IMPROVED SENSITIVITY FOR CULTURING AND IDENTIFYING INFULENZA A SUBTYPES USING MDCK CELLS WITH ENHANCED EXPRESSION OF ALPHA 2, 6 LINKED SIALIC ACID MIXED WITH A-549 CELLS

Introduction:  Rapid sensitive culture of influenza virus from clinical specimens is critical for surveillance and management of outbreaks and individual cases.  The R-mix TOO cell system (Diagnostic Hybrids, International, Athens, OH) has proven to be both rapid and sensitive.  However, in specimens where influenza A titers are low, subtyping is often difficult.  MDCK cells with enhanced expression of alpha 2, 6 linked sialic acid (human influenza receptor) mixed with A-549 cells are shown to be more sensitive for subtyping of influenza virus.  

Method:  MDCK cells with enhanced expression of alpha 2, 6 linked sialic acid were obtained from the Wisconsin Alumni Research Foundation.  They were mixed with A-549 cells to prepare in-house mixed cell culture for respiratory viruses (G-mix).  291 consecutively received respiratory specimens submitted by California  Sentinel Providers in 2007 were tested in R-mix TOO and  frozen aliquots were subsequently tested in G-mix cells.

Results:  Virus was isolated from 222 of 291 (76%) of  specimens in R-mix and 230 of 290 (79%) in G-mix.  In R-mix there were 76 influenza A/H1 viruses, 46 influenza A/H3, 63 influenza A viruses, unable to subtype and 32 influenza B.  In G-mix there were 85 influenza A/ H1, 88 influenza A/H3 10 influenza A, unable to subtype and 32 influenza B. 

Conclusion:  Both R-mix TOO and G-mix are highly sensitive culture systems for influenza virus, as virus was cultured from more than 70% of specimens in each system. This is undoubtedly due to temporal (influenza season) and to the Sentinel provider’s timely collection and handling of specimens.  G-mix were slightly more sensitive for influenza culture and much more sensitive for determination of subtype.  In G-mix 173 of 183 (94.5%) of influenza isolates could be subtyped, while in R-mix 122 of  185 (66 %) were subtyped.  Modified MDCK cells are sensitive for culture of influenza virus and allow for a much higher rate of determining subtype on primary culture than unmodified MDCK.