Since the discovery of White Spot Syndrome Virus (WSSV) in the early 1990s, this virus has killed more farmed shrimp than all other pathogens combined. Naturally, WSSV has also become the most-studied shrimp pathogen, with nearly two thousand published studies on PubMed. Despite this considerable body of information, the exact mechanism by which the virus transmits from host to host remains poorly understood. As a consequence, experimental models to mimic natural transmission under laboratory conditions have remained difficult to establish.

Ghent University (UGent) in Belgium and its spin-off company IMAQUA have been contributing to the research efforts on this topic as well. Following the PhD defence of Natasja Cox (NC), under the promotorship of Professor Dr. Hans Nauwynck (HN) and Dr. João Dantas-Lima (JDL), the three investigators sat down to talk about the past, present and future of WSSV research.

How did the WSSV research start at Ghent University, and what have been the main areas of interest?

HN: The first studies on WSSV in Ghent started in 2002 with the standardization of inoculation procedures and studies on the pathogenesis. This was fundamental work that was essential to allow for experiments to be sufficiently controlled to make them reproducible in time. However, a downside was that artificial infection routes were used to make sure that the shrimp were infected with a known dose.

In order to also study the virus in its hosts when entering via its natural route(s), a long quest ensued during which consecutive projects investigated WSSV entry through wounds, freshly molted cuticle, the midgut, etc., all contributing to the understanding of WSSV infection. A significant breakthrough was achieved when the antennal gland was found to be a portal of entry for the virus from the rearing water. Still, it would take until 2023 for a joint PhD with IMAQUA to lead to the establishment of an inoculation protocol that could reliably reproduce natural infections.

What is IMAQUA and how did the Ghent University join for a PhD project?

JDL: IMAQUA is a contract research organization that was spun off from Ghent University (UGent) in 2015. I founded the company while being a postdoctoral researcher at the lab of Professor Hans Nauwynck. Located in Belgium, IMAQUA provides services to companies looking to test novel feed ingredients, efficacy of genetic selection and health products for shrimp, in particular with activities targeted at shrimp immunity or pathogens.

As there is still great demand for treatments against WSSV, a Baekeland project was conceived by a former PhD student from UGent, Dr. Mathias Corteel, IMAQUA’s R&D manager. Based on his own past work, a new series of experiments was designed on a much larger scale, which had become feasible thanks to IMAQUA’s expanded facilities.

Contrary to the past, when shrimp had to be imported in small quantities, P. vannamei shrimp are now being bred in-house in Belgium and are available in the thousands.

HN: Including Mathias and João, Natasja was my 10th PhD student on the topic of shrimp diseases. It’s a great synergy with this type of Baekeland funding that a PhD student can continue the UGent research line while providing direct solutions for the contract research business of the company.

Natasja, what were the main findings of your work?

NC: The main purpose of our work was to develop standardized and reproducible WSSV infection models that can provide meaningful data on the efficacy of possible mitigation strategies against WSSV.

We took a step back and reassessed the basic concepts for experimentally infecting shrimp by performing a comprehensive study of the existing literature and evaluating the benefits and limitations of different infection model types. Since oral inoculation by feeding on WSSV-infected tissues mimics one of the natural transmission routes of WSSV in shrimp, we subsequently developed several oral inoculation procedures and inoculum types, and we rigorously tested them in individually housed shrimp to develop our model.

A WSSV challenge model via per os route in which shrimp are housed individually has certain advantages. It allows for the accurate collection of research data in a highly controlled scientific setting while increasing the throughput in selecting and developing infection control tools. The clinical outcomes on the level of the individual shrimp can also be evaluated in detail. It is also true, however, that a challenge in a group simulates the on-farm reality of a WSSV outbreak more closely, as it allows for disease transmission between shrimp. That is why a substantial part of our research also focused on the development of a robust WSSV group challenge model. By evaluating different experimental conditions and densities on a large scale, we obtained successful results.

To further characterize these infection models, we then conducted an epidemiological study on the horizontal transmission dynamics of the Thai-1 WSSV strain that is stored at the facilities of IMAQUA. First, we recorded the shedding of the virus from individually housed shrimp, seeing that the peak of virus in the water occurs within 12 hours of the time of death, and by isolating shrimp from the infected population, we were able to pinpoint that transmission to a new host occurs around that period.

In a second set of experiments, we wanted to evaluate the importance of specific environmental components that might be involved in the transmission of WSSV, so we exposed uninfected (sentinel) shrimp to feces and molt skins collected from tanks housing WSSV-infected shrimp. Surprisingly, these materials did not result in the efficient transmission of the infection. However, water from the tanks with a prior WSS outbreak did yield a 100% infection in naïve shrimp. This waterborne transmission had the same efficiency as direct cannibalism on infected tissues. This finding led us to hypothesize that the cannibalism of infected shrimp actually contributes to indirect water-borne WSSV transmission by the spread of free infectious viral particles in the water.

HN: Most of the WSSV studies in the past at UGent were done in setups with individually housed shrimp, this to reduce noise due to secondary infections. However, Natasja has now proven by extensive testing that the probability that a shrimp kept alone in a 10L aquarium could be infected with WSSV by exposing it to infected tissues was lower than when it was housed in a group. Then, through her research, methods were devised to increase the probability of infection in shrimp that were housed individually. When shrimp are housed in groups in 10 or 290L aquaria, the infection dynamics are different, and all animals become infected over time, either directly by viral particles in the inoculum or due to transmission between the animals. These beautiful results were only possible because of the larger scale of the experimental setups at IMAQUA, and the hard and systematic work of Natasja and IMAQUA’s staff.

What are the practical applications of these results?

NC: When inoculating individually housed shrimp with WSSV-infected inoculum, we now have much more control over the challenge dose and probability to establish an infection. These insights have already been integrated into the contract service of IMAQUA to offer more testing possibilities, adapting to the specific research needs of clients. Additionally, we now have a fine-tuned WSSV group infection model that is suited for epidemiological studies. This model comes the closest to the real situation in shrimp ponds, while it has the advantage that the conditions are controlled. As a consequence, our WSSV challenge is much more reproducible between trials compared to field trials while still providing a relevant simulation.

JDL: The application of Natasja’s work will go into all types of WSSV studies, from screening for genetic resistance to testing out interventions that inactivate the virus particles. As part of the review paper, we described flow charts to aid in determining the suitability of the various WSSV challenge models.

What work is still underway at Ghent University and IMAQUA?

HN: At UGent, two more PhDs are in the final stages of completion. One of the topics is focused on the shrimp’s immune cells, the hemocytes. We discovered a subpopulation of hemocytes that are similar to natural killer cells in vertebrates. These cells are upregulating their cytotoxic molecules during WSSV infection. In the other topic, we described a form of behavioral fever in shrimp, where WSSV-infected animals will move to warmer water to slow down the progression of the infection. Additionally, we are still making headway on the development of cell cultures from shrimp, including a patent for a new specialized culture medium. In four years, I will retire, and I will look back with pride on the progress that we made at UGent, together with all the students and collaborators, over the course of the past 22 years.

JDL: At IMAQUA, we currently have three Baekeland students who have started their PhD work, not on WSSV, but on microsporidia and on shrimp immunity. A new type of microsporidia emerged about 10 years ago, the recently renamed Ecytonucleospora hepatopenaei (EHP), which infects the digestive system of P. vannamei. We aim to establish a controlled challenge model at IMAQUA with this pathogen and, in particular, reproduce white feces disease (WFD), which is thought to be a complex disease caused by EHP and other co-factors. The research line on immunity is focused on establishing reference values of several crucial immune parameters, including antiviral. For all three projects, we are continuing the collaboration between IMAQUA and Ghent University with Professor Dr. Annelies Declercq, who took over as director of the Laboratory of Aquaculture & Artemia Reference Center from emeritus Professor Dr. Peter Bossier.

References:

Cox N., De Swaef E., Corteel M., Van Den Broeck W., Bossier P., Dantas-Lima J.J., Nauwynck H.J. The Way of Water: Unravelling White Spot Syndrome Virus (WSSV) Transmission Dynamics in Litopenaeus vannamei Shrimp. Viruses 2023, 15, 1824. doi: 10.3390/v15091824

Cox N., De Swaef E., Corteel M., Van Den Broeck W., Bossier P., Nauwynck H.J., Dantas-Lima J.J. Experimental Infection Models and Their Usefulness for White Spot Syndrome Virus (WSSV) Research in Shrimp. Viruses 2024. 16, 813. doi: 10.3390/v16050813

Cox N. Analyzing transmission dynamics of White Spot Syndrome Virus in Pacific white shrimp. GSA Advocate 16 JAN 2024.

The PhD project received funding through a Baekeland mandate from Flanders Innovation and Entrepreneurship. (HBC.2020.2896).

AUTHORS:

Mathias Corteel, Ph.D., IMAQUA

Natasja Cox, IMAQUA and Laboratory of Virology, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University

Evelien de Swaef, Ph.D., IMAQUA

Annelies Declercq, Ph.D., Laboratory of Aquaculture & Artemia Reference Center, Department of Animal Sciences and Aquatic Ecology, Faculty of Bioscience Engineering, Ghent University

Peter Bossier, Ph.D., Emeritus from Laboratory of Aquaculture & Artemia Reference Center, Department of Animal Sciences and Aquatic Ecology, Faculty of Bioscience Engineering, Ghent University.

Hans Nauwynck, Ph.D., Laboratory of Virology, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University

João Dantas-Lima, Ph.D., IMAQUA, Corresponding author