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Influenza H5N1 and H1N1 virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation.

Chan RW, Yuen KM, Yu WC, Ho CC, Nicholls JM, Peiris JS, Chan MC - PLoS ONE (2010)

Bottom Line: We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of alpha2-6-linked sialic acid receptors and human airway trypsin-like (HAT) protease, in contrast to the low expression in the non-differentiated NHBE cells.The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells.Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the epithelium regeneration, the data generated from the undifferentiated NHBE cultures may also be relevant to disease pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong SAR, People's Republic of China.

ABSTRACT
Influenza H5N1 virus continues to be enzootic in poultry and transmits zoonotically to humans. Although a swine-origin H1N1 virus has emerged to become pandemic, its virulence for humans remains modest in comparison to that seen in zoonotic H5N1 disease. As human respiratory epithelium is the primary target cells for influenza viruses, elucidating the viral tropism and host innate immune responses of influenza H5N1 virus in human bronchial epithelium may help to understand the pathogenesis. Here we established primary culture of undifferentiated and well differentiated normal human bronchial epithelial (NHBE) cells and infected with highly pathogenic influenza H5N1 virus (A/Vietnam/3046/2004) and a seasonal influenza H1N1 virus (A/Hong Kong/54/1998), the viral replication kinetics and cytokine and chemokine responses were compared by qPCR and ELISA. We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of alpha2-6-linked sialic acid receptors and human airway trypsin-like (HAT) protease, in contrast to the low expression in the non-differentiated NHBE cells. When compared to H1N1 virus, the H5N1 virus replicated more efficiently and induced a stronger type I interferon response in the undifferentiated NHBE cells. In contrast, in well differentiated cultures, H5N1 virus replication was less efficient and elicited a lower interferon-beta response in comparison with H1N1 virus. Our data suggest that the differentiation of bronchial epithelial cells has a major influence in cells' permissiveness to human H1N1 and avian H5N1 viruses and the host innate immune responses. The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells. Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the epithelium regeneration, the data generated from the undifferentiated NHBE cultures may also be relevant to disease pathogenesis.

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Virus titer detected in the supernatant of influenza virus infected ud- and wd-NHBE cells.Virus titer of the (A) HK98/H1N1 and (B) VN04/H5N1 was determined after influenza viruses infected in the ud- and wd-NHBE cells from 1 h to 48 h post infection at MOI of 2. (C) The comparison of viral replication kinetic between influenza HK98/H1N1 and VN04/H5N1 viruses in ud- and wd-NHBE at 24 h post infection. The chart showed the mean and the standard error of the virus titer pooled from three independent experiments. Single asterisk indicated statistically significant difference of means with p<0.05, double asterisks indicated statistically significant differences of means with p<0.01. Dotted line represents the detection limit of the TCID50 assay.
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pone-0008713-g005: Virus titer detected in the supernatant of influenza virus infected ud- and wd-NHBE cells.Virus titer of the (A) HK98/H1N1 and (B) VN04/H5N1 was determined after influenza viruses infected in the ud- and wd-NHBE cells from 1 h to 48 h post infection at MOI of 2. (C) The comparison of viral replication kinetic between influenza HK98/H1N1 and VN04/H5N1 viruses in ud- and wd-NHBE at 24 h post infection. The chart showed the mean and the standard error of the virus titer pooled from three independent experiments. Single asterisk indicated statistically significant difference of means with p<0.05, double asterisks indicated statistically significant differences of means with p<0.01. Dotted line represents the detection limit of the TCID50 assay.

Mentions: Infectious virus yield (TCID50) assay was quantitated in the supernatants of HK98/H1N1 and VN04/H5N1 infected ud- and wd-NHBE cell cultures to compare infectious virus yields and the replication kinetics. There was evidence of productive viral replication in HK98/H1N1 infected ud-NHBE and wd-NHBE cells (Figure 5A). The virus titers peaked higher (p = 0.04) and earlier in wd-NHBE compared to ud-NHBE cell cultures. VN04/H5N1 titers in ud-NHBE cells peaked at 24 h post infection with a significant increase in the virus titer (p = 0.006) while there was no convincing evidence of virus replication in VN04/H5N1 virus infected wd-NHBE cells (Figure 5B). Furthermore, at 24 h post infection, the titers of virus in VN04/H5N1 infected ud-NHBE was significantly higher (p = 0.005) than in wd-NHBE.


Influenza H5N1 and H1N1 virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation.

Chan RW, Yuen KM, Yu WC, Ho CC, Nicholls JM, Peiris JS, Chan MC - PLoS ONE (2010)

Virus titer detected in the supernatant of influenza virus infected ud- and wd-NHBE cells.Virus titer of the (A) HK98/H1N1 and (B) VN04/H5N1 was determined after influenza viruses infected in the ud- and wd-NHBE cells from 1 h to 48 h post infection at MOI of 2. (C) The comparison of viral replication kinetic between influenza HK98/H1N1 and VN04/H5N1 viruses in ud- and wd-NHBE at 24 h post infection. The chart showed the mean and the standard error of the virus titer pooled from three independent experiments. Single asterisk indicated statistically significant difference of means with p<0.05, double asterisks indicated statistically significant differences of means with p<0.01. Dotted line represents the detection limit of the TCID50 assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806912&req=5

pone-0008713-g005: Virus titer detected in the supernatant of influenza virus infected ud- and wd-NHBE cells.Virus titer of the (A) HK98/H1N1 and (B) VN04/H5N1 was determined after influenza viruses infected in the ud- and wd-NHBE cells from 1 h to 48 h post infection at MOI of 2. (C) The comparison of viral replication kinetic between influenza HK98/H1N1 and VN04/H5N1 viruses in ud- and wd-NHBE at 24 h post infection. The chart showed the mean and the standard error of the virus titer pooled from three independent experiments. Single asterisk indicated statistically significant difference of means with p<0.05, double asterisks indicated statistically significant differences of means with p<0.01. Dotted line represents the detection limit of the TCID50 assay.
Mentions: Infectious virus yield (TCID50) assay was quantitated in the supernatants of HK98/H1N1 and VN04/H5N1 infected ud- and wd-NHBE cell cultures to compare infectious virus yields and the replication kinetics. There was evidence of productive viral replication in HK98/H1N1 infected ud-NHBE and wd-NHBE cells (Figure 5A). The virus titers peaked higher (p = 0.04) and earlier in wd-NHBE compared to ud-NHBE cell cultures. VN04/H5N1 titers in ud-NHBE cells peaked at 24 h post infection with a significant increase in the virus titer (p = 0.006) while there was no convincing evidence of virus replication in VN04/H5N1 virus infected wd-NHBE cells (Figure 5B). Furthermore, at 24 h post infection, the titers of virus in VN04/H5N1 infected ud-NHBE was significantly higher (p = 0.005) than in wd-NHBE.

Bottom Line: We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of alpha2-6-linked sialic acid receptors and human airway trypsin-like (HAT) protease, in contrast to the low expression in the non-differentiated NHBE cells.The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells.Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the epithelium regeneration, the data generated from the undifferentiated NHBE cultures may also be relevant to disease pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong SAR, People's Republic of China.

ABSTRACT
Influenza H5N1 virus continues to be enzootic in poultry and transmits zoonotically to humans. Although a swine-origin H1N1 virus has emerged to become pandemic, its virulence for humans remains modest in comparison to that seen in zoonotic H5N1 disease. As human respiratory epithelium is the primary target cells for influenza viruses, elucidating the viral tropism and host innate immune responses of influenza H5N1 virus in human bronchial epithelium may help to understand the pathogenesis. Here we established primary culture of undifferentiated and well differentiated normal human bronchial epithelial (NHBE) cells and infected with highly pathogenic influenza H5N1 virus (A/Vietnam/3046/2004) and a seasonal influenza H1N1 virus (A/Hong Kong/54/1998), the viral replication kinetics and cytokine and chemokine responses were compared by qPCR and ELISA. We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of alpha2-6-linked sialic acid receptors and human airway trypsin-like (HAT) protease, in contrast to the low expression in the non-differentiated NHBE cells. When compared to H1N1 virus, the H5N1 virus replicated more efficiently and induced a stronger type I interferon response in the undifferentiated NHBE cells. In contrast, in well differentiated cultures, H5N1 virus replication was less efficient and elicited a lower interferon-beta response in comparison with H1N1 virus. Our data suggest that the differentiation of bronchial epithelial cells has a major influence in cells' permissiveness to human H1N1 and avian H5N1 viruses and the host innate immune responses. The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells. Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the epithelium regeneration, the data generated from the undifferentiated NHBE cultures may also be relevant to disease pathogenesis.

Show MeSH
Related in: MedlinePlus