Enterovirus type 70: receptor interactions and cell entry

Waugh, Sheila M.L. (2007) Enterovirus type 70: receptor interactions and cell entry. PhD thesis, University of Glasgow.

Abstract

Enterovirus type 70 (EV 70) is the causative agent of a highly infectious haemorrhagic conjunctivitis that can rarely be complicated by a polio-like flaccid paralysis, and is one of only two enteroviruses (out of over 90) that infect the eye. EV70 has previously been reported to bind to sialic acid and decay accelerating factor (DAF), however the mechanism by which EV70 enters the cell is unknown. Using a minimally passaged isolate of the J670/71 type strain of EV70 the receptor interactions and cell entry processes of this virus have been investigated. As part of this study, an infectious cDNA and a subgenomic replicon of EV70 were generated. In the work presented here multiple different experimental approaches demonstrated that sialic acid is the primary determinant of EV70 binding and infection and that DAF does not play a significant role. Sialic acid is also responsible for EV70- mediated haemagglutination and binding to a range of non-primate mammalian cells as diverse as Xenopus in origin. On erythrocytes further characterisation showed the receptor to be an alpha2-3 linked sialated GPI-anchored glycoprotein; although binding of EV70 to MRC5 cells was unaffected by the removal of alpha2-3 linked sialic acid from the cell surface, suggesting potential variation in receptor usage. Further studies have demonstrated that EV70 uses clathrin-mediated endocytosis as its initial route of entry to MRC5 cells, and that tyrosine kinases and the actin cytoskeleton are important. The evidence is also suggestive of a role for low pH in exit of the viral genome from late endosomes. The availability of infectious cDNAs for enteroviruses has allowed reverse genetic approaches to be used during analysis of virus structure and function. Prior to this study such reagents did not exist for EV70. Long range PCR was used during the generation of an infectious cDNA for EV70 that, in turn, was used to derive a subgenomic replicon encoding a luciferase reporter gene. The specific infectivity of in vitro generated RNA, and growth characteristics of recovered virus, indicated that the cDNA was truly representative of the EV70 J670/71 stock. These reagents represent invaluable tools for further research into all aspects of the EV70 life cycle.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Virology.
• Subjects: Q Science > QR Microbiology > QR355 Virology
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Evans, Professor David
• Date of Award: 2007
• Depositing User: Enlighten Team
• Unique ID: glathesis:2007-70964
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 08 May 2019 09:21
• Last Modified: 28 May 2021 12:57
• URI: https://theses.gla.ac.uk/id/eprint/70964