Saliva enhances infection of gingival fibroblasts by herpes simplex virus 1

RESEARCH ARTICLE Saliva enhances infection of gingival fibroblasts by herpes simplex virus 1 Yi Zuo1☯†, J. Charles Whitbeck1☯, Gabriel J. Haila2, Abraham A. Hakim3, Paul W. Rothlauf3, Roselyn J. Eisenberg4, Gary H. Cohen1, Claude Krummenacher ID3,5* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Microbiology, School of Dental Medicine University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2 Department of Chemistry and Biochemistry, Rowan University, Glassboro, New Jersey, United States of America, 3 Department of Biological Sciences, Rowan University, Glassboro, New Jersey, United States of America, 4 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 5 Department of Molecular and Cellular Biosciences, Rowan University, Glassboro, New Jersey, United States of America ☯ These authors contributed equally to this work. † Deceased. * Abstract OPEN ACCESS Citation: Zuo Y, Whitbeck JC, Haila GJ, Hakim AA, Rothlauf PW, Eisenberg RJ, et al. (2019) Saliva enhances infection of gingival fibroblasts by herpes simplex virus 1. PLoS ONE 14(10): e0223299. https://doi.org/10.1371/journal.pone.0223299 Editor: Luis M. Schang, Cornell University, UNITED STATES Received: December 12, 2018 Accepted: September 19, 2019 Published: October 3, 2019 Copyright: © 2019 Zuo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This study was supported by Public Health Service grant DE022137 from the National Institute of Dental and Craniofacial Research (to C. K.) (https://www.nidcr.nih.gov/) and the College of Science and Mathematics at Rowan University (https://www.rowan.edu/home/sciencemathematics). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Oral herpes is a highly prevalent infection caused by herpes simplex virus 1 (HSV-1). After an initial infection of the oral cavity, HSV-1 remains latent in sensory neurons of the trigeminal ganglia. Episodic reactivation of the virus leads to the formation of mucocutaneous lesions (cold sores), but asymptomatic reactivation accompanied by viral shedding is more frequent and allows virus spread to new hosts. HSV-1 DNA has been detected in many oral tissues. In particular, HSV-1 can be found in periodontal lesions and several studies associated its presence with more severe periodontitis pathologies. Since gingival fibroblasts may become exposed to salivary components in periodontitis lesions, we analyzed the effect of saliva on HSV-1 and -2 infection of these cells. We observed that human gingival fibroblasts can be infected by HSV-1. However, pre-treatment of these cells with saliva extracts from some but not all individuals led to an increased susceptibility to infection. Furthermore, the active saliva could expand HSV-1 tropism to cells that are normally resistant to infection due to the absence of HSV entry receptors. The active factor in saliva was partially purified and comprised high molecular weight complexes of glycoproteins that included secretory Immunoglobulin A. Interestingly, we observed a broad variation in the activity of saliva between donors suggesting that this activity is selectively present in the population. The active saliva factor, has not been isolated, but may lead to the identification of a relevant biomarker for susceptibility to oral herpes. The presence of a salivary factor that enhances HSV-1 infection may influence the risk of oral herpes and/or the severity of associated oral pathologies. Introduction The highly prevalent herpes simplex virus 1 (HSV-1) is the etiologic agent of oral herpes. In 2015–2016, 48% of American adults were seropositive for HSV-1 [1]. HSV-1 primary infection PLOS ONE | https://doi.org/10.1371/journal.pone.0223299 October 3, 2019 1 / 19 Enhancement of HSV-1 infection by saliva Competing interests: The authors have declared that no competing interests exist. causes gingivostomatitis, which can go unnoticed or cause mucosal ulcerations of various severity [2]. The related HSV-2 is the main agent of herpes genitalis but only rarely causes oral disease [3]. After replication in oral epithelial cells, HSV-1 spreads to innervating sensory neurons, where it establishes latency [4]. This latent phase is punctuated by reactivation episodes during which viral replication in epithelia generates mucocutaneous lesions (cold sores)[2]. Importantly, HSV-1 reactivation often occurs asymptomatically and leads to frequent unnoticed shedding from the oral mucosa [5–7]. For instance, in a cohort of 8 immunocompetent individuals evaluated during 5 consecutive weeks, asymptomatic reactivation was observed at sites throughout the oral cavity at a rate of 27.1% (65/240days) [5]. The variability in frequency of HSV-1 reactivation and severity of herpes diseases is thought to be related to the host immunogenetic factors [8]. Although particular genetic markers have been associated with risks of herpes simplex encephalitis [9], biomarkers associated with risks or severity of oral herpes have not yet been identified [10]. Herpesviruses have been found in healthy and pathological oral tissues, in particular they are associated with periodontal disease (PD)[11]. About 47% of American adults suffer from PD [12]. Subgingival colonization by Gram negative facultative and anaerobic bacteria plays a major role in the development of PD [13]. Interestingly, HSV-1 has been detected in lesions during chronic and aggressive periodontitis [14–17]. The role of HSV-1 in PD pathology remains unclear but several studies associated it with increased severity of lesions [18–20]. Since HSV-1 infection interferes with immune regulators, it may aggravate PD by causing local immunosuppression and inflammation [21, 22]. Oral keratinocytes and epithelial cells, which comprise the main sites of lytic replication during primary and secondary lytic infections, are highly susceptible to HSV-1 infection in vitro [23]. In contrast, gingival fibroblasts, which are normally not exposed in the oral mucosa are less efficiently infected in vitro [24, 25]. Infection of intact oral epithelia ex vivo is inefficient and depends on access to entry receptors on basal keratinocytes [23]. Nectin-1 and HVEM are the main HSV receptors on various oral cells [23, 25]. Interaction of nectin-1, HVEM or 3-O-sulfated heparan sulfate, with HSV glycoprotein D (gD) is an essential step in entry [26, 27]. Receptor-triggered conformational changes in gD initiate the activation of gH/ gL, which in turn activates gB to fuse the viral envelope w (...truncated)