Development of a SYBR green I-based quantitative RT-PCR for Ross River virus: Application in vector competence studies and antiviral drug evaluation.
Ross River virus (RRV) is an emerging Alphavirus and is presently endemic in many parts of Oceania. Keeping in mind its emergence, we developed a molecular detection system and utilized it to study vector competence and evaluate activity of antiviral compounds against RRV. A SYBR Green I-based quantitative RT-PCR for detection of RRV was developed targeting the E2 gene, with a detection limit of 100 RNA copies/reaction. The specificity was confirmed with closely related Alphaviruses and Flaviviruses. The assay was applied to study the vector competence of Indian Aedes aegypti for RRV, which revealed 100% infection and dissemination rate with 75% transmission rate. Viral RNA was found in saliva as early as 3day post infection (dpi). Further application of the assay in antiviral drug evaluation revealed the superior in vitro activity of ribavirin compared to chloroquine in Vero cells. Successful demonstration of this assay to detect RRV in low titre mosquito samples makes it a sensitive tool in vector surveillance. This study also showed that Indian Ae. aegypti are well competent to transmit RRV highlighting the risk of its introduction to naïve territories across continents. Further validation of this assay, revealed its utility in screening of potential antivirals against RRV.
Division of Virology, Defence R and D Establishment, Jhansi Road, Gwalior 474 002, M.P., India. Electronic address: pabandash@drde.drdo.in.
Paban Kumar
PK
Dash
Division of Virology, Defence R and D Establishment, Jhansi Road, Gwalior 474 002, M.P., India.
Ankita
A
Agarwal
Division of Virology, Defence R and D Establishment, Jhansi Road, Gwalior 474 002, M.P., India.
Shashi
S
Sharma
Division of Virology, Defence R and D Establishment, Jhansi Road, Gwalior 474 002, M.P., India.
Amrita
A
Saha
Division of Virology, Defence R and D Establishment, Jhansi Road, Gwalior 474 002, M.P., India.
Gaurav
G
Joshi
Vector Management Division, Defence R and D Establishment, Jhansi Road, Gwalior 474 002, M.P., India.
Natarajan
N
Gopalan
Vector Management Division, Defence R and D Establishment, Jhansi Road, Gwalior 474 002, M.P., India.
Devanathan
D
Sukumaran
Division of Virology, Defence R and D Establishment, Jhansi Road, Gwalior 474 002, M.P., India.
Man Mohan
MM
Parida
D000330:Aedes / Q000821:virology*; D018354:Alphavirus Infections / Q000175:diagnosis / Q000635:transmission / Q000821:virology; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D000998:Antiviral Agents / Q000302:isolation & purification / Q000494:pharmacology; D052160:Benzothiazoles; D002522:Chlorocebus aethiops; D002738:Chloroquine / Q000494:pharmacology; D003959:Diamines; D006801:Humans; D007303:Insect Vectors / Q000821:virology*; D008826:Microbial Sensitivity Tests; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012254:Ribavirin / Q000494:pharmacology; D012398:Ross River virus / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D012463:Saliva / Q000821:virology; D014709:Vero Cells
D000962:Antimalarials; D000998:Antiviral Agents; D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I; D012254:Ribavirin; D002738:Chloroquine
Antiviral; RRV; Real time RT-PCR; Vector competence