Tropism and innate host responses of the 2009 pandemic H1N1 influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract

Abstract

The novel pandemic influenza H1N1 (H1N1pdm) virus of swine origin causes mild disease but occasionally leads to acute respiratory distress syndrome and death. It is important to understand the pathogenesis of this new disease in humans. We compared the virus tropism and host-responses elicited by pandemic H1N1pdm and seasonal H1N1 influenza viruses in ex vivo cultures of human conjunctiva, nasopharynx, bronchus, and lung, as well as in vitro cultures of human nasopharyngeal, bronchial, and alveolar epithelial cells. We found comparable replication and host-responses in seasonal and pandemic H1N1 viruses. However, pandemic H1N1pdm virus differs from seasonal H1N1 influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in its receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection with H1N1pdm. A greater viral replication competence in bronchial epithelium at 33 degrees C may also contribute to the slight increase in virulence of the pandemic influenza virus. In contrast with highly pathogenic influenza H5N1 virus, pandemic H1N1pdm does not differ from seasonal influenza virus in its intrinsic capacity for cytokine dysregulation. Collectively, these results suggest that pandemic H1N1pdm virus differs in modest but subtle ways from seasonal H1N1 virus in its intrinsic virulence for humans, which is in accord with the epidemiology of the pandemic to date. These findings are therefore relevant for understanding transmission and therapy.

Figure 1

Ex vivo organ cultures of conjunctiva infected with (A) VN04/H5N1, (B) NL03/H7N7, and (C) HK09/H1N1pdm at 24 hours postinfection showing positive staining for influenza A nucleoprotein (reddish brown with arrow). Negative staining for influenza A nucleoprotein in (D) HK98/H1N1, (E) OK05/H3N2, and (F) HK09/H1N1 infected conjunctival tissues (G). Conjunctival biopsies infected with 10⁶ TCID₅₀/ml of influenza VN04/H5N1, HK09/H1N1pdm, and NL03/H7N7 viruses demonstrate an increase in viral yield, whereas HK98/98 and OK05/H3N2 demonstrate no productive replication. The chart shows the mean and the SE of the virus titer pooled from three independent experiments. *P < 0.05, **P < 0.01. Horizontal dotted line denotes detection limit of TCID₅₀ assay.

Figure 2

Distribution of Sia on conjunctival tissue ex vivo. Sia in α2-6 linkages were stained using SNA, Sia in α2-3 linkages were stained using MAA, and the Galβ1-3GalNAc were stained using PNA. The conjunctival tissues show negative/weak positive binding of SNA on the conjunctival epithelium (A), whereas there is a strong positive binding of the conjunctival epithelium for MAA (B) and a weak PNA staining (C). The goblet cells of the conjunctival epithelium (B and C) show a strongly positive intracytoplasmic binding for both MAA and PNA (arrow). AEC stain with hematoxylin counterstain.

Figure 3

Human conjunctival tissues were incubated with sialidase, DAS181 (100 U/ml) for 2 hours at 37°C and fixed. Sia in α2-6 linkages (A) was stained in red using SNA, Sia in α2-3 linkages (B) was stained using MAA, and Galβ1-3GalNAc (C), which is exposed after the desialyation, was stained using PNA. D: Viral replication in conjunctival biopsies infected with 10⁶ TCID₅₀/ml of influenza viruses (VN04/H5N1 and HK09/H1N1pdm) with and without DAS181 treatment by TCID₅₀ assay. The chart shows the mean and the SE of the virus titer pooled from three independent experiments. **P < 0.01.

Figure 4

Cellular localization of ex vivo infection of the upper and lower respiratory tract by influenza A viruses (A and B) nasopharynx, (D and E) bronchi, and (G and H) lung infected with influenza HK09/H1N1pdm and HK98/H1N1 viruses, showing positive staining for influenza A nucleoprotein (reddish brown). Viral replication kinetics in ex vivo cultures of (C) nasopharynx, (F) bronchi, and (I) lung biopsies infected with 10⁶ TCID₅₀/ml of influenza viruses by virus titration. The chart showed the mean and the SE of mean of the virus titer pooled from three independent experiments.

Figure 5

Replication kinetics of influenza A viruses in respiratory epithelial cells in vitro by virus titration expressed in TCID₅₀/ml, (A) NPE cells cultured at 33°C, and (C) NPE cells cultured at 37°C; (B) NHBE cells cultured at 33°C and (D) NHBE cultured at 37°C; (E) Type-I like pneumocytes cultured at 37°C. The chart showed the mean and the SE of mean of the virus titer pooled from three independent experiments. *P < 0.05, **P < 0.01.

Figure 6

Cytokine and chemokine gene and protein expression in NHBE after influenza virus infection. The cytokine (A) IFN-β, (B) IL-6, and chemokine (C) RANTES, (D) IP-10, and (E) influenza matrix (M) gene expression from NHBE infected with influenza A viruses at 6 and 24 hours postinfection. Cytokine and chemokine protein secretion (F) IL-6, (G) RANTES, and (H) IP-10 at 24 hours post influenza A virus infection. The graph shows the mean and the SE of mean from three representative experiments. *P < 0.05, ***P < 0.001. Horizontal dotted line denotes detection limit of ELISA assay.

Figure 7

Cytokine and chemokine gene and protein expression in alveolar type I–like pneumocytes after influenza A virus infection. The cytokine (A) IFN-β, (B) IL-6, and chemokine (C) RANTES, (D) IP-10, and (E) influenza matrix (M) gene expression from alveolar type I–like pneumocytes infected with influenza A viruses at 8 hours postinfection. Cytokine and chemokine protein secretion, (F) IL-6, (G) RANTES, and (H) IP-10 at 8 hours postinfection of the pandemic influenza H1N1pdm and seasonal influenza H1N1 viruses. For comparison, the cytokine proteins secretion inducted by the infection of highly pathogenic influenza H5N1 virus in alveolar type I–like pneumocytes are: IL-6 (602 ± 21 pg/ml), RANTES (526 ± 75 pg/ml), and IP-10 (1313 ± 161 pg/ml). The graph shows the mean and the SE of mean from three representative experiments. *P < 0.05.