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

Jiang H, Yu K, Kapczynski DR - Virol. J. (2013)

Bottom Line: 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.CEH can support growth of the tested LPAIVs when with trypsin supplementation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Exotic and Emerging Avian Disease Research Unit, Southeast Poultry Research Laboratory, Agricultural Research Service, USDA, 934 College Station Road, Athens, GA 30605, Greece. darrell.kapczynski@ars.usda.gov.

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.

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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.
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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.

Mentions: To investigate the replication of different virus strains, CEHs were infected with H5N9, H5N3, H7N2 and H9N2 viruses at MOI of 1 and the viral titers in the supernatants were determined as log10EID50/ml. Comparison of the growth characteristics of H5N9, H5N3, H7N2 and H9N2 viruses in CEHs with or without trypsin supplementation in the medium after infection are shown in Figure 1. CEH cannot efficient support growth of the above viruses without supplemental trypsin in the medium, presumably because CEH cannot produce trypsin-like protease. But after with1 μg/ml trypsin supplementation in the medium after infection, viral titers increasing until 24 hpi, especially H7N2 virus has a significant increase. The viral titers for H5N9, H5N3, H7N2 and H9N2 at 24 hpi were 6.8, 6.8, 8.6, and 6.4 log10 EID50 per ml, respectively.


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

Jiang H, Yu K, Kapczynski DR - Virol. J. (2013)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225510&req=5

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.
Mentions: To investigate the replication of different virus strains, CEHs were infected with H5N9, H5N3, H7N2 and H9N2 viruses at MOI of 1 and the viral titers in the supernatants were determined as log10EID50/ml. Comparison of the growth characteristics of H5N9, H5N3, H7N2 and H9N2 viruses in CEHs with or without trypsin supplementation in the medium after infection are shown in Figure 1. CEH cannot efficient support growth of the above viruses without supplemental trypsin in the medium, presumably because CEH cannot produce trypsin-like protease. But after with1 μg/ml trypsin supplementation in the medium after infection, viral titers increasing until 24 hpi, especially H7N2 virus has a significant increase. The viral titers for H5N9, H5N3, H7N2 and H9N2 at 24 hpi were 6.8, 6.8, 8.6, and 6.4 log10 EID50 per ml, respectively.

Bottom Line: 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.CEH can support growth of the tested LPAIVs when with trypsin supplementation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Exotic and Emerging Avian Disease Research Unit, Southeast Poultry Research Laboratory, Agricultural Research Service, USDA, 934 College Station Road, Athens, GA 30605, Greece. darrell.kapczynski@ars.usda.gov.

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.

Show MeSH
Related in: MedlinePlus