Jeannette Kliemt

Novel Lyssavirus in Natterer’s Bat, Germany

A virus isolated from a Natterer’s bat (Myotis nattererii) in Germany was differentiated from other lyssaviruses on the basis of the reaction pattern of a panel of monoclonal antibodies. Phylogenetic analysis supported the assumption that the isolated virus, Bokeloh bat lyssavirus, may represent a new member of the genus Lyssavirus.

Development of a mouse monoclonal antibody cocktail for post-exposure rabies prophylaxis in humans.

As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb) cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP) conditions. Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP and prevention of rabies in humans.

First isolation of EBLV-2 in Germany.

In Europe, rabies in bats is caused by European Bat Lyssavirus (EBLV) type 1 (EBLV-1) or type 2 (EBLV-2) which form two distinct genotypes (gt 5 and 6) within the genus Lyssavirus of the family of Rhadoviridae. Spill-over infections of EBLV in humans have caused fatal rabies encephalitis and highlighted the relevance of this wildlife disease for public health. The vast majority of the 831 European bat rabies cases reported between 1977 and 2006 were identified as EBLV-1. Only few virus isolates originating from Switzerland, The Netherlands and the United Kingdom were characterized as EBLV-2. Here we report the first EBLV-2 case detected in Germany in a Daubentons bat (Myotis daubentonii) in August 2007. The bat showed clinical signs of disorders of the central nervous system and subsequently tested positive for rabies. The virus was isolated and characterized as EBLV-2 based on its antigen pattern and by nucleotide sequencing. Phylogenetic analysis indicated an association to EBLV-2 isolates from Switzerland which correlates with the origin of the bat close to the Swiss border.

Elimination of terrestrial rabies in Germany using oral vaccination of foxes

Oral rabies vaccination (ORV) has become the method of choice in fox rabies control in Europe. During the past three decades fox-mediated rabies virtually disappeared from Western and Central Europe. Following Switzerland, Germany was the second European country to launch ORV field trials on its territory in 1983. This paper provides a historical overview on the emergence of fox rabies in Germany; describing the basic principles and milestones of the German rabies eradication programme and presenting results of two decades of efforts to control the disease in foxes. Also, setbacks as well as country-specific differences and particularities on Germanys long way to rabies elimination in comparison to other European countries are addressed. Since the first field trials in Germany the number of rabies cases steadily decreased from 10 484 in 1983 to three cases recorded in 2006. On February 3rd 2006 the last case of terrestrial rabies in Germany was detected in a fox near the town of Mainz, Rhineland-Palatinate. In 2008, ORV ceased after 25 years and Germany was officially declared as free from terrestrial rabies. The German rabies eradication programme did cost approximately 100 million euro of which 37 million euro were covered by the EU. For the future, efforts should focus on maintaining a rabies free status by implementing measures to prevent reintroduction of terrestrial rabies from endemic countries.

Evaluation of a commercial rabies ELISA as a replacement for serum neutralization assays as part of the pet travel scheme and oral vaccination campaigns of foxes.

EU Regulation 998/2003 requires the serological testing of rabies-vaccinated dogs and cats in approved laboratories using serum neutralization tests prior to movement of pet animals between certain EU member states and before pet animals are imported from unlisted third countries. Serum neutralisation tests are also used for measuring the efficacy of oral rabies vaccination programmes conducted in wild carnivore populations. In this study we evaluated an OIE-listed commercial ELISA as a potential replacement for serum neutralization assays under routine conditions as a diagnostic tool for both the serological testing of dog and cat sera as part of pet travel schemes and for follow-up investigations as part of oral vaccination campaigns. When dog and cat sera were analyzed by ELISA, a sensitivity compared to the standard serological test of 36.9-82.0% and 44.4-88.9%, respectively, was calculated depending on the method used. For fox field samples from oral vaccination areas the sensitivity compared to the Rapid Fluorescent Focus Inhibition Test (RFFIT) was 32.4% (95% CI 24.8-40.0%). In its present format, the ELISA cannot replace standard serological assays neither in the pet travel scheme nor in follow-up investigations of oral vaccination campaigns. The results obtained resemble those of other rabies ELISAs recently evaluated for the same purpose and may therefore exemplify a general misconception (binding versus neutralization) in rabies serology rather than a failure of this ELISA test per se. Also, problems with technical and legislative issues associated with the serological testing of dog and cat sera for non-commercial movement and related to the outcome of this study are addressed.

Detection of European bat lyssavirus 2 (EBLV-2) in a Daubenton's bat (Myotis daubentonii) from Magdeburg, Germany.

In Europe bat rabies in Daubentons bats (Myotisdaubentonii) and in Pond bats (Myotis dasycneme) caused by the European bat lyssavirus 2 (EBLV-2) has been confirmed in less than 20 cases to date. Here we report the second encounter of this virus species in Germany. A Daubentons bat found grounded in the zoological garden in Magdeburg died shortly after. In the frame of a retrospective study the bat carcass was eventually transferred to the national reference laboratory for rabies at the Friedrich-Loeffler-Institute for rabies diagnosis. Lyssavirus was isolated and characterized as EBLV-2.

Rabies Virus Antibodies from Oral Vaccination as a Correlate of Protection against Lethal Infection in Wildlife

Both cell-mediated and humoral immune effectors are important in combating rabies infection, although the humoral response receives greater attention regarding rabies prevention. The principle of preventive vaccination has been adopted for strategies of oral rabies vaccination (ORV) of wildlife reservoir populations for decades to control circulation of rabies virus in free-ranging hosts. There remains much debate about the levels of rabies antibodies (and the assays to measure them) that confer resistance to rabies virus. In this paper, data from published literature and our own unpublished animal studies on the induction of rabies binding and neutralizing antibodies following oral immunization of animals with live attenuated or recombinant rabies vaccines, are examined as correlates of protection against lethal rabies infection in captive challenge settings. Analysis of our studies suggests that, though serum neutralization test results are expected to reflect in vivo protection, the blocking enzyme linked immunosorbent assay (ELISA) result at Day 28 was a better predictor of survival. ELISA kits may have an advantage of greater precision and ability to compare results among different studies and laboratories based on the inherent standardization of the kit format. This paper examines current knowledge and study findings to guide meaningful interpretation of serology results in oral baiting monitoring.

New insights into the genetics of EBLV-1 from Germany.

Previous epidemiological studies on EBLVs indicated a distinct geographical distribution of EBLV-1 in Germany. In this study, 48 isolates were selected to further investigate the spatial and temporal distribution of EBLV-1 variants in Germany. The nucleoprotein-gene (N), the nucleoprotein-phosphoprotein spanning untranslated region (NP-UTR) and the UTR between G- and L-gene of each isolate were sequenced using direct cycle sequencing. Results of the subsequent phylogenetic analysis of the N-gene confirmed previous studies on EBLVs, showing a high sequence identity among German EBLV-1a isolates, and a correlation between genetic and temporal and spatial distance, respectively, was shown. Our results indicate that the GL-UTR is not suitable for phylogenetic analyses. Interestingly, 6 nt insertions in two isolates as well as a single nucleotide insertion in a different isolate were detected in the N-P UTR. Within the UTR between G- and L-gene one isolate showed a 35 nt deletion. The effect of those changes on viral properties remains elusive as such mutations have not been described for lyssaviruses before.