Human noroviruses (HuNoVs) are the most common cause of foodborne illness, with a societal cost of $60 billion and 219,000 deaths/year. The lack of robust small animal models has significantly hindered the understanding of norovirus biology and the development of effective therapeutics. Here we report that HuNoV GI and GII replicate to high titers in zebrafish (Danio rerio) larvae; replication peaks at day 2 post infection and is detectable for at least 6 days. The virus (HuNoV GII.4) could be passaged from larva to larva two consecutive times. HuNoV is detected in cells of the hematopoietic lineage and the intestine, supporting the notion of a dual tropism. Antiviral treatment reduces HuNoV replication by >2 log10, showing that this model is suited for antiviral studies. Zebrafish larvae constitute a simple and robust replication model that will largely facilitate studies of HuNoV biology and the development of antiviral strategies.

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Human noroviruses (HuNoVs) are an important cause of epidemic and endemic acute 21 gastroenteritis worldwide; annually about 700 million people develop a HuNoV infection resulting 22 in ~219,000 deaths and a societal cost estimated at 60 billion US dollars 1 . The lack of robust small 23 animal models has significantly hindered the understanding of norovirus biology and the 24 development of effective therapeutics against HuNoV. Here we report that HuNoV GI and GII 25 replicate to high titers in zebrafish (Danio rerio) larvae; replication peaks at day 2 post infection 26 and is detectable for at least 6 days. HuNoV is detected in cells of the hematopoietic lineage, the 27 intestine, liver and pancreas. Antiviral treatment reduces HuNoV replication by >2 log10, showing 28 that this model is suited for antiviral studies. Downregulation of fucosyltransferase 8 (fut8) in the 29 larvae reduces HuNoV replication, highlighting a common feature with infection in humans. 30 Zebrafish larvae constitute a simple and robust replication model that will largely facilitate studies 31 of HuNoV biology and the development of antiviral strategies. 32 Large outbreaks of norovirus gastroenteritis are frequent and have a significant impact in terms of 33 morbidity, mortality and health care costs, in particular in hospital wards and nursing homes. 34 Chronic norovirus infections present a problem for a large group of immunodeficient patients, who 35 may present with diarrhea for several months. Furthermore, in countries where routine rotavirus 36 vaccination has been implemented, noroviruses are the most common cause of severe childhood 37 diarrhea resulting in important morbidity and mortality 2 . Knowledge on the biology and 38 pathogenesis of human noroviruses largely depends upon the development of robust and 39 physiologically relevant cultivation systems. A number of such model systems have been reported 40 in recent years but still carry important limitations. HuNoV replication has been reported in large 41animals such as chimpanzees, gnotobiotic pigs and calves. However these animals are either not 42 suited for extensive studies or are, in the case of chimpanzees no longer allowed due to ethical 43 reasons 3-5 . Importantly, a HuNoV mouse model was described in BALB/c Rag-γ c-deficient mice, 44 but only a short-lasting replication was achieved, which limits its applications 6 . Standard cell 45 culture models are to date not available, but first steps towards this have been given by establishing 46 that (i) human B-cells are susceptible to HuNoV and that (ii) HuNoV can be cultivated in stem-47