This page has only limited features, please log in for full access.

Dr. Fernando Almazan Toral
Department of Molecular and Cell Biology, National Center for Biotechnology, Darwin 3, 28049 Madrid, Spain

Basic Info


Research Keywords & Expertise

0 Vaccines
0 Virology
0 antivirals
0 Zika virus
0 virus-host interaction

Fingerprints

CoronaVirus
Vaccines
Zika virus
antivirals
virus-host interaction
flavivirus

Honors and Awards

The user has no records in this section


Career Timeline

The user has no records in this section.


Short Biography

The user biography is not available.
Following
Followers
Co Authors
The list of users this user is following is empty.
Following: 0 users

Feed

Journal article
Published: 18 September 2020 in Viruses
Reads 0
Downloads 0

Zika virus (ZIKV) was identified in 1947 in the Zika forest of Uganda and it has emerged recently as a global health threat, with recurring outbreaks and its associations with congenital microcephaly through maternal fetal transmission and Guillain-Barré syndrome. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or antivirals to treat ZIKV infections, which underscores an urgent medical need for the development of disease intervention strategies to treat ZIKV infection and associated disease. Drug repurposing offers various advantages over developing an entirely new drug by significantly reducing the timeline and resources required to advance a candidate antiviral into the clinic. Screening the ReFRAME library, we identified ten compounds with antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV). Moreover, we showed the ability of these ten compounds to inhibit influenza A and B virus infections, supporting their broad-spectrum antiviral activity. In this study, we further evaluated the broad-spectrum antiviral activity of the ten identified compounds by testing their activity against ZIKV. Among the ten compounds, Azaribine (SI-MTT = 146.29), AVN-944 (SI-MTT = 278.16), and Brequinar (SI-MTT = 157.42) showed potent anti-ZIKV activity in post-treatment therapeutic conditions. We also observed potent anti-ZIKV activity for Mycophenolate mofetil (SI-MTT = 20.51), Mycophenolic acid (SI-MTT = 36.33), and AVN-944 (SI-MTT = 24.51) in pre-treatment prophylactic conditions and potent co-treatment inhibitory activity for Obatoclax (SI-MTT = 60.58), Azaribine (SI-MTT = 91.51), and Mycophenolate mofetil (SI-MTT = 73.26) in co-treatment conditions. Importantly, the inhibitory effect of these compounds was strain independent, as they similarly inhibited ZIKV strains from both African and Asian/American lineages. Our results support the broad-spectrum antiviral activity of these ten compounds and suggest their use for the development of antiviral treatment options of ZIKV infection.

ACS Style

Desarey Morales Vasquez; Jun-Gyu Park; Ginés Ávila-Pérez; Aitor Nogales; Juan Carlos De La Torre; Fernando Almazan; Luis Martinez-Sobrido. Identification of Inhibitors of ZIKV Replication. Viruses 2020, 12, 1041 .

AMA Style

Desarey Morales Vasquez, Jun-Gyu Park, Ginés Ávila-Pérez, Aitor Nogales, Juan Carlos De La Torre, Fernando Almazan, Luis Martinez-Sobrido. Identification of Inhibitors of ZIKV Replication. Viruses. 2020; 12 (9):1041.

Chicago/Turabian Style

Desarey Morales Vasquez; Jun-Gyu Park; Ginés Ávila-Pérez; Aitor Nogales; Juan Carlos De La Torre; Fernando Almazan; Luis Martinez-Sobrido. 2020. "Identification of Inhibitors of ZIKV Replication." Viruses 12, no. 9: 1041.

Journal article
Published: 16 January 2020 in Scientific Reports
Reads 0
Downloads 0

Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that has been known to circulate for decades causing mild febrile illness. The more recent ZIKV outbreaks in the Americas and the Caribbean associated with congenital malformations and Guillain-Barré syndrome in adults have placed public health officials in high alert and highlight the significant impact of ZIKV on human health. New technologies to study the biology of ZIKV and to develop more effective prevention options are highly desired. In this study we demonstrate that direct delivery in mice of an infectious ZIKV cDNA clone allows the rescue of recombinant (r)ZIKV in vivo. A bacterial artificial chromosome containing the sequence of ZIKV strain Paraiba/2015 under the control of the cytomegalovirus promoter was complexed with a commercial transfection reagent and administrated using different routes in type-I interferon receptor deficient A129 mice. Clinical signs and death associated with ZIKV viremia were observed in mice. The rZIKV recovered from these mice remained fully virulent in a second passage in mice. Interestingly, infectious rZIKV was also recovered after intraperitoneal inoculation of the rZIKV cDNA in the absence of transfection reagent. Further expanding these studies, we demonstrate that a single intraperitoneal inoculation of a cDNA clone encoding an attenuated rZIKV was safe, highly immunogenic, and provided full protection against lethal ZIKV challenge. This novel in vivo reverse genetics method is a potentially suitable delivery platform for the study of wild-type and live-attenuated ZIKV devoid of confounding factors typical associated with in vitro systems. Moreover, our results open the possibility of employing similar in vivo reverse genetic approaches for the generation of other viruses and, therefore, change the way we will use reverse genetics in the future.

ACS Style

Gines Ávila-Pérez; Aitor Nogales; Jun-Gyu Park; Desarey Morales Vasquez; David A. Dean; Michael Barravecchia; Daniel Perez; Fernando Almazán; Luis Martínez-Sobrido. In vivo rescue of recombinant Zika virus from an infectious cDNA clone and its implications in vaccine development. Scientific Reports 2020, 10, 1 -14.

AMA Style

Gines Ávila-Pérez, Aitor Nogales, Jun-Gyu Park, Desarey Morales Vasquez, David A. Dean, Michael Barravecchia, Daniel Perez, Fernando Almazán, Luis Martínez-Sobrido. In vivo rescue of recombinant Zika virus from an infectious cDNA clone and its implications in vaccine development. Scientific Reports. 2020; 10 (1):1-14.

Chicago/Turabian Style

Gines Ávila-Pérez; Aitor Nogales; Jun-Gyu Park; Desarey Morales Vasquez; David A. Dean; Michael Barravecchia; Daniel Perez; Fernando Almazán; Luis Martínez-Sobrido. 2020. "In vivo rescue of recombinant Zika virus from an infectious cDNA clone and its implications in vaccine development." Scientific Reports 10, no. 1: 1-14.

Journal article
Published: 27 December 2019 in Scientific Reports
Reads 0
Downloads 0

Zika virus (ZIKV) infection is currently one of the major concerns in human public health due to its association with neurological disorders. Intensive effort has been implemented for the treatment of ZIKV, however there are not currently approved vaccines or antivirals available to combat ZIKV infection. In this sense, the identification of virulence factors associated with changes in ZIKV virulence could help to develop safe and effective countermeasures to treat ZIKV or to prevent future outbreaks. Here, we have compared the virulence of two related ZIKV strains from the recent outbreak in Brazil (2015), Rio Grande do Norte Natal (RGN) and Paraiba. In spite of both viruses being identified in the same period of time and region, significant differences in virulence and replication were observed using a validated mouse model of ZIKV infection. While ZIKV-RGN has a 50% mouse lethal dose (MLD50) of ~105 focus forming units (FFUs), ZIKV-Paraiba infection resulted in 100% of lethality with less than 10 FFUs. Combining deep-sequencing analysis and our previously described infectious ZIKV-RGN cDNA clone, we identified a natural polymorphism in the non-structural protein 2 A (NS2A) that increase the virulence of ZIKV. Moreover, results demonstrate that the single amino acid alanine to valine substitution at position 117 (A117V) in the NS2A was sufficient to convert the attenuated rZIKV-RGN in a virulent Paraiba-like virus (MLD50 < 10 FFU). The mechanism of action was also evaluated and data indicate that substitution A117V in ZIKV NS2A protein reduces host innate immune responses and viral-induced apoptosis in vitro. Therefore, amino acid substitution A117V in ZIKV NS2A could be used as a genetic risk-assessment marker for future ZIKV outbreaks.

ACS Style

Gines Ávila-Pérez; Aitor Nogales; Jun-Gyu Park; Silvia Márquez-Jurado; Francisco J. Iborra; Fernando Almazan; Luis Martínez-Sobrido. A natural polymorphism in Zika virus NS2A protein responsible of virulence in mice. Scientific Reports 2019, 9, 1 -17.

AMA Style

Gines Ávila-Pérez, Aitor Nogales, Jun-Gyu Park, Silvia Márquez-Jurado, Francisco J. Iborra, Fernando Almazan, Luis Martínez-Sobrido. A natural polymorphism in Zika virus NS2A protein responsible of virulence in mice. Scientific Reports. 2019; 9 (1):1-17.

Chicago/Turabian Style

Gines Ávila-Pérez; Aitor Nogales; Jun-Gyu Park; Silvia Márquez-Jurado; Francisco J. Iborra; Fernando Almazan; Luis Martínez-Sobrido. 2019. "A natural polymorphism in Zika virus NS2A protein responsible of virulence in mice." Scientific Reports 9, no. 1: 1-17.

Editorial
Published: 14 March 2019 in Viruses
Reads 0
Downloads 0

Zika virus (ZIKV) is an emerging mosquito-borne member of the Flaviviridae family that has historically been known to cause sporadic outbreaks, associated with a mild febrile illness, in Africa and Southeast Asia

ACS Style

Luis Martinez-Sobrido; Fernando Almazán. New Advances on Zika Virus Research. Viruses 2019, 11, 258 .

AMA Style

Luis Martinez-Sobrido, Fernando Almazán. New Advances on Zika Virus Research. Viruses. 2019; 11 (3):258.

Chicago/Turabian Style

Luis Martinez-Sobrido; Fernando Almazán. 2019. "New Advances on Zika Virus Research." Viruses 11, no. 3: 258.

Review
Published: 31 October 2018 in Viruses
Reads 0
Downloads 0

Zika virus (ZIKV) is an emergent mosquito-borne member of the Flaviviridae family that was responsible for a recent epidemic in the Americas. ZIKV has been associated with severe clinical complications, including neurological disorder such as Guillain-Barré syndrome in adults and severe fetal abnormalities and microcephaly in newborn infants. Given the significance of these clinical manifestations, the development of tools and reagents to study the pathogenesis of ZIKV and to develop new therapeutic options are urgently needed. In this respect, the implementation of reverse genetic techniques has allowed the direct manipulation of the viral genome to generate recombinant (r)ZIKVs, which have provided investigators with powerful systems to answer important questions about the biology of ZIKV, including virus-host interactions, the mechanism of transmission and pathogenesis or the function of viral proteins. In this review, we will summarize the different reverse genetic strategies that have been implemented, to date, for the generation of rZIKVs and the applications of these platforms for the development of replicon systems or reporter-expressing viruses.

ACS Style

Ginés Ávila-Pérez; Aitor Nogales; Verónica Martín; Fernando Almazán; Luis Martínez-Sobrido. Reverse Genetic Approaches for the Generation of Recombinant Zika Virus. Viruses 2018, 10, 597 .

AMA Style

Ginés Ávila-Pérez, Aitor Nogales, Verónica Martín, Fernando Almazán, Luis Martínez-Sobrido. Reverse Genetic Approaches for the Generation of Recombinant Zika Virus. Viruses. 2018; 10 (11):597.

Chicago/Turabian Style

Ginés Ávila-Pérez; Aitor Nogales; Verónica Martín; Fernando Almazán; Luis Martínez-Sobrido. 2018. "Reverse Genetic Approaches for the Generation of Recombinant Zika Virus." Viruses 10, no. 11: 597.

Journal article
Published: 07 October 2018 in Viruses
Reads 0
Downloads 0

The recent outbreaks of Zika virus (ZIKV), its association with Guillain–Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies.

ACS Style

Silvia Márquez-Jurado; Aitor Nogales; Ginés Ávila-Pérez; Francisco J. Iborra; Luis Martínez-Sobrido; Fernando Almazán. An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo. Viruses 2018, 10, 547 .

AMA Style

Silvia Márquez-Jurado, Aitor Nogales, Ginés Ávila-Pérez, Francisco J. Iborra, Luis Martínez-Sobrido, Fernando Almazán. An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo. Viruses. 2018; 10 (10):547.

Chicago/Turabian Style

Silvia Márquez-Jurado; Aitor Nogales; Ginés Ávila-Pérez; Francisco J. Iborra; Luis Martínez-Sobrido; Fernando Almazán. 2018. "An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo." Viruses 10, no. 10: 547.

Journal article
Published: 26 January 2018 in Nature Communications
Reads 0
Downloads 0

Fractional killing is the main cause of tumour resistance to chemotherapy. This phenomenon is observed even in genetically identical cancer cells in homogeneous microenvironments. To understand this variable resistance, here we investigate the individual responses to TRAIL in a clonal population of HeLa cells using live-cell microscopy and computational modelling. We show that the cellular mitochondrial content determines the apoptotic fate and modulates the time to death, cells with higher mitochondrial content are more prone to die. We find that all apoptotic protein levels are modulated by the mitochondrial content. Modelling the apoptotic network, we demonstrate that these correlations, and especially the differential control of anti- and pro-apoptotic protein pairs, confer mitochondria a powerful discriminatory capacity of apoptotic fate. We find a similar correlation between the mitochondria and apoptotic proteins in colon cancer biopsies. Our results reveal a different role of mitochondria in apoptosis as the global regulator of apoptotic protein expression. It is unclear what causes variation in cell death in response to chemotherapy. Here, the authors show that cellular mitochondrial content modulates apoptotic protein levels, which in turn regulates response to agents such as TRAIL.

ACS Style

Silvia Márquez-Jurado; Juan Díaz-Colunga; Ricardo Pires das Neves; Antonio Martinez-Lorente; Fernando Almazan; Raúl Guantes; Francisco J. Iborra. Mitochondrial levels determine variability in cell death by modulating apoptotic gene expression. Nature Communications 2018, 9, 1 -11.

AMA Style

Silvia Márquez-Jurado, Juan Díaz-Colunga, Ricardo Pires das Neves, Antonio Martinez-Lorente, Fernando Almazan, Raúl Guantes, Francisco J. Iborra. Mitochondrial levels determine variability in cell death by modulating apoptotic gene expression. Nature Communications. 2018; 9 (1):1-11.

Chicago/Turabian Style

Silvia Márquez-Jurado; Juan Díaz-Colunga; Ricardo Pires das Neves; Antonio Martinez-Lorente; Fernando Almazan; Raúl Guantes; Francisco J. Iborra. 2018. "Mitochondrial levels determine variability in cell death by modulating apoptotic gene expression." Nature Communications 9, no. 1: 1-11.

Erratum
Published: 20 September 2017 in Veterinary Research
Reads 0
Downloads 0

After publication of the article [1], it has been brought to our attention that an acknowledgement has been omitted from the original article. The authors would like to include the following, The authors also thank Prof. En-Min Zhou (Northwest A&F University) and his laboratory for technical support.”

ACS Style

Chengbao Wang; Han Meng; Yujin Gao; Hui Gao; Kangkang Guo; Fernando Almazan; Isabel Sola; Luis Enjuanes; Yanming Zhang; Levon Abrahamyan. Erratum to: Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus. Veterinary Research 2017, 48, 54 .

AMA Style

Chengbao Wang, Han Meng, Yujin Gao, Hui Gao, Kangkang Guo, Fernando Almazan, Isabel Sola, Luis Enjuanes, Yanming Zhang, Levon Abrahamyan. Erratum to: Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus. Veterinary Research. 2017; 48 (1):54.

Chicago/Turabian Style

Chengbao Wang; Han Meng; Yujin Gao; Hui Gao; Kangkang Guo; Fernando Almazan; Isabel Sola; Luis Enjuanes; Yanming Zhang; Levon Abrahamyan. 2017. "Erratum to: Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus." Veterinary Research 48, no. 1: 54.

Short report
Published: 10 August 2017 in Veterinary Research
Reads 0
Downloads 0

In order to gain insight into the role of the transcription regulatory sequences (TRSs) in the regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus (PRRSV), the enhanced green fluorescent protein (EGFP) gene, under the control of the different structural gene TRSs, was inserted between the N gene and 3′-UTR of the PRRSV genome and EGFP expression was analyzed for each TRS. TRSs of all the studied structural genes of PRRSV positively modulated EGFP expression at different levels. Among the TRSs analyzed, those of GP2, GP5, M, and N genes highly enhanced EGFP expression without altering replication of PRRSV. These data indicated that structural gene TRSs could be an extremely useful tool for foreign gene expression using PRRSV as a vector.

ACS Style

Chengbao Wang; Han Meng; Yujin Gao; Hui Gao; Kangkang Guo; Fernando Almazan; Isabel Sola; Luis Enjuanes; Yanming Zhang; Levon Abrahamyan. Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus. Veterinary Research 2017, 48, 1 -7.

AMA Style

Chengbao Wang, Han Meng, Yujin Gao, Hui Gao, Kangkang Guo, Fernando Almazan, Isabel Sola, Luis Enjuanes, Yanming Zhang, Levon Abrahamyan. Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus. Veterinary Research. 2017; 48 (1):1-7.

Chicago/Turabian Style

Chengbao Wang; Han Meng; Yujin Gao; Hui Gao; Kangkang Guo; Fernando Almazan; Isabel Sola; Luis Enjuanes; Yanming Zhang; Levon Abrahamyan. 2017. "Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus." Veterinary Research 48, no. 1: 1-7.

Short communication
Published: 01 January 2016 in Intervirology
Reads 0
Downloads 0

Dengue viruses (DENV) have become the most important arthropod-borne viruses, causing dengue and severe dengue fever in at least 50-100 million cases each year, mainly in tropical and subtropical countries. During recent years, important advances in the molecular biology concerning the life cycle of these viruses have allowed the manipulation and generation of recombinant viruses and replicons with multiple applications, mainly in viral biology and the screening of antiviral compounds. In the present study, we describe the construction of an enhanced green fluorescent protein-bearing DENV replicon under the control of the cytomegalovirus immediate early promoter. Following a rational in silico design and cloning by standard molecular biology techniques, a reporter DENV-2 replicon and a replication-deficient mutant were constructed, and characterized by confocal microscopy and real-time RT-PCR. The results showed successful transcription, translation, and autonomous viral RNA replication of the DENV replicon from its DNA clone. This novel DENV replicon will allow the study of viral replication and testing of antiviral candidates without the need for in vitro transcription.

ACS Style

Jose A. Usme-Ciro; Jaime A. Lopera; Diego Alejandro Alvarez-Diaz; Luis Enjuanes; Fernando Almazán. Generation of a DNA-Launched Reporter Replicon Based on Dengue Virus Type 2 as a Multipurpose Platform. Intervirology 2016, 59, 275 -282.

AMA Style

Jose A. Usme-Ciro, Jaime A. Lopera, Diego Alejandro Alvarez-Diaz, Luis Enjuanes, Fernando Almazán. Generation of a DNA-Launched Reporter Replicon Based on Dengue Virus Type 2 as a Multipurpose Platform. Intervirology. 2016; 59 (5-6):275-282.

Chicago/Turabian Style

Jose A. Usme-Ciro; Jaime A. Lopera; Diego Alejandro Alvarez-Diaz; Luis Enjuanes; Fernando Almazán. 2016. "Generation of a DNA-Launched Reporter Replicon Based on Dengue Virus Type 2 as a Multipurpose Platform." Intervirology 59, no. 5-6: 275-282.

Review
Published: 09 November 2015 in Annual Review of Virology
Reads 0
Downloads 0

Replication of the coronavirus genome requires continuous RNA synthesis, whereas transcription is a discontinuous process unique among RNA viruses. Transcription includes a template switch during the synthesis of subgenomic negative-strand RNAs to add a copy of the leader sequence. Coronavirus transcription is regulated by multiple factors, including the extent of base-pairing between transcription-regulating sequences of positive and negative polarity, viral and cell protein–RNA binding, and high-order RNA-RNA interactions. Coronavirus RNA synthesis is performed by a replication-transcription complex that includes viral and cell proteins that recognize cis-acting RNA elements mainly located in the highly structured 5′ and 3′ untranslated regions. In addition to many viral nonstructural proteins, the presence of cell nuclear proteins and the viral nucleocapsid protein increases virus amplification efficacy. Coronavirus RNA synthesis is connected with the formation of double-membrane vesicles and convoluted membranes. Coronaviruses encode proofreading machinery, unique in the RNA virus world, to ensure the maintenance of their large genome size.

ACS Style

Isabel Sola; Fernando Almazan; Sonia Zuñiga; Luis Enjuanes. Continuous and Discontinuous RNA Synthesis in Coronaviruses. Annual Review of Virology 2015, 2, 265 -288.

AMA Style

Isabel Sola, Fernando Almazan, Sonia Zuñiga, Luis Enjuanes. Continuous and Discontinuous RNA Synthesis in Coronaviruses. Annual Review of Virology. 2015; 2 (1):265-288.

Chicago/Turabian Style

Isabel Sola; Fernando Almazan; Sonia Zuñiga; Luis Enjuanes. 2015. "Continuous and Discontinuous RNA Synthesis in Coronaviruses." Annual Review of Virology 2, no. 1: 265-288.

Journal article
Published: 01 May 2015 in mBio
Reads 0
Downloads 0

A 32-nucleotide (nt) RNA motif located at the 3′ end of the transmissible gastroenteritis coronavirus (TGEV) genome was found to specifically interact with the host proteins glutamyl-prolyl-tRNA synthetase (EPRS) and arginyl-tRNA synthetase (RRS). This RNA motif has high homology in sequence and secondary structure with the gamma interferon-activated inhibitor of translation (GAIT) element, which is located at the 3′ end of several mRNAs encoding proinflammatory proteins. The GAIT element is involved in the translation silencing of these mRNAs through its interaction with the GAIT complex (EPRS, heterogeneous nuclear ribonucleoprotein Q, ribosomal protein L13a, and glyceraldehyde 3-phosphate dehydrogenase) to favor the resolution of inflammation. Interestingly, we showed that the viral RNA motif bound the GAIT complex and inhibited the in vitro translation of a chimeric mRNA containing this RNA motif. To our knowledge, this is the first GAIT-like motif described in a positive RNA virus. To test the functional role of the GAIT-like RNA motif during TGEV infection, a recombinant coronavirus harboring mutations in this motif was engineered and characterized. Mutations of the GAIT-like RNA motif did not affect virus growth in cell cultures. However, an exacerbated innate immune response, mediated by the melanoma differentiation-associated gene 5 (MDA5) pathway, was observed in cells infected with the mutant virus compared with the response observed in cells infected with the parental virus. Furthermore, the mutant virus was more sensitive to beta interferon than the parental virus. All together, these data strongly suggested that the viral GAIT-like RNA motif modulates the host innate immune response. IMPORTANCE The innate immune response is the first line of antiviral defense that culminates with the synthesis of interferon and proinflammatory cytokines to limit virus replication. Coronaviruses encode several proteins that interfere with the innate immune response at different levels, but to date, no viral RNA counteracting antiviral response has been described. In this work, we have characterized a 32-nt RNA motif located at the 3′ end of the TGEV genome that specifically interacted with EPRS and RRS. This RNA motif presented high homology with the GAIT element, involved in the modulation of the inflammatory response. Moreover, the disruption of the viral GAIT-like RNA motif led to an exacerbated innate immune response triggered by MDA5, indicating that the GAIT-like RNA motif counteracts the host innate immune response. These novel findings may be of relevance for other coronaviruses and could serve as the basis for the development of novel antiviral strategies.

ACS Style

Silvia Márquez-Jurado; Aitor Nogales; Sonia Zuñiga; Luis Enjuanes; Fernando Almazan. Identification of a Gamma Interferon-Activated Inhibitor of Translation-Like RNA Motif at the 3′ End of the Transmissible Gastroenteritis Coronavirus Genome Modulating Innate Immune Response. mBio 2015, 6, e00105 -e00105.

AMA Style

Silvia Márquez-Jurado, Aitor Nogales, Sonia Zuñiga, Luis Enjuanes, Fernando Almazan. Identification of a Gamma Interferon-Activated Inhibitor of Translation-Like RNA Motif at the 3′ End of the Transmissible Gastroenteritis Coronavirus Genome Modulating Innate Immune Response. mBio. 2015; 6 (2):e00105-e00105.

Chicago/Turabian Style

Silvia Márquez-Jurado; Aitor Nogales; Sonia Zuñiga; Luis Enjuanes; Fernando Almazan. 2015. "Identification of a Gamma Interferon-Activated Inhibitor of Translation-Like RNA Motif at the 3′ End of the Transmissible Gastroenteritis Coronavirus Genome Modulating Innate Immune Response." mBio 6, no. 2: e00105-e00105.

Protocol
Published: 12 February 2015 in Methods in Molecular Biology
Reads 0
Downloads 0

The large size of the coronavirus (CoV) genome (around 30 kb) and the instability in bacteria of plasmids carrying CoV replicase sequences represent serious restrictions for the development of CoV infectious clones using reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these problems, several approaches have been established in the last 13 years. Here we describe the engineering of CoV full-length cDNA clones as bacterial artificial chromosomes (BACs), using the Middle East respiratory syndrome CoV (MERS-CoV) as a model.

ACS Style

Fernando Almazán; Silvia Márquez-Jurado; Aitor Nogales; Luis Enjuanes. Engineering Infectious cDNAs of Coronavirus as Bacterial Artificial Chromosomes. Methods in Molecular Biology 2015, 1282, 135 -152.

AMA Style

Fernando Almazán, Silvia Márquez-Jurado, Aitor Nogales, Luis Enjuanes. Engineering Infectious cDNAs of Coronavirus as Bacterial Artificial Chromosomes. Methods in Molecular Biology. 2015; 1282 ():135-152.

Chicago/Turabian Style

Fernando Almazán; Silvia Márquez-Jurado; Aitor Nogales; Luis Enjuanes. 2015. "Engineering Infectious cDNAs of Coronavirus as Bacterial Artificial Chromosomes." Methods in Molecular Biology 1282, no. : 135-152.

Journal article
Published: 26 September 2014 in Virus Research
Reads 0
Downloads 0

Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, enteric, hepatic, and central nervous system diseases. The large size of the CoV genome and the instability of some CoV replicase gene sequences during its propagation in bacteria, represent serious obstacles for the development of reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these limitations, several alternatives to more conventional plasmid-based approaches have been established in the last 13 years. In this report, we briefly review and discuss the different reverse genetic systems developed for CoVs, paying special attention to the severe acute respiratory syndrome CoV (SARS-CoV).

ACS Style

Fernando Almazán; Isabel Sola; Sonia Zuñiga; Silvia Marquez-Jurado; Lucia Morales; Martina Becares; Luis Enjuanes. Reprint of: Coronavirus reverse genetic systems: Infectious clones and replicons. Virus Research 2014, 194, 67 -75.

AMA Style

Fernando Almazán, Isabel Sola, Sonia Zuñiga, Silvia Marquez-Jurado, Lucia Morales, Martina Becares, Luis Enjuanes. Reprint of: Coronavirus reverse genetic systems: Infectious clones and replicons. Virus Research. 2014; 194 ():67-75.

Chicago/Turabian Style

Fernando Almazán; Isabel Sola; Sonia Zuñiga; Silvia Marquez-Jurado; Lucia Morales; Martina Becares; Luis Enjuanes. 2014. "Reprint of: Coronavirus reverse genetic systems: Infectious clones and replicons." Virus Research 194, no. : 67-75.

Review
Published: 12 June 2014 in Virus Research
Reads 0
Downloads 0

Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, enteric, hepatic, and central nervous system diseases. The large size of the CoV genome and the instability of some CoV replicase gene sequences during its propagation in bacteria, represent serious obstacles for the development of reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these limitations, several alternatives to more conventional plasmid-based approaches have been established in the last 13 years. In this report, we briefly review and discuss the different reverse genetic systems developed for CoVs, paying special attention to the severe acute respiratory syndrome CoV (SARS-CoV).

ACS Style

Fernando Almazan; Isabel Sola; Sonia Zuñiga; Silvia Márquez-Jurado; Lucia Morales; Martina Becares; Luis Enjuanes. Coronavirus reverse genetic systems: Infectious clones and replicons. Virus Research 2014, 189, 262 -270.

AMA Style

Fernando Almazan, Isabel Sola, Sonia Zuñiga, Silvia Márquez-Jurado, Lucia Morales, Martina Becares, Luis Enjuanes. Coronavirus reverse genetic systems: Infectious clones and replicons. Virus Research. 2014; 189 ():262-270.

Chicago/Turabian Style

Fernando Almazan; Isabel Sola; Sonia Zuñiga; Silvia Márquez-Jurado; Lucia Morales; Martina Becares; Luis Enjuanes. 2014. "Coronavirus reverse genetic systems: Infectious clones and replicons." Virus Research 189, no. : 262-270.

Journal article
Published: 14 December 2013 in Virus Research
Reads 0
Downloads 0

Major progress in Dengue virus (DENV) biology has resulted from the use of infectious clones obtained through reverse genetics. The construction of these clones is commonly based on high- or low-copy number plasmids, yeast artificial chromosomes, yeast-Escherichia coli shuttle vectors, and bacterial artificial chromosomes (BACs). Prokaryotic promoters have consistently been used for the transcription of these clones. The goal of this study was to develop a novel DENV infectious clone in a BAC under the control of the cytomegalovirus immediate-early promoter and to generate a virus with the fusion envelope-green fluorescent protein in an attempt to track virus infection. The transfection of Vero cells with a plasmid encoding the DENV infectious clone facilitated the recovery of infectious particles that increased in titer after serial passages in C6/36 cells. The plaque size and syncytia phenotypes of the recombinant virus were similar to those of the parental virus. Despite the observation of autonomous replication and the detection of low levels of viral genome after two passages, the insertion of green fluorescent protein and Renilla luciferase reporter genes negatively impacted virus rescue. To the best of our knowledge, this is the first study using a DENV infectious clone under the control of the cytomegalovirus promoter to facilitate the recovery of recombinant viruses without the need for in vitro transcription. This novel molecular clone will be useful for establishing the molecular basis of replication, assembly, and pathogenesis, evaluating potential antiviral drugs, and the development of vaccine candidates for attenuated recombinant viruses.

ACS Style

Jose A. Usme-Ciro; Jaime A. Lopera; Luis Enjuanes; Fernando Almazan; Juan C. Gallego-Gomez. Development of a novel DNA-launched dengue virus type 2 infectious clone assembled in a bacterial artificial chromosome. Virus Research 2013, 180, 12 -22.

AMA Style

Jose A. Usme-Ciro, Jaime A. Lopera, Luis Enjuanes, Fernando Almazan, Juan C. Gallego-Gomez. Development of a novel DNA-launched dengue virus type 2 infectious clone assembled in a bacterial artificial chromosome. Virus Research. 2013; 180 ():12-22.

Chicago/Turabian Style

Jose A. Usme-Ciro; Jaime A. Lopera; Luis Enjuanes; Fernando Almazan; Juan C. Gallego-Gomez. 2013. "Development of a novel DNA-launched dengue virus type 2 infectious clone assembled in a bacterial artificial chromosome." Virus Research 180, no. : 12-22.

Journal article
Published: 01 November 2013 in mBio
Reads 0
Downloads 0

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans , indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection. IMPORTANCE Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans , whereas it would only survive a single virus infection cycle in vivo . This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.

ACS Style

Fernando Almazan; Marta L. DeDiego; Isabel Sola; Sonia Zuñiga; Jose L. Nieto-Torres; Silvia Márquez-Jurado; German Andrés; Luis Enjuanes. Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate. mBio 2013, 4, e00650 -13.

AMA Style

Fernando Almazan, Marta L. DeDiego, Isabel Sola, Sonia Zuñiga, Jose L. Nieto-Torres, Silvia Márquez-Jurado, German Andrés, Luis Enjuanes. Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate. mBio. 2013; 4 (5):e00650-13.

Chicago/Turabian Style

Fernando Almazan; Marta L. DeDiego; Isabel Sola; Sonia Zuñiga; Jose L. Nieto-Torres; Silvia Márquez-Jurado; German Andrés; Luis Enjuanes. 2013. "Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate." mBio 4, no. 5: e00650-13.

Review
Published: 01 January 2013 in Virology Journal
Reads 0
Downloads 0

Viral vectors have become the best option for the delivery of therapeutic genes in conventional and RNA interference-based gene therapies. The current viral vectors for the delivery of small regulatory RNAs are based on DNA viruses and retroviruses/lentiviruses. Cytoplasmic RNA viruses have been excluded as viral vectors for RNAi therapy because of the nuclear localization of the microprocessor complex and the potential degradation of the viral RNA genome during the excision of any virus-encoded pre-microRNAs. However, in the last few years, the presence of several species of small RNAs (e.g., virus-derived small interfering RNAs, virus-derived short RNAs, and unusually small RNAs) in animals and cell cultures that are infected with cytoplasmic RNA viruses has suggested the existence of a non-canonical mechanism of microRNA biogenesis. Several studies have been conducted on the tick-borne encephalitis virus and on the Sindbis virus in which microRNA precursors were artificially incorporated and demonstrated the production of mature microRNAs. The ability of these viruses to recruit Drosha to the cytoplasm during infection resulted in the efficient processing of virus-encoded microRNA without the viral genome entering the nucleus. In this review, we discuss the relevance of these findings with an emphasis on the potential use of cytoplasmic RNA viruses as vehicles for the efficient delivery of therapeutic small RNAs.

ACS Style

Jose A Usme-Ciro; Natalia Campillo-Pedroza; Fernando Almazan; Juan C Gallego-Gomez. Cytoplasmic RNA viruses as potential vehicles for the delivery of therapeutic small RNAs. Virology Journal 2013, 10, 185 -185.

AMA Style

Jose A Usme-Ciro, Natalia Campillo-Pedroza, Fernando Almazan, Juan C Gallego-Gomez. Cytoplasmic RNA viruses as potential vehicles for the delivery of therapeutic small RNAs. Virology Journal. 2013; 10 (1):185-185.

Chicago/Turabian Style

Jose A Usme-Ciro; Natalia Campillo-Pedroza; Fernando Almazan; Juan C Gallego-Gomez. 2013. "Cytoplasmic RNA viruses as potential vehicles for the delivery of therapeutic small RNAs." Virology Journal 10, no. 1: 185-185.

Journal article
Published: 01 January 2013 in Veterinary Research
Reads 0
Downloads 0

Here we report the rescue of a recombinant porcine reproductive and respiratory syndrome virus (PRRSV) carrying an enhanced green fluorescent protein (EGFP) reporter gene as a separate transcription unit. A copy of the transcription regulatory sequence for ORF6 (TRS6) was inserted between the N protein and 3′-UTR to drive the transcription of the EGFP gene and yield a general purpose expression vector. Successful recovery of PRRSV was obtained using an RNA polymerase II promoter to drive transcription of the full-length virus genome, which was assembled in a bacterial artificial chromosome (BAC). The recombinant virus showed growth replication characteristics similar to those of the wild-type virus in the infected cells. In addition, the recombinant virus stably expressed EGFP for at least 10 passages. EGFP expression was detected at approximately 10 h post infection by live-cell imaging to follow the virus spread in real time and the infection of neighbouring cells occurred predominantly through cell-to-cell-contact. Finally, the recombinant virus generated was found to be an excellent tool for neutralising antibodies and antiviral compound screening. The newly established reverse genetics system for PRRSV could be a useful tool not only to monitor virus spread and screen for neutralising antibodies and antiviral compounds, but also for fundamental research on the biology of the virus.

ACS Style

Chengbao Wang; Baicheng Huang; Ning Kong; Qiongyi Li; Yuping Ma; Zhijun Li; Jiming Gao; Chong Zhang; Xiangpeng Wang; Chao Liang; Lu Dang; Shuqi Xiao; Yang Mu; Qin Zhao; Yani Sun; Fernando Almazan; Luis Enjuanes; En-Min Zhou. A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit. Veterinary Research 2013, 44, 104 -104.

AMA Style

Chengbao Wang, Baicheng Huang, Ning Kong, Qiongyi Li, Yuping Ma, Zhijun Li, Jiming Gao, Chong Zhang, Xiangpeng Wang, Chao Liang, Lu Dang, Shuqi Xiao, Yang Mu, Qin Zhao, Yani Sun, Fernando Almazan, Luis Enjuanes, En-Min Zhou. A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit. Veterinary Research. 2013; 44 (1):104-104.

Chicago/Turabian Style

Chengbao Wang; Baicheng Huang; Ning Kong; Qiongyi Li; Yuping Ma; Zhijun Li; Jiming Gao; Chong Zhang; Xiangpeng Wang; Chao Liang; Lu Dang; Shuqi Xiao; Yang Mu; Qin Zhao; Yani Sun; Fernando Almazan; Luis Enjuanes; En-Min Zhou. 2013. "A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit." Veterinary Research 44, no. 1: 104-104.

Journal article
Published: 21 March 2012 in Journal of Virology
Reads 0
Downloads 0

The full-length genome of the highly lethal feline infectious peritonitis virus (FIPV) strain DF-2 was sequenced and cloned into a bacterial artificial chromosome (BAC) to study the role of ORF3abc in the FIPV-feline enteric coronavirus (FECV) transition. The reverse genetic system allowed the replacement of the truncated ORF3abc of the original FIPV DF-2 genome with the intact ORF3abc of the canine coronavirus (CCoV) reference strain Elmo/02. The in vitro replication kinetics of these two viruses was studied in CrFK and FCWF-4 cell lines, as well as in feline peripheral blood monocytes. Both viruses showed similar replication kinetics in established cell lines. However, the strain with a full-length ORF3 showed markedly lower replication of more than 2 log10 titers in feline peripheral blood monocytes. Our results suggest that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II FIPV.

ACS Style

Ádám Bálint; Attila Farsang; Zoltán Zádori; Ákos Hornyák; László Dencső; Fernando Almazan; Luis Enjuanes; Sándor Belák. Molecular Characterization of Feline Infectious Peritonitis Virus Strain DF-2 and Studies of the Role of ORF3abc in Viral Cell Tropism. Journal of Virology 2012, 86, 6258 -6267.

AMA Style

Ádám Bálint, Attila Farsang, Zoltán Zádori, Ákos Hornyák, László Dencső, Fernando Almazan, Luis Enjuanes, Sándor Belák. Molecular Characterization of Feline Infectious Peritonitis Virus Strain DF-2 and Studies of the Role of ORF3abc in Viral Cell Tropism. Journal of Virology. 2012; 86 (11):6258-6267.

Chicago/Turabian Style

Ádám Bálint; Attila Farsang; Zoltán Zádori; Ákos Hornyák; László Dencső; Fernando Almazan; Luis Enjuanes; Sándor Belák. 2012. "Molecular Characterization of Feline Infectious Peritonitis Virus Strain DF-2 and Studies of the Role of ORF3abc in Viral Cell Tropism." Journal of Virology 86, no. 11: 6258-6267.