Immunological and biochemical characterisation of the NSP4 protein of human rotavirus strains RV4 and RV5

Rotavirus has been identified as the major aetiological agent of severe dehydrating gastrointestinal disease in the young of humans and animal species. Of particular significance is the pleoitropic properties of the rotavirus non-structural protein 4 (NSP4), the first identified viral enterotoxin. NSP4 has been widely documented to play an integral role in disease pathogenesis and morphogenesis. The induction of cell-mediated and humoral responses to NSP4 has been demonstrated in both humans and in animal models. The enterotoxigenic and cytopathic properties of rotavirus infection has been associated with the inherent ability of extracellular and intracellular NSP4 respectively, to perturb the calcium homeostasis of host cells. This study aimed to investigate the ability of the NSP4 protein of two prototype human rotavirus strains, RV4 and RV5 to mobilise intracellular calcium in vitro and to evoke a humoral response in naturally infected children and vaccine recipients. The baculovirus expression system (BEVS) was used to produce hexa-his-tagged (His6)-NSP4 fusion peptides in Spodoptera frugiperda (Sf-21 cells). The purified proteins were then used in (a) an alpha-NSP4 indirect ELISA and (b) intracellular calcium mobilisation studies. The results obtained from the alpha-NSP4 specific immunoglobulin ELISA demonstrated that natural rotavirus infection and vaccination evoked an NSP4-specific humoral response, which was both heterotypic and/or homotypic. The dominant isotype-specific serum response was to alpha-NSP4 IgG. For children naturally infected with a G1 RV strain, the response was both homotypic and heterotypic, with 80% of samples reactive with NSP4 of genotype B (G1) and 60% to NSP4 genotype A (G2). The alpha-NSP4 IgA response was detected solely for children infected with a strain homotypic to the coating antigen and with a seroconversion rate of 29%. No NSP4-specific IgA was detected for vaccine recipients and the IgG response was less than for naturally infected children. Preliminary data indicated that the proximal location of the His6-tag on NSP4 of the RV4 strain influenced the antigenic properties of the protein. The studies on the effects of NSP4 derived from animal rotavirus strains (predominantly non-human primate strains) and to whole virus on perturbations to intracellular calcium homeostasis has been well established. This study aimed to investigate the ability of human rotaviruses and their corresponding NSP4 to alter the intracellular calcium concentration of cultured renal cells. Two methods for monitoring intracellular calcium concentrations were undertaken; (a) spectrofluorimetric detection of cell populations in suspension and (b) epifluorescence microscopy of cell monolayers. The results acquired from this study demonstrated that SA11 infection and the exogenous application of NSP4 induced changes to the intracellular calcium homeostasis of mammalian cell populations and single cells respectively. An apparent caloric stress effect, arising from the sequential exposure of uninfected mammalian cells to mild hypothermia and hyperthermia was observed to enhance the cytopathic effect of all three strains (SA11, RV4 and RV5) investigated.