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EECG Embarkation: Quantitatively exploring viral evolution in vaccinated hosts

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About the Author:

Isabelle Danforth is an incoming Ph.D. student at the Virginia Institute of Marine Science (VIMS) in Dr. Andrew Wargo’s lab and a Virginia Sea Grant fellow. She received a B.S. in Marine Biology from the University of California, San Diego, and an M.S. in Marine Science from VIMS. Her M.S. research centered on determining the impact of imperfect vaccination on virulence evolution of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). She is fascinated by the evolutionary ecology of aquatic viruses and their impacts on marine ecosystems. Follow Isabelle on Bluesky @isabarbell.bsky.social and visit her blog (@virusea.blog).

The way in which viruses evolve in response to vaccination determines the long-term efficacy of these intervention strategies. This is particularly relevant in aquaculture, where rapid industry expansion has coincided with an increase in viral diseases and a growing reliance on vaccines. In the highly profitable rainbow trout (Oncorhynchus mykiss) industry, infectious hematopoietic necrosis virus (IHNV) poses an existential threat to the future of trout farming and the well-being of feral salmonid populations (Bootland, L.M and Leong, J.C 1999).

Fig. 1: A generalized protocol of the serial passage experiments. 6 experiments were completed in total to optimize the method. (1) The initial cohort of fish (P0) were challenged to a controlled dose of IHNV. (2a) In the cohabitation method, fish were then transferred into tanks containing uninfected fish (P1) to initiate the next infection. (2b) In the homogenization method, fish in P0 were homogenized, and the supernatant was added to a tank of uninfected fish to initiate the next infection. In (3a, 4a), the same cohabitation protocol was followed, and in (3b, 4b), the same homogenization protocol was followed. At all passage steps, the previous cohort of fish was stored at -80˚C for later viral isolation and RNA extraction. Passages were continued for 4-12 passages, depending on the experiment.

Fortunately, several vaccines now exist for IHNV, including a commercially available DNA vaccine. Although this vaccine substantially reduces mortality, it does not eliminate viral shedding or fish-to-fish transmission, allowing the virus to continue circulating among vaccinated fish (Jones et al. 2020; Doumayrou et al. 2025; Corbeil et al. 2000). Under these conditions, selection may favor vaccine escape mutants or increasingly virulent strains that cause greater host mortality (Gandon et al. 2001; McLeod and Gandon 2022). My work has empirically demonstrated that more virulent IHNV isolates experience a fitness advantage in the presence of vaccines, implying that imperfect vaccination may drive the evolution of increased virulence in this system (Danforth et al. in prep.). However, it remains unclear which specific mutations correlate with epidemiologically relevant viral phenotypes, such as virulence, replication rate, and infectivity, in the presence or absence of vaccination.

To address this, I (painstakingly, over many experiments) developed a method of in vivo serial passage to experimentally evolve IHNV in vaccinated or unvaccinated trout. In brief, an initial cohort of fish is infected by exposure to a known dose of IHNV. When fish are shedding at high levels, they are transferred to a new tank containing uninfected fish of the same vaccine status or homogenized to initiate infections in this next cohort. In principle, the previously infected fish will transmit IHNV to the new fish, thereby approximating a natural transmission chain and allowing the virus to adapt as it moves from one group of hosts to the next (Fig. 1 & Fig. 2)

Fig. 2: The serial passage experimental design from our fifth iteration of the protocol. Each tank contains 8 fish per tank, 4 from cohort n (e.g., P0) and 4 from cohort n + 1 (e.g., P1). After a 48-hour cohabitation, the fish in cohort n were euthanized and stored at -80˚C, while those in cohort n + 1 were transferred into tanks containing cohort n + 2.

Thanks to the EECG Research Grant, we will now sequence genomes from fish homogenates collected throughout the passage experiments (capturing the full diversity of variants present in the viral population), as well as from viruses isolated on cells. These data will reveal how vaccination and passage method influence evolutionary rate, transmission bottleneck size, and viral diversity. We will then pair these findings with in vivo experiments measuring virulence, replication rate, and infectivity in vaccinated and unvaccinated fish to elucidate the genomic drivers of IHNV virulence and their adaptive benefit in the presence of imperfect vaccines.

However, such analyses are complicated and the prospect of teaching myself these specialized skills through textbooks and YouTube videos alone was daunting. How does one measure strength of selection in practice? Which program should I use to determine the best-supported phylogenetic tree? How do I even download it? Fortunately, I had the opportunity to attend the Marine Biological Laboratory Molecular Evolution Workshop this summer, where I learned to conduct many of the very analyses this project requires (Fig. 3, hyperlink to course syllabus). The techniques I learned there will now allow us to deeply investigate vaccine-driven viral evolution in our system, with the goal of improving vaccine longevity and efficacy. 

Fig. 3: The author alongside Dr. Jeremy Brown (left), and Dr. Claudia Solis-Lemus (right), who coordinated the MBL Molecular Evolution workshop this year.

Vaccines remain among the most significant biotechnological advancements of the modern era. They are absolutely essential to the continued success of fish aquaculture, which supports millions of people across the world and provides an inexpensive source of proteins for a growing global population. However, the potential for viruses to evade immune responses or increase in virulence threatens the long-term viability of available vaccine formulations (Day et al. 2022; Pamornchainavakul et al. 2025). With the support of the EECG Research Grant, this project will reveal evolution-proof vaccine targets and further demystify the process of viral adaptation in immunized hosts.


References:

Bootland, LM, Leong, JC (1999) Infectious hematopoietic necrosis virus. In Fish Diseases and Disorders. CAB International. 57-112.

Corbeil, S, LaPatra SE, Anderson ED, and Kurath G (2000) Nanogram quantities of a DNA vaccine protect rainbow trout fry against heterologous strains of infectious hematopoietic necrosis virus. Vaccine. 18. 2817–2824. 

Day T, Kennedy DA, Read AF, Gandon S (2022) Pathogen evolution during vaccination campaigns. PLOS Biology. 20. e3001804. 

Doumayrou J, Frazier MG, Brown HN, Wargo AR (2025) Efficacy of three vaccine regimens against infectious hematopoietic necrosis virus transmission potential in rainbow trout. Vaccines. 13. 864. 

Gandon S, Mackinnon MJ, Nee S, Read AF (2001) Imperfect vaccines and the evolution of pathogen virulence. Nature. 414. 751–755. 

Jones DR, Rutan BJ, Wargo AR (2020) Impact of Vaccination and pathogen exposure dosage on shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout. Journal of Aquatic Animal Health. 32. 95–108.

McLeod DV, Gandon S (2022) Effects of epistasis and recombination between vaccine-escape and virulence alleles on the dynamics of pathogen adaptation. Nature Ecology & Evolution. 6. 786–793. 

Pamornchainavakul N, Paploski IAD, Makau DN, et al (2025). Experimental evidence of vaccine-driven evolution of porcine reproductive and respiratory syndrome virus type 2. Virus Evolution. 11. veaf056.

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