A NOVEL SYSTEM FOR IN VITRO TESTING OF POLIOVIRUS NEUROVIRULENCE USING HUMAN EMBRYONIC STEM CELL-DERIVED NEURONS

Yuri Perepliotchikov 1,2 Anna Sloutskin 3 Amos Markus 3 Ronald S. Goldstein 3 Lester M. Shulman 1,2
1Central Virology Laboratory, Public Health Services, Israel Ministry of Health, Sheba Medical Center, Tel Hashomer, Israel
2School of Public Health, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
3Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel

Background: Human-specific neurotropic polioviruses (PV) can destroy nerve cells causing paralysis and sometimes death. Neurovirulence of PV isolates is currently tested in primates and transgenic mice expressing the human poliovirus receptor. Human-specific neurotropic varicella zoster virus infections have been studied in human embryonic stem cell-derived neurons (hESCn).

Aim: To evaluate whether in-vitro poliovirus infection of hESCn can serve as a substitute for testing neurovirulence in live animals.

Methods: hESCn neuronal precursors were co-cultured with stromal cells for ≥ 1 week and terminally differentiated on coverslips or in compartmented microfluidic chambers where axons grew from a cell-body to an axonal compartment through microchannels. hESCn or their axons were infected with non-neurovirulent Sabin 2 polio vaccine (S2PV), and high (HNvPV2) or low (LNvPV2) neurovirulent, serotype-two, vaccine-derived polioviruses (VDPVs) isolated from Israeli sewage. Immunofluorescence staining and qRT-PCR characterized poliovirus presence in cell bodies and released poliovirus, respectively.

Results: All 3 PVs induced productive infections measured by immunofluorescence and qRT-PCR when they were applied to hESCn or their axons in compartmentalized chambers. In both types of infections, VDPVs were detected 27-30 hours earlier than S2PV. Each PV was detected 39 hours earlier when hESCN were infected than when their axons were infected. Appearance times for HNvPV2 and LNvPV2 overlapped for both types of infection.

Conclusion: HNvPV2 and LNvPV2 infections were not distinguishable with the techniques used here, however clear differences in time of appearance of the two neurovirulent type 2 VDPVs compared with non-neurovirulent S2PV were observed after infections of hESCn on coverslips and their axons in microfluidic chambers. Thus this new tissue culture model can potentially provide an ethical alternative for neurovirulence testing in primate and other animals.









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