Project description:Cultures of primary human airway epithelial cells (HAE cells) were exposed to an MDCK equivalent MOI of 0.01 of several swine- and human-origin influenza viruses and RNA was extracted at the 12, 16, and 24 hours post infection. 64 samples consiting of 5 influenza strains, 3 time points and 2 or 3 replicates at each time point. Expression of mock-infected samples measured at each time point.
Project description:Cultures of primary human airway epithelial cells (HAE cells) were exposed to an MDCK equivalent MOI of 0.01 of several swine- and human-origin influenza viruses and RNA was extracted at the 12, 16, and 24 hours post infection.
Project description:Influenza viruses pose a significant burden on global human health. Influenza has a broad cellular tropism in the airway, but how infection of different epithelial cell types impacts replication kinetics and burden in the airways is not fully understood. Using primary human airway cultures, which recapitulate the diverse epithelial cell landscape of the human airways, we investigated the impact of cell type composition on virus tropism and replication kinetics. Cultures were highly diverse across multiple donors and 30 independent differentiation conditions and supported a range of influenza replication. Although many cell types were susceptible to influenza, ciliated and secretory cells were predominantly infected. Despite the strong tropism preference for secretory and ciliated cells, which consistently make up 75% or more of infected cells, only ciliated cells were associated with increased virus production. Surprisingly, infected secretory cells were associated with overall reduced virus output. These data highlight the heterogeneous outcomes of influenza virus infections in primary human airway cultures and the disparate impacts of infected cell identity on burst size, even among preferentially infected cell types.
Project description:Influenza viruses pose a significant burden on global human health. Influenza has a broad cellular tropism in the airway, but how infection of different epithelial cell types impacts replication kinetics and burden in the airways is not fully understood. Using primary human airway cultures, which recapitulate the diverse epithelial cell landscape of the human airways, we investigated the impact of cell type composition on virus tropism and replication kinetics. Cultures were highly diverse across multiple donors and 30 independent differentiation conditions and supported a range of influenza replication. Although many cell types were susceptible to influenza, ciliated and secretory cells were predominantly infected. Despite the strong tropism preference for secretory and ciliated cells, which consistently make up 75% or more of infected cells, only ciliated cells were associated with increased virus production. Surprisingly, infected secretory cells were associated with overall reduced virus output. These data highlight the heterogeneous outcomes of influenza virus infections in primary human airway cultures and the disparate impacts of infected cell identity on burst size, even among preferentially infected cell types.
Project description:To investigate how human airway epithelial cells respond to Influenza or RSV infection, we harvested airway epithelial cells from the mainstream bronchi of human donors and cultured them as previously described (Pickles et al,1998) in a polarized system that resembles the in vivo mucociliary pseudostratified epithelium. Quadruplicate hAEC cultures were infected with 2X105 PFUs Influenza A (Udorn) or with 1x106 PFUs RSV for 2h or mock inoculated and harvested 24h after Influenza infection and 48h after RSV infection. Quadruplicate polarized airway epithelial cell cultures were mock treated or infected with 2x10^5 PFUs of Influenza A (Udorn) for 2h or infected with 1x10^6 PFUs RSV and harvested 24 h post infection for Influenza or 48h post infection for RSV. Total RNA was harvested and gene expression was studied using Genespring GX v7.3.1.
Project description:To investigate how human airway epithelial cells respond to Influenza or RSV infection, we harvested airway epithelial cells from the mainstream bronchi of human donors and cultured them as previously described (Pickles et al,1998) in a polarized system that resembles the in vivo mucociliary pseudostratified epithelium. Quadruplicate hAEC cultures were infected with 2X105 PFUs Influenza A (Udorn) or with 1x106 PFUs RSV for 2h or mock inoculated and harvested 24h after Influenza infection and 48h after RSV infection. Quadruplicate polarized airway epithelial cell cultures were infected with 2x10^5 PFUs of Influenza A (Udorn) for 2h or infected with 1x10^6 PFUs RSV and harvested 24 h post infection for Influenza or 48h post infection for RSV.Duplicate cultures were used as controls for each condition (Two cultures were mock treated mor 2h and harvested after 24h for the Influenza infection and 2 cultures were mock treated for 2h and harvested after 48 hours for the RSV infection.Total RNA was harvested and gene expression was studied using Genespring GX v7.3.1.
Project description:In recent years, the roles of microRNAs playing in the regulation of influenza viruses replication caused researchers' much attenion. However, much work focused on the interactions between human, mice or chicken microRNAs with human or avian influenza viruses rather than the interactions of swine microRNAs and swine influenza viruses. To investigate the roles of swine microRNAs playing in the regulation of swine influenza A virus replication, the microRNA microarray was performed to identify which swine microRNAs were involved in swine H1N1/2009 influenza A virus infection.
Project description:The determinants of influenza transmission remain poorly understood. Swine influenza viruses preferentially attach to receptors found in the upper airways; however, most swine influenza viruses fail to transmit efficiently from swine to humans, and from human-to-human. The pandemic 2009 H1N1 (H1N1pdm) virus was a rare exception of a swine virus that acquired efficient transmissibility from human-to-human, and is reflected in efficient respiratory droplet transmission in ferrets. We hypothesize that virus-induced host responses in the upper airways correlate with airborne transmission in ferrets. To address this question, we used the H1N1pdm virus and swine influenza A/swine/Hong Kong/201/2010 (HK201) virus that has comparable titre in the ferret nasopharynx, but it exhibits differential transmissibility in ferrets via respiratory droplet route. We performed a transcriptomic analysis of tissues from the upper and lower respiratory tract from ferrets infected with either H1N1pdm or HK201 viruses using ferret-specific Agilent oligonucleotide arrays. We found differences in the kinetics of the innate immune response elicited by these two viruses that varied across tissues.
Project description:To investigate how human airway epithelial cells respond to Influenza or RSV infection, we harvested airway epithelial cells from the mainstream bronchi of human donors and cultured them as previously described (Pickles et al,1998) in a polarized system that resembles the in vivo mucociliary pseudostratified epithelium. Quadruplicate hAEC cultures were infected with 2X105 PFUs Influenza A (Udorn) or with 1x106 PFUs RSV for 2h or mock inoculated and harvested 24h after Influenza infection and 48h after RSV infection.
Project description:To investigate how human airway epithelial cells respond to Influenza or RSV infection, we harvested airway epithelial cells from the mainstream bronchi of human donors and cultured them as previously described (Pickles et al,1998) in a polarized system that resembles the in vivo mucociliary pseudostratified epithelium. Quadruplicate hAEC cultures were infected with 2X105 PFUs Influenza A (Udorn) or with 1x106 PFUs RSV for 2h or mock inoculated and harvested 24h after Influenza infection and 48h after RSV infection.