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

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

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

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

Drug-Screening Strategies for Inhibition of Virus-Induced Neuronal Cell Death.

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.

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.

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.

Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition.

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.

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.

Cutting Edge: CCR2 Is Not Required for Ly6Chi Monocyte Egress from the Bone Marrow but Is Necessary for Migration within the Brain in La Crosse Virus Encephalitis.

Inflammatory monocyte (iMO) recruitment to the brain is a hallmark of many neurologic diseases. Prior to entering the brain, iMOs must egress into the blood from the bone marrow through a mechanism, which for known encephalitic viruses, is CCR2 dependent. In this article, we show that during La Crosse Virus-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.

Sexual and Vertical Transmission of Zika Virus in anti-interferon receptor-treated Rag1-deficient mice.

Although Zika virus (ZIKV) is primarily transmitted to humans by the Aedes aegypti mosquito, human-to-human transmission has also been observed from males-to-females as well as mother-to-offspring. In the current study, we studied both sexual transmission (STx) and vertical transmission (VTx) of ZIKV using anti-IFNAR1-treatment of Rag1 -/- (AIR) mice. These mice have suppressed type I IFN responses and lack adaptive immune responses, leading to a prolonged infection prior to clinical disease. STx of ZIKV from infected AIR males to naive Ifnar1 -/- females was observed with greater than 50% incidence, with infection observed in the vaginal tract at early time points. In the case of a resulting pregnancy, virus was also found in the uterus and placental tissue. In additional studies, VTx of virus was observed in AIR female mice. Specifically, peripheral ZIKV infection of pregnant AIR females resulted in detectable virus in brain and/or lymph nodes of fetuses and/or pups. VTx of ZIKV was stochastic, in that not all fetuses/pups within the same dam had detectable virus and infection was not associated with breakdown of maternal/fetal placental barrier. This provides a new model to study the barriers to STx and VTx of ZIKV and the immune responses essential to preventing transmission.

Adaptive Immune Responses to Zika Virus Are Important for Controlling Virus Infection and Preventing Infection in Brain and Testes.

The recent association between Zika virus (ZIKV) and neurologic complications, including Guillain-Barré syndrome in adults and CNS abnormalities in fetuses, highlights the importance in understanding the immunological mechanisms controlling this emerging infection. Studies have indicated that ZIKV evades the human type I IFN response, suggesting a role for the adaptive immune response in resolving infection. However, the inability of ZIKV to antagonize the mouse IFN response renders the virus highly susceptible to circulating IFN in murine models. Thus, as we show in this article, although wild-type C57BL/6 mice mount cell-mediated and humoral adaptive immune responses to ZIKV, these responses were not required to prevent disease. However, when the type I IFN response of mice was suppressed, then the adaptive immune responses became critical. For example, when type I IFN signaling was blocked by Abs in Rag1-/- mice, the mice showed dramatic weight loss and ZIKV infection in the brain and testes. This phenotype was not observed in Ig-treated Rag1-/- mice or wild-type mice treated with anti-type I IFNR alone. Furthermore, we found that the CD8+ T cell responses of pregnant mice to ZIKV infection were diminished compared with nonpregnant mice. It is possible that diminished cell-mediated immunity during pregnancy could increase virus spread to the fetus. These results demonstrate an important role for the adaptive immune response in the control of ZIKV infection and imply that vaccination may prevent ZIKV-related disease, particularly when the type I IFN response is suppressed as it is in humans.

Lymphocytes have a role in protection, but not in pathogenesis, during La Crosse Virus infection in mice.

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.

SARM1, Not MyD88, Mediates TLR7/TLR9-Induced Apoptosis in Neurons.

Neuronal apoptosis is a key aspect of many different neurologic diseases, but the mechanisms remain unresolved. Recent studies have suggested a mechanism of innate immune-induced neuronal apoptosis through the stimulation of endosomal TLRs in neurons. TLRs are stimulated both by pathogen-associated molecular patterns as well as by damage-associated molecular patterns, including microRNAs released by damaged neurons. In the present study, we identified the mechanism responsible for TLR7/TLR9-mediated neuronal apoptosis. TLR-induced apoptosis required endosomal localization of TLRs but was independent of MyD88 signaling. Instead, apoptosis required the TLR adaptor molecule SARM1, which localized to the mitochondria following TLR activation and was associated with mitochondrial accumulation in neurites. Deficiency in SARM1 inhibited both mitochondrial accumulation in neurites and TLR-induced apoptosis. These studies identify a non-MyD88 pathway of TLR7/ TLR9 signaling in neurons and provide a mechanism for how innate immune responses in the CNS directly induce neuronal damage.

Identification of Glial Activation Markers by Comparison of Transcriptome Changes between Astrocytes and Microglia following Innate Immune Stimulation.

The activation of astrocytes and microglia is often associated with diseases of the central nervous system (CNS). Understanding how activation alters the transcriptome of these cells may offer valuable insight regarding how activation of these cells mediate neurological damage. Furthermore, identifying common and unique pathways of gene expression during activation may provide new insight into the distinct roles these cells have in the CNS during infection and inflammation. Since recent studies indicate that TLR7 recognizes not only viral RNA but also microRNAs that are released by damaged neurons and elevated during neurological diseases, we first examined the response of glial cells to TLR7 stimulation using microarray analysis. Microglia were found to generate a much stronger response to TLR7 activation than astrocytes, both in the number of genes induced as well as fold induction. Although the primary pathways induced by both cell types were directly linked to immune responses, microglia also induced pathways associated with cellular proliferation, while astrocytes did not. Targeted analysis of a subset of the upregulated genes identified unique mRNA, including Ifi202b which was only upregulated by microglia and was found to be induced during both retroviral and bunyavirus infections in the CNS. In addition, other genes including Birc3 and Gpr84 as well as two expressed sequences AW112010 and BC023105 were found to be induced in both microglia and astrocytes and were upregulated in the CNS following virus infection. Thus, expression of these genes may a useful measurement of glial activation during insult or injury to the CNS.

Neuropeptide Y Negatively Influences Monocyte Recruitment to the Central Nervous System during Retrovirus Infection.

UNLABELLED: Monocyte infiltration into the CNS is a hallmark of several viral infections of the central nervous system (CNS), including retrovirus infection. Understanding the factors that mediate monocyte migration in the CNS is essential for the development of therapeutics that can alter the disease process. In the current study, we found that neuropeptide Y (NPY) suppressed monocyte recruitment to the CNS in a mouse model of polytropic retrovirus infection. NPY(-/-) mice had increased incidence and kinetics of retrovirus-induced neurological disease, which correlated with a significant increase in monocytes in the CNS compared to wild-type mice. Both Ly6C(hi) inflammatory and Ly6C(lo) alternatively activated monocytes were increased in the CNS of NPY(-/-) mice following virus infection, suggesting that NPY suppresses the infiltration of both cell types. Ex vivo analysis of myeloid cells from brain tissue demonstrated that infiltrating monocytes expressed high levels of the NPY receptor Y2R. Correlating with the expression of Y2R on monocytes, treatment of NPY(-/-) mice with a truncated, Y2R-specific NPY peptide suppressed the incidence of retrovirus-induced neurological disease. These data demonstrate a clear role for NPY as a negative regulator of monocyte recruitment into the CNS and provide a new mechanism for suppression of retrovirus-induced neurological disease. IMPORTANCE: Monocyte recruitment to the brain is associated with multiple neurological diseases. However, the factors that influence the recruitment of these cells to the brain are still not well understood. In the current study, we found that neuropeptide Y, a protein produced by neurons, affected monocyte recruitment to the brain during retrovirus infection. We show that mice deficient in NPY have increased influx of monocytes into the brain and that this increase in monocytes correlates with neurological-disease development. These studies provide a mechanism by which the nervous system, through the production of NPY, can suppress monocyte trafficking to the brain and reduce retrovirus-induced neurological disease.

Age-dependent myeloid dendritic cell responses mediate resistance to la crosse virus-induced neurological disease.

UNLABELLED: La Crosse virus (LACV) is the major cause of pediatric viral encephalitis in the United States; however, the mechanisms responsible for age-related susceptibility in the pediatric population are not well understood. Our current studies in a mouse model of LACV infection indicated that differences in myeloid dendritic cell (mDC) responses between weanling and adult mice accounted for susceptibility to LACV-induced neurological disease. We found that type I interferon (IFN) responses were significantly stronger in adult than in weanling mice. Production of these IFNs required both endosomal Toll-like receptors (TLRs) and cytoplasmic RIG-I-like receptors (RLRs). Surprisingly, IFN expression was not dependent on plasmacytoid DCs (pDCs) but rather was dependent on mDCs, which were found in greater number and induced stronger IFN responses in adults than in weanlings. Inhibition of these IFN responses in adults resulted in susceptibility to LACV-induced neurological disease, whereas postinfection treatment with type I IFN provided protection in young mice. These studies provide a definitive mechanism for age-related susceptibility to LACV encephalitis, where mDCs in young mice are insufficiently activated to control peripheral virus replication, thereby allowing virus to persist and eventually cause central nervous system (CNS) disease. IMPORTANCE: La Crosse virus (LACV) is the primary cause of pediatric viral encephalitis in the United States. Although the virus infects both adults and children, over 80% of the reported neurological disease cases are in children. To understand why LACV causes neurological disease primarily in young animals, we used a mouse model where weanling mice, but not adult mice, develop neurological disease following virus infection. We found that an early immune response cell type, myeloid dendritic cells, was critical for protection in adult animals and that these cells were reduced in young animals. Activation of these cells during virus infection or after treatment with type I interferon in young animals provided protection from LACV. Thus, this study demonstrates a reason for susceptibility to LACV infection in young animals and shows that early therapeutic treatment in young animals can prevent neurological disease.

Age-related differences in neuroinflammatory responses associated with a distinct profile of regulatory markers on neonatal microglia.

BACKGROUND: The perinatal period is one in which the mammalian brain is particularly vulnerable to immune-mediated damage. Early inflammation in the central nervous system (CNS) is linked with long-term impairment in learning and behavior, necessitating a better understanding of mediators of neuroinflammation. We therefore directly examined how age affected neuroinflammatory responses to pathogenic stimuli. METHODS: In mice, susceptibility to neurological damage changes dramatically during the first few weeks of life. Accordingly, we compared neuroinflammatory responses to pathogen associated molecular patterns (PAMPs) of neonatal (two day-old) and weanling (21 day-old) mice. Mice were inoculated intracerebrally with PAMPs and the cellular and molecular changes in the neuroinflammatory response were examined. RESULTS: Of the 12 cytokines detected in the CNS following toll-like receptor 4 (TLR4) stimulation, ten were significantly higher in neonates compared with weanling mice. A similar pattern of increased cytokines in neonates was also observed with TLR9 stimulation. Analysis of cellular responses indicated a difference in microglial activation markers in the CNS of neonatal mice and increased expression of proteins known to modulate cellular activation including CD11a, F4/80 and CD172a. We also identified a new marker on microglia, SLAMF7, which was expressed at higher levels in neonates compared with weanlings. CONCLUSIONS: A unique neuroinflammatory profile, including higher expression of several proinflammatory cytokines and differential expression of microglial markers, was observed in brain tissue from neonates following TLR stimulation. This increased neuroinflammatory response to PAMPs may explain why the developing brain is particularly sensitive to infection and why infection or stress during this time can lead to long-term damage in the CNS.

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.

Toll-like receptor 7 suppresses virus replication in neurons but does not affect viral pathogenesis in a mouse model of Langat virus infection.

Toll-like receptor 7 (TLR7) recognizes guanidine-rich viral ssRNA and is an important mediator of peripheral immune responses to several ssRNA viruses. However, the role that TLR7 plays in regulating the innate immune response to ssRNA virus infections in specific organs such as the central nervous system (CNS) is not as clear. This study examined the influence of TLR7 on the neurovirulence of Langat virus (LGTV), a ssRNA tick-borne flavivirus. TLR7 deficiency did not substantially alter the onset or incidence of LGTV-induced clinical disease; however, it did significantly affect virus levels in the CNS with a log(10) increase in virus titres in brain tissue from TLR7-deficient mice. This difference in virus load was also observed following intracranial inoculation, indicating a direct effect of TLR7 deficiency on regulating virus replication in the brain. LGTV-induced type I interferon responses in the CNS were not dependent on TLR7, being higher in TLR7-deficient mice compared with wild-type controls. In contrast, induction of pro-inflammatory cytokines including tumour necrosis factor, CCL3, CCL4 and CXCL13 were dependent on TLR7. Thus, although TLR7 is not essential in controlling LGTV pathogenesis, it is important in controlling virus infection in neurons in the CNS, possibly by regulating neuroinflammatory responses.