{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,3]],"date-time":"2026-03-03T06:49:36Z","timestamp":1772520576265,"version":"3.50.1"},"reference-count":38,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2025,2,12]],"date-time":"2025-02-12T00:00:00Z","timestamp":1739318400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000925","name":"Australian Government National Health and Medical Research Council (NHMRC) development","doi-asserted-by":"publisher","award":["APP1178896"],"award-info":[{"award-number":["APP1178896"]}],"id":[{"id":"10.13039\/501100000925","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000925","name":"Australian Government National Health and Medical Research Council (NHMRC) development","doi-asserted-by":"publisher","award":["AQIRF067-2020-CV"],"award-info":[{"award-number":["AQIRF067-2020-CV"]}],"id":[{"id":"10.13039\/501100000925","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Research Training Program Stipend from the University of Queensland","award":["APP1178896"],"award-info":[{"award-number":["APP1178896"]}]},{"name":"Research Training Program Stipend from the University of Queensland","award":["AQIRF067-2020-CV"],"award-info":[{"award-number":["AQIRF067-2020-CV"]}]},{"DOI":"10.13039\/501100008914","name":"Advance Queensland Industry Research Fellowship","doi-asserted-by":"publisher","award":["APP1178896"],"award-info":[{"award-number":["APP1178896"]}],"id":[{"id":"10.13039\/501100008914","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100008914","name":"Advance Queensland Industry Research Fellowship","doi-asserted-by":"publisher","award":["AQIRF067-2020-CV"],"award-info":[{"award-number":["AQIRF067-2020-CV"]}],"id":[{"id":"10.13039\/501100008914","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Chimeric orthoflaviviruses derived from the insect-specific Binjari virus (BinJV) offer a promising basis for safe orthoflavivirus vaccines. However, these vaccines have so far only been produced using adherent C6\/36 Aedes albopictus mosquito cell cultures grown in serum-supplemented media, limiting their scalable manufacture. To address this, we adapted C6\/36 cells for serum-free suspension culture using Sf900-III medium, achieving high peak cell densities (up to 2.5 \u00d7 107 cells\/mL). Higher agitation rates reduced cell aggregation, and cryopreservation and direct-to-suspension revival were successful, confirming the adapted line\u2019s stability for research and industrial applications. Despite this, BinJV-based chimeric orthoflaviviruses, including BinJV\/WNVKUN, a candidate vaccine for West Nile virus, and similar vaccines (BinJV\/DENV2 and BinJV\/JEVNSW22) for dengue 2 virus and Japanese encephalitis virus, respectively, exhibited substantially reduced titres in C6\/36 cultures infected in Sf900-III, a phenomenon attributed to the medium\u2019s acidic pH. Switching to the more alkaline, serum-free CD-FortiCHO medium enhanced the replication of these chimeric viruses to peak titres between 1.7 \u00d7 107 and 7.6 \u00d7 109 infectious units per mL whilst preserving viral integrity. These findings suggest that suspension-adapted C6\/36 cultures in CD-FortiCHO medium can support high-yield vaccine production for various orthoflaviviruses and highlight the important role of cell culture media pH for orthoflavivirus bioprocessing. This scalable mosquito cell-based system could reduce production costs and improve vaccine accessibility, supporting efforts to combat arbovirus-related public health challenges.<\/jats:p>","DOI":"10.3390\/v17020250","type":"journal-article","created":{"date-parts":[[2025,2,12]],"date-time":"2025-02-12T06:06:06Z","timestamp":1739340366000},"page":"250","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Serum-Free Suspension Culture of the Aedes albopictus C6\/36 Cell Line for Chimeric Orthoflavivirus Vaccine Production"],"prefix":"10.3390","volume":"17","author":[{"given":"Joshua S.","family":"Dawurung","sequence":"first","affiliation":[{"name":"School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jessica J.","family":"Harrison","sequence":"additional","affiliation":[{"name":"School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3205-4970","authenticated-orcid":false,"given":"Naphak","family":"Modhiran","sequence":"additional","affiliation":[{"name":"School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Roy A.","family":"Hall","sequence":"additional","affiliation":[{"name":"School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia"},{"name":"Australian Infectious Diseases Research Centre, The University of Queensland, St Lucia, QLD 4072, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0139-9829","authenticated-orcid":false,"given":"Jody","family":"Hobson-Peters","sequence":"additional","affiliation":[{"name":"School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia"},{"name":"Australian Infectious Diseases Research Centre, The University of Queensland, St Lucia, QLD 4072, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4327-3418","authenticated-orcid":false,"given":"Henry","family":"de Malmanche","sequence":"additional","affiliation":[{"name":"School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,12]]},"reference":[{"key":"ref_1","first-page":"506","article-title":"Cell cultures derived from larvae of Aedes albopictus (Skuse) and Aedes aegypti (L.)","volume":"36","author":"Singh","year":"1967","journal-title":"Curr. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1099\/0022-1317-40-3-531","article-title":"Isolation of a Singh\u2019s Aedes albopictus cell clone sensitive to Dengue and Chikungunya viruses","volume":"40","author":"Igarashi","year":"1978","journal-title":"J. Gen. Virol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Brackney, D.E., Scott, J.C., Sagawa, F., Woodward, J.E., Miller, N.A., Schilkey, F.D., Mudge, J., Wilusz, J., Olson, K.E., and Blair, C.D. (2010). C6\/36 Aedes albopictus cells have a dysfunctional antiviral RNA interference response. PLoS Neglected Trop. Dis., 4.","DOI":"10.1371\/journal.pntd.0000856"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Morazzani, E.M., Wiley, M.R., Murreddu, M.G., Adelman, Z.N., and Myles, K.M. (2012). Production of virus-derived ping-pong-dependent piRNA-like small RNAs in the mosquito soma. PLoS Pathog., 8.","DOI":"10.1371\/journal.ppat.1002470"},{"key":"ref_5","unstructured":"American Type Culture Collection (2017). Aedes albopictus Clone C6\/36 (ATCC CRL1660TM), ATCC."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Kiesslich, S., and Kamen, A.A. (2020). Vero cell upstream bioprocess development for the production of viral vectors and vaccines. Biotechnol. Adv., 44.","DOI":"10.1016\/j.biotechadv.2020.107608"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"eaax7888","DOI":"10.1126\/scitranslmed.aax7888","article-title":"A recombinant platform for flavivirus vaccines and diagnostics using chimeras of a new insect-specific virus","volume":"11","author":"Harrison","year":"2019","journal-title":"Sci. Transl. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1038\/nm.4253","article-title":"A chikungunya fever vaccine utilizing an insect-specific virus platform","volume":"23","author":"Erasmus","year":"2017","journal-title":"Nat. Med."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Carvalho, V.L., and Long, M.T. (2021). Perspectives on new vaccines against arboviruses using insect-specific viruses as platforms. Vaccines, 9.","DOI":"10.3390\/vaccines9030263"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1016\/j.virol.2008.04.021","article-title":"West Nile virus infection of Drosophila melanogaster induces a protective RNAi response","volume":"377","author":"Chotkowski","year":"2008","journal-title":"Virology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"158","DOI":"10.4269\/ajtmh.1984.33.158","article-title":"Mosquito cell cultures and specific monoclonal antibodies in surveillance for dengue viruses","volume":"33","author":"Gubler","year":"1984","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_12","first-page":"279","article-title":"Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses","volume":"63","author":"Kuno","year":"1985","journal-title":"Bull. World Health Organ."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1007\/s00253-005-1980-8","article-title":"Animal cell cultures: Recent achievements and perspectives in the production of biopharmaceuticals","volume":"68","author":"Butler","year":"2005","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Merten, O.W., Bakker, W.A., Vorlop, J., Reiter, M., Visnovsky, G., J\u00e4ger, V., Merabishvili, M., and Reichl, U. (2014). Virus production under suspension conditions. Industrial Scale Suspension Culture of Living Cells, Wiley.","DOI":"10.1002\/9783527683321.ch14"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1346","DOI":"10.1021\/bp060077c","article-title":"Purification of densonucleosis virus by tangential flow ultrafiltration","volume":"22","author":"Grzenia","year":"2006","journal-title":"Biotechnol. Prog."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1290\/1543-706X(2004)040<0074:POMDVB>2.0.CO;2","article-title":"Production of mosquito densonucleosis viruses by Aedes albopictus C6\/36 cells adapted to suspension culture in serum-free protein-free media","volume":"40","author":"Suchman","year":"2004","journal-title":"In Vitro Cell. Dev. Biol. -Anim."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1007\/BF01578006","article-title":"Suspension culture of Aedes albopictus cells for flavivirus mass production","volume":"12","author":"Morita","year":"1989","journal-title":"J. Tissue Cult. Methods"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1038\/s41541-023-00688-w","article-title":"A chimeric vaccine protects farmed saltwater crocodiles from West Nile virus-induced skin lesions","volume":"8","author":"Habarugira","year":"2023","journal-title":"npj Vaccines"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Vet, L.J., Setoh, Y.X., Amarilla, A.A., Habarugira, G., Suen, W.W., Newton, N.D., Harrison, J.J., Hobson-Peters, J., Hall, R.A., and Bielefeldt-Ohmann, H. (2020). Protective efficacy of a chimeric insect-specific flavivirus vaccine against West Nile virus. Vaccines, 8.","DOI":"10.3390\/vaccines8020258"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Yan, K., Vet, L.J., Tang, B., Hobson-Peters, J., Rawle, D.J., Le, T.T., Larcher, T., Hall, R.A., and Suhrbier, A. (2020). A yellow fever virus 17D infection and disease mouse model used to evaluate a chimeric Binjari-yellow fever virus vaccine. Vaccines, 8.","DOI":"10.3390\/vaccines8030368"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Harrison, J.J., Hobson-Peters, J., Bielefeldt-Ohmann, H., and Hall, R.A. (2021). Chimeric Vaccines Based on Novel Insect-Specific Flaviviruses. Vaccines, 9.","DOI":"10.3390\/vaccines9111230"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1038\/s41541-024-00903-2","article-title":"A chimeric vaccine derived from Australian genotype IV Japanese encephalitis virus protects mice from lethal challenge","volume":"9","author":"Harrison","year":"2024","journal-title":"npj Vaccines"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1175","DOI":"10.1099\/vir.0.82609-0","article-title":"In situ reactions of monoclonal antibodies with a viable mutant of Murray Valley encephalitis virus reveal an absence of dimeric NS1 protein","volume":"88","author":"Clark","year":"2007","journal-title":"J. Gen. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1093\/oxfordjournals.aje.a118408","article-title":"A simple method of estimating fifty per cent endpoints","volume":"27","author":"Reed","year":"1938","journal-title":"Am. J. Epidemiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"831","DOI":"10.1099\/vir.0.027185-0","article-title":"A West Nile virus mutant with increased resistance to acid-induced inactivation","volume":"92","author":"Saiz","year":"2011","journal-title":"J. Gen. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Mart\u00edn-Acebes, M.A., Blazquez, A.-B., de Oya, N.J., Escribano-Romero, E., Shi, P.-Y., and Saiz, J.-C. (2013). A single amino acid substitution in the core protein of West Nile virus increases resistance to acidotropic compounds. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0069479"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1038\/s41541-021-00328-1","article-title":"A chimeric dengue virus vaccine candidate delivered by high density microarray patches protects against infection in mice","volume":"6","author":"Choo","year":"2021","journal-title":"npj Vaccines"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Covarrubias, C.E., Rivera, T.A., Soto, C.A., Deeks, T., and Kalergis, A.M. (2022). Current GMP standards for the production of vaccines and antibodies: An overview. Front. Public Health, 10.","DOI":"10.3389\/fpubh.2022.1021905"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1941","DOI":"10.1099\/0022-1317-67-9-1941","article-title":"The uncoating and infectivity of the flavivirus West Nile on interaction with cells: Effects of pH and ammonium chloride","volume":"67","author":"Gollins","year":"1986","journal-title":"J. Gen. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1006\/viro.1993.1252","article-title":"Selection and partial characterization of dengue 2 virus mutants that induce fusion at elevated pH","volume":"194","author":"Guirakhoo","year":"1993","journal-title":"Virology"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Wagner, J.M., Pajerowski, J.D., Daniels, C.L., McHugh, P.M., Flynn, J.A., Balliet, J.W., Casimiro, D.R., and Subramanian, S. (2014). Enhanced production of Chikungunya virus-like particles using a high-pH adapted spodoptera frugiperda insect cell line. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0094401"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e01274-17","DOI":"10.1128\/JVI.01274-17","article-title":"Novel insect-specific eilat virus-based chimeric vaccine candidates provide durable, mono-and multivalent, single-dose protection against lethal alphavirus challenge","volume":"92","author":"Erasmus","year":"2018","journal-title":"J. Virol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/s44298-024-00052-2","article-title":"Synthetic recovery of Yada Yada virus expands insect-specific alphavirus knowledge and facilitates production of chimeric viruses","volume":"2","author":"Bell","year":"2024","journal-title":"npj Viruses"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Naik, N.G., Lo, Y.-W., Wu, T.-Y., Lin, C.-C., Kuo, S.-C., and Chao, Y.-C. (2018). Baculovirus as an efficient vector for gene delivery into mosquitoes. Sci. Rep., 8.","DOI":"10.1038\/s41598-018-35463-8"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e20220001","DOI":"10.1002\/INMD.20220001","article-title":"Insect-specific viruses used in biocontrol of mosquito-borne diseases","volume":"1","author":"Chen","year":"2023","journal-title":"Interdiscip. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"gix135","DOI":"10.1093\/gigascience\/gix135","article-title":"Analysis of the Aedes albopictus C6\/36 genome provides insight into cell line utility for viral propagation","volume":"7","author":"Miller","year":"2018","journal-title":"Gigascience"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s00253-003-1223-9","article-title":"Insect cell culture for industrial production of recombinant proteins","volume":"62","author":"Ikonomou","year":"2003","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Michl, J., Park, K.C., and Swietach, P. (2019). Evidence-based guidelines for controlling pH in mammalian live-cell culture systems. Commun. Biol., 2.","DOI":"10.1038\/s42003-019-0393-7"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/2\/250\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T16:32:02Z","timestamp":1760027522000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/2\/250"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,12]]},"references-count":38,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2025,2]]}},"alternative-id":["v17020250"],"URL":"https:\/\/doi.org\/10.3390\/v17020250","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,2,12]]}}}