The Preparation of Chicken Kidney Cell Cultures for Virus Propagation

Hennion, Ruth M.; Hill, Gillian

doi:10.1007/978-1-4939-2438-7_6

Cited by 49 publications

(33 citation statements)

References 12 publications

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“…Vero cells were obtained from the central services unit (CSU) at The Pirbright Institute and maintained in Eagle’s minimum essential medium (EMEM, Sigma-Aldrich, St. Louis, MO, USA) containing 10% foetal bovine serum (FBS) and 1% l -Glutamine (Sigma-Aldrich, St. Louis, MO, USA). Chicken kidney (CK) cells were prepared as previously described [ 43 ] from kidneys of 2–3 week old Rhode Island Red (RIR) chickens, reared at The Pirbright Institute. DF-1 cells were obtained from CSU and were maintained in Dulbecco’s minimum essential medium (DMEM) containing 10% FBS and 1% l -Glutamine (Sigma-Aldrich, St. Louis, MO, USA).…”

Section: Methodsmentioning

confidence: 99%

Treatment with Exogenous Trypsin Expands In Vitro Cellular Tropism of the Avian Coronavirus Infectious Bronchitis Virus

Stevenson-Leggett

Keep

Bickerton

2020

Viruses

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The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2′ cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.

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Section: Methodsmentioning

confidence: 99%

Treatment with Exogenous Trypsin Expands In Vitro Cellular Tropism of the Avian Coronavirus Infectious Bronchitis Virus

Stevenson-Leggett

Keep

Bickerton

2020

Viruses

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“…Primary CK cells were produced from 2–3 week-old specific pathogen free (SPF) Rhode Island Red (RIR) chickens44. TOCs were produced from 19 day old SPF embryos2345, or 2–3 week-old SPF RIR chickens, using the same procedure.…”

Section: Methodsmentioning

confidence: 99%

Extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity

Maier

Neuman

Bickerton

et al. 2016

Sci Rep

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Positive-strand RNA (+RNA) viruses rearrange cellular membranes during replication, possibly in order to concentrate and arrange viral replication machinery for efficient viral RNA synthesis. Our previous work showed that in addition to the conserved coronavirus double membrane vesicles (DMVs), Beau-R, an apathogenic strain of avian Gammacoronavirus infectious bronchitis virus (IBV), induces regions of ER that are zippered together and tethered open-necked double membrane spherules that resemble replication organelles induced by other +RNA viruses. Here we compared structures induced by Beau-R with the pathogenic lab strain M41 to determine whether membrane rearrangements are strain dependent. Interestingly, M41 was found to have a low spherule phenotype. We then compared a panel of pathogenic, mild and attenuated IBV strains in ex vivo tracheal organ culture (TOC). Although the low spherule phenotype of M41 was conserved in TOCs, each of the other tested IBV strains produced DMVs, zippered ER and spherules. Furthermore, there was a significant correlation for the presence of DMVs with spherules, suggesting that these structures are spatially and temporally linked. Our data indicate that virus induced membrane rearrangements are fundamentally linked to the viral replicative machinery. However, coronavirus replicative apparatus clearly has the plasticity to function in different structural contexts.

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“…Primary chicken kidney (CK) cells and ex vivo tracheal organ cultures (TOCs) were prepared from 2 to 3-week-old specific pathogen free (SPF) Rhode Island Red (RIR) chickens as described elsewhere [17,18]. Continuous cells lines Vero and DF1 were provided by the cell culture department at The Pirbright Institute.…”

Section: Cells and Virusesmentioning

confidence: 99%

Multiple novel non-canonically transcribed sub-genomic mRNAs produced by avian coronavirus infectious bronchitis virus

Keep

Oade

Lidzbarski-Silvestre

et al. 2020

Journal of General Virology

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Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5′ untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3′-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified.

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The Preparation of Chicken Kidney Cell Cultures for Virus Propagation

Cited by 49 publications

References 12 publications

Treatment with Exogenous Trypsin Expands In Vitro Cellular Tropism of the Avian Coronavirus Infectious Bronchitis Virus

Treatment with Exogenous Trypsin Expands In Vitro Cellular Tropism of the Avian Coronavirus Infectious Bronchitis Virus

Extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity

Multiple novel non-canonically transcribed sub-genomic mRNAs produced by avian coronavirus infectious bronchitis virus

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