|
The Problem Viral infections are common causes of disease outbreaks and mortalities in both wild and captive fish populations. Due to the long history of virus outbreaks there is a substantial body of knowledge about viruses within the field of fish pathology. This project involves research and development studies which create, modify and apply modern biological technologies to increase our understanding of the basic biology, molecular biology, epidemiology, immunology, and host-pathogen interactions of viruses that cause significant disease in salmon and trout in the western U.S. Due to the major significance of infectious hematopoietic necrosis virus (IHNV) as a viral pathogen of salmonid populations of the Pacific Northwest, many of these studies involve IHNV, but other viruses are also being investigated, such as viral hemorrhagic septicemia virus (VHSV). Objectives
Specific objectives addressed include: (1) Characterization of the genomes of various fish viruses to determine gene organization, protein products, and regulatory strategies; (2) generation of molecular clones of fish viruses as tools for novel diagnostic, control, and research applications. (3) development of molecular probe technologies for rapid diagnosis of fish viruses; (4) methods for distinguishing between virulent and non-virulent strains of viruses such as viral hemorrhagic septicemia virus (VHSV); (5) investigation of viral protein interactions at the cellular level; (6) characterization of the immunological basis of protection against viruses in fish hosts; and (7) application of high resolution molecular techniques to virus competition studies that investigate viral ecology and fitness within hosts. Methodology
These various studies all utilize standard methods of molecular biology such as various types of polymerase chain reaction (PCR), extraction of RNA and DNA from cells and tissues, nucleotide sequence analysis, cDNA cloning, hybridization and blotting, restriction digestion analysis, and gel electrophoresis. Specific methods vary according to the needs of the objectives, which are quite diverse. Additional methods in cell culture and tissue harvesting are essential components of these studies. Highlights and Key Findings
The complete genome sequence of the rhabdoviruses spring viremia of carp has been determined and submitted to Genbank. It is part of a manuscript that is submitted for publication. Studies by PCR of IHNV in frozen processed fillets have also been completed and published, indicating no significant risk of transferring IHNV in this form. We are currently conducting preliminary studies for a diagnostic probe to detect the iridovirus viral erythrocytic necrosis (VEN) virus. The study of IHNV population structure within individual fish has been completed and is being written as a manuscript for publication. Our findings indicate that natural IHNV infections in several wild an cultured hosts have extremely low levels of genetic diversity, possibly contributing to the overall low diversity observed for IHNV in the field. Finally, our wetlab system for infection of multiple individual fish has been applied as an in vivo competition system to assess the role of viral fitness and competition in mixed infections. The data from this system can now be used to derive relative fitness ratios between pairs of virus types from the field. A study with two virus types known to co-circulate in the Idaho trout industry has indicated near equal fitness, confirming our hypothesis and suggesting that fitness may play a major role in virus interactions in the field. Modification of the system to test virus types hypothesized to be of unequal fitness are underway. Where Are We Headed In 2003 In the coming year we will continue to develop and improve novel molecular tools and methodologies useful to fish health. This includes tools for known viruses, and for viruses that emerge as health problems in the future. We will apply these tools to investigate both applied and basic questions such as making better diagnostic protocols and understanding the host immune response. We plan to develop a diagnostic probe for the iridovirus VEN, and use it to assess fish and virus movements and temperature effects on virus expession. The manuscript on IHNV population structure in individual fish will be completed and submitted, and more host populations will be assessed for fish-to fish variation. We will increase our efforts in using the wetlab model for IHNV competition within multiple individual fish. To date we have shown that two IHN strains that originated from the same farm in Idaho appear to have the same fitness. We will confirm this by repeating the study and expand the system to test additional virus pairs hypothesized to have unequal fitness. These studies comprise the first in vivo method for assessing virus fitness and ecology within individual hosts. Project Contact Gael Kurath Email: gael_kurath@usgs.gov Publications |
