Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3^−/− and TLR4^−/− mice, i.e. TLR3^−/− mice were highly susceptible to JE, whereas TLR4^−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b⁺Ly-6C^high monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4^−/− mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3^−/− myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4⁺ and CD8⁺ T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b⁺Ly-6C^high monocytes) and CD4⁺Foxp3⁺ Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors. Japanese encephalitis (JE) is major emerging encephalitis, and more than 60% of global population inhabits JE endemic areas. The etiological virus is currently spreading to previously unaffected regions due to rapid changes in climate and demography. However, the impact of TLR molecules on JE progression has not been addressed to date. We found that the distinct outcomes of JE progression occurred in TLR3 and TLR4-dependent manner, i.e. TLR3^−/− mice were highly susceptible, whereas TLR4^−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation manifested by early CD11b⁺Ly-6C^high monocyte infiltration, high expression of proinflammatory cytokines, as well as increased BBB permeability. In contrast, TLR4 ablation provided potent type I IFN innate response in infected mice, as well as in myeloid-derived cells closely associated with strong induction of antiviral ISG genes, and also resulted in enhanced humoral, CD4+, and CD8+ T cell responses along with altered plasmacytoid DC and CD4⁺Foxp3⁺ Treg number. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were coupled with reduced JE lethality. Our studies provide an insight into the role of each TLR molecule on the modulation of JE, as well as its mechanism of neuroinflammation control during JE progression.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic disease, which causes high fever, thrombocytopenia, and death in humans and animals in East Asian countries. The pathogenicity of SFTS virus (SFTSV) remains unclear. We intraperitoneally infected three groups of mice: wild-type (WT), mice treated with blocking anti-type I interferon (IFN)-α receptor antibody (IFNAR Ab), and IFNAR knockout (IFNAR^−/−) mice, with four doses of SFTSV (KH1, 5 × 10⁵ to 5 × 10² FAID₅₀). The WT mice survived all SFTSV infective doses. The IFNAR Ab mice died within 7 days post-infection (dpi) with all doses of SFTSV except that the mice were infected with 5 × 10² FAID₅₀ SFTSV. The IFNAR^−/− mice died after infection with all doses of SFTSV within four dpi. No SFTSV infection caused hyperthermia in any mice, whereas all the dead mice showed hypothermia and weight loss. In the WT mice, SFTSV RNA was detected in the eyes, oral swabs, urine, and feces at 5 dpi. Similar patterns were observed in the IFNAR Ab and IFNAR^−/− mice after 3 dpi, but not in feces. The IFNAR Ab mice showed viral shedding until 7 dpi. The SFTSV RNA loads were higher in organs of the IFNAR^−/− mice compared to the other groups. Histopathologically, coagulation necrosis and mononuclear inflammatory cell infiltration in the liver and white pulp atrophy in the spleen were seen as the main lesions in the IFN signaling lacking mice. Immunohistochemically, SFTSV antigens were mainly detected in the marginal zone of the white pulp of the spleen in all groups of mice, but more viral antigens were observed in the spleen of the IFNAR^−/− mice. Collectively, the IFN signaling-deficient mice were highly susceptible to SFTSV and more viral burden could be demonstrated in various excreta and organs of the mice when IFN signaling was inhibited.

CCR5 is a CC chemokine receptor involved in the migration of effector leukocytes including macrophages, NK, and T cells into inflamed tissues. Also, the role of CCR5 in CD4+Foxp3+ regulatory T cell (Treg) homing has recently begun to grab attention. Japanese encephalitis (JE) is defined as severe neuroinflammation of the central nervous system (CNS) following infection with mosquito-borne flavivirus JE virus. However, the potential contribution of CCR5 to JE progression via mediating CD4+Foxp3+ Treg homing has not been investigated. Infected wild-type (Ccr5+/+) and CCR5-deficient (Ccr5−/−) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, NK- and JEV-specific T cell responses were analyzed. Adoptive transfer of CCR5+CD4+Foxp3+ Tregs was used to evaluate the role of Tregs in JE progression. CCR5 ablation exacerbated JE without altering viral burden in the extraneural and CNS tissues, as manifested by increased CNS infiltration of Ly-6Chi monocytes and Ly-6Ghi granulocytes. Compared to Ccr5+/+ mice, Ccr5−/− mice unexpectedly showed increased responses of IFN-γ+NK and CD8+ T cells in the spleen, but not CD4+ T cells. More interestingly, CCR5-ablation resulted in a skewed response to IL-17+CD4+ Th17 cells and correspondingly reduced CD4+Foxp3+ Tregs in the spleen and brain, which was closely associated with exacerbated JE. Our results also revealed that adoptive transfer of sorted CCR5+CD4+Foxp3+ Tregs into Ccr5−/− mice could ameliorate JE progression without apparently altering the viral burden and CNS infiltration of IL-17+CD4+ Th17 cells, myeloid-derived Ly-6Chi monocytes and Ly-6Ghi granulocytes. Instead, adoptive transfer of CCR5+CD4+Foxp3+ Tregs into Ccr5−/− mice resulted in increased expression of anti-inflammatory cytokines (IL-10 and TGF-β) in the spleen and brain, and transferred CCR5+ Tregs were found to produce IL-10. CCR5 regulates JE progression via governing timely and appropriate CNS infiltration of CD4+Foxp3+ Tregs, thereby facilitating host survival. Therefore, this critical and extended role of CCR5 in JE raises possible safety concerns regarding the use of CCR5 antagonists in human immunodeficiency virus (HIV)-infected individuals who inhabit regions in which both HIV and flaviviruses, such as JEV and West Nile virus, are endemic.

Granulocyte/macrophage colony‐stimulatory factor (GM‐CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen‐presenting cells (APCs) to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells (DCs). This study evaluated the utility of GM‐CSF cDNA as a DNA vaccine adjuvant for glycoprotein B (gB) of pseudorabies virus (PrV) in a murine model. The co‐injection of GM‐CSF DNA enhanced the levels of serum PrV‐specific IgG with a 1.5‐ to 2‐fold increase. Moreover, GM‐CSF co‐injection inhibited the production of IgG2a isotype. However, it enhanced production of an IgG1 isotype resulting in humoral responses biased to the Th2‐type against PrV antigen. In contrast, the co‐administration of GM‐CSF DNA enhanced the T cell‐mediated immunity biased to the Th1‐type, as judged by the significantly higher level of cytokine IL‐2 and IFN‐γ production but not IL‐4. When challenged with a lethal dose of PrV, the GM‐CSF co‐injection enhanced the resistance against a PrV infection. This suggests that co‐inoculation with a vector expressing GM‐CSF enhanced the protective immunity against a PrV infection. This immunity was caused by the induction of increased humoral and cellular immunity in response to PrV antigen.

Cross‐presentation is the pathway by which exogenous antigens are routed for presentation by MHC class I molecules leading to activation of antiviral CD8⁺ T‐cell responses. However, there is little information describing the modulation of cross‐presentation and the impact of pathogen‐derived signals associated with Japanese encephalitis virus (JEV), which is one of the most common causes of encephalitis in humans. In this study, we demonstrate that JEV infection could suppress in vivo cross‐presentation of soluble and cell‐associated antigens, thereby generating weak CD8⁺ T‐cell responses to exogenous antigens, as evaluated by CFSE dilution of adoptively transferred CD8⁺ T cells and in vivo CTL killing activity. Furthermore, CD8α⁺CD11c⁺ dendritic cells (DCs), which are known to be far more efficient at cross‐presenting soluble antigens, played a specific role in contributing to JEV‐mediated inhibition of the cross‐presentation of exogenous antigens through interference with effective antigen uptake. Finally, this study provides evidence that TLR2‐MyD88 and p38 MAPK signal pathway might be involved in JEV‐mediated inhibition of cross‐presentation of soluble and cell‐associated antigens. These observations suggest that the modulation of cross‐presentation of exogenous antigens through TLR signaling has important implications for antiviral immune responses against JEV infection and the development of effective vaccination strategies.