Negeviruses Reduce Replication of Alphaviruses during Coinfection
Negeviruses are a group of insect-specific viruses (ISVs) that have been found in many arthropods. Their presence in important vector species led us to examine their interactions with arboviruses during coinfections. Wild-type negeviruses reduced the replication of several alphaviruses during coinfe...
- Autores:
-
Patterson, Edward Ian
Kautz, Tiffany Faye
Contreras Gutiérrez, María Angélica
Guzmán, Hilda
Tesh, Robert B
Hughes, Grant L.
Forrester, Naomi L.
- Tipo de recurso:
- Article of investigation
- Fecha de publicación:
- 2021
- Institución:
- Tecnológico de Antioquia
- Repositorio:
- Repositorio Tdea
- Idioma:
- eng
- OAI Identifier:
- oai:dspace.tdea.edu.co:tdea/2773
- Acceso en línea:
- https://dspace.tdea.edu.co/handle/tdea/2773
- Palabra clave:
- Alphavirus
Arbovirus
Arboviruses
Chikungunya virus
Vírus de Chikungunya
Negev virus
Negevirus
Insect-specific virus
Paratransgenesis
- Rights
- openAccess
- License
- https://creativecommons.org/licenses/by/4.0/
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| dc.title.none.fl_str_mv |
Negeviruses Reduce Replication of Alphaviruses during Coinfection |
| title |
Negeviruses Reduce Replication of Alphaviruses during Coinfection |
| spellingShingle |
Negeviruses Reduce Replication of Alphaviruses during Coinfection Alphavirus Arbovirus Arboviruses Chikungunya virus Vírus de Chikungunya Negev virus Negevirus Insect-specific virus Paratransgenesis |
| title_short |
Negeviruses Reduce Replication of Alphaviruses during Coinfection |
| title_full |
Negeviruses Reduce Replication of Alphaviruses during Coinfection |
| title_fullStr |
Negeviruses Reduce Replication of Alphaviruses during Coinfection |
| title_full_unstemmed |
Negeviruses Reduce Replication of Alphaviruses during Coinfection |
| title_sort |
Negeviruses Reduce Replication of Alphaviruses during Coinfection |
| dc.creator.fl_str_mv |
Patterson, Edward Ian Kautz, Tiffany Faye Contreras Gutiérrez, María Angélica Guzmán, Hilda Tesh, Robert B Hughes, Grant L. Forrester, Naomi L. |
| dc.contributor.author.none.fl_str_mv |
Patterson, Edward Ian Kautz, Tiffany Faye Contreras Gutiérrez, María Angélica Guzmán, Hilda Tesh, Robert B Hughes, Grant L. Forrester, Naomi L. |
| dc.subject.agrovoc.none.fl_str_mv |
Alphavirus Arbovirus Arboviruses Chikungunya virus Vírus de Chikungunya |
| topic |
Alphavirus Arbovirus Arboviruses Chikungunya virus Vírus de Chikungunya Negev virus Negevirus Insect-specific virus Paratransgenesis |
| dc.subject.proposal.none.fl_str_mv |
Negev virus Negevirus Insect-specific virus Paratransgenesis |
| description |
Negeviruses are a group of insect-specific viruses (ISVs) that have been found in many arthropods. Their presence in important vector species led us to examine their interactions with arboviruses during coinfections. Wild-type negeviruses reduced the replication of several alphaviruses during coinfections in mosquito cells. Negev virus (NEGV) isolates were also used to express green fluorescent protein (GFP) and anti-chikungunya virus (CHIKV) antibody fragments during coinfections with CHIKV. NEGV expressing anti-CHIKV antibody fragments was able to further reduce replication of CHIKV during coinfections, while reductions of CHIKV with NEGV expressing GFP were similar to titers with wild-type NEGV alone. These results are the first to show that negeviruses induce superinfection exclusion of arboviruses and to demonstrate a novel approach to deliver antiviral antibody fragments with paratransgenic ISVs. The ability to inhibit arbovirus replication and express exogenous proteins in mosquito cells makes negeviruses a promising platform for control of arthropod-borne pathogens. IMPORTANCE Negeviruses are a group of insect-specific viruses (ISVs), viruses known to infect only insects. They have been discovered over a wide geographical and species range. Their ability to infect mosquito species that transmit dangerous arboviruses makes negeviruses a candidate for a pathogen control platform. Coinfections of mosquito cells with a negevirus and an alphavirus demonstrated that negeviruses can inhibit the replication of alphaviruses. Additionally, modifying Negev virus (NEGV) to express a fragment of an anti-CHIKV antibody further reduced the replication of CHIKV in coinfected cells. This is the first evidence to demonstrate that negeviruses can inhibit the replication of important arboviruses in mosquito cells. The ability of a modified NEGV to drive the expression of antiviral proteins also highlights a method for negeviruses to target specific pathogens and limit the incidence of vector-borne diseases. |
| publishDate |
2021 |
| dc.date.issued.none.fl_str_mv |
2021 |
| dc.date.accessioned.none.fl_str_mv |
2023-04-11T19:16:42Z |
| dc.date.available.none.fl_str_mv |
2023-04-11T19:16:42Z |
| dc.type.spa.fl_str_mv |
Artículo de revista |
| dc.type.coar.spa.fl_str_mv |
http://purl.org/coar/resource_type/c_2df8fbb1 |
| dc.type.content.spa.fl_str_mv |
Text |
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info:eu-repo/semantics/article |
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http://purl.org/redcol/resource_type/ART |
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info:eu-repo/semantics/publishedVersion |
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0022-538X |
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1098-5514 |
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0022-538X 1098-5514 |
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eng |
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eng |
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12 |
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1 |
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95 |
| dc.relation.ispartofjournal.spa.fl_str_mv |
Journal of Virology |
| dc.relation.references.spa.fl_str_mv |
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Colmant AMG, O'Brien CA, Newton ND, Watterson D, Hardy J, Coulibaly F, Bielefeldt-Ohmann H, Warrilow D, Huang B, Paramitha D, Harrison JJ, Hall RA, Hobson-Peters J. 2020. Novel monoclonal antibodies against Australian strains of negeviruses and insights into virus structure, replication and host -restriction. J Gen Virol 101:440–452. https://doi.org/10.1099/jgv .0.001388. Nabeshima T, Inoue S, Okamoto K, Posadas-Herrera G, Yu F, Uchida L, Ichinose A, Sakaguchi M, Sunahara T, Buerano CC, Tadena FP, Orbita IB, Natividad FF, Morita K. 2014. Tanay virus, a new species of virus isolated from mosquitoes in the Philippines. J Gen Virol 95:1390–1395. https://doi .org/10.1099/vir.0.061887-0. O'Brien CA, McLean BJ, Colmant AMG, Harrison JJ, Hall-Mendelin S, van den Hurk AF, Johansen CA, Watterson D, Bielefeldt-Ohmann H, Newton ND, Schulz BL, Hall RA, Hobson-Peters J. 2017. Discovery and characterisation of castlerea virus, a new species of negevirus isolated in Australia. 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Broad dengue neutralization in mosquitoes expressing an engineered antibody. PLoS Pathog 16:e1008103. https://doi.org/10.1371/journal.ppat.1008103. Buchman A, Gamez S, Li M, Antoshechkin I, Li HH, Wang HW, Chen CH, Klein MJ, Duchemin JB, Crowe JE, Jr, Paradkar PN, Akbari OS. 2020. Broad dengue neutralization in mosquitoes expressing an engineered antibody. PLoS Pathog 16:e1008103. https://doi.org/10.1371/journal.ppat.1008103. Kawakami K, Kurnia YW, Fujita R, Ito T, Isawa H, Asano S, Binh ND, Bando H. 2016. Characterization of a novel negevirus isolated from Aedes larvae collected in a subarctic region of Japan. Arch Virol 161:801–809. https:// doi.org/10.1007/s00705-015-2711-9. Kautz TF, Jaworski E, Routh A, Forrester NL. 2020. A low fidelity virus shows increased recombination during the removal of an alphavirus reporter gene. Viruses 12:660. https://doi.org/10.3390/v12060660 Suzuki Y, Niu G, Hughes GL, Rasgon JL. 2014. A viral over-expression system for the major malaria mosquito Anopheles gambiae. Sci Rep 4:5127. https://doi.org/10.1038/srep05127 Walker T, Jeffries CL, Mansfield KL, Johnson N. 2014. Mosquito cell lines: history, isolation, availability and application to assess the threat of arboviral transmission in the United Kingdom. Parasit Vectors 7:382. https:// doi.org/10.1186/1756-3305-7-382. Gorchakov RV, Tesh RB, Weaver SC, Nasar F. 2014. Generation of an infectious Negev virus cDNA clone. J Gen Virol 95:2071–2074. https://doi.org/ 10.1099/vir.0.066019-0. Levitt NH, Ramsburg HH, Hasty SE, Repik PM, Cole FE, Jr, Lupton HW. 1986. Development of an attenuated strain of chikungunya virus for use in vaccine production. Vaccine 4:157–162. https://doi.org/10.1016/0264 -410X(86)90003-4. Smith DR, Adams AP, Kenney JL, Wang E, Weaver SC. 2008. Venezuelan equine encephalitis virus in the mosquito vector Aedes taeniorhynchus: infection initiated by a small number of susceptible epithelial cells and a population bottleneck. Virology 372:176–186. https://doi.org/10.1016/j .virol.2007.10.011. Kinney RM, Chang GJ, Tsuchiya KR, Sneider JM, Roehrig JT, Woodward TM, Trent DW. 1993. Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 59-noncoding region and the E2 envelope glycoprotein. J Virol 67:1269–1277. https://doi.org/10.1128/JVI.67.3.1269 -1277.1993. |
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Patterson, Edward Ian3196371a-7392-46bb-beb8-36ec6fbf759cKautz, Tiffany Fayed4ba9bb0-bb85-4cdd-a96f-00c8f2a385f0Contreras Gutiérrez, María Angélica7c9504de-e558-4dcd-86c1-81dbc9b08615Guzmán, Hildaca9a5567-397e-474f-bcde-734b8801ed31Tesh, Robert B656a45cb-4a04-41bc-97af-d7a150f98f65Hughes, Grant L.ddcfa300-2714-4c6f-a68b-cfac01af41feForrester, Naomi L.9acf807d-1ea4-4fa0-80b0-077113bffb3e2023-04-11T19:16:42Z2023-04-11T19:16:42Z20210022-538Xhttps://dspace.tdea.edu.co/handle/tdea/27731098-5514Negeviruses are a group of insect-specific viruses (ISVs) that have been found in many arthropods. Their presence in important vector species led us to examine their interactions with arboviruses during coinfections. Wild-type negeviruses reduced the replication of several alphaviruses during coinfections in mosquito cells. Negev virus (NEGV) isolates were also used to express green fluorescent protein (GFP) and anti-chikungunya virus (CHIKV) antibody fragments during coinfections with CHIKV. NEGV expressing anti-CHIKV antibody fragments was able to further reduce replication of CHIKV during coinfections, while reductions of CHIKV with NEGV expressing GFP were similar to titers with wild-type NEGV alone. These results are the first to show that negeviruses induce superinfection exclusion of arboviruses and to demonstrate a novel approach to deliver antiviral antibody fragments with paratransgenic ISVs. The ability to inhibit arbovirus replication and express exogenous proteins in mosquito cells makes negeviruses a promising platform for control of arthropod-borne pathogens. IMPORTANCE Negeviruses are a group of insect-specific viruses (ISVs), viruses known to infect only insects. They have been discovered over a wide geographical and species range. Their ability to infect mosquito species that transmit dangerous arboviruses makes negeviruses a candidate for a pathogen control platform. Coinfections of mosquito cells with a negevirus and an alphavirus demonstrated that negeviruses can inhibit the replication of alphaviruses. Additionally, modifying Negev virus (NEGV) to express a fragment of an anti-CHIKV antibody further reduced the replication of CHIKV in coinfected cells. This is the first evidence to demonstrate that negeviruses can inhibit the replication of important arboviruses in mosquito cells. The ability of a modified NEGV to drive the expression of antiviral proteins also highlights a method for negeviruses to target specific pathogens and limit the incidence of vector-borne diseases.12 páginasapplication/pdfengAmerican Society for MicrobiologyEstados Unidoshttps://creativecommons.org/licenses/by/4.0/Atribución 4.0 Internacional (CC BY 4.0)info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8223947/?report=classicNegeviruses Reduce Replication of Alphaviruses during CoinfectionArtículo de revistahttp://purl.org/coar/resource_type/c_2df8fbb1Textinfo:eu-repo/semantics/articlehttp://purl.org/redcol/resource_type/ARTinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/version/c_970fb48d4fbd8a851214195Journal of VirologyCalisher CH, Higgs S. 2018. The discovery of arthropod-specific viruses in hematophagous arthropods: an open door to understanding the mechanisms of arbovirus and arthropod evolution? 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J Virol 67:1269–1277. https://doi.org/10.1128/JVI.67.3.1269 -1277.1993.AlphavirusArbovirusArbovirusesChikungunya virusVírus de ChikungunyaNegev virusNegevirusInsect-specific virusParatransgenesisLICENSElicense.txtlicense.txttext/plain; charset=utf-814828https://dspace.tdea.edu.co/bitstream/tdea/2773/2/license.txt2f9959eaf5b71fae44bbf9ec84150c7aMD52open accessORIGINALNegeviruses Reduce Replication of Alphaviruses during.pdfNegeviruses Reduce Replication of Alphaviruses during.pdfapplication/pdf2332633https://dspace.tdea.edu.co/bitstream/tdea/2773/1/Negeviruses%20Reduce%20Replication%20of%20Alphaviruses%20during.pdf6998401eb431e2a3282890d399e5bcafMD51open accessTEXTNegeviruses Reduce Replication of Alphaviruses during.pdf.txtNegeviruses Reduce Replication of Alphaviruses during.pdf.txtExtracted texttext/plain45618https://dspace.tdea.edu.co/bitstream/tdea/2773/3/Negeviruses%20Reduce%20Replication%20of%20Alphaviruses%20during.pdf.txtbcef40d370c6b04c5941b95fd5bf70e1MD53open 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 incorporada en las Obras Colectivas.

b.	Distribuir copias o fonogramas de las Obras, exhibirlas públicamente, ejecutarlas públicamente y/o ponerlas a disposición pública, incluyéndolas como incorporadas en Obras Colectivas, según corresponda.

c.	Distribuir copias de las Obras Derivadas que se generen, exhibirlas públicamente, ejecutarlas públicamente y/o ponerlas a disposición pública.
Los derechos mencionados anteriormente pueden ser ejercidos en todos los medios y formatos, actualmente conocidos o que se inventen en el futuro. Los derechos antes mencionados incluyen el derecho a realizar dichas modificaciones en la medida que sean técnicamente necesarias para ejercer los derechos en otro medio o formatos, pero de otra manera usted no está autorizado para realizar obras derivadas. Todos los derechos no otorgados expresamente por el Licenciante quedan por este medio reservados, incluyendo pero sin limitarse a aquellos que se mencionan en las secciones 4(d) y 4(e).

4. Restricciones.
La licencia otorgada en la anterior Sección 3 está expresamente sujeta y limitada por las siguientes restricciones:

a.	Usted puede distribuir, exhibir públicamente, ejecutar públicamente, o poner a disposición pública la Obra sólo bajo las condiciones de esta Licencia, y Usted debe incluir una copia de esta licencia o del Identificador Universal de Recursos de la misma con cada copia de la Obra que distribuya, exhiba públicamente, ejecute públicamente o ponga a disposición pública. No es posible ofrecer o imponer ninguna condición sobre la Obra que altere o limite las condiciones de esta Licencia o el ejercicio de los derechos de los destinatarios otorgados en este documento. No es posible sublicenciar la Obra. Usted debe mantener intactos todos los avisos que hagan referencia a esta Licencia y a la cláusula de limitación de garantías. Usted no puede distribuir, exhibir públicamente, ejecutar públicamente, o poner a disposición pública la Obra con alguna medida tecnológica que controle el acceso o la utilización de ella de una forma que sea inconsistente con las condiciones de esta Licencia. Lo anterior se aplica a la Obra incorporada a una Obra Colectiva, pero esto no exige que la Obra Colectiva aparte de la obra misma quede sujeta a las condiciones de esta Licencia. Si Usted crea una Obra Colectiva, previo aviso de cualquier Licenciante debe, en la medida de lo posible, eliminar de la Obra Colectiva cualquier referencia a dicho Licenciante o al Autor Original, según lo solicitado por el Licenciante y conforme lo exige la cláusula 4(c).

b.	Usted no puede ejercer ninguno de los derechos que le han sido otorgados en la Sección 3 precedente de modo que estén principalmente destinados o directamente dirigidos a conseguir un provecho comercial o una compensación monetaria privada. El intercambio de la Obra por otras obras protegidas por derechos de autor, ya sea a través de un sistema para compartir archivos digitales (digital file-sharing) o de cualquier otra manera no será considerado como estar destinado principalmente o dirigido directamente a conseguir un provecho comercial o una compensación monetaria privada, siempre que no se realice un pago mediante una compensación monetaria en relación con el intercambio de obras protegidas por el derecho de autor.

c.	Si usted distribuye, exhibe públicamente, ejecuta públicamente o ejecuta públicamente en forma digital la Obra o cualquier Obra Derivada u Obra Colectiva, Usted debe mantener intacta toda la información de derecho de autor de la Obra y proporcionar, de forma razonable según el medio o manera que Usted esté utilizando: (i) el nombre del Autor Original si está provisto (o seudónimo, si fuere aplicable), y/o (ii) el nombre de la parte o las partes que el Autor Original y/o el Licenciante hubieren designado para la atribución (v.g., un instituto patrocinador, editorial, publicación) en la información de los derechos de autor del Licenciante, términos de servicios o de otras formas razonables; el título de la Obra si está provisto; en la medida de lo razonablemente factible y, si está provisto, el Identificador Uniforme de Recursos (Uniform Resource Identifier) que el Licenciante especifica para ser asociado con la Obra, salvo que tal URI no se refiera a la nota sobre los derechos de autor o a la información sobre el licenciamiento de la Obra; y en el caso de una Obra Derivada, atribuir el crédito identificando el uso de la Obra en la Obra Derivada (v.g., "Traducción Francesa de la Obra del Autor Original," o "Guión Cinematográfico basado en la Obra original del Autor Original"). Tal crédito puede ser implementado de cualquier forma razonable; en el caso, sin embargo, de Obras Derivadas u Obras Colectivas, tal crédito aparecerá, como mínimo, donde aparece el crédito de cualquier otro autor comparable y de una manera, al menos, tan destacada como el crédito de otro autor comparable.

d.	Para evitar toda confusión, el Licenciante aclara que, cuando la obra es una composición musical:

i.	Regalías por interpretación y ejecución bajo licencias generales. El Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública o la ejecución pública digital de la obra y de recolectar, sea individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, SAYCO), las regalías por la ejecución pública o por la ejecución pública digital de la obra (por ejemplo Webcast) licenciada bajo licencias generales, si la interpretación o ejecución de la obra está primordialmente orientada por o dirigida a la obtención de una ventaja comercial o una compensación monetaria privada.

ii.	Regalías por Fonogramas. El Licenciante se reserva el derecho exclusivo de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, los consagrados por la SAYCO), una agencia de derechos musicales o algún agente designado, las regalías por cualquier fonograma que Usted cree a partir de la obra (“versión cover”) y distribuya, en los términos del régimen de derechos de autor, si la creación o distribución de esa versión cover está primordialmente destinada o dirigida a obtener una ventaja comercial o una compensación monetaria privada.

e.	Gestión de Derechos de Autor sobre Interpretaciones y Ejecuciones Digitales (WebCasting). Para evitar toda confusión, el Licenciante aclara que, cuando la obra sea un fonograma, el Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública digital de la obra (por ejemplo, webcast) y de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, ACINPRO), las regalías por la ejecución pública digital de la obra (por ejemplo, webcast), sujeta a las disposiciones aplicables del régimen de Derecho de Autor, si esta ejecución pública digital está primordialmente dirigida a obtener una ventaja comercial o una compensación monetaria privada.

5. Representaciones, Garantías y Limitaciones de Responsabilidad.
A MENOS QUE LAS PARTES LO ACORDARAN DE OTRA FORMA POR ESCRITO, EL LICENCIANTE OFRECE LA OBRA (EN EL ESTADO EN EL QUE SE ENCUENTRA) “TAL CUAL”, SIN BRINDAR GARANTÍAS DE CLASE ALGUNA RESPECTO DE LA OBRA, YA SEA EXPRESA, IMPLÍCITA, LEGAL O CUALQUIERA OTRA, INCLUYENDO, SIN LIMITARSE A ELLAS, GARANTÍAS DE TITULARIDAD, COMERCIABILIDAD, ADAPTABILIDAD O ADECUACIÓN A PROPÓSITO DETERMINADO, AUSENCIA DE INFRACCIÓN, DE AUSENCIA DE DEFECTOS LATENTES O DE OTRO TIPO, O LA PRESENCIA O AUSENCIA DE ERRORES, SEAN O NO DESCUBRIBLES (PUEDAN O NO SER ESTOS DESCUBIERTOS). ALGUNAS JURISDICCIONES NO PERMITEN LA EXCLUSIÓN DE GARANTÍAS IMPLÍCITAS, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.

6. Limitación de responsabilidad.
A MENOS QUE LO EXIJA EXPRESAMENTE LA LEY APLICABLE, EL LICENCIANTE NO SERÁ RESPONSABLE ANTE USTED POR DAÑO ALGUNO, SEA POR RESPONSABILIDAD EXTRACONTRACTUAL, PRECONTRACTUAL O CONTRACTUAL, OBJETIVA O SUBJETIVA, SE TRATE DE DAÑOS MORALES O PATRIMONIALES, DIRECTOS O INDIRECTOS, PREVISTOS O IMPREVISTOS PRODUCIDOS POR EL USO DE ESTA LICENCIA O DE LA OBRA, AUN CUANDO EL LICENCIANTE HAYA SIDO ADVERTIDO DE LA POSIBILIDAD DE DICHOS DAÑOS. ALGUNAS LEYES NO PERMITEN LA EXCLUSIÓN DE CIERTA RESPONSABILIDAD, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.

7. Término.

a.	Esta Licencia y los derechos otorgados en virtud de ella terminarán automáticamente si Usted infringe alguna condición establecida en ella. Sin embargo, los individuos o entidades que han recibido Obras Derivadas o Colectivas de Usted de conformidad con esta Licencia, no verán terminadas sus licencias, siempre que estos individuos o entidades sigan cumpliendo íntegramente las condiciones de estas licencias. Las Secciones 1, 2, 5, 6, 7, y 8 subsistirán a cualquier terminación de esta Licencia.

b.	Sujeta a las condiciones y términos anteriores, la licencia otorgada aquí es perpetua (durante el período de vigencia de los derechos de autor de la obra). No obstante lo anterior, el Licenciante se reserva el derecho a publicar y/o estrenar la Obra bajo condiciones de licencia diferentes o a dejar de distribuirla en los términos de esta Licencia en cualquier momento; en el entendido, sin embargo, que esa elección no servirá para revocar esta licencia o que deba ser otorgada , bajo los términos de esta licencia), y esta licencia continuará en pleno vigor y efecto a menos que sea terminada como se expresa atrás. La Licencia revocada continuará siendo plenamente vigente y efectiva si no se le da término en las condiciones indicadas anteriormente.

8. Varios.

a.	Cada vez que Usted distribuya o ponga a disposición pública la Obra o una Obra Colectiva, el Licenciante ofrecerá al destinatario una licencia en los mismos términos y condiciones que la licencia otorgada a Usted bajo esta Licencia.

b.	Si alguna disposición de esta Licencia resulta invalidada o no exigible, según la legislación vigente, esto no afectará ni la validez ni la aplicabilidad del resto de condiciones de esta Licencia y, sin acción adicional por parte de los sujetos de este acuerdo, aquélla se entenderá reformada lo mínimo necesario para hacer que dicha disposición sea válida y exigible.

c.	Ningún término o disposición de esta Licencia se estimará renunciada y ninguna violación de ella será consentida a menos que esa renuncia o consentimiento sea otorgado por escrito y firmado por la parte que renuncie o consienta.

d.	Esta Licencia refleja el acuerdo pleno entre las partes respecto a la Obra aquí licenciada. No hay arreglos, acuerdos o declaraciones respecto a la Obra que no estén especificados en este documento. El Licenciante no se verá limitado por ninguna disposición adicional que pueda surgir en alguna comunicación emanada de Usted. Esta Licencia no puede ser modificada sin el consentimiento mutuo por escrito del Licenciante y Usted.
 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