{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,14]],"date-time":"2025-10-14T00:44:38Z","timestamp":1760402678667,"version":"build-2065373602"},"reference-count":38,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2021,4,11]],"date-time":"2021-04-11T00:00:00Z","timestamp":1618099200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"APRC - City University of Hong Kong, New Research Initiatives\/Infrastructure Support from Central","award":["9676013 and 9610451"],"award-info":[{"award-number":["9676013 and 9610451"]}]},{"name":"NIH, World Reference Center for Emerging Viruses and Arboviruses","award":["R24AI120942"],"award-info":[{"award-number":["R24AI120942"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Most alphaviruses are transmitted by mosquitoes and infect a wide range of insects and vertebrates. However, Eilat virus (EILV) is defective for infecting vertebrate cells at multiple levels of the viral life cycle. This host-restriction property renders EILV an attractive expression platform since it is not infectious for vertebrates and therefore provides a highly advantageous safety profile. Here, we investigated the feasibility of versatile EILV-based expression vectors. By replacing the structural genes of EILV with those of other alphaviruses, we generated seven different chimeras. These chimeras were readily rescued in the original mosquito cells and were able to reach high titers, suggesting that EILV is capable of packaging the structural proteins of different lineages. We also explored the ability of EILV to express authentic antigens via double subgenomic (SG) RNA vectors. Four foreign genetic materials of varied length were introduced into the EILV genome, and the expressed heterologous genetic materials were readily detected in the infected cells. By inserting an additional SG promoter into the chimera genome containing the structural genes of Chikungunya virus (CHIKV), we developed a bivalent vaccine candidate against CHIKV and Zika virus. These data demonstrate the outstanding compatibility of the EILV genome. The produced recombinants can be applied to vaccine and diagnostic tool development, but more investigations are required.<\/jats:p>","DOI":"10.3390\/v13040660","type":"journal-article","created":{"date-parts":[[2021,4,12]],"date-time":"2021-04-12T21:47:33Z","timestamp":1618264053000},"page":"660","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["A Productive Expression Platform Derived from Host-Restricted Eilat Virus: Its Extensive Validation and Novel Strategy"],"prefix":"10.3390","volume":"13","author":[{"given":"Lu","family":"Tan","sequence":"first","affiliation":[{"name":"Department of Infectious Diseases and Public Health, Jockey Club of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon Tong, Hong Kong 999077, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yiwen","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases and Public Health, Jockey Club of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon Tong, Hong Kong 999077, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xingxing","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases and Public Health, Jockey Club of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon Tong, Hong Kong 999077, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dal Young","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases and Public Health, Jockey Club of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon Tong, Hong Kong 999077, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,4,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1128\/mr.58.3.491-562.1994","article-title":"The alphaviruses: Gene expression, replication, and evolution","volume":"58","author":"Strauss","year":"1994","journal-title":"Microbiol. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1162","DOI":"10.1016\/j.ibmb.2005.05.008","article-title":"Development and characterization of a double subgenomic chikungunya virus infectious clone to express heterologous genes in Aedes aegypti mosqutioes","volume":"35","author":"Vanlandingham","year":"2005","journal-title":"Insect Biochem. Mol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1016\/0042-6822(89)90526-6","article-title":"In vitro synthesis of infectious venezuelan equine encephalitis virus RNA from a cDNA clone: Analysis of a viable deletion mutant","volume":"171","author":"Davis","year":"1989","journal-title":"Virology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3809","DOI":"10.1128\/jvi.61.12.3809-3819.1987","article-title":"Production of infectious RNA transcripts from Sindbis virus cDNA clones: Mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants","volume":"61","author":"Rice","year":"1987","journal-title":"J. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1016\/0958-1669(94)90063-9","article-title":"Alphavirus expression systems","volume":"5","author":"Liljestrom","year":"1994","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"11371","DOI":"10.1073\/pnas.93.21.11371","article-title":"Alphavirus-based expression vectors: Strategies and applications","volume":"93","author":"Frolov","year":"1996","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"7573","DOI":"10.1016\/j.vaccine.2007.07.061","article-title":"Chimeric Sindbis\/eastern equine encephalitis vaccine candidates are highly attenuated and immunogenic in mice","volume":"25","author":"Wang","year":"2007","journal-title":"Vaccine"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"4309","DOI":"10.1016\/j.vaccine.2009.05.011","article-title":"Chimeric alphavirus vaccine candidates protect mice from intranasal challenge with western equine encephalitis virus","volume":"27","author":"Atasheva","year":"2009","journal-title":"Vaccine"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4363","DOI":"10.1128\/JVI.00065-11","article-title":"Design of chimeric alphaviruses with a programmed, attenuated, cell type-restricted phenotype","volume":"85","author":"Kim","year":"2011","journal-title":"J. Virol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"9249","DOI":"10.1128\/JVI.00844-11","article-title":"Chimeric Chikungunya viruses are nonpathogenic in highly sensitive mouse models but efficiently induce a protective immune response","volume":"85","author":"Wang","year":"2011","journal-title":"J. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"7900","DOI":"10.1128\/jvi.70.11.7900-7909.1996","article-title":"Chimeric Sindbis Ross River viruses to study interactions between alphavirus nonstructural and structural regions","volume":"70","author":"Kuhn","year":"1996","journal-title":"J. Virol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1404","DOI":"10.1128\/JVI.01856-14","article-title":"Eilat Virus Host Range Restriction Is Present at Multiple Levels of the Virus Life Cycle","volume":"89","author":"Nasar","year":"2015","journal-title":"J. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1099\/jgv.0.001344","article-title":"Agua Salud alphavirus defines a novel lineage of insect-specific alphaviruses discovered in the New World","volume":"101","author":"Hermanns","year":"2020","journal-title":"J. Gen. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"14622","DOI":"10.1073\/pnas.1204787109","article-title":"Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication","volume":"109","author":"Nasar","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"570","DOI":"10.1038\/s41586-018-0121-3","article-title":"Mxra8 is a receptor for multiple arthritogenic alphaviruses","volume":"557","author":"Zhang","year":"2018","journal-title":"Nature"},{"key":"ref_16","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_17","doi-asserted-by":"crossref","first-page":"3242","DOI":"10.1016\/j.vaccine.2008.04.006","article-title":"Cellular and humoral immune responses to chimeric EGFP-pseudocapsids derived from the mouse polyomavirus after their intranasal administration","volume":"26","author":"Marek","year":"2008","journal-title":"Vaccine"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"5.4.1","DOI":"10.1002\/cpps.47","article-title":"Overview of the Baculovirus Expression System","volume":"91","author":"Chambers","year":"2018","journal-title":"Curr. Protoc. Protein. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Erasmus, J.H., Needham, J., Raychaudhuri, S., Diamond, M.S., Beasley, D.W., Morkowski, S., Salje, H., Fernandez Salas, I., Kim, D.Y., and Frolov, I. (2015). Utilization of an Eilat Virus-Based Chimera for Serological Detection of Chikungunya Infection. PLoS Negl. Trop Dis., 9.","DOI":"10.1371\/journal.pntd.0004119"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Erasmus, J.H., Seymour, R.L., Kaelber, J.T., Kim, D.Y., Leal, G., Sherman, M.B., Frolov, I., Chiu, W., Weaver, S.C., and Nasar, F. (2018). Novel Insect-Specific Eilat Virus-Based Chimeric Vaccine Candidates Provide Durable, Mono- and Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge. J. Virol., 92.","DOI":"10.1128\/JVI.01274-17"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2035","DOI":"10.1128\/JVI.02990-13","article-title":"Stable, high-level expression of reporter proteins from improved alphavirus expression vectors to track replication and dissemination during encephalitic and arthritogenic disease","volume":"88","author":"Sun","year":"2014","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1186\/s13071-014-0595-2","article-title":"Eilat virus displays a narrow mosquito vector range","volume":"7","author":"Nasar","year":"2014","journal-title":"Parasit. Vectors"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Reynaud, J.M., Kim, D.Y., Atasheva, S., Rasalouskaya, A., White, J.P., Diamond, M.S., Weaver, S.C., Frolova, E.I., and Frolov, I. (2015). IFIT1 Differentially Interferes with Translation and Replication of Alphavirus Genomes and Promotes Induction of Type I Interferon. PLoS Pathog., 11.","DOI":"10.1371\/journal.ppat.1004863"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1006\/viro.1995.1516","article-title":"Double-subgenomic Sindbis virus recombinants expressing immunogenic proteins of Japanese encephalitis virus induce significant protection in mice against lethal JEV infection","volume":"212","author":"Pugachev","year":"1995","journal-title":"Virology"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Kumar, R., Mehta, D., Mishra, N., Nayak, D., and Sunil, S. (2021). Role of Host-Mediated Post-Translational Modifications (PTMs) in RNA Virus Pathogenesis. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22010323"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2472","DOI":"10.1128\/JVI.02073-06","article-title":"The Old World and New World Alphaviruses Use Different Virus-Specific Proteins for Induction of Transcriptional Shutoff","volume":"81","author":"Garmashova","year":"2007","journal-title":"J. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Kim, D.Y., Reynaud, J.M., Rasalouskaya, A., Akhrymuk, I., Mobley, J.A., Frolov, I., and Frolova, E.I. (2016). New World and Old World Alphaviruses Have Evolved to Exploit Different Components of Stress Granules, FXR and G3BP Proteins, for Assembly of Viral Replication Complexes. PLoS Pathog., 12.","DOI":"10.1371\/journal.ppat.1005810"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"5997","DOI":"10.1073\/pnas.85.16.5997","article-title":"Western equine encephalitis virus is a recombinant virus","volume":"85","author":"Hahn","year":"1988","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"4560","DOI":"10.1038\/s41467-018-07008-0","article-title":"Human antibodies targeting Zika virus NS1 provide protection against disease in a mouse model","volume":"9","author":"Bailey","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"14769","DOI":"10.1038\/s41598-017-15039-8","article-title":"A Zika Vaccine Targeting NS1 Protein Protects Immunocompetent Adult Mice in a Lethal Challenge Model","volume":"7","author":"Brault","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Bailey, M.J., Broecker, F., Duehr, J., Arumemi, F., Krammer, F., Palese, P., and Tan, G.S. (2019). Antibodies Elicited by an NS1-Based Vaccine Protect Mice against Zika Virus. MBio, 10.","DOI":"10.1128\/mBio.02861-18"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/j.virusres.2012.10.021","article-title":"African swine fever virus serodiagnosis: A general review with a focus on the analyses of African serum samples","volume":"173","author":"Cubillos","year":"2013","journal-title":"Virus Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1515\/jvetres-2017-0017","article-title":"Roles of African Swine Fever Virus Structural Proteins in Viral Infection","volume":"61","author":"Jia","year":"2017","journal-title":"J. Vet. Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e33","DOI":"10.1017\/S1462399408000859","article-title":"Therapeutic and prophylactic applications of alphavirus vectors","volume":"10","author":"Atkins","year":"2008","journal-title":"Expert Rev. Mol. Med."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2392","DOI":"10.3390\/v6062392","article-title":"Alphavirus-based vaccines","volume":"6","author":"Lundstrom","year":"2014","journal-title":"Viruses"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1379","DOI":"10.1016\/j.immuni.2016.05.006","article-title":"Airway Memory CD4(+) T Cells Mediate Protective Immunity against Emerging Respiratory Coronaviruses","volume":"44","author":"Zhao","year":"2016","journal-title":"Immunity"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"9278","DOI":"10.1128\/JVI.77.17.9278-9286.2003","article-title":"Recombinant Sindbis\/Venezuelan Equine Encephalitis Virus Is Highly Attenuated and Immunogenic","volume":"77","author":"Paessler","year":"2003","journal-title":"J. Virol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0042-6822(03)00120-X","article-title":"Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production","volume":"310","author":"Jarvis","year":"2003","journal-title":"Virology"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/4\/660\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T13:59:28Z","timestamp":1760363968000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/4\/660"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,4,11]]},"references-count":38,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2021,4]]}},"alternative-id":["v13040660"],"URL":"https:\/\/doi.org\/10.3390\/v13040660","relation":{},"ISSN":["1999-4915"],"issn-type":[{"type":"electronic","value":"1999-4915"}],"subject":[],"published":{"date-parts":[[2021,4,11]]}}}