title: Comparative study of influenza virus replication in Vero and MDCK cell lines.
abstract: The choice of a cell line for the production of influenza vaccines is determined by how well the virus is able to replicate and how easily the cell line can be maintained. Madin-Darby canine kidney (MDCK) cells have long been known to successfully support influenza growth. Vero cells are also another well studied candidate cell line. In this work, we have compared these two cell lines for their ability to propagate type A and type B cold-adapted and wild type influenza viruses. The growth of these viruses has been measured as plaque forming units (via plaque assay) as well as viral particle formation (via a novel quantitative RT-PCR assay) to assess the suitability of these cell lines to support the development of live attenuated influenza vaccines. The novel qRT-PCR assay outlined in this work was demonstrated to be an efficient, sensitive and reproducible method for measuring wild type (wt) and cold-adapted (ca) influenza strains. Replicates of six per sample consistently showed an average variation around +/-10%. In this study we have also found qRT-PCR to be a useful method for differentiating between wt and ca influenza strains based on their differing growth characteristics at varying temperatures. This can subsequently be used to assess reassortants prepared from ca donor strains for the purposes of live viral vaccine development. For type A and B influenza viruses studied in this work, MDCK cells supported a more rapid viral growth (measured in terms of genome copies) compared with Vero cells. For the type A viruses studied here, the genome copies: infectious unit (genome copy, gc:infectious unit, iu) ratio was found to be more favorable for Vero cells compared with MDCK cells. For the type B viruses studied in this work, the gc:iu was equivalent in both cell lines tested. Ultimately, however, the use of any new cell line would need to be approved by regulatory agencies prior to its commercial application.
authors.affiliation: Merck & Co., Inc. 770 Sumneytown Pike, WP44L-206B, West Point, PA 19486, USA. rima_youil@merck.com
authors.forename: R
authors.initials: R
authors.lastname: Youil
authors.forename: Q
authors.initials: Q
authors.lastname: Su
authors.forename: T J
authors.initials: TJ
authors.lastname: Toner
authors.forename: C
authors.initials: C
authors.lastname: Szymkowiak
authors.forename: W-S
authors.initials: WS
authors.lastname: Kwan
authors.forename: B
authors.initials: B
authors.lastname: Rubin
authors.forename: L
authors.initials: L
authors.lastname: Petrukhin
authors.forename: I
authors.initials: I
authors.lastname: Kiseleva
authors.forename: A R
authors.initials: AR
authors.lastname: Shaw
authors.forename: D
authors.initials: D
authors.lastname: DiStefano
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004285:Dogs; D009980:Influenza A virus / Q000254:growth & development*; D009981:Influenza B virus / Q000254:growth & development*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014709:Vero Cells; D010948:Viral Plaque Assay; D014776:Virus Cultivation / Q000379:methods*; D014779:Virus Replication