Abstract
Group A rotaviruses are a major cause of diarrhea in the young of many mammalian species. In rotavirus infected piglets mortality can be as high as 60%. Previous research in this laboratory has identified a porcine intestinal GM3 ganglioside receptor that is required for sialic acid-dependent rotavirus recognition of host cells. In addition, we previously demonstrated exogenously added GM3 can competitively inhibit porcine rotavirus binding and infectivity of host cells in vitro. Sialyllactose, the carbohydrate moiety of GM3, is approximately 3 orders of magnitude less effective than GM3 at inhibiting rotavirus binding to cells. Furthermore, production of therapeutic quantities of GM3 ganglioside for use as an oral carbomimetic in swine is cost prohibitive. In an effort to circumvent these problems, a sialyllactose-containing neoglycolipid was synthesized and evaluated for its ability to inhibit rotavirus binding and infectivity of host cells. Sialyllactose was coupled to dipalmitoylphosphatidylethanolamine (PE) by reductive amination and the product (SLPE) purified by HPLC. Characterization of the product showed a single primulin (lipid) and resorcinol (sialic acid) positive band by thin layer chromatography and quantification of phosphate and sialic acid yielded a 1:1 molar ratio. Mass spectroscopy confirmed a molecular weight coinciding with SLPE. Concentration-dependent binding of rotavirus to SLPE was demonstrated using a thin-layer overlay assay. Using concentrations comparable to GM3, SLPE was also shown to inhibit rotavirus binding to host cells by 80%. Furthermore, SLPE was shown to decrease rotavirus infection of host cells by over 90%. Finally, preliminary results of in vivo animal challenge studies using newborn piglets in their natural environment, demonstrated SLPE afforded complete protection from rotavirus disease. The efficacy of SLPE in inhibiting rotavirus binding and infection in vitro and in vivo, coupled with its relatively low-cost, large-scale production capabilities make SLPE a promising candidate for further exploration as a possible prophylactic or therapeutic nutriceutical for combating rotavirus disease in animals. Most importantly, the results presented here provide proof of concept that the nutriceutical approach of providing natural or synthetic dietary receptor mimetics for protection against gastrointestinal virus infectious disease in all species is plausible.
Highlights
Rotaviruses are a major cause of diarrhea in the young of most mammalian species and the most important cause of severe dehydrating diarrhea in children worldwide
We provide proof of concept that an orally administered, synthetic, neoglycolipid can be used as a therapeutic receptor mimetic for the prevention of Group A rotavirus disease in neonatal piglets
We demonstrated the binding affinity for free sialyllactose, the virus binding epitope of GM3, was three orders of magnitude lower than for the intact ganglioside [35]. Based on these results and the desire to produce an efficacious receptor mimetic to protect against rotavirus disease, we set out to synthesize a neoglycolipid, receptor mimetic that could mimic the activity of the natural ganglioside receptor and be produced in large enough quantities for use as a possible nutriceutical
Summary
Rotaviruses are a major cause of diarrhea in the young of most mammalian species and the most important cause of severe dehydrating diarrhea in children worldwide. In excess of 60,000 deaths caused by rotavirus occur in infants and young children annually [1]. Neonatal piglets and calves are very susceptible to naturally occurring rotavirus disease that is often associated with recent weaning. While rotavirus infection is capable of causing significant morbidity and death loss, most infected piglets survive the infection but remain stunted as a result of villous blunting and fusion combined with maldigestion and malabsorption. Rotaviruses display a tropism for the villous enterocytes lining the small intestine. Some minor variations in the pathophysiological responses exist among species, the most prominent and consistent lesion is villous atrophy
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