T84 air‐liquid interface cultures enable isolation of human bocavirus

Schildgen, Verena; Longo, Ylenia; Pieper, Monika; Schildgen, Oliver

doi:10.1111/irv.12567

Cited by 4 publications

(6 citation statements)

References 11 publications

(10 reference statements)

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“…Moreover, in contrast to our data in lung organoids, HBoV2–4 showed a higher activity than HBoV1 or GBoV in colon organoids (Table S2). These experiments were further extended by transducing T84 cells, a human colon cancer cell line that we have previously found to be susceptible to WT HBoV1 infection 49 . In these cells, HBoV2–4 also showed a higher activity than HBoV1, while the best results were obtained with GBoV (Figure S5B; Table S2).…”

Section: Resultsmentioning

confidence: 90%

Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses

Fakhiri

Schneider

Puschhof

et al. 2019

Molecular Therapy - Methods & Clinical Development

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Parvoviruses are highly attractive templates for the engineering of safe, efficient, and specific gene therapy vectors, as best exemplified by adeno-associated virus (AAV). Another candidate that currently garners increasing attention is human bocavirus 1 (HBoV1). Notably, HBoV1 capsids can cross-package recombinant (r)AAV2 genomes, yielding rAAV2/HBoV1 chimeras that specifically transduce polarized human airway epithelia (pHAEs). Here, we largely expanded the repertoire of rAAV/BoV chimeras, by assembling packaging plasmids encoding the capsid genes of four additional primate bocaviruses, HBoV2–4 and GBoV (Gorilla BoV). Capsid protein expression and efficient rAAV cross-packaging were validated by immunoblotting and qPCR, respectively. Interestingly, not only HBoV1 but also HBoV4 and GBoV transduced pHAEs as well as primary human lung organoids. Flow cytometry analysis of pHAEs revealed distinct cellular specificities between the BoV isolates, with HBoV1 targeting ciliated, club, and KRT5+ basal cells, whereas HBoV4 showed a preference for KRT5+ basal cells. Surprisingly, primary human hepatocytes, skeletal muscle cells, and T cells were also highly amenable to rAAV/BoV transduction. Finally, we adapted our pipeline for AAV capsid gene shuffling to all five BoV isolates. Collectively, our chimeric rAAV/BoV vectors and bocaviral capsid library represent valuable new resources to dissect BoV biology and to breed unique gene therapy vectors.

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Section: Resultsmentioning

confidence: 90%

Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses

Fakhiri

Schneider

Puschhof

et al. 2019

Molecular Therapy - Methods & Clinical Development

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“…It has been reported that primary air-liquid interface cell culture, polarized primary human airway epithelial (HAE) from the apical surface, provided productive infection by HBoV1 with disruption of the tight junction barrier, the loss of cilia, and hypertrophy of epithelial cells (Huang et al, 2012). In the contrary, HBoV1 causes a clear cytopathic effect resulting in damage to the three-dimensional CuFi-8 cultures, which first become smaller and finally leaky for basal media (Dijkman et al, 2009), while T84 could serve as a tool for classical virus isolation, although this has to be confirmed by further trials (Schildgen et al, 2018). However, no other common cell line or well-developed animal models support the isolation and culture of HBoV1.…”

Section: Discussionmentioning

confidence: 99%

Human bocavirus 1 is a genuine pathogen for acute respiratory tract infection in pediatric patients determined by nucleic acid, antigen, and serology tests

Zhang

Wang

et al. 2022

Front. Microbiol.

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BackgroundHuman bocavirus 1 (HBoV1), first discovered in 2005, was positive in symptomatic and healthy children and co-detected with other respiratory viruses. It is a long journey to decisively demonstrate the unique viral pathogenic function of acute respiratory tract infection (ARTI) in pediatric patients.MethodsRespiratory specimens collected from pediatric patients with ARTI from January 2017 to December 2021 were screened by a capillary electrophoresis-based multiplex PCR (CEMP) assay, then genotyped by PCR and sequencing for HBoV1. For the antigen test, a part of HBoV1 DNA positive nasopharyngeal aspirates (NPAs) was used as an antigen, while a rabbit anti-HBoV1 DR2 specific to HBoV1 was used as an antibody in the indirect-immunofluorescence assay (IFA). Finally, the levels of IgG specific to HBoV1 in acute and convalescent sera selected retrospectively from only HBoV1 DNA-positive patients were evaluated by IFA.ResultsAmong 9,899 specimens, 681 were positive for HBoV1 DNA (6.88%, 681/9899), which included 336 positives only for HBoV1 (49.34%, 336/681) and 345 (50.66%, 345/681) positives also for other pathogens. In the antigen test, there were 37 among 47 NPAs determined as HBoV1 antigen-positive (78.72%, 37/47), including 18 (48.65%, 18/37) positives solely for HBoV1 DNA. Among 4 pediatric patients with both acute and convalescent sera, there was one positive for HBoV1 antigen (D8873) and 2 lack the antigen results (D1474 and D10792), which showed seroconversion with a ≥ 4-fold increase in IgG levels.ConclusionsThe combination results of nucleic acid, antigen, and serology tests answered that HBoV1 is a genuine pathogen for ARTI in pediatric patients.

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“…In a previous report, the cell line T84 (ATCC™ CCL-248, Manassas, VA, USA) was described to be permissive for HBoV if cultivated as air-liquid interface organoids [21]. The cell line is derived from the lung metastasis of a human large intestine colorectal tumor and can easily be grown as monolayers in DMEM/F12 with 5% (v/v) Fetal Bovine Serum (FBS) and 1% (v/v) of a cell culture-ready penicillin-streptomycin solution (all Gibco/Thermo Fisher, Waltham, MA, USA).…”

Section: Amentioning

confidence: 99%

“…To date, only three cell culture models, namely primary airliquid interface cultures [19], CuFi-8 air-liquid interface cultures [20], and T84 air-liquid interface cultures were described to support HBoV replication [21] While we contributed to the very first primary cell culture of HBoV [19] and have implemented a further cell culture model based on those earlier progress and a reverse genetics system [20]), those earlier cell culture models were cost expensive, took a lot of time before cells could be productively infected, and were limited to small experimental series in sense of the overall volumes and the technical limitations by the air-liquid interface cell culture systems. A novel cell line identified as permissive by colleagues Dirk Grimm and Steeve Boulant from Heidelberg and kindly provided to our lab a few years ago are T84 cells [21], which are permissive for HBoV as air-liquid interface cultures but cost less than CuFi8 and primary cells. T84 cells originate from the lung metastasis of a colorectal tumor and form a pleomorphic and rather ugly monolayer, the latter an observation that triggered our idea that even the monolayer could be permissive for HBoV.…”

Section: Introductionmentioning

confidence: 99%

T84 Monolayer Cell Cultures Support Productive HBoV and HSV-1 Replication and Enable In Vitro Co-Infection Studies

Soldwedel,

Demuth,

Schildgen

2024

Viruses

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Based on several clinical observations it was hypothesized that herpesviruses may influence the replication of human bocaviruses, the second known parvoviruses that have been confirmed as human pathogens. While several cell lines support the growth of HSV-1, HBoV-1 was exclusively cultivated on air–liquid interface cultures, the latter being a rather complicated, slow, and low throughput system. One of the cell lines are T84 cells, which are derived from the lung metastasis of a colorectal tumor. In this study, we provide evidence that T84 also supports HBoV replication when cultivated as monolayers, while simultaneously being permissive for HSV-1. The cell culture model thus would enable co-infection studies of both viruses and is worth being optimized for high throughput studies with HBoV-1. Additionally, the study provides evidence for a supporting effect of HSV-1 on the replication and packaging of HBoV-1 progeny DNA into DNase-resistant viral particles.

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T84 air‐liquid interface cultures enable isolation of human bocavirus

Cited by 4 publications

References 11 publications

Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses

Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses

Human bocavirus 1 is a genuine pathogen for acute respiratory tract infection in pediatric patients determined by nucleic acid, antigen, and serology tests

T84 Monolayer Cell Cultures Support Productive HBoV and HSV-1 Replication and Enable In Vitro Co-Infection Studies

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