The work described in this thesis identifies the structural polypeptide profile of human astrovirus type 1, grown in tissue culture in the presence of trypsin. Four major polypeptides were observed, of molecular weights: 34,000, 33.000, 26,500 and 6-8,000. A fifth of molecular weight 37,000 observed when the virus is prepared by PEG precipitation is considered to represent a precursor polypeptide. Of the four structural polypeptides, designated VP1, VP2, VP3 and VP4 respectively, VP1 and VP2 were found to be immunologically reactive with anti-astrovirus type 1 polyclonal antiserum. Astrovirus type 1 particles were found to have a buoyant density of 1.33-1.34 gm/ral in caesium chloride and a genome approximately 7,200 bases in length, corresponding to a molecular weight of 2.43 x 10° which is assumed to be RNA. This data suggests a relationship between astroviruses and picornaviruses, most especially the enterovirus subgroup, which have similar structural polypeptide and physico-chemical properties to those described above. However, the use of trypsin for the propagation of astrovirus in tissue culture means that further analysis of the structural polypeptides is required to determine whether those observed in this analysis represent four individual proteins, similar to the profile of picornaviruses, or whether some are cleavage products of the larger proteins due to the action of trypsin. The 3' terminal sequence of the astrovirus genome has been cloned and sequenced. It was found to have a poly (A) tail, stop codons in all three reading frames within the last 95 nucleotides and no polyadenylation signals. Several other astrovirus specific clones were obtained, but there is no suggestion as to which regions of the genome these represent. Comparisons between the astrovirus specific clones and the microgenie sequence databank has shown no significant homologies. The highest homology obtained for the 3' terminal sequence when compared to those of other picornaviruses was 47Z with coxsackie A21, this is not taken to confirm a relationship between the viruses. Assay systems were developed for astrovirus during the course of this study. An immunofluorescent end point titration, using type specific antisera, allowed the determination of the infectivity of a virus stock within 48 hours of infection. An immuno dot blot system was developed to assay for amounts of virus protein in samples, such as gradient fractions, and was used to determine the peak banding of virus in sucrose.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:290188 |
| Date | January 1990 |
| Creators | Major, Marian |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/107943/ |
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