{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T03:38:11Z","timestamp":1760240291707,"version":"build-2065373602"},"reference-count":53,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2019,4,29]],"date-time":"2019-04-29T00:00:00Z","timestamp":1556496000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Nucleoprotein (N) is a key element in rabies virus (RABV) replication. To further investigate the effect of N on RABV, we manipulated an infectious cDNA clone of the RABV HEP-Flury to rearrange the N gene from its wild-type position of 1 (N-P-M-G-L) to 2 (P-N-M-G-L), 3 (P-M-N-G-L), or 4 (P-M-G-N-L), using an approach that left the viral nucleotide sequence unaltered. Subsequently, viable viruses were recovered from each of the rearranged cDNA and examined for their gene expression levels, growth kinetics in cell culture, pathogenicity in suckling mice and protection in mice. The results showed that gene rearrangement decreased N mRNA transcription and vRNA replication. As a result, all viruses with rearranged genomes showed worse replication than that of rHEP-Flury in NA cells at a MOI of 0.01, but equivalent or slightly better replication levels at a MOI of 3. Consequently, the lethality in suckling mice infected with N4 was clearly attenuated compared with rHEP-Flury. However, the protection to mice was not enhanced. This study not only gives us insight into the understanding of the phenotype of RABV N gene rearrangement, but also helps with rabies vaccine candidate construction.<\/jats:p>","DOI":"10.3390\/v11050402","type":"journal-article","created":{"date-parts":[[2019,4,29]],"date-time":"2019-04-29T07:01:22Z","timestamp":1556521282000},"page":"402","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Phenotypic Consequence of Rearranging the N Gene of RABV HEP-Flury"],"prefix":"10.3390","volume":"11","author":[{"given":"Mingzhu","family":"Mei","sequence":"first","affiliation":[{"name":"College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China"},{"name":"Guangdong Inspection and Quarantine Technology Center, Guangzhou 510623, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6194-7885","authenticated-orcid":false,"given":"Teng","family":"Long","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qiong","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jing","family":"Zhao","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qin","family":"Tian","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiaojiao","family":"Peng","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jun","family":"Luo","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"He","family":"Jiang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yingyi","family":"Lin","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2896-3629","authenticated-orcid":false,"given":"Zhixiong","family":"Lin","sequence":"additional","affiliation":[{"name":"Guangdong Inspection and Quarantine Technology Center, Guangzhou 510623, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaofeng","family":"Guo","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2019,4,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3501","DOI":"10.1073\/pnas.95.7.3501","article-title":"Gene rearrangement attenuates expression and lethality of a nonsegmented negative strand rna virus","volume":"95","author":"Wertz","year":"1998","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4705","DOI":"10.1128\/JVI.73.6.4705-4712.1999","article-title":"Phenotypic consequences of rearranging the p, m, and g genes of vesicular stomatitis virus","volume":"73","author":"Ball","year":"1999","journal-title":"J. Virol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"7895","DOI":"10.1128\/JVI.74.17.7895-7902.2000","article-title":"Moving the glycoprotein gene of vesicular stomatitis virus to promoter-proximal positions accelerates and enhances the protective immune response","volume":"74","author":"Flanagan","year":"2000","journal-title":"J. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"9121","DOI":"10.1128\/JVI.75.19.9121-9128.2001","article-title":"Rescue of rabies virus from cloned cdna and identification of the pathogenicity-related gene: Glycoprotein gene is associated with virulence for adult mice","volume":"75","author":"Ito","year":"2001","journal-title":"J. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/B978-0-12-387040-7.00001-9","article-title":"Rabies virus transcription and replication","volume":"79","author":"Albertini","year":"2011","journal-title":"Adv. Virus Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1146\/annurev-virology-100114-055157","article-title":"Everything you always wanted to know about rabies virus (but were afraid to ask)","volume":"2","author":"Davis","year":"2015","journal-title":"Annu. Rev. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2909","DOI":"10.1099\/0022-1317-79-12-2909","article-title":"Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures","volume":"79","author":"Iseni","year":"1998","journal-title":"J. Gen. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2001","DOI":"10.1073\/pnas.88.5.2001","article-title":"Rabies virus nucleoprotein expressed in and purified from insect cells is efficacious as a vaccine","volume":"88","author":"Fu","year":"1991","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"9405","DOI":"10.1073\/pnas.0403060101","article-title":"Rabies virus nucleoprotein as a carrier for foreign antigens","volume":"101","author":"Koser","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1111\/1348-0421.12058","article-title":"Importance of rabies virus nucleoprotein in viral evasion of interferon response in the brain","volume":"57","author":"Masatani","year":"2013","journal-title":"Microbiol. Immunol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1016\/j.virusres.2010.09.016","article-title":"Amino acids at positions 273 and 394 in rabies virus nucleoprotein are important for both evasion of host rig-i-mediated antiviral response and pathogenicity","volume":"155","author":"Masatani","year":"2011","journal-title":"Virus Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"4002","DOI":"10.1128\/JVI.02220-09","article-title":"Rabies virus nucleoprotein functions to evade activation of the rig-i-mediated antiviral response","volume":"84","author":"Masatani","year":"2010","journal-title":"J. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.virusres.2007.07.018","article-title":"Glycoprotein gene relocation in rabies virus","volume":"131","author":"Wu","year":"2008","journal-title":"Virus Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"7673","DOI":"10.1128\/JVI.79.12.7673-7681.2005","article-title":"Identification of the rabies virus alpha\/beta interferon antagonist: Phosphoprotein p interferes with phosphorylation of interferon regulatory factor 3","volume":"79","author":"Brzozka","year":"2005","journal-title":"J. Virol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1911","DOI":"10.1128\/JVI.02055-08","article-title":"Attenuation of rabies virus replication and virulence by picornavirus internal ribosome entry site elements","volume":"83","author":"Marschalek","year":"2009","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"558","DOI":"10.1111\/j.1348-0421.2011.00354.x","article-title":"A unique transcription mode of rabies virus high egg passage-flury strain detected in infected baby hamster kidney-21 cells","volume":"55","author":"Morimoto","year":"2011","journal-title":"Microbiol. Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"120","DOI":"10.3389\/fmicb.2017.00120","article-title":"Phenotypic consequences in vivo and in vitro of rearranging the p gene of rabv hep-flury","volume":"8","author":"Mei","year":"2017","journal-title":"Front. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1007\/s13337-014-0220-1","article-title":"Gene order rearrangement of the m gene in the rabies virus leads to slower replication","volume":"25","author":"Yang","year":"2014","journal-title":"Virus Dis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1016\/S0166-0934(02)00249-5","article-title":"An improved method for recovering rabies virus from cloned cdna","volume":"107","author":"Inoue","year":"2003","journal-title":"J. Virol. Methods"},{"key":"ref_20","unstructured":"National Research Council (US) (2011). Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals, National Academies Press (US). [8th ed.]."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1007\/s11262-016-1313-2","article-title":"A recombinant rabies virus carrying gfp between n and p affects viral transcription in vitro","volume":"52","author":"Luo","year":"2016","journal-title":"Virus Genes"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"691","DOI":"10.3389\/fmicb.2015.00691","article-title":"Itraq protein profile analysis of neuroblastoma (na) cells infected with the rabies viruses rhep-flury and hep-dg","volume":"6","author":"Yang","year":"2015","journal-title":"Front. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1704","DOI":"10.1080\/15548627.2016.1196315","article-title":"Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines","volume":"12","author":"Peng","year":"2016","journal-title":"Autophagy"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1016\/j.virol.2014.09.010","article-title":"Recombinant rabies virus expressing ifnalpha1 enhanced immune responses resulting in its attenuation and stronger immunogenicity","volume":"468\u2013470","author":"Wang","year":"2014","journal-title":"Virology"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1128\/JVI.01309-10","article-title":"Rabies virus (rv) glycoprotein expression levels are not critical for pathogenicity of rv","volume":"85","author":"Wirblich","year":"2011","journal-title":"J. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1016\/j.virusres.2006.01.014","article-title":"A highly attenuated rabies virus hep-flury strain reverts to virulent by single amino acid substitution to arginine at position 333 in glycoprotein","volume":"119","author":"Inoue","year":"2006","journal-title":"Virus Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1016\/0092-8674(85)90079-0","article-title":"Role of the nucleocapsid protein in regulating vesicular stomatitis virus rna synthesis","volume":"41","author":"Arnheiter","year":"1985","journal-title":"Cell"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1128\/jvi.49.2.303-309.1984","article-title":"N protein alone satisfies the requirement for protein synthesis during rna replication of vesicular stomatitis virus","volume":"49","author":"Patton","year":"1984","journal-title":"J. Virol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"12074","DOI":"10.1128\/JVI.77.22.12074-12082.2003","article-title":"Dissociation of rabies virus matrix protein functions in regulation of viral rna synthesis and virus assembly","volume":"77","author":"Finke","year":"2003","journal-title":"J. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/B978-0-12-387040-7.00002-0","article-title":"Rabies virus assembly and budding","volume":"79","author":"Okumura","year":"2011","journal-title":"Adv. Virus Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"9837","DOI":"10.1128\/JVI.78.18.9837-9841.2004","article-title":"Fitness analyses of vesicular stomatitis strains with rearranged genomes reveal replicative disadvantages","volume":"78","author":"Novella","year":"2004","journal-title":"J. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1423","DOI":"10.1016\/j.jmb.2004.08.004","article-title":"Molecular basis of fitness loss and fitness recovery in vesicular stomatitis virus","volume":"342","author":"Novella","year":"2004","journal-title":"J. Mol. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"481","DOI":"10.2217\/17460794.3.5.481","article-title":"Concepts in the pathogenesis of rabies","volume":"3","author":"Dietzschold","year":"2008","journal-title":"Future Virol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Ohara, S., Sato, S., Oyama, K., Tsutsui, K., and Iijima, T. (2013). Rabies virus vector transgene expression level and cytotoxicity improvement induced by deletion of glycoprotein gene. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0080245"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3374","DOI":"10.1128\/JVI.76.7.3374-3381.2002","article-title":"Overexpression of the rabies virus glycoprotein results in enhancement of apoptosis and antiviral immune response","volume":"76","author":"Faber","year":"2002","journal-title":"J. Virol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1016\/j.virusres.2005.04.004","article-title":"Replication strategies of rabies virus","volume":"111","author":"Finke","year":"2005","journal-title":"Virus Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"189","DOI":"10.7774\/cevr.2015.4.2.189","article-title":"Application of recombinant adenoviruses expressing glycoprotein or nucleoprotein of rabies virus to korean raccoon dogs","volume":"4","author":"Choi","year":"2015","journal-title":"Clin. Exp. Vaccine Res."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1128\/jvi.50.3.773-778.1984","article-title":"Nucleotide sequence and host la protein interactions of rabies virus leader rna","volume":"50","author":"Kurilla","year":"1984","journal-title":"J. Virol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"43822","DOI":"10.18632\/oncotarget.16517","article-title":"Rabies viruses leader rna interacts with host hsc70 and inhibits virus replication","volume":"8","author":"Zhang","year":"2017","journal-title":"Oncotarget"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"9591","DOI":"10.1128\/JVI.01298-15","article-title":"A dynein light chain 1 binding motif in rabies virus polymerase l protein plays a role in microtubule reorganization and viral primary transcription","volume":"89","author":"Bauer","year":"2015","journal-title":"J. Virol."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Ohara, S., Sota, Y., Sato, S., Tsutsui, K.I., and Iijima, T. (2017). Increased transgene expression level of rabies virus vector for transsynaptic tracing. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0180960"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Liu, X., Yang, Y., Sun, Z., Chen, J., Ai, J., Dun, C., Fu, Z.F., Niu, X., and Guo, X. (2014). A recombinant rabies virus encoding two copies of the glycoprotein gene confers protection in dogs against a virulent challenge. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0087105"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1196\/annals.1299.108","article-title":"Glycoprotein of nonpathogenic rabies viruses is a major inducer of apoptosis in human jurkat t cells","volume":"1010","author":"Lay","year":"2003","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"571","DOI":"10.1080\/13550280500385310","article-title":"Glycoprotein-mediated induction of apoptosis limits the spread of attenuated rabies viruses in the central nervous system of mice","volume":"11","author":"Sarmento","year":"2005","journal-title":"J. Neurovirol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1016\/j.virusres.2006.05.002","article-title":"Rabies virus-induced apoptosis involves caspase-dependent and caspase-independent pathways","volume":"121","author":"Sarmento","year":"2006","journal-title":"Virus Res."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Davis, B.M., Fensterl, V., Lawrence, T.M., Hudacek, A.W., Sen, G.C., and Schnell, M.J. (2017). Ifit2 is a restriction factor in rabies virus pathogenicity. J. Virol., 91.","DOI":"10.1128\/JVI.00889-17"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.virusres.2009.03.014","article-title":"Role of chemokines in the enhancement of bbb permeability and inflammatory infiltration after rabies virus infection","volume":"144","author":"Kuang","year":"2009","journal-title":"Virus Res."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2297","DOI":"10.1007\/s00705-013-1743-2","article-title":"Wild-type rabies virus phosphoprotein is associated with viral sensitivity to type i interferon treatment","volume":"158","author":"Niu","year":"2013","journal-title":"Arch. Virol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"6543","DOI":"10.1128\/JVI.78.12.6543-6555.2004","article-title":"Lyssavirus matrix protein induces apoptosis by a trail-dependent mechanism involving caspase-8 activation","volume":"78","author":"Kassis","year":"2004","journal-title":"J. Virol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"10537","DOI":"10.1128\/JVI.77.19.10537-10547.2003","article-title":"Glycoprotein of nonpathogenic rabies viruses is a key determinant of human cell apoptosis","volume":"77","author":"Prehaud","year":"2003","journal-title":"J. Virol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1016\/S0042-6822(03)00491-4","article-title":"High level of bcl-2 counteracts apoptosis mediated by a live rabies virus vaccine strain and induces long-term infection","volume":"314","author":"Thoulouze","year":"2003","journal-title":"Virology"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"372","DOI":"10.1007\/s00401-005-0006-z","article-title":"Comparative pathogenesis of recombinant rabies vaccine strain sad-l16 and sad-d29 with replacement of arg333 in the glycoprotein after peripheral inoculation of neonatal mice: Less neurovirulent strain is a stronger inducer of neuronal apoptosis","volume":"111","author":"Jackson","year":"2006","journal-title":"Acta Neuropathol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"370","DOI":"10.1007\/s13365-015-0320-8","article-title":"Rabies virus phosphoprotein interacts with mitochondrial complex i and induces mitochondrial dysfunction and oxidative stress","volume":"21","author":"Kammouni","year":"2015","journal-title":"J. Neurovirol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/11\/5\/402\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T12:48:02Z","timestamp":1760186882000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/11\/5\/402"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,4,29]]},"references-count":53,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2019,5]]}},"alternative-id":["v11050402"],"URL":"https:\/\/doi.org\/10.3390\/v11050402","relation":{},"ISSN":["1999-4915"],"issn-type":[{"type":"electronic","value":"1999-4915"}],"subject":[],"published":{"date-parts":[[2019,4,29]]}}}