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 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.

ABSTRACT 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 guts. Cultured guts were metabolically active for 7 d in a 96-well plate at 28°C and were permissive to ZIKV, DENV, Ross River virus, and CHIKV. Ex vivo guts from Culex pipiens mosquitoes were found to be permissive to Usutu virus. Immunofluorescence staining confirmed viral protein synthesis in CHIKV-infected guts 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 and 7-deaza-2′-C-methyladenosine, were able to inhibit CHIKV and ZIKV replication, respectively, in the ex vivo model. Together, our results show that ex vivo guts 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 gut cultures could thus be a new model to study MBVs, offering the advantage of reduced biosafety measures compared to infecting living mosquitoes. IMPORTANCE Mosquito-borne viruses (MBVs) are a significant global health threat since they can cause severe diseases in humans, such as hemorrhagic fever, encephalitis, and chronic arthritis. MBVs rely on the mosquito vector to infect new hosts and perpetuate virus transmission. No therapeutics are currently available. The study of arbovirus infection in the mosquito vector can greatly contribute to elucidating strategies for controlling arbovirus transmission. This work investigated the infection of guts from Aedes aegypti mosquitoes in an ex vivo platform. We found several MBVs capable of replicating in the gut tissue, including viruses of major health importance, such as dengue, chikungunya, and Zika viruses. In addition, antiviral compounds reduced arbovirus infection in the cultured gut tissue. Overall, the gut model emerges as a useful tool for diverse applications such as studying tissue-specific responses to virus infection and screening potential anti-arboviral molecules.

Human norovirus (HuNoV) is the major agent for viral gastroenteritis, causing >700 million infections yearly. Fucose-containing carbohydrates named histo-blood group antigens (HBGAs) are known (co)receptors for HuNoV. Moreover, bacteria of the gut microbiota expressing HBGA-like structures have shown an enhancing effect on HuNoV replication in an in vitro model. Here, we studied the role of HBGAs and the host microbiota during HuNoV infection in zebrafish larvae. Using whole-mount immunohistochemistry, we visualized the fucose expression in the zebrafish gut for the HBGA Lewis X [LeX, α(1,3)-fucose] and core fucose [α(1,6)-fucose]. Costaining of HuNoV-infected larvae proved colocalization of LeX and to a lower extent core fucose with the viral capsid protein VP1, indicating the presence of fucose residues on infected cells. Upon blocking of fucose expression by a fluorinated fucose analogue, HuNoV replication was strongly reduced. Furthermore, by comparing HuNoV replication in conventional and germfree zebrafish larvae, we found that the natural zebrafish microbiome does not have an effect on HuNoV replication, contrary to earlier reports about the human gut microbiome. Interestingly, monoassociation with the HBGA-expressing Enterobacter cloacae resulted in a minor decrease in HuNoV replication, which was not triggered by a stronger innate immune response. Overall, we show here that fucose has an essential role for HuNoV infection in zebrafish larvae, as in the human host, but their natural gut microbiome does not affect viral replication. IMPORTANCE Despite causing over 700 million infections yearly, many gaps remain in the knowledge of human norovirus (HuNoV) biology due to an historical lack of efficient cultivation systems. Fucose-containing carbohydrate structures, named histo-blood group antigens, are known to be important (co)receptors for viral entry in humans, while the natural gut microbiota is suggested to enhance viral replication. This study shows a conserved mechanism of entry for HuNoV in the novel zebrafish infection model, highlighting the pivotal opportunity this model represents to study entry mechanisms and identify the cellular receptor of HuNoV. Our results shed light on the interaction of HuNoV with the zebrafish microbiota, contributing to the understanding of the interplay between gut microbiota and enteric viruses. The ease of generating germfree animals that can be colonized with human gut bacteria is an additional advantage of using zebrafish larvae in virology. This small animal model constitutes an innovative alternative to high-severity animal models.

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) is the most common cause of viral gastroenteritis, causing annually ~700 million infections and resulting in ~200 000 deaths worldwide. HuNoV causes significant morbidity, affects people worldwide and of all age groups, and is responsible for an annual societal burden of US$60 billion. There is no antiviral or vaccine available to treat and/or prevent a HuNoV infection, which is especially necessary for high-risk populations such as young children, the elderly, and immunocompromised. Over the last five years, important progress has been made to develop better in vitro and in vivo models that will aid in the search for new antivirals and vaccines. Potent antivirals against HuNoV are in particular needed to treat chronic infections and as a prophylactic to prevent and control outbreaks, especially in hospitals and long-term care facilities. In this chapter, we review the available in vitro and in vivo models, reported inhibitors of viral replication, and discuss targets that can be explored to design antiviral agents and strategies against this virus.

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 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.