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.

Metal coordinating inhibitors of Rift Valley fever virus replication.

Rift Valley fever virus (RVFV) is a veterinary and human pathogen and is an agent of bioterrorism concern. Currently, RVFV treatment is limited to supportive care, so new drugs to control RVFV infection are urgently needed. RVFV is a member of the order Bunyavirales, whose replication depends on the enzymatic activity of the viral L protein. Screening for RVFV inhibitors among compounds with divalent cation-coordinating motifs similar to known viral nuclease inhibitors identified 47 novel RVFV inhibitors with selective indexes from 1.1-103 and 50% effective concentrations of 1.2-56 µM in Vero cells, primarily α-Hydroxytropolones and N-Hydroxypyridinediones. Inhibitor activity and selective index was validated in the human cell line A549. To evaluate specificity, select compounds were tested against a second Bunyavirus, La Crosse Virus (LACV), and the flavivirus Zika (ZIKV). These data indicate that the α-Hydroxytropolone and N-Hydroxypyridinedione chemotypes should be investigated in the future to determine their mechanism(s) of action allowing further development as therapeutics for RVFV and LACV, and these chemotypes should be evaluated for activity against related pathogens, including Hantaan virus, severe fever with thrombocytopenia syndrome virus, Crimean-Congo hemorrhagic fever virus.

Targeted Mutations in the Fusion Peptide Region of La Crosse Virus Attenuate Neuroinvasion and Confer Protection against Encephalitis.

La Crosse virus (LACV) is a major cause of pediatric encephalitis and aseptic meningitis in the Midwestern, Mid-Atlantic, and Southern United States, where it is an emerging pathogen. The LACV Gc glycoprotein plays a critical role in the neuropathogenesis of LACV encephalitis as the putative virus attachment protein. Previously, we identified and experimentally confirmed the location of the LACV fusion peptide within Gc and generated a panel of recombinant LACVs (rLACVs) containing mutations in the fusion peptide as well as the wild-type sequence. These rLACVs retained their ability to cause neuronal death in a primary embryonic rat neuronal culture system, despite decreased replication and fusion phenotypes. To test the role of the fusion peptide in vivo, we tested rLACVs in an age-dependent murine model of LACV encephalitis. When inoculated directly into the CNS of young adult mice (P28), the rLACV fusion peptide mutants were as neurovirulent as the rLACV engineered with a wild-type sequence, confirming the results obtained in tissue culture. In contrast, the fusion peptide mutant rLACVs were less neuroinvasive when suckling (P3) or weanling (P21) mice were inoculated peripherally, demonstrating that the LACV fusion peptide is a determinant of neuroinvasion, but not of neurovirulence. In a challenge experiment, we found that peripheral challenge of weanling (P21) mice with fusion peptide mutant rLACVs protected from a subsequent WT-LACV challenge, suggesting that mutations in the fusion peptide are an attractive target for generating live-attenuated virus vaccines. Importantly, the high degree of conservation of the fusion peptide amongst the Bunyavirales and, structurally, other arboviruses suggests that these findings are broadly applicable to viruses that use a class II fusion mechanism and cause neurologic disease.

Structural snapshots of La Crosse virus polymerase reveal the mechanisms underlying Peribunyaviridae replication and transcription.

Segmented negative-strand RNA bunyaviruses encode a multi-functional polymerase that performs genome replication and transcription. Here, we establish conditions for in vitro activity of La Crosse virus polymerase and visualize its conformational dynamics by cryo-electron microscopy, unveiling the precise molecular mechanics underlying its essential activities. We find that replication initiation is coupled to distal duplex promoter formation, endonuclease movement, prime-and-realign loop extension and closure of the polymerase core that direct the template towards the active site. Transcription initiation depends on C-terminal region closure and endonuclease movements that prompt primer cleavage prior to primer entry in the active site. Product realignment after priming, observed in replication and transcription, is triggered by the prime-and-realign loop. Switch to elongation results in polymerase reorganization and core region opening to facilitate template-product duplex formation in the active site cavity. The uncovered detailed mechanics should be helpful for the future design of antivirals counteracting bunyaviral life threatening pathogens.

Rottlerin inhibits La Crosse virus-induced encephalitis in mice and blocks release of replicating virus from the Golgi body in neurons.

La Crosse virus (LACV) is a mosquito-borne orthobunyavirus that causes approximately 60 to 80 hospitalized pediatric encephalitis cases in the United States yearly. The primary treatment for most viral encephalitis, including LACV, is palliative care, and specific antiviral therapeutics are needed. We screened the National Center for Advancing Translational Sciences library of 3,833 FDA-approved and bioactive small molecules for the ability to inhibit LACV-induced death in SH-SY5Y neuronal cells. The top three hits from the initial screen were validated by examining their ability to inhibit virus-induced cell death in multiple neuronal cell lines. Rottlerin consistently reduced LACV-induced death by 50% in multiple human and mouse neuronal cell lines with an effective concentration of 0.16-0.69 µg ml-1 depending on cell line. Rottlerin was effective up to 12 hours post-infection in vitro and inhibited virus particle trafficking from the Golgi apparatus to trans-Golgi vesicles. In human inducible pluripotent stem cell-derived cerebral organoids, rottlerin reduced virus production by one log and cell death by 35% compared with dimethyl sulfoxide-treated controls. Administration of rottlerin in mice by intraperitoneal or intracranial routes starting at 3 days post-infection decreased disease development by 30-50%. Furthermore, rottlerin also inhibited virus replication of other pathogenic California serogroup orthobunyaviruses (Jamestown Canyon and Tahyna virus) in neuronal cell lines.

La Crosse Virus Disease in the United States, 2003-2019.

La Crosse virus (LACV) is an arthropod-borne virus that can cause a nonspecific febrile illness, meningitis, or encephalitis. We reviewed U.S. LACV surveillance data for 2003-2019, including human disease cases and nonhuman infections. Overall, 318 counties in 27 states, principally in the Great Lakes, mid-Atlantic, and southeastern regions, reported LACV activity. A total of 1,281 human LACV disease cases were reported, including 1,183 (92%) neuroinvasive disease cases. The median age of cases was 8 years (range: 1 month-95 years); 1,130 (88%) were aged < 18 years, and 754 (59%) were male. The most common clinical syndromes were encephalitis (N = 960; 75%) and meningitis (N = 219, 17%). The case fatality rate was 1% (N = 15). A median of 74 cases (range: 35-130) was reported per year. The average annual national incidence of neuroinvasive disease cases was 0.02 per 100,000 persons. West Virginia, North Carolina, Tennessee, and Ohio had the highest average annual state incidences (0.16-0.61 per 100,000), accounting for 80% (N = 1,030) of cases. No animal LACV infections were reported. Nine states reported LACV-positive mosquito pools, including three states with no reported human disease cases. La Crosse virus is the most common cause of pediatric neuroinvasive arboviral disease in the United States. However, surveillance data likely underestimate LACV disease incidence. Healthcare providers should consider LACV disease in patients, especially children, with febrile illness, meningitis, or encephalitis in areas where the virus circulates and advise their patients on ways to prevent mosquito bites.

Age influences susceptibility of brain capillary endothelial cells to La Crosse virus infection and cell death.

BACKGROUND: A key factor in the development of viral encephalitis is a virus crossing the blood-brain barrier (BBB). We have previously shown that age-related susceptibility of mice to the La Crosse virus (LACV), the leading cause of pediatric arbovirus encephalitis in the USA, was associated with the ability of the virus to cross the BBB. LACV infection in weanling mice (aged around 3 weeks) results in vascular leakage in the olfactory bulb/tract (OB/OT) region of the brain, which is not observed in adult mice aged > 6-8 weeks. Thus, we studied age-specific differences in the response of brain capillary endothelial cells (BCECs) to LACV infection. METHODS: To examine mechanisms of LACV-induced BBB breakdown and infection of the CNS, we analyzed BCECs directly isolated from weanling and adult mice as well as established a model where these cells were infected in vitro and cultured for a short period to determine susceptibility to virus infection and cell death. Additionally, we utilized correlative light electron microscopy (CLEM) to examine whether changes in cell morphology and function were also observed in BCECs in vivo. RESULTS: BCECs from weanling, but not adult mice, had detectable infection after several days in culture when taken ex vivo from infected mice suggesting that these cells could be infected in vitro. Further analysis of BCECs from uninfected mice, infected in vitro, showed that weanling BCECs were more susceptible to virus infection than adult BCECs, with higher levels of infected cells, released virus as well as cytopathic effects (CPE) and cell death. Although direct LACV infection is not detected in the weanling BCECs, CLEM analysis of brain tissue from weanling mice indicated that LACV infection induced significant cerebrovascular damage which allowed virus-sized particles to enter the brain parenchyma. CONCLUSIONS: These findings indicate that BCECs isolated from adult and weanling mice have differential viral load, infectivity, and susceptibility to LACV. These age-related differences in susceptibility may strongly influence LACV-induced BBB leakage and neurovascular damage allowing virus invasion of the CNS and the development of neurological disease.

La Crosse virus spread within the mosquito population in Knox County, TN.

In Appalachia, La Crosse virus (LACV) is a leading pediatric arbovirus and public health concern for children under 16 years. LACV is transmitted via the bite of an infected Aedes mosquito. Thus, it is imperative to understand the dynamics of the local vector population in order to assess risk and transmission. Using entomological data collected from Knox County, Tennessee in 2013, we formulate an environmentally-driven system of ordinary differential equations to model mosquito population dynamics over a single season. Further, we include infected compartments to represent LACV transmission within the mosquito population. Findings suggest that the model, with dependence on degree days and accumulated precipitation, can closely describe field data. This model confirms the need to include these environmental variables when planning control strategies.