Multiple bloodmeals enhance dissemination of arboviruses in three medically relevant mosquito genera.

BACKGROUND: Mosquitoes in nature may acquire multiple bloodmeals (BMs) over the course of their lifetime; however, incorporation of frequent feeding behavior in laboratory vector competence studies is rarely done. We have previously shown that acquisition of a second non-infectious BM can enhance early dissemination of Zika virus (ZIKV), dengue virus, and chikungunya virus in Aedes aegypti and ZIKV in Aedes albopictus mosquitoes, yet it is unknown if other taxonomically-diverse virus-vector pairings show a similar trend under a sequential feeding regimen. METHODS: To test this, we evaluated the impact of a second noninfectious BM on the vector competence of Aedes aegypti and Anopheles quadrimaculatus for Mayaro virus, Culex quinquefasciatus for West Nile virus, Aedes triseriatus for La Crosse virus, and Aedes aegypti for Oropouche virus (OROV). Female mosquitoes were fed BMs containing these viruses and half of them were given a second noninfectious BM at 3 or 4-days post infection. Mosquitoes were harvested at various time points and assayed for virus infection in bodies and disseminated infection in legs by performing reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays. RESULTS: We found that a second noninfectious BM had no impact on midgut infection rates but increased virus dissemination for all but one of the virus-vector pairings- Ae. aegypti and OROV. Unlike the other arboviruses under consideration, which are strictly mosquito-borne, biting midges (Culicoides spp.) serve as the main vector of OROV and this virus rarely disseminated to the mosquito leg tissue in our study. CONCLUSIONS: Taken together, our findings show that sequential blood feeding enhances virus dissemination across diverse arbovirus-vector pairings, representing three mosquito genera and virus families, but a second BM was insufficient to overcome a strong midgut virus escape barrier in a nonnatural virus-vector pairing.

Seropositivity of West Nile virus among acute febrile patients in Ilorin, Nigeria.

INTRODUCTION: West Nile Virus (WNV), a member of Flaviviridae family, is one of the most widely distributed arboviruses in the world. In developing countries like Nigeria, fever resulting from the WNV infection is often presumptively ascribed to malaria or typhoid due to misdiagnosis and low-level awareness of the viral infection. This study determined the prevalence of WNV IgM and IgG antibodies among febrile patients in the Ilorin metropolis. MATERIALS AND METHODS: A total of two hundred (200) blood samples were collected from consenting patients and each serum was screened for anti-WNV IgM and IgG antibodies using indirect enzyme-linked immunosorbent assay (ELISA). Statistical correlation and logistic regression analysis were conducted. RESULTS: Overall, 6% (12/200) anti-WNV IgM seropositivity rate was recorded amongst the acute febrile patients with higher prevalence (6.30%) in females than in males (5.45%). Anti-WNV IgG positivity rate of 52% (104/200) was recorded, with 50.67% positivity rate in males and 38.95% in female participants. The convalescence phase posited by the 5.4% (11/200) co-detection of anti-WNV IgG and IgM antibodies among the participants was recorded. A statistical correlation was noticed with the age and religion of respondents to WNV serological positivity while gender, occupation, use of mosquito nets and formal education had no positive correlation at p < 0.05. However, based on odd ratio at 95% CI and logistic regression coefficients, the evaluated risk factors such as blood transfusion, residency, malaria parasite, and proximity to stagnant water and bush were significant to anti-WNV IgG and IgM positivity. CONCLUSION: The findings of this study show the circulation of WNV in the study area. There is an urgent need for clinicians/physicians to include screening for the West Nile virus in cases of febrile patients before the commencement of treatment.

Regional variation in the landscape ecology of West Nile virus sentinel chicken seroconversion in Florida.

How landscape composition and configuration impact the distribution of multi-vector and multi-host mosquito vector-borne disease systems, such as West Nile virus (WNV), remains challenging because of complex habitat and resource requirements by hosts and vectors that affect transmission opportunities. We examined correlations between landscape composition and configuration and 2018 WNV sentinel chicken seroconversion in Florida, USA across the state and within five National Oceanic Atmospheric Administration (NOAA) bioclimatic regions to understand strength and variation of landscape effects during an elevated transmission year. Although few landscape studies have examined WNV in Florida, we expected higher percentages of residential or medium-developed landscapes and more fragmented landscapes would be positively correlated with WNV seroconversion owing to the main mosquito vector habitats and avian host distributions. However, we expected to find variation in the importance of forest, wetland, and agriculture landscapes across bioclimatic regions in the state. WNV seroconversion rates were calculated using Florida 2018 Department of Health WNV sentinel chicken seroconversion data from 187 flocks maintained by mosquito control programs. Percent land cover and edge density metrics were calculated for multiple land cover classes and within multiple buffer distances from chicken coops using 2019 National Land Cover Data. We used binomial generalized linear mixed effects models to calculate the importance of landscape metrics to WNV seroconversion. We found no statewide predictors of seroconversion, but as expected, the importance of landscape varied across regions. In the north-central part of the state, we found higher seroconversion in less populated suburban areas while higher seroconversion in south-central Florida was correlated with fragmented forested areas within 0.5 km of coops and intact woody wetland areas within 2 km of coops. This work corroborates previous findings that consistent landscape predictors of WNV are difficult to identify across broader geographic areas and sets the stage for additional work that incorporates climate and landscapes interactions for a greater understanding of WNV ecology in this geographic region.

Usutu virus, an emerging arbovirus with One Health importance.

Usutu virus (USUV, Flaviviridae) is an emerging arbovirus that has led to epizootic outbreaks in birds and numerous human neuroinvasive disease cases in Europe. It is maintained in an enzootic cycle with Culex mosquitoes and passerine birds, a transmission cycle that is shared by West Nile virus (WNV) and St. Louis encephalitis virus (SLEV), two flaviviruses that are endemic in the United States. USUV and WNV co-circulate in Africa and Europe, and SLEV and WNV co-circulate in North America. These three viruses are prime examples of One Health issues, in which the interactions between humans, animals, and the environments they reside in can have important health impacts. The three facets of One Health are interwoven throughout this article as we discuss the mechanisms of flavivirus transmission and emergence. We explore the possibility of USUV emergence in the United States by analyzing the shared characteristics among USUV, WNV, and SLEV, including the role that flavivirus co-infections and sequential exposures may play in viral emergence. Finally, we provide insights on the importance of integrated surveillance programs as One Health tools that can be used to mitigate USUV emergence and spread.

A specific domain within the 3' untranslated region of Usutu virus confers resistance to the exonuclease ISG20.

Usutu virus (USUV) and West Nile virus (WNV) are two closely related emerging mosquito-borne flaviviruses. Their natural hosts are wild birds, but they can also cause severe neurological disorders in humans. Both viruses are efficiently suppressed by type I interferon (IFN), which interferes with viral replication, dissemination, pathogenesis and transmission. Here, we show that the replication of USUV and WNV are inhibited through a common set of IFN-induced genes (ISGs), with the notable exception of ISG20, which USUV is resistant to. Strikingly, USUV was the only virus among all the other tested mosquito-borne flaviviruses that demonstrated resistance to the 3'-5' exonuclease activity of ISG20. Our findings highlight that the intrinsic resistance of the USUV genome, irrespective of the presence of cellular or viral proteins or protective post-transcriptional modifications, relies on a unique sequence present in its 3' untranslated region. Importantly, this genomic region alone can confer ISG20 resistance to a susceptible flavivirus, without compromising its infectivity, suggesting that it could be acquired by other flaviviruses. This study provides new insights into the strategy employed by emerging flaviviruses to overcome host defense mechanisms.

Differential susceptibility of human motor neurons to infection with Usutu and West Nile virus.

West Nile virus (WNV) and Usutu virus (USUV) are closely related flaviviruses with differing capacities to cause neurological disease in humans. WNV is thought to use a transneural route of neuroinvasion along motor neurons and causes severe motor deficits. The potential for use of transneural routes of neuroinvasion by USUV has not been investigated experimentally, and evidence from the few clinical case reports of USUV-associated neuroinvasive disease is lacking. We hypothesised that, compared with WNV, USUV is less able to infect motor neurons, and therefore determined the susceptibility of human induced pluripotent stem cell (iPSC)-derived spinal cord motor neurons to infection. Both viruses could grow to high titres in iPSC-derived neural cultures. However, USUV could not productively infect motor neurons due to restriction by the antiviral response, which was not induced upon WNV infection. Inhibition of the antiviral response allowed for widespread infection and transportation of USUV along motor neurons within a compartmented culture system. These results show a stark difference in the ability of these two viruses to evade initiation of intrinsic antiviral immunity. Our data suggests that USUV cannot infect motor neurons in healthy individuals but in case of immunodeficiency may pose a risk for motor-related neurological disease and transneural invasion.

West Nile virus, but not Usutu virus, can productively infect human motor neurons as a possible route of neuroinvasion.

Sentinel chicken surveillance reveals previously undetected circulation of West Nile virus in the Netherlands.

West Nile virus (WNV) was first detected in the Netherlands in 2020, with circulation observed in birds, mosquitoes, and humans in two geographical areas. Usutu virus (USUV) has been circulating in the Netherlands since 2016. Following the detection of WNV in the Netherlands, we investigated the possible use of petting zoos as urban sentinel sites to examine the extent of WNV and USUV circulation around the two WNV outbreak locations. Chickens at petting zoos and in backyards were sampled within a 15-kilometer radius of the confirmed WNV circulation areas at three timepoints over one year (2021-2022). Sera were analysed using a protein microarray for binding antibodies to orthoflavivirus NS1 antigens and reactive samples were confirmed through micro-focus reduction neutralization tests (mFRNT). Furthermore, mosquitoes at sampling locations were collected to assess their blood feeding behaviour. This serosurvey detected the circulation of USUV and WNV in petting zoo and backyard chickens in 2021, both within and outside the 2020 outbreak areas. The WNV circulation was not detected by other existing surveillance schemes in mosquitoes, wild birds, horses and humans. In addition, the results show rapid decay of USUV antibodies in approximately 20 weeks. Our findings support the utility and the added value of petting zoo chickens as sentinels for monitoring USUV and WNV circulation compared to other available methods. Seroconversions observed in petting zoos and backyard chickens living in or near densely populated urban areas further highlighted potential public health risks that went undetected.

[Opsoclonus-myoclonus syndrome associated with West Nile virus]. / Opsoklonus-mioklonus sindrom, obuslovlennyi likhoradkoi Zapadnogo Nila.

Opsoclonus is oculomotor dyskinesia characterized by rapid, repetitive conjugate eye movements that are involuntary, arrhythmic, chaotic, and multidirectional (horizontal, vertical, and torsional components). Most common cause of the symptom is paraneoplastic process. It is combined with myoclonus usually with the development of opsoclonus-myoclonus syndrome. Viral etiology is one of the possible causes of the of this syndrome, which is presented in the following case. A 26-year-old man was admitted to an infectious hospital suspected by encephalitis. After a 2-day febrile fever the patient developed balance problem, nausea, vomiting, tremors in the limbs and head, sensations of jerking of eyeballs. The neurological examination revealed opsoclonus, myoclonic jerking in the limbs, neck and trunk muscles, severe static and dynamic ataxia, there was no consciousness changes or altered mental stature. Cerebrospinal fluid examination revealed a pleocytosis (24 cells), increased protein levels (1.1 g/l). MRI of the brain was normal. After excluding of typical neuroinfections the patient was tested for West Nile fever. Elevated titers of IgG and IgM for West Nile fever virus were detected, as well as positive PCR for virus RNA in the cerebrospinal fluid. Patient was treated by acyclovir, an antibiotic and dexamethasone but severe neurological symptoms were persisted for 2 weeks with inability of sitting and walking. Then the symptoms gradually began to improve, rehabilitation was included with total recovery during the next 2 months. The doctors should be aware for possibility of neuroinvasive form of West Nile fever as the etiology of opsoclonus-myoclonus syndrome.

Mosquito dynamics and their drivers in peri-urban Antananarivo, Madagascar: insights from a longitudinal multi-host single-site survey.

BACKGROUND: Antananarivo, the capital city of Madagascar, is experiencing a steady increase in population growth. Due to the abundance of mosquito vectors in this locality, the population exposed to mosquito-borne diseases is therefore also increasing, as is the risk of epidemic episodes. The aim of the present study was to assess, in a resource-limited setting, the information on mosquito population dynamics and disease transmission risk that can be provided through a longitudinal entomological study carried out in a multi-host single site. METHODS: Mosquitoes were collected every 15 days over 16 months (from January 2017 to April 2018) using six CDC-light traps in a peri-urban area of Antananarivo. Multivariable generalised linear models were developed using indoor and outdoor densities of the predominant mosquito species as response variables and moon illumination, environmental data and climatic data as the explanatory variables. RESULTS: Overall, 46,737 mosquitoes belonging to at least 20 species were collected, of which Culex antennatus (68.9%), Culex quinquefasciatus (19.8%), Culex poicilipes (3.7%) and Anopheles gambiae sensu lato (2.3%) were the most abundant species. Mosquito densities were observed to be driven by moon illumination and climatic factors interacting at different lag periods. The outdoor models demonstrated biweekly and seasonal patterns of mosquito densities, while the indoor models demonstrated only a seasonal pattern. CONCLUSIONS: An important diversity of mosquitoes exists in the peri-urban area of Antananarivo. Some well-known vector species, such as Cx. antennatus, a major vector of West Nile virus (WNV) and Rift-Valley fever virus (RVFV), Cx. quinquefasciatus, a major vector of WNV, Cx. poicilipes, a candidate vector of RVFV and An. gambiae sensu lato, a major vector of Plasmodium spp., are abundant. Importantly, these four mosquito species are present all year round, even though their abundance declines during the cold dry season, with the exception of Cx. quinquefasciatus. The main drivers of their abundance were found to be temperature, relative humidity and precipitation, as well as-for outdoor abundance only-moon illumination. Identifying these drivers is a first step towards the development of pathogen transmission models (R0 models), which are key to inform public health stakeholders on the periods of most risk for vector-borne diseases.

Ocular manifestations of West Nile virus.

PURPOSE OF REVIEW: Over the past two decades, the ophthalmic manifestations of West Nile virus have been more clearly established in the literature. This review aims to summarize its diagnosis and pathogenesis, with a focus on its clinical appearance, characteristic imaging features, and management. RECENT FINDINGS: Ocular manifestations of West Nile virus present early in the disease course and are more common in cases with severe neurological involvement. The use of optical coherence tomography (OCT), optical coherence tomography angiography (OCT-A), fundus autofluorescence (FAF), fluorescein angiogram (FA), and indocyanine green angiography (ICGA) can aid in its diagnosis and management. SUMMARY: West Nile virus infection may present with ocular findings that include anterior uveitis, vitritis, retinitis, chorioretinitis, and optic neuropathy; visual prognosis can range from excellent to poor depending on severity of involvement and the presence of secondary complications, such as occlusive vasculitis and macular ischemia. Diagnosis may be aided by multimodal imaging assessment. The ophthalmologist should have a high clinical suspicion for ocular involvement in cases of severe systemic disease.

Orthoflaviviral Inhibitors in Clinical Trials, Preclinical In Vivo Efficacy Targeting NS2B-NS3 and Cellular Antiviral Activity via Competitive Protease Inhibition.

Orthoflaviviruses, including zika (ZIKV), West Nile (WNV), and dengue (DENV) virus, induce severely debilitating infections and contribute significantly to the global disease burden, yet no clinically approved antiviral treatments exist. This review offers a comprehensive analysis of small-molecule drug development targeting orthoflaviviral infections, with a focus on NS2B-NS3 inhibition. We systematically examined clinical trials, preclinical efficacy studies, and modes of action for various viral replication inhibitors, emphasizing allosteric and orthosteric drugs inhibiting NS2B-NS3 protease with in vivo efficacy and in vitro-tested competitive NS2B-NS3 inhibitors with cellular efficacy. Our findings revealed that several compounds with in vivo preclinical efficacy failed to show clinical antiviral efficacy. NS3-NS4B inhibitors, such as JNJ-64281802 and EYU688, show promise, recently entering clinical trials, underscoring the importance of developing novel viral replication inhibitors targeting viral machinery. To date, the only NS2B-NS3 inhibitor that has undergone clinical trials is doxycycline, however, its mechanism of action and clinical efficacy as viral growth inhibitor require additional investigation. SYC-1307, an allosteric inhibitor, exhibits high in vivo efficacy, while temoporfin and methylene blue represent promising orthosteric non-competitive inhibitors. Compound 71, a competitive NS2B-NS3 inhibitor, emerges as a leading preclinical candidate due to its high cellular antiviral efficacy, minimal cytotoxicity, and favorable in vitro pharmacokinetic parameters. Challenges remain in developing competitive NS2B-NS3 inhibitors, including appropriate biochemical inhibition assays as well as the selectivity and conformational flexibility of the protease, complicating effective antiviral treatment design.

Neuroinflammation-Induced SIADH in West Nile Virus Infection: An Uncommon Neurological and Endocrine Manifestation.

BACKGROUND West Nile virus (WNV) is a vector-borne flavivirus that is typically transmitted by Aedes and Anopheles mosquitos. WNV infection typically presents with symptoms consistent with viral meningitis, which include fever, headache, and meningeal signs. WNV infections are typically self-resolving, with symptoms lasting 3-10 days. Although uncommon, patients with WNV infection can be afflicted with hyponatremia, although the etiology is unclear. Because of encephalitis, neuroinflammation may be involved in the deterioration of adrenal signaling, leading to salt wasting. CASE REPORT We present the case of a 75-year-old man who presented with headache, neck pain, photophobia, and viral illness symptoms. He was found to be profoundly hyponatremic, concerning for SIADH. He had a sodium drop to 117 mmol/L, leading to further lethargy and confusion. He received 3% saline, fluid restriction, and salt tablets, and more common causes of SIADH were ruled out. MRI revealed the absence of the posterior pituitary bright spot. He was subsequently found to have positive WNV titers and improved with continued supportive treatment. CONCLUSIONS WNV presents a unique challenge to providers due to its difficult diagnosis and association with hyponatremia. There is no clear role for specific medical treatment such as corticosteroids vs IV immunoglobulins. Supportive care is recommended for those testing positive for WNV. Clinicians should consider the possibility of viral infections, such as WNV, in patients living in endemic areas who present with unexplained hyponatremia, cognitive symptoms, and relevant history.

Evidence of Lineage 1 and 3 West Nile Virus in Person with Neuroinvasive Disease, Nebraska, USA, 2023.

West Nile virus (WNV) is the most common cause of human arboviral disease in the contiguous United States, where only lineage 1 (L1) WNV had been found. In 2023, an immunocompetent patient was hospitalized in Nebraska with West Nile neuroinvasive disease and multisystem organ failure. Testing at the Centers for Disease Control and Prevention indicated an unusually high viral load and acute antibody response. Upon sequencing of serum and cerebrospinal fluid, we detected lineage 3 (L3) and L1 WNV genomes. L3 WNV had previously only been found in Central Europe in mosquitoes. The identification of L3 WNV in the United States and the observed clinical and laboratory features raise questions about the potential effect of L3 WNV on the transmission dynamics and pathogenicity of WNV infections. Determining the distribution and prevalence of L3 WNV in the United States and any public health and clinical implications is critical.

WNV and SLEV coinfection in avian and mosquito hosts: impact on viremia, antibody responses, and vector competence.

West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are closely related flaviviruses that can cause encephalitis in humans and related diseases in animals. In nature, both are transmitted by Culex, with wild birds, including jays, sparrows, and robins, serving as vertebrate hosts. WNV and SLEV circulate in the same environments and have recently caused concurrent disease outbreaks in humans. The extent that coinfection of mosquitoes or birds may alter transmission dynamics, however, is not well characterized. We therefore sought to determine if coinfection alters infection kinetics and virus levels in birds and infection rates in mosquitoes. Accordingly, American robins (Turdus migratorius), two species of mosquitoes, and vertebrate and invertebrate cells were infected with WNV and/or SLEV to assess how simultaneous exposure may alter infection outcomes. There was variable impact of coinfection in vertebrate cells, with some evidence that SLEV can suppress WNV replication. However, robins had comparable viremia and antibody responses regardless of coinfection. Conversely, in Culex cells and mosquitoes, we saw a minimal impact of simultaneous exposure to both viruses on replication, with comparable infection, dissemination, and transmission rates in singly infected and coinfected mosquitoes. Importantly, while WNV and SLEV levels in coinfected mosquito midguts were positively correlated, we saw no correlation between them in salivary glands and saliva. These results reveal that while coinfection can occur in both avian and mosquito hosts, the viruses minimally impact one another. The potential for coinfection to alter virus population structure or the likelihood of rare genotypes emerging remains unknown.IMPORTANCEWest Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are closely related viruses that are transmitted by the same mosquitoes and infect the same birds in nature. Both viruses circulate in the same regions and have caused concurrent outbreaks in humans. It is possible that mosquitoes, birds, and/or humans could be infected with both WNV and SLEV simultaneously, as has been observed with Zika, chikungunya, and dengue viruses. To study the impact of coinfection, we experimentally infected vertebrate and invertebrate cells, American robins, and two Culex species with WNV and/or SLEV. Robins were efficiently coinfected, with no impact of coinfection on virus levels or immune response. Similarly, in mosquitoes, coinfection did not impact infection rates, and mosquitoes could transmit both WNV and SLEV together. These results reveal that WNV and SLEV coinfection in birds and mosquitoes can occur in nature, which may impact public health and human disease risk.

Drivers and epidemiological patterns of West Nile virus in Serbia.

Background: West Nile virus (WNV) is an emerging mosquito-borne pathogen in Serbia, where it has been detected as a cause of infection in humans since 2012. We analyzed and modelled WNV transmission patterns in the country between 2012 and 2023. Methods: We applied a previously developed modelling approach to quantify epidemiological parameters of interest and to identify the most important environmental drivers of the force of infection (FOI) by means of statistical analysis in the human population in the country. Results: During the study period, 1,387 human cases were recorded, with substantial heterogeneity across years. We found that spring temperature is of paramount importance for WNV transmission, as FOI magnitude and peak timing are positively associated with it. Furthermore, FOI is also estimated to be greater in regions with a larger fraction of older adult people, who are at higher risk to develop severe infections. Conclusion: Our results highlight that temperature plays a key role in shaping WNV outbreak magnitude in Serbia, confirming the association between spring climatic conditions and WNV human transmission risk and thus pointing out the importance of this factor as a potential early warning predictor for timely application of preventive and control measures.

RNase L-induced bodies sequester subgenomic flavivirus RNAs to promote viral RNA decay.

Subgenomic flavivirus RNAs (sfRNAs) are structured RNAs encoded by flaviviruses that promote viral infection by inhibiting cellular RNA decay machinery. Herein, we analyze sfRNA production and localization using single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) throughout West Nile virus, Zika virus, or dengue virus serotype 2 infection. We observe that sfRNAs are generated during the RNA replication phase of viral infection in the cytosol and accumulate in processing bodies (P-bodies), which contain RNA decay machinery such as XRN1 and Dcp1b. However, upon activation of the host antiviral endoribonuclease, ribonuclease L (RNase L), sfRNAs re-localize to ribonucleoprotein complexes known as RNase L-induced bodies (RLBs). RLB-mediated sequestration of sfRNAs reduces sfRNA association with RNA decay machinery in P-bodies, which coincides with increased viral RNA decay. These findings establish a functional role for RLBs in enhancing the cell-mediated decay of viral RNA by sequestering functional viral RNA decay products.

Membrane Retention of West Nile Virus NS5 Depends on NS1 or NS3 for Enzymatic Activity.

West Nile virus (WNV) nonstructural protein 5 (NS5) possesses multiple enzymatic domains essential for viral RNA replication. During infection, NS5 predominantly localizes to unique replication organelles (ROs) at the rough endoplasmic reticulum (RER), known as vesicle packets (VPs) and convoluted membranes (CMs), with a portion of NS5 accumulating in the nucleus. NS5 is a soluble protein that must be in the VP, where its enzymatic activities are required for viral RNA synthesis. However, the mechanistic processes behind the recruitment of NS5 from the cytoplasm to the RER membrane remain unclear. Here, we utilize high-resolution confocal microscopy and sucrose density gradient ultracentrifugation to investigate whether the association of NS5 with other NS proteins contributes to its membrane recruitment and retention. We demonstrate that NS1 or NS3 partially influences the NS5 association with the membrane. We further demonstrate that processed NS5 is predominantly in the cytoplasm and nucleus, indicating that the processing of NS5 from the viral polyprotein does not contribute to its membrane localization. These observations suggest that other host or viral factors, such as the enwrapment of NS5 by the RO, may also be necessary for the complete membrane retention of NS5. Therefore, studies on the inhibitors that disrupt the membrane localization of WNV NS5 are warranted for antiviral drug development.

Characterization of West Nile virus Koutango lineage from phlebotomine sandflies in Kenya.

The West Nile virus (WNV), primarily transmitted by mosquitoes, is one of the most widespread flaviviruses globally, with past outbreaks occurring in the USA and Europe. Recent studies in parts of Africa, including Kenya, have identified the West Nile virus Koutango lineage (WN-KOUTV) among phlebotomine sandfly populations, however, our understanding of this virus remains limited. This study aimed to characterize WN-KOUTV from phlebotomine sandflies. Sandflies were sampled between 12th -16th March 2021 and 16th -20th March 2023 from six villages each in Baringo and Isiolo Counties, using CDC light traps. Female sandflies were taxonomically identified and pooled based on genus and site of collection. Virus isolation was performed in Vero cells. Viral genomes were determined using next-generation sequencing. Phylogenetic and molecular clock analyses were done to decipher the virus's evolutionary relationships. Comparative analyses of amino acid sequences were performed to determine variations. Protein modeling in Pymol was conducted to elucidate variations in key protein regions. Evolutionary pressure analysis investigated the selection pressures on the virus. In vitro experiments were done to investigate the virus growth kinetics in mammalian Vero E6 and mosquito C6/36 cells. We report the isolation of WN-KOUTV from Salabani in Baringo and Aremet in Isiolo, Kenya. The isolated WN-KOUTVs clustered with previously identified WN-KOUTV strains. Comparative analysis revealed a unique amino acid at NS5 653. The WN-KOUTV lineage as a whole is under purifying selective pressure, with diversifying pressure acting at site NS3 267. The current WN-KOUTV replicated in Vero E6 and C6/36 cells comparable to West Nile virus Lineage 1a, isolated from mosquitoes. Subsequent isolations of WN-KOUTV in phlebotomine sandflies suggest potential vectors, however, vector competence studies would confirm this. Replication in mammalian and insect cell lines suggests there may exist a vector/host relationship. We speculate the close genetic relationship of WN-KOUTV strains from East and West Africa may potentially be enabled by bird migratory routes between the two regions. If proven, this could point to a potential future pandemic pathway for this virus.

Host attraction and host feeding patterns indicate generalist feeding of Culex pipiens s.s. and Cx. torrentium.

BACKGROUND: Mosquito host feeding patterns are an important factor of the species-specific vector capacity determining pathogen transmission routes. Culex pipiens s.s./Cx. torrentium are competent vectors of several arboviruses, such as West Nile virus and Usutu virus. However, studies on host feeding patterns rarely differentiate the morphologically indistinguishable females. METHODS: We analyzed the host feeding attraction of Cx. pipiens and Cx. torrentium in host-choice studies for bird, mouse, and a human lure. In addition, we summarized published and unpublished data on host feeding patterns of field-collected specimens from Germany, Iran, and Moldova from 2012 to 2022, genetically identified as Cx. pipiens biotype pipiens, Cx. pipiens biotype molestus, Cx. pipiens hybrid biotype pipiens × molestus, and Cx. torrentium, and finally put the data in context with similar data found in a systematic literature search. RESULTS: In the host-choice experiments, we did not find a significant attraction to bird, mouse, and human lure for Cx. pipiens pipiens and Cx. torrentium. Hosts of 992 field-collected specimens were identified for Germany, Iran, and Moldova, with the majority determined as Cx. pipiens pipiens, increasing the data available from studies known from the literature by two-thirds. All four Culex pipiens s.s./Cx. torrentium taxa had fed with significant proportions on birds, humans, and nonhuman mammals. Merged with the data from the literature from 23 different studies showing a high prevalence of blood meals from birds, more than 50% of the blood meals of Cx. pipiens s.s. were identified as birds, while up to 39% were human and nonhuman mammalian hosts. Culex torrentium fed half on birds and half on mammals. However, there were considerable geographical differences in the host feeding patterns. CONCLUSIONS: In the light of these results, the clear characterization of the Cx. pipiens s.s./Cx. torrentium taxa as ornithophilic/-phagic or mammalophilic/-phagic needs to be reconsidered. Given their broad host ranges, all four Culex taxa could potentially serve as enzootic and bridge vectors.

Larval Competition between Aedes and Culex Mosquitoes Carries over to Higher Arboviral Infection during Their Adult Stage.

The common house mosquito (Culex pipiens) is a native vector for West Nile virus (WNV). Invasive species like the tiger mosquito (Aedes albopictus) and Asian bush mosquito (Aedes japonicus) are rapidly spreading through Europe, posing a major threat as vectors for dengue, chikungunya (CHIKV), and Japanese encephalitis virus (JEV). These mosquitoes share a similar ecological niche as larvae, but the carry-over effects of aquatic larval interactions to the terrestrial adult stage remain largely unknown and their medical relevance requires further investigation. This study examines the context dependency of larval interactions among Aedes albopictus, Aedes japonicus, and Culex pipiens. The survival, development time, growth, and energetic storage were measured in different European populations within density-response (intraspecific) experiments and replacement (interspecific) experiments at 20 °C and 26 °C. Overall, Ae. japonicus was the weakest competitor, while competition between Ae. albopictus and Cx. pipiens varied with temperature. Adults emerging from this larval competition were infected as follows: Culex pipiens with WNV, Ae. albopictus with CHIKV, and Ae. japonicus with JEV. While no JEV infection was observed, mosquitoes experiencing interspecific interactions during their larval stages exhibited higher infection rates and viral RNA titers for CHIKV and WNV. This increased susceptibility to viral infection after larval competition suggests a higher risk of arbovirus transmission in co-occurring populations.