Trent Rector

Date of Award

12-2001

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Advisor

Eric D. Anderson

Second Committee Member

Keith Hutchison

Third Committee Member

John Singer

Abstract

Infectious salmon anemia virus (ISAV) is an emerging pathogen of farmed Atlantic salmon (Salmo salar). The development of an effective ISA virus vaccine is a high priority for salmon producers in the U.S. and elsewhere. The process of developing a recombinant vaccine requires complete genetic characterization of the virus. Toward this end we have cloned, sequenced and determined the organization of the eight segments of single-stranded RNA from ISA virus isolate CCBB. The virus was grown in cell culture and purified by density gradient ultracentrifugation. Viral RNA was isolated from purified ISAV and used in the construction of two different cDNA libraries. After screening the libraries, individual ISA virus-specific cDNA clones were placed into eight groups and the DNA from a representative clone from each group was sequenced. Using Northern blot hybridization results, the eight representative clones were assigned to specific RNA segments of the ISA virus genome and a genetic map of ISA virus strain CCBB was constructed. In addition, N-terminal amino acid sequence analyses of purified ISA virus proteins correlated protein product(s) to specific RNA segments and provided evidence for protein synthesis initiation sites. Finally, Western blot analysis identified viral proteins that were irnmunoreactive with ISA virus-specific serum from mice and Atlantic salmon.

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