Detection of anti-premembrane antibody as a specific marker of four flavivirus serocomplexes and its application to serosurveillance in endemic regions.

In the past few decades, several emerging/re-emerging mosquito-borne flaviviruses have resulted in disease outbreaks of public health concern in the tropics and subtropics. Due to cross-reactivities of antibodies recognizing the envelope protein of different flaviviruses, serosurveillance remains a challenge. Previously we reported that anti-premembrane (prM) antibody can discriminate between three flavivirus infections by Western blot analysis. In this study, we aimed to develop a serological assay that can discriminate infection or exposure with flaviviruses from four serocomplexes, including dengue (DENV), Zika (ZIKV), West Nile (WNV) and yellow fever (YFV) viruses, and explore its application for serosurveillance in flavivirus-endemic countries. We employed Western blot analysis including antigens of six flaviviruses (DENV1, 2 and 4, WNV, ZIKV and YFV) from four serocomplexes. We tested serum samples from YF-17D vaccinees, and from DENV, ZIKV and WNV panels that had been confirmed by RT-PCR or by neutralization assays. The overall sensitivity/specificity of anti-prM antibodies for DENV, ZIKV, WNV, and YFV infections/exposure were 91.7%/96.4%, 91.7%/99.2%, 88.9%/98.3%, and 91.3%/92.5%, respectively. When testing 48 samples from Brazil, we identified multiple flavivirus infections/exposure including DENV and ZIKV, DENV and YFV, and DENV, ZIKV and YFV. When testing 50 samples from the Philippines, we detected DENV, ZIKV, and DENV and ZIKV infections with a ZIKV seroprevalence rate of 10%, which was consistent with reports of low-level circulation of ZIKV in Asia. Together, these findings suggest that anti-prM antibody is a flavivirus serocomplex-specific marker and can be employed to delineate four flavivirus infections/exposure in regions where multiple flaviviruses co-circulate.

Reconstruction of the molecular evolution of Usutu virus in Germany: Insights into virus emersion and circulation.

Usutu virus (USUV) is a mosquito-borne flavivirus that is widely distributed in southern and central Europe. The zoonotic virus circulates primarily between birds and mosquitoes, can, however, in rare cases infect other mammals including humans. In the past, USUV has been repeatedly associated with mass mortalities in birds, primarily blackbirds and owls. Birds commonly succumb either due to the peracute nature of the infection or due to severe encephalitis. In Germany, USUV has spread rapidly since its first detection in 2010 in mosquitoes under the presence of susceptible host and vector species. Nonetheless, there is to date limited access to whole genome sequences resulting in the absence of in-depth phylogenetic and phylodynamic analyses. In this study, 118 wild and captive birds were sequenced using a nanopore sequencing platform with prior target enrichment via amplicons. Due to the high abundancy of Europe 3 and Africa 3 in Germany an ample quantity of associated whole genome sequences was generated and the most recent common ancestor could be determined for each lineage. The corresponding clock phylogeny revealed an introduction of USUV Europe 3 and Africa 3 into Germany three years prior to their first isolation in the avifauna in 2011 and 2014, respectively. Based on the clustering and temporal history of the lineages, evidence exists for the genetic evolution of USUV within Germany as well as new introductions thereof into the country.

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.

Epidemiology, genetic diversity, and evolutionary dynamics of Tembusu virus.

Tembusu virus (TMUV) is an emerging pathogenic flavivirus associated with acute egg-drop and fatal encephalitis in domestic waterfowl. Since its initial identification in mosquitoes in 1955, TMUV has been confirmed to infect ducks, pigeons, sparrows, geese, and chickens, posing a significant threat to the poultry industry. Here, we sequenced two DTMUV strains isolated in 2019 and systematically investigated the possible origin, genetic relationships, evolutionary dynamics, and transmission patterns of TMUV based on complete virus genome sequences in the public database. We found that TMUV can be divided into four major clusters: TMUV, cluster 1, cluster 2, and cluster 3. Interestingly, we found that cluster 2.2 (within cluster 2) is the most commonly involved in interspecies transmission events, and subcluster 2.1.2 (within cluster 2.1) is currently the most prevalent cluster circulating in Asia. Notably, we also identified three positively selected sites in the E and NS1 proteins, which may be involved in virus replication, immune evasion, and host adaptation. Finally, phylogeographic analysis revealed that cluster dispersal originated in Southeast Asia and that short-distance transmission events have occurred frequently. Altogether, these data provide novel insights into the evolution and dispersal of TMUV, facilitating the development of rapid diagnostics, vaccines, and therapeutics against TMUV infection.

Epidemiology, surveillance and diagnosis of Usutu virus infection in the EU/EEA, 2012 to 2021.

BackgroundUsutu virus (USUV) is a flavivirus with an enzootic cycle between birds and mosquitoes; humans are incidental dead-end hosts. In Europe, the virus was first detected in Italy in 1996; since then, it has spread to many European countries.AimWe aimed to report on the epidemiology, surveillance, diagnosis and prevention of USUV infection in humans, mosquitoes and other animals in the European Union/European Economic Area (EU/EEA) from 2012 to 2021.MethodsWe collected information through a literature review, an online survey and an expert meeting.ResultsEight countries reported USUV infection in humans (105 cases, including 12 [corrected] with neurological symptoms), 15 countries in birds and seven in mosquitoes. Infected animals were also found among pets, wild and zoo animals. Usutu virus was detected primarily in Culex pipiens but also in six other mosquito species. Detection of USUV infection in humans is notifiable only in Italy, where it is under surveillance since 2017 and now integrated with surveillance in animals in a One Health approach. Several countries include USUV infection in the differential diagnosis of viral encephalitis and arbovirus infections. Animal USUV infection is not notifiable in any EU/EEA country.ConclusionHuman USUV infections, mainly asymptomatic and, less frequently, with a febrile illness or a neuroinvasive disease, have been reported in several EU/EEA countries, where the virus is endemic. Climate and environmental changes are expected to affect the epidemiology of USUV. A One Health approach could improve the monitoring of its evolution in Europe.

Case report of Usutu virus infection in an immunocompromised patient in Italy, 2022.

Usutu virus (USUV) is an arthropod-borne flavivirus emerged in Africa in 1950s and in Eruope in 1990s causing a massive number of birds' deaths. The role of USUV as human pathogen has been only recently hypothesized and cases of USUV infection in humans remain limited and often related to immunocompromised subjects. Herein, we report a case of USUV meningoencephalitis infection in an immunocompromised patient with no history of previous flavivirus infection. The infection due to USUV evolved rapidly since hospital admission thus resulting fatal in few days after symptoms onset and, although not proven, a suspected bacteria co-infection has been hypothesized. Based on these findings, we suggested that when USUV meningoencephalitis is suspected in countries endemic, careful attention should be applied to neurological syndromes during summer months especially among immunocompromised patients.

Horses as Sentinels for the Circulation of Flaviviruses in Eastern-Central Germany.

Since 2018, autochthonous West Nile virus (WNV) infections have been regularly reported in eastern-central Germany. While clinically apparent infections in humans and horses are not frequent, seroprevalence studies in horses may allow the tracing of WNV and related flaviviruses transmission, such as tick-borne encephalitis virus (TBEV) and Usutu virus (USUV), and consequently help to estimate the risk of human infections. Hence, the aim of our study was to follow the seropositive ratio against these three viruses in horses in Saxony, Saxony Anhalt, and Brandenburg and to describe their geographic distribution for the year 2021. In early 2022, i.e., before the virus transmission season, sera from 1232 unvaccinated horses were tested using a competitive pan-flavivirus ELISA (cELISA). In order to estimate the true seropositive ratio of infection with WNV, TBEV, and USUV for 2021, positive and equivocal results were confirmed by a virus neutralization test (VNT). In addition, possible risk factors for seropositivity using questionnaires were analyzed using logistic regression based on questionnaires similar to our previous study from 2020. In total, 125 horse sera reacted positive in the cELISA. Based on the VNT, 40 sera showed neutralizing antibodies against WNV, 69 against TBEV, and 5 against USUV. Three sera showed antibodies against more than one virus, and eight were negative based on the VNT. The overall seropositive ratio was 3.3% (95% CI: 2.38-4.40) for WNV, 5.6% (95% CI: 4.44-7.04) for TBEV, and 0.4% (95% CI: 0.14-0.98) for USUV infections. While age and number of horses on the holding were factors predicting TBEV seropositivity, no risk factors were discovered for WNV seropositivity. We conclude that horses are useful sentinels to determine the flavivirus circulation in eastern-central Germany, as long as they are not vaccinated against WNV.

JMM Profile: Usutu virus.

Usutu virus (USUV) is an emerging arbovirus belonging to the family Flaviviridae, genus Flavivirus. It is maintained in an enzootic cycle, with mosquitos as the vector and birds as the main amplifying host. Humans, and other mammals such as horses, are dead-end hosts. The virus was originally detected in sub-Saharan Africa, but in the past two decades has spread across Europe. In certain bird species, such as the Eurasian blackbird (Turdus merula), USUV is extremely virulent and can be fatal. Human infection is rare and often asymptomatic, but multiple short-term neurological diseases have been reported, highlighting its public-health risk.

Serological evidence of circulation of West Nile virus in equids in Algerian eastern drylands and its epidemiological risk factors.

In order to determine the prevalence of equine infectious anemia virus (EIAV), Usutu virus (USUV), and West Nile virus (WNV) in eastern Algerian drylands, 340 sera from distinct equids have been collected from 2015 to 2017. Serological analysis for the presence of antibodies against EIAV and flaviviruses was performed using commercially available ELISAs. Sera detected positive, doubtful, or negative close to the doubtful threshold in flavivirus ELISA were tested by the virus neutralization test (VNT), using WNV and USUV strains. The prevalence of WNV antibodies with ELISA was 11.47% (39/340) against 13.53% (46/340) by WNV VNT. EIAV antibodies were not detected in any samples. WNV seroprevalence varies with species, breed and location of horses. Only, one equid was positive for both WNV and USUV neutralizing antibodies. This is the first screening on equids sera of EIAV and USUV in Algeria. This study indicate that WNV and possibly USUV have circulated/are circulating in the Algerian equine population, unlike EIAV does not seem to be present.

European College of Equine Internal Medicine consensus statement on equine flaviviridae infections in Europe.

Horses and other equids can be infected with several viruses of the family Flaviviridae, belonging to the genus Flavivirus and Hepacivirus. This consensus statement focuses on viruses with known occurrence in Europe, with the objective to summarize the current literature and formulate clinically relevant evidence-based recommendations regarding clinical disease, diagnosis, treatment, and prevention. The viruses circulating in Europe include West Nile virus, tick-borne encephalitis virus, Usutu virus, Louping ill virus and the equine hepacivirus. West Nile virus and Usutu virus are mosquito-borne, while tick-borne encephalitis virus and Louping ill virus are tick-borne. The natural route of transmission for equine hepacivirus remains speculative. West Nile virus and tick-borne encephalitis virus can induce encephalitis in infected horses. In the British Isle, rare equine cases of encephalitis associated with Louping ill virus are reported. In contrast, equine hepacivirus infections are associated with mild acute hepatitis and possibly chronic hepatitis. Diagnosis of flavivirus infections is made primarily by serology, although cross-reactivity occurs. Virus neutralization testing is considered the gold standard to differentiate between flavivirus infections in horses. Hepacivirus infection is detected by serum or liver RT-PCR. No direct antiviral treatment against flavi- or hepacivirus infections in horses is currently available and thus, treatment is supportive. Three vaccines against West Nile virus are licensed in the European Union. Geographic expansion of flaviviruses pathogenic for equids should always be considered a realistic threat, and it would be beneficial if their detection was included in surveillance programs.

Evidence for overwintering and autochthonous transmission of Usutu virus to wild birds following its redetection in the United Kingdom.

Usutu virus (USUV) is an emerging zoonotic arbovirus in Europe, where it primarily impacts Eurasian blackbirds (Turdus merula). For mosquito-borne viruses to persist in temperate areas, transovarial transmission in vectors or overwintering in either hosts or diapausing vectors must occur to facilitate autochthonous transmission. We undertook surveillance of hosts and vectors in 2021 to elucidate whether USUV had overwintered in the United Kingdom (UK) following its initial detection there in 2020. From 175 dead bird submissions, we detected 1 case of USUV infection, in a blackbird, from which a full USUV genome was derived. Using a molecular clock analysis, we demonstrate that the 2021 detection shared a most recent common ancestor with the 2020 Greater London, UK, USUV sequence. In addition, we identified USUV-specific neutralizing antibodies in 10 out of 86 serum samples taken from captive birds at the index site, demonstrating in situ cryptic infection and potential sustained transmission. However, from 4966 mosquitoes, we detected no USUV RNA suggesting that prevalence in the vector community was absent or low during sampling. Combined, these results suggest that USUV overwintered in the UK, thus providing empirical evidence for the continued northward expansion of this vector-borne viral disease. Currently, our detection indicates geographically restricted virus persistence. Further detections over time will be required to demonstrate long-term establishment. It remains unclear whether the UK, and by extension other high-latitude regions, can support endemic USUV infection.

West Nile Virus and Usutu Virus: A Post-Mortem Monitoring Study in Wild Birds from Rescue Centers, Central Italy.

West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses that have been associated with neurological diseases in humans and wild birds. Wild bird rescue centers are potential significant hot spots for avian infection surveillance, as recognized in the Italian Integrate National Surveillance Plan for Arboviruses. Here we report the results of a post-mortem active monitoring study conducted from November 2017 to October 2020 on animals hosted in five wild bird rescue centers of Central Italy. Five hundred seventy-six (n = 576) wild birds were tested by real-time polymerase chain reaction (RT-PCR) for the presence of WNV or USUV RNA fragments. No birds tested positive for USUV RNA (n = 0; 0.00%). Evidence of WNV RNA (Ct value = 34.36) was found in one bird (n = 1; 0.17%), an adult little grebe (Tachybaptus ruficollis subsp. ruficollis), that tested WNV positive in December 2019. This study highlights the strategic role of wildlife rescue centers in monitoring both the introduction and circulation of avian emerging zoonotic diseases. In addition, the presence of WNV during the cold season evidences the possible role of birds in overwintering mechanisms in the Italian territory and requires further investigations.

Ivermectin Inhibits the Replication of Usutu Virus In Vitro.

Usutu virus (USUV) is an emerging mosquito-borne arbovirus within the genus Flavivirus, family Flaviviridae. Similar to the closely related West Nile virus (WNV), USUV infections are capable of causing mass mortality in wild and captive birds, especially blackbirds. In the last few years, a massive spread of USUV was present in the avian population of Germany and other European countries. To date, no specific antiviral therapies are available. Nine different approved drugs were tested for their antiviral effects on the replication of USUV in vitro in a screening assay. Ivermectin was identified as a potent inhibitor of USUV replication in three cell types from different species, such as simian Vero CCL-81, human A549 and avian TME R. A 2- to 7-log10 reduction of the viral titer in the supernatant was detected at a non-cytotoxic concentration of 5 µM ivermectin dependent on the applied cell line. IC50 values of ivermectin against USUV lineage Africa 3 was found to be 0.55 µM in Vero CCL-81, 1.94 µM in A549 and 1.38 µM in TME-R cells. The antiviral efficacy was comparable between the USUV lineages Africa 2, Africa 3 and Europe 3. These findings show that ivermectin may be a candidate for further experimental and clinical studies addressing the treatment of USUV disease, especially in captive birds.

Seroprevalence Rates against West Nile, Usutu, and Tick-Borne Encephalitis Viruses in Blood-Donors from North-Western Romania.

INTRODUCTION: West Nile virus (WNV), Usutu virus (USUV), and the tick-borne encephalitis virus (TBEV) are all arboviruses belonging to Flaviviridae family. All are characterized by vectorial transmission and sometimes associated with neuroinvasive infections. The circulation of these viruses is considered endemic in parts of Europe, with human cases reported in many countries. Among hosts, the viruses are vectored by hematophagous arthropods, such as mosquitoes (WNV, USUV) and ticks (TBEV). Considering the currently outdated knowledge regarding the epidemiology of these viruses in Romania, the aim of our study was to assess the seroprevalence rates of WNV, USUV, and TBEV among healthy blood donors in north-western Romania. METHODS: Human blood samples from healthy donors were collected between November 2019 and February 2020 in six counties from the north-western region of Romania. The samples were serologically tested by ELISA and serum neutralization test. RESULTS: Overall, we obtained a seroprevalence of 3.17% for WNV, 0.08% for TBEV, and 0% for USUV. CONCLUSION: Despite the low seroprevalence of WNV, USUV, and TBEV in our study, we highlight the need for continuous nationwide vector and disease surveillance and implementation of control measures. Further research is required for an optimal overview of the epidemiological status of the Romanian population regarding these flaviviruses together with countrywide awareness campaigns.

Bagaza virus and Plasmodium spp. coinfection in red-legged partridges (Alectoris rufa), in Southern Spain 2019.

Flaviviruses such as West Nile (WNV), Usutu (USUV) and Bagaza (BAGV) virus and avian malaria parasites are vector borne pathogens that circulate naturally between avian and mosquito hosts. WNV and USUV and potentially also BAGV constitute zoonoses. Temporal and spatial cocirculation and coinfection with Plasmodium spp., and West Nile virus has been documented in birds and mosquito vectors, and fatally USUV-infected passerines coinfected with Plasmodium spp. had more severe lesions. Also, WNV, USUV and BAGV have been found to cocirculate. Yet little is known about the interaction of BAGV and malaria parasites during consecutive or coinfections of avian hosts. Here we report mortality of free-living red-legged partridges in a hunting estate in Southern Spain that were coinfected with BAGV and Plasmodium spp. The outbreak occurred in the area where BAGV first emerged in Europe in 2010 and where cocirculation of BAGV, USUV and WNV was confirmed in 2011 and 2013. Partridges were found dead in early October 2019. Birds had mottled locally pale pectoral muscles, enlarged, congestive greenish-black tinged livers and enlarged kidneys. Microscopically congestion and predominantly mononuclear inflammatory infiltrates were evident and Plasmodium phanerozoites were present in the liver, spleen, kidneys, muscle and skin. Molecular testing and sequencing detected Plasmodium spp. and BAGV in different tissues of the partridges, and immunohistochemistry confirmed the presence and colocalization of both pathogens in the liver and spleen. Due to the importance of the red-legged partridge in the ecosystem of the Iberian Peninsula and as driver of regional economy such mortalities are of concern. Such outbreaks may reflect climate change related shifts in host, vector and pathogen ecology and interactions that could emerge similarly for other pathogens.

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.

The Evolution, Genomic Epidemiology, and Transmission Dynamics of Tembusu Virus.

Tembusu virus (TMUV) can induce severe egg drop syndrome in ducks, causing significant economic losses. In this study, the possible origin, genomic epidemiology, and transmission dynamics of TMUV were determined. The time to the most recent common ancestor of TMUV was found to be 1924, earlier than that previously reported. The effective population size of TMUV increased rapidly from 2010 to 2013 and was associated with the diversification of different TMUV clusters. TMUV was classified into three clusters (clusters 1, 2, and 3) based on the envelope (E) protein. Subcluster 2.2, within cluster 2, is the most prevalent, and the occurrence of these mutations is accompanied by changes in the virulence and infectivity of the virus. Two positive selections on codons located in the NS3 and NS5 genes (591 of NS3 and 883 of NS5) were identified, which might have caused changes in the ability of the virus to replicate. Based on phylogeographic analysis, Malaysia was the most likely country of origin for TMUV, while Shandong Province was the earliest province of origin in China. This study has important implications for understanding TMUV and provides suggestions for its prevention and control.

Bagaza Virus in Wild Birds, Portugal, 2021.

Bagaza virus emerged in Spain in 2010 and was not reported in other countries in Europe until 2021, when the virus was detected by molecular methods in a corn bunting and several red-legged partridges in Portugal. Sequencing revealed high similarity between the 2021 strains from Portugal and the 2010 strains from Spain.

Combining host and vector data informs emergence and potential impact of an Usutu virus outbreak in UK wild birds.

Following the first detection in the United Kingdom of Usutu virus (USUV) in wild birds in 2020, we undertook a multidisciplinary investigation that combined screening host and vector populations with interrogation of national citizen science monitoring datasets to assess the potential for population impacts on avian hosts. Pathological findings from six USUV-positive wild passerines were non-specific, highlighting the need for molecular and immunohistochemical examinations to confirm infection. Mosquito surveillance at the index site identified USUV RNA in Culex pipiens s.l. following the outbreak. Although the Eurasian blackbird (Turdus merula) is most frequently impacted by USUV in Europe, national syndromic surveillance failed to detect any increase in occurrence of clinical signs consistent with USUV infection in this species. Furthermore, there was no increase in recoveries of dead blackbirds marked by the national ringing scheme. However, there was regional clustering of blackbird disease incident reports centred near the index site in 2020 and a contemporaneous marked reduction in the frequency with which blackbirds were recorded in gardens in this area, consistent with a hypothesis of disease-mediated population decline. Combining results from multidisciplinary schemes, as we have done, in real-time offers a model for the detection and impact assessment of future disease emergence events.

Circulation of zoonotic flaviviruses in wild passerine birds in Western Spain.

We explore the presence of zoonotic flaviviruses (West Nile virus (WNV) and Usutu virus (USUV)) neutralizing antibodies in rarely studied passerine bird species. We report, for the first time in Europe, WNV-specific antibodies in red avadavat and cetti's warbler, and USUV in yellow-crowned bishop. The evidence of WNV and USUV circulating in resident and migratory species has implications for both animal and public health. Future outbreaks in avian reservoir hosts may occur and passerines should be considered as priority target species in flavivirus surveillance programmes.