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SummaryThe use of influenza A virus-inducible reporter gene segments in detecting influenza A virus replication was investigated. The RNA polymerase I promoter/terminator cassette was used to express RNA transcripts encoding green fluorescence protein or firefly luciferase flanked by the untranslated regions of the influenza A/WSN/33 NP segment. Reporter gene activity was detected after reconstitution of the influenza A virus polymerase complex from cDNA or after virus infection, and was influenza A virus-specific. Reporter gene activity could be detected as early as 6 hours post infection and was virus dose-dependent. Inhibitory effects of antibodies or amantadine could be detected a nd quantified rapidly, providing a means of not only identifying influenza A virus -specific replication, but of determining the antigenic subtype as well as antiviral drug susceptibility. Induction of virus-specific reporter genes provides a rapid, sensitive method for detecting virus replication, quantifying virus titers and assessing antiviral sensitivity as well as antigenic subtype.

Influenza A virus interacts with specific types of sialic acid during attachment and entry into susceptible cells. The precise amino acids in the hemagglutinin protein that control sialic acid binding specificity and affinity vary among antigenic subtypes. For H3 subtypes, amino acids 226 and 228 are critical for differentiating between α2,3- and α2,6-linked forms of sialic acid (SA). We demonstrate that position 190 of the HA from A/Udorn/307/72 (H3N2) plays an important role in the recognition of α2,3-SA, as changing the residue from a glutamic acid to an aspartic acid led to alteration of red blood cell hemagglutination and a complete loss of replication in differentiated, murine trachea epithelial cell cultures which express only α2,3-SA. This amino acid change had a minimal effect on virus replication in MDCK cells, suggesting subtle changes in receptor recognition by the H3 hemagglutinin can lead to significant alterations in cell and species tropism.

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BackgroundThe arrival of RNA-seq as a high-throughput method competitive to the established microarray technologies has necessarily driven a need for comparative evaluation. To date, cross-platform comparisons of these technologies have been relatively few in number of platforms analyzed and were typically gene name annotation oriented. Here, we present a more extensive and yet precise assessment to elucidate differences and similarities in performance of numerous aspects including dynamic range, fidelity of raw signal and fold-change with sample titration, and concordance with qRT-PCR (TaqMan). To ensure that these results were not confounded by incompatible comparisons, we introduce the concept of probe mapping directed “transcript pattern”. A transcript pattern identifies probe(set)s across platforms that target a common set of transcripts for a specific gene. Thus, three levels of data were examined: entire data sets, data derived from a subset of 15,442 RefSeq genes common across platforms, and data derived from the transcript pattern defined subset of 7,034 RefSeq genes.ResultsIn general, there were substantial core similarities between all 6 platforms evaluated; but, to varying degrees, the two RNA-seq protocols outperformed three of the four microarray platforms in most categories. Notably, a fourth microarray platform, Agilent with a modified protocol, was comparable, or marginally superior, to the RNA-seq protocols within these same assessments, especially in regards to fold-change evaluation. Furthermore, these 3 platforms (Agilent and two RNA-seq methods) demonstrated over 80 % fold-change concordance with the gold standard qRT-PCR (TaqMan).ConclusionsThis study suggests that microarrays can perform on nearly equal footing with RNA-seq, in certain key features, specifically when the dynamic range is comparable. Furthermore, the concept of a transcript pattern has been introduced that may minimize potential confounding factors of multi-platform comparison and may be useful for similar evaluations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1913-6) contains supplementary material, which is available to authorized users.