Each error bar represents 1 standard error above and below the mean. virus vaccine trials. After malaria, dengue (DEN) is the most important emerging tropical infectious disease. Worldwide it is estimated that 100 million cases of DEN fever occur each year (8). The severe form of the disease, DEN hemorrhagic fever-dengue shock syndrome (DHF/DSS), is one of the most important causes of hospitalization and death among children in Asia. The average case-fatality rate for DHF is 5% when appropriate supportive treatment is given. DEN viruses (serotypes 1 through 4) are mosquito borne and are the causative agents of these diseases. Since the description of DEN etiology in 1944 (27), considerable effort has been put into vaccine development (5, 23); however, significant difficulties have been encountered. Because antibody-dependent enhancement has been associated with DHF/DSS, a tetravalent vaccine is preferable. DEN virus is not normally pathogenic in mice; therefore, an appropriate and useful small animal model has been lacking, despite numerous attempts at development (24). Evidence exists that alpha and beta interferons (IFN-/) and gamma IFN SOD2 (IFN-) might be involved in human DEN infections (16, 18). In addition, exogenously administered IFN appears to protect mice from DEN virus challenge (2). This information suggested to us that mice defective in their IFN response might provide a suitable model for DEN virus infection. Mice with a 129Sv(ev) background that had deficiencies in IFN responses were developed previously (28). When these mice were used, IFN-/ was found to be important in the regulation of vesicular stomatitis virus and Semliki Forest virus replication (22). IFN–deficient mice had increased susceptibility to tuberculosis (3), whereas the functions of IFN-/ and IFN- in combination appeared to be critical for antiviral defense against Theilers (6), vaccinia, and lymphocytic choriomeningitis viruses (28). To determine if any of the IFN transgenic mouse strains were susceptible to DEN virus infection, mice deficient for IFN-/ and – receptors in combination (AG129), as well as mice lacking IFN-/ receptors only (A129) and their wild-type counterparts (WT129), were obtained from B & K Universal, Hull, United Kingdom. Mice without the ability to synthesize IFN- (GKO) (3) and BALB/c controls were also acquired, as a gift. Initially, groups of five AG129 and five WT129 mice aged 4, 6, 8, Orphenadrine citrate and 12 weeks old were inoculated intraperitoneally (i.p.) with 106 PFU of a mouse-adapted DEN 2 virus strain, New Guinea C. AG129 mice of all age groups began to exhibit neurological abnormalities, including hind-leg paralysis and blindness, around day 7 after inoculation, and mice were dead by day 12. In contrast, none of the WT129 mice showed symptoms. To ascertain whether a smaller challenge dose was appropriate for use in adult mice, 10-fold dilutions of virus were introduced i.p. into 8-week-old AG129 mice. Survival times increased as the viral load decreased and ranged from 10 days (106 PFU) to 28 days (103 PFU). For experimental convenience Orphenadrine citrate and maximum stringency, a challenge dose of 106 PFU was used in subsequent studies. To determine which aspect of the IFN response was Orphenadrine citrate critical in protecting these mice from DEN virus infection, animals individually deficient in either IFN-/ (A129) or IFN- (GKO) functions as well as BALB/c controls were subjected to a similar DEN virus challenge. None of these mice exhibited any overt symptoms of illness, indicating that for DEN virus infection, IFN-, -, and – abnormalities in combination were necessary for the mouse-adapted virus to be lethal when the i.p. challenge route was used. The remote possibility exists that IFN–deficient mice with the 129Sv(ev) genetic background differ from the GKO mice used in this study with respect to their susceptibility to DEN 2 virus. Tissue culture-passaged DEN 2 strain 16681 failed to kill 8-week-old AG129 mice (data not shown), suggesting that mouse adaptation was necessary for DEN virus to be lethal. Viremia was monitored in groups of five DEN virus-infected 6-week-old AG129 mice each and in groups of two WT129 mice each. Plaque titrations of virus recovered from the brain, spleen, Orphenadrine citrate and serum were performed (Fig. ?(Fig.1)1) following inoculation with 106 PFU of mouse-adapted DEN virus. Samples Orphenadrine citrate were taken at days 1, 3, 5, and 9 and at days 10 to 12 after inoculation and homogenized with bovine albuminCphosphate-buffered saline (PBS) medium and sterile sand by using a Bellco grinder. Homogenates were centrifuged at 1,500 for 10 min, decanted, and adjusted to make 10% suspensions. Processed tissues were serially diluted and adsorbed for 45 min onto Vero cell monolayers in six-well plates. Infected cells were overlaid with a mixture containing 1% noble agar, 1 medium.