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As an example of the cost-effective large-scale generation of small-interfering RNA (siRNAs), we have created transgenic tobacco plants that produce siRNAs targeted to the mRNA of the non-structural protein NS1 from the influenza A virus subtype H1N1. We have investigated if these siRNAs, specifically targeted to the 5′-portion of the NS1 transcripts (5mNS1), would suppress viral propagation in mammalian cells. Agroinfiltration of transgenic tobacco with an Agrobacterium strain harboring a 5mNS1-expressing binary vector caused a reduction in 5mNS1 transcripts in the siRNA-accumulating transgenic plants. Further, H1N1 infection of siRNA-transfected mammalian cells resulted in significant suppression of viral replication. These results demonstrate that plant-derived siRNAs can inhibit viral propagation through RNA interference and could potentially be applied in control of viral-borne diseases. [Copyright &y& Elsevier]

Influenza A and B viruses are still a major worldwide threat. We demonstrate that influenza B virus infection induces signaling via the Raf/MEK/ERK cascade, a process required for efficient virus production. Expression of dominant-negative Raf and ERK mutants or treatment with a MEK inhibitor (U0126) strongly impaired viral propagation, while selective activation of the pathway resulted in increased virus titers. MEK inhibition appears to interfere with a distinct viral nuclear export process. Most importantly, no resistant virus variants emerged in the presence of U0126 demonstrating that influenza viruses cannot easily adapt to the missing cellular function. [Copyright &y& Elsevier]

Sulfatide, which binds to influenza A viruses and prevents the viral infection, was found to inhibit the sialidase activities of influenza A viruses in a pH-dependent manner. The kinetic parameters of the effect of sulfatide on the sialidase activities of human influenza A viruses using fluorometric assay indicated that sulfatide was a powerful and non-competitive type inhibitor in low-pH conditions. [Copyright &y& Elsevier]

Reliable assays for accurate titration of influenza virus in infectious samples are pivotal to both influenza research and vaccine development. A titration assay adopted commonly for this purpose is the plaque assay on Madin–Darby canine kidney (MDCK) cells, despite it being time and labour consuming. A novel assay is described for titration of influenza viruses based on the detection of intracellular viral nucleoprotein (NP) by fluorescence-activated cell sorting (FACS). By using a panel of viruses of different type, subtype and origin, it is demonstrated that there is a mathematical correlation between titres measured by immunotitration and by classical plaque assay on MDCK cells. Moreover, the availability of NP antibodies specific for type A or type B influenza virus ensures the specificity of the assay. Based on speed, accuracy and specificity, it is concluded that the FACS-based immunotitration of influenza virus represents a valid and efficient alternative to the classical plaque assay. [Copyright &y& Elsevier]