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.

Neuronal maturation reduces the type I IFN response to orthobunyavirus infection and leads to increased apoptosis of human neurons.

BACKGROUND: La Crosse virus (LACV) is the leading cause of pediatric arboviral encephalitis in the USA. LACV encephalitis can result in learning and memory deficits, which may be due to infection and apoptosis of neurons in the brain. Despite neurons being the primary cell infected in the brain by LACV, little is known about neuronal responses to infection. METHODS: Human cerebral organoids (COs), which contain a spectrum of developing neurons, were used to examine neuronal responses to LACV. Plaque assay and quantitative reverse transcription (qRT) PCR were used to determine the susceptibility of COs to LACV infection. Immunohistochemistry, flow cytometry, and single-cell transcriptomics were used to determine specific neuronal subpopulation responses to the virus. RESULTS: Overall, LACV readily infected COs causing reduced cell viability and increased apoptosis. However, it was determined that neurons at different stages of development had distinct responses to LACV. Both neural progenitors and committed neurons were infected with LACV, however, committed neurons underwent apoptosis at a higher rate. Transcriptomic analysis showed that committed neurons expressed fewer interferon (IFN)-stimulated genes (ISGs) and genes involved IFN signaling in response to infection compared to neural progenitors. Furthermore, induction of interferon signaling in LACV-infected COs by application of recombinant IFN enhanced cell viability. CONCLUSIONS: These findings indicate that neuronal maturation increases the susceptibility of neurons to LACV-induced apoptosis. This susceptibility is likely due, at least in part, to mature neurons being less responsive to virus-induced IFN as evidenced by their poor ISG response to LACV. Furthermore, exogenous administration of recombinant IFN to LACV COs rescued cellular viability suggesting that increased IFN signaling is overall protective in this complex neural tissue. Together these findings indicate that induction of IFN signaling in developing neurons is an important deciding factor in virus-induced cell death.

Throw out the Map: Neuropathogenesis of the Globally Expanding California Serogroup of Orthobunyaviruses.

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.

Investigation of Acute Flaccid Paralysis Reported with La Crosse Virus Infection, Ohio, USA, 2008-2014.

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.

The TAM receptor Mertk protects against neuroinvasive viral infection by maintaining blood-brain barrier integrity.

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-ß 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.

Capillaries in the olfactory bulb but not the cortex are highly susceptible to virus-induced vascular leak and promote viral neuroinvasion.

Viral neuroinvasion is a critical step in the pathogenesis of viral encephalitis. Multiple mechanisms of neuroinvasion have been identified, but their relative contribution to central nervous system (CNS) infection remains unclear for many viruses. In this study, we examined neuroinvasion of the mosquito-borne bunyavirus La Crosse (LACV), the leading cause of pediatric viral encephalitis in the USA. We found that the olfactory bulb (OB) and tract were the initial areas of CNS virus infection in mice. Removal of the OB reduced the incidence of LACV-induced disease demonstrating the importance of this area to neuroinvasion. However, we determined that infection of the OB was not due to axonal transport of virus from olfactory sensory neurons as ablation of these cells did not affect viral pathogenesis. Instead, we found that OB capillaries were compromised allowing leakage of virus-sized particles into the brain. Analysis of OB capillaries demonstrated specific alterations in cytoskeletal and Rho GTPase protein expression not observed in capillaries from other brain areas such as the cortex where leakage did not occur. Collectively, these findings indicate that LACV neuroinvasion occurs through hematogenous spread in specific brain regions where capillaries are prone to virus-induced activation such as the OB. Capillaries in these areas may be "hot spots" that are more susceptible to neuroinvasion not only for LACV, but other neurovirulent viruses as well.

Cemeteries are effective sites for monitoring la crosse virus (LACv) and these environments may play a role in LACv infection.

La Crosse encephalitis (LAC) is the leading arboviral disease among children, and was previously limited to the upper Midwest. In 2012 nine pediatric cases of LAC occurred in eastern Tennessee, including one fatal case. In an attempt to identify sites near an active LACv infection and describe the abundance and distribution of potential LACv vectors near a fatal LAC case in the Appalachian region, we initiated an end of season study using a combination of questing and oviposition mosquito traps placed at forty-nine sites consisting of cemeteries and houses within 16 radial kilometers of two pediatric infections. LACv was isolated from three Aedes triseriatus pools collected from cemeteries and spatial clustering analysis identified clusters of Ae. triseriatus and Ae. albopictus populations that overlapped in the same area as the 2012 LACv cases. Results indicate cemeteries are effective sites for monitoring LACv. The role of cemeteries and specific environmental features will be the focus of future investigations.

Innate immune response to La Crosse virus infection.

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.

[Arborviruses of the California encephalitis serogroup 1N Russia and their contribution to infectious pathology].

This review focuses on arborviral infections associated with California serocomplex (Bunyaviridae, Orthobunyavirus). Results of relevant eco-epidemiological and clinical studies in Russia are presented suggesting the ubiquitious nature of diseases caused by viruses of the California encephalitis serocomplex (Inko, Tahyna, Snowshoe Hare). The etiologic structure of these diseases in taiga and mixed woods of the European part and Western Siberia is dominated by the Inco virus and in southern regions by Tahina. The diseases have a well apparent seasonal pattern (July-August) in agreement with the peak summer activity of the pathogens. Two clinical forms of pathology are distinguished, influenza-like and generalized, the latter affecting lungs, kidneys, liver, and CNS. The Inco virus plays a key role in pathogenesis of the generalized form affecting CNS.

La crosse virus encephalitis preceding neurosarcoidosis diagnosed by brain biopsy.

Childhood neurosarcoidosis is rare, and its etiology is unknown. La Crosse virus is one of the most common causes of encephalitis in North America. We report the case of a 12-year-old boy who had La Crosse virus encephalitis and subsequently developed imaging and pathologic findings suggestive of neurosarcoidosis. He presented with acute onset of transient aphasia 4 months after an episode of encephalitis; serology results at the time of aphasia were positive for La Crosse virus. Brain MRI revealed diffuse subcortical nodular lesions. His serum angiotensin-converting enzyme level was elevated, and brain biopsy revealed noncaseating granuloma. It is interesting to note that he has had no recurrence of aphasia; repeat MRI showed significant improvement of nodular lesions without therapy. To our knowledge, this is the first case of La Crosse virus encephalitis associated with neurosarcoid findings.

The incidence risk, clustering, and clinical presentation of La Crosse virus infections in the eastern United States, 2003-2007.

BACKGROUND: Although La Crosse virus (LACV) is one of the most common causes of pediatric arboviral infections in the United States, little has been done to assess its geographic distribution, identify areas of higher risk of disease, and to provide a national picture of its clinical presentation. Therefore, the objective of this study was to investigate the geographic distribution of LACV infections reported in the United States, to identify hot-spots of infection, and to present its clinical picture. METHODS AND FINDINGS: Descriptive and cluster analyses were performed on probable and confirmed cases of LACV infections reported to the Centers for Disease Control and Prevention from 2003-2007. A total of 282 patients had reported confirmed LACV infections during the study period. Of these cases the majority (81 percent) presented during the summer, occurred in children 15 years and younger (83.3 percent), and were found in male children (64.9 percent). Clinically, the infections presented as meningioencephalitis (56.3 percent), encephalitis (20.7 percent), meningitis (17.2 percent), or uncomplicated fever (5 percent). Deaths occurred in 1.9 percent of confirmed cases, and in 8.6 percent of patients suffering from encephalitis. The majority of these deaths were in patients 15 years and younger. The county-level incidence risk among counties (n = 136) reporting both probable and confirmed cases for children 15 years and younger (n = 355) ranged from 0.2 to 228.7 per 100,000 persons. The southern United States experienced a significantly higher (p<0.05) incidence risk during the months of June, July, August, and October then the northern United States. There was significant (p<0.05) clustering of high risk in several geographic regions with three deaths attributed to complications from LAC encephalitis occurring in two of these hot-spots of infections. CONCLUSIONS: Both the incidence risk and case fatality rates were found to be higher than previously reported. We detected clustering in four geographic regions, a shift from the prior geographic distributions, and developed maps identifying high-risk areas. These findings are useful for raising awareness among health care providers regarding areas at a high risk of infections and for guiding targeted multifaceted interventions by public health officials.

Periodic lateralized epileptiform discharges in La Crosse encephalitis, a worrisome subgroup: clinical presentation, electroencephalogram (EEG) patterns, and long-term neurologic outcome.

La Crosse virus encephalitis is the most common mosquito-borne virus in children in the United States. La Crosse virus encephalitis has emerged as a significant health concern due to its potential for acute morbidity, including seizures, alterations in mental status, and, in rare cases, death, as well as the potential for chronic morbidity, including, epilepsy and cognitive and behavioral disorders. The aim of this study is to provide a clinical description of the largest series of children reported with periodic lateralizing epileptiform discharges (PLEDS) associated with La Cross virus encephalitis with reference to their clinical course, seizure type, electroencephalogram (EEG) patterns, and 2- and 10-year long-term neurologic outcome. In addition, to evaluate whether this subset of children may indeed have more severe disease than children with La Crosse virus encephalitis without PLEDS, comparisons are made between the 2 groups on specific variables. All patients presented with fever and disorientation; 6 of the 9 (66%) presented with seizures. PLEDS localized to the temporal lobe in 7 patients (77%). The children with PLEDS had longer intensive care unit stays (6.5 +/- 2.4 vs 3.2 +/- 1.9; P < .0001), a higher rate of intubation (88% vs 20%; P < .001), and a higher rate of cerebral herniation (1%; P < .05) than children with La Crosse virus encephalitis without PLEDS. Follow-up data on the subset with PLEDS also suggest a relatively high rate of epilepsy and behavioral difficulties with hyperactivity symptoms, memory deficits, and school difficulties. The implications for recognition, management, and follow-up of this worrisome subset of patients with La Crosse virus encephalitis are discussed.

Economic and social impacts of La Crosse encephalitis in western North Carolina.

La Crosse encephalitis (LACE), a human illness caused by a mosquito-transmitted virus, is endemic in western North Carolina. To assess the economic and social impacts of the illness, 25 serologically confirmed LACE case patients and/or families were interviewed to obtain information on the economic costs and social burden of the disease. The total direct and indirect medical costs associated with LACE over 89.6 life years accumulated from the onset of illness to the date of interview for 24 patients with frank encephalitis totaled dollar 791,374 (range = dollar 7,521-175,586), with a mean +/- SD per patient cost of dollar 32,974 +/- dollar 34,793. The projected cost of a case with lifelong neurologic sequelae ranged from dollar 48,775 to dollar 3,090,798 (n = 5). For the 25 LACE patients, 55.15 (54.83%) of the 100.59 cumulative life years (CLYs) were impaired to some degree. Disability adjusted life years (DALYs) were calculated to measure the productive life years lost to LACE. Approximately 13.00 DALYs were accumulated over 100.59 CLYs of study. Projected DALYs for case patients (n = 5) with lifelong neurologic sequelae ranged from 12.90 to 72.37 DALYs. An Impact of La Crosse Encephalitis Survey (ILCES) was used to measure the social impact of LACE over time for case patients and their families. The ILCES scores demonstrated that the majority of the social burden of the illness is borne by the five patients with lifelong neurologic sequelae. The socioeconomic burden resulting from LACE is substantial, which highlights the importance of the illness in western North Carolina, as well as the need for active surveillance, reporting, and prevention programs for the infection.

Newly recognized focus of La Crosse encephalitis in Tennessee.

La Crosse virus is a mosquito-borne arbovirus that causes encephalitis in children. Only nine cases were reported in Tennessee during the 33-year period from 1964-1996. We investigated a cluster of La Crosse encephalitis cases in eastern Tennessee in 1997. Medical records of all suspected cases of La Crosse virus infection at a pediatric referral hospital were reviewed, and surveillance was enhanced in the region. Previous unreported cases were identified by surveying 20 hospitals in the surrounding 16 counties. Mosquito eggs were collected from five sites. Ten cases of La Crosse encephalitis were serologically confirmed. None of the patients had been discharged from hospitals in the region with diagnosed La Crosse encephalitis in the preceding 5 years. Aedes triseriatus and Aedes albopictus were collected at the case sites; none of the mosquitos had detectable La Crosse virus. This cluster may represent an extension of a recently identified endemic focus of La Crosse virus infection in West Virginia.

Effects of La Crosse virus infection on pregnant domestic rabbits and mongolian gerbils.

The transplacental transmission of La Crosse virus (LACV) was evaluated in domestic rabbits (Oryctolagus cuniculus) and Mongolian gerbils (Meriones unguiculatis) as a potential mechanism for the maintenance of the virus. Rabbits were infected with LACV at different times of gestation by injection of viral suspensions or by exposure to LACV transovarially (TO) infected Aedes triseriatus. Pregnant gerbils were exposed between 16-24 days of gestation to LACV TO- infected Ae. triseriatus. Our results indicate that LACV can infect gerbils in utero. The LACV was isolated from the brain of suckling gerbils that died 3-5 days after birth from LACV-exposed mothers, representing the first evidence of LACV transplacental transmission. Microgliosis was found histologically in the cerebral cortex. In addition, LACV infection of both pregnant gerbils and rabbits resulted in in utero and neonatal mortality. La Crosse virus was not detected in surviving young of infected rabbits even after immunosuppression by administration of cyclophosphamide. Thus, there was no evidence of persistent infection of rabbits following in utero exposure. Surprisingly, some of the infected pregnant gerbils developed progressive paralysis 9-14-days postexposure, and LACV was isolated from the brains of these animals. Histopathologic studies of these tissue samples showed acute meningoencephalitis. The effects of natural LACV infection should be studied in pregnant amplifying hosts, such as chipmunks and squirrels, and in pregnant women.

Induction of apoptosis by La Crosse virus infection and role of neuronal differentiation and human bcl-2 expression in its prevention.

La Crosse virus causes a highly cytopathic infection in cultured cells and in the murine central nervous system (CNS), with widespread neuronal destruction. In some viral infections of the CNS, apoptosis, or programmed cell death, has been proposed as a mechanism for cytopathology (Y. Shen and T. E. Shenk, Curr. Opin. Genet. Dev. 5:105-111, 1995). To determine whether apoptosis plays a role in La Crosse virus-induced cell death, we performed experiments with newborn mice and two neural tissue culture models. Newborn mice infected with La Crosse virus showed evidence of apoptosis with the terminal deoxynucleotidyl transferase-mediated nicked-end labeling (TUNEL) assay and, concomitantly, histopathological suggestion of neuronal dropout. Infection of tissue culture cells also resulted in DNA fragmentation, TUNEL reactivity, and morphological changes in the nuclei characteristic of apoptotic cells. As in one other system (S. Ubol, P. C. Tucker, D. E. Griffin, and J. M. Hardwick, Proc. Natl. Acad. Sci. USA 91:5202-5206, 1994), expression of the human proto-oncogene bcl-2 was able to protect one neuronal cell line, N18-RE-105, from undergoing apoptosis after La Crosse virus infection and prolonged the survival of infected cells. Nevertheless, expression of bcl-2 did not prevent eventual cytopathicity. However, a human neuronal cell line, NT2N, was resistant to both apoptosis and other types of cytopathicity after infection with La Crosse virus, reaffirming the complexity of cell death. Our results show that apoptosis is an important consequence of La Crosse virus infection in vivo and in vitro.

[The characteristics of the pathogenic properties of new strains of viruses of the California encephalitis antigenic complex isolated in Arkhangelsk Province]. / Kharakteristika patogennykh svoistv novykh shtammov virusov antigennogo kompleksa kaliforniiskogo éntsefalita, izolirovannykh v Arkhangel'skoi oblasti.

Neurotropic properties of Leiv 12724 Ax and Leiv 13004 Ax strains were demonstrated by inoculation of green monkeys, Syrian hamsters and white mice using different routes. The strain Leiv 13004 Ax showed more marked pathogenicity for monkeys and rodents producing lesions in all parts of the brain: temporal, frontal, occipital, cerebellar, medulla oblongata and spinal cord where productive vasculitis, perivascular infiltrations, hemorrhages, and dystrophy of nerve cells were observed. In hamsters, the strains Leiv 13004 Ax and Leiv 12724 Ax inoculated subcutaneously produced latent infection with long-term virus carrier state.