Norwalk virus (NV) has recently been assigned as the prototype species for a new genus in the Caliciviridae provisionally named the Norwalk-like viruses (NLVs). The Norwalk-like human caliciviruses are the major cause of nonbacterial epidemic gastroenteritis that occurs in family, school, institutional, or community-wide outbreaks affecting adults and school-aged children. The NLVs are genetically diverse and cannot be grown in cell culture, which has hampered the ability to detect these agents in gastroenteritis outbreaks. A major accomplishment in our laboratory this year was to adapt and optimize a broadly reactive RT-PCR assay for routine detection and genetic characterization of the NLVs. This assay was used to examine the genetic diversity of viruses in our outbreak collections. Of particular importance, was a study conducted in Maryland nursing homes for the elderly nearly a decade ago. In a collaborative study between NIH and the Maryland State Department of Health (headed at the time by Dr. Kimi Lin), we had previously analyzed sera and stool specimens that were systematically collected from individuals involved in gastroenteritis outbreaks that occurred over an entire winter season. In our previous work, we had shown a strong association of the NLVs with the etiology of the outbreaks by the examination of serum antibody responses to recombinant capsid proteins developed in our laboratory. This year, we analyzed the stool specimens in the twenty outbreaks from the nursing home study and identified approximately 100 NLV-positive samples by RT-PCR. These samples were then characterized at the sequence level. One NLV strain predominated in this setting as an etiologic agent of diarrhea, firmly establishing the NLVs as the predominant cause of outbreaks in the nursing home setting and demonstrating that a major outbreak strain can circulate in a community over a single season. However, remarkably, at least six capsid genetic types (possibly distinct serotypes) were detected in ill individuals in this setting, illustration the complex molecular epidemiology of the NLVs. The implications for vaccine development from this study are directly related to the goals of this project. Efforts continued in the quest for an animal model or a permissive cell culture system. A Norwalk virus-containing stool filtrate was administered orally or intravenously to two juvenile chimpanzees in order to establish an infectious dose titer. The first dose was administered at a 10-5 dilution. A pre-challenge serum sample was collected and then another serum sample was collected approximately 2 weeks later. The animals did not develop illness and also failed to demonstrate a serologic response to Norwalk virus. Stool samples were collected immediately prior to challenge and then daily for approximately 10 days. Each sample from the two chimpanzees was analyzed by RT-PCR for evidence of virus shedding and Norwalk virus was not detected. The titration continued at sequentially lower ten-fold dilutions until the stool filtrate was administered orally to one of the chimpanzees without dilution. At the completion of the study, there was no evidence for Norwalk virus replication in either chimpanzee. These observations were in contrast to earlier work in LID, in which adult chimpanzees showed evidence for infection after challenge with an aliquot of the same filtrate containing Norwalk virus. The reason for the failure in this experiment to infect juvenile chimpanzees is under investigation, and could yield important insight into possible age- or immunity-related host factors related to susceptibility to infection. In collaboration with Dr. Joshua Zimmerberg of the Laboratory of Cellular and Molecular Biophysics, NICHHD, we performed several experiments using the NASA bioreactor technology in an effort to identify a permissive cell culture for Norwalk virus. CaCo-2 and Int407 human cell lines were cultured in this """"""""three-dimensional"""""""" format. A Norwalk virus-positive stool filtrate from a human volunteer was added to the cells and serially passaged up to six times. The presence of virus was monitored by RT-PCR and Western blotting. In all experiments, the virus fell to undetectable limits by the sixth passage, indicating that the virus was not replicating in either cell line. We will continue to explore other cell lines and growth conditions for the NLVs because the growth of these viruses continues to be a much-needed breakthrough.
