National Reference Laboratory for Equine encephalitis viruses (EEEV, VEEV, WEEV)
Institute for Novel and Emerging Infectious Diseases
Markus Keller, Martin H. Groschup
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. During the acute viremic phase of the diseases the detection of viral genome in samples derived from parts of the central nervous system is possible using a nested RT-PCR with primer specific for the individual virus. In addition to this qualitative method for the detection of viral genomes a quantitative real-time RT-PCR has been established in cooperation with Dr. Bernd Hoffmann and Dr. Martin Eiden. This method allows us to make conclusions regarding the virus load in the sent samples. 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. On the other hand a competitive ELISA allowing the detection of a seroconversion in different species has been developed. Both ELISA assays have been used to test horse sera from different parts of Germany. As expected we have not found any positive result. For the routine use of these methods in the national reference laboratory both assays have to be validated with a panel of positive and negative sera whose status has already been determined by other methods (e.g. PRNT). This and the establishment of similar ELISA systems for WEEV are topics of the recent work.
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.