Human Villous Mesenchymal Fibroblasts (HVMF)

In Vitro Competitive Fitness Assays Using Viral Isolates Confirm Replicative Fitness Differences of Recombinant Zika Viruses

RNA viruses evolve rapidly due to their short generation times and high mutation rates. This study investigates the phenotypic evolution of Zika virus during the 2015-2016 epidemic in the Americas, focusing on lineage-specific fitness differences in human cells and mosquitoes.

They obtained Zika virus isolates from Nicaragua (Nica-6547) and Honduras (R103451), which share lineage-defining mutations with clade E, known for high replicative fitness in human primary cells (Fig. 1A-H). They also got isolates from Colombia (FLR) and Panama (PA259359, PA259634, PAN259249, PAN259364), which have mutations specific to clade G, our lowest-performing clade (Fig 1A-H). Additionally, they included isolates from Brazil (Paraiba_01, clade B) and Puerto Rico (PRVABC59, clade I), which has moderate fitness (Fig.1A-H). They used these isolates to infect human villous mesenchymal fibroblasts (HVMF) and retinal pigment epithelial (RPE) to generate in vitro replicative fitness curves. In HVMFs, isolates from Brazil (clade B), Honduras (clade E), and Puerto Rico (clade I) had the highest fitness (Fig 3A). In RPE cells, high-fitness clades did not outperform clade G isolates (Fig. 2B). They conducted competitive fitness assays in HVMFs and RPE cells, competing clade B (Paraiba_01) and two clade E isolates (Nica-6547 and R103451) against three clade G isolates (PA259359, PA259634, PAN259249). Each competition was done at 90%, 50%, and 10% lineage percentages, totaling 27 competitions per cell type. We used deep sequencing to assess lineage frequencies five days post-infection. In RPE cells, Paraiba (clade B), Nicaragua (clade E), and Honduras (clade E) isolates outcompeted clade G isolates (Fig. 2C-E), with average frequency increases of 21% for Paraiba, 18% for Honduras, and 15% for Nicaragua. In HVMF cells, 24/27 competitions showed Paraiba, Nicaragua, and Honduras isolates increasing in frequency (Fig. 2F-H), with average increases of 10% for Paraiba, 7% for Honduras, and 5% for Nicaragua.