Abstract Title
Zebrafish Larvae Support Long-term Passaging of Human Norovirus
Presenter
Lorane Molineaux, KU Leuven
Co-Author(s)
Authors: Lorane Molineaux (1), Emma Roux (1), Loes Vanhoof (1), Xander Vyncke (1), Jana Van Dycke (1), Judith Breuer (2), Joana Rocha-Pereira (1)
(1) KU Leuven - Department of Microbiology, Immunology and Transplantation, Rega Institute, Virus-Host Interactions & Therapeutic Approaches (VITA) Research Group, Leuven, Belgium
(2) Infection, Immunity and Inflammation Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London, London, United Kingdom; Institute of Immunity and Transplantation, University College London, London, UK.
Abstract Category
Entry and Replication
Abstract
In vitro and in vivo cultivation of human norovirus (HuNoV) has been challenging, despite recent advancements. While efficient replication was demonstrated in human intestinal enteroids and zebrafish, long-term passaging has been unsuccessful thus far, suggesting replication is still constrained.
We previously reported robust replication of multiple HuNoV genotypes in zebrafish larvae, and now add the long-term, ongoing passaging of a HuNoV GII.4, reaching 14 successful passages to date. The increase in viral copies per fish (vs. inoculum) varies between 4.6 and 2 log10. In a parallel passaging series, the innate immune-dampening compound ruxolitinib was added to the swimming water. Log10-fold increases ranged between 4.7-1.3, but ceased in passage 14. Thus, ruxolitinib does not clearly enhance GII.4 replication, nor facilitates passaging in zebrafish (with the current strain).
Additionally, a HuNoV GII.3 was passaged 4 times in zebrafish larvae with ruxolitinib (4.1-0.8 log10-fold increase), but showed modest replication (0.9 log10-fold increase) after an infection round without ruxolitinib. This corresponds to an earlier report showing GII.3 replication is impaired by the IFN-mediated response, alleviated by ruxolitinib in our experiments.
Whole-genome sequencing is ongoing to infer key mutations for viral fitness and host adaptation. Overall, zebrafish larvae possess all features required for efficient HuNoV replication, including multiple susceptible organs and cell types (as shown in our other studies). The use of a zebrafish-generated virus inoculum could omit the confounding effects of stool samples as HuNoV source. Likewise, the zebrafish model is suitable to study virus evolutionary dynamics, potentially linked to antiviral pressure.
We previously reported robust replication of multiple HuNoV genotypes in zebrafish larvae, and now add the long-term, ongoing passaging of a HuNoV GII.4, reaching 14 successful passages to date. The increase in viral copies per fish (vs. inoculum) varies between 4.6 and 2 log10. In a parallel passaging series, the innate immune-dampening compound ruxolitinib was added to the swimming water. Log10-fold increases ranged between 4.7-1.3, but ceased in passage 14. Thus, ruxolitinib does not clearly enhance GII.4 replication, nor facilitates passaging in zebrafish (with the current strain).
Additionally, a HuNoV GII.3 was passaged 4 times in zebrafish larvae with ruxolitinib (4.1-0.8 log10-fold increase), but showed modest replication (0.9 log10-fold increase) after an infection round without ruxolitinib. This corresponds to an earlier report showing GII.3 replication is impaired by the IFN-mediated response, alleviated by ruxolitinib in our experiments.
Whole-genome sequencing is ongoing to infer key mutations for viral fitness and host adaptation. Overall, zebrafish larvae possess all features required for efficient HuNoV replication, including multiple susceptible organs and cell types (as shown in our other studies). The use of a zebrafish-generated virus inoculum could omit the confounding effects of stool samples as HuNoV source. Likewise, the zebrafish model is suitable to study virus evolutionary dynamics, potentially linked to antiviral pressure.