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