Noroviruses are members of the Caliciviridae family of positive strand RNA viruses and are one of the leading causes of non-bacterial gastroenteritis in humans. Currently no in vitro cell culture method has been reliably described, which has hindered the progress of norovirus research, leading to little understanding of the replication of these viruses. The purpose of this study was to produce a reliable and sensitive detection assay for noroviruses in oysters, by incorporating a broadly cross-reactive monoclonal antibody. Bivalve shellfish are one of the main transmission routes of noroviruses from the environment to humans, especially when consumed raw. Noroviruses concentrate in the digestive tissue of bivalve shellfish, such as oysters as the shellfish filter feed. In order to develop a detection assay, monoclonal antibodies against the current norovirus surrogate Murine norovirus (MNV, Genogroup V) and prevalent genogroup II noroviruses were produced. Five monoclonal antibodies detected MNV capsid protein and eleven detected GII.4 capsid protein by direct ELISA. Two of the GII.4 monoclonal antibodies showed cross-reactivity, CM160 to Southampton virus (GI) and both CM160 and CM162 detected Desert Shield virus (GII) in ELISA. No broadly cross reactive monoclonal antibody which detected a linear epitope in the capsid protein could be isolated. Alongside development of monoclonal antibodies, chimeric MNV containing a precharacterised human norovirus epitope was created. Monoclonal antibody CM54 had been previously isolated, which detected a linear epitope LEDVRN. This epitope was incorporated into the P2 domain of MNV. LEDVRN is a common epitope to GI capsid proteins but is not present in GII capsid proteins. This was an attempt to side-step the need for MNV/human norovirus cross-reactive monoclonal antibodies, which detected a linear epitope in the P domain of the capsid protein. No viable chimeric virus could be detected after transfection of Raw 264.7 and 293T cells. Detection assays, ELISA, DELFIA and qRT-PCR were compared in the sensitivity of detecting norovirus in oyster digestive tissue, using a Lordsdale (LV) GII.4 monoclonal antibody and GII specific qPCR primers described by Kageyama et al (118). An antigen capture ELISA and DELFIA was developed using hyper-immune rabbit antiserum raised to recombinant LV capsid protein. Both assays were specific for LV in faecal samples. Preliminary experiments indicated that the DELFIA was four fold more sensitive than the ELISA. Ten pacific oysters were analysed, all ten were negative by ELISA, seven positive in the DELFIA and all ten positive by qRT-PCR. This suggests that the majority (70%) of the oysters contained pre-existing norovirus protein or RNA; however these data provide no indication of infectivity. DELFIA demonstrates promise as a more sensitive alternative to the ELISA, while significantly cheaper than qRT-PCR.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:635453 |
| Date | January 2013 |
| Creators | Hadley, Simone |
| Contributors | Clarke, Ian ; Lambden, P. R. |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/373270/ |
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