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West Nile Virus (WNV) and Usutu Virus (USUV) are both neurotropic mosquito-borne viruses belonging to the Flaviviridae family. These closely related viruses mainly follow an enzootic cycle involving mosquitoes as vectors and birds as amplifying hosts, but humans and other mammals can also be infected through mosquito bites. WNV was first identified in Uganda in 1937 and has since spread globally, notably in Europe, causing periodic outbreaks associated with severe cases of neuroinvasive diseases such as meningitis and encephalitis. USUV was initially isolated in 1959 in Swaziland and has also spread to Europe, primarily affecting birds and having a limited impact on human health. There has been a recent expansion of these viruses’ geographic range in Europe, facilitated by factors such as climate change, leading to increased human exposure. While sharing similar biological traits, ecology, and epidemiology, there are significant distinctions in their pathogenicity and their impact on both human and animal health. While WNV has been more extensively studied and is a significant public health concern in many regions, USUV has recently been gaining attention due to its emergence in Europe and the diversity of its circulating lineages. Understanding the pathophysiology, ecology, and transmission dynamics of these viruses is important to the implementation of effective surveillance and control measures. This perspective provides a brief overview of the current situation of these two viruses in Europe and outlines the significant challenges that need to be addressed in the coming years.

Flaviviruses are vector-borne RNA viruses that can emerge unexpectedly in human populations and cause a spectrum of potentially severe diseases including hepatitis, vascular shock syndrome, encephalitis, acute flaccid paralysis, congenital abnormalities and fetal death. This epidemiological pattern has occurred numerous times during the last 70 years, including epidemics of dengue virus and West Nile virus, and the most recent explosive epidemic of Zika virus in the Americas. Flaviviruses are now globally distributed and infect up to 400 million people annually. Of significant concern, outbreaks of other less well-characterized flaviviruses have been reported in humans and animals in different regions of the world. The potential for these viruses to sustain epidemic transmission among humans is poorly understood. In this Review, we discuss the basic biology of flaviviruses, their infectious cycles, the diseases they cause and underlying host immune responses to infection. We describe flaviviruses that represent an established ongoing threat to global health and those that have recently emerged in new populations to cause significant disease. We also provide examples of lesser-known flaviviruses that circulate in restricted areas of the world but have the potential to emerge more broadly in human populations. Finally, we discuss how an understanding of the epidemiology, biology, structure and immunity of flaviviruses can inform the rapid development of countermeasures to treat or prevent human infections as they emerge. Flaviviruses, a group of vector-borne RNA viruses that includes dengue virus, West Nile virus, Zika virus and several lesser-known species, often emerge in human populations and cause epidemics. Here, Pierson and Diamond review the basic biology of these viruses, their life cycles, the diseases they cause and available therapeutic options. They also discuss the global distribution of flaviviruses, with a focus on lesser-known species that have the potential to emerge more broadly in human populations.

Usutu virus (USUV) is an emerging zoonotic flavivirus in Europe and the first zoonotic mosquito-borne virus to be confirmed in animal hosts in the United Kingdom (UK). Repeated detection of USUV in the years following its initial detection in 2020 indicated that the virus is overwintering in the UK and should now be considered endemic within southeast England. Surveillance of avian hosts and mosquito vectors has been insufficient to elucidate the mechanism by which USUV has persisted through temperate winters. Through enhanced targeted vector surveillance at the index site between 2021 and 2024 inclusive, we detected USUV RNA in host-seeking adult female Culex pipiens s.l. as well as in adults reared from field-caught larvae. This is, to our knowledge, the first description of vertical transmission of USUV in an arthropod vector that should consequently be considered a viable mechanism for the persistence of USUV in temperate areas.

Usutu virus (USUV; Flavivirus), a close phylogenetic and ecological relative of West Nile virus, is a zoonotic virus that can cause neuroinvasive disease in humans. USUV is maintained in an enzootic cycle between Culex mosquitoes and birds. Since the first isolation in 1959 in South Africa, USUV has spread throughout Africa and Europe. Reported human cases have increased over the last few decades, primarily in Europe, with symptoms ranging from mild febrile illness to severe neurological effects. In this study, we investigated whether USUV has become more pathogenic during emergence in Europe. Interferon α/β receptor knockout (Ifnar1-/-) mice were inoculated with recent USUV isolates from Africa and Europe, as well as the historic 1959 South African strain. The three tested African strains and one European strain from Spain caused 100% mortality in inoculated mice, with similar survival times and histopathology in tissues. Unexpectedly, a European strain from the Netherlands caused only 12% mortality and significantly less histopathology in tissues from mice compared to mice inoculated with the other strains. Viremia was highest in mice inoculated with the recent African strains and lowest in mice inoculated with the Netherlands strain. Based on phylogenetics, the USUV isolates from Spain and the Netherlands were derived from separate introductions into Europe, suggesting that disease outcomes may differ for USUV strains circulating in Europe. These results also suggest that while more human USUV disease cases have been reported in Europe recently, circulating African USUV strains are still a potential major health concern.

Flaviviruses are a group of positive-sense RNA viruses that are primarily transmitted through arthropod vectors and are capable of causing a broad spectrum of diseases. Many of the flaviviruses that are pathogenic in humans are transmitted specifically through mosquito vectors. Over the past century, many mosquito-borne flavivirus infections have emerged and re-emerged, and are of global importance with hundreds of millions of infections occurring yearly. There is a need for novel, effective, and accessible vaccines and antivirals capable of inhibiting flavivirus infection and ameliorating disease. The development of therapeutics targeting viral entry has long been a goal of antiviral research, but most efforts are hindered by the lack of broad-spectrum potency or toxicities associated with on-target effects, since many host proteins necessary for viral entry are also essential for host cell biology. Mosquito-borne flaviviruses generally enter cells by clathrin-mediated endocytosis (CME), and recent studies suggest that a subset of these viruses can be internalized through a specialized form of CME that has additional dependencies distinct from canonical CME pathways, and antivirals targeting this pathway have been discovered. In this review, we discuss the role and contribution of endocytosis to mosquito-borne flavivirus entry as well as consider past and future efforts to target endocytosis for therapeutic interventions.

Interspecies transmission of viruses poses significant risks to animal and human health. Tembusu virus (TMUV), an emerging flavivirus, is primarily associated with avian diseases. This study reports the first documented natural infection of TMUV in mammals, specifically zoo dolphins in Thailand, offering insights into its evolution, transmission dynamics, and zoonotic potential. In July 2023, three bottlenose dolphins developed neurological symptoms and died. Postmortem analyses, including histopathology, immunohistochemistry, high-throughput sequencing, and transmission electron microscopy, confirmed TMUV infection. Viral loads were high in brain and lung tissues, followed by kidney and spleen whereas the TMUV antigen was identified in only brain tissue. TMUV was localized in neurons and astroglia cells, and immunohistochemistry revealed CD3-positive T lymphocyte perivascular cuffing in the brain. Phylogenetic analysis placed the dolphin TMUV strains within cluster 3, related to strains found in mosquitoes in China. Retrospective analysis of dolphin samples from 2019 confirmed persistent TMUV circulation. Viral isolation on Vero cells showed characteristic cytopathic effects, and transmission electron microscopy revealed enveloped virions. This study highlights the virus’s ability to infect diverse hosts, including mammals. The findings underscore the need for continuous surveillance and a One Health approach to mitigate emerging viral threats.

Background Mosquitoes are efficient vectors of medically significant flaviviruses and serve as hosts for insect-specific flaviviruses (ISFs). Aedes flavivirus (AEFV) is a classical ISF. Given the increasing discovery of ISFs, it is urgent to evaluate the potential risk of ISFs to human health as well as their impact on the transmission of pathogenic flaviviruses. Methods We isolated a strain of AEFV from wild Aedes albopictus populations in Hainan Province, China, using iodixanol density-gradient ultracentrifugation. The infection of the AEFV Hainan strain in Aedes , Culex , and four mammalian cell lines was investigated using fluorescence in situ hybridization (FISH) assays, and relative and absolute quantitative polymerase chain reaction (qPCR). Whether AEFV alters the vector competence of Ae. albopictus for pathogenic arboviruses and the underlying immune mechanisms were explored. Results The AEFV Hainan strain showed close genetic similarity to strains from Yunnan province of China, Thailand, and Peru. This strain was capable of infecting Ae. albopictus and Ae. aegypti but not Culex quinquefasciatus . Cell entry was the critical barrier for AEFV infection in Cx. quinquefasciatus cells. The infection risk of the AEFV Hainan strain in four mammalian cells (BHK-21, Vero, 293 T, and HeLa) was quite low due to the failure of cell entry or extremely limited replication. Prior infection of AEFV was detrimental to the replication of Zika virus and dengue virus serotype 2 in Ae. albopictus through activation of the Janus kinase/signal transducer and activator of transcription, Toll, or RNA interference pathway. Conclusions Our work excludes the risk of the AEFV Hainan strain to human health and highlights its potential as an immune inducer to sabotage Aedes mosquito ability for viral transmission. Graphical Abstract

The surveillance for West Nile virus (WNV) in Catalonia (northeastern Spain) has consistently detected flaviviruses not identified as WNV. With the aim of characterizing the flaviviruses circulating in Catalonia, serum samples from birds and horses collected between 2010 and 2019 and positive by panflavivirus competition ELISA (cELISA) were analyzed by microneutralization test (MNT) against different flaviviruses. A third of the samples tested were inconclusive by MNT, highlighting the limitations of current diagnostic techniques. Our results evidenced the widespread circulation of flaviviruses, in particular WNV, but also Usutu virus (USUV), and suggest that chicken and horses could serve as sentinels for both viruses. In several regions, WNV and USUV overlapped, but no significant geographical aggregation was observed. Bagaza virus (BAGV) was not detected in birds, while positivity to tick-borne encephalitis virus (TBEV) was sporadically detected in horses although no endemic foci were observed. So far, no human infections by WNV, USUV, or TBEV have been reported in Catalonia. However, these zoonotic flaviviruses need to be kept under surveillance, ideally within a One Health framework.

In Europe, many flaviviruses are endemic (West Nile, Usutu, tick-borne encephalitis viruses) or occasionally imported (dengue, yellow fever viruses). Due to the temporal and geographical co-circulation of flaviviruses in Europe, flavivirus differentiation by diagnostic tests is crucial in the adaptation of surveillance and control efforts. Serological diagnosis of flavivirus infections is complicated by the antigenic similarities among the Flavivirus genus. Indeed, most flavivirus antibodies are directed against the highly immunogenic envelope protein, which contains both flavivirus cross-reactive and virus-specific epitopes. Serological assay results should thus be interpreted with care and confirmed by comparative neutralization tests using a panel of viruses known to circulate in Europe. However, antibody cross-reactivity could be advantageous in efforts to control emerging flaviviruses because it ensures partial cross-protection. In contrast, it might also facilitate subsequent diseases, through a phenomenon called antibody-dependent enhancement mainly described for dengue virus infections. Here, we review the serological methods commonly used in WNV diagnosis and surveillance in Europe. By examining past and current epidemiological situations in different European countries, we present the challenges involved in interpreting flavivirus serological tests and setting up appropriate surveillance programs; we also address the consequences of flavivirus circulation and vaccination for host immunity.