Tropism and Infectivity of Influenza Virus, Including Highly Pathogenic Avian H5N1 Virus, in Ferret Tracheal Differentiated Primary Epithelial Cell Cultures

Abstract

Tropism and adaptation of influenza viruses to new hosts is partly dependent on the distribution of the sialic acid (SA) receptors to which the viral hemagglutinin (HA) binds. Ferrets have been established as a valuable in vivo model of influenza virus pathogenesis and transmission because of similarities to humans in the distribution of HA receptors and in clinical signs of infection. In this study, we developed a ferret tracheal differentiated primary epithelial cell culture model that consisted of a layered epithelium structure with ciliated and nonciliated cells on its apical surface. We found that human-like (␣2,6-linked) receptors predominated on ciliated cells, whereas avian-like (␣2,3-linked) receptors, which were less abundant, were presented on nonciliated cells. When we compared the tropism and infectivity of three human (H1 and H3) and two avian (H1 and H5) influenza viruses, we observed that the human influenza viruses primarily infected ciliated cells and replicated efficiently, whereas a highly pathogenic avian H5N1 virus (A/Vietnam/1203/2004) replicated efficiently within nonciliated cells despite a low initial infection rate. Furthermore, compared to other influenza viruses tested, VN/1203 virus replicated more efficiently in cells isolated from the lower trachea and at a higher temperature (37°C) compared to a lower temperature (33°C). VN/1203 virus infection also induced higher levels of immune mediator genes and cell death, and virus was recovered from the basolateral side of the cell monolayer. This ferret tracheal differentiated primary epithelial cell culture system provides a valuable in vitro model for studying cellular tropism, infectivity, and the pathogenesis of influenza viruses.

A Novel A(H7N2) Influenza Virus Isolated from a Veterinarian Caring for Cats in a New York City Animal Shelter Causes Mild Disease and Transmits Poorly in the Ferret Model

“…3) is in agreement with the findings of a prior study showing enhanced H5N1 virus attachment to feline type II pneumocytes in the lower respiratory tract but not to tracheal tissue (54). Thus, the reduced infectivity of NY/108 virus but not NY/107 virus in cultures of FTECs (which possess a greater density of ␣2-6-linked than ␣2-3-linked SA [31]) and the reduced transmissibility of NY/108 virus compared with that of NY/107 virus among ferrets (18) suggest that differences between the HAs of these two LPAI H7N2 viruses may be present. One potential change may be an Ala135Ser substitution in NY/108 virus HA that predicts a 4th N-glycosylation site at position 133, which is not present in NY/107 virus, Tky/VA virus, or the great majority of sequenced North American avian viruses.…”

“…Cells of the human bronchial epithelial cell line Calu-3 (ATCC) were grown on 12-well membrane inserts and cultured as previously described (28). Ferret differentiated primary tracheal epithelial cells (FTECs) were isolated and cultured as previously described (31). Calu-3 cells were cultured under submerged conditions, and ferret primary cells were cultured under air-liquid interface (ALI) conditions.…”

Ferret airway epithelial cell cultures support efficient replication of influenza B virus but not mumps virus

“…3a, b; 4a, b). We confirmed the observations of Zeng et al (2013), that the ciliated cell type in FTE cultures abundantly expressed a2,6 SA, and showed that these cells were the targets of influenza B/Florida/04/2006 (Yamagata lineage) virus infection. Recent reports have described lineage specific SA binding preferences (the Yamagata-like lineage displaying a a2,6 linkage preference and the Victoria-like viruses an a2,6 and a2,3 binding affinity) (Wang et al, 2012).…”

Viral–bacterial interactions in the respiratory tract

“…Death of airway epithelial cells due to viral replication, excessive inflammation and/or loss of repair functions can damage the integrity of the airway epithelium (Herold et al, 2008;Hinshaw et al, 1994;Kash et al, 2011;Schultz-Cherry et al, 2001;Zeng et al, 2013). One consequence of this compromised natural barrier can be invasive bacterial infection.…”