{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T17:52:11Z","timestamp":1785433931575,"version":"3.56.0"},"reference-count":34,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2026,5,24]],"date-time":"2026-05-24T00:00:00Z","timestamp":1779580800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Prevention and Control of Emerging and Major Infectious Diseases-National Science and Technology Major Project","award":["2026ZD01909702"],"award-info":[{"award-number":["2026ZD01909702"]}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32370146"],"award-info":[{"award-number":["32370146"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["32370146"],"award-info":[{"award-number":["32370146"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Guangdong Special Support Plan Provincial Health Commission (HealthTalents) Project","award":["0720240122"],"award-info":[{"award-number":["0720240122"]}]},{"name":"Guangzhou Basic Research Program","award":["2023A03J0810"],"award-info":[{"award-number":["2023A03J0810"]}]},{"name":"Guangdong Province Basic and Applied Basic Research Fund Project","award":["2024A1515010995"],"award-info":[{"award-number":["2024A1515010995"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Marine algal polysaccharides have been widely investigated as antiviral candidates, yet nearly all anti-dengue studies have focused on sulfated species. Whether algal polysaccharides lacking prominent sulfation can inhibit dengue virus (DENV) remains unexplored. Here, we profiled the stage-specific antiviral activity of a heteropolysaccharide (GLHP) from Gracilaria lemaneiformis, whose Fourier-transform infrared (FT-IR) spectrum lacks characteristic sulfate ester absorption bands, against DENV serotype 2 (DENV-2) in Huh7 and BHK-21 cells. GLHP exhibited low cytotoxicity (CC50 exceeding 1000 \u03bcg\/mL in Huh7 cells and approximately 950 \u03bcg\/mL in BHK-21 cells). Time-of-addition analysis revealed that co-inoculation GLHP treatment (Co-inoc.) produced the strongest and most consistent inhibition of intracellular viral RNA, whereas pre-inoculation GLHP treatment (Pre-inoc.) was ineffective, indicating that the antiviral activity is predominantly associated with the virus\u2013cell contact and entry stage. GLHP additionally reduced extracellular progeny virus output under post-inoculation GLHP treatment (Post-inoc.) conditions, and this reduction exceeded the corresponding change in intracellular viral RNA levels, suggesting an additional effect that may involve either a late replication step or secondary entry blockade of progeny virions. Attenuation of virus-induced cytopathic effects under Co-inoc. conditions further supported the antiviral activity. To our knowledge, these findings identify GLHP as the first non-sulfated marine polysaccharide shown to exhibit stage-defined antiviral activity against DENV-2 and support further investigation of its antiviral potential and structural determinants.<\/jats:p>","DOI":"10.3390\/v18060594","type":"journal-article","created":{"date-parts":[[2026,5,25]],"date-time":"2026-05-25T02:32:28Z","timestamp":1779676348000},"page":"594","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Stage-of-Action Characterization of a Non-Sulfated Heteropolysaccharide from Gracilaria lemaneiformis Against Dengue Virus Serotype 2"],"prefix":"10.3390","volume":"18","author":[{"given":"Jiaxin","family":"Dai","sequence":"first","affiliation":[{"name":"School of Public Health, Guangdong Medical University, Dongguan 523808, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingfang","family":"Liu","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingshu","family":"Li","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zihan","family":"He","sequence":"additional","affiliation":[{"name":"School of Public Health, Guangdong Medical University, Dongguan 523808, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kexin","family":"Xi","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8263-3423","authenticated-orcid":false,"given":"Yushan","family":"Jiang","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuenan","family":"Zhang","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kefeng","family":"Wu","sequence":"additional","affiliation":[{"name":"The Marine Biomedical Research Institute of Guangdong Zhanjiang, School of Ocean and Tropical Medicine, Guangdong Medical University, Zhanjiang 524023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bao","family":"Zhang","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Zhao","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weiwei","family":"Xiao","sequence":"additional","affiliation":[{"name":"School of Public Health, Guangdong Medical University, Dongguan 523808, China"},{"name":"BSL-3 Laboratory, Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"504","DOI":"10.1038\/nature12060","article-title":"The Global Distribution and Burden of Dengue","volume":"496","author":"Bhatt","year":"2013","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1016\/S0140-6736(14)60572-9","article-title":"Dengue","volume":"385","author":"Guzman","year":"2015","journal-title":"Lancet"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1016\/S0092-8674(02)00660-8","article-title":"Structure of Dengue Virus: Implications for Flavivirus Organization, Maturation, and Fusion","volume":"108","author":"Kuhn","year":"2002","journal-title":"Cell"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"687","DOI":"10.1139\/cjm-2020-0572","article-title":"Dengue Virus: Epidemiology, Biology, and Disease Aetiology","volume":"67","author":"Roy","year":"2021","journal-title":"Can. J. Microbiol."},{"key":"ref_5","unstructured":"(2026, January 10). WHO Dengue. Available online: https:\/\/www.who.int\/news-room\/fact-sheets\/detail\/dengue-and-severe-dengue."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Shukla, R., Ramasamy, V., Shanmugam, R.K., Ahuja, R., and Khanna, N. (2020). Antibody-Dependent Enhancement: A Challenge for Developing a Safe Dengue Vaccine. Front. Cell Infect. Microbiol., 10.","DOI":"10.3389\/fcimb.2020.572681"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2009","DOI":"10.1056\/NEJMoa1903869","article-title":"Efficacy of a Tetravalent Dengue Vaccine in Healthy Children and Adolescents","volume":"381","author":"Biswal","year":"2019","journal-title":"N. Engl. J. Med."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Obi, J.O., Guti\u00e9rrez-Barbosa, H., Chua, J.V., and Deredge, D.J. (2021). Current Trends and Limitations in Dengue Antiviral Research. Trop. Med. Infect. Dis., 6.","DOI":"10.20944\/preprints202108.0364.v1"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1007\/s40506-023-00263-w","article-title":"Dengue: Update on Clinically Relevant Therapeutic Strategies and Vaccines","volume":"15","author":"Rathore","year":"2023","journal-title":"Curr. Treat. Options Infect. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"888","DOI":"10.1002\/ptr.8408","article-title":"Natural Compounds and Their Analogs as Antivirals Against Dengue Virus: A Review","volume":"39","author":"Komarudin","year":"2025","journal-title":"Phytother. Res."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Geetha Bai, R., and Tuvikene, R. (2021). Potential Antiviral Properties of Industrially Important Marine Algal Polysaccharides and Their Significance in Fighting a Future Viral Pandemic. Viruses, 13.","DOI":"10.3390\/v13091817"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"134903","DOI":"10.1016\/j.foodchem.2022.134903","article-title":"Algal Polysaccharides: Structure, Preparation and Applications in Food Packaging","volume":"405","author":"Thiviya","year":"2023","journal-title":"Food Chem."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Liyanage, N.M., Nagahawatta, D.P., Jayawardena, T.U., Sanjeewa, K.K.A., Jayawrdhana, H.H.a.C.K., Kim, J.-I., and Jeon, Y.-J. (2023). Sulfated Polysaccharides from Seaweeds: A Promising Strategy for Combatting Viral Diseases\u2014A Review. Mar. Drugs, 21.","DOI":"10.3390\/md21090461"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1016\/j.virol.2007.01.043","article-title":"Interference in Dengue Virus Adsorption and Uncoating by Carrageenans","volume":"363","author":"Talarico","year":"2007","journal-title":"Virology"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"135267","DOI":"10.1016\/j.ijbiomac.2024.135267","article-title":"Antiviral Activity of a Polysaccharide from Sargassum Fusiforme against Respiratory Syncytial Virus","volume":"279","author":"Zhang","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"133496","DOI":"10.1016\/j.ijbiomac.2024.133496","article-title":"Inhibition of Dengue Virus Infection in Vitro by Fucoidan and Polysaccharide Extract from Marine Alga Sargassum spp.","volume":"276","author":"Panwong","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/j.antiviral.2005.02.001","article-title":"The Antiviral Activity of Sulfated Polysaccharides against Dengue Virus Is Dependent on Virus Serotype and Host Cell","volume":"66","author":"Talarico","year":"2005","journal-title":"Antivir. Res."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"\u00c1lvarez-Vi\u00f1as, M., Souto, S., Fl\u00f3rez-Fern\u00e1ndez, N., Torres, M.D., Band\u00edn, I., and Dom\u00ednguez, H. (2021). Antiviral Activity of Carrageenans and Processing Implications. Mar. Drugs, 19.","DOI":"10.3390\/md19080437"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1016\/j.ijbiomac.2012.05.028","article-title":"Antiviral Activity against Dengue Virus of Diverse Classes of Algal Sulfated Polysaccharides","volume":"51","author":"Pujol","year":"2012","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1037","DOI":"10.1021\/np050056c","article-title":"Isolation of an Antiviral Polysaccharide, Nostoflan, from a Terrestrial Cyanobacterium, Nostoc Flagelliforme","volume":"68","author":"Kanekiyo","year":"2005","journal-title":"J. Nat. Prod."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"286","DOI":"10.1016\/j.jff.2017.03.062","article-title":"Structural Characterization and Macrophage Immunomodulatory Activity of a Polysaccharide Isolated from Gracilaria lemaneiformis","volume":"33","author":"Ren","year":"2017","journal-title":"J. Funct. Foods"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"112157","DOI":"10.1016\/j.fct.2021.112157","article-title":"Structural Characteristics and Anti-Inflammatory Activity of UV\/H2O2-Treated Algal Sulfated Polysaccharide from Gracilaria lemaneiformis","volume":"152","author":"Gong","year":"2021","journal-title":"Food Chem. Toxicol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1016\/j.ijbiomac.2020.02.168","article-title":"Current Trends in Marine Algae Polysaccharides: The Digestive Tract, Microbial Catabolism, and Prebiotic Potential","volume":"151","author":"Zheng","year":"2020","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"105650","DOI":"10.1016\/j.jff.2023.105650","article-title":"Structural Characterization and Anti-Pigmentation of a Novel Heteropolysaccharide from Gracilaria lemaneiformis via \u03b1-MSH\/MC1R Pathway","volume":"107","author":"Feng","year":"2023","journal-title":"J. Funct. Foods"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"111550","DOI":"10.1016\/j.exger.2021.111550","article-title":"Protective Effects of GLHP from Gracilaria lemaneiformis against UVB-Induced Photodamage in Human Immortalized Keratinocytes Cells and BALB\/c Mice","volume":"155","author":"Ouyang","year":"2021","journal-title":"Exp. Gerontol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Qin, Z., Xie, X., Ye, H., Wu, H., Li, Z., Li, J., He, X., Liang, Z., Liu, X., and Zhu, L. (2025). A Lethal DENV-2 Wild-Type Mouse Model for Mutagenesis Investigations. BMC Biol., 23.","DOI":"10.1186\/s12915-025-02332-6"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"108326","DOI":"10.1016\/j.carres.2021.108326","article-title":"Marine Sulfated Polysaccharides as Potential Antiviral Drug Candidates to Treat Corona Virus Disease (COVID-19)","volume":"505","author":"Andrew","year":"2021","journal-title":"Carbohydr. Res."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Ichiyama, K., Gopala Reddy, S.B., Zhang, L.F., Chin, W.X., Muschin, T., Heinig, L., Suzuki, Y., Nanjundappa, H., Yoshinaka, Y., and Ryo, A. (2013). Sulfated Polysaccharide, Curdlan Sulfate, Efficiently Prevents Entry\/Fusion and Restricts Antibody-Dependent Enhancement of Dengue Virus Infection in Vitro: A Possible Candidate for Clinical Application. PLoS Negl. Trop. Dis., 7.","DOI":"10.1371\/journal.pntd.0002188"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"154","DOI":"10.1007\/s13205-022-03210-6","article-title":"Therapeutic Activity of Fucoidan and Carrageenan as Marine Algal Polysaccharides against Viruses","volume":"12","author":"Oliyaei","year":"2022","journal-title":"3 Biotech"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1093\/jb\/mvj067","article-title":"Identification and Characterization of Carbohydrate Molecules in Mammalian Cells Recognized by Dengue Virus Type 2","volume":"139","author":"Aoki","year":"2006","journal-title":"J. Biochem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1038\/s41421-020-00192-8","article-title":"Sulfated Polysaccharides Effectively Inhibit SARS-CoV-2 in Vitro","volume":"6","author":"Kwon","year":"2020","journal-title":"Cell Discov."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Nanaware, N., Banerjee, A., Mullick Bagchi, S., Bagchi, P., and Mukherjee, A. (2021). Dengue Virus Infection: A Tale of Viral Exploitations and Host Responses. Viruses, 13.","DOI":"10.3390\/v13101967"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.antiviral.2016.08.018","article-title":"Antiviral Activity of Natural and Synthetic \u03b2-Carbolines against Dengue Virus","volume":"134","author":"Quintana","year":"2016","journal-title":"Antiviral Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1016\/S0140-6736(18)32560-1","article-title":"Dengue","volume":"393","author":"Ooi","year":"2019","journal-title":"Lancet"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/6\/594\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,25]],"date-time":"2026-05-25T02:34:28Z","timestamp":1779676468000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/6\/594"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,24]]},"references-count":34,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["v18060594"],"URL":"https:\/\/doi.org\/10.3390\/v18060594","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,24]]}}}