Virology 169:354-364

Virology 169:354-364. Under a tentative cutoff value (0.122) statistically calculated from NS1 antibody levels of horses in an area where JEV is not endemic, a high level of qualitative agreement (85.3%) was obtained between the ELISA and immunostaining methods. A significant correlation coefficient (0.799; < 0.001) was also obtained between the two methods. Three experimentally infected horses seroconverted NU7026 no later on than 13 to 23 days postinfection, whereas 4 field horses infected during an epizootic remained positive for NS1 antibodies for at least 40 weeks. Our results indicate the ELISA used here was sufficiently sensitive to detect subclinical infections in vaccinated equine populations. Japanese encephalitis disease (JEV) is a member of the genus Flavivirus in the family (4). Inside a paradomestic environment, the disease is maintained inside a transmission cycle between amplifier swine and vector mosquitoes (26). Humans NU7026 are infected by bites from infectious mosquitoes and consequently develop neurological diseases at subclinical:medical ratios of 25:1 to 1 1,000:1 (31). This disease is definitely distributed in many areas of Asia and parts of Oceania, with 50,000 human being instances and 10,000 deaths per year reported (5). In Japan, several thousand human being Japanese encephalitis (JE) instances were reported yearly prior to the mid-1960s (8). However, a national JE vaccination system offers successfully controlled the disease, keeping the annual quantity of confirmed human being instances to fewer than 10 during the last decade (28). Horses will also be susceptible to JEV. Although most infective mosquito bites result in subclinical infections, some infected horses develop fatal encephalitis, much like humans. Large epizootics producing several thousands of equine JE instances in one year were reported during the 1930s and 1940s (6). Following a intro of inactivated JE vaccine in 1948, the number of reported instances decreased markedly (22). In recent years, racehorses, which have a substantial economic value, have been vaccinated every year prior to the epizootic time of year in Japan. No JE instances have been reported in racehorses since 1986 (19). The recent reduction in human being and equine JE instances following vaccination shows that vaccination offers contributed to the control of JE (9). On the other hand, the reduction in JE instances might also have been the result, or at least in part, of less contact with infected mosquitoes (8). The use of insecticides and improved irrigation techniques for rice cultivation offers reduced the number of vector mosquitoes. In addition, the structure and locations of pigpens have been changed so that pigs are now less frequently exposed to mosquito bites. As a result of these changes in Japan, concerns about side effects from your inactivated JE vaccine (1, 3, 23) have caused some to query the necessity of continuing vaccination. The most reliable strategy to address this query is to investigate how often humans and equines acquire natural JEV infections. Regrettably, it has been difficult to obtain natural infection rates among vaccinated populations by standard serologic methods, such as neutralization and hemagglutination-inhibiting (HAI) checks, since these methods are not capable of differentiating antibodies induced by natural illness from those induced by vaccination. A sensitive method based on immunochemical staining for the detection of antibodies to nonstructural 1 (NS1) protein of JEV has been established (14). Individuals vaccinated with inactivated JE vaccine consisting of viral structural proteins develop antibodies only to the structural proteins, whereas illness causes development of antibodies to nonstructural proteins in addition to antibodies to structural proteins. Consequently, detection of NS1 antibodies can determine naturally infected individuals among vaccinated populations. By this technique, a small-scale survey revealed relatively high annual illness rates in humans (5 to 10%) (14) and equines (15 to 67%) (13). Based on these results, it seemed desired to carry out a larger-scale survey to show more clearly the situation in respect to exposure of humans and equines to natural illness with JEV. Enzyme-linked immunosorbent assay (ELISA) is simpler than immunostaining and more suitable for larger-scale studies. However, ELISA offers appeared less sensitive; while previous results showed that ELISA could detect NS1 antibody levels induced in sera of medical instances, those of subclinical infections were DLL1 not recognized (27). For this reason, an original immunostaining method that may be used instead of ELISA was previously developed (14). In the present study, we develop an ELISA method that is equally as sensitive as the earlier immunostaining method for detecting NS1 antibodies in horse sera. MATERIALS AND METHODS Antibody production. Monoclonal antibodies to JEV NS1 antigens were generated, based on a method previously explained for generating monoclonal antibodies to NU7026 different antigens (10, 15)..