Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity

Autosomal dominant TLR3 deficiency has been identified as a genetic etiology of childhood herpes simplex virus 1 (HSV-1) encephalitis (HSE). This defect is partial, as it results in impaired, but not abolished induction of IFN-β and -λ in fibroblasts in response to TLR3 stimulation. The apparently normal resistance of these patients to other infections, viral illnesses in particular, may thus result from residual TLR3 responses. We report here an autosomal recessive form of complete TLR3 deficiency in a young man who developed HSE in childhood but remained normally resistant to other infections. This patient is compound heterozygous for two loss-of-function TLR3 alleles, resulting in an absence of response to TLR3 activation by polyinosinic-polycytidylic acid (poly(I:C)) and related agonists in his fibroblasts. Moreover, upon infection of the patient's fibroblasts with HSV-1, the impairment of IFN-β and -λ production resulted in high levels of viral replication and cell death. In contrast, the patient's peripheral blood mononuclear cells responded normally to poly(I:C) and to all viruses tested, including HSV-1. Consistently, various TLR3-deficient leukocytes from the patient, including CD14(+) and/or CD16(+) monocytes, plasmacytoid dendritic cells, and in vitro derived monocyte-derived macrophages, responded normally to both poly(I:C) and HSV-1, with the induction of antiviral IFN production. These findings identify a new genetic etiology for childhood HSE, indicating that TLR3-mediated immunity is essential for protective immunity to HSV-1 in the central nervous system (CNS) during primary infection in childhood, in at least some patients. They also indicate that human TLR3 is largely redundant for responses to double-stranded RNA and HSV-1 in various leukocytes, probably accounting for the redundancy of TLR3 for host defense against viruses, including HSV-1, outside the CNS.     

Heterozygous TBK1 mutations impair TLR3 immunity and underlie herpes simplex encephalitis of childhood

Childhood herpes simplex virus-1 (HSV-1) encephalitis (HSE) may result from single-gene inborn errors of TLR3 immunity. TLR3-dependent induction of IFN-α/β or IFN-λ is crucial for protective immunity against primary HSV-1 infection in the central nervous system (CNS). We describe here two unrelated children with HSE carrying different heterozygous mutations (D50A and G159A) in TBK1, the gene encoding TANK-binding kinase 1, a kinase at the crossroads of multiple IFN-inducing signaling pathways. Both mutant TBK1 alleles are loss-of-function but through different mechanisms: protein instability (D50A) or a loss of kinase activity (G159A). Both are also associated with an autosomal-dominant (AD) trait but by different mechanisms: haplotype insufficiency (D50A) or negative dominance (G159A). A defect in polyinosinic-polycytidylic acid-induced TLR3 responses can be detected in fibroblasts heterozygous for G159A but not for D50A TBK1. Nevertheless, viral replication and cell death rates caused by two TLR3-dependent viruses (HSV-1 and vesicular stomatitis virus) were high in fibroblasts from both patients, and particularly so in G159A TBK1 fibroblasts. These phenotypes were rescued equally well by IFN-α2b. Moreover, the IFN responses to the TLR3-independent agonists and viruses tested were maintained in both patients' peripheral blood mononuclear cells and fibroblasts. The narrow, partial cellular phenotype thus accounts for the clinical phenotype of these patients being limited to HSE. These data identify AD partial TBK1 deficiency as a new genetic etiology of childhood HSE, indicating that TBK1 is essential for the TLR3- and IFN-dependent control of HSV-1 in the CNS.     

Host innate recognition of an intestinal bacterial pathogen induces TRIF-dependent protective immunity

Toll-like receptor 4 (TLR4), which signals through the adapter molecules myeloid differentiation factor 88 (MyD88) and toll/interleukin 1 receptor domain-containing adapter inducing IFN-β (TRIF), is required for protection against Gram-negative bacteria. TRIF is known to be important in TLR3-mediated antiviral signaling, but the role of TRIF signaling against Gram-negative enteropathogens is currently unknown. We show that TRIF signaling is indispensable for establishing innate protective immunity against Gram-negative Yersinia enterocolitica. Infection of wild-type mice rapidly induced both IFN-β and IFN-γ in the mesenteric lymph nodes. In contrast, TRIF-deficient mice were defective in these IFN responses and showed impaired phagocytosis in regional macrophages, resulting in greater bacterial dissemination and mortality. TRIF signaling may be universally important for protection against Gram-negative pathogens, as TRIF-deficient macrophages were also impaired in killing both Salmonella and Escherichia coli in vitro. The mechanism of TRIF-mediated protective immunity appears to be orchestrated by macrophage-induced IFN-β and NK cell production of IFN-γ. Sequential induction of IFN-β and IFN-γ leads to amplification of macrophage bactericidal activity sufficient to eliminate the invading pathogens at the intestinal interface. Our results demonstrate a previously unknown role of TRIF in host resistance to Gram-negative enteropathogens, which may lead to effective strategies for combating enteric infections.     

Selective modulation of TLR4-activated inflammatory responses by altered iron homeostasis in mice

Mice deficient in the hemochromatosis gene, Hfe, have attenuated inflammatory responses to Salmonella infection associated with decreased macrophage TNF-α and IL-6 biosynthesis after exposure to LPS. In this study, we show that the abnormal cytokine production is related to impaired TLR4 signaling. Despite their abnormal response to LPS, Hfe KO macrophages produced amounts of TNF-α similar to those in WT cells after TLR2 stimulation. Consistent with this finding, LPS-induced activation of Mal/MyD88-dependent events was normal in the mutant macrophages. However, LPS-induced IFN-β expression, a TRAM/TRIF-dependent response activated by TLR4, was reduced by Hfe deficiency. This reduction could be replicated in WT macrophages with the use of iron chelators. In contrast, TLR3-activated expression of IFN-β, a TRIF-dependent response, was normal in Hfe KO macrophages and was unaffected by iron chelation. Our data suggest that low intracellular iron selectively impairs signaling via the TLR4/TRAM/TRIF pathway proximal to TRIF and results in reduced LPS-induced cytokine expression. Furthermore, by mimicking the altered iron metabolism associated with Hfe deficiency, we found that 3 different inhibitors of hepcidin attenuated Salmonella-induced and noninfectious enterocolitis. Thus, manipulation of iron homeostasis could represent a new therapeutic approach to controlling inflammation.     

Genetic susceptibility to virus associated encephalitis or encephalopathy

There has been rapid progress in the understanding of the protective mechanism against infection during the past decade. We reviewed the genetic susceptibility in familial or recurrent encephalitis/encephalopathy and subacute sclerosing panencephalitis (SSPE). Recessive mutation of UNC-93B1 and dominant mutation of TLR3 were associated with herpes simplex encephalitis (HSE). Those mutations impair the dsRNA-induced IFN-alpha/beta and IFN-lambda production and predispose to HSE. Dominant mutation of RANBP2 (encoding a nuclear pore protein) was detected in familial or recurrent patients with acute necrotizing encephalopathy. Polymorphisms of MxA, IL-4, IRF1, TLR3, PD1 and TLR3 are probably associated with the development of SSPE. Identification of causing or predisposing genes would enable the early diagnosis and the establishment of effective protection or treatment.     

TLR3 controls constitutive IFN-β antiviral immunity in human fibroblasts and cortical neurons

Human herpes simplex virus 1 (HSV-1) encephalitis can be caused by inborn errors of the TLR3 pathway, resulting in impairment of CNS cell-intrinsic antiviral immunity. Deficiencies of the TLR3 pathway impair cell-intrinsic immunity to vesicular stomatitis virus (VSV) and HSV-1 in fibroblasts, and to HSV-1 in cortical but not trigeminal neurons. The underlying molecular mechanism is thought to involve impaired IFN-α/β induction by the TLR3 recognition of dsRNA viral intermediates or by-products. However, we show here that human TLR3 controls constitutive levels of IFNB mRNA and secreted bioactive IFN-β protein, and thereby also controls constitutive mRNA levels for IFN-stimulated genes (ISGs) in fibroblasts. Tlr3^–/– mouse embryonic fibroblasts also have lower basal ISG levels. Moreover, human TLR3 controls basal levels of IFN-β secretion and ISG mRNA in induced pluripotent stem cell–derived cortical neurons. Consistently, TLR3-deficient human fibroblasts and cortical neurons are vulnerable not only to both VSV and HSV-1, but also to several other families of viruses. The mechanism by which TLR3 restricts viral growth in human fibroblasts and cortical neurons in vitro and, by inference, by which the human CNS prevents infection by HSV-1 in vivo, is therefore based on the control of early viral infection by basal IFN-β immunity.     

LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF

Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-beta (TRIF)-related adaptor molecule (TRAM) is the fourth TIR domain-containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-kappaB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-alpha/beta, regulated on activation, normal T cell expressed and secreted (RANTES), and gamma interferon-inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor-like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.     

Herpes simplex encephalitis in a patient with a distinctive form of inherited IFNAR1 deficiency

Inborn errors of TLR3-dependent IFN-α/β– and IFN-λ–mediated immunity in the CNS can underlie herpes simplex virus 1 (HSV-1) encephalitis (HSE). The respective contributions of IFN-α/β and IFN-λ are unknown. We report a child homozygous for a genomic deletion of the entire coding sequence and part of the 3′-UTR of the last exon of IFNAR1, who died of HSE at the age of 2 years. An older cousin died following vaccination against measles, mumps, and rubella at 12 months of age, and another 17-year-old cousin homozygous for the same variant has had other, less severe, viral illnesses. The encoded IFNAR1 protein is expressed on the cell surface but is truncated and cannot interact with the tyrosine kinase TYK2. The patient’s fibroblasts and EBV-B cells did not respond to IFN-α2b or IFN-β, in terms of STAT1, STAT2, and STAT3 phosphorylation or the genome-wide induction of IFN-stimulated genes. The patient’s fibroblasts were susceptible to viruses, including HSV-1, even in the presence of exogenous IFN-α2b or IFN-β. HSE is therefore a consequence of inherited complete IFNAR1 deficiency. This viral disease occurred in natural conditions, unlike those previously reported in other patients with IFNAR1 or IFNAR2 deficiency. This experiment of nature indicates that IFN-α/β are essential for anti–HSV-1 immunity in the CNS.     

Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence

Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like-interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain-containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain-containing adaptor inducing IFN-beta (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor kappaB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-beta by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence.     

TIR domain--containing adaptors regulate TLR-mediated signaling pathways

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses via signaling pathways mediated by several Toll/IL-1R (TIR) domain-containing adaptors such as MyD88, TIRAP, and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3 and TLR4 that is responsible for type I interferon production in response to double-stranded RNA and LPS, respectively. TIRAP specifically participates in the MyD88-dependent pathways shared by TLR2 and TLR4, and TRAM is essential for the TLR4-mediated MyD88-independent pathway. Thus, TIR domain-containing adaptors play an important role in the TLR mediated signaling pathways.     

Brain immune cells undergo cGAS/STING-dependent apoptosis during herpes simplex virus type 1 infection to limit type I IFN production

Protection of the brain from viral infections involves the type I IFN (IFN-I) system, defects in which render humans susceptible to herpes simplex encephalitis (HSE). However, excessive cerebral IFN-I levels lead to pathologies, suggesting the need for tight regulation of responses. Based on data from mouse models, human HSE cases, and primary cell culture systems, we showed that microglia and other immune cells undergo apoptosis in the HSV-1–infected brain through a mechanism dependent on the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, but independent of IFN-I. HSV-1 infection of microglia induced cGAS-dependent apoptosis at high viral doses, whereas lower viral doses led to IFN-I responses. Importantly, inhibition of caspase activity prevented microglial cell death and augmented IFN-I responses. Accordingly, HSV-1–infected organotypic brain slices or mice treated with a caspase inhibitor exhibited lower viral load and an improved infection outcome. Collectively, we identify an activation-induced apoptosis program in brain immune cells that downmodulates local immune responses.     

HSV-1感染后小胶质细胞炎性因子分泌特点研究

目的：比较单纯疱疹病毒I型（HSV-1）感染小鼠和BV2细胞后,小胶质细胞分泌炎性因子的特征,探讨BV2细胞可否作为小鼠HSV-1性脑炎模型的小胶质细胞功能的模拟研究对象。方法：24只Ba1b／c小鼠分为正常组、HSV-1组、黄芪组及地塞米松组（激素组）各6只;BV2细胞分为同样4组。HSV-1组,黄芪组及激素组小鼠颅内注射HSV-1制造小鼠HSV-1性脑炎（HSE）模型,BV2细胞接种HSV-1制造细胞模型。造模成功后,黄芪组及激素组分别用黄芪及激素作用前后,RT-PCR法检查炎性因子（IL-2、IL-4、IL-10、TNF-α及iNOS）mRNA浓度及细胞的变化。结果：BV2细胞与模型小鼠在病毒感染及相关药物作用前后,炎性因子具有一致的变化趋势。结论：BV2细胞和Balb／c小鼠脑组织具有相似的免疫反应特性,可作为研究单纯疱疹病毒性脑炎小鼠模型中小胶质细胞功能的模拟对象。     

MyD88 but not TRIF is essential for osteoclastogenesis induced by lipopolysaccharide, diacyl lipopeptide, and IL-1alpha

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll-IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88-/-) mice and TRIF-deficient (TRIF-/-) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1alpha stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF-/- mice, but not MyD88-/- mice. These factors stimulated receptor activator of nuclear factor-kappaB ligand mRNA expression in TRIF-/- osteoblasts, but not MyD88-/- osteoblasts. LPS stimulated IL-6 production in TRIF-/- osteoblasts, but not TRIF-/- macrophages. LPS and IL-1alpha enhanced the survival of TRIF-/- osteoclasts, but not MyD88-/- osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88-/- mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover.     

MyD88 inhibition amplifies dendritic cell capacity to promote pancreatic carcinogenesis via Th2 cells

The transition of chronic pancreatic fibroinflammatory disease to neoplasia is a primary example of the paradigm linking inflammation to carcinogenesis. However, the cellular and molecular mediators bridging these entities are not well understood. Because TLR4 ligation can exacerbate pancreatic inflammation, we postulated that TLR4 activation drives pancreatic carcinogenesis. In this study, we show that lipopolysaccharide accelerates pancreatic tumorigenesis, whereas TLR4 inhibition is protective. Furthermore, blockade of the MyD88-independent TRIF pathway is protective against pancreatic cancer, whereas blockade of the MyD88-dependent pathway surprisingly exacerbates pancreatic inflammation and malignant progression. The protumorigenic and fibroinflammatory effects of MyD88 inhibition are mediated by dendritic cells (DCs), which induce pancreatic antigen-restricted Th2-deviated CD4(+) T cells and promote the transition from pancreatitis to carcinoma. Our data implicate a primary role for DCs in pancreatic carcinogenesis and illustrate divergent pathways in which blockade of TLR4 signaling via TRIF is protective against pancreatic cancer and, conversely, MyD88 inhibition exacerbates pancreatic inflammation and neoplastic transformation by augmenting the DC-Th2 axis.     

单纯疱疹病毒性脑炎8例病例分析

目的探讨单纯疱疹病毒性脑炎(HSE)的临床表现、辅助检查、诊断及治疗,提高HSE的早期诊断和治愈率。方法回顾8例HSE患者的临床表现,脑电图、影像学、脑脊液检查结果及治疗,并结合相关文献进行分析。结果 HSE患者均急性或亚急性起病,主要临床表现为发热、头痛、意识及精神障碍。头颅MRI、脑电图检查有利于早期诊断。脑脊液HSV-1 IgM抗体阳性及PCR DNA检测发现该病毒DNA可确诊。结论通过HSE的临床特点,结合脑电图、脑脊液及影像学检查,对HSE作出早期诊断,尽早使用抗病毒治疗是改善预后的关键。     

2011 ESCI Award for Excellence in Basic / Translational Research: innate regulation of adaptive immunity by dendritic cells

Dendritic cells (DC) play a key role in the initiation of adaptive immunity, and the manipulation and/or targeting of DC has great potential for immune intervention. However, clinical applications are hampered by the fact that we still know relatively little about how DC become 'activated' to stimulate and direct T-cell responses. Over the last decade, much emphasis has been placed on dissecting innate signalling pathways that can trigger DC activation and promote T-cell priming. Here, we review work from our laboratory aimed at helping define 'pattern-recognition pathways' involved in DC activation by potential pathogens. One pathway for sensing infection by RNA viruses involves recognition of viral genomes or virally infected cells in endosomal compartments and utilises members of the toll-like receptor (TLRs) family, including TLR9, 7, or 3. RNA virus genomes can additionally be recognised in the cytosol by DExD/H-box helicases such as MDA5 or RIG-I, the latter of which is activated by RNAs bearing 5' tri-phosphates. Finally, a distinct pathway involves cell surface and phagosomal recognition of fungi by C-type lectins, which signal via Syk kinase. Notably, some of these pathways are involved not only in direct sensing of pathogens but also in the recognition of self-alterations that might accompany infection, such as induction of cell death. These studies help build a global picture of the receptors and signalling pathways that regulate DC activation and have applications in immunotherapy of cancer and infectious diseases.     

阿托伐他汀对脂多糖诱导人脐静脉内皮细胞Toll样受体4及其下游信号转导通路的影响

背景：研究表明Toll样受体4参与了动脉粥样硬化的发生和发展,目前Toll样受体4与MyD88依赖性或MyD88非依赖性信号转导通路在动脉粥样硬化发生和发展中的机制尚不明确。目的：观察阿托伐他汀对脂多糖诱导的人脐静脉内皮细胞Toll样受体4及其下游信号转导通路主要元件MyD88、TRAF-6、TRAM及TRIF表达的影响,分析阿托伐他汀防治动脉粥样硬化的机制。方法：体外培养人脐静脉内皮细胞,用脂多糖刺激并加入阿托伐他汀干预24h,收集细胞,用荧光定量PCR方法测定TLR4、MyD88、TRAF-6、TRAM及TRIFmRNA表达;用Westernblotting法测定TLR4、MyD88及TRAF-6蛋白表达。结果与结论：用脂多糖刺激人脐静脉内皮细胞后,引起TLR4、MyD88、TRAF-6、TRAM和TRIF的高表达（P〈0.01）,用阿托伐他汀干预后可显著抑制TLR4、MyD88及TRAF-6的表达（P〈0.01）。提示阿托伐他汀可阻断Toll样受体4高表达,同时阻断Toll样受体4胞内信号转导的MyD88依赖性途径,这可能是阿托伐他汀抗动脉粥样硬化的作用机制之一。     

阿托伐他汀对脂多糖诱导人脐静脉内皮细胞Toll样受体4及其下游信号转导通路的影响

背景:研究表明Toll样受体4参与了动脉粥样硬化的发生和发展,目前Toll样受体4与MyD88依赖性或MyD88非依赖性信号转导通路在动脉粥样硬化发生和发展中的机制尚不明确.目的:观察阿托伐他汀对脂多糖诱导的人脐静脉内皮细胞Toll样受体4及其下游信号转导通路主要元件MyD88、TRAF-6、TRAM及TRIF表达的影响,分析阿托伐他汀防治动脉粥样硬化的机制.方法:体外培养人脐静脉内皮细胞,用脂多糖刺激并加入阿托伐他汀干预24 h,收集细胞,用荧光定量PCR方法测定TLR4、MyD88、TRAF-6、TRAM及TRIF mRNA表达;用Western blotting法测定TLR4、MyD88及TRAF-6蛋白表达.结果与结论:用脂多糖刺激人脐静脉内皮细胞后,引起TLR4、MyD88、TRAF-6、TRAM和TRIF的高表达(P < 0.01),用阿托伐他汀干预后可显著抑制TLR4、MyD88及TRAF-6的表达(P < 0.01).提示阿托伐他汀可阻断Toll样受体4高表达,同时阻断Toll样受体4胞内信号转导的MyD88依赖性途径,这可能是阿托伐他汀抗动脉粥样硬化的作用机制之一.     

Herpes simplex encephalitis: an audit of the use of laboratory diagnostic tests

BACKGROUND: The combination of both PCR and intrathecal antibody studies is recommended to confirm or refute the diagnosis of herpes simplex encephalitis (HSE). AIM: To investigate the pattern of use of laboratory tests in the diagnosis of suspected cases of HSE, and to determine the final diagnosis in cases proven not to be HSE. DESIGN: Structured audit. METHODS: We reviewed the case-notes of all patients who, over a five-year time period, presented with suspected encephalitis; and/or were prescribed aciclovir. Clinical and laboratory criteria were used to categorize the likelihood of HSE. RESULTS: We identified 222 patients: 10 (5%) had definite HSE, 24 (10%) possible HSE, and 144 (65%) a definite alternative diagnosis. In 44 (20%), no final diagnosis was made, but the diagnosis of HSE was excluded. PCR was performed in 68 (31%), intrathecal antibody studies in 24 (11%), and brain biopsy in 17 (8%). A wide range of diseases mimicked HSE, but most common were inflammatory diseases and other infections of the central nervous system. DISCUSSION: Laboratory tests, particularly intrathecal antibody assays, are under-used in the diagnosis of HSE. Although early empirical treatment of suspected HSE is essential, confirmation or exclusion of the diagnosis is equally important to avoid overlooking alternative diagnoses. Identification of the aetiology of encephalitis is of particular importance, given the current concerns of emerging infections and bioterrorism.