{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T18:05:35Z","timestamp":1788458735464,"version":"build-2803163510"},"reference-count":50,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2021,11,16]],"date-time":"2021-11-16T00:00:00Z","timestamp":1637020800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)","award":["022.005.032"],"award-info":[{"award-number":["022.005.032"]}]},{"name":"National Institute Of Allergy And Infectious Diseases of the National Institutes of Health","award":["R01AI151290"],"award-info":[{"award-number":["R01AI151290"]}]},{"DOI":"10.13039\/100009139","name":"Deutsches Zentrum f\u00fcr Infektionsforschung","doi-asserted-by":"publisher","award":["-"],"award-info":[{"award-number":["-"]}],"id":[{"id":"10.13039\/100009139","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100011313","name":"Tokyo Biochemical Research Foundation","doi-asserted-by":"publisher","award":["-"],"award-info":[{"award-number":["-"]}],"id":[{"id":"10.13039\/100011313","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007449","name":"Takeda Science Foundation","doi-asserted-by":"publisher","award":["-"],"award-info":[{"award-number":["-"]}],"id":[{"id":"10.13039\/100007449","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JSPS KAKENHI 24590551"],"award-info":[{"award-number":["JSPS KAKENHI 24590551"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001700","name":"Ministry of Education, Culture, Sports, Science and Technology","doi-asserted-by":"publisher","award":["MEXT KAKENHI JP21H02741"],"award-info":[{"award-number":["MEXT KAKENHI JP21H02741"]}],"id":[{"id":"10.13039\/501100001700","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Primary varicella-zoster virus (VZV) infection leads to varicella and the establishment of lifelong latency in sensory ganglion neurons. Reactivation of latent VZV causes herpes zoster, which is frequently associated with chronic pain. Latent viral gene expression is restricted to the VZV latency-associated transcript (VLT) and VLT-ORF63 (VLT63) fusion transcripts. Since VLT and VLT63 encode proteins that are expressed during lytic infection, we investigated whether pVLT and pVLT-ORF63 are essential for VZV replication by performing VZV genome mutagenesis using CRISPR\/Cas9 and BAC technologies. We first established that CRISPR\/Cas9 can efficiently mutate VZV genomes in lytically VZV-infected cells through targeting non-essential genes ORF8 and ORF11 and subsequently show recovery of viable mutant viruses. By contrast, the VLT region was markedly resistant to CRISPR\/Cas9 editing. Whereas most mutants expressed wild-type or N-terminally altered versions of pVLT and pVLT-ORF63, only a minority of the resulting mutant viruses lacked pVLT and pVLT-ORF63 coding potential. Growth curve analysis showed that pVLT\/pVLT-ORF63 negative viruses were viable, but impaired in growth in epithelial cells. We confirmed this phenotype independently using BAC-derived pVLT\/pVLT-ORF63 negative and repaired viruses. Collectively, these data demonstrate that pVLT and\/or pVLT-ORF63 are dispensable for lytic VZV replication but promote efficient VZV infection in epithelial cells.<\/jats:p>","DOI":"10.3390\/v13112289","type":"journal-article","created":{"date-parts":[[2021,11,17]],"date-time":"2021-11-17T21:32:07Z","timestamp":1637184727000},"page":"2289","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7539-1526","authenticated-orcid":false,"given":"Shirley E.","family":"Braspenning","sequence":"first","affiliation":[{"name":"Department of Viroscience, Erasmus MC, 3015 GD Rotterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1981-0420","authenticated-orcid":false,"given":"Robert Jan","family":"Lebbink","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4292-0599","authenticated-orcid":false,"given":"Daniel P.","family":"Depledge","sequence":"additional","affiliation":[{"name":"Institute of Virology, Hannover Medical School, 30625 Hannover, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claudia M. E.","family":"Schapendonk","sequence":"additional","affiliation":[{"name":"Department of Viroscience, Erasmus MC, 3015 GD Rotterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura A.","family":"Anderson","sequence":"additional","affiliation":[{"name":"Department of Viroscience, Erasmus MC, 3015 GD Rotterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2465-2674","authenticated-orcid":false,"given":"Georges M. G. M.","family":"Verjans","sequence":"additional","affiliation":[{"name":"Department of Viroscience, Erasmus MC, 3015 GD Rotterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7951-6904","authenticated-orcid":false,"given":"Tomohiko","family":"Sadaoka","sequence":"additional","affiliation":[{"name":"Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8393-296X","authenticated-orcid":false,"given":"Werner J. D.","family":"Ouwendijk","sequence":"additional","affiliation":[{"name":"Department of Viroscience, Erasmus MC, 3015 GD Rotterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,11,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"15016","DOI":"10.1038\/nrdp.2015.16","article-title":"Varicella zoster virus infection","volume":"1","author":"Gershon","year":"2015","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"478","DOI":"10.1038\/306478a0","article-title":"Varicella-zoster virus DNA in human sensory ganglia","volume":"306","author":"Gilden","year":"1983","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Depledge, D., Sadaoka, T., and Ouwendijk, W. (2018). Molecular Aspects of Varicella-Zoster Virus Latency. Viruses, 10.","DOI":"10.20944\/preprints201806.0036.v1"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"635","DOI":"10.1056\/NEJM200003023420906","article-title":"Neurologic complications of the reactivation of varicella-zoster virus","volume":"342","author":"Gilden","year":"2000","journal-title":"N. Engl. J. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1526","DOI":"10.1056\/NEJMcp1403062","article-title":"Clinical practice. Postherpetic Neuralgia","volume":"371","author":"Johnson","year":"2014","journal-title":"N. Engl. J. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1019","DOI":"10.1056\/NEJMoa1603800","article-title":"Efficacy of the Herpes Zoster Subunit Vaccine in Adults 70 Years of Age or Older","volume":"375","author":"Cunningham","year":"2016","journal-title":"N. Engl. J. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e01568-20","DOI":"10.1128\/mBio.01568-20","article-title":"Decoding the Architecture of the Varicella-Zoster Virus Transcriptome","volume":"11","author":"Braspenning","year":"2020","journal-title":"Mbio"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.3389\/fmicb.2020.01179","article-title":"Analysis of Virus and Host Proteomes During Productive HSV-1 and VZV Infection in Human Epithelial Cells","volume":"11","author":"Ouwendijk","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1167","DOI":"10.1038\/s41467-018-03569-2","article-title":"A spliced latency-associated VZV transcript maps antisense to the viral transactivator gene 61","volume":"9","author":"Depledge","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"6324","DOI":"10.1038\/s41467-020-20031-4","article-title":"Varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency","volume":"11","author":"Ouwendijk","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Zhang, Z., Selariu, A., Warden, C., Huang, G., Huang, Y., Zaccheus, O., Cheng, T., Xia, N., and Zhu, H. (2010). Genome-wide mutagenesis reveals that ORF7 is a novel VZV skin-tropic factor. PLoS Pathog., 6.","DOI":"10.1371\/journal.ppat.1000971"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1006\/viro.1998.9198","article-title":"Varicella-zoster virus ORF61 deletion mutants replicate in cell culture, but a mutant with stop codons in ORF61 reverts to wild-type virus","volume":"246","author":"Cohen","year":"1998","journal-title":"Virology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e51662","DOI":"10.7554\/eLife.51662","article-title":"Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR\/Cas9","volume":"8","author":"Oh","year":"2019","journal-title":"Elife"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Van Diemen, F.R., Kruse, E.M., Hooykaas, M.J.G., Bruggeling, C.E., Sch\u00fcrch, A.C., van Ham, P.M., Imhof, S.M., Nijhuis, M., Wiertz, E.J.H.J., and Lebbink, R.J. (2016). CRISPR\/Cas9-Mediated Genome Editing of Herpesviruses Limits Productive and Latent Infections. PLoS Pathog., 12.","DOI":"10.1371\/journal.ppat.1005701"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3488","DOI":"10.1128\/JVI.01838-09","article-title":"Characterization of the Varicella-Zoster Virus ORF50 Gene, Which Encodes Glycoprotein M","volume":"84","author":"Sadaoka","year":"2010","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"6943","DOI":"10.1128\/JVI.00407-13","article-title":"Comprehensive Analysis of Varicella-Zoster Virus Proteins Using a New Monoclonal Antibody Collection","volume":"87","author":"Bailer","year":"2013","journal-title":"J. Virol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3730","DOI":"10.4049\/jimmunol.1302307","article-title":"Functional Characterization of Ocular-Derived Human Alphaherpesvirus Cross-Reactive CD4 T Cells","volume":"192","author":"Ouwendijk","year":"2014","journal-title":"J. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","article-title":"1000 Genome Project Data Processing Subgroup The Sequence Alignment\/Map format and SAMtools","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1093\/bioinformatics\/btq033","article-title":"BEDTools: A flexible suite of utilities for comparing genomic features","volume":"26","author":"Quinlan","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4069","DOI":"10.1016\/j.vaccine.2004.03.062","article-title":"Cloning of the varicella-zoster virus genome as an infectious bacterial artificial chromosome in Escherichia coli","volume":"22","author":"Nagaike","year":"2004","journal-title":"Vaccine"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"7720","DOI":"10.1128\/JVI.74.17.7720-7729.2000","article-title":"Fast screening procedures for random transposon libraries of cloned herpesvirus genomes: Mutational analysis of human cytomegalovirus envelope glycoprotein genes","volume":"74","author":"Hobom","year":"2000","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"280","DOI":"10.1016\/j.virol.2010.06.016","article-title":"Characterization of varicella-zoster virus-encoded ORF0 gene\u2014Comparison of parental and vaccine strains","volume":"405","author":"Koshizuka","year":"2010","journal-title":"Virology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1038\/s41587-020-00781-8","article-title":"Targeting herpes simplex virus with CRISPR-Cas9 cures herpetic stromal keratitis in mice","volume":"39","author":"Yin","year":"2021","journal-title":"Nat. Biotechnol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1128\/JVI.02245-13","article-title":"Varicella-Zoster Virus ORF49 Functions in the Efficient Production of Progeny Virus through Its Interaction with Essential Tegument Protein ORF44","volume":"88","author":"Sadaoka","year":"2014","journal-title":"J. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1006\/viro.1997.8652","article-title":"Disruption of the varicella-zoster virus dUTPase and the adjacent ORF9A gene results in impaired growth and reduced syncytia formation in vitro","volume":"234","author":"Ross","year":"1997","journal-title":"Virology"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"5825","DOI":"10.1128\/JVI.00303-08","article-title":"Functions of the ORF9-to-ORF12 gene cluster in varicella-zoster virus replication and in the pathogenesis of skin infection","volume":"82","author":"Che","year":"2008","journal-title":"J. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1038\/nbt.3437","article-title":"Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9","volume":"34","author":"Doench","year":"2016","journal-title":"Nat. Biotechnol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"9024","DOI":"10.1128\/JVI.02666-06","article-title":"Genetic Analysis of Varicella-Zoster Virus ORF0 to ORF4 by Use of a Novel Luciferase Bacterial Artificial Chromosome System","volume":"81","author":"Zhang","year":"2007","journal-title":"J. Virol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Kallimasioti-Pazi, E.M., Thelakkad Chathoth, K., Taylor, G.C., Meynert, A., Ballinger, T., Kelder, M.J.E., Lalev\u00e9e, S., Sanli, I., Feil, R., and Wood, A.J. (2018). Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair. PLoS Biol., 16.","DOI":"10.1101\/267690"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"823","DOI":"10.1126\/science.aac6572","article-title":"Dynamics of CRISPR-Cas9 genome interrogation in living cells","volume":"350","author":"Knight","year":"2015","journal-title":"Science"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1016\/j.stem.2014.10.004","article-title":"Efficient Ablation of Genes in Human Hematopoietic Stem and Effector Cells using CRISPR\/Cas9","volume":"15","author":"Mandal","year":"2014","journal-title":"Cell Stem Cell"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1717","DOI":"10.1111\/febs.12735","article-title":"Dual sgRNAs facilitate CRISPR\/Cas9-mediated mouse genome targeting","volume":"281","author":"Zhou","year":"2014","journal-title":"FEBS J."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"6917","DOI":"10.1128\/JVI.00081-09","article-title":"Enumeration of an extremely high particle-to-PFU ratio for Varicella-zoster virus","volume":"83","author":"Carpenter","year":"2009","journal-title":"J. Virol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e01108-17","DOI":"10.1128\/JVI.01108-17","article-title":"Human Embryonic Stem Cell-Derived Neurons Are Highly Permissive for Varicella-Zoster Virus Lytic Infection","volume":"92","author":"Sadaoka","year":"2018","journal-title":"J. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e12677","DOI":"10.7554\/eLife.12677","article-title":"Nucleosomes impede Cas9 access to DNA in vivo and in vitro","volume":"5","author":"Horlbeck","year":"2016","journal-title":"Elife"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e13450","DOI":"10.7554\/eLife.13450","article-title":"Nucleosome breathing and remodeling constrain CRISPR-Cas9 function","volume":"5","author":"Isaac","year":"2016","journal-title":"Elife"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Finnen, R.L., and Banfield, B.W. (2018). CRISPR\/Cas9 Mutagenesis of UL21 in Multiple Strains of Herpes Simplex Virus Reveals Differential Requirements for pUL21 in Viral Replication. Viruses, 10.","DOI":"10.3390\/v10050258"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"11501","DOI":"10.1128\/JVI.79.17.11501-11506.2005","article-title":"Replication of Varicella-Zoster Virus in Human Skin Organ Culture","volume":"79","author":"Taylor","year":"2005","journal-title":"J. Virol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"e01082-20","DOI":"10.1128\/JVI.01082-20","article-title":"A Novel Human Skin Tissue Model To Study Varicella-Zoster Virus and Human Cytomegalovirus","volume":"94","author":"Lloyd","year":"2020","journal-title":"J. Virol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"8686","DOI":"10.1128\/JVI.00920-16","article-title":"Varicella-Zoster Virus Activates CREB, and Inhibition of the pCREB-p300\/CBP Interaction Inhibits Viral Replication In Vitro and Skin Pathogenesis In Vivo","volume":"90","author":"Sen","year":"2016","journal-title":"J. Virol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"11969","DOI":"10.1073\/pnas.95.20.11969","article-title":"The ORF47 and ORF66 putative protein kinases of varicella-zoster virus determine tropism for human T cells and skin in the SCID-hu mouse","volume":"95","author":"Moffat","year":"1998","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"5607","DOI":"10.1128\/JVI.77.10.5607-5620.2003","article-title":"Mutational Analysis of Open Reading Frames 62 and 71, Encoding the Varicella-Zoster Virus Immediate-Early Transactivating Protein, IE62, and Effects on Replication In Vitro and in Skin Xenografts in the SCID-hu Mouse In Vivo","volume":"77","author":"Sato","year":"2003","journal-title":"J. Virol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"8468","DOI":"10.1128\/JVI.76.16.8468-8471.2002","article-title":"Glycoprotein I of Varicella-Zoster Virus Is Required for Viral Replication in Skin and T Cells","volume":"76","author":"Moffat","year":"2002","journal-title":"J. Virol."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Markus, A., Lebenthal-Loinger, I., Yang, I.H., Kinchington, P.R., and Goldstein, R.S. (2015). An In Vitro Model of Latency and Reactivation of Varicella Zoster Virus in Human Stem Cell-Derived Neurons. PLoS Pathog., 11.","DOI":"10.1371\/journal.ppat.1004885"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"E2403","DOI":"10.1073\/pnas.1522575113","article-title":"In vitro system using human neurons demonstrates that varicella-zoster vaccine virus is impaired for reactivation, but not latency","volume":"113","author":"Sadaoka","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"6065","DOI":"10.1038\/s41467-020-19821-7","article-title":"CRISPR based editing of SIV proviral DNA in ART treated non-human primates","volume":"11","author":"Mancuso","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"41968","DOI":"10.1038\/srep41968","article-title":"A combinational CRISPR\/Cas9 gene-editing approach can halt HIV replication and prevent viral escape","volume":"7","author":"Lebbink","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"13157","DOI":"10.1073\/pnas.1410785111","article-title":"RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection","volume":"111","author":"Wang","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"e02183-18","DOI":"10.1128\/JVI.02183-18","article-title":"Reduction of Kaposi\u2019s Sarcoma-Associated Herpesvirus Latency Using CRISPR-Cas9 To Edit the Latency-Associated Nuclear Antigen Gene","volume":"93","author":"Tso","year":"2019","journal-title":"J. Virol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"4148","DOI":"10.1038\/s41467-020-17936-5","article-title":"Gene editing and elimination of latent herpes simplex virus in vivo","volume":"11","author":"Aubert","year":"2020","journal-title":"Nat. Commun."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/11\/2289\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:30:54Z","timestamp":1760167854000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/11\/2289"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,11,16]]},"references-count":50,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2021,11]]}},"alternative-id":["v13112289"],"URL":"https:\/\/doi.org\/10.3390\/v13112289","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,11,16]]}}}