Studies on the virulence potential of influenza A viruses of poultry by reverse genetics
I. Influence of the polybasic HA cleavage site on virulence.
II. Can other HA subtypes than H5 and H7 generate highly pathogenic strains?
S. Weber, J. Stech, O. Stech, J. Veits, S. Gohrbandt, J. Bogs und T. Mettenleiter
Research of the laboratory focuses on molecular biological investigations on influenza A viruses of poultry. Influenza A viruses belong to the family Orthomyxoviridae. Under the electron microscope they appear as irregularly shaped spherical/ovoid particles with a diameter of 80 to 120 nm or as up to 2000 nm long filamentous structures. Their surface consists of a lipid envelope with surface projections belonging to two types of glycoproteins. Influenza viruses have a single-stranded RNA genome of negative polarity consisting of 8 segments. The virus genome codes for 11 proteins, the most important ones being the glycoproteins haemagglutinin (HA) and neuraminidase (NA). These play a decisive role in the infection of the host cell by the virus and in virus release. 16 haemagglutinin (H1 – H16) and 9 neuraminidase subtypes (N1 – N9) have been described. All highly pathogenic influenza viruses that have been identified so far belong to the subtypes H5 and H7. Depending on the virulence and on the route of infection, infected poultry may show respiratory, gastrointestinal and central nervous disorders. „Highly pathogenic avian influenza viruses“ (HPAIV) are the causative agents of classical fowl plague (FP) and cause severe generalized disease accompanied by massive economic losses. A big problem of influenza viruses is their variability which is due to a high mutation rate in the viral genome. In addition, influenza viruses have the ability to exchange complete genome segments which can lead to sudden modifications in the characteristics of new reassortants. In rare cases, a poultry influenza virus can cross the species barrier and infect mammals including humans. A good example is the so-called ‘bird flu’ HPAIV H5N1. The availability of reverse genetics for influenza A viruses and thus the possibility to manipulate the pathogen’s genome directly have improved the possibilities for investigation of the molecular mechanisms of the infection as precondition for the development of effective control strategies.
We have cloned the sequences coding for typical representants of the 16 haemagglutinin subtypes into the expression vector pHW2000 (Hoffmann et al., PNAS 2000) Each of these 16 different haemagglutinins was equipped with a polybasic cleavage site. By means of the reverse genetic systems for the low pathogenic influenza A virus strains A/duck/Ukraine/1/63 (H3N8) and A/Chicken/Emirates/R66/2002 (H9N2), as well as the highly pathogenic strain A/Swan/Germany/R65/06 (H5N1) we generated virus mutants which express different haemagglutinins on an isogenic background. We hope to gain detailed insight into the molecular biological processes which are of importance for pathogenicity and host specificity.
Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG.(2000). A DNA transfection system for generation of influenza A virus from eight plasmids. PNAS. May 23;97(11):6108-13.