Poliovirus Non-Permissive CD155 Knockout Cells Derived from RD Cell Line Using CRISPR Technology for Handling Poliovirus Potentially Infectious Materials in Virology Laboratories

Background: Destruction of all poliovirus containing materials, and safe and secure handling of retained polioviruses for vaccine production and research will be obligatory to eliminate facility-associated risks. As defined in WHO-GAPIII, poliovirus potentially infectious materials (PIM) include fecal or respiratory samples collected for any purpose in time and geographic area of wild poliovirus or during OPV use and products of such materials from poliovirus permissive cells or animals. Non-polio laboratories culturing viruses from PIM are most affected as cell cultures of human and monkey origin are also poliovirus permissive. Methods: CRISPR gene editing technology was used to knockout poliovirus receptor (PVR/CD155) gene in RD cell line. Poliovirus non-permissive cells were identified by resistance to poliovirus infection, cell surface immunofluorescence and DNA sequencing. PVR knockout RD cell susceptibility was tested using known non-polio enterovirus types. A selected clone (RD-SJ40) was field evaluated for virus isolation from 626 stool samples of AFP cases. Findings: Poliovirus non-permissive cells derived from RD cell line did not show CD155-specific cell surface immunofluorescence. CD155 gene sequencing confirmed nucleotide base pair deletions within exon2 and exon3. The CD155 knockout RD-SJ40 cells did not support growth of poliovirus from positive stool samples. All NPEV types isolated in parental RD cells were also isolated in RD-SJ40. Interpretation: CRISPR correctly edited CD155 gene of RD cells to render them poliovirus non-permissive while susceptibility to NPEV remained unchanged. RD-SJ40 cells are safe for NPEV isolation from poliovirus PIM without derogating GAPIII containment requirements. Funding: Department of Health Research, Govt. of India. Project Grant No.R.12020/08/2018-HR Declaration of Interests: SSN, SS and JD have IPR interests. Other authors have no conflict of interests.