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Human norovirus (HuNoV) is the most common cause of acute gastroenteritis in all age groups. While HuNoV infections are usually acute and self-limiting in healthy individuals, they lead to chronic infections and severe symptoms in immunocompromised people. Currently, no accepted vaccines or antiviral therapies are available for HuNoV infections because development is limited and complicated by the genetic diversity of noroviruses, the emergence of frequent new mutants and recombinants and the lack of in vitro and in vivo models. The overall aim of this master thesis is to increase our knowledge of the intrinsic details of HuNoV replication in vivo through an innovative in house developed model system, namely optically transparent zebrafish larvae. Genotyping using sanger sequencing and RT-qPCR of HuNoV- positive stool samples from the University Hospital of Leuven allowed detection of the most circulating genotypes of HuNoV while mapping their genetic diversity. This showed that the dominant genogroup was HuNoV GII and more specifically the genotype GII.4. The emerging genotype GII.17 was not detected in our study. The replication kinetics of several clinically relevant HuNoV genotypes showed that HuNoV GII.2 [P16], GII.3 [P12] and GII.4 Sydney [P4] replicated well in the zebrafish larval model after measuring viral replication using RT-qPCR at different timepoints post infection. The replication of HuNoV GII.17 [P17] was limited, although it was slightly increased after treatment with ruxolitinib, a selective inhibitor of the JAK/STAT pathway that suppresses the innate immune response. HuNoV GII.4 Sydney [P4] and GII.3 [P12] were passaged from zebrafish larvae to zebrafish larvae in the presence of ruxolitinib to study virus adaptation to the host and eventually produce a HuNoV virus stock. Passaging was successful for HuNoV GII.4 Sydney [P4] and GII.3 [P12] until passage 14 and 5 up to now, respectively (in both cases, the end of this thesis work). The next step will be the complete sequencing of the virus genome of each passage to identify adaptive mutations. In addition, a virus stock can be generated for use in further studies. Alternatively, we used HuNoV-infected zebrafish larvae as inoculum to infect human intestinal enteroids, and observed limited viral replication for both HuNoV GII.4 Sydney [P4] and GII.3 [P12]. Next, we tested a selection of antiviral agents in our zebrafish model using HuNoV GII.4. The active form of remdesivir (GS-441524) or molnupiravir (EIDD-1931) did not lead to a significant reduction in viral load compared to the control. In contrast, pericardial injection of the nucleoside analogs CMX521 and 2CMC led to a significant reduction in viral replication, suggesting their inhibitory effect on HuNoV. As it is still unclear which specific cell types can be infected by HuNoV, we attempted to unravel the cell tropism using a triple immunostaining of transgenic zebrafish larvae expressing fluorescent macrophages. This according to the adapted protocol for primary VP1 antibody, which was optimized as a part of this thesis. The colocalization of the antibodies targeting the macrophages, VP1 and dsRNA intermediate suggested that HuNoV both infects and replicates in intestinal macrophages. The information collected throughout this master’s thesis provides new insights into the intrinsic details of HuNoV replication in vivo. Based on this, more efficient antiviral strategies can be developed against human norovirus.

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Human noroviruses (HuNoVs) are recognised as the major cause of viral gastroenteritis worldwide, accounting for not less than 700 million infections and 219 000 deaths on a yearly basis. HuNoV is linked to sporadic cases of gastroenteritis but can also provoke large outbreaks within communities. Despite great public health, clinical, and economic impacts linked to HuNoV, it remains dramatically understudied and its pathogenesis is still unclear. This is mainly due to the lack of robust in vitro and in vivo replication models, being essential to provide knowledge on the virus biology and for the further development of efficient and specific antiviral treatments or vaccines. Our newly in house developed and robust in vivo model to study HuNoV replication, being zebrafish larvae, provides a first opportunity to study the virus tropism and pathogenesis. We described previously which zebrafish tissues were infected by HuNoV, however, the specific cell types allowing for such an infection remained unclear. In that context, the main objectives of this study were to investigate and define the intestinal host cells and their relative importance, with a special focus on goblet cells and enteroendocrine cells. Their response upon infection was also characterized with the goal of studying their impact on virus dissemination within the host. Flow cytometry results demonstrated the specificity of primary antibodies against HuNoV structural protein VP1 and double-stranded RNA intermediate to successfully label infected goblet cells and enteroendocrine cells derived from zebrafish larvae single cell suspensions. We thereby confirmed the infection of those cell types and provided an estimation of their relative importance as host cells. Regarding the goblet cells response upon infection and particularly their mucins production/secretion, a significant increase in muc 2.1 expression was seen in HuNoV-infected fish. By flow cytometry, a slight increase in the percentage of wheat-germ agglutinin (WGA) positive cells, staining the mucins contained in goblet cells, was also observed in infected fish. Next, to study the role of goblet cells in HuNoV dissemination, retinoic acid treatment was applied to zebrafish larvae. However, we cannot yet determine if the compound can efficiently interfere with goblet cell differentiation, since preliminary data from whole-mount immunostainings after treatment did not show a reduction of the overall WGA signal. Alternatively, a morpholino (MO) knock down of the anterior gradient 2 (Agr2) gene rendering a decreased maturation in goblet cells is currently being explored. However, we are still optimising the doses of MO that confer the expected effect. At this point, there was no significant decrease in WGA signal in whole-mount immunostainings, thus higher MO doses are likely required. The specificity of the antibody against Agr2 in zebrafish larvae lysates was successfully demonstrated via western blot, with the aim to further confirm the effect of the MO. Overall, the information gathered in this research project provides new insights on HuNoV host cell tropism and will help the team to study virus dissemination and pathogenesis in greater detail.