by Dr S. Lindsay and Prof. J. Yin
The well-established plaque assay based on virus culture in static media, has long been used to quantify infective particles. This article describes the authors' work on quantitative virus culture assays in flow environments. These assays are being developed in two modes, one in traditional culture wells and the other in microfluidic devices. Both can be used for efficient drug resistance assays or for drug discovery. The microfluidic assay lends itself more toward drug discovery as it has potential for use as a high-throughput, small-volume drug-screening platform.
Viruses have been a significant threat to human health from the dawn of humanity to the present day. From an ancient Egyptian engraving, we can see a priest with a withered foot characteristic of poliomyelitis. In 412 BC Hippocrates, the Greek physician known as the “father of medicine,” described a major epidemic now thought to be influenza. The growth of medical knowledge since Hippocrates, coupled with a technological explosion over the last century, has given us power to fight back against the virus threat in ways never before imaginable.
The development of vaccines is one of the most important medical accomplishments of modern history, and has prevented countless deaths. Smallpox has been completely eradicated from the wild, polio only remains in small pockets, rabies is no longer a human threat, and the incidence of many other deadly diseases such as measles has been dramatically reduced in many parts of the world.
ANTI-VIRAL VACCINES
Vaccines work by triggering the immune system to recognise viral antigens and produce antibodies against them, so that upon subsequent exposure the immune system will identify the antigen and work quickly to neutralise the virus. Unfortunately vaccination is less effective against rapidly evolving viruses, as the surface antigens are constantly changing. For this reason, despite decades of research, effective vaccines against important viruses such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV) remain elusive. An estimated 33 million people are currently infected with HIV [1], and 170 million with HCV [2]. Influenza, another rapidly mutating virus, infects around 10% of the world’s population every year, killing up to 500,000 [3]. Vaccines against influenza are currently available but need to be redesigned each year to target the newly evolved strains. Scaling a vaccine up to production capacity requires several months, so the vaccines produced for the 'flu season may or may not match the flu viruses actually circulating at the height of the 'flu season. This scale-up lag is also a concern for pandemic influenza. Many public health professionals have warned that “bird 'flu” strains (including H5N1 influenza A) might be able to acquire efficient human-to-human spread and become a highly virulent pandemic virus. Using current technology it would take several months before an effective vaccine could be produced to target the pandemic strain. A general vaccine against H5N1 influenza has been approved for use, but as its effectiveness is highly questionable [4], antiviral drugs look to play a very important role in minimising the pandemic death toll. For this reason, the neuraminidase inhibitor Tamiflu (Roche) is being stockpiled by many governments as part of a pandemic response plan. These drugs are also important in treating seasonal 'flu in high-risk patients like the elderly and immunosuppressed.
The whole issue of anti-viral vaccines and drugs is all the more relevant in the light of the current swine 'flu scare.
ANTI-VIRAL DRUGS
Anti-viral drugs are a powerful tool for saving human lives, but their development has been a challenge for two major reasons. First, because the viral life-cycle relies heavily on host cell processes, compounds that inhibit viral replication are likely to also be toxic to the cells themselves. Second, drug resistance is a major obstacle, especially for rapidly mutating RNA viruses such as HIV and influenza. Even so, various anti-virals targeting these viruses have been developed. For example, HIV is no longer a death sentence for those with access to highly active anti-retroviral treatment (HAART). This involves treating patients with a “cocktail” of three or more drugs from among the five approved classes of antiretroviral drugs. Under HAART therapy, the emergence of a mutant HIV virus simultaneously resistant to all of the drugs is extremely unlikely. Unfortunately, even with this costly treatment the virus is never fully cleared and the lifelong side effects of HAART therapy can be a physical and psychological burden. For influenza, there are only two approved drug classes, neuraminidase inhibitors and M2 ion channel blockers, and drug resistance has been a concern with both. For example, in the current 2008-2009 flu season in the United States the most prevalent strain of influenza (H1N1 influenza A) is resistant to Tamiflu, and the second most prevalent strain (H3N2 influenza A) is resistant to the M2 ion channel blockers. As drug resistance can occur so readily and unpredictably, the discovery of new classes of antiviral drugs for influenza is of great importance [5]. More efficient assays for the diagnosis and surveillance of drug resistant viruses would also be of benefit.
ANTI-VIRAL ASSAYS
Cell culture-based assays must be used to quantify the sensitivity of viruses to drugs or drug candidates. DNA amplification methods are frequently used in viral diagnostics but are less applicable to drug discovery because they cannot predict novel virus inhibitors and novel resistance mechanisms. The principal quantitative culture assay used in virology is the plaque assay. In this method, cells cultured in wells or flasks are inoculated with dilute virus solution and incubated at physiological temperature under semisolid agarose gel. The number of localised areas of cell death, or plaques, under the agarose is visualised by a cell stain and counted [Figure 1]. This count indicates the infectivity (in plaque-forming units per volume) of the original virus-containing sample. This assay can also be carried out in the presence of a drug to determine how much the drug reduces plaque counts relative to parallel infections in the absence of a drug.
The plaque assay, based on virus culture in static media, has remained largely unchanged in more than half a century since its introduction by Dulbecco in 1952 [6]. Our lab is working to extend the culture-based virology toolbox by developing quantitative virus culture assays in flow environments. Coupling the virus spread with fluid flow not only enhances the assay sensitivity, as described below, but may also establish a basis for more physiologically relevant measures since interstitial flows from the vasculature to the lymphatic system result in small but significant flow rates through most tissues in the body [7]. Quantitative flow-based assays are being designed in two modes, one applied in traditional culture wells and the other in microfluidic devices. These can both be used for efficient drug resistance assays or for drug discovery. The microfluidic assay lends itself more toward drug discovery as it has potential for use as a high-throughput small-volume drug-screening platform.
It has long been observed that virus infections, cultured in vitro under liquid media rather than agarose gel, form elongated plaques due to spontaneous flows generated within the well [8-14]. These elongated plaques tend to have a comet-shaped morphology [Figure 2]. The exact mechanism of comet spread remains unclear, but it has been observed that vaccinia virus comets formed in plates tilted at 10 degrees from the horizontal plane tend to spread in the uphill direction [9], and that influenza A comet formation is dependent on virus-receptor binding affinity, with the lower affinity variant preferentially producing comets in vitro, while the higher binding variant produced more circular plaques [12]. The principle of enhanced spread due to flows in tissue culture plates has been used to design qualitative [13] and quantitative [14] assays for drug affect on virus spread. In the latter case, Zhu and Yin found that a quantitative comet assay for the effect of 5-fluorouracil on vesicular stomatitis virus (VSV) had higher sensitivity, by an order of magnitude, than the plaque assay. There are two reasons for the increased sensitivity. The comet infectivity can be more easily quantified based on the infected area, which accounts for both viral fitness and number, unlike simple plaque counts. Also, the flows spread the virus downstream, amplifying the infection signal relative to the plaque assay. One drawback of the comet assays is that the flows are uncontrolled spontaneous flows. In order to create a miniaturised assay with controlled flow properties, Zhu et al [15] used microfluidic channels fabricated from polydimethylsiloxane (PDMS). Cells were grown to confluence within the channels, and a small localised region was infected with a droplet of VSV virus solution through an access port. After one infection cycle, the progeny virions were spread downstream using a bolus flow provided by a surface tension based passive pumping mechanism [16]. After a subsequent virus infection cycle, the infections were quantified based on either protein expression or a cell stain [Figure 3]. Like the comet assay, the microfluidic flow-based assay has greater sensitivity than the plaque assay. In addition, the infection procedure and microfluidic setup are very simple. No tubing or specialised equipment is required, and all liquid handling steps are conducted through simple pipetting.
Current work is being carried out to optimise the flow-based assays for use with influenza virus, and to test the microfluidic system as a high-throughput screening platform. There are several challenges to overcome, including PDMS absorption of small molecules into the bulk of the polymer [17] and characterisation of virus release kinetics in the microfluidic environment, but we believe that these assays will prove useful in influenza surveillance and pandemic preparedness. In general, flow-based assay technology will provide new tools for increased sensitivity, throughput, and physiological relevance in clinical virology and drug discovery.
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THE AUTHORS
Dr S. Lindsay
and
Professor John Yin
Department of Chemical and
Biological Engineering
University of Wisconsin-Madison
1415 Engineering Drive
Madison,
WI 53706-1607,
USA
Correspondence to:
Professor J. Lin,
Tel +1 608 265-3779
Fax +1 608 262-5434
e-mail: yin@engr.wisc.edu
