Rotaviruses are the single most important etiological agent of severe diarrhoea in infants and young children in both developed and developing countries. The World Health Organisation has identified the development of a rotavirus vaccine as a priority area for routine childhood immunisation to control rotavirus infections. However, the candidate vaccine strains have not been very successful. The main aim of this project was to map rotavirus virulence to its gene segments. Such studies can help in developing better vaccines for the control of rotavirus induced diarrhoea. A three step approach was undertaken (i) development of an animal model, (ii) construction and characterisation of reassortants between rotavirus strains of different virulence, (iii) mapping virulence to rotavirus gene segments. The mouse model developed revealed that the outcome of rotavirus infection was influenced by viral dose and viral strain as well as by host age and host strain. Homologous murine rotavirus strain was found to be most virulent. Among the heterologous strains studied, the OSU strain was found to be most virulent and UKtc strain the least virulent. The CD- 1 strain of mouse was found to be the most susceptible to virus infection and C57/BL the least susceptible. A very simple and rapid nucleic acid extraction method has been developed that requires only one centrifugation step and circumvents the use of any hazardous organic chemicals, which can be applied to very large numbers of samples saving time and labour. Rotavirus reassortants were constructed in a variety of ways and their genotype determined from relative mobility of their gene segments on polyacrylamide gels and restriction enzyme digestion of PCR amplified products. Twenty two reassortants (2%) were identified out of more than 1100 progeny clones examined and these reassortants belonged to 15 different genotypes. Possible reasons for obtaining this low number of reassortants are discussed. No reassortant could be identified between a murine rotavirus and other heterologous rotavirus strains. Preliminary sequence of VP7 gene of murine rotavirus strains, EDIM and EBR, was found to be different to the published rotavirus sequences including the recently published five murine rotavirus strains. The virulence mapping studies conducted in mice with some of the 22 reassortants obtained in the present study showed that gene 4 of the OSU and UKtc strains was involved in virulence. Segment 5 of OSU strain and segments 5, and 8 of UKtc strain may also be involved in virulence.
Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:307315 |
Date | January 1995 |
Creators | Kapoor, Sanjay |
Publisher | University of Warwick |
Source Sets | Ethos UK |
Detected Language | English |
Type | Electronic Thesis or Dissertation |
Source | http://wrap.warwick.ac.uk/4250/ |
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