Progress 10/01/03 to 09/30/04
Outputs
Since the last progress report for USDA award 2002-35204-12587, much work has been accomplished towards the completion of both aims 1 and 2. For aim 1 we are currently assessing the relative fitness of three IHNV genotypes known to co-circulate within the rainbow trout farm industry in Idaho. We hypothesize that these viruses will be of equal or nearly equal fitness and therefore exhibit a relative fitness ratio of 1:1. To measure this ratio, a complete fitness assay is performed in two stages. The first is the wetlab portion involving a novel in vivo procedure that allows for viral competitions in isolated individual fish; The following stage is molecular analysis of progeny virus populations using RT-PCR and restriction enzyme digests to quantify relative proportions of two virus genotypes in mixed virus populations. To date, we have completed the wetlab portion of the fitness assay for two new competition pairs B:D and C:D, and we have repeated it for the pair B:C.
Each of these 3 competition experiments contained 60 fish, which has proven to be a sample size large enough for statistical significance. RNA extractions have been conducted for all fish from the 3 experiments (180 fish total) and a preliminary molecular analysis of competition pair C:D confimred co-infections. In the coming month we will complete the progeny analysis of these fitness assays. Toward aim 2 we have completed our first assay assessing the relationship between viral fitness and virulence by competing genotypes of high (220-90) and low (WRAC) virulence. An analysis of 31 fish co-infected with WRAC and 220-90 resulted in a fitness ratio of 1:1.9 revealing a significantly higher fitness for IHNV strain 220-90. This provides the first suggestion that viral fitness correlates with virulence and adds validity to the use of the in vivo competition system for assessing fitness of IHNV genotypes pairs of both equal or unequal fitness. Although optimization of the complete fitness
assay is ongoing, we have been able to demonstrate both the accuracy and reproducibility of this methodology. Multiple laboratory personnel have been trained in conducting in vivo competition assays as well as in the molecular analysis of mixed virus populations. In an attempt to maximize throughput of this assay, we are currently integrating quantitative PCR technology as an alternative method for progeny analyses. During the remaining granting period, we anticipate completing all assays for both aims and we hope to have a high throughput fitness assay system that will ultimately be used to elucidate the role of viral fitness in many important phenomena such as virus geographic distribution patterns, co-circulation, and epidemiology.
Impacts
The work in this proposal involves the first explorations of a completely novel system for assessing viral fitness in a truly in vivo environment, during replication within natural vertebrate hosts. This system may lead to better understanding of virus fitness and how viruses interact when two types of virus co-infect vertebrate hosts. This knowledge will provide insight into the relevance of virus competitions in cell culture systems,which are currently used for many mammalian and human viruses.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
Funding became available for USDA award 2002-35204-12587 on 9/15/2002. In order to complete a previously funded commitment the post-doctoral associate for this project did not begin work toward this award until May 2003, and he did not begin drawing his salary from this award until 8/1/03, at which time he began 100% time commitment. This is reflected in form AD-419. This post-doctoral associate will be responsible for all work on the project, which was revised to the first two scientific aims due to budget limitations. To date the post-doc has been trained in fish husbandry and logistics of conducting in vivo virus competition assays in the wetlab, and in molecular analysis of mixed virus populations in progeny fish by differential restriction enzyme digests and genotype-specific PCR. Preparation of individual virus genotypes selected for competition assays involves propagation of large volume virus stocks in fish cell lines and highly accurate titering of those
stocks. Each genotype is then used individually to derive in vivo growth curves in juvenile rainbow trout under the conditions to be used for the eventual competitions. These growth curves identify any differences in growth kinetics between two genotypes to be competed in vivo, and allows selection of an appropriate time for harvest of each competition, when each member of the genotype pair has reached a plateau of amplification in vivo. This somewhat elaborate preparatory work has been completed for eight of the ten virus genotypes necessary to conduct the competitions in aims 1 and 2, to assess the role of viral fitness in geographic distribution and virulence of IHN virus. The in vivo growth curves indicated that all genotypes tested thus far reach a plateau after 3-5 days of in vivo replication, allowing us to select harvest timepoints suitable for testing genotype pairs in competition assays. Complete fitness assays with IHNV genotypes B and C have been run twice to fulfill the
first of three genotype pairs to be run for research aim 1.1. The results of these assays confirmed our hypothesis that these genotypes are of nearly equal fitness, with relative fitness ratios of 1.03 and 1.10, and also showed that the assay itself is reproducible. The next assays to be conducted will be for aim 2, to test genotype pairs that differ in virulence. A stock of rainbow trout is available for these assays and will reach the required size of 1 g within a month. The most important accomplishments in this first year were completion of training, generation of large scale viral stocks for genotypes needed for assays in both research aims, and demonstration that the different genotypes do not vary dramatically in in vivo replication kinetics. In the coming year we anticipate running most assays for both aims, to test genotype pairs predicted to have both equal and unequal fitness in this novel system. This should determine if our hypotheses regarding fitness are valid, and
inform us about modifications that may be needed to improve this novel assay system.
Impacts
The work in this proposal involves the first explorations of a completely novel system for assessing viral fitness in a truly in vivo environment, during replication within natural vertebrate hosts. The great majority of viral fitness work has been done in cell culture, and there are a very small number of systems for assessing viral fitness in tissue explants, but there are no in vivo models in whole animals that have adaptive immune systems. Thus, the results of the virus competitions here will comprise a foundational advance in understanding viral fitness, and how this parameter functions in complex viral transmission and replication cycles in living hosts. In the future we hope to see this system used to elucidate the role of viral fitness in many important phenomena such as geographic distribution patterns, virus co-circulation or displacement events, virus epidemiology. Ultimately this system may prove to be useful in a predictive sense, if relative fitness
values are found to correlate with viral predominance in the field. At this stage the system provides new insights into how to assess viral fitness in vertebrate hosts, and it may eventually serve as a model for development of additional virus fitness models, such as in mice.
Publications
- No publications reported this period
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