Pathogenesis- Human Caliciviruses in Gnotobiotic Animals

Human enteric caliciviruses (HuCV) are the leading cause of acute epidemic gastroenteritis and they account for 67 percent of the cases of foodborne illness in the U.S. annually. Despite considerable efforts, focused mainly on the prototype Norwalk HuCV, attempts to cultivate the fastidious HuCV in cell culture or to develop an animal disease model have failed. Our lack of understanding of HuCV pathogenesis, replication, serotypes and host immunity impedes development of strategies to prevent HuCV infections. Based on sequence analysis, 3 genogroups of HuCV exist: Norwalk-like viruses (NLV) I and II and Sapporo-like viruses (SLV). Recently we and others found that enteric caliciviruses from pigs (PEC) and calves (BEC) are genetically more closely related to HuCV than to other animal caliciviruses. Moreover, feces from pigs and calves were positive for SLV and NLV II, and NLV I RNA, respectively using primers for HuCV, suggesting the possibility of animal reservoirs for HuCV or zoonotic strains transmissible to humans. Thus our goal is to develop animal models to study the comparative pathogenesis of human and animal caliciviruses, targeting NLV I strains in gnotobiotic (Gn) calves and NLV II and SLV strains in Gn pigs. New strategies we will explore include the use of seronegative, neonatal Gn animal hosts, alternative inoculation routes (intravenous for delivery of lower virus doses versus oral) and pharmacologic immunosuppression to enhance host susceptibility. Parameters to be assessed include host age, clinical signs, viremia, virus shedding, seroconversion and antibody impact on re-exposure, and organs and cell types infected including antigen and lesion distribution. Methods of analysis include electron microscopy, RT PCR and immunofluorescence for virus detection, ELISA (recombinant VLP capsid) for antibody detection and histopathology for lesion examination. Finally our pair of virulent and attenuated, cell-adapted PEC/Cowden and knowledge of their sequence differences (7 total amino acid differences) allows us to explore the molecular basis for PEC virulence. We plan to construct an infectious clone of the attenuated PEC and systematically mutate it to mirror the sequence of the virulent PEC, then test the mutants for pathogenicity in Gn pigs. Our findings should provide new information on the comparative pathogenesis of human and animal caliciviruses, the potential genetic basis for their virulence and a foundation for future studies of host immunity and preventive strategies.