Charakterisierung von reptilienpathogenen Paramyxoviren und Analyse des prokaryotisch exprimierten partiellen Fusionsgens

Viral agents isolated from 18 snakes (families Colubridae, Viperidae, and Crotalidae) showing respiratory symptoms and neuronal diseases were investigated. Identifying the isolates as paramyxoviruses showing characteristic cytopathic effect, was carried out by electronmicroscopy and RNA-PCR of two partial genes (L and F). After sequencing RNA-PCR amplicons, identical sequences were found for the reptilian paramyxoviruses (RPMV) isolated in the same outbreak from different snake species, suggesting they are not host-specific. Sequence alignment of partial L and F genes within the RPMV group revealed sequence homology of at least 79 % for nucleotides (nt), and 94 % for amino acids (aa). Further sequence alignment included the Fer-de-Lance virus (FDLV) as a RPMV representative, the established type species of the paramyxovirus family. The greatest similarity was found to the partial L gene of Sendai virus, with 56 % nt and 61% aa identity. Consequently, the RPMV form a closely related, if somewhat diverse, group, belonging to the family Paramyxoviridae. This result was confirmed by phylogenetic analysis. The RPMV group formed three clusters for the L gene sequence and corresponding clusters for the F gene sequence, leaving the Sendai virus as an outgroup, which was included as a representative of the paramyxovirus family. Another phylogenetic analysis based on partial L and F protein sequences included the FDLV and the established paramyxovirus type species. In this case, the FDLV showed the closest relationship to the Sendai virus. The fusion (F) gene and protein of the reptilian paramyxovirus isolated from the snake Gonosoma oxycephala were also investigated. The sequences of the 1638 nt F gene and its 546 aa F protein were obtained and these sequences were compared to the F protein of the established paramyxovirus type species. The F protein, like its counterparts, was predicted to be glycosylated and to contain a furin cleavage site in the F1 und F2 subunit. Domains were predicted to be the signal-peptide (SP), a hydrophobic fusion peptide, two heptad repeats (HR A and HR B), a leucine zipper motif, and a transmembrane anchor (TM). To further characterize the F protein and identify antigenic determinants, an E. coli expression system and four plasmids containing four various constructs of the F gene were used: the first contained an insert with the entire gene; the second, an insert with the F gene minus the SP and TM; a third construct was inserted with the F2 subunit minus the SP; and the fourth contained an insert with the F1 subunit minus HR B and TM. To monitor expression, an anti-His tag monoclonal antibody in a Western blot (WB) was used. Antigenic epitopes were identified in WB by the use of polyclonal rabbit antiserum, raised against our isolate. No expression was detected for the first construct containing the entire F gene, but all other constructs produced proteins of the predicted molecular weights. The anti-viral rabbit serum detected only the protein encoded by the second construct, that included the predicted furin cleavage site. After treatment of this recombinant protein with furin, the antiserum reacted with only one of the fragments. The reactive fragment was identical to the fourth recombinant protein except for the inclusion of an additional 30 amino acids that contained HR B. The results indicate that HR B of the reptilian paramyxovirus F protein contains a linear antigenic epitope.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Ludwig-Maximilians-Universität

Year

2003

Author dc:creator

• Franke, Julia Margareta

Repository record source_url

https://edoc.ub.uni-muenchen.de/1258/

OAI identifier oai:identifier

oai:edoc.ub.uni-muenchen.de:1258