Date of Award

1999

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Elmer K. Godeny

Abstract

The goal of this project was to determine the genome organization, replication strategy and identification of the large envelope glycoprotein (GL) gene of simian hemorrhagic fever virus (SHFV). This virus is the fourth member of the new virus family Arteriviridae. The 3' end of the RNA genome from the prototype isolate of SHFV was cloned and sequenced. This 6,314 nucleotide (nt) sequence consisted of nine complete overlapping open reading frame (ORFs 2, 2a, 3, 4, 5, 6, 7, 8, and 9). At the 5' end of this sequence is a partial ORF, ORF 1b, and the 3' end is a noncoding region. The sequence data showed that SHFV encodes three additional ORFs as compared to the other arteriviruses. The nine SHFV 3' ORFs were presumed to be translated from separate subgenomic mRNAs (sgRNAs) produced during virus replication. However, RT-PCR analysis using leader- and ORF-specific primers identified only eight sgRNA species. The sequence, 5'-UCNUUAACC-3', was identified as the junction motif between the mRNA body and the 5' leader sequence. An ORF 2b-specific sgRNA was not detected. The SHFV particle consists of two proteins, p15 and p20, and at least two glycoproteins, p42 and p54. Endoglycosidase treatment of purified SHF virions showed that p42 and p54 were actually four separate peptides with molecular masses between 28 and 30 kDa. Thus, the SHFV particle contains at least six structural proteins. In vitro synthesis of the ORF 7 product resulted in a peptide with a molecular mass of 31 kDa; this mass increased to 52 kDa when the in vitro product was glycosylated. However, when the glycosylated ORF 7 product was treated with endoglycosidase, the molecular weight of the peptide shifted to about 29,000. Immunoprecipitation results showed that the ORF 7 product was one of the SHFV GL proteins (p54). Four antigenic epitopes were identified using monoclonal antibodies generated by genetic immunizations of mice with the ORF 7 gene. One of these epitopes was identified as a virus neutralization site. The ORF 7 genes from two additional SHFV isolates were compared to the ORF 7 gene from the prototype isolate. No sequence difference was observed among these three isolates.

ISBN

9780599252493

Pages

115

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