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

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1102
Author(s):  
Phoebe Stevenson-Leggett ◽  
Sarah Keep ◽  
Erica Bickerton
Keyword(s):  
Cell Culture ◽  
Infectious Bronchitis Virus ◽  
Ex Vivo ◽  
Serial Passage ◽  
In Ovo ◽  
Infectious Bronchitis ◽  
Protease Treatment ◽  
Cellular Tropism ◽  
Passage Cell

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.

scholarly journals Carbonized Lysine-Nanogels Protect against Infectious Bronchitis Virus

2021 ◽  
Vol 22 (11) ◽  
pp. 5415
Author(s):  
Ding-Li Chou ◽  
Ju-Yi Mao ◽  
Anisha Anand ◽  
Han-Jia Lin ◽  
John Han-You Lin ◽  
...  
Keyword(s):  
Infectious Bronchitis Virus ◽  
Pseudorabies Virus ◽  
Hatching Rate ◽  
Viral Inhibition ◽  
Infected Chicken ◽  
In Ovo ◽  
Chicken Embryos ◽  
Virus Attachment ◽  
Infectious Bronchitis

In this study, we demonstrate the synthesis of carbonized nanogels (CNGs) from an amino acid (lysine hydrochloride) using a simple pyrolysis method, resulting in effective viral inhibition properties against infectious bronchitis virus (IBV). The viral inhibition of CNGs was studied using both in vitro (bovine ephemeral fever virus (BEFV) and pseudorabies virus (PRV)) and in ovo (IBV) models, which indicated that the CNGs were able to prevent virus attachment on the cell membrane and penetration into the cell. A very low concentration of 30 μg mL−1 was found to be effective (>98% inhibition) in IBV-infected chicken embryos. The hatching rate and pathology of IBV-infected chicken embryos were greatly improved in the presence of CNGs. CNGs with distinctive virus-neutralizing activities show great potential as a virostatic agent to prevent the spread of avian viruses and to alleviate the pathology of infected avian species.

scholarly journals Pustulan Activates Chicken Bone Marrow-Derived Dendritic Cells In Vitro and Promotes Ex Vivo CD4+ T Cell Recall Response to Infectious Bronchitis Virus

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 226
Author(s):  
Frederik T. Larsen ◽  
Bernt Guldbrandtsen ◽  
Dennis Christensen ◽  
Jacob Pitcovski ◽  
Rikke B. Kjærup ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Infectious Bronchitis Virus ◽  
Ex Vivo ◽  
Poultry Industry ◽  
Subunit Vaccines ◽  
Recall Response ◽  
Infectious Bronchitis ◽  
Chicken Bone

Infectious bronchitis virus (IBV) is a highly contagious avian coronavirus. IBV causes substantial worldwide economic losses in the poultry industry. Vaccination with live-attenuated viral vaccines, therefore, are of critical importance. Live-attenuated viral vaccines, however, exhibit the potential for reversion to virulence and recombination with virulent field strains. Therefore, alternatives such as subunit vaccines are needed together with the identification of suitable adjuvants, as subunit vaccines are less immunogenic than live-attenuated vaccines. Several glycan-based adjuvants directly targeting mammalian C-type lectin receptors were assessed in vitro using chicken bone marrow-derived dendritic cells (BM-DCs). The β-1-6-glucan, pustulan, induced an up-regulation of MHC class II (MHCII) cell surface expression, potentiated a strong proinflammatory cytokine response, and increased endocytosis in a cation-dependent manner. Ex vivo co-culture of peripheral blood monocytes from IBV-immunised chickens, and BM-DCs pulsed with pustulan-adjuvanted recombinant IBV N protein (rN), induced a strong recall response. Pustulan-adjuvanted rN induced a significantly higher CD4+ blast percentage compared to either rN, pustulan or media. However, the CD8+ and TCRγδ+ blast percentage were significantly lower with pustulan-adjuvanted rN compared to pustulan or media. Thus, pustulan enhanced the efficacy of MHCII antigen presentation, but apparently not the cross-presentation on MHCI. In conclusion, we found an immunopotentiating effect of pustulan in vitro using chicken BM-DCs. Thus, future in vivo studies might show pustulan as a promising glycan-based adjuvant for use in the poultry industry to contain the spread of coronaviridiae as well as of other avian viral pathogens.

scholarly journals The S2 Subunit of Infectious Bronchitis Virus Beaudette Is a Determinant of Cellular Tropism

2018 ◽  
Vol 92 (19) ◽  
Author(s):  
Erica Bickerton ◽  
Helena J. Maier ◽  
Phoebe Stevenson-Leggett ◽  
Maria Armesto ◽  
Paul Britton
Keyword(s):  
Amino Acids ◽  
Infectious Bronchitis Virus ◽  
Rational Design ◽  
Cultured Cells ◽  
Vero Cells ◽  
Progeny Virus ◽  
Minimum Requirement ◽  
Infectious Bronchitis ◽  
Cellular Tropism

ABSTRACTThe spike (S) glycoprotein of the avian gammacoronavirus infectious bronchitis virus (IBV) is comprised of two subunits (S1 and S2), has a role in virulencein vivo, and is responsible for cellular tropismin vitro. We have previously demonstrated that replacement of the S glycoprotein ectodomain from the avirulent Beaudette strain of IBV with the corresponding region from the virulent M41-CK strain resulted in a recombinant virus, BeauR-M41(S), with thein vitrocell tropism of M41-CK. The IBV Beaudette strain is able to replicate in both primary chick kidney cells and Vero cells, whereas the IBV M41-CK strain replicates in primary cells only. In order to investigate the region of the IBV S responsible for growth in Vero cells, we generated a series of recombinant IBVs expressing chimeric S glycoproteins, consisting of regions from the Beaudette and M41-CK S gene sequences, within the genomic background of Beaudette. The S2, but not the S1, subunit of the Beaudette S was found to confer the ability to grow in Vero cells. Various combinations of Beaudette-specific amino acids were introduced into the S2 subunit of M41 to determine the minimum requirement to confer tropism for growth in Vero cells. The ability of IBV to grow and produce infectious progeny virus in Vero cells was subsequently narrowed down to just 3 amino acids surrounding the S2′ cleavage site. Conversely, swapping of the 3 Beaudette-associated amino acids with the corresponding ones from M41 was sufficient to abolish Beaudette growth in Vero cells.IMPORTANCEInfectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines, both live attenuated and inactivated, are currently grown on embryonated hen's eggs, a cumbersome and expensive process due to the fact that most IBV strains do not grow in cultured cells. The reverse genetics system for IBV creates the opportunity for generating rationally designed and more effective vaccines. The observation that IBV Beaudette has the additional tropism for growth on Vero cells also invokes the possibility of generating IBV vaccines produced from cultured cells rather than by the use of embryonated eggs. The regions of the IBV Beaudette S glycoprotein involved in the determination of extended cellular tropism were identified in this study. This information will enable the rational design of a future generation of IBV vaccines that may be grown on Vero cells.

scholarly journals In Vitro and In Ovo Expression of Chicken Gamma Interferon by a Defective RNA of Avian Coronavirus Infectious Bronchitis Virus

2003 ◽  
Vol 77 (10) ◽  
pp. 5694-5702 ◽  
Author(s):  
Karen Hackney ◽  
Dave Cavanagh ◽  
Pete Kaiser ◽  
Paul Britton
Keyword(s):  
Infectious Bronchitis Virus ◽  
Allantoic Fluid ◽  
Helper Virus ◽  
Gamma Interferon ◽  
Biologically Active ◽  
Site Directed Mutagenesis ◽  
In Ovo ◽  
Infectious Bronchitis

ABSTRACT Coronavirus defective RNAs (D-RNAs) have been used for site-directed mutagenesis of coronavirus genomes and for expression of heterologous genes. D-RNA CD-61 derived from the avian coronavirus infectious bronchitis virus (IBV) was used as an RNA vector for the expression of chicken gamma interferon (chIFN-γ). D-RNAs expressing chIFN-γ were shown to be capable of rescue, replication, and packaging into virions in a helper virus-dependent system following electroporation of in vitro-derived T7 RNA transcripts into IBV-infected cells. Secreted chIFN-γ, under the control of an IBV transcription-associated sequence derived from gene 5 of the Beaudette strain, was expressed from two different positions within CD-61 and shown to be biologically active. In addition, following infection of 10-day-old chicken embryos with IBV containing D-RNAs expressing chIFN-γ, the allantoic fluid was shown to contain biologically active chIFN-γ, demonstrating that IBV D-RNAs can express heterologous genes in vivo.

scholarly journals Gene 5 of the Avian Coronavirus Infectious Bronchitis Virus Is Not Essential for Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8065-8078 ◽  
Author(s):  
Rosa Casais ◽  
Marc Davies ◽  
David Cavanagh ◽  
Paul Britton
Keyword(s):  
Infectious Bronchitis Virus ◽  
Reverse Genetics ◽  
Ex Vivo ◽  
Wild Type Virus ◽  
In Ovo ◽  
Amino Acid Similarity ◽  
Recombinant Viruses ◽  
Infectious Bronchitis ◽  
Ns Gene

ABSTRACT The avian coronavirus Infectious bronchitis virus (IBV), like other coronaviruses, expresses several small nonstructural (ns) proteins in addition to those from gene 1 (replicase) and the structural proteins. These coronavirus ns genes differ both in number and in amino acid similarity between the coronavirus groups but show some concordance within a group or subgroup. The functions and requirements of the small ns gene products remain to be elucidated. With the advent of reverse genetics for coronaviruses, the first steps in elucidating their role can be investigated. We have used our reverse genetics system for IBV (R. Casais, V. Thiel, S. G. Siddell, D. Cavanagh, and P. Britton, J. Virol. 75:12359-12369, 2001) to investigate the requirement of IBV gene 5 for replication in vivo, in ovo, and ex vivo. We produced a series of recombinant viruses, with an isogenic background, in which complete expression of gene 5 products was prevented by the inactivation of gene 5 following scrambling of the transcription-associated sequence, thereby preventing the expression of IBV subgenomic mRNA 5, or scrambling either separately or together of the translation initiation codons for the two gene 5 products. As all of the recombinant viruses replicated very similarly to the wild-type virus, Beau-R, we conclude that the IBV gene 5 products are not essential for IBV replication per se and that they are accessory proteins.

Comparative Evaluation of In Vitro and In Vivo Assays for the Detection of Avian Infectious Bronchitis Virus as a Contaminant of Live Poultry Vaccines

1998 ◽  
Vol 26 (5) ◽  
pp. 629-634
Author(s):  
Emiliana Falcone ◽  
Edoardo Vignolo ◽  
Livia Di Trani ◽  
Simona Puzelli ◽  
Maria Tollis
Keyword(s):  
Infectious Bronchitis Virus ◽  
Serological Response ◽  
Avian Infectious Bronchitis Virus ◽  
Animal Testing ◽  
Rt Pcr ◽  
Pcr Assay ◽  
In Vivo Test ◽  
Infectious Bronchitis

A reverse transcriptase polymerase chain reaction (RT-PCR) assay specific for identifying avian infectious bronchitis virus (IBV) in poultry vaccines, and the serological response to IBV induced by the inoculation of chicks with a Newcastle disease vaccine spiked with the Massachusetts strain of IBV, were compared for their ability to detect IBV as a contaminant of avian vaccines. The sensitivity of the IBV-RT-PCR assay provided results which were at least equivalent to the biological effect produced by the inoculation of chicks, allowing this assay to be considered a valid alternative to animal testing in the quality control of avian immunologicals. This procedure can easily be adapted to detect a number of contaminants for which the in vivo test still represents the only available method of detection.

scholarly journals In Vitro Lung Models and Their Application to Study SARS-CoV-2 Pathogenesis and Disease

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 792
Author(s):  
Natalie Heinen ◽  
Mara Klöhn ◽  
Eike Steinmann ◽  
Stephanie Pfaender
Keyword(s):  
Cell Culture ◽  
Ex Vivo ◽  
Treatment Options ◽  
Pharmaceutical Companies ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Novel Treatment ◽  
Cell Culture Systems ◽  
Drug Efficiency

SARS-CoV-2 has spread across the globe with an astonishing velocity and lethality that has put scientist and pharmaceutical companies worldwide on the spot to develop novel treatment options and reliable vaccination for billions of people. To combat its associated disease COVID-19 and potentially newly emerging coronaviruses, numerous pre-clinical cell culture techniques have progressively been used, which allow the study of SARS-CoV-2 pathogenesis, basic replication mechanisms, and drug efficiency in the most authentic context. Hence, this review was designed to summarize and discuss currently used in vitro and ex vivo cell culture systems and will illustrate how these systems will help us to face the challenges imposed by the current SARS-CoV-2 pandemic.

Some Studies of Infectious Bronchitis Virus in Cell Culture

1965 ◽  
Vol 9 (1) ◽  
pp. 182 ◽  
Author(s):  
C. H. Cunningham ◽  
M. P. Spring
Keyword(s):  
Cell Culture ◽  
Infectious Bronchitis Virus ◽  
Infectious Bronchitis
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