Outbreaks of influenza account for much morbidity during winter months, and result in tens of thousands of deaths each year. The elderly and very young are particularly susceptible to more severe respiratory disease and death due to influenza. These individuals can be vaccinated but because the young are immunologically naïve, and the elderly are immunosenescent, vaccine preparations lack immunogenicity in these population groups [1
]. Antivirals would clearly benefit these individuals and in addition would be of great value to the global population when no suitable vaccine is available to prevent infection [4
]. This is likely the case when there is antigenic shift and a new virus strain emerges that could result in a world-wide pandemic. Pandemics that occurred in 1918, 1957 and 1968 were each the result of the transmission of influenza with a unique HA subtype, with the introduction of H1, H2 and H3 hemagglutinin (HA) gene segments from an avian virus source [5
The avian H5N1 virus that is currently a pandemic threat has resulted in hundreds of human infections, with approximately 60% mortality rate. If such a strain becomes easily transmissible amongst people, there will be extensive death and disease unless a prophylactic vaccine is used or antivirals are administered. The only H5N1 vaccine licensed for emergency use in the United States contains inactivated A/Vietnam/1203/2004. There is no assurance that this vaccine will antigenically match the pandemic H5N1 strain, and so vaccine efficacy cannot be predicted. There is therefore a great need to stockpile effective antiviral drugs. Unfortunately, there are only two classes of antivirals that can be used to treat influenza; adamantanes that inhibit virus replication by blocking the influenza A M2 ion channel and neuraminidase (NA) inhibitors. Of these, the adamantanes are no longer effective against many recent influenza A virus strains [6
] and most H5N1 strains are resistant to this class of drug [8
]. Decreased sensitivity to the second class of antivirals that inhibit NA activity has been noted [9
], and H1N1 viruses that are resistant to one of the two licensed NA inhibitors, oseltamivir, are prevalent in Europe [10
In addition to problems associated with emergence of drug-resistant virus strains, each drug class has potential side effects. While the NA inhibitors were generally thought to have fewer toxic effects than amantadine and rimantadine, oseltamivir is no longer prescribed to children in Japan because of an association with neuropsychiatric disorders that include suicidal behavior, hallucinations and seizures [11
]. Oseltamivir-induced delirium has also been reported in a geriatric patient [12
]. There is clearly a need for licensure of additional inhibitors against influenza, particularly inhibitors to which resistant virus strains are less likely to emerge.
To fill this need, several new candidate antiviral agents have been identified [13
]. In the process to select new candidates, methods targeted to a specific gene product or particular virus replication steps are commonly used; for example, viral RNA transcription [14
]. However, assays that allow for identification of inhibitors with a broad range of targets increase the likelihood of obtaining a product that is effective. Unfortunately these latter viral inhibition assays are usually not suited to high throughput screening (HTS). In this report we describe modifications of the standard virus neutralization assay that facilitates its use in HTS. The key element to this assay is the use of viral NA as a means to quantify virus replication early after infection. This affords higher throughput with excellent signal/noise ratios, providing excellent assay sensitivity. In addition to presenting these properties, we use known influenza virus inhibitors to demonstrate the broad spectrum of antivirals detected by this assay.