Growth and adaptation of Zika virus in mammalian and mosquito cells

Abstract

The recent emergence of Zika virus (ZIKV) in the Americas coincident with increased caseloads of microcephalic infants and Guillain-Barre syndrome has prompted a flurry of research on ZIKV. Much of the research is difficult to compare or repeat because individual laboratories use different virus isolates, growth conditions, and quantitative assays. Here we obtained three readily available contemporary ZIKV isolates and the prototype Ugandan isolate. We generated stocks of each on Vero mammalian cells (ZIKVmam) and C6/36 mosquito cells (ZIKVmos), determined titers by different assays side-by-side, compared growth characteristics using one-step and multi-step growth curves on Vero and C6/36 cells, and examined plaque phenotype. ZIKV titers consistently peaked earlier on Vero cells than on C6/36 cells. Contemporary ZIKV isolates reached peak titer most quickly in a multi-step growth curve when the amplifying cell line was the same as the titering cell line (e.g., ZIKVmam titered on Vero cells). Growth of ZIKVmam on mosquito cells was particularly delayed. These data suggest that the ability to infect and/or replicate in insect cells is limited after growth in mammalian cells. In addition, ZIKVmos typically had smaller, more homogenous plaques than ZIKVmam in a standard plaque assay. We hypothesized that the plaque size difference represented early adaptation to growth in mammalian cells. We plaque purified representative-sized plaques from ZIKVmos and ZIKVmam. ZIKVmos isolates maintained the initial phenotype while plaques from ZIKVmam isolates became larger with passaging. Our results underscore the importance of the cells used to produce viral stocks and the potential for adaptation with minimal cell passages. In addition, these studies provide a foundation to compare current and emerging ZIKV isolates in vitro and in vivo.

Participation of Extracellular Vesicles from Zika-Virus-Infected Mosquito Cells in the Modification of Naïve Cells’ Behavior by Mediating Cell-to-Cell Transmission of Viral Elements

“…The hijacking of mosquito cell membranes by ZIKV could facilitate their escape from host immune responses, promoting the viral elements' spread. We first used the epithelial cells (Vero) from the monkey Cercopithecus aethiops (used as gold standard cells for infection assay) [32]. The presence of lytic plaques ( Figure 4E) in cultures of Vero with the ZIKV C6/36 EVs (lEVs and sEVs) isolates were observed in more concentrated amounts, formed in high or undetermined quantities.…”

“…Cell cultures (mosquito C6/36, human monocytes, or endothelial vascular cells) were seeded in 12-well plate culture (Corning) until confluence. They were then infected with ZIKV at a multiplicity of infection (MOI) of 1, as previously described [31][32][33] and incubated for 2 h at 37 • C in 5% CO 2 . After removal of the viral inoculum, cells were maintained in media supplemented with 5% EV-depleted FBS and incubated for 24,48,72,96, and 120 h. Cytopathic effect formation was observed by light field microscopy (Olympus IX71 inverted microscope; Olympus Corp. Miami, FL, USA).…”

“…As a result of the significance of these data, we also evaluated the possible mammalian naïve cell infection via small/large ZIKV C6/36 EVs. With this aim, first, we used the epithelial cells (Vero) from the monkey Cercopithecus aethiops (used as gold standard cells for infection assay) [32] to perform plaque assays (as described above) in the presence of small and large ZIKV C6/36 EVs (also RNase A + UV-treated and untreated samples). The presence of lytic plaques in ZIKV-infected Vero cells was present in high or undetermined amounts at different dilutions ( Figure 4E).…”

A novel antiviral formulation inhibits a range of enveloped viruses

“…For all in vivo experiments with SFV and ZIKV (Brazil, PE243), viruses were grown once in BHK-21 cells and then passaged once in C6/36 Aedes mosquito cells and titrated before use because mosquito cell-derived virus has distinct glycosylation and because insect cells impose distinct evolutionary constraints on viral progeny (Moser et al, 2018). SFV4 and SFV6 were used at passage 2.…”

Modeling cell infection via virus-producing cells rather than free infectious virus significantly improves fits ofin vitroviral kinetic data

“…Although CHIKV and ZIKV belong to distinct virus families ( Togaviridae and Flaviviridae , respectively), virus particles share common characteristics, such as their positive single-stranded RNA genome and the presence of a lipid envelope derived from the host. Both viruses infect a wide spectrum of mosquito and mammalian cell lines, including Vero cells, mosquito cells Aag2 or C6/36, as well as various human cell lines, including Huh7 [12, 13]. The classical kinetics following infection of a non-lytic virus begins with an eclipse phase in which attachment, entry and the first round of replication and assembly occurs.…”