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.