La Crosse virus (LAC), family Bunyaviridae, is a pathogenic arbovirus recognized as a major cause of pediatric encephalitis in North America. The virus was first identified as a human pathogen in 1960 after its isolation from a 4 year old girl who suffered aseptic encephalitis and later died in La Crosse, Wisconsin. The majority of LAC infections are mild and never reported, however, serologic studies estimate infection rates of 10-30/100,000 in endemic areas. Nonetheless, LAC encephalitis has become the most commonly reported pediatric arboviral encephalitis in the US with 70-130 symptomatic cases a year. Sequence analysis of the complete LAC genomes of low-passage LAC/human/1960, LAC/mosquito/1978, LAC/human/1978, and their respective biological clones, indicate that circulating viruses are genetically stable over time and distance with 100, 99, 98, and 99% amino acid identity for N, NsS, M polyprotein, and L proteins respectively. All three wild type viruses had similar in vitro growth kinetics and phenotypes, growing to titers of 7.2-7.6 logs in Vero cells and 6.8-7.9 logs in C6/36 cells. Cytopathic effects (CPE) of cell rounding and detachment were seen after infection of Vero cells, but no CPE were seen after infection of C6/36 cells. In an effort to develop a laboratory small animal model, neurovirulence (intracerebral inoculation) and peripheral neuroinvasivness (intraperitoneal inoculation) were studied in suckling and weanling Swiss Webster mice. Similar levels of neurovirulence were observed in suckling and weanling mice with a 50% lethal dose of less than 1.7 logs. Interestingly, a terminally diluted clone of the LAC/human/1960 strain was unable to induce clinical disease when inoculated peripherally at a dose of 5 logs compared to a LD50 1-3.08 logs for the two1978 strains or for the uncloned 1960 parental strain. Sequence analysis identified a single, Threonine to Alanine amino acid change at position 148 of the G2 attachment glycoprotein of the attenuated 1960 virus. Efforts are currently underway to develop a reverse genetic system for the characterization of this putative attenuating mutation, and other mutations capable of attenuating LAC are being developed for future vaccine development.