antiviral hammerhead ribozymes are ef
TRANSCRIPT
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 RNACapsid, 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