Biophysical characterization of influenza A virions.

Antigenic drift of the influenza A virus requires that vaccine production is targeted to the strains circulating each year. Live-attenuated influenza A vaccine manufacturing is used to produce intact virions with the surface antigens of the circulating strains. Influenza A typically contains a large percentage (>90%) of non-infective virions. The ribonucleoprotein (RNP) content, virion structure, and aggregation are factors that are thought to have an impact on infectivity. However, these factors are difficult to study because of the intrinsic variability in virion size, shape and overall structural integrity. Negative stain TEM for total particle counts and cryoTEM for detailed size/structural analysis are established benchmark techniques for virus characterization. Other methods may be valuable for certain sample types or circumstances. The aim of this work is to establish a benchmark comparison between orthogonal biophysical techniques for particle counts, population size distribution, structural integrity, and aggregate levels. NTA and FFF-MALS rapidly provided total counts, size distribution, and aggregate/elongated virion content. CryoTEM with size analysis and fraction counting yielded detailed information about the pleomorphism of the sample. The structural integrity of virions was inferred from multi-signal AUC-SV and CryoTEM. The current work provides a comparative assessment and a baseline for the selection of biophysical tools for the determination of particle counts, aggregation and pleomorphic characteristics of influenza A virus.

Comparison of different cell substrates on the measurement of human influenza virus neutralizing antibodies.

Eight cell lines were systematically compared for their permissivity to primary infection, replication, and spread of seven human influenza viruses. Cell lines were of human origin (Caco-2, A549, HEp-2, and NCI-H292), monkey (Vero, LLC-MK2), mink (Mv1 Lu), and canine (MDCK). The influenza viruses included seasonal types and subtypes and a pandemic virus. The MDCK, Caco-2, and Mv1 Lu cells were subsequently compared for their capacity to report neutralization titers at day one, three and six post-infection. A gradient of sensitivity to primary infection across the eight cell lines was observed. Relative to MDCK cells, Mv1 Lu reported higher titers and the remaining six cell lines reported lower titers. The replication and spread of the seven influenza viruses in the eight cell substrates was determined using hemagglutinin expression, cytopathic effect, and neuraminidase activity. Virus growth was generally concordant with primary infection, with a gradient in virus replication and spread. However, Mv1 Lu cells poorly supported virus growth, despite a higher sensitivity to primary infection. Comparison of MDCK, Caco-2, and Mv1 Lu in neutralization assays using defined animal antiserum confirmed MDCK cells as the preferred cell substrate for influenza virus testing. The results observed for neutralization at one day post-infection showed MDCK cells were similar (<1 log(2) lower) or superior (>1 log(2) higher) for all seven viruses. Relative to Caco-2 and Mv1 Lu cells, MDCK generally reported the highest titers at three and six days post-infection for the type A viruses and lower titers for the type B viruses and the pandemic H9N2 virus. The reduction in B virus titer was attributed to the complete growth of type B viruses in MDCK cells before day three post-infection, resulting in the systematic underestimation of neutralization titers. This phenomenon was also observed with Caco-2 cells.