In the national reference laboratory for equine encephalitis diseases caused by the Venezuelan equine encephalitis virus (VEEV), the Eastern equine encephalitis virus (EEEV) and the Western equine encephalitis virus (WEEV) are investigated. These diseases can be combined as viral equine encephalitides. All three viruses belong to the virus family Togovirdae of the genus Alphavirus and are due to their transmission by sanguivorous arthropods members of the Arbovirus group. The ecological reservoir for these viruses is build by birds and in the case of VEEV by small mammalians. These reservoir species develop no clinical symptoms after an infection with these viruses. However, if the virus is distributed by arthropods which take their blood meals on different species, a virus transmission over species frontiers occurs and can result in diseases with a fatal outcome to some extent. So are horses and humans so called dead-end hosts for the Eastern equine encephalitis virus and the Western equine encephalitis virus meaning there is no possibility that these species are origins of further viral transmissions. In these species infections are mostly asymptomatic but in few cases the infection can lead to massive damages of the central nervous system and also to death. For the Venezuelan equine encephalitis virus viral transmission originating from horses and humans has been described whereas in these case the disease shows a clinical milder course.
The national reference laboratory uses a combination of different molecular biological and biochemical methods to identify and differentiate these notifiable diseases. Detection of the viral genome from blood samples is only possible during acute viremia. However, since neurological symptoms only occur after the viruses have infected central nervous tissues, the viral load in the blood has already decreased considerably by this time. Therefore, neurobiological tissue samples are more suitable as starting material for investigations in this case. The viral genome is detected by quantitative RT-PCR (RT-qPCR) using specific primer/probe combinations. In addition to the qualitative detection of the viral genome, this method allows a quantification of the viral load in the sent samples. In addition to this quantitative assessment, the higher specificity of the method as well as its lower sensitivity for contaminations has also contributed to the replacement of the classic method using (semi-) nested RT-PCR for these viruses.
Enabling us to make conclusions about a seroconversion of a possible infected organism two ELISA assay systems for EEEV and VEEV are in establishment. On one hand an IgM-ELISA based on the method used at the OIE reference laboratory in Ames/Iowa has been adopted to the means of our institute. This assay will allow us to detect an acute viral infection in horses by VEEV and EEEV. For the establishment of the assays a small number of sera with limited volumes have been obtained from the OIE reference laboratory in Ames/Iowa. Unfortunately, the routine use of these methods in the national reference laboratory needs a validatioin of both assays with a larger panel of positive and negative sera whose status has already been determined by other methods (e.g. PRNT). This is also true for the establishment of a competitive ELISA allowing the species independent detection of seroconversion in susceptible species. Validation and setting up similar ELISA systems for WEEV are topics of the recent work
Both already established ELISA assays have been used to test horse sera from different parts of Germany. As expected we have not found any positive result since EEEV, VEEV and WEEV are still exotic pathogens in Europe.
Due to the need of inactivated virus antigen for the shown assays, systems using recombinant structural proteins of the viruses are under development. By the development of diagnostic systems for the viral genomes and the seroconversion after an infection the national reference laboratory for equine encephalitis contributes to the surveillance of a possible introduction of equine encephalitis viruses to Germany. Furthermore, these methods enable us to diagnose acute and past infections of the viruses within the scope of investigations necessary in a globalised world with intensive trade of possible hosts especially concerning import regulations of different countries.
- Antigen- and genome detection in sample material
- Antibody detection
- Primary contact for federal, state and European authorities regarding Equine encephalitis viruses diagnostic and control
- Characterization of virus isolates
- Review, standardization and enhancement of diagnostic procedures
- Participation and organization of ring trials (national and international)
- Collaboration with national and international working groups and participation on research projects
- Virus isolation and cultivation in vitro
- Genome detection by RT-PCR (quantitative)
- Characterization of virus strains by genome sequencing
- Quantitative analysis of neutralizing antibodies (serum neutralization assays, plaque reduction assays, surrogate assays)
- Serological analysis of antibodies by ELISA including differentiation of immunoglobulin classes
- Immunofluorescence-based detection of antibodies (iIFA)