Identifier

etd-0711102-124445

Degree

Master of Science (MS)

Department

Plant, Environmental Management and Soil Sciences

Document Type

Thesis

Abstract

Two viral outbreaks occurred 3 years apart (1997 and 2000) in the vivarium at the Pennington Biomedical Research Center. The etiology of both outbreaks was determined by enzyme-linked immunosorbent assay (ELISA) to be sialodacryoadenitis virus. The focus of this work was to fulfill Koch’s postulates and definitively confirm the outbreak etiologies using 4 distinct diagnostic laboratory techniques: cell culture, indirect immunofluorescent assay (IFA), transmission electron microscopy (TEM), and reverse transcriptase polymerase chain reaction (RT-PCR). During each outbreak, infected rats were euthanatized, serum was collected by exsanguination, and infected tissues (submandibular salivary glands, Harderian glands, and lung tissue) were harvested and flash frozen in liquid nitrogen for later extraction and analysis of viral particles (field strains). The field strains were compared to the catalogued type-strain virus from the American Type Culture Collection sialodacryadenitis virus Strain 681 (the ATCC strain). Contrary to ATCC strain viral replication in culture on L2p176 (coronavirus permissive) cells, it was determined that the field strain viruses did not grow well in cell culture, but could be propagated in experimentally infected rats. IFA revealed reactivity between the infected rat serum from each outbreak and the ATCC strain, indicating marked antigenic similarity. The viral morphology, ultrastructural cellular pathology, and location of the stages of replication in field strain infected L2p176 cells examined by TEM were consistent with coronavirus infection. Genetic confirmation of the identity of the field strains was accomplished by RT-PCR. ATCC strain sequence analysis revealed a 412 base pair portion of the spike protein gene (total length: approximately 2000 base pairs) that could be amplified for sequence analysis and comparison. The amplification product, produced from each isolate, was purified, further amplified by bacterial plasmid insertion, and compared to each of the other products and to the published PubMed sequence database. Comparison of the fragments revealed homology with published data on the spike protein gene of sialodacryoadenitis virus. This study lays the groundwork for future analysis of the entire spike protein of the field strains, in comparison with the ATCC strain, to determine genetic bases for variations in infectivity and virulence of sialodacryoadenitis virus isolates.

Date

2002

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

David G. Baker

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