any laboratory worldwide with basic cell culture facilities.

In this project we will assist the World Animal Health Organization's Global Control and Eradication program for PPRV by generating a VSV(PPRV)-based diagnostic test. With this test, we will be able to identify infected animals and investigate populations of wild animals which may be responsible for transmitting this virus to livestock. This information will help ensure that vaccination programmes target susceptible populations in areas with a high risk of transmission and therefore improve the chances of successfully eradicating PPRV.

The eradication of rinderpest virus by vaccination is a global success story. Rinderpest eradication relied upon the combination of effective vaccines and reliable diagnostic tests for the identification of infected animals and routine monitoring of vaccine-induced immune responses. The OIE has declared peste des petits ruminants virus (PPRV) a priority target for global control and eradication. If PPRV control and eradication are to succeed, vaccination should be supported by robust diagnostic tests that are sensitive and rapid, accessible to laboratories worldwide, and which can measure humoral immune responses in diverse species and against the distinct viral lineages in circulation.

The current prescribed test for PPRV is the live virus neutralisation test, a test that is slow, requires BSL-3 biocontainment, and is limited in both sensitivity and flexibility. We have developed a technique in which the PPRV surface glycoproteins are expressed on a replication-defective variant of vesicular stomatitis virus (VSV) to generate VSV(PPRV) pseudotypes. The pseudotype-based test is more rapid and more sensitive than the live virus test and can measure antibody responses against diverse strains of virus representative of the distinct viral lineages in circulation.

In this project, we will optimise and validate the PPRV pseudotype-based test, investigating its utility in the qualitative and quantitative measurement of post-vaccination immune responses and its ability to detect antibodies in exposed animal populations. The modular design of the assay will facilitate the mapping of antigenic epitopes on the PPRV surface glycoproteins, facilitating the rational design of the next generation of broadly acting morbilliviral vaccines. Further, as the test uses freely available reagents and requires only BSL-2 containment, this novel approach to diagnosis will make "gold standard" diagnostic testing for morbillivirus-specific antibodies accessible worldwide.

The outputs of this project will benefit researchers developing PPRV vaccines, livestock-keeping families in PPR-affected areas, the livestock sector in African and Asia, policy-makers concerned with livestock trade and disease control in both PPR-affected areas and disease-free countries, including Europe, and wildlife/conservation agencies. A primary impact of this study will be the global eradication of PPR by vaccination. Our novel test is sensitive and specific, it is rapid, adaptable to diverse strains of virus and scalable for high-throughput screening. The high sensitivity of the test will greatly enhance pre- and post-vaccination sero-surveillance, demonstrating the disease-free status of livestock and the absence of infection in wildlife reservoirs. Our established links with researchers in vaccine development and experts in wildlife conservation will ensure that all our outputs are validated. Our collaborating partners serve on the OIE ad hoc committee for PPR and will advise on test validation to OIE standards. Further, the Pirbright Laboratory is an OIE Reference Laboratory and will perform live virus neutralisation tests in parallel to assist with validation. Once validated, the novel test may then be included in the OIE Terrestrial Manual, a manual that is freely available on-line.

Researchers developing PPRV vaccines will benefit directly from this project. While existing PPRV vaccines induce an active life-long immunity, researchers are developing novel vaccines that circumvent the potential risks associated with live attenuated virus (e.g. excretion and reversion to virulence). Defining the humoral response to PPRV will facilitate the rational design of PPRV vaccines. Our system will enable mapping of antigenic epitopes on the viral surface glycoproteins. Our collaborating partners are active in experimental vaccine development and the use of PPRV vaccines in the field and will help refine the technique to ensure that maximum benefit is achieved.

The project will improve serological diagnosis in the developing world, critical to building capacity for research into emerging morbilliviruses and facilitating research into many other livestock and zoonotic diseases of global concern. Our technologies will enhance accessibility to "gold-standard" diagnostics. The OIE prescribed PPRV test for international trade requires BSL-3 containment and thus is restricted to relatively few institutions globally. Our test may be performed at BSL2. Through our collaborative links in Tanzania (NM-AIST) we have demonstrated that low-cost diagnostic testing can be exported successfully to Africa. Further, we have established links with GALVmed (Global Alliance in Veterinary Medicine), a not for profit organisation for whom PPRV is a primary target for control through improved diagnosis and vaccination.

The pseudotype technique will have a direct impact upon wildlife conservation. PPR is an acute contagious disease and infects not only livestock, but camels, buffalo, gazelles, ibex, gemsbok and even lions. The role of wildlife species in the maintenance of PPRV reservoirs and in viral circulation per se is unclear. Our test facilitates rapid, large scale serosurveys of PPR antibodies in any species. Critically, parallel analyses may be performed simultaneously between PPRV and related morbilliviruses, differentiating PPR-specific responses from co-infections. We have established links with the Wildlife Conservation Society and the International Zoo Veterinary Group to conduct serosurveys in wildlife species. Through our veterinary diagnostic laboratory, we routinely provide diagnostic testing for a range of viral diseases. Accordingly, we have considerable local expertise in test development, validation and performance.