Norwalk-like viruses (NLVs) are a diverse group of single-stranded, nonenveloped, positive-polarity RNA viruses and are the leading cause of epidemic acute gastroenteritis in the United States. In this study, the major capsid gene of Norwalk virus, the prototype NLV, has been cloned and expressed in mammalian cells using a Venezuelan equine encephalitis (VEE) replicon expression system. Upon infection of baby hamster kidney (BHK) cells with VEE replicon particles (VRPs), the Norwalk virus capsid proteins self-assemble to generate high titers of Norwalk virus-like particles (VLPs) that are morphologically and antigenically analogous to wild-type Norwalk virus. Mice inoculated subcutaneously with VRPs expressing the Norwalk virus capsid protein (VRP-NV1) developed systemic and mucosal immune responses to Norwalk VLPs, as well as heterotypic antibody responses to the major capsid protein from another genogroup I NLV strain (NCFL) isolated from a recent outbreak. A second Norwalk virus capsid clone (NV2) containing three amino acid codon mutations from the NV1 clone was also expressed using VEE replicons (VRP-NV2), but upon infection of BHK cells failed to confer VLP self-assembly. Mice inoculated with VRP-NV2 elicited reduced systemic and mucosal immune responses to Norwalk VLPs, demonstrating the importance and potential utility of endogenous VLP presentation for maximum immune induction. Inoculation with either VRP-NV1 or VRP-NV2 resulted in serum antibody responses far superior to the induction in mice dosed orally with VLPs that were prepared using the VEE-NV1 replicon construct, a regimen similar to current models for NLV vaccination. Expression of NLV VLPs in mammalian cells offers a powerful approach for the design of novel NLV vaccines, either alone or in combination with current vaccination models.Norwalk-like viruses (NLVs) are the most frequent cause of epidemic gastroenteritis in the United States, infecting an estimated 23 million people each year, and have accounted for as much as 96% of selected outbreaks of acute, nonbacterial gastroenteritis (20,40,47). Persons of all ages are affected, and it is unclear whether infection typically confers long-term immunity (38). NLV outbreaks commonly occur within families, nursing homes, day care centers, schools, hospitals, and in the military (1,20,40,42). Infection with NLVs usually results in an episode of acute, self-limited gastroenteritis, with symptoms rarely more serious than nausea, vomiting, diarrhea, and lowgrade fever (40), and most outbreaks are likely not reported (47). However, using refined molecular detection methodologies, recent studies have extended the public health significance of NLV infections and suggest that NLVs are a common cause of severe diarrhea in children (22,53). Although further studies are necessary, NLVs may cause as much as 20% of endemic diarrheal disease in family settings in developed countries (42).The mortality attributable to NLV infection remains unclear, but fatalities among infected elderly may not be uncommon. It ...