Poliovirus nonpermissive CD155 knockout cells derived from RD cell line for handling poliovirus potentially infectious materials in virology laboratories

Abstract

Destruction of all poliovirus containing materials, safe and secure handling of retained polioviruses for vaccine production, and research will be obligatory to eliminate facility‐associated risks. Polioviruses and poliovirus potentially infectious materials (PIM) including fecal or respiratory samples requiring containment have been defined in World Health Organization‐Global Action Plan (GAP III) documents. Non‐polio laboratories culturing viruses from PIM are most affected as cell cultures of human and monkey origin are also poliovirus permissive. CRISPR gene‐editing technology was used to knockout the poliovirus receptor (PVR/CD155) gene in the rhabdomyosarcoma (RD) cell line. PVR knockout RD cell susceptibility was tested using known non‐polio enterovirus (NPEV) types. A selected clone (RD‐SJ40) was field evaluated for virus isolation from 626 stool samples of acute flaccid paralysis cases. Poliovirus nonpermissive cells derived from the 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 the growth of poliovirus from positive stool samples. All NPEV types were isolated in RD and RD‐SJ40 cells. CRISPR correctly edited the CD155 gene of RD cells to render them poliovirus nonpermissive while susceptibility to NPEV remained unchanged. RD‐SJ40 cells are safe for NPEV isolation from poliovirus PIM without derogating GAP III containment requirements.