Sampling the complete organ instead of defined parts might affect analysis at both the cellular and transcriptional levels. We defined host responses to H9N2 avian influenza virus (AIV) in trachea and different parts of the lung. Chickens were spray-inoculated with either saline or H9N2 AIV. Trachea and lung were sampled at 1 and 3 days post-inoculation (p.i.) for immunocytochemistry, real-time quantitative RT-PCR and gene-expression profiling. The trachea was divided into upper and lower parts and the lung into four segments, according to anatomy and airflow. Two segments contained the primary and secondary bronchi, cranial versus caudal (parts L1 and L3), and two segments contained the tertiary bronchi, cranial versus caudal (parts L2 and L4). Between the upper and lower trachea in both control and infected birds, minor differences in gene expression and host responses were found. In the lung of control birds, differences in anatomy were reflected in gene expression, and in the lung of infected birds, virus deposition enhanced the differences in gene expression. Differential gene expression in trachea and lung suggested common responses to a wide range of agents and site-specific responses. In trachea, site-specific responses were related to heat shock and lysozyme activity. In lung L1, which contained most virus, site-specific responses were related to genes involved in innate responses, interleukin activity and endocytosis. Our study indicates that the anatomy of the chicken lung must be taken into account when investigating in vivo responses to respiratory virus infections.

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Highly pathogenic H5N1 avian influenza A viruses display a remarkable genetic and antigenic diversity. We examined to what extent genetic distances between several H5N1 viruses from different clades correlate with antigenic differences and vaccine performance. H5-specific antisera were generated, and cross-reactivity and antigenic distances between 12 different viruses were determined. In general, antigenic distances increased proportional to genetic distances although notable exceptions were observed. Antigenic distances correlated better with genetic variation in 27 selected, antigenically-relevant H5 residues, than in the complete HA1 domain. Variation in these selected residues could accurately predict the antigenic distances for a novel H5N8 virus. Protection provided by vaccines against heterologous H5N1 challenge viruses indicated that cross-protection also correlates better with genetic variation in the selected antigenically-relevant residues than in complete HA1. When time is limited, variation at these selected residues may be used to accurately predict antigenic distance and vaccine performance.

In this study a viral infection of a tissue culture model system was compared to an in vivo infection, which is of importance to gauge the utility of the model system. The aim was to characterize early immune responses induced by avian influenza virus using tracheal organ cultures (TOC) as a model system. First, the in vitro system was optimized to ensure that the host transcription responses were only influenced by virus infection and not by differences in viral load. Upper and lower trachea both could be used in the cultures because the virus load was the same. Cilia motility was not affected in non-infected TOC and only slightly in infected TOC at 24h post-inoculation. Gene expression profiles of early immune responses were analyzed in in vitro infected TOC, and were compared to the responses found in in vivo infected trachea. The gene expression profile in infected TOC suggested the up regulation of innate anti-viral responses that were triggered by attachment, entry and uptake of virus leading to several signalling cascades including NF-kappaB regulation. Genes associated with IFN mediated responses were mainly type I IFN related. Overlapping gene expression profiles between non-infected and infected TOC suggested that tissue damage during excision induced wound healing responses that masked early host responses to the virus. These responses were confirmed by real-time quantitative RT-PCR showing up regulation of IL-1beta and IL-6. Microarray analysis showed that gene expression profiles of infected and non-infected TOC had a large overlap. This overlap contained many immune-related genes associated with inflammatory responses, apoptosis and immune system process and development. Infected TOC and in vivo infected trachea shared few significantly differentially expressed genes. The gene expression profile of infected TOC contained fewer genes which were expressed at reduced amplitude of change. Genes that were common between TOC and trachea were associated with early immune responses likely triggered by virus attachment and entry. Most of the genes were associated with IFN-mediated responses, mainly type I IFN related. Our study implicates that although the TOC model is suitable for culturing of virus and lectin or virus binding studies, it is not suitable for measuring early immune responses upon viral infection at host transcriptional level.

Newly hatched chickens are more susceptible to infectious diseases than older birds because of an immature immune system. The aim of this study was to determine to what extent host responses to avian influenza virus (AIV) inoculation are affected by age. Therefore, 1- and 4-week (wk) old birds were inoculated with H9N2 AIV or saline. The trachea and lung were sampled at 0, 8, 16 and 24h post-inoculation (h.p.i.) and gene expression profiles determined using microarray analysis. Firstly, saline controls of both groups were compared to analyse the changes in gene profiles related to development. In 1-wk-old birds, higher expression of genes related to development of the respiratory immune system and innate responses were found, whereas in 4-wk-old birds genes were up regulated that relate to the presence of higher numbers of leukocytes in the respiratory tract. After inoculation with H9N2, gene expression was most affected at 16 h.p.i. in 1-wk-old birds and at 16 and 24h.p.i. in 4-wk-old birds in the trachea and especially in the lung. In 1-wk-old birds less immune related genes including innate related genes were induced which might be due to age-dependent reduced functionality of antigen presenting cells (APC), T cells and NK cells. In contrast cytokine and chemokines gene expression was related to viral load in 1-wk-old birds and less in 4-wk-old birds. Expression of cellular host factors that block virus replication by interacting with viral factors was independent of age or tissue for most host factors. These data show that differences in development are reflected in gene expression and suggest that the strength of host responses at transcriptional level may be a key factor in age-dependent susceptibility to infection, and the cellular host factors involved in virus replication are not.