Transcription factor regulation and cytokine expression following in vitro infection of primary chicken cell culture with low pathogenic avian influenza virus

Abstract

Background: Avian influenza virus (AIV) induced proinflammatory cytokine expression is believed to contribute to the disease pathogenesis following infection of poultry. However, there is limited information on the avian immune response to infection with low pathogenic avian influenza virus (LPAIV).

Methods: To gain a better understanding of the early viral-host interactions of LPAIV in chickens, primary chicken embryo hepatocytes (CEH) were infected with four different LPAIVs of U.S. origin. Kinetics of virus replication, transcription factor (c-Jun, p50 and IRF-3) activation and immune response gene (IL-6, IL-1beta, IFN-alpha and Mx) expression were studied at four different time points (6, 12, 24 and 48 hours) post infection and compared to non-infected controls.

Results: CEH can support growth of the tested LPAIVs when with trypsin supplementation. All four immune response genes tested were upregulated following infection as were transcription factors c-Jun, p50 and IRF-3. Amplification of these genes was dependant on virus replication (e.g. inclusion of trypsin), such that immune response genes and transcription factors were upregulated as viral titers increased.

Conclusion: The results of these studies demonstrate the requirement of virus replication for innate immune regulation and broaden our understanding of transcription factor responses related to LPAIV infection in chickens.

Figure 1

Kinetics of CEH infection by H5N9, H5N3, H7N2 and H9N2 viruses. Cells were infected at MOI of 1 and supplemented with1μg/ml trypsin or without trypsin in the medium. The viral titers in supernatants collected at 6, 12, 24 and 48 hpi were determined as log10EID50/ml. The patterns bars represent the viral titers achieved without the use of supplemental trypsin. The gray bar stacked on top represents the increase in the viral titers with the addition of supplemental trypsin. Error bars show standard deviation of the mean, n = 3. *Indicates the difference (P < 0.05) between the supplemented with and without trypsin group.

Figure 2

Pro-inflammatory IL-6/IL-1β mRNA expression of CEH infection by H5N9, H5N3, H7N2 and H9N2 viruses. Cells were infected for different time periods with LPAIV at MOI of 1 and supplemented with 1 μg/ml trypsin or without trypsin in the medium. Total RNA was isolated and quantitated using QRRT-PCR. The horizontal axis represents virus. The vertical axis represents the fold change. Error bars represent standard deviation across each condition performed in triplicate.

Figure 3

Interferon-α/Mx mRNA expression of CEH infection by H5N9, H5N3, H7N2 and H9N2 viruses. Cells were infected for different time periods with LPAIVs at MOI of 1 and supplemented with 1 μg/ml trypsin or without trypsin in the medium. Total RNA was isolated and quantitated using QRRT-PCR. The horizontal axis represents virus. The vertical axis represents the fold change. Error bars represent standard deviation across each condition performed in triplicate.

Figure 4

C-Jun, p50 and IRF-3 activity in CEH in response to LPAIVs. Cells were infected for different time periods with LPAIVs at MOI of 1 and supplemented with 1 μg/ml trypsin or without trypsin in the medium. Cell lysates (10μg/ml) were tested for binding of the activated c-Jun, IRF-3 and p50 subunits using the Trans-Am ELISA kit. The results are expressed as specific binding (absorbance measured in the presence of the mutated oligonucleotides minus that measured in the presence of the wild-type oligonucleotides) according to the manufacturer’s instructions and are compared to non-infected control.