SPECIFIC RESTRICTIONS IN THE PROGRESSION OF VENEZUELAN EQUINE ENCEPHALITIS VIRUS-INDUCED DISEASE RESULTING FROM SINGLE AMINO-ACID CHANGES IN THE GLYCOPROTEINS

The pathogenesis of Venezuelan equine encephalitis virus (VEE) was exa mined in the mouse model using V3000, a virus derived from a molecular clone of the Trinidad donkey strain of VEE. These results were compar ed in parallel experiments with avirulent mutants of VEE derived by si te-directed mutagenesis of the clone. Adult mice, inoculated subcutane ously in their left rear footpad with V3000, were followed in a time c ourse study for 6 days in which 15 organs were tested for histopatholo gical changes, for the presence of Viral antigen by immunohistochemica l staining, for the presence of viral nucleic acid by in situ hybridiz ation analysis, and for content of Viable virus. Virus was detected in the footpad inoculation site, but until 12 hr postinoculation (pi), t he level of Virus did not suggest early viral replication. By 4 hr pi, however, replication of V3000 was evident in the draining popliteal l ymph node. At this early time point, no virus could be isolated from a ny other organ examined. At 12 hr, a significant serum viremia was obs erved, and Virus was detected at a low level in a number of well vascu larized organs, including spleen, heart, lung, liver, kidney, and adre nal gland. By 18 hr, high virus titers were present in serum and all t he lymphoid organs examined, and these tissues appeared to be the majo r peripheral sites of V3000 replication. Virus in serum and peripheral organs was cleared by 3-4 days pi. In a second phase of the infection , V3000 invaded the central nervous system (CNS), replicated predomina ntly in neurons, and persisted in the brain until death by encephaliti s. Pathologic findings as well as the results of immunocytochemical an d in situ hybridization examination were generally coordinate with vir us titration. A site-directed mutant of V3000, V3010, contained a muta tion in the gene for the E2 glycoprotein at codon 76 (Glu to Lys) whic h rendered it avirulent after footpad inoculation. Detection of V3010 replication in the draining lymph node was sporadic and was sometimes delayed to as long as 3 days pi. Infrequent and/ or delayed virus spre ad to other sites also was observed. Analogous experiments were perfor med with other mutants which were avirulent by the footpad inoculation route: V3014, a mutant differing from V3000 at three loci (E2 Lys 209 , E1 Thr 272, and E2 Asn 239), as well as single-site mutants V3032 (E 2 Lys 209) and V3034 (E1 Thr 272). The mutations in V3014 prevented sp read beyond the draining lymph node. The single-site E2 Lys 209 mutati on allowed spread to the draining lymph node and to other lymphoid org ans without significant serum viremia or invasion of the CNS, and E1 T hr 272 was characterized by near normal peripheral replication, but on ly sporadic replication in the CNS. These results suggest that VEE-ind uced disease results from a sequence of events which can be defined by interdicting the pathogenic pathway with specific mutations in the VE E genome. (C) 1995 Academic Press, Inc.