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Oral inoculation with herpes simplex virus type Journal of 1 infects enteric neuron and mucosal nerve fibers within the gastrointestinal tract in mice. Virology R M Gesser and S C Koo J. Virol. 1996, 70(6):4097. Updated information and services can be found at: Downloaded from http://jvi.asm.org/ on April 10, 2014 by guest http://vi.asm.org/content/70/6/4097 These include: CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» Information about commercial reprint orders: http://journals.asm.org/siteimiscireprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ Journals.ASM.org EFTA_R1_02133695 EFTA02713009 JOURNAL Of VIROLOGY, June 1996, p. 40974102 Vol. 70, No. 6 0022-538XN6/$04.00+0 Copyright C 1996, American Society for Microbiology Oral Inoculation with Herpes Simplex Virus Type 1 Infects Enteric Neurons and Mucosal Nerve Fibers within the Gastrointestinal Tract in Mice R. M. OESSER,12* AND S. C. KOOI Division of Allergy, Itnnuutology. and Infectious Diseases, The Children's Hospital of Philadelphia,' and The Wistar Institute,' Philadelphia, Pennsylvania 19104 Received 27 December 1995/Accepted 28 February 1996 Herpes simplex virus type I (HSV-I) is commonly encountered first during childhood as an oral infection. After this initial infection resolves, the virus remains in a latent form within innervating sensory ganglia for the life of the host. We have previously shown, using a murine model, that IISV-I placed within the lumen of Downloaded from http://jvi.asm.org/ on April 10, 2014 by guest the esophagus gains access to nerves within the gut all and establishes a latent infection in sensory ganglia (nodose ganglia) of the tenth cranial nerve (R. M. Lesser, T. Valyi-Nagy, S. M. Altschuler, and N. W. Fraser, J. Gen. Virol. 75:2379-2386. 1994). Peripheral processes of neurons in these ganglia travel through the vagus nerve and function as primary sensory receptors in most of the gastrointestinal tract, relaying information from the gut wall and mucosal surface to secondary neurons within the brain stem. In the work described here. we further examined the spread of HSV-I through the enteric nervous system after oral inoculation. By immunohistochemistry, IISV-I was found to infect myenteric ganglia in Auerbach's plexus between the inner and outer muscle layers of the gut wall. submucosal ganglia (Meisner's plexus). and periglandular ganglion plexuses surrounding submucosal glands. Virus-infected nerve fibers were also seen projecting through the mucosal layer to interact directly with surface epithelial cells. These intramucosal nerve fibers may be a conduit by which intraluminal virus Is able to gain access to the enteric nervous system from the gastroin- testinal lumen. Oral infection with herpes simplex virus type 1 (HSV-I) the sensory ganglion (nodose ganglion) of the vagus or tenth during childhood causes either an asymptomatic infection or cranial nerve, where it establishes a latent infection (17). This an acute infection of the gums, oral mucosa. and tongue discovery led us to believe that HSV-1 may also be an enteri- termed gingivostomatitis (7, 12, 37). That a considerable per- cally acquired pathogen that is able to breach the mucosal centage of the population is exposed to HSV-1 early in life is surface of the gut to encounter nerve fibers of the enteric attested to by seroepidemiologic studies which demonstrate nervous system. rising levels of seropositivity for HSV during childhood, with The nervous system of the gastrointestinal tract consists of a 70 to 90% positivity by adulthood (7. II, 25, 37). Like other complex intrinsic network of interconnected neurons within alphaherpesviruses (pseudorabies virus, varicella-zoster virus, the gut wall itself (enteric nervous system) (reviewed in refer- and bovine herpcsvirus), HSV-1 exhibits tropism for the pe- ence 15). These vast numbers of enteric neurons direct move- ripheral prortws of nerves which innervate the body surface ment, secretions, and blood flow within the alimentary tract (34, 35). Upon entering a nerve ending, the virus is actively and are regulated by local reflex pathways within the gut as transported to the nerve cell nucleus located in sensory gan- well as by extrinsic innervation from sympathetic, spinal, and glia, often a considerable distance away from the initial site of vagal parasympathetic nerves. Primary sensory neurons in the inoculation (10). Following an acute infection in these ganglia, nodose ganglia, responding to different sensory modalities, the HSV-1 gcnome may remain in a latent form within the send afferent fibers via the vagus nerve to most of the gastro- nuclei of sensory neurons for the life of the host or periodically intestinal wall. Mechanoreceptors (sensitive to stroking)• che- reactivate to cause recrudescent disease or asymptomatic shed- moreceptors (responsive to acidity, hypertonicity, and intralu- ding at the body's surface. While HSV-1 is most commonly minal chemical composition), and thermoreceptors have been thought to latently infect the trigeminal ganglion of the fifth localized to the gut mucosa by functional studies; mechanore- cranial nerve innervating the face and oral mucosa (2, 3), latent ceptors arc also found in the muscular and serosal layers HSV-1 has also been found in humans in the nodose ganglia throughout the gastrointestinal tract (reviewed in references innervating the gastrointestinal tract (36). Furthermore, the 19, 28, and 29). Via these receptors, vagal sensory pathways are virus is a frequent cause of recurrent esophageal mucosal dis- able to respond to and modify changes in the gastrointestinal ease in humans• particularly those who are immunocompro- lumen. mised (1, 8, 13, 26. 38). We have recently shown that HSV-1, In mice after oral inoculation, HSV-1 infects myenteric and orally inoculated into the esophageal lumen of mice, travels to nodose ganglia and spreads to secondary sensory neurons (to which the nodose ganglia centrally project) within the nucleus tractus solitarius of the medulla (17, 18). Because HSV-I de- • Corresponding author. Mailing address: Division of Allergy. Im- livered intraluminally to mice appears to target neurons of the munology and Infectious Diseases. The Children's Hospital of Phila- enteric nervous system and preferentially spread to. infect, and delphia. 34th St. and Civic Center Blvd.. Philadelphia. PA 19104. establish latency in neurons of the vagus nerve sensory path- Phone: (215) 590-4492. Fax: (215) 590-2025. Electronic mail address: ways, we questioned whether terminal vagus nerve fibers were gesser(iremail.chop.edu. involved in the spread of HSV-I after oral inoculation. In the 4097 EFTA_R1_02133696 EFTA02713010 4098 NOTES J. VIROL Downloaded from http://jvi.asm.org/ on April 10, 2014 by guest VAGUS NERVE D FIG. I. Whole-organ indirect immunohistochemistry. The esophagus. stomach, and proximal duodenum were reacted with rabbit polyckmal HSV-I antiserum (Dako) and then with perosidase substrate 3.3.-diaminobauidine tetrahydrochloride as described in reference IS. FISV-l-infected areas are identified by dark staining. (A to CI Tissue from a BAUM mouse 6 days after oral inoculation with I ISV-1 strain 17 4. (A) Dorsal sieve of the stomach, with the attached esophagus and duodenum, showing virus infection in penetrating gastric nerves and the body of the stomach and a concentration of infection in the prepyloric region. (B and Cy Enlarged views of the pstroluodenal junction. The arrows point to infected mycnteric ganglia and internodal fibers within the gastric antrum. In panel C (a New along the greater curvature ante stomach), infected penetrating nerves are apparent in the mesenteric attachment of the ShIlitath and proximal duodenum. (0) At 8 days p.i. with IISV.1 strain 17 r. the esophagus of a SOD mouse has widespread viral infection in mycnteric ganglia and interconnecting fibers. Also shown are infected fibers of the vagus nerve. present study, using immunohistochemistry, we further char- (SCID) mice again showed a limited propensity for the virus to acterize the spread of HSV-1 within neurons of the gut wall. infect mucosal epithelial cells, despite widespread persistent We show that after the placement of HSV-1 into the esopha- infection of neurons and support cells in the enteric nervous geal lumen of mice, the virus spreads via intemodal strands of system. These hosts were unable to clear the primary acute the enteric nervous system, infecting neurons of the myenteric, infection, and yet even in the absence of specific host immu- submucosal, and periglandular plexuses of the esophagus, nity, viral spread in these animals was limited primarily to cells stomach, and duodenum, without significant spread to sur- of the enteric and vagal parasympathetic nervous system (18). rounding tissues. HSV-I-labeled terminal nerve fibers were In the present study, in order to better visualize viral spread also seen penetrating into the lamina propria of the gastric and in the gut, we used a high-titer inoculum of wild-type strains F duodenal mucosa to interact directly with surface epithelial or 17+ and applied the technique of whole-organ immunohis- cells. These new findings in immunocompetent hosts indicate tochemistry to further analyze the entire esophagus, stomach, that HSV-1, previously not considered an cntcric pathogen, and proximal duodenum after intracsophageal inoculation. As spreads considerably within all levels of the enteric nervous described elsewhere (18), this technique allows gross detection system, including nerve fibers directly in contact with the mu- of viral protein expression sites in intact tissues with the aid of cosal epithelium. This discovery raises questions regarding a a dissecting microscope. Immunostained sites can then be se- possible role for HSVs in inflammatory mucosal and functional lected for further histological examination. Controls, consist- disorders of the human gastrointestinal tract. ing of uninfected tissues, and acutely HSV-I-infected tissues HSV-1 Infects enteric ganglia after oral inoculation. In a processed without primary HSV-1 antiserum were consistently previously published study of immunocompetent BALB/c mice negative. orally inoculated with neurovirulent HSV-1 strain 17+. in- BALB/c mice received either 1 x 10° = 13), 3 x 10° (n fected myenteric ganglia were seen in the esophagus and stom- = 15). or 5 x 10° (n = 3) PFU of wild-type IISV-1 strain 17+ ach 4 days postinoculation (p.i.). By immunohistochemistry or F in 200 µI of serum-free medium delivered by intraesoph- with thin tissue sections (5 to 6 pm), these infected ganglia ageal cannulation as previously described (18). HSV-1 immu- were found to represent a small percentage of the total ganglia nostaining was first apparent in fibers of the cervical, thoracic. examined; no infected epithelial cells were apparent (17). Fur- and abdominal portion of the vagus nerve beginning 4 days p.i. ther work with a severely attenuated HSV-I strain (in1814) With polyclonal HSV-I antiserum (Dako, Carpinteria, Calif.) in immunocompromised severe combined immunodeficiency being used, virus-infected nerve fibers were visible along the EFTA_R1_02133697 EFTA02713011 VOL. 70, 1996 NOTES 4099 mesenteric border and in nerves penetrating the serosal sur- face of the gut. Infected enteric ganglia were also first noted at 4 days p.i. Initially, HSV-1 antigen-stained ganglia were most prominent along the lesser curvature of the gastric antrum, extending caudally to include the gastroduodenal junction (Fig. IA to C). Over the following days. up until 9 days p.i., HSV-1 infection in the enteric nervous system continued to progress, involving additional ganglia and intcmodal strands throughout the stomach and proximal duodenum. In immuno- competent BALB/c mice, symptoms of this acute infection resolved by 9 days p.i. or the animals died of encephalitis within 2 weeks of the inoculation. Animals that died 12 to 14 days after the inoculation had no evidence of HSV-I in the gastrointestinal tract (presumably cleared by the immune sys- tem), whereas those that died within the first week p.i. had widespread HSV-1 infection in the enteric nervous system of the esophagus. stomach, and proximal small intestine, as de- tected by immunohistochemistry. When SCID mice (it = 22) Downloaded from http://jvi.asm.org/ on April 10, 2014 by guest were orally infected with HSV-1 strain 17 or F, enteric nervous system infection was once again first apparent 4 days p.i. This infection subsequently spread to involve enteric ganglia in most of the stomach, proximal duodenum, and esophagus (Fig. ID). All SCID animals that succumbed to this infection had widely disseminated enteric HSV-1 infection at the time of death (usually within 10 days p.i.). Some mice, both normal and immunocompromised, remained well after oral inocula- tion, however, without any evidence of enteric infection. We take this to indicate that intraluminal inoculation is a relatively inefficient route of infection for HSV-I, with a considerable portion of the inoculum being inactivated by such nonspecific host defenses as luminal pH. digestive enzymes, and mucosal barriers to infection (18). The myenteric (Auerbach's) plexus is an interconnected net- work of nerve strands and small ganglia (each containing an ,p average of 40 neurons) located between the external longitu- FIG. 2. HSV-I-infected enteric nerves and ganglia in the duodenum of wally dinal and circular muscle coats of the gastrointestinal tract. infected mice. The tissues are sectioned paralkl to the mucosal surface. (A) A Meissner's submucous plexus is found just below the gut mu- SCID mouse at $ days p.i. with strain 17 Two layer. within the intestinal wall cosa. Compared with those of the myenteric plexus, the ganglia arc shown. To the right are infected myenteric ganglia and intemodal fibers (arrowhead) within Auerbach's plexus between the outer muscular walls; to the of the submucous plexus are smaller and contain fewer nerve left arc virus-infected nom fibers (arrow) extending from the deeper myenteric cells; their interconnecting (intemodal) strands also have plexus to surround duodenal Brunner's glands in the submucosa. Slagnification. fewer nerve fibers. Both plexuses are continuous around the )(100. (B) Whole mount of myenteric ganglia of a BALBle mouse at 6 days pa. circumference and along the length of the alimentary tract, with IISV-I strain F. Within an enteric ganglion are both infected (Mack ;mow) essentially forming sheets of intercommunicating neuronal and uninfected (white arrow) neurons. Infected intemodal nerve fibers (arrow- heads) are shown extending towards other ganglia. Magnification, x400. networks running parallel to the gut lumen (reviewed in ref- erence 15). After intraesophageal inoculation, the myenteric and submucosal plexuses of the stomach and duodenum were infected with HSV-1. In whole-mount preparations. infected diately adjacent to Brunner's glands in the submucosa: the neurons and supporting glial cells were clearly apparent. In- processes of these nerves wove in between and just below the fected nerve fibers were seen within myenteric ganglia, travel- glandular epithelium, often surrounding a cluster of glands. A ing through internodal strands to communicate with additional cross section of the duodenum demonstrated infected submu- ganglia. and in groups of nerve bundles within the muscle coats cosal ganglia with connecting internodal strands spreading cir- (Fig. 2). Both uninfected and heavily infected neurons were cumferentially around the intestinal wall, linking infected neu- seen simultaneously within the same enteric ganglion. indicat- rons and ganglia (Fig. 3A to I)). ing to us that spread within the ganglia proceeds most likely by HSV-I labels intramucosal nerve fibers and associated cells. specific transneuronal spread rather than by adjacent cell-to- HSV-1-labeled terminal nerve fibers penetrated into the lam- cell spread. This mode of spread was also made apparent by ina propria of the gastric and duodenal mucosa and into the the lack of infectious spread from cells of the nervous system mucosa of the esophagus. In the stomach and duodenum, the to other tissues of the gastrointestinal wall (17, 18) and was bulk of this activity was centered about the gastroduodenal further underscored by the work of others demonstrating the junction as demarcated by the pyloric sphincter (Fig. 3A and effectiveness of herpesviruses as specific transsynaptic tracing B). In the distal antrum and pyloric region of the stomach, agents (6, 9, 20, 21, 30, 31, 33). immunostained fibers extended to reach the basal surface of Thick tissue sections also revealed diffuse HSV-I infection luminal epithelial cells. In thick tissue sections or whole of the submucosal plexus in the gastric wall, particularly in mounts, these mucosal fibers appeared to connect to submu- neurons and nerve fibers enveloping the gastric glands and in cosal ganglia directly below and with adjacent infected submu- duodenal glands concentrated around the pylorus (Fig. 2A and cosal ganglia via internodal fibers. As was seen in the gastric 3). Within the duodenum, infected neurons were seen imme- mucosa, infected nerve fibers in the duodenum also extended EFTA_R1_02133698 EFTA02713012 4100 NOTES J. VIROI- Eitc.• t •• 1 ,a,. Downloaded from http://jvi.asm.org/ on April 10, 2014 by guest • A .• 4 #---Periglandular runner' 4 elands it 4. I 1. Submucous a .. en *c W brilOintICOUS / I .. C .... ..• ii, AI . tefIC . . 74 .•••••• MG. 3. HSV.1 immunostaining of the ganroduodenal jundion. (A and II) Whole mount,. of the pyloric region from a BAlikc mouse at 7 day, pr. with strain I7-. In panel A, the stomach is to the right of the pylorus and the duodenum is to the left. HSV-l-infected myenteric and submucosal ganglia and fibers (staining black) art shown. Also shown arc infected penetrating nerves (while arrow) cartied along the mesentery and infected nerves penetrating the pyloric sphincter of the stomach. In panel %which is the area enclosed in the box in panel A enlarged, infected submuetsal ganglia (arrowheads) and infected nerve fibers penetrating into the duodenal mucosa (mums) are shown. Magnification. x40 (A) and x2CO (R). (C) A section (25 om thick) of proximal duodenum from a BALER mouse at 5 days p.i. with strain F showing IISV-I•infected ganglia and nerve fibers in the area of duodenal Brunner'sglands. Magnification, x2fXl. (O) A section (7 pm thick) showing IISV.I.infected myenteric and iubmucosal ganglia in the duodenum. Magnification. x MA (E) A section (25 Am thick) showing an infected neuron adjacent to Brumes glands in the submucosa. Magnification. x IOW. towards the lumina! epithelial layer. In the proximal duode- mucosal afferent fibers interact directly with lumina! epithelial num, single and occasionally multiple infected nerve fibers cells and whether sensory nerve endings can directly sample were seen spreading towards the tip of an individual duodenal the intraluminal contents. Recently, Berthoud et al. have villus (Fig. 4). Often these fibers terminated on a single in- shown vagus nerve sensory endings in the rat duodenal mucosa fected mucosal epithelial cell; less often, clusters of infected by injecting the neurotracer Dil directly into the nodose gan- epithelial cells were seen around terminal nerve fibers. Within glia (4). These vagal afferent terminal fibers, originating from the villus lamina propria, labeled nerve fibers were found in sensory neurons in the nodose ganglia, were found to connect contact with virus-infected spindle-shaped cells. These spindle with enteric ganglia in both the myenteric and submucous cells, characterized by an oval nucleus and several slender plexuses and to arborize terminally within the villus lamina dendritic-like processes, were identical to cells that Berthoud propria in close contact with surface epithelial cells. It has been et al. (4) described as being associated with vagus nerve sen- suggested that these terminal nerve endings, positioned as they sory receptors originating from neurons in the nodose ganglia. are, may be able to directly sample the lumen or relay infor- Thus, it is likely that the infected nerve terminals we arc de- mation from surface epithelial cells and essentially function scribing are sensory and also have their origins in the nodose like taste cells of the luminal contents. Similarly, peripheral ganglia. vagal afferent receptors have also been identified in the gastric Vagal sensory receptors are known to respond to intralumi- mucosa. concentrated around the prepyloric region (22, 27). nal changes, transmitting information throughout the enteric We previously showed in immunodeficient (SOD) animals and parasympathetic nervous system; they may also be in- that HSV-I spread in the nervous system is determined by the volved in the uptake and dissemination of orally acquired neu- innervation of the initial inoculation site and that the virus rotropic herpesviruses. We describe here how after intraesoph- dots not spread indiscriminately, for example. by the circula- agcal inoculation, HSV-I spreads to infect multiple layers of tion (18). It is highly likely that virus inoculated in the esoph- the intrinsic enteric nervous system and to infect mucosal ageal lumen also reaches the mucosa of the stomach and small nerve fibers believed to be vagal sensory receptors. Peripheral intestine, where it can apparently access the enteric nerve sensory receptors in the gut consist of free nerve endings rather network; in immunocompetent mice, we have seen significant than specialized receptors, as found elsewhere in the body (5, viral infection in the pyloric region without any apparent in- 16, 22. 23, 27). It has long been questioned whether these fection of the esophageal enteric nerves. The finding of Bud- EFTA_R1_02133699 EFTA02713013 Vol.. 70, 1996 NOTES 4101 A B D pithelial Layer Isent3 Aq pug '01 ipdv uo ffluoluselAfirdmi wo4 pepeolumoa • tj . mina. F1G. 4. Individual duodenal villi dissected out and immunonained for either PGP 93 or HSV.I antigens. (A) Uninfected duodenum reacted with a neuron-specific antibody aping PGP 9-5 (Dako) H4.32). which stains myenteric andant/muck/sal ganglia and interconnecting neuronal fibers. including an elaborate web of nerve fibers in the lamina propria just below the epithelial mface. (Bto Et) Duodenal vith from virus.infected mice immummained with pobrIonal antiserum. Infected submucosal ganglia and neuronal fibers extending into the lamina propria of intestinal villi (arrows) arc shown. Magnification ((or all panels). /400. dingh et al. that infectious virus is shed rectally for weeks after tion, the ulcers in these animals were not directly infected; symptomatic gingivostomatitis in children (7) lends further rather, they were found to overlay virus-infected enteric gan- support to this observation. Virus may breach the mucosal glia. This result suggests that chronic enteric nerve dysfunction surface in a number of places in the gut. Once inside, however, or inflammation may alter the natural mucosal barrier of the it seems apparent that at least regional transsynaptic viral gut, ultimately resulting in epithelial disintegration and ulcer- spread occurs throughout all layers of the intrinsic enteric ation. In less dramatic ways, acute, latent, or reactivated nervous system. Using a neurotropic reovirus strain, Morrison HSV-I enteric nervous system infection may also be involved et al. described the spread, after oral inoculation, to vagal in the pathogenesis of chronic, recurrent functional human efferent areas of the brain stem (dorsal motor nucleus) (24). gastrointestinal disorders. The infected neuronal networks HSV-1 also spreads centripetally to vagal areas of the brain which we describe here arc likely to contribute to such physi- stem, including the dorsal motor nucleus after oral inoculation ologic disorders. (17,18). However, during early HSV-I infection, we clearly see a preference for sensory, fibers which originate in the nodose This work was supported by NMI grant A101106. ganglia and terminate centrally in the nucleus tractus solitarius We thank Elsa Aglow of the Wistar Institute Histotechnology of the brain stem. Furthermore, in the gut. Morrison et al. CORE laboratory and Michael Sidelsky of the Wistar Institute Animal found reovirus-infected neurons only within myenteric ganglia Facility for technical assistance. immediately adjacent to infected lymphoid follicles (Peyer's REFERF.NCES patches) in the ileum, whereas we find HSV-1-infected enteric I. Agba. F. P.. H. H. Lew and T. T. Nostum. 1936. Ilerpetie csophagitis: a neurons predominantly in the proximal duodenum (where Pey- diagnostic challenge in inimunocompromised patients. Mt. J. GagroenteroL er's patches are absent) and throughout all levels of the gut 81:246-253. wall, including the surface mucosa. This finding suggests to us 2. 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