Title:
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Studies on feline calicivirus with particular reference to persistence.
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The molecular evolution of feline calicivirus (FCV) was studied in cell culture and in
persistently infected cats. Sequence analysis of the 5' hypervariable region of the FCV
capsid (5'HVR; located at the 5'end of variable region E), a region known to contain
linear neutralising B cell epitopes, showed FCV existed as a quasispecies which evolved
at the nucleotide and amino acid level during persistent infection. Quasispecies
heterogeneity tended to decrease during the course of persistence. Sequential isolates
from a cat showed marked antigenic variation during the course of persistent infection.
Sequential passage of FCV in cell culture was also associated with sequence evolution of
the 5'HVR. However, these isolates showed no change in antigenicity suggesting that
individual substitutions observed in viruses from cats, but not in viruses from cell
culture, may be responsible for changes in antigenicity. Alternatively, the observed
antigenic changes may be associated with mutations elsewhere in the genome.
In order to identify regions of 'the FCV capsid protein containing linear B-cell epitopes,
two approachesw ere used.F irstly, an expressionl ibrary containingr andom, short (100-
300bp) fragments of an FCV capsid gene was constructed. This library was screened
using polyclonal antisera from a cat that had been challenged experimentally with FCV
to identify immunoreactive clones containing B-cell epitopes. Initial screening identified
five clones that reacted positively to feline antisera in immunoblots. FCV derived
sequencefr om thesec lones all mappedt o the 5'HVR suggestingt his region containst he
immunodominant linear epitopes of the capsid.
The second approach used to identify B-cell epitopes was to map more accurately the
epitope of a neutralising monoclonal antibody (IG9) which had already been shown to lie
in a 37 amino acid region of the 5'HVR (Milton et al. (1992), Journal of General
Virology 73,2435-2439). Replication of plaque purified IG9-sensitive parent virus in
sub-neutralising concentrations of IG9 led to the generation of a neutralisation resistant
escape mutant. Sequence analysis of this mutant and the parent virus revealed a single
non-synonymous nucleotide substitution within the 5'HVR suggesting this residue is
critical to the correct formation of the IG9 epitope.
A method to type FCVs based on sequence analysis of the 5'HVR was established.
Most isolates appeared relatively homogenous. However, some isolates, both from
vaccines and vaccine failures, appeared to contain more than one FCV. Comparison of
5'HVR sequencesfr om different isolatess howed that most isolates were either 0-5.3%
different (related isolates) or 20.7-42.7% different (unrelated isolates). The majority of
the relatedi solatess hareda n epidemiologicall ink, implying they representedis olatest hat
originated from a common source. Comparisons of sequences obtained from vaccine
failures and vaccine virus fell into two similar categories; those with closely related
sequences(0 .0-5.3%) implying a role for the vaccinei n diseasea nd those with divergent
sequences(2 1.3-38.7%)i mplying field virus causedt he disease.
These results were compared with those obtained by using a serotyping method based on
virus neutralisation (VN) which exploits differences in antigenicity between most FCVs
gene (Dawson et al. (1993), Veterinary Record 132,346-350). VN and sequence
analysis gave the same typing result in 65-73% of individual cases. Based on these
results and the difficulty of interpreting VNs, we suggest that molecular based sequence
analysis may be more suitable to the epidemiological investigation of FCV related
disease particularly in the case of vaccine reactions.
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