Human Bocavirus 1 Infection of Well‐Differentiated Human Airway Epithelium

Yan, Ziying; Deng, Xuefeng; Qiu, Jianming

doi:10.1002/cpmc.107

“…Transepithelial electrical resistance (TEER). One hundred microliters of D-PBS was added to the apical chamber to determine the TEER using a Millicell ERS-2 volt-ohm meter (MilliporeSigma, Burlington, MA) following a previously used method (58).…”

Section: Methodsmentioning

confidence: 99%

“…The freshly isolated human bronchial epithelial cells from the donor tissues were seeded onto collagen-coated, semipermeable polycarbonate membrane inserts (0.33 cm 2 , 0.4-μm pore size, Costar Transwell, catalog no. 3413, Corning, New York), and grown at an ALI as previously described ( 58 ). The cultures were maintained in USG medium containing 2% Ultroser G (USG) serum substitute (Pall BioSepra, France).…”

Section: Methodsmentioning

confidence: 99%

Long-Term Modeling of SARS-CoV-2 Infection of In Vitro Cultured Polarized Human Airway Epithelium

Hao

¹

,

Ning

²

,

Küz

³

et al. 2020

mBio

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. Prior studies characterized only short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7 to 10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detected. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of >2.5 × 105 virions per cm2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2. IMPORTANCE The pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to >35 million confirmed cases and >1 million fatalities worldwide. SARS-CoV-2 mainly replicates in human airway epithelia in COVID-19 patients. In this study, we used in vitro cultures of polarized human bronchial airway epithelium to model SARS-CoV-2 replication for a period of 21 to 51 days. We discovered that in vitro airway epithelial cultures endure a long-lasting SARS-CoV-2 propagation with recurrent peaks of progeny virus release at an interval of approximately 7 to 10 days. Our study also revealed that SARS-CoV-2 infection causes airway epithelia damage with disruption of tight junction function and loss of cilia. Importantly, SARS-CoV-2 exhibits a polarity of infection in airway epithelium only from the apical membrane; it infects ciliated and goblet cells but not basal and club cells. Furthermore, the productive infection of SARS-CoV-2 requires a high viral load of over 2.5 × 105 virions per cm2 of epithelium. Our study highlights that the proliferation of airway basal cells and regeneration of airway epithelium may contribute to the recurrent infections.

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“…These polarized HAE-ALI cultures were derived from three independent donors. The freshly isolated human bronchial epithelial cells from the donor tissues were seeded onto collagencoated, semipermeable polycarbonate membrane inserts (0.33 cm 2 , 0.4 μm pore size, Costar Transwell ® , Cat #3413, Corning, New York), and grown at an ALI as previously described 60 .…”

Section: A)mentioning

confidence: 99%

“…100 µl of D-PBS was added to the apical chamber to determine the TEER using a Millicell ERS-2 Voltohmmeter (MilliporeSigma, Burlington, MA) following a previously used method 60 .…”

Section: Transepithelial Electrical Resistance (Teer)mentioning

confidence: 99%

Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

Hao

¹

,

Ning

²

,

Küz

³

et al. 2020

Preprint

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. However, previous studies only characterized short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7-10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detectable. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed Zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of 2.5 × 105 virions per cm2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.

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“…Notably, HEK293 cells do not permit HBoV1 infection. The purified virions are infectious to polarized primary human airway epithelia ( Dijkman et al, 2009 ; Huang et al, 2012 ; Yan et al, 2020 ), which mimics virus infection of the natural host, the human airway epithelia1. HBoV1 directly infects primary human airway epithelia cultured at an air-liquid interface (HAE-ALI) through both the apical and the basolateral surfaces, indicating that the virus receptor is expressed on both the apical (ciliated) and the basal cells ( Figure 6 , Steps 1, 2).…”

Section: Hbov1 Life Cyclementioning

confidence: 99%

Recent Advances in Molecular Biology of Human Bocavirus 1 and Its Applications

Shao

¹

,

Shen

²

,

Wang

³

et al. 2021

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Human bocavirus 1 (HBoV1) was discovered in human nasopharyngeal specimens in 2005. It is an autonomous human parvovirus and causes acute respiratory tract infections in young children. HBoV1 infects well differentiated or polarized human airway epithelial cells in vitro. Unique among all parvoviruses, HBoV1 expresses 6 non-structural proteins, NS1, NS1-70, NS2, NS3, NS4, and NP1, and a viral non-coding RNA (BocaSR), and three structural proteins VP1, VP2, and VP3. The BocaSR is the first identified RNA polymerase III (Pol III) transcribed viral non-coding RNA in small DNA viruses. It plays an important role in regulation of viral gene expression and a direct role in viral DNA replication in the nucleus. HBoV1 genome replication in the polarized/non-dividing airway epithelial cells depends on the DNA damage and DNA repair pathways and involves error-free Y-family DNA repair DNA polymerase (Pol) η and Pol κ. Importantly, HBoV1 is a helper virus for the replication of dependoparvovirus, adeno-associated virus (AAV), in polarized human airway epithelial cells, and HBoV1 gene products support wild-type AAV replication and recombinant AAV (rAAV) production in human embryonic kidney (HEK) 293 cells. More importantly, the HBoV1 capsid is able to pseudopackage an rAAV2 or rHBoV1 genome, producing the rAAV2/HBoV1 or rHBoV1 vector. The HBoV1 capsid based rAAV vector has a high tropism for human airway epithelia. A deeper understanding in HBoV1 replication and gene expression will help find a better way to produce the rAAV vector and to increase the efficacy of gene delivery using the rAAV2/HBoV1 or rHBoV1 vector, in particular, to human airways. This review summarizes the recent advances in gene expression and replication of HBoV1, as well as the use of HBoV1 as a parvoviral vector for gene delivery.

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Human Bocavirus 1 Infection of Well‐Differentiated Human Airway Epithelium

Cited by 12 publications

References 79 publications

Long-Term Modeling of SARS-CoV-2 Infection of In Vitro Cultured Polarized Human Airway Epithelium

Long-Term Modeling of SARS-CoV-2 Infection of In Vitro Cultured Polarized Human Airway Epithelium

Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

Recent Advances in Molecular Biology of Human Bocavirus 1 and Its Applications

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