Influence of Perineurial Cells and Toll-Like Receptors 2 and 9 on Herpes simplex Type 1 Entry to the Central Nervous System in Rat Encephalitis

Herpes simplex encephalitis (HSE) is a rare disease with high mortality and significant morbidity among survivors. We have previously shown that susceptibility to HSE was host-strain dependent, as severe, lethal HSE developed after injection of human Herpes simplex type 1 virus (HSV-1) into the whiskers area of DA rats, whereas PVG rats remained completely asymptomatic. In the present study we investigated the early immunokinetics in these strains to address the underlying molecular mechanisms for the observed difference. The virus distribution and the immunological responses were compared in the whiskers area, trigeminal ganglia and brain stem after 12 hours and the first four days following infection using immunohistochemistry and qRT-PCR. A conspicuous immunopathological finding was a strain-dependent difference in the spread of the HSV-1 virus to the trigeminal ganglia, only seen in DA rats already from 12 hpi. In the whiskers area infected perineurial cells were abundant in the susceptible DA strain after 2 dpi, whereas in the resistant PVG rats HSV-1 spread was confined only to the epineurium. In both strains activation of Iba1+/ED1+ phagocytic cells followed the distribution pattern of HSV-1 staining, which was visible already at 12 hours after infection. Notably, in PVG rats higher mRNA expression of Toll-like receptors (Tlr) -2 and -9, together with increased staining for Iba1/ED1 was detected in the whiskers area. In contrast, all other Tlr-pathway markers were expressed at higher levels in the susceptible DA rats. Our data demonstrate the novel observation that genetically encoded properties of the host nerve and perineurial cells, recruitment of phagocyting cells together with the low expression of Tlr2 and -9 in the periphery define the susceptibility to HSV-1 entry into the nervous system

The Calcitonin Receptor Gene Is a Candidate for Regulation of Susceptibility to Herpes simplex Type 1 Neuronal Infection Leading to Encephalitis in Rat

Herpes simplex encephalitis (HSE) is a fatal infection of the central nervous system (CNS) predominantly caused by Herpes simplex virus type 1. Factors regulating the susceptibility to HSE are still largely unknown. To identify host gene(s) regulating HSE susceptibility we performed a genome-wide linkage scan in an intercross between the susceptible DA and the resistant PVG rat. We found one major quantitative trait locus (QTL), Hse1, on rat chromosome 4 (confidence interval 24.3–31 Mb; LOD score 29.5) governing disease susceptibility. Fine mapping of Hse1 using recombinants, haplotype mapping and sequencing, as well as expression analysis of all genes in the interval identified the calcitonin receptor gene (Calcr) as the main candidate, which also is supported by functional studies. Thus, using unbiased genetic approach variability in Calcr was identified as potentially critical for infection and viral spread to the CNS and subsequent HSE development

Von Willebrand Factor Gene Variants Associate with Herpes simplex Encephalitis

Herpes simplex encephalitis (HSE) is a rare complication of Herpes simplex virus type-1 infection. It results in severe parenchymal damage in the brain. Although viral latency in neurons is very common in the population, it remains unclear why certain individuals develop HSE. Here we explore potential host genetic variants predisposing to HSE. In order to investigate this we used a rat HSE model comparing the HSE susceptible SHR (Spontaneously Hypertensive Rats) with the asymptomatic infection of BN (Brown Norway). Notably, both strains have HSV-1 spread to the CNS at four days after infection. A genome wide linkage analysis of 29 infected HXB/BXH RILs (recombinant inbred lines-generated from the prior two strains), displayed variable susceptibility to HSE enabling the definition of a significant QTL (quantitative trait locus) named Hse6 towards the end of chromosome 4 (160.89-174Mb) containing the Vwf (von Willebrand factor) gene. This was the only gene in the QTL with both cis-regulation in the brain and included several non-synonymous SNPs (single nucleotide polymorphism). Intriguingly, in human chromosome 12 several SNPs within the intronic region between exon 43 and 44 of the VWF gene were associated with human HSE pathogenesis. In particular, rs917859 is nominally associated with an odds ratio of 1.5 (95% CI 1.11-2.02; p-value = 0.008) after genotyping in 115 HSE cases and 428 controls. Although there are possibly several genetic and environmental factors involved in development of HSE, our study identifies variants of the VWF gene as candidates for susceptibility in experimental and human HSE

Sustained Elevation of Kynurenic Acid in the Cerebrospinal Fluid of Patients with Herpes Simplex Virus Type 1 Encephalitis

Herpes simplex virus (HSV) type 1 encephalitis (HSE) is a viral infectious disease with commonly occurring neurodegeneration and neurological/cognitive long-term sequelae. Kynurenic acid (KYNA) is a neuroactive tryptophan metabolite, which is elevated in the cerebrospinal fluid (CSF) during viral infection as a result of immune activation. The aim of the study was to investigate the role of endogenous brain KYNA for the long-term outcome of the disease. CSF KYNA concentration was analyzed in 25 HSE patients along the course of the disease and compared with that of 25 age-matched healthy volunteers. Within 3 weeks of admission CSF KYNA of HSE patients was markedly elevated (median 33.6 nM) compared to healthy volunteers (median 1.45 nM). Following a decline observed after 1–2 months, levels of CSF KYNA were elevated more than 1 year after admission (median 3.4 nM range: 1–9 years). A negative correlation was found between initial CSF KYNA concentrations and severity of the long-term sequelae. This study show a marked elevation in CSF KYNA from patients with HSE, most pronounced during the acute phase of the disease and slowly declining along the recovery. We propose that brain KYNA might potentially protect against neurodegeneration while causing a long-lasting loss in cognitive function associated with the disease

HSV-1 infected congenic lines confirm HSE regulation by <i>Hse1</i>.

<p>A 6.8 Mb region on rat chromosome 4 contains a QTL regulating HSE susceptibility. The black vertical bar represents rat chromosome 4 with a number of microsatellite markers used. The blue vertical bars represent the congenic lines we developed DA.PVG-<i>Hse1</i>, with a PVG fragment (D4Kini3 – D4Rat177) transferred to DA background and DA.PVG-<i>Hse1</i>-R1, with a PVG fragment (D4Kini3 – D4Mgh14) transferred to DA background, containing the region of interest <i>Hse1</i>. All infected rats from these strains were completely protected from HSE development. The green vertical bars represent the chromosome 4 congenic lines R2, R11, R21 (<i>Eae</i>) and R17 (APLEC) with PVG fragments on DA background available in our laboratory, which were used to test for HSE incidence. These strains developed HSE as DA rats. The non-overlapping fragment between DA.PVG-<i>Hse1</i>, R2, R11 and R21 congenic lines delineates the disease regulatory effect of <i>Hse1</i> to a region between D4Kini3 – D4Rat23, which is excluded by the horizontal red dotted lines.</p

Signaling through CalcR inhibits virus replication in HEK293T cells.

<p>HEK293T cells were transfected for 24 hours with plasmids encoding for either RAMP1 alone (A), or in combination with the human CalcR (huCalcR) (B) or rat CalcR (ratCalcR) (C). (A–C) Double staining showed that CalcR was only expressed in RAMP1-positive cells. Albeit detected at a lower level than rat CalcR, huCalcR was distinctly expressed. (D) RAMP1 or RAMP1/huCalcR transfected cells were infected for 30 min with HSV-1 and virus replication was detected 24 h later with antibodies against HSV-1 structural proteins. (E) Ratio between RAMP1 or RAMP1/huCalcR infected and non-infected cells was shown for infection alone, or in the presence of 1 µg/ml or 10 µg/ml rat Amylin. (F) Analysis of the mean fluorescence intensity (MFI) of staining for HSV1 structural proteins in the infected populations. Effect of rat Amylin added prior, during and after infection as compared to incubation post-infection only (G and H). The effect on percentage (G) of RAMP1- or RAMP1/huCalcR-positive and HSV-1-positive cells, or the MFI of the infected population (H) are shown. All analyzes were done in triplicates of transfected and infected wells.</p

Sequences of candidate genes.

<p>Schematic illustration of genomic DNA sequences of the <i>Ccdc132</i>, <i>Calcr</i> and <i>Tfpi2</i> genes showing some of the SNP variations in PVG.A rats. Black boxes represent the different exons in each gene; the arrows below represent SNPs in intronic regions; arrows above represent SNPs in exons and 5′ UTR regions.</p

Whole genome scan and linkage analysis of the different HSE phenotypes tested in F2 (DAxPVG.A).

<p>(A–C) Genome-wide linkage QTL maps of HSE phenotypes (black curve); linkage in males (blue curve) and in females (red curve). Upper horizontal line: significant LOD score genome-wide threshold at <i>P</i>≤0.05. Lower dotted horizontal line: suggestive LOD score genome-wide threshold at <i>P</i>≤0.63. To the right of each graph are the effect plots for each QTL indicating the alleles in both males and females, which drive the HSE phenotypes. DD = DA homozygous; DP = DA/PVG.A heterozygous; PP = PVG.A homozygous. A – incidence of HSE; B – onset of disease; C – weight loss d0–d5.</p

Genome-wide linkage scan of HSE incidence revealed a QTL on chromosome 4 regulating susceptibility.

<p>(A) Genome-wide linkage quantitative trait loci (QTL) map of HSE incidence in F2 (DAxPVG.A). Linkage analysis revealed a main QTL on chromosome 4 and additional significant QTLs on chromosome 3 and 10 (black curve); linkage in males (blue curve) and in females (red curve). The horizontal line: significant LOD score genome-wide threshold at <i>P</i>≤0.05. Lower dotted horizontal line: suggestive LOD score genome-wide threshold at <i>P</i>≤0.63. (B) A genetic map showing the distribution of the 127 microsatellite markers used for linkage analysis of the F2 cohort. (C) HSE main regulating QTL on rat chromosome 4, (<i>Hse1</i>) (black curve); linkage in males (blue curve) and in females (red curve). The horizontal line: significant LOD score genome-wide threshold at <i>P</i>≤0.05. The 20 microsatellite markers used for chromosome 4 linkage analysis are shown in the <i>x</i> axis and the distance between them reflects the recombination fraction in our experimental population rather than physical location. The D4Kini microsatellite makers were designed by our group. The peak marker is D4Kini3 (27.8 Mb). Thick blue horizontal bar above the <i>x</i> axis indicate the confidence interval (CI) of <i>Hse1</i> in males, whereas the thick red horizontal bar above the <i>x</i> axis indicate the CI in females. (D) The effect plots indicate that HSE incidence was driven by DA alleles in both males and females. DD = DA homozygous; DP = DA/PVG.A heterozygous; PP = PVG.A homozygous.</p