A humanized MDCK cell line for the efficient isolation and propagation of human influenza viruses

Takada, Kenjiro; Kawakami, Chiharu; Fan, Shufang; Chiba, Shiho; Zhong, Gongxun; Gu, Chunyang; Shimizu, Kohei; Takasaki, S; Sakai‐Tagawa, Yuko; Lopes, Tiago J. S.; Dutta, Jayeeta; Khan, Zenab; Kriti, Divya; Bakel, Harm van; Yamada, S.; Watanabe, Tokiko; Imai, Masaki; Kawaoka, Yoshihiro

doi:10.1038/s41564-019-0433-6

Cited by 82 publications

(105 citation statements)

References 31 publications

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“…While these cells infect easily and provide a rapid early assessment of the potential efficacy of a therapeutic for IAV, they lack the critical mechanisms and signaling pathways that healthy human airway tissues provide in response to viral infection. Therefore, although these systems are easy to use, they are not often accurate predictors of human clinical efficacy [11]. For coronaviruses, Calu-3 or Vero E6 cells are often used for similar reasons, but again differ by species, cell source, disease state, and innate antiviral signaling pathways [12,13] compared to normal, healthy human conducting airway and alveolar epithelium [14], thus providing an insufficient model.…”

Section: Introductionmentioning

confidence: 99%

High-Throughput Human Primary Cell-Based Airway Model for Evaluating Influenza, Coronavirus, or other Respiratory Virusesin vitro

Gard

Maloney

Cain

et al. 2020

Preprint

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Influenza and other respiratory viruses represent a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as the current COVID-19 crisis. One of the greatest barriers to the development of effective therapeutic agents to treat influenza, coronaviruses, and many other infections of the respiratory tract is the absence of a robust preclinical model. Preclinical studies currently rely on high-throughput, low-fidelity in vitro screening with cell lines and/or low-throughput animal models that often provide a poor correlation to human clinical responses. Here, we introduce a human primary airway epithelial cell-based model integrated into a highthroughput platform where tissues are cultured at an air-liquid interface (PREDICT96-ALI). We present results on the application of this platform to influenza and coronavirus infections, providing multiple readouts capable of evaluating viral infection kinetics and potentially the efficacy of therapeutic agents in an in vitro system. Several strains of influenza A virus are shown to successfully infect the human primary cell-based airway tissue cultured at an air-liquid interface (ALI), and as a proof-of-concept, the effect of the antiviral oseltamivir on one strain of Influenza A is evaluated. Human coronaviruses NL63 (HCoV-NL63) and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for protein priming, and we confirm expression of both in our ALI model. We also demonstrate coronavirus infection in this system with HCoV-NL63, observing sufficient viral propagation over 96 hours post-infection to indicate successful infection of the primary cell-based model. This new capability has the potential to address a gap in the rapid assessment of therapeutic efficacy of various small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

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Section: Introductionmentioning

confidence: 99%

High-Throughput Human Primary Cell-Based Airway Model for Evaluating Influenza, Coronavirus, or other Respiratory Virusesin vitro

Gard

Maloney

Cain

et al. 2020

Preprint

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“…Baloxavir susceptibilities were determined by using a focus reduction assay as previously described [ 1 ] in humanized MDCK cells (i.e., hCK cells), which express high levels of α2,6-sialoglycans and very low levels of α2,3-sialoglycans [ 15 ]. hCK cells were kindly provided by Dr. Yoshihiro Kawaoka (University of Wisconsin–Madison).…”

Section: Methodsmentioning

confidence: 99%

In Vitro Characterization of Multidrug-Resistant Influenza A(H1N1)pdm09 Viruses Carrying a Dual Neuraminidase Mutation Isolated from Immunocompromised Patients

et al. 2020

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Influenza A(H1N1)pdm09 viruses carrying a dual neuraminidase (NA) substitution were isolated from immunocompromised patients after administration of one or more NA inhibitors. These mutant viruses possessed an H275Y/I223R, H275Y/I223K, or H275Y/G147R substitution in their NA and showed enhanced cross-resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir compared to single H275Y mutant viruses. Baloxavir could be a treatment option against the multidrug-resistant viruses because these dual H275Y mutant viruses showed susceptibility to this drug. The G147R substitution appears to stabilize the NA structure, with the fitness of the H275Y/G147R mutant virus being similar or somewhat better than that of the wild-type virus. Since the multidrug-resistant viruses may be able to transmit between humans, surveillance of these viruses must continue to improve clinical management and to protect public health.

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“…hCK cells were maintained in the presence of 2 µg/mL puromycin and 10 µg/mL blasticidin in MEM containing 5% NCS. 9 Human embryonic kidney 293T cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. The cells 108-8639, Japan.…”

Section: Cellsmentioning

confidence: 99%

Antigenic variants of influenza B viruses isolated in Japan during the 2017‐2018 and 2018‐2019 influenza seasons

Kato‐Miyashita

Sakai‐Tagawa

Yamashita

et al. 2020

Influenza Resp Viruses

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Background: Here, we genetically and antigenically analyzed influenza B viruses (IBVs) isolated in Japan during the 2017-2018 and 2018-2019 influenza seasons. Methods: A total of 68 IBVs (61 B/Yamagata/16/88-like [B/Yamagata]-lineage and 7 B/Victoria/2/87-like [B/Victoria]-lineage) were antigenically and genetically characterized by using hemagglutination inhibition (HI) assays and phylogenetic analysis, respectively. The susceptibility of IBVs to neuraminidase (NA) inhibitors was assessed by using a fluorescence-based NA inhibition assay. Results: All 61 B/Yamagata-lineage isolates were genetically closely related to B/ Phuket/3073/2013, the vaccine strain for these two seasons. Eleven B/Yamagatalineage isolates tested were antigenically similar to B/Phuket/3073/2013 by the HI test. Seven B/Victoria-lineage isolates were genetically closely related to B/ Texas/02/2013, the WHO-recommended vaccine strain for the 2017-2018 season;however, they were antigenically distinct from B/Texas/02/2013 with an eightfold or 16-fold difference in HI titer. Of these 7 isolates, 4 possessed a two-aminoacid deletion at positions 162 and 163 in hemagglutinin (HA) and the other 3 had a three-amino-acid deletion at positions 162-164 in HA. Importantly, the variants with the three-amino-acid deletion appeared to be antigenically different from the B/Colorado/06/2017 virus with the two-amino-acid deletion, the vaccine strain for the 2018-2019 season with a fourfold or eightfold difference in HI titer. One B/ Yamagata-lineage isolate carrying a G407S mutation in its NA showed a marked reduction in susceptibility to zanamivir, peramivir, and laninamivir. Conclusions:These results highlight the need for continued monitoring for the prevalence of the antigenic variant with the three-amino-acid deletion and the variant with reduced NA inhibitor susceptibility.

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A humanized MDCK cell line for the efficient isolation and propagation of human influenza viruses

Cited by 82 publications

References 31 publications

High-Throughput Human Primary Cell-Based Airway Model for Evaluating Influenza, Coronavirus, or other Respiratory Virusesin vitro

High-Throughput Human Primary Cell-Based Airway Model for Evaluating Influenza, Coronavirus, or other Respiratory Virusesin vitro

In Vitro Characterization of Multidrug-Resistant Influenza A(H1N1)pdm09 Viruses Carrying a Dual Neuraminidase Mutation Isolated from Immunocompromised Patients

Antigenic variants of influenza B viruses isolated in Japan during the 2017‐2018 and 2018‐2019 influenza seasons

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