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Antiviral Hammerhead ribozymes are ef

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Siddhesh Sapre

Hammerhead ribozymes (hRzs) targeting two regions of the Chikungunya virus (CHIKv) genome were effective at inhibiting CHIKv replication and transmission in cell cultures and transgenic Aedes aegypti mosquitoes. hRz#9 and hRz#14 were the most potent inhibitors of CHIKv, preventing cytopathic effects in transformed Vero cells and blocking CHIKv transmission in the saliva of transgenic mosquitoes. While hRzs show promise as transgenic antiviral molecules for population replacement strategies, further analysis is needed using natural CHIKv strains and mosquito populations.

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Antiviral Hammerhead ribozymes are effective for developing transgenic suppression of Chikungunya Virus in Aedes aegytpi mosquitos Priya Mishra, Coleen Furey, Velmurugan Balaraman and Malcolm J. Fraser Jr. Dept. of Biological Sciences, Eck institute for Global Health, University of Notre Dame Viruses 2016, 8, 163; doi: 10.3390/v8060163 Presented by: Siddhesh U. Sapre M.Sc. Virology (Part II) Roll No. 17 National Institute of Virology, INDIA
CHIKv Family: Togaviridae Genus: Alphavirus Species: Chikungunya Virus Genome: +ve sense RNA (Group IV) Primary vector: Aedes albopictus Other vector(s): Aedes aegypti Picture Credits: ViralZone (ExPASy)
Ribozyme An RNA molecule capable of acting as an enzyme Eg: Leadzyme, Hammerhead ribozyme and Twister ribozyme Robinson R PLoS Biology Vol. 2, No. 1, e28 doi:10.1371/journal.pbio.0020028
Hammerhead Ribozyme (hRz) nsP1-nsP4: Polyprotein encoded by Genomic RNA Capsid, E3, E2, 6K& E1: Polyprotein encoded by sub- genomic RNA hRz #9-#15
Materials and Methods Selection of hRz Target Sites in the CHIKv genome (CLUSTALX, GeneBank database) Plasmid construction Cell Cultures, Transfection, Clonal Generation and Infection RT-PCR Analysis TCID50 Immunofluorescence Assays Caspase 3 Assays Quantitative RT-PCR Generation of hRz Transgenic Mosquito Lines Splinkerette PCR Mosquitoes, Infection and Maintenance Genotyping of Mosquitoes In-situ indirect Immunofluorescence Assays Assays of Virus in Saliva
RT-PCR analysis of transformed Vero cells 130 bp fragment (t-RNAval promoter + ribozyme) for each ribozyme No CPE CPE hRz#9 hRz#14Comparative characteristics of cytopathic effect (CPE) of WT (day 3) and clonal populations (day 7)
TCID50 IFA Analysis of transformed Vero cell clones challenged with 3 different multiplicity of infections (MOIs) of CHIKv
RT +ve RT -ve Control RT-PCR of transgenic mosquitoes RT-PCR and caspase 3 activity of CHIKv challenged transformed Vero cell clones
Saliva titres performed on transgenic mosquitoes challenged with CHIKV- 7 days post blood feeding Indirect IFA performed on transgenic females challenged with CHIKv
Discussion hRzs: extremely effective against CHIKv (in both cell cultures and as transgenic antivirals in mosquitoes) hRz#9 & #14: most effective hRzs for inhibiting CHIKv replication & transmission in transgenic mosquitoes hRzs can be effective as transgenic antiviral molecules and potentially could be useful in permitting replacement of competent vector populations with a CHIKv refractory vector population Antiviral effector molecule remains functionally stable for multiple generations
Limitations At Higher MOIs, the virus seems to out space the levels at which, the hRzs can be expressed Variation among the clonal populations expressing a given hRz suppressor gene attributable to the position effects on transgene expression Heterozygous transgenic mosquitoes are less fit than the native wild-type population Report used 181/25 lab strain of CHIKv & HWE laboratory strain of mosquitoes, additional analysis of these hRzs against natural CHIKv variants in native mosquito strains are needed Only a small percentage of selected cells (30-50%) were actually transformed

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Antiviral Hammerhead ribozymes are ef

  • 1. Antiviral Hammerhead ribozymes are effective for developing transgenic suppression of Chikungunya Virus in Aedes aegytpi mosquitos Priya Mishra, Coleen Furey, Velmurugan Balaraman and Malcolm J. Fraser Jr. Dept. of Biological Sciences, Eck institute for Global Health, University of Notre Dame Viruses 2016, 8, 163; doi: 10.3390/v8060163 Presented by: Siddhesh U. Sapre M.Sc. Virology (Part II) Roll No. 17 National Institute of Virology, INDIA
  • 2. CHIKv Family: Togaviridae Genus: Alphavirus Species: Chikungunya Virus Genome: +ve sense RNA (Group IV) Primary vector: Aedes albopictus Other vector(s): Aedes aegypti Picture Credits: ViralZone (ExPASy)
  • 3. Ribozyme An RNA molecule capable of acting as an enzyme Eg: Leadzyme, Hammerhead ribozyme and Twister ribozyme Robinson R PLoS Biology Vol. 2, No. 1, e28 doi:10.1371/journal.pbio.0020028
  • 4. Hammerhead Ribozyme (hRz) nsP1-nsP4: Polyprotein encoded by Genomic RNA Capsid, E3, E2, 6K& E1: Polyprotein encoded by sub- genomic RNA hRz #9-#15
  • 5. Materials and Methods Selection of hRz Target Sites in the CHIKv genome (CLUSTALX, GeneBank database) Plasmid construction Cell Cultures, Transfection, Clonal Generation and Infection RT-PCR Analysis TCID50 Immunofluorescence Assays Caspase 3 Assays Quantitative RT-PCR Generation of hRz Transgenic Mosquito Lines Splinkerette PCR Mosquitoes, Infection and Maintenance Genotyping of Mosquitoes In-situ indirect Immunofluorescence Assays Assays of Virus in Saliva
  • 6. RT-PCR analysis of transformed Vero cells 130 bp fragment (t-RNAval promoter + ribozyme) for each ribozyme No CPE CPE hRz#9 hRz#14Comparative characteristics of cytopathic effect (CPE) of WT (day 3) and clonal populations (day 7)
  • 7. TCID50 IFA Analysis of transformed Vero cell clones challenged with 3 different multiplicity of infections (MOIs) of CHIKv
  • 8. RT +ve RT -ve Control RT-PCR of transgenic mosquitoes RT-PCR and caspase 3 activity of CHIKv challenged transformed Vero cell clones
  • 9. Saliva titres performed on transgenic mosquitoes challenged with CHIKV- 7 days post blood feeding Indirect IFA performed on transgenic females challenged with CHIKv
  • 10. Discussion hRzs: extremely effective against CHIKv (in both cell cultures and as transgenic antivirals in mosquitoes) hRz#9 & #14: most effective hRzs for inhibiting CHIKv replication & transmission in transgenic mosquitoes hRzs can be effective as transgenic antiviral molecules and potentially could be useful in permitting replacement of competent vector populations with a CHIKv refractory vector population Antiviral effector molecule remains functionally stable for multiple generations
  • 11. Limitations At Higher MOIs, the virus seems to out space the levels at which, the hRzs can be expressed Variation among the clonal populations expressing a given hRz suppressor gene attributable to the position effects on transgene expression Heterozygous transgenic mosquitoes are less fit than the native wild-type population Report used 181/25 lab strain of CHIKv & HWE laboratory strain of mosquitoes, additional analysis of these hRzs against natural CHIKv variants in native mosquito strains are needed Only a small percentage of selected cells (30-50%) were actually transformed