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Detection of Antibodies against Borna Disease Virus and Tick borne Encephalitis Virus in Horses from Spain

Fatima Cruz Lopez†, Susanne Schmid und Matthias König

† Servicio de Vigilancia Sanitaria Equina Centro de Vigilancia Sanitaria Veterinaria (VISAVET) Universidad Complutense Madrid

Borna disease (BoD) is a sporadically occurring, usually fatal disorder caused by a neurotropic RNA virus, the Borna disease virus (BoDV) (Heinig, 1969, Danner, 1982, Rott and Becht, 1995, Richt et al., 2007). BDV infection in horses is endemic in Central Europe (Germany, Austria, Switzerland and Lichtenstein) and there are reports of clinical disease in Israel, Japan, Iran, Australia and the USA (Rott and Becht 1995, Staeheli et al. 2000, Durrwald et al., 2006, Herden and Richt 2009).

Tick borne encephalitis (TBE) is a viral tick-borne infectious disease that occurs in endemic areas across large regions of Europe and Asia(Charrel et al., 2004). It is caused by the Tick borne encephalitis virus (TBEV), which includes three subtypes: western subtype (endemic in central, Eastern and northern Europe), Siberian subtype (endemic in Eastern Europe, Russia and Northern Asia), and far Eastern subtype (endemic in Eastern Russia and some parts of China and Japan) (Gritsun et al., 2003, Suss, 2011).

In Spain, BoDV and TBEV infection are currently regarded as exotic diseases in horses, and serological investigations have been never performed. The aim of this study is to investigate whether there may be serological evidence of BDV and/or TBEV infection in horses in Spain and to determine risk factors for the transmission of this important infection.

Individual serum samples from 500 horses, collected between September 2011 and October 2016 in Spain will be tested by Immunofluorescence (BoDV) and Seroneutralisation (TBEV). Data on factors putatively associated with seropositivity to BVD and/or TBEV will be examined using random effects logistic regression for analysis of clustered data. Furthermore, samples from horses with neurological signs and a negative diagnosis to EHV-1/-4 qPCR and WNV qPCR and IgM ELISA will be tested by PCR in order to determine the presence of BoDV an/or TBEV RNA. Any positives will be further cultivated and characterised in order to compare them to European isolates. The presence of seropositive horses in this study would involve a wider distribution of BoDV and/or TBEV in Europe and therefore would inform about the need for surveillance in this country. The presence of PCR positives among the horses with neurological signs in this study would involve the presence of clinical disease caused by any of these viruses in Spain which, up to present, has never been reported.




Charrel, R.N., Attoui, H., Butenko, A.M., Clegg, J.C., Deubel, V., Frolova, T.V. et al. Tick-borne virus diseases of human interest in Europe. Clin Microbiol Infect. 2004; 10(12):1040-1055.

Danner, K. (1982) Borna-Virus und Borna-Infekionen, Enke Copythek, Stuttgart.

Durrwald, R., Kolodziejek, J. Muluneh, A., Herzog, S. and Nowotny, N. (2006). Epidemiological pattern of classical Borna disease and regional genetic clustering of Borna disease viruses point towards the existence of to-date unknown endemic reservoir host populations. Microbes and Infection 8, 917-929.

Gritsun, T.S., Nuttall, P.A., Gould, E.A. Tick borne flaviviruses. Adv Virus Res. 2003; 61:317-371.

Heinig, A. (1969). Die Bornasche Krankheit der Pferde und Schafe. In: Handbuch der Virusinfektionen bei Tieren, ed: Röhrer, H., Fischer, Jena. p 83-148.

Herden, C. and Richt, J.A. (2009) Equine Borna disease. In Infectious Diseases of the Horse. Eds T. S. Mair, R. E. Hutchison. Equine Veterinary Journal.

Richt, J.A., Grabner, A., Herzog, S., Garten, W., and Herden, C.  (2007) Borna disease  in Equines. In: Equine Infectious diseases. Eds: D.C. Sellon D.C., M. Long, Saunders Elsevier, St. Louis. pp 201-216.

Virusinfektionen bei Tieren, ed: Röhrer, H., Fischer, Jena. p 83-148.

Rott, R., and Becht, H. (1995) Natural and experimental Borna disease in animals. In: Borna disease. Eds:  H. Koprowski H, W.I. Lipkin WI, Springer, Berlin. pp 17-30 (Curr. Top. Immunol. Microbiol. 190).

Staeheli, P., Sauder, C., Hausmann, J., Ehrensperger, F. and Schewemmle, M. (2000) Epidemiology of Borna disease virus. Journal of General Virology 81 (Pt 9), 2123-2135.

Suss, J. Tick-borne encephalitis 2010: Epidemiology, risk areas, and virus strains in Europe and Asia – an overview. Tick-Borne Dis. 2011; 2(1):2-15.

Humoral immune response against Borna Disease Virus

Susanne Schmid, Matthias König, Heinz-Jürgen Thiel

Borna disease, ELISA, serology, BDV

Borna Disease is a non purulent meningoencephalomyelitis with central nervous symptoms in horses and sheep. It is named after a town in Saxony, Germany, where a devastating outbreak took place in the late 19th century. The causative agent is a RNA virus (Borna Disease Virus, BDV) which belongs to the virus family Bornaviridae. he disease is endemic in Germany, Austria, Switzerland and Liechtenstein. Natural hosts are besides horses and sheep also llamas, alpacas and a few other species. Usually only individuals become diseased; the transmission is not completely clarified. Diseased animals show initially unspecific symptoms like hyperthermia, anorexia and colics. These are followed by an encephalomyelitis with ataxia, behavioral disturbances, paralysis and coma. In most cases the disease is lethal; surviving animals show lifelong symptoms like behavioral disturbances. Differential diagnoses are infections with EHV-1, rabies or also intoxication. As there are no pathognomonic symptoms, further investigation in laboratory is needed. As a standard method we use the indirect immunofluorescence out of serum or liquor (CSF) samples where we detect virus specific antibodies. The detection of antibodies in the CSF or a raise of antibody titer in serum samples - together with clinical symptoms - is proving for an acute disease. A disadvantage of the common test system is the species-dependency. As an indirect test you always need anti-species-specific secondary antibodies which make the test quite time-consuming. Additionally interpretation of the test results can be difficult. There might be components in the sera which bind to the cells used in the test and so cause a non specific fluorescence that is almost indistinguishable. In this project it is planned to develop alternative methods for the common serological test. One approach is it to create a competitive Sandwich-ELISA for detecting antibodies in serum and CSF samples. As an advantage to the current test this ELISA could be used for a lot of samples at the same time and it would be species-independent so that you can perform one test for different species. Therefore it is planned to express and purify several viral proteins and check the humoral immune response of different species. Based on these results the proteins will be used as an antigen in the ELISA.

2010 - 2015

Noroviruses and Sapoviruses in farm animals

Barbara Bank-Wolf, Matthias König and Heinz-Jürgen Thiel

Noroviruses and sapoviruses represent distinct genera within the virus family Caliciviridae and are important causative agents of human gastroenteritis. They also occur in farm animals such as cattle and swine (bovine norovirus, porcine norovirus and porcine enteric sapovirus). The classification of animal and human isolates into the same genera as well as the described occurrence of inter- and intragenogroup recombination lead to the question whether virus transmission from animal to man and vice versa takes place.

The relatedness between human and animal isolates was studied. Stool and tissue samples from farm animals (cattle, swine, small ruminants) with and without diarrhea originating from Germany and the Netherlands were tested and accompanied by questionnaires on individual data as well as data about the situation on the farms. These questionnaires were evaluated statistically with regard to the correlation between the presence of noro- or sapovirus infection and specific parameters.

Using different RT-PCRs norovirus was detected in 32 out of 546 samples from calves (5.9%) and sapovirus in 8 of 132 samples from swine (6.1%). 32 stool samples from small ruminants as well as 20 samples from animals of other species gave negative results when tested for noro- and sapoviruses. In addition no positives were detected testing 202 samples from cattle for sapoviruses and 132 samples from swine for noroviruses.

Significant links to age and clinical symptoms were found. In most cases virus detection coincided with occurrence of diarrhea. RT-PCR products were cloned, sequenced and used for phylogenetic analyses. To test for recombinants additional genome parts were analysed. The newly detected virus isolates are more closely related to known animal noro- and sapoviruses than to human isolates. In two samples from swine a double infection with two isolates from different genogroups of porcine enteric sapoviruses could be detected. One of the virus isolates in each sample probably represents a recombinant between members of different sapovirus genera.

The results demonstrate that noro- and sapoviruses occur sporadically in farm animals in Germany and the Netherlands. There are currently no hints for zoonotic transmission of animal noro- and sapoviruses.

Antibody titres of pet animals after vaccination against rabies in view of European regulations concerning animal movement

U.V. Jakel, M. König, H.-J. Thiel

rabies, vaccination, pet animal movement, import regulations

Since the introduction of the pet passport on 01.10.2004 harmonized rules for the non-commercial movement of dogs, cats and ferrets apply within the EU. These rules are specified in the Regulation (EC) No. 998/2003. Apart from clear identification and rabies vaccination measurement of rabies virus neutralizing antibodies by Fluorescent Antibody Virus Neutralization (FAVN) Assay is compulsory for import of these species from non-listed third countries to the EU and for movement of dogs and cats to the rabies free member states UK, Ireland, Malta and Sweden. A considerably high percentage of animals vaccinated corresponding to the manufacturers recommendations fail to achieve the minimum level required of ? 0,5 IU/ml. Our study is aimed to identify possible risk factors for test failure.

We performed a survey by a questionnaire sent to veterinary practitioners to obtain detailed information about animals tested for travel purposes. This included data about breed, age and sex, further medical treatment and rabies vaccination history. A statistical analysis in dependence of test outcome will be performed.

The influence on test outcome of the virus strain used in FAVN test will be studied in a separate laboratory trial.

Final results of our study will be available in 2008.

6/05 - 02/07 extended until 02/08

The project is sponsored by the Federal Ministry of Food, Agriculture and Consumer Protection, Germany
The project is performed in cooperation with the Paul-Ehrlich-Institut, Federal Agency for Sera and Vaccines, Langen, Germany

(1) Institute of Virology, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany
(2) Paul-Ehrlich-Institut, D-63207 Langen, Germany
Accepted for oral presentation at the International Conference "Towards the Elimination of Rabies in Eurasia", Paris, May 2007

Development of antibody titres after vaccination against rabies virus in dogs, cats and ferrets

U.V. Jakel, M. König, H.-J. Thiel

rabies, vaccination, adjuvants, injection site fibrosarcoma

Aim of this project was to evaluate current rabies vaccination protocols in view of travel regulations by performing vaccination experiments in dogs, cats and ferrets. Preliminary data suggest that young dogs vaccinated only once before blood sampling for rabies antibody testing show a higher risk of test failure than dogs that have already received more than one rabies vaccination. Further more the need for use of aluminium adjuvants in rabies vaccines for cats and ferrets was revised as aluminium adjuvants are believed to play a role in the pathogenesis of injection-site associated fibrosarcomas. Different vaccination protocols and different rabies vaccines licensed for human or veterinary use were tetsed. Serum neutralizing antibody titres were repeatedly measured and booster vaccinations administered after different time intervals. Antibody titres after primary and booster vaccinations were compared.

Adjuvanted and non-adjuvanted rabies vaccines were administered to 28 cats in 4 groups. Antibody titres were pursued for 3 years before booster vaccination. 64 ferrets were vaccinated with 5 different rabies vaccines for human or animal use. Different vaccination schemes were applied. Booster vaccination was given 2 years after primary immunization. In dogs two initial rabies vaccinations were administered at intervals of 30 and 120 days in two different groups. Another booster vaccination was given 1 year later. Two rabies vaccines licensed for use in dogs were included.

6/05 - 02/07 extended until 02/08

The project is supported by the Federal Ministry of Food, Agriculture and Consumer Protection, Germany
The project is performed in cooperation with Paul-Ehrlich-Institut, Federal Agency for Sera and Vaccines, Langen, Federal Agency for Risk Assessment, BfR, Berlin, Max-Planck-Institut for Brain Research, Frankfurt/M and Military Dog Training Center, Schule für Diensthundewesen der Bundeswehr, Ulmen

K. Duchow(1), C. Kiefert(2), V. Jakel(3), M. König(3) and K. Cussler(1)
(1) Paul-Ehrlich-Institut, D-63207 Langen, Germany;
(2) Max Planck Institute for Brain Research, D-60496 Frankfurt, Germany;
(3) Institute of Virology, Justus-Liebig-Universität Giessen, D-35392 Giessen
Poster presentation at 1st International Conference on Rabies in Europe, Kiev, 2006

V. Jakel(1), M. König(1), K. Cussler(2), C. Kiefert(3), D. Wolff4, D. Neubert4, H.-J. Thiel(3), K. Duchow(1)
(1) Institute of Virology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany;
(2) Paul-Ehrlich-Institut, 63207 Langen, Germany;
(3) Max Planck Institute for Brain Research, 60496 Frankfurt, Germany;
(4) Federal Institute for Risk Assessment, 14195 Berlin, Germany
Poster presentation at 4th International Veterinary Vaccines and Diagnostics Conference IVVDC 2006, Oslo

Molecular and Biochemical Characterisation of the rabies vaccines "Rabipur" and "Rabivac" and the respective virus strains FluryLEP and PM 1503

Claudia Carolina Lopez Yomayuza, Iris Stallkamp, Matthias König, Heinz-Jürgen Thiel

Rabies Virus, rabies disease, two-dimensional electrophoresis (2-D SDS-PAGE), N-terminal peptide sequencing, Matrix-Assisted Laser Desorption Ionisation Time-of-Flight Mass Spectrometry (MALDI-TOF MS), enzyme immuno assays.

Rabies is an almost invariably fatal viral infection of the central nervous system that affects mammals, including humans. Rabies disease can be prevented through vaccination programs. Current rabies vaccines for humans are well tolerated with rare adverse reactions. Vaccines are extensively tested to ensure quality and safety during vaccine manufacturing, including control of source materials, control of the production process and control of the final bulk material.

Two of the most traded vaccines worldwide for rabies prophylaxis are the purified chick-embryo cell vaccine (PCECV) "Rabipur" and the diploid cell derived vaccine (HDCV) "Rabivac". These vaccines are based on inactivated and purified rabies virus strain FluryLEP and PM 150(3), respectively.

In order to increase the knowledge about the vaccines, the molecular and biochemical characteristics were studied. Genetic modifications of the genome of the rabies virus strain FluryLEP and PM 1503 in comparison to other vaccine strains were analysed, emphasising the viral glycoprotein and nucleoprotein. The content of viral and non viral proteins was studied by using biochemical methodology, including N-terminal amino acid sequencing and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS).

Additionally, a feature of central importance in the evaluation of a vaccine is the potency. So far the potency of rabies vaccine preparations is measured using the national Institute of Health (NIH) Test conducted in mice. This assay is laborious, time-consuming and has high variability. The development of an in vitro potency assay for rabies vaccines will provide the basis to reduce or replace the use of laboratory animals. Moreover, based on the assumption that in order to elicit an effective immune response, the main viral antigen(s) should be presented to the immune system in a conformation resembling the native state in virions, studies of the integrity of virus particles in vaccine preparations could be correlated with potency. Along these lines, enzyme immuno assays for the quantification of viral glycoprotein as well as nucleoprotein in the vaccine preparation were established. On the basis of the relation between both proteins in rabies virions, the relative quantification of intact particles in the vaccines could be possible.

The results supply valuable information with regard to the improvement of the quality control of the vaccine. Molecular characteristics clarify the identity of the vaccine strains and biochemical analyses supply useful information about the purity of the vaccine.

2/2003 - 2/2006

Morphology and Morphogenesis of Pestiviruses

Stefanie Deike, Matthias König and Heinz-Jürgen Thiel

The genus Pestivirus belongs together with the genera Hepacivirus and Flavivirus to the family Flaviviridae. It comprises bovine viral diarrhea virus (BVDV-1 and -2), classical swine fever virus (CSFV) and border disease virus (BDV); these viruses have a significant economical impact on farming worldwide.

Pestiviruses are enveloped RNA viruses. Virions have a size of 40-60nm and an icosahedral particle symmetry is suggested. The envelope consists of a cell derived lipid membrane carrying the viral glycoproteins E(1), E2 and Erns. The nucleocapsid (25-30nm) is composed of the core protein and the ssRNA of positive polarity. Assembly of virions is assumed to take place at intracellular membranes and newly synthesized virions presumably use the cellular secretory pathway to leave the host cell. In comparison with many other virus systems analyses of these processes are impeded by the low rate of virion synthesis.

The aim of this project is to study the events of morphogenesis and virus release of pestivirus infection in cell culture at the ultrastructural level with transmission-electron-microscopy. At first one-step growth curves are generated after synchronized infections and in addition the amount of viral RNA is determined by quantitative real-time RT-PCR. The results of the initial experiments lead to an infection model suitable to study distinct stages of infection on the ultrastructural level. Further analyses on the distribution of viral structural proteins within cells will be done by immunogold-labeling methods.

This project was supported by the "Stiftung der Eheleute Engemann der JLU" from 04/2005 until 09/2007.

07/2004 - 09/2008 (extended)

Characteristics of novel bee disease syndromes - the relevance of the Kashmir bee virus and the acute bee paralysis virus for bee health

Reinhold Siede, Matthias König, Ralph Büchler und Heinz-Jürgen Thiel

real-time PCR, KBV, ABPV, viral loads of Hessian bee colonies, honey bee cells in culture

Bee colonies are worldwide at risk by a disease complex termed Varroosis. The mite Varroa destructor and several bacterial and viral secondary infections are part of the Varroosis complex. The Acute-Bee-Paralysis-Virus (ABPV) and the Kashmir-Bee-Virus (KBV) are associated with V. destructor. Both viruses are small plus-strand RNA viruses of the family Dicistroviridae. The ongoing project investigates the biological significance of both viruses under field conditions. Within a broad survey viral loads of Hessian bee colonies are determined. Possible correlations between virus quantities and the overwintering performance of the colonies will be described. For this purpose quantitative assessment tools are required. A real time PCR based assay has been set up. For the determination of infectious virus particles culture systems of honey bee cells would be helpful. Experiments are conducted to develop such cell lines. Furthermore the permissivity of already established available permanent wasp cell lines to ABPV and KBV will be tested. ABPV and KBV infections are supposed to be promoted by stress factors. A test system with caged bees has been established to check the influence of stressors for the performance of virus infected bees.

10/2004 - 08/2007