West Nile virus spread in Europe: Phylogeographic pattern analysis and key drivers.

BACKGROUND: West Nile virus (WNV) outbreaks in birds, humans, and livestock have occurred in multiple areas in Europe and have had a significant impact on animal and human health. The patterns of emergence and spread of WNV in Europe are very different from those in the US and understanding these are important for guiding preparedness activities. METHODS: We mapped the evolution and spread history of WNV in Europe by incorporating viral genome sequences and epidemiological data into phylodynamic models. Spatially explicit phylogeographic models were developed to explore the possible contribution of different drivers to viral dispersal direction and velocity. A "skygrid-GLM" approach was used to identify how changes in environments would predict viral genetic diversity variations over time. FINDINGS: Among the six lineages found in Europe, WNV-2a (a sub-lineage of WNV-2) has been predominant (accounting for 73% of all sequences obtained in Europe that have been shared in the public domain) and has spread to at least 14 countries. In the past two decades, WNV-2a has evolved into two major co-circulating clusters, both originating from Central Europe, but with distinct dynamic history and transmission patterns. WNV-2a spreads at a high dispersal velocity (88km/yr-215 km/yr) which is correlated to bird movements. Notably, amongst multiple drivers that could affect the spread of WNV, factors related to land use were found to strongly influence the spread of WNV. Specifically, the intensity of agricultural activities (defined by factors related to crops and livestock production, such as coverage of cropland, pasture, cultivated and managed vegetation, livestock density) were positively associated with both spread direction and velocity. In addition, WNV spread direction was associated with high coverage of wetlands and migratory bird flyways. CONCLUSION: Our results suggest that-in addition to ecological conditions favouring bird- and mosquito- presence-agricultural land use may be a significant driver of WNV emergence and spread. Our study also identified significant gaps in data and the need to strengthen virological surveillance in countries of Central Europe from where WNV outbreaks are likely seeded. Enhanced monitoring for early detection of further dispersal could be targeted to areas with high agricultural activities and habitats of migratory birds.

Wildlife susceptibility to infectious diseases at global scales.

Disease transmission prediction across wildlife is crucial for risk assessment of emerging infectious diseases. Susceptibility of host species to pathogens is influenced by the geographic, environmental, and phylogenetic context of the specific system under study. We used machine learning to analyze how such variables influence pathogen incidence for multihost pathogen assemblages, including one of direct transmission (coronaviruses and bats) and two vector-borne systems (West Nile Virus [WNV] and birds, and malaria and birds). Here we show that this methodology is able to provide reliable global spatial susceptibility predictions for the studied host-pathogen systems, even when using a small amount of incidence information (i.e., [Formula: see text] of information in a database). We found that avian malaria was mostly affected by environmental factors and by an interaction between phylogeny and geography, and WNV susceptibility was mostly influenced by phylogeny and by the interaction between geographic and environmental distances, whereas coronavirus susceptibility was mostly affected by geography. This approach will help to direct surveillance and field efforts providing cost-effective decisions on where to invest limited resources.

Fatal West Nile Virus Infection in Horse Returning to United Kingdom from Spain, 2022.

We report fatal West Nile virus (WNV) infection in a 7-year-old mare returning to the United Kingdom from Spain. Case timeline and clustering of virus sequence with recent WNV isolates suggest that transmission occurred in Andalusía, Spain. Our findings highlight the importance of vaccination for horses traveling to WNV-endemic regions.

An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus.

Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2'C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.

A field test of the dilution effect hypothesis in four avian multi-host pathogens.

The Dilution Effect Hypothesis (DEH) argues that greater biodiversity lowers the risk of disease and reduces the rates of pathogen transmission since more diverse communities harbour fewer competent hosts for any given pathogen, thereby reducing host exposure to the pathogen. DEH is expected to operate most intensely in vector-borne pathogens and when species-rich communities are not associated with increased host density. Overall, dilution will occur if greater species diversity leads to a lower contact rate between infected vectors and susceptible hosts, and between infected hosts and susceptible vectors. Field-based tests simultaneously analysing the prevalence of several multi-host pathogens in relation to host and vector diversity are required to validate DEH. We tested the relationship between the prevalence in house sparrows (Passer domesticus) of four vector-borne pathogens-three avian haemosporidians (including the avian malaria parasite Plasmodium and the malaria-like parasites Haemoproteus and Leucocytozoon) and West Nile virus (WNV)-and vertebrate diversity. Birds were sampled at 45 localities in SW Spain for which extensive data on vector (mosquitoes) and vertebrate communities exist. Vertebrate censuses were conducted to quantify avian and mammal density, species richness and evenness. Contrary to the predictions of DEH, WNV seroprevalence and haemosporidian prevalence were not negatively associated with either vertebrate species richness or evenness. Indeed, the opposite pattern was found, with positive relationships between avian species richness and WNV seroprevalence, and Leucocytozoon prevalence being detected. When vector (mosquito) richness and evenness were incorporated into the models, all the previous associations between WNV prevalence and the vertebrate community variables remained unchanged. No significant association was found for Plasmodium prevalence and vertebrate community variables in any of the models tested. Despite the studied system having several characteristics that should favour the dilution effect (i.e., vector-borne pathogens, an area where vector and host densities are unrelated, and where host richness is not associated with an increase in host density), none of the relationships between host species diversity and species richness, and pathogen prevalence supported DEH and, in fact, amplification was found for three of the four pathogens tested. Consequently, the range of pathogens and communities studied needs to be broadened if we are to understand the ecological factors that favour dilution and how often these conditions occur in nature.

Increased genetic diversity and immigration after West Nile virus emergence in American crows: No evidence for a genetic bottleneck.

Infectious diseases can cause steep declines in wildlife populations, leading to changes in genetic diversity that may affect the susceptibility of individuals to infection and the overall resilience of populations to pathogen outbreaks. Here, we examine evidence for a genetic bottleneck in a population of American crows (Corvus brachyrhynchos) before and after the emergence of West Nile virus (WNV). More than 50% of marked birds in this population were lost over the 2-year period of the epizootic, representing a 10-fold increase in adult mortality. Using analyses of single-nucleotide polymorphisms (SNPs) and microsatellite markers, we tested for evidence of a genetic bottleneck and compared levels of inbreeding and immigration in the pre- and post-WNV populations. Counter to expectations, genetic diversity (allelic diversity and the number of new alleles) increased after WNV emergence. This was likely due to increases in immigration, as the estimated membership coefficients were lower in the post-WNV population. Simultaneously, however, the frequency of inbreeding appeared to increase: Mean inbreeding coefficients were higher among SNP markers, and heterozygosity-heterozygosity correlations were stronger among microsatellite markers, in the post-WNV population. These results indicate that loss of genetic diversity at the population level is not an inevitable consequence of a population decline, particularly in the presence of gene flow. The changes observed in post-WNV crows could have very different implications for their response to future pathogen risks, potentially making the population as a whole more resilient to a changing pathogen community, while increasing the frequency of inbred individuals with elevated susceptibility to disease.

Cellular co-infections of West Nile virus and Usutu virus influence virus growth kinetics.

The mosquito-borne flaviviruses West Nile virus (WNV) and Usutu virus (USUV) pose a significant threat to the health of humans and animals. Both viruses co-circulate in numerous European countries including Germany. Due to their overlapping host and vector ranges, there is a high risk of co-infections. However, it is largely unknown if WNV and USUV interact and how this might influence their epidemiology. Therefore, in-vitro infection experiments in mammalian (Vero B4), goose (GN-R) and mosquito cell lines (C6/36, CT) were performed to investigate potential effects of co-infections in vectors and vertebrate hosts. The growth kinetics of German and other European WNV and USUV strains were determined and compared. Subsequently, simultaneous co-infections were performed with selected WNV and USUV strains. The results show that the growth of USUV was suppressed by WNV in all cell lines. This effect was independent of the virus lineage but depended on the set WNV titre. The replication of WNV also decreased in co-infection scenarios on vertebrate cells. Overall, co-infections might lead to a decreased growth of USUV in mosquitoes and of both viruses in vertebrate hosts. These interactions can strongly affect the epidemiology of USUV and WNV in areas where they co-circulate.

Ecological and clinical evidence of the establishment of West Nile virus in a large urban area in Europe, Berlin, Germany, 2021 to 2022.

BackgroundWest Nile virus (WNV), found in Berlin in birds since 2018 and humans since 2019, is a mosquito-borne virus that can manifest in humans as West Nile fever (WNF) or neuroinvasive disease (WNND). However, human WNV infections and associated disease are likely underdiagnosed.AimWe aimed to identify and genetically characterise WNV infections in humans and mosquitoes in Berlin.MethodsWe investigated acute WNV infection cases reported to the State Office for Health and Social Affairs Berlin in 2021 and analysed cerebrospinal fluid (CSF) samples from patients with encephalitis of unknown aetiology (n = 489) for the presence of WNV. Mosquitoes were trapped at identified potential exposure sites of cases and examined for WNV infection.ResultsWest Nile virus was isolated and sequenced from a blood donor with WNF, a symptomatic patient with WNND and a WNND case retrospectively identified from testing CSF. All cases occurred in 2021 and had no history of travel 14 days prior to symptom onset (incubation period of the disease). We detected WNV in Culex pipiens mosquitoes sampled at the exposure site of one case in 2021, and in 2022. Genome analyses revealed a monophyletic Berlin-specific virus clade in which two enzootic mosquito-associated variants can be delineated based on tree topology and presence of single nucleotide variants. Both variants have highly identical counterparts in human cases indicating local acquisition of infection.ConclusionOur study provides evidence that autochthonous WNV lineage 2 infections occurred in Berlin and the virus has established an endemic maintenance cycle.

West Nile virus in the Iberian Peninsula: using equine cases to identify high-risk areas for humans.

BackgroundWest Nile virus (WNV) is a flavivirus with an enzootic cycle between birds and mosquitoes; humans and horses are incidental dead-end hosts. In 2020, the largest outbreak of West Nile virus infection in the Iberian Peninsula occurred, with 141 clusters in horses and 77 human cases.AimWe analysed which drivers influence spillover from the cycle to humans and equines and identified areas at risk for WNV transmission.MethodsBased on data on WNV cases in horses and humans in 2020 in Portugal and Spain, we developed logistic regression models using environmental and anthropic variables to highlight risk areas. Models were adapted to a high-resolution risk map.ResultsCases of WNV in horses could be used as indicators of viral activity and thus predict cases in humans. The risk map of horses was able to define high-risk areas for previous cases in humans and equines in Portugal and Spain, as well as predict human and horse cases in the transmission seasons of 2021 and 2022. We found that the spatial patterns of the favourable areas for outbreaks correspond to the main hydrographic basins of the Iberian Peninsula, jointly affecting Portugal and Spain.ConclusionA risk map highlighting the risk areas for potential future cases could be cost-effective as a means of promoting preventive measures to decrease incidence of WNV infection in Europe, based on a One Health surveillance approach.

West Nile virus in India: An update on its genetic lineages.

West Nile virus (WNV) is a rapidly spreading mosquito-transmitted zoonotic flavivirus. Mosquitoes belonging to the genus Culex are incriminated as the principal vectors of the virus, which causes West Nile fever (WNF) in humans. Manifestations of WNF include a mild, self-limiting, flu-like illness, which in severe cases (rare) may progress to encephalitis, resulting in life-threatening consequences. WNV is geographically distributed worldwide, covering Africa, the Americas, Europe, and Asia (except Antarctica). The virus exists in a bird-mosquito transmission cycle in nature, with humans and horses as incidental/accidental hosts. The virus can infect a large variety of hosts worldwide, i.e., about 300 birds and around 70 different mosquito species belonging to several genera. For a long time, it was believed that WNV was not highly virulent and caused only mild infection globally. However, the recent frequent and increasing incidence of clinically severe WNV infections, such as encephalitis in humans and horses with significant mortality, has been reported in the Americas, Europe, and several East Asian countries. The emergence of lineage 2 strains endemic to Africa, with epidemic potential in humans and horses in Europe, is considered a serious global health concern. Although WNV is known to circulate in India since 1952, its re-emergence with severe neuro-invasive pathogenic potential in humans in Assam, Kerala, West Bengal and Tamil Nadu states signals urgent efforts to understand the dynamics of circulating strains with regard to its vector, hosts, and environment. This could be done by prioritizing "One Health" approach for developing effective preventive and control strategies. In view of the global interest, we present an overview of the circulating genetic lineages of WNV in India in comparison with the global scenario. In addition, we stress on holistic approaches of "One Health" strategy as the current need of the hour for designing effective preventive and control strategies in the country.

Domestic Dogs as Sentinels for West Nile Virus but not Aedes-borne Flaviviruses, Mexico.

We tested 294 domestic pet dogs in Mexico for neutralizing antibodies for mosquito-borne flaviviruses. We found high (42.6%) exposure to West Nile virus in Reynosa (northern Mexico) and low (1.2%) exposure in Tuxtla Gutierrez (southern Mexico) but very limited exposure to Aedes-borne flaviviruses. Domestic dogs may be useful sentinels for West Nile virus.

Mapping the Risk for West Nile Virus Transmission, Africa.

West Nile virus (WNV) is an emergent arthropodborne virus that is transmitted from bird to bird by mosquitoes. Spillover events occur when infected mosquitoes bite mammals. We created a geopositioned database of WNV presence in Africa and considered reports of the virus in all animal components: reservoirs, vectors, and nonhuman dead-end hosts. We built various biogeographic models to determine which drivers explain the distribution of WNV throughout Africa. Wetlands of international importance for birds accounted for the detection of WNV in all animal components, whereas human-related drivers played a key role in the epizootic cases. We combined these models to obtain an integrative and large-scale perspective of the areas at risk for WNV spillover. Understanding which areas pose the highest risk would enable us to address the management of this spreading disease and to comprehend the translocation of WNV outside Africa through avian migration routes.

Effect of Agroecosystems on Seroprevalence of St. Louis Encephalitis and West Nile Viruses in Birds, La Pampa, Argentina, 2017-2019.

In Argentina, the Pampa ecoregion has been almost completely transformed into agroecosystems. To evaluate the environmental (agricultural area, tree coverage, distance to the nearest water body and urban site) and biological (dove, cowbird, and sparrow abundance) effects on free-ranging bird exposure to St. Louis encephalitis virus (SLEV) and West Nile virus (WNV), we used generalized linear mixed models. For 1,019 birds sampled during 2017-2019, neutralizing antibodies were found against SLEV in samples from 60 (5.8%) birds and against WNV for 21 (2.1%). The best variable for explaining SLEV seroprevalence was agricultural area, which had a positive effect; however, for WNV, no model was conclusive. Our results suggest that agroecosystems in the La Pampa ecoregion increase the exposure of avian hosts to SLEV, thus potentially increasing virus activity.

Susceptibility of wild turkeys (Meleagris gallopavo) to experimental West Nile virus infection.

Since the introduction of West Nile virus (WNV) to North America in 1999, WNV is estimated to have contributed to population-level declines in numerous avian species. However, the potential impacts of this virus on many free-ranging upland game bird species, including the wild turkey (Meleagris gallopavo), which is undergoing regional population declines, remain unknown. Herein, two age groups (∼5 to 6 weeks and ∼15 to 16 weeks post-hatch) of juvenile wild turkeys were subcutaneously inoculated with WNV, sampled daily from 1 to 7 days post-inoculation (dpi), and euthanized on 14 dpi. No clinical signs and minimal gross lesions were attributable to WNV infection. Peak viraemia titres were similar between age groups (<101.7 to 104.6 plaque-forming units [PFU]/ml), but the duration of viraemia was longer in the old group (3-4 days) than in the young group (0-3 days). Intermittent oral and/or cloacal viral shedding from 2 to 7 dpi was detected in both age groups. No infectious virus was detected in the heart, brain, kidney, skeletal muscle, spleen, and feathers from WNV-inoculated turkeys euthanized on 14 dpi. All WNV-inoculated birds seroconverted by 14 dpi, as well as two co-housed sham-inoculated birds. The most consistent microscopic lesions among all WNV-inoculated birds were mild lymphoplasmacytic myocarditis and encephalitis. Minimal immunohistochemical labelling was detected in tissues in addition to scant macrophages within the blood, spleen, and bone marrow. These data suggest WNV is unlikely to pose a significant risk to wild turkey populations, although the possibility remains that WNV may indirectly decrease fitness or predispose wild turkeys to other health stressors.RESEARCH HIGHLIGHTS Clinical disease was not observed in wild turkeys experimentally infected with WNV.Pathology attributed to WNV was mild and included brain and heart inflammation.Viraemias suggest WNV-infected wild turkeys do not play a role in WNV transmission.No age-associated differences in WNV clinical disease or pathology were observed.

Mosquito species associated with horses in Madagascar: a review of their vector status with regard to the epidemiology of West Nile fever.

In Madagascar, the high West Nile virus (WNV) antibody prevalence reported in horse populations suggests a high level of vector-horse contact. This study aims to characterize the mosquito species usually involved in WNV transmission in horse stables in Madagascar. Five horse stables were investigated in October and November 2016 in five distinct inland areas. Mosquitoes were collected using double net traps baited with human, poultry or horse as well as light traps. Blood meal identification from engorged females was performed using host-specific PCRs. A total of 2898 adult mosquitoes were collected with Culex (Culex) antennatus (Becker) (40.7%), and Cx. (Cux.) quinquefasciatus Say (14.9%), being the most abundant species. The mosquito abundance varied between horse stables (P < 10-7 ) and depending on the bait used in the double net traps (P < 0.003). Among the 190 tested blood meals, 119 consisted of single blood meals with 85 from horse, 17 from human, 16 from chicken, one from cattle and 71 consisted of mixed blood meals. The mosquito species collected during this study exhibited a generalist feeding behaviour allowing them to act as bridge vectors between different vertebrate hosts involved in WNV transmission cycle. Their vector status with regard to West Nile fever epidemiology is reviewed.

Rapid increase in neuroinvasive West Nile virus infections in humans, Italy, July 2022.

As in 2018, when a large West Nile virus (WNV) epidemic occurred, the 2022 vector season in Italy was marked by an early onset of WNV circulation in mosquitoes and birds. Human infections were limited until early July, when we observed a rapid increase in the number of cases. We describe the epidemiology of human infections and animal and vector surveillance for WNV and compare the more consolidated data of June and July 2022 with the same period in 2018.

One Health surveillance of West Nile and Usutu viruses: a repeated cross-sectional study exploring seroprevalence and endemicity in Southern France, 2016 to 2020.

BackgroundWest Nile virus (WNV) and Usutu virus (USUV), two closely related flaviviruses, mainly follow an enzootic cycle involving mosquitoes and birds, but also infect humans and other mammals. Since 2010, their epidemiological situation may have shifted from irregular epidemics to endemicity in several European regions; this requires confirmation, as it could have implications for risk assessment and surveillance strategies.AimTo explore the seroprevalence in animals and humans and potential endemicity of WNV and USUV in Southern France, given a long history of WNV outbreaks and the only severe human USUV case in France in this region.MethodsWe evaluated the prevalence of WNV and USUV in a repeated cross-sectional study by serological and molecular analyses of human, dog, horse, bird and mosquito samples in the Camargue area, including the city of Montpellier, between 2016 and 2020.ResultsWe observed the active transmission of both viruses and higher USUV prevalence in humans, dogs, birds and mosquitoes, while WNV prevalence was higher in horses. In 500 human samples, 15 were positive for USUV and 6 for WNV. Genetic data showed that the same lineages, WNV lineage 1a and USUV lineage Africa 3, were found in mosquitoes in 2015, 2018 and 2020.ConclusionThese findings support existing literature suggesting endemisation in the study region and contribute to a better understanding of USUV and WNV circulation in Southern France. Our study underlines the importance of a One Health approach for the surveillance of these viruses.

Clinical and pathologic evaluation of chorioretinal lesions in wild owl species.

OBJECTIVE: Investigate histopathology and spectral-domain optical coherence tomography (OCT) imaging of wild owls with chorioretinitis and identify any potential correlation with an infectious etiology. MATERIALS AND METHODS: Ophthalmic examination and retinal OCT imaging were performed on fifteen great horned (Strix varia) and barred (Bubo virginianus) owls (30 eyes) with chorioretinitis and five owls with normal eyes (10 eyes). Testing to investigate the presence of potential infectious diseases included a complete blood count, biochemistry, protein electrophoresis, West Nile virus (WNV) plaque reduction neutralization test, Toxoplasma gondii modified direct agglutination test, WNV RT-PCR, and Avian Influenza RT-PCR. A necropsy was performed on all owls, including ocular histopathology. RESULTS: Fundus lesions included retinal detachment (7/15 owls), depigmented lesions (12/15), pigment clumping (8/15), and retinal tear (4/15). All birds were negative for WNV and Avian Influenza on RT-PCR. Of the owls with chorioretinitis, 3/15 were seropositive for WNV and 7/15 for T. gondii. Optical coherence tomography of 25/30 affected eyes revealed outer retinal lesions (19/25 eyes), retinal detachment (16/25), and retinal tears (3/25). Histopathological examination revealed outer nuclear layer atrophy (19/30 eyes), retinal detachment (18/30), retinal tears (7/30), suprachoroidal hemorrhage (12/30), scleral rupture (3/30), and ossicle fracture (3/30). CONCLUSIONS: Although 20% of birds were seropositive for WNV and 46.6% for T. gondii, histopathologic findings supported that the posterior segment lesions in the study group were likely due to blunt ocular trauma rather than an infectious etiology. The results of OCT imaging and histopathology documented retinal changes most consistent with blunt ocular trauma.

Survival and Release of 5 American Crows (Corvus brachyrhynchos) Naturally Infected With West Nile Virus.

West Nile virus (WNV) has had a significant effect on avian populations in the United States since being first identified in 1999. Avian species in WNV endemic areas do not suffer the same level of mortality that has been reported in birds within the United States since the virus was first identified in North America. Because of their unique susceptibility, American crows (Corvus brachyrhynchos) are often used to monitor the spread and severity of WNV in North America. American crows with WNV infections are received and treated at the Janet L. Swanson Wildlife Hospital (Cornell University, Ithaca, NY, USA) on a regular basis during the summer and fall and have historically had a 100% mortality rate. This report describes WNV-positive American crows that were treated, recovered from the infection, and were subsequently released. The 5 American crows in this case series were tested, when possible, by polymerase chain reaction (PCR) and plaque reduction neutralization on admission and monitored with both PCR and plaque reduction neutralization throughout their rehabilitation process. Four of the 5 birds had a negative PCR test before release, and 1 bird had a "suspect" positive PCR test result before release. One of the crows was confirmed to have survived for at least 2.5 years after release. Viral shedding was documented up to 93 days after initial hospitalization, which is longer than any previous report of WNV shedding in an American crow.

Ecologic Determinants of West Nile Virus Seroprevalence among Equids, Brazil.

Among 713 equids sampled in northeastern Brazil during 2013-2018, West Nile virus seroprevalence was 4.5% (95% CI 3.1%-6.3%). Mathematical modeling substantiated higher seroprevalence adjacent to an avian migratory route and in areas characterized by forest loss, implying increased risk for zoonotic infections in disturbed areas.