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Comparative study of influenza virus replication in MDCK cells and in primary cells derived from adenoids and airway epithelium.

ABSTRACT: Although clinical trials with human subjects are essential for determination of safety, infectivity, and immunogenicity, it is desirable to know in advance the infectiousness of potential candidate live attenuated influenza vaccine strains for human use. We compared the replication kinetics of wild-type and live attenuated influenza viruses, including H1N1, H3N2, H9N2, and B strains, in Madin-Darby canine kidney (MDCK) cells, primary epithelial cells derived from human adenoids, and human bronchial epithelium (NHBE cells). Our data showed that despite the fact that all tissue culture models lack a functional adaptive immune system, differentiated cultures of human epithelium exhibited the greatest restriction for all H1N1, H3N2, and B vaccine viruses studied among three cell types tested and the best correlation with their levels of attenuation seen in clinical trials with humans. In contrast, the data obtained with MDCK cells were the least predictive of restricted viral replication of live attenuated vaccine viruses in humans. We were able to detect a statistically significant difference between the replication abilities of the U.S. (A/Ann Arbor/6/60) and Russian (A/Leningrad/134/17/57) cold-adapted vaccine donor strains in NHBE cultures. Since live attenuated pandemic influenza vaccines may potentially express a hemagglutinin and neuraminidase from a non-human influenza virus, we assessed which of the three cell cultures could be used to optimally evaluate the infectivity and cellular tropism of viruses derived from different hosts. Among the three cell types tested, NHBE cultures most adequately reflected the infectivity and cellular tropism of influenza virus strains with different receptor specificities. NHBE cultures could be considered for use as a screening step for evaluating the restricted replication of influenza vaccine candidates.

SUBMITTER: Ilyushina NA

PROVIDER: S-EPMC3486302 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Comparative study of influenza virus replication in MDCK cells and in primary cells derived from adenoids and airway epithelium.

Ilyushina Natalia A NA Ikizler Mine R MR Kawaoka Yoshihiro Y Rudenko Larisa G LG Treanor John J JJ Subbarao Kanta K Wright Peter F PF

Journal of virology 20120822 21

Although clinical trials with human subjects are essential for determination of safety, infectivity, and immunogenicity, it is desirable to know in advance the infectiousness of potential candidate live attenuated influenza vaccine strains for human use. We compared the replication kinetics of wild-type and live attenuated influenza viruses, including H1N1, H3N2, H9N2, and B strains, in Madin-Darby canine kidney (MDCK) cells, primary epithelial cells derived from human adenoids, and human bronch ...[more]

PMID: 22915797

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Project description:Background Bronchioalveolar stem cells (BASCs) located at the bronchioalveolar-duct junction (BADJ) are stem cells residing in alveoli and terminal bronchioles that can self-renew and differentiate into alveolar type (AT)-1 cells, AT-2 cells, club cells, and ciliated cells. Following terminal-bronchiole injury, BASCs increase in number and promote repair. However, whether BASCs can be differentiated from mouse-induced pluripotent stem cells (iPSCs) remains unreported, and the therapeutic potential of such cells is unclear. We therefore sought to differentiate BASCs from iPSCs and examine their potential for use in the treatment of epithelial injury in terminal bronchioles. Methods BASCs were induced using a modified protocol for differentiating mouse iPSCs into AT-2 cells. Differentiated iPSCs were intratracheally transplanted into naphthalene-treated mice. The engraftment of BASCs into the BADJ and their subsequent ability to promote repair of injury to the airway epithelium were evaluated. Results Flow cytometric analysis revealed that BASCs represented ~?7% of the cells obtained. Additionally, ultrastructural analysis of these iPSC-derived BASCs via transmission electron microscopy showed that the cells containing secretory granules harboured microvilli, as well as small and immature lamellar body-like structures. When the differentiated iPSCs were intratracheally transplanted in naphthalene-induced airway epithelium injury, transplanted BASCs were found to be engrafted in the BADJ epithelium and alveolar spaces for 14 days after transplantation and to maintain the BASC phenotype. Notably, repair of the terminal-bronchiole epithelium was markedly promoted after transplantation of the differentiated iPSCs. Conclusions Mouse iPSCs could be differentiated in vitro into cells that display a similar phenotype to BASCs. Given that the differentiated iPSCs promoted epithelial repair in the mouse model of naphthalene-induced airway epithelium injury, this method may serve as a basis for the development of treatments for terminal-bronchiole/alveolar-region disorders.

Project description:Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37 degrees C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32 degrees C). These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40 degrees C), rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32 degrees C and 37 degrees C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32 degrees C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2) or A/PR/8/34 (H1N1) genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA) and neuraminidase (NA) from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and suggests that adaptation of avian influenza viruses to efficient infection at 32 degrees C may represent a critical evolutionary step enabling human-to-human transmission.

Dataset Information

Comparative study of influenza virus replication in MDCK cells and in primary cells derived from adenoids and airway epithelium.

Publications

Comparative study of influenza virus replication in MDCK cells and in primary cells derived from adenoids and airway epithelium.

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