The overall strategy of this project is to create the knowledge, which is currently lacking, that will promote development of pertinent and informative diagnosis for the identification of AIV isolates posing a major threat to animal and human health. Fundamental knowledge about the importance of both viral and host factors is required to determine the outcome of an AIV infection and the likelihood of transmission within and between species. Although significant advances have been made in defining viral and host determinants of virulence and interspecies transmission (see 7.1.), many questions are only partially answered. What are the molecular elements that determine the relatively large spectrum of virulence of the heterogeneous group of HPAIV H5N1 isolates? Why are AIV much more virulent in domestic chicken when compared to water fowl? Understanding the latter will be essential for a comprehensive view of AI ecology. The factors influencing interspecies transmission are studied in the BVET project 1.05.10 that is complementary to the present project and will therefore not be addressed here. Altogether, the current knowledge does not suffice for a proper management of the AIV crisis, which is not yet controlled. Dangerous AIV strains are likely to be either still present or to be re-introduced into Europe. Consequently, it is very important to employ advanced innovative technologies to develop knowledge, which will permit to understand (i) virus-host interactions, (ii) HPAIV specific virulence factors, (iii) host factors that determine resistance or sensitivity to HPAIV and (iv) virus and host factors that determine species specificity of HPAIV. The latter are of critical importance to assess the probability and danger of transmissibility to mammalian hosts and further spread with pandemic nature.
Consequently, we have decided to focus on two major areas selected with the goal of supporting the AIV Reference Laboratory. These are to establish (i) the molecular tools required to characterise the main viral elements governing virulence by constructing and characterizing cDNA clones of relevant recent H5N1 isolates, and complementary to this, (ii) the experimental in vivo and in vitro systems to analyze AIV virulence in chicken. With these tools in hand we are aiming to identify and characterise viral factors influencing HPAIV virulence. The created knowledge will be applicable in areas directly related to combating AI, including diagnostics and epidemiology. Specifically, these are to improve current molecular diagnostic approaches to classify AIV with respect to their danger for veterinary and public health, and to improve a knowledge-based risk management relying on interdisciplinary expertise in the areas of AIV virulence, virus transmission, species tropism and disease pathogenesis. Without the knowledge from this project, it will be impossible to develop pertinent and informative diagnostic tests allowing us to identify rapidly and accurately AIV isolates posing a major threat to animal and human health.
In order to obtain the promised information of this project, it will be essential to characterise newly engineered AIV in vivo and in vitro by acquiring knowledge on their virological characteristics and their pathogenic potential (see 7.3.1. and 7.3.2). In addition, it is important to emphasize that as a Reference Laboratory for AI, it is not only important to possess molecular diagnostic approaches to enable a rapid diagnosis of newly emerging strains of AIV but also to be able to classify newly emerged AIV strains with respect to their pathogenic potential and virulence. For the general classification of AI into highly pathogenic notifiable AIV (HPNAI) and low pathogenicity notifiable AIV (LPNAI) IVPI tests are performed in chicken (OIE, 2004). Being a lethality test, this procedure does not conform to the principle of reducing animal suffering to a minimum. Furthermore, it does not give any information on pathogenic and virological mechanisms of the infection. Such information would be inestimably important to better understand the basis of AIV virulence, both from the host and the virus point of view. Furthermore, this information will enable the reduction and refinement of animal experimentation in this area. Consequently, we will characterise the parameters of the host response to AIV strains of different pathogenicity using both in vivo and in vitro tests. The approach is to identify the critical in vivo parameters, and on the basis of this establish informative in vitro tests.
The current H5N1 HPAIV appear to be particular virulent when compared to previous viruses and the biological basis of this is far from being understood. In chicken the disease is characterised by generalized systemic infection. The pathohistological characteristics such as coagulopathy - including swelling of the microvascular endothelium, multifocal hemorrhages and thrombosis (Muramoto et al., 2006) - imply the involvement of host factors, in particular a cytokine storm in disease. It is probable that the source of these cytokines would originate from the interaction of the virus with the innate immune system. Innate immune responses induced during acute virus infections represent a double edge sword. On one side, they are essential to control early virus replication before the onset of the adaptive immune response. On the other side they are responsible for severe inflammation and tissue damage resulting, for example, in fatal respiratory distress syndrome. Interestingly, vigorous systemic cytokine responses have been described in human patients infected with highly virulent avian and human influenza virus. However, despite their potential importance for both the innate immune response and disease pathogenesis, nothing is known about cytokine responses induced in HPAIV-infected chicken. As the histopathological characteristics described above are indicating a key role for innate immune responses in the pathogenesis of AIV, we are planning their characterisation.
This will promote a better understanding of disease pathogenesis, and will form an integrated part of our attempts at the IVI to build a knowledge-based risk management with interdisciplinary expertise of the avian influenza problem. To this end, the project will link to the work of the other Departments at the IVI led by Dr. Martin Hofmann (Development) and Dr. Barbara Thür (Diagnostics) which are focussing on establishing the molecular diagnostics and high throughput screening tests for modern, efficient AI diagnosis. The aims of the project will be realized as an interdisciplinary collaboration between the Molecular Virology Laboratory (main applicant and second co-applicant) and the Immunology Laboratory (first co-applicant) within the Research Department (led by Dr. Kenneth C. McCullough, third co-applicant). The project will be complementary to the BVET project 1.05 “Towards identification of influenza A virus strains with pandemic potential in vitro: species-tropism and inflammatory cytokine responses” running in the Immunology Section focussing on aspects of AIV transmission to mammals. It will also be of high value for the project “Constanze”, an international collaboration involving the BVET and the IVI and aimed at studying the ecology of AIV in the region of the lake of Konstanz, in that isolated AIV of interest may be characterised in more detail (see 7.3.3.).
