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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.

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.

A number of viruses, including Herpes Simplex Virus (HSV), West Nile Virus (WNV), La Crosse Virus (LACV), Zika virus (ZIKV) and Tick-borne encephalitis virus (TBEV), have the ability to gain access to the central nervous system (CNS) and cause severe neurological disease or death. Although encephalitis cases caused by these viruses are generally rare, there are relatively few treatment options available for patients with viral encephalitis other than palliative care. Many of these viruses directly infect neurons and can cause neuronal death. Thus, there is the need for the identification of useful therapeutic compounds that can inhibit virus replication in neurons or inhibit virus-induced neuronal cell death. In this paper, we describe the methodology to test compounds for their ability to inhibit virus-induced neuronal cell death. These protocols include the isolation and culturing of primary neurons; the culturing of neuroblastoma and neuronal stem cell lines; infection of these cells with viruses; treatment of these cells with selected drugs; measuring virus-induced cell death using MTT or XTT reagents; analysis of virus production from these cells; as well as the basic understanding in mode of action. We further show direct evidence of the effectiveness of these protocols by utilizing them to test the effectiveness of the polyphenol drug, Rottlerin, at inhibiting Zika virus infection and death of neuronal cell lines.

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.

Anthropogenic land-use change may affect the transmission risk for endemic vector-borne diseases such as La Crosse encephalitis. In this study, we applied a comparative ecological approach to evaluate differences in vector species abundance, gonotrophic status, and environmental variables among six residential habitats (historical case houses) and six paired adjacent forest patches in a La Crosse virus endemic area of North Carolina. A total of 93,158 container Aedes spp. eggs were obtained by ovistrips and 1,040 resting mosquito adults were collected by large-bore aspiration from 10 June through 8 October, 2010. At sites characterized by high densities of artificial containers, the totals of eggs and adult mosquitoes were higher in the peridomestic plots. At sites characterized by lower densities of artificial containers, the totals of eggs and adult mosquitoes were higher in the forested plots. Although Aedes triseriatus, the primary La Crosse virus vector, was more commonly found in forested habitat overall, in sites characterized by high density of artificial containers, it was found in higher abundance in the peridomestic habitat. Similarly, the proportion of gonotrophically active (parous and gravid) mosquitoes was higher in the peridomestic habitat for sites with highartificial container density. Our study suggests that La Crosse virus transmission risk may be higher at peridomestic habitats with higher densities of artificial containers and thus reinforces the importance of public health measures to improve source reduction efforts.

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.

The California serogroup of orthobunyaviruses comprises a group of mosquitoborne viruses, including La Crosse (LACV), snowshoe hare (SSHV), Tahyna (TAHV), Jamestown Canyon (JCV), and Inkoo (INKV) viruses, that cause neurologic disease in humans of differing ages with varying incidences. To determine how the pathogenesis of these viruses differs, we compared their ability to induce disease in mice and replicate and induce cell death in vitro. In mice, LACV, TAHV, and SSHV induced neurologic disease after intraperitoneal and intranasal inoculation, and JCV induced disease only after intranasal inoculation. INKV rarely induced disease, which correlated with less viral antigen in the brain than the other viruses. In vitro, all viruses replicated to high titers; however, LACV, SSHV, and TAHV induced high cell death, whereas JCV and INKV did not. Results demonstrated that CSG viruses differ in neuropathogenesis in vitro and in vivo, which correlates with the differences in pathogenesis reported in humans.

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.

Native to the Eastern United States and Eastern Canada, Aedes triseriatus (eastern tree hole mosquito) is an important vector of La Crosse virus and dog heartworm. Although its range has been well characterized in the United States, few studies have surveyed its distribution within Canada. In this study, mosquitoes were collected from a variety of urban and rural communities throughout Manitoba, Canada between the years of 2018 and 2020. Aedes triseriatus was identified and confirmed molecularly to be present in 13 communities. This includes localities that expand the species known distribution to new northern and western areas, and suggests that past surveillance efforts have not been comprehensive or environmental factors have caused this mosquito species to be present in areas in which it was not found previously. As Canada is showing signs of a changing climate, this may be driving the broader occurrence of Ae. triseriatus.

Domestic arthropod-borne viruses (arboviruses) are single-stranded RNA viruses, the most common of which include the mosquito-borne West Nile virus, St. Louis encephalitis virus, La Crosse virus, Jamestown Canyon virus, and eastern equine encephalitis virus, as well as the tick-borne Powassan virus. Previously considered rare infections, they have been detected with increasing frequency over the past 2 decades. Here, we present an overview of the domestic arboviruses listed above and describe the modalities employed to diagnose infection. Global arboviruses, including dengue virus, Zika virus, and chikungunya virus, have also been increasingly detected in the United States within the last 5 years but are not a focus of this minireview. Typical manifestations of arbovirus infection range from no symptoms, to meningitis or encephalitis, to death. Serologies are the standard means of diagnosis in the laboratory, since most viruses have a short period of replication, limiting the utility of molecular tests. The interpretation of serologies is confounded by antibody cross-reactivity with viruses belonging to the same serogroup and by long-lasting antibodies from prior infections. Next-generation assays have improved performance by increasing antigen purity, selecting optimal epitopes, and improving interpretive algorithms, but challenges remain. Due to cross-reactivity, a positive first-line serology test requires confirmation by either a plaque reduction neutralization test or detection of seroconversion or a 4-fold rise in virus-specific IgM or IgG antibody titers from acute- and convalescent-phase sera. The use of molecular diagnostics, such as reverse transcription PCR or unbiased metagenomic sequencing, is limited to the minority of patients who present with ongoing viremia or central nervous system replication. With the continued expansion of vector range, the diagnosis of domestic arboviruses will become an increasingly important task for generalists and specialists alike.

Background La Crosse virus (LACV) (genus Orthobunyavirus, family Peribunyaviridae) is a mosquito-borne virus that causes pediatric encephalitis and accounts for 50–150 human cases annually in the USA. Human cases occur primarily in the Midwest and Appalachian regions whereas documented human cases occur very rarely in the northeastern USA. Methods Following detection of a LACV isolate from a field-collected mosquito in Connecticut during 2005, we evaluated the prevalence of LACV infection in local mosquito populations and genetically characterized virus isolates to determine whether the virus is maintained focally in this region. Results During 2018, we detected LACV in multiple species of mosquitoes, including those not previously associated with the virus. We also evaluated the phylogenetic relationship of LACV strains isolated from 2005–2018 in Connecticut and found that they formed a genetically homogeneous clade that was most similar to strains from New York State. Conclusion Our analysis argues for local isolation and long-term persistence of a genetically distinct lineage of LACV within this region. We highlight the need to determine more about the phenotypic behavior of these isolates, and whether this virus lineage poses a threat to public health.

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

Schmallenberg virus (SBV) is an insect-transmitted orthobunyavirus that can cause abortions and congenital malformations in the offspring of ruminants. Even though the two viral surface glycoproteins Gn and Gc are involved in host cell entry, the specific cellular receptors of SBV are currently unknown. Using genome-wide CRISPR-Cas9 forward screening, we identified 3′-phosphoadenosine 5′-phosphosulfate (PAPS) transporter 1 (PAPST1) as an essential factor for SBV infection. PAPST1 is a sulfotransferase involved in heparan sulfate proteoglycan synthesis encoded by the solute carrier family 35 member B2 gene (SLC35B2). SBV cell surface attachment and entry were largely reduced upon the knockout of SLC35B2, whereas the reconstitution of SLC35B2 in these cells fully restored their susceptibility to SBV infection. Furthermore, treatment of cells with heparinase diminished infection with SBV, confirming that heparan sulfate plays an important role in cell attachment and entry, although to various degrees, heparan sulfate was also found to be important to initiate infection by two other bunyaviruses, La Crosse virus and Rift Valley fever virus. Thus, PAPST1-triggered synthesis of cell surface heparan sulfate is required for the efficient replication of SBV and other bunyaviruses. IMPORTANCE SBV is a newly emerging orthobunyavirus (family Peribunyaviridae) that has spread rapidly across Europe since 2011, resulting in substantial economic losses in livestock farming. In this study, we performed unbiased genome-wide CRISPR-Cas9 screening and identified PAPST1, a sulfotransferase encoded by SLC35B2, as a host entry factor for SBV. Consistent with its role in the synthesis of heparan sulfate, we show that this activity is required for efficient infection by SBV. A comparable dependency on heparan sulfate was also observed for La Crosse virus and Rift Valley fever virus, highlighting the importance of heparan sulfate for host cell infection by bunyaviruses. Thus, the present work provides crucial insights into virus-host interactions of important animal and human pathogens.

Given the dual life cycle of arboviruses in insect and animal hosts and the importance of serum factors as a first line antiviral defense, we have examined the outcome of interactions between the arbovirus La Crosse Virus (LACV) and human serum. To mimic the life cycle between species, we used LACV derived from insect (I-LACV) and human keratinocyte (HaCaT) cells. Incubation of I-LACV with normal human serum did not result in neutralization, but instead stabilized I-LACV virions and enhanced the amount of infectious virus. Enhanced infectivity was also seen with heat-inactivated serum devoid of complement activity and with serum from a range of animals including mouse, ferret, and non-human primates. Depletion of antibodies from serum resulted in loss of enhancement of infectivity and sucrose gradient sedimentation assays showed IgG co-sedimenting with I-LACV particles. In agreement with our results with I-LACV, HaCaT-derived LACV was not neutralized by complement or antibodies in normal human serum. However, in contrast to I-LACV, HaCaT-derived LACV infectivity was stable when incubated alone and treatment with serum did not enhance infectivity. Our results indicate that LACV derived from insect cells differs substantially from virus derived from human cells, with I-LACV being dependent on serum factors to enhance infectivity. These findings suggest that understanding differential composition of insect versus animal cell-derived LACV may form the foundation for potential new antiviral approaches.

The Toll/IL-1R domain-containing adaptor protein SARM1 is expressed primarily in the brain, where it mediates axonal degeneration. Additional roles for SARM1 in a number of other processes including TLR-signaling, viral infection, chemokine expression, and expression of the proapoptotic protein XAF1 have also been described. Much of the supporting evidence for SARM1 function has been generated by comparing WT C57BL/6 (B6) mice to SARM1-deficient mice backcrossed to the B6 background. Here we show that the Sarm1 gene lies in a gene-rich region encompassing XAF1, and the MIP and MCP chemokine family loci among other genes. Because gene-targeting of SARM1-deficient strains was done with 129 ES cells and these genes are too close to segregate, they remain 129 in sequence. As this could account for phenotypes attributed to SARM1, we generated new knockout mouse strains on a pure B6 background using CRISPR. Experiments in these new strains confirmed the role of SARM1 in axonal degeneration and susceptibility to WNV infection, but not in susceptibility to VSV or LACV infection, or chemokine or Xaf1 expression. Notably, the Xaf1 gene shows sequence variation between B6 and 129, resulting in coding changes and novel splice variants. Given its known role in apoptosis, XAF1 variants may account for some phenotypes described in previously made SARM1-deficient strains. RNAseq in the new strains reveal changes in the mitochondrial electron transport chain and ribosomal proteins, suggesting possible downstream targets of SARM1. Re-evaluation of described phenotypes in these new strains will be critical for defining the function of SARM1.

The mosquito-borne La Crosse virus (LACV; genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales) is prevalent in the United States and can cause severe childhood meningoencephalitis. Its main virulence factor, the nonstructural protein NSs, is a strong inhibitor of the antiviral type I interferon (IFN) system. NSs acts by imposing a global host mRNA synthesis shutoff, mediated by NSs-driven proteasomal degradation of the RPB1 subunit of RNA polymerase II. Here, we show that RPB1 degradation commences as early as 1 h postinfection, and identify the E3 ubiquitin ligase subunit Elongin C (and its binding partners Elongins A and B) as an NSs cofactor involved in RPB1 degradation and in suppression of global as well as IFN-related mRNA synthesis., Mosquito-borne La Crosse virus (LACV; genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales) causes up to 100 annual cases of severe meningoencephalitis in children and young adults in the United States. A major virulence factor of LACV is the nonstructural protein NSs, which inhibits host cell mRNA synthesis to prevent the induction of antiviral type I interferons (IFN-α/β). To achieve this host transcriptional shutoff, LACV NSs drives the proteasomal degradation of RPB1, the large subunit of mammalian RNA polymerase II. Here, we show that NSs acts in a surprisingly rapid manner, as RPB1 degradation was commencing already at 1 h postinfection. The RPB1 degradation was partially dependent on the cellular E3 ubiquitin ligase subunit Elongin C. Consequently, removal of Elongin C, but also of the subunits Elongin A or B by siRNA transfection partially rescued general RNAP II transcription and IFN-beta mRNA synthesis from the blockade by NSs. In line with these results, LACV NSs was found to trigger the redistribution of Elongin C out of nucleolar speckles, which, however, is an epiphenomenon rather than part of the NSs mechanism. Our study also shows that the molecular phenotype of LACV NSs is different from RNA polymerase II inhibitors like α-amanitin or Rift Valley fever virus NSs, indicating that LACV is unique in involving the Elongin complex to shut off host transcription and IFN response. IMPORTANCE The mosquito-borne La Crosse virus (LACV; genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales) is prevalent in the United States and can cause severe childhood meningoencephalitis. Its main virulence factor, the nonstructural protein NSs, is a strong inhibitor of the antiviral type I interferon (IFN) system. NSs acts by imposing a global host mRNA synthesis shutoff, mediated by NSs-driven proteasomal degradation of the RPB1 subunit of RNA polymerase II. Here, we show that RPB1 degradation commences as early as 1 h postinfection, and identify the E3 ubiquitin ligase subunit Elongin C (and its binding partners Elongins A and B) as an NSs cofactor involved in RPB1 degradation and in suppression of global as well as IFN-related mRNA synthesis.

A bite from a La Crosse virus (LACV) infected Aedes mosquito can cause La Crosse encephalitis (LACE), which is a neuro-invasive disease that disproportionately affects children under the age of 16 in Southern Appalachia. The three vectors for LACV are Aedes albopictus (Skuse), Ae. japonicus (Theobald), and Ae. triseriatus (Say). Localized maps of the geographic distribution of vectors are practical tools for mosquito management personnel to target areas with high mosquito abundance. This study hypothesized that LACV vectors have unique species-specific spatial and temporal clusters. To test this, 44 sites were identified in Knox County, Tennessee for their land use/type. At each site, host-seeking mosquitoes were collected approximately every other week from May-October 2018. Spatial clusters of host-seeking mosquito collections for each of the three mosquito species were investigated using Kulldorff's spatial scan statistic, specifying a retrospective space-time Bernoulli model. Most vector clusters were identified in south-central Knox County while the seasonality of clusters varied by mosquito species. Clusters of Ae. albopictus were observed throughout the entire study period while clusters of Ae. japonicus and Ae. triseriatus only occurred May-June. The findings indicate that the relative abundance of LACV vectors were more abundant in south-central Knox County compared to the rest of the county. Of interest, these clusters spatially overlapped with previous LACE diagnosed cases. These findings are useful in guiding decisions on targeted mosquito control in Knox County and may be applied to other counties within Southern Appalachia.

Aedes mosquitoes are vectors for many pathogenic viruses. Cell culture systems facilitate the investigation of virus growth in the mosquito vector. We found Zika virus (ZIKV) growth to be consistent in A. albopictus cells but hypervariable in A. aegypti cell lines. As a potential explanation of this variability, we tested the hypothesis that our cells harbored opportunistic viruses. We screened Aedes cell lines for the presence of insect specific viruses (ISVs), Cell-fusing agent virus (CFAV) and Phasi charoen-like virus (PCLV). PCLV was present in the ZIKV-growth-variable A. aegypti cell lines but absent in A. albopictus lines, suggesting that these ISVs may interfere with ZIKV growth. In support of this hypothesis, PCLV infection of CFAV-positive A. albopictus cells inhibited the growth of ZIKV, dengue virus and La Crosse virus. These data suggest ISV infection of cell lines can impact arbovirus growth leading to significant changes in cell permissivity to arbovirus infection.

The California serogroup (CSG) comprises 18 serologically and genetically related mosquito-borne orthobunyaviruses. Of these viruses, at least seven have been shown to cause neurological disease in humans, including the leading cause of pediatric arboviral encephalitis in the USA, La Crosse virus. Despite the disease burden from these viruses, much is still unknown about the CSG viruses. This review summarizes our current knowledge of the CSG viruses, including human disease and the mechanisms of neuropathogenesis.

Background La Crosse virus (LACV) infection has been shown to manipulate the blood-feeding behaviors of its main vector, Aedes triseriatus. Here, we investigated the effects of virus infection on serotonin and dopamine and their potential roles in host-seeking. In mosquitoes, serotonin depletion has been shown to interfere with blood-feeding but not host-seeking. Dopamine depletion does not affect either blood-feeding or host-seeking; elevations of dopamine, however, has been shown to inhibit host-seeking. The purpose of this study was to determine the effects of LACV infection on the host-seeking behavior of and neurotransmitter levels in Ae. triseriatus. Methods Host-seeking behavior was evaluated using a uni-port olfactometer and a membrane feeder assay. Levels of serotonin and dopamine in infected and control mosquito heads were measured using HPLC-ED. Results Infection with LACV significantly inhibited the activation and attraction of Ae. triseriatus females to a host. A higher proportion of uninfected Ae. triseriatus females were activated by the presence of a host compared to infected mosquitoes and more uninfected mosquitoes were full responders (95.7%) compared to infected ones (91.1%). However, infection with LACV did not significantly affect the landing, probing, or blood-feeding rates of female mosquitoes. LACV-infected mosquitoes had lower serotonin levels than controls (104.5 vs 138.3 pg/head) while the dopamine levels were not affected by infection status (282.3 vs 237 pg/head). Conclusions Our work suggests that virus-induced reduction of serotonin is related to previously reported blood-feeding alterations in LACV-infected mosquitoes and could lead to enhanced transmission and increased vectorial capacity. In addition, some aspects of host-seeking were inhibited by virus infection.

La Crosse virus (LACV) is a mosquito-transmitted arbovirus and the main cause of virus-mediated neurological diseases in children. To date, little is known about the role of C-type lectin receptors (CLRs)—an important class of pattern recognition receptors—in LACV recognition. DC-SIGN remains the only well-described CLR that recognizes LACV. In this study, we investigated the role of additional CLR/LACV interactions. To this end, we applied a flow-through chromatography method for the purification of LACV to perform an unbiased high-throughput screening of LACV with a CLR-hFc fusion protein library. Interestingly, the CARD9-associated CLRs Mincle, Dectin-1, and Dectin-2 were identified to strongly interact with LACV. Since CARD9 is a common adaptor protein for signaling via Mincle, Dectin-1, and Dectin-2, we performed LACV infection of Mincle−/− and CARD9−/− DCs. Mincle−/− and CARD9−/− DCs produced less amounts of proinflammatory cytokines, namely IL-6 and TNF-α, albeit no reduction of the LACV titer was observed. Together, novel CLR/LACV interactions were identified; however, the Mincle/CARD9 axis plays a limited role in early antiviral responses against LACV.

Emergence may involve invasive mosquitoes other than Asian tiger mosquitoes, climate change, and changes in wildlife densities., La Crosse encephalitis is a viral disease that has emerged in new locations across the Appalachian region of the United States. Conventional wisdom suggests that ongoing emergence of La Crosse virus (LACV) could stem from the invasive Asian tiger (Aedes albopictus) mosquito. Efforts to prove this, however, are complicated by the numerous transmission routes and species interactions involved in LACV dynamics. To analyze LACV transmission by Asian tiger mosquitoes, we constructed epidemiologic models. These models accurately predict empirical infection rates. They do not, however, support the hypothesis that Asian tiger mosquitoes are responsible for the recent emergence of LACV at new foci. Consequently, we conclude that other factors, including different invasive mosquitoes, changes in climate variables, or changes in wildlife densities, should be considered as alternative explanations for recent increases in La Crosse encephalitis.

Invasive mosquito species can increase the transmission risk of native mosquito-borne diseases by acting as novel vectors. In this study, we examined the susceptibility of three exotic invasive mosquito species Aedes aegypti (L.), Ae. albopictus (Skuse), and Ochlerotatus japonicus (Theobald) to La Crosse virus (LACV) relative to the native primary vector Ochlerotatus triseriatus (Say). Adult females of the four mosquito species were orally challenged with LACV; incubated for 3, 5, 7, 9, or 11 d; and their midgut infection rates, dissemination rates, and effective vector competence were determined. Overall, Oc. japonicus (2.92) had the highest effective vector competence values, followed by Ae. albopictus (1.55), Ae. aegypti (0.88), and Oc. triseriatus (0.64) . In addition, we assessed the relationship between mosquito size and LACV susceptibility for field-collected Oc. triseriatus and Oc. japonicus . We hypothesized that smaller adults would be more susceptible to LACV; however, our results did not support this hypothesis. Infected Oc. triseriatus tended to be larger than exposed but uninfected females, while infected and uninfected Oc. japonicus were similarly sized. These findings suggest that Oc. japonicus , Ae. albopictus , and Ae. aegypti have significant potential to transmit LACV and more research is needed to uncover their potential role in LACV epidemiology.

Most cases appeared to be subclinical, but a few patients, usually children, required hospitalization., Inkoo virus (INKV) and Chatanga virus (CHATV), which are circulating in Finland, are mosquitoborne California serogroup orthobunyaviruses that have a high seroprevalence among humans. Worldwide, INKV infection has been poorly described, and CHATV infection has been unknown. Using serum samples collected in Finland from 7,961 patients suspected of having viral neurologic disease or Puumala virus infection during the summers of 2001–2013, we analyzed the samples to detect California serogroup infections. IgM seropositivity revealed 17 acute infections, and cross-neutralization tests confirmed presence of INKV or CHATV infections. All children (

Infection with La Crosse virus can cause meningoencephalitis, but it is not known to cause acute flaccid paralysis (AFP). During 2008-2014, nine confirmed or probable La Crosse virus disease cases with possible AFP were reported in Ohio, USA. After an epidemiologic and clinical investigation, we determined no patients truly had AFP.

In the southern Appalachia of the U.S., Aedes mosquitoes maintain and transmit La Crosse virus (LACV) which causes La Crosse encephalitis, a neuroinvasive disease of children. In response to mosquito outbreaks, communities organize prevention, detection, and response measures that are dependent on local characteristics of the mosquito population and the community. Knowing Ae. albopictus is an accessory vector of LACV and a nuisance biter, our objective was to build a system of ordinary differential equations to model dynamics in a single season using our data and readily available environmental variables that can reflect the abundance and activity of Ae. albopictus. Consequently, we built an Ae. albopictus single-season mathematical model for eastern Tennessee to fit our 2013 mosquito collection data in order to understand the population fluctuations. We included precipitation, temperature, and rate of change of temperature in the model because Aedes mosquitoes oviposit desiccant tolerant eggs with peak activity occurring over 26° C and those data are readily available and used frequently as forecast predictors. Our ordinary differential equation model accurately fits the data and facilitates predictions and better understanding of Ae. albopictus populations in southern Appalachia.

The TAM receptors Tyro3, Axl and Mertk are receptor tyrosine kinases that dampen host innate immune responses following engagement with their ligands Gas6 and Protein S, which recognize phosphatidylserine on apoptotic cells. In a form of apoptotic mimicry, many enveloped viruses display phosphatidylserine on the outer leaflet of their membranes, enabling TAM receptor activation and downregulation of antiviral responses. Accordingly, we hypothesized that a deficiency of TAM receptors would enhance antiviral responses and protect against viral infection. Unexpectedly, mice lacking Mertk and/or Axl, but not Tyro3, exhibited greater vulnerability to infection with neuroinvasive West Nile and La Crosse encephalitis viruses. This phenotype was associated with increased blood-brain barrier permeability, which enhanced virus entry into and infection of the brain. Activation of Mertk synergized with interferon-β to tighten cell junctions and prevent virus transit across brain microvascular endothelial cells. Because TAM receptors restrict pathogenesis of neuroinvasive viruses, these findings have implications for TAM antagonists that are currently in clinical development.

La Crosse virus (LACV), a leading cause of arboviral pediatric encephalitis in the United States, is emerging in Appalachia. Here, we report field and laboratory evidence that suggest LACV may be using Culex mosquitoes as additional vectors in this region. This bunyavirus was detected by reverse-transcriptase polymerase chain reaction in two pools of Culex mosquitoes in southwestern Virginia and in six pools in West Virginia. To assess vector competence, we offered LACV blood meals to field-collected Culex restuans Theobald, Cx. pipiens L., and Aedes triseriatus (Say). Both Culex species were susceptible to infection. LACV-positive salivary expectorate, indicative of the ability to transmit, was detected in a small proportion of Cx. restuans (9%) and Cx. pipiens (4%) compared with Ae. triseriatus (40%). In a companion study of Cx. restuans only, we found that adults derived from nutritionally stressed larvae were significantly more likely to disseminate and transmit LACV. Our results indicate a potential role of Culex spp. in LACV dynamics that should be explored further in endemic areas.

La Crosse virus (LACV) is a mosquito-borne virus and a major cause of pediatric encephalitis in the USA. La Crosse virus emerged in Tennessee and other states in the Appalachian region in 1997. We investigated LACV infection rates and seasonal abundances of the native mosquito vector, Aedes triseriatus, and 2 recently introduced mosquito species, Ae. albopictus and Ae. japonicus, in an emerging disease focus in Tennessee. Mosquitoes were collected using multiple trapping methods specific for Aedes mosquitoes at recent human case sites. Mosquito pools were tested via reverse transcriptase-polymerase chain reaction (RT-PCR) of the S segment to detect multiple Bunyamwera and California serogroup viruses, including LACV, as well as real-time RT-PCR of the M segment. A total of 54 mosquito pools were positive, including wild-caught adult females and laboratory-reared adults, demonstrating transovarial transmission in all 3 species. Maximum likelihood estimates (per 1,000 mosquitoes) were 2.72 for Ae. triseriatus, 3.01 for Ae. albopictus, and 0.63 for Ae. japonicus. We conclude that Ae. triseriatus and Ae. albopictus are important LACV vectors and that Ae. japonicus also may be involved in virus maintenance and transmission.

Ochlerotatus triseriatus (Say), the primary vector of La Crosse virus (LAC), develops in a variety of natural and artificial aquatic containers where it often co-occurs with larvae of other mosquito species. We conducted a field study at two woodlots (South Farms and Trelease Woods) in Urbana, IL, to examine how container type influences vector abundance, body size, and susceptibility to LAC. Mosquito pupae were collected from tree holes, plastic bins, and waste tires, and eclosing adults were identified to species morphologically. Oc. triseriatus and Ochlerotatus japonicus (Theobald) females were orally challenged with LAC and midgut infection rate, disseminated infection rate, and body titer were determined by reverse-transcriptase real-time PCR. Oc. triseriatus was the dominant species collected in tree holes while Oc. japonicus and Culex restuans (Theobald) were mostly dominant in artificial containers. Female Oc. triseriatus and Oc. japonicus collected from plastic bins were significantly larger than those collected from tree holes or waste tires. Oc. japonicus females from South Farms were also significantly larger than those from Trelease Woods. Oc. triseriatus females collected from plastic bins and waste tires were significantly more susceptible to LAC infection relative to females collected from tree holes. In addition, Oc. triseriatus females from waste tires had significantly higher LAC titer relative to Oc. triseriatus from tree holes. For each container type and study site, wing length was not correlated to infection or dissemination rates. These findings suggest that the container type in which Oc.triseriatus develop may contribute to the spatial and temporal dynamics of LAC transmission.

Inflammatory monocyte (iMOs) recruitment to the brain is a hallmark of many neurological diseases. Prior to entering the brain, iMOs must first egress into the blood from the bone marrow through mechanism which for known encephalitic viruses is CCR2-dependent. Here, we show that during La Crosse Virus (LACV)-induced encephalitis, egress of iMOs was surprisingly independent of CCR2, with similar percentages of iMOs in the blood and brain of heterozygous and CCR2−/− mice following infection. Interestingly, CCR2 was required for iMO trafficking, from perivascular areas to sites of virus infection within the brain. Thus, CCR2 was not essential for iMO trafficking to the blood or the brain, but was essential for trafficking within the brain parenchyma. Analysis of other Orthobunyaviruses showed that Jamestown Canyon virus also induced CCR2-independent iMO egress to the blood. These studies demonstrate that the CCR2-requirement for iMO egress to the blood is not universal for all viruses.

Orthobunyaviruses include several recently emerging viruses of significant medical and veterinary importance. There is currently very limited understanding on what determines the host species range of these pathogens. In this study we discovered that BST-2/tetherin restricts orthobunyavirus replication in a host-specific manner. We show that viruses with human tropism (Oropouche virus and La Crosse virus) are restricted by sheep BST-2 but not by the human orthologue, while viruses with ruminant tropism (Schmallenberg virus and others) are restricted by human BST-2 but not by the sheep orthologue. We also show that BST-2 blocks orthobunyaviruses replication by reducing the amount of envelope glycoprotein into viral particles egressing from infected cells. This is the first study identifying a restriction factor that correlates with species susceptibility to orthobunyavirus infection. This work provides insight to help us dissect the adaptive changes that bunyaviruses require to cross the species barrier and emerge into new species., Highlights • BST-2 is a determinant of orthobuynyavirus host range. • BST-2 restricts orthobunyavirus replication. • BST-2 reduces the incorporation of envelope glycoprotein into virions.

Background La Crosse Virus (LACV) is a primary cause of pediatric viral encephalitis in the USA and can result in severe clinical outcomes. Almost all cases of LACV encephalitis occur in children 16 years or younger, indicating an age-related susceptibility. This susceptibility is recapitulated in a mouse model where weanling (3 weeks old or younger) mice are susceptible to LACV-induced disease, and adults (greater than 6 weeks) are resistant. Disease in mice and humans is associated with infiltrating leukocytes to the CNS. However, what cell types are infiltrating into the brain during virus infection and how these cells influence pathogenesis remain unknown. Methods In the current study, we analyzed lymphocytes recruited to the CNS during LACV-infection in clinical mice, using flow cytometry. We analyzed the contribution of these lymphocytes to LACV pathogenesis in weanling mice using knockout mice or antibody depletion. Additionally, we studied at the potential role of these lymphocytes in preventing LACV neurological disease in resistant adult mice. Results In susceptible weanling mice, disease was associated with infiltrating lymphocytes in the CNS, including NK cells, CD4 T cells, and CD8 T cells. Surprisingly, depletion of these cells did not impact neurological disease, suggesting these cells do not contribute to virus-mediated damage. In contrast, in disease-resistant adult animals, depletion of both CD4 T cells and CD8 T cells or depletion of B cells increased neurological disease, with higher levels of virus in the brain. Conclusions Our current results indicate that lymphocytes do not influence neurological disease in young mice, but they have a critical role protecting adult animals from LACV pathogenesis. Although LACV is an acute virus infection, these studies indicate that the innate immune response in adults is not sufficient for protection and that components of the adaptive immune response are necessary to prevent virus from invading the CNS.

The mosquito-borne California encephalitis serogroup viruses of the genus Orthobunyavirus (family Bunyaviridae ) include several causative agents of encephalitis in humans. Until recently, Inkoo virus (INKV) was the only orthobunyavirus isolated in Finland, showing high seroprevalence in the population. In this study, we recovered five orthobunyavirus isolates from mosquitoes collected in eastern Finland in the early autumns of 2007 and 2008 by inoculation of Vero cells. The isolates were determined by S, M and L segment sequences to represent the California encephalitis virus species but distinct from INKV (68% polyprotein amino acid (aa) identity). In genetic analyses, isolates clustered together with a number of westernmost Chatanga virus isolates (98% polyprotein aa identity) reported from Russia, forming a distinct phylogroup. However, the sequence homology of this phylogroup to the majority of Chatanga isolates, comprising three different geographically clustered phylogroups, was considerably lower (89–92% polyprotein aa identity). The five new isolates were designated as Mohko isolates of Chatanga virus, according to the village of origin. The isolates were closely related to Snowshoe hare virus (SSHV) and La Crosse virus (LACV) with an aa identity of 87% and 82% within the M segment polyprotein, respectively. The genetic relatedness of Mohko isolates to a number of human pathogenic orthobunyaviruses warrants further investigation on their potential disease associations and further serological analysis is needed to compare them to other Chatanga virus isolates and SSHV to determine their true antigenic relation.

The innate immune system mediates protection against neurotropic viruses capable of infecting the central nervous system (CNS). Neurotropic viruses include herpes simplex virus (HSV), West Nile virus (WNV), rabies virus, La Crosse virus, and poliovirus. Viral infection triggers activation of pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), retinoic acid-inducible gene 1 (RIG-I) like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and cytosolic DNA sensors. Although originally characterised in peripheral immune cells, emerging evidence points to important roles for these PRRs in cells of the CNS. Here, we review recent advances in our understanding of the mechanisms by which these PRRs provide protection against neurotropic viruses, and discuss instances in which these responses become detrimental and cause immunopathology in the CNS.

Mute swans (Cygnus olor) are an invasive species in the United States. The dramatic increase in their populations in localized areas has led to various problems, among them competition with native species and attacks on humans by aggressive swans. However, very little is known about the ability of these swans to transmit pathogens to humans, domestic birds, or wildlife or participate in enzootic maintenance. To learn more about select pathogens that mute swans may harbor, a survey was conducted from April of 2011 to August of 2012 in the Great Lakes region and localized areas of the Atlantic coast, which revealed serologic evidence of arbovirus exposure in mute swans. Of 497 mute swans tested, antibodies were detected for eastern equine encephalitis (4.8%), St. Louis encephalitis (1.4%), West Nile (1.2%), and Turlock (0.6%) viruses. Samples were also tested for evidence of antibodies to La Crosse virus, but none were positive.

Viruses within the Bunyaviridae family are tri-segmented, negative-stranded RNA viruses. The family includes several emerging and re-emerging viruses of humans, animals and plants, such as Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, La Crosse virus, Schmallenberg virus and tomato spotted wilt virus. Many bunyaviruses are arthropod-borne, so-called arboviruses. Depending on the genus, bunyaviruses encode, in addition to the RNA-dependent RNA polymerase and the different structural proteins, one or several non-structural proteins. These non-structural proteins are not always essential for virus growth and replication but can play an important role in viral pathogenesis through their interaction with the host innate immune system. In this review, we will summarize current knowledge and understanding of insect-borne bunyavirus non-structural protein function(s) in vertebrate, plant and arthropod.

We show that interferon-induced transmembrane protein 1 (IFITM-1), IFITM-2, and IFITM-3 exhibit a broad spectrum of antiviral activity against several members of the Bunyaviridae family, including Rift Valley fever virus (RVFV), La Crosse virus, Andes virus, and Hantaan virus, all of which can cause severe disease in humans and animals. We found that RVFV was restricted by IFITM-2 and -3 but not by IFITM-1, whereas the remaining viruses were equally restricted by all IFITMs. Indeed, at low doses of alpha interferon (IFN-α), IFITM-2 and -3 mediated more than half of the antiviral activity of IFN-α against RVFV. IFITM-2 and -3 restricted RVFV infection mostly by preventing virus membrane fusion with endosomes, while they had no effect on virion attachment to cells, endocytosis, or viral replication kinetics. We found that large fractions of IFITM-2 and IFITM-3 occupy vesicular compartments that are distinct from the vesicles coated by IFITM-1. In addition, although overexpression of all IFITMs expanded vesicular and acidified compartments within cells, there were marked phenotypic differences among the vesicular compartments occupied by IFITMs. Collectively, our data provide new insights into the possible mechanisms by which the IFITM family members restrict distinct viruses.

Viral encephalitis represents a significant, and costly, public health threat particularly for high-risk pediatric populations. An emerging mosquito-borne pathogen endemic to the United States, La Crosse virus (LACV) is one of the most common causes of viral encephalitis in children in the United States. However, no licensed therapeutics or vaccines currently exist for treatment. Hampering development efforts, the host response to LACV and its role in disease pathogenesis has only recently been examined. In this review, we discuss the current understanding of innate immune response in the context of viral pathogenesis and host susceptibility to LACV. In addition, we address the need for a clearer understanding of the early host-virus interactions in LACV infections as it relates to viral pathogenesis in the central nervous system.

To monitor mosquito-borne diseases, public health departments conduct mosquito and pathogen surveillance. Our objective was to evaluate mosquito monitoring methods for collecting La Crosse virus (LACV) and West Nile virus (WNV) vectors (Aedes and Culex mosquitoes, respectively) in southern Appalachia. Centers for Disease Control and Prevention (CDC) light traps baited with carbon dioxide (CO(2)), CDC light traps baited with CO(2) and BG lure, BG-Sentinel traps baited with CO(2), gravid traps baited with oak (Quercus)-water infusion, and resting traps were compared in eastern Tennessee in 2013. Traps operated at 8 different urban sites throughout Knox County were randomly assigned to and rotated among 6 plots within each site. Results were specific for each vector; the BG-Sentinel trap was the best method for Aedes triseriatus, the CDC trap baited with CO(2) and BG lure was the best method for Ae. albopictus, and the gravid trap was the best method for Ae. japonicus. Culex erraticus collections varied by week and trapping method, indicating no single method was best, but the questing traps collected more mosquitoes. There was no significant trapping difference for Cx. pipiens complex in this region using the methods tested. The results suggest using a combination of trapping methods when sampling for LACV and/or WNV mosquito vectors in southern Appalachia. Effective trapping methods are necessary to enable accurate surveillance, improve control methods, enhance understanding of dispersal, and use for early detection of vectors and pathogens.

Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was preactivated, was reduced with knockdown of β-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses La Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt-responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed antiviral therapeutics. IMPORTANCE RVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g., the Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. These studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses.

The innate immunity of embryonic stem cells (ESCs) has recently emerged as an important issue in ESC biology and in ESC-based regenerative medicine. We have recently reported that mouse ESCs (mESCs) do not have a functional type I interferon (IFN)-based antiviral innate immunity. They are deficient in expressing IFN in response to viral infection and have limited ability to respond to IFN. Using fibroblasts (FBs) as a cell model, the current study investigated the development of antiviral mechanisms during in vitro differentiation of mESCs. We demonstrate that mESC-differentiated FBs (mESC-FBs) share extensive similarities with naturally differentiated FBs in morphology, marker expression, and growth pattern, but their development of antiviral mechanisms lags behind. Nonetheless, the antiviral mechanisms are inducible during mESC differentiation as demonstrated by the transition of nuclear factor kappa B (NFκB), a key transcription factor for IFN expression, from its inactive state in mESCs to its active state in mESC-FBs and by increased responses of mESC-FBs to viral stimuli and IFN during their continued in vitro propagation. Together with our previously published study, the current data provide important insights into molecular basis for the deficiency of IFN expression in mESCs and the development of antiviral innate immunity during mESC differentiation.

Understanding how midgut microbial communities of field-collected mosquitoes interact with pathogens is critical for controlling vector infection and disease. We used 16S rRNA and internal transcribed spacer sequencing to characterize the midgut bacterial and fungal communities of adult females of Aedes triseriatus and Aedes japonicus collected as pupae in tree holes, plastic bins and waste tires and their response to La Crosse virus (LACV) infection. For both mosquito species and across all habitat and virus treatments, a total of 62 bacterial operational taxonomic units (OTUs) from six phyla and 21 fungal OTUs from two phyla were identified. The majority of bacterial (92%) and fungal (71%) OTUs were shared between the mosquito species; however, several OTUs were unique to each species. Bacterial and fungal communities of individuals that took either infectious or noninfectious bloodmeals were less diverse and more homogeneous compared to those of newly emerged adults. Interestingly, LACV-infected A. triseriatus and A. japonicus had higher bacterial richness and lower fungal richness compared to individuals that took a noninfectious bloodmeal, suggesting that viral infection was associated with an increase in bacterial OTUs and a decrease in fungal OTUs. For both mosquito species, several OTUs were identified that had both high fidelity and specificity to mosquito midguts that were infected with LACV. Overall, these findings demonstrate that bacterial and fungal communities that reside in mosquito midguts respond to host diet and viral infection and could play a role in modulating vector susceptibility to LACV.

Arthropod-borne viruses (arboviruses) cause many diseases worldwide and their transmission is likely to change with land use and climate changes. La Crosse virus is historically transmitted by the native mosquito Aedes triseriatus (Say) in the upper Midwestern U.S., but the invasive congeners Aedes albopictus (Skuse) and Aedes japonicus (Theobald), which co-occur with A. triseriatus in water-holding containers, may be important accessory vectors in the Appalachian region where La Crosse encephalitis is an emerging disease. This review focuses on evidence for how climate, land use, and biological invasions may have direct abiotic and indirect community-level impacts on immature developmental stages (eggs and larvae) of Aedes mosquitoes. Because vector-borne diseases usually vary in space and time and are related to the ecology of the vector species, we propose that the ecology of its mosquito vectors, particularly at their immature stages, has played an important role in the emergence of La Crosse encephalitis in the Appalachian region and represents a model for investigating the effects of environmental changes on other vector-borne diseases. We summarize the health effects of La Crosse virus and associated socioeconomic costs that make it the most important native mosquito-borne disease in the U.S. We review of the transmission of La Crosse virus, and present evidence for the impacts of climate, land use, and biological invasions on Aedes mosquito communities. Last, we discuss important questions about the ecology of La Crosse virus mosquito vectors that may improve our understanding of the impacts of environmental changes on La Crosse virus and other arboviruses.

Aedes albopictus (Skuse) is a potential vector for many arboviruses, including La Crosse virus (LACV), the leading cause of pediatric encephalitis in North America. Bacteria isolated from the midgut and diverticula of field-caught female Ae. albopictus were cultured and identified using 16S ribosomal RNA gene amplification and sequencing. Members of seven and six bacterial families were identified from the midguts and diverticula, respectively, with nearly half of the isolates identified to the family Enterobacteriaceae. Many are related to bacteria identified in other invertebrates, and several may represent previously unknown species or genera. Of the 24 isolated bacteria, 12 (50%) showed a significant reduction in infectivity of LACV for Vero cells. Inhibition of infectivity ranged from 0 to 44% and was not dependent on bacterial classification. The antiviral activity of these bacteria warrants further investigation as an alternate means to interrupt the LACV transmission cycle.

Type I IFN is a major player in innate and adaptive immune responses. Besides, it is involved in organogenesis and tumor development. Generally, IFN responses are amplified by an autocrine loop with IFN-β as the priming cytokine. However, due to the lack of sensitive detection systems, where and how type I IFN is produced in vivo is still poorly understood. In this study, we describe a luciferase reporter mouse, which allows tracking of IFN-β gene induction in vivo. Using this reporter mouse, we reveal strong tissue-specific induction of IFN-β following infection with influenza or La Crosse virus. Importantly, this reporter mouse also allowed us to visualize that IFN-β is expressed constitutively in several tissues. As suggested before, low amounts of constitutively produced IFN might maintain immune cells in an activated state ready for a timely response to pathogens. Interestingly, thymic epithelial cells were the major source of IFN-β under noninflammatory conditions. This relatively high constitutive expression was controlled by the NF Aire and might influence induction of tolerance or T cell development.

The mating efficiencies (the percentage of females inseminated by males) of field-collected and laboratory-colonized Aedes triseriatus (Say) (Diptera: Culicidae) female mosquitoes transovarially infected or uninfected with La Crosse virus (LACV) were compared. The females were placed in cages with age-matched males, and the insemination rates (number of inseminated females of the total number of females examined) were determined daily by detection of sperm in the spermathecae. LACV-infected mosquitoes typically mated earlier than uninfected mosquitoes, i.e., insemination occurred earlier after the mixing of males and females. LACV load was not correlated with increased insemination.

A significant increase in the number of reported cases of La Crosse virus (LACV) infections in eastern Tennessee has occurred in the last ten years. The objective of this study was to determine the abundance and habitat preferences of the potential vectors of LACV in this region. Adult host-seeking mosquitoes were collected using CO(2)-baited CDC light traps and a series of human-landing catches in eastern Tennessee from 2004 to 2006. A total of 4,200 female mosquitoes of 23 species was collected by CO(2)-baited CDC trapping at ten sites during the study period. Aedes albopictus (Skuse) was the most abundant mosquito collected at all sites and vegetation types, with the ratios of total Ae. albopictus to Ae. triseriatus (Say) females collected being 2.1:1 in 2004, 3.8:1 in 2005, and 4.9:1 in 2006. Ten species were collected during a series of human-landing catches made at four different sites; one probable and three confirmed case sites of LACV infections, totaling 528 female mosquitoes. Aedes albopictus was the most abundant species collected, with a 4:1 ratio of Ae. albopictus to Ae. triseriatus females. Aedes albopictus exhibited two clear peaks of "landing" activity, one in the early morning and one in the late afternoon or early evening. Simple and multiple regression analyses of the predictors of the number of mosquitoes collected showed that populations of Ae. albopictus were three times more likely to be collected overall than Ae. triseriatus. Species (Ae. albopictus), vegetation (residential), and the previous cumulative precipitation for the four weeks prior to collection were significantly (P < 0.05) associated with the number of mosquitoes collected by CO(2)-baited CDC trapping. Aedes albopictus was also more likely to be collected than Ae. triseriatus at confirmed cases of LACV infections.

La Crosse virus (LACV) is a mosquito-transmitted member of the Bunyaviridae family that causes severe encephalitis in children. For the LACV nonstructural protein NSs, previous overexpression studies with mammalian cells had suggested two different functions, namely induction of apoptosis and inhibition of RNA interference (RNAi). Here, we demonstrate that mosquito cells persistently infected with LACV do not undergo apoptosis and mount a specific RNAi response. Recombinant viruses that either express (rLACV) or lack (rLACVdelNSs) the NSs gene similarly persisted and were prone to the RNAi-mediated resistance to superinfection. Furthermore, in mosquito cells overexpressed LACV NSs was unable to inhibit RNAi against Semliki Forest virus. In mammalian cells, however, the rLACVdelNSs mutant virus strongly activated the antiviral type I interferon (IFN) system, whereas rLACV as well as overexpressed NSs suppressed IFN induction. Consequently, rLACVdelNSs was attenuated in IFN-competent mouse embryo fibroblasts and animals but not in systems lacking the type I IFN receptor. In situ analyses of mouse brains demonstrated that wild-type and mutant LACV mainly infect neuronal cells and that NSs is able to suppress IFN induction in the central nervous system. Thus, our data suggest little relevance of the NSs-induced apoptosis or RNAi inhibition for growth or pathogenesis of LACV in the mammalian host and indicate that NSs has no function in the insect vector. Since deletion of the viral NSs gene can be fully complemented by inactivation of the host's IFN system, we propose that the major biological function of NSs is suppression of the mammalian innate immune response.