A novel influenza A (H1N1) virus was discovered in Mexico in early 2009 (1). Infections from this strain led to declaration of a pandemic midyear, with about 61 million patients and 13,000 deaths reported by the US Centers for Disease Control (2). Although the pandemic officially ended in August 2010 (3), vaccines are still in demand to protect people against the H1N1 strain that is now expected to circulate seasonally for years to come. To best respond to pandemic outbreaks and annual composition changes, the vaccine industry must be able to produce large quantities of product in a short amount of time (4). New methods for culturing, clone screening, scale-up, and production require analytical methods that can rapidly quantify virus to ensure enhanced productivity. Ideal new virus quantification methods would be fast, precise, robust relative to a wide range of viruses, and would correlate well with established methods (5).

Established methods for influenza virus quantification include viral plaque titer assays, tissue culture infective dose (TCID₅₀) assays, fluorescence focus assays (FFAs), transmission electron microscopy (TEM), and quantitative real-time polymerase chain reaction (qPCR) technology (6). Plaque assays, TCID₅₀, and FFA measure infectious counts, TEM provides total viral particle counts, and qPCR measures total genome copy numbers. Relative errors associated with the plaque assay have been shown to be 10–100%, whereas TCID₅₀ has about a 35% error (7,8). The immunofluorescence FFA assay is useful for cell lines that do not exhibit detectable cytopathic effects, but it requires target-specific antibodies (9). Infectivity assays provide invaluable results, but they are time consuming as well as reagent and personnel-intensive.

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Two primary “physical” methods are used to quantify viruses: TEM and qPCR. Transmission electron microscopy has been used for a number of years and is conceptually straightforward, with results determined from the number, shape, and size of imaged virus particles on a flat surface. But TEM is expensive, its sample preparation is tedious, and the technique requires a skilled operator (10). Quantitative PCR has emerged relatively recently as a useful tool for use with some viruses. Once a well-characterized primer set has been developed, it can be used to quantify the viral nucleic acid concentration of a sample within a matter of hours (11). In addition, qPCR can analyze low virus concentrations due to nucleic acid amplification, and it has been shown to successfully quantify numerous viruses: influenza A and B (12), baculovirus (13,14), hepatitis B virus (15), human immunodeficiency virus (16), and dengue fever virus (17). However, qPCR also requires a skilled operator, and it suffers from myriad potential causes of failure and contamination (18). Given influenza's susceptibility to mutation, PCR primer design is potentially a problem. Genetic change in the priming region could necessitate primer redesign for accurate results over time (19).

Efforts continue toward improving existing assays (20,22), and new analytical methods for virus quantification are being developed to supplement those methods and overcome their limitations. Emerging methods must be evaluated relative to the standard techniques to compare precision, linear dynamic range, and time savings. One new possibility is flow cytometry, which offers the benefit of rapid sample analysis (23). Although this technology traditionally has been used for quantifying surface and intracellular antigens or nucleic acids (24), it has been used in virus quantification by detecting influenza A virus infection of Madin Darby canine kidney (MDCK) cells (25). But the researchers — and nearly all current flow cytometry applications for virus quantification — used indirect detection and quantification of infected cells rather than virus particles. InDevR's Virus Counter (VC) flow cytometer was designed specifically to directly quantify virus particles.

The specialized VC system capitalizes on the benefits of flow cytometry while providing a sensitive detection system that directly quantifies virus particles following a universal staining preparation step (26,27). Other flow-cytometry–based virus quantification protocols require expensive antibodies and a virus-specific, multistep staining protocol that takes hours to perform (23). By contrast, the one-step VC staining process requires only a 30-minute incubation period using inexpensive, commercially available dyes that are nearly universal for all viruses. Once instrument settings have been determined, analysis time is <10 minutes per sample. The bench-top VC instrument and assay represent a new platform for real-time virus quantification.

This instrument quantifies the total number of virus particles per unit volume, which is similar to TEM results. In a VC assay, intact virus particles are quantified in suspension using fluorescence to detect particles with colocalized proteins and nucleic acids. A Combo Dye reagent — consisting of one dye specific for proteins and one specific for nucleic acids — is used to stain samples regardless of virus type. The instrument quantifies virus particles per milliliter (vp/mL) based on the number of events occurring simultaneously in two distinct fluorescence channels and a measured sample flow rate (27,28). Figure 1 is a partial screen capture from InDevR's Virus Counter software, which shows an example of the raw data obtained during VC sample analysis. The software displays photomultiplier tube (PMT) fluorescence signals for protein (red) and nucleic acid (blue) channels in the top and bottom panels, respectively displaying 3 ms and 75 ms segments of data. Threshold values (shown as overlapping horizontal lines in the top panel), are used to discriminate virus events from background noise and signal.

Figure 1: (Click to enlarge)

Here we compare the VC system with qPCR using influenza A/H1N1 samples to evaluate the new instrument and assay's suitability as a rapid alternative for influenza virus quantification.