antiviral hammerhead ribozymes are ef

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