title: Detection of nonhemagglutinating influenza a(h3) viruses by enzyme-linked immunosorbent assay in quantitative influenza virus culture.
abstract: To assess the efficacy of novel antiviral drugs against influenza virus in clinical trials, it is necessary to quantify infectious virus titers in respiratory tract samples from patients. Typically, this is achieved by inoculating virus-susceptible cells with serial dilutions of clinical specimens and detecting the production of progeny virus by hemagglutination, since influenza viruses generally have the capacity to bind and agglutinate erythrocytes of various species through their hemagglutinin (HA). This readout method is no longer adequate, since an increasing number of currently circulating influenza A virus H3 subtype (A[H3]) viruses display a reduced capacity to agglutinate erythrocytes. Here, we report the magnitude of this problem by analyzing the frequency of HA-deficient A(H3) viruses detected in The Netherlands from 1999 to 2012. Furthermore, we report the development and validation of an alternative method for monitoring the production of progeny influenza virus in quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This method is based on the detection of viral nucleoprotein (NP) in virus culture plates by enzyme-linked immunosorbent assay (ELISA), and it produced results similar to those of the hemagglutination assay using strains with good HA activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2), other seasonal A(H1N1), A(H1N1)pdm09, and the majority of A(H3) virus strains isolated in 2009. In contrast, many A(H3) viruses that have circulated since 2010 failed to display HA activity, and infectious virus titers were determined only by detecting NP. The virus culture ELISA described here will enable efficacy testing of new antiviral compounds in clinical trials during seasons in which nonhemagglutinating influenza A viruses circulate.
authors.affiliation: Viroclinics Biosciences B.V., Rotterdam, The Netherlands.
authors.forename: C A
authors.initials: CA
authors.lastname: van Baalen
authors.forename: C
authors.initials: C
authors.lastname: Els
authors.forename: L
authors.initials: L
authors.lastname: Sprong
authors.forename: R
authors.initials: R
authors.lastname: van Beek
authors.forename: E
authors.initials: E
authors.lastname: van der Vries
authors.forename: A D M E
authors.initials: AD
authors.lastname: Osterhaus
authors.forename: G F
authors.initials: GF
authors.lastname: Rimmelzwaan
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D004283:Dog Diseases / Q000175:diagnosis* / Q000188:drug therapy / Q000821:virology; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006385:Hemagglutination Inhibition Tests / Q000379:methods; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000302:isolation & purification; D053122:Influenza A Virus, H3N2 Subtype / Q000187:drug effects / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000188:drug therapy / Q000821:virology; D061985:Madin Darby Canine Kidney Cells; D009426:Netherlands; D009976:Orthomyxoviridae Infections / Q000175:diagnosis* / Q000188:drug therapy / Q000821:virology; D014758:Viral Core Proteins / Q000737:chemistry
chemical_list: D000998:Antiviral Agents; D014758:Viral Core Proteins