scholarly journals The cryo-EM structure of vesivirus 2117 highlights functional variations in entry pathways for viruses in different clades of the Vesivirus genus

2021 ◽  
Author(s):  
Hazel Sutherland ◽  
Michaela J. Conley ◽  
Edward Emmott ◽  
James Streetley ◽  
Ian G. Goodfellow ◽  
...  
Keyword(s):  
Major Capsid Protein ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Outer Face ◽  
Receptor Binding Site ◽  
Amino Acid Insertion ◽  
Positive Sense ◽  
Lost Productivity ◽  
Biopharmaceutical Production ◽  
Immunodominant Epitopes

AbstractVesivirus 2117 is an adventitious agent that has been responsible for lost productivity in biopharmaceutical production following contamination of Chinese hamster ovary cell cultures in commercial bioreactors. A member of the Caliciviridae, 2117 is classified within the Vesivirus genus in a clade that includes canine and mink caliciviruses but is distinct from the vesicular exanthema of swine clade, which includes the extensively studied feline calicivirus (FCV). We have used cryogenic electron microscopy (cryo-EM) to determine the structure of the capsid of this small, icosahedral, positive-sense RNA containing virus. We show that the outer face of the dimeric capsomeres, which contains the receptor binding site and major immunodominant epitopes in all caliciviruses studied thus far, is quite different from that of FCV. This is a consequence of a 22 amino-acid insertion in the sequence of the FCV major capsid protein that forms a ‘cantilevered arm’, which plays an important role in both receptor engagement and undergoes structural rearrangements thought to be important for genome delivery to the cytosol. Our data highlight a potentially important difference in the attachment and entry pathways employed by the different clades of the Vesivirus genus.

scholarly journals The cryo-EM structure of vesivirus 2117 highlights functional variations in entry pathways for viruses in different clades of the Vesivirus genus.

2021 ◽  
Author(s):  
Hazel Sutherland ◽  
Michaela J. Conley ◽  
Edward Emmott ◽  
James Streetley ◽  
Ian G. Goodfellow ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Cell Cultures ◽  
Structural Changes ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Genome Comparison ◽  
Feline Calicivirus ◽  
Outer Face ◽  
Receptor Binding Site ◽  
Virus Attachment

Vesivirus 2117 is an adventitious agent that has been responsible for lost productivity in biopharmaceutical production following contamination of Chinese hamster ovary cell cultures in commercial bioreactors. A member of the Caliciviridae, 2117 is classified within the Vesivirus genus in a clade that includes canine and mink caliciviruses but is distinct from the vesicular exanthema of swine clade, which includes the extensively studied feline calicivirus (FCV). We have used cryogenic electron microscopy (cryo-EM) to determine the structure of the capsid of this small, icosahedral, positive-sense RNA containing virus. We show that the outer face of the dimeric capsomeres, which contains the receptor binding site and major immunodominant epitopes in all caliciviruses studied thus far, is quite different from that of FCV. This is a consequence of a 22 amino-acid insertion in the sequence of the FCV major capsid protein that forms a ‘cantilevered arm’, which plays an important role in both receptor engagement and undergoes structural rearrangements thought to be important for genome delivery to the cytosol. Our data highlight a potentially important difference in the attachment and entry pathways employed by the different clades of the Vesivirus genus. Importance Vesivirus 2117 has caused significant losses in manufacturing of biopharmaceutical products following contamination of cell cultures used in their production. We report the structure of the vesivirus 2117 capsid, the shell which encloses the virus's genome. Comparison of this structure with that of a related Vesivirus - feline calicivirus (FCV), highlighted potentially important differences related to virus attachment and entry. Our findings suggest that these two viruses may bind differently to receptors at the host cell surface. We also show that a region of the capsid protein of FCV that rearranges following receptor engagement, is not present in vesivirus 2117. These structural changes in the FCV capsid have been shown to allow assembly of a portal-like structure, hypothesised to deliver the viral genome to the cell's interior. Our data suggest that the 2117 portal assembly may employ a different means of anchoring to the outer face of the capsid.

scholarly journals Biopharmaceutical-Type Chinese Hamster Ovary Cell Cultivation Under Static Magnetic Field Exposure: A Study of Genotoxic Effect

2021 ◽  
Vol 9 ◽  
Author(s):  
Alina Rekena ◽  
Dora Livkisa ◽  
Edmunds Kamolins ◽  
Juris Vanags ◽  
Dagnija Loca
Keyword(s):  
Magnetic Field ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Micronucleus Assay ◽  
Term Effect ◽  
Genotoxic Effect ◽  
Ovary Cell ◽  
Experimental Setup ◽  
Cell Cultivation ◽  
Biopharmaceutical Production

The lack of a sufficient research base is the reason for the ongoing discussion regarding the genotoxic effect of magnetic field (MF) exposure on mammalian cell cultures. Chinese hamster ovary (CHO) suspension-type cells, which are widely used for biopharmaceutical production, are potentially subjected to an increased MF when cultivated in bioreactors equipped with bottom-placed magnetically coupled stirring mechanisms. The main challenge for conducting research in this field remains the availability of a suitable experimental setup that generates an appropriate MF for the raised research question. In the present study, a simple and cost-effective experimental setup was developed that generated a static MF, similar to what has been modeled in large-scale bioreactors and, at the same time, was suitable for experimental cell cultivation in laboratory conditions. The measured maximum magnetic flux density to which the cells were exposed was 0.66 T. To assess the possible genotoxic effect, cells were continuously subcultivated in laboratory petri dishes for a period of 14 days, corresponding to a typical duration of a biopharmaceutical production process in a conventional fed-batch regime. The genotoxic effect was assessed using the cytokinesis-block micronucleus assay with fluorescent staining. Results showed that a 0.66-T static MF exposure had no significant long-term effect on cell viability and chromosomal damage but demonstrated a short-term effect on cell apoptosis. Significant increase in nuclear bud formation was observed. These findings may encourage other researchers in future studies investigating cellular responses to MF exposure and contribute relevant data for comparison.

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