Endemic La Crosse Virus Neuroinvasive Disease in North Carolina Residents: 2000-2020.

Background: La Crosse virus neuroinvasive disease (LACVND) is the most common cause of arboviral encephalitis in children within the United States; in North Carolina, it is the most prevalent endemic mosquito-borne disease in humans. Methods: Here we report a surveillance summary of confirmed and probable LACVND during 2000-2020 using North Carolina Electronic Disease Surveillance System data, and we describe associated demographic characteristics, spatiotemporal distribution, clinical features, and mortality rates. Results: A total of 355 cases (74.9% confirmed) were reported from 41 North Carolina counties; most cases (92%) occurred in 19 Western North Carolina counties. An average of 17 cases were reported annually with the majority (94%) of cases occurring between mid-June and early October (epiweeks 25-41). The median case age was nine years (range: 1-95 years), 79% were aged ≤ 18 years, and 56.6% were male. Cases commonly presented with headache (95.4%), fever (95.1%), and altered mental status (80.8%); encephalitis (82.0%) and meningitis (45.9%) were frequently diagnosed. Encephalitis was more common in children (87%) than adults (62%) (P < .001). Similarly, seizures were more common in children (54%) than adults (27%) (P < .01). The case fatality rate was 1.4%; however, differences in age-specific rates were observed. Limitations: Changes in case definitions and reporting requirements, missing data, different reporting sources, and the retrospective nature of this study are all important limitations of our study. Conclusion: Given the persistent endemicity of La Crosse virus in Western North Carolina, clinicians and public health providers should consider La Crosse virus disease in all individuals, especially children, with compatible symptoms and a travel history to endemic counties. Prevention measures and educational outreach/public health messaging should focus on caregivers and children in Western North Carolina counties and during times when transmission risk is higher (i.e., summer and early fall).

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.

N4-Hydroxycytidine/molnupiravir inhibits RNA virus-induced encephalitis by producing less fit mutated viruses.

A diverse group of RNA viruses have the ability to gain access to the central nervous system (CNS) and cause severe neurological disease. Current treatment for people with this type of infection is generally limited to supportive care. To address the need for reliable antivirals, we utilized a strategy of lethal mutagenesis to limit virus replication. We evaluated ribavirin (RBV), favipiravir (FAV) and N4-hydroxycytidine (NHC) against La Crosse virus (LACV), which is one of the most common causes of pediatric arboviral encephalitis cases in North America and serves as a model for viral CNS invasion during acute infection. NHC was approximately 3 to 170 times more potent than RBV or FAV in neuronal cells. Oral administration of molnupiravir (MOV), the prodrug of NHC, decreased neurological disease development (assessed as limb paralysis, ataxia and weakness, repeated seizures, or death) by 31% (4 mice survived out of 13) when treatment was started on the day of infection. MOV also reduced disease by 23% when virus was administered intranasally (IN). NHC and MOV produced less fit viruses by incorporating predominantly G to A or C to U mutations. Furthermore, NHC also inhibited virus production of two other orthobunyaviruses, Jamestown Canyon virus and Cache Valley virus. Collectively, these studies indicate that NHC/MOV has therapeutic potential to inhibit viral replication and subsequent neurological disease caused by orthobunyaviruses and potentially as a generalizable strategy for treating acute viral encephalitis.

Fine-scale associations between land cover composition and the oviposition activity of native and invasive Aedes (Diptera: Culicidae) vectors of La Crosse virus.

Land cover is recognized as an important determinant of mosquito community assemblages and pathogen transmission, but few studies have investigated the role of land cover in populations of La Crosse virus (LACV) vectors. La Crosse virus is a zoonotic disease primarily maintained by native Aedes triseriatus, with accessory transmission by invasive vectors Ae. albopictus and Ae. japonicus. The objective of this study was to investigate the association between land cover composition and the egg abundances of LACV vectors in a city with endemic LACV transmission. Oviposition cups were deployed at sites ranging from recreational forests to urban areas. Mixed-effects regression models were used to test the relationships between land cover composition and species abundances. Sites characterized by large percentages of impervious and built surfaces were dominated by Ae. albopictus, but Ae. triseriatus was the most abundant species in forested areas. Aedes japonicus was rare at all sites, but its presence was associated with higher percentages of forested land. These results indicate that forested areas may allow Ae. triseriatus to maintain large populations near urban areas that are dominated by Ae. albopictus. Further research should explore the potential for these land cover-dependent population dynamics to influence LACV transmission cycles.

La Crosse virus encephalitis in children.

PURPOSE OF REVIEW: La Crosse virus encephalitis (LACV-E) is among the most common neuroinvasive arthropod-borne viral infections of childhood in North America. Children are uniquely predisposed to symptomatic disease, whereas symptomatic adult infections remain uncommon. Infection results frequently in neurologic symptomatology including headaches, seizures, and altered mentation, often necessitating hospitalization and significant diagnostic evaluation. The purpose of this review is to provide a contemporary assessment of clinical, laboratory, and neurobehavioral outcomes of children with LACV-E. RECENT FINDINGS: Common clinical manifestations at presentation, specifically seizure activity and altered mental status, are independent predictors of disease severity. Epileptiform discharges on electroencephalogram (EEG) during hospitalization may predict long-term epilepsy diagnosis. Lastly, long-term neurologic sequelae from acute infection is persistent and likely under-recognized among children with LACV-E. SUMMARY: As climate change alters the geographic distribution of mosquito borne illnesses, a possibility of regional expansion of La Crosse virus (LACV) endemicity exists. The above data highlight readily identifiable features and testing modalities for clinicians who may encounter this viral infection. Importantly, an emphasis on long term neurobehavioral follow up is necessary to better identify and provide support of affected individuals. Future research in identifying host immune responses to LACV infection, as well as therapeutic options, are needed.

Persistent spatial clustering and predictors of pediatric La Crosse virus neuroinvasive disease risk in eastern Tennessee and western North Carolina, 2003-2020.

The combined region of eastern Tennessee and western North Carolina has a persistently high risk of pediatric La Crosse virus neuroinvasive disease (LACV-ND). To guide public health intervention in this region, the objectives of this retrospective ecological study were to investigate the geographic clustering and predictors of pediatric LACV-ND risk at the ZIP code tabulation area (ZCTA) level. Data on pediatric cases of LACV-ND reported between 2003 and 2020 were obtained from Tennessee Department of Health and North Carolina Department of Health and Human Services. Purely spatial and space-time scan statistics were used to identify ZCTA-level clusters of confirmed and probable pediatric LACV-ND cases from 2003-2020, and a combination of global and local (i.e., geographically weighted) negative binomial regression models were used to investigate potential predictors of disease risk from 2015-2020. The cluster investigation revealed spatially persistent high-risk and low-risk clusters of LACV-ND, with most cases consistently reported from a few high-risk clusters throughout the entire study period. Temperature and precipitation had positive but antagonistic associations with disease risk from 2015-2020, but the strength of those relationships varied substantially across the study area. Because LACV-ND risk clustering in this region is focally persistent, retroactive case surveillance can be used to guide the implementation of targeted public health intervention to reduce the disease burden in high-risk areas. Additional research on the role of climate in LACV transmission is warranted to support the development of predictive transmission models to guide proactive public health interventions.

LA CROSSE VIRUS VECTOR RESTING BEHAVIORS - FIELD STUDIES WITH PROKOPAK AND RESTING SHELTER COLLECTIONS PROVIDE LOW YIELD.

Resting adult mosquito collections provide opportunities to sample broad physiological conditions (e.g., blood-engorged, gravid, nectar-engorged, and/or parous) that yield important biological information necessary to understand vector and pathogen transmission ecology. In this study, we evaluated Prokopak aspirations of Rhododendron spp. and human-powered pop-up resting shelter collections at 4 residences with historical evidence of proximal La Crosse virus (LACV) transmission from May through September 2022. The goal of this study was to investigate these sampling methods in the context of LACV vector biology-focused principally on Aedes triseriatus (primary LACV vector) and 2 invasive species (Ae. albopictus and Ae. japonicus) that likely serve as secondary LACV vectors. Overall, 304 resting shelters and 80 Prokopak collections yielded a grand total of 33 mosquitoes, of which a third were LACV vectors (Ae. triseriatus [n = 1, 3.0%], Ae. albopictus [n = 4, 12.1%], and Ae. japonicus [n = 6, 18.2%]). Anopheles punctipennis (n = 9, 27.2%) was the most frequently collected species followed by Culex erraticus (n = 7, 21.2%), whereas the least frequently collected species were Ae. triseriatus and Cx. pipiens (n = 1, 3.0%). Despite substantial collection efforts, and concurrent gravid-trap evidence of LACV vectors at the collection sites, Prokopak aspiration of Rhododendron spp. and human-powered pop-up resting shelters did not yield a meaningful number of LACV vectors and thus, as described within, may not be useful adjuncts for the evaluation of LACV ecology and disease risk. Additional approaches to evaluate the resting behavior of these vectors in LACV endemic areas are needed.

C-C motif chemokine receptor 2 and 7 synergistically control inflammatory monocyte recruitment but the infecting virus dictates monocyte function in the brain.

Inflammatory monocytes (iMO) are recruited from the bone marrow to the brain during viral encephalitis. C-C motif chemokine receptor (CCR) 2 deficiency substantially reduces iMO recruitment for most, but not all encephalitic viruses. Here we show CCR7 acts synergistically with CCR2 to control this process. Following Herpes simplex virus type-1 (HSV-1), or La Crosse virus (LACV) infection, we find iMO proportions are reduced by approximately half in either Ccr2 or Ccr7 knockout mice compared to control mice. However, Ccr2/Ccr7 double knockouts eliminate iMO recruitment following infection with either virus, indicating these receptors together control iMO recruitment. We also find that LACV induces a more robust iMO recruitment than HSV-1. However, unlike iMOs in HSV-1 infection, LACV-recruited iMOs do not influence neurological disease development. LACV-induced iMOs have higher expression of proinflammatory and proapoptotic but reduced mitotic, phagocytic and phagolysosomal transcripts compared to HSV-1-induced iMOs. Thus, virus-specific activation of iMOs affects their recruitment, activation, and function.

Biophysical and structural study of La Crosse virus endonuclease inhibition for the development of new antiviral options.

The large Bunyavirales order includes several families of viruses with a segmented ambisense (-) RNA genome and a cytoplasmic life cycle that starts by synthesizing viral mRNA. The initiation of transcription, which is common to all members, relies on an endonuclease activity that is responsible for cap-snatching. In La Crosse virus, an orthobunyavirus, it has previously been shown that the cap-snatching endonuclease resides in the N-terminal domain of the L protein. Orthobunyaviruses are transmitted by arthropods and cause diseases in cattle. However, California encephalitis virus, La Crosse virus and Jamestown Canyon virus are North American species that can cause encephalitis in humans. No vaccines or antiviral drugs are available. In this study, three known Influenza virus endonuclease inhibitors (DPBA, L-742,001 and baloxavir) were repurposed on the La Crosse virus endonuclease. Their inhibition was evaluated by fluorescence resonance energy transfer and their mode of binding was then assessed by differential scanning fluorimetry and microscale thermophoresis. Finally, two crystallographic structures were obtained in complex with L-742,001 and baloxavir, providing access to the structural determinants of inhibition and offering key information for the further development of Bunyavirales endonuclease inhibitors.

Current Status of La Crosse Virus in North America and Potential for Future Spread.

La Crosse virus (LACV) is an arthropod-borne RNA virus with substantial potential for future spread in North America. La Crosse virus is responsible for La Crosse encephalitis, a leading cause of arboviral encephalitis in children in the United States. Primarily transmitted by Aedes triseriatus (Eastern treehole) mosquitos and amplified by small mammal hosts, LACV has caused infections throughout the upper Midwest and, more recently, the mid-Atlantic and southeastern United States. Notably, in recent years, infections have also been identified increasingly in the Appalachian region. Anthropogenic and environmental factors have likely contributed to recent LACV spread, including the introduction of invasive vector species (especially Ae. albopictus), biotic interactions between and among vector and host species, land-use change, habitat disturbance, increased human travel and transport, and rising global temperatures. Prevention and control strategies, such as increased surveillance of vector and host populations, increased awareness among populations at risk for infection, and increased awareness among physicians are needed to limit future spread. Continued climate change with increases in global temperatures and erratic weather patterns may result in the expansion of competent mosquito vector species and thus could facilitate the geographic spread of LACV.

Skin muscle is the initial site of viral replication for arboviral bunyavirus infection.

The first step in disease pathogenesis for arboviruses is the establishment of infection following vector transmission. For La Crosse virus (LACV), the leading cause of pediatric arboviral encephalitis in North America, and other orthobunyaviruses, the initial course of infection in the skin is not well understood. Using an intradermal (ID) model of LACV infection in mice, we find that the virus infects and replicates nearly exclusively within skin-associated muscle cells of the panniculus carnosus (PC) and not in epidermal or dermal cells like most other arbovirus families. LACV is widely myotropic, infecting distal muscle cells of the peritoneum and heart, with limited infection of draining lymph nodes. Surprisingly, muscle cells are resistant to virus-induced cell death, with long term low levels of virus release progressing through the Golgi apparatus. Thus, skin muscle may be a key cell type for the initial infection and spread of arboviral orthobunyaviruses.

Do Aedes triseriatus Respect State Boundaries?: A Paucity of La Crosse Virus in the South Carolina Appalachian Mountains.

Background: La Crosse virus is an important endemic public health concern in the North Carolina Appalachian Mountains; however, human incidence is not commonly noted in this region on the South Carolina side of the mountain range border. No relevant studies have been performed in South Carolina evaluating mosquito vector populations for La Crosse virus (LACV) infection; thus, a pilot mosquito surveillance study was executed in summer 2020. Material and Methods: Vector surveillance occurred at five South Carolina state parks bordering neighboring state endemic counties from May to August 2020. Collections were approved by the state park authority, as noted in Permit No. N-8-20. Results: All three competent mosquito vectors were collected during the study duration; however, these vectors were collected in low abundance: Aedes triseriatus (4.5% of all collected mosquitos); Aedes albopictus (2.0%); Aedes japonicus (1.4%). Principal mosquito vector specimens, Ae. triseriatus, were sent to Centers for Disease Control and Prevention for testing of LACV by real-time reverse transcription PCR-all were negative. Discussion: While entomologic evidence suggests low transmission risk for this arbovirus in the South Carolina Appalachian Mountain region, further eco-epidemiologic investigations are warranted to understand this endemicity variance within a relatively small geographic area.

La Crosse virus neuroinvasive disease: the kids are not alright.

La Crosse virus (LACV) is the most common cause of neuroinvasive mosquito-borne disease in children within the United States. Despite more than 50 years of recognized endemicity in the United States, the true burden of LACV disease is grossly underappreciated, and there remain severe knowledge gaps that inhibit public health interventions to reduce morbidity and mortality. Long-standing deficiencies in disease surveillance, clinical diagnostics and therapeutics, actionable entomologic and environmental risk indices, case response capacity, public awareness, and availability of community support groups clearly frame LACV disease as neglected. Here we synthesize salient prior research and contextualize our findings as an assessment of current gaps and opportunities to develop a framework to prevent, detect, and respond to LACV disease. The persistent burdens of LACV disease clearly require renewed public health attention, policy, and action.

The La Crosse virus class II fusion glycoprotein ij loop contributes to infectivity and replication in vitro and in vivo.

Arthropod-borne viruses (arboviruses) are an emerging and evolving global public health threat, with limited antiviral treatments or vaccines available. La Crosse virus (LACV) from the Bunyavirales order is responsible for pediatric encephalitis cases in the United States, yet little is known about the infectivity of LACV. Given the structural similarities between class II fusion glycoproteins of LACV and chikungunya virus (CHIKV), an alphavirus from the Togaviridae family, we hypothesized that LACV would share similar entry mechanisms with CHIKV. To test this hypothesis, we performed cholesterol-depletion and repletion assays and used cholesterol-modulating compounds to study LACV entry and replication. We found that LACV entry was cholesterol dependent, while replication was less affected by cholesterol manipulation. In addition, we generated single-point mutants in the LACV Gc ij loop that corresponded to known CHIKV residues important for virus entry. We found that a conserved histidine and alanine residue in the Gc ij loop impaired virus infectivity and attenuated LACV replication in vitro and in vivo. Finally, we took an evolution-based approach to explore how the LACV glycoprotein evolves in mosquitoes and mice. We found multiple variants that cluster in the Gc glycoprotein head domain, providing evidence for the Gc glycoprotein as a contributor to LACV adaptation. Together, these results begin to characterize the mechanisms of LACV infectivity and how the LACV glycoprotein contributes to replication and pathogenesis. IMPORTANCE Vector-borne viruses are significant health threats that lead to devastating disease worldwide. The emergence of arboviruses, in addition to the lack of effective antivirals or vaccines, highlights the need to study how arboviruses replicate at the molecular level. One potential antiviral target is the class II fusion glycoprotein. Alphaviruses, flaviviruses, and bunyaviruses encode a class II fusion glycoprotein that contains strong structural similarities at the tip of domain II. Here, we show that the bunyavirus La Crosse virus uses a cholesterol-dependent entry pathway similar to the alphavirus chikungunya virus, and residues in the ij loop are important for virus infectivity in vitro and replication in mice. These studies show that genetically diverse viruses may use similar pathways through conserved structure domains, suggesting that these viruses may be targets for broad-spectrum antivirals in multiple arboviral families.

A Comparison of Oak Leaf and Fescue Hay Infusion-Baited Gravid Trap Collections-An Analysis Steeped in the Context of La Crosse Virus Vector Surveillance Effectiveness.

Neuroinvasive La Crosse virus disease remains the primary cause of pediatric arboviral encephalitis in the USA. In spite of the persistent public health burden, there are limited entomologic surveillance options that target both native and invasive La Crosse virus (LACV) vectors. In this study we used Reiter/Cummings tacklebox gravid traps to compare white oak (Quercus alba) and hay (predominately Festuca arundinacea) infusions within a LACV-endemic area of western North Carolina. Paired gravid traps (approximately 1,728 total trap-hours for each infusion) yielded 485 mosquitoes, with 3 species (Aedes japonicus [n = 265], Ae. triseriatus [n = 156], and Culex restuans [n = 45]) accounting for 96.1% of the total collection. The hay-infusion traps collected 2.5 times more Ae. triseriatus and 1.3 times more Ae. japonicus than the oak-infusion traps. The sum differences in overall collections for these 2 species by infusion type were statistically significant (χ2 = 9.61, df = 1, P = 0.0019). Poisson ratio tests to compare capture rates suggest that hay infusions were more effective for capturing Ae. triseriatus, but that hay and white oak leaf infusions had equivocal capture rates for Ae. japonicus (an invasive LACV accessory vector) and Cx. restuans (an enzootic West Nile virus vector). These results are discussed in the context of operational considerations for LACV vector surveillance.

Identification of age-specific gene regulators of La Crosse virus neuroinvasion and pathogenesis.

One of the key events in viral encephalitis is the ability of virus to enter the central nervous system (CNS). Several encephalitic viruses, including La Crosse Virus (LACV), primarily induce encephalitis in children, but not adults. This phenomenon is also observed in LACV mouse models, where the virus gains access to the CNS of weanling animals through vascular leakage of brain microvessels, likely through brain capillary endothelial cells (BCECs). To examine age and region-specific regulatory factors of vascular leakage, we used genome-wide transcriptomics and targeted siRNA screening to identify genes whose suppression affected viral pathogenesis in BCECs. Further analysis of two of these gene products, Connexin43 (Cx43/Gja1) and EphrinA2 (Efna2), showed a substantial effect on LACV pathogenesis. Induction of Cx43 by 4-phenylbutyric acid (4-PBA) inhibited neurological disease in weanling mice, while Efna2 deficiency increased disease in adult mice. Thus, we show that Efna2 and Cx43 expressed by BCECs are key mediators of LACV-induced neuroinvasion and neurological disease.

Structure, function, and evolution of the Orthobunyavirus membrane fusion glycoprotein.

La Crosse virus, responsible for pediatric encephalitis in the United States, and Schmallenberg virus, a highly teratogenic veterinary virus in Europe, belong to the large Orthobunyavirus genus of zoonotic arthropod-borne pathogens distributed worldwide. Viruses in this under-studied genus cause CNS infections or fever with debilitating arthralgia/myalgia syndromes, with no effective treatment. The main surface antigen, glycoprotein Gc (∼1,000 residues), has a variable N-terminal half (GcS) targeted by the patients' antibody response and a conserved C-terminal moiety (GcF) responsible for membrane fusion during cell entry. Here, we report the X-ray structure of post-fusion La Crosse and Schmallenberg virus GcF, revealing the molecular determinants for hairpin formation and trimerization required to drive membrane fusion. We further experimentally confirm the role of residues in the fusion loops and in a vestigial endoplasmic reticulum (ER) translocation sequence at the GcS-GcF junction. The resulting knowledge provides essential molecular underpinnings for future development of potential therapeutic treatments and vaccines.

Geographically persistent clusters of La Crosse virus disease in the Appalachian region of the United States from 2003 to 2021.

La Crosse virus (LACV) is a mosquito-borne pathogen that causes more pediatric neuroinvasive disease than any other arbovirus in the United States. The geographic focus of reported LACV neuroinvasive disease (LACV-ND) expanded from the Midwest into Appalachia in the 1990s, and most cases have been reported from a few high-risk foci since then. Here, we used publicly available human disease data to investigate changes in the distribution of geographic LACV-ND clusters between 2003 and 2021 and to investigate socioeconomic and demographic predictors of county-level disease risk in states with persistent clusters. We used spatial scan statistics to identify high-risk clusters from 2003-2021 and a generalized linear mixed model to identify socioeconomic and demographic predictors of disease risk. The distribution of LACV-ND clusters was consistent during the study period, with an intermittent cluster in the upper Midwest and three persistent clusters in Appalachia that included counties in east Tennessee / western North Carolina, West Virginia, and Ohio. In those states, county-level cumulative incidence was higher when more of the population was white and when median household income was lower. Public health officials should target efforts to reduce LACV-ND incidence in areas with consistent high risks.

La Crosse Virus Neuroinvasive Disease in Children: A Contemporary Analysis of Clinical/Neurobehavioral Outcomes and Predictors of Disease Severity.

BACKGROUND: La Crosse virus (LACV) is the most common neuroinvasive arboviral infection in children in the United States. However, data regarding predictors of disease severity and neurologic outcome are limited. Additionally, long-term neurologic and neurobehavioral outcomes remain relatively sparse. METHODS: This was a single-center, retrospective cohort study, followed by recruitment for a cross-sectional analysis of long-term neurobehavioral outcomes, among children aged 0-18 years with proven or probable LACV neuroinvasive disease (LACV-ND) between January 2009 and December 2018. Case ascertainment was assured by International Classification of Diseases, Ninth and Tenth Revision, Clinical Modification codes cross-referenced with laboratory results detecting LACV. Demographics, diagnostics, radiographs, and outcomes were evaluated. Recruitment of patients with prior diagnosis of LACV-ND occurred from January 2020 to March 2020, with assessment performed by validated pediatric questionnaires. RESULTS: One-hundred fifty-two children (83 males; median age, 8 years [interquartile range, 5-11.5 years]) were diagnosed with proven (n = 61 [47%]) and probable (n = 91 [60%]) LACV-ND. Sixty-five patients (43%) had severe disease. Altered mental status (AMS) (odds ratio [OR], 6.36 [95% confidence interval {CI}, 2.03-19.95]; P = .0002) and seizures at presentation (OR, 10.31 [95% CI, 3.45-30.86]; P = .0001) were independent predictors of severe disease. Epileptiform discharges on electroencephalogram (EEG) were independently associated with epilepsy diagnosis at follow-up (OR, 13.45 [95% CI, 1.4-128.77]; P = .024). Fifty-four patients were recruited for long-term neurobehavioral follow-up, with frequent abnormal assessments identified (19%-54%) irrespective of disease severity. CONCLUSIONS: Severe disease was observed frequently among children with LACV-ND. Seizures and AMS at presentation were independent predictors of severe disease. EEG may help determine long-term epilepsy risk. Long-term neurobehavioral issues are frequent and likely underrecognized among children with LACV-ND.

Community efforts to monitor and manage Aedes mosquitoes (Diptera: Culicidae) with ovitraps and litter reduction in east Tennessee.

BACKGROUND: East Tennessee (USA) is burdened by mosquito-borne La Crosse virus disease, but minimal resources for mosquito surveillance, management, or related community education exist in the region. To address these needs, we developed a program to train middle and high school educators in basic medical entomology. The educators then used their skills in the classroom to teach students about La Crosse virus disease and conduct mosquito collection experiments. As a case study of a potential application of classroom-collected data, we also partnered with a local non-profit organization to assess the potential for a volunteer litter cleanup to reduce mosquito populations in a Tennessee neighborhood. METHODS: Our first objective was to investigate the ability for educators and their students (schools) to collect high-quality mosquito surveillance data. In 2019 and 2020, we collected Aedes (Diptera: Culicidae) eggs during the same study period as schools and assessed whether data collected by schools reflected the same findings as our own data. Our second objective was to investigate the impact of a volunteer litter cleanup event on Aedes mosquito abundance. In 2021, we collected Aedes eggs before and after a neighborhood trash cleanup while schools conducted their own mosquito egg collections. Using the school collections as non-treatment sites, we used a Before-After-Control-Impact analysis to determine if there was a significant decline in egg abundance after the cleanup. RESULTS: In 2019, mosquito abundance trends were similar between our data and school data but differed significantly during some weeks. After refining our protocols in 2020, school data was highly similar to our data, indicating that schools consistently collected high-quality surveillance data in the program's second year. In 2021, we found a significant decline in Aedes egg abundance after the litter cleanup event in comparison to the schools, but the number of adults reared from those eggs did not differ between sites after the cleanup. CONCLUSION: The results of our work demonstrate the potential for community-driven programs to monitor mosquito abundance trends and for volunteer-based cleanup events to reduce the burden of Aedes mosquitoes. In the absence of infrastructure and resources, academic-community partnerships like the ones evaluated here, provide opportunities to help resource limited areas.