Hello everyone,
does anybody know whether it´s possible to purchase commercially available Influenza A virus which is tagged with any fluorescent dye (i.e. GFP, mCherry, whatever...)? Or does anybody know whether there any other commercially available method to detect and quantify ongoing IAV infection in cultured cells? I would love any suggestion which does not involve Western blotting and plaque assays.
Many thanks in advance!!
Hello, Nicola!
You see in article:
Article Influenza A virus isolation, culture and identification
HA assay
A widely used, inexpensive, rapid and standard screening method for the detection of IAVs is the hemagglutination (HA) assay (originally developed by Hirst40 and Francis et al.41), which is based on the ability of the viral HA protein to bind to and ‘agglutinate’ red blood cells (RBCs). HA binding to RBCs is mediated by α2,3- and/or α2,6-linked sialic acid moieties at the RBC surface. HA assays are performed by mixing dilutions of virus-containing samples with a standard amount of RBCs in microtiter plates and observing agglutination patterns after a period of incubation. Agglutination causes RBCs to form a sheet that settles at the bottom of a well – giving a cloudy appearance – while the lack of agglutination results in RBC settling to the bottom of the well in the form of a point of cells (i.e., a ‘button’) or a circle of cells (i.e., a ‘halo’), surrounded by relatively translucent buffer.
The amount of IAV HA protein in surveillance samples is usually insufficient to yield positive results in the HA assay; however, this assay is effective for screening large numbers of virus cultures before performing additional – more specific – diagnostic analyses (i.e., RT-PCR). Importantly, several factors need to be considered when using this assay, including the following: (1) a positive HA assay indicates the presence of viral HA protein, but does not require infectious virus; (2) some IAVs do not agglutinate RBCs efficiently, even if their infectivity is high; (3) the origin of the RBCs (e.g., chicken, turkey, guinea pig, horse) can significantly affect the HA activity of an IAV, due to species-specific expression patterns of α2,3- and α2,6-linked sialic acid moieties on RBC surfaces42; and (4) hemagglutinin proteins from other viruses (e.g., paramyxoviruses) can induce RBC agglutination, so a positive HA assay result should always be followed with a secondary assay that can specifically identify the IAV. Avian RBCs (e.g., turkey) are preferred for use in HA assays because they are nucleated, heavy, and settle quickly in microtiter plates; thus, negative activity can be clearly differentiated from positive, agglutinating activity. In contrast, lighter, non-nucleated mammalian RBCs (i.e., guinea pig and human RBCs) can form ‘halos’ upon settling, making the differentiation between positive and negative activity potentially less clear. Currently circulating human H3N2 viruses lack the ability to agglutinate chicken RBCs43, and therefore, chicken RBCs should not be used for HA assays for these viruses. In addition, some human IAVs have been shown to agglutinate guinea pig RBCs with high efficiency44. With these points in mind, the source of RBCs for use in HA assays should be carefully selected depending on the viruses that are expected to be present. When the HA assay is used to screen samples for the presence of IAVs, useful controls include cell culture medium or egg fluids without virus (negative control) and a sample known to contain an IAV with efficient agglutinating activity (positive control).
RT-PCR
For specific IAV identification (from original surveillance samples or after virus culture in embryonated chicken eggs or cells), it is preferable to use a method that will detect viruses irrespective of antigenic differences in the HA protein, especially when the HA subtype of the prospective virus is unknown. Although IAV genomic sequences vary widely across subtypes and species-specific virus groups, the ‘M’ gene segment is relatively highly conserved. Therefore, the presence of IAV can be evaluated by using either standard or real-time RT-PCR with primers that recognize the M gene of viruses of all subtypes. Real-time RT-PCR is favored because results can be obtained more rapidly; however, M gene sequences amplified by using standard RT-PCR can be evaluated by using gel electrophoresis with only a modest increase in the time necessary to complete the analysis, provided that the number of samples is relatively low. For quality control purposes, an egg or tissue culture sample known to contain an IAV should be included in all procedures required for the RT-PCR analysis (e.g., RNA extraction, cDNA synthesis, PCR amplification, and agarose gel electrophoresis), so that concerns about procedural efficacy can be excluded when no positive samples are identified. Importantly, care should be taken to avoid cross-contamination between the known positive sample and all other samples (e.g., by using aerosol-resistant micropipette tips and preparing the RT-PCR master mix in a clean location), and all RT-PCR reactions should include a ‘no-template negative’ control to ensure that the reagents used are not a source of contamination.
Other assays for IAV identification
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