Bernhard Dietzschold Thomas Jefferson University

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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. - PowerPoint PPT Presentation

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  • Bernhard DietzscholdThomas Jefferson UniversityParis, FranceMay 2007Dominance of a non-pathogenic over a pathogenic glycoprotein gene in rabies virus

  • The RV G protein plays a major role in the pathogenesis of RVThe 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 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

  • Nucleotide Sequence Analysis of RV GA after the 5th & 10thMouse Passagesingle-base change at nucleotide 639 of G:

    Asn194 Lys194 AAT AAG or AAA

  • Site-directed Mutagenesis of the GA GeneAsn194 [N] Lys194 [K] AAT AAG

  • Nonpathogenic G: GANaa194 = Asn; aa333 = Glu

    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 variantsBecause a RV vaccine candidate containing two GAN genes (SPBNGAN-GAN) 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 inthe serum (B) of mice infected with double-G RV variants

  • ConclusionsThe pathogenicity of an RV containing a GAN and a GAK gene was strongly reduced as compared to that of an RV containing two GAK genesThis 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

  • ConclusionsThe pathogenicity of an RV correlates inversely with its replication rate in tissue cultureVirus production and viral RNA synthesis were markedly higher in SPBNGAN-, SPBNGAK-GAN- and SPBNGAN-GAK-infected neuroblastoma cells than in the SPBNGAK- and SPBNGAK-GAK-infected counterparts, This suggests control of GAN dominance at the level of viral RNA synthesis

  • AcknowledgementsMilosz Faber and Jianwei Li Thomas Jefferson University, Department of Microbiology & Immunology, Philadelphia, PAMarie-Luise FaberMolecular Targeting Technologies, Inc., West Chester, PA Matthias J. SchnellThomas Jefferson University, Department of Microbiology and Immunology, Philadelphia, PA This work is supported by NIH Grants: R01 AI060686-02 R01 AI045097-08

    Overall, relatively low mutation rate unfortunately its pathogenic.No mutation in insert genes; no exchanges in GAG codon encoding Glu333.Mention underlying mutant population GAGA never develops clean population (insert = no mutation)