The influenza virus, a RNA virus, is responsible for the commonly called flu. These viruses cause disease in many vertebrate species, including humans. There are four types of influenza virus – A, B, C and D – but epidemics of human influenza is mainly caused by A and B viruses.

Influenza A virus are by far the biggest threat to human health among all virus types. This group is further divided into subtypes, based on the two viral proteins on the surface of the virus cell: the hemagglutinin (H) and neuraminidase (N). There are 18 different H subtypes and 11 N subtypes and each subtype varies by at least 30% of the amino acid sequence. Different strains are classified based on each specific H and N subtypes. Currently only two subtypes are circulating in humans: H1N1 and H3N2 and in total only four subtypes were ever in circulation in the human population.

Influenza viruses are constantly changing. This change can occur by either antigenic drift or antigenic shift.

Antigenic drift are small constant changes in the genome of the virus that creates viruses that are closely related to one another and therefore share antigenic properties. This means, a healthy immune system infected by the new variant will be able to recognize it and respond. Only when these small changes accumulate with time, a new variant is produced that is no longer recognized by our immune system. This accumulation of genetic mutations is the reason why a person can get sick with the flu multiple times and why the vaccinations need to be updated each year.

Antigenic shift, on the other hand, is a major change in the influenza A viruses, which results in new hemagglutinin or hemagglutinin/neuraminidase surface proteins. These shifts result in new subtypes of the virus and can result in a new subtype that emerges from an animal population that suddenly has the ability to infect the human population. This was exactly what happened in 2009 when an H1N1 virus with a new combination of surface proteins emerged to infect people, who had no protection against the new variant. This caused the pandemic of 2009 swine flu. Luckily, antigenic shift, contrary to antigenic drift, only occurs rarely.

The constant genetic mutations, genomic recombination, and selection pressure ensure the survival of influenza A viruses and its continued threat to mankind. The rapid and threatening evolution of influenza A viruses enables the virus to infect multiple species and the emergence of novel strains is unpredictable and inevitable.


Presently, the only method to prevent large influenza epidemics and pandemics is vaccination. Each year vaccines include influenza A H1N1, H3N2 and one to two influenza B viruses. What strain of the viruses are to be included are determined each year at the beginning of the flu season, by isolating samples from early infected patients.

Characterization of the strains is currently done with resource to either indirect immunologic tests with ferret sera or direct subtyping through genetic analyses. The most sensitive method and the one used by the WHO global influenza surveillance group is the strain characterization through immunized ferrets. These immunologic tests are performed with ferret sera, which is used in hemagglutination inhibition tests. Ferrets, due to their specific immune systems, are able to form subtype-specific antibodies that prevent agglutination of specific influenza subtypes, even in unknown samples. This is currently the most sensitive screening method for antigenic variations of influenza viruses, but it is also time consuming and costly. It involves as well the killing of immunized ferrets.

Direct screening methods are also available for fine characterization of influenza virus variants. Genetic screenings through real-time PCR are one of the standard molecular methods for subtyping in various applications. More recently, sequencing and hybridization capture also constituted alternatives for the direct characterization of influenza variants. These methods are largely used in research, but still play a minor role in practical applications and require a great deal of sample preparation.

Phenotypic commercial detection methods include direct and competitive ELISAs. Antibodies developed by the manufacturers themselves are used to differentiate between different influenza types (A, B or C) or subtypes (e.g. H3, H5). This is currently sufficient for diagnostics in (farm) animals. However, such tests do not allow the fine typing of the variants that currently infect the human population and are therefore ineffective as methods to improve vaccination directives each year.


Due to the constant changing nature of the influenza viruses, the flu vaccine is unable to prevent reoccurring epidemics. Alternatively, artificially produced antibodies that bind to conserved (unchanged) regions of the hemagglutinin proteins have shown success in neutralizing the spread of the virus. However, development, mass production and quality control of antibodies is still expensive and time consuming.

Conversely, short molecules that specifically bind to H or N surface proteins can be massed produced at relatively low costs. These short molecules are called peptides. Peptides are short chains of amino acids linked by amide bonds. In a previous study, peptides were developed to bind specifically to the H-protein of the influenza virus and they showed anti-inflammatory activity. This means, these short molecules are able to recognize specific regions of the virus and bind to them through sequence complementarity.

In this project, we aimed at developing peptides from known anti-influenza antibodies, which would bind specifically with the hemagglutinin surface protein. By slightly changing the amino acid sequence of each peptide, we created molecules with different binding abilities to different virus subtypes.

This library of influenza A hemagglutinin recognizing peptides, will help us develop a diagnostic assay that is able to quickly and reproducibly identify different virus subtypes.

For this, peptides need to be presented to the virus. Through linkers that were added to each peptide sequence, we are able to immobilize the peptides in different presenting surfaces and increase the efficacy of peptide-virus binding, without the need to modify the virus samples beforehand.

Goal of the project

Our goal is to create a standardized method for characterizing influenza A virus subtypes. Our assay is based on a peptide library, where peptides with different binding affinities to different variants of the virus are expected to produce a specific and reproducible binding pattern.

This methodology requires little to no prior virus preparation, is quicker than current characterization methods and is reproducible, with synthetically mass-produced peptides and well-established laboratory methodologies. Another great advantage of this method is the complete independence from using animal testing.