Text by Gilles Gadea & Philippe Desprès
Zika virus (ZIKV) infection is now considered as a major public health issue. Faced with the increasing threat ZIKV represents worldwide, specific antiviral therapies and vaccines are urgently needed to control Zika disease. The development of therapies for ZIKV requires both insights into the viral life cycle and strategies to identify potent antiviral inhibitors. Consequently, it is urgent to dispose of reliable and rapid virus-based assays for the study of the dynamics of viral replication as well as the screening of anti-ZIKV compounds and neutralizing antibodies for vaccine evaluation. Production of a flavivirus reporter virus is known to be time-consuming and technically difficult. To generate a recombinant ZIKV expressing the GFP gene, we decided to apply the performing reverse genetic ISA (for infectious-subgenomic-amplicons) method. ISA was efficient for the rapid production of reliable GFP-expressing mutant of ZIKV with a genomic stability on at least two passages on cultured cells.
The virologists are often confronted to the great difficulties in the production of a reporter gene-containing flavivirus. Recently, the team directed by X. de Lamballerie in Marseille (France) described the elegant ISA method that facilitates the production of infectious RNA virus from genomic DNA material. Through the European PREDEMICS consortium, we were able together to generate molecular clones of various flaviviruses as well as chimeric viruses. A the end of 2015, the decision was taken to implement the ISA method for the rapid production of a molecular clone of ZIKV derived from the first strain MR766 isolated in Uganda in 1947. The success of this strategy allowing the rapid generation of infectious clone of ZIKV in La Reunion raised up the enthusiasm among the team. The sequencing of genomic RNA confirmed that the ISA method was greatly efficient for the production of a recombinant ZIKV. We therefore decided to generate a recombinant ZIKV containing the GFP using an already described strategy in which the reporter gene is expressed as an additional part of the structural protein region. The detection of a strong GFP signal in cells infected with the first virus stocks of ZIKV-GFP raised up a great contentment. We were also gratified when our recombinant ZIKV-GFP was proven to be a reliable molecular tool for the monitoring of viral replication. Today, we are able to provide the set of purified PCR products leading to the rapid production of a recombinant ZIKV-GFP towards the scientists studying the pathogenesis of ZIKV infection.
Detection of GFP fluorescence (green) in cells infected with recombinant ZIKV-GFP. The ZIKV E protein (red) was immunostained using mAb 4G2. Cell in apoptotic state is shown (white arrow). Nuclei are stained in blue. In left, Vero cells infected 24 h. In right, A549 cells infected 48 h.
Introducing the authors
4.Patrick MAVINGUI (head of PIMIT)
Sandra BOS (absent)
About the research
Gilles Gadea, Sandra Bos, Pascale Krejbich-Trotot, Elodie Clain, Wildriss Viranaicken, Chaker El-Kalamouni, Patrick Mavingui, Philippe Desprès
Virology, Volume 497, October 2016, Pages 157–162