Differential lung NK cell responses in avian influenza virus infected chickens correlate with pathogenicity.

Infection of chickens with low pathogenicity avian influenza (LPAI) virus results in mild clinical signs while infection with highly pathogenic avian influenza (HPAI) viruses causes death of the birds within 36-48 hours. Since natural killer (NK) cells have been shown to play an important role in influenza-specific immunity, we hypothesise that NK cells are involved in this difference in pathogenicity. To investigate this, the role of chicken NK-cells in LPAI virus infection was studied. Next activation of lung NK cells upon HPAI virus infection was analysed. Infection with a H9N2 LPAI virus resulted in the presence of viral RNA in the lungs which coincided with enhanced activation of lung NK cells. The presence of H5N1 viruses, measured by detection of viral RNA, did not induce activation of lung NK cells. This suggests that decreased NK-cell activation may be one of the mechanisms associated with the enhanced pathogenicity of H5N1 viruses.

Identification of new populations of chicken natural killer (NK) cells.

Natural killer (NK) cell activity is conserved throughout vertebrate development, but characterization of non-mammalian NK-cells has been hampered by the absence of specific mAbs for these cells. Monoclonal antibodies were generated against in vitro IL-2 expanded sorted CD3-CD8alpha+ peripheral blood lymphocytes, previously described to contain chicken NK-cells. Screening of embryonic and adult splenocytes with hybridoma supernatants resulted in five candidate NK markers. Activation of chicken NK-cells with PMA/Ionomycin or with the NK target cell-line LSCC-RP9 resulted in increased expression of CD107 (LAMP-1) and a newly developed flow cytometry based cytotoxicity assay showed that NK-cells were able to kill target cells. Combining NK markers with functional assays indicated that marker positive cells showed NK-cell function. In conclusion, we generated new monoclonal antibodies and developed two functional assays which will enhance our understanding of the role of NK-cells in healthy and diseased chickens.

Infectious Bronchitis Virus induces acute interferon-gamma production through polyclonal stimulation of chicken leukocytes.

Infectious Bronchitis Virus, a member of the Coronaviridae, is a respiratory pathogen in poultry. We found that in vitro stimulation with IBV resulted in ChIFN-gamma production in splenocytes of both infected birds and uninfected birds. The non-specific stimulation did not occur when other avian viruses or other coronaviruses were used or when mammalian cells were stimulated with IBV. Inactivation of IBV reduced ChIFN-gamma production, but ChIFN-gamma remained elevated compared to unstimulated cells. An increase in ChIFN-gamma mRNA was detected in splenocytes from IBV-infected and uninfected chickens as early as 1 h after stimulation with IBV. These results indicate that IBV acts as a polyclonal stimulus, inducing a rapid production of IFN-gamma even without previous exposure to the virus.