APPLICATION OF BIOLOGICAL AND MOLECULAR TECHNIQUES TO THE DIAGNOSIS AND CONTROL OF AVIAN INFLUENZA AND OTHER EMERGING POULTRY PATHOGENS
Location: Exotic and Emerging Avian Viral Diseases Research Unit
Title: Highly pathogenic H5N1 avian influenza viruses differentially affect gene expression in primary chicken embryo fibroblasts
Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 13, 2008
Publication Date: October 15, 2008
Citation: Sarmento, L., Afonso, C.L., Estevez, C., Wasilenko, J.L., Pantin Jackwood, M.J. 2008. Differential host gene expression in cells infected with highly pathogenic H5N1 avian influenza viruses. Veterinary Immunology and Immunopathology. 125:291-302.
Interpretive Summary: Avian influenza viruses can infect and cause a wide range of disease in poultry, including chickens and turkeys. The most severe form of the disease is referred to as highly pathogenic avian influenza. In order to produce disease in birds avian influenza viruses need to overcome the host response, and the ways how the virus accomplish this is still not clear. In this study chicken embryo fibroblasts (CEF) were infected with two different highly pathogenic avian influenza viruses to compare cell responses to infection. These viruses grew differently in CEF's and three approaches were used to better understand this difference in pathogenicity. First, a complete chicken genome microarray was used to determine differences in early cell gene expression after infection. Second, semi-quantitive RT-PCR, a molecular technique, was performed to check selected genes and to study their expression later in the course of the infection. Third, the same test was used to investigate genes that have been reported to have a role in influenza virus infections. Differences in the expression of immune-related genes were found, which may explain in part the differences in virus replication. The results provide an insight on different mechanisms potentially used by avian influenza viruses to overcome innate host response.
Highly pathogenic avian influenza viruses cause severe clinical disease associated with high mortality in chickens and other gallinaceous species. However, the mechanism by which different strains of avian influenza viruses overcome host response in birds is still unclear. In the present study, chicken embryo fibroblasts (CEF) were infected with two avian influenza viruses with differing replication characteristics, to compare early differences in response to infection that might provide clues to the mechanisms involved in pathogenicity of these viruses. Infection of CEF revealed differences in growth characteristics consistent with their relative phenotype in chickens. Microarray analysis resulted in 191 genes showing differential gene expression by two-fold or more at 4 hours post infection (hpi). Gene ontology revealed that the differentially expressed genes are involved in many vital functional classes including protein metabolism, translation, transcription, host defence/immune response, ubiquitination and the cell cycle. Semi-quantitive RT-PCR was used to validate the differential expression of selected genes associated with host defence and also to investigate whether these genes and/or other genes were induced or suppressed later in the infection. Substantial differences in the expression of IFN-alpha, Mx1 and IL-8 genes were found, which may contribute to the differences in virus replication. All together, our data suggest that avian influenza viruses may use different strategies to evade host response and replicate in host cells.