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.

Favipiravir treatment prolongs the survival in a lethal mouse model intracerebrally inoculated with Jamestown Canyon virus.

BACKGROUND: Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, primarily in North American adults. Currently, there are no available vaccines or specific treatments against JCV infections. METHODOLOGY/PRINCIPAL FINDINGS: The antiviral efficacy of favipiravir (FPV) against JCV infection was evaluated in vitro and in vivo in comparison with that of ribavirin (RBV) and 2'-fluoro-2'-deoxycytidine (2'-FdC). The in vitro inhibitory effect of these drugs on JCV replication was evaluated in Vero and Neuro-2a (N2A) cells. The efficacy of FPV in the treatment of JCV infection in vivo was evaluated in C57BL/6J mice inoculated intracerebrally with JCV, as per the survival, viral titers in the brain, and viral RNA load in the blood. The 90% inhibitory concentrations (IC90) of FPV, RBV, and 2'-FdC were 41.0, 61.8, and 13.6 µM in Vero cells and 20.7, 25.8, and 8.8 µM in N2A cells, respectively. All mice infected with 1.0×104 TCID50 died or were sacrificed within 10 days post-infection (dpi) without treatment. However, mice treated with FPV for 5 days [initiated either 2 days prior to infection (-2 dpi-2 dpi) or on the day of infection (0 dpi-4 dpi)] survived significantly longer than control mice, administered with PBS (p = 0.025 and 0.011, respectively). Moreover, at 1 and 3 dpi, the virus titers in the brain were significantly lower in FPV-treated mice (0 dpi-4 dpi) versus PBS-treated mice (p = 0.002 for both 1 and 3 dpi). CONCLUSIONS/SIGNIFICANCE: Although the intracerebral inoculation route is thought to be a challenging way to evaluate drug efficacy, FPV inhibits the in vitro replication of JCV and prolongs the survival of mice intracerebrally inoculated with JCV. These results will enable the development of a specific antiviral treatment against JCV infections and establishment of an effective animal model.

Double Crossed: A Case of La Crosse Encephalitis.

CASE REPORT: A 10-year-old male with T1DM and recent travel to North Carolina presented to an ED with 1 day of fever, vomiting, and headaches. He was discharged home with the presumptive diagnosis of viral gastroenteritis but returned nine hours later, agitated, and unable to speak. CSF showed pleocytosis. MRI brain was normal, and EEG showed intermittent seizures. He was started on antiepileptics. Antibiotics were discontinued after negative bacterial work-up. Repeat MRI brain one week later showed enhancement in the left cerebral cortex. IVIG was started due to concern for autoimmune encephalitis. Repeat lumbar puncture was positive for La Crosse virus IgM. DISCUSSION: This is the first case of La Crosse encephalitis (LACe) reported in Rhode Island.1 La Crosse virus (LACv) is a ssRNA Bunyavirus transmitted by the eastern tree-hole mosquito typically between July and September. LACv is endemic to the upper Midwestern US and Appalachia. In 2018, 81 of 86 total cases reported by the CDC were pediatric. Children are more likely to present with vomiting, seizures, and focal cortical inflammation or cerebral edema on brain imaging. IgM may be negative early in the disease course. Treatment is antiepileptics and supportive care.

Novel Ionophores Active against La Crosse Virus Identified through Rapid Antiviral Screening.

Bunyaviruses are significant human pathogens, causing diseases ranging from hemorrhagic fevers to encephalitis. Among these viruses, La Crosse virus (LACV), a member of the California serogroup, circulates in the eastern and midwestern United States. While LACV infection is often asymptomatic, dozens of cases of encephalitis are reported yearly. Unfortunately, no antivirals have been approved to treat LACV infection. Here, we developed a method to rapidly test potential antivirals against LACV infection. From this screen, we identified several potential antiviral molecules, including known antivirals. Additionally, we identified many novel antivirals that exhibited antiviral activity without affecting cellular viability. Valinomycin, a potassium ionophore, was among our top targets. We found that valinomycin exhibited potent anti-LACV activity in multiple cell types in a dose-dependent manner. Valinomycin did not affect particle stability or infectivity, suggesting that it may preclude virus replication by altering cellular potassium ions, a known determinant of LACV entry. We extended these results to other ionophores and found that the antiviral activity of valinomycin extended to other viral families, including bunyaviruses (Rift Valley fever virus, Keystone virus), enteroviruses (coxsackievirus, rhinovirus), flavirivuses (Zika virus), and coronaviruses (human coronavirus 229E [HCoV-229E] and Middle East respiratory syndrome CoV [MERS-CoV]). In all viral infections, we observed significant reductions in virus titer in valinomycin-treated cells. In sum, we demonstrate the importance of potassium ions to virus infection, suggesting a potential therapeutic target to disrupt virus replication.

Jamestown Canyon virus encephalitis in a heart transplant patient.

Jamestown Canyon virus (JtCV) is an arbovirus and a member of the California serogroup. To our knowledge, all the cases of JtCV have been reported in immunocompetent patients since it was first detected in 1997. We report a case of JtCV encephalitis in a solid organ transplant patient. A 48-year-old woman from Wisconsin had multiple hospital admissions for symptoms of progressive confusion, visual hallucinations, and inability to perform self-care. Initial evaluation was significant for lymphocytes in cerebrospinal fluid (CSF), and multiple infectious and metabolic causes were excluded. Further investigation found JtCV IgM in serum, and CSF. The patient's clinical course was compatible with JtCV encephalitis, and she was treated with ribavirin in addition to reduction of her immunosuppressive medications. She showed gradual and significant improvement in her mental and functional status. JtCV can cause a variety of symptoms that range from a flu-like syndrome to encephalitis. There have been an increased number of reported cases in recent years which is attributed to increased physician awareness and the availability of laboratory testing. Optimal treatment is still not known.

Activation of the innate signaling molecule MAVS by bunyavirus infection upregulates the adaptor protein SARM1, leading to neuronal death.

La Crosse virus (LACV), a zoonotic Bunyavirus, is a major cause of pediatric viral encephalitis in the United States. A hallmark of neurological diseases caused by LACV and other encephalitic viruses is the induction of neuronal cell death. Innate immune responses have been implicated in neuronal damage, but no mechanism has been elucidated. By using in vitro studies in primary neurons and in vivo studies in mice, we have shown that LACV infection induced the RNA helicase, RIG-I, and mitochondrial antiviral signaling protein (MAVS) signaling pathway, resulting in upregulation of the sterile alpha and TIR-containing motif 1 (SARM1), an adaptor molecule that we found to be directly involved in neuronal damage. SARM1-mediated cell death was associated with induced oxidative stress response and mitochondrial damage. These studies provide an innate-immune signaling mechanism for virus-induced neuronal death and reveal potential targets for development of therapeutics to treat encephalitic viral infections.

Orthobunyavirus entry into neurons and other mammalian cells occurs via clathrin-mediated endocytosis and requires trafficking into early endosomes.

La Crosse virus (LACV) is a leading cause of pediatric encephalitis and aseptic meningitis in the midwestern and southern United States, where it is considered an emerging human pathogen. No specific therapies or vaccines are available for LACV or any other orthobunyaviruses. Inhibition of LACV entry into cells is a potential target for therapeutic intervention, but this approach is limited by our current knowledge of the entry process. Here, we determined that clathrin-mediated endocytosis is the primary mechanism of orthobunyavirus entry and identified key cellular factors in this process. First, we demonstrated that LACV colocalized with clathrin shortly after infection in HeLa cells; we then confirmed the functional requirement of dynamin- and clathrin-mediated endocytosis for orthobunyavirus entry using several independent assays and, importantly, extended these findings to primary neuronal cultures. We also determined that macropinocytosis and caveolar endocytosis, both established routes of virus entry, are not critical for cellular entry of LACV. Moreover, we demonstrated that LACV infection is dependent on Rab5, which plays an important regulatory role in early endosomes, but not on Rab7, which is associated with late endosomes. These findings provide the first description of bunyavirus entry into cells of the central nervous system, where infection can cause severe neurological disease, and will aid in the design and development of antivirals and therapeutics that may be useful in the treatment of LACV and, more broadly, arboviral infections of the central nervous system.

La Crosse viral infection in hospitalized pediatric patients in Western North Carolina.

OBJECTIVE: La Crosse infection, caused by a rare mosquito-transmitted virus, is endemic in Western North Carolina. Given the large number of cases at our institution, our goal was to describe the presentation, management, and clinical course for pediatric patients with this disease. METHODS: We retrospectively reviewed medical records from pediatric patients with antibody-confirmed La Crosse infection admitted to Mission Hospital July 2004 through August 2009. Demographics, clinical characteristics, management methods, length of hospital stay, and complications were analyzed. Regression analysis was used to assess relationships between presentation and clinical course. RESULTS: Forty-seven pediatric patients were identified with antibody-confirmed La Crosse infection. Seventy percent were male, and the median age was 8 years. Admission signs and symptoms included fever (43%), headache (94%), vomiting (78%), altered mental status (58%), and seizures (61%). All patients had pleocytosis on cerebrospinal fluid studies (range 10-1063 cells/mm3). Median length of stay was 5 days. Seizure at admission was associated with an increased length of stay (2.4 additional days, 95% confidence interval 0.7-4.1). Eighteen patients (38%) received intensive care, 7 (19%) received parenteral or enteral (via nasogastric tube) nutrition, and 4 (9%) received mechanical ventilation. No statistically significant associations between presenting signs and symptoms and complications were found. Treatments included antibiotics (87%), antiviral medication (55%), seizure prophylaxis (47%), and isotonic fluids (98%). CONCLUSIONS: Our data reflect few indicators to predict clinical course during hospital stay. Management strategies should include attention to development of seizure activity and preventive measures for syndrome of inappropriate antidiuretic hormone.

Safety and pharmacokinetics of ribavirin for the treatment of la crosse encephalitis.

BACKGROUND: La Crosse viral encephalitis (LACVE) is associated with residual epilepsy and neurocognitive deficits in survivors. This report summarizes 3 phases of clinical studies of children treated with intravenous (IV) ribavirin (RBV), each one exploring a different phase (I, IIA, IIB) of clinical trial development. METHODS: In phase I, 7 children with life-threatening LACVE were treated with emergency use RBV using a moderate IV dose (8.33 mg/kg/dose q 8 hours day 1, 5 mg/kg/dose q 8 hours days 2-10). In phase IIA, 12 children with severe LACVE were enrolled: 8 treated with RBV (same dose as phase I) and 4 with placebo. In phase IIB an escalated dose was used (33 mg/kg dose 1, then 16 mg/kg/dose q 6 hours for 4 days, and 8 mg/kg/dose q 8 hours for 3 days). RESULTS: In a group of 15 children treated in phase I and phase IIA, RBV appeared safe at moderate dose, but based on steady-state RBV levels of 9.3 µM, estimated cerebrospinal fluid levels were less than 20% of the EC50 of RBV for LACVE. At the escalated dose used in phase IIB, adverse events occurred, likely related to RBV, and therefore the trial was discontinued. Nevertheless, valuable pharmacokinetic (PK) and safety data were obtained at moderate dose, with potential treatment implications for other indications. CONCLUSIONS: Although the results do not support the use of RBV for LACVE, this nevertheless is the largest study of antiviral treatment for LACVE to date and the largest pharmacokinetic analysis of IV RBV in children for any indication.

The use of oral ribavirin in the management of La Crosse viral infections.

Since its recognition in 1964, La Crosse (LAC) virus has been recognized as an important cause of pediatric encephalitis in the United States. The annual incidence of this illness is believed to be between 20 and 30 cases per hundred thousand, though most cases remain undiagnosed. It is typically responsible for a relatively mild disease in humans, though a sub-group of patients suffer from life-threatening illness characterized by permanent neuropsychiatric sequelae. There are currently no approved medications to treat LAC viral infections. However, an anti-RNA viral treatment might prove useful in reducing the length and severity of illness while potentially reducing the risk of permanent sequelae. Evidence exists that the use of ribavirin may form the basis of an effective treatment for LAC viral infections. We propose that oral ribavirin may provide a useful treatment to limit the severity and improve the prognosis of those suffering from LAC viral infections.

Severe La Crosse encephalitis with significant neurologic sequelae.

La Crosse encephalitis, a member of the California arbovirus group, is the most common cause of reported mosquito-borne illness in the United States. Approximately 70 cases of La Crosse encephalitis are reported each year. The principal vector is the mosquito Aedes triseriatus. During the summer the virus is amplified horizontally in a cycle among small mammals such as chipmunks and squirrels. Infected female A. triseriatus deposit eggs in the basal holes of hardwood trees, although man-made containers and old tires containing water also supply a suitable breeding site. Some of these eggs infected with La Crosse virus hatch the next spring and give rise to infected adult A. triseriatus, and the host-vector cycle is renewed. Only a minority of children infected with the virus become ill. Clinical disease caused by La Crosse is usually mild, and neurologic sequelae are relatively uncommon. In this report we describe six patients with severe La Crosse meningoencephalitis diagnosed within a 4-week period. All patients required intensive care management, and there was a high rate of neurologic sequelae, suggesting that La Crosse is not necessarily a benign meningoencephalitis.

La Crosse and other forms of California encephalitis.

The California serogroup viruses are mosquito viruses that cause human infections on five continents. They are maintained and amplified in nature by a wide variety of mosquito vectors and mammalian hosts; they thrive in a remarkably wide variety of microclimates (eg, tropical, coastal temperate marshland, lowland river valleys, alpine valleys and highlands, high boreal deserts, and arctic steppes). In 1993, California serogroup viruses caused 71% of all cases of arboviral illness in the United States, principally La Crosse encephalitis. The 30 to 180 annual cases of La Crosse encephalitis represent 8% to 30% of all cases of encephalitis, rendering this illness the most common and important endemic mosquito-borne illness in the USA. Subclinical or mild infections are much more common. Methods and results acquired from intense study of California serogroup viruses have been applied, with benefit, to the study of the ecology and pathogenesis of many more serious human arboviral illnesses. The evolutionary potential of viruses, with particular reference to the development of more virulent strains, has been studied more closely in the California serogroup viruses than in almost any other agent of human disease.

California-La Crosse encephalitis.

La Crosse encephalitis, a mosquito-borne viral disease that can be mistaken for herpes simplex encephalitis, is under-recognized in the United States, despite case reports from 28 states and an incidence in endemic areas (20-30/100,000) exceeding that of bacterial meningitis. The disease recurs every summer in endemic foci in the midwestern and mid-Atlantic United States in areas forested with hardwood trees, which provide breeding sites for the treehole-dwelling mosquito vector, Aedes triseriatus. La Crosse encephalitis should be considered in the child presenting with meningoencephalitis in summer and early fall, particularly for children living in (or recent travel to) endemic areas in mid-Atlantic and midwestern states.

Mechanism of La Crosse virus inhibition by ribavirin.

The effect of ribavirin on the growth and replication of La Crosse virus was examined. The data suggest that low concentrations of ribavirin have a marked effect on the initial steps of La Crosse virus transcription. The therapeutic potential of ribavirin in the treatment of human California encephalitis serotype infections is discussed in light of these findings.

[Antiviral activity of ribamidyl in experimental infections with California encephalitis group viruses]. / Protivovirusnaia aktivnost' ribamidila pri éksperimental'nykh infektsiiakh virusami kompleksa kaliforniiskogo éntsefalita.

A preparation of ribamydil, an analogue of natural nucleosides, synthesized at the Latvian SSR Institute of Organic Chemistry showed a sufficiently high activity against bunyaviruses of California encephalitis complex both in vitro and in vivo. Various modifications of the enzyme immunoassay may be used for control of the effectiveness of treatment with this drug. Some advantages of the subcutaneous route over the intramuscular one were found. Ribamydil may be useful for treatment of infections of California encephalitis complex.