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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.

We have recently established that human norovirus (HuNoV) replicates efficiently in zebrafish larvae after inoculation of a clinical sample into the yolk, providing a simple and robust in vivo system in which to study HuNoV. In this Protocol Extension, we present a detailed description of virus inoculation by microinjection, subsequent daily monitoring and harvesting of larvae, followed by viral RNA quantification. This protocol can be used to study viral replication of genogroup (G)I and GII HuNoVs in vivo within 3–4 d. Additionally, we describe how to evaluate the in vivo antiviral effect and toxicity of small molecules using HuNoV-infected zebrafish larvae, in multi-well plates and without the need for specific formulations. This constitutes a great advantage for drug discovery efforts, as no specific antivirals or vaccines currently exist to treat or prevent norovirus gastroenteritis. This Protocol Extension details the use of zebrafish larvae as a simple and robust in vivo system for studying human norovirus infection, enabling evaluation of the antiviral effects and toxicities of small molecules.

Human norovirus (HuNoV) accounts for over 700 million cases of gastroenteritis annually. Episodes of HuNoV disease are characterized by vomiting and diarrhea as the two most prominent symptoms. Despite its prevalence, our understanding of the pathophysiological mechanisms triggered upon HuNoV infection is limited, mainly due to a lack of suitable animal models. Our aim was to use the recent HuNoV zebrafish larvae model to study the effect of HuNoV infection on intestinal motility and investigate whether one viral protein could act as an enterotoxin, as seen with rotavirus. We studied whether HuNoV infection affects the contraction frequency of the intestinal bulb and the posterior intestine as well as the transit time. Infection of larvae, following injection of a HuNoV GII.4-containing stool sample in the yolk, resulted in an increased contraction frequency in the intestinal bulb. A comparable effect was observed in serotonin-treated larvae, corresponding to the natural function of serotonin. The higher replication efficacy of HuNoV GII.4 likely explains why they have a more marked effect on gut motility, when compared to other genotypes. Additionally, transit time of fluorescent food was prolonged in HuNoV GII.4 infected larvae, suggesting a loss of coordination in bowel movements upon infection. To identify the proteins responsible for the effect, individual HuNoV non-structural proteins and virus-like particles (VLPs) were injected intraperitoneally (ip). VLPs carrying VP1/VP2, but not those with only VP1, induced increased contraction frequencies in the intestinal bulb in a dose-dependent manner. In conclusion, our findings suggest that the viral capsid and potentially the minor capsid protein VP2 play a crucial role in the aetiology of symptoms associated with HuNoV, potentially acting as a viral enterotoxin. This work contributes to the understanding of the pathophysiological mechanisms in HuNoV-induced disease and further attests zebrafish as a valuable HuNoV disease model.

The Rift Valley fever virus (RVFV) causes haemorrhagic fever, encephalitis, and permanent blindness and has been listed by the WHO as a priority pathogen. To study RVFV pathogenesis and identify small-molecule antivirals, we established a novel In Vivo model using zebrafish larvae. Pericardial injection of RVFV resulted in ~4 log10 viral RNA copies/larva, which was inhibited by the antiviral 2′-fluoro-2′-deoxycytidine. The optical transparency of the larvae allowed detection of RVFVeGFP in the liver and sensory nervous system, including the optic tectum and retina, but not the brain or spinal cord. Thus, RVFV-induced blindness likely occurs due to direct damage to the eye and peripheral neurons, rather than the brain. Treatment with the JAK-inhibitor ruxolitinib, as well as knockout of stat1a but not stat1b, enhanced RVFV replication to ~6 log10 viral RNA copies/larva and ultra-bright livers, although without dissemination to sensory neurons or the eye, thereby confirming the critical role of stat1 in RVFV pathogenesis.

Human noroviruses (HuNoVs) are the most common cause of viral gastroenteritis resulting annually in ~219,000 deaths and a societal cost of ~USD 60 billion, and no antivirals or vaccines are available. Here, we assess the anti-norovirus activity of new peptidomimetic aldehydes related to the protease inhibitor rupintrivir. The early hit compound 4 inhibited the replication of murine norovirus (MNV) and the HuNoV GI.1 replicon in vitro (EC50 ~1 µM) and swiftly cleared the HuNoV GI.1 replicon from the cells. Compound 4 still inhibits the proteolytic activity. We selected a resistant GI.1 replicon, with a mutation (I109V) in a highly conserved region of the viral protease, conferring a low yield of resistance against compound 4 and rupintrivir. After testing new derivatives, compound 10d was the most potent (EC50 nanomolar range). Molecular docking indicated that the aldehyde group of compounds 4 and 10d bind with Cys139 in the HuNoV 3CL protease by a covalent linkage. Finally, compound 10d inhibited the replication of HuNoV GII.4 in infected zebrafish larvae, and PK studies in mice showed an adequate profile.

Human norovirus (HuNoV) accounts for over 700 million cases of gastroenteritis annually. Episodes of HuNoV disease are characterized by vomiting and diarrhea as the two most prominent symptoms. Despite its prevalence, our understanding of the pathophysiological mechanisms triggered upon HuNoV infection is limited, mainly due to a lack of suitable animal models. Our aim was to use the recent HuNoV zebrafish larvae model to study the effect of HuNoV infection on intestinal motility and investigate whether one viral protein could act as an enterotoxin, as seen with rotavirus. We studied whether HuNoV infection affects the contraction frequency of the intestinal bulb and the posterior intestine as well as the transit time. Infection of larvae, following injection of a HuNoV GII.4-containing stool sample in the yolk, resulted in an increased contraction frequency in the intestinal bulb. A comparable effect was observed in serotonin-treated larvae, corresponding to the natural function of serotonin. The higher replication efficacy of HuNoV GII.4 likely explains why they have a more marked effect on gut motility, when compared to other genotypes. Additionally, transit time of fluorescent food was prolonged in HuNoV GII.4 infected larvae, suggesting a loss of coordination in bowel movements upon infection. To identify the proteins responsible for the effect, individual HuNoV non-structural proteins and virus-like particles (VLPs) were injected intraperitoneally (ip). VLPs carrying VP1/VP2, but not those with only VP1, induced increased contraction frequencies in the intestinal bulb in a dose-dependent manner. In conclusion, our findings suggest that the viral capsid and potentially the minor capsid protein VP2 play a crucial role in the aetiology of symptoms associated with HuNoV, potentially acting as a viral enterotoxin. This work contributes to the understanding of the pathophysiological mechanisms in HuNoV-induced disease and further attests zebrafish as a valuable HuNoV disease model.

Human noroviruses (HuNoVs) are an important cause of epidemic and endemic acute gastroenteritis worldwide; annually about 700 million people develop a HuNoV infection resulting in ∼219,000 deaths and a societal cost estimated at 60 billion US dollars 1. The lack of robust small animal models has significantly hindered the understanding of norovirus biology and the development of effective therapeutics against HuNoV. 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. HuNoV is detected in cells of the hematopoietic lineage, the intestine, liver and pancreas. Antiviral treatment reduces HuNoV replication by >2 log10, showing that this model is suited for antiviral studies. Downregulation of fucosyltransferase 8 (fut8) in the larvae reduces HuNoV replication, highlighting a common feature with infection in humans. Zebrafish larvae constitute a simple and robust replication model that will largely facilitate studies of HuNoV biology and the development of antiviral strategies.

Aedes aegypti mosquitoes can transmit several arboviruses, including chikungunya virus (CHIKV), dengue virus (DENV), and Zika virus (ZIKV). When blood-feeding on a virus-infected human, the mosquito ingests the virus into the midgut (stomach), where it replicates and must overcome the midgut barrier to disseminate to other organs and ultimately be transmitted via the saliva. Current tools to study mosquito-borne viruses (MBVs) include 2D-cell culture systems and in vivo mosquito infection models, which offer great advantages, yet have some limitations. Here, we describe a long-term ex vivo culture of Ae. aegypti midguts. Cultured midguts were metabolically active for 7 days in a 96-well plate at 28°C and were permissive to ZIKV, DENV, Ross River virus (RRV) and CHIKV. Ex vivo midguts from Culex pipiens mosquitoes were found to be permissive to Usutu virus (USUV). Immunofluorescence staining confirmed viral protein synthesis in CHIKV-infected midguts of Ae. aegypti. Furthermore, fluorescence microscopy revealed replication and spread of a reporter DENV in specific regions of the midgut. In addition, two known antiviral molecules, β-D-N4-hydroxycytidine (NHC) and 7-deaza-2′-C-methyladenosine (7DMA), were able to inhibit CHIKV and ZIKV replication, respectively, in the ex vivo model. Together, our results show that ex vivo midguts can be efficiently infected with mosquito-borne alpha- and flaviviruses and employed to evaluate antiviral drugs. Furthermore, the setup can be extended to other mosquito species. Ex vivo midgut cultures could thus be a new model to study MBVs, offering the advantage of reduced biosafety measures compared to infecting living mosquitoes.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Section on metabolic activity of dissected midguts has been added. Section on ex vivo infection of Culex pipiens midguts has been added. Section on CHIKV viral protein synthesis has been added. Corresponding figures have been updated/added. Authors list updated.

The Rift Valley fever virus (RVFV) is listed by the WHO as priority disease and causes haemorrhagic fever, encephalitis, and permanent blindness. To study RVFV pathogenesis and identify small-molecule antivirals, we established a novel in vivo model using zebrafish larvae. Pericardial injection of RVFV resulted in ~4 log10 viral RNA copies/larva, which was inhibited by antiviral 2'-fluoro-2'-deoxycytidine. The optical transparency of the larvae allowed detection of RVFVeGFP in the liver and sensory nervous system, including the optic tectum and retina, but not the brain or spinal cord. Thus, RVFV-induced blindness likely occurs due to direct damage to the eye and peripheral neurons, rather than the brain. Treatment with JAK-inhibitor ruxolitinib, as well as knockout of stat1a but not stat1b, enhanced RVFV replication to ~6 log10 viral RNA copies/larva and ultra-bright livers, although without dissemination to sensory neurons or the eye, hereby confirming the critical role of stat1 in RVFV pathogenesis. Competing Interest Statement The authors have declared no competing interest.