Although simian hemorrhagic fever virus (SHFV) induces a persistent infection in patas monkeys without clinical illness, this virus causes a fatal hemorrhagic fever in monkeys of the Macaca genus. Once an SHFV- infected animal enters a non-human primate captive colony, the virus is easily spread throughout the colony. The disease is rapid; infected macaques become ill within 1-2 days and die within 7 days post-infection. In past epidemics, up to 100% of susceptible macaques within a given facility have become infected with SHFV. Currently, there is no commercially available diagnostic assay to identify SHFV-infected animals. An efficient diagnostic assay is critically needed to identify SHFV-infected monkeys prior to their entrance into a non-human primate captive colony as well as to identify infected animals within a colony. Most non-human primates currently used in the U.S. are involved in AIDS- related research and reliable diagnostic assays are needed for control of endogenous primate viruses in these research animals. This proposal describes a study to develop enhanced diagnostics for SHFV. In order to develop an efficient diagnostic assay for SHFV, the virus must be characterized at the molecular level. The capsid protein and nonglycosylated envelope protein have already been identified and their genes mapped to the viral genome. The number and molecular weights of the envelope glycoproteins will be determined by SDS-PAGE. Antisera, made in mice against purified SHFV particles, will be used to identify the viral structural proteins which contain the antigenic epitopes. The viral RNA genome will be cloned and sequenced so that the envelope glycoprotein genes can be mapped. The antigenic protein genes from other SHFV isolates, obtained from SHFV epizootics in the U.S., will then be sequenced directly from the RNA genomes to determine the extent of sequence variation between the isolates. An antibody-capture assay will be developed using expressed SHFV fusion proteins synthesized in bacteria as antigen. This assay will be tested against antisera from monkeys infected with each of the various SHFV isolates to determine the detection range of this assay. Additional diagnostics will be developed using conserved sequence regions on the viral genome. A transcription amplification system will be utilized to amplify conserved genomic regions from SHFV present in serum samples an PCR will be used to detect SHFV RNA from paraffin-embedded, infected tissues. PCR products will be identified by Southern blot analysis.
