Association between dietary zinc intake and herpes simplex virus seropositivity in U.S. adults: a retrospective cross-sectional study

Association between dietary zinc intake and herpes simplex virus seropositivity in U.S. adults: a retrospective cross-sectional study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Association between dietary zinc intake and herpes simplex virus seropositivity in U.S. adults: a retrospective cross-sectional study Chunhua Liu, Yingguo Liu, Jingjing Liu, Zhaoyong Lv, Mengpeng Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7036290/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Feb, 2026 Read the published version in BMC Infectious Diseases → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Although recent research has demonstrated an association between serum zinc deficiency and susceptibility to various viral infections, the relationship between dietary zinc intake and herpes simplex virus (HSV) seropositivityremains unclear. Methods: The National Health and Nutrition Examination Survey (NHANES) (2007--2016) provides data on HSV-1 and HSV-2 status and dietary zinc intake. The associationsbetween dietary zinc and HSV-1 and HSV-2 were evaluated via various statistical methods, including multivariate logistic regression, restricted cubic spline analysis, and subgroup analysis. Results: In total, 6,483 individuals were enrolled, with 58.9% (3,817/6,483) testing positive for HSV-1 and 19.3% (1,253/6,483) testing positive for HSV-2. After adjusting for all covariates in the multivariate logistic regression, compared with the lowest zinc intake group (Q1: <7.51 mg/day), the adjusted odds ratios (ORs) for HSV-1 and HSV-2 in the higher-zinc intake groups were as follows: for HSV-1, Q2 (7.51--10.87 mg/day) had an OR of 0.81 (95% confidence interval [CI]: 0.72--0.98, p = 0.027), and Q3 (10.87--15.63 mg/day) had an OR of 0.85 (95% CI: 0.70--1.03, p = 0.093), and Q4 (>15.63 mg/day) had an OR of 0.84 (95% CI: 0.67--1.04, p = 0.114); for HSV-2, Q2 had an OR of 0.80 (95% CI: 0.64–1.00, p = 0.050), Q3 had an OR of 0.81 (95% CI: 0.64--1.02, p = 0.069), and Q4 had an OR of 0.75 (95 Conclusion: Dietary zinc intake exhibited a U-shaped association with HSV-1 and HSV-2 seropositivity, indicating that moderate zinc intake has a protective effect. zinc herpes simplex virus National Health and Nutrition Examination Survey epidemiology Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Herpes simplex virus (HSV), a double-stranded DNA pathogen with a global prevalence, manifests as two primary serotypes: HSV-1 and HSV-2. HSV-1 spreads predominantly through oral‒to‒oral contact, infecting more than 67% of individuals under 50 years of age, whereas HSV-2 is transmitted primarily via sexual routes, affecting approximately 17% of the 15–49 age cohort [ 1 ]. Following initial mucosal invasion, the virus establishes lifelong latency in the sensory ganglia, characterized by periodic reactivation [ 2 ]. Although most outbreaks involve self-limiting mucocutaneous lesions, severe complications may arise, including viral encephalitis, herpetic keratitis (a leading cause of infectious blindness), and increased susceptibility to human immunodeficiency virus (HIV) infection [ 3 – 5 ]. The absence of curative vaccines or virucidal agents combined with asymptomatic viral shedding that facilitates transmission underscores the urgent need to investigate modifiable risk factors, such as dietary patterns, in HSV pathogenesis and epidemiology [ 6 ]. Zinc is a crucial trace element for human growth, development, and maintenance of immune function [ 7 ]. Its immunomodulatory properties extend to both innate and adaptive immunity, wherein it participates in lymphocyte differentiation, cytokine production, and phagocytic activity [ 8 – 10 ]. Zinc status serves as a critical determinant of antiviral immunity, with zinc-deficient populations demonstrating heightened susceptibility to viral infections, including HIV and hepatitis C virus [ 11 , 12 ]. Moreover, emerging evidence has revealed the direct inhibitory effects of zinc on HSV replication through mechanisms involving viral DNA polymerase interference and capsid destabilization [ 13 – 15 ]. Although the interplay between serum zinc status and viral pathogenesis has been extensively investigated, the specific relationship between dietary zinc intake and HSV seropositivity remains unexplored. Therefore, further research is necessary to understand the association between dietary zinc consumption and HSV seropositivity. Data from the National Health and Nutrition Examination Survey (NHANES) were drawn upon in the present study. MATERIALS AND METHODS Study population The NHANES, organized by the United States Centers for Disease Control and Prevention (CDC), serves as a critical surveillance system monitoring population health indicators among noninstitutionalized U.S. residents. In accordance with the approval of the National Center for Health Statistics Research Ethics Committee, the protocol mandates documented consent from all participants prior to enrollment. For our retrospective study that analyzed existing deidentified records, no supplementary ethical clearance or informed consent was necessary according to federal regulations. Publicly accessible datasets spanning 2007–2016 (5 biennial cycles) were retrieved from the official NHANES repository ( https://www.cdc.gov/nchs/nhanes ). Our analytical cohort excluded individuals whose nutritional zinc records were incomplete, whose serological data for herpes simplex virus were unavailable, or whose covariate information was missing. The final sample included complete case data from eligible participants across the designated survey periods. Dietary zinc intake Participants in the NHANES dietary survey reported their food and beverage consumption over a 24-hour period, with data collected between 2006 and 2016 via the automated multiple pass method (AMPM) to ensure precise nutrient calculations on the basis of reported intake. The detailed methodologies are described in the NHANES Dietary Interviewer Procedure Manual. Zinc intake was analyzed as a continuous variable and categorized into quartiles (Q1–Q4). To validate these results, individuals whose reported 24-hour dietary intake aligned with their habitual consumption patterns were prioritized. Specifically, participants were screened via the variable DR1_300, which corresponds to the following question: "Was the amount of food that {you/NAME} ate yesterday much more than usual, usual, or much less than usual?" Only those who responded "usual" were included. Additionally, the zinc intake categories for Q1 were as follows: 15.63 mg/day. HSV antibody measurement To identify HSV-1 and HSV-2 infections, blood samples obtained through venipentesis were analyzed via a solid-phase enzyme immunopoint assay. The publicly released NHANES data file includes HSV-1 data for the 14–49 age group and HSV-2 data for the 18–49 age group. In our study, these two variables were considered independent factors; that is, HSV-1 and HSV-2 infections were defined as positive for their respective viral antibodies, and negative infections were defined as negative for their respective viral antibodies. Records classified as "inconclusive" were excluded from our analysis. Covariates We evaluated several potential covariates on the basis of previous literature [ 16 – 18 ], including age; sex; race and ethnicity; body mass index (BMI); marital status; smoking status; excessive alcohol consumption; number of lifetime sexual partners; acquisition time; and caloric, protein, carbohydrate, sugar, fiber, and fat intake. The categories for race and ethnicity included Mexican American, other Hispanic, non-Hispanic White, non-Hispanic Black, and other races. Marital status was categorized as living alone or being married/living with a partner. BMI was calculated via a standardized approach considering both weight and height. Smoking was defined as having smoked at least 100 cigarettes. Excessive alcohol consumption was defined as periods of drinking 4 or more drinks per day for women and 5 or more for men. Sexual partners were defined as the total number of same-sex or opposite-sex partners with whom any sexual activity occurred. A dietary recall interview was conducted prior to the Mobile Examination Center (MEC) interview to collect participants' nutritional data over a 24-hour period, including the consumption of calories, protein, carbohydrates, sugar, fiber, and fat. Statistical analysis This was a secondary analysis of the datasets available in the public domain. For continuous variables with a normal distribution, we reported the mean and standard deviation (SD). For nonnormally distributed variables, we used medians and interquartile ranges (IQRs). Categorical variables are presented as percentages. To compare differences between groups, we used one-way analysis of variance (ANOVA) for normally distributed data, the Mann-Whitney test for nonnormally distributed data, and the chi‒square test for categorical data. Logistic regression analysis was used to explore the association between dietary zinc intake and HSV seropositivity. Initially, we used univariate logistic regression to identify factors associated with HSV-1 and HSV-2 seropositivity. After controlling for potential confounding factors, we used a multivariate logistic regression model to clarify the relationships between dietary zinc intake and HSV-1 and HSV-2 seropositivity. Model I was adjusted for age, sex, and race. In Model II, we further adjusted for variables such as marital status, smoking status, excessive alcohol consumption, number of lifetime sexual partners, and acquisition time. In Model III, we included all the covariates. Furthermore, restricted cubic spline (RCS) regression analysis was used to assess whether there was a nonlinear relationship between dietary zinc intake and HSV-1 and HSV-2 seropositivity. Finally, a subgroup analysis was conducted to compare the The interaction between dietary zinc intake and HSV-1 and HSV-2 seropositivity across different subgroups was used to evaluate the robustness of the study results. All analyses were conducted via R 4.4.2 statistical software ( http://www.R-project.org ). P values were considered statistically significant if they were less than 0.05. RESULTS Study population A total of 50,588 participants were included in the NHANES conducted between 2007 and 2016. After individuals with missing HSV-1 and HSV-2 data were excluded, 14,599 individuals were included in the study. After selecting those for whom data regarding dietary zinc were available, 9,574 people remained. After further screening for individuals who could recollect the food they consumed the previous day [A1] and excluding individuals with missing covariates, 6,483 people remained. The final population included 2,666 HSV-1-negative and 3,817 HSV-1-positive individuals. For HSV-2, there were 5,230 HSV-2-negative individuals and 1,253 HSV-2-positive individuals. The mean age was 34.52 ± 8.64 years, and 61.4% were male. The mean daily zinc consumption amount was 12.57 ± 8.23 mg (10.12 ± 5.42 mg for women and 14.87 ± 9.64 mg for men) (Fig.1). Baseline characteristics Table 1 summarizes the baseline demographic characteristics stratified by herpes simplex virus (HSV) infection status. Seropositivity for HSV (types 1 and 2) was associated with advanced age, elevated BMI, lower caloric intake, diminished protein and fat consumption, higher binge drinking frequency, increased lifetime sexual partners, female predominance, increased smoking prevalence, reduced zinc intake, and the highest disease incidence during the 2007–2008 survey cycle. Notably, HSV-1 seropositivity was linked to higher cohabitation rates, with the highest seroprevalence observed in Mexican American individuals. In contrast, HSV-2 seropositivity was correlated with a lower prevalence of cohabitation, the highest incidence among non-Hispanic Black populations, reduced carbohydrate and sugar intake, and an elevated frequency of sexual partners annually. Relationship between dietary zinc intake and HSV infection Univariate analysis revealed that both HSV-1 and HSV-2 were associated with age; sex; race; marital status; smoking status; excessive alcohol consumption; lifetime number of sexual partners; year of data collection; and calorie intake, protein intake, dietary fiber intake, zinc intake, and zinc intake quartile groups. Additionally, HSV-1 was specifically associated with carbohydrate intake, whereas HSV-2 was associated with sugar intake (Table 2). In the multivariable-adjusted model evaluating the associations between zinc intake quartiles (low, low-moderate, moderate-high, and high intake) and HSV-1/HSV-2 infections, the following results were observed. Compared with the low-zinc intake group, the low-moderate intake group presented a reduced risk of HSV-1 infection, with an adjusted odds ratio (OR) of 0.84 (95% confidence interval [CI]: 0.70–0.99, p = 0.028), and a statistically significant reduction in HSV-2 infection risk (OR = 0.81, 95% CI: 0.66–0.99, p = 0.039). Moderate–high zinc intake was associated with a decreased risk of HSV-1 infection (OR = 0.84, 95% CI: 0.70–0.99, p = 0.042), but no statistically significant reduction in HSV-2 infection risk was observed (OR = 0.84, 95% CI: 0.68–1.05, p = 0.136). High zinc intake did not significantly reduce the risk of HSV-1 or HSV-2 infection (p > 0.05) (Table 3). These results suggest that low-to-moderate zinc intake may confer a protective effect against both HSV-1 and HSV-2 infections; however, this effect diminishes or becomes insignificant at higher intake levels. Furthermore, restricted cubic spline (RCS) analysis indicated consistent U-shaped trends in the relationships between zinc intake and HSV-1 (p for nonlinearity = 0.005) and HSV-2 (p for nonlinearity < 0.01), supporting a nonlinear dose‒response pattern (Figure 2). Stratified analyses based on additional variables and sensitivity analysis We performed stratified analyses across multiple subgroups to assess potential variations in the association between dietary zinc intake and HSV-1/HSV-2 seropositivity. Stratification according to sex, age and the number of sexual partners revealed no additional interactions (Figure 3). We tested the sensitivity of the model using multiple imputation, and the results showed that both HSV-1 and HSV-2 were consistent with the trends in the original model. The results are shown in Table 4. This sentence was very confusing. Please check that the edit retains your intended meaning. DISCUSSION This cross-sectional study revealed a U-shaped association between dietary zinc intake and HSV-1/HSV-2 seropositivity, indicating that both excessively high and low zinc intake levels lack protective effects against HSV-1 and HSV-2. Stratified and sensitivity analyses further confirmed this conclusion, suggesting that protective effects against HSV-1 and HSV-2 were observed only at moderate zinc intake levels. As an essential trace element in humans, zinc plays a critical role in immune regulation, and its association with HSV infection has been demonstrated in multiple studies [ 19 – 21 ]. In vitro studies have shown that zinc lactate inactivates more than 97% of HSV-1/2 clinical isolates and disrupts the HSV lifecycle by inhibiting viral protein synthesis and potentially interfering with DNA replication [ 22 ]. Clinical trials revealed that systemic zinc supplementation (e.g., daily oral administration of 22.5–50 mg zinc sulfate) significantly reduced the severity of recurrent herpes labialis (50% decrease in episode frequency) and genital herpes (57% reduction in lesion days), with efficacy increasing over time, suggesting a nonplacebo effect [ 23 , 24 ]. Notably, deficient serum zinc levels are strongly correlated with prolonged herpes labialis duration, further supporting the role of zinc in host antiviral defense [ 25 ]. Topical zinc formulations (e.g., zinc oxide/glycine cream or 4% zinc sulfate solution) also have potential for shortening lesion duration, reducing recurrence rates (≤ 6% vs. 80% in controls), and alleviating symptoms, with no significant side effects [ 26 , 27 ]. Zinc oxide nanoparticles were found to directly antagonize HSV-1 infection by suppressing viral genomic replication [ 28 ]. These findings collectively confirm that zinc can directly inhibit the replication of HSV. However, excessive zinc intake also poses risks. Long-term high-dose zinc intake may lead to copper deficiency-related symptoms, including impaired immune function, reduced high-density lipoprotein levels, elevated low-density lipoprotein levels, and neurological symptoms [ 29 , 30 ]. Therefore, maintaining appropriate zinc intake is critically linked to overall health. Consequently, the protective effects of zinc against HSV require further large-scale experimental validation to establish optimal intake thresholds. Strengths and Limitations Our study has several strengths, the most significant of which is the use of data from multiple cycles. This approach helped expand the sample size and enhance the statistical power and generalizability of our findings. Another notable advantage lies in our focus on the continuity of dietary intake. By comparing food consumed daily to usual, we ensured consistency in the assessment of zinc consumption, making the results more reflective of real-world scenarios. Our study employed diverse statistical methodologies, including multivariate logistic regression and stratified analysis. This methodological diversity strengthened the comprehensiveness of our analysis, enabling a thorough investigation of the association between zinc intake and HSV infection. However, this study has several limitations. First, we were unable to determine the timing of the initial HSV infection, which introduces a potential temporal bias between seropositivity and dietary intake. Second, residual confounding effects may persist despite rigorous statistical adjustments. Furthermore, the inherent limitations of the 24-hour dietary recall method, such as recall bias and day-to-day variability, may compromise the reliability and validity of nutritional assessments. Finally, the cross-sectional design precluded the establishment of causal inferences. Future research should prioritize large-scale prospective cohort studies and multicenter randomized controlled trials to elucidate the causal relationship between dietary zinc intake and HSV infection. CONCLUSION Our analysis revealed a U-shaped association between zinc intake and HSV-1 and HSV-2 seropositivity. Although the dual antiviral and immunomodulatory properties of zinc are mechanistically plausible, causality requires confirmation in longitudinal cohorts and randomized controlled trials. If validated, zinc supplementation may emerge as a cost-effective adjunct strategy for HSV prevention. Declarations Acknowledgments We thank Dr. Liu Jie (People's Liberation Army General Hospital, Beijing, China) for his assistance in this revision. Author contributions M.P. Li, and Y.N. Qin share the role of corresponding authors. C.H. Liu proposed the study concepts,performed the data analysis and wrote the manuscript. M.P. Li performed the data analysis and revised the manuscript.J.J. Liu and Z.Y. Lv retrieved and performed the data analysis.Y.G. Liu and Y.N. Qin edited the manuscript and interpreted the results. All the authors read and approved the final manuscript. Funding This work was supported by the Natural Science Foundation of Shandong Province (Grant number: ZR2024ME266) and the Shandong Provincial Science and Technology Office. Data availability All data were sourced from the publicly available NHANES. All statistical code are available by contrating the corresponding authors with proper reason. Clinical Trial Not applicable Ethics approval and consent to participate All data were sourced from the publicly available NHANES, which received informed consent from all participants and was ethically approved by the NCHS Research Ethics Review Board. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Footnotes not applicable Contributor Information Mengpeng Li, Email: [email protected] Yanan Qin, Email: [email protected] References Chaiyakunapruk N, Lee SWH, Kulchaitanaroaj P, et al. Estimated global and regional economic burden of genital herpes simplex virus infection among 15-49 year-olds in 2016. BMC Glob Public Health 2024 ; 2:42. Tognarelli EI, Palomino TF, Corrales N, Bueno SM, Kalergis AM, Gonzalez PA. Herpes Simplex Virus Evasion of Early Host Antiviral Responses. Front Cell Infect Microbiol 2019 ; 9:127. Qiao H, Guo M, Shang J, et al. Herpes simplex virus type 1 infection leads to neurodevelopmental disorder-associated neuropathological changes. PLoS Pathog 2020 ; 16:e1008899. Looker KJ, Welton NJ, Sabin KM, et al. Global and regional estimates of the contribution of herpes simplex virus type 2 infection to HIV incidence: a population attributable fraction analysis using published epidemiological data. 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Int J Nanomedicine 2021 ; 16:8221-33. Sun Y, Wang Y, Wang D, Zhou Q. Dietary zinc intake, supplemental zinc intake and serum zinc levels and the prevalence of kidney stones in adults. J Trace Elem Med Biol 2020 ; 57:126410. Fani M, Khodadad N, Ebrahimi S, et al. Zinc Sulfate in Narrow Range as an In Vitro Anti-HSV-1 Assay. Biol Trace Elem Res 2020 ; 193:410-3. Femiano F, Gombos F, Scully C. Recurrent herpes labialis: a pilot study of the efficacy of zinc therapy. J Oral Pathol Med 2005 ; 34:423-5. Fitzherbert JC. Genital herpes and zinc. Med J Aust 1979 ; 1:399. Ranjbar Z, Zahed M, Ranjbar MA, Shirmardan Z. Comparative study of serum zinc concentration in recurrent herpes labialis patients and healthy individuals. BMC Oral Health 2020 ; 20:296. Godfrey HR, Godfrey NJ, Godfrey JC, Riley D. A randomized clinical trial on the treatment of oral herpes with topical zinc oxide/glycine. Altern Ther Health Med 2001 ; 7:49-56. Mahajan BB, Dhawan M, Singh R. Herpes genitalis - Topical zinc sulfate: An alternative therapeutic and modality. Indian J Sex Transm Dis AIDS 2013 ; 34:32-4. Tavakoli A, Ataei-Pirkooh A, Mm Sadeghi G, et al. Polyethylene glycol-coated zinc oxide nanoparticle: an efficient nanoweapon to fight against herpes simplex virus type 1. Nanomedicine (Lond) 2018 ; 13:2675-90. Doherty K, Connor M, Cruickshank R. Zinc-containing denture adhesive: a potential source of excess zinc resulting in copper deficiency myelopathy. Br Dent J 2011 ; 210:523-5. Schoofs H, Schmit J, Rink L. Zinc Toxicity: Understanding the Limits. Molecules 2024 ; 29. Tables Tables 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Published Journal Publication published 26 Feb, 2026 Read the published version in BMC Infectious Diseases → Version 1 posted Editorial decision: Revision requested 17 Oct, 2025 Reviews received at journal 07 Sep, 2025 Reviews received at journal 16 Aug, 2025 Reviewers agreed at journal 16 Aug, 2025 Reviewers agreed at journal 11 Aug, 2025 Reviewers invited by journal 04 Aug, 2025 Editor invited by journal 09 Jul, 2025 Editor assigned by journal 07 Jul, 2025 Submission checks completed at journal 07 Jul, 2025 First submitted to journal 03 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7036290","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":495570465,"identity":"ba7c5a22-d97a-4560-b9cc-0dbe62dc1acd","order_by":0,"name":"Chunhua Liu","email":"","orcid":"","institution":"Liaocheng People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chunhua","middleName":"","lastName":"Liu","suffix":""},{"id":495570467,"identity":"bda49e97-b5f3-4abc-a196-e461d11755fe","order_by":1,"name":"Yingguo Liu","email":"","orcid":"","institution":"Liaocheng Infectious Diseases Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yingguo","middleName":"","lastName":"Liu","suffix":""},{"id":495570468,"identity":"8359b6e2-53f5-492f-9ec1-189d761d6f85","order_by":2,"name":"Jingjing Liu","email":"","orcid":"","institution":"Liaocheng People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Liu","suffix":""},{"id":495570470,"identity":"4e81a017-bea0-4e96-b8d2-206292aa9b9a","order_by":3,"name":"Zhaoyong Lv","email":"","orcid":"","institution":"Liaocheng People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhaoyong","middleName":"","lastName":"Lv","suffix":""},{"id":495570473,"identity":"c74fe9f2-4897-4961-a4ce-c80a7b223765","order_by":4,"name":"Mengpeng Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBACPmYGBmYGAxs5NvbGxocfiNHCBtZSkGbMz3O42ViCKC0MIC0fDifOnJHeJsBDlBZ27rTHBQaHEzfcfNjGIMFgJ6fbQNBhvNuNZxikG2+4ndj2oIAh2djsAGEt26R5DKxlgVraDSQYDiRuI1ILM+OGmwfbJHhI0OKsOHMGIylaZhiAAjkRGMgGRPiFn//sNumCP6CoPP7w4YcKOzmCWtCAAWnKR8EoGAWjYBTgAACWtTtG6eJougAAAABJRU5ErkJggg==","orcid":"","institution":"Liaocheng People's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Mengpeng","middleName":"","lastName":"Li","suffix":""},{"id":495570475,"identity":"67615b33-3e5f-4360-8a8c-28adc267b84f","order_by":5,"name":"Yanan Qin","email":"","orcid":"","institution":"Liaocheng People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yanan","middleName":"","lastName":"Qin","suffix":""}],"badges":[],"createdAt":"2025-07-03 09:23:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7036290/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7036290/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12879-026-12927-1","type":"published","date":"2026-02-26T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88443510,"identity":"73be0d7f-fe60-4a8c-8e25-685ec09271a3","added_by":"auto","created_at":"2025-08-06 13:16:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":62366,"visible":true,"origin":"","legend":"\u003cp\u003eThe study's flow diagram. HSV, herpes simplex virus; NHANES, National Health and Nutrition Examination Survey.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036290/v1/f67ae6541d7126c59bbb296a.jpg"},{"id":88443513,"identity":"96078630-ef5d-4474-8b7f-c5256983a9cb","added_by":"auto","created_at":"2025-08-06 13:16:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41114,"visible":true,"origin":"","legend":"\u003cp\u003eAssociation between dietary zinc intake and the dietary zinc intake odds ratio (HSV‐1 on the left; HSV‐2 on the right). The solid and dashed lines represent the predicted values and 95% confidence intervals, respectively. They were adjusted for age, sex, race and ethnicity, smoking status, excessive alcohol consumption, sex partner, BMI, marital status, source, calorie consumption, protein consumption, carbohydrate consumption, sugar consumption, fiber consumption, and fat consumption. Only 99% of the data are shown.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036290/v1/4bf5452a0cac3fe95cfc78d6.jpg"},{"id":88443512,"identity":"3637a051-0217-4d5f-ad1e-caace861d792","added_by":"auto","created_at":"2025-08-06 13:16:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":125078,"visible":true,"origin":"","legend":"\u003cp\u003eForest plot of multivariable logistics analysis between dietary zinc intake and HSV-1 or HSV-2. Except for the stratification component itself, each stratification factor was adjusted for age, sex, race and ethnicity, smoking status, excessive alcohol consumption, sex partner, BMI, marital status, source, calorie consumption, protein consumption, carbohydrate consumption, sugar consumption, fiber consumption, fat consumption.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036290/v1/9289c367efec8b8378051882.jpg"},{"id":103765755,"identity":"6a6326f0-16d0-4c20-9623-6674c47773b9","added_by":"auto","created_at":"2026-03-02 16:08:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":889412,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7036290/v1/3573459c-7068-41dd-8db0-bf7cf980722d.pdf"},{"id":88443511,"identity":"d8417f09-5bfa-4374-865a-5787dd344d77","added_by":"auto","created_at":"2025-08-06 13:16:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32633,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7036290/v1/b703bfda2a3dddd702544283.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Association between dietary zinc intake and herpes simplex virus seropositivity in U.S. adults: a retrospective cross-sectional study","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHerpes simplex virus (HSV), a double-stranded DNA pathogen with a global prevalence, manifests as two primary serotypes: HSV-1 and HSV-2. HSV-1 spreads predominantly through oral‒to‒oral contact, infecting more than 67% of individuals under 50 years of age, whereas HSV-2 is transmitted primarily via sexual routes, affecting approximately 17% of the 15\u0026ndash;49 age cohort [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Following initial mucosal invasion, the virus establishes lifelong latency in the sensory ganglia, characterized by periodic reactivation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although most outbreaks involve self-limiting mucocutaneous lesions, severe complications may arise, including viral encephalitis, herpetic keratitis (a leading cause of infectious blindness), and increased susceptibility to human immunodeficiency virus (HIV) infection [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The absence of curative vaccines or virucidal agents combined with asymptomatic viral shedding that facilitates transmission underscores the urgent need to investigate modifiable risk factors, such as dietary patterns, in HSV pathogenesis and epidemiology [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eZinc is a crucial trace element for human growth, development, and maintenance of immune function [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Its immunomodulatory properties extend to both innate and adaptive immunity, wherein it participates in lymphocyte differentiation, cytokine production, and phagocytic activity [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Zinc status serves as a critical determinant of antiviral immunity, with zinc-deficient populations demonstrating heightened susceptibility to viral infections, including HIV and hepatitis C virus [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Moreover, emerging evidence has revealed the direct inhibitory effects of zinc on HSV replication through mechanisms involving viral DNA polymerase interference and capsid destabilization [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although the interplay between serum zinc status and viral pathogenesis has been extensively investigated, the specific relationship between dietary zinc intake and HSV seropositivity remains unexplored.\u003c/p\u003e\u003cp\u003eTherefore, further research is necessary to understand the association between dietary zinc consumption and HSV seropositivity. Data from the National Health and Nutrition Examination Survey (NHANES) were drawn upon in the present study.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cb\u003eStudy population\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe NHANES, organized by the United States Centers for Disease Control and Prevention (CDC), serves as a critical surveillance system monitoring population health indicators among noninstitutionalized U.S. residents. In accordance with the approval of the National Center for Health Statistics Research Ethics Committee, the protocol mandates documented consent from all participants prior to enrollment. For our retrospective study that analyzed existing deidentified records, no supplementary ethical clearance or informed consent was necessary according to federal regulations. Publicly accessible datasets spanning 2007\u0026ndash;2016 (5 biennial cycles) were retrieved from the official NHANES repository (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cdc.gov/nchs/nhanes\u003c/span\u003e\u003cspan address=\"https://www.cdc.gov/nchs/nhanes\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Our analytical cohort excluded individuals whose nutritional zinc records were incomplete, whose serological data for herpes simplex virus were unavailable, or whose covariate information was missing. The final sample included complete case data from eligible participants across the designated survey periods.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDietary zinc intake\u003c/b\u003e\u003c/p\u003e\u003cp\u003eParticipants in the NHANES dietary survey reported their food and beverage consumption over a 24-hour period, with data collected between 2006 and 2016 via the automated multiple pass method (AMPM) to ensure precise nutrient calculations on the basis of reported intake. The detailed methodologies are described in the NHANES Dietary Interviewer Procedure Manual. Zinc intake was analyzed as a continuous variable and categorized into quartiles (Q1\u0026ndash;Q4). To validate these results, individuals whose reported 24-hour dietary intake aligned with their habitual consumption patterns were prioritized. Specifically, participants were screened via the variable DR1_300, which corresponds to the following question: \"Was the amount of food that {you/NAME} ate yesterday much more than usual, usual, or much less than usual?\" Only those who responded \"usual\" were included. Additionally, the zinc intake categories for Q1 were as follows: \u0026lt;7.51 mg/day, Q2: 7.51\u0026ndash;10.87 mg/day, Q3: 10.87\u0026ndash;15.63 mg/day, and Q4: \u0026gt;15.63 mg/day.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHSV antibody measurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify HSV-1 and HSV-2 infections, blood samples obtained through venipentesis were analyzed via a solid-phase enzyme immunopoint assay. The publicly released NHANES data file includes HSV-1 data for the 14\u0026ndash;49 age group and HSV-2 data for the 18\u0026ndash;49 age group. In our study, these two variables were considered independent factors; that is, HSV-1 and HSV-2 infections were defined as positive for their respective viral antibodies, and negative infections were defined as negative for their respective viral antibodies. Records classified as \"inconclusive\" were excluded from our analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCovariates\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe evaluated several potential covariates on the basis of previous literature [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], including age; sex; race and ethnicity; body mass index (BMI); marital status; smoking status; excessive alcohol consumption; number of lifetime sexual partners; acquisition time; and caloric, protein, carbohydrate, sugar, fiber, and fat intake. The categories for race and ethnicity included Mexican American, other Hispanic, non-Hispanic White, non-Hispanic Black, and other races. Marital status was categorized as living alone or being married/living with a partner. BMI was calculated via a standardized approach considering both weight and height. Smoking was defined as having smoked at least 100 cigarettes. Excessive alcohol consumption was defined as periods of drinking 4 or more drinks per day for women and 5 or more for men. Sexual partners were defined as the total number of same-sex or opposite-sex partners with whom any sexual activity occurred. A dietary recall interview was conducted prior to the Mobile Examination Center (MEC) interview to collect participants' nutritional data over a 24-hour period, including the consumption of calories, protein, carbohydrates, sugar, fiber, and fat.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThis was a secondary analysis of the datasets available in the public domain. For continuous variables with a normal distribution, we reported the mean and standard deviation (SD). For nonnormally distributed variables, we used medians and interquartile ranges (IQRs). Categorical variables are presented as percentages. To compare differences between groups, we used one-way analysis of variance (ANOVA) for normally distributed data, the Mann-Whitney test for nonnormally distributed data, and the chi‒square test for categorical data. Logistic regression analysis was used to explore the association between dietary zinc intake and HSV seropositivity. Initially, we used univariate logistic regression to identify factors associated with HSV-1 and HSV-2 seropositivity. After controlling for potential confounding factors, we used a multivariate logistic regression model to clarify the relationships between dietary zinc intake and HSV-1 and HSV-2 seropositivity. Model I was adjusted for age, sex, and race. In Model II, we further adjusted for variables such as marital status, smoking status, excessive alcohol consumption, number of lifetime sexual partners, and acquisition time. In Model III, we included all the covariates.\u003c/p\u003e\u003cp\u003eFurthermore, restricted cubic spline (RCS) regression analysis was used to assess whether there was a nonlinear relationship between dietary zinc intake and HSV-1 and HSV-2 seropositivity. Finally, a subgroup analysis was conducted to compare the\u003c/p\u003e\u003cp\u003eThe interaction between dietary zinc intake and HSV-1 and HSV-2 seropositivity across different subgroups was used to evaluate the robustness of the study results.\u003c/p\u003e\u003cp\u003eAll analyses were conducted via R 4.4.2 statistical software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.R-project.org\u003c/span\u003e\u003cspan address=\"http://www.R-project.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). P values were considered statistically significant if they were less than 0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eStudy population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 50,588 participants were included in the NHANES conducted between 2007 and 2016. After individuals with missing HSV-1 and HSV-2 data were excluded, 14,599 individuals were included in the study. After selecting those for whom data regarding dietary zinc were available, 9,574 people remained. After further screening for individuals who could recollect the food they consumed the previous day\u003ca\u003e[A1]\u003c/a\u003e and excluding individuals with missing covariates, 6,483 people remained. The final population included 2,666 HSV-1-negative and 3,817 HSV-1-positive individuals. For HSV-2, there were 5,230 HSV-2-negative individuals and 1,253 HSV-2-positive individuals. The mean age was 34.52 ± 8.64 years, and 61.4% were male. The mean daily zinc consumption amount was 12.57 ± 8.23 mg (10.12 ± 5.42 mg for women and 14.87 ± 9.64 mg for men) (Fig.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBaseline characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 1 summarizes the baseline demographic characteristics stratified by herpes simplex virus (HSV) infection status. Seropositivity for HSV (types 1 and 2) was associated with advanced age, elevated BMI, lower caloric intake, diminished protein and fat consumption, higher binge drinking frequency, increased lifetime sexual partners, female predominance, increased smoking prevalence, reduced zinc intake, and the highest disease incidence during the 2007–2008 survey cycle. Notably, HSV-1 seropositivity was linked to higher cohabitation rates, with the highest seroprevalence observed in Mexican American individuals. In contrast, HSV-2 seropositivity was correlated with a lower prevalence of cohabitation, the highest incidence among non-Hispanic Black populations, reduced carbohydrate and sugar intake, and an elevated frequency of sexual partners annually.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelationship \u003c/strong\u003e\u003cstrong\u003ebetween\u003c/strong\u003e\u003cstrong\u003e dietary zinc intake and HSV \u003c/strong\u003e\u003cstrong\u003einfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnivariate analysis revealed that both HSV-1 and HSV-2 were associated with age; sex; race; marital status; smoking status; excessive alcohol consumption; lifetime number of sexual partners; year of data collection; and calorie intake, protein intake, dietary fiber intake, zinc intake, and zinc intake quartile groups. Additionally, HSV-1 was specifically associated with carbohydrate intake, whereas HSV-2 was associated with sugar intake (Table 2).\u003c/p\u003e\n\u003cp\u003eIn the multivariable-adjusted model evaluating the associations between zinc intake quartiles (low, low-moderate, moderate-high, and high intake) and HSV-1/HSV-2 infections, the following results were observed. Compared with the low-zinc intake group, the low-moderate intake group presented a reduced risk of HSV-1 infection, with an adjusted odds ratio (OR) of 0.84 (95% confidence interval [CI]: 0.70–0.99, p = 0.028), and a statistically significant reduction in HSV-2 infection risk (OR = 0.81, 95% CI: 0.66–0.99, p = 0.039). Moderate–high zinc intake was associated with a decreased risk of HSV-1 infection (OR = 0.84, 95% CI: 0.70–0.99, p = 0.042), but no statistically significant reduction in HSV-2 infection risk was observed (OR = 0.84, 95% CI: 0.68–1.05, p = 0.136). High zinc intake did not significantly reduce the risk of HSV-1 or HSV-2 infection (p \u0026gt; 0.05) (Table 3).\u003c/p\u003e\n\u003cp\u003eThese results suggest that low-to-moderate zinc intake may confer a protective effect against both HSV-1 and HSV-2 infections; however, this effect diminishes or becomes insignificant at higher intake levels. Furthermore, restricted cubic spline (RCS) analysis indicated consistent U-shaped trends in the relationships between zinc intake and HSV-1 (p for nonlinearity = 0.005) and HSV-2 (p for nonlinearity \u0026lt; 0.01), supporting a nonlinear dose‒response pattern (Figure 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStratified analyses based on additional variables and sensitivity analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed stratified analyses across multiple subgroups to assess potential variations in the association between dietary zinc intake and HSV-1/HSV-2 seropositivity. Stratification according to sex, age and the number of sexual partners revealed no additional interactions (Figure 3). We tested the sensitivity of the model using multiple imputation, and the results showed that both HSV-1 and HSV-2 were consistent with the trends in the original model. The results are shown in Table 4.\u003c/p\u003e\n\u003cp\u003eThis sentence was very confusing. Please check that the edit retains your intended meaning.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis cross-sectional study revealed a U-shaped association between dietary zinc intake and HSV-1/HSV-2 seropositivity, indicating that both excessively high and low zinc intake levels lack protective effects against HSV-1 and HSV-2. Stratified and sensitivity analyses further confirmed this conclusion, suggesting that protective effects against HSV-1 and HSV-2 were observed only at moderate zinc intake levels.\u003c/p\u003e\u003cp\u003eAs an essential trace element in humans, zinc plays a critical role in immune regulation, and its association with HSV infection has been demonstrated in multiple studies [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In vitro studies have shown that zinc lactate inactivates more than 97% of HSV-1/2 clinical isolates and disrupts the HSV lifecycle by inhibiting viral protein synthesis and potentially interfering with DNA replication [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Clinical trials revealed that systemic zinc supplementation (e.g., daily oral administration of 22.5\u0026ndash;50 mg zinc sulfate) significantly reduced the severity of recurrent herpes labialis (50% decrease in episode frequency) and genital herpes (57% reduction in lesion days), with efficacy increasing over time, suggesting a nonplacebo effect [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Notably, deficient serum zinc levels are strongly correlated with prolonged herpes labialis duration, further supporting the role of zinc in host antiviral defense [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Topical zinc formulations (e.g., zinc oxide/glycine cream or 4% zinc sulfate solution) also have potential for shortening lesion duration, reducing recurrence rates (\u0026le;\u0026thinsp;6% vs. 80% in controls), and alleviating symptoms, with no significant side effects [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Zinc oxide nanoparticles were found to directly antagonize HSV-1 infection by suppressing viral genomic replication [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These findings collectively confirm that zinc can directly inhibit the replication of HSV. However, excessive zinc intake also poses risks. Long-term high-dose zinc intake may lead to copper deficiency-related symptoms, including impaired immune function, reduced high-density lipoprotein levels, elevated low-density lipoprotein levels, and neurological symptoms [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, maintaining appropriate zinc intake is critically linked to overall health. Consequently, the protective effects of zinc against HSV require further large-scale experimental validation to establish optimal intake thresholds.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStrengths and Limitations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur study has several strengths, the most significant of which is the use of data from multiple cycles. This approach helped expand the sample size and enhance the statistical power and generalizability of our findings. Another notable advantage lies in our focus on the continuity of dietary intake. By comparing food consumed daily to usual, we ensured consistency in the assessment of zinc consumption, making the results more reflective of real-world scenarios. Our study employed diverse statistical methodologies, including multivariate logistic regression and stratified analysis. This methodological diversity strengthened the comprehensiveness of our analysis, enabling a thorough investigation of the association between zinc intake and HSV infection.\u003c/p\u003e\u003cp\u003eHowever, this study has several limitations. First, we were unable to determine the timing of the initial HSV infection, which introduces a potential temporal bias between seropositivity and dietary intake. Second, residual confounding effects may persist despite rigorous statistical adjustments. Furthermore, the inherent limitations of the 24-hour dietary recall method, such as recall bias and day-to-day variability, may compromise the reliability and validity of nutritional assessments. Finally, the cross-sectional design precluded the establishment of causal inferences. Future research should prioritize large-scale prospective cohort studies and multicenter randomized controlled trials to elucidate the causal relationship between dietary zinc intake and HSV infection.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eOur analysis revealed a U-shaped association between zinc intake and HSV-1 and HSV-2 seropositivity. Although the dual antiviral and immunomodulatory properties of zinc are mechanistically plausible, causality requires confirmation in longitudinal cohorts and randomized controlled trials. If validated, zinc supplementation may emerge as a cost-effective adjunct strategy for HSV prevention.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Liu Jie (People\u0026apos;s Liberation Army General Hospital, Beijing, China) for his assistance in this revision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.P. Li, and Y.N. Qin share the role of corresponding authors. C.H. Liu proposed the\u0026nbsp;study concepts,performed the\u0026nbsp;data analysis and wrote the manuscript. M.P. Li performed the\u0026nbsp;data analysis and revised the manuscript.J.J. Liu and Z.Y. Lv retrieved and performed the\u0026nbsp;data analysis.Y.G. Liu and Y.N. Qin edited the manuscript and interpreted the results. All the\u0026nbsp;authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Shandong Province (Grant number: ZR2024ME266) and the Shandong Provincial Science and Technology Office.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were sourced from the publicly available NHANES. All statistical code are available by contrating the corresponding authors with proper reason.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were sourced from the publicly available NHANES, which received informed consent from all participants and was ethically approved by the NCHS Research Ethics Review Board.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFootnotes\u003c/h2\u003e\n\u003cp\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributor Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMengpeng Li, Email: [email protected]\u003c/p\u003e\n\u003cp\u003eYanan Qin, Email: [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChaiyakunapruk N, Lee SWH, Kulchaitanaroaj P, et al. 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Dietary zinc intake and incident chronic kidney disease. Clin Nutr \u003cstrong\u003e2021\u003c/strong\u003e; 40:1039-45.\u003c/li\u003e\n\u003cli\u003eLin Y, Kang D, Chen Q. Association between dietary zinc intake and asthma in overweight or obese children and adolescents: A cross-sectional analysis of NHANES. World Allergy Organ J \u003cstrong\u003e2025\u003c/strong\u003e; 18:101044.\u003c/li\u003e\n\u003cli\u003eHouston DMJ, Bugert JJ, Denyer SP, Heard CM. Correction: Potentiated virucidal activity of pomegranate rind extract (PRE) and punicalagin against Herpes simplex virus (HSV) when co-administered with zinc (II) ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV. PloS one \u003cstrong\u003e2017\u003c/strong\u003e; 12:e0188609.\u003c/li\u003e\n\u003cli\u003eMelk MM, El-Hawary SS, Melek FR, et al. Antiviral Activity of Zinc Oxide Nanoparticles Mediated by Plumbago indica L. Extract Against Herpes Simplex Virus Type 1 (HSV-1). Int J Nanomedicine \u003cstrong\u003e2021\u003c/strong\u003e; 16:8221-33.\u003c/li\u003e\n\u003cli\u003eSun Y, Wang Y, Wang D, Zhou Q. Dietary zinc intake, supplemental zinc intake and serum zinc levels and the prevalence of kidney stones in adults. J Trace Elem Med Biol \u003cstrong\u003e2020\u003c/strong\u003e; 57:126410.\u003c/li\u003e\n\u003cli\u003eFani M, Khodadad N, Ebrahimi S, et al. Zinc Sulfate in Narrow Range as an In Vitro Anti-HSV-1 Assay. Biol Trace Elem Res \u003cstrong\u003e2020\u003c/strong\u003e; 193:410-3.\u003c/li\u003e\n\u003cli\u003eFemiano F, Gombos F, Scully C. Recurrent herpes labialis: a pilot study of the efficacy of zinc therapy. J Oral Pathol Med \u003cstrong\u003e2005\u003c/strong\u003e; 34:423-5.\u003c/li\u003e\n\u003cli\u003eFitzherbert JC. Genital herpes and zinc. Med J Aust \u003cstrong\u003e1979\u003c/strong\u003e; 1:399.\u003c/li\u003e\n\u003cli\u003eRanjbar Z, Zahed M, Ranjbar MA, Shirmardan Z. Comparative study of serum zinc concentration in recurrent herpes labialis patients and healthy individuals. BMC Oral Health \u003cstrong\u003e2020\u003c/strong\u003e; 20:296.\u003c/li\u003e\n\u003cli\u003eGodfrey HR, Godfrey NJ, Godfrey JC, Riley D. A randomized clinical trial on the treatment of oral herpes with topical zinc oxide/glycine. Altern Ther Health Med \u003cstrong\u003e2001\u003c/strong\u003e; 7:49-56.\u003c/li\u003e\n\u003cli\u003eMahajan BB, Dhawan M, Singh R. Herpes genitalis - Topical zinc sulfate: An alternative therapeutic and modality. Indian J Sex Transm Dis AIDS \u003cstrong\u003e2013\u003c/strong\u003e; 34:32-4.\u003c/li\u003e\n\u003cli\u003eTavakoli A, Ataei-Pirkooh A, Mm Sadeghi G, et al. Polyethylene glycol-coated zinc oxide nanoparticle: an efficient nanoweapon to fight against herpes simplex virus type 1. Nanomedicine (Lond) \u003cstrong\u003e2018\u003c/strong\u003e; 13:2675-90.\u003c/li\u003e\n\u003cli\u003eDoherty K, Connor M, Cruickshank R. Zinc-containing denture adhesive: a potential source of excess zinc resulting in copper deficiency myelopathy. Br Dent J \u003cstrong\u003e2011\u003c/strong\u003e; 210:523-5.\u003c/li\u003e\n\u003cli\u003eSchoofs H, Schmit J, Rink L. Zinc Toxicity: Understanding the Limits. Molecules \u003cstrong\u003e2024\u003c/strong\u003e; 29.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"zinc, herpes simplex virus, National Health and Nutrition Examination Survey, epidemiology","lastPublishedDoi":"10.21203/rs.3.rs-7036290/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7036290/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eAlthough recent research has demonstrated an association between serum zinc deficiency and susceptibility to various viral infections, the relationship between dietary zinc intake and herpes simplex virus (HSV) seropositivityremains unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e The National Health and Nutrition Examination Survey (NHANES) (2007--2016) provides data on HSV-1 and HSV-2 status and dietary zinc intake. The associationsbetween dietary zinc and HSV-1 and HSV-2 were evaluated via various statistical methods, including multivariate logistic regression, restricted cubic spline analysis, and subgroup analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eIn total, 6,483 individuals were enrolled, with 58.9% (3,817/6,483) testing positive for HSV-1 and 19.3% (1,253/6,483) testing positive for HSV-2. After adjusting for all covariates in the multivariate logistic regression, compared with the lowest zinc intake group (Q1: \u0026lt;7.51 mg/day), the adjusted odds ratios (ORs) for HSV-1 and HSV-2 in the higher-zinc intake groups were as follows: for HSV-1, Q2 (7.51--10.87 mg/day) had an OR of 0.81 (95% confidence interval [CI]: 0.72--0.98, \u003cem\u003ep \u003c/em\u003e= 0.027), and Q3 (10.87--15.63 mg/day) had an OR of 0.85 (95% CI: 0.70--1.03, \u003cem\u003ep\u003c/em\u003e = 0.093), and Q4 (\u0026gt;15.63 mg/day) had an OR of 0.84 (95% CI: 0.67--1.04, \u003cem\u003ep\u003c/em\u003e = 0.114); for HSV-2, Q2 had an OR of 0.80 (95% CI: 0.64–1.00, \u003cem\u003ep\u003c/em\u003e = 0.050), Q3 had an OR of 0.81 (95% CI: 0.64--1.02, \u003cem\u003ep\u003c/em\u003e = 0.069), and Q4 had an OR of 0.75 (95\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eDietary zinc intake exhibited a U-shaped association with HSV-1 and HSV-2 seropositivity, indicating that moderate zinc intake has a protective effect.\u003c/p\u003e","manuscriptTitle":"Association between dietary zinc intake and herpes simplex virus seropositivity in U.S. adults: a retrospective cross-sectional study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-06 13:16:29","doi":"10.21203/rs.3.rs-7036290/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-17T13:18:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-08T02:39:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-16T19:58:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"250106343398050822385540408121393600154","date":"2025-08-16T14:23:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328785941382025610688172294195059462431","date":"2025-08-11T22:56:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-04T17:52:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-09T05:08:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-07T07:28:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-07T07:27:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2025-07-03T09:18:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ebe1146a-c3e3-4867-8cd3-ac1f07df6ab6","owner":[],"postedDate":"August 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:05:59+00:00","versionOfRecord":{"articleIdentity":"rs-7036290","link":"https://doi.org/10.1186/s12879-026-12927-1","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2026-02-26 15:57:48","publishedOnDateReadable":"February 26th, 2026"},"versionCreatedAt":"2025-08-06 13:16:29","video":"","vorDoi":"10.1186/s12879-026-12927-1","vorDoiUrl":"https://doi.org/10.1186/s12879-026-12927-1","workflowStages":[]},"version":"v1","identity":"rs-7036290","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7036290","identity":"rs-7036290","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}