The primary objective of this proposal is to characterize the molecular and genetic composition of Flanders virus (FV), a member of the Hart Park Serogroup of the Rhabdoviridae. FV has been shown to have 8 virus-specific proteins and at least 6mRNAs. A cDNA library will be prepared from FV-specific polyadenylated RNA and FV-specific clones selected and grouped by reciprocal-cross hybridization and Northern blots. Selected clones will be utilized in hybrid-selection studies to determine the protein-coding assignment of each unique mRNA. FV genes will be mapped and the intergenic regions identified utilizing available readthrough RNAs as well as by primer extension directly from the genome RNA from synthetic oligodeoxynucleotide primers. Two techniques will be used to sequence the genome 3'end; first, two-dimensional oligonucleotide fingerprint analysis and second, by polyadenylation of the 3'-end and extension of an oligo (dT) primer into the 3'end. RNA from infected cells will be analyzed by RNase protection assay to determine if a plus strand leader RNA is produced. Selected genes such as any unique genes not represented in the vesiculovirus or lyssavirus will be sequenced. A large number of """"""""bullet-shaped"""""""" viruses recovered from arthropods and vertebrates have been assigned to the Rhabdoviridae yet remain uncharacterized and their potential as human and/or animal pathogens unknown. Hart Park Serogroup viruses have been isolated from mosquito species known to feed on humans and antibodies to two members of the group have been detected in humans in Africa. The Rhabdovirus family includes a number of viruses known to be etiological agents of disease in humans and animals yet essentially only the Vesiculoviruses and Lyssaviruses have been intensively studied. Recent findings of more than 5 genetic elements or gene products in specific rhabdoviruses, including FV, emphasize the need for detailed molecular and genetic studies of other Rhabdoviruses. If, in fact, Hart Park Serogroup viruses encode 8 proteins on 6 or 7 mRNAs the mechanism(s) of transcription, translation, or protein processing could provide valuable new insights into the regulation of gene expression in negative-stranded RNA viruses and form a basis for further assessment of the interrelatedness among them, particularly the Rhabdoviruses, Paramyxoviruses, and Filoviruses.
