Isolation and genetic characterization of human coronavirus NL63 in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells

Table 1 Indications of more than one virus in contaminated RPTEC

Test performed	Cell line^f
	CV-1	HEK-293	LLC-MK2	MDCK	MDCK-London	Mv1 Lu	RPTEC	Vero E6	WI-38
Microscopy, 12 hr post-seed	NA^a	NA	NA	NA	NA	NA	Owl eye nuclei; enlarged cells	NA	NA
IFA, 48 hr post-seed	NA	NA	NA	NA	NA	NA	CMV positive	NA	NA
PCR, 48 hr post-seed	NA	NA	NA	NA	NA	NA	CMV positive^g	NA	NA
BGM cytotoxicity	No effect	NT^c	No effect	No effect	NT	NT	NA	No effect	No effect
Bioactive agent release assay	Vacuolation 12 hpi, 37°C	Vacuolation 12 hpi, 37°C	Vacuolation 12 hpi, 37°C	NT	NT	NT	NA	Vacuolation 12 hpi, 37°C	C.R., Sw. & Vac.^h 12 hpi, 37°C; cell death 48 hpi
Subcultures, 5 d post-seed of RPTEC	Vacuolation 12 hpi; CPE 6 dpi^b	CPE 7 dpi^d	Vacuolation 12 hpi; CPE 5 dpi^e	CPE 6 dpi^e	CPE 6 dpi^e	CPE 6 dpi^e	NA	Vacuolation 12 hpi; CPE 6 dpi^b	C.R. Sw. & vac.^h 12 hpi, 37°C; cell death 48 hpi
Subcultures, freeze-thaw 7d post-seed of RPTEC	CPE 6 dpi^b	CPE 7 dpi^d	CPE 5 dpi^e	CPE 6 dpi^e	CPE 6 dpi^e	CPE 5 dpi^e	NA	CPE 6 dpi^e	No CPE 30 dpi

^a NA, not applicable.
^b Vacuolation, formation of syncytia, rounding of the cells and detachment or cytolysis, at 34° and 37°C.
^c NT, not tested.
^d CPE consisting of rounding of the cells and detachment or cytolysis. Tested at 37°C [HEK-293 have reduced adherence at lower temperatures].
^e Vacuolation, formation of syncytia, focal rounding of the cells, formation of striations, or formation of syncytia followed by cytolysis, at 34° and 37°C., with or without TPCK-trypsin.
^f No CPE were detected in A549, NIH/3 T3, and BHK-21 cells for 1 month post-exposure to RPTEC-derived material.
^g PCR negative for HHV-1, HHV-2, BKV, and SV40.
^h C.R., Sw. & Vac., cell rounding, swelling and vacuolation.

ISSN: 1743-422X