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On the Cherokee Indian Reservation and surrounding area of western North Carolina, an area-wide serosurvey was conducted to determine the prevalence of neutralizing antibody to La Crosse (LAC) virus. A questionnaire was used to identify risk factors important in exposure to virus-infected mosquitoes in populations near the reservation. Of 1,008 serum samples tested, 9.6% were positive for LAC virus antibody. For samples solely collected from on (n = 311) or off (n = 697) the reservation, the prevalence of seropositive samples was 20.6% on the reservation and only 4.7% off the reservation. Seropositivity increased directly with age, indicating that transmission of LAC virus was highly endemic. Age and location residence (on versus off the reservation) were significant risk factors for exposure to LAC virus. Persons on the reservation were 5.5 times more likely to have been exposed to LAC virus than were people who reside off the reservation. An additive increase in risk of 1.5 times over each age group was found, so that the oldest age group (≥ 75 years) was 7.5 times more likely to have been exposed to LAC virus than was the youngest age group (< 1–14 years).