title: Isolation of viruses from clinical specimens in microtitre plates with cells inoculated in suspension.
abstract: Virus isolation is essential for the provision of a full diagnostic virology service. Present methods are time consuming, expensive and relatively inflexible for routine use. Our objective was to audit our existing virus isolation system and to develop a sensitive, flexible virus isolation system which could be adapted for use in a busy routine laboratory which is required to provide a service for a wide range of clinical situations. We carried out a pilot study which compared conventional roller tube monolayer cultures to a microplate system using cells inoculated in suspension and showed that the microplate method using extra cell lines could provide a more sensitive system for virus isolation. This system was adapted for routine use using six cell lines inoculated in suspension and the results are presented for 2610 specimens for virus isolation and 972 for Clostridium difficile toxin (CDT) detection. There were 516 viruses isolated and 229 specimens positive for CDT using this system. Polioviruses (92), echoviruses (35), coxsackieviruses (15) and untyped enteroviruses (13) were isolated in RMK, E6-vero and RD cells. Adenoviruses (137) were isolated in HEp2 and E6-vero cells. Herpes simplex virus (HSV) was isolated from 149 specimens in E6-vero, FCL and HFF9 cells. Myxoviruses (38) and paramyxoviruses were isolated in RMK cells. HEp2 was the only cell line necessary to isolate the 33 respiratory syncytial viruses (RSV). Cytomegaloviruses (CMV) (2) and varicella zoster (1) virus (VZV) were isolated only in the human fibroblast cell line HFF9. Rubella virus was isolated from a baby with congenital rubella in RMK, E6-vero and additionally in BGM cells. In conclusion, the use of cells inoculated in suspension in microtitre plates for virus isolation was sensitive and convenient. It allowed the use of six cell lines for routine virus isolation without using additional laboratory staff time. It improved turnaround times. It was also safer microbiologically than conventional isolation in tube monolayers. The precise identification of virus isolates was simplified.
authors.affiliation: Regional Virus Laboratory, Royal Hospitals Trust, Belfast, UK.
authors.forename: H J
authors.initials: HJ
authors.lastname: O'Neill
authors.forename: J D
authors.initials: JD
authors.lastname: Russell
authors.forename: D E
authors.initials: DE
authors.lastname: Wyatt
authors.forename: C
authors.initials: C
authors.lastname: McCaughey
authors.forename: P V
authors.initials: PV
authors.lastname: Coyle
mesh_terms: D000256:Adenoviridae / Q000302:isolation & purification*; D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured / Q000821:virology; D002522:Chlorocebus aethiops; D016360:Clostridioides difficile / Q000737:chemistry; D004770:Enterovirus / Q000302:isolation & purification*; D005347:Fibroblasts / Q000821:virology; D006564:Herpesviridae / Q000302:isolation & purification*; D006801:Humans; D009975:Orthomyxoviridae / Q000302:isolation & purification*; D010865:Pilot Projects; D012136:Respiratory Syncytial Viruses / Q000302:isolation & purification*; D018108:Respirovirus / Q000302:isolation & purification*; D012412:Rubella virus / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014709:Vero Cells / Q000821:virology
chemical_list: D001427:Bacterial Toxins