Transcript Slide 1

Dominance of a non-pathogenic
over a pathogenic glycoprotein gene
in rabies virus
Bernhard Dietzschold
Thomas Jefferson University
Paris, France
May 2007
The RV G protein plays a major
role in the pathogenesis of RV
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The RV G is not only the major antigen
responsible for the induction of protective
immunity, but is also a major contributor to the
pathogenicity of the virus.
To abolish the pathogenicity, the recombinant
RVs have been constructed to carry the G gene
of SADB19 in which Arg333 is replaced by
Glu333.
The Glu333 G protein, referred to as GAN,
renders the virus non-pathogenic for adult mice
after i.c. infection
An ARG  ILE
mutation in the RV G
results in
a loss of
pathogenicity
for adult mice
The non-pathogenic phenotype
associated with GAN is not stable
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After several passages of SPBNGAN in
mice, an Asn → Lys mutation arose at
position 194 of GAN resulting in GAK,
which was associated with a reversion
to the pathogenic phenotype.
Reversion to the pathogenic phenotype
in GA variants after Virus Passage
in suckling mice
Mortality*
Virus Stock
SPBNGA
SPBNGA-GA
SPBNGA-Cyto-C
SPBN**
0 Passage
0/10
0/10
0/10
9/10
(10%)
(10%)
(10%)
(90%)
*Mice were infected i.c. with 104 FFU
** SPBN, leathal for adult mice, serves as a control
***ND = not done
10th Mouse
Passage
1/10 (10%)
0/10 (10%)
4/10 (40%)
ND***
Nucleotide
Sequence
Analysis
of RV GA after
the 5th & 10th
Mouse Passage
single-base change at
nucleotide 639 of G:
Asn194  Lys194
AAT
AAG
or
AAA
Site-directed Mutagenesis
of the GA Gene
Asn194 [N]  Lys194 [K]
AAT
AAG
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Nonpathogenic G: GAN
– aa194 = Asn; aa333 = Glu
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Pathogenic G: GAK
– aa194 = Lys; aa 333= Glu
Mortality (A),
clinical score (B),
and body weight (C)
of Swiss-Webster
mice infected i.c.
with different ratios
of SPBNGAN and
SPBNGAK
Effect of Asn → Lys mutation on the
pathogenicity of double G variants
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Because a RV vaccine candidate
containing two GAN genes (SPBNGANGAN) exhibits increased
immunogenicity in vivo as compared
to the single GAN construct, we tested
whether the presence of two GAN
genes might also enhance the
probability of reversion to
pathogenicity
Construction schematic of recombinant RVs
containing one or two modified G genes encoding
either an Asn (GAN) or a Lys (GAK) at position 194
Mortality (A),
clinical score (B),
and body weight (C)
of Swiss-Webster
mice infected i.c.
with SPBNGAN-GAN,
SPBNGAK-GAK,
SPBNGAK-GAN, or
SPBNGAN-GAK
Synthesis of
genomic RV RNA
(A) in the brain
and induction of
RV VNA titers in
the serum (B) of
mice infected with
double-G RV
variants
Conclusions
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The pathogenicity of an RV containing
a GAN and a GAK gene was strongly
reduced as compared to that of an RV
containing two GAK genes
– This indicates that GAN is dominant in
determining the pathogenicity phenotype
of the RV.
Single-step virus
growth curves (A)
and mitochindrial
respiration (B)
in NA cells infected
with double G RV
variants
Transcription of
viral mRNA (A)
and viral genomic
RNA (B) in NA cells
infected with the
single or double G
recombinant RVs
Conclusions
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The pathogenicity of an RV correlates
inversely with its replication rate in tissue
culture
Virus production and viral RNA synthesis
were markedly higher in SPBNGAN-,
SPBNGAK-GAN- and SPBNGAN-GAK-infected
neuroblastoma cells than in the SPBNGAKand SPBNGAK-GAK-infected counterparts,
– This suggests control of GAN dominance at the
level of viral RNA synthesis
Acknowledgements
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Milosz Faber and Jianwei Li
Thomas Jefferson University, Department of Microbiology & Immunology,
Philadelphia, PA
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Marie-Luise Faber
Molecular Targeting Technologies, Inc., West Chester, PA
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Matthias J. Schnell
Thomas Jefferson University, Department of Microbiology and Immunology,
Philadelphia, PA
This work is supported by NIH Grants:
R01 AI060686-02
R01 AI045097-08