{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T16:22:32Z","timestamp":1791390152319,"version":"4.3.3"},"reference-count":55,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2022,12,20]],"date-time":"2022-12-20T00:00:00Z","timestamp":1671494400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000774","name":"Defense Threat Reduction Agency (DTRA)","doi-asserted-by":"publisher","award":["W911QY-15-1-0019"],"award-info":[{"award-number":["W911QY-15-1-0019"]}],"id":[{"id":"10.13039\/100000774","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000774","name":"Defense Threat Reduction Agency (DTRA)","doi-asserted-by":"publisher","award":["NIH\/NIAID R01AI22186"],"award-info":[{"award-number":["NIH\/NIAID R01AI22186"]}],"id":[{"id":"10.13039\/100000774","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The Department of Defense recently began an effort to improve and standardize virus challenge materials and efficacy determination strategies for testing therapeutics and vaccines. This includes stabilization of virus genome sequences in cDNA form where appropriate, use of human-derived virus isolates, and noninvasive strategies for determination of challenge virus replication. Eventually, it is desired that these approaches will satisfy the FDA \u201cAnimal Rule\u201d for licensure, which substitutes animal efficacy data when human data are unlikely to be available. To this end, we created and examined the virulence phenotype of cDNA clones of prototypic human infection-derived strains of the alphaviruses, Venezuelan (VEEV INH9813), eastern (EEEV V105) and western (WEEV Fleming) equine encephalitis viruses, and created fluorescent and luminescent reporter expression vectors for evaluation of replication characteristics in vitro and in vivo. Sequences of minimally passaged isolates of each virus were used to synthesize full-length cDNA clones along with a T7 transcription promoter-based bacterial propagation vector. Viruses generated from the cDNA clones were compared with other \u201cwild type\u201d strains derived from cDNA clones and GenBank sequences to identify and eliminate putative tissue culture artifacts accumulated in the cell passaged biological stocks. This was followed by examination of aerosol and subcutaneous infection and disease in mouse models. A mutation that increased heparan sulfate binding was identified in the VEEV INH9813 biological isolate sequence and eliminated from the cDNA clone. Viruses derived from the new human isolate cDNA clones showed similar mouse virulence to existing clone-derived viruses after aerosol or subcutaneous inoculation.<\/jats:p>","DOI":"10.3390\/v15010005","type":"journal-article","created":{"date-parts":[[2022,12,20]],"date-time":"2022-12-20T04:45:53Z","timestamp":1671511553000},"page":"5","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["In Vitro and In Vivo Phenotypes of Venezuelan, Eastern and Western Equine Encephalitis Viruses Derived from cDNA Clones of Human Isolates"],"prefix":"10.3390","volume":"15","author":[{"given":"Christina L.","family":"Gardner","sequence":"first","affiliation":[{"name":"Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chengqun","family":"Sun","sequence":"additional","affiliation":[{"name":"The Center for Vaccine Research and Department of Immunology, The University of Pittsburgh, Pittsburgh, PA 15261, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew D.","family":"Dunn","sequence":"additional","affiliation":[{"name":"The Center for Vaccine Research and Department of Immunology, The University of Pittsburgh, Pittsburgh, PA 15261, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"Jr.","given":"Theron C.","family":"Gilliland","sequence":"additional","affiliation":[{"name":"The Center for Vaccine Research and Department of Immunology, The University of Pittsburgh, Pittsburgh, PA 15261, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Derek W.","family":"Trobaugh","sequence":"additional","affiliation":[{"name":"Elanco Animal Health, Greenfield, IN 46140, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yutaka","family":"Terada","sequence":"additional","affiliation":[{"name":"The Center for Vaccine Research and Department of Immunology, The University of Pittsburgh, Pittsburgh, PA 15261, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0076-9023","authenticated-orcid":false,"given":"Douglas S.","family":"Reed","sequence":"additional","affiliation":[{"name":"The Center for Vaccine Research and Department of Immunology, The University of Pittsburgh, Pittsburgh, PA 15261, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0857-2973","authenticated-orcid":false,"given":"Amy L.","family":"Hartman","sequence":"additional","affiliation":[{"name":"The Center for Vaccine Research and Department of Infectious Diseases and Microbiology, Graduate School of Public Health, The University of Pittsburgh, Pittsburgh, PA 15261, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4506-7842","authenticated-orcid":false,"given":"William B.","family":"Klimstra","sequence":"additional","affiliation":[{"name":"The Center for Vaccine Research and Department of Immunology, The University of Pittsburgh, Pittsburgh, PA 15261, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,20]]},"reference":[{"key":"ref_1","unstructured":"Knipe, D., and Howley, P. (2013). Alphaviruses. Fields Virology, Lippincott Wiliams & Wilkins. [6th ed.]."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1146\/annurev.ento.49.061802.123422","article-title":"Venezuelan equine encephalitis","volume":"49","author":"Weaver","year":"2004","journal-title":"Annu. Rev. Entomol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Vittor, A.Y., Armien, B., Gonzalez, P., Carrera, J.P., Dominguez, C., Valderrama, A., Glass, G.E., Beltran, D., Cisneros, J., and Wang, E. (2016). Epidemiology of Emergent Madariaga Encephalitis in a Region with Endemic Venezuelan Equine Encephalitis: Initial Host Studies and Human Cross-Sectional Study in Darien, Panama. PLoS Negl. Trop. Dis., 10.","DOI":"10.1371\/journal.pntd.0004554"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1016\/j.vetmic.2009.08.023","article-title":"Encephalitic alphaviruses","volume":"140","author":"Zacks","year":"2010","journal-title":"Vet. Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1016\/j.virol.2008.08.012","article-title":"Western Equine Encephalitis submergence: Lack of evidence for a decline in virus virulence","volume":"380","author":"Forrester","year":"2008","journal-title":"Virology"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1283","DOI":"10.1126\/science.158.3806.1283","article-title":"Arbovirus infections of laboratory workers. Extent of problem emphasizes the need for more effective measures to reduce hazards","volume":"158","author":"Hanson","year":"1967","journal-title":"Science"},{"key":"ref_7","first-page":"46","article-title":"Clinical aspects of human Venezuelan equine encephalitis in Texas","volume":"10","author":"Bowen","year":"1976","journal-title":"Bull. Pan Am. Health Organ."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"790","DOI":"10.1177\/0300985810372508","article-title":"Review Paper: Pathology of animal models of alphavirus encephalitis","volume":"47","author":"Steele","year":"2010","journal-title":"Vet. Pathol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Rusnak, J.M., Glass, P.J., Weaver, S.C., Sabourin, C.L., Glenn, A.M., Klimstra, W., Badorrek, C.S., Nasar, F., and Ward, L.A. (2019). Approach to Strain Selection and the Propagation of Viral Stocks for Venezuelan Equine Encephalitis Virus Vaccine Efficacy Testing under the Animal Rule. Viruses, 11.","DOI":"10.3390\/v11090807"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/0042-6822(91)90114-Q","article-title":"Attenuating mutations in the E2 glycoprotein gene of Venezuelan equine encephalitis virus: Construction of single and multiple mutants in a full-length cDNA clone","volume":"183","author":"Davis","year":"1991","journal-title":"Virology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"49","DOI":"10.4269\/ajtmh.2001.64.49","article-title":"Comparative neurovirulence of attenuated and non-attenuated strains of Venezuelan equine encephalitis virus in mice","volume":"64","author":"Ludwig","year":"2001","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"410","DOI":"10.1177\/030098589102800509","article-title":"Pathogenesis of Venezuelan equine encephalitis virus infection in mice and hamsters","volume":"28","author":"Jackson","year":"1991","journal-title":"Vet. Pathol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1006\/viro.2001.0878","article-title":"Immunopathogenesis and immune modulation of Venezuelan equine encephalitis virus-induced disease in the mouse","volume":"284","author":"Charles","year":"2001","journal-title":"Virology"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e00226-17","DOI":"10.1128\/JVI.00226-17","article-title":"Antibody preparations from human transchromosomic cows exhibit prophylactic and therapeutic efficacy versus Venezuelan equine encephalitis virus","volume":"91","author":"Gardner","year":"2017","journal-title":"J. Virol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"11300","DOI":"10.1128\/JVI.79.17.11300-11310.2005","article-title":"Variation in interferon sensitivity and induction among strains of eastern equine encephalitis virus","volume":"79","author":"Aguilar","year":"2005","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"16026","DOI":"10.1073\/pnas.1110617108","article-title":"Heparan sulfate binding by natural eastern equine encephalitis viruses promotes neurovirulence","volume":"108","author":"Gardner","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1038\/nature12869","article-title":"RNA viruses can hijack vertebrate microRNAs to suppress innate immunity","volume":"506","author":"Trobaugh","year":"2013","journal-title":"Nature"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1007\/BF01320552","article-title":"An attenuated variant of Eastern encephalitis virus: Biological properties and protection induced in mice","volume":"47","author":"Brown","year":"1975","journal-title":"Arch. Virol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1848","DOI":"10.1099\/vir.0.008656-0","article-title":"Virulence variation among isolates of western equine encephalitis virus in an outbred mouse model","volume":"90","author":"Logue","year":"2009","journal-title":"J. Gen. Virol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1186\/s12985-015-0386-1","article-title":"Eastern equine encephalitis virus in mice I: Clinical course and outcome are dependent on route of exposure","volume":"12","author":"Honnold","year":"2015","journal-title":"Virol. J."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"7357","DOI":"10.1128\/JVI.72.9.7357-7366.1998","article-title":"Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor","volume":"72","author":"Klimstra","year":"1998","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"12077","DOI":"10.1128\/JVI.01406-14","article-title":"Combined alphavirus replicon particle vaccine induces durable and cross-protective immune responses against equine encephalitis viruses","volume":"88","author":"Reed","year":"2014","journal-title":"J. Virol."},{"key":"ref_23","first-page":"144","article-title":"Twenty-eight alphavirus strains isolated from mosquitoes and ticks captured from Hainan Island, China","volume":"11","author":"Chen","year":"1997","journal-title":"Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi = Zhonghua Shiyan He Linchuang Bingduxue Zazhi = Chin. J. Exp. Clin. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"18S","DOI":"10.4269\/ajtmh.1987.37.18S","article-title":"The ecology of western equine encephalomyelitis virus in the Central Valley of California, 1945\u20131985","volume":"37","author":"Hardy","year":"1987","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"240","DOI":"10.4269\/ajtmh.1997.57.240","article-title":"Mouse and baby chicken virulence of enzootic strains of western equine encephalomyelitis virus from California","volume":"57","author":"Hardy","year":"1997","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"447","DOI":"10.4269\/ajtmh.1988.38.447","article-title":"Reevaluation of the western equine encephalitis antigenic complex of alphaviruses (family Togaviridae) as determined by neutralization tests","volume":"38","author":"Calisher","year":"1988","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e00243-15","DOI":"10.1128\/genomeA.00243-15","article-title":"Complete coding sequences of eastern equine encephalitis virus and venezuelan equine encephalitis virus strains isolated from human cases","volume":"3","author":"Yu","year":"2015","journal-title":"Genome Announc."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"436","DOI":"10.1016\/S0140-6736(96)02275-1","article-title":"Re-emergence of epidemic Venezuelan equine encephalomyelitis in South America. VEE Study Group","volume":"348","author":"Weaver","year":"1996","journal-title":"Lancet"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"509","DOI":"10.4049\/jimmunol.87.5.509","article-title":"Studies on the virus of Venezuelan equine encephalomyelitis. II. Modification by specific immune serum of response of central nervous system of mice","volume":"87","author":"Berge","year":"1961","journal-title":"J. Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Mossel, E.C., Ledermann, J.P., Phillips, A.T., Borland, E.M., Powers, A.M., and Olson, K.E. (2013). Molecular determinants of mouse neurovirulence and mosquito infection for Western equine encephalitis virus. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0060427"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1190","DOI":"10.3201\/eid1708.050841","article-title":"Venezuelan equine encephalitis virus transmission and effect on pathogenesis","volume":"12","author":"Smith","year":"2006","journal-title":"Emerg. Infect. Dis."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3563","DOI":"10.1128\/JVI.02494-06","article-title":"Heparan sulfate binding can contribute to the neurovirulence of neuroadapted and nonneuroadapted Sindbis viruses","volume":"81","author":"Ryman","year":"2007","journal-title":"J. Virol."},{"key":"ref_33","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_34","doi-asserted-by":"crossref","unstructured":"Ma, H., Albe, J.R., Gilliland, T., McMillen, C.M., Gardner, C.L., Boyles, D.A., Cottle, E.L., Dunn, M.D., Lundy, J.D., and Salama, N. (2022). Long-term persistence of viral RNA and inflammation in the CNS of macaques exposed to aerosolized Venezuelan equine encephalitis virus. PLoS Pathog., 18.","DOI":"10.1371\/journal.ppat.1009946"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/j.virol.2011.12.020","article-title":"Interferon-alpha\/beta deficiency greatly exacerbates arthritogenic disease in mice infected with wild-type chikungunya virus but not with the cell culture-adapted live-attenuated 181\/25 vaccine candidate","volume":"425","author":"Gardner","year":"2012","journal-title":"Virology"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1981","DOI":"10.1128\/jvi.70.3.1981-1989.1996","article-title":"Deduced consensus sequence of Sindbis virus strain AR339: Mutations contained in laboratory strains which affect cell culture and in vivo phenotypes","volume":"70","author":"McKnight","year":"1996","journal-title":"J. Virol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1718","DOI":"10.1128\/JVI.76.4.1718-1730.2002","article-title":"Positively charged amino acid substitutions in the e2 envelope glycoprotein are associated with the emergence of venezuelan equine encephalitis virus","volume":"76","author":"Brault","year":"2002","journal-title":"J. Virol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1006\/viro.2000.0546","article-title":"Mutations in the E2 glycoprotein of Venezuelan equine encephalitis virus confer heparan sulfate interaction, low morbidity, and rapid clearance from blood of mice","volume":"276","author":"Bernard","year":"2000","journal-title":"Virology"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"454","DOI":"10.3201\/eid1403.070816","article-title":"Molecular epidemiology of eastern equine encephalitis virus, New York","volume":"14","author":"Young","year":"2008","journal-title":"Emerg. Infect. Dis."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"338","DOI":"10.1016\/j.virol.2009.05.030","article-title":"Type I interferon induction is correlated with attenuation of a South American eastern equine encephalitis virus strain in mice","volume":"390","author":"Gardner","year":"2009","journal-title":"Virology"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Trobaugh, D.W., Sun, N., Bhalla, C.L., Gardner, M., and Dunn, W.B. (2019). Klimstra Cooperativity between the 3\u2019 untranslated region microRNA binding sites is critical for virulence of eastern equine encephalitis virus. PLoS Pathog., 15.","DOI":"10.1101\/649525"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"e01223-19","DOI":"10.1128\/MRA.01223-19","article-title":"Complete Coding Sequence of Western Equine Encephalitis Virus Strain Fleming, Isolated from a Human Case","volume":"9","author":"Burke","year":"2020","journal-title":"Microbiol. Resour. Announc."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Albe, J.R., Ma, H., Gilliland, T.H., McMillen, C.M., Gardner, C.L., Boyles, D.A., Cottle, E.L., Dunn, M.D., Lundy, J.D., and O\u2019Malley, K.J. (2021). Physiological and immunological changes in the brain associated with lethal eastern equine encephalitis virus in macaques. PLoS Pathog., 17.","DOI":"10.1371\/journal.ppat.1009308"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Ma, H., Lundy, J.D., Cottle, E.L., O\u2019Malley, K.J., Trichel, A.M., Klimstra, W.B., Hartman, A.L., Reed, D.S., and Teichert, T. (2020). Applications of minimally invasive multimodal telemetry for continuous monitoring of brain function and intracranial pressure in macaques with acute viral encephalitis. PLoS ONE, 15.","DOI":"10.1101\/2020.04.15.042606"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Ma, H., Lundy, J.D., O\u2019Malley, K.J., Klimstra, W.B., Hartman, A.L., and Reed, D.S. (2019). Electrocardiography Abnormalities in Macaques after Infection with Encephalitic Alphaviruses. Pathogens, 8.","DOI":"10.20944\/preprints201910.0121.v1"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"e001726","DOI":"10.1099\/jgv.0.001726","article-title":"Glycosaminoglycan binding by arboviruses: A cautionary tale","volume":"103","author":"Alcorn","year":"2022","journal-title":"J. Gen. Virol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"7349","DOI":"10.1128\/JVI.72.9.7349-7356.1998","article-title":"Binding of Sindbis virus to cell surface heparan sulfate","volume":"72","author":"Byrnes","year":"1998","journal-title":"J. Virol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"10128","DOI":"10.1128\/JVI.76.20.10128-10137.2002","article-title":"Adaptation of alphaviruses to heparan sulfate: Interaction of Sindbis and Semliki forest viruses with liposomes containing lipid-conjugated heparin","volume":"76","author":"Smit","year":"2002","journal-title":"J. Virol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"8582","DOI":"10.1128\/JVI.00937-13","article-title":"Natural variation in the heparan sulfate binding domain of the eastern equine encephalitis virus E2 glycoprotein alters interactions with cell surfaces and virulence in mice","volume":"87","author":"Gardner","year":"2013","journal-title":"J. Virol."},{"key":"ref_50","first-page":"201","article-title":"RNA virus quasispecies: Significance for viral disease and epidemiology","volume":"3","author":"Duarte","year":"1994","journal-title":"Infect. Agents Dis."},{"key":"ref_51","first-page":"99","article-title":"Viral quasispecies and the problem of vaccine-escape and drug-resistant mutants","volume":"48","author":"Domingo","year":"1997","journal-title":"Prog. Drug Res."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1128\/MMBR.05023-11","article-title":"Viral quasispecies evolution","volume":"76","author":"Domingo","year":"2012","journal-title":"Microbiol. Mol. Biol. Rev. MMBR"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"2620","DOI":"10.1128\/JVI.01784-07","article-title":"Alpha\/beta interferon inhibits cap-dependent translation of viral but not cellular mRNA by a PKR-independent mechanism","volume":"82","author":"Tesfay","year":"2008","journal-title":"J. Virol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"3587","DOI":"10.1128\/JVI.72.5.3587-3594.1998","article-title":"Foot-and-mouth disease virus virulent for cattle utilizes the integrin alpha(v)beta3 as its receptor","volume":"72","author":"Neff","year":"1998","journal-title":"J. Virol."},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Gardner, C.L., Hritz, J., Sun, C., Vanlandingham, D.L., Song, T.Y., Ghedin, E., Higgs, S., Klimstra, W.B., and Ryman, K.D. (2014). Deliberate attenuation of chikungunya virus by adaptation to heparan sulfate-dependent infectivity: A model for rational arboviral vaccine design. PLoS Negl. Trop. Dis., 8.","DOI":"10.1371\/journal.pntd.0002719"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/1\/5\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:44:37Z","timestamp":1760147077000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/1\/5"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,12,20]]},"references-count":55,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2023,1]]}},"alternative-id":["v15010005"],"URL":"https:\/\/doi.org\/10.3390\/v15010005","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,12,20]]}}}