Elsevier

Vaccine

Volume 36, Issue 22, 24 May 2018, Pages 3140-3145
Vaccine

Propagation of Brazilian Zika virus strains in static and suspension cultures using Vero and BHK cells

https://doi.org/10.1016/j.vaccine.2017.03.018Get rights and content
Under a Creative Commons license
open access

Highlights

  • Brazilian Zika virus isolates propagate similar in Vero and BHK-21 cells.

  • ZIKV production in BHK-21SUS cells possible.

  • Process intensification using ATF perfusion shows the potential of BHK-21SUS cells.

  • Non-optimized perfusion process already yields 3.9 × 107 PFU/mL.

Abstract

The recent spread of Zika virus (ZIKV) in the Americas and the Pacific has reached alarming levels in more than 60 countries. However, relatively little is known about the disease on a virological and epidemiological level and its consequences for humans. Accordingly, a large demand for in vitro derived Brazilian ZIKV material to support in vitro and in vivo studies has arisen. However, a prompt supply of ZIKV and ZIKV antigens cannot be guaranteed as the production of this virus typically using Vero or C6/36 cell lines remains challenging.

Here we present a production platform based on BHK-21 suspension (BHK-21SUS) cells to propagate Brazilian ZIKV at larger quantities in perfusion bioreactors. Scouting experiments performed in tissue culture flasks using adherent BHK-21 and Vero cells have demonstrated similar permissivity and virus yields for four different Brazilian ZIKV isolates. The cell-specific yield of infectious virus particles varied between respective virus strains (1–48 PFU/cell), and the ZIKV isolate from the Brazilian state Pernambuco (ZIKVPE) showed to be a best performing isolate for both cell lines. However, infection studies of BHK-21SUS cells with ZIKVPE in shake flasks resulted in poor virus replication, with a maximum titer of 8.9 × 103 PFU/mL. Additional RT-qPCR measurements of intracellular and extracellular viral RNA levels revealed high viral copy numbers within the cell, but poor virus release. Subsequent cultivation in a perfusion bioreactor using an alternating tangential flow filtration system (ATF) under controlled process conditions enabled cell concentrations of about 1.2 × 107 cells/mL, and virus titers of 3.9 × 107 PFU/mL. However, while the total number of infectious virus particles was increased, the cell-specific yield (3.3 PFU/cell) remained lower than determined in adherent cell lines. Nevertheless, the established perfusion process allows to provide large amounts of ZIKV material for research and is a first step towards process development for manufacturing inactivated or live-attenuated ZIKV vaccines.

Keywords

Brazilian Zika virus
BHK-21
Suspension cell line
High cell density
Perfusion process
Process intensification

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