Severity of enteric nervous system neuropathy during Herpes simplex virus type 1 infection is related to viral replication

Background. Recently, we have shown that HSV1 establishes a latent infection in the mouse enteric nervous system (ENS), leading to gut dysmotility with no signs of illness.(1) Aim. Considering that ICP27, a HSV1 immediate early gene, is involved in viral DNA replication (2), this study aimed to assess the effects of HSV-1 replication on ENS morphology and intestinal contractility in mice infected with wild type (WT) and ICP27-null (KO) HSV1. Methods. In C57/Bl6 mice (males, body weight=24±2 g) infection with WT or KO HSV1 was established by intranasal viral dose (103 pfu) followed 4 weeks (W) later by an intragastric inoculum (IG; 108 pfu).1 Mice (n=6-8) were sacrificed 1, 4 and 8W after IG and the presence of WT and KO HSV1 infection was shown by PCR amplification of HSV1-tk gene in brain and myenteric ganglia (MG). In isolated ileal segments changes in tension were recorded isometrically following electric field stimulation (EFS, 1-40 Hz). Alterations of ENS morphology of whole mount preparations of ileal longitudinal muscle-myenteric plexus (LMMPs), immunostained with HuC/D, peripherin, or nNOS (neuronal Nitric Oxide Synthetase) was assessed by confocal microscopy. For each experimental point 5 LMMPs were examined and 5 microscopic fields were captured to evaluate immunostained cells numbers. Cholinergic network was investigated by analyzing LMMPs, biochemically stained with acetylcholinesterase (AChE) and evaluated in 8 random fields by optical microscopy. Data are reported as percentage of mean ± standard error and statistically analyzed by unpaired Student’s t test. P<0.05 were considered statistically significant. Results. In the brain and ENS WT and KO HSV1 established a latent infection shown by the presence of viral tk-DNA. In the ENS of WT HSV1 infected mice analyses of LMMPs double-immunostained with HuC/D and nNOS revealed an altered number of HuC/D+ and nNOS+ cells at 1W (-37±12% and +60±20%, respectively; P<0.05 vs sham) and 8W (-32±10% and +99±21%, respectively; P<0.05 vs sham) after IG infection whereas KO HSV1 infection reduced the number of HuC/D+ cells at 1W PI (-43±10%) with no changes in the number of nNOS+ neural cells. By performing peripherin labelling an irregular distribution and interrupted neuronal processes was found in MG after 8W of WT HSV1 infection. AChE staining revealed a significant reduction in the density of cholinergic processes after 1W of WT HSV1 infection. In KO HSV1 infected mice no changes in peripherin immunoreactivity were found at 1-8 W PI whereas AChE distribution showed a reduction at 1W and a significant increase at 4 and 8W PI in cholinergic fibers density. Neurally-mediated contractions induced by EFS (20 Hz) were significantly reduced at 1W (-52±17%; P<0.05 vs sham) and increased at 8W (+43±20%; P<0.05 vs sham) post WT HSV1 infection. Similar contractile alterations were found also in KO HSV1 infected mice. Conclusions. HSV1 infection determines alterations of morphology and neurochemical code in the ENS. Viral ICP27 protein-dependent replication appears to be essentially involved in the severity of ENS neuropathy development. References (1) Brun et al. Gastroenterology 2010;138:1790-801 (2) Uprichard et al. J Virol. 1996;70:1969–80