Generation and characterization of genetically and antigenically diverse infectious clones of dengue virus serotypes 1–4

ABSTRACT

Dengue is caused by four genetically distinct viral serotypes, dengue virus (DENV) 1-4. Following transmission by Aedes mosquitoes, DENV can cause a broad spectrum of clinically apparent disease ranging from febrile illness to dengue hemorrhagic fever and dengue shock syndrome. Progress in the understanding of different dengue serotypes and their impacts on specific host-virus interactions has been hampered by the scarcity of tools that adequately reflect their antigenic and genetic diversity. To bridge this gap, we created and characterized infectious clones of DENV1–4 originating from South America, Africa, and Southeast Asia. Analysis of whole viral genome sequences of five DENV isolates from each of the four serotypes confirmed their broad genetic and antigenic diversity. Using a modified circular polymerase extension reaction (CPER), we generated de novo viruses from these isolates. The resultant clones replicated robustly in human and insect cells at levels similar to those of the parental strains. To investigate in vivo properties of these genetically diverse isolates, representative viruses from each DENV serotype were administered to NOD Rag1^−/−, IL2rg^null Flk2^−/− (NRGF) mice, engrafted with components of a human immune system. All DENV strains tested resulted in viremia in humanized mice and induced cellular and IgM immune responses. Collectively, we describe here a workflow for rapidly generating de novo infectious clones of DENV – and conceivably other RNA viruses. The infectious clones described here are a valuable resource for reverse genetic studies and for characterizing host responses to DENV in vitro and in vivo.

Keywords:

• Dengue
• dengue virus
• host tropism
• animal models
• host response

Acknowledgements

We thank Drs. Robert Tesh and Kenneth Plante at the World Reference Centre for Emerging Viruses and Arboviruses (WRCEVA) for providing Dengue viruses, Christina J. DeCoste and Katherine Rittenbach from the Flow Cytometry Resource Facility for support and advice on flow experiment, members of the Genomics Core Facility at Princeton University for performing the Next-Generation Sequencing experiment, and all the Ploss Lab members, especially Emily Mesev for critical discussions and technical advice.

Disclosure statement

No potential conflict of interest pertaining to this study was reported by the authors.

Additional information

Funding

This work was supported in part by grants from the National Institutes of Health (R01 AI138797, R01 AI107301, R01 AI146917, R01 AI153236), a Research Scholar Award from the American Cancer Society (RSG-15-048-01-MPC) a Burroughs Wellcome Fund Award for Investigators in Pathogenesis (101539) and the Centre for Health and Wellbeing at Princeton University (all to A.P.). Services, results and/or products in support of the research project were generated by the Rutgers Cancer Institute of New Jersey Flow Cytometry and Cell Sorting Shared Resource, supported, in part, with funding from Cancer Institute of New Jersey (NCI-CCSG P30CA072720-5921). T.T. is a recipient of postdoctoral fellowship awards from the Uehara Memorial Foundation and the Japanese Society for the Promotion of Science. J.Z. received a predoctoral fellowship from the China Scholarship Council. M.P.S. is supported by the NIGMS of the National Institutes of Health under grant number T32GM007388 and a HMEI-STEP fellowship from the from the High Meadows Environmental Institute at Princeton University. None of the funders have influence on the design of the study and/or interpretation of the data.