Characterisation of norovirus isolates obtained from Children with acute gastroenteritis

Thesis with publications.

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Life Sciences, Faculty of Science Technology and Engineering, La Trobe University, Bundoora.

Noroviruses are the most common cause of gastroenteritis. Human noroviruses are classified into more than 26 genotypes based on sequence of the capsid or RNA-dependent RNA polymerase (RdRp) gene. Genotype II.4 is the most commonly identified norovirus genotype in adults, however, genotype distribution in children is variable. This dissertation aimed to conduct a molecular epidemiological study of norovirus strains causing paediatric gastroenteritis, while investigating the evolutionary mechanisms employed by paediatric-associated strains, and identifying antibody-binding epitopes on the capsid protein. Stool samples were collected from 272 Australian children hospitalised with gastroenteritis during 2006 to 2008. Using RT-PCR, norovirus was detected in 35% of samples, with GII.4 (49%) and GII.3 recombinant strains (46%) identified as the most prevalent genotypes. Due to the high incidence of recombination and paediatric association of GII.3 strains, the evolutionary mechanisms were investigated. The capsid gene of 6 GII.3 norovirus paediatric strains were sequenced and aligned with 66 GII.3 capsid sequences from GenBank. Phylogenetic analysis predicted the GII.3 capsid gene to have evolved via five genetic lineages and the clustering of GII.3 capsid sequences was associated with intergenic recombination. Rate analyses suggested that the switching of RdRp genes may aid in increased evolution rate and adaptability. Six antibody-binding epitopes were identified on the GII.3 capsid using immunoprecipitation, mass spectrometry and Mimotopes. The epitopes were highly conserved, except for two within the P2 domain that were in close association with human attachment factor binding sites. There were several sites within these two epitopes that varied according to lineage, suggesting a possible role of immune pressure in GII.3 evolution. Interestingly, GII.3 noroviruses exhibited broad intergenotype cross-reactivity. This study highlighted the importance of GII.3 norovirus as a cause of paediatric disease. Furthermore, this study characterised major evolutionary mechanisms and immunogenic regions important to the persistence and spread of this virus.